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Stimulation of benzodiazepine receptors in the dorsal hippocampus and median raphé reveals differential GABAergic control in two animal tests of anxiety
Abstract
The effects of pharmacological challenges to the benzodiazepine receptors in the dorsal hippocampus and median raphé nucleus were investigated in the social interaction and the elevated plus-maze tests of anxiety in rats. In the social interaction test, bilateral administration of midazolam (1 and 2 μg), into the dorsal hippocampus had anxiolytic effects; flumazenil (500 ng) was silent, but was able to antagonize the anxiolytic effects of midazolam (2 μg). In the social interaction test, midazolam was also anxiolytic when infused into the median raphé nucleus; flumazenil (100 and 500 ng) increased locomotor activity, but did not change anxiety measures. As an anatomical control, midazolam (1 and 2 μg) was infused into the adjacent pontine reticular nucleus, and was without effect. In contrast to the social interaction test, local infusion of midazolam (1 and 2 μg) and flumazenil (100 and 500 ng) into either the dorsal hippocampus or the median raphé nucleus failed to change anxiety measures in the elevated plus-maze (trials 1 and 2). These results show that stimulation of the benzodiazepine receptors in the hippocampus or the median raphé nucleus leads to anxiolytic effects in the social interaction test, but not in the elevated plus-maze. It would therefore appear that the two tests detect different types of anxiety that are differentially modulated by GABAA-benzodiazepine receptors in the dorsal hippocampus and the median raphé nucleus.
... Local infusions of benzodiazepines into the amygdala have anxiolytic effects on measures of freezing, shock probe avoidance or the elevated plus-maze (see Davis 2000 for review). Anxiety as measured by the social interaction tests is also reduced following infusion of benzodiazepines into the dorsal hippocampal formation [Gonzalez et al. 1998]. ...
... The differential outcome of unilateral pathology, depending on whether it is developmental or acquired in adulthood, may reflect the greater plasticity of the young brain, which in turn may facilitate reorganisation and compensation [Teuber 1975;Goldman 1979]. It should be noted that the site and size of the lesion plays a critical role, with some systems showing greater resilience than others [Teuber 1975]. ...
... In contrast to unilateral damage suffered in adulthood, comparable unilateral damage resulting from early onset neurodevelopmental or acquired pathology rarely leads to chronic and selective deficits in cognitive and behavioural functioning [Shallice 1988;Warrington and Warrington 1990;Vargha-Khadem et al. 1994]. The absence of such deficits is believed to be due to the increased plasticity of the immature brain which supports functional reorganisation of tissue in the homologous regions contralateral to the damaged side [Teuber 1975;Goldman 1979]. It is only when early damage to the neural substrates subserving a cognitive or behavioural system is bilateral that profound and chronic impairments are documented. ...
Autism is a neurodevelopmental psychiatric syndrome characterised by impairments in three domains: social interaction, communication, and restricted and repetitive behaviours and interests. The neuropathology associated with Autism is unclear. A recently developed animal model implicates the medial temporal lobes in Autism. Bachevalier and colleagues have demonstrated that bilateral ablation of the medial temporal lobes of neonatal monkeys leads to the development of a constellation of symptoms similar to those of Autism. This thesis investigates the hypothesis that medial temporal lobe abnormality is responsible for some of the cognitive and behavioural impairments seen in individuals with Autism. A number of different techniques are used to compare the brain structure and cognitive and behavioural function of children with Autism with those of normal controls. The neuropsychological investigations described in Part I revealed an impairment in episodic memory and no evidence of impaired semantic memory or recognition memory. Additionally, a protocol of tests of executive functions revealed a deficit in a task sensitive to the functioning of the orbitofrontal cortex and a motor checklist revealed co-ordination difficulties consistent with cerebellar abnormality. Part II describes investigations of brain structure, using a variety of magnetic resonance techniques. A new analysis technique was developed specifically to examine bilateral abnormalities in developmental disorders. This technique revealed bilateral abnormalities in the amygdala, hippocampal formation, orbitofrontal cortex, superior temporal gyrus and cerebellum in children with Autism. In Part III, the functional integrity of three event related potentials was investigated. These components were selected as they have been shown to be disrupted by medial temporal lobe abnormality. The results suggest that any functional abnormality of these waveforms is of a more subtle nature than could be detected through the paradigms used in this thesis. In summary, convergent evidence of abnormality in the medial temporal lobes, orbitofrontal cortex and the cerebellum in Autism was obtained.
... The lack of sensitivity to benzodiazepines on trial 2 is not because the animals have habituated to the apparatus. There is no habituation to the corticosterone response (File et al. 1994;Holmes et al. 1998) and the behavioural measures are either unchanged (Pellow et al. 1985;File 1990;Taukulis and McKay 1992) or show further reductions in time spent on the open arms, indicating increased anxiety (Rodgers et al. 1992;Rodgers and Shepherd 1993;Treit et al. 1993;Fernandes and File 1996;File et al. 1998). Because of the insensitivity to benzodiazepines on trial 2, and the importance of the fear of heights on this trial, it was proposed that the nature of anxiety evoked was similar to a simple, or specific, phobia (File and Zangrossi 1993;File et al. 1996). ...
... Previously, we have shown that rats that received lidocaine injections into the basolateral amygdala (thus inducing a temporary functional deactivation), immediately after trial 1 in the plus-maze, responded with an anxiolytic response to chlordiazepoxide when tested 48 hr later on trial 2 (File et al. 1998). Those that received a sham lesion after trial 1 showed the usual lack of response to chlordiazepoxide on trial 2. Thus the basolateral amygdala plays a crucial role in the consolidation of information that leads to the formation of a different type of fear and subsequent insensitivity to benzodiazepines on trial 2. ...
... There is certainly no habituation to anxiety or to the corticosterone stress response (File et al. 1994;Holmes et al. 1998). The lack of response to the test dose of chlordiazepoxide is also not due to locomotor habituation, as there was no change in the number of closed arm entries from trial 1 to trial 2. Numerous experiments have found insensitivity to the anxiolytic effects of chlordiazepoxide on trial 2, without any locomotor habituation (e.g., Gonzalez and File 1997;File et al. 1998). ...
Trial 2 in the elevated plus-maze provides an animal model of specific phobia (fear of heights). On this trial, rats no longer respond to benzodiazepines. The present experiment examined the role of the dorsomedial hypothalamus in mediating insensitivity to chlordiazepoxide on trial 2. Rats received a 5 min exposure to the maze, undrugged. Forty-eight hours later, rats injected with control infusions into the dorsomedial hypothalamus showed the usual lack of response to chlordiazepoxide (5 mg/kg, i.p.). However, those receiving lidocaine injections (40 micrograms/microliter in a volume of 0.2 microliter) in the dorsomedial hypothalamus (producing functional inactivation), immediately before trial 2, responded with an anxiolytic response to chlordiazepoxide, characterised by an increased percentage of time on the open arms and by an increased number of entries into, and time spent on, the distal portions of the open arms. Since the lidocaine injections were without anxiolytic effects, our results suggest that this region of the hypothalamus regulates the functional state of benzodiazepine receptors in other brain regions.
... A study of R. rosea preparation Rhodax ® on humans with generalized anxiety disorders also indicated anxiolytic activity, although the cohort size was too small to draw definite conclusions [22]. Pharmacological studies have shown that modulation of specific combinations of subunits of GABA A receptors is a major factor in mediating anxiolytic responses in the EPM [23,24], SI [25][26][27], and fear responses in the CER [28]. Surprisingly, despite the presence of clear anxiolytic effects in the EPM and CER, the binding affinity of R. rosea extract to the GABA A -BZD receptor is low, except at high concentrations, which may not be biologically significant. ...
... Flavonoids in R. rosea such as kaempferol [33] have been shown to exert anxiolytic effects through the GABA A receptor [34], as have terpenes such as geraniol [35], which potentiate the GABA A receptor response to the GABA neurotransmitter [36]. Modulation of the GABA A receptor is not the primary mode of anxiolytic action of R. rosea; alternate mechanisms include serotonergic involvement, particularly in mediating "risk assessment" behavior in EPM including unprotected head dips [37][38] and in social interaction [25,27]. Long-term treatment with R. rosea restores levels of serotonin in the hippocampus of depressive rats [14] and reduces depression-like symptoms after nicotine withdrawal in dependent rats [39]. ...
Rhodiola rosea is a medicinal plant used by the indigenous Inuit people of Nunavik and Nunatsiavut, Eastern Canada, as a mental and physical rejuvenating agent. This traditional use led to the present investigation of R. rosea in the context of anxiety disorders. An alcohol extract of R. rosea roots was characterized phytochemically and orally administered for three consecutive days to Sprague-Dawley rats at 8 mg/kg, 25 mg/kg, and 75 mg/kg body weight. The rats were subjected to three behavioral paradigms of anxiety, including the elevated plus maze, social interaction, and contextual conditioned emotional response tests. Rhodiola rosea showed dose-dependent anxiolytic activity in the elevated plus maze and conditioned emotional response tests, with moderate effects in the higher-anxiety SI test. The active dose varied according to the anxiety test. In order to elucidate a mechanism, the extract was further tested in an in vitro GABAA-benzodiazepine receptor-binding assay, where it demonstrated low activity. This study provides the first comparative assessment of the anxiolytic activity of Nunavik R. rosea in several behaviour models and suggests that anxiolytic effects may be primarily mediated via pathways other than the GABAA-benzodiazepine site of the GABAA receptor.
... Similar to the original fear conditioning, extinction requires acquisition, consolidation and receptor in fear/anxiety have reported conflicting results, perhaps due to differences in paradigms and drug dosages (Gonzalez et al. 1998;Nevins andAnthony 1994, Yoshioka et al. 1995). Here we report that over-expession of the 5-HT3 receptor is anxiolytic in the both the elevated plus-maze and novelty exploration. ...
... While fearful reactions are generally considered to be controlled by the amygdala (Davis and Whalen 2001; Rogan and LeDoux 1996), the hippocampal formation also is important in anxious behaviors (Crestani et al. 1999;Gonzalez et al. 1998;McNaughton and Gray 2000). In fact, the hippocampal formation is thought to be capable of coding critical aspects of anxiety, allowing for the integration of anxiety states and learning cues. ...
Alcohol use has been identified as an important factor in aggressive, or violent, behavior in humans. Alcohol not only increases the incidence but also the severity of violent attacks. Several clinical studies have reported the observation that highly aggressive individuals display a serotonin-deficient trait. A number of studies indicate that the 5HT(sub 3) receptor system mediates alcohol consumption and the subjective effects of alcohol. The 5HT(sub 3) receptor is unique in the serotonin receptor family in that it is a cation channel and modulates the release of a number of other neurotransmitters, including GABA and dopamine. Thus, the 5-HT(sub 3) receptor is likely to play a critical role influencing alcohol consumption, which appears to involve dopamine and influencing both natural and alcohol-heightened aggression through the GABAA receptor system. We have developed a 5-HT(sub 3) receptor over-expressing mouse to study the role of this receptor in alcohol drinking and aggression. We hypothesize that 5HT(sub 3) receptor over-expression decreases alcohol preference and aggressive activity through a 5HT(sub 3) receptor sensitive mechanism increasing the release of GABA. We will evaluate the transgene on 3 different inbred strains (C57, DBA, 129) who differ in alcohol preference in a two-bottle choice design and aggression using intruder- aggression test. We will test the ability of 5HT(sub 3) receptor antagonists to block and GABA receptor agonist to mimic the phenotypic effects of over-expression. These mice should prove useful in testing hypothesis regarding the role 5-HT(sub 3) receptors play in alcohol abuse and alcohol-heightened aggression.
... Mounting evidence indicates that pro-inflammatory cytokines contribute to the etio-pathophysiology of depression (File et al., 2000;Gonzalez et al., 1998). Chronic stress has been found to produce excess inflammatory cytokines and to cause glucocorticoid resistance as a result of impaired GR function (Funder, 1997). ...
Prior studies suggest that individual differences in stress responses contribute to the pathogenesis of neuropsychiatric disorders. In the present study, we investigated the role of small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) in mediating stress responses to chronic social defeat stress (CSDS). We found that mRNA and protein levels of PIAS 1 were decreased in the hippocampus of high-susceptibility (HS) mice but not in low-susceptibility (LS) mice after CSDS. Local overexpression of PIAS1 in the hippocampus followed by CSDS exposure promoted stress resilience by attenuating social avoidance and improving anxiety-like behaviors. Viral-mediated gene transfer to generate a conditional knockdown of PIAS1 in the hippocampus promoted social avoidance and stress vulnerability after subthreshold microdefeat. HS mice displayed decreased levels of glucocorticoid receptor (GR) expression, and GR SUMOylation in the hippocampus was associated with stress vulnerability. Furthermore, cytokine/chemokine levels were changed predominantly in the hippocampus of HS mice. These results suggest that hippocampal PIAS1 plays a role in the regulation of stress susceptibility by post-translational modification of GRs.
... GABAergic inhibitor neurons have been implicated in the induction of LTP [21]. Our previous studies have indicated that enhanced GABAergic neurotransmission after stroke might play a role in cognitive deficits and anxiolytic response [13,22,23]. The study implicated a potential role of the ERK/MAPK signaling pathway in the hippocampal LTP induction and spatial learning and memory impairments [13], which has also been reported by other laboratories [24][25][26]. ...
Stroke leads to devastating outcomes including impairments of sensorimotor and cognitive function that may be long lasting. New intervention strategies are needed to overcome the long-lasting effects of ischemic injury. Previous studies determined that treatment with 5-methoxyindole-2-carboxylic acid (MICA) conferred chemical preconditioning and neuroprotection against stroke. The purpose of the current study was to determine whether the preconditioning can lead to functional improvements after stroke (done by transient middle cerebral artery occlusion). After 4 weeks of MICA feeding, half the rats underwent ischemic injury, while the other half remained intact. After one week recovery, all the rats were tested for motor and cognitive function (rotorod and water maze). At the time of euthanasia, measurements of long-term potentiation (LTP) were performed. While stroke injury led to motor and cognitive dysfunction, MICA supplementation did not reverse these impairments. However, MICA supplementation did improve stroke-related impairments in hippocampal LTP. The dichotomy of the outcomes suggest that more studies are needed to determine optimum duration and dosage for MICA to lead to substantial motor and cognitive improvements, along with LTP change and neuroprotection.
... All drugs were dissolved in saline (except TRX which was dissolved in artificial cerebrospinal fluid prepared as described by Wang et al. [24] and were infused at room temperature). The dose of scopolamine used in this study was determined from a preliminary study based on the doses used by Izquierdo et al. [25] and Gonzalez et al. [26]. The other doses used were determined based on pilot experiments and on previous studies showing the effect of TRX on learning, memory and other behavioral and physiological variables. ...
Alzheimer's disease (AD) is a neurodegenerative disorder associated with cognitive impairment and cholinergic neuronal death, characteristic of the effect of time on biochemical neuronal function. The use of medicinal plants as an alternative form of prevention, or even as a possible treatment of AD, is therefore interesting areas of research, since the standard drugs have many side effects. Taraxerol (TRX) is a triterpene that has been isolated from several plant species, and its various pharmacological properties have already been identified, such the acetylcholinesterase (AChE) inhibition activity in vitro. There is a lack of information in literature that confirms the effect of TRX in an animal AD-like model. Seeking to fill this gap in the literature, in the present work we assessed the effect of TRX on AChE activity in the animals' encephalon and hippocampus. We also investigated the effect of TRX (1.77 µM/side, 0.5μl) isolated from leaves of Eugenia umbelliflora Berg. on aversive memory impairments induced by scopolamine (2 µg/side, 0.5 µL) infused into rat hippocampus, and the effect of TRX (0.89 and 1.77 µM/side, 0.5μl) on aversive memory impairments induced by streptozotocin (STZ) (2.5 mg/mL, 2.0 µL) infused i.c.v. into mice, using the step-down inhibitory avoidance task. We found that TRX significantly inhibited AChE activity in the animal's hippocampus. Furthermore, TRX significantly improved scopolamine and STZ-induced memory impairment. Taking together, these results confirms its AChE activity inhibition in animals and indicate that TRX has anti-amnesic activity that may hold significant therapeutic value in alleviating certain memory impairments observed in AD.
... extracts induce physiological changes at targets of anxiety and stress. Evidence of anxiolytic activity of Souroubea spp. in unconditioned (EPM, SI) and conditioned (CER, FPS) tests suggested that Souroubea extracts and (or) compounds may be modulating anxiety responses by acting as agonists at the GABA A -BZD receptor (17)(18)(19). Mullally et al. (16) showed that in the presence of a GABA A -BZD receptor-specific antagonist, flumazenil, the anxiolytic activity of extracts of Souroubea as well as methyl-BA were markedly attenuated in the EPM, and to a lesser degree, in the CER (Fig. 5). ...
As part of our ongoing research into botanical therapies for anxiety disorders, the neotropical vine Souroubea sympetala was chosen for study as a phytochemical discovery strategy focusing on rare Central American plant families. When orally administered to male Sprague-Dawley rats, the crude plant extract, its ethyl acetate fraction, supercritical carbon dioxide fraction, or its isolated triterpenes reduced anxiety and/or fear-related behavior in standardized behavioral models. Pharmacological studies showed that the extracts acted at the benzodiazepine GABAA receptor and reduced corticosterone levels. A preparation containing Souroubea fortified with a second triterpene containing plant, Platanus occidentalis, was shown to be safe in a 28-day feeding trial with beagles at 5 times the intended dose. Subsequent trials with beagles in a thunderstorm model of noise aversion showed that the material reduced anxiety behaviors and cortisol levels in dogs. The formulation has been released for the companion animal market in Canada and the USA under the Trademark “Zentrol.” Ongoing research is exploring the use of the material in treatment of anxiety and post-traumatic stress in humans.
... To analyze the putative role of the GABA A /BDZ receptor as a target for the anxiolytic-like action of rutin, it was administered following a systemic (5 mg/kg, intraperitoneal, − 15 min, n = 6) (Fernandez-Guasti and Lopez-Rubalcava, 1998) or an intra-amygdala (0.5 nmol/4 µl, intracerebral, − 1 min, n = 6) (Gonzalez et al., 1998) pretreatment with flumazenil (a competitive BDZ receptor antagonist). In addition, picrotoxin [a chloride channel GABA A antagonist, 0.3 nmol/4 µl, intracerebral, − 1 min (Lopez et al., 2007)] was administered intraamygdala. ...
Rutin is a bioflavonoid found in medicinal plants used to reduce anxiety. Evidence is lacking of rutin's anxiolytic-like activity, putative mechanism(s) of action, and neural sites of effects. The basolateral amygdala (BLA) is the main brain region that regulates anxiety, through GABAA/benzodiazepine (BDZ) receptors, which are modulated by flavonoids. Therefore, the main aim of this study was to investigate whether the anxiolytic-like effect of rutin involves GABAA/BDZ receptors in the BLA. Rutin was administered systemically (30-1000 mg/kg, intraperitoneal) or microinjected into the BLA (16 nmol/4 µl, intracerebral), and its effects were assessed in the elevated plus-maze and open-field tests. Diazepam (1 mg/kg, intraperitoneal, or 7 nmol/4 µl, respectively) was used as a positive control. The mechanism of action was studied using flumazenil (BDZ antagonist, 5 mg/kg, intraperitoneal, or 7 nmol/4 µl, intracerebral) or picrotoxin (chloride channel GABAA antagonist, 0.3 nmol/4 µl, intracerebral). Rutin, administered systemically or intra-amygdala, induced anxiolytic-like responses, similar to those of diazepam. The effect of diazepam was completely blocked by flumazenil, which also partly antagonized the effects of systemic rutin. By contrast, flumazenil exerted no effect and picrotoxin had only a partial action when rutin was infused in the BLA. These results suggest that the anxiolytic-like effect of rutin in the BLA involves GABAergic neurotransmission that is not associated with BDZ receptors.
... The prototypical anxiolytic agents and benzodiazepines (BZPs), diazepam and clorazepate, at doses corresponding to those expressing their anxiolytic properties , markedly suppressed FCX levels of 5-HT, dopamine and NAD (Table 3). These findings are analogous to those obtained in the hippocampus and other subcortical tissues and reflect the facilitation of GABA A receptor-mediated mechanisms inhibitory to monoaminergic pathways (Pan and Williams, 1989;Miczek et al., 1995;Broderick, 1997;Millan et al., 1997a;Broderick et al., 1998;Gonzalez et al., 1998;Millan et al., 1999b). In fact, it has been suggested that a reinforcement in GABAergic transmission might counteract depressive states (Lloyd et al., 1989;Petty et al., 1995). ...
The highly-interconnected and neurochemically-rich frontal cortex plays a crucial role in the regulation of mood and cognition, domains disrupted in depression and other central nervous system disorders, and it is an important site of action for their therapeutic control. For improving our understanding of the function and dysfunction of the frontal cortex, and for identifying improved treatments, quantification of extracellular pools of neuromodulators by microdialysis in freely-moving rodents has proven indispensable. This approach has revealed a complex mesh of autoreceptor and heteroceptor interactions amongst monoaminergic pathways, and led from selective 5-HT reuptake inhibitors to novel classes of multi-target drugs for treating depression like the mixed α2-adrenoceptor/5-HT reuptake inhibitor, S35966, and the clinically-launched vortioxetine and vilazodone. Moreover, integration of non-monoaminergic actions resulted in the discovery and development of the innovative melatonin receptor agonist/5-HT2C receptor antagonist, Agomelatine. Melatonin levels, like those of corticosterone and the “social hormone”, oxytocin, can now be quantified by microdialysis over the full 24 h daily cycle. Further, the introduction of procedures for measuring extracellular histamine and acetylcholine has provided insights into strategies for improving cognition by, for example, blockade of 5-HT6 and/or dopamine D3 receptors. The challenge of concurrently determining extracellular levels of GABA, glutamate, d-serine, glycine, kynurenate and other amino acids, and of clarifying their interactions with monoamines, has also been resolved. This has proven important for characterizing the actions of glycine reuptake inhibitors that indirectly augment transmission at N-methyl-d-aspartate receptors, and of “glutamatergic antidepressants” like ketamine, mGluR5 antagonists and positive modulators of AMPA receptors (including S47445). Most recently, quantification of the neurotoxic proteins Aβ42 and Tau has extended microdialysis studies to the pathogenesis of neurodegenerative disorders, and another frontier currently being broached is microRNAs. The present article discusses the above themes, focusses on recent advances, highlights opportunities for clinical “translation”, and suggests avenues for further progress.
... Also, the predication that variable phytochemisty would lead to a different profile of activity in the behavioural test was supported. have shown that modulation of the GABA A receptor via agonists is a major factor in modulating the anxiety-like response behaviour in the EPM (Gonzalez, Ouagazzal, & File 1998;Menard & Treit 1999;Rezayat et al. 2005). But there are studies that also indicate also a serotoninergic involvement in mediating EPM anxiety-like behavior (Grundmann et al. 2007;Rodgers, Cole, & Davies 1994;Rodgers, Cutler, & Jackson 1997 (Gonzalez, Andrews, & File 1996). ...
... In addition the latter study failed to examine subjective anxiety levels and hence it is unclear if theanine has direct effects on anxiety. However, given that L-theanine increases both serotonin and GABA (Kimura & Murata, 1971;Yokogoshi et al., 1998a,b), and both these neurotransmitters play an important role in the pathophysiology of anxiety disorders and anxiolytic effects (Gonzalez et al., 1998;Graeff, 2002;Kent et al., 2002), it is possible L-theanine may have anxiolytic effects. ...
L-theanine (N-ethyl-L-glutamine) or theanine is a major amino acid uniquely found in green tea. L-theanine has been historically reported as a relaxing agent, prompting scientific research on its pharmacology. Animal neurochemistry studies suggest that L-theanine increases brain serotonin, dopamine, GABA levels and has micromolar affinities for AMPA, Kainate and NMDA receptors. In addition has been shown to exert neuroprotective effects in animal models possibly through its antagonistic effects on group 1 metabotrophic glutamate receptors. Behavioural studies in animals suggest improvement in learning and memory. Overall, L-theanine displays a neuropharmacology suggestive of a possible neuroprotective and cognitive enhancing agent and warrants further investigation in animals and humans.
... Hippocampus is an important component of neuronal circuitry controlling anxiety-related behaviors (Andrews et al., 1997;Gonzalez et al., 1998). Although the direct link between hippocampal MF plasticity and anxiety-like behavior has not been reported, studies have shown a positive correlation between SP-MF sprouting and increased anxiety-like behavior in inbred (Prior et al., 1997), as well as in outbred rats (de Oliveira et al., 2008;Oztan et al., 2011), suggesting that the MF system may contribute to modulation of anxiety-like responses. ...
Experimentally naïve outbred rats display varying rates of locomotor reactivity in response to the mild stress of a novel environment. Namely, some display high rates (HR) whereas some display low rates (LR) of locomotor reactivity. Previous reports from our laboratory show that HRs, but not LRs, develop locomotor sensitization to a low dose nicotine challenge and exhibit increased social anxiety-like behavior following chronic intermittent nicotine training. Moreover, the hippocampus, specifically hippocampal Y2 receptor (Y2R)-mediated neuropeptide Y signaling is implicated in these nicotine-induced behavioral effects observed in HRs. The present study examines the structural substrates of the expression of locomotor sensitization to a low dose nicotine challenge and associated social anxiety-like behavior following chronic intermittent nicotine exposure during adolescence in the LRHR hippocampi. Our data showed that the expression of locomotor sensitization to the low dose nicotine challenge and the increase in social anxiety-like behavior were accompanied by an increase in mossy fiber terminal field size, as well as an increase in spinophilin mRNA levels in the hippocampus in nicotine pre-trained HRs compared to saline pre-trained controls. Furthermore, a novel, selective Y2R antagonist administered systemically during 1 wk of abstinence reversed the behavioral, molecular and neuromorphological effects observed in nicotine-exposed HRs. These results suggest that nicotine-induced neuroplasticity within the hippocampus may regulate abstinence-related negative affect in HRs, and implicate hippocampal Y2R in vulnerability to the behavioral and neuroplastic effects of nicotine in the novelty-seeking phenotype.
... Ethological differences among animal models are crucial for detecting particular anxiety traits. The animal models may generate different kinds of fear, as suggested by pharmacological studies [13,18,19,20] and factor analysis [12,46]. Therefore, the fact that the anxiolysis induced by T. gondii was detected only by the plus-maze on W3-PI indicates that the plus-maze is more sensitive to the effect of toxoplasma infection, possibly because this model closely reflects "thigmotaxis" (an anti-predator reflex triggered by the open spaces) [24,49,55]. ...
It has been suggested that the parasite Toxoplasma gondii reduces the fear of rodents toward their feline predators, which may lead to an augmented rate of predation and multiplication of the parasite through an increased number of life cycles. To investigate whether T. gondii infection induces selective effects on behavior associated with anxiety, Wistar rats were inoculated i.p. with several doses of T. gondii tachyzoites and tested in two animal tests of anxiety. In the third week following inoculation, rats infected with 100 and 1000 tachyzoites increased plus-maze open arm exploration in a dose-related manner. However, no effect was detected in either social interaction levels or motor activity measures. In the seventh week after inoculation, rats infected with 100 and 1000 tachyzoites showed increased open arm exploration and social investigation without change on any motor activity measures. However, rats infected with a higher dose (1500 tachyzoites) showed a drop in locomotion. These data support the hypothesis that T. gondii impairs mechanism of warning as a function of reduced anxiety. The pattern of brain colonization by the parasite and the host immune response suggests that the predominant invasion to limbic areas works as a natural anxiolytic mechanism.
... The hippocampus has also been implicated in the control of anxiety and is thought to play a possible role in social behavioral responses in the social interaction task. Stimulation of benzodiazepine receptors via bilateral injection of midalzolam into the dorsal hippocampus significantly increased social interaction in rats (Gonzalez et al., 1998). Lesione studies showed varied responses as male rats lesioned with ibotenic acid in the ventral or dorsal hippocampus at 8 weeks of age showed no changes in social interaction, while male rats lesioned at 8 months of age produced a significant increase in social interaction (Becker et al., 1999;Deacon et al., 2002). ...
Negative symptoms (e.g., asociality and anhedonia) are a distinct symptomatic domain that has been found to significantly affect the quality of life in patients diagnosed with schizophrenia. Additionally, the primary negative symptom of asociality (i.e., withdrawal from social contact that derives from indifference or lack of desire to have social contact) is a major contributor to poor psychosocial functioning and has been found to play an important role in the course of the disorder. Nonetheless, the pathophysiology underlying these symptoms is unknown and currently available treatment options (e.g., antipsychotics and cognitive-behavioral therapy) fail to reliably produce efficacious benefits. Utilizing rodent paradigms that measure social behaviors (e.g., social withdrawal) to elucidate the neurobiological substrates that underlie social dysfunction and to identify novel therapeutic targets may be highly informative and useful to understand more about the negative symptoms of schizophrenia. Accordingly, the purpose of this review is to provide an overview of the behavioral tasks for assessing social functioning that may be translationally relevant for investigating negative symptoms associated with schizophrenia.
... As observed with these lesion studies, local facilitation of GABA A -mediated inhibitory neurotransmission in the MRN, through microinjection of benzodiazepine (BZD) receptor agonists, also causes anxiolysis, but this seems to be dependent on the experimental model used. Thus, at the same doses, intra-MRN administration of midazolam was anxiolytic in the social interaction test, but ineffective in the elevated plus maze (Gonzalez et al., 1998). ...
Although the role of the median raphe nucleus (MRN) in the regulation of anxiety has received less attention than that of the dorsal raphe nucleus (DRN) there is substantial evidence supporting this function. Reported results with different animal models of anxiety in rats show that whereas inactivation of serotonergic neurons in the MRN causes anxiolysis, the stimulation of the same neurons is anxiogenic. In particular, studies using the elevated T-maze comparing serotonergic interventions in the MRN and in the DRN indicate that the former affect only the inhibitory avoidance task, which has been related to generalized anxiety. In contrast, similar operations in the DRN change both the inhibitory avoidance and the one-way escape task, the latter being representative of panic disorder. Simultaneous injections of 5-HT-acting drugs in the MRN and in the dorsal hippocampus (DH) suggest that the MRN-DH pathway mediates the regulatory function of the MRN in anxiety. Overall, the results discussed in this review point to a relevant role of the MRN in the regulation of anxiety, but not panic, through the 5-HT pathway that innervates the DH.
... Our finding is in agreement with the observation that GABA function is enhanced in the brain area adjacent to stroke damage (Clarkson et al., 2010). Increased GABAergic neurotransmission in the hippocampus might also explain the anxiolytic effect induced by tMCAO (Gonzalez et al., 1998). ...
Vascular dementia ranks as the second leading cause of dementia in the United States. However, its underlying pathophysiological mechanism is not fully understood and no effective treatment is available. The purpose of the current study was to evaluate long-term cognitive deficits induced by transient middle cerebral artery occlusion (tMCAO) in rats and to investigate the underlying mechanism. Sprague-Dawley rats were subjected to tMCAO or sham surgery. Behavior tests for locomotor activity and cognitive function were conducted at 7 or 30 days after stroke. Hippocampal long term potentiation (LTP) and involvement of GABAergic neurotransmission were evaluated at 30 days after sham surgery or stroke. Immunohistochemistry and Western blot analyses were conducted to determine the effect of tMCAO on cell signaling in the hippocampus. Transient MCAO induced a progressive deficiency in spatial performance. At 30 days after stroke, no neuron loss or synaptic marker change in the hippocampus were observed. LTP in both sides of the hippocampus was reduced at 30 days after stroke. This LTP impairment was prevented by blocking GABAA receptors. In addition, ERK activity was significantly reduced in both sides of the hippocampus. In summary, we identified a progressive decline in spatial learning and memory after ischemic stroke that correlates with suppression of hippocampal LTP, elevation of GABAergic neurotransmission, and inhibition of ERK activation. Our results indicate that the attenuation of GABAergic activity or enhancement of ERK/MAPK activation in the hippocampus might be potential therapeutic approaches to prevent or attenuate cognitive impairment after ischemic stroke.
... Ethological differences among animal models are crucial for detecting particular anxiety traits. The animal models may generate different kinds of fear, as suggested by pharmacological studies [13,18,19,20] and factor analysis [12,46]. Therefore, the fact that the anxiolysis induced by T. gondii was detected only by the plus-maze on W3-PI indicates that the plus-maze is more sensitive to the effect of toxoplasma infection, possibly because this model closely reflects " thigmotaxis " (an anti-predator reflex triggered by the open spaces) [24,49,55]. ...
It has been suggested that the parasite Toxoplasma gondii reduces the fear of rodents toward their feline predators, which may lead to an augmented rate of predation and multiplication of the parasite through an increased number of life cycles. To investigate whether T. gondii infection induces selective effects on behavior associated with anxiety, Wistar rats were inoculated i.p. with several doses of T. gondii tachyzoites and tested in two animal tests of anxiety. In the third week following inoculation, rats infected with 100 and 1000 tachyzoites increased plus-maze open arm exploration in a dose-related manner. However, no effect was detected in either social interaction levels or motor activity measures. In the seventh week after inoculation, rats infected with 100 and 1000 tachyzoites showed increased open arm exploration and social investigation without change on any motor activity measures. However, rats infected with a higher dose (1500 tachyzoites) showed a drop in locomotion. These data support the hypothesis that T. gondii impairs mechanism of warning as a function of reduced anxiety. The pattern of brain colonization by the parasite and the host immune response suggests that the predominant invasion to limbic areas works as a natural anxiolytic mechanism.
... Full agonists of the benzodiazepine receptor (e.g., diazepam) produced reliable anxiolytic effects in several of the tests when microinfused into the dorsal or median raphe (Table 2). Flumazenil, a benzodiazepine receptor antagonist, produced no effects by itself but reversed many of the anxiolytic effects found with full agonists (Gonzalez et al., 1998;Gonzalez and File, 1997). As might be expected, the benzodiazepine inverse agonist β-CCM produced an anxiogenic effect when infused into the dorsal raphe (Hindley et al., 1985). ...
Intra-cerebral (i.c.) microinfusion of selective receptor agonists and antagonists into behaving animals can provide both neuroanatomical and neurochemical insights into the neural mechanisms of anxiety. However, there have been no systematic reviews of the results of this experimental approach that include both a range of unconditioned anxiety reactions and a sufficiently broad theoretical context. Here we focus on amino acid, monoamine, cholinergic and peptidergic receptor ligands microinfused into neural structures previously implicated in anxiety, and subsequent behavioral effects in animal models of unconditioned anxiety or fear. GABAA receptor agonists and glutamate receptor antagonists produced the most robust anxiolytic-like behavioral effects, in the majority of neural substrates and animal models. In contrast, ligands of the other receptor systems had more selective, site-specific anti-anxiety effects. For example, 5-HT1A receptor agonists produced anxiolytic-like effects in the raphe nuclei, but inconsistent effects in the amygdala, septum, and hippocampus. Conversely, 5-HT3 receptor antagonists produced anxiolytic-like effects in the amygdala but not in the raphe nuclei. Nicotinic receptor agonists produced anxiolytic-like effects in the raphe and anxiogenic effects in the septum and hippocampus. Unexpectedly, physostigmine, a general cholinergic agonist, produced anxiolytic-like effects in the hippocampus. Neuropeptide receptors, although they are popular targets for the development of selective anxiolytic agents, had the least reliable effects across different animal models and brain structures, perhaps due in part to the fact that selective receptor ligands are relatively scarce. While some inconsistencies in the microinfusion data can easily be attributed to pharmacological variables such as dose or ligand selectivity, in other instances pharmacological explanations are more difficult to invoke: e.g., even the same dose of a known anxiolytic compound (midazolam) with a known mechanism of action (the benzodiazepine–GABAA receptor complex), can selectively affect different fear reactions depending upon the different subregions of the nucleus into which it is infused (CeA versus BLA). These particular functional dissociations are important and may depend on the ability of a GABAA receptor agonist to interact with distinct isoforms and combinations of GABAA receptor subunits (e.g., α1-6, β1-3, ϒ1-2, δ), many of which are unevenly distributed throughout the brain. Although this molecular hypothesis awaits thorough evaluation, the microinfusion data overall give some support for a model of “anxiety” that is functionally segregated along different levels of a neural hierarchy, analogous in some ways to the organization of sensorimotor systems.
... BMI and muscimol were dissolved in artificial cerebrospinal fluid; flumazenil, U0126 and KN62 were dissolved in 0.1% DMSO in saline (0.9% NaCl) before infusion. Fifteen minutes before the retention trial, mice were carefully restrained by hand and bilaterally infused with BMI (0.25, 0.5, or 1 mg/ml/side, Luft et al, 2004), flumazenil (0.5, 1, or 2 mg/ml/side, Gonzalez et al, 1998), muscimol (0.25, 0.5, or 1 mg/ml/side, Holt andMaren, 1999), diazepam (10, 20, or 40 mg/ml/side, Wesołowska et al, 2006), U0126 (0.25, 0.5, or 1 nmol/ml/side, Kim et al, 2009), KN62 (1 mg/ml/side, Ahi et al, 2004) or vehicle through injector cannulae (30 G) extended 1.0 mm beyond the tips of guide cannulae. The infusion volume was 1 ml/side, and the infusion rate was 0.5 ml/min. ...
Available evidence strongly suggests that the γ-aminobutyric acid type A (GABA(A)) receptor has a crucial role in memory retrieval. However, the signaling mechanisms underlying the role of GABA(A) receptor modulation in memory retrieval are unclear. We conducted one-trial passive avoidance task with pre-retention trial drug administration in the hippocampus to test the effects of GABA(A) receptor modulation on memory retrieval. We further tested the co-involvement of signaling molecules: extracellular signal-regulated kinase (ERK), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), and cAMP responsive element-binding protein (CREB). First, we observed that the phosphorylation of hippocampal ERK was required for memory retrieval during the task. Accordingly, to investigate whether GABA(A) receptor activation or inhibition induces ERK phosphorylation during memory retrieval, drugs that target the GABA(A) receptor were administered into the hippocampus before the retention trial. Muscimol, a GABA(A) receptor agonist, and diazepam, an agonist to benzodiazepine-binding site of GABA(A) receptor, blocked retention trial-induced ERK phosphorylation and impaired memory retrieval. Furthermore, co-treatment with sub-effective dose of U0126, a mitogen-activated protein kinase inhibitor, blocked the upregulation of ERK phosphorylation and impaired memory retrieval, and bicuculline methiodide (BMI), a GABA(A) receptor antagonist, increased ERK phosphorylation induced by the retention trial and facilitated memory retrieval. Finally, the effects of BMI were blocked by the co-application of a sub-effective dose of U0126. These results suggest that GABA(A) receptor-mediated memory retrieval is closely related to ERK activity.
... These different patterns illustrate the ability of microstructural analysis to distinguish between different types of effects on ingestive behavior that could not be detected by examining intake data alone. Since intra-MR injections of benzodiazepines have been shown to produce several behavioral effects (Gonzalez, Quagazzal & File, 1998;Sainati & Lorens, 1983), which are presumably mediated through enhanced GABAergic transmission, an interesting possibility is that the MR may be one of the sites at which systemically administered benzodiazepines act to influence feeding. ...
Previous studies have shown that microinjections of the GABA-A agonist muscimol into the median raphe nucleus (MR) result in large increases in the intake of solid foods. In the current study, we used microstructural techniques to characterize the effects of intra-MR muscimol injections on the consumption of either a 0.05 M or a 0.29 M sucrose solution. After injections of either saline or muscimol, animals consumed more of the 0.29 M than the 0.05 M solution, an effect which resulted primarily from increases in the initial rate of consumption with no change in the rate at which licking decayed across the test session. In contrast, intra-MR muscimol injections had little effect on the initial licking rate, but greatly increased meal duration, indicating that this treatment affected ingestion in a different way than did altering the sucrose concentration. Muscimol injections produced a significantly larger increase in the intake of the 0.29 M than of the 0.05 M solution. Intra-MR muscimol injections did not alter the within burst rate of licking, suggesting that they did not affect the functioning of the licking pattern generator. In contrast, these injections did increase the number of licks contained within "clusters," that is groups of licks separated from each other by intervals of more than 0.5 sec. These findings show that inactivation of the MR produces a powerful effect on the intake of liquid diets, and that the nature of this effect is different from that produced here by changes in sucrose concentration and from those reported after pharmacological manipulations of a number of other brain systems. We additionally discuss several theoretical issues arising in the interpretation of microstructural data. (PsycINFO Database Record (c) 2011 APA, all rights reserved).
... Understanding how specific raphe circuits and neuron populations control particular behaviors requires a mechanistic description at the cellular and circuit level. Many investigators have proposed that the different subfields of DR and MR have differential roles in terms of mediating stress, anxiety and depression (Adell et al., 1997;Andrade and Graeff, 2001;Andrade et al., 1999;Andrews et al., 1997;Andrews et al., 1994;File and Gonzalez, 1996;Gonzalez and File, 1997;Gonzalez et al., 1998;Graeff et al., 1996;Lowry, 2002). Raphe circuits involve direct modulation of the HPA axis as well as indirectly mediated influences orchestrated by raphe projections to other limbic structures (e.g., the hippocampus, amygdala, and medial prefrontal cortex) and brainstem areas that regulate the autonomic nervous system. ...
The median (MR) and dorsal raphe (DR) nuclei contain the majority of the 5-hydroxytryptamine (5-HT, serotonin) neurons that project to limbic forebrain regions, are important in regulating homeostatic functions and are implicated in the etiology and treatment of mood disorders and schizophrenia. The primary synaptic inputs within and to the raphe are glutamatergic and GABAergic. The DR is divided into three subfields, i.e., ventromedial (vmDR), lateral wings (lwDR) and dorsomedial (dmDR). Our previous work shows that cell characteristics of 5-HT neurons and the magnitude of the 5-HT(1A) and 5-HT(1B) receptor-mediated responses in the vmDR and MR are not the same. We extend these observations to examine the electrophysiological properties across all four raphe subfields in both 5-HT and non-5-HT neurons. The neurochemical topography of glutamatergic and GABAergic cell bodies and nerve terminals were identified using immunohistochemistry and the morphology of the 5-HT neurons was measured. Although 5-HT neurons possessed similar physiological properties, important differences existed between subfields. Non-5-HT neurons were indistinguishable from 5-HT neurons. GABA neurons were distributed throughout the raphe, usually in areas devoid of 5-HT neurons. Although GABAergic synaptic innervation was dense throughout the raphe (immunohistochemical analysis of the GABA transporters GAT1 and GAT3), their distributions differed. Glutamate neurons, as defined by vGlut3 anti-bodies, were intermixed and co-localized with 5-HT neurons within all raphe subfields. Finally, the dendritic arbor of the 5-HT neurons was distinct between subfields. Previous studies regard 5-HT neurons as a homogenous population. Our data support a model of the raphe as an area composed of functionally distinct subpopulations of 5-HT and non-5-HT neurons, in part delineated by subfield. Understanding the interaction of the cell properties of the neurons in concert with their morphology, local distribution of GABA and glutamate neurons and their synaptic input, reveals a more complicated and heterogeneous raphe. These results provide an important foundation for understanding how specific subfields modulate behavior and for defining which aspects of the circuitry are altered during the etiology of psychological disorders.
... By the same token, the same NPY mechanism in the hippocampus could simultaneously regulate anxiety-like behavior. Dorsal hippocampus is an important component of neuronal circuitry controlling anxiety-related behaviors [1,14], where NPY overexpression is shown to induce an anxiolytic-like effect in the open field and the EPM tests [30]. Hence observed increase in abstinence-related social anxiety-like behavior in nicotine pre-trained HRs may be associated with an anxiogenic neuronal phenotype partly marked by a deficit in hippocampal NPY signaling. ...
An outbred rat model of novelty-seeking phenotype has predictive value for the expression of locomotor sensitization to nicotine. When experimentally naïve rats are exposed to a novel environment, some display high rates of locomotor reactivity (HRs, scores ranking at top 1/3rd of the population), whereas some display low rates (LRs, scores ranking at bottom 1/3rd of the population). Basally, HRs display lower anxiety-like behavior compared to LRs along with higher neuropeptide Y (NPY) mRNA in the amygdala and the hippocampus. Following an intermittent behavioral sensitization to nicotine regimen and 1 wk of abstinence, HRs show increased social anxiety-like behavior in the social interaction test and robust expression of locomotor sensitization to a low dose nicotine challenge. These effects are accompanied by a deficit in NPY mRNA levels in the medial nucleus of the amygdala and the CA3 field of the hippocampus, and increases in Y2R mRNA levels in the CA3 field and corticotropin releasing factor (CRF) mRNA levels in the central nucleus of the amygdala. Systemic and daily injections of a Y2R antagonist, JNJ-31020028, during abstinence fully reverse nicotine-induced social anxiety-like behavior, the expression of locomotor sensitization to nicotine challenge, the deficit in the NPY mRNA levels in the amygdala and the hippocampus, as well as result an increase in Y2R mRNA levels in the hippocampus and the CRF mRNA levels in the amygdala in HRs. These findings implicate central Y2R in neuropeptidergic regulation of social anxiety in a behavioral sensitization to nicotine regimen in the LRHR rats.
... 3,5,6 However, there are several published data that have demonstrated the role of CA1 neural systems in the modulation of anxiety behaviors. 1,[7][8][9] Previous studies have indicated that the serotonergic system of the dorsal hippocampal and in particular, the postsynaptic 5-HT1A receptors, mediate an anxiogenic response, whereas the dorsal hippocampal cholinergic tone mediates an anxiolytic response. 1,2 One report shows that opioids are involved in diverse functions, e.g. ...
In the present study, we investigated the possible influence of the opioidergic system of the dorsal hippocampus on anxiety-like behaviors.
Elevated plus-maze, which is one of the methods used for testing anxiety, was used in the present study. Rats were anesthetized with ketamine and xylazine and special cannulas were inserted stereotaxically into the CA1 region of the dorsal hippocampus. After 1 week of recovery, the effects of intra-CA1 administration of morphine (0.25, 0.5, 1 and 2 µg/rat; 1 µl/rat; 0.5 µl/in each side), naloxone (2, 4, 6 and 8 µg/rat), enkephalin (1, 2, 5 and 10 µg/rat) and naltrindole (0.25, 0.5, 1 and 2 µg/rat) on percentage open arm time (%OAT) and percentage open arm entries (%OAE) were determined.
Bilateral administration of morphine into CA1 decreases %OAT and %OAE, indicating an anxiogenic-like effect. Intra-CA1 injection of naloxone, an opioid receptor antagonist, increased both %OAT and %OAE, parameters of anxiolytic-like behavior. Bilateral administration of δ-opioid receptor agonist, [D-Pen(2,5) ]-enkephalin acetate hydrate into the CA1, induced an anxiolytic-like effect. Furthermore, intra-CA1 injection of δ-opioid receptor antagonist, naltrindole hydrochloride, increased anxiety-related behaviors.
The results of the present study demonstrate that activation of μ-opioid receptors in this area produce an anxiogenic response while activation of δ-opioid receptors produces an anxiolytic response.
... The absence of N/OFQ effects was somewhat unexpected given the role of the hippocampus in modulation of fear/anxiety states (Engin and Treit, 2008) and the high expression of NOP receptor in this brain structure (Darland et al., 1998). Despite its potent suppressive actions on hippocampal synaptic activity, N/OFQ peptide seems to differ from other inhibitory neurotransmitters, such as GABA, in that its action cannot be detected under anxiogenic-provoking situations (Gonzalez et al., 1998). It may be argued that the doses used in this study are not optimal for detecting anxiolytic-like effects of N/OFQ since changes in anxiety and fear learning were found with lower dose-ranges in other structures such as the amygdala (Roozendaal et al., 2007;Uchiyama et al., 2008). ...
Nociceptin/orphanin-FQ (N/OFQ) peptide and its receptor (NOP: N/OFQ opioid peptide receptor) are highly expressed in the hippocampus, but their functional role remains poorly understood. We recently showed that hippocampal N/OFQ inhibits learning and memory abilities in mice. Here, we investigated whether the endogenous peptide also regulated emotional responses at the level of the hippocampus. Bilateral infusions of the selective NOP receptor antagonist, UFP-101 (1-3 nmol/side), into the dorsal hippocampus produced antidepressant-like effects in the mouse forced swim and tail suspension tests comparable with those obtained with the prototypical antidepressant, fluoxetine (10-30 mg/kg, intraperitoneal). In the light-dark test, neither UFP-101 (1-3 nmol/side) nor N/OFQ peptide (1-3 nmol/side) modified anxiety measures when injected at behaviorally active doses in the dorsal hippocampus. These findings show a clear dissociation in the involvement of hippocampal N/OFQ system in anxiety- and despair-related behaviors. We conclude that the dorsal hippocampus is a brain region in which there is an important N/OFQ modulation of mnemonic processes and adaptive emotional responses associated to despair states.
... Corroborating this hypothesis, the fact that Fos expression in the dorsal hippocampus was unchanged by midazolam suggests that the dose used in the present study did not affect memory consolidation between sessions, and the animal recognized the EPM during the retest session. Previous studies have demonstrated no significant effects of midazolam injections into the dorsal hippocampal in rats subjected to test and retest sessions in the EPM (Gonzalez et al., 1998), and lesions of this structure had no effects on any standard behavioral category in the EPM (Treit and Menard, 1997). Regarding the PVN, we have shown recently that the same dose of midazolam that decreased plasma corticosterone levels (Albrechet- Souza et al., 2007) did not change Fos expression in this nucleus in the present study. ...
Prior experience with the elevated plus maze (EPM) increases the avoidance of rodents to the open arms and impairs the anxiolytic-like effects of benzodiazepines on the traditional behaviors evaluated upon re-exposure to the maze, a phenomenon known as one-trial tolerance. Risk assessment behaviors are also sensitive to benzodiazepines. During re-exposure to the maze, these behaviors reinstate the information-processing initiated during the first experience, and the detection of danger generates stronger open-arm avoidance. The present study investigated whether the benzodiazepine midazolam alters risk assessment behaviors and Fos protein distribution associated with test and retest sessions in the EPM. Naive or maze-experienced Wistar rats received either saline or midazolam (0.5 mg/kg i.p.) and were subjected to the EPM. Midazolam caused the usual effects on exploratory behavior, increasing exploratory activity of naive rats in the open arms and producing no effects on these conventional measures in rats re-exposed to the maze. Risk assessment behaviors, however, were sensitive to the benzodiazepine during both sessions, indicating anxiolytic-like effects of the drug in both conditions. Fos immunohistochemistry showed that midazolam injections were associated with a distinct pattern of action when administered before the test or retest session, and the anterior cingulate cortex, area 1 (Cg1), was the only structure targeted by the benzodiazepine in both situations. Bilateral infusions of midazolam into the Cg1 replicated the behavioral effects of the drug injected systemically, suggesting that this area is critically involved in the anxiolytic-like effects of benzodiazepines, although the behavioral strategy adopted by the animals appears to depend on the previous knowledge of the threatening environment.
... Moreover, experimental research indicates that the dorsal hippocampus modulates several anxiety-like responses. For example, Gonzalez et al. [41] found anxiolytic effects in the social interaction test after microinjection of benzodiazepinic sites in GABA A into the dorsal hippocampus. Additionally, Rezayat et al. [42] showed an interaction between GABA and cholecystokinin during modulation anxiety in the elevated plus-maze after injections of agonists and antagonists of both neurotransmitters into the dorsal hippocampus. ...
Selection for contextual fear conditioning is an important behavioral paradigm for studying the role of genetic variables and their interaction with the surrounding environment in the etiology and development of anxiety disorders. Recently, a new line of animals selectively bred for high levels of freezing in response to contextual cues previously associated with footshock was developed from a Wistar population. The purpose of the present study was to evaluate the emotional and cognitive aspects of this new line of animals, which has been named Carioca High-Freezing (CHF). For the characterization of anxious behavior, CHF and control animals were tested in the elevated plus-maze (EPM) and the social interaction test. CHF animals were significantly more anxious than control rats in terms of both the number of entries into EPM open arms and the percentage of time spent in these arms. The time spent in social interaction behavior was also significantly decreased. No statistical differences were found in locomotor activity, as measured by both the number of entries into the closed arms of the EPM and the number of crossings into the social interaction test arena. No differences between CHF and control groups were found in the depression forced swimming test, suggesting that the anxiety trait selected in the CHF line did not interact with affective disorders traits such as those for depression. Cognitive aspects of the CHF rats were evaluated in the object recognition task. Results from this test indicated no difference between the two groups. The present study also encompassed histological analysis of the dorsal hippocampus from CHF and control animals. Results revealed an absence of qualitative and quantitative differences between these two groups of animals in cells located in the dentate gyrus, CA1, and CA3 areas. Therefore, future studies are required to further investigate the possible neural mechanisms involved in the origin and development of the anxious phenotype observed in this model.
... influence of anxiolytic and antipsychotic agents upon FCX levels of dopamine, NAD and 5-HT The prototypical anxiolytic agents and benzodiazepines (BZPs), diazepam and clorazepate, at doses corresponding to those expressing their anxiolytic properties (Millan et al., 1997a), markedly suppressed FCX levels of 5-HT, dopamine and NAD (Table 3). These findings are analogous to those obtained in the hippocampus and other subcortical tissues and reflect the facilitation of GABA A receptor-mediated mechanisms inhibitory to monoaminergic pathways (Pan and Williams, 1989; Miczek et al., 1995; Broderick, 1997; Millan et al., 1997a; Broderick et al., 1998; Gonzalez et al., 1998; Millan et al., 1999b). In fact, it has been suggested that a reinforcement in GABAergic transmission might counteract depressive states (Lloyd et al., 1989; Petty et al., 1995). ...
The frontal cortex (FCX) plays a key role in processes that control mood, cognition and motor behaviour, functions which are compromised in depression, schizophrenia and other psychiatric disorders. In this regard, there is considerable evidence that a perturbation of monoaminergic input to the FCX is involved in the pathogenesis of these states. Correspondingly, the modulation of monoaminergic transmission in the FCX and other corticolimbic structures plays an important role in the actions of antipsychotic and antidepressant agents. In order to further understand the significance of monoaminergic systems in psychiatric disorders and their treatment, it is essential to characterize mechanisms underlying their modulation. Within this framework, the present commentary focuses on our electrophysiological and dialysis analyses of the complex and reciprocal pattern of auto- and heteroreceptor mediated control of dopaminergic, noradrenergic and serotonergic transmission in the FCX. The delineation of such interactions provides a framework for an interpretation of the influence of diverse classes of antidepressant agent upon extracellular levels of dopamine, noradrenaline and serotonin in FCX. Moreover, it also generates important insights into strategies for the potential improvement in the therapeutic profiles of antidepressant agents.
... These results are in agreement with our own previous observations and those of others suggesting that the EPMZ and SI tests measure different dimensions of anxiety-like behavior in rats (e.g., File, 1992;Ramos et al., 1997;Cecchi et al., 2002). Stimulation of benzodiazepine receptors in the hippocampus or median raphe nucleus caused anxiolytic effects in the SI test, but not the EPMZ (Gonzalez et al., 1998). Nicotine administration in the dorsal hippocampus produced anxiogeniclike behavior in the SI test, but was without effect in the EPMZ (File et al., 2000). ...
The brain noradrenergic system is activated by stress, and modulates the activity of forebrain regions involved in behavioral and neuroendocrine responses to stress, such as the lateral bed nucleus of the stria terminalis (BSTL). This region of the limbic forebrain receives dense noradrenergic innervation, and has been implicated in both anxiety and regulation of the hypothalamic–pituitary–adrenal axis. We hypothesized that stress-induced release of norepinephrine in the BSTL modulates anxiety-like behavioral responses to stress and activation of the hypothalamic–pituitary–adrenal stress axis.
... An alternative explanation is that differences between plus-or zero-maze experiments and other paradigms may simply result from variability in the former. Although intrahippocampal midazolam was reported to be inactive in the plus-maze (Gonzalez, Ouagazzal, & File, 1998), a more recent study showed that it was active (Menard & Treit, 2001). It is possible, therefore, given the variability of such behavioral tests, that under suitable circumstances the plus-or zero-maze might be sensitive to hippocampal manipulations. ...
Rats with cytotoxic lesions of the hippocampus were given 3 anxiety tests: social interaction with a novel rat, the elevated zero-maze (a modification of the plus-maze), and hyponeophagia (eating familiar and novel foods in a novel place). Marked anxiolytic effects were seen in the social interaction and hyponeophagia tests, but not on the zero-maze. These results confirm and extend previous experiments that used traditional lesion techniques. The zero-maze result was consistent with other experiments using the plus-maze, in which intrahippocampal administrations of pharmacological agents were not anxiolytic, although variability in ethological tests may also be a factor. As the hyponeophagia test used an elevated apparatus, as in the zero- and plus-mazes, the lack of a lesion effect in the zero-maze was unlikely to have been due to an inability to relieve height-induced anxiety.
Temporal lobe epilepsy (TLE), which is one of the most common neurological diseases, is accompanied by a high incidence of psychiatric disorders. Among these psychiatric disorders, anxiety is one of the major psychiatric comorbidities in epilepsy patients. However, anxiety in epilepsy patients often remains unrecognized and untreated. It is believed that the inhibitory networks of γ-aminobutyric acid (GABA) neurotransmission play pivotal roles in the modulation of emotion and mood responses in both physiological and pathological conditions. The impairment of neurotransmission mediated by GABAergic signaling is related to the pathophysiology of anxiety. However, it remains unclear whether and how GABAergic signaling modulates anxiety responses in the context of an epileptic brain. In the present study, we sought to determine the role of inhibitory networks of GABAergic signaling in the anxiety-like behavior of epileptic mice. Our results show epileptic mice exhibited increased anxiety-like behavior, and this increased anxiety-like behavior was accompanied by a decrease in GABAergic interneurons and an increase in GABA type A receptor (GABA A R) β3 subunit (GABRB3) expression in the hippocampus. Furthermore, the activation of GABA A Rs produced an anxiolytic-like effect, while the inhibition of GABA A Rs elicited an anxiogenic-like effect in the epileptic mice, suggesting that the alteration of GABAergic signaling is associated with anxiety-like behavior in epileptic mice. Thus, targeting GABAergic signaling in the epileptic brain may provide an effective anxiolytic treatment in epilepsy patients.
The role of 5-HT2C receptors (5-HT2CRs) in the regulation of anxiety has been widely acknowledged. However, conflicting results have been reported on whether stimulation of these receptors increases or decreases anxiety. We here investigated the role of 5-HT2CRs of the dorsal hippocampus (DH) in the mediation of anxiety- or panic-associated defensive behaviors and in the anxiolytic effect of the tricyclic antidepressant imipramine. In the Vogel conflict test, administration of the mixed 5-HT2CR agonist mCPP into the DH of male Wistar rats was anxiogenic, whereas infusions of the more selective agonists MK-212 and RO-600175 were anxiolytic. The 5-HT2CR antagonist SB-242084, on the other hand, was anxiogenic. A sub-effective dose of this antagonist blocked the anxiolytic effect of RO-600175, but not the increase in anxiety observed with mCPP, indicating that the latter effect was not due to 5-HT2CR activation. In full agreement with these findings, MK-212 and RO-600175 in the DH also inhibited inhibitory avoidance acquisition in the elevated T-maze, whereas SB-242084 caused the opposite effect. None of these drugs interfered with escape expression in this test, which has been associated with panic. Chronic administration of imipramine (15 mg/kg, ip, 21 days) caused an anxiolytic effect in the elevated T-maze and light-dark transition tests, which was not blocked by previous infusion of SB-242084 into the DH. Therefore, facilitation of 5-HT2CR-mediated neurotransmission in the DH decreases the expression of anxiety-, but not panic-related defensive behaviors. This mechanism, however, is not involved in the anxiolytic effect caused by imipramine.
Panic disorder (PD) is a highly incapacitating psychiatric disorder. Its wide range of somatic and psychological symptoms makes it plausible that a number of different brain structures and circuits are likely to mediate this condition. In this chapter we highlight the possible contributions of the hippocampus, a key brain region involved in the regulation of cognition (learning/memory), mood and defensive responses (fear/anxiety), for the pathophysiology of PD. This chapter will present the anatomy of the hippocampus and highlight its role in emotional regulation, so that an understanding of the involvement of the hippocampus in PD can be drawn. Evidence from both animal and human findings on this topic will be approached. Particularly, the capacity of the hippocampus to continually generate newly functional neurons throughout life, a phenomenon called adult hippocampal neurogenesis, will be pointed as part of the key future directions for the study of the neurobiological basis of PD.
Complete and dorsal hippocampal lesions impaired spatial performance on 2 working memory tasks: rewarded alternation on the T maze and matching to position in the water maze. In contrast, ventral hippocampal lesions had no effect on these tasks, even when task difficulty was increased by the introduction of delays. Ventral lesions did resemble complete lesions in reducing anxiety in 3 commonly used tests of anxiety (social interaction, plus-maze, and hyponeophagia). Dorsal lesions also appeared to be anxiolytic in the social interaction and plus-maze tests, but they did not affect hyponeophagia. Complete- and dorsal-lesioned rats displayed hyperactivity, whereas ventral-lesioned rats did not. These results show a double dissociation between dorsal and ventral hippocampal lesions (hyponeophagia vs. spatial memory), suggesting differentiation of function along the septotemporal axis of this structure.
The gastro-intestinal tract hosts a complex microbial community, the gut microbiota, whose collective genome coding capacity vastly exceeds that of the host genome. The involvement of the gut microbiota in various aspects of the host physiology, such as the nutritional metabolism and the immunity, has long been studied. In contrast, the possible action of the gut microbiota on brain development and functioning is a new line of research, still poorly explored. In this context, we performed a first general study of the effect of gut microbiota on the brain by comparing the sensory-motor functions, the anxiety-like behaviour, the activation of the hypothalamic-pituitary-adrenal axis and the brain monoamine profile in germ-free and conventional F344 rats. The results show that, in this particularly stress-sensitive strain, absence of gut microbiota exacerbates the anxiety-like behaviour and neuroendocrine response to stress, and reduces brain dopamine metabolism. To investigate the means by which the microbiota can affect the brain, a second study was conducted, targeting a specific bacterial metabolite, indole, whose oxidative derivatives, produced by the liver, are known to have neuroactive properties. Indole is a natural metabolite of the gut microbiota, whoseoverproduction could occur during a microbiota dysbiosis. Two conditions of overproduction, namely chronic and acute, were modelled. In both cases, significant changes in the behaviour of the host were observed. In chronic overproduction, indole promotes anxiety- and depressive-like behaviours, while acute overproduction has a marked sedative effect. From a mechanistic point of view, we confirm that indole can act on the central nervous system through its oxidized derivatives and show for the first time that it can also act by activating the brain nuclei of the vagus nerve.
In clinical populations, prevalence rates for a number of anxiety disorders differ between males and females and gonadal hormones are thought to contribute to these differences. While these hormones have been shown to modulate the anxiolytic effects of the benzodiazepine agonist diazepam in some models, findings are inconsistent. Here, we tested for sex differences in response to anxiogenic stimuli following a 30-min diazepam (1.0mg/kg) pre-treatment in male and female rats showing high (HAn) and low (LAn) anxiety-like behavior on the elevated plus maze. Acute diazepam administration resulted in decreased anxiety-like behavior only in HAn males as demonstrated by a significant increase in percent open arm time in the elevated plus maze (EPM). Immunohistochemical analysis for parvalbumin (PV; a calcium-binding protein that selectively stains GABAergic neurons) in central amygdala (CeA), caudate putamen (CPu) and the hippocampus indicated the number of GABAergic interneurons in these areas differed across sex and anxiety trait. In the CPu, females had significantly more PV-immunoreactive (IR) cells than males, and LAn females had greater PV-IR neurons than HAn females. In the CeA, males displayed an increased number of PV-IR neurons compared to females, with no differences found between LAn and HAn. Further, trait differences were evident in the CA2 region of the hippocampus, regardless of sex. Taken together, these data suggest that gonadal hormones and trait anxiety may influence the sensitivity to the anti-anxiety effects of diazepam and these differences may be due in part to the distribution of GABA-containing interneurons.
Rationale: The benzodioxane, S15535, possesses low intrinsic activity and marked selectivity at 5-HT1A receptors, hippocampal populations of which are implicated in anxious states. Objective: Herein, we examined its potential anxiolytic actions in relation to its influence upon extracellular levels of 5-HT in the dorsal hippocampus of freely-moving rats. Its effects were compared with those of other anxiolytic agents: the 5-HT1A agonists, buspirone and 8-hydroxy-2-(di-n-propylamino)-tetralin HBr (8-OH-DPAT), the 5-HT2C antagonist, SB206,553 and the benzodiazepine, diazepam. Methods: Potential anxiolytic actions were evaluated in the Vogel conflict paradigm (increase in punished responses) and the social interaction (SI) test (increase in active SI) in rats. Extracellular levels of 5-HT were determined by microdialysis. Results: In analogy to diazepam, S15535 increased punished responses in the Vogel test. This action was dose dependently expressed over a broad (16-fold) dose range. Buspirone and 8-OH-DPAT were likewise active, but yielded highly biphasic dose–response curves. SB206,553 was dose dependently active in this model. In the SI test, S15535 similarly mimicked the anxiolytic-like effect of diazepam and was active over a broad dose range. Buspirone and 8-OH-DPAT again showed biphasic dose–response curves, as did SB206,553. In both the Vogel and SI tests, the anxiolytic-like effects of S15535 were abolished by the selective 5-HT1A receptor antagonist, WAY100,635, which was inactive alone. S15535 exerted its anxiolytic-like effects with a more pronounced separation to motor-disruptive doses than the other drugs. Finally, S15535 suppressed dialysate levels of 5-HT in the dorsal hippocampus, an action abolished by WAY100,635. Buspirone, 8-OH-DPAT and diazepam, but not SB206,553, also reduced 5-HT levels. Conclusion: Likely reflecting its distinctive ability to selectively and preferentially activate pre- versus postsynaptic 5-HT1A receptors, S15535 suppresses hippocampal 5-HT release and displays marked anxiolytic-like effects over a broad dose range in the relative absence of motor perturbation.
1.1. Different animal tests model different anxiety disorders. Thus, the social interaction test is a model of generalised anxiety disorder, plus-maze Trial 1 models elements of panic disorder and Trial 2 in the elevated plus-maze is a model of specific phobia.2.2. Studies of the neuroanatomical and neurochemical pathways controlling behaviour in these different tests provides information on the neurobiological mechanisms modulating anxiety disorders.3.3. In the social interaction test, nicotine and 8-OH-DPAT had anxiogenic effects when injected into the dorsal hippocampus or the lateral septum.4.4. These ligands were without effect on Trial 1 in the plus-maze when injected into the dorsal hippocampus, but had anxiogenic effects when injected into the lateral septum.5.5. On Trial 2 in the elevated plus-maze, nicotine had an anxiolytic effect, but 8-OH-DPAT had an aaxiogenic effect when injected into the dorsal hippocampus.
A review of the literature suggests that the dorsal hippocampal serotonergic system, and, in particular, the postsynaptic 5-HT1A receptor, mediates an anxiogenic response, whereas endogenous dorsal hippocampal cholinergic tone mediates an anxiolytic response. Accordingly, it has been shown that direct dorsal hippocampal administration of the 5-HT1A receptor agonist, 8-OH-DPAT, the nicotinic receptor antagonist, mecamylamine, and the M1 muscarinic receptor antagonist, pirenzepine, all have anxiogenic effects in rats tested in the social interaction test. It is therefore surprising that nicotine also has an anxiogenic effect in this test following dorsal hippocampal administration. However, the anxiogenic effects of mecamylamine and nicotine in the dorsal hippocampus are blocked by coadministration of the 5-HT1A receptor antagonist, WAY 100635, suggesting that both of these compounds act by enhancing hippocampal serotonergic transmission, thereby stimulating postsynaptic 5-HT1A receptors. This conclusion is supported by the observation that both nicotine and mecamylamine stimulate basal [3H]-5-HT release from dorsal hippocampal slices. A possible mechanism by which nicotinic receptor ligands modulate hippocampal 5-HT release is discussed, and it is proposed that the dorsal hippocampal serotonergic and cholinergic systems are tightly coupled and function antagonistically in the modulation of anxiety, as measured in the social interaction test. These systems are relatively unimportant in controlling behaviour on trial 1 in the plus-maze. On trial 2 in the elevated plus-maze, a model of specific phobia, the endogenous cholinergic system, nicotine, and the M1 receptor agonist, McN-A-343, all mediate an anxiolytic effect, whereas stimulation of 5-HT1A receptors mediates an anxiogenic effect. It is proposed that the hippocampus may predominantly control the avoidance components of phobic anxiety, with other regions, such as the dorsomedial hypothalamus, controlling the escape components.
Abnormal behaviors and death associated with the use of oseltamivir (Tamiflu) have emerged as a major issue in influenza patients taking the drug. Here, we investigated the mechanisms underlying the effects of oseltamivir on the behavior of mice using light-dark and open-field preference tests. Oseltamivir (75 and 150 mg/kg, intraperitoneally (i.p.)) alone affected neither time spent in the open area in the light-dark preference test nor ambulation in the open-field test at 2h post-injection. However, a non-selective adenosine A(1)/A(2) receptor antagonist, caffeine (10mg/kg, i.p.) in combination with oseltamivir (150 mg/kg, i.p.) increased time spent in the open area in the light-dark preference test. This enhancement was not inhibited by a benzodiazepine receptor antagonist, flumazenil (10-20mg/kg, subcutaneously (s.c.)). Enhancement of ambulation in the open-field test was also observed when caffeine (10mg/kg, i.p.) was combined with oseltamivir (150 mg/kg, i.p.). This enhancement was inhibited by a dopamine D(2) receptor antagonist, haloperidol (0.1mg/kg, s.c.). Furthermore, an adenosine A(2) receptor antagonist, SCH58261 (3mg/kg, i.p.) in combination with oseltamivir (150 mg/kg, i.p.) increased ambulation in the open-field test, while an adenosine A(1) receptor antagonist, DPCPX (1-3mg/kg, i.p.) did not. These findings suggest that the actions of oseltamivir may involve the dopamine and adenosine systems. Our findings suggest that due to the interaction between central blockade of adenosine A(2) receptors by caffeine, and oseltamivir-induced behavioral changes, patients being treated with oseltamivir should be closely monitored.
Rationale: Lead-exposure during developmental periods may alter reinforcing patterns of drugs of abuse in adulthood. Anxiety related mechanisms may also influence drug intake. Interactions between the two altering factors may exist. Objectives: The present study examined the effects of perinatal lead-exposure on cocaine self-administration after a GABAA antagonist pre-treatment. Methods: Female rats were exposed to a regimen of 16 mg lead daily for 30 days prior to breeding with un-exposed males. This continued throughout gestation and lactation until postnatal day (PND) 21. On PND 63, animals were implanted with indwelling jugular catheters. After a 7 day recovery period, animals were trained to self-administer 0.50 mg/kg cocaine intravenously [IV]. After stable responding had been established, testing procedures began using combinations of 0.03 and 0.06 mg/kg cocaine [IV] and 0.00, 0.50, 1.00 and 2.00 mg/kg bicuculline (a GABAA antagonist) intraperitoneal [IP]. Results: Bicuculline pre-treatment caused directionally opposite effects in both treatment groups (Group 0-Lead and Group 16-Lead) at the 0.06 mg/kg cocaine dose. Group 0-Lead animals showed an increase in self-administration, while Group 16-Lead animals showed a decrease in responding on the active (cocaine) lever. Results at the 0.03 mg/kg cocaine dose showed no discernable pattern. Group 0-Lead animals decreased in active lever responding at the 2.00 mg/kg bicuculline dose. Group 16-Lead animals showed no differences in responding at any dose of bicuculline. Conclusions: These data further suggest the influential role of GABA in mediating cocaine reward and the ability of developmental lead-exposure to alter mechanisms mediating drug responsiveness even after exposure has terminated.
One of the most profound properties of central neuropeptide Y (NPY) is its anxiolytic, or anti-anxiety, effect. This has been demonstrated repeatedly in a number of animal models. In addition, stressors affect NPY expression in the central nervous system, with acute and repeated (chronic) stress having differential effects. Here, a brief summary of some work performed in our laboratory is presented that supports a role for NPY in regulation of stress responses and behaviors.
The elevated plus-maze provides a test situation in which distinctive states of anxiety are elicited on trials 1 and 2 and the dorsal hippocampus has previously been shown to mediate the anxiogenic effects of (-)-nicotine in the social interaction test.
To determine the effects of a wide dose range of (-)-nicotine on trial 1 and 2 in the plus-maze after systemic administration and whether the dorsal hippocampus is a site mediating the anxiogenic effect of nicotine.
(-)-Nicotine (0.001, 0.005, 0.01, 0.05, 0.1, 0.5 and 1 mg/kg) was injected IP 30 min before testing for 5 min in the plus-maze. Rats receiving dorsal hippocampal infusions received bilateral infusions of 0.5 microl of artificial CSF or (-)-nicotine (0.1, 1, 4 or 8 microg). The needle was left in place for 50 s after injection and testing took place 3 min later. Rats tested on trial 1 were naive to the plus-maze, those tested on trial 2 had received a previous 5-min undrugged exposure to the maze 48 h earlier.
Low doses of (-)-nicotine (0.001, 0.005, 0.01, 0.05 and 0.1 mg/kg, IP) were without effect on either trial, but higher doses (0.5 and 1 mg/kg, IP) had anxiogenic effects on both trials, as shown by decreases in percentage time spent and percentage entries onto the open arms. Infusion of (-)-nicotine (0.1, 1, 4 and 8 microg) bilaterally into the dorsal hippocampus was without effect on trial 1, but 1 microg had an anxiolytic effect on trial 2, shown by an increased percentage time spent on the open arms.
The results on both trials in the plus-maze after systemic administration of nicotine add to previous reports from the social interaction test that high doses of nicotine have anxiogenic effects. However, the effects of nicotine in the dorsal hippocampus are different in all three anxiety tests (anxiogenic in social interaction, ineffective on trial 1, anxiolytic on trial 2) showing that nicotinic cholinergic control in this brain region may vary depending on the state and/or type of anxiety generated by the test. The brain region(s) underlying the anxiogenic effects of IP nicotine on both trials in the plus-maze remain to be identified.
Nitric oxide (NO) is a neuromodulator and an intercellular and retrograde messenger that mediates several functions in the central nervous system. The effects of ethanol (EtOH) on neuronal NO-dependent pathways have been the focus of recent research. Most studies have concentrated on the actions of chronic EtOH exposure. In this study, we examined the role of NO-dependent pathways in the acute actions of EtOH.
We used the elevated plus-maze test to study the role of NO-dependent pathways in the behavior of rats treated with acute EtOH. We tested the effects of 7-nitroindazole, a reversible competitive inhibitor of nitric oxide synthase. We also studied the effects of the cyclic guanylate monophosphate (cGMP) analog, 8-Bromoguanosine cyclic 3',5'-monophosphate sodium salt, and the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside.
When injected by either intraperitoneal (6 mg/kg) or intrahippocampal (20 nmol) routes, 7-nitroindazole increased the percentage of open arm entries and time spent in open arms for rats injected with EtOH (1.0 g/kg, ip). This dose of EtOH did not produce an anxiolytic effect when administered alone. Additional experiments were performed with a dose of 1.2 g/kg of EtOH (ip), which produced an anxiolytic effect. Intrahippocampal administration of the cGMP analog, 8-Bromoguanosine cyclic 3',5'-monophosphate sodium salt (40 nmol), or the NO donors S-nitroso-N-acetylpenicillamine (20 or 40 nmol) or sodium nitroprusside (20 or 40 nmol) blocked the anxiolytic effect of this dose of EtOH.
These results indicate that inhibition of NO-dependent pathways enhances, whereas stimulation of these pathways decreases, the efficacy of EtOH to produce anxiolytic effects in rats. We postulate that NO-dependent increases in guanylate cyclase activity and cGMP levels oppose the anxiolytic effects produced by acute EtOH administration.
The effects of nicotine administration into the dorsal hippocampus and lateral septum provide further evidence that different neurochemical and neuroanatomical substrates control behaviour in different animal tests. Thus, in the social interaction test (a model of generalised anxiety disorder), bilateral administration of nicotine (1-4 microg) into both regions has anxiogenic effects in test conditions that generate moderate anxiety. The anxiogenic effects are mediated by a nicotine-evoked increase in 5-hydroxytryptamine (5-HT) release and are reversed by co-administration of the 5-HT(1A) receptor antagonist, N-(2-(6-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)-cyclohex -ane carboxamide trichloride (WAY 100,635). On trial 1 in the elevated plus-maze (which models the escape components of panic disorder), nicotine is without effect when administered to the dorsal hippocampus, but has anxiogenic effects after lateral septal administration. On trial 2 in the elevated plus-maze (a model of specific phobia), nicotine (1 microg) has anxiolytic effects when administered to the dorsal hippocampus, but is ineffective (4 and 8 microg) in the lateral septum.
In the present work, we studied the effects of benzodiazepine (BZ) receptor antagonist, flumazenil, and of the agonist, diazepam, on social interaction-induced transient changes in defensive burying (DB). Enhanced defensive burying was observed after 1.5 min of social interaction experience, while a longer social interaction experience, 15 min, inhibited the expression of burying behavior. Defensive burying and social interaction paradigms have been used for the screening of compounds with anxiolytic potential and, more extensively, to study the neurobiology of anxiety. To elucidate the participation of the BZ receptor on transient changes induced by intervals of social interaction experience, its receptor antagonist, flumazenil (2.5, 5, and 10 mg/kg) was intraperitoneally injected (IP). Flumzenil enhanced in a dose-dependent manner, the blocking effect of the saline IP injection on facilitated DB in 1.5-min social interaction-experienced subjects. In addition, flumazenil enlarged in a dose-dependent manner the blocking effect of saline IP on defensive burying levels in animals exposed to social interaction experience for 15 min. To analyze the presumed participation of the BZ receptor mediating enhanced burying behavior levels in subjects exposed to 1.5 min of social interaction, a suboptimal dose of diazepam (0.25 mg/kg) was administered. Diazepam enhanced the saline IP elicited defensive burying reduction. Results are discussed in terms of the suggested BZ receptor mediation on transient changes in defensive burying elicited by social interaction experience.
In the elevated plus-maze test of anxiety, nicotine (0.1 mg/kg sc; 30 min after injection) had a significant anxiogenic effect, shown by specific decreases in the percentage of time spent on the open arms and in the percentage of open-arm entries. Tolerance developed to this anxiogenic effect after 7 days of nicotine treatment (0.1 mg/kg/day). Five minutes after an acute injection, nicotine (0.1 mg/kg) was ineffective, but after 7 days of treatment a significant anxiolytic effect, shown by specific increases in the percentage of time spent on the open arms and in the percentage of open-arm entries, emerged. After 14 days of nicotine treatment, tolerance developed to this anxiolytic effect. There was a complete dissociation between the effects of nicotine on the measures of anxiety, and on the locomotor activity as measured by closed-arm entries. No changes in closed-arm entries were found after acute administration of nicotine, but rats tested 30 min after their 7th injection made significantly fewer, and those tested 5 min after their 14th injection made significantly more, entries than their respective controls. Rats that were tested after 24 h withdrawal from six daily nicotine injections showed a significant anxiogenic effect. A low dose of nicotine (5 ng) injected into the dorsal hippocampus was without effect in vehicle pretreated rats, but it was able to reverse the anxiogenic effect found after 24 h of withdrawal from 6 days of nicotine treatment.
In the social interaction test of anxiety, microinjections of midazolam (2-8 microg) into the dorsal hippocampus or dorsal raphé nucleus significantly increased the time spent in active social interaction, without changing locomotor activity, thus indicating specific anxiolytic effects. However, tolerance developed to these effects in rats that had been pre-treated for 6 days with (-)-nicotine (0.1 mg/kg/day; subcutaneous). Thus, cross-tolerance to the anxiolytic effects of midazolam develops rapidly following a short period of treatment with a low dose of nicotine, which contrasts with the more slowly developing tolerance (about 3 weeks) that develops after benzodiazepine treatment. Following 6 days of nicotine treatment there was a significant reduction in [(3)H]flunitrazepam binding at 2 and 10 nM in the hippocampus, but no change in the midbrain. The decrease in benzodiazepine binding could explain tolerance to the effects of midazolam when administered to the dorsal hippocampus, but other mechanisms, such as indirect effects on the serotonergic (5-HT) system, might be involved in tolerance to the effects of dorsal raphé nucleus administration.
The influence of peripheral benzodiazepine receptor ligands Ro5-4864 (0.05 or 1.0 mg/kg, i.p.) or PK11195 (0.05 or 1.0 mg/kg, i.p.) on the anxiolytic effect of ethanol (1.2 g/kg; 14% p/v; i.p.) was investigated in rats tested on the elevated plus-maze. Other animals were injected through intrahippocampal administrations of the ligands (0.5 or 1.0 nmol/0.5 &mgr;l) before ethanol (1.2g/kg; 14% p/v; i.p.) and submitted to the elevated plus-maze test. The results showed that the systemic administration of either ligands 24 hours before the ethanol treatment resulted in a reduced anxiolytic effect of this drug. Only PK11195 reversed the effect of ethanol after intrahippocampal injection. These data suggest that peripheral benzodiazepine receptors play a role in ethanol anxiolysis.
1. The available evidence suggests that stress induced release of acetylcholine (ACh) in the brain has a significant role in mediating neuroendocrine, emotional, and physiological responses to stress. Recent findings also suggest that stress indirectly (via acetylcholine) and nicotine directly stimulates the HPA axis through activation of nAChRs. 2. Our working hypothesis is that under stressful conditions, nicotinic receptor antagonists, such as mecamylamine, should act to attenuate the activation of the HPA axis and exhibit anxiolytic behavioral effects. The purpose of this study was to determine whether or not mecamylamine would: a) produce anxiolytic effects in rats on the elevated plus maze and b) blunt the plasma corticosterone response to predator stress in rats. 3. Results suggested that mecamylamine has anxiolytic properties under stressful conditions. In the EPM experiment, mecamylamine (0.3 mg/kg) produced increased time spent in the open arms. Similarly, in the predator stressor experiment, mecamylamine blunted the stress-induced plasma corticosterone response, with the lowest dose of mecamylamine (0.1 mg/kg). 4. These findings may have important therapeutic implications since clinical observations have shown that low doses of mecamylamine reduce tension and anxiety in patients with Tourette syndrome.
Effects of septal and amygdaloid lesions were compared in 2 models of rat “anxiety.” Septal lesions decreased burying behavior in the “shock-probe burying test” and increased open-arm exploration in the “elevated plus-maze test,” whereas amygdaloid lesions produced neither of these anxiolytic effects. However, amygdaloid lesions increased rats' contacts of the electrified probe, an anxiolytic effect not produced by septal lesions. Each of these distinct, anxiolytic effects of septal or amygdaloid lesions were displayed together in animals with lesions of both structures. FurtherXSmore, the magnitude of these anxiolytic effects after combined lesions was comparable to their magnitude after individual lesions. Taken together, these results suggest the amygdala and the septum independently control the expression of different fear-related behaviors.
The purpose of this study was to determine the roles of the presynaptic 5-hydroxytryptamine 1A (5-HT 1A ) receptors in the median raphénucleus (MRN) and of the postsynaptic 5-HT 1A receptors in its projection area of the dorsal hippocampus in the social interaction and elevated plus-maze tests of anxiety. Direct administration of the 5-HT 1A receptor agonist (±)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT, 200 ng) into the MRN had significant anxiolytic effects in all three test situations examined (social interaction, plus-maze trials 1 and 2). These anxiolytic effects were antagonized by a silent dose (200 ng) of the 5-HT 1A receptor antagonist WAY 100635, confirming that they were mediated by 5-HT 1A receptors. In contrast, after bilateral administration to the dorsal hippocampus, 8-OH-DPAT (100 ng) had significant anxiogenic effects in the social interaction test and in plus-maze trial 2. These anxiogenic effects were antagonized by silent doses of 5-HT 1A receptor antagonists [(+)WAY 100135, 10 mg/kg s.c., and intrahippocampal (±)tertatolol, 3 μg, respectively], confirming mediation by 5-HT 1A receptors. In rats naive to the plus-maze, neither 8-OH-DPAT (50, 100, or 200 ng) nor the 5-HT 1A receptor antagonist (±)tertatolol (3 μg) had any significant effect when administered to the dorsal hippocampus. This demonstrates that previous experience of a rat in the plus-maze has a major effect on the sensitivity of dorsal hippocampal 5-HT 1A receptors, as we have demonstrated previously for the 5-HT 1A receptors in the dorsal raphé nucleus. Overall, our results provide evidence that stimulation of the presynaptic 5-HT 1A receptors in the MRN results in an anxiolytic action, whereas stimulation of the post-synaptic 5-HT 1A receptors in its projection area results in an anxiogenic effect. These results are consistent with an overall reduction in 5-HT neurotransmission in the dorsal hippocampus having an anxiolytic effect, and they explain the relatively weak anxiolytic profile detected when 5-HT 1A receptor agonists are given systemically.
The function of the central cannabinoid receptor (CB1) was investigated by invalidating its gene. Mutant mice did not respond to cannabinoid drugs, demonstrating the exclusive
role of the CB1 receptor in mediating analgesia, reinforcement, hypothermia, hypolocomotion, and hypotension. The acute effects of opiates
were unaffected, but the reinforcing properties of morphine and the severity of the withdrawal syndrome were strongly reduced.
These observations suggest that the CB1 receptor is involved in the motivational properties of opiates and in the development of physical dependence and extend the
concept of an interconnected role of CB1 and opiate receptors in the brain areas mediating addictive behavior.
Changes in release and uptake of [3H]5-HT and [14C]GABA were compared in slices taken from the hippocampus and frontal cortex of rats, left undisturbed in their home-cages, or exposed for 5 min to the elevated plus-maze or social interaction tests of anxiety. Exposure to the plus-maze decreased cortical GABA function (shown by decreased release) and increased hippocampal 5-HT function (shown by increased K(+)-evoked release but more markedly by decreased uptake). Compared with undisturbed home cage controls, only the high light, familiar condition of the social interaction test resulted in a significant increase in K(+)-evoked release of both [3H]5-HT and [14C]GABA from the hippocampus. All four social interaction test conditions resulted in increases in cortical uptake of [3H]5-HT and all but the high light, unfamiliar condition increased cortical uptake of [14C]GABA. Analysing the two factors manipulated in the social interaction test, unfamiliarity with the test arena resulted in increased uptake of hippocampal [3H]5-HT and decreased cortical [14C]GABA, whereas an increase in the level of light decreased the cortical uptake of [14C]GABA. The results show that changes in presynaptic function occur rapidly in response to a brief exposure to animal tests of anxiety. However, only the increased hippocampal release of 5-HT is likely to be causally linked to anxiety and the results show that this cannot be the sole explanation.
The purpose of this study was to determine the roles of the presynaptic 5-hydroxtryptamine1A (5-HT1A) receptors in the median raphé nucleus (MRN) and of the postsynaptic 5-HT1A receptors in its projection area of the dorsal hippocampus in the social interaction and elevated plus-maze tests of anxiety. Direct administration of the 5-HT1A receptor agonist (+/-)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT, 200 ng) into the MRN had significant anxiolytic effects in all three test situations examined (social interaction, plus-maze trails 1 and 2). These anxiolytic effects were antagonized by a silent dose (200 ng) of the 5-HT1A receptor antagonist WAY 100635, confirming that they were mediated by 5-HT1A receptors. In contrast, after bilateral administration to the dorsal hippocampus, 8-0H-DPAT (100 ng) had significant anxiogenic effects in the social interaction test and in plus-maze trial 2. These anxiogenic effects were antagonized by silent doses of 5-HT1A receptor antagonists [(+)WAY 100135, 10 mg/kg s.c., and intrahippocampal (+/-)tertatolol, 3 microg, respectively], confirming mediation by 5-HT1A receptors. In rats naive to the plus-maze, neither 8-OH-DPAT (50, 100, or 200 ng) nor the 5-HT1A receptor antagonist (+/-)tertatolol (3 microgram) had any significant effect when administered to the dorsal hippocampus. This demonstrates that previous experience of a rat in the plus-maze has a major effect on the sensitivity of dorsal hippocampal 5-HT1A receptors, as we have demonstrated previously for the 5-HT1A receptors in the dorsal raphé nucleus. Overall, our results provide evidence that stimulation of the presynaptic 5-HT1A receptors in the MRN results in an anxiolytic action, whereas stimulation of the post-synaptic 5-HT1A receptors in its projection area results in an anxiogenic effect. These results are consistent with an overall reduction in 5-HT neurotransmission in the dorsal hippocampus having an anxiolytic effect, and they explain the relatively weak anxiolytic profile detected when 5-HT1A receptor agonists are given systemically.
A single 5 min exposure to the elevated plus-maze test of anxiety renders animals insensitive to the anxiolytic effects of the benzodiazepines in this test. The purpose of the present experiments was to explore whether this phenomenon resulted from a change in the functional state of benzodiazepine receptors in the dorsal raphe nucleus. The benzodiazepine receptor agonist midazolam (0.5, 1, and 2 microg) and antagonist flumazenil (100 and 500 ng) were directly administered to the dorsal raphe nucleus in rats either naive to, or with one previous 5 min exposure of, the elevated plus-maze. In naive rats, midazolam produced significant anxiolytic effects at all doses, and flumazenil was silent. In plus-maze-experienced rats, midazolam no longer had anxiolytic effects in the plus-maze, but flumazenil did, indicating that the previous experience of the maze had changed the state of the benzodiazepine receptor. This changed receptor function generalized to the social interaction test. Thus, in naive animals tested in high light, midazolam (0.5, 1, and 2 microg) had significant anxiolytic effects and flumazenil (100 and 500 ng) was silent, whereas in plus-maze-experienced rats both midazolam (1 microg) and flumazenil (500 ng) had significant anxiolytic effects. Extensive analysis of locomotor activity in both tests showed that the changed responsivity to midazolam could not be explained by habituation, because on none of the measures used was there any difference in motor activity scores between plus-maze-naive and experienced rats.
On the basis of our previous series of experiments we had postulated that the increased anxiety that occurred during diazepam withdrawal was mediated by increased 5-HT release in the hippocampus. The present series of experiments provide evidence for a major role of the median raphé nucleus (MRN) dorsal hippocampal pathway. Rats were treated once daily for 21 days with diazepam (2 mg/kg IP) and then tested after 24 h withdrawal in the social interaction test of anxiety. Relative to chronically vehicle treated animals, those withdrawn from diazepam were significantly more anxious and had significantly greater K(+)-evoked release of [3H]-5-hydroxytryptamine (5-HT) from slices of dorsal and of ventral regions of the hippocampus. Estimation of extracellular concentrations of 5-HT within the dorsal hippocampus, using in-vivo microdialysis, showed doubling in the levels of 5-HT in the rats withdrawn from chronic diazepam treatment. This just failed to reach significance, but 33% of the rats showed dramatic increases (650%). It was not possible to test these animals in the social interaction test, but it is proposed that only the diazepam-withdrawn rats with raised extracellular levels of 5-HT would have displayed increased anxiety. 5-HT1A receptor agonists injected into the MRN decrease the MRN firing rate, and hence the release of 5-HT in the dorsal hippocampus. As a further test of our hypothesis, we examined the effects of MRN injection of the 5-HT1A receptor agonist, 8-OH DPAT, on animals withdrawn from diazepam and tested in the low light familiar condition of the social interaction test. 8-OH DPAT (50-200 ng) dose-dependently reversed the anxiogenic effect of diazepam withdrawal, while having no effects in chronic vehicle-treated animals. These results provide clear evidence that the MRN-dorsal hippocampal 5-HT pathway is at least one of the pathways playing an important role in mediating diazepam withdrawal-induced anxiety.
Fear reactions of rats given bilateral lesions to the septum, hippocampus, or amygdala were compared with those of rats given sham lesions, in 2 animal models of anxiety: the shock-probe burying test and the elevated plus-maze test. Septal lesions produced anxiolytic effects in both tests (i.e., an increase in open-arm activity and a decrease in burying), whereas hippocampal and amygdaloid lesions produced neither of these effects. On the other hand, hippocampal and amygdaloid lesions impaired rats' passive avoidance of the electrified shock-probe, whereas septal lesions did not. These dissociations suggest that limbic structures such as the septum, amygdala, and hippocampus exert parallel but distinct control over different fear reactions.
Dorsal hippocampal cholinergic modulation of behavior in different tests of anxiety was investigated by direct injection of the muscarinic M1 and M2 receptor antagonists, pirenzepine and gallamine, and the nicotinic receptor antagonist mecamylamine. In the social interaction test, the anxiogenic effect of pirenzepine (30-100 ng) provided evidence for a tonic cholinergic anxiolytic action mediated by postsynaptic M1 receptors. The anxiogenic action of mecamylamine (30 and 100 ng) was most likely mediated by its action of presynaptic nicotinic receptors to reduce acetylcholine release. Gallamine (10-1,000 ng) was without effect, suggesting that M2 receptors in this brain region do not play a significant role in this behavioral test. On Trial 1 in the elevated plus-maze, the receptor antagonists were without any effect, but in those with a previous 5-min experience of the plus-maze pirenzepine and mecamylamine had anxiogenic effects in the dose range of 30-300 ng; gallamine (100 and 300 ng) was without significant effect.
1 Pairs of male rats were placed in a test box for 10 min and the time they spent in active social interaction was scored. Maximum active interaction was found when the rats were tested under low light in a box with which they were familiar. When the light level was increased or when the box was unfamiliar active social interaction decreased. 2 Exploration (time spent sniffing objects) decreased in the same way in relation to test conditions as did social interaction. As these decreased, defecation, and freezing increased. 3 Anosmic controls showed that the decrease in social interaction across test conditions could not be attributed to olfactory changes in the partner. 4 Chlordiazepoxide (5 mg/kg) given chronically prevented or significantly reduced the decrease in social interaction that occurred in undrugged rats as the light level or the unfamiliarity of the test box was increased. Controls showed that this effect could not be entirely attributed to chlordiazepoxide acting selectively to increase low levels of responding. 5 The effect of chronic chlordiazepoxide contrasts with its action when given acutely; in the latter case it has only sedative effects. 6 Whether this test can be used as an animal model of anxiety is discussed and this test is compared with existing tests of anxiety.
The differential projections from the dorsal raphe and median raphe nuclei of the midbrain were autoradiographically traced in the rat brain after 3H-proline micro-injections. Six ascending fiber tracts were identified, the dorsal raphe nucleus being the sole source of four tracts and sharing one with the median raphe nucleus. The tracts can be classified as those lying within the medial forebrain bundle (dorsal raphe forebrain tract and the median raphe forebrain tract) and those lying entirely outside (dorsal raphe arcuate tract, dorsal raphe periventricular tract, dorsal raphe cortical tract, and raphe medial tract). The dorsal raphe forebrain tract lies in the ventrolateral aspect of the medial forebrain bundle (MFB) and projects mainly to lateral forebrain areas (e.g., basal ganglion, amygdala, and the pyriform cortex). The median raphe forebrain tract lies in the ventromedial aspect of the MFB and projects to medial forebrain areas (e.g., cingulate cortex, medial septum, and hippocampus). The dorsal raphe cortical tract lies ventrolaterally to the medial longitudinal fasciculus and projects to the caudate-putamen and the parieto-temporal cortex. The dorsal raphe periventricular tract lies immediately below the midbrain aqueduct and projects rostrally to the periventricular region of the thalamus and hypothalamus. The dorsal raphe arcuate tract curves laterally from the dorsal raphe nucleus to reach the ventrolateral edge of the midbrain and projects to ventrolateral geniculate body nuclei and the hypothalamic suprachiasmatic nuclei. Finally, the raphe medial tract receives fibers from both the median and dorsal raphe nuclei and runs ventrally between the fasciculus retroflexus and projects to the interpeduncular nucleus and the midline mammillary body.
Further studies were done to test whether the fiber tracts travelling in the MFB contained 5-HT. Unilateral (left) injections of 5,7-dihydroxytryptamine (5 μgm/400 nl) 18 days before midbrain raphe microinjections of 3H-proline produced a reduction in the grain concentrations in all the ascending fibers within the MFB. Furthermore, pharmacological and behavioural evidence was obtained to show that the 5-HT system had been unilaterally damaged; these animals displayed preferential ipsilateral turning in a rotameter which was strongly reversed to contralateral turning after 5-hydroxytryptophan administration.
The results show that DR and MR nuclei have numerous ascending projections whose axons contain the transmitter 5-HT. The results agree with the neuroanatomical distribution of the 5-HT system previously determined biochemically, histochemically, and neurophysiologically. The midbrain serotonin system seems to be organized by a series of fiber pathways. The fast transport rate in these fibers was found to be about 108 mm/day.
To investigate whether an elevated plus-maze consisting of two open and two closed arms could be used as a model of anxiety in the mouse, NIH Swiss mice were tested in the apparatus immediately after a holeboard test. Factor analysis of data from undrugged animals tested in the holeboard and plus-maze yielded three orthogonal factors interpreted as assessing anxiety, directed exploration and locomotion. Anxiolytic drugs (chlordiazepoxide, sodium pentobarbital and ethanol) increased the proportion of time spent on the open arms, and anxiogenic drugs (FG 7142, caffeine and picrotoxin) reduced this measure. Amphetamine and imipramine failed to alter the indices of anxiety. The anxiolytic effect of chlordiazepoxide was reduced in mice that had previously experienced the plus-maze in an undrugged state. Testing animals in the holeboard immediately before the plus-maze test significantly elevated both the percentage of time spent on the open arms and the total number of arm entries, but did not affect the behavioral response to chlordiazepoxide. The plus-maze appears to be a useful test with which to investigate both anxiolytic and anxiogenic agents.
A novel test for the selective identification of anxiolytic and anxiogenic drug effects in the rat is described, using an elevated + -maze consisting of two open arms and two enclosed arms. The use of this test for detecting such drug effects was validated behaviourally, physiologically, and pharmacologically. Rats made significantly fewer entries into the open arms than into the closed arms, and spent significantly less time in open arms. Confinement to the open arms was associated with the observation of significantly more anxiety-related behaviours, and of significantly greater plasma corticosterone concentrations, than confinement to the closed arms. Neither novelty nor illumination was a significant contributor to the behaviour of the rats on the + -maze. A significant increase in the percentage of time spent on the open arms and the number of entries into the open arms was observed only within clinically effective anxiolytics (chlordiazepoxide, diazepam and, less effectively, phenobarbitone). Compounds that cause anxiety in man significantly reduced the percentage of entries into, and time spent on, the open arms (yohimbine, pentylenetetrazole, caffeine, amphetamine). Neither antidepressants nor major tranquilisers had a specific effect. Exposure to a holeboard immediately before placement on the + -maze showed that behaviour on the maze was not clearly correlated either with exploratory head-dipping or spontaneous locomotor activity.
As reported in the preceding study, the ability of certain antipsychotic and adrenolytic agents to inhibit the spontaneous firing of serotonergic 5HT neurons in the dorsal raphe nucleus appeared to be related to adrenergic blocking efficacy. However, the interaction between adrenergic and serotonergic systems was apparently indirect. In this phase of the study we investigated the hypothesis that another transmitter system could mediate this interaction. We examined the effects of two inhibitory amino acid transmitters (GABA and glycine) for possible effects on dorsal raphe cell firing using single cell recording and microiontophoretic techniques. In addition, the ability of the GABA antagonist, picrotoxin and the glycine antagonist, strychnine to reverse the effects of the antipsychotic and alpha-blocking drugs on dorsal raphe firing was tested. Both GABA and glycine were found to inhibit raphe cell firing selectively, allowing for a possible neurotransmitter function for these amino acids within the dorsal raphe nucleus. However, picrotoxin but not strychnine was found to reverse the effects of the antipsychotic and alpha-blocking drugs on raphe firing. Based on these results, we propose that the adrenergic input may influence 5HT neurons indirectly via a GABAergic interneuron or interposed GABA neuron.
Based on evidence that the dorsal raphe nucleus (DR) has specific and independent receptors for 5HT, GABA and glycine (Gallager and Aghajanian, 1976; Wang and Aghajanian, 1977), alterations in the firing rate of DR neurons following the administration of benzodiazepines (BZ) were evaluated to determine whether they were the result of a direct interaction with 5HT receptors or due to interactions of these drugs with GABA and/or glycine. The effects of BZs after both direct and systemic application were tested in rats using microiotophoretic and single-cell recording techniques. Although the BZs did not alter the spontaneous firing rate of the DR, both the systemic and iontophoretic administration of these drugs were found to potentiate the inhibitory response produced by GABA. The data suggest that this potentiation is mediated postsynaptically. Since the effects of BZs on the spontaneous activity of the DR are only apparent following pretreatments with AOAA, it is speculated that these drugs may only have pronounced effects when GABAergic input is prominent.
Pairs of male rats were placed in a test box for 10 min and the time they spent in active social interaction was scored. Maximum active interaction was found when the rats were tested under low light in a box with which they were familiar. When the light level was increased or when the box was unfamiliar active social interaction decreased.
Exploration (time spent sniffing objects) decreased in the same way in relation to test conditions as did social interaction. As these decreased, defecation, and freezing increased.
Anosmic controls showed that the decrease in social interaction across test conditions could not be attributed to olfactory changes in the partner.
Chlordiazepoxide (5 mg/kg) given chronically prevented or significantly reduced the decrease in social interaction that occurred in undrugged rats as the light level or the unfamiliarity of the test box was increased. Controls showed that this effect could not be entirely attributed to chlordiazepoxide acting selectively to increase low levels of responding.
The effect of chronic chlordiazepoxide contrasts with its action when given acutely; in the latter case it has only sedative effects.
Whether this test can be used as an animal model of anxiety is discussed and this test is compared with existing tests of anxiety.
GABAA receptors are ligand-gated Cl- ion channels and the site of action of a variety of pharmacologically and clinically important drugs. In this review evidence is summarized indicating that these drugs, by interacting with several distinct binding sites at these receptors, allosterically modulate GABA-induced Cl- ion flux. Other results indicate that the affinity, as well as the modulatory efficacy of drugs, changes with receptor composition. A though investigation of the pharmacological properties of the individual binding sites on different GABAA receptor subtypes could open new avenues for selective modulation of GABAA receptors in different brain regions.
The purpose of the present study was two-fold. Firstly, to present a more comprehensive analysis of the disinhibitory effects of 5-HT1A receptor agonists after discrete dorsal raphe (DRN) injections (Higgins et al. 1988). Secondly, the effects of the 5-HT1B receptor agonist CGS12066B and the 5-HT1B/1C agonist mCPP were examined following injection into this nucleus. The increases in social interaction (SI) induced by intra-raphe injections of 8-OH DPAT (0.02-1 micrograms), buspirone (0.04-0.2 microgram), ipsapirone (0.2 microgram) and gepirone (0.2-1 micrograms) under a high light unfamiliar paradigm (HLU) were typically due to increased bout frequency, duration and a higher incidence of sniff, follow, allogroom behaviour. These increases were qualitatively similar to those seen in control animals tested under low light/familiar (LLF) conditions, thus supporting the belief that the drug-induced increases in SI reflected decreases in anxiety. Furthermore, at doses effective under the HLU condition, 8-OH DPAT, buspirone and gepirone failed to modify SI under conditions of minimal suppression (LLF paradigm). At doses which significantly increased punished responding in a water-lick conflict test 8-OH DPAT, ipsapirone and gepirone tended to also increase unpunished rates of drinking. However, in drug untreated rats, prior habituation to the test apparatus also increased unpunished drinking, suggesting some neophobia-induced suppression. At a comparatively high dose, the 5-HT1B agonist CGS12066B (2.5 micrograms), but not the putative 5-HT1B/1C agonist mCPP (0.5-12.5 micrograms), increased SI under the HLU condition.(ABSTRACT TRUNCATED AT 250 WORDS)
The purpose of the present experiment was to determine how a rat's prior history (of repeated gentle handling and/or of the elevated plus-maze apparatus) modified the behavioural and neurochemical response to chlordiazepoxide. In handled animals one previous exposure to the plus-maze rendered the rats insensitive to the anxiolytic effects of chlordiazepoxide in this test. This phenomenon of 'one-trial tolerance' was not seen in unhandled rats and thus both prior handling and prior maze experience were necessary to abolish the behavioural response to chlordiazepoxide. The effects of chlordiazepoxide on K(+)-evoked [14C]GABA (gamma-aminobutyric acid) release were also modified by the rat's past history. The drug-induced reduction of GABA release in the cortex was abolished by prior plus-maze experience; whereas handling modified chlordiazepoxide's effects on GABA release in the hippocampus (the drug decreased release in unhandled rats and increased release in those given repeated gentle handling). Thus an anxiolytic response to chlordiazepoxide in the plus-maze was accompanied by reduced GABA release in both cortex and hippocampus. The 5-HT system (5-hydroxytryptamine) also proved sensitive to the rats' past history. The effects of chlordiazepoxide on K(+)-evoked [3H]5-HT release from the hippocampus depended on both prior handling and plus-maze experience and could be predicted from the undrugged level of evoked release; when this was low, chlordiazepoxide increased it, when it was high, chlordiazepoxide reduced it. These results raise the possibility that the beneficial effects of a benzodiazepine may depend on the baseline condition of the animals.
Recent studies have shown that brief exposure to an elevated plus-maze (EPM) produces non-opioid antinociception in male mice. The present experiments were designed to assess the effects of diazepam on this phenomenon. When acutely administered, low doses (0.5-1.0 mg/kg) of diazepam failed to produce an anxiolytic profile and exerted rather inconsistent effects on EPM-induced elevations in tail-flick latencies. In EPM-experienced mice, chronic treatment with higher doses of diazepam (2-4 mg/kg, 8 days) produced a weak anxiolytic action and inhibited the early phase of EPM antinociception only. However, in EPM-naive mice, 8-day diazepam pretreatment exerted a marked anxiolytic effect and completely eliminated the antinociceptive response to the maze. Together, these data support the view that anxiety is a key factor in certain forms of adaptive pain inhibition and suggest a possible mediational role for benzodiazepine receptors. Our findings also show that prior exposure to the EPM, rather than chronic handling/injection, greatly reduces the anti-anxiety effect of diazepam. Furthermore, since re-exposure to the maze, per se, decreased time spent on the open arms and central platform, a shift in behavioural baseline ("retest anxiogenesis") may have contributed to the weak behavioural effects of diazepam in test-experienced animals. Importantly, as chronic treatment with diazepam did not influence this anxiogenic-like retest profile, our data suggest that a single prior experience of the EPM may radically alter the nature of the anxiety reaction provoked by this test.
Chlordiazepoxide (CDP 7.5 mg/kg) had a significant anxiolytic effect in rats tested on the plus-maze for the first time. On a second trial the control scores did not change, but those of the CDP group did and they no longer differed from controls. Rats previously tested undrugged or after flumazenil (4 mg/kg) also failed to show an anxiolytic response to CDP. Thus this phenomenon of one-trial tolerance depended on prior experience with the plus-maze. It also depended on CDP acting at the benzodiazepine receptors on trial 2, since the joint administration of CDP and flumazenil on trial 2 reversed the phenomenon.
Quantitative autoradiography of rat brain coronal sections show that maximum enhancement (more than 80%) of [3H]flunitrazepam binding by GABA occurs in brain regions particularly rich in type I benzodiazepine receptors (inferior colliculus, medium raphe, central gray and substantia nigra); conversely, brain areas where type II predominates show the lowest enhancement by GABA (about 50%). These results, suggesting that the coupling of GABA receptors with type I sites is more efficient than that with type II sites, are at variance with those reported on GABA-benzodiazepine receptors expressed in transfected cells, where the greater GABA potentiation of benzodiazepine binding is due to a subtype of the type II site containing the alpha 3 subunit of the GABAA receptor. One possible explanation of these discrepancies is that the type II receptors found in type I-enriched tissues (inferior colliculus, median raphe, central gray and substantia nigra) are associated with the alpha 3-subunit, while the type II sites present in limbic and cortical regions represent a subpopulation carrying the alpha 2-subunit of the GABAA receptor, with lower GABA potentiation.
The behavioural and biochemical response to the 5-HT1-like receptor compounds, 5-carboxamidotryptamine (5-CT), 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), buspirone and ipsapirone and the GABA agonist, muscimol, injected into the dorsal raphé nucleus (DRN) are reported. All compounds increased social interaction under high light, unfamiliar conditions and increased punished responding in a Vogel conflict test. At doses ranging from 5-25 times greater than those which were effective in these anxiety models, muscimol, 5-CT and 8-OH-DPAT induced a marked hypothermia and a flattening of body posture. Buspirone, on the other hand, failed to induce a significant reduction in core temperature or produce a marked flattening of posture in all animals, even at doses 100 times those effective in anxiety models. Following injection of muscimol, 5-CT, 8-OH-DPAT and buspirone into the dorsal raphé nucleus, all tended to reduce the 5-HIAA:5-HT ratios in the frontal cortex, hippocampus and hypothalamus. These findings, together with available electrophysiological data suggest that these behavioural responses are a consequence of a depression of the firing of cells in the dorsal raphé nucleus, with a corresponding decrease in functional activity of 5-HT in the forebrain.
The effects of benzodiazepine- and GABAA-receptor agonists and antagonists on the release and metabolism of 5-HT were measured in the ventral hippocampus of freely moving rats using microdialysis. Systemic injections of the benzodiazepine agonists, flurazepam and diazepam reduced the levels of 5-HT while the partial inverse agonist, FG 7142 (N-methyl-beta-carboline-3-carboxamide), had no effect. Local perfusion of flurazepam through the dialysis probe also decreased the release of 5-HT in the ventral hippocampus, an effect which was completely blocked by the benzodiazepine antagonist, Ro15-1788 (flumazenil). Local application of the GABAA agonist muscimol had no effect on the release of 5-HT, while the antagonist picrotoxin, administered locally, caused a 4-fold enhancement of release of 5-HT. Picrotoxin also resulted in a complete block of the inhibitory effect of flurazepam on release of 5-HT. None of these drugs caused significant changes in the levels of the metabolite of 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) in the ventral hippocampus. These results suggest that the inhibitory effect of flurazepam on the release of 5-HT is mediated by benzodiazepine/GABAA receptors in the hippocampus and that GABA exerts a tonic inhibitory effect on the release of 5-HT in the region of the brain.
A two‐compartment exploratory test was used to assess the role of central 5‐hydroxytryptaminergic neurones in the anxiolytic activity of buspirone in rats.
Buspirone 0.1 mg kg ⁻¹ , administered subcutaneously 15 min before testing, significantly increased black‐white transitions (BWT) in control rats but had no effect in animals injected intra‐cerebroventricularly one week before with 150 μg 5,7‐dihydroxytryptamine (in 20 μl).
Infusion of buspirone in the median raphe (but not in the dorsal raphe) significantly enhanced BWT, at doses from 1 μg to 10 μg (in 0.5 μl). Buspirone 5 and 10 μg, but not 1 μg, administered in the median raphe, significantly enhanced motor activity of rats during the first 10 min of testing in the activity cages.
The effect on BWT of 5 μg buspirone in the median raphe was completely antagonized in animals which had received either 5,7‐dihydroxytryptamine intraventricularly, 150 μg (in 20 μl), one week before or an infusion of 0.1 μg (in 0.5 μl) (−)‐propranolol in the same area 5 min before. (−)‐Propranolol infused in the median raphe did not modify the effect of buspirone on locomotion.
Infusion of 5 μg buspirone (in 0.5 μl) in the median raphe significantly enhanced punished responses in a conflict test with no effect on unpunished responding. Buspirone infused in the dorsal raphe had no effect on punished or unpunished responding over a wide dose range.
The results indicate that at the relatively low dose used in the present study buspirone produces an anxiolytic effect by acting on central 5‐hydroxytryptaminergic neurones. It is likely that activation of 5‐hydroxytryptamine 1A ‐receptors in the median raphe is involved.
Interactions between the benzodiazepines (BZs) chlordiazepoxide (CDP) and midazolam (MDZ), the BZ antagonist R0 15-1788, the inverse BZ receptor agonists CGS 8216 and FG 7142, γ-aminobutyrate (GABA), serotonin (5-HT), the 5-HT2 antagonist methysergide and the putative 5-HT agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) were investigated using peripheral and intra-amygdaloid treatments. A multiple schedule consisting of rewarded, nonrewarded (Time out: TO) and conflict periods was used to compare in parallel effects on successive discrimination between rewarded and nonrewarded periods and punished responding. The three components were presented in both a fixed order (Experiment 1) and a random order (Experiments 2 and 3).
Intra-amygdaloid treatments with GABA and the BZs selectively increased rates of punished responding. CDP given systemically, on the other hand, increased both TO and conflict rates, suggesting an additional impairment of discrimination, which was more marked in the random than the fixed order condition. R0 15-1788, CGS 8216 and FG 7142 given by both routes counteracted the anti-conflict effects of CDP given centrally or systemically. However increases in TO rates induced by IP CDP were antagonized only by IP treatments with these compounds. The two inverse agonists, but not R0 15-1788, also counteracted increases in punished responding which were found after intra-amygdaloid GABA infusions. In Experiments 2 and 3 where baseline rates of pressing in Conflict periods were sufficiently high to detect decreases, CGS 8216 and FG 7142 reduced responding below control level, suggesting a specific anxiogenic activity. Evidence for effects of R0 15-1788 by itself was inconclusive. 5-HT injected into the amygdala also reduced punished responding below control level, whereas methysergide increased it with both central and peripheral treatment. Effects of 8-OH-DPAT varied according to route of administration. With IP treatment Conflict rates were increased, but after amygdaloid infusion both TO and Conflict rates were marginally reduced below control level, with a more consistent depression of punished responding.
These results provide evidence that effects of BZs on punished responding are mediated by a GABAergic system which includes the lateral/basolateral amygdala, but which does not participate in BZ-induced disruption of discrimination. They also indicate that the antagonistic effects of CGS 8216 and FG 7142 involve a decrease in GABA transmission, and that these compounds may also be anxiogenic. Finally, the results suggest that 5-HT utilizes the same system for regulating resistance to punishment, but plays no significant part in reward-nonreward successive discrimination, which is impaired after systemic BZs.
The effects of a selective serotonin 1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), were studied in two animal models of anxiety. Peripherally injected 8-OH-DPAT in doses ranging from 0.125 to 2.0 mg/kg did not increase black-white transitions (BWT) and black square entries (BSE) in a two-compartment exploratory test or punished responding in a test of conditioned suppression of drinking. With 2.0 mg/kg 8-OH-DPAT BSE and unpunished responding were reduced. In an investigation of the drinking time of water-deprived rats, naive or habituated to the test environment, 1.0 and 2.0 mg/kg 8-OH-DPAT increased the drinking time of naive rats but 2.0 mg/kg 8-OH-DPAT reduced that of habituated animals. In animals deprived of water for 48 h or subjected to immobilization stress for 2 h, 1.0 mg/kg 8-OH-DPAT increased BWT and BSE values in the two-compartment exploratory test. Infusions of 5 micrograms/0.5 microliter 8-OH-DPAT in the nucleus raphe medianus increased BWT and BSE values in the exploratory test and punished responding in the test of conditioned suppression of drinking, whereas the same dose of 8-OH-DPAT injected in the nucleus raphe dorsalis had no effect on punished but suppressed unpunished responding. The effects of 8-OH-DPAT are only detectable in the appropriate experimental conditions. When injected systemically, the effects are evident when a state of arousal of the animals contributes to the overall behavioural output. 8-OH-DPAT shows effects comparable to those of established anxiolytics such as benzodiazepines and barbiturates when it is injected in the nucleus raphe medianus, but not in the dorsalis.(ABSTRACT TRUNCATED AT 250 WORDS)
The social interaction test in rats provides a method for detecting anxiolytic activity that does not use food or water deprivation, or electric shock, and therefore obviates difficulties of interpretation that might arise from drug-induced changes in motivation. Since social interaction is measured under more than one test condition any overall increase or decrease in social behaviour can be detected independently from the drug x test condition interaction that characterizes an anxiolytic drug. The Geller-Seifter conflict test was designed with two schedules of reinforcement for the same reasons. Any candidate test for anxiolytic action that examines drug effects under only one experimental condition is open to misinterpretation and may also prove unreliable if the critical experimental factors ( e.g. the level of food deprivation or the shock intensity) are changed. The testing procedure in the social interaction test is relatively time consuming in terms of observer-hours, but no lengthy pretraining of the animals is required. There is no way of fully automating the scoring and therefore it is important that the observers do not know the experimental group of the rats that they are scoring, and that tape recordings are made so that the scores can be checked. It has not so far been fruitful to analyze drug effects on every individual social behaviour, but this method does allow changes in individual behaviours to be detected. By entering the data directly into a computer we are now able to store the frequency and duration of each behaviour as well as the sequence of behaviours. It will then be possible to determine whether a detailed analysis of drug effects on the patterning of social behaviours will prove a useful addition to the social interaction test
Different technical, physiological and biochemical aspects concerning the study of the release of 5-HT are discussed herein. Isotopic methods are the most suitable techniques since these allow the release of 3H-5-HT to be measured after having determined the identity of the labelled compounds formed from 3H-tryptophan by co-chromatography. Under these conditions, the 3H-amine released in the superfusates comes from serotoninergic nerve endings, since tryptophan hydroxylase is exclusively localized in serotoninergic neurons. Moreover, it appears that newly synthesized 5-HT is preferentially released. The release of 5-HT is dependent on neuronal activity, but is not always linked to the synthesis of 5-HT. The increase in the firing rate of serotoninergic cell bodies by a local application of glutamate in the area of the nucleus raphe dorsalis induces a marked increase n the release of 5-HT in the caudate nucleus; an opposite effect is observed after cooling this region. The local depolarization of serotoninergic terminals located in the caudate nucleus increases the release of this amine. This effect is blocked by TTX. LSD reduces the stimulating effect of KCl, thus indicating that the release of 5-HT can be controlled at a presynaptic level. In addition, the release of the amine is dependent on the presence of calcium. Serotoninergic neuronal activity can be controlled at the preterminal or at the cell body levels by the activity of other neuronal systems. The effects of the release of dopamine from dendrites, and that of GABA in the substantia nigra are reported herein. Furthermore, changes in the activity of the dopaminergic, gabaergic and serotoninergic systems innervating the nucleus raphe dorsalis modulate the release of 5-HT, measured both in the caudate nucleus and in the nucleus raphe magnus. Finally, it has been reported that the release of 5-HT can be estimated in the raphe nuclei dorsalis and magnus. It has been shown that the amounts of 3H-5-HT continuously formed from 3H-TRP and released in the nucleus raphe dorsalis are much greater than those estimated in the caudate nucleus or in the substantia nigra. Although the quantities of endogenous 5-HT measured in the nucleus raphe dorsalis are the highest in the brain, this structure presents only a few serotoninergic nerve endings. This raises the question of the origin of the 5-HT released in serotoninergic nuclei. A possible dendritic release of 5-HT is discussed.
Chlordiazepoxide and GABA (5 X 10(-5) M; 0.2 microliters) applied into the nucleus raphe dorsalis significantly reduced behavioral suppression induced in rats by a 10-min presentation of a signal previously associated with the punishment of lever pressing for food. As compared to saline control rats, this reduction was of the same order of magnitude as that obtained after intraperitoneal administration of chlordiazepoxide (8 mg X kg-1). Chlordiazepoxide or GABA (5 X 10(-5) M) failed to alter response maintained under non-aversive conditions. These data suggest the involvement of GABAergic neurons of the raphe dorsalis in the 'disinhibitory' effects of benzodiazepines.
The effects of GABA and diazepam on the spontaneous and potassium-evoked release of 3H-5-HT from rat hippocampal synaptosomes, preloaded by incubation with 3H-5-HT, have been investigated. Both GABA and diazepam independently increased the spontaneous release of 5-HT whereas they blocked the calcium-dependent, potassium-evoked release. However diazepam antagonised the GABA-mediated increase in spontaneous release but enhanced the inhibitory effects of GABA on potassium-stimulated release. The results suggest that 5-HT release in the hippocampus is under the presynaptic control of GABA and that diazepam may selectively enhance the inhibitory effect of GABA on the evoked release of 5-HT.
The behavioural response of rats in the high light unfamiliar condition of the social interaction test of anxiety was observed following direct administration of the 5-HT1A receptor agonist, (+/-)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT, 50, 100 or 200 ng) or antagonist tertatolol (3 micrograms) into the median raphe nucleus or dorsal hippocampus. In the median raphe nucleus, 8-OH-DPAT (200 ng) significantly increased social interaction without changing locomotor activity; lower doses were inactive. In the dorsal hippocampus, bilateral injection of 8-OH-DPAT (100 ng) significantly decreased social interaction, without effect on locomotor activity; both 50 and 100 ng significantly changed grooming. Tertatolol had no effect on social interaction following administration to the median raphe nucleus, but significantly increased locomotor activity. Bilateral injection of tertatolol into the dorsal hippocampus decreased social interaction and changed grooming. These effects are similar to those of 8-OH-DPAT suggesting tertatolol may have 5-HT1A receptor agonist properties. In conclusion, the findings of this study demonstrate that 5-HT1A somatodendritic autoreceptors and post-synaptic receptors mediate anxiolytic and anxiogenic effects, respectively, in the social interaction test.
Microinfusions of the benzodiazepine anxiolytic midazolam into the central or basolateral amygdaloid nuclei produced different anxiolytic effects in two tests of rat 'anxiety'. Infusions into the basolateral nucleus impaired open-arm avoidance in the elevated plus-maze test, but did not impair shock-probe avoidance in the shock-probe burying test. In contrast, infusions into the central nucleus impaired shock-probe avoidance, but did not impair open-arm avoidance. Both of these site-specific, midazolam-induced anxiolytic effects were blocked by a pre-infusion of the benzodiazepine receptor antagonist Ro 15-1788 (flumazenil). None of the treatments affected defensive burying. These results suggest that benzodiazepine receptors in the central and basolateral amygdaloid nuclei differentially mediate the anti-anxiety effects of benzodiazepine anxiolytics.
A single prior undrugged exposure to the elevated plus-maze has been reported to reduce open arm activity on retest and to attenuate/abolish the anxiolytic response to benzodiazepines at retest intervals ranging from 48 h to 14 days. The present study was designed to examine the generality of these findings by comparing the effects of prior maze experience on baseline behaviour and response to diazepam in two murine models of anxiety. Parallel experiments were conducted in which DBA/2 mice were exposed/not exposed to the plus-maze, treated daily with saline or diazepam (2-4 mg/kg daily for 8 days) and then tested on either the elevated plus-maze or in the light/dark test of exploration. Results show that, in both tests, diazepam reduced behavioural indices of anxiety in maze-naive mice only. However, interpretation of this apparent loss of diazepam efficacy is at least partially confounded by the observation that maze experience per se altered baseline behaviour in both procedures, reducing open arm activity in the plus-maze and increasing light compartment activity in the light/dark test. The apparent elimination of an anxiolytic response to diazepam in two animal models of anxiety by prior plus-maze experience is discussed in relation to experience-related baseline shifts in behaviour.
A factor analysis of the scores from rats given two trials in the elevated plus-maze showed that four independent factors emerged. Measures of anxiolytic activity on trial 1 (number of open arm entries and time spent on open arms) loaded on factor 1, measures of anxiolytic activity on trial 2 loaded on factor 2, the measure of general activity (number of closed arm entries) on both trials loaded on factor 3, and a measure of decision time (time spent in central square) for both trials loaded on factor 4. The independence of trials 1 and 2 anxiety measures raises the possibility that the state of anxiety/fear on the second trial in the plus-maze is qualitatively different from that on trial 1. This difference is reflected in the loss of anxiolytic action of diazepam (2 mg/kg) on trial 2. However, this occurs only when the trials are short (5 min); when they are longer (10 min) diazepam retains anxiolytic efficacy. It is concluded that during a brief (5 min) trial in the plus-maze rats acquire a specific phobic anxiety, which is relatively resistant to benzodiazepines. With a longer exposure to the plus-maze this form of fear extinguishes.
Diazepam (5 mg/kg) increased the number of shocks accepted by rats on two successive trials in the punished drinking test. Thus, the phenomenon of "one trial tolerance" to the anxiolytic effects of benzodiazepines in the elevated plus-maze does not generalise to this other animal test of anxiety. FG 7142 (20 mg/kg) and prior exposure to the odour of a cat had significant anxiogenic effects on two successive trials in the plus-maze. Thus the phenomenon of "one trial tolerance" does not generalise to these anxiogenic effects in the plus-maze. Furthermore, chlordiazepoxide retained its ability to counteract the anxiogenic effects in the plus-maze of prior exposure to cat odour, over successive trials. On the basis of these and previous experiments it is suggested that the state of anxiety generated on trial 2 in the plus-maze is close to a phobic state, against which benzodiazepines are relatively ineffective. Chlordiazepoxide (5 and 10 mg/kg) was also ineffective against the behavioural responses of rats during exposure to cat odour, another possible animal test of phobia. This contrasted with its efficacy against the anxiogenic effects of cat odour that subsequently generalised to and could be detected in the plus-maze.
In rats, the 5-HT1A receptor full agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and the 5-HT1A receptor partial agonists ipsapirone and buspirone dose dependently and completely inhibited shock-induced ultrasonic vocalization after systemic injection and after microinjection into the dorsal raphe nucleus, a brain region rich in somatodendritic 5-HT1A receptors. As compared with injection into the dorsal raphe nucleus, ipsapirone and 8-OH-DPAT were significantly less potent after microinjection into the lateral ventricle or the median raphe nucleus. Depletion of brain 5-HT (5-hydroxytryptamine) by means of 5,7-dihydroxytryptamine or parachlorophenylalanine inhibited ultrasonic vocalization. In lesioned rats, however, ipsapirone (i.p. or dorsal raphe nucleus) and 8-OH-DPAT (dorsal raphe nucleus) retained their ability to inhibit ultrasonic vocalization and, in non-lesioned rats, bilateral injection of ipsapirone, buspirone and 8-OH-DPAT into the dorsal hippocampus and the amygdala - two brain regions rich in postsynaptic 5-HT1A receptors - also inhibited ultrasonic vocalization. In a Geller-Seifter conflict test, i.p. and local injection of 8-OH-DPAT in the dorsal raphe nucleus and the hippocampus selectively enhanced punished responding. It is suggested that both presynaptic and (possibly to a lesser extent) postsynaptic 5-HT1A receptors are involved in the anxiolytic effects of ipsapirone, buspirone, and 8-OH-DPAT.
The effect of intra-hippocampal (dentate gyrus) microinjections of the benzodiazepine, midazolam (10 micrograms per site, bilaterally), was examined in the Vogel conflict test in serotonin depleted rats (pretreated with p-chlorophenylalanine). p-Chlorophenylalanine and midazolam, when given separately, significantly disinhibited rat conflict behavior. Midazolam retained its anticonflict potency in the p-chlorophenylalanine pretreated animals. These and other data indicate: (i) the specific involvement of limbic serotonin in emotional control, (ii) a dissociation of the effects of serotonin depletion and intra-hippocampal benzodiazepine on conflict behavior, and (iii) the important role of the hippocampus in processing emotional input.
In naive rats benzodiazepines have clear anxiolytic effects in the elevated plus-maze, but in rats with previous plus-maze experience benzodiazepines are ineffective. This phenomenon does not depend on the drug state on trial 1 or on the inter-trial interval and generalises across mazes of different material; it is dependent on experience of the open arm. The phenomenon of "one-trial tolerance" to the anxiolytic effect of benzodiazepines is not seen in other animal tests; and there is no equivalent phenomenon of "one-trial withdrawal" or of tolerance to anxiogenic effects in the plus-maze. The phenomenon is not seen in unhandled rats, in rats given longer trials in the plus-maze, or in those given an amnesic treatment on trial 1. It is suggested that during the first 5 min in the elevated plus-maze the rats are acquiring a fear of heights and it is this phobic anxiety state that is measured during the second 5-min trial. Thus the nature of the anxiety generated by the maze is different on trials 1 and 2. The results of a factor analysis study confirm this conclusion.
Effects of septal and amygdaloid lesions were compared in 2 models of rat "anxiety." Septal lesions decreased burying behavior in the "shock-probe burying test" and increased open-arm exploration in the "elevated plus-maze test," whereas amygdaloid lesions produced neither of these anxiolytic effects. However, amygdaloid lesions increased rats' contacts of the electrified probe, an anxiolytic effect not produced by septal lesions. Each of these distinct, anxiolytic effects of septal or amygdaloid lesions were displayed together in animals with lesions of both structures. Furthermore, the magnitude of these anxiolytic effects after combined lesions was comparable to their magnitude after individual lesions. Taken together, these results suggest the amygdala and the septum independently control the expression of different fear-related behaviors.
The quest for the mutation responsible for Huntington's disease (HD) has required an exceptionally detailed analysis of a large part of 4p16.3 by molecular genetic techniques, making this stretch of 2.2 megabases one of the best characterized regions of the human genome. Here we describe the construction of a cosmid and P1 clone contig spanning the region containing the HD gene, and the establishment of a detailed, high resolution restriction map. This ordered clone library has allowed the identification of several genes from the region, and has played a vital role in the recent identification of the Huntington's disease gene. The restriction map provides the framework for the detailed analysis of a region extremely rich in coding sequences. This study also exemplifies many of the strategies to be used in the analysis of larger regions of the human genome.
Blockade of GABAergic inhibition in the region of the anterior basolateral amygdala (BLA) of rats elicits physiologic changes associated with a defense reaction. The present study was undertaken to determine whether GABA receptors in the BLA might be involved in regulating experimental anxiety using the social interaction (SI) and conflict test. Guide cannulae were stereotaxically implanted bilaterally in the BLA of rats for intracerebral microinjections. In the BLA, injection of the GABAA receptor antagonists bicuculline methiodide (BMI) and picrotoxin (PIC) produced anxiogenic-like effects in the SI paradigm, as did BMI injection using the conflict paradigm. Injection of the GABAA agonist muscimol (MUS) into the central nucleus of the amygdala (Ce) produced anxiolytic-like effects in the SI test. Microinjection of MUS, baclofen (GABAB agonist), 2OH-saclofen (GABAB antagonist) or strychnine (glycine antagonist) into the BLA or BMI into the Ce elicited no change in experimental anxiety as measured by the SI test. These results suggest that endogenous GABA acts tonically at GABAA receptors in the BLA to inhibit anxiety responses.
There are conflicting results on the function of 5-HT in anxiety and depression. To reconcile this evidence, Deakin and Graeff have suggested that the ascending 5-HT pathway that originates in the dorsal raphe nucleus (DRN) and innervates the amygdala and frontal cortex facilitates conditioned fear, while the DRN-periventricular pathway innervating the periventricular and periaqueductal gray matter inhibits inborn fight/flight reactions to impending danger, pain, or asphyxia. To study the role of the DRN 5-HT system in anxiety, we microinjected 8-OH-DPAT into the DRN to inhibit 5-HT release. This treatment impaired inhibitory avoidance (conditioned fear) without affecting one-way escape (unconditioned fear) in the elevated T-maze, a new animal model of anxiety. We also applied three drug treatments that increase 5-HT release from DRN terminals: 1) intra-DRN microinjection of the benzodiazepine inverse agonist FG 4172, 2) intra-DRN microinjection of the excitatory amino acid kainic acid, and 3) intraperitoneal injection of the 5-HT releaser and uptake blocker D-fenfluramine. All treatments enhanced inhibitory avoidance in T-maze. D-Fenfluramine and intra-DRN kainate also decreased one-way escape. In healthy volunteers, D-fenfluramine and the 5-HT agonist mCPP (mainly 5-HT2C) increased, while the antagonists ritanserin (5-HT2A/2C) and SR 46349B (5-HT2A) decreased skin conductance responses to an aversively conditioned stimulus (tone). In addition, D-fenfluramine decreased, whereas ritanserin increased subjective anxiety induced by simulated public speaking, thought to represent unconditioned anxiety. Overall, these results are compatible with the above hypothesis. Deakin and Graeff have suggested that the pathway connecting the median raphe nucleus (MRN) to the dorsal hippocampus promotes resistance to chronic, unavoidable stress. In the present study, we found that 24 h after electrolytic lesion of the rat MRN glandular gastric ulcers occurred, and the immune response to the mitogen concanavalin A was depressed. Seven days after the same lesion, the ulcerogenic effect of restraint was enhanced. Microinjection of 8-OH-DPAT, the nonselective agonist 5-MeO-DMT, or the 5-HT uptake inhibitor zimelidine into the dorsal hippocampus immediately after 2 h of restraint reversed the deficits of open arm exploration in the elevated plus-maze, measured 24 h after restraint. The effect of the two last drugs was antagonized by WAY-100135, a selective 5-HT1A receptor antagonist. These results are compatible with the hypothesis that the MRN-dorsal hippocampus 5-HT system attenuates stress by facilitation of hippocampal 5-HT1A-mediated neurotransmission. Clinical implications of these results are discussed, especially with regard to panic disorder and depression.
In order to investigate the role of the 5-HT1A receptors of the amygdala in modulating anxiety, rats were implanted with bilateral cannulae aimed at the basolateral nucleus of the amygdala complex and infused with either artificial cerebrospinal fluid (aCSF) or the selective 5-HT1A receptor agonist 8-OH-DPAT (50-200 ng) and tested in two animal models of anxiety. In the elevated plus-maze test, no significant effects were detected in this dose range. In contrast, 8-OH-DPAT caused an overall reduction in levels of social investigation, thus indicating anxiogenic actions in the social interaction test. At 50 ng, 8-OH-DPAT had a selective action on anxiety, while at 200 ng there was a concomitant reduction in locomotor activity and, in some animals, signs of the 5-HT1A syndrome. Evidence that the anxiogenic effect of 8-OH-DPAT (50 ng) was due to activation of 5-HT1A receptors came from the finding that (-)-tertatolol, a 5-HT1A receptor antagonist, reversed this effect at a dose (1.5 micrograms) which was silent when given alone. The benzodiazepine receptor agonist, midazolam (1 and 2 micrograms) was bilaterally administered into the basolateral nucleus of the amygdala and evoked clear-cut anxiolytic effects in the social interaction test. These data indicate that the agonist activation of post-synaptic 5-HT1A receptors in the basolateral nucleus of the amygdala may produce anxiogenic effects, while agonist activation of BDZ receptors in the same areas evokes anxiolytic effects. Our results from the social interaction test are similar to those previously reported from tests of anxiety using punished paradigms, but contrast with those found in the elevated plus-maze. Thus, it is concluded that either the two tests have different sensitivities to midazolam and 8-OH-DPAT or more intriguingly, the tests are evoking fundamentally different states of anxiety, with that evoked by the plus-maze being mediated via brain areas or receptors different from those studied here.
Extracellular 5‐hydroxytryptamine (5‐HT) was determined in dorsal raphe nucleus (DRN), median raphe nucleus (MRN) and nucleus accumbens by use of microdialysis in unanaesthetized rats.
Infusion of the γ‐aminobutyric acid (GABA) A receptor agonist muscimol into DRN and MRN resulted in decreased 5‐HT in DRN and MRN, respectively. Muscimol infusion into nucleus accumbens had no effect on 5‐HT.
Infusion of the GABA A receptor antgonist bicuculline into DRN resulted in increased DRN and nucleus accumbens 5‐HT. Bicuculline infusion into MRN had no effect on 5‐HT. This suggests that endogenous GABA had a tonic, GABA A receptor‐mediated inhibitory effect on 5‐HT in DRN, but not in MRN.
Infusion of the GABA B receptor agonist baclofen into DRN produced a decrease in DRN 5‐HT. Baclofen infusion into nucleus accumbens resulted in decreased nucleus accumbens 5‐HT. This suggests that GABA B receptors are present in the area of cell bodies and terminals of 5‐hydroxytryptaminergic neurones.
Infusion of the GABA B receptor antagonists phaclofen and 2‐hydroxysaclofen had no effect on midbrain raphe and forebrain 5‐HT. This suggests that GABA B receptors did not contribute to tonic inhibition of 5‐HT release.
In conclusion, 5‐HT release is physiologically regulated by distinct subtypes of GABA receptors in presynaptic and postsynaptic sites.
Previous work has implicated the amygdala, especially the central and basolateral nuclei, in the anxiolytic effects of ben zodiazepines in animal models of anxiety. However there are contradictory findings on the relative importance of each nucleus. The experiments reported here show that midazolam (1.0 [mu]g) injected bilaterally into the basolateral nucleus significantly increased open arm activity on the elevated plus-maze. In contrast midazolam (1.0 [mu]g) had no effect when injected into the central nucleus. These results suggest that the basolateral nucleus is one substrate mediating anxiolytic effects. However, a brief review of the literature supports the suggestion that both nuclei can be involved, but findings will depend upon the particular paradigm used.
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