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The distribution of fos immunoreactivity in rat brain following freezing and escape responses elicited by electrical stimulation of the inferior colliculus
Brain Research 950 (2002) 186–194 www.elsevier.com / locate / bres
Research report
The distribution of fos immunoreactivity in rat brain following freezing and escape responses elicited by electrical stimulation of the inferior colliculus Marisol R. Lamprea a , Fernando P. Cardenas a , Daniel Machado Vianna a , a b ˜ a ,* Vanessa M. Castilho , Sara Eugenia Cruz-Morales , Marcus L. Brandao a
´ Laboratorio de Psicobiologia, FFCLRP, Campus USP, Av. Bandeirantes 3900, 14049 -901 Ribeirao Preto, SP, Brazil Laboratory of Psychopharmacology, FES-Iztacala, UNAM, P.O. Box 314, Tlanepantla, Edo. Mexico 54090, Mexico
b
Accepted 6 May 2002
Abstract Several sources of evidence indicate that the inferior colliculus also integrates acoustic information of an aversive nature besides its well-known role as a relay station for auditory pathways. Gradual increases of the electrical stimulation of this structure cause in a hierarchical manner alertness, freezing and escape behaviors. Independent groups of animals implanted with bipolar electrodes into the inferior colliculus received electrical stimulation at one of these aversive thresholds. Control animals were submitted to the same procedure but no current was applied. Next, analysis of Fos protein expression was used to map brain areas activated by the inferior colliculus stimulation at each aversive threshold and in the controls. Whereas alertness elicited by stimulation of the inferior colliculus did not cause any significant labeling in any structure studied in relation to the respective control, electrical stimulation applied at the freezing threshold increased Fos-like immunoreactivity in the central amygdaloid nucleus and entorhinal cortex. In contrast, escape response enhanced Fos-like immunoreactivity in the nucleus cuneiform and the dorsal periaqueductal gray matter of the mesencephalon. This evidence supports the notion that freezing and escape behaviors induced by electrical stimulation of the inferior colliculus activate different neural circuitries in the brain. Both defensive behaviors caused significant expression of c-fos in the frontal cortex, hippocampus and basolateral amygdaloid nucleus. This indistinct pattern of c-fos distribution may indicate a more general role for these structures in the 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 of aversive 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 We have shown that alertness, freezing or escape behaviors may be generated at the level of the dorsal periaqueductal gray (dPAG) of the rat depending on the intensity of the electrical stimulation applied to this region [9,12,14]. Nowadays, there is general agreement that animal models of anxiety differ according to the an*Corresponding author. Fax: 155-16-633-1609. ˜ E-mail address: [email protected] (M.L. Brandao).
xiogenic stimulus presented to the animals and with the behaviour taken as an index of anxiety. Therefore, distinct kinds of aversive stimuli are supposed to generate different types of fear [6,8,40,41,43]. Indeed, we have recently shown that distinct types of freezing are elaborated in the periaqueductal gray matter; contextual conditioned freezing is related to circuits in its ventral part and unconditioned freezing to its dorsal area [86]. By the same token, electrical and chemical stimulation of the inferior colliculus also cause defensive behavior characterized by alertness, freezing and escape responses accompanied by
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M.R. Lamprea et al. / Brain Research 950 (2002) 186–194
increases in mean blood pressure, tachycardia, and other autonomic responses common to the defense reaction such as piloerection, micturition and defecation [10,11,15]. It has been shown that the neurocircuitry in the inferior colliculus responsible for the production of aversive states is regulated by a large number of neurotransmitters [11,12,14]. Moreover, we reported that aversive stimulation of the inferior colliculus enhances dopamine release in the prefrontal cortex, a structure normally activated during threatening situations [24]. Nevertheless, no attempt has been made so far to establish the anatomical connections and functional significance of the several components of the defense reaction generated at the level of the inferior colliculus. Defensive strategies as distinct as risk assessment, avoidance, escape and fight are likely to be organized by different neural networks [5,7,8,32,40–42]. Therefore, it is expected that the set of brain structures activated by freezing response induced by electrical stimulation of the inferior colliculus should be different from that activated by escape behavior so-induced. To test this prediction, Fos protein immunoreactivity was presently measured in serial sections of the brain following expression of either freezing or escape induced by electrical stimulation of the inferior colliculus. Sham-stimulated animals of the control group were exposed to the same procedure. It has been chosen because it can be controlled by the experimenter, the novelty of the exposition and the locomotor activity of the animal while exploring the arena between stimulations. Fos protein is the product of the proto-oncogene c-fos and was shown to be synthesized in neurons as a consequence of increases in intracellular calcium, cyclic AMP, or other second messengers [61,62,77]. Since this protein remains in the cell nucleus for a short period of time, its detection has been extensively used to map neuronal bodies activated by specific stimuli [30,71,82]. This increase in Fos protein expression has been shown to occur as a result of neuronal activation by a wide range of stimuli, such as convulsing agents [26,61], electrical current [51,72,79], neurotransmitters [70,83], immobilization stress [44,46], pain [17,45], injection of hypertonic saline [22,76] and exposure to threatening stimuli of the elevated plus-maze or T-maze [78,80].
2. Materials and methods
2.1. Animals Naive male Wistar rats weighing 240–260 g were used. Animals were kept under controlled temperature (2262 8C) and a 12-h light:dark cycle (lights on at 07:00 h). They were housed two per cage and had free access to food and water throughout the experiment. The experiments reported in this article were performed in compliance with the recommendations of SBNeC (Brazilian Society of Neuroscience and Behavior), which are based
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on the US National Institutes of Health Guide for Care and Use of Laboratory Animals.
2.2. Surgery The animals were anaesthetized with tribromoethanol (250 mg / kg, i.p.) and placed in a stereotaxic frame (David Kopf, USA). A brain electrode was implanted in the midbrain, aimed at the inferior colliculus. The electrode was made of stainless steel wire, 160 mm in diameter, insulated except at the cross section of the tip. The upper incisor bar was set 3.3 mm below the interaural line so that the skull was horizontal between bregma and lambda. The electrode was introduced vertically using the following coordinates with the lambda serving as the reference for each plane: postero-anterior, 1.2 mm; medio-lateral, 1.5 mm; and dorso-ventral, 4.5 mm [64]. The electrode was fixed to the skull by means of acrylic resin and three stainless steel screws. The electrode wire was connected to a connector so that it could be plugged into an amphenol socket at the end of a flexible electrical cable used for brain stimulation.
2.3. Apparatus One week after surgery, the rats were placed in an open field which was a circular enclosure 60 cm in diameter and 50 cm high. The rat was placed in the arena and had its brain electrode connected to a flexible wire cable, allowing ample movement inside the box. The cable, in turn, was connected to the stimulator by means of a mercury swivel mounted on the top of the experimental chamber. The rats were allowed a 15 min period of habituation in the enclosure. The brain was stimulated electrically by means of a sine wave stimulator [58]. The stimulation current was monitored by measuring the voltage drop across a 1 kV resistor with an oscilloscope (Philips, USA). Brain stimulation (AC, 60 Hz, 15 s) was presented at 1 min intervals with the current intensity increasing by steps of 5 mA for measurements of the alertness, freezing and escape thresholds. Alertness threshold was operationally defined as the lowest intensity producing episodes of movement arrest with head orientation in two consecutive series of electrical stimulation. Freezing threshold was operationally defined as lowest intensity producing stop of the ongoing behavior in two consecutive series of electrical stimulation accompanied by at least two of the following autonomic responses: piloerection, defecation, micturition and exhophtalmus. Escape threshold was operationally defined as the lowest current intensity that produced running or jumping in two successive ascending series of electrical stimulation. Animals with an escape threshold above 120 mA (peak-topeak) were discarded from the experiment. The adequacy of the current intensity levels for the aversive responses studied herein was verified on the basis of previous studies from this laboratory [59,13,85].
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2.4. Experimental procedure The animals were randomly allocated to the alertness (n56), freezing (n56), escape (n56) or sham-stimulated groups (n56). Animals of all four groups were placed in the arena and allowed 15 min for habituation. Next, the aversive thresholds were determined in only one series of ascending intensity of electrical stimulation. then submitted to its respective experimental session. On the next day, the electrical stimulation was applied at the aversive threshold determined the day before and afterwards their brains were processed in parallel, as described below.
2.5. Fos protein immunohistochemistry Two hours after the electrical stimulation procedure, the animals were deeply anaesthetized with urethane (1.25 g / kg; Sigma, USA) and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (pH 7.4). Brains were removed, immersed (4 8C) in the above fixative for 2 h and then kept in 30% sucrose in 0.1 M PBS until soaked. They were then quickly frozen in isopentane (240 8C) and sliced by the use of a cryostat (215 8C). Two adjacent series of 40-mm thick brain slices were obtained, having as reference the following AP coordinates: Bregma 13.2, 10.7, 20.3, 21.3, 21.8, 23.3, 24.8, 26.3, 27.8, 28.3, 29.8 mm. One series was Nissl stained and used for neuroanatomical comparison purposes. The other series was collected in 0.1 M PBS and subsequently processed freefloating according to the avidine–biotine procedure, using the Vectastain ABC Elite peroxidase rabbit IgG kit (Vector, USA, Ref. PK 6101). All reactions were carried out under agitation, at room temperature. The slices were first incubated with 1% H 2 O 2 for 10 min, washed four times with 0.1 M PBS (5 min each) and then incubated overnight with the primary Fos polyclonal antibody (Santa Cruz, USA, SC-52) at a concentration of 1 / 20 000 in 0.1 M PBS enriched with 4% normal goat serum and 0.2% Triton-X. Slices were again washed three times (5 min each) with 0.1 M PBS and incubated for 1 h with biotinylated goat antirabbit antibody (25 ml from the kit for 10 ml of 0.1 M PBS enriched with 4% normal goat serum and 0.2% Triton-X). After another series of three 5-min washings in 0.1 M PBS, they were incubated for 1 h with the avidine– peroxidase solution (two drops of each solution labeled A and B of the kit for 25 ml of 0.1 M PBS) and three times washed in 0.1 M PBS (5 min per wash). The slices were finally allowed to remain for 5 min in a solution of 3,39-di-aminobenzidine (DAB, 0.02%) to which hydrogen peroxide (0.04%) was added just prior to use and were washed twice with 0.1 M PBS.
2.6. Quantification of fos-positive cells Sections were mounted on gelatin-coated slides, dehydrated and coverslipped for observation and cell counting
under bright-field microscopy in brain structures earlier shown to be activated by aversive conditions as reported by other studies [24,53,72,73,78,80]. The nomenclature and nuclear boundaries utilized were based on the atlas of Paxinos and Watson [64] and the planes of analyzed sections were standardized as far as possible. Neuronal nuclei expressing levels of DAB reaction product above tissue background were automatically counted by a computerized image analysis system (Image Pro Plus 4.0, Media Cybernetics, USA), according to method used previously in this laboratory [80]. Briefly, mounted sections of the tissue were observed using a light microscope (Olympus BX-50) equipped with a video camera module (Hamatsu Photonics C2400) and coupled to a computerized image analysis system (Image Pro Plus 5.0, Media Cybernetics, USA). Counting of fos-positive cells was performed at a magnification 3100, in one field per area encompassing the entire brain region included in quantification. An area of the same shape and size per brain region was used for each rat. The same light and threshold conditions were employed for all sections. Fos staining could vary from one area to another. However, in order to ensure accuracy of measurement and avoid variations among same areas in different subjects, the background of every area was measured and digitally subtracted from the area under examination. Accordingly, the threshold conditions were set for each area and maintained for all subjects [80]. All brain regions were bilaterally counted in various sections for each rat depending on the size of the structure. After that, counts for each region were averaged over the sections. Nuclei were counted individually and expressed as number of Fos-positive nuclei per 0.1 mm 2 [68].
2.7. Statistics Statistical analysis of thresholds (alertness, freezing and escape) and immunohistochemical data were subject to one-way ANOVA followed by Duncan’s test. The results are presented as mean6S.E.M.
3. Results
3.1. Behavioural effects The tips of the electrodes were situated inside the central nucleus of the inferior colliculus. A representative site with a bipolar electrode in the inferior colliculus is shown elsewhere [21]. The intensity of the electric current applied to the inferior colliculus of the animals to induce alertness, freezing and escape responses was 31.6666.62, 61.6769.50 and 99.17611.14 mA (peak-to-peak), respectively. ANOVA performed on these data revealed that the differences between these aversive thresholds were highly significant (F2,15 513.29, P,0.001). Post-hoc analysis
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indicated that there is a statistically significant difference among the groups.
3.2. Fos protein expression Immunoreactive cells exhibited a dark nucleus in neuronal nuclei expressing clearly upon the surrounding background tissue. In the control group, clusters of Fos-like immunoreactive cells could already be identified in some brain regions, namely the frontal cortex (Fig. 1A), the dorsal periaqueductal gray matter of the mesencephalon (Fig. 3A) and the inferior colliculus (Fig. 3C). The results obtained with quantitative analyses of Foslike immunoreactivity in the studied brain regions after induction of freezing and escape responses in rats stimulated in the inferior colliculus are summarized in Table 1. As no significant differences could be detected between the non-operated and alertness groups for all structures studied here these data were pooled together in just one control group. Statistical comparison of these data by means of one-way ANOVA indicated that there was a significant difference in Fos protein expression in the frontal cortex (F2,12 511.45, P,0.01), basolateral amygdala (F2,15 57.39, P,0.01), entorhinal cortex (F2,17 54.18, P,0.05), dorsal hippocampus (F2,17 57.36, P,0.01), central amygdaloid nuclei (F2,15 53.76, P,0.05), dorsal periaqueductal gray matter of the mesencephalon (F2,16 5315.17, P,0.001), cuneiform nucleus (F2,16 56.55, P,0.01) and central nucleus of the inferior colliculus (F2,15 54.67, P,0.05). Marginal significance was obtained in the septum (F2,11 5 3.18, P50.08) and the ventral periaqueductal gray (F2,17 5
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3.07, P50.07). No significant fos expression could be detected in the remaining structures listed in Table 1. Post-hoc comparisons (Duncan’s test, P,0.05) indicated that the freezing and escape groups exhibited a higher density of Fos-like immunoreactive cells in the frontal cortex (Fig. 1A), basolateral amygdaloid nuclei (Fig. 1B) and in dorsal hippocampus in relation to their respective control groups. In animals submitted to the electrical stimulation of the inferior colliculus at the freezing threshold, post-hoc analysis showed that Fos-like immunoreactivity was significantly enhanced in the entorhinal cortex (Fig. 2A) and central amygdaloid nucleus (Fig. 2B) in relation to the control group. Fos-like immunoreactivity in the escape group was significantly increased in dPAG (Fig. 3A), cuneiform nucleus (Fig. 3B) and inferior colliculus (Fig. 3C) in relation to the control group.
4. Discussion In the present work, stepwise increases in the electrical stimulation of the inferior colliculus elicits first alertness, then freezing and finally escape accompanied by autonomic responses, such as piloerection, exophtalmos, micturition and defecation. These data are in agreement with results obtained earlier in our laboratory, suggesting that the inferior colliculus is also part of a brain aversion system [10–12,14]. The inferior colliculus establishes direct or indirect connections with several structures containing neural sub-
Fig. 1. Photomicrographs of Fos-like immunoreactive cells (dark dots) in coronal sections through brain regions with significant increase in Fos expression after performance of both freezing (middle panel) and escape (right panel) responses after electrical stimulation of the inferior colliculus, in relation to the control group (left panel). FrCx, frontal cortex; BlA, basolateral amygdaloid nucleus. Scale bar5200 mm.
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Table 1 Number of Fos-like immunoreactive nuclei per 0.1 mm 2 (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 Frontal cortex Entorhinal cortex Dorsal Hippocampus Septum Septohypothalamic nucleus Central amygdaloid nucleus Basolateral amygdaloid nucleus Medial amygdaloid nucleus Bed N of stria terminalis Preoptic nucleus Anterior hypothalamic nucleus Dorsomedial hypothalamus Suprachiasmatic nucleus Lateral hypothalamus Periv. hypothalamus Supamammilary nucleus Substantia nigra, pars reticulata Ventral PAG Dorsal PAG Superior colliculus Cuneiform nucleus Dorsal raphe n. Locus coeruleus Central n. inferior colliculus a
Control 165.48639.68 28.4364.73 7.0861.87 10.5762.94 92.03621.81 24.2663.72 24.4463.27 35.9264.33 31.9465.63 61.0567.76 46.8566.08 76.28612.60 31.2967.74 50.3363.53 5.1461.96 108.6568.89 2.1560.40 20.7962.45 18.0763.46 28.7367.93 24.9364.80 30.3464.18 50.5463.84 78.87611.01
Freezing 616.96645.72 48.9767.54 a 18.4665.40 a 25.3066.95 82.71630.27 47.3363.17 a 41.6564.08 a 46.7666.54 57.78616.14 69.05612.23 56.81611.67 69.60610.93 24.8868.38 52.7466.43 7.3162.48 96.73617.04 4.6662.46 35.3765.27 16.5063.39 35.3063.43 38.0365.11 25.4663.84 64.8766.86 85.25620.28
Escape a
374.386109.41 a 43.9566.93 22.0161.44 a 18.9264.21 72.75627.88 32.04610.71 48.1869.40 a 51.03610.55 42.00615.05 51.29612.31 44.55610.19 83.55623.91 28.31613.21 54.56611.44 5.3261.57 144.95627.86 0.3960.18 31.9467.99 49.2165.51 a 37.5563.56 56.4468.88 a 33.1964.66 69.65611.04 151.66613.44 a
P,0.05, in relation to the control group (Duncan’s test).
strates of fear [14]. The present findings clearly show increased fos-expression in the basolateral amygdaloid nuclei after electrical stimulation of the inferior colliculus
at the freezing and escape thresholds. Reported evidence indicates that basolateral amygdala is a point of convergence 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 expression specifically after performance of freezing (middle panel) induced by electrical stimulation of the inferior colliculus, in relation to the control group (left panel). Fos expression in the group that received electrical stimulation of the inferior colliculus at the escape threshold is also indicated. EntCx, entorhinal cortex; 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 expression after 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, central nucleus of the inferior colliculus. Scale bar5200 mm.
to impart emotional value to sensory stimulation [19,25,32,33,35,48,54,55,57]. Accordingly, increased expression of immediate early genes in the amygdala has been reported using several paradigms of aversive conditioning [3,5,62,66,69]. It has been suggested that the involvement of the inferior colliculus in fear processes can occur through its anatomical and functional connections with the amygdala [14,56]. Marked increases in c-fos expression in the freezing and escape groups were also observed in the prefrontal cortex. These findings are not surprising as cortical projections are also activated by a wide variety of aversive stimulation [1,28,34,39,84]. These cortical areas are clearly connected to the inferior colliculus [2,16,23,38,60]. One of these pathways is provided by projections from the central nucleus of the inferior colliculus to the prefrontal cortex through the medial geniculate nucleus, amygdala and dorsomedial thalamus [16,38]. It has been shown that this circuit is concerned with the processing of auditory information of aversive nature, which triggers fear-like behaviors [14,24].
According to the hypothesis under testing, the freezing and escape responses were expected to activate different sets of brain structures and this prediction was confirmed by the present results. Only central amygdaloid nucleus, dorsal hippocampus and entorhinal cortex were labeled by c-fos immunoreactivity following freezing responses induced by electrical stimulation of the inferior colliculus. Although marginally significant, the ventral periaqueductal gray also showed considerable labeling after freezing but not after escape response induced by stimulation of the inferior colliculus. These data are in line with several studies that show that this structure is crucial for passive defensive responses [32,35]. However, as we have already shown that lesion of the ventral part of the periaqueductal gray, increased the freezing and escape thresholds determined by stimulation of the inferior colliculus (although the effects reached marginal significance), we should not discard the possibility that the inferior colliculus could be part of a defense system that could have the ventral periaqueductal gray as one of its output [56].
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On the other hand, immediate threat causes escape behavior, which seems to be mediated by the dorsal periaqueductal gray, hypothalamus and amygdala [40,41]. The only regions selectively activated by escape were the dorsal periaqueductal gray matter, cuneiform nucleus and the inferior colliculus itself. Electrical or chemical stimulation of the dorsal periaqueductal gray has been shown to elicit an explosive flight reaction that resembles a panic attack [9,40,41,47]. Also, neurosurgical patients reported feelings of terror and impending death after electrical stimulation of this region [63]. Several studies using c-fos have shown that the dorsal periaqueductal gray is activated in stressful situations [3,4,20,49,50,67,72,73], inclusive after intraperitoneal injection of drugs capable of eliciting panic symptoms [81]. Moreover, it was shown that disruption of tonic GABAergic inhibition in the dPAG elicits a constellation of behavioural and physiological responses that resembles a human panic attack [9,12,14]. The cuneiform nucleus seems to be the main output from the dPAG [18]. Electrical and chemical stimulation of the cuneiform nucleus causes clear defense reactions resembling that produced by activation of neural substrates of fear in the dPAG or inferior colliculus [27,73]. Also, neurons of cuneiform nucleus are activated by exposure of rats to cat odor [29] and show an increase of c-fos expression with stimulation of the dPAG and medial hypothalamus [72,73]. Expectedly, the central nucleus of the inferior colliculus was also significantly labeled after stimulation of this structure at the escape threshold. The inferior colliculus establishes reciprocal anatomical connections with the dorsal periaqueductal gray [52,60]. Along with this latter structure our data also point to the cuneiform nucleus as a good candidate for the output of escape responses generated at this level. In the present experiment, we found no difference in Fos protein expression between freezing, escape and control groups in the anterior, dorsomedial and preoptic nuclei of the hypothalamus. These findings were unexpected since the hypothalamus is one of the major recipients of information processed in the amygdala [65]. Defense reactions seem to be organized by both anterior [36,37,74,75] and medial hypothalamic nuclei [36,37] and these structures are activated after exposure of rodents to natural predators [20] or agonistic encounters [49,50]. Other studies have employed analysis of c-fos gene expression to identify brain structures activated by exposure to the elevated plus-maze [31,78]. An interesting finding of one of these studies was the remarkable fos expression of the central nucleus of the inferior colliculus [78]. This finding fits quite well within the framework of our hypothesis that the inferior colliculus has neural substrates for more than one kind of fear, since the elevated plus-maze has been considered a ‘mixed’ model in the sense that it may generate unconditioned and conditioned responses to threatening stimuli [43]. Because of some known limitations of the c-fos tech-
nique the present results should be interpreted cautiously. In fact, this technique does not allow for distinguishing between retrograde activation of afferent connections and feedforward efferent connections to the structure stimulated. Thus, we cannot discard the possibility that the electrical stimulation of the inferior colliculus could therefore produce distant fos-expression by antidromic then orthodromic activation of branched afferent collaterals. Also, the different levels of electrical stimulation produced different behavioral reactions, that is, the different groups of animals were exposed to different levels of external and internal sensory information, simply as a result of different stimulus-evoked movement. These confounding differences could also cause differential c-fos expression between the groups. Furthermore, the fact that high levels of IC stimulation failed to evoke fos-expression in some structures activated by lower levels of stimulation, for instance central nucleus of the amygdala, may be easier to understand in terms of indirect rather than direct mechanisms. This latter methodological limitation of this technique could explain why lesions of the central amygdala increase the threshold for both freezing and escape behaviors [56] and c-fos expression in this nucleus was only increased after freezing, as noted in the present study. On the other hand, autonomic reactions could not be easily evoke as a probable explanation for such differences, at least those recorded in this study, piloerection, exophtalmos, micturition and defecation were present as accompanied responses of both behavioral reactions, freezing and escape. In conclusion, the present neuroanatomical evidence adds to earlier reported behavioural results indicating that freezing and escape responses represent two types of fear with separate neural circuits in the brain. Our data also point out to the modulatory role played by the basolateral amygdala, dorsal hippocampus and the prefrontal cortex in the integration of aversive information generated at the level of the inferior colliculus.
Acknowledgements Research supported by FAPESP (Proc No. 98 / 11187-2 and 02 / 03705-0) and CNPq (Proc No. 94 / 5933-2). F. Cardenas, M.R. Lamprea and V.M. Castilho are recipients of doctor scholarships from FAPESP. D.M. Vianna is recipient of a doctor scholarship from CNPq. S.E. CruzMorales had a sabbatical leave supported by CONACYT ´ and DGAPA, UNAM, Mexico.
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Research report
The distribution of fos immunoreactivity in rat brain following freezing and escape responses elicited by electrical stimulation of the inferior colliculus Marisol R. Lamprea a , Fernando P. Cardenas a , Daniel Machado Vianna a , a b ˜ a ,* Vanessa M. Castilho , Sara Eugenia Cruz-Morales , Marcus L. Brandao a
´ Laboratorio de Psicobiologia, FFCLRP, Campus USP, Av. Bandeirantes 3900, 14049 -901 Ribeirao Preto, SP, Brazil Laboratory of Psychopharmacology, FES-Iztacala, UNAM, P.O. Box 314, Tlanepantla, Edo. Mexico 54090, Mexico
b
Accepted 6 May 2002
Abstract Several sources of evidence indicate that the inferior colliculus also integrates acoustic information of an aversive nature besides its well-known role as a relay station for auditory pathways. Gradual increases of the electrical stimulation of this structure cause in a hierarchical manner alertness, freezing and escape behaviors. Independent groups of animals implanted with bipolar electrodes into the inferior colliculus received electrical stimulation at one of these aversive thresholds. Control animals were submitted to the same procedure but no current was applied. Next, analysis of Fos protein expression was used to map brain areas activated by the inferior colliculus stimulation at each aversive threshold and in the controls. Whereas alertness elicited by stimulation of the inferior colliculus did not cause any significant labeling in any structure studied in relation to the respective control, electrical stimulation applied at the freezing threshold increased Fos-like immunoreactivity in the central amygdaloid nucleus and entorhinal cortex. In contrast, escape response enhanced Fos-like immunoreactivity in the nucleus cuneiform and the dorsal periaqueductal gray matter of the mesencephalon. This evidence supports the notion that freezing and escape behaviors induced by electrical stimulation of the inferior colliculus activate different neural circuitries in the brain. Both defensive behaviors caused significant expression of c-fos in the frontal cortex, hippocampus and basolateral amygdaloid nucleus. This indistinct pattern of c-fos distribution may indicate a more general role for these structures in the 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 of aversive 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 We have shown that alertness, freezing or escape behaviors may be generated at the level of the dorsal periaqueductal gray (dPAG) of the rat depending on the intensity of the electrical stimulation applied to this region [9,12,14]. Nowadays, there is general agreement that animal models of anxiety differ according to the an*Corresponding author. Fax: 155-16-633-1609. ˜ E-mail address: [email protected] (M.L. Brandao).
xiogenic stimulus presented to the animals and with the behaviour taken as an index of anxiety. Therefore, distinct kinds of aversive stimuli are supposed to generate different types of fear [6,8,40,41,43]. Indeed, we have recently shown that distinct types of freezing are elaborated in the periaqueductal gray matter; contextual conditioned freezing is related to circuits in its ventral part and unconditioned freezing to its dorsal area [86]. By the same token, electrical and chemical stimulation of the inferior colliculus also cause defensive behavior characterized by alertness, freezing and escape responses accompanied by
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03036-6
M.R. Lamprea et al. / Brain Research 950 (2002) 186–194
increases in mean blood pressure, tachycardia, and other autonomic responses common to the defense reaction such as piloerection, micturition and defecation [10,11,15]. It has been shown that the neurocircuitry in the inferior colliculus responsible for the production of aversive states is regulated by a large number of neurotransmitters [11,12,14]. Moreover, we reported that aversive stimulation of the inferior colliculus enhances dopamine release in the prefrontal cortex, a structure normally activated during threatening situations [24]. Nevertheless, no attempt has been made so far to establish the anatomical connections and functional significance of the several components of the defense reaction generated at the level of the inferior colliculus. Defensive strategies as distinct as risk assessment, avoidance, escape and fight are likely to be organized by different neural networks [5,7,8,32,40–42]. Therefore, it is expected that the set of brain structures activated by freezing response induced by electrical stimulation of the inferior colliculus should be different from that activated by escape behavior so-induced. To test this prediction, Fos protein immunoreactivity was presently measured in serial sections of the brain following expression of either freezing or escape induced by electrical stimulation of the inferior colliculus. Sham-stimulated animals of the control group were exposed to the same procedure. It has been chosen because it can be controlled by the experimenter, the novelty of the exposition and the locomotor activity of the animal while exploring the arena between stimulations. Fos protein is the product of the proto-oncogene c-fos and was shown to be synthesized in neurons as a consequence of increases in intracellular calcium, cyclic AMP, or other second messengers [61,62,77]. Since this protein remains in the cell nucleus for a short period of time, its detection has been extensively used to map neuronal bodies activated by specific stimuli [30,71,82]. This increase in Fos protein expression has been shown to occur as a result of neuronal activation by a wide range of stimuli, such as convulsing agents [26,61], electrical current [51,72,79], neurotransmitters [70,83], immobilization stress [44,46], pain [17,45], injection of hypertonic saline [22,76] and exposure to threatening stimuli of the elevated plus-maze or T-maze [78,80].
2. Materials and methods
2.1. Animals Naive male Wistar rats weighing 240–260 g were used. Animals were kept under controlled temperature (2262 8C) and a 12-h light:dark cycle (lights on at 07:00 h). They were housed two per cage and had free access to food and water throughout the experiment. The experiments reported in this article were performed in compliance with the recommendations of SBNeC (Brazilian Society of Neuroscience and Behavior), which are based
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on the US National Institutes of Health Guide for Care and Use of Laboratory Animals.
2.2. Surgery The animals were anaesthetized with tribromoethanol (250 mg / kg, i.p.) and placed in a stereotaxic frame (David Kopf, USA). A brain electrode was implanted in the midbrain, aimed at the inferior colliculus. The electrode was made of stainless steel wire, 160 mm in diameter, insulated except at the cross section of the tip. The upper incisor bar was set 3.3 mm below the interaural line so that the skull was horizontal between bregma and lambda. The electrode was introduced vertically using the following coordinates with the lambda serving as the reference for each plane: postero-anterior, 1.2 mm; medio-lateral, 1.5 mm; and dorso-ventral, 4.5 mm [64]. The electrode was fixed to the skull by means of acrylic resin and three stainless steel screws. The electrode wire was connected to a connector so that it could be plugged into an amphenol socket at the end of a flexible electrical cable used for brain stimulation.
2.3. Apparatus One week after surgery, the rats were placed in an open field which was a circular enclosure 60 cm in diameter and 50 cm high. The rat was placed in the arena and had its brain electrode connected to a flexible wire cable, allowing ample movement inside the box. The cable, in turn, was connected to the stimulator by means of a mercury swivel mounted on the top of the experimental chamber. The rats were allowed a 15 min period of habituation in the enclosure. The brain was stimulated electrically by means of a sine wave stimulator [58]. The stimulation current was monitored by measuring the voltage drop across a 1 kV resistor with an oscilloscope (Philips, USA). Brain stimulation (AC, 60 Hz, 15 s) was presented at 1 min intervals with the current intensity increasing by steps of 5 mA for measurements of the alertness, freezing and escape thresholds. Alertness threshold was operationally defined as the lowest intensity producing episodes of movement arrest with head orientation in two consecutive series of electrical stimulation. Freezing threshold was operationally defined as lowest intensity producing stop of the ongoing behavior in two consecutive series of electrical stimulation accompanied by at least two of the following autonomic responses: piloerection, defecation, micturition and exhophtalmus. Escape threshold was operationally defined as the lowest current intensity that produced running or jumping in two successive ascending series of electrical stimulation. Animals with an escape threshold above 120 mA (peak-topeak) were discarded from the experiment. The adequacy of the current intensity levels for the aversive responses studied herein was verified on the basis of previous studies from this laboratory [59,13,85].
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2.4. Experimental procedure The animals were randomly allocated to the alertness (n56), freezing (n56), escape (n56) or sham-stimulated groups (n56). Animals of all four groups were placed in the arena and allowed 15 min for habituation. Next, the aversive thresholds were determined in only one series of ascending intensity of electrical stimulation. then submitted to its respective experimental session. On the next day, the electrical stimulation was applied at the aversive threshold determined the day before and afterwards their brains were processed in parallel, as described below.
2.5. Fos protein immunohistochemistry Two hours after the electrical stimulation procedure, the animals were deeply anaesthetized with urethane (1.25 g / kg; Sigma, USA) and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (pH 7.4). Brains were removed, immersed (4 8C) in the above fixative for 2 h and then kept in 30% sucrose in 0.1 M PBS until soaked. They were then quickly frozen in isopentane (240 8C) and sliced by the use of a cryostat (215 8C). Two adjacent series of 40-mm thick brain slices were obtained, having as reference the following AP coordinates: Bregma 13.2, 10.7, 20.3, 21.3, 21.8, 23.3, 24.8, 26.3, 27.8, 28.3, 29.8 mm. One series was Nissl stained and used for neuroanatomical comparison purposes. The other series was collected in 0.1 M PBS and subsequently processed freefloating according to the avidine–biotine procedure, using the Vectastain ABC Elite peroxidase rabbit IgG kit (Vector, USA, Ref. PK 6101). All reactions were carried out under agitation, at room temperature. The slices were first incubated with 1% H 2 O 2 for 10 min, washed four times with 0.1 M PBS (5 min each) and then incubated overnight with the primary Fos polyclonal antibody (Santa Cruz, USA, SC-52) at a concentration of 1 / 20 000 in 0.1 M PBS enriched with 4% normal goat serum and 0.2% Triton-X. Slices were again washed three times (5 min each) with 0.1 M PBS and incubated for 1 h with biotinylated goat antirabbit antibody (25 ml from the kit for 10 ml of 0.1 M PBS enriched with 4% normal goat serum and 0.2% Triton-X). After another series of three 5-min washings in 0.1 M PBS, they were incubated for 1 h with the avidine– peroxidase solution (two drops of each solution labeled A and B of the kit for 25 ml of 0.1 M PBS) and three times washed in 0.1 M PBS (5 min per wash). The slices were finally allowed to remain for 5 min in a solution of 3,39-di-aminobenzidine (DAB, 0.02%) to which hydrogen peroxide (0.04%) was added just prior to use and were washed twice with 0.1 M PBS.
2.6. Quantification of fos-positive cells Sections were mounted on gelatin-coated slides, dehydrated and coverslipped for observation and cell counting
under bright-field microscopy in brain structures earlier shown to be activated by aversive conditions as reported by other studies [24,53,72,73,78,80]. The nomenclature and nuclear boundaries utilized were based on the atlas of Paxinos and Watson [64] and the planes of analyzed sections were standardized as far as possible. Neuronal nuclei expressing levels of DAB reaction product above tissue background were automatically counted by a computerized image analysis system (Image Pro Plus 4.0, Media Cybernetics, USA), according to method used previously in this laboratory [80]. Briefly, mounted sections of the tissue were observed using a light microscope (Olympus BX-50) equipped with a video camera module (Hamatsu Photonics C2400) and coupled to a computerized image analysis system (Image Pro Plus 5.0, Media Cybernetics, USA). Counting of fos-positive cells was performed at a magnification 3100, in one field per area encompassing the entire brain region included in quantification. An area of the same shape and size per brain region was used for each rat. The same light and threshold conditions were employed for all sections. Fos staining could vary from one area to another. However, in order to ensure accuracy of measurement and avoid variations among same areas in different subjects, the background of every area was measured and digitally subtracted from the area under examination. Accordingly, the threshold conditions were set for each area and maintained for all subjects [80]. All brain regions were bilaterally counted in various sections for each rat depending on the size of the structure. After that, counts for each region were averaged over the sections. Nuclei were counted individually and expressed as number of Fos-positive nuclei per 0.1 mm 2 [68].
2.7. Statistics Statistical analysis of thresholds (alertness, freezing and escape) and immunohistochemical data were subject to one-way ANOVA followed by Duncan’s test. The results are presented as mean6S.E.M.
3. Results
3.1. Behavioural effects The tips of the electrodes were situated inside the central nucleus of the inferior colliculus. A representative site with a bipolar electrode in the inferior colliculus is shown elsewhere [21]. The intensity of the electric current applied to the inferior colliculus of the animals to induce alertness, freezing and escape responses was 31.6666.62, 61.6769.50 and 99.17611.14 mA (peak-to-peak), respectively. ANOVA performed on these data revealed that the differences between these aversive thresholds were highly significant (F2,15 513.29, P,0.001). Post-hoc analysis
M.R. Lamprea et al. / Brain Research 950 (2002) 186–194
indicated that there is a statistically significant difference among the groups.
3.2. Fos protein expression Immunoreactive cells exhibited a dark nucleus in neuronal nuclei expressing clearly upon the surrounding background tissue. In the control group, clusters of Fos-like immunoreactive cells could already be identified in some brain regions, namely the frontal cortex (Fig. 1A), the dorsal periaqueductal gray matter of the mesencephalon (Fig. 3A) and the inferior colliculus (Fig. 3C). The results obtained with quantitative analyses of Foslike immunoreactivity in the studied brain regions after induction of freezing and escape responses in rats stimulated in the inferior colliculus are summarized in Table 1. As no significant differences could be detected between the non-operated and alertness groups for all structures studied here these data were pooled together in just one control group. Statistical comparison of these data by means of one-way ANOVA indicated that there was a significant difference in Fos protein expression in the frontal cortex (F2,12 511.45, P,0.01), basolateral amygdala (F2,15 57.39, P,0.01), entorhinal cortex (F2,17 54.18, P,0.05), dorsal hippocampus (F2,17 57.36, P,0.01), central amygdaloid nuclei (F2,15 53.76, P,0.05), dorsal periaqueductal gray matter of the mesencephalon (F2,16 5315.17, P,0.001), cuneiform nucleus (F2,16 56.55, P,0.01) and central nucleus of the inferior colliculus (F2,15 54.67, P,0.05). Marginal significance was obtained in the septum (F2,11 5 3.18, P50.08) and the ventral periaqueductal gray (F2,17 5
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3.07, P50.07). No significant fos expression could be detected in the remaining structures listed in Table 1. Post-hoc comparisons (Duncan’s test, P,0.05) indicated that the freezing and escape groups exhibited a higher density of Fos-like immunoreactive cells in the frontal cortex (Fig. 1A), basolateral amygdaloid nuclei (Fig. 1B) and in dorsal hippocampus in relation to their respective control groups. In animals submitted to the electrical stimulation of the inferior colliculus at the freezing threshold, post-hoc analysis showed that Fos-like immunoreactivity was significantly enhanced in the entorhinal cortex (Fig. 2A) and central amygdaloid nucleus (Fig. 2B) in relation to the control group. Fos-like immunoreactivity in the escape group was significantly increased in dPAG (Fig. 3A), cuneiform nucleus (Fig. 3B) and inferior colliculus (Fig. 3C) in relation to the control group.
4. Discussion In the present work, stepwise increases in the electrical stimulation of the inferior colliculus elicits first alertness, then freezing and finally escape accompanied by autonomic responses, such as piloerection, exophtalmos, micturition and defecation. These data are in agreement with results obtained earlier in our laboratory, suggesting that the inferior colliculus is also part of a brain aversion system [10–12,14]. The inferior colliculus establishes direct or indirect connections with several structures containing neural sub-
Fig. 1. Photomicrographs of Fos-like immunoreactive cells (dark dots) in coronal sections through brain regions with significant increase in Fos expression after performance of both freezing (middle panel) and escape (right panel) responses after electrical stimulation of the inferior colliculus, in relation to the control group (left panel). FrCx, frontal cortex; BlA, basolateral amygdaloid nucleus. Scale bar5200 mm.
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Table 1 Number of Fos-like immunoreactive nuclei per 0.1 mm 2 (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 Frontal cortex Entorhinal cortex Dorsal Hippocampus Septum Septohypothalamic nucleus Central amygdaloid nucleus Basolateral amygdaloid nucleus Medial amygdaloid nucleus Bed N of stria terminalis Preoptic nucleus Anterior hypothalamic nucleus Dorsomedial hypothalamus Suprachiasmatic nucleus Lateral hypothalamus Periv. hypothalamus Supamammilary nucleus Substantia nigra, pars reticulata Ventral PAG Dorsal PAG Superior colliculus Cuneiform nucleus Dorsal raphe n. Locus coeruleus Central n. inferior colliculus a
Control 165.48639.68 28.4364.73 7.0861.87 10.5762.94 92.03621.81 24.2663.72 24.4463.27 35.9264.33 31.9465.63 61.0567.76 46.8566.08 76.28612.60 31.2967.74 50.3363.53 5.1461.96 108.6568.89 2.1560.40 20.7962.45 18.0763.46 28.7367.93 24.9364.80 30.3464.18 50.5463.84 78.87611.01
Freezing 616.96645.72 48.9767.54 a 18.4665.40 a 25.3066.95 82.71630.27 47.3363.17 a 41.6564.08 a 46.7666.54 57.78616.14 69.05612.23 56.81611.67 69.60610.93 24.8868.38 52.7466.43 7.3162.48 96.73617.04 4.6662.46 35.3765.27 16.5063.39 35.3063.43 38.0365.11 25.4663.84 64.8766.86 85.25620.28
Escape a
374.386109.41 a 43.9566.93 22.0161.44 a 18.9264.21 72.75627.88 32.04610.71 48.1869.40 a 51.03610.55 42.00615.05 51.29612.31 44.55610.19 83.55623.91 28.31613.21 54.56611.44 5.3261.57 144.95627.86 0.3960.18 31.9467.99 49.2165.51 a 37.5563.56 56.4468.88 a 33.1964.66 69.65611.04 151.66613.44 a
P,0.05, in relation to the control group (Duncan’s test).
strates of fear [14]. The present findings clearly show increased fos-expression in the basolateral amygdaloid nuclei after electrical stimulation of the inferior colliculus
at the freezing and escape thresholds. Reported evidence indicates that basolateral amygdala is a point of convergence 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 expression specifically after performance of freezing (middle panel) induced by electrical stimulation of the inferior colliculus, in relation to the control group (left panel). Fos expression in the group that received electrical stimulation of the inferior colliculus at the escape threshold is also indicated. EntCx, entorhinal cortex; 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 expression after 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, central nucleus of the inferior colliculus. Scale bar5200 mm.
to impart emotional value to sensory stimulation [19,25,32,33,35,48,54,55,57]. Accordingly, increased expression of immediate early genes in the amygdala has been reported using several paradigms of aversive conditioning [3,5,62,66,69]. It has been suggested that the involvement of the inferior colliculus in fear processes can occur through its anatomical and functional connections with the amygdala [14,56]. Marked increases in c-fos expression in the freezing and escape groups were also observed in the prefrontal cortex. These findings are not surprising as cortical projections are also activated by a wide variety of aversive stimulation [1,28,34,39,84]. These cortical areas are clearly connected to the inferior colliculus [2,16,23,38,60]. One of these pathways is provided by projections from the central nucleus of the inferior colliculus to the prefrontal cortex through the medial geniculate nucleus, amygdala and dorsomedial thalamus [16,38]. It has been shown that this circuit is concerned with the processing of auditory information of aversive nature, which triggers fear-like behaviors [14,24].
According to the hypothesis under testing, the freezing and escape responses were expected to activate different sets of brain structures and this prediction was confirmed by the present results. Only central amygdaloid nucleus, dorsal hippocampus and entorhinal cortex were labeled by c-fos immunoreactivity following freezing responses induced by electrical stimulation of the inferior colliculus. Although marginally significant, the ventral periaqueductal gray also showed considerable labeling after freezing but not after escape response induced by stimulation of the inferior colliculus. These data are in line with several studies that show that this structure is crucial for passive defensive responses [32,35]. However, as we have already shown that lesion of the ventral part of the periaqueductal gray, increased the freezing and escape thresholds determined by stimulation of the inferior colliculus (although the effects reached marginal significance), we should not discard the possibility that the inferior colliculus could be part of a defense system that could have the ventral periaqueductal gray as one of its output [56].
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On the other hand, immediate threat causes escape behavior, which seems to be mediated by the dorsal periaqueductal gray, hypothalamus and amygdala [40,41]. The only regions selectively activated by escape were the dorsal periaqueductal gray matter, cuneiform nucleus and the inferior colliculus itself. Electrical or chemical stimulation of the dorsal periaqueductal gray has been shown to elicit an explosive flight reaction that resembles a panic attack [9,40,41,47]. Also, neurosurgical patients reported feelings of terror and impending death after electrical stimulation of this region [63]. Several studies using c-fos have shown that the dorsal periaqueductal gray is activated in stressful situations [3,4,20,49,50,67,72,73], inclusive after intraperitoneal injection of drugs capable of eliciting panic symptoms [81]. Moreover, it was shown that disruption of tonic GABAergic inhibition in the dPAG elicits a constellation of behavioural and physiological responses that resembles a human panic attack [9,12,14]. The cuneiform nucleus seems to be the main output from the dPAG [18]. Electrical and chemical stimulation of the cuneiform nucleus causes clear defense reactions resembling that produced by activation of neural substrates of fear in the dPAG or inferior colliculus [27,73]. Also, neurons of cuneiform nucleus are activated by exposure of rats to cat odor [29] and show an increase of c-fos expression with stimulation of the dPAG and medial hypothalamus [72,73]. Expectedly, the central nucleus of the inferior colliculus was also significantly labeled after stimulation of this structure at the escape threshold. The inferior colliculus establishes reciprocal anatomical connections with the dorsal periaqueductal gray [52,60]. Along with this latter structure our data also point to the cuneiform nucleus as a good candidate for the output of escape responses generated at this level. In the present experiment, we found no difference in Fos protein expression between freezing, escape and control groups in the anterior, dorsomedial and preoptic nuclei of the hypothalamus. These findings were unexpected since the hypothalamus is one of the major recipients of information processed in the amygdala [65]. Defense reactions seem to be organized by both anterior [36,37,74,75] and medial hypothalamic nuclei [36,37] and these structures are activated after exposure of rodents to natural predators [20] or agonistic encounters [49,50]. Other studies have employed analysis of c-fos gene expression to identify brain structures activated by exposure to the elevated plus-maze [31,78]. An interesting finding of one of these studies was the remarkable fos expression of the central nucleus of the inferior colliculus [78]. This finding fits quite well within the framework of our hypothesis that the inferior colliculus has neural substrates for more than one kind of fear, since the elevated plus-maze has been considered a ‘mixed’ model in the sense that it may generate unconditioned and conditioned responses to threatening stimuli [43]. Because of some known limitations of the c-fos tech-
nique the present results should be interpreted cautiously. In fact, this technique does not allow for distinguishing between retrograde activation of afferent connections and feedforward efferent connections to the structure stimulated. Thus, we cannot discard the possibility that the electrical stimulation of the inferior colliculus could therefore produce distant fos-expression by antidromic then orthodromic activation of branched afferent collaterals. Also, the different levels of electrical stimulation produced different behavioral reactions, that is, the different groups of animals were exposed to different levels of external and internal sensory information, simply as a result of different stimulus-evoked movement. These confounding differences could also cause differential c-fos expression between the groups. Furthermore, the fact that high levels of IC stimulation failed to evoke fos-expression in some structures activated by lower levels of stimulation, for instance central nucleus of the amygdala, may be easier to understand in terms of indirect rather than direct mechanisms. This latter methodological limitation of this technique could explain why lesions of the central amygdala increase the threshold for both freezing and escape behaviors [56] and c-fos expression in this nucleus was only increased after freezing, as noted in the present study. On the other hand, autonomic reactions could not be easily evoke as a probable explanation for such differences, at least those recorded in this study, piloerection, exophtalmos, micturition and defecation were present as accompanied responses of both behavioral reactions, freezing and escape. In conclusion, the present neuroanatomical evidence adds to earlier reported behavioural results indicating that freezing and escape responses represent two types of fear with separate neural circuits in the brain. Our data also point out to the modulatory role played by the basolateral amygdala, dorsal hippocampus and the prefrontal cortex in the integration of aversive information generated at the level of the inferior colliculus.
Acknowledgements Research supported by FAPESP (Proc No. 98 / 11187-2 and 02 / 03705-0) and CNPq (Proc No. 94 / 5933-2). F. Cardenas, M.R. Lamprea and V.M. Castilho are recipients of doctor scholarships from FAPESP. D.M. Vianna is recipient of a doctor scholarship from CNPq. S.E. CruzMorales had a sabbatical leave supported by CONACYT ´ and DGAPA, UNAM, Mexico.
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