Group Title: Molecular Pain 2007, 3:10
Title: Inward currents induced by ischemia in rat spinal cord dorsal horn neurons
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Title: Inward currents induced by ischemia in rat spinal cord dorsal horn neurons
Series Title: Molecular Pain 2007, 3:10
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Creator: Chen M
Tao YX
Gu JG
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Molecular Pain


B
BiolMed Central


Research


Inward currents induced by ischemia in rat spinal cord dorsal horn
neurons
Meng Chen', Yuan-Xiang Tao2 and Jianguo G Gu*1


Address: 'Department of Oral and Maxillofacial Surgery, McKnight Brain Institute and College of Dentistry, University of Florida, Gainesville,
Florida, 32610, USA and 2Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, 355 Ross, 720
Rutland Ave., Baltimore, Maryland 21205, USA
Email: Meng Chen meng.chen@duke.edu; Yuan-Xiang Tao ytau@jhmi.edu; Jianguo G Gu* jgu@dental.ufl.edu
* Corresponding author


Published: 25 April 2007
Molecular Pain 2007, 3:10 doi:10.1 186/1744-8069-3-10


Received: 21 March 2007
Accepted: 25 April 2007


This article is available from: http://www.molecularpain.com/content/3/1/10
2007 Chen et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Hypoxia and ischemia occur in the spinal cord when blood vessels of the spinal cord are
compressed under pathological conditions such as spinal stenosis, tumors, and traumatic spinal
injury. Here by using spinal cord slice preparations and patch-clamp recordings we investigated the
influence of an ischemia-simulating medium on dorsal horn neurons in deep lamina, a region that
plays a significant role in sensory hypersensitivity and pathological pain. We found that the
ischemia-simulating medium induced large inward currents in dorsal horn neurons recorded. The
onset of the ischemia-induced inward currents was age-dependent, being onset earlier in older
animals. Increases of sensory input by the stimulation of afferent fibers with electrical impulses or
by capsaicin significantly speeded up the onset of the ischemia-induced inward currents. The
ischemia-induced inward currents were abolished by the glutamate receptor antagonists CNQX
(20 [tM) and APV (50 [tM). The ischemia-induced inward currents were also substantially inhibited
by the glutamate transporter inhibitor TBOA (100 [tM). Our results suggest that ischemia caused
reversal operation of glutamate transporters, leading to the release of glutamate via glutamate
transporters and the subsequent activation of glutamate receptors in the spinal dorsal horn
neurons.


Background
Glutamate is the principle neurotransmitter that mediates
sensory transmission in the spinal cord dorsal horn.
Under physiological conditions, glutamate is released
synaptically by primary afferent fibers, descending termi-
nals from supraspinal regions, and excitatory intemeu-
rons in the spinal cord dorsal horn [1]. The synaptically
released glutamate is rapidly taken up through glutamate
transporters located at presynaptic terminals, postsynaptic
cells, and on the surrounding glia cells [2-5]. These trans-
porters keep extracellular glutamate at low levels to ensure
high fidelity sensory transmission, to limit nonspecific


neuronal excitation and hyperactivity, and to prevent exci-
tatory toxicity [3,6].

Increased glutamate concentrations in extracellular spaces
can occur as a consequence of CNS tissue injury, which in
turn can produce neuronal hyperactivity and secondary
neuronal tissue damage due to excitatory toxicity [7]. It
has been shown that extracellular glutamate levels
increased significantly in the brain following ischemic
and hypoxic injury [8,9]. In the spinal cord, ischemia and
hypoxia can occur under a number of pathological condi-
tions including traumatic spinal cord injury, tumors


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within the spinal cord, spinal stenosis, cardiac arrest, mas-
sive hemorrhagic shock, and surgical procedures [10-14].
These conditions often cause spinal blood vessel compres-
sion, resulting in spinal cord ischemia and hypoxia. Simi-
lar to the brain, spinal cord ischemia and hypoxia also can
result in the increases of extracellular glutamate levels to
cause neuronal excitatory toxicity in the spinal cord.
When these pathological processes occur in the dorsal
horn of the spinal cord, sensory functions may be signifi-
cantly altered to result in pathological pain states.

Leak of glutamate from damaged cells and release of
glutamate from synaptic sites were thought to contribute
to the elevation of extracellular glutamate concentrations
under pathological conditions. However, studies have
suggested that a change of glutamate transporter function
plays a critical role in the sustained elevation of extracel-
lular glutamate levels during ischemia and hypoxia [9,15].
Under physiological conditions, glutamate transporters
co-transport one glutamate molecule and 3 Na+ ions into
the cell to maintain the concentration gradient of micro-
molar extracellular glutamate against millimolar intracel-
lular glutamate [16,17]. This active transport function is
supported by the transmembrane ion gradients estab-
lished by Na+-K+ ATPase [16,17]. Under pathological con-
ditions, for example, during brain ischemia and hypoxia,
ATP is depleted and Na+-K+ ATPase function is impaired.
This subsequently results in the loss of transmembrane
ion gradients and thereby reducing the driving force for
the active uptake of glutamate from extracelular glutamate
[15]. In fact, studies using brain tissues suggested that the
depletion of intracellular energy not only compromises
glutamate uptake, but also can result in glutamate release
through glutamate transporter system due to the reversal
operation of the glutamate transporters [9].

In the present study, we tested the hypothesis that
ischemic condition results in the reversal operation of
glutamate transport system to cause glutamate release and
subsequent excitation of sensory neurons in the spinal
cord dorsal horn. The study may have implications in
pathological pain states associated with ischemic and
hypoxic conditions in the spinal cord dorsal horn [5].

Results
The ischemic condition was produced by the bath appli-
cation of a modified Kreb's solution that did not contain
glucose and was bubbled with N2 gas to deoxygenate the
solution. The bath solution also contained 1 mM sodium
cyanide to block glycolysis and oxidative phosphorylation
[9]. When this ischemia-simulating medium [18,19] was
perfused to the spinal cord slice preparations, we recorded
large inward currents (ischemia-induced inward currents)
from lamina V neurons of the spinal cord dorsal horn (Fig
1A). The onset time of the ischemia-induced inward cur-


rents showed large variations. When animals at the age of
6 days old were used, the onset of the ischemia-induced
inward currents was at 22 1 min (n = 6, Figure 1A&1B).
The ischemia-induced inward currents reached peak levels
rapidly and then gradually decayed to baseline levels in
recordings when the spinal cord slices were prepared from
these young animals (Figure 1A). The onset time of the
ischemia-induced inward current became shorter when
older animals were used. For animals at the age of 10 days
old, the onset of the ischemia-induced inward currents
was at 18.5 0.5 min (n = 6). When animals at the age of
20 days old were used, the onset time was 8 0.4 min (n
= 6), 3 times shorter than the onset time for animals at the
age of 6 days old (Figure 1). We observed that, in older
animals, the ischemia-induced inward currents usually
did not return to baseline and membrane seals on patch-
clamp electrodes were eventually lost (Figure 1A). The
age-dependence in the onset of the ischemia-induced
inward currents was observed in the range from 4 days to
20 days old (Figure 1B); animals over 20 days old were
not examined in this study. The peak amplitude of the
ischemia-induced inward currents, on the other hand, was
not found to be age-dependence. Overall, the peak ampli-
tude was 720 38 pA (n = 43)

We tested whether sensory input affects the ischemia-
induced inward currents. Sensory input into spinal cord
dorsal horn was elicited by electrical stimulation of pri-
mary afferent fibers at the intensity of 100 pA and fre-
quency of 100 Hz. In this set of experiments, all the spinal
cord slices were prepared from animals at the age of 9 days
old. When primary afferent fibers were not stimulated
electrically, the onset of the ischemia-induced inward cur-
rents was at 20 + 0.6 min (n = 6) following the application
of ischemic solution (Figure 2A&2C). On the other hand,
the onset time was 16 0.3 (n = 4) min when primary
afferent fibers were repeatedly stimulated at the frequency
of 100 Hz (Figure 2B&2C), significantly shorter than the
onset time when primary afferent fibers were not stimu-
lated (P < 0.05, Figure 2C). There was no significant differ-
ences in the peak current amplitude between the
experiments without electrical stimulation (1400 350
pA, n = 6) and the experiments with electrical stimulation
(1359 345 pA, n= 4).

We tested whether stimulation of TRPV1-expressing noci-
ceptive afferent fibers with capsaicin also affect ischemia-
induced inward currents. In this set of experiments, all the
spinal cord slices were prepared from animals at the age of
7 days. In experiments when capsaicin was not applied,
ischemia-induced inward currents had onset time of 22 +
1.8 min (n = 6) following the application of the ischemic
Kreb's solution (Figure 3A&3C). When capsaicin (2 [tM)
was included in the ischemic solution, the onset time of
the inward currents was 18 0.2 min (n = 4; Fig 3B&3C),


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Ischemia


6 days old



10 days old



20 days old


200 pA


10 20
Time (min)


30

-25

20

C 15

S10
o.


4 6 8 10 12 14 16 18 20
Age (days)

Figure I
Ischemia-induced inward currents in spinal cord dorsal horn neurons of rats at different ages. A). Three sample
traces show inward currents induced by ischemia in dorsal horn neurons from rats at the age of 6 days old (top trace), 10 days
old (middle trace), and 20 days old (bottom trace). The horizontal bar above the traces indicates the time period when
ischemia bath solution was perfused to the spinal cord slice preparations. B). The relationship between ages of rats and onset
time of ischemia-induced inward currents. Numbers above the curve indicates the number of animals. All recordings were
made from lamina V dorsal horn neurons.



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A


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A Ischemia


07





0


I 200 pA


17 18 19 20 21 22
Time (min)


Ischemia/electrical stimulation


I 200 pA


0


Ischemia

Ischemia/
Stimulation


17 18 19 20 21 22
Time (min)


-p


0 5 10 15 20
Onset Time (min)

Figure 2
Shortening of the onset time of ischemia-induced inward currents by capsaicin stimulation. A). A sample trace
shows ischemia-induced inward current in a lamina V neuron of a rat at the age of 9 days old. B). A sample trace shows
ischemia-induced inward currents in a lamina V neuron of another rat at the age of 9 days old. In this experiment, focal stimu-
lation (100 [tA, 100 Hz) was applied to dorsal root entry zone while ischemic bath solution was applied to the spinal cord slice
preparation. Note that in this sample trace, stimulation artifacts were superimposed on the inward currents. C). Pooled
results from experiments represented in A (open bar, n = 6) and B (solid bar, n = 4). All animals used were at the age of 9 days
old.


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significantly shorter than the onset time of the experi-
ments when capsaicin was absent (P < 0.05, Figure 3C).
There was no significant differences in the peak current
amplitude between the experiments without capsaicin
stimulation (805 395 pA, n = 6) and the experiments
with capsaicin stimulation (765 120 pA, n = 4).

We explored mechanisms by which ischemia induced
inward currents. In this set of experiments, all animals
used were at the age of 10 days old. The ischemia-induced
inward currents had an onset time of ~17 min (Figure 4A,
also see Figure 1B) with peak currents of 646 150 pA (n
= 4). In the presence of glutamate receptor antagonists
CNQX (20 |pM, for non-NMDA receptors) and APV (50
[tM, for NMDA receptors), ischemia-induced inward cur-
rents were almost completely abolished (16 2 pA, n = 6,
p > 0.05; Figure 4B&4D). In the presence of DL-threo-P-
benzyloxyaspartate (TBOA, 100 [tM), a potent glutamate
transporter inhibitor, the ischemia-induced inward cur-
rents were also largely inhibited (Figure 4C&4D). The
ischemia-induced inward currents were 77 36 pA (n = 9)
in the presence of TBOA, only 12% of the ischemia-
induced inward currents in those experiments when
TBOA was absent (Figure 4D).

We determined whether ischemic conditions have a sig-
nificant effect on inward currents evoked by exogenously
applied glutamate. In this set of experiments, glutamate
(500 [tM, 1 min) was first applied for 1 min under the nor-
mal Kreb's bath condition. We then switched the normal
bath solution to ischemic bath solution. Following a 10-
min perfusion of ischemic bath solution, a second appli-
cation of glutamate (500 [tM, 1 min) was tested. This
application was made prior to the onset of the ischemia-
induced inward currents (Figure 5A). We found that the
inward current evoked by the second glutamate applica-
tion was 250 + 65% of the inward current induced by the
first glutamate application (p < 0.05, n = 6) (Figure
5A&5C). In contrast, when tissues were maintained at
normal Kreb's bath solution, the inward current elicited
by the second application of glutamate was usually
slightly smaller than the current elicited by the first gluta-
mate application (78 8%, p > 0.05, n = 6, Figure
5B&5C).

Discussion
In this study, we show that ischemia induced significant
inward currents in dorsal horn neurons of the spinal cord
and that the inward currents could be completely blocked
by ionotropic glutamate receptor inhibitors as well as by
glutamate transporter inhibitors. This observation is con-
sistent with a previous study using brain slice preparations
showing that glutamate transporter inhibitors blocked
ischemia-induced inward currents [9]. These findings sug-
gest that ischemia may cause reversal operation of gluta-


mate transporters, leading to the release of glutamate via
glutamate transporters and the subsequent activation of
glutamate receptors in the neurons of the spinal dorsal
horn and brain. Our study, however, cannot exclude other
mechanisms that may contribute to ischemia-induced
membrane depolarization as shown in hippocampal neu-
rons [18,19].

We observed that the onset of ischemia-induced inward
currents took many minutes. This delay in the onset sug-
gests that intracellular energy depletion is a slow process
under our experimental conditions and transporter
reversal does not occur before intracellular energy is sub-
stantially depleted. Interestingly, we have found that the
onset times of ischemia-induced currents are age-depend-
ent, being shorter in older animals and longer in younger
ones. One explanation for the age-dependence is that the
rates of intracellular energy depletion may be different
between younger and older animals under ischemic con-
ditions. The onset times of ischemia-induced currents
were shortened when primary afferent fibers were stimu-
lated electrically or with the noxious stimulant capsaicin.
This is mort likely due to fast energy depletion when pri-
mary afferent fibers were stimulated. In addition to the
demonstration of ischemia-induced inward currents, we
have also showed that inward currents evoked by exoge-
nous glutamate were significantly larger under ischemia
condition than under normal condition. This is probably
because the actual concentrations of exogenous glutamate
that reached the recorded neurons were higher under
ischemia condition than under normal condition. This
result suggest that prior to the reversal operation of gluta-
mate transporters, glutamate uptake is severely compro-
mised under ischemia condition. Glutamate transporters
are expressed on both neuronal and glia cells in the spinal
cord dorsal horn [4,5]. To date, five subtypes of glutamate
transporters have been cloned: GLAST (EAAT1), GLT-1
(EAAT2), EAAC-1 (EAAT3), EAAT4 and EAAT5 [6].
GLAST, and GLT-1 are predominantly localized in astro-
cytes [2,20,21], while EAAC-1, EAAT4, and EAAT5
appears to be mostly neuronal [22-25]. While both neuro-
nal and glial glutamate transporters actively participate in
the uptake of extracellular glutamate [26,27]. Glutamate-
induced excitatory toxicity under ischemia conditions
appears to be mainly due to the impairment of glial gluta-
mate transporters [3,28,29].

Previous studies have demonstrated that TBOA blocks
glutamate uptake under physiological conditions [30].
We showed that ischemia-induced glutamate release was
inhibited by glutamate transporter inhibitor TBOA in the
present study. These results together suggest that TBOA bi-
directionally blocks glutamate transporters. Effects of
TBOA on sensory behaviors have previously been studied
in both normal animals and animals with pathological


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Ischemia


"r-/


1300 pA


14 16 18
Time (min)


20 22 24


Ischemia/Capsaicin


0
----4y/t




;--


1300 pA


14 16 18
Time (min)


20 22 24


C


Ischemia

Ischemia/
Capsaicin


r *


0 5 10 15 20 25
Onset Time (min)

Figure 3
Shortening of the onset time of ischemia-induced inward currents by electrical stimulation. A). A sample trace
shows ischemia-induced inward current in a lamina V neuron of a rat at the age of 9 days old. B). A sample trace shows
ischemia-induced inward currents in a lamina V neuron of another rat at the age of 9 days old. In this experiment, capsaicin (2
[tM) was applied in ischemic bath solution to the spinal cord slice preparation. C). Pooled results from experiments repre-
sented in A (open bar, n = 6) and B (solid bar, n = 4). All animals used were at the age of 9 days old.


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Ischemia

*mm//i L | n


I 100 pA
2 min


C


Ischemia/CNQX+APV


- ---/-, _1
15 min
2 min



Ischemia/TBOA


1
15 min


V.------


D 1000o

< 800

c 600

) 400
-

I 200

0


Ischemia Ischemia
CNQX
APV


Figure 4
Blocking of ischemia-induced inward currents by ionotropic glutamate receptor antagonists and glutamate
transporter inhibitors. A). A sample trace shows ischemia-induced inward currents from a lamina V neuron of a rat at the
age of 10 days old. B). A sample trace shows the block of ischemia-induced inward currents by the glutamate receptor antago-
nists CNQX (20 [tM) and APV (50 [tM). The recording was made from a rat different from A. C). Inhibition of ischemia-
induced inward currents by the glutamate transporter inhibitor TBOA (100 [tM). The recording was made from a rat different
from A and B. D). Summary of ischemia-induced inward currents in the absence (first bar, n = 4), in presence of CNQX and
APV (n = 6), or in the presence of TBOA (n = 9). All recordings were made from lamina V neurons of the rats at the age of 10
days old.



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2 min


**


Ischemia
TBOA


Molecular Pain 2007, 3:10


z W"' I






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Glu Glu
Ischemia


B
Normal
bath







C


Glu


Glu


l[ First application
I Second application


Control


_ 100 pA
2 min





-J 200 pA
2 min


*

T


Ischemia


Figure 5
Inward currents evoked by exogenous glutamate under ischemia conditions. A). A sample trace shows inward cur-
rents elicited by two applications of exogenous glutamate. The first application was made in normal bath solution and the sec-
ond application was made 10 min following the perfusion of ischemia bath solution. An arrow indicates the start of the
ischemia-induced inward currents. B). A sample trace shows inward currents elicited by two applications of exogenous gluta-
mate in normal bath solution. C). Summary of the changes of glutamate-evoked currents in normal bath solution (first set of
bars) and in ischemic bath solution (second set of bars). The current amplitudes were normalized. Open bars represent the
currents evoked by the first application recordings of glutamate (n = 6); black bars represent the currents evoked by the sec-
ond applications of glutamate (n = 6).


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pain conditions. In normal animals, intrathecal injection
ofTBOA was shown to induce nociceptive behaviors, such
as licking, shaking, and caudally directed biting [31].
These effects were thought due to the block of glutamate
uptake by TBOA, which subsequently results in the eleva-
tion of extracellular glutamate levels to cause hyperactivity
in the spinal cord dorsal horn neurons [31]. Interestingly,
under pathological pain conditions, glutamate trans-
porter inhibitors were found to produce anti-nociceptive
effects. For example, glutamate transporter inhibitors
were shown to attenuate the induction of allodynia
induced by PGE2, PGF2a, and NMDA [32], to reduced
formalin-induced nociceptive responses, and to attenuate
Complete Freund's adjuvant (CFA)-evoked thermal
hyperalgesia [5,33]. In addition, transient knockdown of
spinal GLT-1 led to significant reduction of nociceptive
behavior in the formalin model [5,33]. It has been pro-
posed that pathological pain conditions may cause a
depletion of intracellular energy in the spinal cord dorsal
horn, which subsequently reverses glutamate transporters
to release glutamate and to produce hyperactivity in the
spinal cord dorsal horn neurons [5]. Therefore, by block-
ing glutamate release from its transporters, glutamate
transporter inhibitors may execute an anti-nociceptive
effect under pathological conditions [5]. The present in
vitro study supports the rationales for the use of glutamate
transporter inhibitors.

Methods
Principles of laboratory animal care (NIH publication No.
86-23, revised 1985) were followed in all the experiments
described in the present study. Spinal cord slice prepara-
tions and patch-clamp recordings were described in
details in our previous studies [34]. In brief, Sprague Daw-
ley rats at the postnatal age of 4-21 days were used. Trans-
verse spinal cord slices were prepared from lumbar
enlargement of the spinal cords. The thickness of each
slice was 400 |tm. The spinal cord slices were maintained
in a basket submerged in ~200 ml Krebs solution at 24 C.
The Krebs solution contained (in mM): NaCl 117, KC1
3.6, CaC12 2.5, MgCl2 1.2, NaH2PO4 1.2, NaHCO3 25 and
glucose 11; the solution was saturated with 95 % 02 and
5% CO2 and had pH of 7.4. In each experiment, a spinal
cord slice was transferred to a recording chamber and
placed on the stage of an upright IR-DIC microscope. The
slice was perfused at ~10 ml/min with the Krebs solution
and all experiments were performed at room temperature
(24 C). To induce ischemia, the slices were perfused by a
ischemic bath solution, which is modified Kreb's solution
that had no glucose (replaced with 7 mM sucrose) and
contained 1 mM sodium cyanide [9], and the bath solu-
tion was deoxygenated by continuously bubbling with
100% nitrogen.


Individual neurons were identified under an IR-DIC
microscope with a 40x water immersion objective. Whole
cell patch-clamp recordings were made in deep lamina
(lamina V) neurons with electrodes that were filled with
an internal solution contained (mM): Cs2SO4 110, TEA-C1
5, CaC12 0.5, MgCl2 2, EGTA 5, HEPES 5, pH 7.3. The
resistance of electrodes was ~5 MQ when filled with the
internal solution. The access resistance was below 30 MQ
and was not compensated. Signals was amplified and fil-
tered at 2 kHz (Axopatch 200B) and sampled at 5 kHz
using pCLAMP 7.0 (Axon Instruments). In all recordings,
neurons were voltage-clamped at -30 mV. In some experi-
ments, primary afferent fibers were stimulated by focal
electrical stimulation at the dorsal root entry zone or by
chemical stimulation with capsaicin. For the focal electri-
cal stimulation, the stimulation intensity was 100 pA and
the stimulation frequency was 100 Hz. For chemical stim-
ulation, capsaicin at the concentration of 2 [tM was bath
applied to stimulate capsaicin-sensitive nociceptive affer-
ent fibers. Pharmacological tests, including the tests of
CNQX (20 [tM), APV (50 [tM), and TBOA (100 [tM) were
performed by applications of these compound through
bath solution. Unless otherwise indicated, all recordings
were performed in the presence of 20 [tM bicucullin and
2 jM strychnine.

Data were presented as mean + SEM. Student's t-tests were
used for statistical analysis and significance was consid-
ered at the level of the p < 0.05.

Competing interests
The authors) declare that they have no competing inter-
ests.

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