. . _ 3..fl...%§.é§fifi3 ..A... .. ......owl.u-n.....nm&n P1 ... . uh! fl. ..m.w§_fiam {me fax“? ..~ . 3.1.5 1 .u . .53.».er . .av .....kuuwah. r. .3: v.» .n. .mx‘m L7: . v r 6%.. RUM I «a. = . x .15 ‘ . wizuwzrwv! .. '1i_ 1‘???" . . K (I flew” 3mm.» 4 .3 .. ... IV (luL --..Xa 1 1.: I. li“fl.."»1t3l...l» ”ya! ~ . . .0. 3&1. , . “tr! .. 2....13 tiny... :14? . Al. . lurk). N3: ..v...h...vLu4.l ... . C1! . . magi. '0' a J @nunrflcfidn . fir. ..hru ..al-dmvflrn? 04.5".» ,. 4., . a... ' i '1' . wit. 4,22% :01 fl '2’ \ v I“ it 5 E 9 [d 'I £1 3g is 5‘ i .. “Volt: .\.ni \ ‘~0liu 1.73).”: O 'c‘l - ‘ W "95...... f a I ' ‘1' , h...“ . - with}; o! , .v 0.. It...” WIOJWM . Amuuffi‘, x. .,v u& .91 JIM luv L . ... wlc‘.:l..\‘ v .. t .- Vn' if .n n II . ‘n ‘ rune?! Adah.» e. A . :. 3!... kn... {..UPHD .3 .1 . III... 1:0,. in... .5 $5.15.; [..J‘V‘A .1 k v; . . as H . . \s . ,.A .. A M ‘ )Aadfiflrut. . . . . . , A 1 . .. .“A 1*.Wfldun1hatlflvafi3‘flfi i} I! ‘5‘. . y .. r - v (t ‘1» D killxiMJkAflvu.fiod-va.bf A rival" if. :(wlp . 44.: . .1 r1 . .‘ s0!.uV«d¢ .lerth'tuo fv ‘ . I.LIAVH;.1|:. \‘ ol!‘ to‘ It.’ .. llao ls}|¥n.tl.|i!l. illifl .I A... A 45.1.2.3“ o. ....) A .. LINPJrllyvvlvtrtzttndlufi)’. a . -lul Ito!‘ 1.md.wunm.m .. .. .A A. . . . . . . . . . . .rfil,r¢lui}.l-i$..th h1h2v1x! .A .. A . A . V . . ..u . . A A, A {‘11. . , ... .A . ... ., . A .y . ‘ . 7. . ..(O‘tls. Jilhi'i .v. 1 .,. . v .3 ..A , ‘ T .. A . . . y . : . l‘isti'lvztflfi‘" .l. . .A. . . . , , . I A Kilt . ‘VI ‘rv‘D‘vll {fil‘fi I. A . , ; V .. .. . . ... . . V . . A . d. . . ‘ . . . . . . v. ... . . ‘ A ,.,\4dll1.n:\llll..(t'l|ti,|9|. :l‘vAi .ad: . , .. . C y A‘ A A .: (“\‘33Al . . . . , V7. , t. Atl ‘Ilvq Hikw vl . .‘v. . . . , . A . . . .I‘E.L lt.nl"!¢.|’ ... b.\v.¢ 00.1! ...."N‘v. . . ..V A . . .. .. . . . y n 0 . , . . A A .. . , . . . . . )1 tn.yc)leLLtllafa|IIsIt-VHVJ-H :8 ~ I p: L; I‘ {i :10 :n I 1: t V I Alf-.. ..L..II‘ 1. . 1.)!n0t. vifiw.‘ ? r .A'\ 4‘ .Ilw A‘b‘ . . . (A w I . I fly . ‘ v .1: . ...EéI-lJ- 5v, .1 ., .3... HA1 L. - 1.. .- . ...t. 2 ...A. ...:wtni- Liuu‘fifi . A . -y .ullAu..olI., '1'"; llllllflllllllllilliHUNlllllllmllllllllllllllllllilllllifl 293 01570 69 This is to certify that the thesis entitled The ontogeny of brown adipose tissue in Microtus ochrogaster in various thermal and sociaT environments. presented by Bradiey A1an White has been accepted towards fulfillment of the requirements for Magefis— degree in loo-109+— Major professor 7/15/96 Date 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution v f v ‘D V f LIBRARY Michigan State University PLACE IN RETURN BOX to remove thie checkout from your record. TO AVOID FINES return on or before dete due. DATE DUE DATE DUE DATE DUE MSU IeAn Afflnnetive Action/Ewe! Opportunity Inetituion Wane-u THE ONTOGENY OF BROWN ADIPOSE TISSUE IN.MICROTUS OCHROGASTER IN VARIOUS THERMAL AND SOCIAL ENVIRONMENTS BY Bradley A. White A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1996 ABSTRACT THE ONTOGENY OF BROWN ADIPOSE TISSUE IN MICROTUS OCHROGASTER IN VARIOUS THERMAL AND SOCIAL ENVIRONMENTS BY Bradley A. White Short- and long—term brown adipose tissue (BAT) responses to postnatal cold exposure (PCB) and the effects of paternal care during PCE were examined in multiple BAT deposits in prairie voles (Microtus ochrogaster). PCE enhanced BAT recruitment in nestlings but not adults. Recruitment was higher when fathers were absent (versus present) during PCE. Responses in various BAT deposits were correlated regardless of treatments, suggesting that measures of single BAT deposits reflect responses in total BAT; however, deposits differed in relative lipid content. Prairie voles showed a short-term BAT recruitment response to PCE similar to that seen in murine rodents. However, prairie voles did not exhibit long-term BAT recruitment (into adulthood) in response to postnatal PCE. Nestling BAT recruitment was curtailed when paternal care was available during PCE, suggesting a possible mechanism for enhanced development observed in this species when paternal care is available. To my parents, Gary White and Beverly Berini, with love. iii ACKNOWLEDGMENTS First I would like to thank Dr. Richard Hill for providing tireless support, direction, advice, and encouragement throughout my graduate studies. I feel most fortunate to have been befriended by such a sincere, generous, and inspiring individual and his family. This work has benefitted tremendously from the critical insight and suggestions provided by my committee members, Drs. Kay Holekamp, Laura Smale, and Dale Romsos. Dr. Scott Winterstein contributed helpful thoughts on my statistical analyses. I wish to thank Gregorios Papakostas for being an intellectually stimulating and tolerant officemate, and for helping to maintain my sanity and spirit through frequent outdoor adventures. I owe thanks to Michael Sanregret, Brian Wagner, and Paula Hildebrandt for their cheerful, reliable assistance with colony maintenance. This work would not have been possible without support from the Department of Zoology, the Ecology and Evolutionary Biology Program, and the M.S.U. Museum. The students and staff of these units have made innumerable contributions to my graduate studies. In particular, I thank Dr. Donald Hall for his contagious enthusiasm for biology and dedication to an enriching graduate student experience. iv TABLE OF CONTENTS LIST OF TABLES ........................................... vii LIST OF FIGURES ......................................... viii INTRODUCTION ............................................... 1 Postnatal Nonshivering Thermogenesis and BAT ........................................ 2 BAT Thermogenesis Mechanisms .......................... 3 BAT Recruitment and Its Regulation .................... 6 Recruitment Stimuli and Time Scales ................... 8 Measurement of BAT Recruitment ....................... 11 Lipid Levels and Cold-induced Recruitment ............ 12 Defining Cold Exposure ............................... 14 Prior Studies of BAT in Microtine Rodents ............ 15 Prairie Voles: Natural History ....................... 16 EFFECTS OF LOW AMBIENT TEMPERATURE ON BROWN ADIPOSE TISSUE DEVELOPMENT .................. 17 I. Short-term Effects of Postnatal Cold Exposure: Objectives and Experimental Design ........................................ 17 II. Long-term Effects of Postnatal Cold Exposure: Objectives and Experimental Design ........................................ 18 Methods: Short- and Long-term Studies ................ 19 Results .............................................. 25 I. Short-term Study ............................. 25 II. Long—term Study ............................. 63 V Discussion ........................................... 84 I. Short—term Study ............................. 84 II. Long-term Study ............................. 93 EFFECTS OF PATERNAL CARE ON BAT DEVELOPMENT .............. 100 General Overview of the Problem: Postnatal BAT Recruitment in Response to Paternal Care .................... 100 Objectives and Experimental Design .................. 103 Methods ............................................. 104 Results ............................................. 109 Discussion .......................................... 122 RESPONSE RELATIONSHIPS BETWEEN BAT DEPOSITS .............. 127 General Overview of the Problem ..................... 127 Objectives .......................................... 129 Methods ............................................. 130 Results ............................................. 131 Discussion .......................................... 171 LIST OF REFERENCES ....................................... 173 vi Table Table Table Table Table LIST OF TABLES Results from the short—term study ............... 27 Results from the long-term study ................ 65 Results from the paternal-care study ........... 110 Results from the response-relationships study for weight-specific lipid free BAT dry weight (LFDW/g) ........................ 133 Results from the response-relationships study for lipid weight per lipid-free BAT dry weight (LW/LFDW) ....................... 144 vii LIST OF FIGURES Figure 1 - Schematic depiction of metabolic processes in a brown adipocyte .................. 5 Figure 2 - Interscapular weight-specific lipid-free BAT dry weight (LFDW/g) for cold-treated and control animals of pairs in the short-term study .......................................... 29 Figure 3 - Residual weight-specific lipid—free BAT dry weight (LFDW/g) for cold-treated and control animals of pairs in the short-term study .................. 31 Figure 4 — Squamosal weight-specific lipid-free BAT dry weight (LFDW/g) for cold-treated and control animals of pairs in the short-term study .................. 33 Figure 5 - Interscapular BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated and control animals of pairs in the short-term study .................. 35 Figure 6 - Residual BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated and control animals of pairs in the short-term study .................. 37 Figure 7 - Squamosal BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated and control animals of pairs in the short-term study .................. 39 Figure 8 - Short-term study: Interscapular BAT lipid—free dry weight (LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight ............................ 42 viii Short—term study: Residual BAT lipid—free dry weight (LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight ........................... 44 Figure 9 Figure 10 - Short—term study: Squamosal BAT lipid-free dry weight (LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight ........................... 46 Figure 11 - Short-term study: Interscapular BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight ........................................ 48 Figure 12 - Short-term study: Residual BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight .......... 50 Figure 13 - Short-term study: Squamosal BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight .......... 52 Figure 14 - Short-term study: Interscapular BAT lipid weight (LW) for cold-treated (solid circles) and control animals (open circles) plotted against lipid—free dry weight (LFDW) ............................. 55 Figure 15 - Short-term study: Residual BAT lipid weight (LW) for cold-treated (solid circles) and control animals (open circles) plotted against lipid-free dry weight (LFDW) ................................. 57 Figure 16 — Short-term study: Squamosal BAT lipid weight (LW) for cold—treated (solid circles) and control animals (open circles) plotted against lipid—free dry weight (LFDW) ............................. 59 Figure 17 - Short-term study: Body weight at three ages in cold-treated (white bars) and control animals (black bars) .............. 62 ix Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure l8 19 20 21 22 23 24 25 26 27 Interscapular weight-specific lipid-free BAT dry weight (LFDW/g) for cold-treated and control animals of pairs in the long-term study ............................... 67 Squamosal weight-specific lipid-free BAT dry weight (LFDW/g) for cold-treated and control animals of pairs in the long—term study ............................... 69 Interscapular BAT lipid weight per lipid-free BAT dry weight (LW/LFDW) for cold-treated and control animals of pairs in the long-term study .................. 71 Squamosal BAT lipid weight per lipid-free BAT dry weight (LW/LFDW) for cold-treated and control animals of pairs in the long-term study .................. 73 Long-term study: Interscapular BAT lipid—free dry weight (LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight ........................... 76 Long-term study: Interscapular BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight ........................... 78 Long—term study: Interscapular BAT lipid weight (LW) for cold-treated (solid circles) and control animals (open circles) plotted against lipid-free dry weight (LFDW) ............................. 80 Long-term study: Squamosal BAT lipid weight (LW) for cold-treated (solid circles) and control animals (open circles) plotted against lipid-free dry weight (LFDW) ............................. 82 Interscapular weight-specific lipid-free BAT dry weight (LFDW/g) for father-absent (FA) and father-present (FP) litters of pairs in the paternal-care study ............. 112 Squamosal weight-specific lipid-free BAT dry weight (LFDW/g) for father-absent (FA) and father-present (FP) litters of pairs in the paternal-care study ............. 114 Figure 28 - Interscapular lipid weight per lipid-free BAT dry weight (LW/LFDW) for father-absent (FA) and father—present (FP) litters of pairs in the paternal-care study ........................................ 116 Figure 29 — Squamosal lipid weight per lipid—free BAT dry weight (LW/LFDW) for father-absent (FA) and father-present (FP) litters of pairs in the paternal-care study ............. 118 Figure 30 - Short—term study: Correlation between weight-specific lipid-free BAT dry weight (LFDW/g) values for squamosal and interscapular BAT in cold—treated (closed circles) and control animals (open circles) ............................... 135 Figure 31 - Short-term study: Correlation between weight—specific lipid—free BAT dry weight (LFDW/g) values for squamosal and residual BAT in cold-treated (closed circles) and control animals (open circles) ............................... 137 Figure 32 - Short-term study: Correlation between weight-specific lipid-free BAT dry weight (LFDW/g) values for interscapular and residual BAT in cold-treated (closed circles) and control animals (open circles) ..................................... 139 Figure 33 - Long-term study: Correlation between weight—specific lipid-free BAT dry weight (LFDW/g) values for interscapular and squamosal BAT in cold-treated (closed circles) and control animals (open circles) ..................................... 141 Figure 34 — Paternal-care study: Correlation between weight-specific lipid-free BAT dry weight (LFDW/g) values for interscapular and squamosal BAT in father-absent (closed circles) and father-present litters (open circles) ........ 143 Figure 35 - Short—term study: Correlation between lipid weight per lipid-free BAT dry weight (LW/LFDW) values for interscapular and residual BAT in cold-treated (closed circles) and control animals (open circles) ..................................... 146 xi Figure 36 — Short-term study: Correlation between lipid weight per lipid-free BAT dry weight (LW/LFDW) values for interscapular and squamosal BAT in cold-treated (closed circles) and control animals (open circles) ............................... 148 Figure 37 - Short-term study: Correlation between lipid weight per lipid-free BAT dry weight (LW/LFDW) values for residual and squamosal BAT in cold-treated (closed circles) and control animals (open circles) ..................................... 150 Figure 38 - Long-term study: Correlation between lipid weight per lipid-free BAT dry weight (LW/LFDW) values for interscapular and squamosal BAT in cold-treated (closed circles) and control animals (open circles) ..................................... 152 Figure 39 - Paternal—care study: Correlation between lipid weight per lipid-free BAT dry weight (LW/LFDW) values for interscapular and squamosal BAT in father-absent (closed circles) and father-present litters (open circles) ........ 154 Figure 40 - Short-term study: Lipid weight (LW) plotted against lipid-free dry weight (LFDW) for interscapular (closed circles) and squamosal BAT (open circles) for control animals ........... 156 Figure 41 — Short-term study: Lipid weight (LW) plotted against lipid-free dry weight (LFDW) for interscapular (closed circles) and squamosal BAT (open circles) for cold-treated animals ............ 158 Figure 42 — Long-term study: Lipid weight (LW) plotted against lipid—free dry weight (LFDW) for interscapular (closed circles) and squamosal BAT (open circles) for control animals ........... 160 Figure 43 - Long-term study: Lipid weight (LW) plotted against lipid-free dry weight (LFDW) for interscapular (closed circles) and squamosal BAT (open circles) for cold-treated animals ...................................... 162 xii Figure 44 - Paternal-care study: Lipid weight (LW) plotted against lipid—free dry weight (LFDW) for interscapular (closed circles) and squamosal BAT (open circles) for father-absent (FA) litters ................................. 164 Figure 45 — Paternal-care study: Lipid weight (LW) plotted against lipid-free dry weight (LFDW) for interscapular (closed circles) and squamosal BAT (open circles) for father-present (FP) litters ................................. 166 Figure 46 - Short—term study: Lipid weight (LW) plotted against lipid-free dry weight (LFDW) for interscapular (closed circles), squamosal (open circles) and residual (triangles) BAT for control animals ...................... 168 Figure 47 - Short—term study: Lipid weight (LW) plotted against lipid-free dry weight (LFDW) for interscapular (closed circles), squamosal (open circles) and residual (triangles) BAT for cold-treated animals ................. 170 xiii INTRODUCTION Very little is known about the influences of low ambient temperature on postnatal brown adipose tissue (BAT) development in nonmurine altricial rodents. However, studies on murine rodents and other mammals have shed much light on BAT recruitment, and several predictions can be made for microtine rodents regarding 1) how BAT might respond over short (preweaning) and long (adult) time scales to cold exposure during the postnatal period, 2) how the presence or absence of paternal care during cold exposure might affect nestling BAT responses, and 3) how interrelated these responses might be as expressed in the various BAT deposits of an individual animal. Before elaborating on these predictions, I will present a general overview of the function, mechanisms, and process of BAT recruitment, focusing on altricial rodents. Recruitment time scales in nestlings and adults, methods of measuring BAT recruitment, the state of knowledge of microtine BAT responses, and the natural history of Microtus ochrogaster -- the focus of the studies presented here -— will also be briefly reviewed. First, BAT responses to postnatal cold exposure will be examined in preweanling prairie voles in the short-term study and in adult prairie voles in the long-term study. Second, BAT responses to paternal care during cold exposure will be examined in preweanling prairie voles in the paternal-care study. Finally, responses of various BAT deposits will be compared in the study of response relationships between BAT deposits. POSTNATAL NONSHIVERING THERMOGENESIS AND BAT Studies of isolated nestlings have shown that a remarkable enhancement of thermoregulatory capacity occurs in small-bodied rodents during the postnatal period. The lack of a developed musculoskeletal system, high surface- area-to-volume ratio, and high rates of movement-related heat loss preclude shivering from playing a major role in thermoregulation in neonatal rodents (Jansky, 1973; Gordon, 1993). Experiments with surgical and pharmacological treatments to block shivering and nonshivering mechanisms have shown that nonshivering thermogenesis (NST) predominates during the postnatal period (e.g., Brueck and Wuennenberg, 1966). Brown adipose tissue (BAT) is the principal site of nonshivering thermogenesis (NST) in mammals. BAT THERMOGENES I S ME CHANI SMS The mechanisms of BAT thermogenesis have been well studied in laboratory rodents and reviewed recently by Himms-Hagen (1990) and Cannon (1995) (Figure 1). Norepinephrine (NE) stimulation of beta3-adrenergic receptors on the surface of brown adipocytes elevates cyclic-AMP (cAMP) via G-proteins, activating protein kinases, which phosphorylate hormone-sensitive lipase (HSL). Intracellular triacylglycerol is hydrolyzed by activated HSL to free fatty acids (FFA), which enter the mitochondria and undergo beta-oxidation to produce acetyl- CoA and reduced cofactors NADH and FADHZ. Oxidation of these cofactors via the electron transport chain, in addition to those produced elsewhere, such as by acetyl— CoA oxidation in the tricarboxylic acid cycle, creates a proton electrochemical gradient between the mitochondrial matrix and intermembrane space. ATP synthetase content is unusually low in BAT mitochondria (Houstek et al., 1995), and this proton gradient is largely dissipated by a protein found only in the BAT inner mitochondrial membrane, called uncoupling protein (UCP). UCP, also known as thermogenin, is unique to BAT. Its primary function is to allow protons accumulated in the intermembrane space to bypass ATP synthetase in returning to the mitochondrial matrix, thereby uncoupling Figure 1. Schematic depiction of metabolic processes in a brown adipocyte. Figure 1 wig 88055? nE.< ?\>.§&2(\ C02: 3915 I 08 DEBS N 2828230 I managed...— mEmaE. .m ease mDD._.w 2mm.._.-._.mOIw l v.0 od Nd wd ad 0... N... v._. 0;. 'IVWINV GELVEHl-G'IOO NI (5/5‘”) IEDIMClzl'l 30 Figure 3. Residual weight-specific lipid-free BAT dry weight (LFDW/g) for cold-treated and control animals of pairs in the short—term study. The line marked "null" indicates expected location of points for each pair if there were no differences among cold- treated and control nestlings for LFDW/g. 31 Figure 3 ._<_2_z< ._oEzoo 2. Base 939... m P 1 u q _ as, m_095 2mm...-...mOIm 'IVWINV causal-moo NI (5/5w) 9/MCI:I'| 32 Figure 4. Squamosal weight-specific lipid-free BAT dry weight (LFDW/g) for cold—treated and control animals of pairs in the short-term study. The line marked "null" indicates expected location of points for each pair if there were no differences among cold- treated and control nestlings for LFDW/g. 33 5.0 0.0 0.0 #0 0.0 N0 ..DD._.m 2mm...-...mOIm 4L 0.0 00 N0 0.0 v0 0.0 6.0 no no 0.0 0._. 'IVININV GHlVBHl-O'IOO NI (5/5‘“) IEDIMCH'I 34 Figure 5. Interscapular BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated and control animals of pairs in the short-term study. The line marked "null" indicates expected location of points for each pair if there were no differences among cold-treated and control nestlings for LW/LFDW. 35 Figure 5 ._095 2mm._.-._.mOIm ‘IVWINV causal-moo NI (5/5) MGd‘I/M'i 36 Figure 6. Residual BAT lipid weight per lipid—free dry weight (LW/LFDW) for cold-treated and control animals of pairs in the short-term study. The line marked "null" indicates expected location of points for each pair if there were no differences among cold-treated and control nestlings for LW/LFDW. 37 ._095 EmeLEOIw 'IVININV ClEllVEJHl-CI'IOO NI (5/5) MGd‘l/M'i 38 Figure 7. Squamosal BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated and control animals of pairs in the short—term study. The line marked "null" indicates expected location of points for each pair if there were no differences among cold-treated and control nestlings for LW/LFDW. 39 Figure 7 ._DD._.m meHFmOIm 'IVWINV causal-moo NI (5/5) MCH'l/M'I 40 The hypothesis that nestling lipid-free BAT dry weight is dependent on nestling body weight was generally not supported. Interscapular BAT LFDW in control nestlings showed a significant relation to BW (r2=0.15, p<0.05), as did residual BAT LFDW in cold-treated nestlings (r2=0.52, p=0.02). Otherwise, LFDW showed no significant relation to BW. As Figures 8-10 illustrate, there was a tendency for lower BW to result in lower LFDW in controls for all fat types. This tendency was eliminated, however, in interscapular and squamosal BAT in cold—exposed nestlings, where relatively high LFDW weights were observed in small animals as compared to the regression line of controls extrapolated into the low BW range. The lower BW range was occupied only by cold- treated nestlings, resulting in a wider BW range for cold- treated animals (cold-treated: 11.82 to 24.07 9, versus controls: 16.44 to 24.01 g). The hypothesis that lipid weight per lipid-free BAT weight varies as a function of body weight was supported for cold-treated but not control nestlings. As shown in Figures 11-13, in all three deposits under cold-exposure conditions, a similar positive relationship existed between LW/LFDW and BW (interscapular BAT: r2=0.47, p<0.001; residual BAT: r2=0.67, p<0.01; squamosal BAT r2=0.45, p=0.01). There was not a significant dependence of LW/LFDW on BW in control individuals, however. The data 41 Figure 8. Short-term study: Interscapular BAT lipid-free dry weight (LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight. A9 Eon; >08 Figure 8 42 >DD._.m mehIhmOIm 0N VN NN 0N 0 P 0 F v F N _. 0 F O O I o I . I. .2280 . I 6| I o I 0930-200 ll I .53 m<43d<0mmmhz_ 000.0 0 5.0 N _.0.0 v _.0.0 0 _.0.0 0 5.0 0N0.0 NN0.0 ¢N0.0 0N0.0 (5) M0:I'I 43 Figure 9. Short-term study: Residual BAT lipid-free dry weight (LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight. 44 Figure 9 av Eon; >000 00 «N 00 ow 00 or «F NF 3.5000 .IIOII uwumwbIEOO IIIQI ._.00._.w EmmhIkmOT—m 0 F 00.0 I v0.0 I 00.0 I 00.0 I no.0 L 00.0 I 00.0 (5) MCH‘I 45 Figure 10. Short-term study: Squamosal BAT lipid-free dry weight (LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight. 46 Figure 1 0 A3 50.0; >000 00 em mm 00 3 o F 3 N F _ a _ q _ q _ i d u _ q — i _ ‘0‘]: .2250 i¢II . 000000-200 IIoII ...(m I_00._.m EmmHIhmOIw 0.. l l l l N00.0 V000 000.0 000.0 0 5.0 N 5.0 v 5.0 0 5.0 (5) MCI:I'I 47 Figure 11. Short-term study: Interscapular BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold- treated (solid circles) and control animals (open circles) plotted against body weight. 48 Figure 1 1 0N 30 5.0.02, >000 <0 mm 00 0 F o F fi I — d — d — q — 3.5000 .IIOII Omummbugoo IOII ._.00._.m 200500010 0F (5/5) MClzl‘llM'i 49 Figure 12. Short-term study: Residual BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight. 3:10.02, >000 00 a mm 00 m: 0 F 3 N F — 4 _ 1 _ q _ q _ . — . _ q 5 Figure 1 2 50 _05:00 .léll o 005005-200 IT ...(m I_<00_mmm >00._.m 5.00.70.00.10 (5/5) MCI:I‘|/M'l 51 Figure 13. Short-term study: Squamosal BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight. 0:10.02, >000 3 ...I. 0N VN NN ON 05 or 3 NF 05 w _ . _ . _ . _ a _ q a . _ a _ . l o “H o I F I N I m 2 5 1 L 2 O O .9250 .IéII I m 002000-200 lol. 0 I 0 k0D._.m EmmHIhmOIm (5/5) MCId'I/M'I 53 for control nestlings encompassed only the upper portion of the BW range of cold-treated nestlings. Figures 11-13 show much overlap between cold-treated and control nestling LW/LFDW for all three BAT deposits within the BW range of controls. The hypothesis that BAT lipid.weight is a function of lipid-free BAT dry weight was supported in most deposits, regardless of postnatal treatment. As can be seen in Figures 14-16, LW was dependent upon LFDW in all deposits except squamosal BAT from cold—treated nestlings (cold- treated interscapular BAT: 2=0.22, p=0.02; cold-treated residual BAT: r2=0.77, p<0.01; control interscapular BAT: r2=0.34, p<0.01; control residual BAT: r2=0.44, p=0.03; control squamosal BAT: r2=0.31, p<0.01). A POSTERIORI COMPARISONS: Several variables were examined together in the absence of preformed hypotheses to determine whether propositions could be posed after studying their relationships. Paired comparisons were performed on body weights at ages 2, 10-11, and 19-20 d between cold-treated and control nestlings in each pair. The effects of sex on BW, LFDW/g, and LW/LFDW were examined by one—way ANOVA on unpaired data. The relationship between litter size and these variables was determined by linear regression 54 Figure 14. Short-term study: Interscapular BAT lipid weight (LW) for cold-treated (solid circles) and control animals (Open circles) plotted against lipid-free dry weight (LFDW). 55 Figure 1 4 _ _ 0N0.0 VN0.0 NN0.0 0N0.0 05.0 05 d 30 2,0": A q _ . _ .0 V50 N50 05.0 000.0 .9250 0280-200 .IIAVIIII ll ._.00._.w 5.00.70.00.00 00.0 No.0 #00 00.0 00.0 0 5.0 N ...0 v 5.0 (5) M1 56 Figure 15. Short-term study: Residual BAT lipid weight (LW) for cold-treated (solid circles) and control animals (open circles) plotted against lipid-free dry weight (LFDW). 57 Figure 1 5 58 Figure 16. Short—term study: Squamosal BAT lipid weight (LW) for cold-treated (solid circles) and control animals (open circles) plotted against lipid-free dry weight (LFDW). .930“: 05.0 10.0 06.0 20.0 08.0 08.0 — . _ q d. Figure 1 6 59 0980-260 ITI ._.00._.w 200500010 V000 N000 .2250 . I éI I I 00.0 5.0 N00 000 V0.0 000 00.0 (5) M1 60 analysis on unpaired data, with litter size treated as the independent variable. Cold exposure resulted in lower nestling growth rates. Growth rate was faster for control than for cold- treated members of sibling pairs. Pairing accounted for most of the early variation in BW (ages 2 and 10-11 d), but the effects of cold-exposure increased over time, such that control nestlings weighed significantly more than cold—treated littermates by 19-20 d (Figure 17). Nestling body weight at 21 days was not affected by‘ sex. Due to heteroscedasticity, a Mann-Whitney U nonparametric test was used in lieu of a one-way ANOVA. Nestling BW was not affected by sex. Nestling body weight at 21 days was not a function of litter size. BW was found not to vary significantly as a function of litter size. Variance in nestling weight-specific lipid-free BAT dry weight was not explained by litter size. Variation in LFDW/g for all fat and treatment types was not dependent on litter size. 61 Figure 17. Short-term study: Body weight at three ages in cold—treated (white bars) and control animals (black bars). *** indicates significant difference in body weight at p < 0.001. 62 Figure 1 7 0NI0 .. .283 00< :0 F N «ca (OVNO I. v? .9250 I 8.82-28 Hm. >00...m EmmhIhmOIm 05 NF 0.. 0— ,- 0N (5)1H0I3M mos 63 Sex had no effect on nestling weight-specific lipid- free BAT dry weight. For all deposit types, LFDW/g was not significantly affected by sex. Lipid weight per lipid-free BAT weight in nestlings did not vary as a function of litter size. Litter size did not explain a significant portion of variance in LW/LFDW. Nestling lipid weight per lipid-free BAT weight was not affected by sex. LW/LFDW values were not significantly affected by sex. II. LONG-TERM STUDY A PRIORI COMPARISONS: The following results respond to the a priori hypotheses presented in the OBJECTIVES section. The hypothesis that postnatal cold exposure increases the weight-specific lipid-free BAT dry weight in adults was not supported. LFDW/g data were log-transformed prior to analysis. By 20 weeks of age, no detectable differences existed among the 46 pairs of siblings in postnatal—cold-treated and control groups in LFDW/g (Table 2). As can be seen in Figures 18 and 19, in the adults, 64 the numbers of pairs exhibiting a higher LFDW/g value in the postnatal—cold-treated individual (interscapular BAT: 25 of 46, about 54%; squamosal BAT: 24 of 46, about 52%) approximately equalled the numbers of pairs in which LFDW/g was higher in the control individual. Pairing accounted for a significant component of variation in squamosal BAT LFDW/g at 20 weeks (Table 2). The hypothesis that adults exposed to cold during the postnatal period have lipid weight per lipid-free BAT dry weight values equal to individuals in the postnatal control group was supported. No significant differences were detected between treatment groups for LW/LFDW for either BAT deposit type (Table 2). Figures 20 and 21 demonstrate that the numbers of pairs exhibiting higher values for LW/LFDW for either treatment were approximately equal; for both interscapular and squamosal BAT, 22 (about 48%) of 46 pairs had higher adult LW/LFDW for cold-treated nestlings. LW/LFDW pairing effects were significant for both deposit types (Table 2). The hypothesis that adult lipid-free BAT dry weight is dependent on adult body weight was supported, regardless of treatment. For both interscapular (cold— treated: r2=0.40, p<0.01; control: r2=0.38, p<0.01) and squamosal BAT (cold-treated: r2=0.37, p<0.01; control: r2=0.18, p<0.01), LFDW depended significantly on BW. Data 65 Table 2. Results from the long—term study. N is the number of pairs of animals (cold—treated and control) analyzed. Standard deviation (SD) is given in parentheses. The probability for treatment ("trtment") is the likelihood of no difference between cold-treated and control animals. That for pairs is the likelihood that none of the overall variance was attributable to differences among pairs. LFDW/g = weight—specific lipid-free BAT dry weight. LW/LFDW = lipid weight per lipid-free BAT dry weight. Mean (SD) Probability Variable N Cold-treated Control Trtment Pairs LFDW/g m Interscapular 46 0.36 (0.11) 0.35 (0.09) 0.707 0.19 Squamosal 46 0.17 (0.04) 0.17 (0.05) 0.506 <0.01 LW/LFDW 19.19.). Interscapular 46 11.67 (4.14) 12.02 (3.49) 0.605 0.02 Squamosal 46 5.37 (2.12) 5.19 (1.90) 0.595 <0.01 66 Figure 18. Interscapular weight-specific lipid-free BAT dry weight (LFDW/g) for cold—treated and control animals of pairs in the long-term study. The line marked "null" indicates expected location of points for each pair if there were no differences among cold-treated and control nestlings for LFDW/g. 67 Figure 1 8 ._<_2_z< 0000200 2_ .005. 0.2.0"... 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 . . _ . . q . u _ . _ d _ 0_0D._.w 5.0050200 0.0 'IVININV 03030-0100 NI (fi/fiw) 9/MGd1 68 Figure 19. Squamosal weight-specific lipid-free BAT dry weight (LFDW/g) for cold-treated and control animals of pairs in the long—term study. The line marked "null" indicates expected location of points for each pair if there were no differences among cold— treated and control nestlings for LFDW/g. 69 Figure 1 9 v0 ._<_2_z< 55200 2_ .002. 0.2.0“: 00 N0 50 0.0 _ _ . _ . _ 0.0 @A/ e e L ...o u n . . . e o o .0 o o o co 0 o o e “on L N0 0 e 00 o o o o o o 0 0.0 0.60 004.0 “.0 0000.205. ......00 2. ...<0 440052000 >0D...0 5.00...-0200 'iVININV causal-moo NI (5/5w) 9/MCH‘I 70 Figure 20. Interscapular BAT lipid weight per lipid—free BAT dry weight (LW/LFDW) for cold-treated and control animals of pairs in the long-term study. The line marked "null" indicates expected location of points for each pair if there were no differences among cold-treated and control nestlings for LW/LFDW. 71 Figure 20 0<_2_z< 000200 2_ .00. 2.055.. 00 0. 0. E 00 0. 0 0 v . J — . _ a d 0 a 7 u q — 0 . q . O //9¢ O O O o o o o 0 ~ 0 .0. 0 0 O O O O. ‘ O O 0.. O O O O O O O O. 0 0.00._.0 5.00.7020; 00 00 0N ‘IVWINV GSlVBHl-G‘IOO NI (5/5) MG:I‘|/M'I 72 Figure 21. Squamosal BAT lipid weight per lipid-free BAT dry weight (LW/LFDW) for cold-treated and control animals of pairs in the long-term study. The line marked "null" indicates expected location of points for each pair if there were no differences among cold-treated and control nestlings for LW/LFDW. 73 Figure 21 ._<_2_z< 0000200 2_ .00. 2.0002... 0 F 0 0 k 0 0 v 0 m 0 Q coo. .0 e o 0000. o e o 00 o o o e e o o o o I oo o o e o. e 05?. IO<0 ”.0 00005.05. 0.00 2. ...<0 I.<0O.2<000 >00...0 5.00.7020; FommN‘DmeN'I—o FF 'IVININV CEIlVElHl-G‘IOO NI (5/5) MCH'I/M1 74 for interscapular BAT LFDW are shown in Figure 22 (squamosal BAT showed a similar pattern). The hypothesis that lipid weight per lipid-free BAT weight varies as a function of body weight in adults was supported for both treatments. For both deposit types there was a significant functional dependence of LW/LFDW on BW. Figure 23 shows this relationship for interscapular BAT LW/LFDW (cold-treated: r2=0.26, p<0.01; control: r2=0.14, p=0.01), which was related to body weight much like squamosal BAT LW/LFDW (cold-treated: r2=0.40, p<0.01; controls: r2=0.17, p<0.01). The hypothesis that BAT lipid weight depends on lipid-free BAT dry weight was supported, irrespective of treatment. As can be seen in Figures 24 and 25, a significant amount of variation in LW was explained by LFDW in both interscapular (cold-treated: r2=0.62, p<0.01; control: r2=0.67, p<0.01) and squamosal BAT (cold-treated: r2=0.66, p<0.01; control: r2=0.45, P<0.01). A POSTERIORI COMPARISONS: Several variables were examined together in the absence of preformed hypotheses to determine whether propositions could be posed after studying their relationships. Paired comparisons were performed on body weights (BW) at 20 75 Figure 22. Long-term study: Interscapular BAT lipid-free dry weight (LFDW) for cold-treated (solid circles) and control animals (open circles) plotted against body weight. 76 .0. 2.002. >000 00 00 00 0... 0N Figure 22 .2250 000000-200 IT ...<0 000._.0 5.00...-0204 00.0 L 5.0 I N00 I 00.0 I 20.0 L 00.0 (5) MCI:l"l 77 Figure 23. Long-term study: Interscapular BAT lipid weight per lipid-free dry weight (LW/LFDW) for cold- treated (solid circles) and control animals (open circles) plotted against body weight. 78 Figure 23 00 .0. 21002. >000 00 00 00 00 0N . . q a — 4 _ . .2250 10!. 002020-200 II.) h00...0 5.00...-0204 (O'd'N 0N N.. 5... 05 05 NN (5/5) MCH‘I/M'i 79 Figure 24. Long-term study: Interscapular BAT lipid weight (LW) for cold-treated (solid circles) and control animals (open circles) plotted against lipid-free dry weight (LFDW). 80 Figure 24 v0.0 000 N00 5.0 _05000 .IIOII o 005005-200 ll ._.00...0 5.00...-vaOI. 00.0 (5) M1 81 Figure 25. Long—term study: Squamosal BAT lipid weight (LW) for cold-treated (solid circles) and control animals (open circles) plotted against lipid—free dry weight (LFDW). 82 Figure 25 .0. 2.0“: 25.0 050 N50 :00 05.0 000.0 000.0 N000 000.0 000.0 200.0 000.0 1 _ _ d — I — u — (1 — d di 1 - d d 1 . .2250 000000-200 .IIOII ll) ._.00._.0 20005020.. 5.0 N00 000 V0.0 000 00.0 50.0 00.0 00.0 0 ...0 F ...0 (5) M1 83 weeks. The effects of sex on BW, LFDW/g, and LW/LFDW were examined by one-way ANOVA on unpaired data. As earlier noted, potential relationships between litter size and these variables were not examined because litters sometimes lived with other, related litters between 30 d and 20 weeks of age. Adult body weight was not affected by postnatal cold exposure. No significant effect of treatment on BW was found in paired comparisons between cold-exposed and control adults. Mean BW for 20-week-old cold-treated individuals (mean = 45.23 g +/- 9.85 S.D.) was not different from BW of controls at 20 weeks (mean = 44.33 g +/- 9.14 S.D.). Body weight by 20 weeks of age was affected by sex. Males (mean = 48.68 +/- 7.24 g) weigh significantly more than females (mean 40.58 +/- 9.35 9) when examined by a Wilcoxon Rank Sum W test (p<0.0001). Sex had an effect on adult weight-specific lipid free BAT dry weight for interscapular but not squamosal BAT. LFDW/g was significantly higher in females than in males for interscapular BAT (p=0.012). Although squamosal BAT showed the same trend (females: mean = 0.1803 mg/g +/- 0.0528; males: mean = 0.1633 mg/g +/- 0.384), it was not significant when examined by Wilcoxon Rank Sum W test. 84 Lipid weight per lipid-free BAT weight was not affected by sex. Due to variance heterogeneity, interscapular lipid weight per lipid-free BAT dry weight was examined nonparametrically and found not to be significantly affected by sex by Wilcoxon Rank Sum W test. Likewise, sex had no effect on squamosal lipid content per lipid-free deposit weight, as determined by one-way ANOVA. DISCUSSION I. SHORT-TERM STUDY As appears to be true of other altricial rodent species examined previously, 21-day-old nestling prairie voles exposed to cold during the postnatal period have higher weight-specific lipid-free BAT weights than those maintained at nest temperature. Because no BAT measurements were taken at an earlier stage of development under these environmental conditions, it is unclear whether the difference at 21 d reflects enhanced recruitment or interruption of involution in M1 ochrogaster, although studies on altricial murine rodent nestlings suggest the difference reflects mostly the latter. In postnatal rats at regular laboratory temperatures, wet BAT weights expressed per neonate surface area (i.e., 85 per gram body weight0'67) peak around 5 d, followed by rapid decline (Nedergaard et al., 1986). This initial decline in wet BAT weight, however, primarily reflects a depletion of BAT lipid stores in response to cold exposure at birth; more direct measurements of thermogenic activity (e.g., cytochrome c oxidase activity) suggest that postnatal BAT involution occurs more gradually, coinciding with the onset of shivering around 9 d (Mouroux et al., 1990; Gordon, 1993). Postnatal prairie voles in this study visibly shivered following cold exposure at approximately 8 days of age (occasionally seen as well in controls at about the same age). Thus if involution occurs concomitantly with shivering onset in prairie voles, as seen in murine rodents, then the difference in weight-specific lipid-free BAT dry weights between cold-treated and control littermates in the present study probably reflects involution in control nestlings which has been at least partially delayed in cold—exposed littermates. Regarding nestling lipid weight per lipid—free BAT dry weight, the results of the present study again provide only a static view of differences between cold-exposed and control nestlings, but are consistent with previous findings of lower BAT lipid content associated with higher thermogenic activity in response to cold rearing (e.g., 86 Senault et al., 1982, Mouroux et al, 1990). It is unclear why squamosal BAT deposits did not exhibit this pattern; the retention of lipid in squamosal BAT deposits relative to lipid-free dry weight in cold—treated nestlings in this study (also seen in litter average responses to postnatal cold exposure during paternal absence, as discussed later in the study of paternal care effects on BAT development), may reflect underlying functional specialization between different BAT deposits (discussed later in the study of response relationships among deposits). In general, lipid—free BAT dry weight appears not to depend on nestling body weight. This was an unexpected result which might raise the question of whether weight- specific measurements of lipid-free BAT dry weight are an appropriate variable for assessing recruitment responses in nestlings. However, there is a trend for control nestlings of lower body weight to have lower lipid-free BAT dry weight, and the fact that the regression is significant for only interscapular BAT may be partly a function of the narrow range of body weight values observed for nestlings in the control group and the small sample size for residual BAT; both factors would reduce the power to detect a regression which deviates from a slope of zero (Figure 9). 87 Other factors are likely to operate in cold-treated individuals to mask an underlying relationship between lipid-free BAT dry weight and body weight. Cold-treated nestlings span a wider body weight range (Figure 9). Compared to the regression for controls, small cold- treated nestlings often appear to have relatively high lipid-free interscapular and squamosal BAT dry weights (Figures 8 and 10, respectively), reducing the slope of the regression to a nonsignificant value. In other words, for the low ambient temperature applied in the present study, the positive relationship between body weight and the absolute dry weight of lipid-free BAT may be counterbalanced by an inverse relationship between sensitivity to cold (and thus the magnitude of BAT recruitment) and body weight. Both inter- and intraspecifically, smaller mammals tend to show larger BAT-mediated responses to cold (Horwitz, 1989) due to increased rates of heat loss across a relatively large surface area and greater limits on insulation (Cannon, 1995). Smaller nestling voles therefore may develop relatively large interscapular and squamosal BAT deposits in response to cold. However, a single measure of lipid- free BAT dry weight does not provide information on the relative extent of BAT recruitment for a given nestling. Furthermore, the residual BAT lipid-free BAT dry weight is explained to a significant extent by the nestling's body 88 weight, although this may reflect a difference between deposits in response to cold. The dependence of lipid weight per lipid-free BAT weight on body weight in cold-treated nestlings, with larger animals displaying higher BAT lipid content, could be interpreted as further evidence for relatively enhanced thermogenic activity in smaller individuals. Alternatively, heavier individuals might have similar thermogenic demands but relatively large lipid stores. Body size differences may have less of an effect on lipid stores in the absence of thermal stress, resulting in a nonsignificant relationship between lipid weight per lipid-free BAT dry weight and body weight for nestlings treated at nest temperatures. Additionally, the narrow range of body weight values for control animals may have limited the power of the regression slope test. This latter explanation seems more likely in light of the significant relationship found between lipid content per lipid-free deposit and body weight in adults in the absence of cold-treatment effects, discussed below. In most instances, lipid content is a function of BAT deposit size. Although cold-exposed nestling BAT generally becomes more depleted of lipid than BAT in controls, the weight of lipid per lipid-free tissue dry weight varies in a predictable fashion irrespective of 89 treatment; larger deposits have more lipid than smaller deposits. Interestingly, periodic cold exposure resulted in lower body weight between 19 and 20 days of age, suggesting that either nestling vole growth becomes limited by energy supplies during cold exposure, or that periodic hypothermia slows growth by thermally slowing key biochemical processes. As will be discussed, these hypotheses are not mutually exclusive. Perrigo and Bronson (1985) found that adult house mice (MUS domesticus, which, like nestling prairie voles, lack the energy-conserving mechanisms of torpor and food hoarding) direct assimilated energy preferentially towards meeting primary demands of thermoregulation, maintenance, and food-gathering, before investing surplus in growth, fat storage, and other secondary demands. The growth (or maintenance) of BAT in cold-exposed nestling voles in the present study was observed simultaneously with lower body weights, suggesting that limited energy supplies might be allocated to maintenance of thermoregulation preferentially instead of growth in cold-stressed nestlings. If energy supply in the form of milk was inadequate in the present study to meet both primary and secondary demands, nutritional supplementation would be expected to curtail the weight loss by permitting 90 allocation of extra assimilated energy to growth. As will be discussed, however, nestling energy supply appeared to be unlimited by about 16 days of age. A second hypothesis to explain the concurrent reduction of body weight and increase in lipid-free BAT dry weight is that biochemical processes which regulate postnatal body growth are more inhibited by low body temperatures than are processes which regulate postnatal BAT development. Isolated neonatal altricial rodents, which cannot defend their body temperature in the face of severe thermal stress, are subject to what Barcroft termed the "tyranny of the Arrhenius equation" (in Hill and Wyse, 1989); their metabolic rates fluctuate with ambient temperature. In spite of an adequate resource supply to meet the energetic requirements of body growth, a reduction of molecular kinetic energy due to lower body temperature could slow rate-limiting enzyme—catalyzed reactions necessary for growth, resulting in stunting. At the same time, stimulation of NE release in response to low body temperature could result in enhanced BAT recruitment. Presumably, a failure of nutritional supplementation to equalize growth rates among cold- treated and control nestlings would indicate that those systems which regulate body growth in nestling prairie voles are unable to acclimate to hypothermia (e.g., by quantitative or kinetic modulation of key enzymes). 91 The two hypotheses are not mutually exclusive. The latter hypothesis might secondarily entail cold-induced energy limitation: in spite of adequate energy supply, energy assimilation processes may be slowed by low body temperature. The resulting energy limitation could result in preferential energy allocation to thermoregulation as in the first hypothesis. Furthermore, the two proposed growth-stunting mechanisms might exert their effects at different stages of postnatal development. At the end of the three hour cold treatment, nestlings generally were inactive and felt cool to the touch until about 10 days of age, whereas by about 16 days of age, they typically were active and felt warm to the touch after cold treatment (personal observation). Thus, nestling prairie voles in this study appeared to be unable to defend body temperature before 10 days of age during periodic exposures to low ambient temperature. This inability might result in the slowing of biochemical processes in the early postnatal period, stunting growth. Furthermore, as nestlings develop the ability to defend body temperature during cold exposure, energy costs should increase. This increase in primary energy demands (i.e. thermoregulation) later in the postnatal period might result in the allocation of limited energy away from secondary demands, including growth. 92 Since no significant differences in body weight were observed until 19 to 20 days of age, it appears unlikely that biochemical processes affecting growth are slowed substantially in response to cold exposure during early postnatal development in M: ochrogaster. The remaining hypothesis (that energy limitation results in allocation of assimilated energy away from growth) requires further examination, however, because nestling voles were observed to consume solid food -- an unlimited energy supply —- by about 13 days of age. Thus, it remains unclear why cold-treated nestlings show a reduction in body weight by 19 to 20 days of age. Food quality and the ontogeny of digestive physiology might be important; nestlings may not be able to assimilate enough energy from solid foods by 20 days of age to sustain body growth under thermally stressful conditions. It is also possible that cold exposure negatively affects energy intake in nestling voles by influencing feeding performance, appetite, or satiety. Quantification of energy consumed and assimilated by nestlings would provide insight into these factors. A posteriori examination revealed no significant effects of sex and litter size on any of the variables examined in this study. Because measurements were taken on prereproductive nestlings in the short-term study, the 93 lack of sex effects is not surprising. It is possible that the litter size range observed for M1 ochrogaster in the present study (2 to 8 nestlings per litter) was too narrow to permit detection of litter size effects, as might be seen in rodent species which bear larger litters (e.g., ML domesticus). II. LONG-TERM STUDY Contrary to the results of previous studies on lab rats and mice (Lynch et al., 1976; Lacy et at., 1978; Doi and Kuroshima, 1979), no lasting effect of early cold exposure is observed in M1 ochrogaster lipid-free BAT dry weight in response to postnatal cold exposure. This unexpected result may reflect idiosyncrasies in method between the present study and previous studies, or real variation between murine and microtine rodent recruitment and involution responses. Both possibilities shall be considered. The cold-treatment temperature used in the present study (10°C) was higher than that applied in previous research on lasting cold effects on NST thermoregulatory patterns (5°C: Lynch et al., 1976; Lacy et al., 1978; Doi and Kuroshima 1979), and exposures were periodic, as opposed to the continuous exposure used by Lynch et al. (1976) and Lacy et a1. (1978). However, nestling mice 94 were housed with mothers, littermates, and nesting material in previous studies (Lynch et al., 1976; Lacy et al., 1978), which likely decreased the cold stress experienced by the pups. (It is unclear whether rats used in the investigation by Doi and Kuroshima in 1979 were treated in isolation or with littermates; the authors merely state that litters were separated into two groups for daily treatment at 5 or 25°C.) Furthermore, it was shown earlier in the short—term study that 10°C is sufficient to cause significant recruitment in nestling prairie voles. Differences in the length of postnatal treatment (25 days: Lynch et al., 1976; 70 days: Lacy et al., 1978; 14 days: Doi and Kuroshima, 1979; versus 20 days in the present study) and the age at examination (50— 56 days: Lynch et al., 1976; 115 days: Lacy et al., 1978; 133 days: Doi and Kuroshima, 1979; versus 137-143 days in the present study) may have affected the present outcome, although these differences would appear to be too insignificant to fully account for the lack of detectable response in adult prairie voles. For instance, the length of treatment and age at dissection of prairie voles were within a week of those used by Doi and Kuroshima (1979). It is possible, albeit unlikely, that the ambient temperature at which prairie voles were housed until 20 weeks of age (21°C) may have masked some of the effects of early cold exposure in one of two ways. Incidental mild 95 hypothermia induced by colony room temperature, which is usually below the thermoneutral zone of small mammals, can confound BAT studies by stimulating recruitment in supposedly non-cold-treated animals (Himms-Hagen, 1986). Wunder et al. (1977) found that the thermoneutral zone for adult prairie voles varies between 27 and 34°C. Although littermates and nesting material with which weaned voles were housed after postnatal treatment probably raised the effective ambient temperature, controls in the long-term study may have been partially cold acclimated, which could have masked lasting differences caused by early environmental temperature, if these differences were weak. Of course, such weak lasting effects of early cold would probably be biologically unimportant. On the other hand, if postnatal cold exposure enhances the capacity to respond to cold later in life by preparing BAT for a more rapid or complete recruitment in response to subsequent cold, the temperature to which adults were acclimated may have been too close to thermoneutrality to detect such a predisposition. It seems unlikely that such methodological idiosyncrasies affected the results of the present study, however, since lasting recruitment responses were observed in previous studies when rats or lab mice were housed at similar ambient temperatures in the weeks prior to dissection. Therefore, long-term BAT responses to 96 postnatal cold stress in the present study do not appear to differ from past investigations due to idiosyncrasies of method. It is not possible to determine from the results presented whether murine rodents examined in the previous studies exhibited greater differences at the time of weaning in recruitment between postnatal cold-treated versus control animals than M: ochrogaster did in the present study. If preweaning responses to cold are greater in murine than in microtine rodents, this difference may partly explain the persistences of early— cold-exposure effects observed in murine but not microtine rodents. On the other hand, it is possible that BAT recruitment in M1 ochrogaster differs from that in lab rats and mice in that it does not exhibit permanent changes in response to early cold. Lynch et al. (1976) and Lacy et al. (1978) were interested in delineating genetic and environmentally- imposed thermoregulatory components of an individual’s phenotype so that quantitative genetic analyses could be applied to the genetic components to study evolutionary adaptation. In this context, irreversible developmental responses observed in lipid-free BAT dry weight were viewed mainly as nuisances to investigators wishing to perform comparative evolutionary studies on wild-caught 97 species. However, as shown in the present study, the flexibility of BAT recruitment and involution may vary among species. It would seem energetically costly, and thus maladaptive, for BAT to be maintained permanently in an active state in response to early cold stress when it can be recruited later in life when necessary. Thus the permanent response observed in lab rats and mice but not in prairie voles may indicate that arvicoline rodents have evolved energy-saving (and thus adaptive) phenotypic plasticity in a thermoregulatory trait which displays developmental irreversibility in murine rodents. As was expected, postnatal cold exposure had no lasting effect on lipid weight per lipid-free BAT dry weight in either interscapular or squamosal deposits in adult prairie voles. Although this prediction was based on the rapid replenishment of lipid stores observed previously in BAT of cold-acclimated adult rats (Cameron and Smith, 1964), lipid levels in adult prairie voles reared with periodic exposure to cold may equal those of adults reared at warmer temperatures due to the lack of permanent BAT recruitment effects in this species as described above. Adult lipid-free BAT dry weight depends on adult body weight. This relationship is clearly displayed in adults in the long—term study, in contrast to nestlings in the 98 short-term study, probably because the sample size was larger and because the effects of early cold—exposure did not significantly influence the relationship (Figure 22). Thus, in the absence of treatment effects, heavier adult voles have heavier lipid-free BAT deposits. Likewise, the amount of lipid per lipid-free BAT deposit varies as a function of adult body weight in this species. As was presupposed, larger adult prairie voles have more lipid per BAT deposit than smaller animals when acclimated as adults to ambient temperatures near thermoneutrality, regardless of postnatal thermal conditions. A predisposition towards lipid storage might have resulted concurrently in higher body weight and higher lipid weight per lipid-free BAT dry weight. Body composition analyses could be conducted to test this hypothesis, which would be supported by evidence of relatively high lipid levels in heavier individuals. As seen in the short-term study, the lipid weight of a BAT deposit increases with lipid-free BAT dry weight in adult prairie voles, irrespective of rearing temperature. This observation might reflect a relationship between the lipid content of BAT cells and their volume at a given level of thermogenic activity in a well-provisioned adult animal, such that an increase in cell number or size leads to a rise in lipid-free BAT dry weight and total lipid 99 content. Body weight, however, should affect this relationship, and presumably accounts for some of the variation seen in Figures 24 and 25. In contrast to findings from the short-term study, a posteriori observation revealed that adult body weight was not affected by postnatal cold exposure, but was affected by sex. The latter finding corroborates the observations of sex effects on body weight in mice by Lacy et al. (1978); male M1 domesticus were found to weigh more than females. Unlike the present study, however, Lacy et al. found no relationship between body weight and lipid-free BAT dry weight, which led them to believe that sex differences they observed in thermoregulatory traits reflected fundamental differences between male and female thermoregulatory mechanisms. Lacy et al. found that females scored higher than males on NST indices including weight-specific lipid-free BAT dry weight of the interscapular deposit. Although lipid-free BAT dry weight was found to increase with body weight in the present study, adult male prairie voles, which were heavier than adult females, had less weight-specific lipid-free BAT in the interscapular deposit than did females (squamosal BAT showed a similar, though nonsignificant, trend). This discovery supports and extends to adult microtine rodents 100 the interpretation of Lacy et al. (1978) that sex affects thermoregulation via mechanisms other than the effect sex has on body weight. The amount of lipid per lipid-free BAT weight, on the other hand, was found not to be affected by sex for either interscapular or squamosal deposits. Thus in spite of the fact that adult female prairie voles were smaller and had relatively high levels of thermogenically active BAT (measured as weight-specific lipid—free BAT dry weight), the lipid concentration in BAT of adult females was not depleted to a greater extent than males under the conditions of the present study. It would be interesting to determine whether adult females are able to maintain BAT lipid stores to the same extent as males under thermally or energetically stressful conditions as well. EFFECTS OF PATERNAL CARE ON BAT DEVELOPMENT GENERAL OVERVIEW OF THE PROBLEM: POSTNATAL BAT RECRUITMENT IN RESPONSE TO PATERNAL CARE The goal of this study was to determine whether postnatal brown adipose tissue (BAT) recruitment is reduced in altricial nestlings when paternal care is available during periodic cold exposure in a free-ranging 101 microtine rodent, the prairie vole (Microtus ochrogaster). This temperate-zone species is likely to experience thermal stress during development. Recent studies have shown that paternal care in this species facilitates offspring development, but the mechanisms by which paternal care aids postnatal development are unclear. It has been demonstrated in the first section of this thesis that BAT recruitment in nestling prairie voles is enhanced by periodic exposure to low ambient temperatures during the first 20 days postpartum. For studies of paternal care, BAT recruitment may thus be useful as an index of both thermoregulatory ontogeny (specifically, NST) and thermal stress experienced by nestlings in the presence or absence of their father during periodic postnatal cold exposure. MONOGAMY AND PATERNAL CARE IN MICROTINE RODENTS An especially intriguing aspect of the biology of M2 ochrogaster is its monogamous mating system, in which both parents share responsibilities of rearing young. Juveniles remain in the natal nest after weaning and also assist in rearing the next litter (Gruder-Adams and Getz, 1985; Wang and Novak, 1992; Solomon, 1994). Alloparental care by juveniles results in higher body weights and earlier eye opening in the next litter (Solomon, 1991; Solomon, 1994). 102 Wang and Novak (1992) recently demonstrated that paternal care facilitates development of lab-reared neonatal prairie voles maintained on ad lib food, at 20°C ambient temperature. They found that nestlings raised with both parents ate solid food and exited the nest sooner than those raised in the absence of fathers, even when juveniles were present. Although not a statistically significant difference, eye opening tended to occur earlier in nestlings with fathers present. Several entire litters died in the absence of the father and were excluded from analysis. The remaining litters showed no differences in either nestling weight at 20 days, or pup mortality. Paternal care seems likely to have more profound effects on nestling survivorship under natural conditions, which might result in selection for paternal investment, a concept known as the Male Care Hypothesis (Kleiman, 1977). It was demonstrated by Gubernick et al. (1993) that, in cold (8.5-10.50C) conditions or when parents were required to forage for food (by means of a dispenser linked to a running wheel), nestling survival in the California mouse (Peromyscus californicus) was substantially reduced when the father was absent. P. californicus, like M1 ochrogaster, is noteworthy for exhibiting exceptional levels of paternal care relative to other species of its genus. 103 Developmental acceleration by paternal care may be mediated by thermoregulatory enhancement. Thus a goal of this study was to evaluate the influence of the father’s presence on the development of BAT in M3 ochrogaster. Because brown adipose tissue simultaneously affects thermoregulation and responds to thermal stimuli in quantifiable ways, it provides a means to investigate whether thermoregulatory aspects of paternal care may impinge on the development of thermoregulatory systems in postnatal nestlings. If energy supplies limit growth and maturation among altricial rodents such that tradeoffs in energy allocation exist, the effect of paternal care on BAT recruitment might have profound effects on other developmental parameters. OBJECTIVES AND EXPERIMENTAL DESIGN To test the hypothesis that the absence of the father during exposure to periodic postnatal cold has a stimulatory effect on BAT recruitment in nestlings, I exposed lab-reared M} ochrogaster litters from the day after birth through 20 days of age to periodic low ambient temperatures, with or without the father present. Litter averages for several variables were examined. I compared lipid—free, weight-specific BAT weights and lipid weights per lipid free dry BAT weight in the two treatments. Based on earlier observations, I hypothesized that the 104 average lipid-free dry BAT weight per gram body weight for litters with fathers absent would be higher than the average litter value with fathers present (controls). I further hypothesized, based on the earlier studies on lipid content and postnatal cold exposure, that father- absent litters would have lower average lipid weights per lipid-free dry BAT weight than would controls. I also examined several presuppositions underlying these hypotheses. I presumed that lipid—free BAT dry weight and lipid weight per lipid-free BAT weight would correlate positively with body weight. I also presumed that the lipid content of a BAT deposit would correlate positively with its lipid—free dry weight. METHODS ANIMAL SUBJECTS AND HUSBANDRY The animals used were laboratory—born prairie voles (M2 ochrogaster) from a colony at Michigan State University established in 1991 from animals originally captured near Urbana, Illinois. Throughout the study, all breeding pairs were housed together at 21°C on a 16:8 light:dark cycle in plastic cages (38x33x16 cm) provided with a standardized volume (about 960 mL) of corn cob bedding (Andersons Industrial Products) and a standardized 105 weight (44-46 g) of aspen laboratory wood shavings (Northeastern Products Corp.). Parents typically built nests from the shavings. Food (Teklad Rodent Diet 8640 and Rabbit Diet) and water were provided ad lib. PROCEDURE Two litters from each breeding unit (mother and father) were assigned to father-present (FP) or father- absent (FA) treatment groups. These two litters were paired in a randomized complete block design to control for variation among breeding units. To control for possible parity effects, the order of assignment (i.e. FP first or FA first) was determined randomly, such that about half of the pairs (13 out of 28) were assigned FA treatment first, and the other half FP first. Most litters in the study for both FA and PP treatments (23 of 25 pairs) were from multiparous parents. Fathers from FA- assigned litters were removed from the cage to a holding chamber provided with ad lib food and water during cold treatment, whereas fathers from FP-assigned litters were left in the cage during cold treatment. To control for possible disturbance effects, fathers from FP—assigned litters were handled briefly and returned to the cage prior to and following cold treatment. For both treatment groups, the entire cage (including the mother) was exposed from 1 day of age through 15 d for 5 h/d during the 106 lights-on period to 10°C. During all non-treatment hours, both parents remained in the cage with the litter. Attempts were made at all times to minimize disturbance. Two incubators were used in this study for cold treatments. However, all litters from a given breeding unit were treated in a single incubator, so that incubator differences would be controlled by the blocked design. At 16 d of age, litters were euthanized with C02, weighed and sexed, and BAT deposits were removed under a dissecting microscope, placed in numbered glass vials and frozen for later drying, lipid extraction and weighing. All nestlings in a litter were dissected up to a total of four; litters with more than four young were briefly sorted by sex just prior to the first dissection, and four animals were chosen at random while balancing the numbers of each sex whenever possible. A total of 210 animals was dissected. Mean litter size was 4.94 (+/- 1.53 SD). The interscapular and squamo-occipito—cervical deposits of BAT were located as described by Rauch and Hayward (1969) and dissected from each animal (the squamo-occipito-cervical deposit is hereafter called simply squamosal). BAT samples were dried in a drying oven at 55°C until they reached a constant weight (about 24 hours). Lipids were extracted from the tissue samples per Lacy et al. (1978) using two changes of anhydrous ethyl ether (J. T. Baker, Inc.). It was determined in preliminary trials that two 107 changes of ether produce a constant lipid-free dry weight which remains unaltered after additional ether changes. DESIGN AND ANALYSIS Litters from the same parents and representing each treatment were paired in a randomized complete block design (n=27 pairs). Several litters could not be paired and thus were left out of the analysis. To avoid non- orthogonality effects caused by unequal litter sizes (disproportional, unequal subclasses), paired comparisons were performed on litter means. Thus, for all variables, the average response of the litter was examined in this study, as opposed to individual responses or variability of individual responses within litters. Paired comparisons were performed using a two-way ANOVA to permit investigation of blocking effects in addition to treatment effects. Normality was examined using normal probability plots, and homogeneity of variances was evaluated using Cochran’s C and Bartlett-Box F tests (SPSS PC+). Data were transformed using natural logarithms when such transformations were found to enhance normality and variance homogeneity. Outliers were identified by Grubb’s test per Sokal and Rohlf (1995) and removed prior to analysis. Data which remained heteroscedastic or non- normal after these attempts were analyzed using a Wilcoxon 108 matched-pairs signed-ranks test, a nonparametric test for paired—comparisons (SPSS PC+). The weight-specific, lipid-free dry BAT weight (LFDW/g), and the lipid weight per lipid-free BAT weight (LW/LFDW) were compared between treatments within pairs using the MANOVA procedure in SPSS PC+ (unless a nonparametric test had to be used). Scattergrams were plotted to facilitate visual assessment of differences between father-absent and father-present litters within pairs of litters. Least squares linear regression analysis was used to test presuppositions by examination of functional relationships between body weight and both lipid-free BAT dry weight and lipid weight per lipid-free BAT weight, and between the lipid content of a BAT deposit and lipid-free dry weight. Because rigorous use of linear regression implies assumptions which are often not met in functions among physiological variables (e.g., "independent" variables are often not fixed), correlation analysis was also employed to evaluate the degree of association among variables. Because the correlation results generally were commensurate with the results of the regression analyses, only the regression results are reported. 109 RESULTS A PRIORI COMPARISONS The following results respond to the a priori hypotheses presented in the OBJECTIVES section. The hypothesis that paternal absence during periodic postnatal cold-exposure increases the weight-specific lipid-free BAT dry weight in litters was supported. Paternal absence had a significant stimulatory effect on LFDW/g litter averages for both interscapular and squamosal BAT deposits, as seen in Table 3. Figures 26 and 27 indicate that about 77 percent (20 of 26, one outlier pair removed) of the litter pairs had higher LFDW/g values for father-absent than father-present treatments for interscapular BAT, and about 67 percent (18 of 27) had higher father-absent that father-present LFDW/g for squamosal BAT. Significant variance existed among pairs of litters for interscapular and squamosal BAT LFDW/g (Table 3). The hypothesis that paternal absence results in a depletion of nestling BAT lipid per lipid-free BAT dry weight, measured as litter averages, was only partly supported. Results are presented in Table 3 and Figures 28 and 29. Interscapular BAT LW/LFDW was significantly 110 Table 3. Results from the paternal-care study. N is the number of pairs of litters (father-absent and father- present) analyzed. Standard deviation (SD) is given in parentheses. The probability for treatment ("trtment") is the likelihood of no difference between father-absent (FA) and father—present (FP) litters. That for pairs is the likelihood that none of the overall variance was attributable to differences among pairs. NPAR indicates that the Wilcoxon test (a nonparametric test) was used; this test yields no probability for pairs. LFDW/g = weight- specific lipid-free BAT dry weight. LW/LFDW = lipid weight per lipid—free BAT dry weight. Mean (SD) Probability Variable N FA FP Trtment Pairs LFDW/g ImeLq). Interscapular 26 0.67 (0.12) 0.57 (0.14) 0.001 <0.03 Squamosal 27 0.43 (0.06) 0.38 (0.08) 0.009 0.03 LW/ LFDW lgLal Interscapular 26 4.33 (1.01) 4.98 (1.09) 0.016 <0.lO Squamosal 28 3.01 (0.62) 3.26 (1.09) <0.388 NPAR 111 Figure 26. Interscapular weight-specific lipid-free BAT dry weight (LFDW/g) for father-absent (FA) and father-present (FP) litters of pairs in the paternal-care study. The line marked "null" indicates expected location of points for each pair if there were no differences among FA and PP litters for LFDW/g. 112 Figure 26 «fit...— hzwmmmn—imlhxl Z. 33:: 0\>>Du3 o. _. md md No @d md v.0 md Nd to 0.0 a a _ . _ . _ q 1 . 4 q _ d 4 _ //94r m_095 mm<0-._DD._.m mm<0-._>D..._._\>>._ n m m w m N F _ . _ . _ . a . _ . I . _ m_095 mm<0-._DD._.m wm<0-._>._.m_m ZO_._.<._wm >QD._.m 2mm...-...mOIw Nd Vd Nd _.d md md 0d wd dd 0... 1V8 'Ivsowvnos 80:! (ti/Btu) S/MCH'I 136 Figure 31. Short-term study: Correlation between weight— specific lipid—free BAT dry weight (LFDW/9) values for squamosal and residual BAT in cold-treated (closed circles) and control animals (open circles). 137 Figure 31 55 ._DDNm 2mm...-...mOIw 1V8 1vncnsau 30:: (film!) o/Mcm 138 Figure 32. Short-term study: Correlation between weight- specific lipid-free BAT dry weight (LFDW/g) values for interscapular and residual BAT in cold—treated (closed circles) and control animals (open circles). 139 Figure 32 bad m<1_Dn_>n_..._1_ N. P F. _. o. _. ad ad Nd md md ed _ . _ . _ . _ . _ . 4 . _ . _ O O o N o o o 1 O O O O O O C e e 1 m C. O O 1 v 0 7.35.500 O “Ogden—-200 o ._.n_D._.m EmwN-._.mOIw 1V8 1vncusau HO:| (fi/fiw) 9/MCH'I 140 Figure 33. Long-term study: Correlation between weight- specific lipid-free BAT dry weight (LFDW/g) values for interscapular and squamosal BAT in cold—treated (closed circles) and control animals (open circles). 141 Em m>Nmm ZO_._.<._mm >DD._.w SEW—.-.OZOJ 1V8 1vsowvnos HOd (fi/fiw) O/MCH'I 142 Figure 34. Paternal—care study: Correlation between weight-specific lipid-free BAT dry weight (LFDW/g) values for interscapular and squamosal BAT in father-absent (closed circles) and father-present litters (open circles). 143 Em $425232. mo“. see 939: 4 o. F ad ad Nd md md vd md Nd 3 _ . _ . _ . _ . _ . _ . _ . _ . m U o g H o o % e o o e o 8 o a. o 00 be. e oo 00 o o e e o o 00 O O 0’ 0 0 e e o o o . Emmma 55m... 0 o o o Emmom 55m“. . ._.>._.mm ZO_N<._mm >DDNm mm<0-1_>._.wm ZO_._.<._mm >DDNw EEmNNmOIm 1V8 1vnolsaa 80:! (5/5)Mc1:rI/M'I 147 Figure 36. Short-term study: Correlation between lipid weight per lipid—free BAT dry weight (LW/LFDW) values for interscapular and squamosal BAT in cold- treated (closed circles) and control animals (open circles). 148 Figure 36 ...(m m>Dm._\>>1_ N 0 m V m — d — 4 — E q + — 4 .0 .0 o oo o o .2280 0030:1200 ._.095 2mm...-...mOIm 1V8 'Ivsowvnos 80:! (5/5)Mc1;n/M1 149 Figure 37. Short—term study: Correlation between lipid weight per lipid-free BAT dry weight (LW/LFDW) values for residual and squamosal BAT in cold- treated (closed circles) and control animals (open circles). 150 Figure 37 Em ._<:0_mmm mo“. 35 305%.. o m w m 0 O 00 O O O a 00 e O O O O O O .9200 o . 0280-200 . ...(m 1_>._.m_m ZO_._.<._mm >095 EEMN-._.EOIw 1V8 1vsowvnos 80:! (5/5) MCI:I'|/M'i 151 Figure 38. Long-term study: Correlation between lipid weight per lipid-free BAT dry weight (LW/LFDW) values for interscapular and squamosal BAT in cold- treated (closed circles) and control animals (open circles). 152 N43 m<1_3n_>0n_1_:s1. 3 mm cu m. m. I S S m m m 3 I . _ 1 q . _ . _ . _ . _ _ _ m U 00 1 .m. 00 F. e e 0%00 See ”a 1 O 0 .0000 b000 O I oo o o e o e o o o o o 1 e ee 0 o 1 o m Ame 00W 00 o e e 1 o 8 o o m o 1 e o o 1 o e 00 e o 1 e . J oo o .2200 . . . 8522-260 ._.095 SENEOZOJ OGQNOLDVO‘JN F F F 1V8 'lVSOWVi'iOS 110:! (5/5) Mflzl'l/M‘l 153 Figure 39. Paternal—care study: Correlation between lipid weight per lipid-free BAT dry weight (LW/LFDW) values for interscapular and squamosal BAT in father-absent (closed circles) and father-present litters (open circles). 154 Figure 39 Em $505055. mo“. 33 2,2230 m N 0 m w m _ a A q — . _ . _ . d o 0 C O o o o O O O 000 00 00 00 e e e O O O o O O. O ‘ 0000 o 60 o o 2.0020 522...“. 0 E300 00.2mm. . ...(m 120053500 0Z< m<1_Dm>._.wm ZO_._.<1_m_¢ >09_.m mm<0-1_00 mwmu-de “.0 29.—.029... < m< ...Igm; 0E: mm._O> 400N200 ”>09_..w Samba-.0010 cod 5d mod vod mod Nod mod mod (5) M1 157 Figure 41. Short-term study: Lipid weight (LW) plotted against lipid—free dry weight (LFDW) for interscapular (closed circles) and squamosal BAT (open circles) for cold-treated animals. .0. 2.0“... mNod mNod FNod o—od Nwod mad m—od ..wod mood Nood mood . . _ u _ . q . q q _ . _ . — a — . q Figure 41 O 158 . ._.00 wwmu-Qafi ”.0 ZOE-023“. < m< N..—OE; 0E... wm1.0> 0m.._.095 EmmN-NEOIw l ood Nod vod ood mod 0 Pd N ...o v ...o (5) M‘l 159 Figure 42. Long—term study: Lipid weight (LW) plotted against lipid-free dry weight (LFDW) for interscapular (closed circles) and squamosal BAT (open circles) for control animals. 160 Figure 42 .9 2,0“: omod mNod ONod m 5.0 o 5.0 mood _ o o§emmvbwowéo o badooaooo Ed .mmoegam . :0 5.3000220. 110111 + 0.500000 sz0wmn=0 Z. NICE; >00 00000.0... ".0 200.023”. 4. m< 000.03 0.0.1. mm..O> 1.000.200 H>0D._.m 5.00.76201. l oood od Nd md vd md (5)M'1 161 Figure 43. Long—term study: Lipid weight (LW) plotted against lipid-free dry weight (LFDW) for interscapular (closed circles) and squamosal BAT (open circles) for cold-treated animals. 162 a. 2.0“: 3.0 8.0 No.0 5.0 _ 4 _ . _ F19ure 43 0 0.99 £0 . ood N00 00000.0... 00 200-0230 < w< 0.10.03 0.0.... m01_O> 00N<00._.-01.OO H>095 200-7020.. (5)M'1 163 Figure 44. Paternal-care study: Lipid weight (LW) plotted against lipid-free dry weight (LFDW) for interscapular (closed circles) and squamosal BAT (open circles) for father-absent (FA) litters. 164 Figure 44 a. so“: 0Nod 05.0 05.0 v—od N_-o.o owod wood wood wood Nood 4 . . _ my - 0 O O . O -..00 00000.0... 00 200-0200 < 0< 0.10.03 0.0... 000NN... ...Z000<-00I-_.<0 U>09..0 0000 0000-0.0... 0O ZOE-02:0 < 04. 0.10.02, 0.0.... 0000-00.. 0200000001520 H>09F0 00(0-..00 00000.0... 00 20.0.0230 < 0< 0.10.0.5 0.0... 00..O> 400N200 n>09..0 200050-0010 ood mod o ...o m to ONd mNd omd (5) M1 169 Figure 47. Short-term study: Lipid weight (LW) plotted against lipid—free dry weight (LFDW) for interscapular (closed circles), squamosal (open circles) and residual (triangles) BAT for cold- treated animals. 170 a. 2.2. od ...o Nd 0d vd 7 mod mod no.0 00.0 mod vod mod No.0 5.0 00.0 4 _ _ _ . . . _ _ _ . _ . _ . _ q . _ e m g .H 4 4\\ O \ \ \ ‘\ <\\ ‘\\ \\4 \\ t 4 \\ \ \\\ \ Em 3.6.9.0 .-...- 4 N00 00000.0: 00 ZOE-02.1.0 < 0< 0.1.0.03 0.0... 00..O> 00._-<00-..-0._OO H>09_-0 2000-00010 0.0 (5) M‘I 171 examining scattergrams for the a priori comparisons just described, it was noticed that lipid weight relative to lipid-free BAT dry weight appeared to differ consistently between deposit types across the short-term, long-term, and paternal—care studies. To explore this possibility, regressions of LW against LFDW for different deposit types were plotted together. As seen in Figures 40-45, in every treatment in each of the three studies, interscapular tissue seemed to have higher LW per LFDW than squamosal tissue. Residual BAT may be even higher in lipid content than interscapular BAT per LFDW (Figures 46-47). DISCUSSION In an investigation of changes in BAT in response to various ambient temperatures in adult albino mice, Heldmaier (1974) observed differences in response between deposits dissected from four regions. Because he measured weights on freshly dissected tissue, however, it is difficult to interpret whether these differences represent regional BAT thermogenic specialization, or differences in lipid content or degree of hydration. The results of the present study indicate that responses in the various deposits are associated; therefore, changes in weight- specific lipid-free BAT dry weight and lipid weight per lipid-free BAT dry weight measured in the interscapular deposit probably are indicative of responses in other BAT 172 deposits in the cervical and thoracic regions. Associations among BAT deposit responses are seen in both nestlings and adults, and do not appear to depend on the degree of thermal stress in M3 ochrogaster. However, a few differences among deposits were observed as well. Interscapular tissue appears to contain an intermediate concentration of lipid per lipid—free BAT dry weight in prairie voles, regardless of age and thermal conditions. Furthermore, during cold stress in nestlings, squamosal BAT exhibits greater retention of lipid content per lipid-free BAT dry weight than interscapular BAT. These differences may reflect unique processes and functional specializations in different BAT deposits which await further study. LIST OF REFERENCES LIST OF REFERENCES Ailhaud, G., P. Grimaldi and R. Negrel. 1992. Cellular and molecular aspects of adipose tissue development. Annu. Rev. NUtr. 12: 207-233. Anderson, M. P., and J. C. Rauch. 1984. Seasonal changes in white and brown adipose tissue in Clethrionomys gapperi (red—backed vole) and in.Microtus pennsylvanicus (meadow vole). Cbmp. Biochem. Physiol. 79A(2): 305-310. Bartness, T. J., J. M. Hamilton, G. N. Wade, and B. D. Goldman. Regional differences in fat pad responses to short days in Siberian hamsters. Am. J. Physiol. 257 (26): R1533-R1540. Bertin, R., F. DeMarco, and R. Portet. 1992. Perinatal changes in the thyroxine S’deiodinase activity in the brown adipose tissue of rats; effects of rearing temperature. J. Therm. Biol. 17(4-5): 257—261. Brueck, K., and B. Wuennenberg. 1966. Influence of ambient temperature in the process of replacement of nonshivering by shivering thermogenesis during postnatal development. FEd. Proc. 25(4): 1332-1337. Cameron I. L., and R. E. Smith. 1964. Cytological responses of brown fat tissue in cold-exposed rats. J. Cell. Biol. 23: 89-100. Cannon, B. 1995. The mammalian perogative: sympathetically controlled thermogenesis. verh. Dtsch. Zool. Ges. 88.2: 191-201. Carneheim, C., J. Nedergaard, and B. Cannon. 1984. Beta- adrenergic stimulation of lipoprotein lipase in rat brown adipose tissue during acclimation to cold. Am. J. Physiol. 246: E327—E333. Chaffee, R. R. J., and J. C. Roberts. 1971. Temperature acclimation in birds and mammals. Ann. Rev. Physiol. 33: 155-197. 173 174 Desautels, M., G. Zoror-Behrens, and J. Himms-Hagen. 1978. Increased purine nucleotide binding, altered polypeptide composition, and thermogenesis in brown adipose tissue mitochondria of cold- acclimated rats. can. J3 Biochem. 56: 378-383. Doi, D., and A. Kuroshima. 1979. Lasting effect of infantile cold exposure on cold tolerance in adult rats. Jap. J. Physiol. 29: 139-150 Feil, S. and J. Rafael. 1994. Effect of acclimation temperature on the concentration of uncoupling protein and GDP binding in rat brown fat mitochondria. Eur. J. Biochem. 219: 681-691. Feist, D. D. and M. Rosenmann. 1976. Norepinephrine thermogenesis in seasonally acclimatized and cold acclimated red-backed voles in Alaska. can. J; Physiol. Pharmacol. 54: 146-153. Gemmel, R. T., A. W. Bell, and G. Alexander. 1978. Morphology of adipose cells in lambs at birth and during subsequent transition of brown to white adipose tissue in cold and warm conditions. Am. J1 Anat. 133: 143-164. Gordon, C. J. 1993. Temperature Regulation in Laboratory Rodents. Cambridge University Press. New York. 276 PP- Gruder-Adams, S., and L. L. Getz. 1985. Comparison of the mating system and paternal behavior in.Microtus ochrogaster and.Microtus pennsylvanicus. J..Mammal. 66: 165-167. Heldmaier, G. 1975a. The effect of short daily cold exposures on the development of brown adipose tissue in mice. J. COmp. Physiol. 98: 161-168. ------- 1975b. The influence of the social thermoregulation on the cold-adaptive growth of BAT in hairless and furred mice. Pflugers Arch. 355: 261-266. ------- and K. Hoffman. 1974. Melatonin stimulates growth I of brown adipose tissue. 247 (5438): 224-225. Hill, R.W. and G. A. Wyse. 1989. Animal Physiology. Harper and Row, Publishers, Inc. New York. 656 pp. Hill, R. W. 1992. The altricial/precocial contrast in the thermal relations and energetics of small mammals. In Mammalian Energetics: Interdisciplinary Views of .Metabolism and Reproduction. (T. E. Tomasi and T. H. 175 Horton, eds.), pp. 122-159. Cornell University Press., Ithaca. Himms-Hagen, J. 1986. Brown adipose tissue and cold acclimation. In Brown Adipose Tissue (P. Trayhurn and D. G. Nicholls, eds.), pp. 214-268. Edward Arnold Ltd., London. ------- 1990. Brown adipose tissue thermogenesis: interdisciplinary studies. FASEB J. 4: 2890-2898. Hissa, R. 1968. Postnatal development of thermoregulation in the Norwegian lemming and the golden hamster. Ann. Zool. Fenn. 5: 345-379. Horwitz, B. A. 1989. Biochemical mechanisms and control of cold-induced cellular thermogenesis in placental mammals. lg Advances in Cbmparative and Environmental Physiology (4): Animal Adaptation to Cold. (L. C. H. Wang, ed.), pp. 83-116. Houstek, J., U. Andersson, P. Tvrdik, J. Nedergaard, and B. Cannon. 1995. The expression of subunit c correlates with and thus may limit the biosynthesis of the mitochondrial F(sub-O)F(sub-1)-ATPase in brown adipose tissue. J. Biol. Chem. 270 (13): 7689-7694. Jansky, L. (1973). Non-shivering thermogenesis and its thermoregulatory significance. Biol. Rev. 48: 85-132. Karasov, W. H., and J. M. Diamond. 1988. Interplay between physiology and ecology in digestion. BioScience 38(9): 602-611. Keller, B. L., and C. L. Krebs. 1970. Microtus population biology. III. Reproductive changes is fluctuating populations of M1 ochrogaster and M3 pennsylvanicus in southern Indiana. 1965-1967. Ecol..Monogr. 40: 263- 294. Kleiman, D. G. 1977. Monogamy in mammals. Q. Rev. Biol. 52: 39-69. Lacy, R. C., C. B. Lynch, and G. R. Lynch. 1978. Developmental and adult acclimation effects of ambient temperature on temperature regulation of mice selected for high and low levels of nest-building. J. COmp. Physiol. 123: 185-192. Lynch, C. B. 1992. Clinal variation in cold adaptation in MUS domesticus: verification of predictions from laboratory populations. Am. Nat. 139(6): 1219-1236. 176 Lynch, G. R. 1973. Seasonal changes in thermogenesis, organ weights, and body composition in the white- footed mouse, Peromyscus leucopus. Oecologia 13: 363- 376. ------- , C. B. Lynch, M. Dube, and C. Allen. 1976. Early cold exposure: effects on behavioral and physiological thermoregulation in the house mouse, MUS musculus. Physiol. Zool. 49: 191—199 McDevitt, R. M. and J. R. Speakman. 1994. Long photophase is not a sufficient stimulus to reduce thermogenic capacity in winter-acclimatized short-tailed field voles (Microtus agrestis) during long-term cold acclimation. J. Comp. Physiol. B. 164: 159-164. Milner, R. E., A. Geloen, and P. Trayhurn. 1990. A dot immunobinding assay for the rapid quantification of uncoupling protein in brown adipose tissue mitochondria. Trans. Biochem. Soc. 18(4): 547 Mitchell, J. R. D., A. Jacobsson, T. G. Kirchgessner, M. C. Schotz, B. Cannon, and J. Nedergaard. 1992. Regulation of expression of the lipoprotein lipase gene in brown adipose tissue. Am. J. Physiol. 263 (26): 500-506. Nadeau, J. H. 1985. Ontogeny. In Biology of New WCrld .Microtus. (R. E. Tamarin, ed.), pp. 254-285. American Society of Mammalogists, Pennsylvania. Nedergaard, J., E. Connolly, and B. Cannon. 1986. Brown adipose tissue in the mammalian neonate. lg Brown Adipose Tissue (P. Trayhurn and D. G. Nicholls, eds.), Pp. 152-213. Edward Arnold Ltd., London. ------- , L. Unelius, A. Jacobsson, M. Muhleisen, P. Svoboda, and B. Cannon. 1993. Why are brown—fat cells from cold-acclimated animals not better? In Life in the Cbld: Ecological, Physiological, and Mblecular .Mechanisms (C. Carey, G. L. Florant, B. A. Wunder, and B. Horwitz, eds.) pp. 344—359. Westview Press, Boulder. ------- , and B. Cannon. 1994. Brown Adipose Tissue: Receptors and recruitment. In Temperature Regulation: Recent Physiological and Pharmacological Advances (A. S. Milton, ed.), pp. 345-350. Burkhauser Verlag, Basel. ------- , D. Herron, A. Jacobsson, S. Rehnmark, and B. Cannon. 1995. Norepinephrine as a morphogen?: its unique interaction with brown adipose tissue. Int. J. Dev. Biol. 39: 827-837. 177 Nicholls, D. G. 1976. Hamster brown-adipose-tissue mitochondria: purine nucleotide control of the ion conductance of the inner membrane, the nature of the nucleotide binding site. Eur. J. Biochem. 62: 223- 228. Obregon, M. J., A. Jacobsson, T. Kirchgessner, M. C. Schotz, B. Cannon, and J. Nedergaard. 1989. Postnatal recruitment of brown adipose tissue is induced by the cold stress experienced by the pups. An analysis of mRNA levels for thermogenin and lipoprotein lipase. Biochem. J. 259: 341-346. Perrigo, G. and F. H. Bronson. 1985. Behavioral and physiological responses of female house mice to foraging variation. Physiol. Behav. 34: 437-440. Rauch, J. C., and J. S. Hayward. 1969. Topography and vascularization of brown fat in a small nonhibernator (deer mouse, Peromyscus maniculatus). can. J. Zool. 47: 1301-1314. Reeves, G. D. 1994. Influences of photoperiod and ambient temperature on maternal-pup interactions in the meadow vole (Mficrotus pennsylvanicus). Ph.D. Dissertation. The University of Michigan. 196 pp. Rose, R. K., and M. S. Gaines. 1978. The reproductive cycle of.Microtus ochrogaster in eastern Kansas. Ecol. Monogr. 48: 21-42. Rothwell, N. J., and M. J. Stock. 1986. Brown adipose tissue and diet-induced thermogenesis. I; Brown Adipose Tissue (P. Trayhurn and D. G. Nicholls, eds.), pp. 269-298. Edward Arnold Ltd., London. Senault, C., M. Solier, M. Beauvallet, and R. Portet. 1982. Cold-induced changes in fatty acid composition of rat brown fat during the perinatal period. EXperientia 38: 585-587. Solomon, N. G. 1991. Current indirect fitness benefits associated with philopatry in juvenile prairie voles. Behav. Ecol. Sociobiol. 29: 277-282. ------- 1994. Anim. Behav. 48: 331-341. Symonds, M. E., J. J. Bryandt, L. Clarke, C. J. Darby, and M. A. Lomax. 1992. Effect of maternal cold exposure on brown adipose tissue and thermogenesis in the neonatal lamb. J. Physiol. 455: 487-502. 178 ------- , and M. A. Lomax. 1992. Maternal and environmental influences on thermoreguation in the neonate. Proc. NUtri. Soc. 51: 165—172. Tarkkonen, H. and H. Julku. 1968. Brown adipose tissue in young mice: activity and role in thermoregulation. EXperientia 24: 798—799. Thomas, J. A., and E. C. Birney. 1979. Parental care and mating system of the prairie vole,.Microtus ochrogaster. Behav. Ecol. Sociobiol. 5: 171-186. Trayhurn, P., M. Ashwell, G. Jennings, D. Richard, and D. M. Stirling. 1987. Effect of warm or cold exposure on GDP binding and uncoupling protein in rat brown fat. Am. J. Physiol. 252: E237-E243. ------- , and R. E. Milner. 1989. A commentary on the interpretation of in vitro biochemical measures of brown adipose tissue thermogenesis. can. J. Physiol. Pharmacol. 67: 811-819. ------- 1993. Species distribution of brown adipose tissue: characterization of adipose tissues from uncoupling protein and its mRNA. In Life in the Cbld: Ecological, Physiological, and Mblecular.Mechanisms (C. Carey, G. L. Florant, B. A. Wunder, and B. Horwitz, eds.) pp. 361-368. Westview Press, Boulder. Trier, T. M. 1994. Ecological implications of diet-induced thermogenesis in the prairie vole, Microtus ochrogaster. Ph.D. dissertation. Michigan State University. 167 pp. Wang, Z., and M. A. Novak. 1992. Influence of the social environment on parental behavior and pup development of meadow voles (Microtus pennsylvanicus) and prairie voles (M1 ochrogaster). J. Comp. Psychol. 106(2): 163- 171. Weidemann, M. J., Erdelt, H., and M. Klingenberg. 1970. Adenine nucleotide translocation of mitochondria: identification of carrier sites. Eur. J. Biochem. 16: 313:-335. Wunder, B.A., Dobkin, D. S., and R. D. Gettinger. 1977. Shifts of thermogenesis in the prairie vole (Microtus ochrogaster). Oecologia. 29: 11-26. Yonetani, T. and G. S. Ray. 1965. Studies on cytochrome oxidase. VI. Kinetics of the aerobic oxidation of ferrocytochrome c by cytochrome oxidase. J. Biol. Chem. 240(8): 3392-3398. nICHIan smTE UNIV. LIBRARIES 1111111H111HIHIW111111111111111111111 31293015706918