Sex-dependent pronociceptive role of spinal α5 -GABAA receptor and its epigenetic regulation in neuropathic rodents.

Extrasynaptic α5 -subunit containing GABAA (α5 -GABAA ) receptors participate in chronic pain. Previously, we reported a sex difference in the action of α5 -GABAA receptors in dysfunctional pain. However, the underlying mechanisms remain unknown. The aim of this study was to examine this sexual dimorphism in neuropathic rodents and the mechanisms involved. Female and male Wistar rats or ICR mice were subjected to nerve injury followed by α5 -GABAA receptor inverse agonist intrathecal administration, L-655,708. The drug produced an antiallodynic effect in nerve-injured female rats and mice, and a lower effect in males. We hypothesized that changes in α5 -GABAA receptor, probably influenced by hormonal and epigenetic status, might underlie this sex difference. Thus, we performed qPCR and western blot. Nerve injury increased α5 -GABAA mRNA and protein in female dorsal root ganglia (DRG) and decreased them in DRG and spinal cord of males. To investigate the hormonal influence over α5 -GABAA receptor actions, we performed nerve injury to ovariectomized rats and reconstituted them with 17ß-estradiol (E2). Ovariectomy abrogated L-655,708 antiallodynic effect and E2 restored it. Ovariectomy decreased α5 -GABAA receptor and estrogen receptor α protein in DRG of neuropathic female rats, while E2 enhanced them. Since DNA methylation might contribute to α5 -GABAA receptor down-regulation in males, we examined CpG island DNA methylation of α5 -GABAA receptor coding gene through pyrosequencing. Nerve injury increased methylation in male, but not female rats. Pharmacological inhibition of DNA methyltransferases increased α5 -GABAA receptor and enabled L-655,708 antinociceptive effect in male rats. These results suggest that α5 -GABAA receptor is a suitable target to treat chronic pain in females.

Involvement of the median preoptic nucleus in blood pressure control.

Studies have demonstrated that median preoptic nucleus (MnPO) neurons play a role in organizing the cardiovascular responses induced by changes in the circulating blood volume. The present study examined whether the MnPO controls cardiovascular function. Male Wistar normotensive (NT) rats and spontaneously hypertensive rats (SHRs; 250-300 g) were anesthetized with urethane (1.2 g kg(-1), i.v.) and instrumented for recordings of mean arterial blood pressure (MAP) and renal blood flow (RBF). The renal vascular conductance (RVC) was calculated as the RBF:MAP ratio and was expressed as a percentage of the baseline value. In the NT rats (n=6), MnPO inhibition produced a MAP reduction (-8.1±1.1 mmHg, p<0.05). In the SHRs (n=6), the MAP response to MnPO inhibition was significantly greater (-22.3±4 mmHg, p<0.05) than in the NT rats. Furthermore, the increase in the RVC was higher in the SHRs (10.9±3.3%, p<0.05). Histological analyses confirmed that the injection sites were confined to the MnPO. We conclude that the MnPO is involved in the tonic regulation of blood pressure in NT rats. Moreover, the greater cardiovascular response to MnPO inhibition observed in the SHRs strongly suggests that the MnPO may contribute to the pathophysiology of essential hypertension.

Differential behavioral profile induced by the injection of dipotassium chlorazepate within brain areas that project to the nucleus accumbens septi.

BACKGROUND: The effect of the agonism on γ-aminobutyric acid (GABA) receptors was studied within medial prefrontal cortex (mPFC), amygdala (AMY) and ventral hipocampus (VH) in the plus-maze test in male rats bilaterally cannulated. These structures send glutamatergic projections to the nucleus accumbens septi (NAS), in which interaction and integration between these afferent pathways has been described. In a previous study of our group, blockade of glutamatergic transmission within NAS induced an anxiolytic like effect. METHODS: Three rat groups received either saline or dipotassium chlorazepate (1 or 2 µg/1 µl solution) 15 min before testing. Time spent in the open arms (TSOA), time per entry (TPE), extreme arrivals (EA), open and closed arms entries (OAE, CAE) and relationship between open- and closed-arms quotient (OCAQ) were recorded. RESULTS: In the AMY injected group TSOA, OAE and EA were increased by the higher doses of dipotassium chlorazepate (p < 0.01). In the mPFC, TPE was decreased by both doses (p < 0.05). Injection within ventral hippocampus (VH) decreased TSOA, OAE and OCAQ with lower doses (p < 0.05). When the three studied saline groups were compared, TSOA, OAE, EA and OCAQ were enhanced in the VH group when compared to mPFC and AMY (p < 0.001). Insertion of inner canula (p < 0.001, p < 0.01, p < 0.01) and saline injection showed an increasing significant difference (p < 0.001 in all cases) with the action of guide cannula alone within VH in TSOA, OAE and EA. CONCLUSION: We conclude that the injection of dipotassium chlorazepate has a differential effect depending of the brain area, leading to facilitatory and inhibitory effects on anxiety processing.

Baclofen into the lateral parabrachial nucleus induces hypertonic sodium chloride intake during cell dehydration.

BACKGROUND: Activation of GABA(B) receptors with baclofen into the lateral parabrachial nucleus (LPBN) induces ingestion of water and 0.3 M NaCl in fluid replete rats. However, up to now, no study has investigated the effects of baclofen injected alone or combined with GABA(B) receptor antagonist into the LPBN on water and 0.3 M NaCl intake in rats with increased plasma osmolarity (rats treated with an intragastric load of 2 M NaCl). Male Wistar rats with stainless steel cannulas implanted bilaterally into the LPBN were used. RESULTS: In fluid replete rats, baclofen (0.5 nmol/0.2 µl), bilaterally injected into the LPBN, induced ingestion of 0.3 M NaCl (14.3 ± 4.1 vs. saline: 0.2 ± 0.2 ml/210 min) and water (7.1 ± 2.9 vs. saline: 0.6 ± 0.5 ml/210 min). In cell-dehydrated rats, bilateral injections of baclofen (0.5 and 1.0 nmol/0.2 µl) into the LPBN induced an increase of 0.3 M NaCl intake (15.6 ± 5.7 and 21.5 ± 3.5 ml/210 min, respectively, vs. saline: 1.7 ± 0.8 ml/210 min) and an early inhibition of water intake (3.5 ± 1.4 and 6.7 ± 2.1 ml/150 min, respectively, vs. saline: 9.2 ± 1.4 ml/150 min). The pretreatment of the LPBN with 2-hydroxysaclofen (GABA(B) antagonist, 5 nmol/0.2 µl) potentiated the effect of baclofen on 0.3 M NaCl intake in the first 90 min of test and did not modify the inhibition of water intake induced by baclofen in cell-dehydrated rats. Baclofen injected into the LPBN did not affect blood pressure and heart rate. CONCLUSIONS: Thus, injection of baclofen into the LPBN in cell-dehydrated rats induced ingestion of 0.3 M NaCl and inhibition of water intake, suggesting that even in a hyperosmotic situation, the blockade of LPBN inhibitory mechanisms with baclofen is enough to drive rats to drink hypertonic NaCl, an effect independent of changes in blood pressure.

Endocannabinoid/GABA interactions in the entopeduncular nucleus modulates alcohol intake in rats.

Alcohol use disorder is a compulsive behavior driven by motivational systems and by a poor control of consummatory behavior. The entopeduncular nucleus (EP) seems to be involved in the regulation of executive mechanisms, hence, in the expression of behavior. Endocannabinoids (eCB) are involved in alcohol intake mechanisms. The eCB receptor name cannabinoid receptor 1 (CB1R) is expressed in the EP in GABAergic terminals. The role of the eCB system (eCBs) of the EP in the modulation of alcohol seeking and intake behavior is unknown. Therefore, we decided to investigate the role of the eCBs and its interaction with GABA transmission in rat EP, in the regulation of alcohol intake behavior. Rats were submitted to a 10-day period of moderate alcohol (10% in tap water) ingestion. No tap water was available. On day 11, either anandamide (AEA, CB1 receptor agonist), AM251 (CB1R inverse agonist), baclofen (BAC, GABAB receptor agonist), or CGP35348 (GABAB receptor antagonist) was administered into the EP. One bottle of water and one of alcohol (10% in water) were available ad libitum for the following 24 h, and consumption was quantified at the end of this period. Results show that administration of AEA into the EP decreased alcohol consumption while AM251 and BAC administered independently increased alcohol consumption. AEA prevented the increase induced by AM251 or BAC. Likewise, CGP35348 prevented alcohol ingestion induced by AM251. These data suggest that eCBs dysfunction in the EP may be playing a crucial role in the abuse and dependence of alcohol and other drugs.

Enhancing a declarative memory in humans: the effect of clonazepam on reconsolidation.

A consolidated memory recalled by a specific reminder can become unstable (labile) and susceptible to facilitation or impairment for a discrete period of time. This labilization phase is followed by a process of stabilization called reconsolidation. The phenomenon has been shown in diverse types of memory, and different pharmacological agents have been used to disclose its presence. Several studies have revealed the relevance of the GABAergic system to this process. Consequently, our hypothesis is that the system is involved in the reconsolidation of declarative memory in humans. Thus, using our verbal learning task, we analyzed the effect of benzodiazepines on the re-stabilization of the declarative memory. On Day 1, volunteers learned an association between five cue- response-syllables. On Day 2, the verbal memory was labilized by a reminder presentation, and then a placebo capsule or 0.25 mg or 0.03 mg of clonazepam was administered to the subjects. The verbal memory was evaluated on Day 3. The volunteers who had received the 0.25 mg clonazepam along with the specific reminder on Day 2, exhibited memory improvement. In contrast, there was no effect when the drug was given without retrieval, when the memory was simply retrieved instead of being reactivated or when short-term memory testing was performed 4 h after reactivation. We discuss the GABAergic role in reconsolidation, which shows a collateral effect on other memories when the treatment is aimed at treating anxiety disorders. Further studies might elucidate the role of GABA in the reconsolidation process associated with dissimilar scenarios. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Differential roles of the dorsal hippocampal regions in the acquisition of spatial and temporal aspects of episodic-like memory.

Episodic memory refers to the recollection of what, where and when an event occurred. Computational models suggest that the dentate gyrus (DG) and the CA3 hippocampal subregions are involved in pattern separation and the rapid acquisition of episodes, while CA1 is involved in the formation of a temporal context. Most of the studies performed to test this hypothesis failed to simultaneously address the aspects of episodic memory. Recently, a new task of object recognition was validated in rats. In the first sample trial, the rat is exposed to four copies of an object. In second sample, the rat is exposed to four copies of a different object. In the test trial, two copies of each of the previous objects are presented. One copy of the object used in sample trial one is located in a different place, and it is expected to be the most explored. Our goal was to evaluate whether the pharmacological inactivation of the dorsal DG/CA3 and CA1 subregions could differentially impair the acquisition of the task. Inactivation of the DG/CA3 subregions impaired the spatial discrimination, while the temporal discrimination was preserved. Rats treated with muscimol in CA1 explored all the objects equally well, irrespective of place or presentation time. Our results are consistent with computational models that postulate a role for DG/CA3 in rapid encoding and in spatial pattern separation, and a role for CA1 in the in the formation of the temporal context of events and as well as in detecting spatial novelty.

GABAergic processes within the median preoptic nucleus promote NREM sleep.

GABAergic mechanisms in the preoptic region of the hypothalamus (POA) have been implicated in the generation and maintenance of NREM (quiet) sleep. We recently reported that neurons in the median peptic nucleus (MnPN) in the POA of the cat are selectively activated during NREM sleep. In the present study, we explored the hypothesis that NREM sleep is controlled by GABAergic mechanisms within the MnPN. Consequently, adult cats were utilized to determine GABA immunorreactivity within the MnPN and to examine the effects on sleep of the microinjection of a GABA(A) agonist (muscimol) and a GABA(A) antagonist (bicuculline) into this area. GABAergic neurons were present throughout the MnPN. Compared with control microinjections, after the application of muscimol, the time spent in NREM sleep (59.8±7.5 min) and REM sleep (6.9±4.7 min) decreased compared with control microinjections (103.8±5.2 and 20.2±4.3 min, respectively; P<0.005). In contrast, bicuculline microinjections increased only NREM sleep time (103.0±23.0 vs 77.7±23.7 min; P<0.05). These results demonstrate that GABAergic processes within the MnPN are involved in the generation and maintenance of sleep, especially NREM sleep.

GABA in the central amygdaloid nucleus modulates the electrolyte excretion and hormonal responses to blood volume expansion in rats.

We investigated the involvement of GABAergic mechanisms of the central amygdaloid nucleus (CeA) in unanesthetized rats subjected to acute isotonic or hypertonic blood volume expansion (BVE). Male Wistar rats bearing cannulas unilaterally implanted in the CeA were treated with vehicle, muscimol (0.2 nmol/0.2 microL) or bicuculline (1.6 nmol/0.2 microL) in the CeA, followed by isotonic or hypertonic BVE (0.15 or 0.3 M NaCl, 2 mL/100 g body weight over 1 min). The vehicle-treated group showed an increase in sodium excretion, urinary volume, plasma oxytocin (OT), and atrial natriuretic peptide (ANP) levels compared to control rats. Muscimol reduced the effects of BVE on sodium excretion (isotonic: 2.4 +/- 0.3 vs vehicle: 4.8 +/- 0.2 and hypertonic: 4.0 +/- 0.7 vs vehicle: 8.7 +/- 0.6 microEq.100 g-1.40 min-1); urinary volume after hypertonic BVE (83.8 +/- 10 vs vehicle: 255.6 +/- 16.5 microL.100 g-1.40 min-1); plasma OT levels (isotonic: 15.3 +/- 0.6 vs vehicle: 19.3 +/- 1 and hypertonic: 26.5 +/- 2.6 vs vehicle: 48 +/- 3 pg/mL), and ANP levels (isotonic: 97 +/- 12.8 vs vehicle: 258.3 +/- 28.1 and hypertonic: 160 +/- 14.6 vs vehicle: 318 +/- 16.3 pg/mL). Bicuculline reduced the effects of isotonic or hypertonic BVE on urinary volume and ANP levels compared to vehicle-treated rats. However, bicuculline enhanced the effects of hypertonic BVE on plasma OT levels. These data suggest that CeA GABAergic mechanisms are involved in the control of ANP and OT secretion, as well as in sodium and water excretion in response to isotonic or hypertonic blood volume expansion.

GABA and opioid mechanisms of the central amygdala underlie the withdrawal-potentiated startle from acute morphine.

Anxiety is an affective symptom common to withdrawal from acute or chronic opiate treatment. Although the potentiation of the acoustic startle reflex has been proposed as an index of increased anxiety, there are variable effects of the opiate withdrawal on the startle reflex in chronic dependence models. On the other hand, withdrawal from acute morphine treatment consistently potentiates the acoustic startle reflex, a response that seems to be mediated by the central nucleus of the amygdala (CeA). However, the underlying neurochemical mechanisms have not been elucidated yet. In the present study, we firstly made a comparison between the effects of the withdrawal from both acute and chronic treatments with morphine on the motor activity and the anxiety-like behavior of rats tested in two experimental models, the acoustic startle reflex and the open-field tests. Our second objective was to investigate the role of GABAergic and opioid mechanisms of the CeA in the modulation of the withdrawal-potentiated startle as a measure of anxiety induced by morphine withdrawal. For the production of chronic dependence, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of this treatment, independent groups were probed in the startle reflex and open-field tests. For the acute dependence model, groups of rats were tested in the open field and startle tests under control conditions and under withdrawal from a single injection of morphine (10 mg/kg; s.c.) precipitated by naltrexone injections (0.1 mg/kg; s.c.). The results obtained showed that withdrawal from chronic and acute morphine treatments produced anxiety-like behavior in the open field test, although the anxiogenic-like effects could not be dissociated from the motor effects in the acute dependence model. On the other hand, only the withdrawal from acute morphine treatment significantly potentiated the startle response. Next, we examined the effects of intra-CeA microinjections of muscimol-a GABA(A) receptors agonist-and DAMGO-a mu-opioid receptors agonist-on the potentiated startle induced by acute morphine withdrawal. The results obtained showed that intra-CeA injections of muscimol (1 nmol) and DAMGO (0.5 and 1 nmol) significantly inhibited this response. These findings suggest that the acute dependence model is more suitable to study the aversive effects of morphine withdrawal on the acoustic startle response than the chronic opiate dependence model. Besides, mechanisms mediated by mu- and GABA(A)-receptors in the CeA appear to exert an inhibitory influence on the anxiety-like behavior induced by withdrawal from acute morphine treatment.

GABA in the central amygdaloid nucleus modulates the electrolyte excretion and hormonal responses to blood volume expansion in rats

We investigated the involvement of GABAergic mechanisms of the central amygdaloid nucleus (CeA) in unanesthetized rats subjected to acute isotonic or hypertonic blood volume expansion (BVE). Male Wistar rats bearing cannulas unilaterally implanted in the CeA were treated with vehicle, muscimol (0.2 nmol/0.2 µL) or bicuculline (1.6 nmol/0.2 µL) in the CeA, followed by isotonic or hypertonic BVE (0.15 or 0.3 M NaCl, 2 mL/100 g body weight over 1 min). The vehicle-treated group showed an increase in sodium excretion, urinary volume, plasma oxytocin (OT), and atrial natriuretic peptide (ANP) levels compared to control rats. Muscimol reduced the effects of BVE on sodium excretion (isotonic: 2.4 ± 0.3 vs vehicle: 4.8 ± 0.2 and hypertonic: 4.0 ± 0.7 vs vehicle: 8.7 ± 0.6 µEq·100 g-1·40 min-1); urinary volume after hypertonic BVE (83.8 ± 10 vs vehicle: 255.6 ± 16.5 µL·100 g-1·40 min-1); plasma OT levels (isotonic: 15.3 ± 0.6 vs vehicle: 19.3 ± 1 and hypertonic: 26.5 ± 2.6 vs vehicle: 48 ± 3 pg/mL), and ANP levels (isotonic: 97 ± 12.8 vs vehicle: 258.3 ± 28.1 and hypertonic: 160 ± 14.6 vs vehicle: 318 ± 16.3 pg/mL). Bicuculline reduced the effects of isotonic or hypertonic BVE on urinary volume and ANP levels compared to vehicle-treated rats. However, bicuculline enhanced the effects of hypertonic BVE on plasma OT levels. These data suggest that CeA GABAergic mechanisms are involved in the control of ANP and OT secretion, as well as in sodium and water excretion in response to isotonic or hypertonic blood volume expansion.

Sodium intake by hyperosmotic rats treated with a GABA(A) receptor agonist into the lateral parabrachial nucleus.

Inhibitory mechanisms in the lateral parabrachial nucleus (LPBN) and central GABAergic mechanisms are involved in the regulation of water and NaCl intake. Besides increasing fluid depletion-induced sodium intake, the activation of GABA(A) receptors with muscimol into the LPBN also induces ingestion of 0.3 M NaCl in normonatremic, euhydrated rats. It has been suggested that inhibitory mechanisms activated by osmotic signals are blocked by GABA(A) receptor activation in the LPBN, thereby increasing hypertonic NaCl intake. Therefore, in the present study we investigated the effects of muscimol injected into the LPBN on water and 0.3 M NaCl intake in hyperosmotic cell-dehydrated rats (rats treated with an intragastric load of 2 M NaCl). Male Wistar rats with stainless steel cannulas implanted bilaterally into the LPBN were used. In euhydrated rats, muscimol (0.5 nmol/0.2 microl), bilaterally injected into the LPBN, induced ingestion of 0.3 M NaCl (24.6+/-7.9 vs. vehicle: 0.5+/-0.3 ml/180 min) and water (6.3+/-2.1 vs. vehicle: 0.5+/-0.3 ml/180 min). One hour after intragastric 2 M NaCl load (2 ml), bilateral injections of muscimol into the LPBN also induced 0.3 M NaCl intake (22.1+/-5.2 vs. vehicle: 0.9+/-0.8 ml/210 min) and water intake (16.5+/-3.6 vs. vehicle: 7.8+/-1.8 ml/210 min). The GABA(A) antagonist bicuculline (0.4 nmol/0.2 microl) into the LPBN reduced the effect of muscimol on 0.3 M NaCl intake (7.1+/-2.1 ml/210 min). Therefore, the activation of GABA(A) receptors in the LPBN induces ingestion of 0.3 M NaCl by hyperosmotic cell-dehydrated rats, suggesting that plasma levels of renin or osmolarity do not affect sodium intake after the blockade of LPBN inhibitory mechanisms with muscimol.

GABAA and GABAB agonist microinjections into medial accumbens shell increase feeding and induce anxiolysis in an animal model of anxiety.

This study investigated the effect of GABAA (muscimol, MUSC) and GABAB (baclofen, BACL) agonist receptors microinjected into medial accumbens shell on feeding and the level of fear in free-feeding rats submitted to the elevated plus-maze (EPM), an animal model of anxiety. Bilateral microinjections of either MUSC (128 pmol/0.2 microl/side) or BACL (128 and 256 pmol/0.2 microl/side) induced an anxiolytic-like effect since they decreased the occurrence of risk assessment, a defensive behaviour positively correlated with the animal anxiety level. Bilateral BACL microinjection (128 pmol), but not MUSC, also increased the head-dipping frequency over the open arms of the EPM, another representative behaviour of anxiety, but negatively correlated with it. In addition to anxiolysis, the present study also showed that the microinjection of MUSC and BACL agonists into rostral sites of the medial Acb shell (AP, +1.2 to +1.6) increased food intake significantly whereas drinking behaviour kept unchanged. Both doses of MUSC and BACL also decreased feeding latency. BACL but not MUSC dose-dependently increased food length. The data indicated a putative role of GABA receptors (especially GABAB) in the medial Acb shell for anxiety modulation in rats.

Involvement of GABA A receptor-associated chloride channels in the peripheral antinociceptive effect induced by GABA A receptor agonist muscimol.

The effect of chloride and potassium channel blockers on the antinociception induced by GABA(A) receptor agonist muscimol was investigated using the paw pressure test. Muscimol (1, 2, 4, 8 ng/paw) elicited a peripheral antinociceptive effect that was antagonized by bicuculline (10, 20, 40, 80 ng/paw), suggesting a specific effect. The muscimol effect was reverted by the chloride channel coupled GABA(A) receptor blocker, picrotoxin (0.4, 0.6, 0.8, 2 microg/paw). Potassium channel blockers did not modify the peripheral antinociception induced by muscimol. This study provides evidence that the peripheral antinociceptive effect of muscimol results from the activation of GABA(A) receptor-associated chloride channels.

Cardiovascular effects produced by activation of GABA receptors in the rostral ventrolateral medulla of conscious rats.

The rostral ventrolateral medulla (RVLM) has been proposed as a region playing a major role in the tonic and reflex control of sympathetic vasomotor activity and blood pressure. Pharmacological activation of GABA(A) receptors with muscimol in the RVLM of anesthetized rats results in a large fall in mean arterial pressure (MAP), heart rate (HR) and sympathetic activity. In this study we evaluated the effects of activation of GABA receptors in the RVLM of conscious, freely moving rats. Bilateral microinjections of muscimol into the RVLM of conscious rats produced a large fall in MAP (-38+/-4 mm Hg, n=7) when compared with saline injections (NaCl 0.9%, 7+/-1 mm Hg, n=4). The decrease in MAP evoked by muscimol was accompanied by a significant increase in HR (muscimol 69+/-13 bpm vs. vehicle -33+/-12 bpm, P<0.05), an effect that was completely abolished by beta1 adrenergic receptor blockade. Conversely, bilateral microinjections of GABA(B) agonist, baclofen, evoked a pressor response, but in this case, the increase was not significantly different from that evoked by vehicle injections. These results 1) indicate that GABA(A) receptors have a powerful influence on the resting activity of RVLM neurons in conscious rats; 2) indicate that a compensatory sympathetic-mediated tachycardia is present after inhibition of RVLM neurons in conscious rats; 3) confirm and extend previous findings showing that RVLM neurons are critical for blood pressure maintenance even in normal non-anesthetized conditions.

Effects of medial amygdala inactivation on a panic-related behavior.

In the last years, the role played by the medial nucleus of the amygdala (MeA) in the modulation of fear- and anxiety-related behaviors has been increasingly investigated. This nucleus plays an important role in the processing of predator odor-induced defensive reactions, i.e. freezing and risk-assessment behaviors. Immunohistochemical evidence also indicates that the MeA may be involved in the regulation of escape, a defensive behavior related to panic attacks. In this study, we further addressed this question by investigating the effects of the reversible inactivation of the nucleus on escape behavior generated in male Wistar rats by two different aversive stimuli, electrical stimulation of the dorsal periaqueductal gray matter (dPAG) and exposure to one of the open arms of the elevated T-maze. Results showed that intra-MeA administration of either the reversible sodium channel blocker lidocaine (34 nmol/0.2 microl) or the GABA(A) receptor agonist muscimol (0.22 nmol/0.2 microl) raised the threshold of aversive electrical stimulation, increasing the amount of current that applied to the dPAG evokes escape, an antiaversive effect. Local microinjection of muscimol (0.22 nmol/0.2 microl) inhibited escape behavior in the elevated T-maze, also suggesting an antiaversive effect. In this latter test, muscimol did not affect inhibitory avoidance, a behavior associated with generalized anxiety disorder. Muscimol effect in the elevated T-maze was independent of changes in general exploratory activity as measured in an open-field. Taken together, our data corroborate previous evidences suggesting that the MeA is involved in the modulation of escape. Dysfunction of this regulatory mechanism may be of relevance in the genesis/maintenance of panic disorder.

Role of CGRP and GABA in the hypotensive effect of intrathecally administered anandamide to anesthetized rats.

In urethane-anesthetized rats the intrathecal (i.t.) injection of 100 nmol anandamide produced a hypotensive effect (-19.3+/-1.6 mm Hg; n=6) that was mimicked by i.t. administration of 0.25 nmol calcitonin gene-related peptide (CGRP; -26.2+/-1.8 mm Hg, n=4). Both effects were antagonized either by the CGRP receptor antagonist CGRP(8-37) (5 nmol; i.t.) or by the gamma-aminobutyric acid (GABA)(A) receptor antagonist bicuculline (8.8 nmol, i.t) or by the GABA(B) receptor antagonist 2-hydroxy saclofen (110 nmol; i.t.). On the contrary, blockade of spinal CGRP receptors by CGRP(8-37) did not modify the hypotensive response to either the GABA(A)-receptor agonist muscimol (8.8 nmol; i.t.) or the GABA(B)-receptor agonist baclofen (100 nmol; i.t). This result suggests a unidirectional effect of CGRP on the GABAergic system. The response to anandamide remained unaltered after acute inhibition of nitric oxide (NO) synthase activity by either i.t. (1 micromol) or i.v. (10 mg/kg) injection of N(G)-nitro-L-arginine methyl ester (L-NAME), but increased significantly after long-term L-NAME administration (70 mg/kg/day; four weeks; p.o.), thus suggesting compensatory changes in cardiovascular homeostasis. It is proposed that the hypotensive effect of anandamide in urethane-anesthetized rats could involve the release of CGRP followed by the release of GABA in the spinal cord. NO does not appear to have a direct participation in the spinal mechanisms involved in the decrease of the blood pressure caused by anandamide.

GABA receptor agonists in the medial preoptic area and maternal behavior in lactating rats.

We studied the effects of injecting agonists of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) muscimol (GABA-A receptor agonist) and baclofen (GABA-B receptor agonist) in the medial preoptic area (MPOA) and neighboring brain regions, the bed nucleus of the stria terminalis (BNST), and lateral preoptic area (LPO) on maternal behavior. Lactating female rats were implanted with bilateral cannulae in the MPOA/BNST on day 1 postpartum. On day 5, a maternal behavior test was conducted in the home cage after females received injections of muscimol or baclofen (0, 12.5, 50 or 200 ng per side). On day 7, after MPOA/BNST injections, a second maternal behavior test was conducted with pups placed at the end of a T-runway projecting from the home cage. Finally, after injections on day 9 maternal aggression, olfaction, and locomotor behavior were tested. The GABA receptor agonists injected in the MPOA/BNST produced dose-dependent deficits in all components of maternal behavior, including maternal aggression, except licking. Muscimol produced deficits in the active component, nest building at lower doses than baclofen, both agonists produced deficits in retrieving, while baclofen produced deficits in passive components (hovering and crouching over pups) at lower doses than muscimol. Both GABA receptor agonists increased locomotor activity and reduced olfactory responsiveness but these were only correlated with deficits in retrieving and crouching in baclofen-treated females.

Conditioned and unconditioned fear organized in the periaqueductal gray are differentially sensitive to injections of muscimol into amygdaloid nuclei.

The lateral and basolateral nuclei of the amygdala (LaA and BLA, respectively) serve as a filter for unconditioned and conditioned aversive information that ascends to higher structures from the brainstem, whereas the central nucleus of the amygdala (CeA) is considered to be the main output for the defense reaction. It has been shown that the dorsal periaqueductal gray (dPAG) is activated by threatening stimuli and has important functional links with the amygdala through two-way anatomical connections. In this work, we examined the influence of chemical inactivation of these nuclei of amygdala on the freezing and escape responses induced by electrical stimulation through electrodes implanted in the dPAG of Wistar rats. Each rat also bore a cannula implanted in the LaA, BLA or CeA for injections of muscimol (0.5 microg/0.5 microL) or its vehicle. The duration of freezing behavior that outlasts electrical stimulation of the dPAG was also measured. On the following day, these animals were submitted to a contextual fear-conditioning using foot shocks as unconditioned stimulus. Conditioned freezing to contextual cues previously associated with foot shocks was also inhibited by injections of muscimol into these amygdaloid nuclei. The contextual conditioned freezing behavior is generated in the neural circuits of conditioned fear in the amygdala. The data obtained also show that injections of muscimol into the three amygdaloid nuclei did not change the aversive threshold of freezing, but disrupted the dPAG post-stimulation freezing. Previous findings that the latter freezing results directly from dPAG stimulation and that it is not sensitive to a context shift suggest that it is unconditioned in nature. Thus, the amygdala can affect some, but not all, aspects of unconditioned freezing. Post-stimulation freezing may reflect the process of transferring aversive information from dPAG to higher brain structures.

The inactivation of the basolateral nucleus of the rat amygdala has an anxiolytic effect in the elevated T-maze and light/dark transition tests.

Pharmacological evidence indicates that the basolateral nucleus of the amygdala (BLA) is involved in the mediation of inhibitory avoidance but not of escape behavior in the elevated T-maze test. These defensive responses have been associated with generalized anxiety disorder (GAD) and panic disorder, respectively. In the present study, we determined whether the BLA plays a differential role in the control of inhibitory avoidance and escape responses in the elevated T-maze. Male Wistar rats (250-280 g, N = 9-10 in each treatment group) were pre-exposed to one of the open arms of the maze for 30 min and 24 h later tested in the model after inactivation of the BLA by a local injection of the GABA A receptor agonist muscimol (8 nmol in 0.2 microL). It has been shown that a prior forced exposure to one of the open arms of the maze, by shortening latencies to withdrawal from the open arm during the test, improves the escape task as a behavioral index of panic. The effects of muscimol in the elevated T-maze were compared to those caused by this GABA agonist in the avoidance reaction generated in the light/dark transition test. This defensive behavior has also been associated with GAD. In the elevated T-maze, intra-BLA injection of muscimol impaired inhibitory avoidance (control: 187.70 +/- 14.90 s, muscimol: 37.10 +/- 2.63 s), indicating an anxiolytic effect, without interfering with escape performance. The drug also showed an anxiolytic effect in the light/dark transition test as indicated by the increase in the time spent in the lighted compartment (control: 23.50 +/- 2.45 s, muscimol: 47.30 +/- 4.48 s). The present findings point to involvement of the BLA in the modulation of defensive responses that have been associated with GAD.