Alterations in behavioral responses to stressors following excitotoxin lesions of dorsomedial hypothalamic regions

BRAIN RESEARCH ELSEVIER

Brain Research 633 (1994) 151-161

Research Report

Alterations in behavioral responses to stressors following excitotoxin lesions of dorsomedial hypothalamic regions Jon R. Inglefield a, Steven B. Schwarzkopf b, Carol K. Kellogg c,. Departments of a Neurobiology and Anatomy, b Psychiatry, and c Psychology, Room 186, Meliora Hall, University of Rochester, Rochester, N Y 14627, USA

(Accepted 17 August 1993)

Abstract

The dorsomedial hypothalamus is important for regulation of cardiovascular responses associated with emotional arousal. This region has also been identified as a component of neural circuitry involved in fear/anxiety, yet clear evidence as to the effects of lesioning on stress-related behaviors is missing. In this study, we lesioned the dorsomedial hypothalamic region with the neurotoxin, ibotenic acid (IBO; 2.0/zg in 0.2/zl), and studied the impact on spontaneous and unlearned behavioral responses to stressors. In the open field test, we observed non-generalized increases in motility parameters in the IBO rats with the differences occurring in the latter two-thirds of the test. In the elevated plus-maze, the IBO rats displayed a classic anxiolytic response with a greater proportion of entries into (and greater time spent in) the open arms of the maze. In the environmentspecific social interaction (SI) test, the IBO rats showed a normal familiar/unfamiliar environment discrimination with respect to Total SI; however, the composition of the behaviors ('curiosity' vs. physical contact) by the IBO rats was markedly altered, with there being a 2-fold increase in non-violent physical interactions. Additionally, the differences in these traditional indices of anxiety were associated with lesioned animals exhibiting greater acoustic startle responsiveness than controls as a function of prepulse intensity. Overall, the results following IBO lesions indicate an altered responsiveness to sudden stressors, particularly as relates to novelty or exploration-oriented behaviors. The hypothalamic lesion may, therefore, have resulted in a disinhibition of normally suppressed responding to innate fear or challenging stimuli. This study contributes to those that have begun to define neural interactions that are essential for integrated stress responses. Key words: Autonomic nervous system; Anxiety; Acoustic startle response; Elevated plus-maze; Locomotor activity; Social

interaction; Stress

I. Introduction

'Escape behaviors' and cardiovascular activation occur following manipulation of two adjoining hypothalamic nuclei, the paraventricular nucleus (PVN) [19,28,31,59] and the dorsomedial hypothalamic nucleus (DMH) [4,15,30,47,59]. Earlier evidence from some studies indicated that the posterior hypothalamus was an active site [24,45,46], but it was later determined that the D M H was mostly responsible [9,48]. The D M H and PVN have reciprocal connections with one another [26,54]; indeed the D M H gives a major projection to the PVN. The existence of intrahypotha-

* Corresponding author. Fax: (1) (716) 271-3043. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(93)E1184-5

lamic neural connections between regions involved in responses to stressors suggests that these nuclei may be coordinating one another's reactions to challenges in homeostatic processes. Also, these nuclei are central to the extensively studied 'fear circuitry', which courses from the amygdala through the hypothalamus to the midbrain central grey and surrounding tegmentum [33]. Recent evidence underlined the functional connectivity of this circuit by demonstrating a rapid elevation in the levels of c-Fos protein in response to conditioned or unconditioned aversive stimuli, with increases observed in several areas, including the amygdala, PVN, D M H and midbrain central grey [37,58]. There is compelling evidence that the affective state that normally (i.e. in an unlesioned state) results from activity of the amygdala-medial hypothalamus circuit is aversive in nature [33]. If the efferent signal from the amygdala is aver-

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sive, t h e n l e s i o n i n g / removal of a target could lead to alterations in behavioral responses to stressors. In addition to the well-established c o n s e q u e n c e s of psychological stress on a u t o n o m i c function, activation of the sympathetic nervous system would seem capable of modifying an arousal state [41]. H e n c e , lesions in areas involved in a u t o n o m i c responses to stressors may b l u n t the p a r t i c i p a t i o n of some physiologic responses in modifying e m o t i o n a l states, t h e r e b y altering behavioral responses to stressors. T h e p r e s e n t study b e g a n to investigate these hypotheses. T h e 7 - a m i n o b u t y r i c acid ( G A B A ) / b e n z o d i a z e p i n e ( B D Z ) receptor complex is an essential c o m p o n e n t in stress-related behaviors [10]. R e s e a r c h has shown altered b i n d i n g of ligands to a n d modified f u n c t i o n i n g of the cerebral cortical G A B A / B D Z receptor complex in response to e n v i r o n m e n t a l challenges [1,18,57]. T h e biologic m e c h a n i s m s m e d i a t i n g the impact of the envir o n m e n t a l challenge u p o n the receptor complex are presently u n k n o w n . W e have recently reported, however, on the i m p o r t a n c e of h y p o t h a l a m i c f u n c t i o n to stressor-induced responsiveness of the G A B A / B D Z receptor complex in the cerebral cortex [21]. In that study, lesioning c a t e c h o l a m i n e r g i c terminals (using 6O H D A ) in the h y p o t h a l a m u s led to altered f u n c t i o n i n g of the G A B A / B D Z receptor complex in the cerebral cortex in response to restraint without c h a n g i n g the robust release of corticosterone (CS). Additionally, the 6 - O H D A lesion gave evidence for a change in mechanisms m e d i a t i n g behavioral responses to a challenge: specifically, the rats with h y p o t h a l a m i c lesions showed altered behavior in a social i n t e r a c t i o n (SI) test. We hypothesized that b e c a u s e the h y p o t h a l a m u s is important to s u d d e n stressor-induced changes in the G A B A A receptor in the cerebral cortex a n d since behavior to an e n v i r o n m e n t a l challenge was altered, there may be a n e u r a l circuit involving the h y p o t h a l a m u s and cortical regions that is responsible for transfer of i n f o r m a t i o n regarding stressors. To c o n t i n u e to investigate the theory that the hyp o t h a l a m u s has a role in i n t e g r a t i n g responses to stressors by acting t h r o u g h central n e r v o u s system pathways to inform higher b r a i n centers, the p r e s e n t study was designed to focus o n indices that study i n n a t e aspects of fear or anxiety (i.e. u n l e a r n e d , s p o n t a n e o u s behaviors). W e e x a m i n e d w h e t h e r localized lesioning of n e u rons in the dorsal medial h y p o t h a l a m i c region would alter any of several behavioral indices of anxiety, (namely: elevated plus-maze, e n v i r o n m e n t - s p e c i f i c social interaction, or exploratory behaviors) or responses to acoustic startle. T h e acoustic startle reflex ( A S R ) is an easily q u a n t i f i e d reflex muscle c o n t r a c t i o n m a d e in response to a s u d d e n , i n t e n s e noise burst, resulting in a t e m p o r a r y c r o u c h e d posture. I n t e n s e acoustic stimuli may be classified as stressors since they elicit corticosterone release [13], a classic h o r m o n a l index of a

stressed state. The A S R is known to be e n h a n c e d in situations that were previously c o n d i t i o n e d to induce fear [8]. This evidence supports the modification of the A S R by n e u r a l systems i m p o r t a n t to stress responses. and so the response to startle of I B O - l e s i o n e d and control rats was also compared.

2. Materials and methods 2.1. Animals

Male rats of the Long-Evans Blue Spruce strain used in these studies were either bred in the University of Rochester vivarium from stock obtained from Harlan Sprague-Dawley or obtained directly from Harlan Sprague-Dawley (Altamont, NY). Equal numbers of rats from the two sources were apportioned to control and lesioned groups. They were housed individually until use at 90-100 days of age. All animals were maintained on a 12-12 h light-dark cycle under ad lib access to food and water (unless being prepared for surgery). 2.2. Surgical procedures

Surgery on 40 adult rats of the same age and weight was performed under ketamine (60 mg/kg) and xylazine (12.5 mg/kg) anesthesia. Neuronal cell bodies in dorsomedial hypothalamic regions were lesioned using stereotaxic injection of the neurotoxin, ibotenic acid (IBO; 2.0/zg/0.2 ~1). After drilling the skull openings, injections were made bilaterally using a Kopf stereotaxic device and 10 /zl Hamilton syringe according to the coordinates from Paxinos and Watson [34]: -2.8 mm anterior-posterior, +0.5 mm mediallateral, and - 8 mm dorsal-ventral. The neurotoxin was dissolved in a 50 mM sodium phosphate-buffered saline (pH 7.2) and injected slowly over a period of 1-2 rain. Sham-operated control animals received identical surgery with the exception that the 30 gauge needle was not lowered into the brain. Confounds of non-specific tissue damage were unlikely because a small needle of the size used causes very little physical damage. Wounds were closed using stainless steel surgical staples. A Nissl stain was performed at the conclusion of these experiments to verify the site and extent of the lesions. Staining for cytochrome oxidase (Sigma) was also done to assess lesion extent. The lesioned animals displayed no appreciable changes in body weight compared to controls up to 6 weeks post-surgery. 2.3. Behat,ioral testing

The behavioral measures were assessed 3 weeks following lesioning and after 1 week of gentle handling. Each rat underwent the behavioral tests in the following order with the noted between-session interval: social interaction (SI; 3 days), motility followed by ASR (3 days), and finally, elevated plus-maze. Because the SI test requires suitable number of pairs of rats for each of four different testing conditions (see below), it was necessary to prepare additional animals for this test alone. A total of 40 rats (10 sham and 10 ibotenate pairs) were used for this test. For the other behavioral measures, a much smaller sample size (10 control and 15 lesioned rats) was suitable. All rats that were tested in the motility, ASR, and elevated plus-maze tests were also included in the SI study. 2.4. Social interaction test

The amount of time pairs of untreated male rats spend in SI is a function of their familiarity with the test environment, such that SI is

J.R. Inglefield et al. / Brain Research 633 (1994) 151-161 greater when the environment is familiar [14,39]. In control animals, decreased SI in an unfamiliar environment is accompanied by an increase in stress-like reactions, such as increased corticosterone (CS) levels [14]. Pairs of rats were matched for body weight (within each pair, the weights did not differ by more than 10-15 g), surgical procedure, and experience (i.e. familiarity with the test environment). Individually housed rats were handled on each of 5 days preceding the SI test. On each of the 2 days before the test, rats to be tested in the familiar environment were transported to the test room and placed singly in the test arena for 7.5 min. Rats that were to be tested in the unfamiliar environment were transported to the test room on these 2 days but kept in their home cage. On the test day, a pair of rats were placed in the arena (a 41 x 25 x 30 cm Plexiglas cage located in a sound isolation chamber with an illumination intensity of 30-32 lux) for 7.5 min. All testing was done between 09.00 and 10.00 h. Behavior of 20 pairs of rats was recorded on video tape using a TV monitor and VCR. The tapes were later viewed and SI behavior was scored by an experienced individual blind to the condition being scored. Two categories of behavior were measured: Explorative-contact ('curiosity'), which included sniffing and following, and vigorous-contact (physical), which included pushing, walking over, wrestling and grooming of the other. The total time spent in interaction as well as the proportion of the total time spent in the two separate categories was recorded. The total score was multiplied by two in order to reflect the contribution of both rats to the score.

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trials (18 of each trial type) were used in the analyses. All acoustic stimuli were white noise bursts of 30 ms duration having rapid rise and fall times (less than 1 ms). Each trial type was presented in random order with inter-trial intervals ranging from 8-23 s (average 15 s); testing took approximately 20 min. The average startle response over the 100 ms recording window (in arbitrary units) was calculated for each stimulus type for the entire session, with median PA amplitude also calculated for each 5-min block in order to assess any change in startle amplitude over time.

2. 7. Elevated plus-maze The maze, elevated to a height of 50 cm, consisted of two open arms (50 cm long by 10 cm wide) and two enclosed arms (50 cm long by 10 cm wide by 40 cm high) with an open roof. The maze is arranged so that the two enclosed arms are opposite to one another. The testing room was quiet and dimly lit at a level of 25 lux. At the beginning of the test, each rat was separately placed into the center zone of the maze. The number of entries into the open and closed arms and the time spent on the open and closed arms was tabulated for 5 min. The criterion used to determine an entry was that the head and both forelimbs were situated in the new arm.

2.8. Data analysis 2.5. Exploratory (autotracker) testing The testing apparatus consisted of a Plexiglas platform (37 x 37 cm), with 12-cm walls and covered with a perforated Plexiglas cover (Columbus Instruments, Inc., Columbus, OH). Fifteen infra-red photobeams per horizontal axis detected horizontal movement. The apparatus was situated on the floor of the testing chamber. Software allowed the calculation of the animal's position, distance traveled, time spent in locomotion, time spent resting, and time spent in repetitive small displacement motor activity (idle investigation, grooming and stereotypy; which were termed 'non-locomotor movements'). Digital sampling occurred every 0.1 s and data was stored on a computer disk for off line analysis. Animals were brought to the testing area more than 10 min prior to actual testing and placed in a quiet room in an attempt to decrease the effect of transport on the animals' open field behavior. Testing began with each animal being placed in the center of the platform. Movement was assessed, as detailed above, for a 10-min period. Distance traveled, time in locomotion, resting time, and non-locomotor movements were calculated for each animal both by minute and for the total time.

2.6. Acoustic startle response Acoustic startle testing was carried out using SR-Lab testing equipment (San Diego Instruments, Inc., San Diego, CA), including: startle chamber (Plexiglas cylinder, 8.2 cm diameter, mounted on Plexiglas frame), piezoelectric sensor attached to the bottom of Plexiglas frame, rectifier and amplification unit, computer interface, analog to digital circuitry with 12 bit resolution, and Radio Shack super tweeter. Piezoelectric output over 100 ms post-stimulus, representing whole body startle of the animal, was used as the raw dependent measure. Testing began with a 5-min period during which animals were exposed to 70 dB white noise. Following this period, 75 startle stimuli were delivered, consisting of a 116 dB startle stimulus alone (pulse alone, [PAl) or one of three prepulse-pulse pairs (116 dB startle stimulus preceded by 100 ms with prepulses of 75 dB [PP75-P], 80 dB [PP80-P], or 85 dB [PP85-P]). The first three adaptive trials (PA) were not used in the analysis, as is customary. The following 72

Analysis of variance (ANOVA) with repeated measures was used to assess locomotion and acoustic startle behaviors. This analysis assessed both repeated factors, between subjects factors, and possible interactions. In the social interaction study, lesion status, behavior composition ('curiosity' or physical), and environment (familiar or unfamiliar) were the independent variables for the ANOVA. Motility data was analyzed using Period 1, 2, and 3 as the repeated measure, lesion status as the between factor in comparing total distance traveled. This enabled us to assess time effects, lesion effects, and potential interactions of lesion x time. The effects of prepulses were assessed with trial type (PA, PP75-P, PP80-P, and PP85-P) as the repeated factor and lesion status as the between-subjects factor. All analyses were performed using a standard statistical package [40] utilizing post-hoc comparisons where appropriate. The elevated plus-maze data as well as overall locomotion data for the 10-min tests were assessed using the Student's t-test. The alpha level for significance was P _<0.05.

3. Results 3.1. H i s t o l o g y An example of a representative lesion placed within t h e d o r s o m e d i a l h y p o t h a l a m u s is i l l u s t r a t e d in Fig. 1. T h e a r e a o f n e u r o n a l cell d e a t h (as v i e w e d b y N i s s l s t a i n in Fig. 1 A ) c o r r e s p o n d e d t o a n a r e a o f a t t e n u a t e d c y t o c h r o m e o x i d a s e l e v e l s (Fig. 1B), a n d in t h i s c a s e extended

from the

DMH

into the nearby, dorsally-

situated, dorsal area. On average, there was complete elimination of neurons within a radius of 200-250/xm o f t h e i n j e c t i o n site, a n d c l o s e l y a d j o i n i n g r e g i o n s exh i b i t e d d i s t i n c t n e u r o n loss a n d gliosis. I n s o m e c a s e s , l o s s o f c e l l s in t h e D M H w a s a c c o m p a n i e d b y cell loss in t h e d o r s a l a r e a . I n o t h e r a n i m a l s , t h e r e w a s o n l y u n i l a t e r a l cell l o s s in t h e D M H ; o n t h e o t h e r s i d e , t h e

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lesion was situated in the dorsal area a n d / o r had cell loss extending into the zona incerta, nucleus reuniens of the thalamus or lateral hypothalamus. Any animal

that had a bilaterally misplaced lesion was eliminated from the analyses, leaving 11 lesioned rats and 10 controls that underwent all tests. In the S1 study, a total of 8 lesioned and 10 control pairs of rats were included. Those animals having unilateral lesions in the D M H with a lesion on the other side extending into the dorsal area (5 rats), the LH a n d / o r zona incerta (5 rats) or the thalamic reuniens nucleus ( 1 rat) had very similar behaviors to those bilaterally lesioned in the DMH, and thus were included in the study. Fig. 2 depicts the injection sites at the level of the medial hypothalamus for those I I rats that received ibotenie acid and underwent all tests.

3.2. Exploratory behat,ior Fig. 3A shows the mean ( + S . E . M . ) for total distance traveled by group for the 10-min open-field test. The lesioned rats displayed a statistically significant increase in the total distance traveled (Student's t-test, t2~ = 2.21, P < 0.05). The 10-rain test period was divided into three 3-4-rain periods in order to assess the effect of lesion when the environment was most novel vs. least novel (Fig. 3B). A comparison between the periods revealed a Group × Time Period interaction (repeated measures two-way A N O V A , FL6s = 3.81, P < 0.05). Post-hoc testing showed that the lesioned animals exhibited an increased distance traveled in the latter two time periods (Fig. 3B; one-way ANOVA, Scheff~ analysis for each, P < 0.05). With regard to other behaviors recorded in the exploratory chamber, there were significantly more movements of the non-locomotor type exhibited by the lesioned group for the 10-rain test. (control had 640 + 27 vs. lesioned 723 _+ 26 p h o t o b e a m breaks in 10-min test; Student's t-test, t21 = 2.2, P < 0.05). Non-locomotor movements are those where the animal is engaged in small movements but covered little or no distance. The behaviors that would be recorded are grooming, stereotypic movements, and turning around. The autotracker equipment, however, is not able to distinguish between these various movements, and thus no break-

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Fig. 1. A: photomicrograph of a Nissl stained section illustrating a representative lesion site in the DMH on the left side. The lesion also impacted the dorsal area (DA; see B) Ibotenate (2 p,g in 0.2 ~,1) was delivered ( * = site of injection) into the medial hypothalamus. In this rat, the D M H and dorsal area on the right are apparently intact at this level of hypothalamus. This level corresponds to - 2 . 8 0 mm AP. B: cytochrome oxidase stain of same lesioned site illustrating extent of neuronal damage as depicted by lightened area surrounding needle mark (* = site of injection). This demonstrates the limited extent of the typical lesion. The extent of the other lesions were in accord with that depicted here, extending a radius of 200-250 p,m from the injection site. IIIv., 3rd ventricle; rot, mammillothalamic tract. Bars = 100 p,m.

J.R. Inglefield et aL /Brain Research 633 (1994) 151-161

down of non-locomotor movements into first, middle, and last 3-min periods was done.

3. 3. Elevated plus-maze As indicated in Fig. 4, IBO-lesioned animals made a significantly greater proportion of entries into the open arms compared to controls (t2~ = 4.69, P < 0.01)). The lesioned rats also spent a considerably greater percentage of time exploring the open arms (t21 = 3.30, P < 0.01; Fig. 4). The lesion also resulted in a significant increase in the total number of entries from 10.3 + 1.2 to 17.5 + 0.9 (Table 1, t21 = 4.75 P < 0.01). This increase appears due largely to the increase in the number of open arm entries by the lesioned animals. The lesion increased open arm entries by 146% but increased closed arm entries only 31% (Table 1). There was no difference in the total combined time spent in the open and closed arms between the two groups. The increased number of entries into open arms by the lesioned rats was not continuous nor did it appear to

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be rushed, but rather entries appeared to be intermittent, and were performed in a calm manner. In contrast, the control rats frequently went into one of the closed arms at the onset of the test, and would traverse rapidly to the other enclosed arm with only a brief pause in one of the open arms.

3.4. Social interaction test The impact of ibotenic acid lesions in the medial hypothalamus on environment-specific SI is depicted in Fig. 5. Overall, there was a significant environmental effect in both groups of rats (two-way A N O V A , Fl,14 = 18.7, P < 0.01) (Fig. 5A); namely, animals spent a significantly greater total time in SI in the familiar than in the unfamiliar environment (posthoc t-test). Control: t 8 = 3.4, P < 0.01 and IBO-lesioned rats: t 8 = 2.8, P < 0.05). There was no significant effect of lesion or Lesion x Environment interaction. However, there was a significant effect of the lesion on the composition of the SI behaviors regardless of

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Fig. 2. Localization of the bilateral injection sites in the medial hypothalamus for the 11 rats having lesions that underwent testing in all of the behavioral paradigms. Each triangle depicts a single site of ibotenate injection on the corresponding frontal planes of the Paxinos and Watson atlas. For inclusion in the data analysis, those few cases where the lesion site on one side was outside the dorsal medial hypothalamic region (e.g. lateral hypothalamus), the lesion on the opposite side was always properly situated. For clarity, the abbreviations of the pertinent structures are given: DA, dorsal area of hypothalamus; DMH, dorsomedial hypothalamic nucleus; F, fornix; LH, lateral hypothalamus; ME, median eminence; ML, medial lemniscus; MT, mammillothalamic tract; OPT, optic tract; PH, posterior hypothalamus region; VMH, ventromedial hypothalamic nucleus; ZI, zona incerta.

.l.R. lngle[ield et al. / Brain Research 033 (1994) 151 - 161

156

Fable 1 Elevated-plus maze behavior in rats with ibotenate lesions in dorsalmedial hypothalamus. Mean ( + S E M ) number of entries into and time spent (see) in {)pen arms or ch)sed arms for the 5 minute tesl

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environment (as revealed by a significant Lesion × Composition interaction (Fig. 5B) (two-way ANOVA, F~,28 = 7.33, P < 0.01)). Physical interactions (allogrooming, walking over, and sideways pushing) were significantly greater across the environments in animals with IBO lesions. Lesion × Composition × Environment interaction was not significant. 3.5. Acoustic startle response

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Fig. 6A represents the startle response to pulse alone (PA) and the three prepulse-pulse (PP-P) trial

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Fig. 3. A: total distance traveled for the 10-min test in a novel environment (autotracker). The total distance traveled by IBO animals was significantly greater than the control animals. B: time course of the distance traveled in the autotracker test for control and IBO rats. The 10-min test was broken down into 3-4-rain periods to assess effects when environment was most novel vs. least novel. Overall, there was a Lesion × Period interaction, P < 0.05. The differences emerged in the latter two time periods (* P < 0.05 relative to control for each post-hoc comparison), suggesting the animals in the two groups differentially acclimated to the novel environment. Error bars are S.E.M.

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Fig. 4. Effect of IBO lesions in the dorsomedial hypothalamus on performance in the elevated plus-maze. Rats tested in the elevated plus-maze one month following IBO lesions into the DMH exhibited a greater percentage of entries into the open arms, and spent a greater proportion of the total time in the open arms. Error bars are S.E.M. (* P < 0.05 for each respective comparison).

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Fig. 5. Total social interaction (A) and composition of social interaction behavior (B) as a function of IBO lesion of the DMH or control (sham). Testing was conducted 4 weeks after surgery. Pairs of rats were tested for 7.5 min in either a familiar (F) or unfamiliar (UF) environment. In A, * indicates a significant (P < 0.05) difference between familiar and unfamiliar environments, for respective group. Analysis indicated that behavioral composition (B) varied significantly as a function of the lesion; namely, physical interactions were greater and exploratory interactions were less in IBO-lesioned rats compared to controls (P < 0.05). The sample size was 4-5 pairs per environment and group. Error bars are S.E.M..

J.R. Inglefield et al. / Brain Research 633 (1994) 151-161

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startle amplitude across time was performed. Fig. 6B indicates the breakdown of the median PA trial type across the three 5-min blocks. Neither the controls nor IBO group exhibited decreases in startle amplitude over the 15 min of startle testing. Interestingly, there was a trend for a Group effect when collapsing across Blocks (Repeated Measures ANOVA, F ] : 2 = 3 . 3 2 , 0.10 > P > 0.05), but no Group × Block interaction. The increasing PA startle amplitudes with each successive time block in the IBO group was likely responsible for the considerable variance observed overall for PA in the IBO group (Fig. 6A).

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Fig. 6. A: the relative acoustic startle amplitudes for each trial type (pulse alone [PA] and prepulse-pulse pairs [PP-P]) for control and IBO animals. In each of the PP-P trial types, the IBO animals had significantly greater startle responses (P <0.05). Analysis of the relative reduction in startle amplitude (prepulse inhibition; calculated for each of the three PP-P trial types using the formula: [(1 - PP-P/PA)*100]) showed a trend (0.10 > P > 0.05) for the control animals to have greater PPI than IBO-treated animals (not shown). B: time course of startle amplitudes to above PA trials for control and IBO animals. Each block was 5 min in duration and Block 1 followed a 5-min acclimatization period, during which the animals were exposed to 70 dB white noise. The increase in the response of the IBO group as the test continued helps to explain the considerable variance in the PA trials in (A). All values are mean-tS.E.M. (* P < 0.05 for each respective post-hoc comparison).

types by group. IBO-lesioned animals exhibited greater startle responsiveness over all trials than controls (Repeated measures ANOVA, Main Effect for Group, F1,13 = 5.9, P < 0.05). Pairwise post-hoc comparisons showed significant group differences for all PP-P trials (Fig. 6A). The relative reduction in startle amplitude (prepulse inhibition, PPI) for each of the PP-P trial types showed the control animals to have a trend for greater PPI than IBO treated animals (Repeated measures ANOVA, Fl,13 = 3.54, 0.10 > P > 0.05; data not shown). There was not a significant Trial Type by Group interaction for A S k . In view of the considerable variance in the IBO pulse alone (PA) data (Fig. 6A), an analysis of PA

Taken together, the effects of small IBO lesions placed in the regions of dorsomedial hypothalamus suggest that the lesions may have led to a disinhibition of normally suppressed responding to innate fear or challenging stimuli. The lesion increased locomotion in a novel environment, increased performance on the open arms of the elevated plus-maze, and increased the acoustic startle response. It also led to an altered composition of behavior in the test of social interaction. These effects most likely are related to disruption of neuronal cell bodies, rather than fibers of passage, considering the small amount of IBO that was used [42,51]. The motility testing findings indicate greater overall locomotion in the 1BO-treated animals than controls. Enhanced ambulation of rats that are inexperienced with the open field situation has been previously seen following administration of a benzodiazepine (BDZ), an anxiolytic drug [6]. However, the finding of greater exploratory behavior is not very specific in terms of anxiety-related behaviors, since several other factors that are not directly linked to anxiolysis can affect this behavior (e.g. dopaminergic system activation, see [25]). The lesioned animals showed motoric behavior analogous to controls early in the testing session, but further analysis revealed significant differences between the groups in the latter two periods of the 10-min test. Thus when the situation was the most novel (i.e. the first 3 min of the session), both groups had similar ambulatory scores. But as time progressed the lesioned rats showed a different response to the situation. None of the lesioned animals' behavior was such that it would be described as flight-oriented nor was there any jumping; rather, they seemed to frequently move across the center to explore other sides of the chamber or they engaged in grooming, looking around, stereotypy, etc. Interestingly, increased ambulation in an open field test following specific ibotenic acid lesions of the parvocellular component of the PVN has also been reported [19]. This similarity in behavioral effects seen

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in an open field test regardless of whether the lesion was situated within the PVN or D M H is striking and argues for the dual involvement of these connected nuclei in exploration-oriented behaviors. Excessive grooming, which detracts from locomotor activity, has been shown to occur following intraventricular administration of C R F [11], a peptide with behavioral arousal-inducing effects [5]. On the other hand, stereotypy can result from striatal activation, and increased striatal dopamine turnover is predictive of enhanced exploratory behavior in rats [53]. Such behaviors as self-grooming and small repetitive movements (stereotypy) were combined under the non-locomotor movement category by the autotracker equipment. The inability to distinguish between these movements, which may reflect a different emotional valence, precludes any conclusions being drawn from this particular data. The clearest indicator of a possible anxiolytic effect of the lesion comes from the elevated plus-maze results. The elevated plus-maze has been established as a sensitive index of the level of anxiety in rats [35]. Treatment with anxiolytic drugs (such as BDZs) increases performance (increased percentage of number of entries and of time) on the open-arms [35]. Our results show that small excitotoxic lesions in areas of the dorsomedial hypothalamus mediate a similar antianxiety effect that would not seem to be attributed to an increase in general activity. The fact that we observed the total number of entries to be significantly increased in IBO rats might suggest an enhanced spontaneous motor activity (as we saw in the open field test). While locomotor activity was not measured in this test, the finding that the lesion increased open arm entries by 146% but increased closed arm entries only 31% would seem to argue against an increase in overall general activity as responsible for the effects observed in this test. Interestingly, Pesold and Treit have found that extensive excitatory amino acid (EAA) lesions of the posterior septum also lead to anti-anxiety effects on the elevated plus-maze [36]. Notably, rats with this lesion spent much more of the total time on the open arms and a greater percentage of entries in the open arms (70-75% for both indices) as compared to that observed with our lesioned rats (47% of total entries and 36% of total time in open arms). They concluded that neurons in the posterior septum play a primary role in mediating anxiety in this test. Of particular interest is the recent finding that one of the few projections of the D M H to higher brain centers is to the lateral septum [56]. In light of our current findings, this suggests that one function of this connection between the D M H and septum may be to modulate fear reactions. Unlike the results on the plus-maze, testing for environment-specific social interaction (SI) did not re-

veal an anxiolytic effect of the IBO lesion; lesioned animals did not demonstrate greater interaction in the unfamiliar environment than controls. Administration of anxiolytic drugs is known to increase SI in the unfamiliar environment [14]. The lesions did, however, alter the behavioral composition of SI. While the finding of a greater time spent in vigorous-contact (physical) interactions by the IBO rats as compared to controls cannot be readily interpreted, similar behavioral composition has been observed in previous studies. Firstly, rats having hypothalamic 6 - O H D A lesions demonstrated an increased physical (non-violent) behavior relative to sham-operated controls [21]. Secondly, enhanced physical interactions have also been observed in juvenile (day 28) and young adolescent (day 35) rats compared to young adult rats [38]. This type of behavior resembles the 'rough and tumble' play behavior described by Meaney and Stewart [29] in male rats between 26 and 40 days of age. The fact that matching findings from the SI test are observed after placing 6 - O H D A lesions in the PVN or lesioning neurons in the dorsomedial hypothalamus suggests that these interconnected nuclei may serve in a similar capacity in terms of transmitting information to higher brain centers for 'translation' into behavioral changes. The question arises as to whether the increases in adult physical interactions (especially in the unfamiliar environment) might relate to an anxiogenic effect of the lesion. This would not seem to be the case since physical interactions do not increase in the unfamiliar environment in intact, adult male rats [38]. Also, intraventricular infusion of a 'stress transmitter,' corticotropin releasing factor, has been shown to significantly decrease Total SI in the familiar environment, and this effect can be antagonised by pre-treatment with a B D Z anxiolytic [12]. However, in our study, the IBO rats in the familiar environment spent an identical amount of time engaged in Total SI compared to control rats in the same environment. This indicates that the increased time spent in physical interactions by the IBO rats probably was not the result of an anxiogenic effect of the lesion. The acoustic startle findings demonstrated a significant enhancement of startle responsiveness in lesioned rats with a trend for the relative efficacy of prepulses to inhibit the startle response (prepulse inhibition, PPI) to be reduced in IBO animals. An enhancement in startle amplitude has been cited most often in its relation to conditioned fear [7,8] and background noise [22]; both being stressors. Sudden dark onset, a possible ecologically-relevant visual stimulus, has also been shown to potentiate startle amplitude [20]. Modification of responses to these facilitators of startle can be effected by administering the anxiolytic drug, diazepam, which blunts the facilitated ASR [8,20,22].

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Since, in the present study, the startle stimulus was presented against a 70 dB white noise background, it could be postulated that a greater response in the basal startle amplitude for the IBO-treated rats reflects a diminution of anxiolytic influences, a supposition opposite in direction to our above findings. However, the interpretation that increased startle response always reflects an increased stress state is not uniformly upheld in all reports. Sorenson and Swerdlow [49] reported a significant decrement in the ASR amplitude when the rats were exposed to repetitive tail pinch (a stimulus that elicits an increase in plasma corticosterone (CS), a classic index of a stress state). Another study that employed a startle paradigm similar to that used here observed an inverse relationship between startle response amplitude and plasma CS levels [17]. Thus, in this situation, although counter to the theory that a potentiated startle response is directly related to heightened fear (stress) state, it is possible startle amplitude is not a direct index of stress state. Rather, it may instead reflect a general change in the animal's attentiveness to external stimuli. The fact that startle amplitude tended to increase in the IBO rats over time and did not change over the 20-min test period in the controls (Fig. 6B), further supports an increased state of arousal or attention in the lesioned group. This may have been the result of a disinhibition, an interpretation that would also support the findings from the other behavioral tests carried out in this study. Interestingly, the trend in the IBO group for a reduction in PPI is in agreement with findings from a recent study reporting that less PPI strongly predicted greater exploratory behavior and increased time spent in the open arms of the elevated plus-maze [43]. It is plausible that a sudden loud noise activates neural circuits that involve the D M H and dorsal area, and that lie outside the primary (brainstem) ASR pathway. The present results are in agreement with relationships found between mesolimbic dopaminergic transmission and l o c o m o t o r / s t a r t l e phenomena. There is substantial evidence that increased dopaminergic transmission can increase startle [7,44]. Furthermore, changes in locomotion and modification of the ASR are thought to be mediated through ventral striatal pathways [52,53]. These results, in conjunction with the fact that there are likely efferents from the dorsal medial hypothalamic region to the striatum (see [33]), suggest that the IBO lesion may have perturbed such a connection. Alternatively, besides the striatum, the midbrain central grey receives an input from the D M H [27,56], and the central grey is capable of modifying the ASR substantially [3]. Hence, alterations in the ASR may also be the result of a disruption of these other pathways. This region of the hypothalamus that was lesioned is clearly important for regulation of physiological and

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behavioral responses that are associated with emotional arousal [9,15,30,48]. It is particularly interesting that our findings further point to the medial hypothalamus as not merely an effector system for physiological or neuroendocrine regulation, but also as a component of an ascendant system to influence functioning of higher brain centers. Since there is reason to believe behavioral and autonomic responses to stressors would occur in a coordinated manner, it can be postulated from the aggregation of these findings that the D M H (along with the nearby dorsal area) is situated within several diverging functional systems so that it may coordinate the actions of specific brain nuclei and thereby be involved in both behavioral and autonomic responses to a challenge (see ref. 9). In support of this, the dorsomedial hypothalamus has, besides the striatal and lateral septal projections already mentioned, modest ascendant projections to the hippocampus, amygdala, and frontal and cingulate cortices [23,55]. Interestingly, those behaviors from rats having lesions on one side that were situated in the dorsal area or lateral hypothalamic-perifornical region were similar to those of the bilaterally DMH-lesioned animals. These areas have demonstrated cardiovascular control functions [16,50]. It may be that the lesion impinged on a portion of a population of melanocyte-concentrating hormone (MCH) neurons that are situated in the hypothalamic dorsal area and dorsal lateral hypothalamus [2,32]. These neurons have prominent terminal fields in all of the regions that the D M H innervates and, coincidentally, are postulated to have functional roles in arousal and sensorimotor integration [2]. In summary, from this set of experiments it would appear that lesions in the dorsomedial hypothalamic region disrupt behavioral responses to novel and challenging environments in a disinhibitory manner. Thus, there would seem to be neural systems that can rapidly convey stressor-related information from the hypothalamus to higher brain centers to invoke changes in behavior and the present evidence suggests the dorsal medial hypothalamus is a key component in that system. Acknowledgements.

This work was supported by U.S. Public Health Service Grants DA07080 and MH00651. We wish to thank Jennifer Coleman and G. Peter Bowen for expert technical assistance on the autotracker and acoustic startle equipment, and G. Peter Bowen as well as Drs. Joe DiMicco and Jim Ison for discussion of the manuscript.

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