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Cognitive, Affective, & Behavioral Neuroscience 2001, 1 (3), 287-296

Inhibiting metabolism in the hippocampus of rats in behavioral estrus decreases behaviors and enhances exploratory and antinociceptive behaviors

MADELINE E. RHODES and CHERYL A. FRYE State University of New York, Albany, New York

Blocking progesterone’s metabolism to 5a -pregnan-3a -ol-20-one (3a ,5a -THP) with finasteride, a 5a -reductase inhibitor, and effects on anxiolytic, exploratory, and antinociceptive behaviors of rats in behavioral estrus were examined. Rats in behavioral estrus received finasteride systemically (SC), to the hippocampus, or to control implant sites, the nucleus accumbens (NA) or ventral tegmental area (VTA), and were tested in horizontal crossing, open-field, elevated plus-maze, emergence, holeboard, social interaction, tailflick, pawlick, and defensive freezing tasks. Finasteride, SC or intrahippocampally, reduced 3a ,5a -THP in the hippocampus relative to vehicle implants or finasteride to the NA or VTA. Systemic or intrahippocampal finasteride decreasedcentral entries in the open field and open-arm time on the elevated plus-maze and increasedfreezing in response to shock relative to vehicle. Finasteride to the hippocampus decreased emergence latencies and increased social interaction, pawlick, and tail- flick latencies relative to all other groups. Finasteride to the hippocampus of rats in behavioral estrous decreased anxiolysis and enhanced exploration and analgesia. In summary, these data demonstrate that decreasesin anxiolytic behavior of behavioral estrous rats can be produced by reductions in 3a ,5a - THP in the hippocampus, which suggest that elevations in 3a ,5a -THP in the hippocampus may give rise to anxiolysis seen during behavioral estrus.

There are sex differences, as well as differences across Guasti et al., 1999; Fernandez-Guasti & Picazo, 1990; the ovarian cycle, in rodents on performance in Frye et al., 2000). tasks and responsiveness to noxious stimuli. Neonatally Evidence suggests that progestins modulate sex and feminized male and female rats spend more time on the ovarian cycle differences in anxiety behavior and respon- open arms of the elevated plus-maze and exhibitless con- siveness to noxious stimuli in rodents. Ovariectomy of ditioned defensive burying, as adults, than do neonatally rats makes the anxiety and reactivity behaviorof females androgenized female or male rats (Fernandez-Guasti, more male typical. Ovariectomy reduces open-arm time Martinez-Mota, Estrada-Camarena, Contreras, & Lopez- of females in the elevated plus-maze and produces conflict Rubalcava,1999; Fernandez-Guasti & Picazo, 1990, 1999; behavior that is comparable to that of males (Fernandez- Johnston& File, 1991; Zimmerberg & Farley, 1993). The Guasti & Picazo, 1990; Rodriguez-Sierra, Howard, Pol- anxiety behavior of proestrous females tends to be most lard, & Hendricks, 1984). Progesterone administration to different from that of males, whereas the performance of ovariectomizedfemales significantly increases open-arm diestrous females is more male typical.Proestrous females time in the elevated plus-maze relative to that seen fol- spend more time on the open arms of the elevated plus- lowing vehicle administration(Bitran, Shiekh, & McLeod, maze than do diestrous female or male rats (Fernandez- 1995), produces a decrease in conditioned defensive Guasti et al., 1999; Frye, Petralia, & Rhodes, 2000; Mora, behavior (Martinez-Mota, Estrada-Camarena, Lopez- Dussaubat, & Diaz-Veliz, 1996; Vinogradova, 1999). Pro- Rubalcava, Contreras, & Fernandez-Guasti,2000), and fa- estrous females have a longer latency to commence bury- cilitates lickingof an electrified water source in the Geller- ing an electrified prod and, once initiated,they bury it for Siefter conflict test (Rodriguez-Sierra, Hagley, & less time than do diestrous female or male rats (Fernandez- Hendricks, 1986; Rodriguez-Sierra et al., 1984). These data suggestthat progestinscan have salient activationalef- fects on anxiety behavior and reactivity to noxiousstimuli. This research was supported by Grant 96-10 from The Whitehall Over the estrous cycle, progestin levels vary concomi- Foundation and by Grants 95-14463 and 98-96263 from the National tant with the occurrence of physiological changes within Science Foundation.The technical assistance provided by Melissa Nu- the hippocampus. There is increased physiological activ- bruegge, Zoe Orecki, Erik Rist, and April Smith is greatly appreciated. Correspondenceshouldbe addressed to C. A. Frye, Department of Psy- ity (Kawakami, Terasawa, & Ibuki, 1970), enhancedplas- chology,University of Albany–SUNY,1400 WashingtonAve., Albany, ticity (Terasawa & Timiras, 1968),facilitatedlong-term po- NY 12222 (e-mail: [email protected]). tentiation(Korol et al., 1994; Warren, Humphreys, Juraska,

287 Copyright 2001 Psychonomic Society, Inc. 288 RHODES AND FRYE

& Greenough, 1995), and greater synaptic density (Gould, AP = -3.8, ML = ±2.0, DV = -2.0), the nucleus accumbens (NA; n = Woolley, Frankfurt, & McEwen, 1990) observed in the 13; from bregma AP= +1.7, ML= ±1.5, DV= -6.0), or the ventral tegmental area (VTA; n = 9; from bregma AP= -5.3, ML= ±0.4, DV = hippocampus during proestrus when estradiol and pro- - gesterone levels are increased relative to other phases of 7.0). Cannulae consisted of 23-ga thin wall stainless steel guide tub- ing with 30-ga removable inserts, made to extend 3 mm beyond the the cycle. end of the guide cannulae. Prior to behavioral testing, control inserts Progestins may have effects in the hippocampus to either were tamped in or were left empty. Inserts for the mediate anxiety and responsiveness to noxious stimuli experimental condition were tamped in finasteride. Immediately prior in rodents. First, there is evidence to suggest that the 5a- to application, we verified that the inserts contained a 1-mg implant of reduced progesterone metabolite 5a-pregnan-3a-ol-20- finasteride by checking the mass of the filled insert and/or confirm- one (3a,5a-THP) may mediate performance on some ing, with a dissecting microscope, that the end of the insert was filled. anxiety tasks. Systemic administration of 3a,5a-THP to Determination of Behavioral Estrous both males and females produces a reduction in condi- Daily screening was performed on all rats between 0700 and tioned defensive burying (Fernandez-Guasti & Picazo, 0800 h to determine whether the rats were in behavioral estrus. The 1999). Second, administrationof progestins to the hippo- rats were placed in an arena with a male that was allowed to mount campus, or areas connected to the hippocampus,appears once. If lordosis was exhibited, the female was considered in behav- to be sufficient to increase anxiolyticbehaviorin rodents. ioral estrus. (Note that behavioral estrus, rather than examination of Injections of pregnanalone, a prohormone for 3a,5a- vaginal epithelium, was used as an indicator of hormonal status of the rats in order to reduce vaginocervical stimulation, which occurs when THP, to the hippocampus increase the amount of time collecting vaginal cytology of rats and which might have had an ef- spent in the open arms of the elevated plus-maze and de- fect on performance in the behavioral measures examined.) Once a crease both the latency to burial of an electrified prod female exhibited a consistent pattern of behavioral estrus every 4–5 and the duration of burying behavior (Bitran, Dugan, days, it was tested on the subsequent occurrence of behavioral estrus. Renda, Ellis, & Foley, 1999). Infusions of 3a,5a-THP to Each rat was tested in the complete battery of tests, described below, the central nucleus of the amygdala increase open-arm during one test session, on the day that it was in behavioral estrus. time in the elevated plus-maze and punished responding Behavioral Testing in the Geller-Seifter conflict test (Akwa, Purdy, Koob, & There is no single measure of anxiolytic behavior in rodents; Britton, 1999). Third, progesterone and 3a,5a-THP lev- therefore, a battery of tests, each of which are considered an index of els are increased in the hippocampus coincident with anxiety behavior, are often used in conjunction to examine factors that anxiety reduction and decreased defensive responses in influence anxiety behavior (Johnston & File, 1991; Pellow, Chopin, proestrous rats relative to females in other phases of the File, & Briley, 1985). The order in which these tests were adminis- tered was not counterbalanced; hence, the effects of one or more of cycle or male rodents. Proestrous females spend more time these tasks may have influenced the rats’ behavior on subsequent on the open arms of the elevated plus-maze and spend tasks. All rats were exposed to the tests in the same order, so any order less time freezing in response to shock from an electri- effects of the tests would be expected to be uniform across groups. fied prod than do females in other phases of the estrous The rats in behavioral estrus that had not undergone surgery (n = cycle or male rats (Frye et al., 2000). Together, these data 40) were randomly assigned to receive systemic vehicle (sesame oil, are consistent with the notion that proestrous changes in 0.2 cc SC, n = 18) or systemic finasteride (50 mg/kg SC, n =22)2h prior to behavioral testing. The rats that had chronic guide cannulae some anxiety and defensive behaviors may be influenced and were in behavioral estrus were randomly assigned to receive ve- by 3a,5a-THP levels in the hippocampus. hicle (cholesterol) or no implants to the hippocampus (hippocampal The present experiment tested the hypothesis that vehicle, n = 9) or bilateral 1-mg implants of finasteride to the hippo- blocking the metabolism of progesterone to 3a,5a-THP campus (n =15),NA(n = 13), or VTA (n = 9) 2 h prior to behavioral in the hippocampus would decrease anxiolyticbehaviors testing. Implants of finasteride to the VTA of behavioral estrous ro- of proestrous rats. dents according to this paradigm have been demonstrated to signifi- cantly reduce midbrain levels of 5a-reduced progestins (Frye & Vongher, in press) and the number of 5a-reductase immunoreactive METHOD neurons in the VTA (Frye, 2001a). Each rat was individually tested through the entire test battery dur- These methods were preapproved by the Institutional Animal ing one test session by one of four observers (interrater reliability Care and Use Committee. was greater than 95%) that were blind to the hypothesized outcome of the study. The rats were assigned to groups with counterbalanc- Animals and Housing ing on and between days, in order to minimize any possible order Female Long-Evans rats (n=86), approximately 55 days of age, effects. Up to 4 rats were tested daily, each for 60 min, in the same were obtained from Taconic Farms (Germantown, NY). Rats with brightly lit room adjacent to the animal housing facility, between guide cannulae were individually housed and those receiving sys- 0800 and 1200 h, in the behavioral tasks described below. Each rat temic injections were group housed (4 per cage) in polycarbonate was tested individually and never observed other rats performance cages (45 ´ 24 ´ 21 cm) in a temperature-controlled room (21º ± in these tasks. Each rat was exposed to the tasks consecutively in the 1º C) in the laboratory animal care facility. The rats were main- following order without any rest periods: horizontal crossing, open tained on a 12:12-h reversed light cycle (lights off 8:00 a.m.) with field, elevated plus-maze, emergence, holeboard, social interaction, access to Purina Rat Chow and tap water in their home cages. tailflick, pawlick, and defensive freezing. Apparatus for all of the tasks were present in the room for the entire test period. Surgery Some rats (n = 46) were anesthetized with Rhompun (60 mg/kg) Emergence Test and Ketaset (80 mg/kg) and stereotaxically implanted with bilateral The emergence test, which has been demonstrated to be sensitive guide cannulae aimed at the hippocampus (n=24; from bregma to “anxiety” of rats, was utilized as a measure of exploratory behav- PROGESTIN METABOLISM, ANXIETY, AND HIPPOCAMPUS 289 ior (Frye et al., 2000; Holson, Ali, Scallet, Slikker, & Paule, 1989; amount of time spent on the open arms was recorded during the test Morley & McGregor, 2000). The rats were placed in a closed opaque by an observer. The rats were considered to be in either the closed cylinder (21 ´ 7 ´ 7 cm) that was set in an open field and secured or the open arms of the maze, and open-arm time was recorded only to prevent rolling. The lid of the cylinder was removed and the la- when the rat had all four paws on the open arm of the elevated plus- tency for the rat to emerge completely (all four paws) from the maze. cylinder into the center of a brightly lit open field was recorded (maximum latency 15 min). Defensive Freezing The defensive freezing test has been validated as a model that is Social Interaction sensitive to compounds that alter anxiety (Frye et al., 2000; Gallo Performance in the social interaction task is also considered a dis- & Smith, 1993; Treit, 1985; Treit, Pinel, & Fibiger, 1981). The test criminable assay for anxiety and exploratory behavior (Cheeta, chamber (26.0 ´ 21.2 ´ 24.7 cm) utilized for the defensive freezing Irvine, Kenny, & File, 2001; Frye et al., 2000). The social interaction procedure was constructed of clear Plexiglas. A pedestal (2.5 cm in test was conducted in an open-field apparatus (60 ´ 60 ´ 35 cm) sim- diameter and 10.0 cm in height) was placed in the right rear corner ilar to that used for the holeboard test. As per Ge, Barnes, Costall, of the chamber, 3.0 cm from the back wall and 2.5 cm from the right and Naylor (1997), each member of a pair of rats was placed in op- wall. Wood-chip bedding was placed 5.0 cm deep from the bottom posite corners of the open field and then left undisturbed for 10 min of the chamber, such that the pedestal extended 4.5 cm above the while the total duration of time (in seconds) that the test rat engaged material. The pedestal was wrapped by two wires that were con- the stimulus animal in crawling over and under partner, sniffing of nected to a shock source (Lafayette Model A615B, Lafayette, IN) set partner, following with contact, genital investigation of partner, tum- to deliver 6.66 mA of unscrambled shock when the animal placed bling, boxing and grooming was recorded. Stimulus rats were con- both forepaws on the pedestal. The shock was terminated by the ex- specifics in diestrus that were randomly selected from a pool of rats perimenter after initial contact with the probe by the rat. The dura- that were not behaviorally tested in this study. tion of freezing behavior manifest in response to the shock stimu- lus from the electrified prod was recorded for 15 min. Tailflick The tailflick paradigm, as described by D’Amour and Smith Horizontal Crossings (1941) and Frye and Duncan (1994, 1995), is used to assess noci- The horizontal crossing task has previously been used as an index ception in rodents. This measurement is sensitive to differences in of motor behavior (Brudzynski & Krol, 1997; Frye et al., 2000). nociception produced by analgesic and anxiolytic and hor- Briefly, the rats were placed in a brightly-lit 39 ´ 39 ´ 30 cm Digis- mones. The rats were handled, covered with a towel, placed on the can Optical Animal Activity Monitor (Accuscan Instruments Inc., platform of the tailflick apparatus (San Diego Instruments, San Columbus, OH). The total number of beam breaks that occurred in Diego, CA), and held in place while their tails were smoothed above a 5-min test was mechanically recorded. the radiant heat source. The mean latency of three tailflick trials was used as an index of nociception (maximum latency 10 sec). Holeboard Performance in a holeboard task is considered a measure of motor Pawlick and exploratory behavior (File, Pellow, & Braestrup, 1985; Frye The latency for a rat to lick its hindpaw, a recuperative behavior, et al., 2000; Zangrossi & File, 1992). The holeboard was an open in response to being placed on a heated surface is a measure that is field (60 ´ 60 ´ 35 cm) divided into nine equal squares. At the four sensitive to - and hormone-induced variations in nociception intersecting grid lines were equally spaced holes (each 3.8 cm in and anxiety (Bardo & Valone, 1994; Frye et al., 2000; Smythe, Mc- diameter). As per Zangrossi and File (1992), the rats were placed in Cormick, Rochford, & Meaney, 1994). The pawlick apparatus con- the box, and the incidence of head dips into the holes and total num- sisted of a slide-warming tray (Fisher Scientific, Swanee, GA, ber of squares entered were recorded for a period of 5 min. Model No. 77), on which a clear, floorless, plastic chamber (28.5 ´ 17.5 ´ 12.5 cm) was placed. The plastic chamber was used to con- Tissue Collection fine the rat to the hotplate surface that was heated to 53º C. Utiliz- Brains were rapidly removed, and the hippocampus was dissected ing this procedure, the rats were placed in the chamber on the hot- bilaterally, placed in dry ice, and stored at -70°C until radioim- plate, and the latency to lick the hindpaw was recorded (maximum munoassay for 3a,5a-THP.Tissues were collected from several rats latency 180 sec). that were behaviorally tested and received systemic vehicle (n = 2), systemic finasteride (n = 6), hippocampal vehicle (n = 3), or hip- Open Field pocampal finasteride (n = 3). A number of rats that did not undergo The open field is a conflict task commonly used to assess anxi- behavioral testing had tissue collected in the following experimental olytic behavior of rodents (Blizard, Lippman, & Chen, 1975; Frye conditions: systemic vehicle (n = 3), hippocampal vehicle (n = 3), et al., 2000). The open field box (76 ´ 57 ´ 35 cm) used had a 48- or hippocampal finasteride (n = 3). Consistent with previous find- square grid floor (6 ´ 8 squares, 9.5 cm/side) and an overhead light ings (Frye et al., 2000), there were no signif icant differences in illuminating the central squares (all but the 24 perimeter squares were 3a,5a-THP levels between rats that were behaviorally tested and considered central). As per McCarthy, Felzenberg, Robbins, Pfaff, rats that were not. Not all rats had tissues collected; this was, in part, and Schwartz-Giblin (1995), the rats were placed in the open field an effort to minimize the number of animals utilized for this exper- and observed for 5 min. During this time, the number of central and iment, which was a close approximation of previous studies from peripheral squares entered was recorded by an observer. our laboratory (Frye, 2001a; Frye, Bayon, Pursnani, & Purdy, 1998; Frye & Vongher, 1999, in press). Elevated Plus-Maze The elevated plus-maze has been previously shown to be a valid Radioimmunoassay of Brain 3a,5a-THP measure of anxiolytic and anxiogenic drug effects (Frye et al., 2000; 3a,5a-THP was measured according to previously established Pellow, et al., 1985). The elevated plus-maze consisted of four arms, methods (Finn & Gee, 1994; Frye & Bayon, 1999; Frye et al., 1998; 49 cm long and 10 cm wide, elevated 50 cm off the ground. Two Purdy et al., 1990). Briefly, steroids were extracted from homoge- arms were enclosed by walls 30 cm high, and the other two arms nized brain samples in 50% MeOH, 1% acetic acid through a series were exposed. As per Pellow and File (1986) and Dunn, Copeland, of centrifugation and filtrations. 300 ml of 0.1 M phosphate assay Reed, and Frye (1998), the rats were placed at the junction of the buffer (pH = 7.4) was added to test tubes containing steroid extracts open and closed arms of the maze and were observed for 5 min. The and equilibrated. 290 RHODES AND FRYE

Figure 1. The panels represent the following: emergence latencies (sec ± SEM) of rats administered systemic vehicle (open bars; n = 18), hippocampal vehicle (diagonally striped bars; n = 9), systemic finasteride (black bars; n = 22), or hippocampal finasteride (horizontally striped bars; n =15);mean number of squares entered in the holeboard task (± SEM) of rats administered systemic vehicle, hip- pocampal vehicle, systemic finasteride, or hippocampal finasteride; mean tailflick latencies (sec ± SEM ) of rats administered systemic vehicle, hippocampal vehicle, systemic finasteride, or hip- pocampal finasteride; and mean pawlick latencies (secs ± SEM) of rats administered systemic vehi- cle, hippocampal vehicle, systemic finasteride, or hippocampal finasteride. Bars with different let- ters are significantly different from each other ( p < .05).

The antibody, purchased from R. H. Purdy (Veterans Medical Af- hoc tests to determine differences among groups. Alpha level for fairs, La Jolla, CA), is very specific to 3a,5a-THP (Finn & Gee, the determination of statistical significance was p £ .05. 1994). The 1: 5,000 dilution of this antibody bound between 40% and 3 60% of [ H] 3a,5a-THP (NET-1047, 51.3 ci/mmol; New England RESULTS Nuclear, Boston). The standard curve was prepared in duplicate with a range of nine concentrations from 50 to 8,000 pg/ml (total volume 950 ml). Emergence Test Incubation at 4ºC for 24 h was terminated by charcoal separation Finasteride to the hippocampus significantly reduced of bound and free. Sample tube concentrations were calculated the latency to emerge from the opaquecylinder[F(1,60) = using the logit-log method of Rodbard and Hutt’s (1974) interpola- 8.03, p = .01] relative to the emergence latencies of all tion of the standards and correction for recovery. The minimum de- other groups (see Figure 1). tectable limit of the assay was 100 pg. The intraassay and interas- say coefficients of variance were 12% and 15%. The emergence latencies of the rats that received fi- nasteride either to the hippocampus or to the NA were Statistical Analyses significantly [F(2,34) = 22.23, p £ .01] lower than those Two-way analyses of variance (ANOVAs) were used to examine of the rats that received finasteride to the VTA (see effects of drug treatment (finasteride vs. vehicle) and route of ad- Table 1). ministration (systemic vs. intrahippocampal) on the various behav- ioral and endocrine indices. One-way ANOVAs were used to ex- amine effects of CNS site of finasteride implants (hippocampus, Social Interaction NA, or VTA) on the various behavioral indices. Where appropriate, Finasterideto the hippocampussignificantly[F(1,60)= ANOVAs were followed by Fisher’s least significant difference post 11.89, p < .01] increased the total duration of time spent PROGESTIN METABOLISM, ANXIETY, AND HIPPOCAMPUS 291

Table 1 Mean ± SEM Emergence Latencies, Duration of Social Interaction, Tailflick and Pawlick Latencies, Central Open-Field Squares Entered, Open-Arm Time in the Plus-Maze, Time Spent in Defensive Freezing, Number of Beam Breaks, Total Holeboard Squares Entered, and Hippocampal 3a,5a-THP Concentrations of Rats Administered Finasteride to the Hippocampus, NA, or VTA Duration Open Field Open-Arm Horizontal Holeboard Emergence of Social Tailflick Pawlick (No. of Time in Defensive Crossings (No. of 3a,5a- Latency Interaction Latency Latency Central PlusMaze Freezing (No. of Squares THP n (sec) (sec) (sec) (sec) Entries) (sec) (sec) Beam Breaks) Entered) (ng/g) Hippocampus 15 38 ± 6a 127 ± 11a 4 ± 0.4a 168 ± 7a 10 ± 2a 43 ± 9a 307 ± 59 957 ± 94 55 ± 7a 5 ± 0.5a NA 13 113 ± 67a 271 ± 8b 2 ± 0.1b 114 ± 16b 23 ± 3b 17 ± 4b 239 ± 99 908 ± 63 84 ± 6b 10 ± 0.7b VTA 9 678 ± 128b 182 ± 13c 2 ± 0.1b 98 ± 19b 6 ± 3a 1 ± 1b 164 ± 92 754 ± 91 37 ± 9a 11 ± 0.5b Note—Significant differences across groups for each behavioral task are represented by different letters (in superscripts) for groups that are sig- nificantly different from one another.

in contact with a conspecific in genital investigation, squares entered in the open field relative to that seen sniffing, crawling over and under, tumbling, boxing and after finasteride to the VTA or hippocampus(see Table 1). grooming time relative to that of all other groups (see Figure 1). Elevated Plus-Maze Finasteride administration to the various CNS sites re- Administration of finasteride, systemically or intra- sulted in significantdifferences [F(2,34) = 52.04,p £ .01] hippocampally,significantly[F(1,60) = 15.15, p <.01]re- in duration of social interaction.Finasteride to the hippo- duced the amountof time spent on the open arms of the el- campus resulted in less social interaction than did finas- evated plus-maze relative to that seen after administration teride to the NA or VTA. Finasteride to the NA produced of vehicle (see Figure 2). more social interactionthan did finasteride to the VTA or Finasterideto the hippocampussignificantly[F(2,34)= hippocampus (see Table 1). 8.73, p £ .01] increased the amount of time in the open arms of the elevated plus-maze relative to that seen after Tailflick administration of finasteride to the NA or the VTA (see Finasteride to the hippocampus(3.6 ± 0.4 sec) signifi- Table 1). cantly [F(1,60) = 4.06, p = .05] increased tailflick laten- cies relative to vehicleto the hippocampus(2.8 ± 0.1 sec), Freezing Behavior but did not significantly increase tailflick latencies rela- Finasteride significantly [F(1,60) = 5.22, p = .03] in- tive to systemic vehicle (3.2 ± 0.2 sec) or systemic finas- creased the total duration of time spent freezing following teride (3.0 ± 0.1 sec; see Figure 1). shock relative to that seen following vehicleadministration Implants of finasteride to the hippocampussignificantly (see Figure 2). [F(2,34) = 6.12, p £ .01] increased tailflick latencies rela- There were no significant differences across groups for tive to implants of finasteride to the NA or VTA (see time spent freezing in response to shock following admin- Table 1). istrationof finasterideto the hippocampus,NA, or VTA (see Table 1). Pawlick Finasteride to the hippocampus (167.5 ± 7.0 sec) sig- Horizontal Crossings nificantly [F(1,60) = 4.00, p = .05] increased pawlick la- Manipulations directly to the hippocampus with im- tencies relative to vehicle to the hippocampus (144.4 ± plants of either vehicle or finasteride significantly in- 10.1 sec), but did not significantly increase pawlick la- creased the rats’ total number of beam breaks (927 ± 76) tencies relative to systemic vehicle (156.6 ± 7.4 sec) or relative to that of the rats that did not receive hippocampal systemic finasteride (148.2 ± 6.4 sec; see Figure 1). implants but that received systemic injections (779 ± 64) Implants of finasteride to the hippocampussignificantly [F(1,60) = 4.36, p = .04]. [F(2,34) = 7.49, p £ .01] increased pawlick latencies rela- Although there were no significant differences on the tive to implants of finasteride to the NA or VTA (see numberof beam breaksas a functionof the CNS siteto which Table 1). finasteride was applied, the rats that received finasteride to the NA or the hippocampushad a greater numberof beam Open Field breaks than did the rats that received finasterideto the VTA The rats administeredfinasteride, either systemicallyor (see Table 1). to the hippocampus,entered significantly [F(1,60) = 6.93, p = .01] fewer central squares in the open field than did the Holeboard rats administered vehicle (see Figure 2). Manipulations directly to the hippocampus with im- Administration of finasteride to the NA significantly plants of either vehicle or finasteride significantly in- [F(2,34) = 8.17, p £ .01] increased the number of central creased the rats’ total number of squares entered in the 292 RHODES AND FRYE

Figure 2. The panels represent the following: mean number of squares entered in the open field (±SEM) of rats administered systemic vehicle (open bars; n = 18), hippocampal vehicle (diagonally striped bars; n = 9), systemic finasteride (black bars; n = 22), or hippocampal finasteride (horizon- tally striped bars; n = 15); mean duration of time spent in the open arms of the elevated plus-maze (sec ± SEM) of rats administered systemic vehicle, hippocampal vehicle, systemic finasteride, or hip- pocampal finasteride; mean time spent freezing in the defensive freezing task (sec ± SEM) of rats ad- ministered systemic vehicle, hippocampal vehicle, systemic finasteride, or hippocampal finasteride; hippocampal levels of 3a,5a-THP in rats administered systemic vehicle (open bars; n = 4), hip- pocampal vehicle (diagonally striped bars; n = 6), systemic finasteride (black bars; n = 6), or hip- pocampal finasteride (horizontally striped bars; n = 6). Bars with different letters are significantly different from each other ( p < .05). holeboard task (54 ± 7) relative to that of the rats that did Implants of finasterideto the hippocampussignificantly not receive hippocampal implants but that received sys- [F(2,14) = 31.9, p £ .01] reduced hippocampal 3a,5a- temic injections (40 ± 6) [F(1,60) = 4.00, p = .05]. THP concentrations relative to those produced by finas- The number of squares entered in the holeboardtask by teride implants to the NA or VTA (see Table 1). the rats that received finasteride to the NA was signifi- cantly increased [F(2,34) = 9.84, p £ .01] relative to that of DISCUSSION the rats that received finasteride to the VTA or hippocam- pus (see Table 1). The present findings supported our hypothesis that blocking the metabolism of progesterone to 3a,5a-THP in Radioimmunoassay of Brain 3a ,5a-THP the hippocampus would decrease anxiolytic behaviors of The rats administered finasteride, either systemically rats in behavioral estrus. Finasteride (systemically or in- or intrahippocampally,had significantlylower [F(1,18)= trahippocampally)decreased entries into central squares in 122.4, p £ .01] hippocampal 3a,5a-THP concentrations the open field, reduced open-arm time on the elevated plus- than did the rats administered vehicle either systemically maze, and increased freezing in response to shock relative or intrahippocampally (see Figure 2). to that seen in vehicle-administered rats. However, finas- PROGESTIN METABOLISM, ANXIETY, AND HIPPOCAMPUS 293 teride to the hippocampusenhancedexploratorybehavior, azepines at enhancing GBR function (Majewska, Harri- as indicated by shorter emergence latencies and more son, Schwartz, Barker, & Paul, 1986; McAuley, Reynolds, time spent in social interactionrelativeto all other groups, Kroboth, Smith, & Kroboth, 1995; Wu, Gibbs, & Farb, and also increased analgesia, as demonstrated by longer 1990). It may be through actions at GBRs, that 3a,5a- tailflick and pawlick latencies. Both finasteride regimens THP has its anxiolyticeffects. For example, - employed significantly decreased 3a,5a-THP in the hip- induced (pregnanolone is a prohormone of 3a,5a-THP) pocampus relative to vehicle administration. Taken to- increases in open-arm time in the elevated plus-maze are gether, these data indicate that finasteride application to attenuated by coadministration of , a GBR an- the hippocampus,which decreased 3a,5a-THP levels, was tagonist(Bitran et al., 1995). Our present findings that the sufficient to attenuateanxiolyticbehaviorsand had some 5a-reductase inhibitor attenuates anxiolytic behavior of enhancing effects on exploratorybehavior and analgesia. rats in behavioralestrus in the open-field, plus-maze, and The present findingsare consistentwith the findings of defensive freezing paradigms, taken together with previ- previousstudiesthat have demonstratedthat metabolism, ous findings that GBRs are a substrate for some of 3a,5a- or biosynthesis, of 3a,5a-THP may mediate progestin- THP’sanxiolyticeffects, suggest that these anxiolyticbe- inducedanxiolysis.For example,exogenousadministration haviors observed on behavioral estrus may be due in part of progesterone or 3a,5a-THP induces anxiolyticbehav- to 3a,5a-THP’s actions at GBRs in the hippocampus. ior in male or female rodents in the open field (McCarthy The actions of 3a,5a-THP at GBRs in the hippocampus et al., 1995) and in the plus-maze (Bitran, Hilvers, & Kel- may be sufficient to mediate some anxiolytic behaviors. logg,1991; Mora et al., 1996), conditioneddefensivebury- First, GBRs localized to the hippocampus are sensitive to ing (Gallo & Smith, 1993; Picazo, Fernandez-Guasti, adrenal (Orchinik, Weiland, & McEwen, 1994) and go- Lemus, & Garcia, 1998), and the Geller-Seifter and Vogel nadal (Gould et al., 1990) hormones. Second, progestins conflict paradigms (Brot, Akwa, Purdy, Koob, & Britton, can have effects on anxiety behaviors following direct ap- 1997;Carboni,Wieland,Lan, & Gee, 1996).Endogenous plication to the hippocampus. 3a,5a-THP administered changes in 3a,5a-THP over the estrous cycle coincide to the hippocampus increases open-arm time in the ele- with differences on several measures of anxiety.Proestrous vated plus-maze, and this effect is attenuated by pre- rats show more activity in a brightly lit open field and in treatment with the GBR antagonist picrotoxin (Bitran the elevated plus-maze and show less aversion to noxious et al., 1999). Third, enhancingbiosynthesisof 3a,5a-THP stimuli, as well as increases in plasma and hippocampal in the hippocampus by the activation of peripheral ben- 3a,5a-THP,relative to females in other phases of the es- zodiazepine receptors produces anxiolyticeffects in rats trous cycle or males (Fernandez-Guasti & Picazo, 1992; (Bitran et al., 2000). Fourth, the decrease in anxiolyticbe- Frye et al., 2000). Enhancingbiosynthesisof 3a,5a-THP haviors in the present experiment were producedsimilarly in the hippocampus increases open-arm time in the ele- by either systemic or intrahippocampaladministration of vated plus-maze (Bitran, Foley, Audette, Leslie, & Frye, finasteride, both of which reduced levels of 3a,5a-THP in 2000). Furthermore, blocking metabolism of progesterone the hippocampus.This suggeststhat when absolute levels to 3a,5a-THP attenuates the aforementioned anxiolytic of 3a,5a-THP in the hippocampus are reduced, anxi- effects. Coadministration of progesterone and a 5a-reduc- olytic behaviorsare decreased. Furthermore, the behavioral tase inhibitor attenuates progesterone-induced increases and biochemical effects of intrahippocampal blockade of in open-arm time in the elevated plus-maze (Bitran et al., 3a,5a-THP were no different from systemic administra- 1995). Finally,instigatingwithdrawal from 3a,5a-THP by tion, which suggest that the actions of 3a,5a-THP in the blocking progesterone’smetabolism with administration hippocampus were sufficient to mediate the anxiolytic of the 3-hydroxysteroid dehydrogenase inhibitor, in- effects seen here. Notably, administration of systemic fi- domethacin, increases anxiogenicconditioneddefensive nasteride never produced significant differences in anx- burying behaviorrelative to that seen in rats administered iolytic behavior when intrahippocampal finasteride ad- chronic progesterone and vehicle (Gallo & Smith, 1993). ministration did not significantly change behavior. This The progestin-facilitated anxiolysis described above suggests that the hippocampus is important for mediat- may be mediated through 3a,5a-THP’s actions at g- ing the anxiolytic behaviors measured in the present ex- aminobutyric acid (GABA)A/ periment. However, it should be noted that there may be complexes (GBRs). Although progesterone has a high redundant mechanisms and brain areas through which affinity for intracellular progestin receptors (PRs), there progestins can effect anxiolytic behaviors. For example, is evidence that 3a,5a-THP’s anxiolyticeffects do not re- rats administered3a,5a-THP to the central nucleusof the quire actions at PRs. 3a,5a-THP is devoid of affinity for amygdala have increased open-arm entries and open-arm intracellular PRs (Smith et al., 1974). Administration of time in the elevated plus-maze relative to vehicle-infused the PR blocker RU38486 does not prevent progesterone- controls (Akwa et al., 1999). induced increases in plus-maze behavior (Bitran et al., In addition to supporting previous findings regarding 1995). The most effective endogenous modulator of proestrous-induced anxiolysis in the open-field, plus- GBRs is 3a,5a-THP (Schwartz, 1988). Progesterone it- maze, and defensive freezing behaviors in rodents, the self is only weakly effective at enhancing the function of present results also provide some intriguing insights re- GBRs, but 3a,5a-THP is more potent than benzodi- garding proestrous-enhanced exploratory and analgesia 294 RHODES AND FRYE behavior.Proestrus is associated with increases in social receptors in the VTA or the NA evoked dose-dependent interaction and decreased emergence latencies (Frye increases in motor activity (Swanson & Kalivas, 2000). et al., 2000). In the present experiment, administration Also, steroids in the VTA and the NA modulate other be- of finasteride to the hippocampus enhanced exploration, haviors characterized by dramatic changes in motoricity, as demonstrated by significantly decreasing the emer- such as social behavior (Frye, 2001b) and place learning gence latencies and increasing the duration of time rats (Frye, Rhodes, Rosellini, & Svare, 2001). spent in interaction with a conspecific. Proestrous- and In summary, these data demonstrate that anxiolytic be- progesterone-induced increases in tailflick latencies are havior of rats in behavioral estrous can be decreased by also well established (Frye, Bock, & Kanarek, 1992; producing reductions in 3a,5a-THP in the hippocampus. Frye & Duncan, 1994, 1995). Finasteride to the hippo- Notably, the exploration and analgesia of the rats in be- campus further increased analgesia, as was seen by the havioral estrus that had finasteride applied to the hippo- significantly greater tailflick and pawlick latencies that campus were amplified, suggesting that progestin’s ac- occurred only with finasteride to the hippocampus (not tions in the hippocampusmay mitigate behavioralestrous- to the NA or VTA). An interpretation of these findings inducedchangesin these behaviors.Finally,these findings, is that 3a,5a-THP in the hippocampus is not the prog- in combination with our previous reports on cyclic vari- estin that modulates physiological changes in explo- ations in anxiety behavior coinciding with changes in ration or analgesia over the estrous cycle. 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