<%BANNER%>

Rats Exhibit Behavioral Despair and Hormonal Alterations after Social Defeat Stress: Implications for Major Depression

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 E20101208_AAAASG INGEST_TIME 2010-12-09T04:44:33Z PACKAGE UFE0017942_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 47830 DFID F20101208_AACLLR ORIGIN DEPOSITOR PATH stone_k_Page_30.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
c02582ff2f32139af0570ae12416f3c2
SHA-1
763a50fbd2551ae4438d60e92e49a169999297b2
75614 F20101208_AACLGU stone_k_Page_16.jp2
be7862dc59435cb11b8a48a8a5bbb2df
6dff227d833794a135db0bd1d9aab4696cba26dd
2102 F20101208_AACLQP stone_k_Page_19.txt
283cc48f7d57cc345790035ab355400d
ac733c9b14ac81fcd705419db4f53e07fe0798dc
54128 F20101208_AACLLS stone_k_Page_31.jpg
359d3b184c66d5fa17d287f6b4bb6fb3
dd3b3194837a7233de00ba825ce24f27b035f8ff
65555 F20101208_AACLGV stone_k_Page_27.jpg
f18d97161f20d26ef868aa1ab1516160
d3faade3c7be7062b068658e767ba02519f27cb1
2076 F20101208_AACLQQ stone_k_Page_20.txt
a13eb665e6c1266c474cb75ef52a58bc
7dc9d9fb3f37b51a0f238fdca85e626b19433395
46871 F20101208_AACLLT stone_k_Page_32.jpg
77eb2dcd490c0189a71dac2858905450
00fb233ba34e32f950097131d5dcc9b9591223b1
65314 F20101208_AACLGW stone_k_Page_29.jp2
1dd3f69577acd3058cf6566c39825d32
e3b138c8a4f5cb96882f3472f35b0a843fc10fe8
1967 F20101208_AACLQR stone_k_Page_21.txt
daf6a3df490f595792f324cba8bab3b7
dd78c590d3fbadfdcebad654ec0b29adae204fce
74797 F20101208_AACLLU stone_k_Page_33.jpg
7ef78b50662caf360a8b93b72bf02407
6dd718a8f26b246265e4a49c365370868e9c1615
11647 F20101208_AACLGX stone_k_Page_26.QC.jpg
d075208cc8be0a47aa96bd3c00e1dee6
720009dddab3d199ff79582d9f2397c887378956
1840 F20101208_AACLQS stone_k_Page_24.txt
85c18ce824e8e68898e431a9213e7d55
242e9f5ca6d3984a2ac22639ac13001818df6bc0
38843 F20101208_AACLLV stone_k_Page_34.jpg
7a2aa39a42c49d19ec0653ba83875512
effb707690256a5e25d7ddfc95f8b00b02141f5b
1053954 F20101208_AACLGY stone_k_Page_16.tif
9f0738194680018230ee6b8eeab23ab1
17a6d0fac2e964727f710e90ae8623158f10f9fe
365 F20101208_AACLQT stone_k_Page_25.txt
6faa6e0618216842b165c9d09ea3f8bb
c79d049cf6fb40899e5fa7ac51c12cbc5079b7ec
50321 F20101208_AACLLW stone_k_Page_36.jpg
79db11ef5909f2d8923fcbd58ba7b4ec
b829398a285b721a36300f92b0f71a8f4acec7b6
25287 F20101208_AACLGZ stone_k_Page_01.jpg
99ce48b858c7e956a25c494c5f3a86d3
a8d9727bc233853c2207ec9bde069a45c25af08a
51886 F20101208_AACLLX stone_k_Page_37.jpg
4497d5ae616914e9b8a972f24b3ecabe
7e7d346744d90ce80bb585a77e01e267dba5e74b
489 F20101208_AACLQU stone_k_Page_26.txt
be598496fba95452d6646d80693411b0
0ae18e27e62de193b9d1bce51b21d3f07c4a55bb
70805 F20101208_AACLLY stone_k_Page_41.jpg
7c5151125e64e28663cd486ef6a02238
0505d627768b48af30b1c1286931e867af02c3f5
1873 F20101208_AACLQV stone_k_Page_27.txt
41574650e7783473009f610df19f1fcb
8eb26c060b8394114fe80a7b100efd08789e4483
5550 F20101208_AACLJA stone_k_Page_07.QC.jpg
6504e9cbd9fe34f0075472060f9aca6f
0b60bc7ecff95915995c6d32cc2ba198d5b5f078
73097 F20101208_AACLLZ stone_k_Page_43.jpg
a23b372fa61f6eab791e45d514f62ca6
f4c55a3bc64c1b75f1c795d0afae1186eea98e85
1118 F20101208_AACLQW stone_k_Page_28.txt
34048e7c4332a4f7cd389fbad7ff97bd
025c0474a4bdc739d176b8bb607dc6d1b55498d2
22193 F20101208_AACLJB stone_k_Page_48.jpg
96a3cb450f4b025c2b557e1454636198
0e81451688bb28ef29a54d2432aefc52dda77d15
1519 F20101208_AACLQX stone_k_Page_29.txt
ad7997f30006cfc3329ad273448ca930
32fff74935b74912adac9127af3cf7622f467942
22774 F20101208_AACLJC stone_k_Page_13.QC.jpg
60662ac0f251cedc0c5b3152f2a7f52f
90593926985532b9f11719d91195bcdd65e21cf7
F20101208_AACLOA stone_k_Page_25.tif
4c4502dc107759a3e854651abc488a3d
f315f81271bac56ce340a154aff09002522927d9
917 F20101208_AACLQY stone_k_Page_30.txt
14b0a675b4becfce9a2128adf2d1f35e
f65ca700a93602eeea84afb7feaee5b210f86642
522047 F20101208_AACLJD stone_k.pdf
072692f474c0a45614f59e6ca9c081af
48cd5c7e5ca1c0a9da0f3caa226dc4f890a64976
F20101208_AACLOB stone_k_Page_26.tif
67cc6d4a8dfa8396cbdca17252b27501
38f797c4c3632409f4a190791156c9335e6be8bc
955 F20101208_AACLQZ stone_k_Page_31.txt
db4eb106c82dc3c9a2b901a1c2dc23ab
a6fd6111cb2c60839eea0db4241655eafa0bf589
2233 F20101208_AACLJE stone_k_Page_47.txt
9a0bcc8d5923e065fafbf00ded85052a
6a7e6635c86c3d99f3647597af86b41fe002f35a
F20101208_AACLOC stone_k_Page_27.tif
860c2801027080da80254a99ef9eac58
c329325da535d6b107406a6fff380a7498d35a8a
20654 F20101208_AACLJF stone_k_Page_39.QC.jpg
473ef7e93890b55133fccdfd7905a89b
b47aac212ff21f17b369f90539aa57c9f61dd0bc
F20101208_AACLOD stone_k_Page_28.tif
cd7f7ccaf0b25a1d4bbf1a48ec7809b8
cc18617bf45daf67f22eabb245eec628efacb557
22718 F20101208_AACLJG stone_k_Page_24.QC.jpg
84291f0253c963a9fdd6086a7c8251fc
6f20b5521e7ba021baa3307912d355b6006cf0ee
21237 F20101208_AACLTA stone_k_Page_10.QC.jpg
b1ccce36fa2ed260ce40420ffa8a1790
e597896df861f35c1f40f30d2b888418c7ac1914
F20101208_AACLOE stone_k_Page_29.tif
75c50d5e0e8847dcaa67ccb1c7a8726f
31e6f28bd774fb9dadc8b60e15e6f8b27a508454
63851 F20101208_AACLJH stone_k_Page_39.jpg
ae804cef37e523daebc322a2ff0ecc28
74f09341d621f9771a43855ad98f2ad9fa70bda3
5943 F20101208_AACLTB stone_k_Page_11thm.jpg
61b28c99c8cfa71e49d6d867a5343d71
bdba23bf4d11b73d0a04dc3a242c3476bf6314f2
25271604 F20101208_AACLOF stone_k_Page_31.tif
a4e03f2e6efcea755cb5358f21c281fe
81e366914d47c8f91ec017f9b92289330884f970
52935 F20101208_AACLJI stone_k_Page_20.pro
c576713e03659beb2ace0e30d065b7b4
38f5150f5af7afda465d78807d7f1dec58878bf5
6381 F20101208_AACLTC stone_k_Page_12thm.jpg
2bc545f6cc6709310412ebba8eb4a35b
35a652a4524b4f4bbfc90823b5288d28db9877fe
F20101208_AACLOG stone_k_Page_33.tif
f87e710d1cf1865b530c24d43195817a
119594d22a5ba23e8c6e5b2bf36c60816e4bd833
49926 F20101208_AACLJJ stone_k_Page_12.pro
eef06ff554fea6f240f1b6e2f2afc8f9
8e08fe0f8e096d2ebbe558d8b8bd4c35bd00abf4
6038 F20101208_AACLTD stone_k_Page_14thm.jpg
ebccec6671173658c17b1d06704b7b8a
1c631e09645bb4e8ba8556236229cfe39118d86d
F20101208_AACLOH stone_k_Page_35.tif
bbeb9e51e873630521d54e58c28e9ed1
f84456e9ac624ee77ed2df9b8027a1f36046faeb
6531 F20101208_AACLJK stone_k_Page_18thm.jpg
05f7225bb688b981eb81dae670910c4a
f8b4bbb9a84a2c021d407710551b939010425815
6464 F20101208_AACLTE stone_k_Page_15thm.jpg
1bce06b0daefc71dd93fa1cdb1a99bfc
8c6a525de4d27de3e3524f094707ba5a66d481cd
8423998 F20101208_AACLOI stone_k_Page_36.tif
ca787af8f5ab828e0912424bf65feec7
e16d029d653e2ac07181d35d2f994f5bf5b7f267
4789 F20101208_AACLJL stone_k_Page_37thm.jpg
4d33d01ac2bb91d1ec1caebd1697d4de
5fb49040f13d3c8be9b7f0a5e1728cd5765fc262
5688 F20101208_AACLTF stone_k_Page_17thm.jpg
22b37c07266d9b3b06ff9c466f17b7cd
15f99bce282c321699873d3c68317cceeee15502
F20101208_AACLOJ stone_k_Page_37.tif
f784d49157949873fea7f97812a1e596
a78cad58ed60b36491e244547b1e787269ceb171
45634 F20101208_AACLJM stone_k_Page_10.pro
48506824f902e2d241e418d7feb76a56
110bcb82821c69dabb6b0eb92cae6a0fab13f5b2
6643 F20101208_AACLTG stone_k_Page_19thm.jpg
99e97bf722cb5a582d032e768ff963e5
af3bde682cac9814b9c3193b299bee159300f190
F20101208_AACLOK stone_k_Page_38.tif
007424506dfe013e10e14a3cb07cec52
60f0883c20ec4c63eefb588fea9a2d3950256262
6625 F20101208_AACLTH stone_k_Page_20thm.jpg
cc862f924f4323fb9f758728a4e375bd
d90cb64c74d02fcdb9518634eaa5841f28cfda7e
F20101208_AACLOL stone_k_Page_39.tif
1fb3692626036bf4e355f6c54338ec06
08d3ba91cacfaace3c60b19c339c10215a3bcfc5
4302 F20101208_AACLJN stone_k_Page_34thm.jpg
f09880f7b5d685f687952588a752258a
50b10412a9bdd12e00fbc610697cabd862c3dabd
6495 F20101208_AACLTI stone_k_Page_21thm.jpg
5d765771a2df258105ce1440ca0b8124
74030d21e20a9cd0814be0ecc2d0acb5797bf2a1
F20101208_AACLOM stone_k_Page_40.tif
467ae03a2d76cff4986dbd39490a58d0
efde91528dbd17f5956293b8aa157e5dd138813b
17254 F20101208_AACLJO stone_k_Page_38.QC.jpg
9d5466858e5f0aaa948fb4a895f0bd44
ae16f8286a9dd0772fee6aad8722b5bb77103606
6499 F20101208_AACLTJ stone_k_Page_25.QC.jpg
6537225d18af69767223c4b677074840
25c36f058cc72f8f89bec3041b0ec3bd90ff8f2e
F20101208_AACLON stone_k_Page_42.tif
c73a70326336c4d284c53037bf72066f
221279e7f8ff647d7b20b40e66c203415a70c2f1
23281 F20101208_AACLJP stone_k_Page_25.jp2
db1a3f769dd20894d61fab68a0a13257
8e5609c5bff6323aceeffb9485bf21a178668ecc
6384 F20101208_AACLTK stone_k_Page_41thm.jpg
5df314cb0f9ff428e9fe2fe853e3304a
53f51666729936393ebc32bd2af2a92b91d01ab4
F20101208_AACLOO stone_k_Page_43.tif
1b624c5050ae686c156dfcc4576e7865
55cd34aae107398bb5cd2f960105951875d43510
8683 F20101208_AACLJQ stone_k_Page_07.pro
b068ed2759c9e59e7e43a472b7fa11a7
09aca5a4a91f134ddadd545cf111e4c25d686384
6358 F20101208_AACLTL stone_k_Page_42thm.jpg
337716b026c194db32f6b628f3fb6a47
685dd222ff9a21aa1481908148665f64d13cc5b4
F20101208_AACLOP stone_k_Page_44.tif
1e02bbfa649c0c3eef9d939246e99f55
c72b6a0051fac1c54e316f1b42319c658c1ab6b8
26149 F20101208_AACLJR stone_k_Page_01.jp2
51a226710708078823601e78854c749d
d52d85d0a5207f67e9ba8c590f2e3bb16f036226
6592 F20101208_AACLTM stone_k_Page_43thm.jpg
a5cf857f54eee5e8d4925bb4322c95ac
47c280a2f40c31532b822aad846ff4242f346861
F20101208_AACLOQ stone_k_Page_45.tif
772b33a2c69be6f136336d17f4acd0fb
7a86818a7987af38c3c287c06656e42a2f8d1abd
59223 F20101208_AACLJS stone_k_Page_09.jpg
6c964f003319218a035b9c7ab116c7a6
020716f38681f86c4bb9b5dcb4aba8a8668eed7e
5233 F20101208_AACLTN stone_k_Page_44thm.jpg
76338d9a99c97efdacb9d65a24ffcdb4
a5fa4bd70886383108eaf0aa468995464793ac04
F20101208_AACLOR stone_k_Page_47.tif
ef4aca8998109149e3b9b78a629b7d1e
b69c882e8796a0f45af2106634682c815964a425
2021 F20101208_AACLJT stone_k_Page_22.txt
8b4524ebcff8cc2d8e1d0ea5defe8850
8747a2455118d119e166e03131fa1fd049696092
6088 F20101208_AACLTO stone_k_Page_45thm.jpg
05627bcdc35cfe4011b48b8c460210c4
ab1ea1f0de7971a576c22d0ff193cd167543923e
40833 F20101208_AACLJU stone_k_Page_35.jpg
2a48ac33e5c29a85d796b3d9fd8538ec
8351d04f5daa9ad6ada243d7896179f7127801bf
6365 F20101208_AACLTP stone_k_Page_46thm.jpg
0d1f5cfd29ca8561e4f267cfc6466d6a
9474b00dfb643ab0f70dd1283bdf1d62b8e2589c
8433376 F20101208_AACLOS stone_k_Page_38a.tif
059834a2c0d730ebd50d06dbdb314b01
df688f2b5b68e7f88cc16780c0641a55b95c4b92
6488 F20101208_AACLJV stone_k_Page_40thm.jpg
97e7fe9be6385716c8e133707fa44b57
ef3f9b1237f4f482e2de3bf25fb58b20fca5a579
6179 F20101208_AACLTQ stone_k_Page_47thm.jpg
3c5e8bc42b61029c0a3642154d1dcb64
ab00a0143a9a00adbc5b65f357a0c123ab724646
9045 F20101208_AACLOT stone_k_Page_01.pro
0f80ec3f1cc35c3932f34571840db5e9
f7a35570f739583f8a0b59191eee2b27ca6a6eba
24847 F20101208_AACLJW stone_k_Page_46.QC.jpg
e8589be3e260734acd4e52f23a2ec09a
9a26a1a18c2698f2b6514b2e68738ff9cb2d65f6
2425 F20101208_AACLTR stone_k_Page_48thm.jpg
53520c6d17f4da9ce683b1634c93a344
305101c7e799c2703a7a5cd1630d04b4a59ea34a
1196 F20101208_AACLOU stone_k_Page_02.pro
18d097e04b4ced38787eb1c6bd9990a8
5557e0eaea404a53b57602e3ab314b40eb4963e4
2419 F20101208_AACLJX stone_k_Page_46.txt
d306ef5f1457518d02426b6138c34823
78ebc839847eab16538032b041c95238a7de2121
59007 F20101208_AACLTS UFE0017942_00001.mets FULL
388d1214c53087e3cac19d519b5c328b
eea84aa56f075ca912fdd4487eabe90c1b53b82c
8780 F20101208_AACLOV stone_k_Page_03.pro
bfb062102b8daae2b970dbb36fb394ff
9d9d2f28bbbacc6f6b514561b5b4202424881009
988 F20101208_AACLHA stone_k_Page_36.txt
bc4a43c956402d0b62916a16aec843b6
52696b6f1f7e566c935162cc8123ccd770459cd5
F20101208_AACLJY stone_k_Page_46.tif
ad1922e41eb0fe55b0a3302de0fcb2c6
1d593c7a8c73c3c26bdd595a5787b85d4741a294
16833 F20101208_AACLOW stone_k_Page_04.pro
019ce020a8374f914ac5276a27920dd0
c3de1e852854296cc0618ceda12252b57ba5b57c
72955 F20101208_AACLHB stone_k_Page_40.jpg
9f56ab0e614e27d58060240df559d2f0
1fdc21ec021658fc1a9a64259122a916a98aa529
100257 F20101208_AACLJZ stone_k_Page_10.jp2
32fbf86285e439f1c160b7945fb5822e
3b0bc0516d6cefc62ab589bb3066048116292cbf
57518 F20101208_AACLOX stone_k_Page_05.pro
78f2b4dc94dd112df479bc9bfb9c6d01
5b07e44cd21f5faba5b8d401d600515edede7ff2
483 F20101208_AACLHC stone_k_Page_01.txt
9844afa41990d6a7356c1f468462e32e
086121aea2941d8956387396ab199deb3d087b48
7781 F20101208_AACLOY stone_k_Page_06.pro
bf2afdc63c01519ebb7da92ad631c73f
b2771d30ba74bd0121f901641047bb426ddbf54b
34539 F20101208_AACLHD stone_k_Page_26.jp2
cce6c9e13e021d48e343f2025392e7f9
95ec635cad91034fa987cfef5b9fd49bd8953f11
57619 F20101208_AACLMA stone_k_Page_44.jpg
326cf550c6375542ae0f3f2a1514e3fb
a6b3b83e629d1323690e1257882305375a76827c
27033 F20101208_AACLOZ stone_k_Page_08.pro
756dacea8c27cc929c651d7e7501afc7
fcb59a6ab1138f332ad6cf82bb67cc87722fe6c0
F20101208_AACLHE stone_k_Page_21.tif
53813e9e6c55bd0c71160f8ced2c45be
413d0380568bf147ef25176d1222e0aa1131b583
85002 F20101208_AACLMB stone_k_Page_46.jpg
39401e7f86d772fe7028bfa1c22dc22c
99caa9676d899be439e7a9c7770d85de318d3cd8
106902 F20101208_AACLHF stone_k_Page_13.jp2
430506a6404355ba88d0c629fd92c78b
286d538fcdb082e2be1170a109316d1010bf06ae
81043 F20101208_AACLMC stone_k_Page_47.jpg
903078bb1d1a4839c36ac3bcd6019f70
bae99e872d22fe3a8a9c8013506d661c37c38fa4
47227 F20101208_AACLHG stone_k_Page_23.pro
852043aca6336264f469d42a29e092fd
704f24be3b19174f58052850c9e6895e102c19a7
484 F20101208_AACLRA stone_k_Page_32.txt
50d7aa73b071e3959e7100ecf03370f8
72e55c1f2930713c8350d988412bf307da6eebd8
6451 F20101208_AACLMD stone_k_Page_38a.jpg
a46ddb986f23eb2facced9975b9a75f3
e561b3cf07a45e6338f2902d330785fa2dd9ea28
69393 F20101208_AACLHH stone_k_Page_18.jpg
144ddcf5e51272edeb2b27e472af2ebc
b03ab78e618bd055d963436cdb82fc2dabb8d75e
2112 F20101208_AACLRB stone_k_Page_33.txt
c1c687562b6986c39f438ebc5c8cd9c4
4b2bfae3a57f8dda1a8100aa489aa38087e95288
21533 F20101208_AACLME stone_k_Page_03.jp2
00b0c8829265f9a4ebf4049bce15a32a
d31bd8f43ca98e4bff8f658dca860a7f73a6b744
F20101208_AACLHI stone_k_Page_30.tif
0457d72beb2875195814ea6e2436c95d
d2b8edef6cda029df06aeb208993ba81f90bc647
762 F20101208_AACLRC stone_k_Page_34.txt
663f1e38773b9d0b2ed66bea065ea305
796572ac5412bfc37ef8885b0130746f7cef7f52
40021 F20101208_AACLMF stone_k_Page_04.jp2
e34864db19376e711e3dc6062b35ca42
212449491038c39022b4cbc159b20b7c4730eb7d
1938 F20101208_AACLHJ stone_k_Page_38a.QC.jpg
9932127fc5619208205a2d5537b781c4
d14e55de3de533d35c1e7d929731a7ed23bb4563
1358 F20101208_AACLRD stone_k_Page_37.txt
769e0dd6a71d95891c908269d84fe334
f86e621b0d5834e5e7e138263b2c3bcfb1381b16
1051969 F20101208_AACLMG stone_k_Page_05.jp2
4ff495ff1a436d255216d28532d3bf49
80137d859944f6b3f0b0afbbade5af13b8f5641e
F20101208_AACLHK stone_k_Page_02.jp2
ab0638cee5f42d7e2059d80e18c067ea
4dc99a302b7aba196acc6232a2b436e3b4ad3769
1852 F20101208_AACLRE stone_k_Page_39.txt
afc835294a62df8d88718542f9496fb6
12e1858bd85774dbb4ee28ec05471f7125ceae18
288978 F20101208_AACLMH stone_k_Page_06.jp2
31ba3c7e5afc355ef6470fd8218ba769
d83c72684813a34d86945dc1e73fbfd10a153693
2034 F20101208_AACLRF stone_k_Page_40.txt
876e7a2ef98495371ba24fe37fb79396
a22f1e05ae3fd4e388d7c3aad7fb103c9b45e894
273510 F20101208_AACLMI stone_k_Page_07.jp2
dcf84a1a8636d637b1ab4d3c94f549e0
f5408ae67a4560db626debd3f101bc1389b33f7a
48483 F20101208_AACLHL stone_k_Page_42.pro
566845317fe8cd7ed0a5ff40a051939e
0f641ade47979db10484ffd6c28b070a2c19443b
2025 F20101208_AACLRG stone_k_Page_41.txt
9d515de2af3e694f1bd5f78584e64ce7
a4425a231edc9b584fd0fecfea6fa6e6839f9b40
989602 F20101208_AACLMJ stone_k_Page_08.jp2
df065630b13690f2c7f8d1bb22118562
8503371f91957d5dc1ad985b3b73e3fec80e3bbe
887 F20101208_AACLHM stone_k_Page_38.txt
72c2bf2d5a13ff34108bfeb92c500cd0
cab6de5e7b3a0ef2e808096afb7a6004f8823090
1913 F20101208_AACLRH stone_k_Page_42.txt
4828a38f6aaa7abb311d01913f59a473
73d1aca85588aefe17daa6655d83e530b5807ec1
84541 F20101208_AACLMK stone_k_Page_09.jp2
538e93d7d938365a1fa99b0374082585
58e65a255b8fbdfbfdb33899adebe1e535499e11
107436 F20101208_AACLHN stone_k_Page_21.jp2
7ac596ac47d9296a2740bc0df33156a0
f9c309d6fb541fdc741b89d858f9595c97d3c6d2
2096 F20101208_AACLRI stone_k_Page_43.txt
f9ba0ce664712a29a3bde99eca4351bc
cdcc8e1c191d88f1bed016881698df433135a32b
97143 F20101208_AACLML stone_k_Page_11.jp2
53edb9af93643055a38a55ee17610c5b
afb6760bfbd6db85e210a50aa779f29b4ca193f8
F20101208_AACLHO stone_k_Page_06.tif
7ea70c23a4a63a461aacf845cf4e7ea8
c262033b7cb5d629f6beef9f539f7bc60b081c7f
1571 F20101208_AACLRJ stone_k_Page_44.txt
0271cfce98557277b611d09f1b319445
cbe9affef82b4edd0d92274880b8d1dc9ab55231
108101 F20101208_AACLMM stone_k_Page_12.jp2
a4689dc4dc6e74d328c30ad9fe592bd2
3d5cbb31fb487b5cd1635be242ce056a3327c99a
2254 F20101208_AACLRK stone_k_Page_45.txt
4ce35e41369e0969518a0901478befb5
747367a2b64146362b752c615fef556d811c2584
101781 F20101208_AACLMN stone_k_Page_14.jp2
e6be3ab33840c97d7affe8ed6ea3c27a
4cd443aaf5e8d29446f3d038e89bd9e787c1b432
16561 F20101208_AACLHP stone_k_Page_37.QC.jpg
52f9e57987700a68b31d23fcb4f4e12a
bf4c066b12f077c2473f703063b4fd2a149a3f7d
442 F20101208_AACLRL stone_k_Page_48.txt
66784e2ed325833f8483933e09f8aab4
0878730c32dfc2dcaac9a99c10a6f650bf25c636
93587 F20101208_AACLMO stone_k_Page_17.jp2
5ce52807ecb078ceb2a9f4bb859ce245
b6c10e311a62f31b56987af997629b68b0cd7b76
1922 F20101208_AACLHQ stone_k_Page_06thm.jpg
f8701354b6bbf1209839773e0fc362ea
7ce5ea92806ce35df599d558d6627619242f3329
3 F20101208_AACLRM stone_k_Page_38a.txt
bc949ea893a9384070c31f083ccefd26
cbb8391cb65c20e2c05a2f29211e55c49939c3db
105624 F20101208_AACLMP stone_k_Page_18.jp2
0143e8ee32217273d1395fa2c7d48054
37efb76df494460530d042fe5da283762eef3ec6
69263 F20101208_AACLHR stone_k_Page_42.jpg
da07b6ab3059a06588a2f0fb71db9dab
5ec0ddf36198ab31678b1a8c7827dfb55e877539
5825 F20101208_AACLRN stone_k_Page_03.QC.jpg
f080f41cb652b22d147eb6f8369fbdb0
e8fcf0c91a95a662ee6ed1e0496e4b9d9ddec489
78329 F20101208_AACLHS stone_k_Page_45.jpg
c06550ec81ffcd1be7b3cb9fa3bc8732
52f27f44da7a3ec95cffa1da5342bd97346df113
9987 F20101208_AACLRO stone_k_Page_04.QC.jpg
bd231f9a874eec308f9109403564db19
f71fd03c0eb582ff434d43e18cbcadfb2dbfbe7c
107127 F20101208_AACLMQ stone_k_Page_22.jp2
f36358ba1294def8347a869d2118ea65
6576562e59f6ace5f55f88199b3f8d88f8322acc
114173 F20101208_AACLHT stone_k_Page_19.jp2
954a78693256b92a9d2df63d63f52078
4a1b14b1c5726b21edecea3c97811802372bb51b
16688 F20101208_AACLRP stone_k_Page_05.QC.jpg
ed873bb4ca51f5bb3d88fb611ede2d7c
62834399b6a13749df1bd887f31370e3d785110e
107947 F20101208_AACLMR stone_k_Page_23.jp2
3e8d1c9d77fee4ce125ab9445e674692
cf30aa295a65daf288d6e431ff95e7b52afa0d61
45883 F20101208_AACLHU stone_k_Page_34.jp2
78e89b069211a736ff7cd18f48319c13
aaeb85772ab62189aad36642d307563b8d6f70f7
5178 F20101208_AACLRQ stone_k_Page_06.QC.jpg
0186d6120505adee7e5525a389d1f567
5f49d4a0fbd85a45c83368131f709cdaa9df4595
105358 F20101208_AACLMS stone_k_Page_24.jp2
4c7ef2cfcf03246d8279cf2ce70c490c
f7524fa11c5a256219c2cdeb78c7cf1f04b90010
70859 F20101208_AACLHV stone_k_Page_12.jpg
9d20e70b68814f1c00ad25168e897272
329185f99d823896208cd274222363e39e473e33
12399 F20101208_AACLRR stone_k_Page_08.QC.jpg
8b80de01b27bd328345b3e3b6113c540
4e4b712582afdb1772ba360674749a74a10bf906
95926 F20101208_AACLMT stone_k_Page_27.jp2
d3a9a20ba01bbcd0b3d44482f4e072ca
8dd403b3e04cf4645dbc0046edc94c76342de8ed
5183 F20101208_AACLHW stone_k_Page_31thm.jpg
f6ef8b7115f07a6b45b21e487324d82b
8c326f0f1ed3b3e017cb5d1eb35390983a232884
6107 F20101208_AACLRS stone_k_Page_10thm.jpg
14261469ce7e24acd38b2e73d4563b47
b23a989b684dcdd91623d3650879112ecd2ebccc
61533 F20101208_AACLMU stone_k_Page_30.jp2
a7765ea5d696fa9603437b45f9b593e4
23cba6de98cc9eb72116ba6eedcd970deda58eb9
44341 F20101208_AACLHX stone_k_Page_28.jpg
9af5a44074a05d689d1033e298fec916
7358e5f6c0058acef1f7be3c28d343438b6e026d
20666 F20101208_AACLRT stone_k_Page_11.QC.jpg
8ef2b4fcc972c1fcd621827a186a32d7
1dc1abe0aa27ee3db6720e9d6caf6570424d6768
605534 F20101208_AACLMV stone_k_Page_31.jp2
142b8b46f1e487d4449fe55f38a4e012
91eaa6ce005838548b6e19abf29ad24fc1a5e938
4780 F20101208_AACLHY stone_k_Page_16thm.jpg
a23d18d828b4b24c8dd0842f2d0d131b
414af58380c48b0998a861c695df23c6e69b2322
23225 F20101208_AACLRU stone_k_Page_12.QC.jpg
e8f0a25d9482c6b2505490fe3904e715
cba5f72578ca0544f85b26e77c1f8fdb3a51b75b
491108 F20101208_AACLMW stone_k_Page_32.jp2
4cc6cc5965f2142cf2fa4a3df4d302c2
32e9d55ee1dbb3e190f21fdb32f9cea5a7f4c345
6084 F20101208_AACLHZ stone_k_Page_24thm.jpg
69ccf6f15048ac902bdecd36b6c9f06d
7675340b3c76eb3b04b477dcf43a853ae1dcf708
50218 F20101208_AACLMX stone_k_Page_35.jp2
4fa56739f563bd84505b8dd2a774347d
7e0098db5fa50649ebc77b81b8a20e9ade27d4d4
21255 F20101208_AACLRV stone_k_Page_14.QC.jpg
3c0ae74b3f1876095fbe19d15c3f6b8c
f2b2ffe12312604b327a647a8f4020bf38ee5971
71814 F20101208_AACLKA stone_k_Page_22.jpg
6cf5c110a1c94d21d0cdc7c7a86e6473
7ba485890f05f58b171dfbcdedf4364861e4ab52
614023 F20101208_AACLMY stone_k_Page_36.jp2
65f2996958f41a565ad164348e1a1eac
c46eaec02f2ed3d71edca543ac417161d8ab7346
23460 F20101208_AACLRW stone_k_Page_15.QC.jpg
6ed437b291dfea31c84a9286fe8f11d6
e370d2cae7e9d054674f5f45309d0f6a6cae3817
1386 F20101208_AACLKB stone_k_Page_02thm.jpg
024b341f752ca5204fae9afe76d11603
680d152fcf658a84528eefbaa8120d1233406fce
805389 F20101208_AACLMZ stone_k_Page_38.jp2
81c302094185c3680985aeb481797288
824f0ee44727747f4b2ba9fcea7cd12da6cca0f9
16791 F20101208_AACLRX stone_k_Page_16.QC.jpg
ecbfea181fefb873185a634136a8e9f5
32d0ec67cc1e287da267d7486089243f594f21b3
24056 F20101208_AACLKC stone_k_Page_43.QC.jpg
8639e66923e06ae47f5aefbe7d8280e8
955c488dbb85b64603e9b261b8f77fd6da223de5
44115 F20101208_AACLKD stone_k_Page_39.pro
5d68a3da578b8290182d1cf1fff7ebdf
baa31db39d276cf4e9cba3e5b669fce8d8da008c
45125 F20101208_AACLPA stone_k_Page_11.pro
dbb6dc4f63c28d6e586d24a10aa67d08
d0d3bdd45d5b4cb3ac061d2e4e6865c98bd5d21e
20240 F20101208_AACLRY stone_k_Page_17.QC.jpg
97ee9362e61cf1e0363224a9225ff11a
a674b2630c1e1d73032e2b6219ab16e995b508e4
63233 F20101208_AACLKE stone_k_Page_28.jp2
8bcb9b4b1ca12b1effda5453e0afc0b6
5cb5924362e4359e618cf4c35e5f55aee3edc29e
48967 F20101208_AACLPB stone_k_Page_13.pro
703fabc74b742669e02dc30f612189a7
f0b9753402e3674655a7291aa9b0f10aba22e0b8
24918 F20101208_AACLRZ stone_k_Page_19.QC.jpg
f8053aa7ed04965136f1c9e5e2b6df6e
875f499321a6cf295082a3d0ed301b1e32929d09
6500 F20101208_AACLKF stone_k_Page_13thm.jpg
849305b57ef7a9761803c9e6d58887a6
e536e6bafb8700f4b50e3284c82dd2723945e663
51215 F20101208_AACLPC stone_k_Page_15.pro
d80cb307de7ca047daed3b48f1e7d2f3
bc031ea14ceb651786f3d044df5b4a46bb01b950
23485 F20101208_AACLKG stone_k_Page_40.QC.jpg
dc784da9effe55502374147697340704
31ce1d3e53576e768457e05ec2c21a2df0af6d6e
33626 F20101208_AACLPD stone_k_Page_16.pro
5904fb4ceaeda7f93668aff760faf5e6
1b673261dfdf9aa9c680812455b06162fd25c105
54358 F20101208_AACLKH stone_k_Page_47.pro
daf3b6771ad60eac0a93907f6da2283e
22ab6ebe632f534d1c62cfc61a1f75919ddcfac6
42625 F20101208_AACLPE stone_k_Page_17.pro
d4510ca6ca031111718f172fbd05fb50
4d1924d85cd83d90818c2644b9dc01c3a59306d9
13035 F20101208_AACLKI stone_k_Page_34.pro
743466f43f0feafc97ca6b0c2bc5a5cc
c51ff567fddbb5e57527d8974f206deda5436210
48290 F20101208_AACLPF stone_k_Page_18.pro
bcee66ece7b8ae0eb8b65b0a94e7fb24
06718db0be8e1a1e784ad7de3a4d1bdf6e866e58
5873 F20101208_AACLKJ stone_k_Page_39thm.jpg
18707a80f35fd30a2b4b9406f9eca4b1
e6e4efebc7279dd10bd6f0ae4eb69c96918904af
53681 F20101208_AACLPG stone_k_Page_19.pro
b5c16b197ae02c6fe66b9149f1e1af13
554778c8e52af581af358eac91559847f4e07ba0
2492 F20101208_AACLKK stone_k_Page_01thm.jpg
a725ccf9e199781706390db155a26329
1e8c73adfbc96420366bf944df1c92fd570ea152
49753 F20101208_AACLPH stone_k_Page_21.pro
d54f339b9f46216e74892dda9e58da40
e6e3200ed27e664fe8f3aa68a91e03f4598014ea
3291 F20101208_AACLKL stone_k_Page_02.QC.jpg
a567073b9c9301c078b47818da8e5bd4
56aebe22d18f72b93349a91ef88de9569b3422ab
46270 F20101208_AACLPI stone_k_Page_24.pro
abb15bbb2bdcf8da8d95ce59d236ba37
f96250e3aad452f5545e9880bf65286a34a98ae1
9086 F20101208_AACLPJ stone_k_Page_25.pro
73d86d0f44aa52219ee921f027719f70
d8b76a813314767b52fd65c2bdb035e9187bef4a
110338 F20101208_AACLKM stone_k_Page_15.jp2
80baf2008db5963d782243ad3361c351
075b8a5555f300ecc6dd2a347b06e1c7dd55d877
12150 F20101208_AACLPK stone_k_Page_26.pro
c4257e05ce19b2258b130c50d083d87f
a2c01793c4a708b7e81d3bfaa09f56fe7e968a04
74969 F20101208_AACLKN stone_k_Page_20.jpg
21400ccb05e6f5af1d15398ab0ebe68b
b2eed640fe34b21d3f5a308c244eddd0ef07a103
44991 F20101208_AACLPL stone_k_Page_27.pro
5ab3f673744864e68e4cb32faf8a1a5d
2c8f49966c06aaf04bcac3073d6666a0dc2a70b4
27860 F20101208_AACLPM stone_k_Page_28.pro
9c60014871a6e55557f34c52dc2d7ee4
9cd52f8b539c98e7d13e32eff4d4eac60489b6cc
23595 F20101208_AACLKO stone_k_Page_21.QC.jpg
d9c8b119b66f6130d109d9397aeed552
a4faefe940356e57d7f6c3ecdd1c91f3be0ae52e
20824 F20101208_AACLPN stone_k_Page_30.pro
36967dd38c6afe96b4d6a3a8ab57c2ed
c139c8e0178e58d3c28f44bd18de93b0b7788a84
46617 F20101208_AACLKP stone_k_Page_14.pro
d4c116344f29a50cd350a19f856ad5c9
6cc2a4d2d704d8a3abd174fb4b91788411e38c5e
21200 F20101208_AACLPO stone_k_Page_31.pro
69eb259d0afd2518908ded42dc2edca3
7fcbbe269600534d29f5f8c68ece95722f1e576d
F20101208_AACLKQ stone_k_Page_20.tif
747b8d1c0714f423dde55493e869d829
ceb895f91a6a24de3f0c1b4039442e537018366c
8953 F20101208_AACLPP stone_k_Page_32.pro
8bf4efb95983f623e17019232c24bd65
b7c4757a6dfcb777c743c249e460e14fef624ed0
12968 F20101208_AACLKR stone_k_Page_34.QC.jpg
dd59ed189ef97dbd957f6daa895f5c92
2b69d48400f30939c03b70ed856f8bded5ca0b3a
52947 F20101208_AACLPQ stone_k_Page_33.pro
cb127de4cf09b68da40e3a87c17736ec
5949a6478daca644df08e521de506c20ed2d3369
49549 F20101208_AACLKS stone_k_Page_22.pro
7464f0aea202983e87bc2479db58cf5d
ca1074dda42b2f17fdf696b68c6c89b8b8be8749
13947 F20101208_AACLPR stone_k_Page_35.pro
4665c744feb53832bb63b456c0cee735
dd77d3ab5dcca880c90242530bd223d8072d918b
741 F20101208_AACLKT stone_k_Page_35.txt
08d8f72531d859941bdc3ca3d01ef5b6
2c52cf1dfc84f36ba07b1bc2bbeeb88cd09ac64a
22044 F20101208_AACLPS stone_k_Page_36.pro
a4a69ebb7b61f3d649439267378c4cb9
d4a24bfe4d6ca955e0a37887d433cd2f9dac482f
71266 F20101208_AACLKU stone_k_Page_13.jpg
93ffb29e9439ffc847212f057bf2227f
45a0ae855bc7e68d82f2d654a58f480907df025b
97206 F20101208_AACLKV stone_k_Page_39.jp2
9b452a49f3a2e4dee5ef82365d7bc36a
ed8c42aac88a3d4c10e1bafcc98c525bbf72b850
32869 F20101208_AACLPT stone_k_Page_37.pro
037381ecd4d1d721510665d3d5845905
e32d935d2d9d6d291da936f91f6f033bdf1a1ada
23132 F20101208_AACLKW stone_k_Page_22.QC.jpg
d3e4bbb0483f041a63cf47abc3e0271a
1b604e4c33f23f5452d7c9cde779abd0d7c6dbd1
19288 F20101208_AACLPU stone_k_Page_38.pro
8c69a9808f819cc5d52397a6f8365c75
975f6310f7e5c1bbdf8814a7f2ac4a8c1b681ea8
720 F20101208_AACLKX stone_k_Page_04.txt
48ff4f8842f388b1ed75e15edd579bb5
0bdf5d5a1380b5ab768a4a582e65b884a82b13ec
50986 F20101208_AACLPV stone_k_Page_40.pro
bae19c1fe31abe0a5e243b55facc59c5
b2d7bebeba1fed2483e51a8ed963e9ad63908e50
15398 F20101208_AACLIA stone_k_Page_36.QC.jpg
c33da9438a05064f2c5872e13d12e8b1
0361fb7223d91f997c85814e10c3c14c047f17b1
18564 F20101208_AACLKY stone_k_Page_09.QC.jpg
394a8cd4cd1bf88d5277df40dc302524
5d472a27075aea22464d84dc755892d30e9a08ec
50736 F20101208_AACLPW stone_k_Page_41.pro
af47d6458ef643579291a2239c97c535
8a65c047e17cd42e2a9ad44dd973cb39e33fef87
74117 F20101208_AACLIB stone_k_Page_37.jp2
c417c3382f67be6b74cae7be50bef997
92acd2fb99eca7c2d8198581cfe30f35f1f7d29f
76017 F20101208_AACLKZ UFE0017942_00001.xml
fbb90cb67679fac86521e762ce51024d
968fd01ce1a25ccd272512d7ba7f5140e84d5cfc
52093 F20101208_AACLPX stone_k_Page_43.pro
d6893b451cc513173bb184d96d3a5204
b724c1bf7d0f014eca62cda0219dd20d96c14bca
38264 F20101208_AACLIC stone_k_Page_09.pro
84b14a947f687e5fdfe54de9674990a0
9f0840aaa67f96390559ee2ed7d1858d9767c7bd
111464 F20101208_AACLNA stone_k_Page_40.jp2
9c5bde69db1f914f7b14d79cad4b74b9
eb68b7f56a6493649c6a7cfcfb4f77916661d2e1
38589 F20101208_AACLPY stone_k_Page_44.pro
38065e443e7f96a66b7a7ad6b40b2214
e15b81102fc1af9b82cc5a243b5608c4e4f4bc58
55468 F20101208_AACLID stone_k_Page_45.pro
10230f568d9096b3826d2ed047fb57ac
519cec4c89250cd3239cdcea3cec797f70a51d41
109789 F20101208_AACLNB stone_k_Page_41.jp2
02a65bd9b594914e23391003ff76306e
171a690534f241194cfc1f7c35264f2afbcc10c0
58933 F20101208_AACLPZ stone_k_Page_46.pro
bda9ec083bd6ea12938378192699db57
5a697ef7e808ba6795f9a10bff48fa6ce5662e45
F20101208_AACLIE stone_k_Page_32.tif
d9ae20bdfa781cf503a38dc1ae904df4
c1b5e9c0db7ab019fc37d2d94ff774ac37a8a1c3
105262 F20101208_AACLNC stone_k_Page_42.jp2
74a513c4c559397e933b1001f9590e66
3847d3bb1c3d614ba69d29402e574b96526338af
F20101208_AACLIF stone_k_Page_18.tif
cad8746c0f591f4ec86709acbff7f78a
b158021cf4e0dc334b54f0b4b10f5828dc070e10
111438 F20101208_AACLND stone_k_Page_43.jp2
39c7421cfaab1bb35c0178e5914eb021
f4796ac2295139af54ee70cc9cd82ed245a09569
F20101208_AACLIG stone_k_Page_48.tif
47ead8c67ee898e32d4e79ce48e1809c
4fe306250331ea3f7c25d8073549b6faee86dbac
24626 F20101208_AACLSA stone_k_Page_20.QC.jpg
31cdd7985bcf8465c038bfe579764db5
6e244f282a2055c6ed8f797c993bbf20f38dbaa2
86887 F20101208_AACLNE stone_k_Page_44.jp2
402f640a10ec04cf7ba598e65fb093a5
2a8ad09d396d0f71ff24b38a83172f4921e717b5
19354 F20101208_AACLIH stone_k_Page_03.jpg
d8648866ecf13fb6914c2c889effada0
6b43266e88db8623039331b3ffd9b465f7bb7f16
2272 F20101208_AACLSB stone_k_Page_25thm.jpg
6d30ea2d2fa8ce71bb33f7f124f524f3
441614ee2d391c3c489ecd827bc134e76c17c6e2
119576 F20101208_AACLNF stone_k_Page_45.jp2
a35b3c46e2e5e34e481bb1cffcd3bd24
86fc6c0bc45860611239301865e7eeaf74f97bb8
14937 F20101208_AACLII stone_k_Page_30.QC.jpg
0593e80146841c07cf9406e40b56e568
7bf43a320583e1b5506452c8fdec897a33c03dbd
3397 F20101208_AACLSC stone_k_Page_26thm.jpg
4efd063909a5f03db79db8d414c66646
785d3b3bd12f481d0c7e4b71696ff6589e820453
120861 F20101208_AACLNG stone_k_Page_47.jp2
23a9ee365bca80d683a743632e6dd0b0
1fd17b20b3d1c8d6be5d096cf421e91de044aec7
23300 F20101208_AACLIJ stone_k_Page_41.QC.jpg
f2b18bcb3d7f48baae423a2639acebc1
e3bceef022e83b4b049ea4c8d7b4eefc056ef51b
21123 F20101208_AACLSD stone_k_Page_27.QC.jpg
f34f8b9955304fc3dc862dd81b5e821a
476ff949549e3d2d030b388668249ba3ffcf2d43
3552 F20101208_AACLNH stone_k_Page_38a.jp2
17431b3cf496028b592635f7a3dd6641
608c968231b04e7e20442afe54bdb7dd9d32c2e3
65824 F20101208_AACLIK stone_k_Page_10.jpg
f8b980047ab985986e22225a3ae1c92c
b40b72d0f2038df727d30c4841b8a4cbdde274ba
5946 F20101208_AACLSE stone_k_Page_27thm.jpg
73b4d16a7fdb7e2a55b007493734ef12
bb1c9256668201e418117c78c108444c320d4938
5165 F20101208_AACLIL stone_k_Page_09thm.jpg
f3dc62d70e792555c0fa1212bcdc5ddb
cc525cbd9405a3951a438919d0c0f716a14e3777
14392 F20101208_AACLSF stone_k_Page_28.QC.jpg
9186086e7e84a009c0f682596112ab1d
df694d2295fe3eb63de7fbce33ff03348ce8600a
F20101208_AACLNI stone_k_Page_01.tif
b91f3e50158a6b2ac6b8e90e346b971e
e91c91634b0d4583e686d047708754549743500a
4157 F20101208_AACLSG stone_k_Page_28thm.jpg
fec2ac4db7344ac5bb6558f7cea43fd8
6b9dbf0f2ff04eea00ee202d2a3e49b8d3e2f9b0
F20101208_AACLNJ stone_k_Page_02.tif
e955a0d9c7fa4fc9df37f1b2334be6c6
d5b475c8acb627fc9f8687185a741d45ee47397b
6349 F20101208_AACLIM stone_k_Page_23thm.jpg
ad0481565dc927d5b9e75d0d0cb15e26
da1942cb3cb8bf3097d9537b443e6941df4dc3d4
16049 F20101208_AACLSH stone_k_Page_29.QC.jpg
00a0640e9aad8ec9d4f4ba7452c5eea6
40d87a89f6ce999761383d530cfa3241df084e92
F20101208_AACLNK stone_k_Page_03.tif
8fa337e34e1d2d45bd9a889714d86370
d0375602cb439463de9e9f5884c1022a1c070ee8
26072 F20101208_AACLIN stone_k_Page_48.jp2
af75a8f2b57954366d5e67c6180bb214
df85f1df7f9f6b74ae1527fb33022c93ccb3f3ee
5042 F20101208_AACLSI stone_k_Page_29thm.jpg
2c7e5ffb70d0b04756d241bc8cd1910d
e7f5debd4a7b6eb51a2cbb5eac83892b3bc47b17
F20101208_AACLNL stone_k_Page_04.tif
b722d4ec74c717b69eaf682b74b35750
917cf9e58a377a83962a2628f78ac44428e2693b
112956 F20101208_AACLIO stone_k_Page_33.jp2
fc6caea572eee9b688ea499c264eb8ec
6875ff2a29f7210810895ccf33b4440de80e8b42
16268 F20101208_AACLSJ stone_k_Page_31.QC.jpg
5869a8f1b88597aec2e43b979fbc6656
226b6859fa421c2b6829b9ecd42be6398532a64e
F20101208_AACLNM stone_k_Page_05.tif
0a52b2edc4adbc8964f395b2ac331a0e
8668ab7310c63016bf241f5e0c9ed017e2cf4de1
F20101208_AACLIP stone_k_Page_12.txt
9b228410114d4409f4913e5b588e71a5
f946be6a984ed2ec7328cf5d44d623f3f6e3c347
15155 F20101208_AACLSK stone_k_Page_32.QC.jpg
7df154532c15694965b15d6f8be94540
4f88a855c25a489e484d22dcb29c69cf4de2ac6e
F20101208_AACLNN stone_k_Page_07.tif
8db8a865492cc638bdddc8737923383b
83a57a67b831612bc881f94b898ad5f369f8fb29
F20101208_AACLIQ stone_k_Page_41.tif
2608a1dfbb6d555b11e70dec13b38fa1
c1e8c1277eae722e8f5a91e7e9de28f4a330beff
4680 F20101208_AACLSL stone_k_Page_32thm.jpg
cda5283a9b9b4a105540ca0c1d5fbf05
41c57d0adae473fd24c03f8769b62ac8772bd30c
F20101208_AACLNO stone_k_Page_08.tif
33a6a0201084adeb5594bb6b651668a8
3c8c36ea33e628ecd5302e4865800552e77849f7
23273 F20101208_AACLIR stone_k_Page_45.QC.jpg
0ea90892f14469d81004b81769bf5873
aaa4210538781f8c01b89c646b098906fef6653e
6751 F20101208_AACLSM stone_k_Page_33thm.jpg
4cb2f36b129d0ddeef7c45a60e488c5b
cfcad282f7bebf1b8cad69901cf5001afbe60606
F20101208_AACLNP stone_k_Page_09.tif
960199ca6cc858b6a75ef2b51cabf175
05a4d99163232ec3dfff59650d3ec8f22962d002
1096 F20101208_AACLIS stone_k_Page_08.txt
e46c5499794feb34e06a0c88539694cc
decb7fbe21b744f3fd1869730efb79c38f5050be
4179 F20101208_AACLSN stone_k_Page_35thm.jpg
7e596d2828bd54f20158f02920aa3744
7306f85dcd9443e60fb970372bab771d20221211
F20101208_AACLNQ stone_k_Page_10.tif
bace6b08114ae895d4eb5f3015971249
79fb0786c331b0be2f0ed4ab59c183066d029047
22778 F20101208_AACLIT stone_k_Page_18.QC.jpg
b7089dbab4a28398f768e62ff3292f2d
9ffc61ff6899c60acbe2917eaab68690df4c5ed0
4465 F20101208_AACLSO stone_k_Page_36thm.jpg
cf143eb4102f8268d061d1e88b3400d4
154335ccb29a76f8f43a81de2b6626744d3a8791
51156 F20101208_AACLIU stone_k_Page_29.jpg
36270986a221d9f6f11611a6753b3492
a3234e7be8ed62101d98e8a9305485f1a3a3766a
4661 F20101208_AACLSP stone_k_Page_38thm.jpg
52df9a6dfc8a04bf37cbf47722a600b6
96ef0e4a3794fd986b83e47b774ab22f329d387d
F20101208_AACLNR stone_k_Page_11.tif
8815ffb0052199b6570609d2b3b5cd1e
f603149fa8bc4e1adadac0fe3108d8ba10ad1673
25170 F20101208_AACLIV stone_k_Page_29.pro
957a8c4f8d42010c95019409694f40e9
7e28ddd99d0b2aa63e62dc5d13f36a9493fb538f
22175 F20101208_AACLSQ stone_k_Page_42.QC.jpg
9e178ba1de6b37b73636919fd70cee31
6a674cf0607c4fc6ac3bfd2255eb01b75eed4a77
F20101208_AACLNS stone_k_Page_13.tif
5712bc8e2381df0bd070cda438e470c4
fd98ec63cdf38da850b54ae546660b56893b5b28
4521 F20101208_AACLIW stone_k_Page_30thm.jpg
006be5571694ff6c3c92125e3527e098
66e14ac74927e32e3a9618529f9eea376d2906c1
18933 F20101208_AACLSR stone_k_Page_44.QC.jpg
bb5a351dcb0f7eb4e4c551f1a76415bc
057501eea2a3867aaffcbc504e6bd8ef5c6caf07
F20101208_AACLNT stone_k_Page_14.tif
230bba03537f9edb3031c65f04921fca
2a9552f4ac94700d3d1df7cfb65a57676a662570
127640 F20101208_AACLIX stone_k_Page_46.jp2
9b881bcbc67a094fe3fefaa5539551d4
61b4617e2f6fdde11f4a826d8b9b13ee9f388b16
22735 F20101208_AACLSS stone_k_Page_47.QC.jpg
8936912c1c68240b5bbc42d3ab552bba
c344b8733fb188a75d1539cc13eb6eddbf23543a
F20101208_AACLNU stone_k_Page_15.tif
0cffc68997588d12963272d7d1f26904
1d5c5cce5a407a84374e4265277aaf6e0d3b2506
24723 F20101208_AACLIY stone_k_Page_33.QC.jpg
75138655c1163486bbb62f3ce08b11cb
aea390bf0515725d4d9866071b1172a0384bdd26
7178 F20101208_AACLST stone_k_Page_48.QC.jpg
70565318f31b23012da63af0e9f1eac0
d82aa6cab475a20d8eff9ee71feaa44c52a61f85
F20101208_AACLNV stone_k_Page_17.tif
21fcc57497083328ebee50b63d304dd2
2c8767c4edc28f475452d93e072bd30d140ab7cd
113200 F20101208_AACLIZ stone_k_Page_20.jp2
1cf37f64330706957a9ee6ebedf5e78f
d339b44ca38c65062fab0fb7343d7e8fa83ad399
7775 F20101208_AACLSU stone_k_Page_01.QC.jpg
13734882d488351da73a5f54f1079143
08c02d5399afe91fb4b3470edbeb3d1aea848284
F20101208_AACLNW stone_k_Page_19.tif
4ae55736febc7bec9b76d28491f6bf58
518eea7f4a45ee45913c4ced16175efb7c2d5c05
2039 F20101208_AACLSV stone_k_Page_03thm.jpg
950481c593f941a76b39f08cf759cbc4
a2ecff6b1074d1c1bad65e9676d83fe350dafac0
F20101208_AACLNX stone_k_Page_22.tif
a72bfd2bb8c80dce2bad6ed388af6db1
abb620d58fc0359b0426f6afe3bb0298ed71b08c
F20101208_AACLNY stone_k_Page_23.tif
92651f710e631631ede6b7af052a3db1
ffbc65423518344ea4618286a041777d773da2eb
3114 F20101208_AACLSW stone_k_Page_04thm.jpg
941fb0341de9c0ab7be486a15af8c397
1a6a032e2f91a5aecca7366926d008e04380d600
F20101208_AACLNZ stone_k_Page_24.tif
a9a9ed5abb083ac76f67f09f85eec4e8
9ff106dc82cc8ed22d839f8931ceb98116fa82f1
4426 F20101208_AACLSX stone_k_Page_05thm.jpg
e212263826d252146303c6c4de35a30a
55473115057e62cc67f74d03b16617934edbaeae
10286 F20101208_AACLLC stone_k_Page_02.jpg
383ab74eff0387fc75d81c6ef290572e
c5dd0659028d2f63527b3110ea91c45a5c9f4cca
9821 F20101208_AACLQA stone_k_Page_48.pro
ea07e43c4ebb34d586fe2365981d53dd
3e1b9b23108ccf4fd848df1c527c5910176d36d8
2022 F20101208_AACLSY stone_k_Page_07thm.jpg
d11c84b8d370499f128401ddcfd102de
d567938a72006f5e651747b2845008ad996c42f7
30969 F20101208_AACLLD stone_k_Page_04.jpg
b9fb94d2eea60c2ad4e20a3e0feed709
991059960e3dbebe937ae0ded0d35a0b01696d13
235 F20101208_AACLQB stone_k_Page_38a.pro
0705dfbf70cb4ae4b4adc6e5e9e3c689
d98c473bd03b678321d2148300076c0d87934b2b
3578 F20101208_AACLSZ stone_k_Page_08thm.jpg
2f8929d6019cde3ac3765ab6884f019f
7834a1321612d1a9dea39dd5ff5d1e419b445163
65471 F20101208_AACLLE stone_k_Page_05.jpg
7ce3182f616a7527d01a31271654fa63
fddae12436e831690921b917c6a7d2225b55e5e7
111 F20101208_AACLQC stone_k_Page_02.txt
cfd7b3242a147bb827175c183f7dddc2
9a58fe47e1822640b6a694d898cb92eee2473979
18506 F20101208_AACLLF stone_k_Page_06.jpg
2e82c3a2b1c4715dd20e8b6b08537557
906a2a7c5e3d2e565fdd26949d9247c3e00babd6
433 F20101208_AACLQD stone_k_Page_03.txt
7ac839a906657e418df5e47464421a84
25217d4e86d4cddd9eb8857f62aacb879c2e05c0
40372 F20101208_AACLLG stone_k_Page_08.jpg
79a41d1a4637eaf60e7fd6014aa3a3b6
2ca82ff4174538655a250ea342c24bd6e3384af8
1897 F20101208_AACLGJ stone_k_Page_23.txt
17f689e11d99d481b322527708a4ee1a
27d0bfe37de2bbdb2b403e55df5a17281bb0eb4e
64387 F20101208_AACLLH stone_k_Page_11.jpg
c07339d554abc5ed3088f59c1c4be191
d2e2cc5b87ae86ad2eac8c47f785ecd5fc57f80d
2469 F20101208_AACLQE stone_k_Page_05.txt
992faf34e0a9eef84b16e4df6871ac6c
8b071651fa9849d8a9ce583d675e8d9dd737f760
66524 F20101208_AACLLI stone_k_Page_14.jpg
35e33796ab91ea0c33be06e11f046d8e
b57bf98208df9a416b1bb5cd0d77956a8b878470
6606 F20101208_AACLGK stone_k_Page_22thm.jpg
2b805fc19bb48283fd96bd89ab738e06
eded25c9fa49191ce8a8880d95635f143d3fc975
327 F20101208_AACLQF stone_k_Page_06.txt
9a218bf2e3364a51c7685c8e603e3fe8
70aa789ed508697d4366ea07b97a0d601014d5d0
50181 F20101208_AACLLJ stone_k_Page_16.jpg
815cadf3823588cc9825811f62fd5fcf
8fb8b12e3cdca9b9e2d32a525cc8dbe46576f84e
1811 F20101208_AACLGL stone_k_Page_17.txt
6f30226fe7373084c7c27490071c8be4
55eae2fff7cec691781980321d5663866d0539a6
420 F20101208_AACLQG stone_k_Page_07.txt
adc6c6da28780cb3bd5d8174321bd4e4
07a11c5a77d9d778dd713a81aa24837582320f81
63476 F20101208_AACLLK stone_k_Page_17.jpg
cd9d0ca2bb5ffee3f5ca1bc95bf991d5
06b97ac10152efed8e09fa17d4ea79a3906d6774
71858 F20101208_AACLGM stone_k_Page_15.jpg
265d09e1866a98d01c2e8775d0690b53
c2dd4f59ad062b7c2f57467ca69c3eed8f72b8bf
1679 F20101208_AACLQH stone_k_Page_09.txt
77357700d074d4b5f5c4590a4bb0eeb0
1be69370c00c2d2acbee4fec780af21fc5c68abd
76022 F20101208_AACLLL stone_k_Page_19.jpg
9049873f391316ea80bdaf3d4a6436cd
e8286fb3caac49c18aaaa2f06892d3403601fd40
F20101208_AACLGN stone_k_Page_12.tif
97d166502419c71086ca655243f261bd
c16d0a2b9f1fac1660686d31337e74f889796973
1805 F20101208_AACLQI stone_k_Page_10.txt
8ac1fe8cc53879eb6765f6cbd40f24c3
fa60b771154c7a4afc16eb8a9bceeb2c9cac4366
70561 F20101208_AACLLM stone_k_Page_21.jpg
7f3ab49f322c36aa2ecbf0c75f856635
d8723f793d854307863b29aabd83d399fb4fbc5e
13676 F20101208_AACLGO stone_k_Page_35.QC.jpg
e4c0daa6ac8783a57a4ccf45cdb3e4bf
7db74b99e2d4a51cb5595d2477a9f746b110cebe
1860 F20101208_AACLQJ stone_k_Page_11.txt
81a2396df1c75e37c1ef26e8c3e170fd
9a2ea12a7f4a56b5ca0f94957ac736f8aa94ef9d
69842 F20101208_AACLLN stone_k_Page_23.jpg
c6dda791efc719236323a97af2ee828d
c97540caebc841db066f21a8425f44162a680319
60405 F20101208_AACLGP stone_k_Page_38.jpg
194f2e06b09f4d96db661b9ac2ead793
2f466fe67b82054af4f383be1164a883a2d7dc26
1934 F20101208_AACLQK stone_k_Page_13.txt
41738c37e665020d455d9d25e4400c19
5d87509ed14f895775b67644df61ec4b18e4dfa5
68516 F20101208_AACLLO stone_k_Page_24.jpg
2b85ddf85a1b88a9b5804dcd5dc90964
2673eba9ad8b71e439fc9aca9314462a41579e64
764 F20101208_AACLGQ stone_k_Page_38athm.jpg
cf03b1f5d80caf6cff087a97f6d295b6
7ec011c7c1ce1586078ec2fcb6e3510cf7166bb4
1848 F20101208_AACLQL stone_k_Page_14.txt
5d994b71d5693d90363f251bac22cdd3
3d5fedfbb99e72360db94c0d4c3f19bb16e42393
F20101208_AACLGR stone_k_Page_34.tif
619cfc5888364e828e819d5ccff88e85
153264e5dbf59c09482a2d038420a69e370589c5
2017 F20101208_AACLQM stone_k_Page_15.txt
c32667eda8706f624ef3041c22e6f8dc
af86e31da5c9cd6c3829a205b7a2e35b207debc6
20047 F20101208_AACLLP stone_k_Page_25.jpg
adfbdde05747c6d6b57bf35efcf3e904
7f3a05e6d654c595a0c754d67e247a2a1d2f1c84
22728 F20101208_AACLGS stone_k_Page_23.QC.jpg
96184c4c3a3fb4151e4f169bf12d6b89
349f5ecd0a9295077f3c5e4510009bbcf922294f
1344 F20101208_AACLQN stone_k_Page_16.txt
56ada76e7d1b16b2c4fc96a1b69b331b
1a067047c9f11caee8375e2f9825926f2c6c0c6a
35622 F20101208_AACLLQ stone_k_Page_26.jpg
687201951afb369cb46d319b725e034f
03f2cef53a4d8a8a241d3200da916da6c82516a5
17454 F20101208_AACLGT stone_k_Page_07.jpg
ea6ace5879cf7a890b9285a701cbcdf2
10de82e098ab9093b50cb4bed034f75e0ba3c36f
1985 F20101208_AACLQO stone_k_Page_18.txt
e11e0c7d7ca718c2d03bba6dae936b21
6f61d904ac84c94f31f74addbacd651269e195de



PAGE 1

RATS EXHIBIT BEHAVIORAL DESPA IR AND HORMONAL ALTERATIONS AFTER SOCIAL DEFEAT STRESS: IM PLICATIONS FOR MAJOR DEPRESSION By KRISTEN L. STONE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

PAGE 2

Copyright 2006 by Kristen L. Stone

PAGE 3

To my loving husband (Justin), my wonderful parents (Linda, Kenneth, and Donna), and my brothers and sisters (Kim, Rick, Minet, Roger, and Jay). Thank you for your uninterrupted love and support. Also to my extended family (both bi ological and in-law ) and my friends. Thank you for your continued encouragement and guidance.

PAGE 4

iv ACKNOWLEDGMENTS I would like to thank my supervisory committee members (Dr. Mohamed Kabbaj from Florida State University; Dr. Neil Ro wland; and Dr. Linda Hermer-Vazquez) for their time and patience. I would like to ex tend special gratitude to my advisor (Dr. Darragh Devine) for his persistent guidance ; and to Andrea Naccarato for her diligent work with the Porsolt swim test s. I would also like to th ank my mentors in Gainesville (especially Clare Mathes) for their support during this long journey of higher education. Lastly, and most importantly, I would like to thank God for the breadth of knowledge He has bestowed upon me.

PAGE 5

v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ix CHAPTER 1 INTRODUCTION........................................................................................................1 2 METHODS...................................................................................................................7 Animals........................................................................................................................ .7 Surgical Procedures......................................................................................................8 Experimental Procedures..............................................................................................8 General Social Defeat Stress Procedure................................................................8 Social Defeat Stress Regi men: Experiment 1a.....................................................9 Social Defeat Stress Regi men: Experiment 1b...................................................10 Social Defeat Stress and Porsolt Swim Test: Experiment 2...............................11 Behavioral Assays......................................................................................................12 Histological Assays....................................................................................................12 Statistical Analyses.....................................................................................................13 3 RESULTS...................................................................................................................17 Experiment 1a: Six Daily Sessi ons of Social Defeat Stress......................................17 Experiment 1b: Twelve Intermittent Sessions of Social Defeat Stress.....................23 Experiment 2: Social Defeat St ress Followed by Porsolt Swim Testing...................27 Inter-Observer Reliability...........................................................................................27 4 DISCUSSION.............................................................................................................29 Daily Social Defeat Stress..........................................................................................29 Intermittent Social Defeat Stress................................................................................30 Relevance to Previous Work......................................................................................31

PAGE 6

vi Porsolt Swim Test.......................................................................................................33 Conclusions and Future Directions.............................................................................34 LIST OF REFERENCES...................................................................................................35 BIOGRAPHICAL SKETCH.............................................................................................38

PAGE 7

vii LIST OF TABLES Table page 2-1 Experiment 1a, group assignments...........................................................................16 2-2 Experiment 1b, group assignments..........................................................................16 2-3 Experiment 2, group assignments............................................................................16

PAGE 8

viii LIST OF FIGURES Figure page 3-1 Social defeats per daily session................................................................................19 3-2 Intruder behavior during daily social defeat sessions..............................................20 3-3 Circulating hormones after daily social defeat stress...............................................21 3-4 Effects of daily social def eat stress on glandular masses.........................................22 3-5 Social defeats per intermittent session.....................................................................24 3-6 Intruder behaviors during intermittent social defeat sessions..................................25 3-7 Circulating hormones after inte rmittent social defeat stress....................................26 3-8 Effects of intermittent social defeat stress on glandular masses..............................26 3-9 Immobility during the Porsolt swim test..................................................................28 3-10 Immobility measured in 5-minute bi ns during the Porsolt swim test......................28

PAGE 9

ix Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science RATS EXHIBIT BEHAVIORAL DESPA IR AND HORMONAL ALTERATIONS AFTER SOCIAL DEFEAT STRESS: IM PLICATIONS FOR MAJOR DEPRESSION By Kristen L. Stone December 2006 Chair: Darragh P. Devine Major Department: Psychology Chronic emotional stress plays a pivotal role in the origin of many psychiatric disorders, including major depression. Acco rdingly, we investigat ed the behavioral, hormonal, and glandular effects of both repe ated and acute emoti onal stress using the social defeat model in rats. We compared one week of da ily social defeat exposure to one month of intermittent exposure to exam ine the effectiveness of massed versus intermittent stressors. In two experiments, nave male intruder rats were each exposed to dominant male resident rats. Each resident and intruder pair was allowed to interact for 5 minutes or until the intruder exhibited submissive supine posture three times. The intruder was then briefly removed, placed into a protective wire mesh cage, and returned to the residents cage until a total of 10 minutes elapsed from initial entry. Additional control rats were not exposed to social defeat stress. For the first experiment, the in truder rats were killed 10 minutes, 30 minutes, or 24 hours after the la st social defeat session and plasma

PAGE 10

x corticosterone and adrenocorticotropic horm one concentrations were assayed. For the second experiment, the previously-stressed ra ts were tested with a 15-minute Porsolt forced swim test. The intruder rats exhibited more freezing behavior and less exploratory locomotion across consecutive social defeat sessions resembling the loss of interest found in depressed patients. Exposure to social def eat stress produced signifi cant elevations in circulating hormones 10 minutes and 30 minutes after the session, when compared with concentrations in the control rats. The repeat edly stressed rats also exhibited higher basal concentrations of circulati ng corticosterone 24 hours late r, mimicking the augmented circulating hormones found in clinically depr essed patients. These results were evident after six daily exposures; however, basal horm one concentrations were not significantly elevated with the extended regimen of one social defeat session every 72 hours. Inconsistent thymus involution in the chronically stressed ra ts in both regimens suggests that a longer, more intense daily stress re gimen may be necessary to alter glandular masses. Exposure to chronic so cial defeat stress also produc ed a significan t increase in total immobility time during the forced swim test when compared with the immobility times for the rats that were exposed to a si ngle acute social defeat session and with the immobility times for the rats that were not exposed to social defeat stress, thus representing behavioral desp air in the chronically stressed animals. Overall, the behavioral, hormonal, and glandul ar alterations that occurred af ter repeated social defeat stress resemble some of the sympto ms of major depression in humans.

PAGE 11

1 CHAPTER 1 INTRODUCTION Annually, approximately 6.6% of the na tional adult population suffers from depression (Kessler et al., 2003). It is a wide-spread illness that interferes w ith the ability to eat, sleep, work, and enjoy formerly pleasur able activities. The economic impact of this devastating disorder is high, but the co st in human distress cannot be estimated. The 4th Edition of the Diagnostic and St atistical Manual of Mental Disorders (1994) defines depression according to the follo wing criteria, with at least five of the symptoms present on a daily basis for at leas t 2 weeks: depressed mood, loss of interest or pleasure, significant wei ght loss or gain, insomnia or hypersomnia, psychomotor agitation or retardation, fati gue, undue guilt and/or fee lings of worthlessness, indecisiveness, recurrent thoughts of death, and significant distress or impairment in social or occupational functioning. These sy mptoms must represent a change from the individuals normal level of interpersonal function. Major depression is also clinically characterized by altered hormonal function stemming from ongoing elevations in overall organism distress. It has been well established that chronic emotional stress pl ays a pivotal role in the genesis of many psychiatric disorders such as depression (for review, see Agid et al., 2000). Chronic stress weighs on the physiological systems that maintain homeostasis and produces changes in the operating limits of those hormonal systems. Allostatic load, or the strain from the elevated activity of systems under major stress, can predispose an animal to many psychiatric disorders, including depr ession (McEwen and Stellar, 1993). The

PAGE 12

2 concept of allostatic load suggests that there is a fixed state in which enduring environmental challenges are balanced by a hormonal response that is raised above normal, basal levels. Patients with majo r depression, regardless of age, show higher 24-hour average cortisol levels when compar ed to normal subjects (Linkowski et al., 1985). Additionally, depressed patients reach the nadir of their daily cortisol cycle two to three hours before control subjects (Pfohl et al., 1985) This imbalance between activation and recovery of the stress response is implicated in the inability to maintain homeostasis, thus leading to neuroendocri ne maladjustment and heightened risk for depression (De Kloet, 2003). Stressful stimuli are categorized into two de scriptive classes. Systemic stressors, such as exposure to heat or cold, present immediate threat s to somatic homeostasis while processive stressors emphasize higher leve l cognitive processing (Herman and Cullinan, 1997). Common processive stresso rs include instability in th e social hierarchy and loss of environmental control. In accordance with the emotional nature of processive stress, it is particularly implicated in a variety of psychiatric diso rders including depression (for review, see Anisman and Matheson, 2005). Inputs from the brainstem (if the stressor is systemic in nature) and cortical and limbic structures (if the stressor is processive in nature) converge at the paraventricular nucleus (PVN) of the hypothalamus where parv ocellular neurons project to the median eminence. From there, corticotrophin -releasing hormone (CRH) and arginine vasopressin (AVP) are released into th e hypophyseal portal circulation, stimulating adrenocorticotropic hormone (ACTH) release from the anterior pituitary corticotrope cells into the general circulat ion. The release of ACTH then stimulates the synthesis and

PAGE 13

3 release of glucocorticoids from the adrenal cortex. Cortisol is the main glucocorticoid in humans and corticosterone (CORT) is the ma in glucocorticoid in rats. The elevated circulating levels of glucocor ticoids decrease the further ac tivity of the HPA axis through exertion of negative feedback on neuros ecretory cells of the hypothalamus and corticotrope cells of the pituitar y (for review, see Whitnall, 1993). Altered regulation of hormona l activity in depressed pati ents is thought to be a result of increased activity of specific CRH-c ontaining neurons in the PVN. The average total number of such neurons is up to four times higher in depressed patients when compared to normal control subjects. Also, co-localization of AVP in CRH-expressing neurons has been indicated as an index for stress-activated neuronal activity. The average number of neurons co-expressing both CRH and AVP in depressed patients is up to three times higher than those for normal s ubjects. These results suggest that increased expression of CRHand AVP-containing neur ons in the PVN may cause at least a fraction of the collective symptomatology of depression (Raadsheer et al., 1994). When physically or emotionally stresse d, non-human animals endure physiological responses that lead to beha vioral and hormonal impairment which may be fundamentally similar to the impairment seen in human stress-induced psychopathology. Behavioral responses to stressful stimuli (including in creased drug-taking propensity, decreased performance in learning tasks, sleep distur bances, and unsocial behavior) have been observed in a variety of species (for review, see Amiel-Tison et al., 2004). Also, there is significant evolutionary homol ogy in stress-regulating pep tides, such as CRH and CRHrelated molecules (Chang and Hsu, 2004). Thus, the use of an animal model for

PAGE 14

4 processive stress that produces both behavioral and hormonal e ffects is a logical approach to the study of human st ress-induced disorders. The effects of chronic and acute processi ve stress have been studied in rats (Simpkiss and Devine, 2003). Experimentally nave rats were exposed to a chronic variable stress (CVS) regimen of twice daily stressors for fifteen days. The stressors included novel environment, switched cage mates, forced swim, light open field, intermittent white noise, and intermittent footshock, administered on a random intermittent schedule. The CVS regimen wa s unsuccessful in producing elevations in basal circulating concentrations of ACTH and CORT, or in hormonal response to an acute stressor. The rats showed a blunt ed ACTH response, but no altered CORT response. Another model for emotional stress, social defeat, has also produced significant elevations in circulating CO RT during and after acute and repeated exposure to the stressful stimulus (Sgoifo et al., 1996). The procedure, developed by Miczek (1979), is designed to model social stress. A male intruder rat is expo sed to social stress when it is placed into the home cage of a larger male resident rat. The resident rat exhibits dominant behavior toward the intruder rat by displaying assertive posture, standing over the intruder. The intruder submits by displaying supine posture, positioned beneath the resident. The further effects of repeated social defeat stress on behavioral and hormonal (HPA axis) responses have been studied in rats (Lopes and Devine, 2004). In a preliminary study, a repeatedly stressed group of intruder rats s howed significantly elevated circulating CORT concentrations 24 hours after their final so cial defeat session,

PAGE 15

5 when compared to the concentr ations in the unstressed contro l animals. This suggests an enduring change in the circadian regulation of the HPA axis following chronic social defeat. Overall, the effects of six days of so cial defeat stress exceed ed the results attained with the fifteen days of CVS previously described. The brain sites activated by social defeat have been studied using c-fos immunohistochemistry. The immediate-early gene (IEG) c-fos is expressed in many cells in the brain, but typically at very low basal levels. C-fos and other IEGs are intracellular signaling mechanisms that regulate gene transcription and expression of various neuropeptides and trophic molecules in res ponse to stress (for re view, see Sabban and Kvetnansky, 2001). Various stressful stim uli can initiate increased levels of c-fos mRNA expression, lasting for minutes to hours. For instance, social defeat produces elevated c-fos expression in limbic, limbic-associated, a nd brainstem sites in both hamsters and rats one hour after a single de feat session (Kollack-Walker et al., 1997; Martinez et al., 1998). These results point to the brain structur es that are important in the processing of emotionally stressful events. However, after re peated social defeat sessions in rats, the pattern of neuronal activity was modified, de spite the fact that intruder submissive behavior persisted across trials. C-fos mRNA expression endured for many of these limbic and brainstem nuclei, while other limbic and brainstem regions exhibited a decrease in the social defeat-induced c-fos mRNA expression. The Porsolt swim test, originally describe d by Porsolt and collea gues (1978), is the most commonly utilized behavioral test for screening antidepressant treatments in rats and has been used to evaluate the behavior al effects of stress exposure. Immobility during the inescapable swim is measured as an indicator of be havioral despair or

PAGE 16

6 depressive-like behavior. Re gimens of chronic stress or repeated administration of gluccocorticoids increase the amount of time an animal spends immobile, decrease the latency to immobility, and decrease the am ount of time engaged in active swimming (Molina et al., 1994; Johnson et al., 2006) Antidepressant drugs, on the other hand, ameliorate these effects. For example, de sipramine (a tricyclic antidepressant) and fluoxetine (a selective serotoni n reuptake inhibitor) reduce immobility and increase the amount of time a stressed animal will struggle to escape the forced swim (Molina et al., 1994; Lucki et al., 2001). Based on the results of investigations usi ng social defeat stress and the Porsolt swim test, we have begun to further charac terize the social defeat model of emotional stress in rats. We have evaluated the impact of both acute and re peated social defeat stress on regulation of ACTH and CORT at va rious times after the stressor. We have also compared six daily social defeat sessions to one month of stress every third day to examine the effectiveness of massed and inte rmittent stress exposure. In addition, we exposed socially defeated rats to the Pors olt swim test in order to characterize the enduring behavioral effects of social defeat stress.

PAGE 17

7 CHAPTER 2 METHODS Animals Ninety male Long-Evans (LE) rats and twel ve female LE rats were purchased from Harlan Co. of Indianapolis, IN. Twenty of the male Long Evans (LE) rats (225-250 g) were used as unhandled controls. Fifty-eight of the male LE rats (225-250 g) were used as intruders. Twelve of the male LE rats (300-325 g) were used as residents and the twelve female LE rats (225-250 g) were used as housing mates for the residents. The weight ranges indicate weights at the time the rats were purchased. The intruders weighed 275-325 g and the residents weighed 500800 g at the time of the experiments. Six of the unhandled control rats were used in Experiment 1a, six were used in Experiment 1b, and eight were used in Experime nt 2. Thirty of the intruder rats were used in Experiment 1a, twelve were used in Experiment 1b, and sixteen were used in Experiment 2. The resident males and the females were used in multiple experiments. The rats were housed in polycarbonat e cages (43 x 21.5 x 25.5 cm) with sexmatched and weight-matched pairings for five to seven days of acclimation to a 12hr/12hr light/dark cycle (light s on at 7:00a.m.). Standard chow (LabDiet 5001) and tap water were available ad libitum Temperature and humidity in the housing facility were controlled to 22.72C 0.94C and 54.1% 14.6%, respectively. The intruder rats were exposed to social defeat stress and remained pair-housed throughout the experiment. The control rats we re not exposed to social defeat stress and were pair-housed throughout the experiment. The resident rats were vasectomized,

PAGE 18

8 singly-housed, and given 10 days to recover fr om the surgery. The residents were then pair-housed in a separate housing room, each wi th a female, for 2 weeks prior to and then throughout the experiment. All procedures were pre-approved by the Institutional Animal Care and Use Committee at the Un iversity of Florida and conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Surgical Procedures After a week of acclimation to the hous ing facility, the resident rats were anaesthetized with ketamine:xylazine (50 mg/kg:5 mg/kg, i.p.). Seda tion was verified by testing the pedal withdrawal reflex 10 mi n after injection and every 10 min during the procedure. After sedation was verified, each ra t was shaved from the rostral edge of the scrotum to the caudal abdomen. The area was then washed with Betadine three times. Surgery began with a 2 cm ventral midline incision, just above the scrotal area. Through the incision, the vas deferens was lo cated and externalized. A 0.25 cm section of each duct was removed with a micro-cautery knife. The muscle walls were sutured and the external incision was closed using st erilized staples that were removed ten days after the surgery. Each surgic al procedure lasted 15-30 min. Each rat was given a 1 mL injection of 0.9% warm saline and ketorolac subcutaneously and then placed into a post-ope rative Plexiglas cage, heated by an electric heating pad. Following observation of locomoti on and urination, each rat was returned to its individual home cage and was monito red daily throughout the recovery period. Experimental Procedures General Social Defeat Stress Procedure At the beginning of the 10-min social def eat session, the female rat paired with one resident rat was taken out of the cage and pl aced into an empty, identical cage. The

PAGE 19

9 intruder rat was then placed into the cage of the resident (direct interaction). The session was then closely monitored for submissive, supine posture expressed by the intruder. Each time the intruder exhibited a supine postu re with the resident physically contacting the intruder for 2 sec or more, one defeat was counted. After three defeats or a total of 5 min elapsed (whichever came first), the intr uder was taken out of the residents cage momentarily and quickly placed into a 10 x 10 x 15 cm double-walled protective wire mesh cage. The intruder (within the wire mesh cage) was then placed back into the residents cage for the remainder of the 10min session (indirect interaction). The two intruder rats in each housing pair were run in simultaneous defeat sessions in the cages of two resident males that were placed side by side. Each pair of cage-mates received the same treatment. Each social defeat sessi on was run between 8:00a .m. and 10:00a.m., and was videotaped for further analysis. Social Defeat Stress Regi men: Experiment 1a Thirty-six nave male LE rats were assigned to six experimental groups (Table 2-1). The control rats from Group 1 (n = 6) rema ined in their original cages throughout the experiment and were rapidly decapitated between 8:00a.m. and 10:00a.m. on the final experimental day. These rats were exposed to no chronic and no acute stress (NC/NA). The intruders from Group 2 (n = 6) were expos ed to social defeat stress once every 24 h across six experimental days and were kill ed by rapid decapitation between 8:00a.m. and 10:00a.m., 24 h after their final social defeat se ssion. These rats were exposed to chronic, but not acute stress (C/NA). The intruders fr om Group 3 (n = 6) were only exposed to social defeat stress on the final experiment al day and were then immediately decapitated, 10 min after the start of the social defeat sess ion (t = 10 min). These rats were exposed to no chronic, just acute stress (NC/A-10). Th e intruders from Group 4 (n = 6) were only

PAGE 20

10 exposed to social defeat stress on the final experimental day and were rapidly decapitated 30 min after the start of the social defeat sess ion (t = 30 min). These rats were exposed to no chronic, just acute stress (NC/A-30). The intruders from Group 5 (n = 6) were exposed to social defeat stress once every 24 h over the course of six experimental days and were killed by rapid decapitation 10 min af ter the start of the social defeat session on the sixth day (t = 10 min). These rats were exposed to chronic and acute stress (C/A-10). The intruders from Group 6 (n = 6) were expos ed to social defeat stress once every 24 h over the course of six experimental days and were killed by rapid decapitation 30 min after the start of the social de feat session on the sixth day (t = 30 min). These rats were exposed to chronic and acute st ress (C/A-30). Each intruder fr om the repeatedly stressed groups was placed in the cage of a different resident each day of his chronic stress routine. Social Defeat Stress Regi men: Experiment 1b Eighteen nave male LE rats were assigne d to three experimental groups (Table 2-2). The unhandled control rats from Group 1 (n = 6) remained in their original cages throughout the experiment and were killed by rapid decapitation between 8:00a.m. and 10:00a.m. on the final experimental day. Th ese rats were exposed to no chronic and no acute stress (NC/NA). The intrud ers from Group 2 (n = 6) were exposed to social defeat stress once every 72 h across thirty-four expe rimental days and were rapidly decapitated between 8:00a.m. and 10:00a.m., 24 h after thei r final social defeat session. These rats were exposed to chronic, but not acute stress (C/NA). The intruders from Group 3 (n = 6) were exposed to social defeat stress on ce every 72 h across thirty-four experimental days and were killed by rapid decapitation 30 min after the start of the social defeat session on the final day (t = 30 min). These ra ts were exposed to ch ronic and acute stress

PAGE 21

11 (C/A). All intruder rats from Experiment 1b were exposed to a total of twelve social defeat sessions. Each intruder from the repe atedly stressed groups was placed in the cage of a different resident each da y of his chronic stress routine. Social Defeat Stress and Porsolt Swim Test: Experiment 2 Twenty-four nave male LE rats were assi gned to three experimental groups (Table 2-3). The unhandled control rats from Group 1 (n = 8) remained in their original cages throughout the social defeat portion of the expe riment. These rats were exposed to no chronic and no acute social de feat stress (NC/NA). The intr uders from Group 2 (n = 8) were only exposed to social defeat stress on th e final day of the social defeat portion of the experiment. These rats were exposed to no chronic, just acut e stress (NC/A). The intruders from Group 3 (n = 8) were repeatedly exposed to social defeat stress over the course of five days, once every 24 h. These ra ts were exposed to ch ronic and acute stress (C/A). Each repeatedly stressed intruder wa s placed in the cage of a different resident each day of his chronic stress routine. All of the rats from each of the three groups were then exposed to the Porsolt swim test. Twenty-four hours after the final social defeat session, each rat from Experiment 2 was individually removed from its home cag e and placed into a plastic cylinder filled with approximately 25 cm of clean tap water at 24-27 C. The depth of water allowed each rat to reach the bottom of the cylinder with its tail, wi th enough head room that the rat was unable to escape from the tank. The water in each cylinder was changed between trials. Each rat was subjected, one at a tim e, to a 15-min swim session then carefully dried and returned to its home cage. Each Porsolt swim session was run between 8:00a.m. and 10:00a.m., and was vi deotaped for further analysis.

PAGE 22

12 Behavioral Assays Number of defeats, latency to first de feat, freezing behavior, and exploratory locomotion were scored from the recorded soci al defeat sessions. Freezing behavior was recorded whenever the intruder remained motionl ess for at least 2 sec, and is reported as percent time during the direct interaction between the intruder and the resident. Exploratory locomotion was recorded every time the intruder crossed with all four paws from one third of the cage into the adjacent th ird of the cage. Vertical lines were drawn on the video image of each residents home cage and exploratory locomotion was scored from the videotapes. Exploratory locomotion is reported as lines crossed per minute of direct interaction time. Two tr ained observers scored the soci al defeat stress videos from Experiments 1a and 1b and inter-observer reliability was assessed. Immobility was recorded during the Porsolt swim tests whenever the rat balanced on its tail, completely motionless, or exhi bited only slight forepaw movement for a minimum of 2 sec. This behavior is repor ted as total time spent immobile during the swim test. Two trained observers scored th e swim test videos from Experiment 2 and inter-observer reliability was assessed. Histological Assays Immediately after decapitation in experi ments 1a and 1b, 6 mL of trunk blood from each rat was collected in pol ypropylene tubes on ice with 600 l of Na2EDTA at 20 g/ L. The blood samples were then immedi ately centrifuged at 1000 rcf for 5 min and the plasma fraction frozen in 300 l aliquots at C. The brain from each intruder was quickly removed, frozen in 2-methylbutane at -40C, and stored at -80C for future analysis of molecular variable s involved in HPA axis function. The thymus and adrenal glands were removed from each rat and frozen separately at 80C. These glands were

PAGE 23

13 later weighed to determine the health and st ress status of each rat at time of death. Plasma ACTH and CORT concentrations fr om the rats were later analyzed with radioimmunoassay (RIA). ACTH RIAs were run with kits from Alpco Diagnostics (Salem, NH) and CORT RIAs were run with kits from Diagnostic Products Corporation (Los Angeles, CA). Statistical Analyses Potential between-groups differences in number of defeats and latency to first defeat were analyzed using two one-way anal yses of variance (ANOVAs) to compare the five groups of intruders (i.e. three repeatedly stre ssed, and two just acutely stressed) in Experiment 1a during their firs t exposure to social defeat stress. A 3x6 (group x session) repeated-measures ANOVA (for Experiment 1a ) and a 2x12 (group x session) repeatedmeasures ANOVA (for Experiment 1b) were used to examine potential differences in number of defeats between groups and across experimental sessions for the repeatedly stressed groups. A 3x6 (group x session) repeated-measures ANOVA (for Experiment 1a) and a 2x12 (group x session ) repeated-measures ANOVA (for Experiment 1b) were used to examine potential differences in late ncy to first defeat between groups and across experimental sessions for th e repeatedly stressed groups. Potential between-groups differences in freezing responses were analyzed using a one-way ANOVA to compare freezing behavior between the five groups in Experiment 1a during their first exposure to social defeat stress. A 3x6 (group x session) repeatedmeasures ANOVA (for Experiment 1a) and a 2x12 (group x session ) repeated-measures ANOVA (for Experiment 1b) were used to examine potential freezing behavior differences between groups and across experime ntal sessions for the repeatedly stressed groups.

PAGE 24

14 Potential between-groups differences in exploratory locomotion were analyzed using a one-way analysis of variance ( ANOVA) to compare exploratory locomotion between the five groups in Experiment 1a dur ing their first exposur e to social defeat stress. A 3x6 (group x session) repeated-m easures ANOVA (for Experiment 1a) and a 2x12 (group x session) repeated-measures ANOVA (for Experiment 1b) were used to examine potential exploratory locomotion differences between groups and across experimental sessions for th e repeatedly stressed groups. Potential between-groups differences in plasma ACTH concentrations and in plasma CORT concentrations were anal yzed with one-way ANOVAs in each of Experiments 1a and 1b. Also, potential betw een-groups differences in adrenal weights and in thymus weights were analyzed with one-way ANOVAs in each of Experiments 1a and 1b. The results were further analyzed using pre-planned Newman-Keuls multiple comparison tests for all significant ANOVAs. Potential between-groups differences in immobility duration scores for the 15-min Porsolt swim test session in Experiment 2 were analyzed using a one-way ANOVA. The results were further analyzed using Newman-K euls multiple comparison tests. The data were then analyzed in 5-min bins using a 3x3 (group x bin) repeated-measures ANOVA. The results were further analyzed using Newman-Keuls multiple comparison tests, making pair-wise comparisons between each stressed group and the control group, for each bin. Inter-observer agreement was assessed for freezing behavior in Experiments 1a and 1b by comparing total times recorded by each trained observer for each social defeat session. Inter-observer agreement was also assessed for exploratory locomotion in

PAGE 25

15 Experiments 1a and 1b by comparing total number of line crossings recorded by each trained observer for each social defeat se ssion. Inter-observer agreement was assessed for immobility duration scores for the Porso lt swim test from Experiment 2 by comparing total immobility times recorded by each trained observer for each test session.

PAGE 26

16 Table 2-1. Experiment 1a, group assignments Group Repeated Stress Acute Stress Kill Time NC/NA C/NA X t = 24 h NC/A-10 X t = 10 min NC/A-30 X t = 30 min C/A-10 X X t = 10 min C/A-30 X X t = 30 min Table 2-2. Experiment 1b, group assignments Group Repeated Stress Acute Stress Kill Time NC/NA C/NA X t = 24 h C/A X X t = 30 min Table 2-3. Experiment 2, group assignments Group Repeated Stress Acute Stress Porsolt Swim Test NC/NA X NC/A X X C/A X X X

PAGE 27

17 CHAPTER 3 RESULTS Experiment 1a: Six Daily Sess ions of Social Defeat Stress During their first exposure to social defeat stress, the ra ts from all five of the groups in Experiment 1a did not show statisti cally different numbers of defeats (F (4, 25) = 0.2419, p < 0.9118; Fig. 3-1A) nor latencies to first defeat (F (4, 25) = 0.7308, p < 0.5795; Fig. 3-1B). Each of the three repeatedly stressed groups (C/NA, C/A-10, and C/A-30), showed no significant between-group s difference, time effect, or group by time interaction effect in numbe r of defeats (F (2, 15) = 1.603, p < 0.2340; F (5, 15) = 0.3463, p < 0.8831; F (10, 15) = 0.3139, p < 0.9753; Fig. 3-1A) nor latencies to first defeat across the six experimental sessions (F ( 2, 15) = 1.571, p < 0.2401; F (5, 15) = 0.2106, p < 0.9570; F (10, 15) = 0.5255, p < 0.8669; Fig. 3-1B). During their first exposure to social defeat stress, the ra ts from all five of the groups did not show statistically different amounts of freezing behavior (F (4, 25) = 0.1757, p < 0.8405; Fig. 3-2A). Each of the th ree repeatedly stressed groups showed significantly increased freezing behavior across the six experimental sessions (F (5, 75) = 17.08, p < 0.0001; Fig. 32A), reaching asymptote by day 3, with no significant betweengroups differences or group by time interaction effect. During their first exposure to social defeat stress, the ra ts from all five groups did not show statistically different amounts of exploratory locomotion (F (4, 25) = 0.5711, p < 0.5767; Fig. 3-2B). Each of the three repeat edly stressed groups showed significantly decreased exploratory locomotion across the si x experimental sessions (F (5, 75) = 11.76,

PAGE 28

18 p < 0.0001; Fig. 3-2B), reaching asymptote by da y 4, with no significant between-groups differences or group by time interaction effect. Exposure to acute social de feat (NC/A and C/A) produced significant elevations in circulating ACTH concentrations when compar ed with the ACTH concentrations in the control rats (NC/NA) (F (5, 30) = 3.874, p < 0.0079; Fig. 3-3A). Circulating ACTH concentrations were not elevated in the chroni cally, but not acutely, st ressed rats (C/NA). Exposure to social defeat stress for al l groups (including C/NA) produced significant elevations in circulating CORT concentrations when compared with the CORT concentrations in the control rats (F (5, 30) = 20.79, p < 0.0001; Fig. 3-3B). Exposure to social defeat significantly d ecreased thymus masses in Groups C/A-10 and C/A-30, but not for the other groups (F (2, 15) = 5.651, p < 0.0148; Fig. 3-4A). There were no significant diffe rences in adrenal masses betw een the rats in the stressed groups and the rats in the control gr oup (F (5, 30) = 0.9299, p < 0.4756; Fig. 3-4B).

PAGE 29

19 1 2 3 4 5 6 0 1 2 3 C/NA NC/A-10 NC/A-30 C/A-10 C/A-30 Experimental DayNumber of Defeats 1 2 3 4 5 6 0 25 50 75 100 125 C/NA NC/A-10 NC/A-30 C/A-10 C/A-30 Experimental DayLatency to First Defeat (sec)A B Figure 3-1. Social defeats per daily session. The rats exposed to repeated social defeat stress (C/NA, C/A-10, C/A-30) exhibite d equivalent (A) number of defeats per session and (B) latency to firs t defeat per sess ion across the 6 experimental sessions. The rats that received only one acu te defeat session (NCA-10 and NCA-30) were stressed at the same time as the final stress session for the chronically stressed groups, and so the values for these groups are illustrated on day 6. Values expre ssed are group means SEM (n = 6 rats per group). Abbreviations: C/NA = ch ronic stress/no acute stress, NC/A = no chronic stress/acute stress, C/A = chronic stress/acute stress.

PAGE 30

20 0 1 2 3 4 5 6 0 10 20 30 40 50 60 70 80 90 100 C/NA NC/A-10 NC/A-30 C/A-10 C/A-30 Experimental DayTotal Time Freezing (% direct interaction time) 0 1 2 3 4 5 6 0 1 2 3 4 5 6 7 8 9 10 11 C/NA NC/A-10 NC/A-30 C/A-10 C/A-30 Experimental DayLine Crossings per MinuteA B Figure 3-2. Intruder behavior during daily soci al defeat sessions. The rats exposed to repeated social defeat stress exhibited (A) increases in fr eezing behavior and (B) decreases in exploratory locomotion across the 6 experimental sessions. The rats that received only one acute de feat session were stressed at the same time as the final stress session for the ch ronically stressed groups, and so the values for these groups are illustrate d on day 6. Values expressed are group means SEM (n = 6 rats per group).

PAGE 31

21 NC /NA C/N A N C/A-1 0 NC / A -3 0 C/A 10 C/A-3 0 0 50 100 150 200* * n.s. n.s.n.s. n.s.ACTH Concentration (pg/ml) NC/NA C / N A N C /A-10 N C /A-30 C / A -1 0 C / A -3 0 0 100 200 300 400 500* * * ++ +n.s.CORT Concentration (ng/ml)A B Figure 3-3. Circulating hormones after daily social defeat st ress. The rats exposed to social defeat stress had elevated circ ulating concentrations of (A) ACTH and (B) CORT when compared to basal con centrations in control rats. Values expressed are group means SEM (n = 6 rats per group). Significant differences between the socially defeat ed rats and the unhandled control rats (NC/NA) are expressed as [* ] p < 0.05. Significant di fferences in pre-planned comparisons between the stressed groups of rats are expressed as [+] p < 0.05, with lines connecting the groups th at were compared. Additional abbreviation: NC/NA = no chr onic stress/no acute stress.

PAGE 32

22 N C /N A C/ N A N C /A1 0 N C /A3 0 C /A1 0 C /A3 0 0 100 200 300 400 500 600* *Thymus Weight (mg) NC/ NA C/ NA NC/A-10 NC/A-30 C/ A1 0 C/ A3 0 0 10 20 30 40 50 60Adrenal Weight (mg)A B Figure 3-4. Effects of daily soci al defeat stress on glandular masses. Social defeat stress (A) significantly decreased thymus masse s for 2 of the repeatedly stressed groups (C/A-10 and C/A-30), but (B) did not significantly affect adrenal gland masses for any group. Values expressed are group means SEM (n = 6 rats per group). Significant differences between the socially defeated rats and the control rats are expressed as [*] p < 0.05.

PAGE 33

23 Experiment 1b: Twelve Intermittent Sessions of Social Defeat Stress There were no significant between-groups differences, time effect, or group by time interaction effect in num ber of defeats (F (1, 10) = 0.0787, p < 0.7847; F (11, 10) = 0.9032, p < 0.5399; F (11, 10) = 0.8203, p < 0.6199; Fig. 3-5A) nor latency to first defeat (F (1, 10) = 0.4653, p < 0.5107; F (11, 10) = 0.8400, p < 0.6007; F (11, 10) = 0.3414, p < 0.9743; Fig. 3-5B) for the repeatedly stressed groups (C/NA and C/A) in Experiment 1b. Freezing behavior significantly increased for both of the repeatedly stressed groups across the twelve experimental sessions (F (11, 10) = 6.224, p < 0.0001; Fig. 3-6A), with no significant between-groups differences or group by time interaction effect. Exploratory locomotion signifi cantly decreased for both of the repeatedly stressed groups across the twelve experimental se ssions (F (11, 10) = 7.252, p < 0.0001; Fig. 3-6B), with no significant between-groups differences or group by time interaction effect. Acute exposure to social defeat stress pr oduced significant elevations in circulating ACTH concentrations when compared with th e basal ACTH concentra tions in the control rats (F (2, 15) = 4.525, p < 0.0290; Fig. 3-7A). Acut e exposure to social defeat stress also produced significant elevations in circulating CORT concentrations when compared with the basal CORT concentrati ons in the control rats (F (2, 15) = 5.853, p < 0.0132; Fig. 3-7B). These elevations in circulating hormones were limite d to the rats that were stressed acutely before termination (C/A) the basal concentrations in the other chronically stressed rats (C/ NA) did not significantly differ from the basal concentrations in the control rats (NC/NA). There were no significant differences in thymus masses (F (2, 15) = 0.04457, p < 0.9565; Fig. 3-8A) or adrenal masses (F ( 2, 15) = 1.826, p < 0.1951; Fig. 3-8B) between the rats in the socially defeated groups and the rats in the control group.

PAGE 34

24 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 C/NA C/A Experimental DayNumber of Defeats 1 2 3 4 5 6 7 8 9 10 11 12 0 25 50 75 100 125 C/NA C/A Experimental DayLatency to First Defeat (sec)A B Figure 3-5. Social defeats per intermittent session. The rats exposed to repeated social defeat stress (C/NA and C/A) exhibite d equivalent (A) number of defeats per session and (B) latency to first defeat per session across the 12 experimental sessions. Values expressed are group means SEM (n = 6 rats per group).

PAGE 35

25 0 1 2 3 4 5 6 7 8 9 10 11 12 0 10 20 30 40 50 60 70 80 90 100 C/NA C/A Defeat SessionTotal Time Freezing (% direct interaction time) 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 C/NA C/A Defeat SessionLine Crossings per MinuteA B Figure 3-6. Intruder behaviors during intermittent social defeat sessions. The rats exposed to repeated social defeat stress exhibited ( A ) increases in freezing behavior and ( B ) decreases in exploratory lo comotion across 12 experimental sessions. Values expressed are group means SEM (n = 6 rats per group).

PAGE 36

26 NC/ NA C/NA C /A 0 50 100 150 200*ACTH Concentration (pg/ml) NC/ NA C/ NA C/ A 0 100 200 300 400 500*CORT Concentration (ng/ml)A B Figure 3-7. Circulating hormones after interm ittent social defeat stress. Immediately after, but not 24 h later, th e rats exposed to repeated social defeat stress had elevated circulating concentrations of (A) ACTH and (B) CORT when compared to basal concentrations in the control rats (NC/NA). Values expressed are group means SEM (n = 6 rats per group). Significant differences between the socially defeat ed rats and the unhandled control rats are expressed as [*] p < 0.05. NC/NA C /N A C/A 0 100 200 300 400 500 600Thymus Weight (mg) NC/NA C / NA C / A 0 10 20 30 40 50 60Adrenal Weight (mg)A B Figure 3-8. Effects of intermittent social de feat stress on glandular masses. Repeated social defeat stress had no effect on (A) thymus masses or (B) adrenal gland masses. Values expressed are group m eans SEM (n = 6 rats per group).

PAGE 37

27 Experiment 2: Social Defeat Stre ss Followed by Porsolt Swim Testing Exposure to chronic social de feat stress (C/A) produced a significant increase in total immobility time during the 15-min forced swim when compared with the immobility times for the control rats that were not exposed to social defe at stress (NC/NA) (F (2, 21) = 5.363, p < 0.0131; Fig. 3-9). When the data we re analyzed in 5-min bins, there was a significant between-groups effect as well as a significant tim e effect (F (2, 42) = 2.424, p < 0.0150; F (2, 42) = 72.74, p < 0.0001; Fig. 3-10), but no significant group by time interaction effect (F ( 4, 42) = 1.270, p < 0.2970; Fig. 3-10). Inter-Observer Reliability The two observer's recordings of total time freezing for the social defeat sessions from Experiments 1a and 1b combined differed by less than 20 sec for 94% of the sessions and never differed by more than 28 sec. The two observer's recordings of exploratory locomotion during social defeat sessions were identical in 70% of the sessions and never differed by more than 3 lines crossed. The two observer's recordings of total immobility time during the Porsolt swim test differed by less than 40 sec in 92% of the sessions and never differed by more than 43 sec.

PAGE 38

28 NC/NANC/AC/A 0 100 200 300 400 500 600 700 800 900*Total Immobility Time (sec) Figure 3-9. Immobility during the Porsolt swim test. The rats exposed to repeated social defeats stress (Chronic) e xhibited increased immobility for the duration of the Porsolt forced swim test, when comp ared to the immobility times for the intruder rats exposed to acute social de feat stress (Acute) a nd the control rats (No SD). Values expressed are group means SEM (n = 8 rats per group). Significant differences between the soci ally defeated rats and the unhandled control rats are expressed as [*] p < 0.05. Additional abbreviation: No SD = no social defeat stress. 1 2 3 0 100 200 300 NC/NA NC/A C/A * *+Total Immobility Time (sec) Figure 3-10. Immobility measured in 5-minute bins during the Porsolt swim test. There were significant main effects for time and between-groups difference when comparing the socially defeated groups (Acute and Chronic) to the control group (No SD). Values expressed ar e group means SEM (n = 8 rats per group). Significant differences between the chronically def eated rats and the controls are expressed as [*] p < 0.05. Significant differences between the acutely defeated rats and the controls are expressed as [+] p < 0.05.

PAGE 39

30 CHAPTER 4 DISCUSSION Overall, repeated social de feat stress produced behavioral, hormonal, and glandular changes that model some of th e symptoms that are seen in depressed humans. Behavioral despair, HPA axis dysregulation, and thymus involution were found in rats that were exposed to daily social stress; and these effect s were greater overall than the effects that were seen in the rats that were stressed intermittently. Daily Social Defeat Stress The equivalent mean freezing scores and the equivalent mean locomotion scores during the first social defeat se ssion indicate that all five gr oups that experienced social defeat stress were comparable at the be ginning of the experiment. The equivalent progressive increases in freezing behavior a nd the equivalent decreases in exploratory locomotion for the three repeatedly stressed groups indicate that these groups underwent comparable stress-induced behavioral change s across social defeat sessions. These behaviors, however, did not diminish the pe rsistent displays of dominant behavior (consistent number of defeats and equivalent la tencies to first defeat) by the resident rats. After 3-4 days of stress exposure, the behavi oral curves for both freezing behavior and exploratory locomotion reached asymptote, suggesting that the behavioral changes associated with daily social defeat stress do not progress beyond the first few exposures. The equivalent mean ACTH concentra tions and the equivalent mean CORT concentrations 10 min after the onset of st ress between the groups with and without a history of stress suggest that there is simila r activation of the HPA axis for these groups.

PAGE 40

31 However, the lower CORT concentrations 30 min after the onset of stress in the repeatedly stressed group compared to the CO RT concentrations in the acutely stressed group suggests a sensitization in the ne gative feedback regulation of CORT. Decreased thymus mass and/or increased adrenal mass has been shown to result from chronic stress regimens involving physic al and/or psychologi cal stressors (for examples, see Blanchard et al., 1998; Bryant et al., 1991; Simpkiss and Devine, 2003) as well as from major depression in victims of suicide (Szigethy et al ., 1994). Given that two of the three groups that were stressed daily (C/A-10 and C/A-30) exhibited thymus involution while one (C/NA) did not, it cannot be concluded that the daily stress regimen consistently caused a decrease in thymus ma ss. These findings, along with adrenal mass equivalency across groups regard less of number of stress e xposures, suggest that the chronic social defeat stress regimen of six da ily exposures may not have been adequate to consistently alter glandular masses. A l onger or unpredictable social defeat stress regimen could possibly generate consistent thymus involution and adrenal hypertrophy. Perhaps an additional stressor for the intruders, such as isolation housing to ensure more timid behavior (Kabbaj et al., 2000), could cont ribute to the effectiven ess of the regimen. Also, using a restraint stress harness instead of the prot ective wire mesh cage could elevate the efficiency of the regimen by producing an inescapable condition for the intruders. Intermittent Social Defeat Stress The equivalent progressive increases in freezing behavior and the equivalent progressive decreases in exploratory loco motion in the intermittent stress regimen indicate that the repeated ly stressed groups underwent comparable stress-induced behavioral changes. However, the escala tion of freezing behavior and the decline of

PAGE 41

32 exploratory locomotion occurred more gradua lly when the social defeat sessions were 72 hours apart than when the social defeat sess ions occurred daily. This suggests that the intermittent stress regimen was not as effici ent at inducing these behavioral changes, although the overall magnitudes of the beha vioral effects were eventually roughly equivalent. Once again, the intruders' behavioral adapta tions did not decrease the displays of agonistic behavior from the re sidents (consistent nu mber of defeats and equivalent latencies to first defeat across groups and days). The equivalence in basal CORT concentr ations as well as the equivalence in glandular masses between the chronically st ressed animals and the control animals indicates that the temporally spaced regime n was not potent enough to sufficiently alter hormonal or glandular basal states. Even though the more temporally spaced chronic stress regimen was able to pr oduce equivalent effects with re gards to behavioral changes (albeit, more slowly), it was less effec tive than the daily regimen in producing the hormonal and glandular changes that correlate with some of the symptoms of major depression. The results indicate, therefore, that there may be a critical window for vulnerability to an additional stressorit is possible that the rats were able to partially recover from the initia l stressor by the next exposure in the more temporally spaced chronic regimen. Relevance to Previous Work Overall, these results confirm and extend a previous report that daily social defeat is an effective emotional stressor for male rats. In a study by Haller and colleagues (1999), intruder rats were exposed to reside nt rats for 4 hours on four consecutive days, producing increased basal CORT concentrations in the intruders. The daily social defeat stress regimen used in our study effectivel y increased basal CORT concentrations as

PAGE 42

33 well, with briefer stressful interactions than previously reported. However, in the same study, Haller and colleagues found increased ad renal mass with no decrease in thymus mass, results opposite to those seen after our similar daily chr onic stress regimen. Perhaps it is necessary for a daily chronic stre ss regimen to extend past four (or six) days to generate reliable glandular changes. The HPA axis response to an acute stre ssor largely depends upon whether the acute stressor is heterotypic or homotypic. In a study by Armario and colleagues (2004), investigators reported that when previously -stressed rats (via immobilization) were presented with a heterotypic stressor (for ced swimming), a minor sensitization was observed. Also, following exposure to a se vere stressor (such as shock, restraint, immobilization, or large doses of endotoxin) there was habituation of the HPA axis response to a homotypic stressor. These resu lts differ from the results obtained after daily social defeat stress. Fo llowing our repeated daily social defeat stress regimen, there was a robust activation but rapid shutdown of CORT release. Most probably, the difference is due to the fact that Armari o and colleagues utiliz ed a single stressor followed by either homotypic or heterotypic chal lenges. Our model of social defeat does not appear to function as a homotypic st ress regimen. There are several variables involved in the direct interacti on phase of social defeat stre ss (physical contact, olfactory cues, and ultra-sonic vocalizations) as well as the introduction of a di fferent resident rat with each exposure. Conceivably, the use of a different resident for each stress exposure and the potentially different threats imposed by these different reside nts creates a novel, therefore heterotypic, situation with each session.

PAGE 43

34 In general, the behavioral changes in the intruder rats after rep eated social defeat stress model some of the symptoms expresse d in clinically depre ssed patients, including impairment in social or occupational func tioning and loss of interest (DSM IV, 1994). Also, the elevated basal CORT concentrations after daily social de feat stress during the nadir of the daily cycle c onfirm previous findings from our laboratory (Lopes and Devine, 2004) and mimic the augmented ci rculating hormones found in clinically depressed patients (Linkowski et al., 1985; Pfohl et al., 1985). Porsolt Swim Test Based on the combined results from the daily and the intermittent stress regimens, a daily social defeat stress regimen was us ed to for the Porsolt study to optimize the potential to produce behavi oral despair. The fact that ac ute social defeat stress did not produce as much immobility as repeated st ress indicates that th e neuronal plasticity caused by repeated exposure to social stress (demonstrated through hormonal and behavioral plasticity) is a determining factor in the expression of behavioral despair. A 5-min swim test is commonly used for det ection of behavioral despair (Gavioli et al., 2003; Hinojosa et al., 2006; Porsolt et al ., 1978; Rygula et al., 2005). Interestingly, when analyzed in 5-min bins, the data suggest that in all the bins th e repeatedly stressed rats exhibited more immobility than the c ontrols did, and in one bin (5-10 min) the acutely defeated rats also exhibited more immo bility than the contro ls did. Therefore, a 10-min session may be necessary to closely examine the subtle differences between groups that have a less severe stress history and non-stre ssed control rats. Overall, these results model the behavior al despair, or low mood and anhedonia, found in clinically depresse d patients (for review, s ee Harrison, 2002). Also, the significantly elevated immobility times for the repeatedly stressed rats confirm and

PAGE 44

35 extend the results from a previous report. In a study by Rygula a nd colleagues (2005), investigators used a more extensive stress re gimen of hour-long daily sessions of social defeat for five straight weeks to elicit be havioral despair in s ubsequent Porsolt swim testing. The daily social def eat stress regimen used in our study effectively increased immobility in the repeatedly stressed rats as we ll, with briefer stressful interactions and a shorter regimen than previously reported. Conclusions and Future Directions In conclusion, the behavioral, hormona l, and glandular changes produced by repeated social defeat closely resemble many of the psychopathol ogical symptoms in patients with major depression. The daily so cial defeat stress regimen provides an interesting and effec tive model for stressinduced psychopathology. The effect of repeated social defeat on the overall circadian rhythm was not evaluated, nor was the persistence of the altered regulation across days, weeks, or months. Both of these topics may be interes ting issues for future studies. Also, potential adaptations in stress-related molecules (CRH and AVP in the hypothalamus, CRH1 and V1b in the pituitary and amygdala, and minera locorticoid receptor and gluccocorticoid receptor in the hippocampus) after social def eat should be investigated. These studies will enhance our growing knowledge of the neurobiological basis for stress-induced psychopathology (e.g., major depression).

PAGE 45

36 LIST OF REFERENCES Agid O, Kohn Y, Lerer B (2000). Environm ental stress and psychiatric illness. Biomedicine and Pharmacotherapy, 54: 135-141. Amiel-Tison C, Cabrol D, Denver R, Jarreau P, Papiernik E, Piazza PV (2004). Fetal adaptation to stress part II. Evolutio nary aspects; stress-induced hippocampal damage; long-term effects on behavior; consequences on adult health. Early Human Development, 78: 81-94. Anisman H, Matheson K (2005). Stress, depr ession, and anhedonia: Caveats concerning animal models. Neuroscience and Biobehavioral Reviews 29: 525-546. Armario A, Valles A, Dal-Zotto S, Marqu ez C, Belda X (2004). A single exposure to severe stressors causes long-term desensit ization of the physio logical response to the homotypic stressor. Stress 7 (3): 157-172. Blanchard RJ, Nikulina JN, Sakai RR, McKittri ck C, McEwen B, Blanchard DC (1998). Behavioral and endocrine change fo llowing chronic predatory stress. Physiology and Behavior 63 (4): 561-569. Bryant HU, Bernton EW, Kenner JR, Holaday JW (1991). Role of adrenal cortical activation in the immunos uppressive effects of chr onic morphine treatment. Endocrinology, 128 (6): 3253-3258. Chang CL, Hsu SYT (2004). Ancient evolu tion of stress-regulating peptides in vertebrates. Peptides 25: 1681-1688. De Kloet ER (2003). Hormones, brain and stress. Endocrine Regulations, 37: 51-68. Diagnostic and statistical manua l of mental disorders, 4th ed. (1994). Washington, DC: American Psychiatry Association. Gavioli EC, Marzola G, Guerri ni R, Bertorelli R, Zucchin i S, De Lima TCM, Rae GA, Salvadori S, Regoli D, Calo G (2003). Blockade of nocicep tin/orphanin FQ-NOP receptor signaling produces antidepressant -like effects: Pharmacological and genetic evidences from the m ouse forced swimming test. European Journal of Neuroscience 17: 1987-1990. Haller J, Fuchs E, Halasz J, Makara GB (1999). Defeat is a major stressor in males while social instability is mainly stressful for females: Towards the development of a social stress model in female rats. Brain Research Bulletin, 50 (1): 33-39.

PAGE 46

37 Harrison PJ (2002). The neuropath ology of primary mood disorder. Brain, 125: 14281449. Herman JP, Cullinan WE (1997). Neurocircuit ry of stress: Central control of the hypothalamo-pituitary-adrenocortical axis. Trends in Neuroscience 20 (2): 78-84. Hinojosa FR, Spricigo L, Izidio G, Bruske GR, Lopes DM, Ramos A (2006). Evaluation of two genetic animal models in behavi oral tests of anxiet y and depression. Behavioural Brain Research, 168: 127-136. Johnson SA, Fournier NM, Kalynchuk LE ( 2006). Effect of different doses of corticosterone on depressive-like behavi or and HPA axis responses to a novel stressor. Behavioural Brain Research 168: 280-288. Kabbaj M, Devine DP, Savage VR, Akil H (2000). Neurobiological correlates of individual differences in novelty-seeking behavior in the rat: Differential expression of stress-related molecules. The Journal of Neuroscience 20 (18): 6983-6988. Kessler RC, Berglund P, Demler O, Jin R, Ko retz Z, Merikangas KR, Rush AJ, Walters EE, Wang PS (2003). The epidemiology of major depressive disorder. Journal of the American Medical Association 289 (23): 3095-3105. Kollack-Walker S, Watson SJ, Akil H (1997). Soci al stress in hamsters: Defeat activates specific neurocircuits within the brain. The Journal of Neuroscience 17 (22): 8842-8855. Linkowski P, Mendlewicz J, Leclercq R, Brasseur M, Hubain P, Golstein J, Copinschi G, Van Cauter E (1985). The 24hour profile of adrenocorti cotropin and cortisol in major depressive illness. Journal of Clinical E ndocrinology and Metabolism 61 (3): 429-438. Lopes KO, Devine DP (2004). Elevation of HPA axis activity in response to repeated social defeat stress in rats. Society for Neuroscience Abstracts 30: 426.12. Lucki I, Dalvi A, Mayorga AJ (2001). Sens itivity to the effect s of pharmacologically selective antidepressants in different strains of mice. Psychopharmacology 155: 315-322. Martinez M, Phillips PJ, Herbert J (1998). Adaptation in patterns of c-fos expression in the brain associated with exposure to either single or repeated social stress in male rats. European Journal of Neuroscience 10: 20-33. McEwen BS, Stellar E (1993). Stress and the individual. Archives of Internal Medicine 153: 2093-2101.

PAGE 47

38 Miczek KA (1979). A new test for aggression in rats without aver sive stimulation: Differential effects of d -amphetamine and cocaine. Psychopharmacology 60: 253-259. Molina VA, Heyser CJ, Spear LP (1994). Chronic variable stress enhances the stimulatory action of a low dose of mo rphine: Reversal by desipramine. European Journal of Pharmacology 260: 57-64. Pfohl B, Sherman B, Schlechte J, Stone R (1985). Pituitary-ad renal axis rhythm disturbances in psychiatric depression. Archives of General Psychiatry 42: 897903. Porsolt RD, Anton G, Blavet N, Jalfre M ( 1978). Behavioural desp air in rats: A new model sensitive to antidepressant treatments. European Journal of Pharmacology 47: 379-391. Raadsheer FC, Hoogendijk WJG, Stam FC, T ilders FJH, Swaab DF (1994). Increased numbers of corticotrophin-releasing hormone expressing neurons in the hypothalamic paraventricular nucl eus of depressed patients. Clinical Neuroendocrinology 60: 436-444. Rygula R, Abumaria N, Flugge G, Fuchs E, Ruther E, Havemann-Reinecke U (2005). Anhedonia and motivational defi cits in rats: Impact of chronic social stress. Behavioural Brain Research, 162: 127-134. Sabban EL, Kvetnansky R (2001). Stress-tri ggered activation of gene expression in catecholaminergic systems: Dynamic s of transcriptional events. TRENDS in Neurosciences 24 (2): 91-98. Sgoifo A, De Boer SF, Haller J, Koolhaas JM (1996). Individual differences in plasma catecholamine and corticosterone stress res ponses of wild-type rats: Relationship with aggression. Physiology and Behavior, 60 (6): 1403-1407. Simpkiss JL, Devine DP (2003). Responses of th e HPA axis after chronic variable stress: Effects of novel and familiar stressors. Neuroendocrinology Letters Nos. 1/2 24: 97-103. Szigethy E, Conwell Y, Forbes NT, Cox C, and Caine ED (1994). Adrenal weight and morphology in victims of completed suicide. Biological Psychiatry, 36 (6): 374380. Whitnall MH (1993). Regulation of the hypot halamic corticotrophin-releasing hormone neurosecretory system. Progress in Neurobiology 40: 573-629.

PAGE 48

39 BIOGRAPHICAL SKETCH Kristen L. Stone graduated in May 2004 fr om the University of Central Florida (Orlando) with her Bachelor of Science degree in psychol ogy. She began her graduate education in the Psychology Department at th e University of Florida (Gainesville) in August 2004, working toward her Master of Science degree, in the Behavioral Neuroscience program.


Permanent Link: http://ufdc.ufl.edu/UFE0017942/00001

Material Information

Title: Rats Exhibit Behavioral Despair and Hormonal Alterations after Social Defeat Stress: Implications for Major Depression
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0017942:00001

Permanent Link: http://ufdc.ufl.edu/UFE0017942/00001

Material Information

Title: Rats Exhibit Behavioral Despair and Hormonal Alterations after Social Defeat Stress: Implications for Major Depression
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0017942:00001


This item has the following downloads:


Full Text











RATS EXHIBIT BEHAVIORAL DESPAIR AND HORMONAL ALTERATIONS
AFTER SOCIAL DEFEAT STRESS: IMPLICATIONS FOR MAJOR DEPRESSION
















By

KRISTEN L. STONE


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

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Kristen L. Stone





























To my loving husband (Justin),
my wonderful parents (Linda, Kenneth, and Donna),
and my brothers and sisters (Kim, Rick, Minet, Roger, and Jay).
Thank you for your uninterrupted love and support.

Also to my extended family (both biological and in-law) and my friends.
Thank you for your continued encouragement and guidance.
















ACKNOWLEDGMENTS

I would like to thank my supervisory committee members (Dr. Mohamed Kabbaj

from Florida State University; Dr. Neil Rowland; and Dr. Linda Hermer-Vazquez) for

their time and patience. I would like to extend special gratitude to my advisor (Dr.

Darragh Devine) for his persistent guidance; and to Andrea Naccarato for her diligent

work with the Porsolt swim tests. I would also like to thank my mentors in Gainesville

(especially Clare Mathes) for their support during this long j ourney of higher education.

Lastly, and most importantly, I would like to thank God for the breadth of knowledge He

has bestowed upon me.




















TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. .................... iv


LI ST OF T ABLE S ............ ...... .___ ............... vii...


LIST OF FIGURES ........._.._.._ ....__. ..............viii...


AB STRAC T ................ .............. ix


CHAPTER


1 INTRODUCTION ................. ...............1.......... ......


2 IVETHODS .............. ...............7.....


Animal s............... ...............7.

Surgical Procedures .............. ...............8.....
Experimental Procedures .................. ........... ...............8.......
General Social Defeat Stress Procedure ................. ............. ......... .......8
Social Defeat Stress Regimen: Experiment la............... ...............9...
Social Defeat Stress Regimen: Experiment lb............... .. ...............10
Social Defeat Stress and Porsolt Swim Test: Experiment 2 ............... ... ............11
Behavioral Assays .............. ...............12....
Histological As says .............. ...............12....
Statistical Analyses ................. ...............13.................

3 RE SULT S ................. ...............17.......... .....


Experiment la: Six Daily Sessions of Social Defeat Stress .............. ....................17
Experiment lb: Twelve Intermittent Sessions of Social Defeat Stress .....................23
Experiment 2: Social Defeat Stress Followed by Porsolt Swim Testing...................27
Inter-Ob server Reliability ................. ...............27................

4 DI SCUS SSION ................. ...............29................


Daily Social Defeat Stress .............. ...............29....
Intermittent Social Defeat Stress .............. ...............30....
Relevance to Previous Work .............. ...............3 1....













Porsolt Swim Test............... .... ...............33
Conclusions and Future Directions............... ...............3


LIST OF REFERENCES ............. ...... .__ ...............35..


BIOGRAPHICAL SKETCH .............. ...............38....


















LIST OF TABLES

Table pg

2-1 Experiment la, group assignments............... ..............1

2-2 Experiment lb, group assignments .............. ...............16....

2-3 Experiment 2, group assignments .............. ...............16....


















LIST OF FIGURES


Figure pg

3-1 Social defeats per daily session.. ............. ...............19.....

3-2 Intruder behavior during daily social defeat sessions. ............. .....................2

3-3 Circulating hormones after daily social defeat stress ................. ........__. ........21

3-4 Effects of daily social defeat stress on glandular masses. .............. ...................22

3-5 Social defeats per intermittent session. ............. ...............24.....

3-6 Intruder behaviors during intermittent social defeat sessions. ............. .................25

3-7 Circulating hormones after intermittent social defeat stress. ............. ..................26

3-8 Effects of intermittent social defeat stress on glandular masses. ............. ...... ..........26

3-9 Immobility during the Porsolt swim test. .............. ...............28....

3-10 Immobility measured in 5-minute bins during the Porsolt swim test. .....................28
















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

RATS EXHIBIT BEHAVIORAL DESPAIR AND HORMONAL ALTERATIONS
AFTER SOCIAL DEFEAT STRESS: IMPLICATIONS FOR MAJOR DEPRESSION

By

Kristen L. Stone

December 2006

Chair: Darragh P. Devine
Major Department: Psychology

Chronic emotional stress plays a pivotal role in the origin of many psychiatric

disorders, including major depression. Accordingly, we investigated the behavioral,

hormonal, and glandular effects of both repeated and acute emotional stress using the

social defeat model in rats. We compared one week of daily social defeat exposure to

one month of intermittent exposure to examine the effectiveness of massed versus

intermittent stressors.

In two experiments, naive male intruder rats were each exposed to dominant male

resident rats. Each resident and intruder pair was allowed to interact for 5 minutes or

until the intruder exhibited submissive supine posture three times. The intruder was then

briefly removed, placed into a protective wire mesh cage, and returned to the resident' s

cage until a total of 10 minutes elapsed from initial entry. Additional control rats were

not exposed to social defeat stress. For the first experiment, the intruder rats were killed

10 minutes, 30 minutes, or 24 hours after the last social defeat session and plasma









corticosterone and adrenocorticotropic hormone concentrations were assayed. For the

second experiment, the previously-stressed rats were tested with a 15-minute Porsolt

forced swim test.

The intruder rats exhibited more freezing behavior and less exploratory locomotion

across consecutive social defeat sessions, resembling the loss of interest found in

depressed patients. Exposure to social defeat stress produced significant elevations in

circulating hormones 10 minutes and 30 minutes after the session, when compared with

concentrations in the control rats. The repeatedly stressed rats also exhibited higher basal

concentrations of circulating corticosterone 24 hours later, mimicking the augmented

circulating hormones found in clinically depressed patients. These results were evident

after six daily exposures; however, basal hormone concentrations were not significantly

elevated with the extended regimen of one social defeat session every 72 hours.

Inconsistent thymus involution in the chronically stressed rats in both regimens suggests

that a longer, more intense daily stress regimen may be necessary to alter glandular

masses. Exposure to chronic social defeat stress also produced a significant increase in

total immobility time during the forced swim test when compared with the immobility

times for the rats that were exposed to a single acute social defeat session and with the

immobility times for the rats that were not exposed to social defeat stress, thus

representing behavioral despair in the chronically stressed animals. Overall, the

behavioral, hormonal, and glandular alterations that occurred after repeated social defeat

stress resemble some of the symptoms of maj or depression in humans.















CHAPTER 1
INTTRODUCTION

Annually, approximately 6.6% of the national adult population suffers from

depression (Kessler et al., 2003). It is a wide-spread illness that interferes with the ability

to eat, sleep, work, and enj oy formerly pleasurable activities. The economic impact of

this devastating disorder is high, but the cost in human distress cannot be estimated.

The 4th Edition of the Diagnostic and Statistical Manual of Mental Disorders

(1994) defines depression according to the following criteria, with at least Hyve of the

symptoms present on a daily basis for at least 2 weeks: depressed mood, loss of interest

or pleasure, significant weight loss or gain, insomnia or hypersomnia, psychomotor

agitation or retardation, fatigue, undue guilt and/or feelings of worthlessness,

indecisiveness, recurrent thoughts of death, and significant distress or impairment in

social or occupational functioning. These symptoms must represent a change from the

individual' normal level of interpersonal function.

Maj or depression is also clinically characterized by altered hormonal function

stemming from ongoing elevations in overall organism distress. It has been well

established that chronic emotional stress plays a pivotal role in the genesis of many

psychiatric disorders such as depression (for review, see Agid et al., 2000). Chronic

stress weighs on the physiological systems that maintain homeostasis and produces

changes in the operating limits of those hormonal systems. Allostatic load, or the strain

from the elevated activity of systems under maj or stress, can predispose an animal to

many psychiatric disorders, including depression (McEwen and Stellar, 1993). The









concept of allostatic load suggests that there is a fixed state in which enduring

environmental challenges are balanced by a hormonal response that is raised above

normal, basal levels. Patients with major depression, regardless of age, show higher

24-hour average cortisol levels when compared to normal subj ects (Linkowski et al.,

1985). Additionally, depressed patients reach the nadir of their daily cortisol cycle two to

three hours before control subj ects (Pfohl et al., 1985). This imbalance between

activation and recovery of the stress response is implicated in the inability to maintain

homeostasis, thus leading to neuroendocrine maladjustment and heightened risk for

depression (De Kloet, 2003).

Stressful stimuli are categorized into two descriptive classes. Systemic stressors,

such as exposure to heat or cold, present immediate threats to somatic homeostasis while

processive stressors emphasize higher level cognitive processing (Herman and Cullinan,

1997). Common processive stressors include instability in the social hierarchy and loss

of environmental control. In accordance with the emotional nature of processive stress, it

is particularly implicated in a variety of psychiatric disorders including depression (for

review, see Anisman and Matheson, 2005).

Inputs from the brainstem (if the stressor is systemic in nature) and cortical and

limbic structures (if the stressor is processive in nature) converge at the paraventricular

nucleus (PVN) of the hypothalamus where parvocellular neurons proj ect to the median

eminence. From there, corticotrophin-releasing hormone (CRH) and arginine

vasopressin (AVP) are released into the hypophyseal portal circulation, stimulating

adrenocorticotropic hormone (ACTH) release from the anterior pituitary corticotrope

cells into the general circulation. The release of ACTH then stimulates the synthesis and









release of glucocorticoids from the adrenal cortex. Cortisol is the main glucocorticoid in

humans and corticosterone (CORT) is the main glucocorticoid in rats. The elevated

circulating levels of glucocorticoids decrease the further activity of the HPA axis through

exertion of negative feedback on neurosecretory cells of the hypothalamus and

corticotrope cells of the pituitary (for review, see Whitnall, 1993).

Altered regulation of hormonal activity in depressed patients is thought to be a

result of increased activity of specific CRH-containing neurons in the PVN. The average

total number of such neurons is up to four times higher in depressed patients when

compared to normal control subjects. Also, co-localization of AVP in CRH-expressing

neurons has been indicated as an index for stress-activated neuronal activity. The

average number of neurons co-expressing both CRH and AVP in depressed patients is up

to three times higher than those for normal subj ects. These results suggest that increased

expression of CRH- and AVP-containing neurons in the PVN may cause at least a

fraction of the collective symptomatology of depression (Raadsheer et al., 1994).

When physically or emotionally stressed, non-human animals endure physiological

responses that lead to behavioral and hormonal impairment which may be fundamentally

similar to the impairment seen in human stress-induced psychopathology. Behavioral

responses to stressful stimuli (including increased drug-taking propensity, decreased

performance in learning tasks, sleep disturbances, and unsocial behavior) have been

observed in a variety of species (for review, see Amiel-Tison et al., 2004). Also, there is

significant evolutionary homology in stress-regulating peptides, such as CRH and CRH-

related molecules (Chang and Hsu, 2004). Thus, the use of an animal model for










processive stress that produces both behavioral and hormonal effects is a logical approach

to the study of human stress-induced disorders.

The effects of chronic and acute processive stress have been studied in rats

(Simpkiss and Devine, 2003). Experimentally naive rats were exposed to a chronic

variable stress (CVS) regimen of twice daily stressors for fifteen days. The stressors

included novel environment, switched cage mates, forced swim, light open Hield,

intermittent white noise, and intermittent footshock, administered on a random

intermittent schedule. The CVS regimen was unsuccessful in producing elevations in

basal circulating concentrations of ACTH and CORT, or in hormonal response to an

acute stressor. The rats showed a blunted ACTH response, but no altered CORT

response.

Another model for emotional stress, social defeat, has also produced significant

elevations in circulating CORT during and after acute and repeated exposure to the

stressful stimulus (Sgoifo et al., 1996). The procedure, developed by Miczek (1979), is

designed to model social stress. A male "intruder" rat is exposed to social stress when it

is placed into the home cage of a larger male "resident" rat. The resident rat exhibits

dominant behavior toward the intruder rat by displaying assertive posture, standing over

the intruder. The intruder submits by displaying supine posture, positioned beneath the

resident.

The further effects of repeated social defeat stress on behavioral and hormonal

(HPA axis) responses have been studied in rats (Lopes and Devine, 2004). In a

preliminary study, a repeatedly stressed group of intruder rats showed significantly

elevated circulating CORT concentrations 24 hours after their Einal social defeat session,









when compared to the concentrations in the unstressed control animals. This suggests an

enduring change in the circadian regulation of the HPA axis following chronic social

defeat. Overall, the effects of six days of social defeat stress exceeded the results attained

with the fifteen days of CVS previously described.

The brain sites activated by social defeat have been studied using c-fos

immunohi stochemi stry. The immediate-early gene (IEG) c-fos is expressed in many cells

in the brain, but typically at very low basal levels. C-fos and other IEGs are intracellular

signaling mechanisms that regulate gene transcription and expression of various

neuropeptides and trophic molecules in response to stress (for review, see Sabban and

Kvetnansky, 2001). Various stressful stimuli can initiate increased levels of c-fos mRNA

expression, lasting for minutes to hours. For instance, social defeat produces elevated

c-fos expression in limbic, limbic-associated, and brainstem sites in both hamsters and

rats one hour after a single defeat session (Kollack-Walker et al., 1997; Martinez et al.,

1998). These results point to the brain structures that are important in the processing of

emotionally stressful events. However, after repeated social defeat sessions in rats, the

pattern of neuronal activity was modified, despite the fact that intruder submissive

behavior persisted across trials. C-fos mRNA expression endured for many of these

limbic and brainstem nuclei, while other limbic and brainstem regions exhibited a

decrease in the social defeat-induced c-fos mRNA expression.

The Porsolt swim test, originally described by Porsolt and colleagues (1978), is the

most commonly utilized behavioral test for screening antidepressant treatments in rats

and has been used to evaluate the behavioral effects of stress exposure. Immobility

during the inescapable swim is measured as an indicator of behavioral despair or










depressive-like behavior. Regimens of chronic stress or repeated administration of

gluccocorticoids increase the amount of time an animal spends immobile, decrease the

latency to immobility, and decrease the amount of time engaged in active swimming

(Molina et al., 1994; Johnson et al., 2006). Antidepressant drugs, on the other hand,

ameliorate these effects. For example, desipramine (a tricyclic antidepressant) and

fluoxetine (a selective serotonin reuptake inhibitor) reduce immobility and increase the

amount of time a stressed animal will struggle to escape the forced swim (Molina et al.,

1994; Lucki et al., 2001).

Based on the results of investigations using social defeat stress and the Porsolt

swim test, we have begun to further characterize the social defeat model of emotional

stress in rats. We have evaluated the impact of both acute and repeated social defeat

stress on regulation of ACTH and CORT at various times after the stressor. We have

also compared six daily social defeat sessions to one month of stress every third day to

examine the effectiveness of massed and intermittent stress exposure. In addition, we

exposed socially defeated rats to the Porsolt swim test in order to characterize the

enduring behavioral effects of social defeat stress.















CHAPTER 2
IVETHOD S

Animals

Ninety male Long-Evans (LE) rats and twelve female LE rats were purchased from

Harlan Co. of Indianapolis, INT. Twenty of the male Long Evans (LE) rats (225-250 g)

were used as unhandled controls. Fifty-eight of the male LE rats (225-250 g) were used

as intruders. Twelve of the male LE rats (300-325 g) were used as residents and the

twelve female LE rats (225-250 g) were used as housing mates for the residents. The

weight ranges indicate weights at the time the rats were purchased. The intruders

weighed 275-325 g and the residents weighed 500-800 g at the time of the experiments.

Six of the unhandled control rats were used in Experiment la, six were used in

Experiment lb, and eight were used in Experiment 2. Thirty of the intruder rats were

used in Experiment la, twelve were used in Experiment lb, and sixteen were used in

Experiment 2. The resident males and the females were used in multiple experiments.

The rats were housed in polycarbonate cages (43 x 21.5 x 25.5 cm) with sex-

matched and weight-matched pairings for five to seven days of acclimation to a

12hr/12hr light/dark cycle (lights on at 7:00a.m.). Standard chow (LabDiet 5001) and tap

water were available ad libitum. Temperature and humidity in the housing facility were

controlled to 22.720C & 0.940C and 54.1% + 14.6%, respectively.

The intruder rats were exposed to social defeat stress and remained pair-housed

throughout the experiment. The control rats were not exposed to social defeat stress and

were pair-housed throughout the experiment. The resident rats were vasectomized,










singly-housed, and given 10 days to recover from the surgery. The residents were then

pair-housed in a separate housing room, each with a female, for 2 weeks prior to and then

throughout the experiment. All procedures were pre-approved by the Institutional

Animal Care and Use Committee at the University of Florida and conducted in

accordance with the Guide for the Care and Use of Laboratory Animals.

Surgical Procedures

After a week of acclimation to the housing facility, the resident rats were

anaesthetized with ketamine:xylazine (50 mg/kg:5 mg/kg, i.p.). Sedation was verified by

testing the pedal withdrawal reflex 10 min after injection and every 10 min during the

procedure. After sedation was verified, each rat was shaved from the rostral edge of the

scrotum to the caudal abdomen. The area was then washed with Betadine three times.

Surgery began with a 2 cm ventral midline incision, just above the scrotal area.

Through the incision, the vas deferens was located and externalized. A 0.25 cm section

of each duct was removed with a micro-cautery knife. The muscle walls were sutured

and the external incision was closed using sterilized staples that were removed ten days

after the surgery. Each surgical procedure lasted 15-30 min.

Each rat was given a 1 mL inj section of 0.9% warm saline and ketorolac

subcutaneously and then placed into a post-operative Plexiglas cage, heated by an electric

heating pad. Following observation of locomotion and urination, each rat was returned to

its individual home cage and was monitored daily throughout the recovery period.

Experimental Procedures

General Social Defeat Stress Procedure

At the beginning of the 10-min social defeat session, the female rat paired with one

resident rat was taken out of the cage and placed into an empty, identical cage. The









intruder rat was then placed into the cage of the resident (direct interaction). The session

was then closely monitored for submissive, supine posture expressed by the intruder.

Each time the intruder exhibited a supine posture with the resident physically contacting

the intruder for 2 sec or more, one defeat was counted. After three defeats or a total of

5 min elapsed (whichever came first), the intruder was taken out of the resident' s cage

momentarily and quickly placed into a 10 x 10 x 15 cm double-walled protective wire

mesh cage. The intruder (within the wire mesh cage) was then placed back into the

resident' s cage for the remainder of the 10-min session (indirect interaction). The two

intruder rats in each housing pair were run in simultaneous defeat sessions in the cages of

two resident males that were placed side by side. Each pair of cage-mates received the

same treatment. Each social defeat session was run between 8:00a.m. and 10:00a.m., and

was videotaped for further analysis.

Social Defeat Stress Regimen: Experiment la

Thirty-six naive male LE rats were assigned to six experimental groups (Table 2-1).

The control rats from Group 1 (n = 6) remained in their original cages throughout the

experiment and were rapidly decapitated between 8:00a.m. and 10:00a.m. on the final

experimental day. These rats were exposed to no chronic and no acute stress (NC/NA).

The intruders from Group 2 (n = 6) were exposed to social defeat stress once every 24 h

across six experimental days and were killed by rapid decapitation between 8:00a.m. and

10:00a.m., 24 h after their final social defeat session. These rats were exposed to chronic,

but not acute stress (C/NA). The intruders from Group 3 (n = 6) were only exposed to

social defeat stress on the final experimental day and were then immediately decapitated,

10 min after the start of the social defeat session (t = 10 min). These rats were exposed to

no chronic, just acute stress (NC/A-10). The intruders from Group 4 (n = 6) were only










exposed to social defeat stress on the final experimental day and were rapidly decapitated

30 min after the start of the social defeat session (t = 30 min). These rats were exposed to

no chronic, just acute stress (NC/A-30). The intruders from Group 5 (n = 6) were

exposed to social defeat stress once every 24 h over the course of six experimental days

and were killed by rapid decapitation 10 min after the start of the social defeat session on

the sixth day (t = 10 min). These rats were exposed to chronic and acute stress (C/A-10).

The intruders from Group 6 (n = 6) were exposed to social defeat stress once every 24 h

over the course of six experimental days and were killed by rapid decapitation 30 min

after the start of the social defeat session on the sixth day (t = 30 min). These rats were

exposed to chronic and acute stress (C/A-30). Each intruder from the repeatedly stressed

groups was placed in the cage of a different resident each day of his chronic stress

routine.

Social Defeat Stress Regimen: Experiment lb

Eighteen naive male LE rats were assigned to three experimental groups (Table

2-2). The unhandled control rats from Group 1 (n = 6) remained in their original cages

throughout the experiment and were killed by rapid decapitation between 8:00a.m. and

10:00a.m. on the final experimental day. These rats were exposed to no chronic and no

acute stress (NC/NA). The intruders from Group 2 (n = 6) were exposed to social defeat

stress once every 72 h across thirty-four experimental days and were rapidly decapitated

between 8:00a.m. and 10:00a.m., 24 h after their final social defeat session. These rats

were exposed to chronic, but not acute stress (C/NA). The intruders from Group 3 (n = 6)

were exposed to social defeat stress once every 72 h across thirty-four experimental

days and were killed by rapid decapitation 30 min after the start of the social defeat

session on the final day (t = 30 min). These rats were exposed to chronic and acute stress










(C/A). All intruder rats from Experiment lb were exposed to a total of twelve social

defeat sessions. Each intruder from the repeatedly stressed groups was placed in the cage

of a different resident each day of his chronic stress routine.

Social Defeat Stress and Porsolt Swim Test: Experiment 2

Twenty-four naive male LE rats were assigned to three experimental groups (Table

2-3). The unhandled control rats from Group 1 (n = 8) remained in their original cages

throughout the social defeat portion of the experiment. These rats were exposed to no

chronic and no acute social defeat stress (NC/NA). The intruders from Group 2 (n = 8)

were only exposed to social defeat stress on the final day of the social defeat portion of

the experiment. These rats were exposed to no chronic, just acute stress (NC/A). The

intruders from Group 3 (n = 8) were repeatedly exposed to social defeat stress over the

course of five days, once every 24 h. These rats were exposed to chronic and acute stress

(C/A). Each repeatedly stressed intruder was placed in the cage of a different resident

each day of his chronic stress routine. All of the rats from each of the three groups were

then exposed to the Porsolt swim test.

Twenty-four hours after the final social defeat session, each rat from Experiment

2 was individually removed from its home cage and placed into a plastic cylinder filled

with approximately 25 cm of clean tap water at 24-270C. The depth of water allowed

each rat to reach the bottom of the cylinder with its tail, with enough head room that the

rat was unable to escape from the tank. The water in each cylinder was changed between

trials. Each rat was subjected, one at a time, to a 15-min swim session then carefully

dried and returned to its home cage. Each Porsolt swim session was run between

8:00a.m. and 10:00a.m., and was videotaped for further analysis.









Behavioral Assays

Number of defeats, latency to first defeat, freezing behavior, and exploratory

locomotion were scored from the recorded social defeat sessions. Freezing behavior was

recorded whenever the intruder remained motionless for at least 2 sec, and is reported as

percent time during the direct interaction between the intruder and the resident.

Exploratory locomotion was recorded every time the intruder crossed with all four paws

from one third of the cage into the adj acent third of the cage. Vertical lines were drawn

on the video image of each resident' s home cage and exploratory locomotion was scored

from the videotapes. Exploratory locomotion is reported as lines crossed per minute of

direct interaction time. Two trained observers scored the social defeat stress videos from

Experiments la and lb and inter-observer reliability was assessed.

Immobility was recorded during the Porsolt swim tests whenever the rat balanced

on its tail, completely motionless, or exhibited only slight forepaw movement for a

minimum of 2 sec. This behavior is reported as total time spent immobile during the

swim test. Two trained observers scored the swim test videos from Experiment 2 and

inter-observer reliability was assessed.

Histological Assays

Immediately after decapitation in experiments la and lb, 6 mL of trunk blood from

each rat was collected in polypropylene tubes on ice with 600 Cl1 of Na2EDTA at

20 Cpg/CLL. The blood samples were then immediately centrifuged at 1000 ref for 5 min

and the plasma fraction frozen in 300 Cl1 aliquots at -800C. The brain from each intruder

was quickly removed, frozen in 2-methylbutane at -400C, and stored at -800C for future

analysis of molecular variables involved in HPA axis function. The thymus and adrenal

glands were removed from each rat and frozen separately at -800C. These glands were









later weighed to determine the health and stress status of each rat at time of death.

Plasma ACTH and CORT concentrations from the rats were later analyzed with

radioimmunoassay (RIA). ACTH RIAs were run with kits from Alpco Diagnostics

(Salem, NH) and CORT RIAs were run with kits from Diagnostic Products Corporation

(Los Angeles, CA).

Statistical Analyses

Potential between-groups differences in number of defeats and latency to first

defeat were analyzed using two one-way analyses of variance (ANOVAs) to compare the

five groups of intruders (i.e. three repeatedly stressed, and two just acutely stressed) in

Experiment la during their first exposure to social defeat stress. A 3x6 (group x session)

repeated-measures ANOVA (for Experiment la) and a 2xl2 (group x session) repeated-

measures ANOVA (for Experiment lb) were used to examine potential differences in

number of defeats between groups and across experimental sessions for the repeatedly

stressed groups. A 3x6 (group x session) repeated-measures ANOVA (for Experiment

la) and a 2xl2 (group x session) repeated-measures ANOVA (for Experiment lb) were

used to examine potential differences in latency to first defeat between groups and across

experimental sessions for the repeatedly stressed groups.

Potential between-groups differences in freezing responses were analyzed using a

one-way ANOVA to compare freezing behavior between the five groups in Experiment

la during their first exposure to social defeat stress. A 3x6 (group x session) repeated-

measures ANOVA (for Experiment la) and a 2xl2 (group x session) repeated-measures

ANOVA (for Experiment lb) were used to examine potential freezing behavior

differences between groups and across experimental sessions for the repeatedly stressed

groups.









Potential between-groups differences in exploratory locomotion were analyzed

using a one-way analysis of variance (ANOVA) to compare exploratory locomotion

between the five groups in Experiment la during their first exposure to social defeat

stress. A 3x6 (group x session) repeated-measures ANOVA (for Experiment la) and a

2xl2 (group x session) repeated-measures ANOVA (for Experiment lb) were used to

examine potential exploratory locomotion differences between groups and across

experimental sessions for the repeatedly stressed groups.

Potential between-groups differences in plasma ACTH concentrations and in

plasma CORT concentrations were analyzed with one-way ANOVAs in each of

Experiments la and lb. Also, potential between-groups differences in adrenal weights

and in thymus weights were analyzed with one-way ANOVAs in each of Experiments la

and lb. The results were further analyzed using pre-planned Newman-Keuls multiple

comparison tests for all significant ANOVAs.

Potential between-groups differences in immobility duration scores for the 15-min

Porsolt swim test session in Experiment 2 were analyzed using a one-way ANOVA. The

results were further analyzed using Newman-Keuls multiple comparison tests. The data

were then analyzed in 5-min bins using a 3x3 (group x bin) repeated-measures ANOVA.

The results were further analyzed using Newman-Keuls multiple comparison tests,

making pair-wise comparisons between each stressed group and the control group, for

each bin.

Inter-observer agreement was assessed for freezing behavior in Experiments la and

lb by comparing total times recorded by each trained observer for each social defeat

session. Inter-observer agreement was also assessed for exploratory locomotion in










Experiments la and lb by comparing total number of line crossings recorded by each

trained observer for each social defeat session. Inter-observer agreement was assessed

for immobility duration scores for the Porsolt swim test from Experiment 2 by comparing

total immobility times recorded by each trained observer for each test session.











Table 2-1. Experiment la, group assignments
Group Repeated Stress Acute Stress Kill Time
NC/NA
C/NA X t = 24 h
NC/A-10 X t =10 min
NC/A-30 X t = 30 min
C/A-10 X X t =10 min
C/A-30 X X t = 30 min

Table 2-2. Experiment lb, group assignments
Group Repeated Stress Acute Stress Kill Time
NC/NA
C/NA X t = 24 h
C/A X X t = 30 min

Table 2-3. Experiment 2, group assignments
Group Repeated Stress Acute Stress Porsolt Swim Test
NC/NA X
NC/A X X
C/A X X X















CHAPTER 3
RESULTS

Experiment la: Six Daily Sessions of Social Defeat Stress

During their first exposure to social defeat stress, the rats from all Eive of the

groups in Experiment la did not show statistically different numbers of defeats (F (4, 25)

= 0.2419, p < 0.9118; Fig. 3-1A) nor latencies to Birst defeat (F (4, 25) = 0.7308, p <

0.5795; Fig. 3-1B). Each of the three repeatedly stressed groups (C/NA, C/A-10, and

C/A-30), showed no significant between-groups difference, time effect, or group by time

interaction effect in number of defeats (F (2, 15) = 1.603, p < 0.2340; F (5, 15) = 0.3463,

p < 0.8831; F (10, 15) = 0.3139, p < 0.9753; Fig. 3-1A) nor latencies to first defeat across

the six experimental sessions (F (2, 15) = 1.571, p < 0.2401; F (5, 15) = 0.2106, p <

0.9570; F (10, 15) = 0.5255, p < 0.8669; Fig. 3-1B).

During their first exposure to social defeat stress, the rats from all Eive of the

groups did not show statistically different amounts of freezing behavior (F (4, 25) =

0.1757, p < 0.8405; Fig. 3-2A). Each of the three repeatedly stressed groups showed

significantly increased freezing behavior across the six experimental sessions (F (5, 75) =

17.08, p < 0.0001; Fig. 3-2A), reaching asymptote by day 3, with no significant between-

groups differences or group by time interaction effect.

During their first exposure to social defeat stress, the rats from all five groups did

not show statistically different amounts of exploratory locomotion (F (4, 25) = 0.5711i, p

< 0.5767; Fig. 3-2B). Each of the three repeatedly stressed groups showed significantly

decreased exploratory locomotion across the six experimental sessions (F (5, 75) = 11.76,









p < 0.0001; Fig. 3-2B), reaching asymptote by day 4, with no significant between-groups

differences or group by time interaction effect.

Exposure to acute social defeat (NC/A and C/A) produced significant elevations in

circulating ACTH concentrations when compared with the ACTH concentrations in the

control rats (NC/NA) (F (5, 30) = 3.874, p < 0.0079; Fig. 3-3A). Circulating ACTH

concentrations were not elevated in the chronically, but not acutely, stressed rats (C/NA).

Exposure to social defeat stress for all groups (including C/NA) produced significant

elevations in circulating CORT concentrations when compared with the CORT

concentrations in the control rats (F (5, 30) = 20.79, p < 0.0001; Fig. 3-3B).

Exposure to social defeat significantly decreased thymus masses in Groups C/A-10

and C/A-30, but not for the other groups (F (2, 15) = 5.651, p < 0.0148; Fig. 3-4A).

There were no significant differences in adrenal masses between the rats in the stressed

groups and the rats in the control group (F (5, 30) = 0.9299, p < 0.4756; Fig. 3-4B).













,3-


2- -5- C/NA
-9- NCIA-10
.0 -- NCIA-30
E 1
3- C/A-10
-A- C/A-30

1 2 3 4 5 6
Experimental Day




s 125




o 75
-5- C/NA
,, I +-/-- NCIA-10
9 I T-+-- NCIA-30
o"25-1 -6 C/A-10
I I-- C/A-30

1 2 3 4 5 6
Experimental Day


Figure 3-1. Social defeats per daily session. The rats exposed to repeated social defeat
stress (C/NA, C/A-10, C/A-30) exhibited equivalent (A) number of defeats
per session and (B) latency to first defeat per session across the 6
experimental sessions. The rats that received only one acute defeat session
(NCA-10 and NCA-30) were stressed at the same time as the final stress
session for the chronically stressed groups, and so the values for these groups
are illustrated on day 6. Values expressed are group means + SEM (n = 6 rats
per group). Abbreviations: C/NA = chronic stress/no acute stress, NC/A = no
chronic stress/acute stress, C/A = chronic stress/acute stress.












15' 1 0 0

90-
Ss-


S70-

a60-

50-

40-

S30-

20-

10-
.

0-


-5- C/NA
-V NC/A-10
-9 NC/A-30
-6 C/A-10
-A- C/A-30


Exp~erimental Day


56


-5- C/NA
-V NC/A-10
-9- NC/A-30
-6 C/A-10
-A- C/A-30


Exp~erimental Day


I I


Figure 3-2. Intruder behavior during daily social defeat sessions. The rats exposed to
repeated social defeat stress exhibited (A) increases in freezing behavior and
(B) decreases in exploratory locomotion across the 6 experimental sessions.
The rats that received only one acute defeat session were stressed at the same
time as the final stress session for the chronically stressed groups, and so the
values for these groups are illustrated on day 6. Values expressed are group
means + SEM (n = 6 rats per group).











n.s.
n.s. n.s.


A






o
1--


T


n.s.


Je

I


~ ~


n.s.
+ +


B






o


1--i

O


Je
I


~ ~


Figure 3-3. Circulating hormones after daily social defeat stress. The rats exposed to
social defeat stress had elevated circulating concentrations of (A) ACTH and
(B) CORT when compared to basal concentrations in control rats. Values
expressed are group means & SEM (n = 6 rats per group). Significant
differences between the socially defeated rats and the unhandled control rats
(NC/NA) are expressed as [*] p < 0.05. Significant differences in pre-planned
comparisons between the stressed groups of rats are expressed as [+] p < 0.05,
with lines connecting the groups that were compared. Additional
abbreviation: NC/NA = no chronic stress/no acute stress.






















300-`
200- c









1--~
100-O p'




Biur 60-4 fet di sca ee srs gadl mse Sca ee te

50- infcnl dcesdtyu ase o ftereetdysrse
40us(/-0adCA3) u B i ntsgiiatyafc dea ln







Figre -4.Efect gofp) daniyscial deffeatsress oewen glndla mcasss.Soia defeated ras dtres


control rats are expressed as [*] p < 0.05.









Experiment lb: Twelve Intermittent Sessions of Social Defeat Stress

There were no significant between-groups differences, time effect, or group by time

interaction effect in number of defeats (F (1, 10) = 0.0787, p < 0.7847; F (11, 10) =

0.9032, p < 0.5399; F (11, 10) = 0.8203, p < 0.6199; Fig. 3-5A) nor latency to first defeat

(F (1, 10) = 0.4653, p < 0.5107; F (11, 10) = 0.8400, p < 0.6007; F (11, 10) = 0.3414, p <

0.9743; Fig. 3-5B) for the repeatedly stressed groups (C/NA and C/A) in Experiment lb.

Freezing behavior significantly increased for both of the repeatedly stressed groups

across the twelve experimental sessions (F (11, 10) = 6.224, p < 0.0001; Fig. 3-6A), with

no significant between-groups differences or group by time interaction effect.

Exploratory locomotion significantly decreased for both of the repeatedly stressed

groups across the twelve experimental sessions (F (11, 10) = 7.252, p < 0.0001; Fig.

3-6B), with no significant between-groups differences or group by time interaction effect.

Acute exposure to social defeat stress produced significant elevations in circulating

ACTH concentrations when compared with the basal ACTH concentrations in the control

rats (F (2, 15) = 4.525, p < 0.0290; Fig. 3-7A). Acute exposure to social defeat stress also

produced significant elevations in circulating CORT concentrations when compared with

the basal CORT concentrations in the control rats (F (2, 15) = 5.853, p < 0.0132; Fig.

3-7B). These elevations in circulating hormones were limited to the rats that were

stressed acutely before termination (C/A) the basal concentrations in the other

chronically stressed rats (C/NA) did not significantly differ from the basal concentrations

in the control rats (NC/NA).

There were no significant differences in thymus masses (F (2, 15) = 0.04457, p <

0.9565; Fig. 3-8A) or adrenal masses (F (2, 15) = 1.826, p < 0. 1951; Fig. 3-8B) between

the rats in the socially defeated groups and the rats in the control group.
















3-

O 2-
O




-t- C/NA
-A- C/A


0 12 3 4


5 6 7
Exp~erimental Day


8 9 10 11 12


S100-





050-


S25-



1 2 3 4 5 6 7 8 9 10 11 12
Experimental Day


-t- C/NA
-A- C/A


Figure 3-5. Social defeats per intermittent session. The rats exposed to repeated social
defeat stress (C/NA and C/A) exhibited equivalent (A) number of defeats per
session and (B) latency to first defeat per session across the 12 experimental
sessions. Values expressed are group means + SEM (n = 6 rats per group).






















































11 I I f I -5- C/NA- /

0 1 2 3 4 5 6 7 8 9 10 11 12
Defeat Session


Figure 3-6. Intruder behaviors during intermittent social defeat sessions. The rats
exposed to repeated social defeat stress exhibited (A) increases in freezing
behavior and (B) decreases in exploratory locomotion across 12 experimental
sessions. Values expressed are group means + SEM (n = 6 rats per group).


-5- C/NA
-A- C/A


I I I I I I
012345


Ii I
Defeat Session


IIb I' I












200-
-

S150-


S100-
o

S50-
-


-r


Figure 3-7. Circulating hormones after intermittent social defeat stress. Immediately
after, but not 24 h later, the rats exposed to repeated social defeat stress had
elevated circulating concentrations of (A) ACTH and (B) CORT when
compared to basal concentrations in the control rats (NC/NA). Values
expressed are group means & SEM (n = 6 rats per group). Significant
differences between the socially defeated rats and the unhandled control rats
are expressed as [*] p < 0.05.


B60,

501


-r


O~L~LL~L


I

~


Figure 3-8. Effects of intermittent social defeat stress on glandular masses. Repeated
social defeat stress had no effect on (A) thymus masses or (B) adrenal gland
masses. Values expressed are group means & SEM (n = 6 rats per group).


I I









Experiment 2: Social Defeat Stress Followed by Porsolt Swim Testing

Exposure to chronic social defeat stress (C/A) produced a significant increase in

total immobility time during the 15-min forced swim when compared with the immobility

times for the control rats that were not exposed to social defeat stress (NC/NA) (F (2, 21)

= 5.363, p < 0.0131; Fig. 3-9). When the data were analyzed in 5-min bins, there was a

significant between-groups effect as well as a significant time effect (F (2, 42) = 2.424, p

< 0.0150; F (2, 42) = 72.74, p < 0.0001; Fig. 3-10), but no significant group by time

interaction effect (F (4, 42) = 1.270, p < 0.2970; Fig. 3-10).

Inter-Observer Reliability

The two observer's recordings of total time freezing for the social defeat sessions

from Experiments la and lb combined differed by less than 20 sec for 94% of the

sessions and never differed by more than 28 sec. The two observer's recordings of

exploratory locomotion during social defeat sessions were identical in 70% of the

sessions and never differed by more than 3 lines crossed.

The two observer's recordings of total immobility time during the Porsolt swim test

differed by less than 40 sec in 92% of the sessions and never differed by more than 43 sec.














a, 700 1





NC-N 600- C/
Fiur 50-9.Imblt uigteProtsi et h asepsdt eetdsc
deet 400-(hoic xiie ncesdimbiiyfrteduaino h
O osl ocdsi et hncmae oteimblt ie o h





E 300-J J

-200-

E 100-




Figure 3-90. Immobility esr i -nu bs during the Porsolt swim test. The rt xoe o eetdsca
defeat stresscn (Chrnic exhibitedo tinceased immeegobilit dfor teduainc ofhen

intpruder rts xoe oaue socially defeated stress (Acute n hoi) and the control rt
go (No SD). Values expressed are group means & SEM (n = 8 rats pergru)
gop Significant differences between the socinially defeated rats and theunade
controls are expressed as [*] p < 0.05. Addnitioanal abbrevia btioen: No D
nosciael defeated satrss.dtecnrl r xpesda + .5


















CHAPTER 4
DISCUSSION

Overall, repeated social defeat stress produced behavioral, hormonal, and glandular

changes that model some of the symptoms that are seen in depressed humans. Behavioral

despair, HPA axis dysregulation, and thymus involution were found in rats that were

exposed to daily social stress; and these effects were greater overall than the effects that

were seen in the rats that were stressed intermittently.

Daily Social Defeat Stress

The equivalent mean freezing scores and the equivalent mean locomotion scores

during the first social defeat session indicate that all five groups that experienced social

defeat stress were comparable at the beginning of the experiment. The equivalent

progressive increases in freezing behavior and the equivalent decreases in exploratory

locomotion for the three repeatedly stressed groups indicate that these groups underwent

comparable stress-induced behavioral changes across social defeat sessions. These

behaviors, however, did not diminish the persistent displays of dominant behavior

(consistent number of defeats and equivalent latencies to first defeat) by the resident rats.

After 3-4 days of stress exposure, the behavioral curves for both freezing behavior and

exploratory locomotion reached asymptote, suggesting that the behavioral changes

associated with daily social defeat stress do not progress beyond the first few exposures.

The equivalent mean ACTH concentrations and the equivalent mean CORT

concentrations 10 min after the onset of stress between the groups with and without a

history of stress suggest that there is similar activation of the HPA axis for these groups.









However, the lower CORT concentrations 30 min after the onset of stress in the

repeatedly stressed group compared to the CORT concentrations in the acutely stressed

group suggests a sensitization in the negative feedback regulation of CORT.

Decreased thymus mass and/or increased adrenal mass has been shown to result

from chronic stress regimens involving physical and/or psychological stressors (for

examples, see Blanchard et al., 1998; Bryant et al., 1991; Simpkiss and Devine, 2003) as

well as from major depression in victims of suicide (Szigethy et al., 1994). Given that

two of the three groups that were stressed daily (C/A-10 and C/A-30) exhibited thymus

involution while one (C/NA) did not, it cannot be concluded that the daily stress regimen

consistently caused a decrease in thymus mass. These findings, along with adrenal mass

equivalency across groups regardless of number of stress exposures, suggest that the

chronic social defeat stress regimen of six daily exposures may not have been adequate to

consistently alter glandular masses. A longer or unpredictable social defeat stress

regimen could possibly generate consistent thymus involution and adrenal hypertrophy.

Perhaps an additional stressor for the intruders, such as isolation housing to ensure more

timid behavior (Kabbaj et al., 2000), could contribute to the effectiveness of the regimen.

Also, using a restraint stress harness instead of the protective wire mesh cage could

elevate the efficiency of the regimen by producing an inescapable condition for the

intruders .

Intermittent Social Defeat Stress

The equivalent progressive increases in freezing behavior and the equivalent

progressive decreases in exploratory locomotion in the intermittent stress regimen

indicate that the repeatedly stressed groups underwent comparable stress-induced

behavioral changes. However, the escalation of freezing behavior and the decline of










exploratory locomotion occurred more gradually when the social defeat sessions were

72 hours apart than when the social defeat sessions occurred daily. This suggests that the

intermittent stress regimen was not as efficient at inducing these behavioral changes,

although the overall magnitudes of the behavioral effects were eventually roughly

equivalent. Once again, the intruders' behavioral adaptations did not decrease the

displays of agonistic behavior from the residents (consistent number of defeats and

equivalent latencies to first defeat across groups and days).

The equivalence in basal CORT concentrations as well as the equivalence in

glandular masses between the chronically stressed animals and the control animals

indicates that the temporally spaced regimen was not potent enough to sufficiently alter

hormonal or glandular basal states. Even though the more temporally spaced chronic

stress regimen was able to produce equivalent effects with regards to behavioral changes

(albeit, more slowly), it was less effective than the daily regimen in producing the

hormonal and glandular changes that correlate with some of the symptoms of maj or

depression. The results indicate, therefore, that there may be a critical window for

vulnerability to an additional stressor--it is possible that the rats were able to partially

recover from the initial stressor by the next exposure in the more temporally spaced

chronic regimen.

Relevance to Previous Work

Overall, these results confirm and extend a previous report that daily social defeat

is an effective emotional stressor for male rats. In a study by Haller and colleagues

(1999), intruder rats were exposed to resident rats for 4 hours on four consecutive days,

producing increased basal CORT concentrations in the intruders. The daily social defeat

stress regimen used in our study effectively increased basal CORT concentrations as










well, with briefer stressful interactions than previously reported. However, in the same

study, Haller and colleagues found increased adrenal mass with no decrease in thymus

mass, results opposite to those seen after our similar daily chronic stress regimen.

Perhaps it is necessary for a daily chronic stress regimen to extend past four (or six) days

to generate reliable glandular changes.

The HPA axis response to an acute stressor largely depends upon whether the acute

stressor is heterotypic or homotypic. In a study by Armario and colleagues (2004),

investigators reported that when previously-stressed rats (via immobilization) were

presented with a heterotypic stressor (forced swimming), a minor sensitization was

observed. Also, following exposure to a severe stressor (such as shock, restraint,

immobilization, or large doses of endotoxin), there was habituation of the HPA axis

response to a homotypic stressor. These results differ from the results obtained after

daily social defeat stress. Following our repeated daily social defeat stress regimen, there

was a robust activation but rapid shutdown of CORT release. Most probably, the

difference is due to the fact that Armario and colleagues utilized a single stressor

followed by either homotypic or heterotypic challenges. Our model of social defeat does

not appear to function as a homotypic stress regimen. There are several variables

involved in the direct interaction phase of social defeat stress (physical contact, olfactory

cues, and ultra-sonic vocalizations) as well as the introduction of a different resident rat

with each exposure. Conceivably, the use of a different resident for each stress exposure

and the potentially different threats imposed by these different residents creates a novel,

therefore heterotypic, situation with each session.










In general, the behavioral changes in the intruder rats after repeated social defeat

stress model some of the symptoms expressed in clinically depressed patients, including

impairment in social or occupational functioning and loss of interest (DSM IV, 1994).

Also, the elevated basal CORT concentrations after daily social defeat stress during the

nadir of the daily cycle confirm previous findings from our laboratory (Lopes and

Devine, 2004) and mimic the augmented circulating hormones found in clinically

depressed patients (Linkowski et al., 1985; Pfohl et al., 1985).

Porsolt Swim Test

Based on the combined results from the daily and the intermittent stress regimens, a

daily social defeat stress regimen was used to for the Porsolt study to optimize the

potential to produce behavioral despair. The fact that acute social defeat stress did not

produce as much immobility as repeated stress indicates that the neuronal plasticity

caused by repeated exposure to social stress (demonstrated through hormonal and

behavioral plasticity) is a determining factor in the expression of behavioral despair.

A 5-min swim test is commonly used for detection of behavioral despair (Gavioli et

al., 2003; Hinojosa et al., 2006; Porsolt et al., 1978; Rygula et al., 2005). Interestingly,

when analyzed in 5-min bins, the data suggest that in all the bins the repeatedly stressed

rats exhibited more immobility than the controls did, and in one bin (5-10 min) the

acutely defeated rats also exhibited more immobility than the controls did. Therefore, a

10-min session may be necessary to closely examine the subtle differences between

groups that have a less severe stress history and non-stressed control rats.

Overall, these results model the behavioral despair, or low mood and anhedonia,

found in clinically depressed patients (for review, see Harrison, 2002). Also, the

significantly elevated immobility times for the repeatedly stressed rats confirm and









extend the results from a previous report. In a study by Rygula and colleagues (2005),

investigators used a more extensive stress regimen of hour-long daily sessions of social

defeat for five straight weeks to elicit behavioral despair in subsequent Porsolt swim

testing. The daily social defeat stress regimen used in our study effectively increased

immobility in the repeatedly stressed rats as well, with briefer stressful interactions and a

shorter regimen than previously reported.

Conclusions and Future Directions

In conclusion, the behavioral, hormonal, and glandular changes produced by

repeated social defeat closely resemble many of the psychopathological symptoms in

patients with major depression. The daily social defeat stress regimen provides an

interesting and effective model for stress-induced psychopathology.

The effect of repeated social defeat on the overall circadian rhythm was not

evaluated, nor was the persistence of the altered regulation across days, weeks, or

months. Both of these topics may be interesting issues for future studies. Also, potential

adaptations in stress-related molecules (CRH and AVP in the hypothalamus, CRH1 and

Vlb in the pituitary and amygdala, and mineralocorticoid receptor and gluccocorticoid

receptor in the hippocampus) after social defeat should be investigated. These studies

will enhance our growing knowledge of the neurobiological basis for stress-induced

psychopathology (e.g., maj or depression).
















LIST OF REFERENCES


Agid O, Kohn Y, Lerer B (2000). Environmental stress and psychiatric illness.
Bionzedicine andPharnacotherapy,, 54: 135-141.

Amiel-Tison C, Cabrol D, Denver R, Jarreau P, Papiernik E, Piazza PV (2004). Fetal
adaptation to stress part II. Evolutionary aspects; stress-induced hippocampal
damage; long-term effects on behavior; consequences on adult health. Early
Human Development, 78: 81-94.

Anisman H, Matheson K (2005). Stress, depression, and anhedonia: Caveats concerning
animal model s. Neuroscience and Biobehavioral Reviews, 29: 525-546.

Armario A, Valles A, Dal-Zotto S, Marquez C, Belda X (2004). A single exposure to
severe stressors causes long-term desensitization of the physiological response to
the homotypic stressor. Stress, 7 (3): 157-172.

Blanchard RJ, Nikulina JN, Sakai RR, McKittrick C, McEwen B, Blanchard DC (1998).
Behavioral and endocrine change following chronic predatory stress. Physiology
andBehavior, 63 (4): 561-569.

Bryant HU, Bernton EW, Kenner JR, Holaday JW (1991). Role of adrenal cortical
activation in the immunosuppressive effects of chronic morphine treatment.
Endocrinology, 128 (6): 3253-3258.

Chang CL, Hsu SYT (2004). Ancient evolution of stress-regulating peptides in
vertebrates. Peptides, 25: 1681-1688.

De Kloet ER (2003). Hormones, brain and stress. Endocrine Regulations, 37: 51-68.

Diagnostic and statistical manual of mental disorders, 4th ed. (1994). Washington, DC:
American Psychiatry Association.

Gavioli EC, Marzola G, Guerrini R, Bertorelli R, Zucchini S, De Lima TCM, Rae GA,
Salvadori S, Regoli D, Calo G (2003). Blockade of nociceptin/orphanin FQ-NOP
receptor signaling produces antidepressant-like effects: Pharmacological and
genetic evidences from the mouse forced swimming test. European Journal of
Neuroscience, 17: 1987-1990.

Haller J, Fuchs E, Halasz J, Makara GB (1999). Defeat is a maj or stressor in males while
social instability is mainly stressful for females: Towards the development of a
social stress model in female rats. Brain Research Bulletin, 50 (1): 33-39.










Harrison PJ (2002). The neuropathology of primary mood disorder. Brain, 125: 1428-
1449.

Herman JP, Cullinan WE (1997). Neurocircuitry of stress: Central control of the
hypothalamo-pituitary-adrenocortical axis. Trends in Neuroscience, 20 (2): 78-84.

Hinojosa FR, Spricigo L, Izidio G, Bruske GR, Lopes DM, Ramos A (2006). Evaluation
of two genetic animal models in behavioral tests of anxiety and depression.
Behavioural Brain Research, 168: 127-136.

Johnson SA, Fournier NM, Kalynchuk LE (2006). Effect of different doses of
corticosterone on depressive-like behavior and HPA axis responses to a novel
stressor. Behavioural Brain Research, 168: 280-288.

Kabbaj M, Devine DP, Savage VR, Akil H (2000). Neurobiological correlates of
individual differences in novelty-seeking behavior in the rat: Differential
expression of stress-related molecules. The Journal ofNeuroscience, 20 (18):
6983-6988.

Kessler RC, Berglund P, Demler O, Jin R, Koretz Z, Merikangas KR, Rush AJ, Walters
EE, Wang PS (2003). The epidemiology of major depressive disorder. Journal of
the American M\~edical Association, 289 (23): 3095-3 105.

Kollack-Walker S, Watson SJ, Akil H (1997). Social stress in hamsters: Defeat activates
specific neurocircuits within the brain. The Journal ofNeuroscience, 17 (22):
8842-8855.

Linkowski P, Mendlewicz J, Leclercq R, Brasseur M, Hubain P, Golstein J, Copinschi G,
Van Cauter E (1985). The 24-hour profile of adrenocorticotropin and cortisol in
major depressive illness. Journal of Clinical Endocrinology and Metabolism, 61
(3): 429-438.

Lopes KO, Devine DP (2004). Elevation of HPA axis activity in response to repeated
social defeat stress in rats. Society for Neuroscience Abstracts, 30: 426.12.

Lucki I, Dalvi A, Mayorga AJ (2001). Sensitivity to the effects of pharmacologically
selective antidepressants in different strains of mice. Psychopharmacology, 155:
315-322.

Martinez M, Phillips PJ, Herbert J (1998). Adaptation in patterns of c-fos expression in
the brain associated with exposure to either single or repeated social stress in male
rats. European Journal ofNeuroscience, 10: 20-33.

McEwen BS, Stellar E (1993). Stress and the individual. Archives oflnternal2\~edicine,
153: 2093-2101.










Miczek KA (1979). A new test for aggression in rats without aversive stimulation:
Differential effects of d-amphetamine and cocaine. Psychopharmacology, 60:
253-259.

Molina VA, Heyser CJ, Spear LP (1994). Chronic variable stress enhances the
stimulatory action of a low dose of morphine: Reversal by desipramine. European
Journal ofPharmacology, 260: 57-64.

Pfohl B, Sherman B, Schlechte J, Stone R (1985). Pituitary-adrenal axis rhythm
disturbances in psychiatric depression. Archives of GeneralIPsychiatry, 42: 897-
903.

Porsolt RD, Anton G, Blavet N, Jalfre M (1978). Behavioural despair in rats: A new
model sensitive to antidepressant treatments. European Journal ofPharmacology,
47: 379-391.

Raadsheer FC, Hoogendijk WJG, Stam FC, Tilders FJH, Swaab DF (1994). Increased
numbers of corticotrophin-releasing hormone expressing neurons in the
hypothalamic paraventricular nucleus of depressed patients. Clinical
Neuroendocrinology, 60: 436-444.

Rygula R, Abumaria N, Flugge G, Fuchs E, Ruther E, Havemann-Reinecke U (2005).
Anhedonia and motivational deficits in rats: Impact of chronic social stress.
Behavioural Brain Research, 162: 127-134.

Sabban EL, Kvetnansky R (2001). Stress-triggered activation of gene expression in
catecholaminergic systems: Dynamics of transcriptional events. 7RENDS in
Neurosciences, 24 (2): 91-98.

Sgoifo A, De Boer SF, Haller J, Koolhaas JM (1996). Individual differences in plasma
catecholamine and corticosterone stress responses of wild-type rats: Relationship
with aggression. Physiology and Beehavior, 60 (6): 1403-1407.

Simpkiss JL, Devine DP (2003). Responses of the HPA axis after chronic variable stress:
Effects of novel and familiar stressors. Neuroendocrinology Letters Nos. 1/2, 24:
97-103.

Szigethy E, Conwell Y, Forbes NT, Cox C, and Caine ED (1994). Adrenal weight and
morphology in victims of completed suicide. BiologicalPsychiatry, 36 (6): 374-
380.

Whitnall MH (1993). Regulation of the hypothalamic corticotrophin-releasing hormone
neurosecretory system. Progress in Neurobiology, 40: 573-629.
















BIOGRAPHICAL SKETCH

Kristen L. Stone graduated in May 2004 from the University of Central Florida

(Orlando) with her Bachelor of Science degree in psychology. She began her graduate

education in the Psychology Department at the University of Florida (Gainesville) in

August 2004, working toward her Master of Science degree, in the Behavioral

Neuroscience program.