Galanin subtype 1 and subtype 2 receptors mediate opposite anxiety-like effects in the rat dorsal raphe nucleus.

About 40% of the dorsal raphe nucleus (DRN) neurons co-express serotonin (5-HT) and galanin. Serotonergic pathways from the DRN to the amygdala facilitate learned anxiety, while those from the DRN to the dorsal periaqueductal grey matter (DPAG) impair innate anxiety. Previously, we showed that galanin infusion in the DRN of rats induces anxiolytic effect by impairing inhibitory avoidance without changing escape behaviour in the elevated T-maze (ETM). Here, we evaluated: (1) which galanin receptors would be involved in the anxiolytic effect of galanin in the DRN of rats tested in the ETM; (2) the effects of galanin intra-DRN on panic-like behaviours evoked by electrical stimulation of the DPAG. The activation of DRN GAL1 receptors by M617 (1.0 and 3.0nmol) facilitated inhibitory avoidance, whereas the activation of GAL2 receptors by AR-M1896 (3.0nmol) impaired the inhibitory avoidance in the ETM, suggesting an anxiogenic and an anxiolytic-like effect respectively. Both agonists did not change escape behaviour in the ETM or locomotor activity in the open field. The anxiolytic effect of AR-M1896 was attenuated by the prior administration of WAY100635 (0.18nmol), a 5-HT1A antagonist. Galanin (0.3nmol) administered in the DRN increased discreetly flight behaviours induced by electrical stimulation of the DPAG, suggesting a panicolytic effect. Together, our results showed that galanin mediates opposite anxiety responses in the DRN by activation of GAL1 and GAL2 receptors. The anxiolytic effect induced by activation of Gal2 receptors may depend on serotonergic tone. Finally, the role of galanin in panic related behaviours remains uncertain.

Galanin microinjection into the dorsal periaqueductal gray matter produces paradigm-dependent anxiolytic effects.

Galanin is a peptide that is present in the central nervous system in mammals, including rodents and humans. The actions of galanin are mediated by three types of metabotropic receptors: GAL1, GAL2, and GAL3. GAL1 and GAL3 increase K(+) efflux, and GAL2 increases intracellular Ca(2+) levels. The distribution of galanin and its receptors suggests its involvement in fear and/or anxiety. The periaqueductal gray matter (PAG) is a key mediator of defensive behaviors that is both targeted by galaninergic projections and supplied with GAL1 receptors and, less markedly, GAL2 receptors. We examined the effects of galanin microinjections in the dorsal PAG (dPAG) on the performance of rats in different models of anxiety. Male Wistar rats (n=7-12) were implanted with guide cannulae in the dPAG. They received microinjections of either galanin (0.3, 1.0, and 3.0 nmol) or vehicle and were tested in the Vogel conflict test (VCT), elevated plus maze (EPM), and elevated T-maze (ETM). Rats that were tested in the ETM were further evaluated for exploratory activity in the open field test (OFT). Galanin microinjections had no effects on anxiety-like behavior in the EPM or VCT or exploratory activity in the EPM or OFT. In the ETM, however, microinjections of 3 nmol galanin impaired learned anxiety (i.e., avoidance of the open arms) without changing unconditioned fear (i.e., escape from the open arms). The present data suggest that galanin transmission in the dPAG inhibits the acquisition of anxiety-like responses in the ETM.

Serotonin in the dorsal periaqueductal gray inhibits panic-like defensive behaviors in rats exposed to acute hypoxia.

It has been proposed that spontaneous panic attacks are the outcome of the misfiring of an evolved suffocation alarm system. Evidence gathered in the last years is suggestive that the dorsal periaqueductal gray (dPAG) in the midbrain harbors a hypoxia-sensitive suffocation alarm system. We here investigated whether facilitation of 5-HT-mediated neurotransmission within the dPAG changes panic-like defensive reactions expressed by male Wistar rats submitted to a hypoxia challenge (7% O2), as observed in other animal models of panic. Intra-dPAG injection of 5-HT (20 nmol), (±)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT) (8 nmol), a 5-HT1A receptor agonist, or (±)-2,5-dimethoxy-4-iodo amphetamine hydrochloride (DOI) (16 nmol), a preferential 5-HT2A agonist, reduced the number of upward jumps directed to the border of the experimental chamber during hypoxia, interpreted as escape attempts, without affecting the rats' locomotion. These effects were similar to those caused by chronic, but not acute, intraperitoneal administration of the antidepressant fluoxetine (5-15 mg/kg), or acute systemic administration of the benzodiazepine receptor agonist alprazolam (1-4 mg/kg), both drugs clinically used in the treatment of panic disorder. Our findings strengthen the view that the dPAG is a key encephalic area involved in the defensive behaviors triggered by activation of the suffocation alarm system. They also support the use of hypoxia-evoked escape as a model of respiratory-type panic attacks.

Role of pulmonary stretch receptors and sympathetic system in the inhibition of reflex bradycardia produced by chemical stimulation of the periaqueductal gray matter of the rat.

The present study examined the role of the sympathetic system and pulmonary afferent feedback in the baroreflex inhibition by chemical stimulation of the dorsal periaqueductal gray matter (DPAG) of the anesthetized rat. The baroreflex bradycardia was induced by phenylephrine infusions (PHE, 50 µg/ml/min, i.v.) given either alone or combined with glutamate microinjections (GLU, 10 nmol/100 nl) into the DPAG. GLU microinjections alone produced marked increases in respiratory amplitude (67±19%), but barely changed the respiratory frequency (15±3 cpm) and blood pressure (14±2 mm Hg), and did not affect the heart rate. In contrast, the same injections produced a 92% inhibition of PHE-induced bradycardia (from -62 to -5 bpm). Because GLU microinjections per se had little effects on blood pressure, the baroreflex inhibition should be credited to the deactivation of both the vagal and sympathetic reflex pathways at the medulla. Indeed, the baroreflex was inhibited in only 47% following the DPAG stimulation of atenolol-treated rats. The GLU-evoked inhibition of baroreflex was also correlated with concomitant increases in respiratory amplitude. The role of pulmonary feedback in baroreflex inhibition was thus examined before and after the neuromuscular blockade of atenolol-treated rats. In spontaneously breathing rats, GLU microinjections reversed PHE-induced bradycardia to tachycardia, thereby producing a 153% inhibition of reflex bradycardia (from -38 bpm to +20 bpm). In contrast, the baroreflex inhibition was attenuated in only 53% after neuromuscular blockade (from -34 to -16 bpm). Data are the first evidence of the contribution of pulmonary stretch receptor feedback in DPAG-evoked inhibition of reflex bradycardia.

Evidence of a suffocation alarm system within the periaqueductal gray matter of the rat.

Dyspnea, hunger for air, and urge to flee are the cardinal symptoms of panic attacks. Patients also show baseline respiratory abnormalities and a higher rate of comorbid and antecedent respiratory diseases. Panic attacks are also precipitated by infusion of sodium lactate and inhalation of 5% CO2 in predisposed patients but not in healthy volunteers or patients without panic disorder. Accordingly, Klein [Klein (1993) Arch Gen Psychiatry 50:306-317] suggested that clinical panic is the misfiring of an as-yet-unidentified suffocation alarm system. In rats, selective anoxia of chemoreceptor cells by potassium cyanide (KCN) and electrical and chemical stimulations of periaqueductal gray matter (PAG) produce defensive behaviors, which resemble panic attacks. Thus, here we examined the effects of single or combined administrations of CO2 (8% and 13%) and KCN (10-80 µg, i.v.) on spontaneous and PAG-evoked behaviors of rats either intact or bearing electrolytic lesions of PAG. Exposure to CO2 alone reduced grooming while increased exophthalmus, suggesting an arousal response to non-visual cues of environment. Unexpectedly, however, CO2 attenuated PAG-evoked immobility, trotting, and galloping while facilitated defecation and micturition. Conversely, KCN produced all defensive behaviors of the rat and facilitated PAG-evoked trotting, galloping, and defecation. There were also facilitatory trends in PAG-evoked exophthalmus, immobility, and jumping. Moreover, whereas the KCN-evoked defensive behaviors were attenuated or even suppressed by discrete lesions of PAG, they were markedly facilitated by CO2. Authors suggest that the PAG harbors an anoxia-sensitive suffocation alarm system which activation precipitates panic attacks and potentiates the subject responses to hypercapnia.

Functional specializations within the tectum defense systems of the rat.

Here we review the differential contribution of the periaqueductal gray matter (PAG) and superior colliculus (SC) to the generation of rat defensive behaviors. The results of studies involving sine-wave and rectangular pulse electrical stimulation and chemical (NMDA) stimulation are summarized. Stimulation of SC and PAG produced freezing and flight behaviors along with exophthalmus (fully opened bulged eyes), micturition and defecation. The columnar organization of the PAG was evident in the results obtained. Defecation was elicited primarily by lateral PAG stimulation, while the remaining defensive behaviors were similarly elicited by lateral and dorsolateral PAG stimulation, although with the lowest thresholds in the dorsolateral column. Conversely, the ventrolateral PAG did not appear to participate in unconditioned defensive behaviors, which were only elicited by high intensity stimulation likely to encroach on adjacent regions. In the SC, the most important differences relative to the PAG were the lack of stimulation-evoked jumping in both intermediate and deep layers, and of NMDA-evoked galloping in intermediate layers. Therefore, we conclude that the SC may be only involved in the increased attentiveness (exophthalmus, immobility) and restlessness (trotting) of prey species exposed to the cues of a nearby predator. These responses may be distinct from the full-blown flight reaction that is mediated by the dorsolateral and lateral PAG. However, other evidences suggest the possible influences of stimulation schedule, environment dimensions and rat strain in determining outcomes. Overall our results suggest a dynamically organized representation of defensive behaviors in the midbrain tectum.

Organization of electrically and chemically evoked defensive behaviors within the deeper collicular layers as compared to the periaqueductal gray matter of the rat.

Stimulation of the periaqueductal gray matter (PAG) and the deeper layers of superior colliculus (SC) produces both freezing (tense immobility) and flight (trotting, galloping and jumping) behaviors along with exophthalmus (fully opened bulging eyes) and, less often, micturition and defecation. The topography of these behaviors within the distinct layers of SC remains unclear. Therefore, this study compared the defensive repertoire of intermediate (ILSC) and deep (DLSC) layers of SC to those of dorsolateral periaqueductal gray matter (DLPAG) and lateral periaqueductal gray matter (LPAG) [Neuroscience 125 (2004) 71]. Electrical stimulation was carried out through intensity- (0-70 microA) and frequency-varying (0-130 Hz) pulses. Chemical stimulation employed a slow microinfusion of N-methyl-d-aspartic acid (NMDA, 0-2.3 nmol, 0.5 nmol/min). Probability curves of intensity-, frequency- and NMDA-evoked behaviors, as well as the unbiased estimates of median stimuli, were obtained by threshold logistic analysis. Compared with the PAG, the most important differences were the lack of frequency-evoked jumping in both layers of SC and the lack of NMDA-evoked galloping in the ILSC. Moreover, although galloping and jumping were also elicited by NMDA stimulation of DLSC, effective doses were about three times higher than those of DLPAG, suggesting the spreading of the injectate to the latter structure. In contrast, exophthalmus, immobility and trotting were evoked throughout the tectum structures. However, whatever the response and kind of stimulus, the lowest thresholds were always found in the DLPAG and the highest ones in the ILSC. Besides, neither the appetitive, nor the offensive, muricide or male reproductive behaviors were produced by any kind of stimulus in the presence of appropriate targets. Accordingly, the present data suggest that the deeper layers of SC are most likely involved in the increased attentiveness (exophthalmus, immobility) or restlessness (trotting) behaviors that herald a full-blown flight reaction (galloping, jumping) mediated in the PAG.

Organization of single components of defensive behaviors within distinct columns of periaqueductal gray matter of the rat: role of N-methyl-D-aspartic acid glutamate receptors.

The periaqueductal gray matter (PAG) is functionally organized in longitudinal columns arranged along the aqueduct. Stimulation of lateral and dorsal columns produces a complex set of unconditioned behaviors named the 'defense reaction.' Overt responses in rats comprise a tense immobile display, fully opened eyes (herein named exophthalmus), trotting, galloping, jumping, micturition and defecation. Besides, the PAG is rich in glutamate and respective receptors, including the N-methyl-d-aspartic acid (NMDA) type. Therefore, the present study employed regression analysis to map out electrically and NMDA-induced single components of defensive behaviors produced by stepwise increasing stimulation of PAG. Data confirmed the defensive nature of PAG-evoked responses. Neither the appetitive, nor offensive, mouse-killing or male reproductive behaviors were produced by stimulation of PAG in presence of appropriate targets. Threshold and dose-response logistic analyses largely corroborated the columnar organization of PAG-evoked responses. Thus, whereas the defecation was restricted to PAG lateral column, exophthalmus, micturition and somatic defensive responses were similarly organized in dorsolateral and lateral, but not in the ventrolateral column. Moreover, thresholds of dorsolateral and lateral repertoires were strictly hierarchical, with exophthalmus, immobility, trotting, galloping and jumping appearing in this very order. However, the defensive responses of PAG dorsolateral column required NMDA doses significantly lower than those of lateral PAG. Accordingly, NMDA receptors within the dorsolateral PAG are likely to play a major role in the initiation of PAG-evoked defensive responses. In contrast, the present data do not support the organization of unconditioned defensive behaviors in ventrolateral PAG. The neuroanatomical substrate of each response and the role of PAG and NMDA receptors are discussed in relation to the present data. Further, this is the first report on PAG columnar organization of single components of defensive behaviors.

Long-term effects of clomipramine and fluoxetine on dorsal periaqueductal grey-evoked innate defensive behaviours of the rat.

RATIONALE: The defensive responses induced by electrical stimulation of dorsal periaqueductal grey (DPAG) of the rat have been proposed as a model of panic attacks in humans. OBJECTIVE: The present experiments were carried out to evaluate the long-term effects of clinically effective panicolytics on these responses. METHODS: Rats that had electrodes implanted into the DPAG were treated for 21 days with clomipramine (CLM; 5, 10 and 20 mg/kg), fluoxetine (FLX; 1 and 5 mg/kg) or saline. Long-term effects were assessed prior to the treatment to avoid acute drug effects. Threshold logistic functions of defensive responses were compared by likelihood ratio coincidence tests. RESULTS: CLM attenuated DPAG-evoked defensive behaviours at a time-course similar to that observed in panic therapy. Administration of 10 mg/kg CLM for 21 days produced significant increases in the thresholds of immobility (24%), running (49%) and jumping (45%). Splitting of running into single responses disclosed selective threshold increases of galloping (75%) and trotting (138%) with 5 and 10 mg/kg, respectively. Thresholds of micturition were markedly increased (87%) by 5 mg/kg CLM. In turn, FLX (1 mg/kg) virtually abolished the galloping response. No threshold changes were observed following the long-term administration of the higher dose of either CLM or FLX. Saline-treated rats had a significant increase (35%) in galloping thresholds only. CONCLUSIONS: The present data partially validate the DPAG-evoked defence reaction of the rat as a model of panic attacks in humans. Attenuation of galloping by lower doses of FLX and CLM also suggests the prominent modulation of this response by serotonin.

The brain decade in debate: III. Neurobiology of emotion.

This article is a transcription of an electronic symposium in which active researchers were invited by the Brazilian Society of Neuroscience and Behavior (SBNeC) to discuss the advances of the last decade in the neurobiology of emotion. Four basic questions were debated: 1) What are the most critical issues/questions in the neurobiology of emotion? 2) What do we know for certain about brain processes involved in emotion and what is controversial? 3) What kinds of research are needed to resolve these controversial issues? 4) What is the relationship between learning, memory and emotion? The focus was on the existence of different neural systems for different emotions and the nature of the neural coding for the emotional states. Is emotion the result of the interaction of different brain regions such as the amygdala, the nucleus accumbens, or the periaqueductal gray matter or is it an emergent property of the whole brain neural network? The relationship between unlearned and learned emotions was also discussed. Are the circuits of the former the underpinnings of the latter? It was pointed out that much of what we know about emotions refers to aversively motivated behaviors, like fear and anxiety. Appetitive emotions should attract much interest in the future. The learning and memory relationship with emotions was also discussed in terms of conditioned and unconditioned stimuli, innate and learned fear, contextual cues inducing emotional states, implicit memory and the property of using this term for animal memories. In a general way it could be said that learning modifies the neural circuits through which emotional responses are expressed.

The brain decade in debate: III. Neurobiology of emotion

This article is a transcription of an electronic symposium in which active researchers were invited by the Brazilian Society of Neuroscience and Behavior (SBNeC) to discuss the advances of the last decade in the neurobiology of emotion. Four basic questions were debated: 1) What are the most critical issues/questions in the neurobiology of emotion? 2) What do we know for certain about brain processes involved in emotion and what is controversial? 3) What kinds of research are needed to resolve these controversial issues? 4) What is the relationship between learning, memory and emotion? The focus was on the existence of different neural systems for different emotions and the nature of the neural coding for the emotional states. Is emotion the result of the interaction of different brain regions such as the amygdala, the nucleus accumbens, or the periaqueductal gray matter or is it an emergent property of the whole brain neural network? The relationship between unlearned and learned emotions was also discussed. Are the circuits of the former the underpinnings of the latter? It was pointed out that much of what we know about emotions refers to aversively motivated behaviors, like fear and anxiety. Appetitive emotions should attract much interest in the future. The learning and memory relationship with emotions was also discussed in terms of conditioned and unconditioned stimuli, innate and learned fear, contextual cues inducing emotional states, implicit memory and the property of using this term for animal memories. In a general way it could be said that learning modifies the neural circuits through which emotional responses are expressed

The brain decade in debate: II. Panic or anxiety? From animal models to a neurobiological basis.

This article is a transcription of an electronic symposium sponsored by the Brazilian Society of Neuroscience and Behavior (SBNeC). Invited researchers from the European Union, North America and Brazil discussed two issues on anxiety, namely whether panic is a very intense anxiety or something else, and what aspects of clinical anxiety are reproduced by animal models. Concerning the first issue, most participants agreed that generalized anxiety and panic disorder are different on the basis of clinical manifestations, drug response and animal models. Also, underlying brain structures, neurotransmitter modulation and hormonal changes seem to involve important differences. It is also common knowledge that existing animal models generate different types of fear/anxiety. A challenge for future research is to establish a good correlation between animal models and nosological classification.

The brain decade in debate: II. Panic or anxiety? From animal models to a neurobiological basis

This article is a transcription of an electronic symposium sponsored by the Brazilian Society of Neuroscience and Behavior (SBNeC). Invited researchers from the European Union, North America and Brazil discussed two issues on anxiety, namely whether panic is a very intense anxiety or something else, and what aspects of clinical anxiety are reproduced by animal models. Concerning the first issue, most participants agreed that generalized anxiety and panic disorder are different on the basis of clinical manifestations, drug response and animal models. Also, underlying brain structures, neurotransmitter modulation and hormonal changes seem to involve important differences. It is also common knowledge that existing animal models generate different types of fear/anxiety. A challenge for future research is to establish a good correlation between animal models and nosological classification

Modeling panic attacks.

The isomorphism of dorsal periaqueductal gray-evoked defensive behaviors and panic attacks was appraised in the present study. Thresholds of electrically induced immobility, trotting, galloping, jumping, exophthalmus, micturition and defecation were recorded before and after acute injections of anxiolytic, anxiogenic and antidepressant drugs. Antidepressant effects were further assessed 24h after injections of 7-14- and 21-day treatments. Chronic administration of clomipramine (CLM, 5-10mg/kg) a clinically effective antipanic drug increased the thresholds of immobility (24%), trotting (138%) galloping (75%), jumping (45%) and micturition (85%). The 21-day treatment with fluoxetine (FLX, 1mg/kg) virtually abolished galloping without changing the remaining responses. Galloping thresholds were also increased by 5mg/kg acute injections of CLM (19%) and FLX (25%). In contrast, chronically administered maprotiline (10mg/kg), a noradrenaline (NE) selective reuptake inhibitor, selectively increased the thresholds of immobility (118%). Diazepam (1.8mg/kg) and midazolam (MDZ, 2.5mg/kg) failed in attenuating the somatic defensive responses. Yet, the sedative dose of MDZ (5mg/kg) attenuated immobility. The panicogenic drug, pentylenetetrazole (50mg/kg), markedly decreased the thresholds of galloping (-51%) and micturition (-66%). These results suggest that whereas immobility is a NE-mediated attentional response, galloping is the panic-like behavior best candidate.

L-type calcium channels selectively control the defensive behaviors induced by electrical stimulation of dorsal periaqueductal gray and overlying collicular layers.

The present study reports the involvement of L-type calcium channels in the control of defensive behaviors produced by electrical stimulation of dorsal periaqueductal gray and overlying collicular layers. Rats that had chemitrodes in the dorsal midbrain and which stimulation produced freezing or flight behaviors with less than 55 microA were selected for drug experiments. Stimulation was repeated the day after the screening session 20 min following the microinjection into the dorsal periaqueductal gray of 15 nmol of either verapamil, a selective L-type calcium channel antagonist, or cobalt chloride (CoCl(2)), a calcium-specific channel modulator. Post-drug sessions were performed 48 h after. Threshold functions were obtained by logistic fitting of accumulated response frequencies. Verapamil and CoCl(2) significantly attenuated the output of immobility, exophthalmus, running and jumping. Although to a lesser degree, verapamil also attenuated defecation. Because CoCl(2) had no effect on defecation, the attenuation of this response by verapamil suggests a non-specific action of this drug. Neither verapamil nor CoCl(2) changed the output of micturition. Finally, whereas there was a complete recovery of defensive thresholds following the microinjection of verapamil, the attenuating effects of CoCl(2) were still present 48 h after. These results support an important role of L-type calcium channels in the neurogenesis of dorsal periaqueductal gray-evoked immobility, exophthalmus, running and jumping, but not defecation and micturition responses.

Micturition and defensive behaviors are controlled by distinct neural networks within the dorsal periaqueductal gray and deep gray layer of the superior colliculus of the rat.

Electrical stimulation of the dorsal periaqueductal gray (DPAG) or the deep gray layer of the superior colliculus (DGSC) of rats placed in an open-field elicited either a display of tense immobility, accompanied by exophthalmus and/or defecation and micturition, or running and jumping responses. Threshold curves of each response were obtained for each structure by the logistic fitting of accumulated response frequencies. DPAG and DGSC threshold curves were compared by likelihood-ratio coincidence tests. The output of micturition was significantly higher following the stimulation of DPAG (P < 0.0005). In contrast, no differences were found for the remaining responses. These data support previous studies in anaesthetized cats suggesting the critical involvement of DPAG in the control of micturition. Furthermore, they also suggest that topographically distinct neural networks within the DPAG and DGSC control micturition and the other defensive behaviors.

Cardiovascular changes following acute and chronic chemical lesions of the dorsal periaqueductal gray in conscious rats.

This study was carried out to investigate the effects of chemical lesions of dorsal periaqueductal gray (DPAG) on resting arterial pressure (AP) and heart rate (HR) as well as on cardiac baroreflex of conscious normotensive rats. Lesions were performed by bilateral microinjections of 150 mM NMDA into the DPAG (DPAG-lesion group). Controls were similarly injected with 165 mM NaCl (DPAG-sham group). Animals with chronic lesions confined only to the superior colliculus (SC-lesion group) were also used as controls of DPAG-lesion. Cardiovascular parameters were recorded 1 or 7 days after the microinjections of NMDA in acute and chronic groups, respectively. Cardiac baroreflex was assessed by measuring the HR responses to the intravenous injection of phenylephrine or sodium nitroprusside. Baroreflex was estimated by sigmoidal curve fitting of HR responses. An increased baroreflex gain was observed in chronic DPAG-lesion rats compared to both DPAG-sham (p < 0.01) and SC-lesion (p < 0.05) chronic groups. The chronic DPAG-lesion group showed also an elevation of both the tachycardia (p < 0.05) and bradycardia (p < 0.01) plateaus compared to chronic DPAG-sham rats, while the SC-lesion group showed an elevation of the bradycardia plateau only (p < 0.01). Similar results on baroreflex function were observed following acute lesion of the DPAG, i.e. an increase in baroreflex gain (p < 0.01) and the elevation of both tachycardia (p < 0.05) and bradycardia plateaus (p < 0.01) compared to the acute DPAG-sham group. Resting AP and HR did not differ among the chronic groups. In contrast, the acute lesion of the DPAG produced a reduction in AP (p < 0.01) accompanied by an increase in HR (p < 0.01). The present data suggest that the DPAG is involved in the tonic and reflex control of AP and HR in conscious rats. In addition, the SC seems to contribute to the baroreflex cardioinhibition.

NMDA-coupled periaqueductal gray glycine receptors modulate anxioselective drug effects on plus-maze performance.

The present study was carried out to investigate a possible interaction between the effects of anxiety modulating drugs which act at the GABA-A receptor complex and selective N-methyl-D-aspartic acid (NMDA) coupled glycine receptor (GLY-B receptor) ligands within the dorsal periaqueductal gray (DPAG). The plus-maze performance of rats pretreated with diazepam (0.37 and 0.75 mg/kg, i.p.) or pentylenetetrazole (15 and 30 mg/kg, i.p.), standard anxiolytic and anxiogenic drugs respectively, was assessed following intra-periaqueductal injections of either glycine (0.2 M, 0.4 microl/30 s, i.c.) or its competitive antagonist, 7-chlorokynurenic acid (7ClKYN, 0.02 M, 0.4 microl/30 s, i.c.). Whilst diazepam produced a typical anxiolytic effect in intracranially-injected CSF rats, increasing open arm exploration, pentylenetetrazole displayed an opposite anxiogenic profile. Either anxiogenic or anxiolytic effects were seen in peripherally-injected vehicle rats following intra-periaqueductal injections of glycine or 7ClKYN, respectively. Intra-periaqueductal injection of glycine markedly attenuated the anxiolytic effect of diazepam. Moreover, while the anxiogenic effects of pentylenetetrazole were barely changed by glycine, they were markedly attenuated by intra-periaqueductal injection of 7ClKYN. Interaction of diazepam and 7ClKYN produced non-selective sedative-like effects which masked any possible anxiolytic action. Accordingly, the present results suggest that the NMDA-coupled glycine receptors located in the DPAG interfere with anxioselective effects of GABA-A acting drugs on the elevated plus-maze. In spite of the prevailing notion that the NMDA coupled glycine receptor is saturated at in vivo brain concentrations of glycine, our results also suggest that either unoccupied or low-affinity GLY-B receptors are likely to be activated by glycine injection into DPAG.

Alcohol dependence induced in rats by semivoluntary intermittent intake

The objetive of the present experiment was to assess ethyl alcohol (ETOH) dependence brought about by a semivoluntary intermittent intake regimen in rats. Male Wistar rats weighing 150-250 g at the onset of the experiment were assigned to the following groups: 0 percent ETOH (N = 11), 5 percent ETOH (N = 20), 20 percent ETOH (N = 20) and 40 percent ETOH (N = 18). ETOH solutions were offered at the end of the day and overnight from Monday to Friday, and throughout weekends, for 90 days. The concentration of the ETOH solutions was increased in a stepwise fashion allowing the rats to get used to the taste of alcohol. Reposition of pure water was permitted during 1-h water drinking periods in the morning. Daily volume intake (+ SEM) averaged 25.4 + 0.4 ml (0 percent ETOH), 23.8 + 0.6 ml (5 percent ETOH), 17.6 + 0.7 ml (20 percent ETOH) and 17.5 + 0.6 ml (40 percent ETOH). ETOH consumption differed significantly (P<0.05) among groups, averaging 4.4 + 0.2 g Kg(-1) day(-1) (5 percent ETOH), 10.3 + 0.3 g Kg(-1) day(-1) (20 percent ETOH) and 26 + 1.2 g kg(-1) day(-1) (40 percent ETOH). Furthermore, ETOH detection in plasma 10-12h after offering the solution indicated that its consumption in the 40 percent ETOH group was sufficient to override its metabolism. Overt signs of ETOH dependence, such as increased thirst, hyperactivity, puffing, hair ruffling and startle responsiveness as well as reduced drowsiness, were significantly increased in the 20 percent and 40 percent ETOH groups compared to the 0 percent and 5 percent groups. Accordingly, the model described here proved to be a useful tool for the evaluation of subtle or moderate behavioral and physical consequences of long-term ETOH intake.

Alcohol dependence induced in rats by semivoluntary intermittent intake.

The objective of the present experiment was to assess ethyl alcohol (ETOH) dependence brought about by a semivoluntary intermittent intake regimen in rats. Male Wistar rats weighing 150-250 g at the onset of the experiment were assigned to the following groups: 0% ETOH (N = 11), 5% ETOH (N = 20), 20% ETOH (N = 20) and 40% ETOH (N = 18). ETOH solutions were offered at the end of the day and overnight from Monday to Friday, and throughout weekends, for 90 days. The concentration of the ETOH solutions was increased in a stepwise fashion allowing the rats to get used to the taste of alcohol. Reposition of pure water was permitted during 1-h water drinking periods in the morning. Daily volume intake (+/- SEM) averaged 25.4 +/- 0.4 ml (0% ETOH), 23.8 +/- 0.6 ml (5% ETOH), 17.6 +/- 0.7 ml (20% ETOH) and 17.5 +/- 0.6 ml (40% ETOH). ETOH consumption differed significantly (P < 0.05) among groups, averaging 4.4 +/- 0.2 g kg-1 day-1 (5% ETOH), 10.3 +/- 0.3 g kg-1 day-1 (20% ETOH) and 26 +/- 1.2 kg-1 day-1 (40% ETOH). Furthermore, ETOH detection in plasma 10-12 h after offering the solution indicated that its consumption in the 40% ETOH group was sufficient to override its metabolism. Overt signs of ETOH dependence, such as increased thirst, hyperactivity, puffing, hair ruffling and startle responsiveness as well as reduced drowsiness, were significantly increased in the 20% and 40% ETOH groups compared to the 0% and 5% groups. Accordingly, the model described here proved to be a useful tool for the evaluation of subtle or moderate behavioral and physical consequences of long-term ETOH intake.