Oral Administration of Efavirenz Dysregulates the Tph2 Gene in Brain Serotonergic Areas and Alters Weight and Mood in Mice.

Most HIV-antiretroviral drugs have adverse effects. Efavirenz (EFV) is an example of a drug with neuropsychiatric effects, such as anxiety, depression, and suicidal thoughts, in people living with HIV (PLWH). The mechanisms by which EFV causes neuropsychiatric alterations in PLWH are complex, multifactorial, and not fully understood, although several studies in animals have reported changes in brain energy metabolism, alterations in monoamine turnover, GABA, and glutamate levels, and changes in 5-HT receptors. In this report, we studied the effects of EFV on the serotonergic system in healthy mice, specifically, whether EFV results in alterations in the levels of the tryptophan hydroxylase 2 (Tph2) gene in the brain. EFV (10 mg/kg) and distilled water (1.5 µL/kg) (control group) were orally administered to the mice for 36 days. At the end of the treatment, Tph2 expression levels in mouse brains were measured, and mood was evaluated by three trials: the forced swim test, elevated plus maze, and open field test. Our results revealed dysregulation of Tph2 expression in the brainstem, amygdala, and hypothalamus in the EFV group, and 5-HT levels increased in the amygdala in the EFV group. In the behavioral tests, mice given EFV exhibited a passive avoidance response in the forced swim test and anxiety-like behavior in the elevated plus maze, and they lost weight. Herein, for the first time, we showed that EFV triggered dysregulation of the Tph2 gene in the three serotonergic areas studied; and 5-HT levels increased in the amygdala using the ELISA method. However, further studies will be necessary to clarify the increase of 5-HT in the amygdala as well as understand the paradoxical decrease in body weight with the simultaneous increase in food consumption. It will also be necessary to measure 5-HT by other techniques different from ELISA, such as HPLC.

Membrane Heteroreceptor Complexes as Second-Order Protein Modulators: A Novel Integrative Mechanism through Allosteric Receptor-Receptor Interactions.

Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) together with the proximity ligation method revealed the existence of G-protein-coupled receptors, Ionotropic and Receptor tyrosine kinase heterocomplexes, e.g., A2AR-D2R, GABAA-D5R, and FGFR1-5-HT1AR heterocomplexes. Molecular integration takes place through allosteric receptor-receptor interactions in heteroreceptor complexes of synaptic and extra-synaptic regions. It involves the modulation of receptor protomer recognition, signaling and trafficking, as well as the modulation of behavioral responses. Allosteric receptor-receptor interactions in hetero-complexes give rise to concepts like meta-modulation and protein modulation. The introduction of receptor-receptor interactions was the origin of the concept of meta-modulation provided by Katz and Edwards in 1999, which stood for the fine-tuning or modulation of nerve cell transmission. In 2000-2010, Ribeiro and Sebastiao, based on a series of papers, provided strong support for their view that adenosine can meta-modulate (fine-tune) synaptic transmission through adenosine receptors. However, another term should also be considered: protein modulation, which is the key feature of allosteric receptor-receptor interactions leading to learning and consolidation by novel adapter proteins to memory. Finally, it must be underlined that allosteric receptor-receptor interactions and their involvement both in brain disease and its treatment are of high interest. Their pathophysiological relevance has been obtained, especially for major depressive disorder, cocaine use disorder, and Parkinson's disease.

Evidence for the existence of facilitatory interactions between the dopamine D2 receptor and the oxytocin receptor in the amygdala of the rat. Relevance for anxiolytic actions.

Introduction: The amygdala is a limbic region of high value for understanding anxiety and its treatment. Dopamine D2 receptors (D2Rs) and oxytocin receptors (OXTRs) have both been shown to participate in modulating anxiety involving effects in the amygdala. The goal is to understand if D2R-OXTR heterocomplexes exist in the central amygdala and if, through enhancing allosteric receptor-receptor interactions, may enhance anxiolytic actions. Methods: The methods used involve the shock-probe burying test, the in situ proximity ligation assay (PLA), image acquisition and analysis, and the BRET2 assay. Bilateral cannulas were introduced into the amygdala, and the effects of the coadministration of oxytocin and the D2R-like agonist quinpirole into the amygdala were studied. Results: The combination treatment enhanced the anxiolytic effects compared to the single treatment. The D2R/D3R antagonist raclopride blocked the effects of the combination treatment of oxytocin and the D2R agonist, although oxytocin is regarded as a distinct modulator of fear-mediating anxiolytic effects. In situ PLA results indicate the existence of D2R-OXTR heteroreceptor complexes and/or the co-location of OXTR and D2R within the same cell membrane nanodomains in the central amygdala. With BRET2, evidence is given for the existence of D2R-OXTR heteromers in HEK293 cells upon co-transfection. Discussion: The enhanced behavioral effects observed upon co-treatment with OXTR and D2R agonists may reflect the existence of improved positive receptor-receptor interactions in the putative D2R-OXTR heterocomplexes in certain neuronal populations of the basolateral and central amygdala. The D2R-OXTR heterocomplex, especially upon agonist co-activation in the central amygdala, may open a new pharmacological venue for the treatment of anxiety.

Dysfunctional Heteroreceptor Complexes as Novel Targets for the Treatment of Major Depressive and Anxiety Disorders.

Among mental diseases, major depressive disorder (MDD) and anxiety deserve a special place due to their high prevalence and their negative impact both on society and patients suffering from these disorders. Consequently, the development of novel strategies designed to treat them quickly and efficiently, without or at least having limited side effects, is considered a highly important goal. Growing evidence indicates that emerging properties are developed on recognition, trafficking, and signaling of G-protein coupled receptors (GPCRs) upon their heteromerization with other types of GPCRs, receptor tyrosine kinases, and ionotropic receptors such as N-methyl-D-aspartate (NMDA) receptors. Therefore, to develop new treatments for MDD and anxiety, it will be important to identify the most vulnerable heteroreceptor complexes involved in MDD and anxiety. This review focuses on how GPCRs, especially serotonin, dopamine, galanin, and opioid heteroreceptor complexes, modulate synaptic and volume transmission in the limbic networks of the brain. We attempt to provide information showing how these emerging concepts can contribute to finding new ways to treat both MDD and anxiety disorders.

The oxytocin receptor represents a key hub in the GPCR heteroreceptor network: potential relevance for brain and behavior.

In the last 10 years, it has become increasingly clear that large numbers of axon collaterals extend from the oxytocin (OXT) hypothalamic axons, especially the parvocellular components, to other brain regions. Consequently, the OXT signaling system forms, like other monoamine axons, a rich functional network across several brain regions. In this manuscript, we review the recently indicated higher order G-protein coupled heteroreceptor complexes of the oxytocin receptor (OXTR), and how these, via allosteric receptor-receptor interactions modulate the recognition, signaling, and trafficking of the participating receptor protomers and their potential impact for brain and behavior. The major focus will be on complexes of the OXTR protomer with the dopamine D2 receptor (D2R) protomer and the serotonin 2A (5-HT2AR) and 2C (5-HT2CR) receptor protomers. Specifically, the existence of D2R-OXTR heterocomplexes in the nucleus accumbens and the caudate putamen of rats has led to a postulated function for this heteromer in social behavior. Next, a physical interaction between OXTRs and the growth hormone secretagogue or ghrelin receptor (GHS-R1a) was demonstrated, which consequently was able to attenuate OXTR-mediated Gαq signaling. This highlights the potential of ghrelin-targeted therapies to modulate oxytocinergic signaling with relevance for appetite regulation, anxiety, depression, and schizophrenia. Similarly, evidence for 5-HT2AR-OXTR heteromerization in the pyramidal cell layer of CA2 and CA3 in the dorsal hippocampus and in the nucleus accumbens shell was demonstrated. This complex may offer new strategies for the treatment of both mental disease and social behavior. Finally, the 5-HT2CR-OXTR heterocomplexes were demonstrated in the CA1, CA2, and CA3 regions of the dorsal hippocampus. Future work should be done to investigate the precise functional consequence of region-specific OXTR heteromerization in the brain, as well across the periphery, and whether the integration of neuronal signals in the brain may also involve higher order OXTR-GHS-R1a heteroreceptor complexes including the dopamine (DA), noradrenaline (NA) or serotonin (5-HT) receptor protomers or other types of G-protein coupled receptors (GPCRs).

Increase of 5-HT levels is induced both in mouse brain and HEK-293 cells following their exposure to a non-viral tryptophan hydroxylase construct.

Tryptophan hydroxylase type 2 (Tph2) is the rate-limiting enzyme for serotonin (5-HT) biosynthesis in the brain. Dysfunctional Tph2 alters 5-HT biosynthesis, leading to a deficiency of 5-HT, which could have repercussions on human behavior. In the last decade, several studies have associated polymorphisms of the TPH2 gene with suicidal behavior. Additionally, a 5-HT deficiency has been implicated in various psychiatric pathologies, including alcoholism, impulsive behavior, anxiety, and depression. Therefore, the TPH2 gene could be an ideal target for analyzing the effects of a 5-HT deficiency on brain function. The aim of this study was to use the construct pIRES-hrGFP-1a-Tph2-FLAG to treat CD1-male mice and to transfect HEK-293-cells and then to evaluate whether this treatment increases 5-HT production. 5-HT levels were enhanced 48 h post-transfection, in HEK-293 cells. Three days after the ocular administration of pIRES-hrGFP-1a-Tph2-FLAG to mice, putative 5-HT production was significantly higher than in the control in both hypothalamus and amygdala, but not in the brainstem. Further research will be needed on the possible application of this treatment for psychiatric diseases involving a Tph2 dysfunction or serotonin deficiency.

The Balance of MU-Opioid, Dopamine D2 and Adenosine A2A Heteroreceptor Complexes in the Ventral Striatal-Pallidal GABA Antireward Neurons May Have a Significant Role in Morphine and Cocaine Use Disorders.

The widespread distribution of heteroreceptor complexes with allosteric receptor-receptor interactions in the CNS represents a novel integrative molecular mechanism in the plasma membrane of neurons and glial cells. It was proposed that they form the molecular basis for learning and short-and long-term memories. This is also true for drug memories formed during the development of substance use disorders like morphine and cocaine use disorders. In cocaine use disorder it was found that irreversible A2AR-D2R complexes with an allosteric brake on D2R recognition and signaling are formed in increased densities in the ventral enkephalin positive striatal-pallidal GABA antireward neurons. In this perspective article we discuss and propose how an increase in opioid heteroreceptor complexes, containing MOR-DOR, MOR-MOR and MOR-D2R, and their balance with each other and A2AR-D2R complexes in the striatal-pallidal enkephalin positive GABA antireward neurons, may represent markers for development of morphine use disorders. We suggest that increased formation of MOR-DOR complexes takes place in the striatal-pallidal enkephalin positive GABA antireward neurons after chronic morphine treatment in part through recruitment of MOR from the MOR-D2R complexes due to the possibility that MOR upon morphine treatment can develop a higher affinity for DOR. As a result, increased numbers of D2R monomers/homomers in these neurons become free to interact with the A2A receptors found in high densities within such neurons. Increased numbers of A2AR-D2R heteroreceptor complexes are formed and contribute to enhanced firing of these antireward neurons due to loss of inhibitory D2R protomer signaling which finally leads to the development of morphine use disorder. Development of cocaine use disorder may instead be reduced through enkephalin induced activation of the MOR-DOR complex inhibiting the activity of the enkephalin positive GABA antireward neurons. Altogether, we propose that these altered complexes could be pharmacological targets to modulate the reward and the development of substance use disorders.

The Role of Central Serotonin Neurons and 5-HT Heteroreceptor Complexes in the Pathophysiology of Depression: A Historical Perspective and Future Prospects.

Serotonin communication operates mainly in the extracellular space and cerebrospinal fluid (CSF), using volume transmission with serotonin moving from source to target cells (neurons and astroglia) via energy gradients, leading to the diffusion and convection (flow) of serotonin. One emerging concept in depression is that disturbances in the integrative allosteric receptor-receptor interactions in highly vulnerable 5-HT1A heteroreceptor complexes can contribute to causing major depression and become novel targets for the treatment of major depression (MD) and anxiety. For instance, a disruption and/or dysfunction in the 5-HT1A-FGFR1 heteroreceptor complexes in the raphe-hippocampal serotonin neuron systems can contribute to the development of MD. It leads inter alia to reduced neuroplasticity and potential atrophy in the raphe-cortical and raphe-striatal 5-HT pathways and in all its forebrain networks. Reduced 5-HT1A auto-receptor function, increased plasticity and trophic activity in the midbrain raphe 5-HT neurons can develop via agonist activation of allosteric receptor-receptor interactions in the 5-HT1A-FGFR1 heterocomplex. Additionally, the inhibitory allosteric receptor-receptor interactions in the 5-HT1AR-5-HT2AR isoreceptor complex therefore likely have a significant role in modulating mood, involving a reduction of postjunctional 5-HT1AR protomer signaling in the forebrain upon activation of the 5-HT2AR protomer. In addition, oxytocin receptors (OXTRs) play a significant and impressive role in modulating social and cognitive related behaviors like bonding and attachment, reward and motivation. Pathological blunting of the OXTR protomers in 5-HT2AR and especially in 5-HT2CR heteroreceptor complexes can contribute to the development of depression and other types of psychiatric diseases involving disturbances in social behaviors. The 5-HTR heterocomplexes are novel targets for the treatment of MD.

Conventional and Novel Pharmacological Approaches to Treat Dopamine-Related Disorders: Focus on Parkinson's Disease and Schizophrenia.

The dopaminergic system integrated by cell groups distributed in several brain regions exerts a modulatory role in brain. Particularly important for this task are the mesencephalic dopamine neurons, which from the substantia nigra and ventral tegmental area project to the dorsal striatum and the cortical/subcortical limbic systems, respectively. Dopamine released from these neurons operates mainly via the short distance extrasynaptic volume transmission and activates five different dopaminergic receptor subtypes modulating synaptic GABA and glutamate transmission. To accomplish this task dopaminergic neurons keep mutual modulating interactions with neurons of other neurotransmitter systems, including allosteric receptor-receptor interactions in heteroreceptor complexes. As a result of its modulatory role dopaminergic mechanisms are involved in either the etiology or physiopathology of many brain diseases such as Parkinsons disease and schizophrenia. The aim of this work is to review some novel and conventional approaches that either have been used or are currently employed to treat these diseases. Particular attention is paid to the approaches derived from the knowledge recently acquired in the realm of receptor-receptor interactions taking place through multiple dopamine heteroreceptor complexes in the plasma membrane. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Brain Dopamine Transmission in Health and Parkinson's Disease: Modulation of Synaptic Transmission and Plasticity Through Volume Transmission and Dopamine Heteroreceptors.

This perspective article provides observations supporting the view that nigro-striatal dopamine neurons and meso-limbic dopamine neurons mainly communicate through short distance volume transmission in the um range with dopamine diffusing into extrasynaptic and synaptic regions of glutamate and GABA synapses. Based on this communication it is discussed how volume transmission modulates synaptic glutamate transmission onto the D1R modulated direct and D2R modulated indirect GABA pathways of the dorsal striatum. Each nigro-striatal dopamine neuron was first calculated to form large numbers of neostriatal DA nerve terminals and then found to give rise to dense axonal arborizations spread over the neostriatum, from which dopamine is released. These neurons can through DA volume transmission directly influence not only the striatal GABA projection neurons but all the striatal cell types in parallel. It includes the GABA nerve cells forming the island-/striosome GABA pathway to the nigral dopamine cells, the striatal cholinergic interneurons and the striatal GABA interneurons. The dopamine modulation of the different striatal nerve cell types involves the five dopamine receptor subtypes, D1R to D5R receptors, and their formation of multiple extrasynaptic and synaptic dopamine homo and heteroreceptor complexes. These features of the nigro-striatal dopamine neuron to modulate in parallel the activity of practically all the striatal nerve cell types in the dorsal striatum, through the dopamine receptor complexes allows us to understand its unique and crucial fine-tuning of movements, which is lost in Parkinson's disease. Integration of striatal dopamine signals with other transmitter systems in the striatum mainly takes place via the receptor-receptor interactions in dopamine heteroreceptor complexes. Such molecular events also participate in the integration of volume transmission and synaptic transmission. Dopamine modulation of the glutamate synapses on the dorsal striato-pallidal GABA pathway involves D2R heteroreceptor complexes such as D2R-NMDAR, A2AR-D2R, and NTSR1-D2R heteroreceptor complexes. The dopamine modulation of glutamate synapses on the striato-entopeduncular/nigral pathway takes place mainly via D1R heteroreceptor complexes such as D1R-NMDAR, A2R-D1R, and D1R-D3R heteroreceptor complexes. Dopamine modulation of the island/striosome compartment of the dorsal striatum projecting to the nigral dopamine cells involve D4R-MOR heteroreceptor complexes. All these receptor-receptor interactions have relevance for Parkinson's disease and its treatment.

Differential activation of arginine-vasopressin receptor subtypes in the amygdaloid modulation of anxiety in the rat by arginine-vasopressin.

RATIONALE: The amygdala plays a paramount role in the modulation of anxiety and numerous studies have shown that arginine vasopressin (AVP) elicits anxiogenic effects following either its systemic or septal administration. OBJECTIVES: The aim of this paper was to study the involvement of vasopressinergic neurotransmission in the amygdaloid modulation of unconditioned anxiety and to ascertain whether or not AVP receptor subtypes may have a differential role in this modulation. METHODS: Anxiety behavior was evaluated both in Shock-Probe Burying Test and Light-Dark Box following the bilateral microinfusion of AVP alone or AVP together with either AVP 1a or AVP 1b receptor antagonists into the central amygdala (CeA). RESULTS: AVP microinfusion elicited at low (1 ng/side) but not at high doses (10 ng/side) anxiogenic-like responses in the Shock-Probe Burying Test but not in the Light-Dark Box. SSR149415, an AVP 1b antagonist unlike Manning compound, an AVP 1a antagonist, fully prevented AVP effects in the Shock-Probe Burying Test when it was administered simultaneously with AVP. In addition, oxytocin receptor blockade also failed to affect AVP effects. No effects of any AVP antagonist by itself were observed in both anxiety paradigms. CONCLUSIONS: Our results indicate that AVP 1b receptor contribute to the amygdaloid modulation of anxiety at least in the context of the Shock-Probe Burying Test since no effects were noticed in the Light-Dark Box. It remains to the future to ascertain whether AVP receptor subtypes have indeed differential actions either in the modulation of global or specific features of unconditioned anxiety.

Dopamine D1 receptor activity is involved in the increased anxiety levels observed in STZ-induced diabetes in rats.

Epidemiological surveys have indicated that anxiety disorders are more frequent in diabetic patients than in the general population. Similar results have been shown in animal studies using the streptozotocin (STZ)-induced diabetes model. The mechanisms underlying this relationship are not clearly understood, but it has been suggested that alterations in the dopaminergic neurotransmission, which plays an important role in the amygdaloid modulation of fear and anxiety, may be involved. The aim of this study was to ascertain whether or not the amygdaloid DA D1 receptors are involved in the increase of anxiety-like behavior observed in "diabetic" animals. Adult Wistar male rats were injected with STZ (50mg/kg, i.p.) in two consecutive days and subjected to the Shock-Probe Burying Test 10days after the beginning of treatment. STZ-treated rats showed a significant increase in immobility/freezing behavior whereas no effects were elicited in latency to bury, burying behavior itself and the number of shocks received during testing as compared with non-diabetic controls. These results suggest the triggering of a passive coping response in the STZ-treated rats. Interestingly, immobility/freezing behavior was reversed following the intra-amygdaloid dopamine D1 receptor blockade by the local microinfusion of SCH23390 (100ng/side). Autoradiographic experiments showed a selective increase of [(3)H]-SCH23390 binding in the ventral intercalated paracapsular islands of STZ-treated rats when compared to the non-treated control group. Our results suggest that a hyperdopaminergic state involving DA D1 receptors within the amygdala may have a role in the increase of anxiety observed in diabetic rats.

Signaling in dopamine D2 receptor-oxytocin receptor heterocomplexes and its relevance for the anxiolytic effects of dopamine and oxytocin interactions in the amygdala of the rat.

Dopamine D2 receptor (D2R)-oxytocin receptor (OTR) interactions exist within heterocomplexes with facilitatory effects on D2R recognition and Gi/o coupling. In this work the hypothesis is tested using cotransfected HEK293 cells whether allosteric reciprocal D2R-OTR interactions can enhance signaling of D2R-OTR heterocomplexes along the CREB, MAPK and PLC pathways and whether the anxiolytic effects of OT may involve facilitatory D2R-OTR interactions within the central amygdaloid nucleus (CeA). Oxytocin enhanced the D2-like agonist quinpirole induced inhibition of the AC-PKA-pCREB signaling cascade and increased its signaling over the RAS-MAPK-pELK pathway. Quinpirole enhanced the oxytocin induced increases in the activity of the PLCbeta-IP3-calcineurin and RAS-MAPK-pELK cascades. Bilateral infusion of oxytocin (0.9-150ng/side) into the CeA of the rat elicited anxiolytic effects in the Shock-Probe Burying test, an unconditioned model of fear/anxiety. This action was not observed when oxytocin (25ng/side) was simultaneously co-infused with raclopride (neither 250 nor 500ng/side), a D2/D3 antagonist, into the CeA. Based on the current findings, the blockade of the anxiolytic effects of oxytocin by the simultaneous intra-CeA administration of raclopride can be explained by a lack of facilitatory protomer interactions in D2R-OTR heterocomplexes. Dysfunction and/or disruption of such interactions in the central amygdala may lead to anxiety development. Restoration of such interactions may represent a new strategy for development of novel anxiolytic drugs.

Role of thirst and visual barriers in the differential behavior displayed by streptozotocin-treated rats in the elevated plus-maze and the open field test.

Conflicting results have been obtained by several groups when studying the effects of streptozotocin (STZ)-treated rats in the elevated plus-maze (EPM). Since thirst is a prominent feature in STZ-induced diabetic-like condition, we studied whether the walls of the closed arms of the EPM, by limiting the search for water in the environment, may contribute to the observed differential behavioral outcomes. The aim of this study was to ascertain whether visual barriers within the EPM have an influence on the behavior of STZ-treated rats in this test of anxiety. A striking similarity between STZ-treated (50 mg/kg, i.p., in two consecutive days) and water deprived rats (72 h) was found in exploratory behavior in the EPM, showing an anxiolytic-like profile. However the anxiolytic response of STZ-treated rats exposed to the EPM shifts into an anxiogenic profile when they are subsequently tested in the open-field test, which unlike the EPM is devoid of visual barriers. Likewise, water deprived rats (72 h) also showed an anxiogenic profile when they were exposed to the open-field test. Our results indicate that experimental outcomes based on EPM observations can be misleading when studying physiological or pathological conditions, e.g. diabetes, in which thirst may increase exploratory behavior.

The intercalated paracapsular islands as a module for integration of signals regulating anxiety in the amygdala.

The intercalated paracapsular (IPC) islands are clusters of dopamine-D1-and µ-opioid 1-receptor rich GABAergic neurons which surround the rostral half of the basolateral complex of the amygdala (BLA) giving rise to several subgroups which can be further subdivided. IPC cells are small-sized and have an axonal and dendritic pattern which differs according to the group they belong. Functionally, IPC neurons are endowed with unique properties that set them apart from other amygdaloid interneurons and allow them to participate in integrative functions. Consistent with this role IPC cells usually remain confined within the amygdala where they receive BLA and cortical inputs and interact synaptically with each other. They project into both the central (CeA) and medial (MeA) amygdaloid nuclei. Their main effect at the network level seems to control the trafficking of nerve impulses to the main input (BLA) and output (CeA) stations of the amygdala. Such a task seems to be accomplished by providing feedforward inhibition to BLA neurons from putative inputs of the medial prefrontal cortex (mPFC) and to CeA from both mPFC and BLA projections. Current experimental evidence will be discussed suggesting that through feedforward inhibitory effects on specific amygdaloid nuclei IPC neurons participate in the maintenance of basal anxiety as well as in the modulation of unconditioned and conditioned fear, and in the process of fear extinction. This article is part of a Special Issue entitled: Brain Integration.

GABA(A) ρ receptor mechanisms in the rat amygdala and its role in the modulation of fear and anxiety.

RATIONALE: Accumulating evidence for the presence of GABA(A) ρ receptors within the amygdala which differ from other members of the GABA(A) receptor family in both subunit composition and functional properties has been recently obtained. OBJECTIVES: This work was conducted to study whether GABA(A) ρ receptors may have a putative role in the amygdaloid modulation of fear and anxiety. RESULTS: It was found that the bilateral intra-amygdaloid administration (6-240 pmol/side) of (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid, a selective GABA(A) ρ receptor antagonist, reduced dose-dependently the exploration of the open arms of the elevated plus-maze without affecting locomotion and increased the plasma levels of corticosterone. In contrast, bicuculline in the dose range used (1.8-60 pmol/side) induced seizures, but had no effects on the exploration of the maze. CONCLUSIONS: It is suggested that GABA(A) ρ receptors may have a role in the amygdaloid modulation of fear and anxiety.

Wiring and volume transmission in rat amygdala. Implications for fear and anxiety.

The amygdala plays a key role in anxiety. Information from the environment reaches the amygdaloid basolateral nucleus and after its processing is relayed to the amygdaloid central nucleus where a proper anxiogenic response is implemented. Experimental evidence indicates that in this information transfer a GABAergic interface controls the trafficking of impulses between the two nuclei. Recent work indicates that interneuronal communication can take place by classical synaptic transmission (wiring transmission) and by volume transmission in which the neurotransmitter diffuses and flows through the extracellular space from its site of release and binds to extrasynaptic receptors at various distances from the source. Based on evidence from our laboratory the concept is introduced that neurotransmitters in the amygdala can modulate anxiety involving changes in fear learning and memories by effects on receptor mosaics in the fear circuits through wiring and volume transmission modes of communication.

Role of the amygdaloid cholecystokinin (CCK)/gastrin-2 receptors and terminal networks in the modulation of anxiety in the rat. Effects of CCK-4 and CCK-8S on anxiety-like behaviour and [3H]GABA release.

The amygdala plays a key role in fear and anxiety. The intercalated islands are clusters of glutamate-responsive GABAergic neurons rich in cholecystokinin (CCK)-2 receptors which control the trafficking of nerve impulses from the cerebral cortex to the central nucleus of amygdala. In this study, the nature of the CCK-glutamate-GABA interactions within the rat rostral amygdala, and their relevance for anxiety, were studied. CCK/gastrin-like immunoreactive nerve terminals were found to be mainly restricted to the paracapsular intercalated islands and the rostrolateral part of the main intercalated island. Behaviourally, the bilateral microinjection of CCK-4 (0.043-4.3 pmol/side) or CCK-8S (4.3 pmol/side) into the rostrolateral amygdala reduced the open-arm exploration in the elevated plus-maze without affecting locomotion. In contrast, neither CCK-4 nor CCK-8S (0.043-4.3 pmol/side) had any effects in the shock-probe burying test as compared with their saline-treated controls. Biochemically, CCK-4 (0.3 and 1.5 microm), unlike CCK-8S, enhanced significantly the K(+)-stimulated release of [(3)H]GABA from amygdala slices. These effects were fully prevented by prior superfusion of the slices with either the selective CCK-2 receptor antagonist CR2945 (3 microm), or 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), 10 microm, a glutamatergic (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist. It is suggested that CCK modulates glutamate-GABA mechanisms by acting on CCK-2 receptors via volume transmission occurring at the level of the basolateral amygdaloid nucleus and/or by synaptic or perisynaptic volume transmission in the region of the rostrolateral main and paracapsular intercalated islands, resulting in subsequent disinhibition of the central amygdaloid nucleus and anxiety or panic-like behaviour.

Anxiolytic-like effects of the selective metabotropic glutamate receptor 5 antagonist MPEP after its intra-amygdaloid microinjection in three different non-conditioned rat models of anxiety.

The intercalated islands, clusters of dopamine D1-rich GABAergic neurons, are interposed between the basolateral and central nuclei of the amygdala, and control the traffic of nerve impulses between these two structures. Metabotropic glutamate receptor 5- (mGluR5)-like immunoreactivity was studied by immunohistochemistry in this part of the amygdala and was found to be mainly restricted to the central and basolateral nuclei and to a lesser extent to the medial paracapsular intercalated islands. The role of the metabotropic glutamate receptor 5 in the modulation of anxiety has been studied in this region by microinjection of small volumes of the mGluR5 antagonist 2-methyl-6(phenylethenyl) pyridine (MPEP), with restricted diffusion from its injection site, into the rostral amygdala near the basolateral and central amygdaloid nuclei and the intercalated islands, and the behavior of the animals was evaluated using three non-conditioned models of anxiety. Anxiolytic-like effects were observed after MPEP administration in all tests used. In the White and Black Box test, MPEP (2 nmol per side) significantly increased the time spent in the white compartment of the box. In line with these results, MPEP (8 nmol per side) increased the exploration of the open arms of the Elevated Plus-Maze. Burying behavior latency was increased and burying behavior itself was decreased in the Shock-Probe Burying test. It is suggested that anxiolytic effects of MPEP may be mediated by blockade of mGluR5 in the basolateral and/or central amygdaloid nuclei, reducing glutamate transmission in the basolateral amygdaloid nuclei and glutamate output from the central amygdala.

Anxiolytic effects of intra-amygdaloid injection of the D1 antagonist SCH23390 in the rat.

The intercalated islands are intra-amigdaloid clusters of D1 receptor rich GABAergic neurons, which control impulse traffic between the basolateral complex and the central nucleus of the amygdala. As dopaminergic transmission within the amygdala may play a role in anxiety, the effect of the D1 antagonist SCH23390 microinjected mainly close to the rostral intercalated islands in rats was studied, using the White and Black Box test. SCH23390 reduced anxiety by an increase in the latency of the first entry into the black compartment and by an increase in the total time spent in the white compartment of the White and Black Box test, while there was no significant modification of locomotion. It is suggested that blockade of D1 receptors in the rostral intercalated islands may reduce anxiety through a reduction of GABA-mediated dishinibition of the central amygdaloid nucleus.