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The discovery of receptor-receptor interactions in the early 1980s, together with a more accurate focusing of allosteric mechanisms in proteins, expanded the knowledge on the G protein-coupled receptor (GPCR)-mediated signaling processes. GPCRs were seen to operate not only as monomers, but also as quaternary structures shaped by allosteric interactions. These integrative mechanisms can change the function of the GPCRs involved, leading to a sophisticated dynamic of the receptor assembly in terms of modulation of recognition and signaling. In this context, the heterodimeric complex formed by the adenosine A2A and the dopamine D2 receptors likely represents a prototypical example. The pharmacological evidence obtained, together with the tissue distribution of the A2A-D2 heteromeric complexes, suggested they could represent a target for new therapeutic strategies addressing significant disorders of the central nervous system. The research findings and the perspectives they offer from the therapeutic standpoint are the focus of the here presented discussion.

New findings show existence of FGFR1-5-HT1A heteroreceptor complexes in 5-HT nerve cells of the dorsal and median raphe nuclei of the rat midbrain and hippocampus. Synergistic receptor-receptor interactions in these receptor complexes indicated their enhancing role in hippocampal plasticity. The existence of FGFR1-5-HT1A heteroreceptor complexes also in midbrain raphe 5-HT nerve cells open up the possibility that antidepressant drugs by increasing extracellular 5-HT levels can cause an activation of the FGF-2/FGFR1 mechanism in these nerve cells as well. Therefore, the agonist modulation of the FGFR1-5-HT1A heteroreceptor complexes and their specific role is now determined in rat medullary raphe RN33B cells and in the caudal midline raphe area of the midbrain rich in 5-HT nerve cells. The combined i.c.v. treatment with FGF-2 and the 5-HT1A agonist 8-OHDPAT synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and in the RN33B cells. Cotreatment with FGF2 and the 5-HT1A agonist induced RN33B cell differentiation as seen from development of an increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TMV but not by TMII of the 5-HT1A receptor. Taken together, the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A heteroreceptor complex in the midbrain raphe 5-HT nerve cells appears to have also a trophic role in the central 5-HT neuron systems besides playing a key role in reducing the firing of these neurons.

The central nervous system (CNS) has been proposed to be formed by functional modules (FMs) that are structurally organized as “Russian dolls”, which form transient assemblies, according to the different integrative tasks that networks within the CNS have to carry out. Integration is achieved by means of communication processes among and within FMs. Two major types of communication processes occur in the CNS, wiring transmission (WT) and volume transmission (VT). WT involves classical synaptic transmission via diffusion of a neurotransmitter across the synaptic cleft, while VT is transmission via a neuroactive substance that is carried by the extracellular and cerebrospinal fluids to distant targets. Recently, evidence has been obtained for the existence of tunneling nanotubes that mediate WT and microvesicles that mediate VT, allowing horizontal transfer of receptors, RNAs, and micro-RNAs. The recognition and decoding process at the target level involves receptor heteromers (receptor mosaics) that are generated by direct receptor–receptor interactions, as an emergent property of this system. Receptor mosaics allow the integration of incoming information at the plasma membrane level. These new aspects of the structural and functional organization of the CNS open up a new field of investigation in the physiology and pathology of the CNS.

This review is focused on the D2 heteroreceptor complexes within the ventral striatum with their receptor-receptor interactions and relevance for the treatment of schizophrenia. A "guide-and-clasp" manner for receptor-receptor interactions is proposed where "adhesive guides" may be amino acid triplet homologies, which were determined for different kinds of D2 heteroreceptor complexes. The first putative D2 heteroreceptor complex to be discovered in relation to schizophrenia was the A2A-D2 heteroreceptor complex where antagonistic A2A-D2 receptor-receptor interactions were demonstrated after A2A agonist treatment in the ventral striatum. The A2A agonist CGS 21680 with atypical antipsychotic properties may at least in part act by increasing β-arrestin2 signaling over the D2 protomer in the A2A-D2 heteroreceptor complex in the ventral striatum. The antagonistic NTS1-D2 interactions in the NTS1-D2 heteroreceptor complex in the ventral striatum are proposed as one molecular mechanism for the potential antipsychotic effects of NT. Indications were obtained that the psychotic actions of the 5-HT2AR hallucinogens LSD and DOI can involve enhancement of D2R protomer signaling via a biased agonist action at the 5-HT2A protomer in the D2-5-HT2A heteroreceptor complex in the ventral striatum. Facilitatory allosteric D2likeR-OTR interactions in heteroreceptor complexes in nucleus accumbens may have a role in social and emotional behaviors. By blocking the D2 protomers of these heteroreceptor complexes, antipsychotics can fail to reduce the negative symptoms of schizophrenia. The discovery of different types of D2 heteroreceptor complexes gives an increased understanding of molecular mechanisms involved in causing schizophrenia and new strategies for its treatment and understanding the side effects of antipsychotics.

G protein-coupled receptors (GPCRs) play critical roles in cellular processes and signaling and have been shown to form heteromers with diverge biochemical and/or pharmacological activities that are different from those of the corresponding monomers or homomers. However, despite extensive experimental results supporting the formation of GPCR heteromers in heterologous systems, the existence of such receptor heterocomplexes in the brain remains largely unknown, mostly because of the lack of appropriate methodology. Herein, we describe the in situ proximity ligation assay procedure underlining its high selectivity and sensitivity to image GPCR heteromers with confocal microscopy in brain sections. We describe here how the assay is performed and discuss advantages and disadvantages of this method compared with other available techniques.

A large body of evidence indicates that G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signaling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signaling molecules. This integrative phenomenon is reciprocal and can place also RTK signaling downstream of GPCR. Formation of either stable or transient complexes by these two important classes of membrane receptors is involved in regulating all aspects of receptor function, from ligand binding to signal transduction, trafficking, desensitization, and downregulation among others. Functional phenomena can be modulated with conformation-specific inhibitors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses or by means of small interfering heteroreceptor complex interface peptides. The bioluminescence resonance energy transfer (BRET) technology has emerged as a powerful method to study the structure of heteroreceptor complexes closely associated with the study of receptor-receptor interactions in such complexes. In this chapter, we provide an overview of different BRET(2) assays that can be used to study the structure of GPCR-RTK heteroreceptor complexes and their functions. Various experimental designs for optimization of these experiments are also described.

Publisher Summary In the chapter, aspects of the information handling in the central nervous system (CNS) are analyzed in the frame of the so called volume transmission (VT)— that is, of a widespread mode of intercellular communication that occurs in the extracellular fluid (ECF) of the brain and in the cerebrospinal fluid (CSF). Five aspects are examined, the first three on the basis of deductions from the available evidence and from the characteristics of the VT, the fourth on the basis of recent experimental evidence gathered in the laboratory, and the last one on the basis of the analogy between VT communication channels and the connection devices in the B-type unorganized machine. The chapter concludes that learning in the CNS networks can take place not only by instructions acting on the cells (classically by changing the weights— that is, efficacies of the synapses in the circuit, but also by changing the characteristics of the extracellular matrix, for example, by focal changes in its capability of transmitting or destroying a signal flowing through it.

Publisher Summary This chapter discusses clearance mechanisms of volume transmission (VT) signals— namely, clearance over the brain-blood barrier and over the leaky brain-CSF (cerebrospinal-fluid) interface as well as through receptor mediated uptake into other discrete nerve cell systems through internalization. Evidence are presented that protein kinase C may be an important regulator of the tight and adherens junctions of the brain-CSF and CSF-blood interfaces, as well as of endocrine-like VT. Moreover, endothelial nitric oxide may be a regulator of the clearance of VT signals over the brain-blood barrier. Evidence has been obtained that exogenous β- endorphin and galanin locally and in CSF can undergo rapid somatodendritic internalization into discrete populations of interneuronal populations of the neostriatum and hippocampus, underlining their potential role in CSF signaling. These results support the possibility that at least in these specific interneurons β-endorphin and galanin and/or their active fragments can act as transcellular signals by effects on gene regulation and/or by rapid dendritic and somatic release into the surrounding neuropil to activate their respective high affinity peptide receptors.

Publisher Summary This chapter discusses the neurochemical and behavioral studies on L-dopa toxicity in the model of manganese kesioned nigrostriatal pathway in the rat. Evidence was obtained with in vitro studies of a toxic action of L-dopa on neuroblastoma cells, evaluated as [ 3 H]-thymidine uptake and cell survival. It was found that the chronic administration of L-dopa to normal adult rats or mice did not lead to nigral cell disappearance and did not endanger the survival of foetal ventral mesencephalic grafts inplanted in 6-OHDA lesioned striatum. Accordingly, studies performed on humans did not give evidence of the histological signs of L-dopa toxicity in normal subjects or in Parkinsonian patients. However, some clinical trials suggest that the wearing-off phenomenon, tardive dyskinesia, and on–off phenomena are the possible side effects of long-term L-dopa treatment. Apart from human studies, experiments on the in vivo toxicity of L-dopa were performed in unlesioned mice or rats and on foetal grafts in 6-OHDA lesioned rats suggesting a lack of any effect of L-dopa chronic administration on cell survival.

Publisher Summary This chapter focuses on the analysis of glucocorticoid receptors (GR)-immunoreactive nerve cell and glial cell populations in the pre- and postnatal period, in adult life, and in aging emphasizes the involvement of these receptors in the trophic regulation of neuronal networks. The nuclear GR immunoreactivity in glial cells is not as strong as in the nerve cells but is clearly demonstrable within the astroglia. Evidence suggests that prenatal corticosterone affects locomotor activity of the late prepubertal male and female rats. It has been reported that adrenalectomy in adulthood can protect against agerelated hippocampal changes and that increases in the corticosterone serum levels can result in neurotoxic effects on hippocampal nerve cells. Future work will determine the differential trophic effects of glucocorticoids in various nerve cell populations and their interaction with mineralocorticoids in this regulation.

The vigilance promoting drug Modafinil has been demonstrated to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity (40 mg/kg sc) in the nigrostriatal dopamine (DA) system of the black mouse. 16, 29 A single dose of Modafinil (30 mg/kg ip, at different time intervals before and after MPTP) significantly counteracted the MPTP-induced depletion of striatal dopamine (DA) as analyzed 14 days later. A similar treatment protocol showed that Modafinil significantly counteracted the paradoxical MPTP-induced increase in locomotion and motility. Chronic administration of Modafinil (30 mg/kg ip daily for 14 days) led to a more pronounced counteraction of the MPTP-induced decrease in striatal DA and also attenuated the MPTP-induced increase in DA turnover. After a similar chronic treatment with Modafinil (10–100 mg/kg) the disappearance of tyrosine hydroxylase immunoreactive neurons in substantia nigra appeared to be dose-dependently counteracted. In summary, biochemical, behavioral and morphological evidence indicates a potential neuroprotective role of Modafinil, which could eventually lead to new pharmacological strategies in Parkinson's disease, once the mechanism of action has been established.

Publisher Summary This chapter describes the morphological and functional adaptation of basal ganglia neurons after cerebral ischemia. A series of neurological and behavioral impairments have been associated with brain injury that follows spontaneous or experimental cerebral ischemia. In 1979, a procedure—the 4-vessel occlusion method—of transient forebrain ischemia in the rat was proposed as a model of cardiac arrest. Reproducible focal neuronal damage in the dorsolateral part of the striatum as well as in various tele-diencephalic areas was obtained with this method. Recently, another reliable method for the induction of striatal ischemic damage in rats was obtained by local injection of endothelin-1 (ET-1), a potent vasoconstrictor agent. The hypoxicischemic damage induced by these procedures differently affects the various cell populations of the brain, in particular neurons. The chapter summarizes some of the results obtained by studying morphological and functional recovery after ischemic striatal injury induced by the 4-vessel occlusion or ET-1 intrastriatal injection in the rat.

Publisher Summary This chapter focuses on the immunocytochemical studies on glucocorticoid receptor (GR). The localization of the GR-immunoreactive neurons in the rat central nervous system has been studied using an anti-GR monoclonal mouse antibody, MAb 7, directed against an epitope in the N-terminal domain of the rat GR. Subjective analyses reporting various degrees of intensity/density of GR immunoreactivity can be improved by computer-assisted image analysis to provide semiquantitative microdensitometric evaluations. An in vitro study using BUGR-2 revealed the presence of GR immunoreactivity in primary cultures of mixed glial cells and within glial cell lines. Some of the discrepancies may be explained by technical circumstances such as different species, cell types, anti-GR antibodies, fixation/permeabilization procedures, and detection techniques.

Publisher Summary In many vertebrate classes, mammals included, the pineal complex acts as a biological clock converting the information about environmental lighting into a specific hormonal output. The glands of reptiles and amphibians are directly photosensitive, but during the phylogenesis, the pineal has been transformed into a pure neuroendocrine organ, which receives lighting information from the eyes through a complex neuronal pathway. Nerve impulses, stimulated by light impinging on the retinas and carrying the coded photic information, travel via the optic nerve, optic chiasm, and—following decussation to the hypothalamic suprachiasmatic nucleus—a structure that seems to represent the main autonomous oscillator generating circadian rhythmicity. The effects of environmental lighting on pineal indoleamine metabolism and melatonin biosynthesis result from a light-induced decrease and a dark-induced increase in the firing rate of the adrenergic fibers innervating the gland. The presence of specific protein substrates for the cyclic adenosine monophosphate (cAMP)-, calcium/calmodulin (Ca/CM)-, and calcium/phosphatidylserine (Ca/PS)-dependent protein kinases in the pineal gland has been reported. Three major proteins of 59, 56, and 35 kDa are phosphorylated by the cAMP-dependent enzyme.

The present paper supports the hypothesis that hypothalamic dopamine (DA), noradrenaline (NA) and adrenaline (A) nerve terminal systems play an important role in the control of the secretion of LH, prolactin, TSH, GH and ACTH secretion. These catecholamine (CA) systems are not only influenced pre-synaptically by steroidal and hypophyseal hormones, but estrogen also produces a marked desensitization of α-adrenergic receptors as revealed by a marked reduction in the affinity of 3H-clonidine for its binding sites in the hypothalamus following estrogen treatment of castrated female rats for three days. Under these conditions estrogen exerts inhibitory feedback action on LH secretion.