Minerva Crespo-Ramírez

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.

Distribution of dopamine D2-like receptors in the rat amygdala and their role in the modulation of unconditioned fear and anxiety

Amygdaloid dopamine D(2) receptors play an important role in the modulation of fear/anxiety. Their topographical distribution within the amygdala is however unclear, and their role in unconditioned fear/anxiety remains largely unknown. The aim of this paper was to study the intra-amygdaloid distribution of D(2) receptors and to ascertain their role in unconditioned anxiety. Chemical anatomical studies in the rat, using D(2) and D(3)in situ hybridization, quantitative receptor autoradiography with either [(3)H]raclopride or [(125)I]sulpiride, and D(2)-like immunocytochemistry showed that the highest density of dopamine D(2) receptors is present in the central amygdaloid nucleus, particularly within its latero-capsular division, in which a D(2) but not a D(3) mRNA signal was observed. However, although at considerably reduced densities dopamine D(2) receptors were also found in other locations within the amygdala, including the basolateral nucleus. Behaviorally, the infusion of raclopride (0.75-4 μg/side) in the area of the central amygdaloid nucleus resulted at low doses in the appearance of anxiogenic-like effects in the Shock-Probe Burying test, whereas no effects of raclopride treatment were found at any dose in the Elevated Plus-Maze and the Open-Field test. Our results indicate that amygdaloid dopamine D(2)-like receptors have a topographically differentiated distribution within the rat amygdala, the major location being in the central amygdaloid nucleus. D(2)-like receptors play a role in the modulation of anxiety responses involving a potential differential function of D(2)-like receptors in the central amygdaloid nucleus versus the basolateral amygdaloid nucleus.

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.

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 µm), unlike CCK‐8S, enhanced significantly the K+‐stimulated release of [3H]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 µm), or 6,7‐dinitroquinoxaline‐2,3(1H,4H)‐dione (DNQX), 10 µm, a glutamatergic (+/–)‐α‐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.

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.

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.

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 Parkinsons 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 Parkinsons disease and its treatment.