Fos-like immunoreactivity in the brain associated with freezing or escape induced by inhibition of either glutamic acid decarboxylase or GABAA receptors in the dorsal periaqueductal gray

GABAergic neurons exert tonic control over the neural substrates of aversion in the dorsal periaqueductal gray (dPAG). It has been shown that electrical stimulation of this region at freezing or escape thresholds activates different neural circuits in the brain. Since electrical stimulation activates cell bodies and fibers of passage, it is necessary to use chemical stimulation that activates only post-synaptic receptors. To investigate this issue further, reduction of GABA transmission was performed with local injections of either the GABA-A receptor antagonist bicuculline or the glutamic acid decarboxylase (GAD) inhibitor semicarbazide into the dorsolateral periaqueductal gray (dlPAG). Local infusions of semicarbazide (5.0 microg/0.2 microl) or bicuculline (40 ng/0.2 microl) into this region caused freezing and escape, respectively. The results obtained showed that freezing behavior induced by semicarbazide was associated with an increase in Fos expression in the laterodorsal nucleus of the thalamus (LD) and ventrolateral periaqueductal gray (vlPAG), while bicuculline-induced escape was related to widespread increase in Fos labeling, notably in the columns of the periaqueductal gray, hypothalamus nuclei, the central amygdaloid nucleus (Ce), the LD, the cuneiform nucleus (CnF) and the locus coeruleus (LC). Thus, the present data support the notion that freezing and escape behaviors induced by GABA blockade in the dlPAG are neurally segregated: freezing activates only structures that are mainly involved in sensory processing, and bicuculline-induced escape activates structures involved in both sensory processing and motor output of defensive behavior. Therefore, the freezing elicited by activation of dlPAG appears to be related to the acquisition of aversive information, whereas most brain structures involved in the defense reaction are recruited during escape.     

Neural segregation of Fos-protein distribution in the brain following freezing and escape behaviors induced by injections of either glutamate or NMDA into the dorsal periaqueductal gray of rats

Freezing and escape responses induced by gradual increases in the intensity of the electrical current applied to dorsal regions of the periaqueductal gray (dPAG) cause a distinct pattern of Fos distribution in the brain. From these studies, it has been suggested that a pathway involving the dPAG itself, dorsomedial hypothalamus and the cuneiform nucleus (CnF) would mediate responses to immediate danger and another one involving the amygdala and ventrolateral periaqueductal gray (vlPAG) would mediate cue-elicited responses. As electrical stimulation activates body cells and fibers of passage the need of studies with chemical stimulation of only post-synaptic fibers of the dPAG is obvious. To examine further this issue we measured Fos protein expression in brain areas activated by stimulation of the dPAG with glutamate (5 nmol/0.2 microL) and N-methyl-D-aspartate (NMDA) at doses that provoke either freezing (4 nmol/0.2 microL) or escape (7 nmol/0.2 microL) responses, respectively. The results showed that glutamate-induced freezing caused a selective increase in Fos expression in the superior and inferior colliculi as well as in the laterodorsal nucleus of the thalamus. On the other hand, NMDA-induced escape led to widespread increases in Fos labeling in almost all structures studied. Differently from glutamate, NMDA at doses provoking freezing caused significant increase of Fos labeling in the dPAG and CnF. Therefore, the present data support the notion that freezing behavior induced by activation of either non-NMDA or NMDA receptors in the dorsolateral periaqueductal gray (dlPAG) is neurally segregated: glutamate activates only structures that are mainly involved in the sensorial processing and NMDA-induced freezing structures involved in the motor output of defensive behavior. Therefore, the freezing elicited by the activation of non-NMDA receptors seem to be related to the acquisition of aversive information, whereas that resulting from the activation of NMDA receptors could serve as a preparatory response for flight.     

Distribution of Fos immunoreactivity in the rat brain after freezing or escape elicited by inhibition of glutamic acid decarboxylase or antagonism of GABA-A receptors in the inferior colliculus

It has been shown that electrical stimulation of the central nucleus of the inferior colliculus (IC) at freezing or escape thresholds activates different neural circuits in the brain. Since electrical stimulation activates cell bodies and fibers of passage it is necessary to use chemical stimulation that activates only post-synaptic receptors. To examine this issue in more detail, we took advantage of the fact that GABAergic neurons exert tonic control over the neural substrates of aversion in the IC. Reduction of GABA transmission in this structure was performed with the use of semicarbazide - an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) - and the GABA-A receptor antagonist bicuculline. Depending on the dose employed local infusions of semicarbazide (6.0 microg/0.2 microl) or bicuculline (40 ng/0.2 microl) into this region caused freezing and escape, respectively. The results obtained showed that freezing behavior induced by semicarbazide was associated with an increase in Fos expression in the dorsomedial column of the PAG (dmPAG) only, while bicuculline-induced escape was related to widespread increase in Fos labeling, notably in the periaqueductal gray, hypothalamus nuclei, amygdaloid nuclei, the laterodorsal nucleus of thalamus (LD), the cuneiform nucleus (CnF) and the locus coeruleus (LC). Thus, the present data support the notion that freezing and escape behaviors induced by GABA blockade in the IC are neurally segregated: acquisition of aversive information of acoustic nature from the IC probably uses the dmPAG column as a relay station to higher brain centers whereas bicuculline-induced escape activates structures involved in both sensory processing and motor output of defensive behavior. These results support the existence of distinct neural circuits mediating the sensory and motor responses of the defense reaction. The extent of the brain activation during freezing appears to be limited to the anatomical connections of the dmPAG, whereas an overall activation of the limbic system predominates during escape behavior induced by IC stimulation.     

Defensive freezing evoked by electrical stimulation of the periaqueductal gray: comparison between dorsolateral and ventrolateral regions.

Previous reports indicated that ventrolateral periaqueductal gray (vlPAG) plays a role in the expression of freezing behavior whereas dorsolateral periaqueductal gray (dlPAG) is involved on both freezing and active forms of defensive behaviors. In order to evaluate the role of each of these areas in the occurrence of defensive reactions, rats were electrically stimulated either in the dlPAG or vlPAG with different stimulus frequencies. Stepwise increases in the electrical stimulation of both dlPAG or vlPAG induced initially freezing and then a jumping response. Freezing induced by vlPAG stimulation had a tendency to disappear when the stimulation was turned off whereas freezing induced by dlPAG stimulation remained high in the absence of the stimulation. These results suggest that dlPAG and vlPAG are involved on defensive freezing probably through different neural circuitries.     

The distribution of fos immunoreactivity in rat brain following freezing and escape responses elicited by electrical stimulation of the inferior colliculus

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.     

Tracing from the dorsal premammillary nucleus prosencephalic systems involved in the organization of innate fear responses

The dorsal premammillary nucleus (PMd) is thought to play a critical role in the expression of fear responses to environmental threats. We have previously reported that, during an encounter with a predator, the PMd presents an impressive increase in Fos levels and cell body-specific chemical lesions therein virtually eliminated the expression of escape and freezing responses. Therefore, the PMd may be viewed as a strategic starting point to delineate prosencephalic circuits seemingly critical for the organization of innate fear responses. In the present review, we provide a comprehensive examination of the neural circuits putatively involved in influencing this hypothalamic site, and supplement this analysis with recent observations from our laboratory on the expression of Fos protein in the central nervous system of rats exposed to a live predator.     

Blockade of NMDA receptors and nitric oxide synthesis in the dorsolateral periaqueductal gray attenuates behavioral and cellular responses of rats exposed to a live predator

Innate fear stimulus induces activation of neurons containing the neuronal nitric oxide synthase enzyme (nNOS) in defensive‐related brain regions such as the dorsolateral periaqueductal gray (dlPAG). Intra‐dlPAG administration of nitric oxide synthase (NOS) inhibitors and glutamate antagonists induce anxiolytic‐like responses. We investigated the involvement of nitric oxide (NO) and glutamate neurotransmission in defensive reactions modulated by dlPAG. We tested if intra‐dlPAG injections of the selective nNOS inhibitor, N‐propyl‐L ‐arginine (NP), or the glutamate antagonist, AP7 (2‐amino‐7‐phosphonoheptanoic acid), would attenuate behavioral responses and cellular activation induced by predator exposure (cat). Fos‐like immunoreactivity (FLI) was used as a marker of neuronal functional activation, whereas nNOS immunohistochemistry was used to identify NOS neurons. Cat exposure induced fear responses and an increase of FLI in the dlPAG and dorsal premammillary nucleus (PMd). NP and AP7 attenuated the cat‐induced behavioral responses. Whereas NP tended to attenuate FLI in the dlPAG, AP7 induced a significant reduction in cellular activation of this region. The latter drug, however, increased FLI and double‐labeled cells in the PMd. Cellular activation of this region was significantly correlated with time spent near the cat (r = 0.7597 and 0.6057 for FLI and double‐labeled cells). These results suggest that glutamate/NO‐mediated neurotransmission in the dlPAG plays an important role in responses elicit by predator exposure. Blocking these neurotransmitter systems in this brain area impairs defensive responses. The longer time spent near the predator that follows AP7 effect could lead to an increased cellular activation of the PMd, a more rostral brain area that has also been related to defensive responses. © 2009 Wiley‐Liss, Inc.     

Distinct Fos expression in the brain following freezing behavior elicited by stimulation with NMDA of the ventral or dorsal inferior colliculus

The inferior colliculus (IC) is an important relay station for ascending auditory information to the medial geniculate nucleus (MGN) and temporal cortex. It has been reported that the ventral (ICv) and dorsal (ICd) regions of the IC are involved with the defensive reaction and audiogenic seizures, respectively. As freezing is the first response induced by stimulation of these IC nuclei with increasing doses of N-methyl-d-aspartate (NMDA), a question that arises is whether or not fear and audiogenic seizures generated at the IC level are interrelated processes. To address this issue, the Fos distribution in selected limbic structures following injections of NMDA into the ICv or ICd at freezing (7 nmol)- and escape (20 nmol)-producing doses was examined. Freezing behavior induced by intra-ICd NMDA caused an increase of Fos expression in the MGN, superior colliculus, dorsal columns of the periaqueductal gray and locus coeruleus while freezing induced by intra-ICv NMDA caused a significant Fos immunoreactivity in the prelimbic (PrL) and cingulate (Cg) cortices, basolateral and medial nuclei of the amygdala, ventrolateral periaqueductal gray, cuneiform nucleus and locus coeruleus. Escape behavior induced by NMDA injections into both nuclei caused a widespread Fos labeling in all limbic structures examined in this study. These results suggest that distinct circuits underlie the freezing behavior generated at the level of ICd and ICv. This is the first study to map Fos distribution associated with the stimulation of the ICv and ICd, regions supposed to be involved with fear and audiogenic seizures, respectively.     

Stimulation of 5-HT1A or 5-HT2A receptors in the ventrolateral periaqueductal gray causes anxiolytic-, but not panicolytic-like effect in rats

Evidences from studies using electrical or chemical stimulation of the midbrain periaqueductal gray (PAG) suggest that whereas the dorsal PAG is critical for the regulation of panic-related defensive behaviors, the ventrolateral PAG (vlPAG) modulates generalized anxiety-related responses. In the present study we evaluated whether the activation of 5-HT1A and 5-HT2A/2C receptors in the ventrolateral column of the periaqueductal gray (vlPAG) causes differential effects on an anxiety- and a panic-related defensive behavior, respectively, inhibitory avoidance and escape, in male Wistar rats submitted to the elevated T-maze. Our results showed that intra-vlPAG injection of the endogenous agonist serotonin, the 5-HT1A/7 agonist 8-OH-DPAT or 5-HT2A/2C agonist DOI impaired the acquisition of inhibitory avoidance, without interfering with escape performance. The same selective anxiolytic effect was also observed after local administration of the benzodiazepine receptor agonist midazolam. Moreover, as shown by the results of antagonism studies, 5-HT2A receptors are recruited for the anxiolysis caused by serotonin and DOI, while 5-HT1A receptors account for the effect of 8-OH-DPAT. In conclusion, our data show that the activation of 5-HT1A and 5-HT2A receptors in the vlPAG affects defensive responses related to generalized anxiety, but not panic disorder.     

Dorsolateral and ventral regions of the periaqueductal gray matter are involved in distinct types of fear

Stepwise increases in the electrical stimulation of the dorsolateral periaqueductal gray (dlPAG) produces alertness, then freezing and finally escape. This paper examines whether this freezing is (i) caused by Pavlovian fear conditioning to the contextual cues present during stimulation and (ii) the result of the stimulation of neurons located inside the dlPAG or elsewhere. To this end, freezing behavior was assessed in rats exposed either to the same or a different environment (context shift test) following the application of either footshocks or stimulation of the dlPAG at the freezing threshold. Rats submitted to footshocks presented freezing to the context 24 h later whereas rats submitted to the dlPAG stimulation showed freezing only immediately after the stimulation, regardless of the context. In the second experiment, aversive states generated by activation of the dlPAG were assessed either by measuring the thresholds for freezing and escape responses or the duration of these responses following microinjections of semicarbazide inside the dlPAG. The duration of freezing behavior was also measured in rats submitted to a contextual fear-conditioning paradigm using footshocks as unconditioned stimulus. Lesions of the ventral periaqueductal gray (vPAG) disrupted conditioned freezing to contextual cues associated to footshocks but vPAG lesions did not change the threshold of either freezing or escape responses elicited by electrical stimulation of the dlPAG. Lesions of the vPAG did not change the amount of freezing or escape responses produced by microinjections of semicarbazide into the dlPAG. These results indicate that stimulation of dlPAG neurons produce freezing behavior independent of any contextual fear conditioning and add to previously reported evidence showing that the vPAG is a critical structure for the expression of conditioned fear. In contrast, the neural substrate of unconditioned dlPAG stimulation-induced freezing is likely to elaborate unconditioned fear responses to impending danger, which have been implicated in panic disorder.     

Predatory hunting and exposure to a live predator induce opposite patterns of Fos immunoreactivity in the PAG

Considering the periaqueductal gray's (PAG) general roles in mediating motivational responses, in the present study, we compared the Fos expression pattern in the PAG induced by innate behaviors underlain by opposite motivational drivers, in rats, namely, insect predation and defensive behavior evoked by the confrontation with a live predator (a cat). Exposure to the predator was associated with a striking Fos expression in the PAG, where, at rostral levels, an intense Fos expression was found largely distributed in the dorsomedial and dorsolateral regions, whereas, at caudal levels, Fos-labeled cells tended to be mostly found in the lateral and ventrolateral columns, as well as in the dorsal raphe nucleus. Quite the opposite, insect predation was associated with increased Fos expression predominantly in the rostral two thirds of the lateral PAG, where the majority of the Fos-immunoreactive cells were found at the oculomotor nucleus levels. Remarkably, both exposure to the cat and insect predation upregulated Fos expression in the supraoculomotor region and the laterodorsal tegmental nucleus. Overall, the present results clearly suggest that the PAG activation pattern appears to reflect, at least partly, the animal's motivational status. It is well established that the PAG is critical for the expression of defensive responses, and, considering the present findings, it will be important to investigate how the PAG contributes to the expression of the predatory behavior, as well.     

Hypothalamic sites responding to predator threats--the role of the dorsal premammillary nucleus in unconditioned and conditioned antipredatory defensive behavior

In this study we provide a comprehensive analysis of the hypothalamic activation pattern during exposure to a live predator or an environment previously associated with a predator. Our results support the view that hypothalamic processing of the actual and the contextual predatory threats share the same circuit, in which the dorsal premammillary nucleus (PMd) plays a pivotal role in amplifying this processing. To further understand the role of the PMd in the circuit organizing antipredatory defensive behaviors, we studied rats with cytotoxic PMd lesions during cat exposure and examined the pattern of behavioral responses as well as how PMd lesions affect the neuronal activation of the systems engaged in predator detection, in contextual memory formation and in defensive behavioral responses. Next, we investigated how pharmacological blockade of the PMd interferes with the conditioned behavioral responses to a context previously associated with a predator, and how this blockade affects the activation pattern of periaqueductal gray (PAG) sites likely to organize the conditioned behavioral responses to the predatory context. Behavioral observations indicate that the PMd interferes with both unconditioned and conditioned antipredatory defensive behavior. Moreover, we have shown that the PMd influences the activation of its major projecting targets, i.e. the ventral part of the anteromedial thalamic nucleus which is likely to influence mnemonic processing, and PAG sites involved in the expression of antipredatory unconditioned and conditioned behavioral responses. Of particular relevance, this work provides evidence to elucidate the basic organization of the neural circuits integrating unconditioned and contextual conditioned responses to predatory threats.     

Antidepressant treatment reduces Fos-like immunoreactivity induced by swim stress in different columns of the periaqueductal gray matter

Antidepressant treatment attenuates behavioral changes induced by uncontrollable stress. The periaqueductal gray matter (PAG) is proposed to be a brain site involved in the behavioral responses to uncontrollable stress and antidepressant effects. The main goal of the present study was to investigate the effect of antidepressant treatment on the pattern of neural activation of the PAG along its mediolateral and rostrocaudal subregions after a forced swim stress episode. Male Wistar rats were sub-acutely treated with desipramine (a selective noradrenaline re-uptake blocker, three injections of 10 mg/kg in 24 h) or clomipramine (a non-selective serotonin and noradrenaline re-uptake blocker, three injections of 10 mg/kg in 24 h) and submitted to the forced swimming test (FST). Two hours after the test their brain were removed for Fos immunohistochemistry. Fos-like immunoreactivity (FLI) in rostral, intermediate and caudal portions of dorsomedial (dmPAG), dorsolateral (dlPAG), lateral (lPAG) and ventrolateral (vlPAG) PAG were quantified by a computerized system. The FST session increased FLI in most parts of the PAG. Previous treatment with desipramine or clomipramine reduced FLI in all columns of the PAG. FLI in the PAG correlated positively with to the immobility time and negatively with to climbing behavior scored during the test. These results indicate that neurons in the PAG are activated by uncontrollable stress. Moreover, inhibitory action of antidepressants on this activity may be associated with the anti-immobility effects of these drugs in the FST.     

Conditioned place aversion organized in the dorsal periaqueductal gray recruits the laterodorsal nucleus of the thalamus and the basolateral amygdala

The amygdala-ventral periaqueductal gray circuit is crucial for the expression of contextual conditioned fear. However, little is known about the neural circuits activated when the stimulation of the dorsal periaqueductal gray (dPAG) is used as unconditioned stimulus (US) in conditioned fear paradigms. The present paper examines the Fos-protein distribution in the brain of rats submitted to a conditioned place aversion (CPA) paradigm using the dPAG chemical stimulation with semicarbazide (SMC), an inhibitor of the GABA synthesizing enzyme, as US and the quadrant of an arena where the drug was injected as the paired neutral stimulus. Our results show that CPA associated with SMC injections caused a significant Fos labeling in the laterodorsal nucleus of the thalamus (LD), basolateral nucleus of amygdala (BLA) and in the dorsomedial PAG (dmPAG). This pattern of brain activation is clearly different from the neural substrates of the classical fear conditioning reported in the literature. Moreover, this paper shows that CPA with the use of chemical stimulation of the dPAG could be used as an experimental model of panic disorder with agoraphobia in the extent that panic attacks repeatedly associated with specific contexts may turn in this condition in the clinics. This condition activates the BLA probably through the LD. Besides, it indicates that the dPAG can be the link between amygdala and the brainstem motor regions that controls CPA when dPAG stimulation is used as US instead of footshocks. From this evidence we suggest that a loop dPAG-LD-BLA-dPAG is activated during the panic disorder with agoraphobia.     

Differential effects of NK1 receptors in the midbrain periaqueductal gray upon defensive rage and predatory attack in the cat

This study utilized anatomical and behavioral-pharmacological methods to determine the role of NK(1)-Substance P receptors in the midbrain periaqueductal gray (PAG) in defensive rage behavior in cats. For behavioral pharmacological experiments, monopolar stimulating electrodes were implanted in the medial hypothalamus for elicitation of defensive rage behavior and cannula-electrodes were implanted in the PAG for microinjections of receptor compounds. Microinjections of the NMDA antagonist, AP-7 (2 nmol), into the dorsal PAG blocked defensive rage elicited by medial hypothalamic stimulation, thus establishing the PAG as a synaptic region that receives hypothalamic inputs linked to defensive rage behavior. Microinjections of the NK(1) agonist, GR73632, into the same injection sites facilitated defensive rage in a dose-dependent manner, and also induced spontaneous hissing in five cats. The effects of GR73632 were reduced by pretreatment of the PAG with the NK(1) antagonist, GR82334 (16 nmol), microinjected into the same sites. Microinjections of GR73632 (8 nmol) into the PAG also suppressed predatory attack elicited by stimulation of the lateral hypothalamus. Immunohistochemical methods utilized to detect Substance P and Fos immunoreactivity revealed that neurons in the PAG activated after defensive rage-inducing medial hypothalamic stimulation lie in the same region as Substance-P-immunoreactive processes. Fos immunoreactivity was highest in the dorsomedial aspect of the rostral PAG after medial hypothalamic stimulation. Cats that were unstimulated or that exhibited predatory attack after lateral hypothalamic stimulation had low c-fos expression levels in the PAG. Substance P immunoreactivity was high throughout the dorsal PAG. The results indicate that NK(1) receptors in the PAG potentiate defensive rage and suppress predatory aggression in the cat.     

High-frequency stimulation of the dorsolateral periaqueductal gray and ventromedial hypothalamus fails to inhibit panic-like behaviour

Electrical stimulation of the dorsolateral periaqueductal gray (dlPAG) and one of its target structures, the ventromedial hypothalamus (VMH), produces a typical behaviour in rats consisting of vigorous running and jumping which is known as “escape behaviour”. Escape behaviour in rodents closely mimics panic attacks in humans. Since electrical stimulation at higher frequencies generally inhibits the stimulated region, we tested in this study the hypothesis that deep brain stimulation (DBS) of the dlPAG and VMH at higher frequencies (>100 Hz) would not induce escape behaviour. More specifically, we evaluated whether experimental DBS could be used to inhibit panic-like behaviour. Rats underwent implantation of DBS-electrodes at the level of the dlPAG and VMH and the effects of various stimulation parameters were assessed. In addition, we studied the neural activation pattern resulting from DBS of the dlPAG and VMH using c-Fos immunohistochemistry. We found that stimulation amplitude is the most important stimulation parameter in the induction of escape behaviour. Remarkably, stimulation frequency (1–300 Hz) had no effect on stimulation-induced escape behaviour and therefore it was not possible to prevent the induction of escape behaviour with higher frequencies. The neuronal activation pattern resulting from dlPAG and VMH DBS was similar. These findings suggest that DBS of the dlPAG and VMH induces panic-related behaviours even at higher frequencies.     

The role of mu and kappa opioid receptors within the periaqueductal gray in the expression of conditional hypoalgesia

The periaqueductal gray (PAG) is a midbrain structure involved in the modulation of pain and expression of classically conditioned fear responses. Non-selective opioid antagonists applied to the PAG block the expression of hypoalgesia in rats exposed to a Pavlovian signal for shock. This study was conducted to determine the anatomical and pharmacological specificity of the PAG's role in conditional hypoalgesia. Rat subjects received injections of either the mu opioid antagonist CTAP (6.6 nMol), the kappa opioid antagonist Nor-binaltorphimine (Nor-BNI, 6.6 nMol) or saline. Injections were made into either the dorsolateral (dlPAG) or ventrolateral (vlPAG) PAG prior to the presentation of an auditory stimulus that had previously been paired with foot shock while measuring nociception with the radiant heat tail flick (TF) test. Elevation in TF latency in response to the auditory stimulus was blocked only by administration of CTAP into the vlPAG. These results suggest that conditional hypoalgesia (CHA) is subserved by mu but not kappa opioid receptors located in the vlPAG but not the dlPAG.     

Sensing danger through the olfactory system: the role of the hypothalamic dorsal premammillary nucleus

The dorsal premammillary nucleus (PMd) has a critical role on the expression of defensive responses to predator odor. Anatomical evidence suggests that the PMd should also modulate memory processing through a projecting branch to the anterior thalamus. By using a pharmacological blockade of the PMd with the NMDA-receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5), we were able to confirm its role in the expression of unconditioned defensive responses, and further revealed that the nucleus is also involved in influencing associative mechanisms linking predatory threats to the related context. We have also tested whether olfactory fear conditioning, using coffee odor as CS, would be useful to model predator odor. Similar to cat odor, shock-paired coffee odor produced robust defensive behavior during exposure to the odor and to the associated context. Shock-paired coffee odor also up-regulated Fos expression in the PMd, and, as with cat odor, we showed that this nucleus is involved in the conditioned defensive responses to the shock-paired coffee odor and the contextual responses to the associated environment.     

Orbitomedial prefrontal cortical projections to distinct longitudinal columns of the periaqueductal gray in the rat

We utilised retrograde and anterograde tracing procedures to study the origin and termination of prefrontal cortical (PFC) projections to the periaqueductal gray (PAG) in the rat. A previous study, in the primate, had demonstrated that distinct subgroups of PFC areas project to specific PAG columns. Retrograde tracing experiments revealed that projections to dorsolateral (dlPAG) and ventrolateral (vlPAG) periaqueductal gray columns arose from medial PFC, specifically prelimbic, infralimbic, and anterior cingulate cortices. Injections made in the vlPAG also labeled cells in medial, ventral, and dorsolateral orbital cortex and dorsal and posterior agranular insular cortex. Other orbital and insular regions, including lateral and ventrolateral orbital, ventral agranular insular, and dysgranular and granular insular cortex did not give rise to appreciable projections to the PAG. Anterograde tracing experiments revealed that the projections to different PAG columns arose from specific PFC areas. Projections from the caudodorsal medial PFC (caudal prelimbic and anterior cingulate cortices) terminated predominantly in dlPAG, whereas projections from the rostroventral medial PFC (rostral prelimbic cortex) innervated predominantly the vlPAG. As well, consistent with the retrograde data, projections arising from select orbital and agranular insular cortical areas terminated selectively in the vlPAG. The results indicate: (1) that rat orbital and medial PFC possesses an organisation broadly similar to that of the primate; and (2) that subdivisions within the rat orbital and medial PFC can be recognised on the basis of projections to distinct PAG columns.     

c-Fos expression increase in NADPH-diaphorase positive neurons after exposure to a live cat

The present study investigated if NOS positive neurons localized in regions related to defensive reactions are activated after exposure to an innate fear stimulus (a live cat). Male Wistar rats were exposed to a live or a toy cat for 10 min and 2h later had their brains removed and processed for c-Fos immunohistochemistry (a marker of neuronal functional activation) and NADPH-diaphorase (NADPH-d; used to detect the presence of NOS neurons) histochemistry. Cat exposure induced a small (11%) to moderate (50%) significant increase in the percentage of double-stained cells (c-Fos+NADPH-d positive neurons) in the anteromedial bed nucleus of stria terminalis (BSTMA), medial amygdala (MeA), parvocellular paraventricular (pPVN), lateral (LH) and dorsal premammillary (PMd) hypothalamic nuclei, dorsolateral periaqueductal grey (dlPAG) and dorsal raphe nucleus (DRN). This increase was attenuated in the PMd, DRN and dlPAG by i.c.v. injection of AP7 (5 nmol/2 microl), an NMDA receptor antagonist. The drug increased the percentage of time the rats remained close to the cat in the observation box. The results suggest that exposure to a live predator activates neurons containing NOS in brain areas related to defensive reactions. They also indicate that this effect probably involves activation of NMDA glutamate receptors.