<%BANNER%>

Comparison of Lever Press and Nose Poke Operants for an Analysis of Food Intake and Meal Patterns in Mice

Permanent Link: http://ufdc.ufl.edu/UFE0021862/00001

Material Information

Title: Comparison of Lever Press and Nose Poke Operants for an Analysis of Food Intake and Meal Patterns in Mice
Physical Description: 1 online resource (56 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: leverpress, meal, mice, nosepoke, patterns
Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Much research in the field and in laboratory studies has focused on behavioral economics of food intake in several species. Operants such as lever press, nose poke, or key peck have been used to generate demand functions that express the relationship between the cost of food and the amount of food consumed. There have been very few such studies of motivated food seeking and demand in mice, and none has examined systematically consummatory cost or meal patterns. Using albino (CD1) male mice, the present study compares food intake and meal patterns across a series of ratio consummatory schedules. Two operants, lever press and nose poke, were compared in a between groups design. A closed economy was used in which the mice were in the test chambers for 23 h/day and earned all of their food via the operant under four fixed (FR5, FR10, FR25, FR50), variable (VR10, VR20, VR50) and progressive (PR1.25, PR1.5, PR1.75) ratios. When averaged across all schedules, mice in the nose poke group consumed more food. Mice were run for 4 days at each ratio; there were no systematic differences between the first and last day indicating that behavioral adjustments to schedule changes occurred very rapidly. Meal number significantly differed when two criteria for the definition of 'a meal' (15 and 30 min) were used.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Rowland, Neil E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021862:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021862/00001

Material Information

Title: Comparison of Lever Press and Nose Poke Operants for an Analysis of Food Intake and Meal Patterns in Mice
Physical Description: 1 online resource (56 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: leverpress, meal, mice, nosepoke, patterns
Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Much research in the field and in laboratory studies has focused on behavioral economics of food intake in several species. Operants such as lever press, nose poke, or key peck have been used to generate demand functions that express the relationship between the cost of food and the amount of food consumed. There have been very few such studies of motivated food seeking and demand in mice, and none has examined systematically consummatory cost or meal patterns. Using albino (CD1) male mice, the present study compares food intake and meal patterns across a series of ratio consummatory schedules. Two operants, lever press and nose poke, were compared in a between groups design. A closed economy was used in which the mice were in the test chambers for 23 h/day and earned all of their food via the operant under four fixed (FR5, FR10, FR25, FR50), variable (VR10, VR20, VR50) and progressive (PR1.25, PR1.5, PR1.75) ratios. When averaged across all schedules, mice in the nose poke group consumed more food. Mice were run for 4 days at each ratio; there were no systematic differences between the first and last day indicating that behavioral adjustments to schedule changes occurred very rapidly. Meal number significantly differed when two criteria for the definition of 'a meal' (15 and 30 min) were used.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Rowland, Neil E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021862:00001


This item has the following downloads:


Full Text





COMPARISON OF LEVER PRESS AND NOSE POKE OPERANTS FOR AN ANALYSIS OF
FOOD INTAKE AND MEAL PATTERNS IN MICE





















By

DENIZ ATALAYER


A THESIS PRESENTED HERE TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF
FLORIDA INT PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2008



































O 2008 Deniz Atalayer





































To my mom, dad, and my advisor Dr. Neil E. Rowland












TABLE OF CONTENTS


page


LIST OF TABLES .........__... ......._. ...............5....


LIST OF FIGURES .............. ...............6.....


AB S TRAC T ......_ ................. ............_........7


CHAPTER


1 INTRODUCTION ................. ...............8.......... ......


2 MATERIALS AND METHODS .............. ...............19....


Subj ects ................. ...............19.................
Apparatus ................. ...............19.................
Procedure ................. .... ...............2

Analyses and Data Acquisition............... ..............2

3 RE SULT S .............. ...............23....


FR Schedules .............. ...............23....
VR Schedules............... ...............2
PR Schedules ........................... ... ............2

Comparison Between Schedule Types .............. ...............25....
Last Phase ............. ...... ...............25...


4 DI SCUS SSION ............. ...... .__ ...............47..


LIST OF REFERENCES ............. ...... .__ ...............52..


BIOGRAPHICAL SKETCH .............. ...............56....










LIST OF TABLES


Table


page


1-1 Earlier Studies on Meal Patterning In Mice ................. ...............16......_.._..

3-1 The average for total pellets per day for main procedure and last phase of scheduling
(m ean~s.d.) .............. ...............46....










LIST OF FIGURES


Figure page


1-1 The demand function. ............. ...............17.....

1-2 The demand function. ............. ...............18.....

3-1 Total pellets per day by operant with FR schedules. ............. ...............27.....

3-2 Total pellets per day by operant across FR schedules. ............. ...............28.....

3-3 Daily number of meals with 15min MMI by operants across FR schedules. .................. ..29

3-4 Daily number of meals with 30min MMI by operants across FR schedules. .................. ..30

3-5 Meal size with 15min MMI by operant across FR schedules ................. .....................31

3-6 Meal size with 30Omin MMI by operant across FR schedules ................. .....................3 2

3-7 Total pellets per day by operant across VR schedules............... ...............3

3-8 Daily numbers of meals with 15min MMI by operants across VR schedules. ................3 4

3-9 Daily number of meals with 30min MMI by operants across VR schedules. .................35

3-10 Meal size with 15min MMI by operant across VR schedules. ............. .....................3

3-11 Meal size with 30min MMI by operant across VR schedules. ............. .....................3

3-12 Total pellets per day by operant across PR schedules. ............. ...............38.....

3-13 Daily number of meals with 15min MMI by operants across PR schedules. .................. ..39

3-14 Daily number of meals with 30min MMI by operants across PR schedules. .................. ..40

3-15 Meal size with 15min MMI by operant across PR schedules ................. ............... .....41

3-16 Meal size with 30min MMI by operant across PR schedules ................. ............... .....42

3-17 Compari son of PR1.5 and PR1.5 schedules with 30Omin resetting criteria ................... .....43

3-18 Compari son of PR1.5 and PR1.5 schedules with 30Omin resetting criteria ................... .....44

3-19 Compari son of PR1.5 and PR1.5 schedules with 30Omin resetting criteria ................... .....45









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Master of Science

COMPARISON OF LEVER PRESS AND NOSE POKE OPERANTS FOR AN ANALYSIS OF
FOOD INTAKE AND MEAL PATTERNS IN MICE

By

Deniz Atalayer

May 2008

Chair: Neil E. Rowland
Major: Psychology

Much research in the field and in laboratory studies has focused on behavioral economics

of food intake in several species. Operants such as lever press, nose poke, or key peck have been

used to generate demand functions that express the relationship between the cost of food and the

amount of food consumed. There have been very few such studies of motivated food seeking and

demand in mice, and none has examined systematically consummatory cost or meal patterns.

Using albino (CDI) male mice, the present study compares food intake and meal patterns across

a series of ratio consummatory schedules. Two operants, lever press and nose poke, were

compared in a between groups design. A closed economy was used in which the mice were in

the test chambers for 23 h/day and earned all of their food via the operant under four fixed (FR5,

FR10, FR25, FR50), variable (VR10, VR20, VR50) and progressive (PR1.25, PR1.5, PR1.75)

ratios. When averaged across all schedules, mice in the nose poke group consumed more food.

Mice were run for 4 days at each ratio; there were no systematic differences between the first and

last day indicating that behavioral adjustments to schedule changes occurred very rapidly. Meal

number significantly differed when two criteria for the definition of 'a meal' (15 and 30 min)

were used.









CHAPTER 1
INTTRODUCTION

Obesity is a serious threat to human health and has a growing incidence in the recent

decades. According to the most recent WHO report, at least 400 million adults globally are obese

(World Health Organization, Obesity: 2005). Different approaches including use of animal

models have been developed to study eating and yield insights into the etiology of obesity. From

the early studies, it has been known that homeostasis has a crucial role in the regulation of food

intake and energy balance (Levin, 2002; Teitelbaum, 1964; Berthoud, 2002; Woods, 1991,

1998). In addition, there have been advances in molecular biology and genetics that have

identified a large number of genes responsible for satiety and hunger (Lubrano-Berthelier et al.,

2003a, 2003b; Branson, 2003). It has been estimated that 40 to 70 percent of the variation in

obesity-related phenotypes in humans is heritable (Comuzzie and Allison, 1998). However, it has

a complex etiology and is a multifactorial phenomenon (Blundell and Cooling, 2000; Erlanson-

Albertsson, 2005) that arises from the interactions of multiple genes and brain neural systems

(Gelegen et al. 2006; King, 2006; Petrovich and Gallagher, 2007), external factors from

environment (Rolls et al., 2002; French, 2003), and behavior (Stellar, 1954; Saelens and Epstein,

1996; Drewnowski, 1995; Lowe and Butryn, 2007).

Today it is highly recognized that there are many central and peripheral factors involved in

energy homeostasis and regulation of food intake, and understanding these mechanisms should

lead to effective treatments in the control of obesity. For example, some studies reported that

obesity in United States is to a great extent an economical issue (Drewnowski, 2004).

Specifically, the cost of energy-dense foods is often low, and it has been shown that dietary

energy density influences the regulation of food intake and body weight (Drewnowski, 2004;

2003). Thus, feeding behavior is highly influenced by the economic structure of the current









environment in which the individual lives and economic analysis provides a tool for

understanding this type of behavior. My goal in this study is to combine and examine the

economic concepts within the context of nutritional homeostasis.

Earlier studies with physiological, genetic or homeostatic animal models have failed to

provide a systematic protocol to examine whether food intake changes under controlled

laboratory conditions. Recently, the principles of economics, which relate the commodity to its

price, have been applied to the field of behavior. In the case of eating, it has been shown food

intake does change under different conditions of food availability (Sumpter et al., 1999; Rovee-

Collier et al., 1982; Hursh, 1980; Hursh et al., 1988).

Economics is considered a science of highly organized human behavior and is defined as

the computational analysis of anticipated cost and benefits (Hursh, 1984). In most research

protocols with animal models investigating eating in terms of economics of behavior, the price is

defined as responses required per reinforcement or reward (e.g., food, water). These designs

employ various experimental protocols that require some effort (cost) to obtain the reward. For

example, on a ratio schedule, a specified number of responses are required to obtain a particular

commodity or reinforcement. The schedules used in the present study are:

* FIXED RATIO (FR). The ratio in which each unit of the item costs a Eixed amount (e.g.,
number of responses),

* VARIABLE RATIO (VR). The ratio in which each food item costs a mean amount but the
actual cost of each item varies around that mean, and

* PROGRESSIVE RATIO (PR). The ratio in which successive food items within an episode of
feeding become progressively more costly.

FR schedules are believed to be the most direct and are the most commonly used method

to set the price of a commodity (Bauman et al., 1996; Hursh, 1984). By using different

schedules, researchers aim to understand how animal's preference for food is shaped by the









different economic environments and by extrapolation to the human condition, how an

individual's food preferences may be affected in these economies where different types of food

are usually available at all times.

In order to survive animals must learn how to search for food. They often perform a series

of behaviors traveling, catching and consuming this food. Earlier studies on behavioral

economics found that feeding patterns vary according to the cost of access to the food (Collier,

1985; Collier et al., 1986, Hursh, 1980). There are at least three definable costs related to eating

behavior (Morato et al., 1995):

* Cost of procuring access to food -procurement cost (travel effort and/or time)

* Cost of consuming food -consummatory cost (the cost within the patch such as digging or
climbing for food items, sucking, catching, holding etc., which are the equivalent of
operant schedules in an experimental setting in most studies)

* Cost of proce ssing food (phy si ologi c consequences/digesti on)

Thus from the point of view of economics of behavior, the apparent question is how the

animals will change the rate or amount of responding as the required effort to access to the

commodity, the food is increased (Hursh, 1980). It is recognized that answer is a product of a

complex interaction between the availability and/or cost of the food and hunger state of the

organism, however it can be defined and summarized in a demand function which relates the

price and food intake (Figure 1-1). The slope of the demand function is determined by the

amount of the effort that an animal will work to obtain a commodity as the price for that

commodity increases. The theory of economics claims that the consumption of most goods will

decrease as price increases (Hursh et al., 1988).

Morato et al. (1995) compared the food intake in rats under low vs. medium vs. high

procurement cost that were varied across days, and showed that when the price of procurement

was stable for more than about three days, rats ate less frequent and relatively larger meals when










the price was high than the lower prices (Figure 1-2). Many behavioral economists working with

animal models claim that animals optimize their food intake in accordance with 3 goals. First,

animals adjust their eating behavior to maintain the daily food intake. Second, by eating less

frequent, animals limit the time and energy spent for foraging. Third by adjusting the meal size,

animals limit the physiological cost of processing ingested food. Thus, an optimal meal pattern

represents a compromise between eating as infrequently as possible so as to minimize foraging

cost, and eating meals as small as possible so as to minimize processing cost (Collier et al., 1986;

Morato et al., 1995; Stephens and Krebs, 1995).

Morato et al. (1995) showed that for high cost requirement (FR400) condition rats did save

foraging cost by reducing their food intake. Some rats did not consume anything at all on those

`expensive' days, and thus did avoid paying the high procurement price. Some rats however,

continued to eat on high-price days but their meals were not large enough to compensate for their

reduced meal frequency, so their total food intake fluctuated. A new meal frequency was

established on the first day that the new price was set but meal size seemed to change more

slowly. A similar result was also found when change in caloric density was used rather than

change in procurement cost change (Le Magnen, 1992). Rats can use this strategy to increase

their efficiency with which they exploit resources. For example, if the

procurement/consummatory cost is lower during the day than at night, rats will switch from a

nocturnal to diurnal feeding (Jensen et al., 1983). As mentioned above, when a day of high cost

is followed by a day of low cost rather than by another high cost day, intake is reduced or even

eliminated on high cost day and the deficit is made up entirely on the low cost day (Morato et al.,

1995). Since the predictability of the environment affected the meal patterns in rats, the present










study also tested this in mice by using random vs. ascending order reinforcement schedule ratios

(variable vs. progressive ratio schedules).

Open and closed economies. The literature of behavioral economics has offered two

different settings for analysis of eating behavior -open and closed economies- and these lead to

sometimes disparate results (Timberlake and Peden, 1987; Bauman, 1991; Killeen, 1995). In a

closed economy all of the commodity is earned in the experimental session which often is in

force all of the time. In an open economy protocol, a commodity may be earned during the

experimental session, which is time-limited, but in the case of food, additional food is usually

offered outside of the economy. For example, an animal may receive supplemental free rations

after a session to ensure that it maintains a specific body weight. Further, an open economy

usually requires food deprivation prior to the experimental session. Hursh (1980) demonstrated

that in an open economy as the rate of reinforcement schedule decreased, response rate slightly

decreased too. In contrast, in a closed economy, the response rate increased remarkably (Killeen,

1995). Hursh (1980) explains this dilemma by suggesting that in a closed economy, the

commodity and costs are present at all times and animals can only obtain food as a consequence

of the scheduled ratio of reinforcements. On the other hand, Killeen (1995) proposes that the

trade between the animal and the environment is guided to a great extent by the deprivation level

of the organism which he argues that in a closed economy setting it is not taken into much

consideration. Open economy experiments effectively study single meals, thus to acquire an

eating episode in a given experimental session, food deprivation prior to the session is essential.

In a closed economy protocol, no food restriction is imposed by the experimenter; any change in

meal taking is thus a direct consequence of the economic structure. However, it is known that

food restriction increases food consumption and also reinforcing value of food (Raynor and










Epstein, 2003). Collier (1985) argues that humans live in a closed economy as the food is almost

always available, thus closed economy protocols may provide more realistic models for human

eating behavior.

It has been shown that the meal size and subsequent interval to the next initiation of eating

were positively correlated (LeMagnen, 1992); such a correlation which cannot be studied in a

single meal session of the open economy environment. On the other hand, closed economy

settings are concerned with sequence of meals or meal patterns. The present experiments thus

employ a closed economy setting for studying meal patterning in mice.

Determining the relationship between food consumption and the price for food (demand function)

is the one of the main foci of the economics of ingestive behavior. To acquire such demand

functions, systematic analyses on meal patterns have been done with various animal models, with

various schedules of reinforcement. To reach a consistency between days for a day-to-day meal

pattern for each animal, researchers use time frames ranging from several days to weeks for each

schedule of reinforcement (Morato et al., 1995, Collier et al., 1986). To obtain a demand curve for

the rats, Hursh (1980) reported that it required 5 to 6 months. They adapted a rapid method for

determining demand curves but it still required 40 days. Of great significance to this project, it has

been reported in a study using rats with a lever press operant that a demand function can be

generated in a much shorter period (Raslear et al. 1988). They suggested that in an environment

where the cost for food is changing and unpredictable, rats are able to adjust their food intakes

very rapidly probably less than one day.

We do not know whether all these findings on rats reflect the facts about mice there has not

been much consistency on the field about meal patterns in mice. Studies suggested that the

average number of meals per day shows substantial variation for different animal species










(Berthoud, 2002). When several factors are controlled, such as stress, existence of predators or

social competitors, light-dark cycle and given adequate food, species-specific meal patterns seem

to become apparent (Madden, 2005).

The initiations and terminations and of meal to meal intervals (MMI) are called "the meal

pattern" of rodents. However, defining a meal within each session, and differentiating a pause

within a meal (intra-meal intervals) from an MMI require a suitable time resolution (typically <5

min). 2 min after animals stopping eating, the probability of animals resume eating within the

next 10 min, was found to be maximal. That probability decreases from 10 min to 40 min, and is

minimal at 40 min and increases again for longer (because the animal is probably hungry again).

Thus a meal has been defined as an eating episode initiated after at least 40 min of non-eating

period (Kissileff, 1970; LeMagnen, 1992; Clifton, 2000). In mice, spontaneous food intake

occurs in number of eating bouts separated by periods of non-eating or inter-meal or meal-to-

meal intervals that typically are at least 30 min in length (Clifton, 2000). However, there is a

drastic discrepancy between different laboratories for the number of meals taken per day in

experiments with mice (Petersen and McCarthy, 1981; Gannon et al., 1992; Strohmayer and

Smith, 1987; Vaughan and Rowland, 2003; Fox and Byerly, 2004; Richard and Low, 2007).

Table 1-1 summarizes these results for operant and non-operant conditions.

Although rats typically do not show so much between-laboratory variability in results,

some studies with rats have suggested differences with use of a nose poke compared with a more

traditional lever press operant (Ettenberg et al., 1981). Also it has been claimed that changes in

the meal patterns in different studies with rats might arise from the effects of the experimental

manipulations; such as single vs. multiple presentation of food, method of choosing between two

options, water availability, ratio or interval schedules etc. (Clifton et al., 1984). In their study,









dose-dependent reduction in response rates was not found when nose poke operant was used.

According to their report even at the highest doses, rats continued to respond. Thus, they

concluded the effect of the drugs on responding rate was partially a result of the instrumental

paradigm that has been employed in the experimental design.

Roche and Timberlake (1998) emphasized the importance of designing protocols that

examine the natural behavioral traits and behavioral hierarchies that may be species rather than

responses that are completely arbitrary. It has been demonstrated that there is a species-typical

perceptual/motor organization in rats related to common maze equipment, such as straight alleys,

and radial arm mazes and in many studies it is suggested that the operant has a great deal of

importance (Roche and Timberlake, 1998; Schindler et al., 1993; Marusich and Branch, 2006). It

is has been suggested that the baseline level of nose poke operant response was high and

acquisition with food reinforcement occurred rapidly, particularly when compared with a lever

press operant response. Therefore, the nose-poke response appeared to be particularly useful for

the study of the acquisition of operant responses (Schindler et al., 1993).

Thus, we compared two different operants, nose poke which has not been much used and

lever press which has been most commonly used in these types of protocols. For analytic

simplification in defining the parameters of feeding behavior in this particular species, we

designed a parametric study with an operant (nose poke) that to our knowledge has not been used

previously in an eating task in mice and using various environmental conditions of food

availability.









Table 1-1. Earlier Studies on Meal Patterning In Mice


MOUSE
STRAIN
Small 'S' and
large 'L' inbred
SWR/J

C57BL/6J
(lean and ob/ob)

C57BL/6J


C57BL/6J
(lean and ob/ob)

129/B6 (wild type
for BNDF +/-)
129/B6 (wild type
for BNDF +/-)
129/B6 (wild type
for MC4R +/-)

129/B6 (wild type
for MC4R +/-)

C57BL/6J (wild
type)


OPERANT

Overhead door
panel
Recess at the floor
level
Sipper spout in
cage

Lever press


Lever press and
food receptacle

Pellet removal from
trough
Liquid diet from
0.02ml dipper
Lever press and
food receptacle
(procurement cost)
Lever press and
food receptacle
(progressive ratio)
Lever press
(procurement cost-
FRs)


DIET

Powdered
rodent chow
Powdered
rodent chow
Liquid diet
EC116

Noyes 20mg
pellets

Noyes 20mg
pellets

BioSery 20mg
pellets
Isocal-High fat
liquid
Noyes 20mg
pellets

Noyes 20mg
pellets

BioSery 20mg
pellets


#OF
MEAL S/DAY
12 (5min)

36 (5min)

50(male)
30(female)
(1 and 5min)
~8 (24 h food
access through
FR schedules)
2-10, function of
access cost
(10min)
~12 (18 hr food
access)
~15 (18 hr food
access)
2-7, function of
procurement cost
(10min)
25-50, function of
20 vs 3min reset
criterion
(4g per day)
(735sec)


REFERENCE

Petersen and
McCarthy (1981)
Gannon et al
(1992)
Strohmayer and
Smith (1987)

Vaughan and
Rowland (2001)

Vaughan and
Rowland (2003)

Fox and Byerly
(2004)
Fox and Byerly
(2004)
Vaughan et al
(2005)

Vaughan et
al(2006)

Richard and Low
(2007)




























30-


o 25-


o 20-


15-


10-



0 24 6 810

cost


Figure 1-1. The demand function. The figure summarizes the relation between the cost and
demand. The slope of the function is determined by the amount of the effort that a
person or an animal will work to obtain a commodity as the price for that commodity
increases (the numbers do not indicate any real data; the graph was drawn for
conceptual purposes).






















17




















-* Meal num ber
-0 Meal size


Low


Med


High


Relative procurement cost


Figure 1-2. The demand function. The slope of the demand function is determined by the
amount of the effort that an animal will work to obtain a commodity as the price for
that commodity increases. The theory of economics claims that the consumption of
most goods will decrease as price increases (the numbers do not indicate any real
data; the graph was drawn for conceptual purposes).









CHAPTER 2
MATERIALS AND METHODS

Subj ects

A total of sixteen male albino (CD I) mice, initially about 3 months of age were used. We

did not include female mice in our design to avoid the possibility that estrous cyclicity would

interact with our analysis of meal patterns. The average weight of the animals was 39.712.7g at

the beginning and 45.814g at the end of the experiments (Mean a s.d.).

During the experimental periods, mice lived in the operant chambers for 23 hr per day. The

mice were weighed daily during a 1 hr cleaning period and were kept in empty holding cages.

The operant chambers were wiped with 70% ethanol solution and distilled water each time

before the mice were placed. When not in experiments, mice were housed in a standard,

polycarbonate cages (separate vivaria) with Purina Chow pellets and tap water available ad

libitum and a 12:12 light cycle in place (lights on 0700). During the experiments, mice obtained

20 mg; complete nutritional mouse pellets (Research Diets Inc) when they completed an imposed

cost determined by the reinforcement schedule. Our preliminary studies demonstrated that the

spillage with this type of pellets was typically very little. Tap water was available freely from a

sipper tube.

The Psychology department vivaria are part of the centralized University of Florida

Animal Care program with full AAALAC accreditation. Animal use is approved by a campus-

wide IACUC and is compliant with the recommendation of the Guide for the Care and Use of

Laboratory Animals (1996).

Apparatus

Sixteen operant chambers (Med Associates: 13xl3xl2 cm with Plexiglas and metal walls

and stainless steel grill floor plus solid nesting platform) enclosed in ventilated, noise attenuating









cabinets with the same 12:12 light cycle as the vivarium (a 15 watt bulb in a standard light

fixture run from a 24 hr timer) were used in the experimental procedures in the present study. All

chambers were equipped with one lever press and one nose poke operant device, located 2cm

above the floor, situated on one wall on either side of a food aperture. Water was supplied from

sipper tubes mounted on the wall opposite side to the food magazine and the two operant

devices. In each chamber and for a given mouse only one manipulandum was active during the

whole experimental protocol.

A record of the total pellets obtained by mice and number of responses (nosepoking and

leverpressing) were acquired by the MEP-PC IV computer software (1VED Associates, St.

Albans, VT). The computer recordings allowed an accurate analysis of the number of meals and

the amount eaten at each meal. Data were accumulated in each 15 min (for FR and VR) and 5

min (for PR) time bins for each 23 hr period.

Procedure

To investigate whether the form of the operant (nosepoking vs. leverpressing) influences

the economic analysis of meal patterning, mice were divided randomly in two groups of 8, with

one group obtaining food pellets by pressing the lever and the other group by nosepoking.

Prior to the study, to habituate mice to the operant chambers and to the novel pellets, a 1 hr

training period was applied with free food was available in the food magazine of the operant

chambers without any cost. Later, a fixed ratio-1 (FR1) where a pellet was delivered as a

consequence of one response on the active manipulandulum, was used as a magazine training for

a day or two to acclimate mice to the operant conditioning protocol. For the training, a mouse

was considered to have learned the conditioning paradigm if they earned enough pellets to

maintain their body weight. No food deprivation protocol was used prior to the experimental










sessions. After they successfully learned to press the lever or nose poke, animals were exposed to

several reinforcement schedules as the experimental design.

Experimental sessions lasted 23 hr. A short protocol (4 days with each ratio) was used

because previous studies in our laboratory (Vaughan et al., 2006) have indicated that mice adjust

to changes in ratios within a day or so. Mice were exposed to an incrementing series of fixed

ratios (FR1, FR5, FR10, FR25, FR50) and then, variable ratios (VR10, VR20, VR50), and finally

progressive ratios (PR1.25, PR1.5, PR1.75). In the VR, the actual ratios selected randomly by the

program were VR10; 1, 5, 10, 15, 19, for VR20; 2, 10, 20, 30, 38, for VR50; 5, 25, 50, 75, 95. In

the PR, the number of responses required for the next (n+1)th pellet in a series, Rn+1= Rn x 1.25

and Rn+1= Rn x 1.5 and Rn+1= Rn x 1.75 (Rn = nth response requirement). The resulting number

was rounded to the nearest integer, giving the following sequences: for PR1.25; 1, 2, 2, 2, 2, 4, 4,

5, 6, 8, 10, 12, 15, for PR1.5; 1, 2, 2, 3, 5, 8, 11, 17, 26, 38, 58, 86, 130 and PR1.75; 2, 3, 5, 9,

16, 29, 50, 88, 154, 269, 471, 825. Further, in the PR series, whenever 15 min elapsed without a

response the ratio was reset to the initial value of the particular schedule. This reset allows the

animals to effectively quit eating when the unit cost of a pellet becomes too high and shift to

another "patch" (in this case with a 15 min temporal boundary). For comparison, we additionally

ran a PR 1.5 reinforcement schedule using a 30 min reset criterion.

In the final phase of the experiment, after all the above schedules were completed, to

determine whether mice can follow schedule changes even more rapidly as well as to determine

whether differences that we observed across schedules were not merely due to experience or age,

each reinforcement schedule was employed for one day consecutively.

Analyses and Data Acquisition

In the present experiment, two different meal-to-meal interval (MMI) criteria (15 and 30

min) were applied. The raw data from the computer recordings, showed how many responses









were made and the number of pellets earned at each 15 min (for FRs and VRs) and 5 min (for

PRs) throughout the whole 23 hr period (1380 min) each day. Non-responding (non-eating)

episodes were showed as zero for each 15min time bin in the computer software and a minimum

of 15 or 30 min was used to distinguish separate meal events. After the numbers of meals for

each mouse were counted by the experimenter for each day of each schedule from the raw data,

the mean meal size was derived by dividing the number of total pellets by the number of meals

for the particular day. With one exception, no systematic difference was noted across the four

days for pellet intake per day. Thus, the mean for number of meals, total pellets earned and meal

sizes were computed for each mouse and for each reinforcement schedule by averaging them

over for four days. Parameters were analyzed for significance with SPSS computer software by

using repeated measures analysis of variance (ANOVA), with the operant (nosepokers vs.

leverpressers) as between-subj ect variable and schedules as within-subj ect variable. Analyses

within each ratio schedule type (FR, VR and PR), One-way ANOVAs were used to measure the

significance of each variable. Independent t-tests were used where necessary. In all cases, p<

0. 05 p was considered significant. Graphs are drawn using Sigma-plot computer software.









CHAPTER 3
RESULTS

When averaged across all of the ratio schedules, mice consumed (Mean & S.D.)

259.5 & 56.7 pellets per day (Figure 3-1) distributed as either 20 & 7.5 or 11 & 3.2 meals at the 15

and 30 min MMI, respectively. The corresponding meal sizes were 15 & 6 and 28 & 17.8 pellets.

Since each pellet is 20mg, this corresponds to mean meal sizes approximately of 0.2 and 0.4 g.

With the exception of FR10 [F(3,60)= 8,193; p < 0.01~], under each schedule, meal

numbers, meal sizes and the number of pellets did not differ significantly across the four days of

each schedule. Thus, data were averaged across 4 days to give a single datum for each mouse.

When averaged across schedules, nosepokers (NP) consumed significantly more pellets

than leverpressers (LP) (Mean & S.D.; NP: 274.5 & 51.7, LP: 244.6 & 58). ANOVA analyses

resulted in a group effect for the operant [F(1,174) = 13.086; p<0. 01].

FR Schedules

The number of pellets taken in the FR phases is shown in Figure 3-2, the meal numbers in

Figure 3-3a, b and the meal sizes in Figure 3-5, 3-6. Total pellets earned per day differed

significantly across the four FR (FR5, FR10, FR25, FR50) schedules [F(3,60)= 5.763; p < 0. 05].

Post-hoc Bonferroni test showed that mice at FR50 mice consumed fewer pellets compared to

their intake on the other three FR schedules (Figure 3-2).

Across FR schedules, there were significant differences between LP and NP for total

pellets [F(1,62)=7.818; p < 0.01~] (Figure 3-2), meal numbers at 30min MMI [F(1,62)=4.802; p

< 0. 05] (Figure 3-4), and meal size at 15min MMI [F(1,62)=10.763; p < 0.01~] (Figure 3-5).

Post-hoc t-tests showed that, NP consumed more number of pellets daily [t (62) = -2.796; p <

0. 01], had more meals with the 30 min MMI definition criterion [t (62) = -2. 191; p < 0. 05,] and

larger meals with the 15 min MMI criterion [t (62) = -3.281; p < 0. 01].









VR Schedules

The number of pellets taken in the VR phases is shown in Figure 3-7, the meal numbers in

Figure 3-8, 3-9 and the meal sizes in Figure 3-10, 3-11. Total pellets earned per day differed

significantly across the three VR (VR10, V20, VR50) schedules [F(2,45)=15.728; p < 0. 01].

Post-hoc Bonferroni test showed that mice at VR50 mice consumed fewer pellets compared to

their intake on the other two VR schedules (Figure 3-7).

Across VR schedules, there was no significant difference in the number of pellets taken by

NP vs. LP (Figure 3-7). However, the type of operant showed a significant between-subj ects

effect on meal number [F(1,46)=5.213; p < 0. 05] (Figure 3-8) and meal size under with 15min

IV1VI definition criterion [F(1,46)=7. 123; p < 0.01~] (Figure 3-10). The t-test revealed that

nosepokers took fewer but larger meals than leverpressers [for meal numbers; t (46) = 2.283; p <

0. 05, for meal size; t (46) = -2.669; p < 0. 01].

PR Schedules

The number of pellets taken in the PR phases is shown in Figure 3-12, the meal numbers in

Figure 3-13, 3-14 and the meal sizes in Figure 3-15, 3-16. Total pellets earned per day differed

significantly across the four PR (PR1.25, PR1.5, PR1.75, PR1.5/30Omin) schedules

[F(3,60)= 8.500; p < 0.01~]. Post-hoc Bonferroni test showed that at PR1.25 mice consumed more

pellets compared to their intake on the other three PR schedules (Figure 3-12).

In PR schedules, operant type displayed a significant between-subj ects effect for total

pellets earned per day [F(1,62)=6.640; p < 0. 05] (Figure 3-12), and meal number with 30min

IV1VI [F(1,62)=5.676; p < 0. 05] (Figure 3-9b). Meal sizes did not differ significantly

(Figure 3-15, 3-16).

The comparison between 15 min and 30 min reset criteria applied in PR1.5 schedules did

not seem to make any difference for total pellets (Figure 3-17), number of meals (Figure 3-18) or









meal size (Figure 3-19) per day except when 30Omin MMI used [F(1,30)=15.896; p < 0.01~]. A

follow-up independent t-test showed when 30min MMI used, mice had more meals per day with

the 30 than the 15 min reset criterion [t(30)= -3.987; p < 0. 01].

Comparison Between Schedule Types

Daily pellet intake showed significant variation between three types of ration

[F(2,173)=17.846; p < 0.01~]. A follow up post-hoc analysis showed that mice took more pellets

per day under PR schedules compared to FR and VR schedules. PR schedules also resulted in

more number of meal intakes but only when 15Smin MMI was used [F(2, 173)=233.987; p <

0. 01].

Last Phase

The last phase of the experiment that each schedule were employed for one day in the

exact same order with the whole procedure, showed no significant difference on the total number

of pellets earned daily when averaged over the schedules. However, when one to one

comparisons between the number of pellets computed by the average of previous four days for a

schedule and the number of pellets per day as the last phase for the same schedule, some reached

the significance [for FR5: t(1 5)=-3.629; p < 0. 01, for VR50: t(15)=5.385; p < 0. 01, for PR1.25:

t(15)=2.474; p < 0. 05, and for PR1.75: t(15)=3.142; p < 0.01~]. Table 3 summarizes the result.

In addition, mice obtained more number of pellets [F(2,173)= 5.667; p < 0. 01] and more

number of meals per day [for 15min MMI F(2,173)=79.679; p < 0. 01 and for 30Omin MMI

F(2, 173)=4.3 56; p < 0. 05] in PR schedules compared to FR and VR schedules in this last phase

of consecutive one-day employment of the each schedule.

When 30Omin MMI was used to define 'a meal', there was a significant between-subj ects

effect of operants [F(1,174)= 8.75 8; p < 0. 05]. Independent t-test conducted as a follow-up

showed that nose pokers had more meals per day [t (174) = -2.959; p < 0. 05]. However, when









15min MMI criteria was used to define a meal, the group effect of operants did not reach

significance. The difference between the meal sizes nose pokers and lever pressers on the other

hand, reached statistical significance for 15min MMI but not for 30min MMI definition criteria

[F(1,174)=13.143; p 0. 01] The t-test analysis concluded that nosepokers took larger meals

when 15min MMI definition criteria was used [t (174) = -3.625; p 0. 01].

Regardless of the operants, defining a meal by 15Smin MMI vs. 30min MMI resulted in

different number of meals and the meal size per meal. Statistical significance were at

F(1,3 50)=210.049; p 0. 01 for number of meals and F(1,3 50)= 86.93 3;

p 0. 01 for meal size. Mice ate significantly fewer and larger meals when 30min MMI used

compared to 15Smin MMI [t (3 50) = 14.493; p 0. 01 and t (3 50) = -9.324; p 0. 01 respectively].












400.00




y 300.00




O 200.00

*

100.00


- *


T


T


FR5 FR10 FR25 FR50 VR10 VR20 VR50 PR1.25 PR1.5 PR1.75 PR1.5-
30min
schedules


Figure 3-1. Total pellets per day by operant with FR schedules.













SLeverpressers
[ "I Nosepokers


300 -


250 -


200 -


150 -


100 -


TI'


FR10


01


FR5


FR25


FR50


Figure 3-2. Total pellets per day by operant across FR schedules.





















18 _0N

16-

14-

a, 12-



10 -



6-

4-

2-

0
FR5 FR10 FR25 FR50




Figure 3-3. Daily number of meals with 15min MMI by operants across FR schedules.






















14 _0N


12-


mI 10-


S8-


S6-


4-


2-


0
FR5 FR10 FR25 FR50




Figure 3-4. Daily number of meals with 30min MMI by operants across FR schedules.

















30-

I NP

25-



20-







15



10




FR5 FR10 FR25 FR50



Figure 3-5. Meal size with 15min MMI by operant across FR schedules.



















I NP


30-










20






FR5 FR10 FR25 FR50



Figure 3-6. Meal size with 30min MMI by operant across FR schedules.



















350 -M L
I NP

300


250






.03 150


100




50


VR10 V"R20 VR50



Figure 3-7. Total pellets per day by operant across VR schedules.
















SLP
I NP


01


VR10


VR20


VR50


Figure 3-8. Daily numbers of meals with 15Smin MMI by operants across VR schedules.
















SLP
I NP


TI-r


01


VR20


VR10


VR50


Figure 3-9. Daily number of meals with 30min MMI by operants across VR schedules.

















30-

I INP
25-



20-







15



10




VR10 VR20 VR50



Figure 3-10. Meal size with 15min MMI by operant across VR schedules.















SLP
I NP


01


VR10


VR20 VR50


Figure 3-11. Meal size with 30min MMI by operant across VR schedules.

















400 -






300 -






200 -






'100 -


0


PR1.25


PR1.5


PR1 .75 PR1 .5 wrl30~min


Figure 3-12. Total pellets per day by operant across PR schedules.
















I NP
35-


30-


25-


E 20-






5 -5


10




PR1.25 PR1.5 PR1.75 PR1.5 wl30min



Figure 3-13. Daily number of meals with 15min MMI by operants across PR schedules.


















18, I NP

16-


14-

cn 12-


E 10-

,8-


S6-








PR1.25 PR1.5 PR1.75 PR1.5 wl30min



Figure 3-14. Daily number of meals with 30min MMI by operants across PR schedules.


















18 -M L
I INP
16-

14-

12-




6 -o




4-

2-

0
PR1.25 PR1.5 PR1.75 PR1.5 wl30min



Figure 3-15. Meal size with 15min MMI by operant across PR schedules.



















SLP
I NP


80-











60
PR .5P 15 R .5 P 1. l0 i


Fiue3-6 ea iewih3mi M b prntarssP chdls

















350~ I NP


300-


250-


a, 200-


B 150-


100-




50


PR1.5 PR1.5 wl30min



Figure 3-17. Comparison of PR1.5 and PR1.5 schedules with 30min Program resetting criteria
(daily number of pellets).



















M LPmealsize 15MMI
I LPmealsize 30MMI
M NPmealsize 15MMI
I NPmealsize 30MMI


PR1.5 PR1.5 wl30min


Figure 3-18.
(meal size).


Comparison of PR1.5 and PR1.5 schedules with 30min program resetting criteria
















M LPmeal# 15MMI
I LPmeal# 30MMI
30 -1 I NPmeal# 15MMI
I NPmeal# 30MMI

25-


S20-


8 15-





10



PR1.5 PR1.5 wl30min



Figure 3-19. Comparison of PR1.5 and PR1.5 schedules with 30min program resetting criteria
(daily meal numbers).
















Table 3.1 The average for total pellets per day for main procedure and last phase of scheduling
(mean~s.d.)


schedules


Main procedure


Last phase of
schedules
308163
270140
226150
188161
297138
254156
160156
308155
279135
263141
256157


FR5
FR10
FR25
FR50
VR10
VR20
VR50
PR1.25
PR1.5
PR1.75
PR1.5-30MIN


251140
261130
227168
190163
293145
268134
215140
325151
279133
286124
275142










CHAPTER 4
DISCUSSION

The main focus of the present study was to design an instrumental conditioning paradigm

that would allow us to conduct a systematic analysis for meal patterns in mice as a function of

effort and with and explicit comparison of two different operants used.

Mice consumed ~250 pellets (~5g/day) but the meal distribution was critically dependent

on the MMI criterion: at the 15min MMI, the overall mean was ~28 meals with a meal size of

~10 p-ellets (~0.2g), whereas at the 30 min MMI the overall mean was ~15 meals with a meal

size of ~18 pellets (~0.4g) a day, regardless of the operant. The number of meals and average

total intake consumed (grams) per day did show consistency with most of the previous research

in the literature (Table 1). This might suggest that using different operants and MMI criteria for

defining a meal have reasonable effects on the results for eating behavior analysis in mice

(Kissileff, 1970).

One of the findings of the present study was that for each reinforcement schedule, the meal

pattern of mice did not significantly differ across four days of each schedule with the exception

of FR10. However, the statistical significance for the FR10 condition disappeared when the data

from first day was excluded from the four days of FR10 schedule and analysis was conducted

across the second, third and fourth days. It might be possible that mice had difficulty adjusting to

the novel eating condition as the first day of FR10 schedule was the first day that they

encountered a cost that required a relatively more effort compared to free access or FR1 or FR5

schedules. This particular effect for the initial FR10 schedule shown in this study was also found

in other studies examining meal patterns in mice (Richard and Low, 2007).

The rapid adaptation of mice to the changing schedules, usually 1 but at most 2 days

shown here agree with a study by Raslear et al. (1988) with rats in an operant task that showed a









stable relationship between food consumption and price for food throughout seven consecutive

days after schedule change. Thus, our findings in mice, along with Raslear' s results in rats

indicate that for consummatory costs, rodents have a substantial capability to adapt rapidly the

changes in the schedules of reinforcement. Future use of short-term protocols like the procedure

in this study should allow further research using anti-obesity drugs that have short half-lives

and/or to shorten the time period that is needed to complete an economic profile.

We have presented a parametric study with an animal model to examine the role that

operant plays on meal pattern analysis in mice. Hence, nosepokers obtained considerably more

pellets per day when averaged across all schedules. Also, when analyzed separately, in each

group of ratio schedule (FR, VR and PR) operant type appeared to be an important factor

affecting the total number of pellets eaten. This showed that in reinforcement schedule

paradigms, using different operants has a crucial effect on the results and the nose poke operant

is particularly useful in this type of research. These findings agree with some of the studies with

rats comparing nose poke and lever press operants (Schindler et al., 1993; Ettenberg et al., 1981;

David et al., 2001). Schindler et al. (1993) suggested that acquisition of the nose poke response

in rats occurred much more rapidly than of other operants. In addition they reported that if there

is no experimenter intervention such as shaping, acquisition of lever pressing response occurs

rather slowly. Thus, a nose poke operant might be useful whenever short-term procedures are to

be used (Schindler et al., 1993). On the other hand some studies with rats also indicated that the

type of operant (lever press vs. nose poke) did not have a significant effect on the acquisition of

intravenous heroin/cocaine self-administration or dose-related responding (David et al., 2001).

However, in that particular study, only two types and low ratio schedules (FR1 and FR3) were









used. In order to make an accurate comparison, more variety of reinforcement schedules is

required.

Eating behavior in animals occurs in episodes (Collier and Johnson, 2004). The size of

each bout depends heavily on the eating environment of the animal such as the availability of

food resources and effort that requires consuming the particular resource. Adjusting the meal size

for required costs for food is part of the economizing strategy of the animal in eating behavior. In

the present design, meal size was affected by the operant type, as nose pokers ate bigger meals

regardless of the different schedule requirements. It has been shown that rats increased their meal

size as they decreased the frequency of meals (decreasing meal numbers) in a compensatory

fashion as the required cost to access to food increased (Mathis et al., 1995; Collier et al., 1998;

Collier et al., 2002). However, total number of pellets earned per day showed significant changes

under each schedule which indicated that for our results daily intake was not unaffected by the

changes in the schedules. In other words, daily intake was not maintained as it was suggested in

the literature (Morato et al., 1995; Collier et al., 1998). Nevertheless, it was also claimed that at

the highest costs, animals made a sacrifice by decreasing their food intake, thereby avoid paying

the expensive price (Morato et al., 1995). Thus, at the highest costs for each ratio in our

experiment, it could be argued that mice avoided excessive consummatory cost by sacrificing

some of their intakes and reduced the number of pellets earned a day. This is, of course, the

defining feature of a demand function. The schedule-associated decrease in food intake was

apparent in FR and VR schedules as at FR50 and VR50 mice consumed less pellets. This was

also in agreement with a recent mice meal pattern study suggesting that FR40 cost was not

enough to alter the meal pattern of mice from the baseline (Johnson and Low, 2007). For PRs,

significance was found at the lowest PRs as PR1.25 was resulted in more pellet intake per day.









PR schedules were found to result in higher number of pellets earned per day when

compared to the FRs and PRs. However, since a considerable amount of time (~5 months)

elapsed between the first day of FR5 and the first day of PR schedules, it is reasonable to argue

that PR schedules did result in more number of pellets per day because of that amount of the time

that has passed. In an effort to control this issue, after the last PR schedule, we conducted each

schedule for only one day in the same order to see if the intake was comparable with the previous

4-day of intake. When averaged across all eleven reinforcement schedules of one day, the total

pellets earned per day did not differ from the previous 4-day scheduling when they are averaged

across schedules. On the other hand, comparing each schedule separately with the 4-day average

of the same schedule resulted in a significant difference between the pellets obtained per day for

some of the reinforcement schedules (FR5, VR50, PR1.25 and PR1.75). However, opposite to

what we wanted to control as a potential confounding of increase in intake as the time passed, the

number of pellets decreased-not increased when compared to the previous 4-days of scheduling.

Thus, the argument for the possible confounding effect of the time passage on the higher number

of pellets for the PR schedules was eliminated. It can be concluded that PR schedules resulted in

significantly more number of pellets compared to the FR and VR schedules.

In addition, in this one day protocol of each reinforcement schedule, the comparison

between the three reinforcement schedules (FR, VR, PR) in terms of total pellets and meal

numbers per day, agree with the results from the same type of comparison of FR, VR and PR of

the main 4-day procedure. Thus, mice took more pellets and meals (both with 15min MMI and

30min MMI) daily in PR schedules compared to FR and VR schedules also at the 1-day phase of

the experiment.










Our data revealed that the criterion used for the definition of a meal' has an important

effect on the results. Mice appeared to take considerably more meals when 15min was used as

MMI compared to 30min MMI. Reciprocally, 30min MMI resulted in larger meal sizes when

compared to 15min MMI. The group effect of the operants also differed when different MMIs

were used. All these comparisons in the food intake parameters between the two MMIs indicated

that defining a meal' has a crucial influence on the detail of the meal pattern of animal models.

Although it would be hard to determine which of the MMIs is a better representation for 'a meal'

for this strain of mice, 15min MMI seemed to concur with some of the earlier studies with mice

(Petersen and McCarthy, 1981; Vaughan and Rowland, 2003; Fox and Byerly, 2004; Vaughan et

al., 2005; Richard and Low, 2007) although strain differences cannot be excluded as a source of

variance.

Comparison between 30min vs. 15min reset criteria applied separately on PR1.5 schedules

indicated no different meal patterns. Since the first few pellets in each meal were the cheapest,

mice ate many small meals by quitting eating as the cost increased and letting the program reset

itself to the lower initial cost. However, mice maintained the total pellet intake same in both

PR1.5 schedules.

Future directions. In our study we looked at the consummatory cost, which is the

equivalent of the near-the-patch cost. We are currently conducting a foraging cost design study

by applying both Collier' s two costs; procurement and consummatory costs with the same strain

of mice. We may further look at the genetic models to see how these meal patterns change in

genetically mutated obese mice as an implication for the human obese models.









LIST OF REFERENCES

Bauman R. (1991). An experimental analysis on the cost of the food in a closed economy. JExp
Anal Behav, 56(1: 33-50.

Bauman R.A., Raslear T.G., Hursh S.R., Shurtleff D., Simmons L. (1996). Substitution and
caloric regulation in a closed economy. JExp Anal Behav, 65(2): 401-422.

Berthoud H-R. ( 2002). Multiple neural systems controlling food intake and body weight.
Neuroiscience and Behavioral Reviews, 26: 393-428.

Branson R, Potoczna N, Kral JG, Lentes KU, Hoehe MR & Horber FF. (2003). Binge eating as
a major phenotype of Melanocortin 4 receptor gene mutations. New Englan2d J2ed, 348:
1096-1103.

Blundell J.E., Cooling J. (2000). Routes to obesity: phenotypes, food choices and activity. British
Journal ofNutrition, 83(6):33-38.

Clifton P.G., Poplewell D.A., Burton M.J. (1984). Feeding rate and meal patterns in the
laboratory rat. Physiology & Behavior, 32(3):360-374.

Clifton P.G. (2000). Meal patterning in rodents: psychopharmacological and neuroanatomical
studies. Neuroscience and Biobehavioral Reviews. 24:213-222.

Collier G.H. (1985). Satiety: an ecological perspective. Brain Res Bull. 14(6):693-700.

Collier G.H., Johnson D.F., Hill W.L., Kaufman L.W. (1986). The economics of the law of
effect. JExp Anal Behav, 46(2):113-136.

Collier G.H., Johnson D.F., Berman J (1998). Patch choice as a function of procurement cost and
encounter rate. JExp Anal Behav, 69(1:5-16.

Collier G.H., Johnson D.F., Mathis C. (2002). The currency of procurement cost. JExp Anal
Behav, 78 ():3 1-61.

Collier G.H., Johnson D.F.(2004). The paradox of satiation. Physiol Behav. 82(1:149-53

Comuzzie A.G., Allison D.B. (1998). The search for human obesity genes. Science, 280:1374
13 77

David J., Polis I., McDonald J., Gold L.H. (2001). Intravenous self-administration of
heroin/cocaine combinations speedballl) using nose-poke or lever-press operant
responding in mice. Behavioral Pharmacology, 12;25-34.

Drewnowski, A. (1995). Energy intake and sensory properties of food. The American Journal of
Clinical Nutrition, 62 (5): 1081-1085.

Drewnowski A. (2003). The role of energy density. Lipids, 38(2):109-115.










Drewnowski A. (2004). Obesity and the food environment Dietary energy density and diet
costs. American Journal of Preventive M~edicine, 2 7(3):154-1 62.

Erlanson-Albertsson C. (2005). How palatable food disrupts appetite regulation. Basic and'
Clinical Pharmacology and' Toxicology, 97:61-73.

Ettenberg A., Koob G.F., Bloom F.E. (1981). Response artifact in the measurement of
neuroleptic-induced anhedonia. Science, New Series, 213(4505):357-359.

Fox E. A. and Byerly M.S. (2004). A mechanism underlying mature-onset obesity: evidence
from the hyperphagic phenotype of brain-derived neurotrophic factor mutants. Am J
Physiol Regul Integr Comp Physiol, 286:994-1004.

French S.A. (2003). Pricing effects on food choices. J. Nutr. 133:841-843.

Gannon K.S., Smith J.C., Henderson R., Hendrick P. (1992). A system for studying the
microstructure of ingestive behavior in mice. Physiol Behav, 51(3):515-21.

Gelegen C., Collier D.A., Campbell I.C., Oppelaar H., Kas M.J.H. (2006). Behavioral,
physiological, and molecular differences in response to dietary restriction in three inbred
mouse strains. Am JPhysiol Endocrinol Metab, 291:E574-E581.

Guide for the Care and Use of Laboratory Animals (1996). Institute of laboratory animal
resources commission on life sciences national research council. National Academy Press.
Washington, D.C.

Hursh S.R. (1980). Economic concept for the analysis of behavior. JExp Anal Behav, 34(2):219-
238.

Hursh S.R. (1984). Behavioral economics. JExp AnalBehav, 42(3): 435-452.

Hursh S.R., Raslear T.G., Shurtleff D., Bauman R.A., Simmons L. (1988). A cost-benefit
analysis of demand for food. J Exp Anal Behav, 50(3):414-440.

Jensen G.B., Collier G.H., Medvin M.V. (1983). A cost-benefit analysis of nocturnal feeding in
the rat. Physiol Behav., 31(4):555-9.

Killeen P.R. (1995). Economics, ecologists, and mechanics: The dynamics of responding under
conditions of varying motivation. JExp Anal Behav, 64:405-431.

King B.M. (2006). The rise, fall, and resurrection of the ventromedial hypothalamus in the
regulation of feeding behavior and body weight. Physiol. Behav.,87:221-244.

Kissileff H.R. (1970). Free feeding in normal and 'recovered lateral' rats monitored by a pellet-
detecting eatometer. Physiol. Behav., 5(2):163-1 74.

LeMagnen J. (1992). Neurobiology of feeding and nutrition. Academic Press, San Diego.










Levin B.E. (2002). Glucosensing neurons do more than just sense glucose. International Journal
of Obesity. 25(5):68-72.

Lowe M.R. and Butryn M.L. (2007). Hedonic hunger: A new dimension of appetite. Physiol.
Behav., 91:432-439.

Lubrano-Berthelier C., Cavazos M., Dubern B. (2003a). Molecular genetics of human obesity-
associated MC4R mutations. Ann N. Y. Acad' Sci 994: 49-57.

Lubrano-Berthelier C., Cavazos M., Le Stunff C., Haas K., Shapiro A., Zhang S., Bougneres P.,
Vaisse C. (2003b). The human MC4R promoter: Characterization and role in obesity.
Diabetes. 52:2996-3000.

Madden G.J., Dake J.M., Mauel E.C., Rowe R.R. (2005). Labor supply consumption of food in a
closed economy under a range of fixed -and rando- ratio schedules : test of unit price. J
Exp Anal Behav, 83(2): 99-118.

Marusich J.A. and Branch M.N. (2006). Stability of cocaine dose-response functions at different
inter-dose intervals. Pharmacology Biochemistry and Behavior, 84(2); 360-369

Mathis C.E., Johnson D.F., and Collier G.C. (1995). Procurement time as a determinant of meal
frequency and meal duration. JExp Anal Behav. 63(3): 295-311.

Morato S., Johnson D.F., Collier G. (1995). Feeding patterns of rats when food-access cost is
alternately low and high. Physiology and Behavior, 57(2): 21-26.

Petersen S. and McCarthy J.C., (1981). Correlated changes in feeding behavior on selection for
large and small body size in mice. Behavior Genetics. 11(1):57-64.

Petrovich G.D. and Gallagher M. (2007). Control of food consumption by learned cues: A
forebrain-hypothalamic network. Physiology and Behavior, 91:397-403.

Raslaer T.G., Bauman R.A., Hursh S.R., Shurtleff D., Simmons L. (1988). Rapid demand curves
for behavioral economics. Animal Learning & Behavior, 16 (3): 330-339.

Raynor H.A. and Epstein L.H. (2003). The relative reinforcing value of food under differing
levels of food deprivation and restriction. Appetite, 40:15-24.

Richard C.D. and Low M.J. (2007). Drinking-explicit meal pattern analysis in mice: an
ethological perspective. Am JPhysiol Regul Integr Comp Physiol, 278: 797-805

Roche J.R., Timberlake W. (1998). The influence of artificial paths and landmarks on the
foraging behavior of Norway rats. Animal Learning & Behavior, 26:76-84.

Rolls B.J., Morris E.L., Roe L. S. (2002). Portion size of food affects energy intake in normal-
weight and overweight men and women. Am J Clin Nutr, 76:1207-13.










Rovee-Collier C.K., Clapp B.A., Collier G.H. (1982). The economics of food choice in chicks.
Physiol Behav, 28(6):1097-1102.

Saelens B.E. and Epstein L.H. (1996). Reinforcing value of food in obese and non-obese women.
Appetite, 27:41-50

Shindler C.W., Thorndike E.B., Goldberg S.R. (1993). Acquisition of a nose-poke response in
rats as an operant. Bull Psychon Soc, 31: 291-294.

Stellar E. (1954). The physiology of motivation. PhysiolReview. 101(2): 301-311

Stephens D.W., Krebs J.R. (1986). Foraging theory. Princeton University Press, New Jersey.

Strohmayer A.J. and Smith G.P. (1987). The meal pattern of genetically obese (ob/ob) mice.
Appetite, 8(2):111-23.

Sumpter C.E., William T., Foster T.M. (1999). The effect of differing response types and price
manipulations on demand measures. JExp Anal Behav, 71:329-354.

Teitelbaum P. (1964). "Appetite". Proceedings of the American Philosophical Society. 108(6):
464-472.

Timberlake W., Peden B.F. (1987). On the distinction between open and closed economies. J
Exp Anal Behav, 48(1):35-60.

Vaughan C.H., Rowland N.E. (2001). Operant conditioning utilizing mice in a foraging
paradigm. Appetite, 37 (2): 169.

Vaughan C.H., Rowland N.E. (2003). Meal patterns of lean and leptin-deficient obese mice in a
simulated foraging environment. Physiology Behav, 79(2):2 75-9.

Vaughan C.H., Marcus M.C., Haskell-Luevano C., Rowland N.E. (2005). Meal patterns and
foraging in melanocortin receptor knockout mice. Physiology Behav, 84:129-133.

Vaughan C.H., Marcus M.C., Haskell-Luevano C., Rowland N.E. (2006). Food motivated
behavior of melanocortin-4 receptor knockout mice under a progressive ratio schedule.
Peptides, 27:2829-2835.

Woods, S.C. (1991). The eating paradox: How we tolerate food. Physiological Review, 98(4):
488-505.

Woods, S.C., Seeley, R.J., Daniel Porte Jr., Schwartz, M.W. (1998). Signals that regulate food
intake and energy homeostasis. Science, 280: 1378-1383.









BIOGRAPHICAL SKETCH

Deniz Atalayer graduated from Bogazici University-Istanbul-Turkiye in 2004 with a

Bachelor of Science degree in psychology. Her interest in neuroscience began in the last two

years of undergraduate as she worked in a psychobiology lab specifically conducting research on

circadian rhythmicity with rats. She also worked in a behavioral analysis/learning lab on sexual

preference with quails. After completing her bachelor' s degree in psychology, she began to seek

for a graduate degree combining the behavioral work and neuroscience research. In fall 2005, she

was admitted to the behavioral neuroscience program in psychology department at the University

of Florida, and started to work with Dr. Neil Rowland. Her field of study includes eating

behavior and obesity research both from genetic, neurological, physiological, and

neuroeconomical perspectives. She defended her master' s thesis in fall 2007 which concerns the

effects of the use of different operants in the meal pattern analysis on mice, and she is currently

seeking candidacy to pursue a Ph.D. in the same program.





PAGE 1

1 COMPARISON OF LEVER PRESS AND NOSE POKE OPERANTS FOR AN ANALYSIS OF FOOD INT AKE AND MEAL PATTERNS IN MICE By DENIZ ATALAYER A THESIS PRESENTED HERE TO THE GRADU ATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Deniz Atalayer

PAGE 3

3 To my mom, dad, and my advisor Dr. Neil E. Rowland

PAGE 4

4 TABLE OF CONTENTS page LIST OF TABLES................................................................................................................. ..........5 LIST OF FIGURES.........................................................................................................................6 ABSTRACT.....................................................................................................................................7 CHAP TER 1 INTRODUCTION....................................................................................................................8 2 MATERIALS AND METHODS........................................................................................... 19 Subjects...................................................................................................................................19 Apparatus................................................................................................................................19 Procedure................................................................................................................................20 Analyses and Data Acquisition............................................................................................... 21 3 RESULTS...............................................................................................................................23 FR Schedules..........................................................................................................................23 VR Schedules..........................................................................................................................24 PR Schedules..........................................................................................................................24 Comparison Between Schedule Types................................................................................... 25 Last Phase...............................................................................................................................25 4 DISCUSSION.........................................................................................................................47 LIST OF REFERENCES...............................................................................................................52 BIOGRAPHICAL SKETCH.........................................................................................................56

PAGE 5

5 LIST OF TABLES Table page 1-1 Earlier Studies On M eal Patterning In Mice ...................................................................... 16 3-1 The average for total pellets per day for m ain procedure and last phase of scheduling (means.d.).................................................................................................................... ....46

PAGE 6

6 LIST OF FIGURES Figure page 1-1 The demand function........................................................................................................ .17 1-2 The demand function........................................................................................................ .18 3-1 Total pellets per day by ope rant with FR schedules. .........................................................27 3-2 Total pellets per day by ope rant across FR schedules. ...................................................... 28 3-3 Daily number of meals with 15min MMI by operants across FR schedules. .................... 29 3-4 Daily number of meals with 30min MMI by operants across FR schedules. .................... 30 3-5 Meal size with 15min MMI by operant across FR schedules............................................ 31 3-6 Meal size with 30min MMI by operant across FR schedules............................................ 32 3-7 Total pellets per day by ope rant across VR schedules. ...................................................... 33 3-8 Daily numbers of meals with 15min MMI by operants across VR schedules................... 34 3-9 Daily number of meals with 30min MMI by operants across VR schedules. ...................35 3-10 Meal size with 15min MMI by operant across VR schedules........................................... 36 3-11 Meal size with 30min MMI by operant across VR schedules........................................... 37 3-12 Total pellets per day by ope rant across PR schedules. ...................................................... 38 3-13 Daily number of meals with 15min MMI by operants across PR schedules. .................... 39 3-14 Daily number of meals with 30min MMI by operants across PR schedules. .................... 40 3-15 Meal size with 15min MMI by operant across PR schedules............................................ 41 3-16 Meal size with 30min MMI by operant across PR schedules............................................ 42 3-17 Comparison of PR1.5 and PR1.5 schedul es with 30m in resetting criteria........................ 43 3-18 Comparison of PR1.5 and PR1.5 schedul es with 30m in resetting criteria........................ 44 3-19 Comparison of PR1.5 and PR1.5 schedul es with 30m in resetting criteria........................ 45

PAGE 7

7 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science COMPARISON OF LEVER PRESS AND NOSE POKE OPERANTS FOR AN ANALYSIS OF FOOD INTAKE AND MEAL PATTERNS IN MICE By Deniz Atalayer May 2008 Chair: Neil E. Rowland Major: Psychology Much research in the field and in laborato ry studies has focused on behavioral economics of food intake in several species. Operants such as lever press, nose poke, or key peck have been used to generate demand functions that express the relationship between th e cost of food and the amount of food consumed. There ha ve been very few such studies of motivated food seeking and demand in mice, and none has examined systemati cally consummatory cost or meal patterns. Using albino (CD1) male mice, the present study compares food intake an d meal patterns across a series of ratio consummatory schedules. Two operants, lever pr ess and nose poke, were compared in a between groups design. A closed economy was used in which the mice were in the test chambers for 23 h/day and earned all of their food via the operant under four fixed (FR5, FR10, FR25, FR50), variable (VR10, VR20, VR 50) and progressive (PR1.25, PR1.5, PR1.75) ratios. When averaged across all schedules, mi ce in the nose poke group consumed more food. Mice were run for 4 days at each ratio; there we re no systematic differences between the first and last day indicating that behavior al adjustments to schedule change s occurred very rapidly. Meal number significantly differed when two criteria for the definition of `a meal` (15 and 30 min) were used.

PAGE 8

8 CHAPTER 1 INTRODUCTION Obesity is a serious thre at to human health and has a growing in cidence in the recent decades. According to the most recent WHO report, at least 400 million adults globally are obese (World Health Organization, Obesity: 2005) Different approaches including use of animal models have been developed to study eating and yield insights into the etiology of obesity. From the early studies, it has been known that homeostas is has a crucial role in the regulation of food intake and energy balance (Levin, 2002; Teitelbaum, 1964; Berthoud, 2002; Woods, 1991, 1998). In addition, there have been advances in molecular biology and genetics that have identified a large number of genes responsible fo r satiety and hunger (Lubr ano-Berthelier et al., 2003a, 2003b; Branson, 2003). It has been estimated that 40 to 70 percent of the variation in obesity-related phenotypes in humans is heritabl e (Comuzzie and Allison, 1998). However, it has a complex etiology and is a multifactorial phenomenon (Blundell and Cooling, 2000; ErlansonAlbertsson, 2005) that arises from the interactio ns of multiple genes and brain neural systems (Gelegen et al. 2006; King, 2006; Petrovich a nd Gallagher, 2007), exte rnal factors from environment (Rolls et al., 2002; French, 2003), a nd behavior (Stellar, 1954; Saelens and Epstein, 1996; Drewnowski, 1995; Lowe and Butryn, 2007). Today it is highly recognized th at there are many central and peripheral factors involved in energy homeostasis and regulati on of food intake, and understa nding these mechanisms should lead to effective treatments in the control of obesity. For example, some studies reported that obesity in United States is to a great ex tent an economical issue (Drewnowski, 2004). Specifically, the cost of energy-dense foods is often low, and it has been shown that dietary energy density influences the regulation of f ood intake and body weight (Drewnowski, 2004; 2003). Thus, feeding behavior is highly influe nced by the economic structure of the current

PAGE 9

9 environment in which the individual lives a nd economic analysis provides a tool for understanding this type of beha vior. My goal in this study is to combine and examine the economic concepts within the context of nutritional homeostasis. Earlier studies with physiological, genetic or homeostatic animal models have failed to provide a systematic protocol to examine wh ether food intake changes under controlled laboratory conditions. Recently, the principles of economics, which relate the commodity to its price, have been applied to the field of behavior. In the case of eating, it has been shown food intake does change under different conditions of food availability (S umpter et al., 1999; RoveeCollier et al., 1982; Hursh, 1980; Hursh et al., 1988). Economics is considered a science of highly or ganized human behavior and is defined as the computational analysis of anticipated cost and benefits (Hursh, 1984). In most research protocols with animal models investigating eating in terms of economics of behavior, the price is defined as responses required per reinforcem ent or reward (e.g., food, water). These designs employ various experimental protocols that requir e some effort (cost) to obtain the reward. For example, on a ratio schedule, a specified number of responses are required to obtain a particular commodity or reinforcement. The sche dules used in the present study are: FIXED RATIO (FR). The ratio in which each unit of the item costs a fixed amount (e.g., number of responses), VARIABLE RATIO (VR). The ratio in which each food item costs a mean amount but the actual cost of each item varies around that mean, and PROGRESSIVE RATIO (PR). The ratio in which successive food items within an episode of feeding become progressively more costly. FR schedules are believed to be the most di rect and are the most commonly used method to set the price of a comm odity (Bauman et al., 1996; Hu rsh, 1984). By using different schedules, researchers aim to understand how an imals preference for food is shaped by the

PAGE 10

10 different economic environments and by extr apolation to the human condition, how an individuals food preferences may be affected in these economies where different types of food are usually available at all times. In order to survive animals mu st learn how to search for food. They often perform a series of behaviors traveling, catchi ng and consuming this food. Ea rlier studies on behavioral economics found that feeding patterns vary accordi ng to the cost of access to the food (Collier, 1985; Collier et al., 1986, Hursh, 1980). There are at least three definable co sts related to eating behavior (Morato et al., 1995): Cost of procuring access to food -procure ment cost (travel effort and/or time) Cost of consuming food consummatory cost (the cost within the patc h such as digging or climbing for food items, sucking, catching, hol ding etc., which are the equivalent of operant schedules in an experime ntal setting in most studies) Cost of processing food (physiologic consequences/digestion) Thus from the point of view of economics of behavior, the apparent question is how the animals will change the rate or amount of responding as the required effort to access to the commodity, the food is increased (Hursh, 1980). It is recognized that answ er is a product of a complex interaction between the availability and/or cost of the food and hunger state of the organism, however it can be defined and summar ized in a demand function which relates the price and food intake (Figure 1-1). The slope of the demand function is determined by the amount of the effort that an animal will work to obtain a commodity as the price for that commodity increases. The theory of economics clai ms that the consumption of most goods will decrease as price increas es (Hursh et al., 1988). Morato et al. (1995) compared the food inta ke in rats under low vs. medium vs. high procurement cost that were varied across days, and showed that when th e price of procurement was stable for more than about three days, rats ate less frequent and relatively larger meals when

PAGE 11

11 the price was high than the lower prices (Figur e 1-2). Many behavioral economists working with animal models claim that animals optimize their food intake in accordance with 3 goals. First, animals adjust their eating behavior to mainta in the daily food intake. Second, by eating less frequent, animals limit the time and energy spent for foraging. Third by adjusting the meal size, animals limit the physiological cost of processing ingested food. Thus, an optimal meal pattern represents a compromise between eating as infrequently as possi ble so as to minimize foraging cost, and eating meals as small as possible so as to minimize processing cost (Collier et al., 1986; Morato et al., 1995; Stephens and Krebs, 1995). Morato et al. (1995) showed that for high cost requirement (FR400) condition rats did save foraging cost by reducing their food intake. Some rats did not consume anything at all on those `expensive` days, and thus did avoid paying the high procurement price. Some rats however, continued to eat on high-price days but their meal s were not large enough to compensate for their reduced meal frequency, so their total food in take fluctuated. A ne w meal frequency was established on the first day that the new price was set but meal size seemed to change more slowly. A similar result was also found when cha nge in caloric density was used rather than change in procurement cost change (Le Magnen, 1992). Rats can use this strategy to increase their efficiency with which they expl oit resources. For example, if the procurement/consummatory cost is lower during the day than at night, rats will switch from a nocturnal to diurnal feeding (Jen sen et al., 1983). As mentioned above, when a day of high cost is followed by a day of low cost rather than by another high cost day, intake is reduced or even eliminated on high cost day and the deficit is ma de up entirely on the low cost day (Morato et al., 1995). Since the predictability of the environment affected the meal patterns in ra ts, the present

PAGE 12

12 study also tested this in mice by using random vs. ascending order reinforcement schedule ratios (variable vs. progressive ratio schedules). Open and closed economies. The literature of behavior al economics has offered two different settings for analysis of eating behavior -open and closed economiesand these lead to sometimes disparate results (Timberlake and Peden, 1987; Bauman, 1991; Killeen, 1995). In a closed economy all of the commodity is earned in the experimental session which often is in force all of the time. In an open economy pr otocol, a commodity may be earned during the experimental session, which is time-limited, but in the case of food, additional food is usually offered outside of the economy. For example, an animal may receive supplemental free rations after a session to ensure that it maintains a specific body weight. Further, an open economy usually requires food deprivati on prior to the experimental session. Hursh (1980) demonstrated that in an open economy as the rate of reinforcement schedule decreased, response rate slightly decreased too. In contrast, in a closed economy, the response rate increased remarkably (Killeen, 1995). Hursh (1980) explains th is dilemma by suggesting that in a closed economy, the commodity and costs are present at all times and animals can onl y obtain food as a consequence of the scheduled ratio of reinforcements. On the other hand, Killeen (1995) proposes that the trade between the animal and the environment is guided to a great extent by the deprivation level of the organism which he argues that in a closed economy setting it is not taken into much consideration. Open economy experiments effec tively study single meals, thus to acquire an eating episode in a given experimental session, food deprivation prior to th e session is essential. In a closed economy protocol, no food restriction is imposed by the experimenter; any change in meal taking is thus a direct consequence of the economic structure. However, it is known that food restriction increases food consumption and also reinforcing value of food (Raynor and

PAGE 13

13 Epstein, 2003). Collier (1985) argues that humans liv e in a closed economy as the food is almost always available, thus closed economy protocol s may provide more realistic models for human eating behavior. It has been shown that the meal size and subsequent interval to the next initiation of eating were positively correlated (LeM agnen, 1992); such a correlation which cannot be studied in a single meal session of the open economy environment. On the other hand, closed economy settings are concerned with sequ ence of meals or meal patterns. The present experiments thus employ a closed economy setting for studying meal patterning in mice. Determining the relationship be tween food consumption and the price for food (demand function) is the one of the main foci of the economics of ingestive behavior. To acquire such demand functions, systematic analyses on meal patterns ha ve been done with various animal models, with various schedules of reinforcement. To reach a consistency between days for a day-to-day meal pattern for each animal, researchers use time fram es ranging from several days to weeks for each schedule of reinforcement (Morato et al., 1995, Co llier et al., 1986). To obtain a demand curve for the rats, Hursh (1980) reported th at it required 5 to 6 months. They adapted a rapid method for determining demand curves but it still required 40 da ys. Of great significance to this project, it has been reported in a study using rats with a le ver press operant that a demand function can be generated in a much shorter period (Raslear et al. 1988). They suggested that in an environment where the cost for food is changi ng and unpredictable, ra ts are able to adjust their food intakes very rapidly probably less than one day. We do not know whether all these findings on rats reflect the facts about mice there has not been much consistency on the field about meal patterns in mice. Studies suggested that the average number of meals per da y shows substantial variation for different animal species

PAGE 14

14 (Berthoud, 2002). When several fact ors are controlled, such as stress, existence of predators or social competitors, light-dark cy cle and given adequate food, speci es-specific meal patterns seem to become apparent (Madden, 2005). The initiations and terminations and of meal to meal intervals (MMI) are called the meal pattern of rodents. However, defining a meal within each session, and differentiating a pause within a meal ( intra -meal intervals) from an MMI require a suitable time resolution (typically <5 min). 2 min after animals stopping eating, the proba bility of animals resu me eating within the next 10 min, was found to be maximal. That probab ility decreases from 10 min to 40 min, and is minimal at 40 min and increases again for longer (because the animal is probably hungry again). Thus a meal has been defined as an eating episod e initiated after at leas t 40 min of non-eating period (Kissileff, 1970; LeMagnen, 1992; Clifto n, 2000). In mice, spontaneous food intake occurs in number of eating bouts separated by periods of non-eating or inter-meal or meal-tomeal intervals that typically are at least 30 min in length (Clifton, 2000). However, there is a drastic discrepancy between different laborator ies for the number of meals taken per day in experiments with mice (Petersen and McCart hy, 1981; Gannon et al., 1992; Strohmayer and Smith, 1987; Vaughan and Rowland, 2003; Fox a nd Byerly, 2004; Richard and Low, 2007). Table 1-1 summarizes these results fo r operant and non-operant conditions. Although rats typically do not show so much between-laboratory variability in results, some studies with rats have suggested differences with use of a nose poke compared with a more traditional lever press operant (Ettenberg et al., 1 981). Also it has been claimed that changes in the meal patterns in different st udies with rats might arise from the effects of the experimental manipulations; such as single vs. multiple presen tation of food, method of choosing between two options, water availability, ratio or interval sc hedules etc. (Clifton et al., 1984). In their study,

PAGE 15

15 dose-dependent reduction in re sponse rates was not found when nose poke operant was used. According to their report even at the highest doses, rats continued to respond. Thus, they concluded the effect of the dr ugs on responding rate was partially a result of th e instrumental paradigm that has been employe d in the experimental design. Roche and Timberlake (1998) emphasized the importance of designing protocols that examine the natural behavioral traits and behavior al hierarchies that may be species rather than responses that are completely arbi trary. It has been demonstrated that there is a species-typical perceptual/motor organization in rats related to common maze equipm ent, such as straight alleys, and radial arm mazes and in many studies it is suggested that the operant has a great deal of importance (Roche and Timberlake, 1998; Schindler et al., 1993; Marusich and Branch, 2006). It is has been suggested that the baseline le vel of nose poke operant response was high and acquisition with food reinforcement occurred rapi dly, particularly when compared with a lever press operant response. Therefore, the nose-poke response appeared to be particularly useful for the study of the acquisition of operant responses (Schindler et al., 1993). Thus, we compared two different operants, no se poke which has not been much used and lever press which has been most commonly used in these types of protocols. For analytic simplification in defining the pa rameters of feeding behavior in this particular species, we designed a parametric study with an operant (nose poke) that to our knowledge has not been used previously in an eating task in mice and using various environmental conditions of food availability.

PAGE 16

16 Table 1-1. Earlier Studies On Meal Patterning In Mice MOUSE STRAIN OPERANT DIET #OF MEALS/DAY REFERENCE Small `S` and large `L` inbred Overhead door panel Powdered rodent chow 12 (5min) Petersen and McCarthy (1981) SWR/J Recess at the floor level Powdered rodent chow 36 (5min) Gannon et al (1992) C57BL/6J (lean and ob/ob) Sipper spout in cage Liquid diet EC116 50(male) 30(female) (1 and 5min) Strohmayer and Smith (1987) C57BL/6J Lever press Noyes 20mg pellets ~8 (24 h food access through FR schedules) Vaughan and Rowland (2001) C57BL/6J (lean and ob/ob) Lever press and food receptacle Noyes 20mg pellets 2-10, function of access cost (10min) Vaughan and Rowland (2003) 129/B6 (wild type for BNDF +/-) Pellet removal from trough BioServ 20mg pellets ~12 (18 hr food access) Fox and Byerly (2004) 129/B6 (wild type for BNDF +/-) Liquid diet from 0.02ml dipper Isocal-High fat liquid ~15 (18 hr food access) Fox and Byerly (2004) 129/B6 (wild type for MC4R +/-) Lever press and food receptacle (procurement cost) Noyes 20mg pellets 2-7, function of procurement cost (10min) Vaughan et al (2005) 129/B6 (wild type for MC4R +/-) Lever press and food receptacle (progressive ratio) Noyes 20mg pellets 25-50, function of 20 vs 3min reset criterion Vaughan et al(2006) C57BL/6J (wild type) Lever press (procurement costFRs) BioServ 20mg pellets (4g per day) (735sec) Richard and Low (2007)

PAGE 17

17 cost 024681 0demand 5 10 15 20 25 30 35 Figure 1-1. The demand functi on. The figure summarizes the re lation between the cost and demand. The slope of the function is determined by the amount of the effort that a person or an animal will work to obtain a commodity as the price for that commodity increases (the numbers do not indicate any real data; the graph was drawn for conceptual purposes).

PAGE 18

18 Relative procurement cost Low Med HighMeal number/day or size (g) 0 2 4 6 8 10 12 Meal number Meal size Figure 1-2. The demand function. The slope of the demand function is determined by the amount of the effort that an animal will wo rk to obtain a commodity as the price for that commodity increases. The theory of economics claims that the consumption of most goods will decrease as price increases (the numbers do not indicate any real data; the graph was drawn fo r conceptual purposes).

PAGE 19

19 CHAPTER 2 MATERIALS AND METHODS Subjects A total of sixteen m ale albino (CD1) mice, in itially about 3 months of age were used. We did not include female mice in our design to avoid the possibility that estrous cyclicity would interact with our analys is of meal patterns. Th e average weight of the animals was 39.7.7g at the beginning and 45.8g at the end of the experi ments (Mean s.d.). During the experimental periods, mice lived in th e operant chambers for 23 hr per day. The mice were weighed daily during a 1 hr cleaning period and were kept in empty holding cages. The operant chambers were wiped with 70% et hanol solution and distilled water each time before the mice were placed. When not in e xperiments, mice were housed in a standard, polycarbonate cages (separate vivaria) with Puri na Chow pellets and ta p water available ad libitum and a 12:12 light cycle in place (light s on 0700). During the experiments, mice obtained 20 mg; complete nutritional mouse pellets (Research Diets Inc) when they completed an imposed cost determined by the reinforcement schedule. Our preliminary studies demonstrated that the spillage with this type of pellets was typically very little. Tap water was available freely from a sipper tube. The Psychology department vivaria are part of the centralized Univ ersity of Florida Animal Care program with full AAALAC accred itation. Animal use is approved by a campuswide IACUC and is compliant with the recomme ndation of the Guide for the Care and Use of Laboratory Animals (1996). Apparatus Sixteen operant cham bers (Med Associates : 13x13x12 cm with Plexig las and metal walls and stainless steel grill floor plus solid nesting platform) enclosed in ventilated, noise attenuating

PAGE 20

20 cabinets with the same 12:12 light cycle as the vivarium (a 15 watt bulb in a standard light fixture run from a 24 hr timer) were used in the experimental procedures in the present study. All chambers were equipped with one lever press and one nose poke operant device, located 2cm above the floor, situated on one wall on either si de of a food aperture. Water was supplied from sipper tubes mounted on the wall opposite side to the food magazine and the two operant devices. In each chamber and for a given mous e only one manipulandum was active during the whole experimental protocol. A record of the total pellets obtained by mice and number of responses (nosepoking and leverpressing) were acquired by the MEP-PC IV computer so ftware (MED Associates, St. Albans, VT). The computer recordings allowed an accurate analysis of the number of meals and the amount eaten at each meal. Data were accu mulated in each 15 min (for FR and VR) and 5 min (for PR) time bins for each 23 hr period. Procedure To investigate whether the for m of the operant (nosepoking vs. leverpressing) influences the economic analysis of meal patterning, mice we re divided randomly in two groups of 8, with one group obtaining food pellets by pressing the lever and th e other group by nosepoking. Prior to the study, to habituate mi ce to the operant chambers and to the novel pellets, a 1 hr training period was applied with free food was av ailable in the food magazine of the operant chambers without any cost. Later, a fixed ratio-1 (FR1) where a pellet was delivered as a consequence of one response on the active manipul andulum, was used as a magazine training for a day or two to acclimate mice to the operant conditioning protocol. For the training, a mouse was considered to have learned the conditioning paradigm if they earned enough pellets to maintain their body weight. No food deprivation protocol was used prior to the experimental

PAGE 21

21 sessions. After they successfully learned to press the lever or nose poke, animals were exposed to several reinforcement schedules as the experimental design. Experimental sessions lasted 23 hr. A short protocol (4 days with each ratio) was used because previous studies in our laboratory (Vau ghan et al., 2006) have indicated that mice adjust to changes in ratios within a day or so. Mice we re exposed to an incrementing series of fixed ratios (FR1, FR5, FR10, FR25, FR50) and then, va riable ratios (VR10, VR20, VR50), and finally progressive ratios (PR1.25, PR1.5, PR1.75). In the VR the actual ratios se lected randomly by the program were VR10; 1, 5, 10, 15, 19, for VR20; 2, 10, 20, 30, 38, for VR50; 5, 25, 50, 75, 95. In the PR, the number of responses required for the next (n+1)th pellet in a series, Rn+1= Rn x 1.25 and Rn+1= Rn x 1.5 and Rn+1= Rn x 1.75 (Rn = nth response requirement). The resulting number was rounded to the nearest integer, giving the following sequences: for PR1.25; 1, 2, 2, 2, 2, 4, 4, 5, 6, 8, 10, 12, 15, for PR1.5; 1, 2, 2, 3, 5, 8, 11, 17, 26, 38, 58, 86, 130 and PR1.75; 2, 3, 5, 9, 16, 29, 50, 88, 154, 269, 471, 825. Further, in the PR se ries, whenever 15 min elapsed without a response the ratio was reset to the initial value of the pa rticular schedule. This reset allows the animals to effectively quit eating when the unit co st of a pellet becomes too high and shift to another patch (in this case with a 15 min te mporal boundary). For comp arison, we additionally ran a PR 1.5 reinforcement schedule using a 30 min reset criterion. In the final phase of the e xperiment, after all the above schedules were completed, to determine whether mice can follow schedule changes even more rapidly as well as to determine whether differences that we obser ved across schedules were not merely due to experience or age, each reinforcement schedule was employed for one day consecutively. Analyses and Data Acquisition In the present experim ent, two different meal -to-meal interval (MMI) criteria (15 and 30 min) were applied. The raw data from the co mputer recordings, show ed how many responses

PAGE 22

22 were made and the number of pellets earned at each 15 min (for FRs and VRs) and 5 min (for PRs) throughout the whole 23 hr period (1380 mi n) each day. Non-responding (non-eating) episodes were showed as zero for each 15min time bin in the computer software and a minimum of 15 or 30 min was used to distinguish separate meal events. After the numbers of meals for each mouse were counted by the experimenter for each day of each schedule from the raw data, the mean meal size was derived by dividing the nu mber of total pellets by the number of meals for the particular day. With one exception, no syst ematic difference was noted across the four days for pellet intake per day. Thus, the mean for number of meals, total pellets earned and meal sizes were computed for each mouse and for each reinforcement schedule by averaging them over for four days. Parameters were analyzed for significance with SPSS computer software by using repeated measures analysis of varian ce (ANOVA), with the operant (nosepokers vs. leverpressers) as betwee n-subject variable and schedules as within-subject variable. Analyses within each ratio schedule type (FR, VR and PR), One-way ANO VAs were used to measure the significance of each variable. Independent ttests were used where necessary. In all cases, p< 0.05 p was considered significant. Graphs are drawn using Sigma-plot computer software.

PAGE 23

23 CHAPTER 3 RESULTS When averaged across all of the ratio schedules, mice consumed (Mean S.D.) 259.5 56.7 pellets per day (Figure 3-1) distributed as either 20 7.5 or 11 3.2 meals at the 15 and 30 min MMI, respectively. The corresponding meal sizes were 15 6 and 28 17.8 pellets. Since each pellet is 20mg, this corresponds to m ean meal sizes approximately of 0.2 and 0.4 g. With the exception of FR10 [ F (3,60) =8,193; p < 0.01 ], under each schedule, meal numbers, meal sizes and the number of pellets did not differ si gnificantly across th e four days of each schedule. Thus, data were averaged across 4 days to give a single datum for each mouse. When averaged across schedules, nosepokers (NP) consumed significantly more pellets than leverpressers (LP) (Mean S.D. ; NP: 274.5 51.7, LP: 244.6 58). ANOVA analyses resulted in a group effect for the operant [ F (1,174) = 13.086; p<0.01]. FR Schedules The num ber of pellets taken in the FR phases is shown in Figure 3-2, the meal numbers in Figure 3-3a, b and the meal si zes in Figure 3-5, 3-6. Total pe llets earned per day differed significantly across the four FR (FR5, FR10, FR25, FR50) schedules [ F (3,60) =5.763; p < 0.05 ]. Post-hoc Bonferroni test showed that mice at FR50 mice consumed fewer pellets compared to their intake on the other three FR schedules (Figure 3-2). Across FR schedules, there were significant differences between LP and NP for total pellets [ F (1,62) =7.818; p < 0.01 ] (Figure 3-2), meal numbers at 30min MMI [F (1,62) =4.802; p < 0.05 ] (Figure 3-4), and meal size at 15min MMI [F (1,62) =10.763; p < 0.01 ] (Figure 3-5). Post-hoc t -tests showed that, NP consumed more number of pellets daily [ t (62) = -2.796; p < 0.01], had more meals with the 30 min MMI definition criterion [ t (62) = -2.191; p < 0.05 ,] and larger meals with the 15 min MMI criterion [ t (62) = -3.281; p < 0.01 ].

PAGE 24

24 VR Schedules The num ber of pellets taken in the VR phases is shown in Figure 3-7, the meal numbers in Figure 3-8, 3-9 and the meal si zes in Figure 3-10, 3-11. Total pellets earned per day differed significantly across the three VR (VR10, V20, VR50) schedules [ F (2,45) =15.728; p < 0.01 ]. Post-hoc Bonferroni test showed that mice at VR50 mice consumed fewer pellets compared to their intake on the other two VR schedules (Figure 3-7). Across VR schedules, there was no significant di fference in the number of pellets taken by NP vs. LP (Figure 3-7). However, the type of operant showed a significant between-subjects effect on meal number [ F (1,46) =5.213; p < 0.05 ] (Figure 3-8) and meal size under with 15min MMI definition criterion [ F (1,46) =7.123; p < 0.01 ] (Figure 3-10). The t-test revealed that nosepokers took fewer but larger meals th an leverpressers [for meal numbers; t (46) = 2.283; p < 0.05, for meal size; t (46) = -2.669; p < 0.01 ]. PR Schedules The num ber of pellets taken in the PR phases is shown in Figure 3-12, the meal numbers in Figure 3-13, 3-14 and the meal sizes in Figure 3-15, 3-16. Total pellets earned per day differed significantly across the four PR (PR1.25, PR1.5, PR1.75, PR1.5/30min) schedules [ F (3,60) =8.500; p < 0.01 ]. Post-hoc Bonferroni test showed that at PR1.25 mi ce consumed more pellets compared to their intake on the other three PR schedules (Figure 3-12). In PR schedules, operant type displayed a si gnificant between-subjects effect for total pellets earned per day [ F (1,62) =6.640; p < 0.05 ] (Figure 3-12), and meal number with 30min MMI [ F (1,62) =5.676; p < 0.05 ] (Figure 3-9b). Meal si zes did not differ signi ficantly (Figure 3-15, 3-16). The comparison between 15 min and 30 min reset criteria applied in PR1.5 schedules did not seem to make any difference for total pellets (Figure 3-17), number of meals (Figure 3-18) or

PAGE 25

25 meal size (Figure 3-19) per day except when 30min MMI used [ F (1,30) =15.896; p < 0.01 ]. A follow-up independent t-test show ed when 30min MMI used, mice had more meals per day with the 30 than the 15 min reset criterion [ t (30)= -3.987; p < 0.01 ]. Comparison Between Schedule Types Daily pellet intake showed significant variation betw een three types of ration [ F (2,173) =17.846; p < 0.01 ]. A follow up post-hoc analysis s howed that m ice t ook more pellets per day under PR schedules compared to FR and VR schedules. PR schedules also resulted in more number of meal intakes but only when 15min MMI was used [F (2,173) =233.987; p < 0.01]. Last Phase The last ph ase of the experiment that each schedule were employed for one day in the exact same order with the whole procedure, sh owed no significant difference on the total number of pellets earned daily when averaged ove r the schedules. However, when one to one comparisons between the number of pellets comput ed by the average of previous four days for a schedule and the number of pellets per day as the last phase for the same schedule, some reached the significance [for FR5: t (15) =-3.629; p < 0.01 for VR50: t (15) =5.385; p < 0.01 for PR1.25: t (15) =2.474; p < 0.05 and for PR1.75: t (15) =3.142; p < 0.01 ]. Table 3 summarizes the result. In addition, mice obtained more number of pellets [ F (2,173) =5.667; p < 0.01 ] and more number of meals per day [for 15min MMI F (2,173) =79.679; p < 0.01 and for 30min MMI F (2,173) =4.356; p < 0.05 ] in PR schedules compared to FR a nd VR schedules in this last phase of consecutive one-day employment of the each schedule. When 30min MMI was used to define `a m eal`, there was a significant between-subjects effect of operants [ F (1,174) =8.758; p < 0.05 ]. Independent t -test conducted as a follow-up showed that nose pokers ha d more meals per day [ t (174) = -2.959; p < 0.05 ]. However, when

PAGE 26

26 15min MMI criteria was used to define a m eal, the group effect of operants did not reach significance. The difference between the meal si zes nose pokers and lever pressers on the other hand, reached statistical significance for 15mi n MMI but not for 30min MMI definition criteria [ F (1,174) =13.143; p < 0.01 ] The t-test analysis concluded that nosepokers took larger meals when 15min MMI definiti on criteria was used [t (174) = -3.625; p < 0.01 ]. Regardless of the operants, de fining a meal by 15min MMI vs 30min MMI resulted in different number of meals a nd the meal size per meal. Sta tistical significance were at F (1,350) =210.049; p < 0.01 for number of meals and F (1,350) =86.933; p < 0.01 for meal size. Mice ate significantly fewer and larger meals when 30min MMI used compared to 15min MMI [ t (350) = 14.493; p < 0.01 and t (350) = -9.324; p < 0.01 respectively].

PAGE 27

27 schedulesPR1.530min PR1.75 PR1.5 PR1.25 VR50 VR20 VR10 FR50 FR25 FR10 FR5 Mean totalpellets400.00 300.00 200.00 100.00 0.00 Figure 3-1. Total pellets per da y by operant with FR schedules.

PAGE 28

28 Figure 3-2. Total pellets per da y by operant across FR schedules.

PAGE 29

29 FR5 FR10FR25FR50number of meals 0 2 4 6 8 10 12 14 16 18 20 LP NP Figure 3-3. Daily number of meals with 15min MMI by operants across FR schedules.

PAGE 30

30 FR5 FR10FR25FR50number of meals 0 2 4 6 8 10 12 14 16 LP NP Figure 3-4. Daily number of meals with 30min MMI by operants across FR schedules.

PAGE 31

31 FR5 FR10FR25FR50meal size 0 5 10 15 20 25 30 LP NP Figure 3-5. Meal size with 15min MMI by operant across FR schedules.

PAGE 32

32 FR5 FR10FR25FR50meal size 0 10 20 30 40 LP NP Figure 3-6. Meal size with 30min MMI by operant across FR schedules.

PAGE 33

33 Figure 3-7. Total pellets per da y by operant across VR schedules.

PAGE 34

34 VR10 VR20 VR50number of meals 0 2 4 6 8 10 12 14 16 18 20 LP NP Figure 3-8. Daily numbers of meals with 15min MMI by operants across VR schedules.

PAGE 35

35 VR10 VR20 VR50number of meals 0 2 4 6 8 10 12 14 LP NP Figure 3-9. Daily number of meals with 30min MMI by operants across VR schedules.

PAGE 36

36 VR10 VR20 VR50meal size 0 5 10 15 20 25 30 LP NP Figure 3-10. Meal size with 15min MMI by operant across VR schedules.

PAGE 37

37 VR10 VR20 VR50meal size 0 5 10 15 20 25 30 35 LP NP Figure 3-11. Meal size with 30min MMI by operant across VR schedules.

PAGE 38

38 Figure 3-12. Total pellets per da y by operant across PR schedules.

PAGE 39

39 PR1.25PR1.5PR1.75PR1.5 w/30minnumber of meals 0 5 10 15 20 25 30 35 LP NP Figure 3-13. Daily number of meals with 15min MMI by operants across PR schedules.

PAGE 40

40 PR1.25PR1.5PR1.75PR1.5 w/30minnumber of meals 0 2 4 6 8 10 12 14 16 18 LP NP Figure 3-14. Daily number of meals with 30min MMI by operants across PR schedules.

PAGE 41

41 PR1.25PR1.5PR1.75PR1.5 w/30minmeal size 0 2 4 6 8 10 12 14 16 18 LP NP Figure 3-15. Meal size with 15min MMI by operant across PR schedules.

PAGE 42

42 PR1.25PR1.5PR1.75PR1.5 w/30minmeal size 0 20 40 60 80 100 LP NP Figure 3-16. Meal size with 30min MMI by operant across PR schedules.

PAGE 43

43 PR1.5 PR1.5 w/30minnumber of pellets 0 50 100 150 200 250 300 350 LP NP Figure 3-17. Comparison of PR1.5 and PR1.5 schedul es with 30min Program resetting criteria (daily number of pellets).

PAGE 44

44 PR1.5 PR1.5 w/30minmeal size 0 10 20 30 40 50 LPmealsize 15MMI LPmealsize 30MMI NPmealsize 15MMI NPmealsize 30MMI Figure 3-18. Comparison of PR1.5 and PR1.5 schedul es with 30min program resetting criteria (meal size).

PAGE 45

45 PR1.5 PR1.5 w/30minnumber of meals 0 5 10 15 20 25 30 LPmeal# 15MMI LPmeal# 30MMI NPmeal# 15MMI NPmeal# 30MMI Figure 3-19. Comparison of PR1.5 and PR1.5 schedul es with 30min program resetting criteria (daily meal numbers).

PAGE 46

46 Table 3.1 The average for total pellets per day fo r main procedure and last phase of scheduling (means.d.) schedules Main procedure Last phase of schedules FR5 25140 30863 FR10 26130 27040 FR25 22768 22650 FR50 19063 18861 VR10 29345 29738 VR20 26834 25456 VR50 21540 16056 PR1.25 32551 30855 PR1.5 27933 27935 PR1.75 28624 26341 PR1.5-30MIN 27542 25657

PAGE 47

47 CHAPTER 4 DISCUSSION The m ain focus of the present study was to de sign an instrumental conditioning paradigm that would allow us to conduct a systematic analys is for meal patterns in mice as a function of effort and with and explicit comparis on of two different operants used. Mice consumed ~250 pellets (~5g /day) but the meal distribu tion was critically dependent on the MMI criterion: at the 15m in MMI, the overall mean was ~28 meals with a meal size of ~10 p-ellets (~0.2g), whereas at the 30 min MMI the overall mean was ~15 meals with a meal size of ~18 pellets (~0.4g) a day, regardless of the operant. The number of meals and average total intake consumed (grams) per day did show c onsistency with most of the previous research in the literature (Table 1). This might suggest that using differe nt operants and MMI criteria for defining a meal have reasonable effects on the results for eati ng behavior analysis in mice (Kissileff, 1970). One of the findings of the present study was th at for each reinforcement schedule, the meal pattern of mice did not significantly differ across four days of each schedule with the exception of FR10. However, the statistical significance fo r the FR10 condition disappeared when the data from first day was excluded from the four da ys of FR10 schedule and analysis was conducted across the second, third and fourth days. It might be possible that mice had difficulty adjusting to the novel eating condition as the first day of FR10 schedule wa s the first day that they encountered a cost that required a relatively more effort compared to free access or FR1 or FR5 schedules. This particular effect for the initia l FR10 schedule shown in this study was also found in other studies examinin g meal patterns in mice (Richard and Low, 2007). The rapid adaptation of mice to the changing schedules, usually 1 but at most 2 days shown here agree with a study by Rasl ear et al. (1988) with rats in an operant task that showed a

PAGE 48

48 stable relationship between food consumption and price for food throughout seven consecutive days after schedule change. Thus, our findings in mice, along with Rasl ear`s results in rats indicate that for consummatory costs, rodents ha ve a substantial capability to adapt rapidly the changes in the schedules of reinforcement. Future use of short-term prot ocols like the procedure in this study should allow furthe r research using antiobesity drugs that have short half-lives and/or to shorten the time period that is needed to co mplete an economic profile. We have presented a parametric study with an animal model to examine the role that operant plays on meal pattern analysis in mice. Hence, nosepokers obtained considerably more pellets per day when averaged across all sche dules. Also, when analyzed separately, in each group of ratio schedule (FR, VR and PR) operant type appeared to be an important factor affecting the total number of pellets eaten. This showed that in reinforcement schedule paradigms, using different operant s has a crucial effect on the re sults and the nose poke operant is particularly useful in this type of research. These findings agree with some of the studies with rats comparing nose poke and lever press operants (Schindler et al., 1993; Ettenberg et al., 1981; David et al., 2001). Schindler et al. (1993) suggested that acquisition of the nose poke response in rats occurred much more rapidly than of othe r operants. In addition they reported that if there is no experimenter intervention such as shapi ng, acquisition of lever pressing response occurs rather slowly. Thus, a nose poke operant might be useful whenever short-term procedures are to be used (Schindler et al., 1993). On the other hand some studies w ith rats also indicated that the type of operant (lever press vs. nose poke) did not have a signifi cant effect on the acquisition of intravenous heroin/cocaine self -administration or dose-related responding (David et al., 2001). However, in that particular study, only two type s and low ratio schedules (FR1 and FR3) were

PAGE 49

49 used. In order to make an accurate comparison, more variety of reinforcement schedules is required. Eating behavior in animals o ccurs in episodes (Collier and Johnson, 2004). The size of each bout depends heavily on the eating environment of the animal such as the availability of food resources and effort that requires consuming the particular resource. Adjusting the meal size for required costs for food is part of the economizi ng strategy of the animal in eating behavior. In the present design, meal size was affected by the operant type, as nose po kers ate bigger meals regardless of the different schedule requirements. It has been shown that ra ts increased their meal size as they decreased the fre quency of meals (decreasing meal numbers) in a compensatory fashion as the required cost to access to food in creased (Mathis et al., 199 5; Collier et al., 1998; Collier et al., 2002). However, total number of pellets earned per day showed significant changes under each schedule which indicated that for our results daily intake was not unaffected by the changes in the schedules. In other words, daily in take was not maintained as it was suggested in the literature (Morato et al., 1995; Collier et al., 1998). Nevertheless, it was also claimed that at the highest costs, animals made a sacrifice by decreasing their food intake, thereby avoid paying the expensive price (Morato et al., 1995). Thus, at the highest costs for each ratio in our experiment, it could be argued that mice avoide d excessive consummatory cost by sacrificing some of their intakes and reduced the number of pellets earned a day. This is, of course, the defining feature of a demand function. The sche dule-associated decrease in food intake was apparent in FR and VR schedules as at FR50 and VR50 mice c onsumed less pellets. This was also in agreement with a recent mice meal pattern study suggesting that FR40 cost was not enough to alter the meal pattern of mice from the baseline (Johnson and Low, 2007). For PRs, significance was found at the lowest PRs as PR1.25 was resulted in more pellet intake per day.

PAGE 50

50 PR schedules were found to result in highe r number of pellets earned per day when compared to the FRs and PRs. However, sinc e a considerable amount of time (~5 months) elapsed between the first day of FR5 and the first day of PR schedules, it is reasonable to argue that PR schedules did result in more number of pellets per day because of that amount of the time that has passed. In an effort to control this issue, after the last PR schedule, we conducted each schedule for only one day in the same order to see if the intake was comparable with the previous 4-day of intake. When averaged across all elev en reinforcement schedule s of one day, the total pellets earned per day did not differ from the pr evious 4-day scheduling when they are averaged across schedules. On the other hand, comparing each schedule separately with the 4-day average of the same schedule resulted in a significant diffe rence between the pellets obtained per day for some of the reinforcement schedules (FR 5, VR50, PR1.25 and PR1.75). However, opposite to what we wanted to control as a potential confoundi ng of increase in intake as the time passed, the number of pellets decreased-not increased when compared to the previous 4-days of scheduling. Thus, the argument for the possible confounding e ffect of the time passage on the higher number of pellets for the PR schedules was eliminated. It can be concluded that PR schedules resulted in significantly more number of pellets compared to the FR and VR schedules. In addition, in this one day protocol of each reinforcement schedule, the comparison between the three reinforcement schedules (FR, VR, PR) in terms of total pellets and meal numbers per day, agree with the results from the same type of comparison of FR, VR and PR of the main 4-day procedure. Thus, mice took more pellets and meals (both with 15min MMI and 30min MMI) daily in PR schedules compared to FR and VR schedules also at the 1-day phase of the experiment.

PAGE 51

51 Our data revealed that the criterion used fo r the definition of ` a meal` has an important effect on the results. Mice appeared to take c onsiderably more meals when 15min was used as MMI compared to 30min MMI. Reciprocally, 30mi n MMI resulted in larger meal sizes when compared to 15min MMI. The group effect of the operants also differed when different MMIs were used. All these comparisons in the food intake parameters between the two MMIs indicated that defining `a meal` has a cruc ial influence on the detail of the meal pattern of animal models. Although it would be hard to determine which of th e MMIs is a better representation for `a meal` for this strain of mice, 15min MMI seemed to concur with some of the earlier studies with mice (Petersen and McCarthy, 1981; Vaughan and Ro wland, 2003; Fox and Byerly, 2004; Vaughan et al., 2005; Richard and Low, 2007) although strain differences cannot be excluded as a source of variance. Comparison between 30min vs. 15min reset criteria applied separately on PR1.5 schedules indicated no different meal patte rns. Since the first few pellets in each meal were the cheapest, mice ate many small meals by quitting eating as th e cost increased and letting the program reset itself to the lower initial cost. However, mice ma intained the total pellet intake same in both PR1.5 schedules. Future directions In our study we looked at the c onsummatory cost, which is the equivalent of the near-the-patch cost. We ar e currently conducting a foraging cost design study by applying both Colliers two cost s; procurement and consummatory costs with the same strain of mice. We may further look at the genetic models to see how th ese meal patterns change in genetically mutated obese mice as an im plication for the human obese models.

PAGE 52

52 LIST OF REFERENCES Baum an R. (1991). An experimental analysis on the cost of the food in a closed economy. J Exp Anal Behav, 56(1): 33-50. Bauman R.A., Raslear T.G., Hursh S.R., Shur tleff D., Simmons L. (1996). Substitution and caloric regulation in a closed economy. J Exp Anal Behav, 65(2): 401-422. Berthoud H-R. ( 2002). Multiple neural system s controlling food intake and body weight. Neuroiscience and Behavioral Reviews, 26: 393-428. Branson R, Potoczna N, Kral JG, Lentes KU, Ho ehe MR & Horber FF. (2003). Binge eating as a major phenotype of Melanocortin 4 receptor gene mutations. New England J Med, 348: 1096-1103. Blundell J.E., Cooling J. (2000). Routes to obesity: phenotypes, food choices and activity. British Journal of Nutrition, 83(6):33-38. Clifton P.G., Poplewell D.A., Burton M.J. (1984 ). Feeding rate and meal patterns in the laboratory rat. Physiology & Behavior, 32(3):360-374. Clifton P.G. (2000). Meal patterning in rodents: psychopharmacological and neuroanatomical studies. Neuroscience and Biobehavioral Reviews. 24:213-222. Collier G.H. (1985). Satiety: an ecological perspective. Brain Res Bull. 14(6):693-700. Collier G.H., Johnson D.F., Hill W.L., Kaufman L.W. (1986). The economics of the law of effect. J Exp Anal Behav, 46(2):113-136. Collier G.H., Johnson D.F., Berman J (1998). Patch choice as a function of procurement cost and encounter rate. J Exp Anal Behav,69(1):5-16. Collier G.H., Johnson D.F., Mathis C. (2002). The currency of procurement cost. J Exp Anal Behav,78(1):31-61. Collier G.H., Johnson D.F.(2004). The paradox of satiation. Physiol Behav.82(1):149-53 Com uzzie A.G., Allison D.B. (1998). The search for human obesity genes. Science, 280:1374 1377. David J., Polis I., McDonald J., Gold L.H. (2001). Intravenous se lf-administration of heroin/cocaine combinations (speedball ) using nose-poke or lever-press operant responding in mice. Behavioral Pharmacology, 12;25-34. Drewnowski, A. (1995). Energy intake and sensor y properties of food. The American Journal of Clinical Nutrition, 62 (5): 1081-1085. Drewnowski A. (2003). The role of energy density. Lipids, 38(2):109-115

PAGE 53

53 Drewnowski A. (2004). Obesity and the food envi ronment Dietary energy density and diet costs. American Journal of Preventive Medicine, 27(3):154-162. Erlanson-Albertsson C. (2005). How palatable food disrupts appetite regulation. Basic and Clinical Pharmacology and Toxicology, 97:61-73. Ettenberg A., Koob G.F., Bloom F.E. (1981). Response artifact in the measurement of neuroleptic-induced anhedonia. Science, New Series, 213(4505):357-359. Fox E. A. and Byerly M.S. (2004). A mechan ism underlying mature-onset obesity: evidence from the hyperphagic phenotype of brain-deri ved neurotrophic factor mutants. Am J Physiol Regul Integr Comp Physiol, 286:994-1004. French S.A. (2003). Pricing effects on food choices. J. Nutr. 133:841-843. Gannon K.S., Smith J.C., Henderson R., Hendr ick P. (1992). A syst em for studying the microstructure of ingestive behavior in mice. Physiol Behav, 51(3):515-21. Gelegen C., Collier D.A., Campbell I.C., Oppe laar H., Kas M.J.H. (2006). Behavioral, physiological, and molecular differences in resp onse to dietary restri ction in three inbred mouse strains. Am J Physiol Endocrinol Metab, 291:E574-E581. Guide for the Care and Use of Laboratory An imals (1996). Institute of laboratory animal resources commission on life scienc es national research council. National Academy Press. Washington, D.C. Hursh S.R. (1980). Economic concept for the analysis of behavior. J Exp Anal Behav, 34(2):219238. Hursh S.R. (1984). Behavioral economics. J Exp Anal Behav, 42(3): 435. Hursh S.R., Raslear T.G., Shurtleff D., Baum an R.A., Simmons L. (1988). A cost-benefit analysis of demand for food. J Exp Anal Behav, 50(3):414-440. Jensen G.B., Collier G.H., Medvin M.V. (1983). A cost-benefit analysis of nocturnal feeding in the rat. Physiol Behav., 31(4):555-9. Killeen P.R. (1995). Economics, ecologists, a nd mechanics: The dynamics of responding under conditions of varying motivation. J Exp Anal Behav, 64:405-431 King B.M. (2006). The rise, fall, and resurrectio n of the ventromedial hypothalamus in the regulation of feeding behavior and body weight. Physiol. Behav.,87:221-244. Kissileff H.R. (1970). Free feeding in normal and r ecovered lateral rats monitored by a pelletdetecting eatometer. Physiol. Behav., 5(2):163-174. LeMagnen J. (1992). Neurobiology of feeding a nd nutrition. Academic Press, San Diego.

PAGE 54

54 Levin B.E. (2002). Glucosensing neurons do more than just sense glucose. International Journal of Obesity. 25(5):68-72. Lowe M.R. and Butryn M.L. (2007). Hedoni c hunger: A new dimension of appetite. Physiol. Behav., 91:432-439. Lubrano-Berthelier C., Cavazos M., Dubern B. (2003a). Molecular genetics of human obesityassociated MC4R mutations. Ann N.Y. Acad Sci 994: 49-57. Lubrano-Berthelier C., Cavazos M., Le Stunff C ., Haas K., Shapiro A., Zhang S., Bougneres P., Vaisse C. (2003b). The human MC4R promoter: Ch aracterization and role in obesity. Diabetes. 52:2996-3000. Madden G.J., Dake J.M., Mauel E.C., Rowe R.R. (2005). Labor supply consumption of food in a closed economy under a range of fixed and randoratio schedules : test of unit price. J Exp Anal Behav, 83(2): 99-118. Marusich J.A. and Branch M.N. (2006). Stability of cocaine doseresponse functions at different inter-dose intervals. Pharmacology Biochemistry and Behavior 84(2) ; 360-369 Mathis C.E., Johnson D.F., and Collier G.C. (1995) Procurement time as a determinant of meal frequency and meal duration. J Exp Anal Behav. 63(3): 295. Morato S., Johnson D.F., Collier G. (1995). Feedi ng patterns of rats when food-access cost is alternately low and high. Physiology and Behavior, 57(2): 21-26. Petersen S. and McCarthy J.C., (1981). Correlated changes in feeding behavior on selection for large and small body size in mice. Behavior Genetics. 11(1):57-64. Petrovich G.D. and Gallagher M. (2007). Cont rol of food consumption by learned cues: A forebrain-hypothalamic network. Physiology and Behavior, 91:397-403. Raslaer T.G., Bauman R.A., Hursh S.R., Shurtle ff D., Simmons L. (1988). Rapid demand curves for behavioral economincs. Animal Learning & Behavior, 16 (3): 330-339. Raynor H.A. and Epstein L.H. (2003). The rela tive reinforcing value of food under differing levels of food depriv ation and restriction. Appetite, 40:15-24 Richard C.D. and Low M.J. (2007). Drinking-e xplicit meal pattern analysis in mice: an ethological perspective. Am J Physiol Regul Integr Comp Physiol, 278: 797-805 Roche J.R., Timberlake W. (1998). The influenc e of artificial paths and landmarks on the foraging behavior of Norway rats. Animal Learning & Behavior, 26:76-84. Rolls B.J., Morris E.L., Roe L.S. (2002). Portion size of food affects energy intake in normalweight and overwei ght men and women. Am J Clin Nutr, 76:1207-13.

PAGE 55

55 Rovee-Collier C.K., Clapp B.A., Collier G.H. (1982). The economics of food choice in chicks. Physiol Behav, 28(6):1097-1102 Saelens B.E. and Epstein L.H. (1996). Reinforcing value of food in obese and non-obese women. Appetite, 27:41-50 Shindler C.W., Thorndike E.B., Goldberg S.R. (1993). Acquisition of a nose-poke response in rats as an operant. Bull Psychon Soc, 31: 291-294. Stellar E. (1954). The physiology of motivation. Physiol Review. 101(2): 301-311 Stephens D.W., Krebs J.R. (1986). Foraging theo ry. Princeton University Press, New Jersey. Strohmayer A.J. and Smith G.P. (1987). The me al pattern of genetical ly obese (ob/ob) mice. Appetite, 8(2):111-23. Sumpter C.E., William T., Foster T.M. (1999). The effect of differing response types and price manipulations on demand measures. J Exp Anal Behav, 71:329-354. Teitelbaum P. (1964). Appetite. Proceedings of the American Philosophical Society. 108(6): 464-472. Timberlake W., Peden B.F. (1987). On the dis tinction between open and closed economies. J Exp Anal Behav, 48(1):35-60. Vaughan C.H., Rowland N.E. (2001). Operant conditioning utilizing mice in a foraging paradigm. Appetite, 37 (2): 169 Vaughan C.H., Rowland N.E. (2003). Meal patterns of lean and leptin-defic ient obese mice in a simulated foraging environment. Physiology Behav,79(2):275-9. Vaughan C.H., Marcus M.C., Haskell-Luevano C., Rowland N.E. (2005). Meal patterns and foraging in melanocortin receptor knockout mice. Physiology Behav,84:129-133. Vaughan C.H., Marcus M.C., Haskell-Lueva no C., Rowland N.E. (2006). Food motivated behavior of melanocortin-4 receptor knockout mice under a pr ogressive ratio schedule. Peptides, 27:2829-2835. Woods, S.C. (1991). The eating pa radox: How we tolerate food. Physiological Review, 98(4): 488-505. Woods, S.C., Seeley, R.J., Daniel Porte Jr., Sc hwartz, M.W. (1998). Signals that regulate food intake and energy homeostasis. Science, 280: 1378-1383.

PAGE 56

BIOGRAPHICAL SKETCH Deniz Atalayer graduated from Bogazici Un iversity-Istanbul-Turkiye in 2004 with a Bachelor of Science degree in psychology. Her in terest in neuroscience began in the last two years of undergraduate as she worked in a ps ychobiology lab specifically conducting research on circadian rhythmicity with rats. She also worked in a behavioral anal ysis/learning lab on sexual preference with quails. After co mpleting her bachelors degree in psychology, she began to seek for a graduate degree combining the behavioral work and neuros cience research. In fall 2005, she was admitted to the behavioral neuroscience program in psychology department at the University of Florida, and started to work with Dr. Neil Rowland. Her field of study includes eating behavior and obesity resear ch both from genetic, neur ological, physiological, and neuroeconomical perspectives. She defended her mast ers thesis in fall 2007 which concerns the effects of the use of different operants in the meal pattern analysis on mice, and she is currently seeking candidacy to pursue a Ph.D. in the same program.


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101108_AAAACY INGEST_TIME 2010-11-09T02:34:13Z PACKAGE UFE0021862_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 2160 DFID F20101108_AACFTW ORIGIN DEPOSITOR PATH atalayer_d_Page_48.txt GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
eeba72d197b918e1994e7b8a58672cbb
SHA-1
7b41aab1ce127b694cc955bde9808bbada8f5ecf
20895 F20101108_AACFMA atalayer_d_Page_45.jpg
fb228a63cb241379c8d97d90dd081a1c
59cead982e8f5070c25eccc263c2842770e9ec12
25271604 F20101108_AACFOY atalayer_d_Page_16.tif
b6fbfbc8e9853fb3d02e337d82a3be43
71418df1ebfbffb98286ab16b3fe94c9bc344245
2183 F20101108_AACFTX atalayer_d_Page_49.txt
714e75caf00d9210f1733a833e507574
ddcb794a5d4447a84036e8ffe42152e13ca8841d
29039 F20101108_AACFMB atalayer_d_Page_46.jpg
ce10237e2a7d255bd12d091f6d471dfe
9126833be6e33b7de490ba4ec8bee2695dec1c23
1053954 F20101108_AACFOZ atalayer_d_Page_17.tif
85313f2e02fb1ef6f6506f1ef81d7dc8
958a9234f2fbba85e7247bb8d723c0bfc03809be
2052 F20101108_AACFTY atalayer_d_Page_50.txt
8092a9c3f969c93991961c4aede8b205
6edfcbdff738e266090870d4cf730bae44d3a02c
83945 F20101108_AACFMC atalayer_d_Page_47.jpg
29c054a55ed4e24299c50c7526f710cd
9346cb5e5b96ead4c6de1378779a401e0f6a7c47
51194 F20101108_AACFRA atalayer_d_Page_20.pro
55ef54fbd773333cde6ea6b1435bf435
b18a18bc2761c35621d1b6db324905a1bfeb9274
2507 F20101108_AACFTZ atalayer_d_Page_53.txt
47e59fbb2fa9484905f1d6284be43684
bb69e3d4c5da88bbbc4a77c95a54866883cfd13b
86209 F20101108_AACFMD atalayer_d_Page_48.jpg
c2e20ab5886a1e86a7b7d5c92634ef20
7e20adca6b05b7575bcaa43f30e41a738ede7bbd
56680 F20101108_AACFRB atalayer_d_Page_21.pro
739ee7571e88582f0dfbbd25336cfac3
0d105d72e80ace52b4a7ceec03801761b772f8f9
86788 F20101108_AACFME atalayer_d_Page_49.jpg
381af2d962c64cc5eb37ed9aad5be056
bfd0f6598da3075f57c8694f08abe35c8fd14f6f
36400 F20101108_AACFRC atalayer_d_Page_22.pro
be5ce41e0a9aa5b202eb35e582f401c6
9c92f9625e386121e0a8b7f38e9c64a2131a2368
83662 F20101108_AACFMF atalayer_d_Page_50.jpg
7e0dc9da03404f30c0e994a9aa705cad
2f02b2b7b0f8f12067c7d05a782ede72ad9d5df4
50111 F20101108_AACFRD atalayer_d_Page_23.pro
4fe4836edf264127bb3668378504e32c
c0390efb9ce0132ace8d1d93436fbb82069cfa6f
76318 F20101108_AACFMG atalayer_d_Page_51.jpg
7ccdba44e9eca05a5ae00422065a8fab
e05fa9ccbc7a906a1974da2c5790a4d1b1dc1cb0
7762 F20101108_AACFWA atalayer_d_Page_28.QC.jpg
a3eb8861c529e14154cacbdc7289abed
13a6dc2d04b7491234c694e8eb5428c55b5c6346
50290 F20101108_AACFRE atalayer_d_Page_24.pro
dcd531c735c48b7efaf5119381329717
05be882b13c8c61eaf535e39db0f7e620ccc160c
90692 F20101108_AACFMH atalayer_d_Page_52.jpg
5b437f4fc5063279a9ac6a172ea54c97
5d7eccd6dab84e1d07d618261bfd4963e6ad1fb9
2841 F20101108_AACFWB atalayer_d_Page_28thm.jpg
ee414284cd312726a4c1aa88dd028ce7
3397dca0ea9763bc9e50fde88e8ea204579bdaa1
23197 F20101108_AACFRF atalayer_d_Page_26.pro
35a8633782baf26203fbc70285f6c713
bb1486de747307c8659572486e258ebf1c554d5f
95861 F20101108_AACFMI atalayer_d_Page_53.jpg
bc40a963dd2d13ad3e7d76ddcae858f3
cf42a2863d530cdc3e5427eec8e79be2c17ad83b
6658 F20101108_AACFWC atalayer_d_Page_29.QC.jpg
e3d2cd6479644f0328c57c51f971c889
00218bccbbb011b29761bb28a36bf184ae28f476
4920 F20101108_AACFRG atalayer_d_Page_28.pro
59d8a5e27cbffb531f932650729ca07e
58e6f98a637d13dfc4473ec6055dac9fa9f66822
97343 F20101108_AACFMJ atalayer_d_Page_54.jpg
fd6533fa74d0bf7db6d47458c732b17d
87f8894bb1d776c926e8c3fb2338063af164f7f3
2182 F20101108_AACFWD atalayer_d_Page_29thm.jpg
d0d1ef60536f6d193671926777408d88
c416920a41da42b66c9cb5457a1cbef082856512
3854 F20101108_AACFRH atalayer_d_Page_30.pro
ef1f48cf106264042ec48f58fc818952
0323bf5dc9097a53854e4cb8434ffa053ee61ac9
79600 F20101108_AACFMK atalayer_d_Page_55.jpg
7ded5a37b63e23ce08f6ff737b41c6f3
0327a8ec67cab7a8d6a9b398bff2f34dea7b9cfc
6367 F20101108_AACFWE atalayer_d_Page_30.QC.jpg
c1759ac6af92f6dbbb5b5108c24eb3b9
8cd28570dbfe8fc2905c52fd680d61fa8f761fa9
3250 F20101108_AACFRI atalayer_d_Page_31.pro
d081ec6c6ec5a520a05e9e0f5fb5efea
9e3b59d5d3812f30378ff3bef4f05195781ed621
50826 F20101108_AACFML atalayer_d_Page_56.jpg
a92caf4b9a37cb84d9b0f209e628f649
0fb80c7dc5572fbb12e4fd97611f45f23c74db5c
2082 F20101108_AACFWF atalayer_d_Page_30thm.jpg
e2f01380f66cae2077217c9f0324ea87
d8b31b4f22bf7fe8f614e7577367cf295b01d27a
2917 F20101108_AACFRJ atalayer_d_Page_32.pro
d6c3a3b8c591c471139d4b8a5029557c
d00f5440c1451c150d754e4e87c0d319138900b3
263602 F20101108_AACFMM atalayer_d_Page_01.jp2
aa16d0c4f433b5757653469fd1adead4
cd4ea12763de7249f014739fa25a1f4a8ed44180
5814 F20101108_AACFWG atalayer_d_Page_31.QC.jpg
602c2a9c56d35fe07d4e26462f25c44b
05984bee2e5c416b45c7f0e5df2ed2b21dc6ee3f
4867 F20101108_AACFRK atalayer_d_Page_33.pro
f9e5d6f196d4ba7537fa26fc804401d9
e582ecb7db38419af67c829c16ebea1ece0316fd
24432 F20101108_AACFMN atalayer_d_Page_02.jp2
d0f0e8f6b4eab395cd35525de5aa4a5b
d58f5b8212d575285b485aeee80eec46737b687f
5960 F20101108_AACFWH atalayer_d_Page_32.QC.jpg
5ed565dd708b59ffb0ad649df5838144
2ffee1c541e753443fc9da74758394697604d03e
3557 F20101108_AACFRL atalayer_d_Page_34.pro
6ec5749530f6840d5839a8fad2625346
ecc7ec2dba3523398eabd923d262a3ae53b61bfa
42519 F20101108_AACFMO atalayer_d_Page_03.jp2
9689c090e1c5a36b4923c79cae19faea
e0f3ed05238fddf983f3233dab899b69e35ad455
1991 F20101108_AACFWI atalayer_d_Page_32thm.jpg
1ac18b245ef8cc03a98df2bff43b8413
c9dcfc9157279a335c9c5d32d8e2569c4aef3fb1
1051978 F20101108_AACFMP atalayer_d_Page_04.jp2
b24ca4ad94307cd3366f544698a0f024
e79992f7e14e4ba6fb2d782a7e1ceffc48406c50
7578 F20101108_AACFWJ atalayer_d_Page_33.QC.jpg
000ebaa5dc8292a2ff118637acc697d6
8436af1245939c2eefc1ec21db9d3c817206bbed
3712 F20101108_AACFRM atalayer_d_Page_35.pro
3236d9c60773b2dc340ab8e0b92d70ee
81afd44cd549e984f2eb0bf7fbcff0ff61cdd83d
293540 F20101108_AACFMQ atalayer_d_Page_05.jp2
cd273074a1b76a0b8d07c7438370856c
55da6096f9641960bdda9eda651e0e98979167f2
2790 F20101108_AACFWK atalayer_d_Page_33thm.jpg
5c4b654016a61ad650e0b1278c426388
79da170b6bbb3f01a16a6f6f377a239ea0b2ff29
3162 F20101108_AACFRN atalayer_d_Page_36.pro
b67f6dae032daf4322419ffbcb888f12
0d9068610165276cc3673fb1b91c126dd2bfe701
1051976 F20101108_AACFMR atalayer_d_Page_06.jp2
50d227f17dd0ed15e192c2fb80852073
d74ad7406be4592fb4bad606b475bde04a8b5e4f
6255 F20101108_AACFWL atalayer_d_Page_34.QC.jpg
ee43b34cac5f8d65def8dc94dc6d1e4d
799473857a46794dfae8acadcfaf1674d5ffe27b
3116 F20101108_AACFRO atalayer_d_Page_37.pro
454dedee1ec3ee84262e00caf7affb5b
d097ae33520e95e9fbc584db2b6e3c07610398f4
1011750 F20101108_AACFMS atalayer_d_Page_07.jp2
115f2136fa5a0d0f2259b5e974f70b9c
fd334d58455bf1ca0287c11d7e5a32832bfa0b54
2153 F20101108_AACFWM atalayer_d_Page_34thm.jpg
1e867d4fa9bae1214003da0f619957b5
b6f60252300ed55b88d930e67424f34fa7740c21
4116 F20101108_AACFRP atalayer_d_Page_38.pro
0198088dcb9a3f2fc6f8083d616d47f2
4a85499271aff3653d6cca20d14912b496d72036
1051971 F20101108_AACFMT atalayer_d_Page_08.jp2
7e499bf58cc516184ef7b146b4ae4258
51e9af5f51349d148246235924910ec3d17f3fc9
2119 F20101108_AACFWN atalayer_d_Page_35thm.jpg
cec3b5e3ffa156f1c0074e42690f7d23
be473f64297dd20296b9eb14f65c9780eb39eaae
4204 F20101108_AACFRQ atalayer_d_Page_39.pro
c7c2015aaac25e2d8427cc8c4cebd390
37d1a41edb8e4bde9e68f218d2b1b6e5eada3ad9
1051870 F20101108_AACFMU atalayer_d_Page_09.jp2
4e0379217c956cdbe316ec4a7741e614
7149095842731c60e9d2fdca4a9fa426e43181b7
5729 F20101108_AACFWO atalayer_d_Page_36.QC.jpg
8cad83232e6531f145516c6547218b9a
5aa27e5d67cbf17f6c28b00f3b01db3d30777524
4389 F20101108_AACFRR atalayer_d_Page_40.pro
e287005d5d7434f439de42f699a5a79b
bf8bf6a937445455a9b0f49061c6238f681a6a97
2025 F20101108_AACFWP atalayer_d_Page_36thm.jpg
ea6c55fa2a0218b90f9e3e3cce65a667
0888fb34c496ad476b877c522123e21ff4c42c9f
5183 F20101108_AACFRS atalayer_d_Page_41.pro
72d602917be91e3922149cca60ce9865
6781f7050c0fd90700aae67b51734b1404845ff9
1051935 F20101108_AACFMV atalayer_d_Page_10.jp2
a1b0948f6558ebf0e9269144239c6b5a
044d7bdd0d28c367b5b2e4f210cee4c5e406a11b
5997 F20101108_AACFWQ atalayer_d_Page_37.QC.jpg
0a28d36caed536c53d3ec5791ed0b46c
f9296474580c391f4af639f1b1b94bd7393bd11f
2791 F20101108_AACFRT atalayer_d_Page_42.pro
893b7eaeb44fb07cc6708968c92bf23a
fe96e3d32036b795017db83da64bb9cbf80ca20e
1051921 F20101108_AACFMW atalayer_d_Page_12.jp2
69d3e1526515f941d717f6175d9174d6
82fac643ae3e9cf7a1359f549aec4ca4bb58b348
F20101108_AACFWR atalayer_d_Page_37thm.jpg
a2c7867ba71ce71be97fdd52ad301742
4b43ea1a1a6321b1394ed66c7dd3314f8314ed24
5097 F20101108_AACFRU atalayer_d_Page_43.pro
87fd5fcec0b0caade0f9ff1f48ed27de
d4a0ee3f00df5360aea9bab8327e197563c82309
F20101108_AACFMX atalayer_d_Page_13.jp2
bdc1b87a17b81f9dec49f223bfe35974
fc855301e9641ea08c125ad8b0f9bd7e3ed3c626
8653 F20101108_AACFWS atalayer_d_Page_38.QC.jpg
3eef8440ea6d49100a9b429ab0b63504
9dcebe1d122124d6196062c39ec0306932776b8c
5755 F20101108_AACFRV atalayer_d_Page_44.pro
709dcc24425ae71bf9ca691e9c550189
100c174acb05d5e221b7863c0a3f9d8fc735220e
1051980 F20101108_AACFMY atalayer_d_Page_14.jp2
31521170fe64f1888bd116b10bd9e43d
adc03ee73e7316e62dc0edf0ac10197404fbbf27
3248 F20101108_AACFWT atalayer_d_Page_38thm.jpg
70e6df83047c04d0b71e7d201e04f282
67760f260d4ef526188d0cf6131d6b3a54e7c6af
6147 F20101108_AACFRW atalayer_d_Page_45.pro
8f7d0d958180587e4ef6805215bb1462
bf2ef6b4d8b80602621d5b5e70bbe19cbad32331
1872 F20101108_AACFKA atalayer_d_Page_51.txt
931dbf4e1fee915419f38b23f42829b5
bcd0a011f7f776c141fe022f55cb68e77e4ce3c6
1021541 F20101108_AACFMZ atalayer_d_Page_15.jp2
28c5341186838c727fefd5d62e3939f5
4d8f433dcb2fc7a3be012c488f82f0d558a323f2
6803 F20101108_AACFWU atalayer_d_Page_39.QC.jpg
17626ac0480371abb57637a990ee1a3c
ecad385030e448797e361b251ed740addffa468d
10939 F20101108_AACFRX atalayer_d_Page_46.pro
3cb5741446e018594bf8416df9dbe862
b0597d3548f186cd97151973692c444408f57bdf
5615 F20101108_AACFKB atalayer_d_Page_42.QC.jpg
620b95c9e1f881651fd6b64dcaf15e41
e309d1e9c4616e0a6907b230af90a518c035b181
2234 F20101108_AACFWV atalayer_d_Page_39thm.jpg
abc20ff8366411023a43c3d9a06a9343
b90a593d15c220d484570aca67bf00caf09524db
52484 F20101108_AACFRY atalayer_d_Page_47.pro
5202a6e1ff39c1091a027763bddfe7af
495bf3543a9e376d48ac56af5b5ddda126106cd3
7530 F20101108_AACFKC atalayer_d_Page_14thm.jpg
49d2cf2cf58d6d49c316ae48330f67fe
d35ef3449ad828925b46e145c0be94f4f55642c8
6502 F20101108_AACFWW atalayer_d_Page_40.QC.jpg
6ef9d461be877ab19de2a0aae65d85ee
bb36649df19ce3bac772bf83cb4be9561cc6bd56
F20101108_AACFPA atalayer_d_Page_18.tif
42f6447800148a0a40c5002c0ec37dbf
8207d225a8943130f5af8d4de31e1e9212b20b7b
55072 F20101108_AACFRZ atalayer_d_Page_48.pro
e73afdd5cfb46a02efe45e6f0ed442de
780619fbf288d202ba6f13ba4f0b4272cab21bad
19987 F20101108_AACFKD atalayer_d_Page_22.QC.jpg
187468cdb8550495f2f6ab10bf29cede
408054d6143d314bd81398cdfaf910716b161eda
2163 F20101108_AACFWX atalayer_d_Page_40thm.jpg
e7d8b450bb07dddf9b1718c858faff42
253ba1d639373c339d77a6fc35071fa5b0ac5f29
F20101108_AACFPB atalayer_d_Page_19.tif
650fb5051c2648073cbbd53796efb5a5
11ddd457774aa9d1064b4a415c5a784ef3e6a55b
2312 F20101108_AACFKE atalayer_d_Page_52.txt
7760d0afbcd69212247d685e66675004
49a6c4fad3f8d70874ac558f610e43eccfa0ea03
6156 F20101108_AACFWY atalayer_d_Page_41.QC.jpg
43ceebe9c9cb57da666e62d8943368d2
fa6934bbe5db082a550b578213395df24523004c
F20101108_AACFPC atalayer_d_Page_20.tif
a3c0d096472cc552c462b74e8de47194
86006803d2fc68f93c8af60023a189698793a928
1051982 F20101108_AACFKF atalayer_d_Page_11.jp2
2520cad34cfbe1d348e337495f5683a1
d2dcec9ac757bd1866702e1b0e095c2deb625442
2568 F20101108_AACFUA atalayer_d_Page_54.txt
7b43fe576f7c52b71e222c76c5b447be
ab2ebbaa5534b956fd487593a09270e3e8596025
2098 F20101108_AACFWZ atalayer_d_Page_41thm.jpg
ebbcda425698f4ea3e2e6db2faadb8ed
acbba8f6904bfc31769e3795c02afa314a26ea3d
F20101108_AACFPD atalayer_d_Page_21.tif
e81f6981e3833cd07b90aa8f0d8f3ca3
85d530b7a6c25988ea7a76250ac546d4dd6ee579
75742 F20101108_AACFKG atalayer_d_Page_19.jpg
bc1ea0fb790397775c7359791e1f0d8d
65d370b3e3f784c535c7c245417bc9488ce3c2cb
2075 F20101108_AACFUB atalayer_d_Page_55.txt
ab31fd8153c6337f26b8bc884bb1415b
0ead1f2330733be0f0b7c02a6846b0d9fb460c99
F20101108_AACFPE atalayer_d_Page_22.tif
7b8afec84a0db309aef076ac667bf5bc
c0d9cdaa0832ad534c8068278a8eb1803f7ae948
5601 F20101108_AACFKH atalayer_d_Page_27.pro
843647272860e47842697e73ced41f6c
1ee3926ddcb2ce0b08026f89f99fce53c5a7afd7
179457 F20101108_AACFUC atalayer_d.pdf
1fc359019dbdf5a00459fb0555966b9e
0fe9758c45cd098f8916c2bdc093b3c77127165c
F20101108_AACFPF atalayer_d_Page_23.tif
7c5222aaa45e708f719063b2016d0999
206622a6d2ee6b82bbf4cb6319d1dfdcba5c13ed
1921 F20101108_AACFKI atalayer_d_Page_07.txt
bc9b7612159207b2bb1f86a0d210336a
0e621b52193304ee97dc0a2daeb80becf9c8d7ff
2328 F20101108_AACFUD atalayer_d_Page_01thm.jpg
eee06bab2710a03f25a6c865a910b6d3
e2971b276c82eca6a555b44e159bab84fffc40ad
F20101108_AACFPG atalayer_d_Page_24.tif
e716d166bcd3056f37dae59887326e59
54b5a3fa624cdcd12338ffd0500d7b6eef548dae
17639 F20101108_AACFKJ atalayer_d_Page_05.jpg
4fef35ed334a434f16c5f116b99ecee3
bf30fb449c2db45085c641d9f6c70d618f45ddd1
7973 F20101108_AACFUE atalayer_d_Page_01.QC.jpg
c1ca49c6f46e4ca33c89673bbc844793
87500ddd50205332c0b1541d87487d277eb0a6dc
F20101108_AACFPH atalayer_d_Page_26.tif
d97c1bf9e856599dd696d176ec430cb4
9036c64b9c8ee7e6996c32d42109fb10fb2e6449
F20101108_AACFKK atalayer_d_Page_55.tif
ea9a191937a1f1833c605d4fc6e62022
3bbd86ff15fd5acb859a8ce2f51292e321701b3b
3068 F20101108_AACFUF atalayer_d_Page_02.QC.jpg
4d4299f2e73f130a85e0bda44cb5d5c3
119d22938dedbad7dc4df9e4f0a89900508b1640
F20101108_AACFPI atalayer_d_Page_28.tif
ac874aca1eb7c058c98c3c601ccfe040
86f1dbe2e692c431c1563a57f4f98360e082397a
84704 F20101108_AACFKL atalayer_d_Page_09.jpg
067b45e99c52c04acbbcf0df3d232632
98d6084d7b278b0b26f8d1eb42eab20a314874ad
1324 F20101108_AACFUG atalayer_d_Page_02thm.jpg
5d48cb31eadc9af550e1d06f8f0fd79d
4cfdc952189a0ca3a849b248a142e66234ec5ae7
8423998 F20101108_AACFPJ atalayer_d_Page_29.tif
4a849ac2f9e2e8c37882899eed0b156f
7ab78e0f2fc0c62de1907c3b7373006a3b462b18
129537 F20101108_AACFKM atalayer_d_Page_30.jp2
1f22e0e4de6d7b8c3c94459480c6a5a6
b8144b64c5148e7a5ede3a1979b437f293719142
3389 F20101108_AACFUH atalayer_d_Page_03.QC.jpg
50404f609f11c2c147f2c92e29ac4ec2
a1595a3fae76d8fb58a591e28aa5a8f26f66abbc
F20101108_AACFPK atalayer_d_Page_30.tif
9f8a2152cbf10233bba3cd8f4fa15408
2e72945a3c4fe1ae42bcb9c8563acf68b8875a1f
47098 F20101108_AACFKN atalayer_d_Page_51.pro
ec59810c86b918b574588dce14db2169
08d96d59b25d532077dc5dff41eef064cc72c401
F20101108_AACFPL atalayer_d_Page_31.tif
588c435be62f1b5e4025a60eda488870
c0a21e8a4de2eaca3385e7ab86cdd80df465ea41
87254 F20101108_AACFKO UFE0021862_00001.xml FULL
b9bbe02a5e6a904daebcb4dcf2644e7f
bca6ce1a3c0a8330b2badd1a5848afe57cccb491
1524 F20101108_AACFUI atalayer_d_Page_03thm.jpg
5cc3177ec633b6eebf26debcbca5101e
465867bf65145bdd12789841e018ab6830d9bb98
F20101108_AACFPM atalayer_d_Page_32.tif
4515edc742af274e03f5a762525d724f
d852c982efb146a21583422361a4986688e36ad9
3845 F20101108_AACFUJ atalayer_d_Page_04thm.jpg
6dd3e9f4504523e2f8867cb5cd5eeff8
ee51d9180fd5c9c791a8c3d153bee0e928c25c8e
F20101108_AACFPN atalayer_d_Page_33.tif
6f2dbd58a43b4918a9fd22f48564def7
970074a6f4c01e95877e6d28d1690aeecc23be5a
5544 F20101108_AACFUK atalayer_d_Page_05.QC.jpg
8d547edca0b817e08126b8edfe97fcad
e719d3c8ce00d994e607cf65f38d8b67f931e9ba
F20101108_AACFPO atalayer_d_Page_34.tif
7e1f0bccd866315f2538171e342dfbda
9ab763c5f2b594289762dd4ab4274e5e4923a936
27215 F20101108_AACFKR atalayer_d_Page_01.jpg
a4f2a42eb3a651e3412650b9cf05e08c
f011ef64e0021061ff156987cfdd6b4cdf2a1197
1890 F20101108_AACFUL atalayer_d_Page_05thm.jpg
5804a3f903b5ace9bcf3ad4a6c05b179
1ba073392735bddbf2b0d680f1e4a6ea2b4be5a7
F20101108_AACFPP atalayer_d_Page_35.tif
7076af9b37900a14348bf4a040ed2340
30f270c019ba2673107c4315ab9a94f0f6a565ea
9940 F20101108_AACFKS atalayer_d_Page_02.jpg
47ce048af1d5cc68a6d2d96e1d545339
e45d04a04a5abfcb59ba4c9c6aa9506af12eb9fd
23939 F20101108_AACFUM atalayer_d_Page_06.QC.jpg
35e6377e42ccb7a57e5da7f9ade89c55
ae6a71353aed6fec174393ebc30259da558e68bd
F20101108_AACFPQ atalayer_d_Page_36.tif
6ef130c5e0000dc59ed0eb268306b107
f6e64581457a31645b9e8e55c7f4aa2a6957519a
6290 F20101108_AACFUN atalayer_d_Page_06thm.jpg
39c28544b339950cbccb1b873a41cb5c
2d498194f59f18389aef89abeb1ca3e5bfd7fc86
F20101108_AACFPR atalayer_d_Page_37.tif
83bc747ba5b0ec2efd417acb20c0c6e0
f8e430f8a6408373b65d1c938013bd9984dff967
11200 F20101108_AACFKT atalayer_d_Page_03.jpg
cee6879111509431721bf81c8b043039
cc15cfaec0bb2f0393e331033e3ce00ba2fe6c81
22907 F20101108_AACFUO atalayer_d_Page_07.QC.jpg
27543d1c1d5fe46f28e7ca2c398357d3
dec9c81d5b4924bd39669633f0a26472263bd3cc
F20101108_AACFPS atalayer_d_Page_38.tif
394ece921318db2761b210c1bb165764
06e31d2eefd754c765198dc5db85d74ee6cc0abd
47735 F20101108_AACFKU atalayer_d_Page_04.jpg
192594a830f36f6ef974dfe93c306d0a
1b791635e7f129eceac5803afea1aabb8a9c79db
6380 F20101108_AACFUP atalayer_d_Page_07thm.jpg
a2ae848a213e36f2dac4fa4544e3a624
489147d491bd3d34429b0b8aba16a0a9ac1ab8a5
F20101108_AACFPT atalayer_d_Page_40.tif
19e449b4327777337b7995946a79d710
65db4152dbe46b1c67ab557632cc7da9721c34d5
79899 F20101108_AACFKV atalayer_d_Page_06.jpg
6e1c1eefb51a257cb27ef305a702416c
1d910fe9ea65992659e0d9108ba1fa447fd5e800
26973 F20101108_AACFUQ atalayer_d_Page_08.QC.jpg
2999e52a982c02e941ef784e4d4b7c9b
cf4cf538c10db6e30a1c2328be3ca6c5a4c16200
F20101108_AACFPU atalayer_d_Page_41.tif
6d650a1af70f0a4fe176d5b454c1b7d8
4516006a05d53c9183c48dee79b57e1f230ac250
75306 F20101108_AACFKW atalayer_d_Page_07.jpg
8656d54ee5ebd6cbd37870a5fa2a6835
4ade592212d588765266d43dabdec5158886f2f8
7349 F20101108_AACFUR atalayer_d_Page_08thm.jpg
4bddfd7f9e2daa850b00db4e24227862
5978abfd217e9fef2d8acdd733ef968d424efdcc
F20101108_AACFPV atalayer_d_Page_42.tif
79d97a3a4796b8c559e409e1b092d0cc
a8c98870b322e406185bccfb4c543c7253e144c4
85721 F20101108_AACFKX atalayer_d_Page_10.jpg
91a4e5b88f236debf7585e5062832f4e
26ae2505563495b833da88ab703b9984774942c2
26955 F20101108_AACFUS atalayer_d_Page_09.QC.jpg
be7ee0db4d5804609bc069ec310681e5
84a4437db2fa5c1e55934bd0796fd4c9bd6b8eb4
F20101108_AACFPW atalayer_d_Page_43.tif
2bb6033a224ccf0afd7056400f19a215
59601e63d10e74515547e216a3cd3257be998540
87543 F20101108_AACFKY atalayer_d_Page_12.jpg
6ba0dc136a5c61cbd81a382258d21116
bf0654db0f7355bb38601ed667e825b10987b65d
7451 F20101108_AACFUT atalayer_d_Page_09thm.jpg
41fa3c7849eb7abeef1cac6ae706e641
1f46b82a9e3d5ece5e6b583702d74df389bc62b5
F20101108_AACFPX atalayer_d_Page_44.tif
d1c6c3215f81076a4a914fcb0de79891
58151d535c5492425edce59804a15d6c5eebdb1e
84525 F20101108_AACFKZ atalayer_d_Page_13.jpg
834fca8f896a58ac5859822defac8b6b
498ca7c35ba18b3685e604a4424d9047484d081c
27378 F20101108_AACFUU atalayer_d_Page_10.QC.jpg
cb7f1a351260c9d5ff6ff08aea553c78
f9d8a5fdfcd858eac8fd32a572761b07c5b086b2
7441 F20101108_AACFUV atalayer_d_Page_10thm.jpg
70c747ad782a6490d2c18c6a0c529c30
c59c39cde8dc2078facfc9833c87d9dccc58b967
F20101108_AACFPY atalayer_d_Page_45.tif
3dfbc9355da915028d157fa810eb081e
a5ed6c69188099c10ab21919a852efd1de552d74
26588 F20101108_AACFUW atalayer_d_Page_11.QC.jpg
35a8aecf5905a4ba434f1974cf507618
4e836a647cce0d454ca7379dd9c94183e497e6e5
893313 F20101108_AACFNA atalayer_d_Page_16.jp2
cfb2e8ddc51adaba2a3c7f79e3df4ef3
5a3bf62452b08b27a032dc627f4ac62e165d1e40
F20101108_AACFPZ atalayer_d_Page_46.tif
39ebd6709ae6a821fae576b8600fdbab
b6ff4d29494710976c732130f7213a90bfbd6cd1
7288 F20101108_AACFUX atalayer_d_Page_11thm.jpg
205aa4c84547489c1e72deb6e1c51837
c55e1fd58e829944bdac287e882c9ead949eefdf
29034 F20101108_AACFNB atalayer_d_Page_17.jp2
91400ad7a5c99145cea1df7bea2e80eb
82d0ea84123987decff874b258437f7202e0a376
27441 F20101108_AACFUY atalayer_d_Page_12.QC.jpg
2d10a5e30ddc1bc780369c3076fd79ab
76970541dd3369d7a15742cbfde40497e258322e
1047460 F20101108_AACFNC atalayer_d_Page_19.jp2
25b216568d36cf71703417631eb6d131
16b4eea8fd984ffa659e10e673f0a050e41d54c1
55756 F20101108_AACFSA atalayer_d_Page_49.pro
aa1804e0073ebf60ad5a353ba7dc66bb
a683f81c020b71b9702f599bce7ecb5e5392b0f4
7330 F20101108_AACFUZ atalayer_d_Page_12thm.jpg
2ea6f34a12de19baa78e7bc12559bd1b
2563dd4b276ba0ee2744fce18476f8e7b29b298a
1051949 F20101108_AACFND atalayer_d_Page_20.jp2
1a8eb84f593b2634a72aadf23b244441
c6910d05d1ecac8ef0217ee78baec25726b2c6e9
51948 F20101108_AACFSB atalayer_d_Page_50.pro
8996867956720d12f9bed76e11d0bc67
cbc24f182a9192d4b3f680d330edfbb6ea45f660
1051940 F20101108_AACFNE atalayer_d_Page_21.jp2
d895a404b5673697e2d3cdb165c38669
42e21974d5961860b042e2e85ef01c50777e2944
56845 F20101108_AACFSC atalayer_d_Page_52.pro
f467776bbd05f96541188f03af7001d5
3a79d3ea9044081bf2ee1c50167892374e614b3e
838412 F20101108_AACFNF atalayer_d_Page_22.jp2
84cb0485d82844e4b02157b0085f78ad
0da34e333fcecb5785a367d26522243f4a2b6684
2012 F20101108_AACFXA atalayer_d_Page_42thm.jpg
7f3d26be269d8a34e423a112b3a2c06f
70b33183ba5c72e5d579b86d5c7bc404d36cc8b7
62524 F20101108_AACFSD atalayer_d_Page_53.pro
1cb2de4d0706a6a5dc93fb381026b37f
b3881f12474e9b930ffe320a763a407ec74621aa
1051974 F20101108_AACFNG atalayer_d_Page_23.jp2
71f9b91a19fbee7dc352ffb9e751f39b
a07d2e5d5f2b8e6ea188950c856b3aa43e3ff1b6
6406 F20101108_AACFXB atalayer_d_Page_43.QC.jpg
a06108ab0643363d8440dc939b0f73cb
59742d68abb7c4326f5dcaba3984e63bec377b04
63890 F20101108_AACFSE atalayer_d_Page_54.pro
244045e6d195cd43fe8cd0866898c8f5
1266ec0ea435526a51a8085137758b9cba3bb45d
1051984 F20101108_AACFNH atalayer_d_Page_24.jp2
de7ff58980fe4c11aeff46a2b01a0f19
e52fd2f957950ada3af24e5c8ffa2086caf1612b
2195 F20101108_AACFXC atalayer_d_Page_43thm.jpg
7f13ec301d6122f3ad15b9a0b4a9d762
dd6a9e82a977d3f4b34780b9da6e015ad0831911
51160 F20101108_AACFSF atalayer_d_Page_55.pro
cfeaeb320da489ba504d4a16d4c9ad14
665061dfcdc4abbc914b287cc22f084596a7c05f
1051947 F20101108_AACFNI atalayer_d_Page_25.jp2
001b5a37e30124117cd4c755436c2179
fc608b8a6d1dcce1cbc1c5b6fb1f5d9545996d4c
6333 F20101108_AACFXD atalayer_d_Page_44.QC.jpg
1a9c9084cf35c1b683d82762de7132dc
237780e1235b47a365b9e771bad2fd4795f6c9ad
29091 F20101108_AACFSG atalayer_d_Page_56.pro
f9e59bad2e95175ac08270c1b0a58b21
0c6e64fb7210c89fe119b6992669c492e7f83f32
525770 F20101108_AACFNJ atalayer_d_Page_26.jp2
46fa476b27ba26e95df94fcb66059241
32b5b09a7a17f38086089cf796ffe09f5a5c29f0
524 F20101108_AACFSH atalayer_d_Page_01.txt
2074da02d894e2b81f1fe47d33db1521
675d0e792b7c3444bda5347de08ec3d294315831
183294 F20101108_AACFNK atalayer_d_Page_27.jp2
d5e00aa561e4fdbd0a768cf21a46259d
5c59e8134def1dadc9a4a97cae4d8186bf1ac300
2035 F20101108_AACFXE atalayer_d_Page_44thm.jpg
15cea973ed489118f87ffbb0355e5103
f975722289eeabab979871432454871138a1c368
90 F20101108_AACFSI atalayer_d_Page_02.txt
32efbaa5d57f6ecefa4ab6f7915555b1
3bc94196d4a478c0b05aaa48ee034f51be19a825
144699 F20101108_AACFNL atalayer_d_Page_28.jp2
344462a573e51892fdeada9ede7af746
ecb1dec9546510e3bec5c55e91dc27f51d18a06a
7351 F20101108_AACFXF atalayer_d_Page_45.QC.jpg
a58e0be75961e4e4da1de3edea48143d
e17e66c2c1a94ce8aafb4b47ec760ec0b604de3d
1663 F20101108_AACFSJ atalayer_d_Page_04.txt
4719c0cf628b4fdbb9f56224f4922773
d33b711e6e57d5a61ab08af171c61783a3246f58
113564 F20101108_AACFNM atalayer_d_Page_31.jp2
8d2908e2bd32d51bb4b6dd5fe5a77c72
a7d30f198d44b89dae6b3b4274e34029a161dbfd
2388 F20101108_AACFXG atalayer_d_Page_45thm.jpg
ad992ba15e19fc34eb0c2308ef95d7c4
fce7a5ff1eda57ee043fb4228b11fc0dfea30eec
349 F20101108_AACFSK atalayer_d_Page_05.txt
746d66dbf4c91391d5fe063b47c6b81b
18458e37f42fad6b587859c0432596962f82c5d5
113972 F20101108_AACFNN atalayer_d_Page_32.jp2
964876f33ebd88c123cbce7d1b875813
9332e5ea8d11b839facd3611b8c7b91b87d74532
8252 F20101108_AACFXH atalayer_d_Page_46.QC.jpg
e501c5ecf431c6dd689ee36fa6d9b599
d5daec576b118f163383d8fe240fadfd13b5b264
2191 F20101108_AACFSL atalayer_d_Page_08.txt
154dce716fe13784e82b7e8290933734
155e0cd7408ed2f17d5c9520c56856af449b3d22
140608 F20101108_AACFNO atalayer_d_Page_33.jp2
395b3b9b4e590b89ea28c0268d0e889d
162e876a91b48f25bde24efe95eb28492eb4bf10
25920 F20101108_AACFXI atalayer_d_Page_47.QC.jpg
bee7e8491581c84c9ec17ad00d65cd2c
703b88dac6703093f1d7546ad85219b75f0a9da8
2157 F20101108_AACFSM atalayer_d_Page_09.txt
0489cca2c410946e7f05315edbfe2413
7745323e9593dd34e446bfccb5c128fa37253f2b
127209 F20101108_AACFNP atalayer_d_Page_34.jp2
85a4404cd16b7fb68a3624dad6cce011
4943f5a679eb85fd095d559405ed80015f095cbc
7092 F20101108_AACFXJ atalayer_d_Page_47thm.jpg
5747d2e0ce8181a523f52cdeefe1fb96
f3b0cdf9f79fc1870e8145e6382bafda3e7ce34b
2180 F20101108_AACFSN atalayer_d_Page_10.txt
faff880a42099d1c49a07be301a7242a
1005b1bdc2020f96be488da7fe9d7ee872d3dc7e
121226 F20101108_AACFNQ atalayer_d_Page_35.jp2
b0607398c83ca736a27ae6345262604c
eea989e214e871991f050c65d705bb22b160043e
26826 F20101108_AACFXK atalayer_d_Page_48.QC.jpg
cb4aee061b90f21005f00a5bec845f57
fe9b5f0a84991658d70409c7906f57cf1a81fc9e
2106 F20101108_AACFSO atalayer_d_Page_11.txt
d8ad3d027024989c9aaff6bf0c3352dd
02f6d9f3245db9665ca02b856fb8faa9c88efeb2
110012 F20101108_AACFNR atalayer_d_Page_36.jp2
c9feb149438637b86c65207d204f19dd
053288313f12d3e4c448655759d6f6c242fd0e2c
7359 F20101108_AACFXL atalayer_d_Page_48thm.jpg
75c5a581c07088646edb737f1d761078
f70a19517a5ee2b6ecf54af30d4a3a62161e0eb8
2136 F20101108_AACFSP atalayer_d_Page_13.txt
d91ecdfde2ad62103bee5447254a990c
0b4d60cc537e902fd00b2840f2dbe5dbe4cb292a
113595 F20101108_AACFNS atalayer_d_Page_37.jp2
de179fe20550a25bca0e2a8a4bc65243
cd4d81b9f499d87aedb0cf24e718ea2582b7879b
27453 F20101108_AACFXM atalayer_d_Page_49.QC.jpg
b3d9adf0638c8d7017e6694ba9d31b3b
34516585ba2f44e637081ef3ed0c1b385539ee24
2066 F20101108_AACFIV atalayer_d_Page_25.txt
ab6632ba0275b10ca8b0ec382e57b72d
891a6a51c1529d76d0738a19f3b4df375efe5d7b
2190 F20101108_AACFSQ atalayer_d_Page_14.txt
90ec6618701e7cfae074c35f3b6e1f63
a308be152e284dcd10d0993b42f3f601d1fdabaf
155290 F20101108_AACFNT atalayer_d_Page_38.jp2
03854442b6dd62185199cbd3d0cf6fc6
9d6292a8b6e6ab0b9ee94793ea3b66e44f28ffe0
7334 F20101108_AACFXN atalayer_d_Page_49thm.jpg
5a98cb61b311f0e1784e839a0d8f25b0
53d2c6a804ed198dd342c042cb5c8e37003bbba1
7705 F20101108_AACFIW atalayer_d_Page_21thm.jpg
2f8a5886e576d1d85756297ceaed296b
a484d90243b95e68514ef415fef6e1cc5d2391ab
1805 F20101108_AACFSR atalayer_d_Page_15.txt
bd529ab102b56cbe5b740569028cba16
3953b65c30702f652305b189cad522ac9aa64f80
139960 F20101108_AACFNU atalayer_d_Page_39.jp2
1f395f12eaa6f0db32672c5d0dc54d4e
53fbcb9d9860c746efbf133781c1b2f21e3858f4
26595 F20101108_AACFXO atalayer_d_Page_50.QC.jpg
3ef0edecc1ec079a4ae02f26cc442fae
e22f8610e8581dc976cd73bf819990b8ddd3909d
1595 F20101108_AACFSS atalayer_d_Page_16.txt
e71204ae9550719ccc68b60bac690ff1
b6d1288360930c7300c80c424547b4a7b52492b4
137851 F20101108_AACFNV atalayer_d_Page_40.jp2
da090dbc1e5cca29da1a9be942e2620a
4806f0e2e69d9b50b1e7a08c0618a40865a50c40
7323 F20101108_AACFXP atalayer_d_Page_50thm.jpg
a192b32f623c5776d6077e5007e32cd5
09248413b897a133c4ef66ae3609fe91b1267ebf
2215 F20101108_AACFIX atalayer_d_Page_06.txt
ec13c5b9d800568e014b4afe2d2c42e2
6353fafbff8034d70dfbcf6bd99811a66650f1c2
792 F20101108_AACFST atalayer_d_Page_17.txt
1cb73a7186bfa3c5424f083f9d17ad48
ed422185ee998dffb39119140252790d9387b54a
23942 F20101108_AACFXQ atalayer_d_Page_51.QC.jpg
6f17a2871b7a60050d1f73435ddbdb69
0671ceffdff28ad4f69f9fc2538b8c713f9c2d1e
6163 F20101108_AACFIY atalayer_d_Page_35.QC.jpg
f11163504bb08a0e1832714dd98dd0e7
a835721e41ae620d2bc5769aa8c56d94b477c0c3
563 F20101108_AACFSU atalayer_d_Page_18.txt
0e716a626882db9c9ca8c6a36d74e1f4
ffa8e402a26a70ab7fec8abc6ae04e9818c046f1
123801 F20101108_AACFNW atalayer_d_Page_41.jp2
674418f2ec21b410563ca49dc7c437be
ffaad2c67c965ecd3c47bf4f4515af5ec7f49d0b
7006 F20101108_AACFXR atalayer_d_Page_51thm.jpg
1f7e14f1813114c802a3ce698d8bf5fa
d0a03354c950f03d06e3ea0f4a2811e7182befcb
50719 F20101108_AACFIZ atalayer_d_Page_25.pro
937c9ce0d82f4c104ebce7cc52fc821a
b9564206980d1a7e8e4acd10082a847f4236bcb5
1976 F20101108_AACFSV atalayer_d_Page_19.txt
b7479bf06cabf8a1bd4ca9683375f9e8
e6ecd65db5c22b2bc7b2bcfce8d5800c6fed8c7c
115505 F20101108_AACFNX atalayer_d_Page_42.jp2
df35f7f52524c3b084955d165eefc213
3654f2dc0bb2132c6e5cc9fa0dcb8bec642ca822
26476 F20101108_AACFXS atalayer_d_Page_52.QC.jpg
0a78c761804787201d52260849233c5c
ed1bd20bf25c7a5ca8507ed75aa6aad3fd02cdaa
2057 F20101108_AACFSW atalayer_d_Page_20.txt
428bc7bf57cddcead5febdb255cd428b
e9545d39699de578a854a13d65215c7717ee627e
73256 F20101108_AACFLA atalayer_d_Page_15.jpg
85c55561170f78b45316203d37358799
0c081a630948f77041d5b6f22c19334e9cc8ccc9
142917 F20101108_AACFNY atalayer_d_Page_43.jp2
8d967d0b729856dc5fb9f055db105ce0
afd6cf40d48f31d6beb8ab5a644a9f79214663fa
7433 F20101108_AACFXT atalayer_d_Page_52thm.jpg
fd69b852f1d345c1c4bdcf4eacafb670
fe981976bdc0466dbb5c0305c4e9e2f059925284
2262 F20101108_AACFSX atalayer_d_Page_21.txt
ecaa16fe80b1ee00aa4c8e7c368d5202
0784e0a294c4353407e58f7e17c98e4d53f5a88c
68494 F20101108_AACFLB atalayer_d_Page_16.jpg
bbabb350deabea6582f0ab2658bf3dc7
734b47403bb69423e9f18f1617b2193ed99b6b8b
159191 F20101108_AACFNZ atalayer_d_Page_44.jp2
a5a1fd7fc1b2f07c277b3b10bb1729be
79d4264c3c2c4514d2fefa6425265adc073bb529
28517 F20101108_AACFXU atalayer_d_Page_53.QC.jpg
fd70d9b363b771510e84a48d5a21e792
b74f2d98b150bb4293c984db66271a6a2e7f3d0e
1440 F20101108_AACFSY atalayer_d_Page_22.txt
c8493835c77c1895ac6b21b6ddba6b84
474c9084c448b83df02561d016478b226761c96f
24175 F20101108_AACFLC atalayer_d_Page_17.jpg
24b14a6715c1cd133f3a74e2a531c90c
ed65c35797e27b7f54cac7bf5d63d6676b6a53ee
28306 F20101108_AACFXV atalayer_d_Page_54.QC.jpg
f1de7a0f2efa79abd7cf4ae7a1ec61a6
b9d0b9238d87dbef05e3e5e35ce04bfa200200e6
2122 F20101108_AACFSZ atalayer_d_Page_23.txt
fd7cc18810a20b309cf962f33400353b
8ff1aa11850b0416cf245b00cd641a9c6eec2589
30791 F20101108_AACFLD atalayer_d_Page_18.jpg
0eecc1d3231388333a3831f334ae53e0
1e78a67ebf21d4daaad2eda8be999032851aa384
7567 F20101108_AACFXW atalayer_d_Page_54thm.jpg
c492fa803ac493b1c0fddda30ef3f4bc
75e815f107cb905d73b600131e16470a82b90149
F20101108_AACFQA atalayer_d_Page_47.tif
c84300e2a3c975549fdb8c418755a61d
656f2094772c00e438df394ac63cabec96691df0
82893 F20101108_AACFLE atalayer_d_Page_20.jpg
b4b8936376d6b7b5e5592678a6c0e2d9
c9145c46282724ee33aad41ce5648e0c095a8f8d
24369 F20101108_AACFXX atalayer_d_Page_55.QC.jpg
f3a47e6abead540819e2a19e6b91b324
a56d45e7c67796dc1a1ac56f59369b3d2decfb3c
F20101108_AACFQB atalayer_d_Page_48.tif
f809973e8e7bfa5889f0f239cbe78893
b1c9f56cdb1cdf7dcd4f1a4ed628e1c8d1ac4899
88300 F20101108_AACFLF atalayer_d_Page_21.jpg
857c5e1fc6cab280c97199504cacdddf
6aa12af9850c668b0b07f3f1bba3375f6f26faaf
6908 F20101108_AACFXY atalayer_d_Page_55thm.jpg
6bc78b50efdd550167c54ecf6431bc6c
f2107d6f5876af54715e13646b97dbbfc7a1d42e
F20101108_AACFQC atalayer_d_Page_49.tif
45870120cd3a0f1864b837cb99d03ad4
7d2655f34738c599eb0a3c7942401cfe872845cc
61350 F20101108_AACFLG atalayer_d_Page_22.jpg
ea02c3cbf3a2d3892d80c9d35a785218
25c7fcfdfc82491399463640ae8652c3d0b71547
27338 F20101108_AACFVA atalayer_d_Page_13.QC.jpg
a3c3e964dfdb7785c4f28191e3442a5d
77b74710860a995bd77e79a2e2b10d01b2043e74
15515 F20101108_AACFXZ atalayer_d_Page_56.QC.jpg
96cfc7782f7a1c9f3858e6a3b62ecfba
cd73d0bcc387f79bc6884103020f0a9150f9c360
F20101108_AACFQD atalayer_d_Page_50.tif
3c9d5bbc6e8b4d4fd7c52a394d0ffad4
c15ddea5801f9f9428a5b8d822c6d0ed666dda12
80820 F20101108_AACFLH atalayer_d_Page_23.jpg
047ca71daf9dccda1589cbb6b02639e0
537d08c4c073933239158f53d1f100cb22b81d6a
7283 F20101108_AACFVB atalayer_d_Page_13thm.jpg
9d40290db92f1b7f41bf2ab7afe1079b
82661711cfa629e2e88f1241a2e8aeb96108dcee
F20101108_AACFQE atalayer_d_Page_52.tif
2454ee6a549f2213c6b6cc74a7ae4e1b
50ee2d8f641d867cb275d3e059085d13bd63c9ec
81063 F20101108_AACFLI atalayer_d_Page_25.jpg
080f6c9edeb6ff123e488af161dc2b73
036083e2fd79724e8a446a9296a0c86a8430dd54
27892 F20101108_AACFVC atalayer_d_Page_14.QC.jpg
2812eb1b64847f6ddeb8fb68285546eb
90215e84887e8960a15d64891d375e10509d80cb
F20101108_AACFQF atalayer_d_Page_53.tif
09bc32bf7bbb3125e947cc6a42ead17d
51651b4011cf6200eb0181237af8060fc3ee1f0f
42680 F20101108_AACFLJ atalayer_d_Page_26.jpg
a072c7218dbd1edb0c3009ea493a5950
1d876496c34158f251657fb06576ecff91d9db95
23577 F20101108_AACFVD atalayer_d_Page_15.QC.jpg
3341a032eac64c1583e50af7796a72c0
b2e6f478babd67f7e4a73e1068951d165a301615
F20101108_AACFQG atalayer_d_Page_54.tif
1838239576980faf66350849223f336a
e540f519056ee5872534f0319afea566bac3b612
26285 F20101108_AACFLK atalayer_d_Page_27.jpg
c31a0b062b6669b4c058166c1c548417
1ff1c4f2189173997514765b6bea983e04dd3c6a
6379 F20101108_AACFVE atalayer_d_Page_15thm.jpg
e9860a12a1793666025fbb0d678823d0
5a45a1c2ee43b352e046c14ab98b91f19f52dd46
F20101108_AACFQH atalayer_d_Page_56.tif
f46c470d7af1eb6795f772f998598e67
2ea8124e813449d079690faae5d7c72f21688cf9
21478 F20101108_AACFLL atalayer_d_Page_28.jpg
d2d9f985a189a5ebbfe6ad2a15d9cd59
83b0db49a709c00f00e31014d0f788307a43414a
21436 F20101108_AACFVF atalayer_d_Page_16.QC.jpg
7e590fef18df15dccc196b35a6fafa90
a944e33715a9858a47f5f3606ed4f1f73690c6da
8790 F20101108_AACFQI atalayer_d_Page_01.pro
bbe9a4be64c1cb06c2c85e4000f259a1
26afbab3cdf3cd57915523c936376afd245998c7
18321 F20101108_AACFLM atalayer_d_Page_29.jpg
75591e2fcd78c9b4e87ee2872b29044d
4258e0f3630c8251ff3f8456f58c3ac5f5e87fa3
5443 F20101108_AACFVG atalayer_d_Page_16thm.jpg
5ca32d77293cc826139b1f4b9c34b1c3
5e20ae982f810dfa9fc415556a4d281121de6ef1
831 F20101108_AACFQJ atalayer_d_Page_02.pro
e16eb613209c2b6883e6564da81576f2
ef8e4100263bcd16008793b841db94a390cb1618
17618 F20101108_AACFLN atalayer_d_Page_30.jpg
b4c3d8bdd1e835ccff85d3b7434bbed9
18dc4054addccb0689f88d4ddc268668ab4415cc
7282 F20101108_AACFVH atalayer_d_Page_17.QC.jpg
d07c7138307ca128111edc4aab25be4c
b34046dbd87fea47a63bff4fc54bf250b1128e90
1585 F20101108_AACFQK atalayer_d_Page_03.pro
9e640fce06b5911b0438a5ad2b421596
5efa1db31448e361cd470576ed091af2ad65b6a3
15885 F20101108_AACFLO atalayer_d_Page_31.jpg
8c5a3a4bea03523bc7c7941c3ba99f31
145913ddb9770744ec52b96839e746f281be7fe8
2569 F20101108_AACFVI atalayer_d_Page_17thm.jpg
d973b55fc895b7fb6419cdc6a710028d
c8be1c2c7b9bc7037854aea8f4ce3ec888df2264
37843 F20101108_AACFQL atalayer_d_Page_04.pro
f6483a2fe26a28aadc2a54a375caaaca
c5d4e61a9d8db4a27d9e7ddf6e0bc79ef9bc4ba1
16514 F20101108_AACFLP atalayer_d_Page_32.jpg
76db1c5f1328bb0d28e3654a934efb5a
5a97c725d09c595affbce4d35b8c07ce3c546011
9920 F20101108_AACFVJ atalayer_d_Page_18.QC.jpg
43a4715d04c81af87eb661cbe2c7f712
47514165d995bc2555827023fcb641ee0d09eaf6
7960 F20101108_AACFQM atalayer_d_Page_05.pro
b69de6e443e28dc9fc199c7c690b60bf
3380ae94c50e6f6d69475d6e50de773a85fdfcb6
21130 F20101108_AACFLQ atalayer_d_Page_33.jpg
61f29ec7faa52df5b7189bceb14faa6d
3281d5ea929abaee8e7864ba005c6e66015a5af3
3242 F20101108_AACFVK atalayer_d_Page_18thm.jpg
e4e1736f1e0aaa89a63ef1dc36a349f4
94c144f4a1967e9764ccadf5502f987b74efb9ba
55762 F20101108_AACFQN atalayer_d_Page_06.pro
9bcf1a1a379ac230d5cf41ccee50e8a1
aef59b98bff286f55b4a95a35067fc42ef6633ad
17252 F20101108_AACFLR atalayer_d_Page_34.jpg
6e4748d7d855db62ac5d7457aa7b4b23
11b0639e0c65c4d2dc70e71cc453aa767007c113
23888 F20101108_AACFVL atalayer_d_Page_19.QC.jpg
a870165dd5d15e107ca150f038328892
2f457fd133666991bd7a5cdd01266e94f4f614e5
43832 F20101108_AACFQO atalayer_d_Page_07.pro
da341d8b5e93335d57651b3fae8ead14
a246eaac66d995f8f0d13bcfde3295dab9e757fb
17068 F20101108_AACFLS atalayer_d_Page_35.jpg
381b11ce161ee997dbc975ca5d9bbd2f
c9b41402913a0eaa62b2d6cbab7ab0bf0e967786
6524 F20101108_AACFVM atalayer_d_Page_19thm.jpg
730c034605f4e2464339bc3bd4579bd9
de488e23afd08e2ec2716abf67aaa6ecddb19f8b
53900 F20101108_AACFQP atalayer_d_Page_08.pro
a586181f91c53140c387710f43fc5adf
90076c650e7bf99cd3d6f0779470af6b14e59664
15942 F20101108_AACFLT atalayer_d_Page_36.jpg
8c0df4332d020305473f05acd7884a27
18b44e2793882da651b456d07a194ede0e63ccf4
26590 F20101108_AACFVN atalayer_d_Page_20.QC.jpg
3c6c531949110b35eb1b4b9541dd0163
2e78fcebc158abcf802833c816f35dea9e93e4a1
54342 F20101108_AACFQQ atalayer_d_Page_09.pro
edfb09eebb72e029d8cbad3138508c15
f7ce315b2a59d0d3757e3061f7c9a74cee59e55e
6821 F20101108_AACFVO atalayer_d_Page_20thm.jpg
c1a1f9147b21b3cec096d6c3870ed7d1
4edc2cdfed90f3c601fa2648e6655fc35ebff2c3
55106 F20101108_AACFQR atalayer_d_Page_10.pro
8a6a5d64a53091dec2503e62d9f1c42b
19c575fc80a077125faa2142ef730f5cce1cf1fa
28170 F20101108_AACFVP atalayer_d_Page_21.QC.jpg
70bdb15b39ffd0cae67c3a855d698493
b11c8689174ee8a4801f04f3c3719699d931db4b
53627 F20101108_AACFQS atalayer_d_Page_11.pro
bdcc2fdc4ba2e0bc6a345dafff2cc971
640cfdde98233969f3712976ab408c55bf75315b
16429 F20101108_AACFLU atalayer_d_Page_37.jpg
b5aa702d77952cb8f50591d9639f4f92
a23c932d507a537c9fdadc99b87a62fc2373ffd0
5567 F20101108_AACFVQ atalayer_d_Page_22thm.jpg
b6e3a4f6bcdf43cc59a685e3a3d55fb1
8856e9a01cec795fc0a4747d3e1fb26eeb9063a3
55943 F20101108_AACFQT atalayer_d_Page_12.pro
e22eb16580b4f3c88732f8681e0885ae
1010d038e4e34a8d584fc79c0dcc0081ab741bc5
23511 F20101108_AACFLV atalayer_d_Page_38.jpg
757157be13014ccc8da0cb4b0506e263
4932d49e834ae4c2bbc287ad73a4f4a81cfb67cc
25636 F20101108_AACFVR atalayer_d_Page_23.QC.jpg
7c270e5320202f212b9e1c6d28c8acbd
d60d3496f2266a1d70e867f2683cbc78617b8889
53623 F20101108_AACFQU atalayer_d_Page_13.pro
6a95c1452863cfc410c9acc1294877b2
f70e379ac03f74ec510dd5393420fdae956179c9
18135 F20101108_AACFLW atalayer_d_Page_40.jpg
013ae792810aea639d1311beabd06bf9
49be3678476f09aa344389d5e31c02d9d183ad53
7154 F20101108_AACFVS atalayer_d_Page_23thm.jpg
95ac7562a57fba2f8fa290e336764f78
759037bf9e34f6534750336ba4712bb358a31fda
55783 F20101108_AACFQV atalayer_d_Page_14.pro
e431b66a39e4eb3ba8988d30259ef6be
c6684aa30c3a85d7d0bfa290eb417ff3de6ab366
15409 F20101108_AACFLX atalayer_d_Page_42.jpg
b3905413d97d72e1569942835ddf3c3d
82dd4d727be9d8ca69214c22f70cc7a4b08d0707
24802 F20101108_AACFVT atalayer_d_Page_24.QC.jpg
f33c07a92def61e387d10e35ab0aaa3f
d1b580f7dd32e7d23e3dfebf7502bdb4b40a78b3
45285 F20101108_AACFQW atalayer_d_Page_15.pro
3935d68e110476b3aaa23e23b2b8dd5e
906d27589c7e063c7c12f0406fdf69ce873a8b8f
26211 F20101108_AACFJA atalayer_d_Page_25.QC.jpg
cffcf239e3cec92a493f1ca6830f74ff
e53f584b6dab48878b82396b6c87981fe494a285
17545 F20101108_AACFLY atalayer_d_Page_43.jpg
69d714adb5eeaa629a698cfc2e0c22b4
0e6ff66b7cec90a911ad3dc53c264b18807064c7
6923 F20101108_AACFVU atalayer_d_Page_24thm.jpg
ccfb5ec3c19b9fa6abe7653b9fbcaeea
33071f48c7d880dd82f497a227d783d283b47d0c
38393 F20101108_AACFQX atalayer_d_Page_16.pro
2637751d9c56cf04fa45efb9fe878289
b9c7dc3616ac9ae6cdbc7b1e8400924273080537
F20101108_AACFJB atalayer_d_Page_25.tif
0a4cb4a7bb25126ce030aff5169a33cc
fe76952d9e2fa67d556e925b1b0d09d88970bd4d
18297 F20101108_AACFLZ atalayer_d_Page_44.jpg
08bf7d834acbbc629332ad1ce646d0a6
3dda30e46358eacc23d3e3f6e20963daa6f55799
7432 F20101108_AACFVV atalayer_d_Page_25thm.jpg
28b87ce6659ff6971c3af9b528233a1e
d61daaadcbb696b411563183a60dc4744232cda4
11147 F20101108_AACFQY atalayer_d_Page_17.pro
ca4efa48f93cb9bd94ec19b0e2f0f162
d28e82f13abb8bbcb26fc9ef964cd7dd12ada54b
87930 F20101108_AACFJC atalayer_d_Page_14.jpg
c8241c2b0ea373514a473dea0e169b3a
2443f14140807934639d23e324e1c27a04db7114
13373 F20101108_AACFVW atalayer_d_Page_26.QC.jpg
711bae423f393a18a1619e752ee7e794
3729e8f8545a56b172c8143f196564e1fa7268dc
170075 F20101108_AACFOA atalayer_d_Page_45.jp2
810ef3e12abd3572e1a94026a9de8788
fce5e399869526e74b5e633b400db3ed223e517d
F20101108_AACFJD atalayer_d_Page_51.tif
4fb0f54cbec332a0c0fecb3443df8fd9
314f477a97e2fead4117e07c0662d02ecb0f9fcf
4009 F20101108_AACFVX atalayer_d_Page_26thm.jpg
d56fcab31854de97bfe5de879559c6b6
f4b859b272aec8df3dcdf2e3bdbe729e4cda4af7
289559 F20101108_AACFOB atalayer_d_Page_46.jp2
8b3ca9d77e94a4a2d62bb5b9560383ce
6c4c862c2b30ddaca129d8000fa362343145d5a1
12544 F20101108_AACFQZ atalayer_d_Page_18.pro
40987ad48ae312ab2798a91d6dcd1070
1c93864b63d3ed48bff51bfe573c06da67b6dd4d
18805 F20101108_AACFJE atalayer_d_Page_39.jpg
82598f3a4f2e5474756a56cace1cbdb2
de2ad95dc7ebe20e70dd7da20c4a2b927ac78a51
9298 F20101108_AACFVY atalayer_d_Page_27.QC.jpg
2c27d71a6fcaef22e428a9cfc35d84bd
d061a3a6417d5c65e4acc169bfa6a57c0ad9f8fa
1051962 F20101108_AACFOC atalayer_d_Page_47.jp2
66e28c5fd43e068607d64b083abc2a6d
46180519c0600053effd27a829dae7a22bf0c37d
79419 F20101108_AACFJF atalayer_d_Page_24.jpg
42babc24b06a10ffa1c5240bb5bb2caa
520206349a23a847e6ca02c3aa03e27c025fe0c7
3486 F20101108_AACFVZ atalayer_d_Page_27thm.jpg
8f44336cde62085f5fdee51781c07b77
1e33ba75fde8544e31897aa36c123173a80cf405
F20101108_AACFOD atalayer_d_Page_48.jp2
6cf6cce3df91b7c828b40b9e2d0f395c
158db0ea46d5f79b738374bcc2eeddf066325eee
37733 F20101108_AACFJG atalayer_d_Page_18.jp2
d23240a0a2b15dd8d613590948597a93
4b2134652142466901f1fa21814e22a72ca2e389
2069 F20101108_AACFTA atalayer_d_Page_24.txt
da7c903e66c6495224949cb4d7e94c03
aab1689552b123d38bfc0d850b9fff4ffef4e9b7
1051922 F20101108_AACFOE atalayer_d_Page_49.jp2
9ef043f2a064b02ff83ff656f6aa5716
e3405215b91d0a7cfb97d18a72dedbb70f6f4266
F20101108_AACFJH atalayer_d_Page_27.tif
842cdacf2a2acc5f1ca8ca1bea1e4833
5f8f0b38a849f87c37bd962826a9366802dda115
943 F20101108_AACFTB atalayer_d_Page_26.txt
79341c89b65d8e1345f519076b5b99fc
b0eef2066859211d6dfa5e012d66d3495985b210
1051983 F20101108_AACFOF atalayer_d_Page_50.jp2
894747bd7f0c9ec8781e351df65814c4
1c437093e22b7f07ebaed183c208b6029dca440b
4590 F20101108_AACFYA atalayer_d_Page_56thm.jpg
4c88cff5da1cf09734227f5d41d005c0
cad14e35165c6d863a9dcd20fca4272a82d77a42
1190 F20101108_AACFJI atalayer_d_Page_56.txt
71cd80917d5a90d45023ed9216e6888a
84b8026b974b40c529713d678007786de2bbb64d
435 F20101108_AACFTC atalayer_d_Page_27.txt
9c5f822aad347db6405243783d403b18
08177c2a610119bb2fca65b5547b575a9c42aeb6
1051928 F20101108_AACFOG atalayer_d_Page_51.jp2
9216c0f47b85d6213576bb0b73e6cd1f
43dbfcbfc15505e9498926e4ba2065611339d3c6
2061 F20101108_AACFJJ atalayer_d_Page_31thm.jpg
b0d0662d105ffb164e0eb0223f9f82a1
ad165b6a5ad496473a6bb0bc82d093221bbc801e
240 F20101108_AACFTD atalayer_d_Page_28.txt
b8888247f246a00e70f9ee39e7657f9d
33822331633b9d89f80f42d33d511c2fcb498755
1051960 F20101108_AACFOH atalayer_d_Page_52.jp2
74e7488fe431f22f056f5f837db38807
a90c6fdc117e1cd6107554e79b1ce1314981b5b1
67765 F20101108_AACFYB UFE0021862_00001.mets
dfe25830c899e2a3ec4a08332857aa4f
90a52605ab3444e6af11f15642e9a3728d32a600
3969 F20101108_AACFJK atalayer_d_Page_29.pro
fc5e469829953011b1c8fc0772ccc790
c349f108ca76d7cf6439bee82975110782a33870
363 F20101108_AACFTE atalayer_d_Page_29.txt
a05eefeed0e164e77484e1689a3e6297
a30b8c05a993cc21ef4bd2d014f379220e7df663
1051931 F20101108_AACFOI atalayer_d_Page_54.jp2
8bc7af86b7122daae7e83deace1e57b5
50b42717401dd04758f8b9cb48a3745181996911
14622 F20101108_AACFJL atalayer_d_Page_04.QC.jpg
d6d80b0f894ebf1b1249e6ef74f56046
37a16cf2a676959b60c63a916dd1d28b055b8121
338 F20101108_AACFTF atalayer_d_Page_30.txt
64373d4f674f566505d5d20740e522e5
4c32f9381682e3424cb2fcc94b4b4a2de2f7f98c
1051951 F20101108_AACFOJ atalayer_d_Page_55.jp2
4d2a171ad913e400c9ac03cbacda5527
b35c251ecaaff4a51eda56d7674e29ea12c4f784
123 F20101108_AACFJM atalayer_d_Page_03.txt
8b44cf33458efa10013c9f5531849cef
57864b65ded39621f7bebffa50379db231142169
347 F20101108_AACFTG atalayer_d_Page_31.txt
c91b69df74fc5169600e6dd3018b809e
3732febc5398b5adec43458040d3fd8a4568baee
F20101108_AACFOK atalayer_d_Page_01.tif
2b7cdf412055df13861fd3926eaa9da7
56505dcb02d6c76394f4f2b8114e16704bcd50ad
132860 F20101108_AACFJN atalayer_d_Page_29.jp2
4d4328762489544609d85e3f6f8d196a
ef37e1e19aa8d85b1ec770efc457417bcb371eb2
294 F20101108_AACFTH atalayer_d_Page_32.txt
8e04aa945d3a48e3386d804afcd2e13e
93d971f1a9b5909afaa012067d946d1c6f0a6242
F20101108_AACFOL atalayer_d_Page_02.tif
e6ed85b47d4d661882f743e97f4e3a34
aba139fd68a41b8d0328087da9b3c0e0b46176fc
558 F20101108_AACFJO atalayer_d_Page_45.txt
020d1c16bd7ec2bb10ef07abff21eb79
a1fb307fdcacab533c6960b378f4713845dc6ca1
364 F20101108_AACFTI atalayer_d_Page_33.txt
e745d808fbb6a4595751857770ac4e56
ffc16870c205104c0ec81d7e22df694154c14251
F20101108_AACFOM atalayer_d_Page_03.tif
2cf12226d79c81e1dc767ca685461f7b
aebee2cdb1fdc5b171e8a1eb4db4f676ec44b3d0
45923 F20101108_AACFJP atalayer_d_Page_19.pro
51cbddda30172b5dc3749f051c338003
19a769b46ff8c3dafab7adc40b6667da56439a96
171 F20101108_AACFTJ atalayer_d_Page_34.txt
ca1576a4381be8a723e664281f0e76ff
dc6a43a77ca6b6f800302b2b5250fd6235aeb560
F20101108_AACFON atalayer_d_Page_04.tif
60b17c30a2b812bf80365b633d7344f4
a24346689dfdf906988060429b0fde9729f5ea9f
86099 F20101108_AACFJQ atalayer_d_Page_08.jpg
cf66a6524f486b88eea6d5b352c51110
b97c105de8677dc979240688feb5c42c676a1aca
179 F20101108_AACFTK atalayer_d_Page_35.txt
ef1a7a41be9a3ef56ce4f75f2b10f41d
5046185ec6dc1efa47fbc1dddef10f192b5278b2
F20101108_AACFOO atalayer_d_Page_05.tif
ded1aa223dabb2614209a9aa6f664e0a
ee1fa8664265f6912dfc8bb0eddcaf855e3f79c3
F20101108_AACFJR atalayer_d_Page_11.tif
9376c656eb97f361f728e7f2309e7e9f
bba5c3e213f495549d1388d1c3363a02be87f9fd
341 F20101108_AACFTL atalayer_d_Page_36.txt
32ad05fcfa43a5d50469dc981979f96e
2ec72a5ab735a9bbdc9f3d2d810d24d7f670e576
F20101108_AACFOP atalayer_d_Page_06.tif
7bc0e66456184b66dfd23bd7c97868f9
7744bb4ba1b77fe5a3857a6fc09ae454ca18013e
150 F20101108_AACFTM atalayer_d_Page_37.txt
31d2bc5eac85b44abed931fb02a0be63
1cc8dcbe48471b613a95a7fd1db32873200910f6
16843 F20101108_AACFJS atalayer_d_Page_41.jpg
42cbb1e199c7f7fb552e336a385439a4
33ba9a32d9feb92fb2d1e872088a4676d1403479
228 F20101108_AACFTN atalayer_d_Page_38.txt
9470b77e0c7fb054cda90082efb8e926
9c3900c544b109948e4f07dca971e9a43fd99d92
F20101108_AACFOQ atalayer_d_Page_07.tif
e2fda9e7e0cc0f722305b40852d4fecd
cf617a5aa1f12adecbd45e35900cb7a68981c9c2
668963 F20101108_AACFJT atalayer_d_Page_56.jp2
c77c7e634090915246e9e06fe1ad2205
7e319dcd016dcfa7f12f17cd50a5461ec50f5184
387 F20101108_AACFTO atalayer_d_Page_39.txt
676c9346e49217814ff187d6eeefa871
7a098d7ad1a6d2e94859b862ef46890499c0cd0f
F20101108_AACFOR atalayer_d_Page_08.tif
d27f6b501c84a3a18ddd38566e3098f3
92539f02bea20fc40ca9ef3b2ec3fe11abf0cc9e
2769 F20101108_AACFJU atalayer_d_Page_46thm.jpg
50e8322ba8dbe0a5b88f2f09713c42b6
9fdfb29ddfb537269019955595dc56e7965e1cee
346 F20101108_AACFTP atalayer_d_Page_40.txt
6e6bb5ce339d58b3b082312e5add8f6d
a66f42b06ecaec37f7900942dbff29f8803a773f
F20101108_AACFOS atalayer_d_Page_09.tif
5c19028d635549cf6d18687f3f7a2e97
b7f536fab385e0bba4f28480a6e5c67fffadf41a
F20101108_AACFJV atalayer_d_Page_53thm.jpg
120512900128f55d52803f1f7c57c3a1
b97f84bd402b5abbbf2b9f0ce2d2d661e427f0ee
448 F20101108_AACFTQ atalayer_d_Page_41.txt
420c634043b58476649e1e9a388cfa7c
3bcf105f2473ffc0909b59282f8ee232fe729f13
F20101108_AACFOT atalayer_d_Page_10.tif
04ece88f6d96a8ea0e2561653e7f1092
ace86efd0b54b9b57c08efad55f2c7b883322439
F20101108_AACFJW atalayer_d_Page_39.tif
70770fbd0570eba13c411a8b9d600f60
6ac29be7992f79a38a70a84d73d3a8da8dfe91ab
186 F20101108_AACFTR atalayer_d_Page_42.txt
417da5ae4a06baf44b4d91d184b463d8
e6f2c43a89bfe9a677c7c8921fe9ccc907ef1b7b
F20101108_AACFOU atalayer_d_Page_12.tif
e01df69af3f074b14f2a7ba65811fc25
1a193d398b71bd91c3a2584c8ab0fa4e9b419c44
84140 F20101108_AACFJX atalayer_d_Page_11.jpg
9db85f967bb4b07765266639f3085796
2c87eefa130bdcdeb285129c7b3c5907885bdadd
366 F20101108_AACFTS atalayer_d_Page_43.txt
1c57e860ea92e8ddf4c9b71f52d8bbd6
edeabc50f89b437dfa085452335c1380c027c846
F20101108_AACFOV atalayer_d_Page_13.tif
a102172d08144873b5d8f9fa510b2043
f7b9c870443d35b1a57ad704b528b8a90c4af583
F20101108_AACFJY atalayer_d_Page_12.txt
cb406d666c3681b62b16de2b2dfd29d8
02293a79d3573663a557ef03748fe81a98ad5dcf
261 F20101108_AACFTT atalayer_d_Page_44.txt
8251c645b0707c69ddd3d6869d1efa6b
b90a44fcdf94065b9115b8f10ede13569121ef25
F20101108_AACFOW atalayer_d_Page_14.tif
12459781e835b3f14fd234404f5eee63
8f6de5c062d0f64afe7c11082d8146b1701bf1dc
F20101108_AACFJZ atalayer_d_Page_53.jp2
898b354aee4c94213beda03f3b2ac700
df91e7a65e73ea162de9c110812efa2cca1033b9
465 F20101108_AACFTU atalayer_d_Page_46.txt
1015374be54cc53968b08de319374579
12eb4ffbb901750eb355e1ddc93bc6a9da9991d3
2145 F20101108_AACFTV atalayer_d_Page_47.txt
0256a227004e19534f11edd9eb50bf03
d8d1a8819282b49cb11884ecd107ea6c75264564
F20101108_AACFOX atalayer_d_Page_15.tif
cb0d4e40bfe4507d87599c843f03115e
3d7d48f40a1b1b8eceb453b0c5eb52925e9f4451