Role of nitric oxide on defensive behavior and long-term aversive learning induced by chemical stimulation of the dorsolateral periaqueductal gray matter.

The midbrain periaqueductal gray matter, especially the dorsolateral portion (dlPAG), coordinates immediate defensive responses (DR) to threats, but also ascends forebrain information for aversive learning. The synaptic dynamics in the dlPAG regulate the intensity and type of behavioral expression, as well as long-term processes such as memory acquisition, consolidation, and retrieval. Among several neurotransmitters and neural modulators, nitric oxide seems to play an important regulatory role in the immediate expression of DR, but it remains unclear if this gaseous on-demand neuromodulator contributes to aversive learning. Therefore, the role of nitric oxide in the dlPAG was investigated, during conditioning in an olfactory aversive task. The behavioral analysis consisted of freezing and crouch-sniffing in the conditioning day after glutamatergic NMDA agonist injection into the dlPAG. Two days later, rats were re-exposed to the odor cue and avoidance was measured. 7NI, a selective neuronal nitric oxide synthase inhibitor (40 and 100 nmol), injected before NMDA (50 pmol) impaired immediate DR and consequent aversive learning. The scavenging of extrasynaptic nitric oxide by C-PTIO (1 and 2 nmol) induced similar results. Moreover, spermine NONOate, a nitric oxide donor (5, 10, 20, 40, and 80 nmol), produced DR by itself, but only the low dose also promoted learning. The following experiments utilized a fluorescent probe, DAF-FM diacetate (5 µM), directly into the dlPAG, to quantify nitric oxide in the three previous experimental situations. Nitric oxide levels were increased after NMDA stimulation, decreased after 7NI, and increased after spermine NONOate, in line with alterations in defensive expression. Altogether, the results indicate that nitric oxide plays a modulatory and decisive role in the dlPAG regarding immediate DR and aversive learning.

Dexamethasone impairs encoding and expression of aversive conditioning promoted by pentylenetetrazole.

Behavioral and neuroendocrine responses following threatening situations promote the release of corticosterone, which is known to modulate trauma-related learning and memory process. However, it remains unknown whether the aversive learning generated by interoceptive fear conditioning is affected by glucocorticoid modulation. Therefore, the present study aimed to investigate the role of dexamethasone suppression in encoding and expression of pentylenetetrazole-induced olfactory fear conditioning (OFC) and in contextual second-order conditioning promoted by the conditioned odor. Adult male Long-Evans rats were treated with dexamethasone 60 min before the encoding or the expression in both OFC and contextual second-order conditioning. Dexamethasone treatment impaired encoding and expression of the OFC, but failed to impair encoding and expression of the contextual second-order conditioning. Altogether, our results show that although OFC and thereafter contextual second-order conditioning may allow the study of traumatic memories, each order of conditioning seems to present specific features related to their pharmacological modulation. These findings highlight the importance of addressing the role of neuromodulatory systems in first-order and second-order conditioning to gain a better understanding of these phenomena and support future therapies related to traumatic memories.

Inactivation of the dorsolateral periaqueductal gray matter impairs the promoting influence of stress on fear memory during retrieval.

Exposure to stressful conditions induces long-lasting neurobiological changes in selected brain areas, which could be associated with the emergence of negative emotional responses. Moreover, the interaction of a stressful experience and the retrieval of an established fear memory trace enhance both fear expression and fear retention. Related to this, the stimulation of the dorsolateral part of the mesencephalic periaqueductal gray matter (dlPAG) prior to retrieval potentiates a fear memory trace previously acquired. Therefore, the question that arises is whether the dlPAG mediates the increased fear expression and fear retention after retrieval. Rats were subjected to a contextual fear conditioning paradigm using a single footshock, and 1 day later, rats were subjected to a stressful situation. As previously reported, there was an increase of freezing response only in those rodents that were re-exposed to the associated context at 1 and 5 days after stress exposure. Muscimol intra-dlPAG prior to the restraint event prevented such increase. Conversely, Muscimol intra-dlPAG infusion immediately after the stress experience had no effect on the resulting fear memory. When the neuroendocrine response to stress was explored, intra-dlPAG infusion of muscimol prior to stress decreased Fos expression in the paraventricular nucleus and serum corticosterone levels. Moreover, this treatment prevented the enhancement of the density of hippocampal "mature" spines associated with fear memory. In conclusion, the present results suggest that the dlPAG is a key neural site for the negative valence instruction necessary to modulate the promoting influence of stress on fear memory.

Periaqueductal gray glutamatergic, cannabinoid and vanilloid receptor interplay in defensive behavior and aversive memory formation.

Stimulation of the midbrain periaqueductal gray matter (PAG) in humans elicits sensations of fear and impending terror, and mediates predator defensive responses in rodents. In rats, pharmacological stimulation of the dorsolateral portion of the PAG (dlPAG) with N-Methyl-d-Aspartate (NMDA) induces aversive conditioning that acts as an unconditioned stimulus (US). In the present work, we investigated the interplay between the vanilloid TRPV1 and cannabinoid CB1 receptors in the NMDA-dlPAG defensive response and in subsequent aversive learning. Rats were subjected to dlPAG NMDA infusion in an olfactory conditioned stimulus (CS) task allowing the evaluation of immediate and long-term defensive behavioral responses during CS presentation. The results indicated that an intermediate dose of NMDA (50 pmol) induced both immediate and long-term effects. A sub-effective dose of NMDA (25 pmol) was potentiated by the TRPV1 receptor agonist capsaicin (CAP, 1 nmol) and the CB1 receptor antagonist, AM251 (200 pmol). CAP (10 nmol) or the combination of CAP (1 nmol) and AM251 (200 pmol) induced long-term effects without increasing immediate defensive responses. The glutamate release inhibitor riluzole (2 or 4 nmol) and the AMPA/kainate receptor antagonist DNQX (2 or 4 nmol) potentiated the immediate effects but blocked the long-term effects. The results showed that immediate defensive responses rely on NMDA receptors, and aversive learning on the fine-tuning of TRPV1, CB1, metabotropic glutamate and AMPA receptors located in pre- and postsynaptic membranes. In conclusion, the activity of the dlPAG determines core affective aspects of aversive memory formation controlled by local TRPV1/CB1 balance.

The periaqueductal gray and primal emotional processing critical to influence complex defensive responses, fear learning and reward seeking.

The periaqueductal gray (PAG) has been commonly recognized as a downstream site in neural networks for the expression of a variety of behaviors and is thought to provide stereotyped responses. However, a growing body of evidence suggests that the PAG may exert more complex modulation of a number of behavioral responses and work as a unique hub supplying primal emotional tone to influence prosencephalic sites mediating complex aversive and appetitive responses. Of particular relevance, we review how the PAG is involved in influencing complex forms of defensive responses, such as circa-strike and risk assessment responses in animals. In addition, we discuss putative dorsal PAG ascending paths that are likely to convey information related to threatening events to cortico-hippocampal-amygdalar circuits involved in the processing of fear learning. Finally, we discuss the evidence supporting the role of the PAG in reward seeking and note that the lateral PAG is part of the circuitry related to goal-oriented responses mediating the motivation to hunt and perhaps drug seeking behavior.

Acquisition and expression of fear memories are distinctly modulated along the dorsolateral periaqueductal gray axis of rats exposed to predator odor.

The dorsolateral region of the midbrain periaqueductal gray (dlPAG) modulates both innate and conditioned fear responses. However, the contribution of the rostrocaudal portions of the dlPAG to defense reactions and aversive memories remains unclear. Here, we sought to investigate the effects of N-methyl-d-aspartate (NMDA) receptor blockade within rostral or caudal dlPAG of rats exposed to innate and learned fear to cat odor. For this, adult male Wistar rats were microinjected with the NMDA antagonist D-2-amino-5-phosphono-pentanoate (AP5; 3 or 6nmol/0.2µl) into the rostral or caudal dlPAG before and after the exposure to the cat odor or to the context paired with the predator odor. The results demonstrated that cat odor exposure induced unconditioned defensive behaviors as well as contextual fear. AP5 microinjected in the rostral dlPAG reduced the defensive responses to cat odor and impaired the acquisition, but not consolidation of contextual fear. On the other hand, AP5 infused within the caudal dlPAG promoted long-lasting reduction of contextual fear expression. Altogether, our data suggest that NMDA receptors mediate a functional dichotomy in the rostrocaudal axis of dlPAG regulating unconditioned and conditioned defensive reactions to predatory cues.

Olfactory instruction for fear: neural system analysis.

Different types of predator odors engage elements of the hypothalamic predator-responsive circuit, which has been largely investigated in studies using cat odor exposure. Studies using cat odor have led to detailed mapping of the neural sites involved in innate and contextual fear responses. Here, we reviewed three lines of work examining the dynamics of the neural systems that organize innate and learned fear responses to cat odor. In the first section, we explored the neural systems involved in innate fear responses and in the acquisition and expression of fear conditioning to cat odor, with a particular emphasis on the role of the dorsal premammillary nucleus (PMd) and the dorsolateral periaqueductal gray (PAGdl), which are key sites that influence innate fear and contextual conditioning. In the second section, we reviewed how chemical stimulation of the PMd and PAGdl may serve as a useful unconditioned stimulus in an olfactory fear conditioning paradigm; these experiments provide an interesting perspective for the understanding of learned fear to predator odor. Finally, in the third section, we explored the fact that neutral odors that acquire an aversive valence in a shock-paired conditioning paradigm may mimic predator odor and mobilize elements of the hypothalamic predator-responsive circuit.

Paradoxical mineralocorticoid receptor-mediated effect in fear memory encoding and expression of rats submitted to an olfactory fear conditioning task.

There is general agreement that the substantial modification in memory and motivational states exerted by corticosteroids after a traumatic experience is mediated in complementary manner by the mineralocorticoid (MR) and glucocorticoid (GR) receptors. Here we tested the hypothesis that pharmacological manipulation of MR activity would affect behavioral strategy and information storage in an olfactory fear conditioning (OFC) task. Male Wistar rats were submitted to the OFC with different training intensities. We observed that following high intensity OFC acquisition, a set of defensive coping strategies, which includes avoidance and risk assessment behaviors, was elicited when subjects were exposed to the conditioned stimulus (CS) 48 h later. In addition, following either OFC acquisition or retrieval (CS-I test) a profound corticosterone secretion was also detected. Systemic administration of the MR antagonist spironolactone altered the behavioral coping style irrespective the antagonist was administered 60 min prior to the acquisition or before the retrieval session. Surprisingly, the MR agonist fludrocortisone given 60 min prior to acquisition or retrieval of OFC had similar effects as the antagonist. In addition, post-training administration of fludrocortisone, following a weak training procedure, facilitated the consolidation of OFC. Fludrocortisone rather than spironolactone reduced serum corticosterone levels, suggesting that, at least in part, the effects of the MR agonist may derive from additional GR-mediated HPA-axis suppression. In conclusion, the present study suggests the involvement of the MR in the fine-tuning of behavioral adaptation necessary for optimal information storage and expression, as revealed by the marked alterations in the risk assessment behavior.

Enhanced noradrenergic activity potentiates fear memory consolidation and reconsolidation by differentially recruiting α1- and ß-adrenergic receptors.

Consolidation and reconsolidation are phases of memory stabilization that diverge slightly. Noradrenaline is known to influence both processes, but the relative contribution of α1- and ß-adrenoceptors is unclear. The present study sought to investigate this matter by comparing their recruitment to consolidate and/or reconsolidate a contextual fear memory trace under enhanced noradrenergic activity induced by yohimbine. We report that this α2-adrenoceptor antagonist was able to potentiate fear memory trace consolidation or reconsolidation when administered immediately after acquisition or retrieval, respectively, resulting in increased freezing expression. In either case, generalization of this response to an unpaired context was also seen when it achieved a ceiling level in the paired context. These effects endured for over 7 d and relied on action at central rather than peripheral sites, but were prevented when a memory trace was not acquired, when memory reactivation was omitted, or when administration of yohimbine was delayed until 6 h after acquiring or retrieving the memory trace. The ß-adrenoceptor antagonist propranolol was able to prevent the above-mentioned effects of yohimbine, while pretreatment with the α1-adrenoceptor antagonist prazosin blocked only its facilitating effects on memory reconsolidation. These results highlight a differential participation of α1- and ß-adrenoceptors in fear memory processing. Moreover, it was shown that the α2-adrenoceptor agonist clonidine, as opposed to yohimbine, mitigates fear expression by weakening memory consolidation or reconsolidation.

Anxiogenic-like profile of Wistar adult rats based on the pilocarpine model: an animal model for trait anxiety?

RATIONALE: There is extensive evidence indicating the influence of seizures on emotional responses observed in human and animals, but so far few studies are focusing on the behavioral profile of animals that do not have seizures despite being treated with convulsant agents. OBJECTIVES: We aimed to establish the behavioral profile, biochemical, and electrographic features of rats submitted to the pilocarpine model of temporal lobe epilepsy METHODS: Rats treated with pilocarpine (20 to 350 mg/kg, i.p.) that did not develop status epilepticus or spontaneous recurrent seizures were evaluated 1 month later in the elevated plus maze (EPM), T-maze (ETM), open-field (OF), and step-down avoidance tests. Electroencephalographic (EEG), glutamate uptake, and hippocampal neuronal death assays were also performed RESULTS: Pilocarpine (150 or 350 mg/kg) promoted anxiogenic-like effects in rats evaluated in the EPM, ETM, and OF tests, whereas only the highest dose evoked spike-wave discharges during EEG recordings. Hippocampal theta rhythm was increased by pilocarpine 150 or 350 mg/kg and only the highest dose reduced the L-[(3)H]-glutamate uptake and cell viability on hippocampal slices. CONCLUSIONS: Subconvulsant doses of pilocarpine promote long-lasting alterations on neural circuitry, reflected by an increased theta activity in the hippocampus and an anxiety-like profile of rats evaluated 1 month after the treatment which is independent of seizure occurrence and is not related to changes in glutamate uptake or hippocampal damage. These results prompt us to suggest that a systemic administration of subconvulsant doses of pilocarpine could be useful as a new tool to model trait anxiety in rats.

Dorsolateral periaqueductal gray stimulation prior to retrieval potentiates a contextual fear memory in rats.

The association of a neutral context with an aversive stimulus, such as foot-shock, result in a contextual fear memory. A growing number of evidence have revealed that prior exposure to diverse threatening situations facilitates the encoding of fear memory during acquisition and such reports support the widespread notion that emotionally arousal results in stronger and long-lasting memories. However, few studies have investigated if a threatening experience can affect the recall and the persistence of such fear memory trace. To test the hypothesis that an emotionally negative experience could modify the retrieval of a memory and potentiate the expression of a fear memory, the present study used the chemical stimulation (microinjection of NMDA) of the dorsolateral periaqueductal gray matter (dlPAG) of rats in order to induce an aversive emotional state. Such stimulation was performed one day after a weak fear training protocol, and the fear expression was analyzed in subsequent re-exposures to the conditioned context. The results showed that the negative emotional state induced by the dlPAG stimulation enhanced the fear memory trace when this trace was reactivated one day after this aversive experience. Additionally, the potentiation of the fear response was contingent to the associated context since no potentiation was evident when NMDA-stimulated animals were subsequently placed in a non-associated context. Finally, the model suggests that the enhancement of fear responses is long-lasting since NMDA-treated animals performed a robust fear response six days after memory retrieval.

The dorsolateral periaqueductal gray and its role in mediating fear learning to life threatening events.

The dorsolateral column of the periaqueductal gray (dlPAG) integrates aversive emotional experiences and represents an important site responding to life threatening situations, such as hypoxia, cardiac pain and predator threats. Previous studies have shown that the dorsal PAG also supports fear learning; and we have currently explored how the dlPAG influences associative learning. We have first shown that N-methyl-D-aspartate (NMDA) 100 pmol injection in the dlPAG works as a valuable unconditioned stimulus (US) for the acquisition of olfactory fear conditioning (OFC) using amyl acetate odor as conditioned stimulus (CS). Next, we revisited the ascending projections of the dlPAG to the thalamus and hypothalamus to reveal potential paths that could mediate associative learning during OFC. Accordingly, the most important ascending target of the dlPAG is the hypothalamic defensive circuit, and we were able to show that pharmacological inactivation using ß-adrenoceptor blockade of the dorsal premammillary nucleus, the main exit way for the hypothalamic defensive circuit to thalamo-cortical circuits involved in fear learning, impaired the acquisition of the OFC promoted by NMDA stimulation of the dlPAG. Moreover, our tracing study revealed multiple parallel paths from the dlPAG to several thalamic targets linked to cortical-hippocampal-amygdalar circuits involved in fear learning. Overall, the results point to a major role of the dlPAG in the mediation of aversive associative learning via ascending projections to the medial hypothalamic defensive circuit, and perhaps, to other thalamic targets, as well. These results provide interesting perspectives to understand how life threatening events impact on fear learning, and should be useful to understand pathological fear memory encoding in anxiety disorders.

Acquisition of Pavlovian fear conditioning using ß-adrenoceptor activation of the dorsal premammillary nucleus as an unconditioned stimulus to mimic live predator-threat exposure.

In the present work, we sought to mimic the internal state changes in response to a predator threat by pharmacologically stimulating the brain circuit involved in mediating predator fear responses, and explored whether this stimulation would be a valuable unconditioned stimulus (US) in an olfactory fear conditioning paradigm (OFC). The dorsal premammillary nucleus (PMd) is a key brain structure in the neural processing of anti-predatory defensive behavior and has also been shown to mediate the acquisition and expression of anti-predatory contextual conditioning fear responses. Rats were conditioned by pairing the US, which was an intra-PMd microinjection of isoproterenol (ISO; ß-adrenoceptor agonist), with amyl acetate odor-the conditioned stimulus (CS). ISO (10 and 40 nmol) induced the acquisition of the OFC and the second-order association by activation of ß-1 receptors in the PMd. Furthermore, similar to what had been found for contextual conditioning to a predator threat, atenolol (ß-1 receptor antagonist) in the PMd also impaired the acquisition and expression of OFC promoted by ISO. Considering the strong glutamatergic projections from the PMd to the dorsal periaqueductal gray (dPAG), we tested how the glutamatergic blockade of the dPAG would interfere with the OFC induced by ISO. Accordingly, microinjections of NMDA receptor antagonist (AP5, 6 nmol) into the dPAG were able to block both the acquisition, and partially, the expression of the OFC. In conclusion, we have found that PMd ß-1 adrenergic stimulation is a good model to mimic predatory threat-induced internal state changes, and works as a US able to mobilize the same systems involved in the acquisition and expression of predator-related contextual conditioning.

Role of beta-adrenergic receptors in the ventromedial prefrontal cortex during contextual fear extinction in rats.

It has been reported that stress-related activation of the noradrenergic system strengthens the formation of aversive memories and that beta-adrenergic receptors seem to be involved in this emotional memory processing. In this study, the effects of beta-adrenergic compounds on the extinction of contextual conditioned fear responses were evaluated. Rats were trained with footshock in a conditioning box. In the 3 days following the training, the animals were re-exposed to the apparatus and received either a single or repeated intraperitoneal injections of the beta-adrenergic antagonist propranolol, the beta-adrenergic agonist isoproterenol, or saline 30 min before (acquisition of extinction) or immediately after (consolidation of extinction) the extinction sessions. A drug-free session was performed on the last day. While repeated isoproterenol treatment facilitated the consolidation of contextual fear extinction, repeated propranolol administration impaired the acquisition and the consolidation of this process. Further, the role of ventromedial prefrontal cortex (vmPFC) in the extinction of contextual conditioned fear was tested with an immunohistochemistry assay. Our results show a reduction in Fos-protein expression between the first and the last extinction session. In a follow-up experiment, intra-vmPFC microinjection of isoproterenol before the first extinction session facilitated the extinction of contextual fear. This facilitation was antagonized by pre-treatment with atenolol, suggesting that this change is mediated by beta-1-adrenergic activity. Our results reinforce the role of the vmPFC in fear extinction mechanisms, suggesting that vmPFC-beta-1-adrenergic receptor activation underlies part of the facilitation of the fear extinction processes.

Impairment of contextual conditioned fear extinction after microinjection of alpha-1-adrenergic blocker prazosin into the medial prefrontal cortex.

Long-lasting memories of aversive or stressful events have been associated with the noradrenergic system activation. Alpha-1-adrenergic antagonist prazosin has successfully been used in the last years to treat anxiety disorders related to aversive memories recurrence in humans. Contextual conditioned fear extinction paradigm in rats has been used to better understand the mechanisms involved in the attenuation of defensive behaviour after a traumatic situation. Here we investigated the effects of systemic administration of prazosin in the fear extinction processes. Rats were previously paired in a contextual fear conditioning box (1 footshock, 1 mA, 2s duration), further returning to the same box during three consecutive days receiving an intraperitoneal injection of vehicle or prazosin 30 min before (acquisition of extinction; 0.1 or 0.5mg/kg) or immediately after (consolidation of extinction, 0.5 or 1.5mg/kg) each extinction session (10 min). On the last day, all animals were re-exposed undrugged to the apparatus. Since the medial prefrontal cortex (mPFC) has been described as a key structure in the modulation of conditioned fear extinction, the effects of intra-mPFC microinjection (0.2 microl per side) of vehicle (PBS) or prazosin (0.75 or 2.5 nmol) in the acquisition of fear extinction (10 min before extinction session 1) were further evaluated. Subjects were drug-free re-exposed to the same box in the next day (extinction session 2). The percentage of freezing time was used as the memory retention parameter. The results showed that either systemic or intra-mPFC-alpha-1-adrenergic blockade increased the freezing time in the last extinction sessions, suggesting impairment of the extinction of contextual conditioned fear in rats.

The dorsal periaqueductal gray modulates the increased fear-like behavior exhibited by experienced rats in the elevated plus-maze.

Elevated plus-maze (EPM) experienced rats show reduced open arms exploration in a subsequent EPM exposure, expressing the increased open arms avoidance which is characteristic of anxiety/fear-like behavior. The midbrain dorsal periaqueductal gray (dPAG) is an important integrative area of the neuroaxis able to control the motivational states of an animal. It has been shown that dPAG participates in the mediation of anxiety/fear-like behavior elicited in the EPM. The present study was outlined to evaluate the dPAG-NMDA-receptor contribution to the increased open arms avoidance found in EPM experienced rats. In addition, a possible mnemonic effect, due to the dPAG-NMDA-receptor blockade, was tested in the step-down inhibitory avoidance task (SD). Male Wistar rats received dPAG infusion of NMDA antagonists (AP5 or ifenprodil) before being submitted to the EPM test and retest sessions, or the SD training and test sessions. Both NMDA-receptor antagonists infused in the dPAG, reduced the fear-like behavior exhibited in the EPM by increasing the open arms exploration in both naïve and EPM experienced rats. In addition, the dPAG-NMDA-receptor blockade also reduced the latency to SD during the retrieval without interfering with the acquisition or the consolidation of the inhibitory avoidance. These results suggest that the NMDA-receptor dPAG activation could underlie the defensive response evoked in the EPM test and retest and also the retrieval of aversive memories involved in the SD. The results support the notion that the dPAG is a key structure in the modulation of the best behavioral strategy to cope with aversive contexts.

Pentylenetetrazole as an unconditioned stimulus for olfactory and contextual fear conditioning in rats.

The association of five footshocks with a neutral odor is able to establish an olfactory fear conditioning in rats. The present study sought to investigate whether the systemic administration of pentylenetetrazole (PTZ; 3.75-15 mg/kg) would turn the coffee odor in a conditioned stimulus in the fear conditioning paradigm. The results showed that rats started to display risk assessment and avoidance after PTZ (15 mg/kg)-coffee odor pairing. When three mild footshocks (0.4 mA for 2 s) were delivered during this pairing, the conditioned response exhibited was greater than before. In both cases, however, pretreatment with the benzodiazepine midazolam (MDZ. 0.5 mg/kg i.p.) fully counteracted the expression of these defensive behaviors. Moreover, after being paired with 15 mg/kg of PTZ alone or combined with footshocks, the coffee odor was able to promote a new fear conditioning related to the context where it was re-exposed. The present findings point out the usefulness of PTZ as an unconditioned stimulus to promote fear conditioning to olfactory and contextual cues in rats.

New perspectives on beta-adrenergic mediation of innate and learned fear responses to predator odor.

In the present study, we investigated the role of noradrenergic transmission in unconditioned and conditioned responses to predatory threats. First, we examined the effects of systemically injected beta-blockers on unconditioned and contextual conditioned response to cat odor. The centrally acting beta-blocker (propranolol) was able to impair unconditioned responses, as well as the acquisition of the contextual fear to cat odor; however, the peripherally acting (nadolol) was not effective. Next, we examined the neural substrate underlying the noradrenergic modulation of the defensive response to cat odor and focused on the dorsal premammillary nucleus (PMd), because it represents the hypothalamic site most responsive to predatory threats and, at the same time, presents a dense plexus of noradrenergic fibers. We were able to see that propranolol significantly reduced PMd-Fos expression in response to cat odor and that beta-adrenoceptor blockade in the PMd, before cat odor exposure, reduced defensive responses to the cat odor and to the cat odor-related environment. We have also shown that beta-adrenoceptor blockade in the PMd, before the exposure to cat odor-related context, impaired the contextual conditioned responses. Overall, the present results provide convincing evidence suggesting that central noradrenergic mediation is critical for the expression of unconditioned and contextual conditioned antipredatory responses. We have further shown that the PMd appears to be an important locus to mediate these beta-adrenoceptor effects.

Interplay between glutamate and serotonin within the dorsal periaqueductal gray modulates anxiety-related behavior of rats exposed to the elevated plus-maze.

Several neurotransmitters, including glutamate and serotonin, modulate defensive behaviors related to anxiety in the rat dorsal periaqueductal gray (PAG). Although both glutamate N-methyl-d-aspartic acid (NMDA) and serotonin type 1-A (5-HT(1A)) receptors have been shown to interfere with these subtle responses, such as inhibitory avoidance, a possible interaction between them remains to be examined. To address this issue, the present study investigated whether the activation or the blockage of 5-HT(1A) receptors located in the dorsal PAG would interact with NMDA function in animals exposed to the elevated plus-maze task. The effect of the NMDA (25 pmol) was evaluated in rats pretreated with the 5-HT(1A) receptor antagonist WAY-100135 (2.0 or 5.0 nmol). In addition, the effect of the NMDA (100 pmol) was evaluated in rats pretreated with the 5-HT(1A) receptor agonist 8-OH-DPAT (2.0 or 8.0 nmol). Intra-dorsal PAG injection of NMDA (25 pmol) increased inhibitory avoidance behavior. This anxiogenic-like effect of the NMDA was counteracted by the pretreatment with WAY-100135 (5.0 nmol). Although 100 pmol of NMDA failed to increase inhibitory avoidance in the vehicle-pretreated group, in rats pretreated with 8-OH-DPAT this NMDA dose produced an anxiogenic-like effect. These results suggest that 5-HT(1A) and NMDA receptors interact in the dorsal PAG to modulate the anxiety-related behavior.

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.