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DIFFERENTIAL REGIONAL AND CELLULAR DISTRIBUTION OF DOPAMINE D2-LIKE RECEPTORS : AN IMMUNOCYTOCHEMICAL STUDY OF SUBTYPE-SPECIFIC ANTIBODIES IN RAT AND HUMAN BRAIN

Dopamine D2‐like receptors (D2, D3, and D4) are major targets for action of typical and atypical neuroleptics, commonly used in the treatment of schizophrenia. To understand their individual functional contribution, subtype‐selective anti‐peptide antibodies were raised against D2, D3, and D4 receptor proteins. The antibodies were shown to be specific on immunoblots of rat brain membranes and immunoprecipitated the solubilized native dopamine receptors in an antibody concentration‐dependent manner. In addition, they also bind selectively to the respective recombinant D2, D3, and D4 receptor membrane proteins from cDNA transfected cells. Immunolocalization studies show that the D2‐like receptor proteins had differential regional and cellular distribution in the cerebral cortex, hippocampus, basal ganglia, cerebellum, and midbrain, thus providing anatomical substrate for area‐specific regulation of the dopamine neurotransmission. In cortical neurons, D4 receptor protein was found in both pyramidal and nonpyramidal cells, whereas D2 and D3 seem to be mostly associated with nonpyramidal interneurons. In rat hippocampus, the expression pattern of D2‐like receptors (D4>D3>D2) mirrored that obtained with immunoprecipitation studies. D2 and D4 receptor immunolabeling was observed in the thalamic reticular nucleus, which was negative for the D3 subtype. Species differences were also observed; for example, the D4 subtype receptor is the most highly expressed protein in the rat cortex, whereas it is significantly less in human cortex. Differential patterns of D2, D3, and D4 receptor expression in rat and human brain should shed light on the therapeutic actions of neuroleptic drugs and may lead to the development of more specifically targeted antipsychotic drugs. J. Comp. Neurol. 402:353–371, 1998.

Molecular phenotype of rat striatal neurons expressing the dopamine D5 receptor subtype

Dopamine is one of the principal neurotransmitters in the basal ganglia, where it plays a critical role in motor control and cognitive function through its interactions with the specific dopamine receptors D1 to D5. Although the activities mediated by most dopamine receptor subtypes have already been determined, the role of the D5 receptor subtype in the basal ganglia has still not been established. Furthermore, it is often difficult to distinguish between dopamine D5 and D1 receptors as they are stimulated by the same ligands, and they have a similar molecular structure and pharmacology. In an effort to understand the differences between these two receptor subtypes, we have studied the distribution of neurons containing D5 receptors in the striatum, and their molecular phenotype. As a result, we show that the D5 receptor subtype is present in two different populations of striatal neurons, projection neurons and interneurons. Overall, the abundance of this receptor subtype in the striatum is low, particularly in striatal projection neurons of both the direct and indirect projection pathways. In contrast, the expression of D5 receptors in striatal interneurons (cholinergic, somatostatin‐ or parvalbumin‐positive neurons) is high, while low to moderate expression was observed in calretinin‐positive neurons. Our results demonstrate the presence of D5 receptors in all the striatal cell populations so far described, although at different intensities in each. The fact that a large number of striatal neurons express the D5 receptor subtype suggests that this receptor fulfils an important function in the process of integrating information in the striatum.

Dopamine D4 receptors are heterogeneously distributed in the striosomes/matrix compartments of the striatum.

Two important aspects of striatal function, exploratory behaviour and motor co‐ordination, require the integrity of the dopamine D4 receptor subtype. These receptors are also implicated in the pathophysiology of certain neuropsychiatric disorders. However, the distribution of D4 receptors in the striatum has not yet been described and this situation impairs our understanding of the anatomical substrate in which D4 receptors function. We developed a D4 receptor‐specific anti‐body that has permitted us to investigate the regional and cellular localization of the receptor in the neostriatum of the rat, mouse, cat and monkey. The subcellular distribution and the synaptic organization of this receptor were also determined in the rat striatum. We found moderate levels of D4 receptor expression in the caudoputamen and lower levels in the nucleus accumbens. These receptors were expressed in cell bodies and in the neuropil and were heterogeneously distributed among different striatal compartments, being more abundant in striosomes than in the matrix. At the subcellular level, the receptor immunoreactivity was mainly localized to dendritic shafts and spines. The prominent immunoreactivity observed in the striosomes indicates that integrative processes involved in D4‐mediated limbic behaviours occurs through the striosomes rather than accumbens, whereas the motor behaviour is based in the striatal matrix.

Expression of D4 dopamine receptors in striatonigral and striatopallidal neurons in the rat striatum

Recent studies have reported the regional distribution of D(4) dopamine receptors in the rat striatum at the cellular and subcellular levels. However, the precise identity of the striatal neurons that express these receptors remains unknown. We have studied the expression of D(4) receptors in the striatal interneurons as well as in the output regions of the striatum using immunohistochemistry. Furthermore, we have evaluated the contribution of the striatum to D(4) receptor immunoreactivity in these areas by means of ibotenic acid lesion of the striatum. D(4) receptors were observed in the substantia nigra pars reticulata (SNr), the entopeduncular nucleus (EP) and the globus pallidus (GP), and they were found, using electron microscopy, to be located presynaptically. D(4) immunoreactivity in the striatal output nuclei was observed to dramatically decrease following lesion of the striatum with ibotenic acid. Striatal interneurons were not found to express D(4) receptors. These results demonstrate that D(4) receptors are located almost exclusively in striatal projection neurons, in both striatonigral and striatopallidal neurons.

Galanin–neuropeptide Y (NPY) interactions in central cardiovascular control: involvement of the NPY Y1 receptor subtype

The interactions between neuropeptide Y (NPY), specifically through NPY Y1 and Y2 receptor subtypes, and galanin [GAL(1–29)] have been analysed at the cardiovascular level. The cardiovascular effects of intracisternal coinjections of GAL(1–29) with NPY or NPY Y1 or Y2 agonists, as well as quantitative receptor autoradiography of the binding characteristics of NPY Y1 and Y2 receptor subtypes in the nucleus of the solitary tract (NTS), in the presence or absence of GAL(1–29), have been investigated. The effects of coinjections of GAL(1–29) and the NPY Y1 agonist on the expression of c‐FOS immunoreactivity in the NTS were also studied. The coinjection of NPY with GAL(1–29) induced a significant vasopressor and tachycardic action with a maximum 40% increase (P < 0.001). The coinjection of the NPY Y1 agonist and GAL(1–29) induced a similar increase in mean arterial pressure and heart rate as did NPY plus GAL(1–29), actions that were not observed with the NPY Y2 agonist plus GAL(1–29). GAL(1–29), 3 nm, significantly and substantially (by ∼ 40%) decreased NPY Y1 agonist binding in the NTS. This effect was significantly blocked (P < 0.01) in the presence of the specific galanin antagonist M35. The NPY Y2 agonist binding was not modified in the presence of GAL(1–29). At the c‐FOS level, the coinjection of NPY Y1 and GAL(1–29) significantly reduced the c‐FOS‐immunoreactive response induced by either of the two peptides. The present findings suggest the existence of a modulatory antagonistic effect of GAL(1–29) mediated via galanin receptors on the NPY Y1 receptor subtype and its signalling within the NTS.

Dopamine D4 receptor activation decreases the expression of μ‐opioid receptors in the rat striatum

The dopaminergic and opioid peptide systems interact in many nuclei of the brain. In the striatum, dopamine/opioid peptide interactions modulate locomotor and motivated behaviors as well as reward, motivational, and tolerance processes in opiate dependence. Dopamine D4 receptors (D4 R) and μ‐opioid receptors (MOR) are highly concentrated in the striosomes (islands) of the striatum, suggesting the existence of receptor–receptor interactions between them. In the present work we studied the role of D4 R in modulating MOR expression in the islands by using immunohistochemistry and image analysis. The activation of D4 R by the agonist PD168,077 (1 mg/kg) decreased MOR immunoreactivity (IR) in the striosomes 6 hours after drug treatment. MOR IR levels had recovered 12 hours later. Treatment with a D4 R antagonist (L745,870, 1mg/kg) blocked downregulation of MOR IR, showing that the D4 R agonist effects observed were specific. Furthermore, treatment with the D2/D3 receptor agonist quinpirol (1 mg/kg) and D2/D3 receptor antagonist raclopride (1 mg/kg) had no effect in MOR IR, suggesting that D4 R is the only D2‐like receptor producing an MOR downregulation in the islands. The decreases of MOR IR in the striosomes suggest that D4 R activation may reduce MOR signaling. Increasing evidence has demonstrated that the islands in the striatum play a critical role in habit acquisition during drug addiction. D4 R/MOR interactions could be crucial in such processes. J. Comp. Neurol. 502:358–366, 2007.

Immunocytochemical localization of somatostatin in the cerebral cortex of lizards

The distribution of somatostatin-like immunoreactivity in the cerebral cortex of two lizards has been studied. Results are similar in both species. Somatostatin-positive neurons show variable morphology; they are bipolar, multipolar or pyramidal cells. Their distribution within the cerebral cortex is not homogeneous: they tend to be found in the innermost cortical layer, the deep plexiform layer, where they constitute a constant population in the region of the dorsomedial cortex. All immunoreactive processes observed in the cerebral cortex belong to somatostatin cortical neurons since no immunoreactive fiber is found to reach or leave the telencephalic cortex. Most of the dendrites are smooth or sparsely spinous. Axons are abundant in the deep plexiform layer; in the dorsomedial cortex a prominent terminal field appears in the outermost region of the superficial plexiform layer, which may arise from the neuronal somatostatin immunoreactive population found deeper in the same cortex.

A tangential neuronal migration in the olfactory bulbs of adult lizards

TRITIATED thymidine ([3H]thymidine) autoradiography at different times after experiments showed that postnatally generated neurones in the adult lizard olfactory bulbs are not generated in a local germinative zone but in the ventricular zone (VZ) of the cerebral hemispheres, which is a long distance from the olfactory bulbs. The new cells originating in the VZ of rostral telencephalon migrate to both main and accessory olfactory bulbs through the olfactory peduncle, along a highly restricted pathway orthogonal to the orientation of radial glial fibres. Our results suggest that the olfactory bulb interneurones of adult lizards may follow a tangential migration route from their germinative zone, as is the case in mammals.

Effect of acute and continuous morphine treatment on transcription factor expression in subregions of the rat caudate putamen. Marked modulation by D4 receptor activation.

Acute administration of the dopamine D(4) receptor (D(4)R) agonist PD168,077 induces a down-regulation of the μ opioid receptor (MOR) in the striosomal compartment of the rat caudate putamen (CPu), suggesting a striosomal D(4)R/MOR receptor interaction in line with their high co-distribution in this brain subregion. The present work was designed to explore if a D(4)R/MOR receptor interaction also occurs in the modulation of the expression pattern of several transcription factors in striatal subregions that play a central role in drug addiction. Thus, c-Fos, FosB/ΔFosB and P-CREB immunoreactive profiles were quantified in the rat CPu after either acute or continuous (6-day) administration of morphine and/or PD168,077. Acute and continuous administration of morphine induced different patterns of expression of these transcription factors, effects that were time-course and region dependent and fully blocked by PD168,077 co-administration. Moreover, this effect of the D(4)R agonist was counteracted by the D(4)R antagonist L745,870. Interestingly, at some time-points, combined treatment with morphine and PD168,077 substantially increased c-Fos, FosB/ΔFosB and P-CREB expression. The results of this study give indications for a general antagonistic D(4)R/MOR receptor interaction at the level of transcription factors. The change in the transcription factor expression by D(4)R/MOR interactions in turn suggests a modulation of neuronal activity in the CPu that could be of relevance for drug addiction.

Serotonin innervation of the cerebral cortex in lizards

The serotonin (5-HT) innervation of the cerebral cortex in two species of lizards has been studied. Results show no differences between both species. Most of the cerebral cortex of these lizards is innervated by serotoninergic fibers, which are fine and varicose. Their density varies greatly from one cortical region to another: the areas with higher density of serotoninergic fibers and terminals are parts of the medial and dorsal cortices. There is a laminar pattern of distribution of serotoninergic fibers. In the medial cortex, 5-HT fibers are found preferentially in both plexiform layers just above and below the cellular layer. In the dorsomedial cortex, there is an immunoreactive plexus in the outermost third of the superficial plexiform layer and another in the depth of the layer, whereas 5-HT fibers are distributed evenly in the deep plexiform layer of this cortex. In the pars medialis of the dorsal cortex, serotoninergic fibers are abundant in all layers, whereas in the pars lateralis, fibers are found predominantly in the external third of the superficial plexiform layer. The lateral cortex is almost devoid of immunoreactive fibers. These results show a different organization of the cortical serotonin innervation between lizards and turtles.