Brain Research 950 (2002) 186–194www.elsevier.com/ locate/bres

Research report

T he distribution of fos immunoreactivity in rat brain followingfreezing and escape responses elicited by electrical stimulation of the

inferior colliculusa a aMarisol R. Lamprea , Fernando P. Cardenas , Daniel Machado Vianna ,

a b a,*˜Vanessa M. Castilho , Sara Eugenia Cruz-Morales , Marcus L. Brandaoa ´Laboratorio de Psicobiologia, FFCLRP, Campus USP, Av. Bandeirantes 3900, 14049-901 Ribeirao Preto, SP, BrazilbLaboratory of Psychopharmacology, FES-Iztacala, UNAM, P.O. Box 314, Tlanepantla, Edo. Mexico 54090,Mexico

Accepted 6 May 2002

Abstract

Several sources of evidence indicate that the inferior colliculus also integrates acoustic information of an aversive nature besides itswell-known role as a relay station for auditory pathways. Gradual increases of the electrical stimulation of this structure cause in ahierarchical manner alertness, freezing and escape behaviors. Independent groups of animals implanted with bipolar electrodes into theinferior colliculus received electrical stimulation at one of these aversive thresholds. Control animals were submitted to the sameprocedure but no current was applied. Next, analysis of Fos protein expression was used to map brain areas activated by the inferiorcolliculus stimulation at each aversive threshold and in the controls. Whereas alertness elicited by stimulation of the inferior colliculus didnot cause any significant labeling in any structure studied in relation to the respective control, electrical stimulation applied at the freezingthreshold increased Fos-like immunoreactivity in the central amygdaloid nucleus and entorhinal cortex. In contrast, escape responseenhanced Fos-like immunoreactivity in the nucleus cuneiform and the dorsal periaqueductal gray matter of the mesencephalon. Thisevidence supports the notion that freezing and escape behaviors induced by electrical stimulation of the inferior colliculus activatedifferent neural circuitries in the brain. Both defensive behaviors caused significant expression of c-fos in the frontal cortex, hippocampusand basolateral amygdaloid nucleus. This indistinct pattern of c-fos distribution may indicate a more general role for these structures inthe modulation of fear-related behaviors. Therefore, the present data bring support to the notion that amygdala, dorsal hippocampus,entorhinal cortex, frontal cortex, dorsal periaqueductal gray matter and cuneiform nucleus altogether play a role in the integration ofaversive states generated at the level of the inferior colliculus. 2002 Elsevier Science B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: Stress

Keywords: Fos expression; Freezing; Escape; Fear; Inferior colliculus; Amygdala; Periaqueductal gray matter

1 . Introduction xiogenic stimulus presented to the animals and with thebehaviour taken as an index of anxiety. Therefore, distinct

We have shown that alertness, freezing or escape kinds of aversive stimuli are supposed to generate differentbehaviors may be generated at the level of the dorsal types of fear [6,8,40,41,43]. Indeed, we have recentlyperiaqueductal gray (dPAG) of the rat depending on the shown that distinct types of freezing are elaborated in theintensity of the electrical stimulation applied to this region periaqueductal gray matter; contextual conditioned freez-[9,12,14]. Nowadays, there is general agreement that ing is related to circuits in its ventral part and uncon-animal models of anxiety differ according to the an- ditioned freezing to its dorsal area [86]. By the same token,

electrical and chemical stimulation of the inferior col-liculus also cause defensive behavior characterized by*Corresponding author. Fax:155-16-633-1609.

˜E-mail address: mbrandao@usp.br(M.L. Brandao). alertness, freezing and escape responses accompanied by

0006-8993/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 02 )03036-6

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increases in mean blood pressure, tachycardia, and other on the US National Institutes of Health Guide for Care andautonomic responses common to the defense reaction such Use of Laboratory Animals.as piloerection, micturition and defecation [10,11,15]. Ithas been shown that the neurocircuitry in the inferior

2 .2. Surgerycolliculus responsible for the production of aversive statesis regulated by a large number of neurotransmitters

The animals were anaesthetized with tribromoethanol[11,12,14]. Moreover, we reported that aversive stimula-

(250 mg/kg, i.p.) and placed in a stereotaxic frame (Davidtion of the inferior colliculus enhances dopamine release in

Kopf, USA). A brain electrode was implanted in thethe prefrontal cortex, a structure normally activated during

midbrain, aimed at the inferior colliculus. The electrodethreatening situations [24]. Nevertheless, no attempt has

was made of stainless steel wire, 160mm in diameter,been made so far to establish the anatomical connections

insulated except at the cross section of the tip. The upperand functional significance of the several components of

incisor bar was set 3.3 mm below the interaural line so thatthe defense reaction generated at the level of the inferior

the skull was horizontal between bregma and lambda. Thecolliculus.

electrode was introduced vertically using the followingDefensive strategies as distinct as risk assessment,

coordinates with the lambda serving as the reference foravoidance, escape and fight are likely to be organized by

each plane: postero-anterior, 1.2 mm; medio-lateral, 1.5different neural networks [5,7,8,32,40–42]. Therefore, it is

mm; and dorso-ventral, 4.5 mm [64]. The electrode wasexpected that the set of brain structures activated by

fixed to the skull by means of acrylic resin and threefreezing response induced by electrical stimulation of the

stainless steel screws. The electrode wire was connected toinferior colliculus should be different from that activated

a connector so that it could be plugged into an amphenolby escape behavior so-induced. To test this prediction, Fos

socket at the end of a flexible electrical cable used forprotein immunoreactivity was presently measured in serial

brain stimulation.sections of the brain following expression of either freez-ing or escape induced by electrical stimulation of theinferior colliculus. Sham-stimulated animals of the control 2 .3. Apparatusgroup were exposed to the same procedure. It has beenchosen because it can be controlled by the experimenter, One week after surgery, the rats were placed in an openthe novelty of the exposition and the locomotor activity of field which was a circular enclosure 60 cm in diameter andthe animal while exploring the arena between stimulations. 50 cm high. The rat was placed in the arena and had itsFos protein is the product of the proto-oncogene c-fos and brain electrode connected to a flexible wire cable, allowingwas shown to be synthesized in neurons as a consequence ample movement inside the box. The cable, in turn, wasof increases in intracellular calcium, cyclic AMP, or other connected to the stimulator by means of a mercury swivelsecond messengers [61,62,77]. Since this protein remains mounted on the top of the experimental chamber. The ratsin the cell nucleus for a short period of time, its detection were allowed a 15 min period of habituation in thehas been extensively used to map neuronal bodies acti- enclosure. The brain was stimulated electrically by meansvated by specific stimuli [30,71,82]. This increase in Fos of a sine wave stimulator [58]. The stimulation current wasprotein expression has been shown to occur as a result of monitored by measuring the voltage drop across a 1 kVneuronal activation by a wide range of stimuli, such as resistor with an oscilloscope (Philips, USA). Brain stimula-convulsing agents [26,61], electrical current [51,72,79], tion (AC, 60 Hz, 15 s) was presented at 1 min intervalsneurotransmitters [70,83], immobilization stress [44,46], with the current intensity increasing by steps of 5mA forpain [17,45], injection of hypertonic saline [22,76] and measurements of the alertness, freezing and escape thres-exposure to threatening stimuli of the elevated plus-maze holds. Alertness threshold was operationally defined as theor T-maze [78,80]. lowest intensity producing episodes of movement arrest

with head orientation in two consecutive series of electricalstimulation. Freezing threshold was operationally defined

2 . Materials and methods as lowest intensity producing stop of the ongoing behaviorin two consecutive series of electrical stimulation accom-

2 .1. Animals panied by at least two of the following autonomic re-sponses: piloerection, defecation, micturition and exhop-

Naive male Wistar rats weighing 240–260 g were used. htalmus. Escape threshold was operationally defined as theAnimals were kept under controlled temperature lowest current intensity that produced running or jumping(2262 8C) and a 12-h light:dark cycle (lights on at 07:00 in two successive ascending series of electrical stimulation.h). They were housed two per cage and had free access to Animals with an escape threshold above 120mA (peak-to-food and water throughout the experiment. The experi- peak) were discarded from the experiment. The adequacyments reported in this article were performed in com- of the current intensity levels for the aversive responsespliance with the recommendations of SBNeC (Brazilian studied herein was verified on the basis of previous studiesSociety of Neuroscience and Behavior), which are based from this laboratory [59,13,85].

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2 .4. Experimental procedure under bright-field microscopy in brain structures earliershown to be activated by aversive conditions as reported

The animals were randomly allocated to the alertness by other studies [24,53,72,73,78,80]. The nomenclature(n56), freezing (n56), escape (n56) or sham-stimulated and nuclear boundaries utilized were based on the atlas ofgroups (n56). Animals of all four groups were placed in Paxinos and Watson [64] and the planes of analyzedthe arena and allowed 15 min for habituation. Next, the sections were standardized as far as possible. Neuronalaversive thresholds were determined in only one series of nuclei expressing levels of DAB reaction product aboveascending intensity of electrical stimulation. then sub- tissue background were automatically counted by a com-mitted to its respective experimental session. On the next puterized image analysis system (Image Pro Plus 4.0,day, the electrical stimulation was applied at the aversive Media Cybernetics, USA), according to method usedthreshold determined the day before and afterwards their previously in this laboratory [80]. Briefly, mounted sec-brains were processed in parallel, as described below. tions of the tissue were observed using a light microscope

(Olympus BX-50) equipped with a video camera module2 .5. Fos protein immunohistochemistry (Hamatsu Photonics C2400) and coupled to a comput-

erized image analysis system (Image Pro Plus 5.0, MediaTwo hours after the electrical stimulation procedure, the Cybernetics, USA). Counting of fos-positive cells was

animals were deeply anaesthetized with urethane (1.25 performed at a magnification3100, in one field per areag/kg; Sigma, USA) and transcardially perfused with 0.9% encompassing the entire brain region included in quantifi-saline followed by 4% paraformaldehyde in 0.1 M phos- cation. An area of the same shape and size per brain regionphate-buffered saline (PBS) (pH 7.4). Brains were re- was used for each rat. The same light and thresholdmoved, immersed (48C) in the above fixative for 2 h and conditions were employed for all sections. Fos stainingthen kept in 30% sucrose in 0.1 M PBS until soaked. They could vary from one area to another. However, in order towere then quickly frozen in isopentane (240 8C) and sliced ensure accuracy of measurement and avoid variationsby the use of a cryostat (215 8C). Two adjacent series of among same areas in different subjects, the background of40-mm thick brain slices were obtained, having as refer- every area was measured and digitally subtracted from theence the following AP coordinates: Bregma13.2, 10.7, area under examination. Accordingly, the threshold con-20.3, 21.3, 21.8, 23.3, 24.8, 26.3, 27.8, 28.3, 29.8 ditions were set for each area and maintained for allmm. One series was Nissl stained and used for neuro- subjects [80]. All brain regions were bilaterally counted inanatomical comparison purposes. The other series was various sections for each rat depending on the size of thecollected in 0.1 M PBS and subsequently processed free- structure. After that, counts for each region were averagedfloating according to the avidine–biotine procedure, using over the sections. Nuclei were counted individually and

2the Vectastain ABC Elite peroxidase rabbit IgG kit (Vector, expressed as number of Fos-positive nuclei per 0.1 mmUSA, Ref. PK 6101). All reactions were carried out under [68].agitation, at room temperature. The slices were firstincubated with 1% H O for 10 min, washed four times2 2 2 .7. Statisticswith 0.1 M PBS (5 min each) and then incubated overnightwith the primary Fos polyclonal antibody (Santa Cruz,

Statistical analysis of thresholds (alertness, freezing andUSA, SC-52) at a concentration of 1/20 000 in 0.1 M PBS

escape) and immunohistochemical data were subject toenriched with 4% normal goat serum and 0.2% Triton-X.

one-way ANOVA followed by Duncan’s test. The resultsSlices were again washed three times (5 min each) with 0.1

are presented as mean6S.E.M.M PBS and incubated for 1 h with biotinylated goatantirabbit antibody (25ml from the kit for 10 ml of 0.1 MPBS enriched with 4% normal goat serum and 0.2%Triton-X). After another series of three 5-min washings in 3 . Results0.1 M PBS, they were incubated for 1 h with the avidine–peroxidase solution (two drops of each solution labeled A 3 .1. Behavioural effectsand B of the kit for 25 ml of 0.1 M PBS) and three timeswashed in 0.1 M PBS (5 min per wash). The slices were The tips of the electrodes were situated inside the centralfinally allowed to remain for 5 min in a solution of nucleus of the inferior colliculus. A representative site with3,39-di-aminobenzidine (DAB, 0.02%) to which hydrogen a bipolar electrode in the inferior colliculus is shownperoxide (0.04%) was added just prior to use and were elsewhere [21]. The intensity of the electric current appliedwashed twice with 0.1 M PBS. to the inferior colliculus of the animals to induce alertness,

freezing and escape responses was 31.6666.62,2 .6. Quantification of fos-positive cells 61.6769.50 and 99.17611.14mA (peak-to-peak), respec-

tively. ANOVA performed on these data revealed that theSections were mounted on gelatin-coated slides, dehy- differences between these aversive thresholds were highly

drated and coverslipped for observation and cell counting significant (F 513.29, P,0.001). Post-hoc analysis2,15

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indicated that there is a statistically significant difference 3.07,P50.07). No significant fos expression could beamong the groups. detected in the remaining structures listed in Table 1.

Post-hoc comparisons (Duncan’s test,P,0.05) indi-3 .2. Fos protein expression cated that the freezing and escape groups exhibited a

higher density of Fos-like immunoreactive cells in theImmunoreactive cells exhibited a dark nucleus in neuro- frontal cortex (Fig. 1A), basolateral amygdaloid nuclei

nal nuclei expressing clearly upon the surrounding back- (Fig. 1B) and in dorsal hippocampus in relation to theirground tissue. In the control group, clusters of Fos-like respective control groups.immunoreactive cells could already be identified in some In animals submitted to the electrical stimulation of thebrain regions, namely the frontal cortex (Fig. 1A), the inferior colliculus at the freezing threshold, post-hocdorsal periaqueductal gray matter of the mesencephalon analysis showed that Fos-like immunoreactivity was sig-(Fig. 3A) and the inferior colliculus (Fig. 3C). nificantly enhanced in the entorhinal cortex (Fig. 2A) and

The results obtained with quantitative analyses of Fos- central amygdaloid nucleus (Fig. 2B) in relation to thelike immunoreactivity in the studied brain regions after control group. Fos-like immunoreactivity in the escapeinduction of freezing and escape responses in rats stimu- group was significantly increased in dPAG (Fig. 3A),lated in the inferior colliculus are summarized in Table 1. cuneiform nucleus (Fig. 3B) and inferior colliculus (Fig.As no significant differences could be detected between the 3C) in relation to the control group.non-operated and alertness groups for all structures studiedhere these data were pooled together in just one controlgroup. Statistical comparison of these data by means of4 . Discussionone-way ANOVA indicated that there was a significantdifference in Fos protein expression in the frontal cortex In the present work, stepwise increases in the electrical(F 511.45,P,0.01), basolateral amygdala (F 57.39, stimulation of the inferior colliculus elicits first alertness,2,12 2,15

P,0.01), entorhinal cortex (F 54.18, P,0.05), dorsal then freezing and finally escape accompanied by au-2,17

hippocampus (F 57.36, P,0.01), central amygdaloid tonomic responses, such as piloerection, exophtalmos,2,17

nuclei (F 53.76, P,0.05), dorsal periaqueductal gray micturition and defecation. These data are in agreement2,15

matter of the mesencephalon (F 5315.17, P,0.001), with results obtained earlier in our laboratory, suggesting2,16

cuneiform nucleus (F 56.55, P,0.01) and central nu- that the inferior colliculus is also part of a brain aversion2,16

cleus of the inferior colliculus (F 54.67, P,0.05). system [10–12,14].2,15

Marginal significance was obtained in the septum (F 5 The inferior colliculus establishes direct or indirect2,11

3.18,P50.08) and the ventral periaqueductal gray (F 5 connections with several structures containing neural sub-2,17

Fig. 1. Photomicrographs of Fos-like immunoreactive cells (dark dots) in coronal sections through brain regions with significant increase in Fos expressionafter performance of both freezing (middle panel) and escape (right panel) responses after electrical stimulation of the inferior colliculus, in relation to thecontrol group (left panel). FrCx, frontal cortex; BlA, basolateral amygdaloid nucleus. Scale bar5200 mm.

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Table 12Number of Fos-like immunoreactive nuclei per 0.1 mm (mean6S.E.M.) in different brain regions of animals submitted to the electrical stimulation of the

inferior colliculus at either freezing or escape thresholds

Areas Control Freezing Escapea aFrontal cortex 165.48639.68 616.96645.72 374.386109.41

aEntorhinal cortex 28.4364.73 48.9767.54 43.9566.93a aDorsal Hippocampus 7.0861.87 18.4665.40 22.0161.44

Septum 10.5762.94 25.3066.95 18.9264.21Septohypothalamic nucleus 92.03621.81 82.71630.27 72.75627.88

aCentral amygdaloid nucleus 24.2663.72 47.3363.17 32.04610.71a aBasolateral amygdaloid nucleus 24.4463.27 41.6564.08 48.1869.40

Medial amygdaloid nucleus 35.9264.33 46.7666.54 51.03610.55Bed N of stria terminalis 31.9465.63 57.78616.14 42.00615.05Preoptic nucleus 61.0567.76 69.05612.23 51.29612.31Anterior hypothalamic nucleus 46.8566.08 56.81611.67 44.55610.19Dorsomedial hypothalamus 76.28612.60 69.60610.93 83.55623.91Suprachiasmatic nucleus 31.2967.74 24.8868.38 28.31613.21Lateral hypothalamus 50.3363.53 52.7466.43 54.56611.44Periv. hypothalamus 5.1461.96 7.3162.48 5.3261.57Supamammilary nucleus 108.6568.89 96.73617.04 144.95627.86Substantia nigra, pars reticulata 2.1560.40 4.6662.46 0.3960.18Ventral PAG 20.7962.45 35.3765.27 31.9467.99

aDorsal PAG 18.0763.46 16.5063.39 49.2165.51Superior colliculus 28.7367.93 35.3063.43 37.5563.56

aCuneiform nucleus 24.9364.80 38.0365.11 56.4468.88Dorsal raphe n. 30.3464.18 25.4663.84 33.1964.66Locus coeruleus 50.5463.84 64.8766.86 69.65611.04

aCentral n. inferior colliculus 78.87611.01 85.25620.28 151.66613.44a P,0.05, in relation to the control group (Duncan’s test).

strates of fear [14]. The present findings clearly show at the freezing and escape thresholds. Reported evidenceincreased fos-expression in the basolateral amygdaloid indicates that basolateral amygdala is a point of conver-nuclei after electrical stimulation of the inferior colliculus gence for conditioned and unconditioned stimuli and seems

Fig. 2. Photomicrographs of Fos-like immunoreactive cells (dark dots) in coronal sections through brain regions with significant increase in Fos expressionspecifically after performance of freezing (middle panel) induced by electrical stimulation of the inferior colliculus, in relation to the control group (leftpanel). Fos expression in the group that received electrical stimulation of the inferior colliculus at the escape threshold is also indicated. EntCx, entorhinalcortex; CeA, central amygdaloid nucleus. Scale bar5200 mm.

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Fig. 3. Photomicrographs of Fos-like immunoreactive cells (dark dots) in coronal sections through brain regions with significant increase in Fos expressionafter performance of escape responses (right panel) induced by electrical stimulation of the inferior colliculus, in relation to the control group (left panel).Fos expression in the freezing group is also illustrated (middle panel). dPAG, dorsal peraqueductal gray matter; Cnf, cuneiform nucleus; CIC, centralnucleus of the inferior colliculus. Scale bar5200 mm.

to impart emotional value to sensory stimulation According to the hypothesis under testing, the freezing[19,25,32,33,35,48,54,55,57]. Accordingly, increased ex- and escape responses were expected to activate differentpression of immediate early genes in the amygdala has sets of brain structures and this prediction was confirmedbeen reported using several paradigms of aversive con- by the present results. Only central amygdaloid nucleus,ditioning [3,5,62,66,69]. It has been suggested that the dorsal hippocampus and entorhinal cortex were labeled byinvolvement of the inferior colliculus in fear processes can c-fos immunoreactivity following freezing responses in-occur through its anatomical and functional connections duced by electrical stimulation of the inferior colliculus.with the amygdala [14,56]. Marked increases in c-fos Although marginally significant, the ventral periaqueductalexpression in the freezing and escape groups were also gray also showed considerable labeling after freezing butobserved in the prefrontal cortex. These findings are not not after escape response induced by stimulation of thesurprising as cortical projections are also activated by a inferior colliculus. These data are in line with severalwide variety of aversive stimulation [1,28,34,39,84]. These studies that show that this structure is crucial for passivecortical areas are clearly connected to the inferior col- defensive responses [32,35]. However, as we have alreadyliculus [2,16,23,38,60]. One of these pathways is provided shown that lesion of the ventral part of the periaqueductalby projections from the central nucleus of the inferior gray, increased the freezing and escape thresholds de-colliculus to the prefrontal cortex through the medial termined by stimulation of the inferior colliculus (althoughgeniculate nucleus, amygdala and dorsomedial thalamus the effects reached marginal significance), we should not[16,38]. It has been shown that this circuit is concerned discard the possibility that the inferior colliculus could bewith the processing of auditory information of aversive part of a defense system that could have the ventralnature, which triggers fear-like behaviors [14,24]. periaqueductal gray as one of its output [56].

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On the other hand, immediate threat causes escape nique the present results should be interpreted cautiously.In fact, this technique does not allow for distinguishingbehavior, which seems to be mediated by the dorsalbetween retrograde activation of afferent connections andperiaqueductal gray, hypothalamus and amygdala [40,41].feedforward efferent connections to the structure stimu-The only regions selectively activated by escape were thelated. Thus, we cannot discard the possibility that thedorsal periaqueductal gray matter, cuneiform nucleus andelectrical stimulation of the inferior colliculus could there-the inferior colliculus itself. Electrical or chemical stimula-fore produce distant fos-expression by antidromic thention of the dorsal periaqueductal gray has been shown toorthodromic activation of branched afferent collaterals.elicit an explosive flight reaction that resembles a panicAlso, the different levels of electrical stimulation producedattack [9,40,41,47]. Also, neurosurgical patients reporteddifferent behavioral reactions, that is, the different groupsfeelings of terror and impending death after electricalof animals were exposed to different levels of external andstimulation of this region [63]. Several studies using c-fosinternal sensory information, simply as a result of differenthave shown that the dorsal periaqueductal gray is activatedstimulus-evoked movement. These confounding differ-in stressful situations [3,4,20,49,50,67,72,73], inclusiveences could also cause differential c-fos expression be-after intraperitoneal injection of drugs capable of elicitingtween the groups. Furthermore, the fact that high levels ofpanic symptoms [81]. Moreover, it was shown that disrup-IC stimulation failed to evoke fos-expression in sometion of tonic GABAergic inhibition in the dPAG elicits astructures activated by lower levels of stimulation, forconstellation of behavioural and physiological responsesinstance central nucleus of the amygdala, may be easier tothat resembles a human panic attack [9,12,14]. Theunderstand in terms of indirect rather than direct mecha-cuneiform nucleus seems to be the main output from thenisms. This latter methodological limitation of this tech-dPAG [18]. Electrical and chemical stimulation of thenique could explain why lesions of the central amygdalacuneiform nucleus causes clear defense reactions resem-increase the threshold for both freezing and escape be-bling that produced by activation of neural substrates ofhaviors [56] and c-fos expression in this nucleus was onlyfear in the dPAG or inferior colliculus [27,73]. Also,increased after freezing, as noted in the present study. On

neurons of cuneiform nucleus are activated by exposure ofthe other hand, autonomic reactions could not be easily

rats to cat odor [29] and show an increase of c-fosevoke as a probable explanation for such differences, at

expression with stimulation of the dPAG and medialleast those recorded in this study, piloerection, exophtal-

hypothalamus [72,73]. Expectedly, the central nucleus ofmos, micturition and defecation were present as accom-

the inferior colliculus was also significantly labeled afterpanied responses of both behavioral reactions, freezing and

stimulation of this structure at the escape threshold. Theescape.

inferior colliculus establishes reciprocal anatomical con-In conclusion, the present neuroanatomical evidence

nections with the dorsal periaqueductal gray [52,60].adds to earlier reported behavioural results indicating that

Along with this latter structure our data also point to thefreezing and escape responses represent two types of fear

cuneiform nucleus as a good candidate for the output ofwith separate neural circuits in the brain. Our data also

escape responses generated at this level.point out to the modulatory role played by the basolateral

In the present experiment, we found no difference in Fosamygdala, dorsal hippocampus and the prefrontal cortex in

protein expression between freezing, escape and controlthe integration of aversive information generated at the

groups in the anterior, dorsomedial and preoptic nuclei oflevel of the inferior colliculus.

the hypothalamus. These findings were unexpected sincethe hypothalamus is one of the major recipients of in-formation processed in the amygdala [65]. Defense re-

A cknowledgementsactions seem to be organized by both anterior[36,37,74,75] and medial hypothalamic nuclei [36,37] and

Research supported by FAPESP (Proc No. 98/11187-2these structures are activated after exposure of rodents to

and 02/03705-0) and CNPq (Proc No. 94/5933-2). F.natural predators [20] or agonistic encounters [49,50].

Cardenas, M.R. Lamprea and V.M. Castilho are recipientsOther studies have employed analysis of c-fos gene

of doctor scholarships from FAPESP. D.M. Vianna isexpression to identify brain structures activated by expo-

recipient of a doctor scholarship from CNPq. S.E. Cruz-sure to the elevated plus-maze [31,78]. An interesting

Morales had a sabbatical leave supported by CONACYTfinding of one of these studies was the remarkable fos

´and DGAPA, UNAM, Mexico.expression of the central nucleus of the inferior colliculus[78]. This finding fits quite well within the framework ofour hypothesis that the inferior colliculus has neural

R eferencessubstrates for more than one kind of fear, since theelevated plus-maze has been considered a ‘mixed’ model

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