Lack of effects of clomipramine on Fos and NADPH-diaphorase double-staining in the periaqueductal gray after exposure to an innate fear stimulus

Lack of effects of clomipramine on Fos and NADPH-diaphorase double-staining in the periaqueductal gray after exposure to an innate fear stimulus - nitric oxide (NO) acts as a neurotransmitter in the rat dorsolateral periaqueductal gray (dIPAG), a midbrain structure that modulates fear and defensive behavior. Since defensive reactions can be alleviated by anxiolytic/anti-panic drugs, the present study tested the effect of clomipramine, a serotonin re-uptake inhibitor, on the activation of NO-producing neurons in the dlPAG of rats exposed to a live predator. Double staining was performed using Fos immunohistochemistry and NADPH-diaphorase as techniques to mark neural activation and to detect NO-producing neurons, respectively. Male Wistar rats received acute or chronic (21 days) injections of saline or clomipramine (10 or 20 mg/kg/day) and were exposed to a live cat. The animals exhibited a robust defensive reaction accompanied by an increase in the number of Fos- and doublestained neurons in the dlPAG, suggesting that cat exposure activates NO-producing neurons. Such effects were not significantly attenuated by clomipramine treatments. The intensity of fear reaction correlated with the intensity of neural staining in the dlPAG, regardless the drug treatment. Thus, the present results reinforce the hypothesis that NO may coordinate defensive responses in the dIPAG and indicate that this mechanism may not be modulated by a serotonin re-uptake inhibitor. (C) 2008 Elsevier Inc. All rights reserved.

Activation of CB1 cannabinoid receptors in the dorsolateral periaqueductal gray reduces the expression of contextual fear conditioning in rats

Rationale Conditioned fear to context causes freezing and cardiovascular changes in rodents and has been used to measure anxiety. It also activates the dorsolateral column of the periaqueductal gray (dlPAG). Microinjections of cannabinoid agonists into the dlPAG produced anxiolytic-like effects in the elevated plus maze, but the effects of these treatments on fear conditioning remains unknown. Objective The objective of this study was to verify if intra-dlPAG injection of the CB1 cannabinoid receptor agonist anandamide (AEA) or the anandamide transport inhibitor AM404 would attenuate behavioral (freezing) and cardiovascular (increase of arterial pressure and heart rate) responses of rats submitted to a contextual fear-conditioning paradigm. Materials and methods Male Wistar rats with cannulae aimed at the dlPAG were re-exposed to a chamber where they had received footshocks 48 h before. Fifteen minutes before the test, the animals received a first intra-dlPAG injection of vehicle or AM251, a CB1 receptor antagonist (100 pmol/200 nl), followed 5 min later by vehicle, AEA (5 pmol/200 nl) or AM404 (50 pmol/200 nl). Freezing and cardiovascular responses were recorded for 10 min. Results Freezing and cardiovascular responses were reduced by administration of either AEA or AM404 into the dlPAG before re-exposition to the aversively conditioned context. These effects were abolished when the animals were locally pretreated with AM251. The latter drug, even at a higher dose (300 pmol), was ineffective when administered alone into the dlPAG. Conclusion The results suggest that facilitation of endocannabinoid-mediated neurotransmission in the dlPAG, through activation of local CB1 receptors, attenuates the expression of contextual fear responses.

DIFFERENT ROLE OF THE VENTRAL MEDIAL PREFRONTAL CORTEX ON MODULATION OF INNATE AND ASSOCIATIVE LEARNED FEAR

Reversible inactivation of the ventral portion of medial prefrontal cortex (vMPFC) of the rat brain has been shown to induce anxiolytic-like effects in animal models based on associative learning. The role of this brain region in situations involving innate fear, however, is still poorly understood, with several contradictory results in the literature. The objective of the present work was to verify in male Wistar rats the effects of vMPFC administration of cobalt chloride (CoCl(2)), a selective inhibitor of synaptic activity, in rats submitted to two models based on innate fear, the elevated plus-maze (EPM) and light-dark box (LOB), comparing the results with those obtained in two models involving associative learning, the contextual fear conditioning (CFC) and Vogel conflict (VCT) tests. The results showed that, whereas CoCl(2) induced anxiolytic-like effects in the CFC and VCT tests, it enhanced anxiety in rats submitted to the EPM and LOB. Together these results indicate that the vMPFC plays an important but complex role in the modulation of defensive-related behaviors, which seems to depend on the nature of the anxiety/fear inducing stimuli. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Cannabinoid CB(1) receptors in the medial prefrontal cortex modulate the expression of contextual fear conditioning

The ventral portion of the medial prefrontal cortex (vMPFC) has been related to the expression of contextual fear conditioning. This study investigated the possible involvement of CB(1) receptors in this aversive response. Male Wistar rats were submitted to a contextual aversive conditioning session and 48 h later re-exposed to the aversive context in which freezing and cardiovascular responses (increase of arterial pressure and heart rate) were recorded. The expression of CB(1) receptor-mRNA in the vMPFC was also measured using real time-PCR. In the first experiment intra-vMPFC administration of the CB(1) receptor agonist anandamide (AEA, 5 pmol/200 nl) or the AEA transport inhibitor AM404 (50 pmol/200 nl) prior to re-exposure to the aversive context attenuated the fear-conditioned responses. These effects were prevented by local pretreatment with the CB(1) receptor antagonist AM251 (100 pmol/200 nl). Using the same conditioning protocol in another animal group, we observed that CB(1) receptor mRNA expression increased in the vMPFC 48 h after the conditioning session. Although AM251 did not cause any effect by itself in the first experiment, this drug facilitated freezing and cardiovascular responses when the conditioning session employed a lesser aversive condition. These results indicated that facilitation of cannabinoid-mediated neurotransmission in the vMPFC by local CB(1) receptor activation attenuates the expression of contextual fear responses. Together they suggest that local endocannabinoid-mediated neurotransmission in the vMPFC can modulate these responses.

Involvement of the lateral septal area in the expression of fear conditioning to context

Considering the evidence that the lateral septal area (LSA) modulates defensive responses, the aim of the present study is to verify if this structure is also involved in contextual fear conditioning responses. Neurotransmission in the LSA was reversibly inhibited by bilateral microinjections of cobalt chloride (CoCl(2), 1 mM) 10 min before or after conditioning or 10 min before re-exposure to the aversively conditioned chamber. Only those animals that received CoCl(2) before re-exposure showed a decrease in both cardiovascular and behavioral conditioned responses. These results suggest that the LSA participates in the expression, but not acquisition or consolidation, of contextual fear conditioning.

Involvement of the prelimbic prefrontal cortex on cannabidiol-induced attenuation of contextual conditioned fear in rats

Rationale: Systemic administration of cannabidiol (CBD), a non-psychotomimetic component of Cannabis sativa, is able to attenuate cardiovascular and behavioral (freezing) changes induced by re-exposure to a context that had been previously paired with footshocks. The brain sites mediating this effect, however, remain unknown. The medial prefrontal cortex (mPFC) has been related to contextual fear conditioning. Objectives: (1) To verify, using c-Fos immunocytochemistry, if the mPFC is involved in the attenuation of contextual fear induced by systemic administration of CBD; (2) to investigate if direct microinjections of CBD into mPFC regions would also attenuate contextual fear. Results: Confirming previous results systemic administration of CBD (10 mg/kg) decreased contextual fear and associated c-Fos expression in the prefrontal cortex (prelimbic and infralimbic regions). The drug also attenuated c-Fos expression in the bed nucleus of the stria terminalis (BNST). Direct CBD (30 nmol) microinjection into the PL prefrontal cortex reduced freezing induced by re-exposure to the aversively conditioned context. In the infralimbic (IL) prefrontal cortex, however, CBD (30 nmol) produced an opposite result, increasing the expression of contextual fear conditioning. This result was confirmed by an additional experiment where the conditioning session was performed under a less aversive protocol. Conclusion: These results suggest that the PL prefrontal cortex may be involved in the attenuation of contextual fear induced by systemic injection of CBD. They also support the proposition that the IL and PL play opposite roles in fear conditioning. A possible involvement of the BNST in CBD effects needs to be further investigated. (C) 2009 Elsevier B.V. All rights reserved.

GABA(A) receptor blockade in dorsomedial and ventromedial nuclei of the hypothalamus evokes panic-like elaborated defensive behaviour followed by innate fear-induced antinociception

Dysfunction in the hypothalamic GABAergic system has been implicated in panic syndrome in humans. Furthermore, several studies have implicated the hypothalamus in the elaboration of pain modulation. Panic-prone states are able to be experimentally induced in laboratory animals to study this phenomenon. The aim of the present work was to investigate the involvement of medial hypothalamic nuclei in the organization of panic-like behaviour and the innate fear-induced oscillations of nociceptive thresholds. The blockade of GABA(A) receptors in the neuronal substrates of the ventromedial. or dorsomedial hypothalamus was followed by elaborated defensive panic-like reactions. Moreover, innate fear-induced antinociception was consistently elicited after the escape behaviour. The escape responses organized by the dorsomedial and ventromedial hypothalamic nuclei were characteristically more elaborated, and a remarkable exploratory behaviour was recorded during GABA(A) receptor blockade in the medial hypothalamus. The motor characteristic of the elaborated defensive escape behaviour and the patterns of defensive alertness and defensive immobility induced by microinjection of the bicuculline either into the dorsomedial. or into the ventromedial hypothalamus were very similar. This was followed by the same pattern of innate fear-induced antinociceptive response that lasted approximately 40 min after the elaborated defensive escape reaction in both cases. These findings suggest that dysfunction of the GABA-mediated neuronal system in the medial hypothalamus causes panic-like responses in laboratory animals, and that the elaborated escape behaviour organized in both dorsomedial and ventromedial hypothalamic nuclei are followed by significant innate-fear-induced antinociception. Our findings indicate that the GABA(A) receptor of dorsomedial and ventromedial hypothalamic nuclei are critically involved in the modulation of panic-like behaviour. (C) 2009 Elsevier B.V. All rights reserved.

Escitalopram prolonged fear induced by simulated public speaking and released hypothalamic-pituitary-adrenal axis activation

Simulated public speaking (SPS) test is sensitive to drugs that interfere with serotonin-mediated neurotransmission and is supposed to recruit neural systems involved in panic disorder. The study was aimed at evaluating the effects of escitalopram, the most selective serotonin-selective reuptake inhibitor available, in SPS. Healthy males received, in a double-blind, randomized design, placebo (n = 12), 10 (n = 17) or 20 (n = 14) mg of escitalopram 2 hours before the test. Behavioural, autonomic and neuroendocrine measures were assessed. Both doses of escitalopram did not produce any effect before or during the speech but prolonged the fear induced by SPS. The test itself did not significantly change cortisol and prolactin levels but under the higher dose of escitalopram, cortisol and prolactin increased immediately after SPS. This fear-enhancing effect of escitalopram agrees with previously reported results with less selective serotonin reuptake inhibitors and the receptor antagonist ritanserin, indicating that serotonin inhibits the fear of speaking in public.

Outcome analysis of immediately placed, immediately restored implants in the esthetic area: The clinical relevance of different interimplant distances

Background: The purpose of this study was to compare and evaluate bone and soft tissue levels between immediately placed, immediately restored implants positioned in the esthetic anterior region with different interimplant distances (IID). Methods: Forty-nine patients requiring multiple implant restorations in the anterior regions received 152 implants, which were restored immediately. Periapical radiographs and digital images of 99 interimplant sites were taken at the regular follow-up examinations at 0, 6, 12, and 24 months after surgery. They were digitally recorded and analyzed. The presence of the interproximal papilla was assessed and compared to the distances between the bone crest and the contact point between the natural teeth and the restoration crowns. Results: Implants with an IID <2 mm seemed to lose less bone laterally. When the IID was <2 mm, vertical crestal bone loss was significantly greater than in the group with IID >4 mm. The percentage of the interproximal papilla presence decreased when the distance between the bone crest and the contact point between the two restoration crowns was >6 mm and when two implants were placed at a distance >= 4 mm. Conclusions: To guarantee a better esthetic result in immediately placed, immediately restored implants, the contact point between the two prosthetic crowns should be placed at 3 to 4 mm, and never >6 mm, from the bone peak. Two adjacent implants should be placed at a distance >2 and <4 mm.

Contextual, but not auditory, fear conditioning is disrupted by neurotoxic selective lesion of the basal nucleus of amygdala in rats

The basolateral amygdala complex (BLA) is involved in acquisition of contextual and auditory fear conditioning. However, the BLA is not a single structure but comprises a group of nuclei, including the lateral (LA), basal (BA) and accessory basal (AB) nuclei. While it is consensual that the LA is critical for auditory fear conditioning, there is controversy on the participation of the BA in fear conditioning. Hodological and neurophysiological findings suggest that each of these nuclei processes distinct information in parallel; the BA would deal with polymodal or contextual representations, and the LA would process unimodal or elemental representations. Thus, it seems plausible to hypothesize that the BA is required for contextual, but not auditory, fear conditioning. This hypothesis was evaluated in Wistar rats submitted to multiple-site ibotenate-induced damage restricted to the BA and then exposed to a concurrent contextual and auditory fear conditioning training followed by separated contextual and auditory conditioning testing. Differing from electrolytic lesion and lidocaine inactivation, this surgical approach does not disturb fibers of passage originating in other brain areas, restricting damage to the aimed nucleus. Relative to the sham-operated controls, rats with selective damage to the BA exhibited disruption of performance in the contextual, but not the auditory, component of the task. Thus, while the BA seems required for contextual fear conditioning, it is not critical for both an auditory-US association, nor for the expression of the freezing response. (C) 2009 Elsevier Inc. All rights reserved.

Stem anatomy of Rhipsalis (Cactaceae) and its relevance for taxonomy

Several species of the genus Rhipsalis (Cactaceae) are extremely important as ornamentals and are endangered in their natural habitat. However, only a few studies have addressed its taxonomy, morphology (including anatomy), phylogeny and evolutionary history. Consequently, the limited knowledge of the genus coupled with the problematic delimitation of species had led to problems in the identification of taxa. In the current work six species of Rhipsalis, R. cereoides, R. elliptica, R. grandiflora, R. paradoxa, R. pentaptera and R. teres were studied to evaluate the relevance of anatomical characters for the taxonomy of the genus. An anatomical characterization of the primary structure of the stem of Rhipsalis is provided highlighting the differences between species. Features of the stem epidermis are found to discriminate best between species and therefore provide clear and useful characters for the separation of species.

Sexual System, Sex Ratio, and Group Living in the Shrimp Thor amboinensis (De Man): Relevance to Resource-Monopolization and Sex-Allocation Theories

The sexual system of the symbiotic shrimp Thor amboinensis is described, along with observations on sex ratio and host-use pattern of different populations. We used a comprehensive approach to elucidate the previously unknown sexual system of this shrimp. Dissections, scanning electron microscopy, size-frequency distribution analysis, and laboratory observations demonstrated that T amboinensis is a protandric hermaphrodite: shrimp first mature as males and change into females later in life. Thor amboinensis inhabited the large and structurally heterogeneous sea anemone Stichoclactyla helianthus in large groups (up to 11 individuals) more frequently than expected by chance alone. Groups exhibited no particularly complex social structure and showed male-biased sex ratios more frequently than expected by chance alone. The adult sex ratio was male-biased in the four separate populations studied, one of them being thousands of kilometers apart from the others. This study supports predictions central to theories of resource monopolization and sex allocation. Dissections demonstrated that unusually large males were parasitized by an undescribed species of isopod (family Entoniscidae). Infestation rates were similarly low in both sexes (approximate to 11%-12%). The available information suggests that T. amboinensis uses pure search promiscuity as a mating system. This hypothesis needs to be formally tested with mating behavior observations and field measurements on the movement pattern of both sexes of the species. Further detailed studies on the lifestyle and sexual system of all the species within this genus and the development of a molecular phylogeny are necessary to elucidate the evolutionary history of gender expression in the genus Thor.

Acquisition of Pavlovian Fear Conditioning Using beta-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; beta-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 beta-1 receptors in the PMd. Furthermore, similar to what had been found for contextual conditioning to a predator threat, atenolol (beta-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 beta-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. Neuropsychopharmacology (2011) 36, 926-939; doi:10.1038/npp.2010.231; published online 5 January 2011

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.

Dissecting the brain`s fear system reveals the hypothalamus is critical for responding in subordinate conspecific intruders

Effective defense against natural threats in the environment is essential for the survival of individual animals. Thus, instinctive behavioral responses accompanied by fear have evolved to protect individuals from predators and from opponents of the same species (dominant conspecifics). While it has been suggested that all perceived environmental threats trigger the same set of innately determined defensive responses, we tested the alternate hypothesis that different stimuli may evoke differentiable behaviors supported by distinct neural circuitry. The results of behavioral, neuronal immediate early gene activation, lesion, and neuroanatomical experiments indicate that the hypothalamus is necessary for full expression of defensive behavioral responses in a subordinate conspecific, that lesions of the dorsal premammillary nucleus drastically reduce behavioral measures of fear in these animals, and that essentially separate hypothalamic circuitry supports defensive responses to a predator or a dominant conspecific. It is now clear that differentiable neural circuitry underlies defensive responses to fear conditioning associated with painful stimuli, predators, and dominant conspecifics and that the hypothalamus is an essential component of the circuitry for the latter two stimuli.