Dopamine D4 Receptor Is a Regulator of Morphine-Induced Plasticity in the Rat Dorsal Striatum.

Long-term exposition to morphine elicits structural and synaptic plasticity in reward-related regions of the brain, playing a critical role in addiction. However, morphine-induced neuroadaptations in the dorsal striatum have been poorly studied despite its key function in drug-related habit learning. Here, we show that prolonged treatment with morphine triggered the retraction of the dendritic arbor and the loss of dendritic spines in the dorsal striatal projection neurons (MSNs). In an attempt to extend previous findings, we also explored whether the dopamine D4 receptor (D4R) could modulate striatal morphine-induced plasticity. The combined treatment of morphine with the D4R agonist PD168,077 produced an expansion of the MSNs dendritic arbors and restored dendritic spine density. At the electrophysiological level, PD168,077 in combination with morphine altered the electrical properties of the MSNs and decreased their excitability. Finally, results from the sustantia nigra showed that PD168,077 counteracted morphine-induced upregulation of µ opioid receptors (MOR) in striatonigral projections and downregulation of G protein-gated inward rectifier K+ channels (GIRK1 and GIRK2) in dopaminergic cells. The present results highlight the key function of D4R modulating morphine-induced plasticity in the dorsal striatum. Thus, D4R could represent a valuable pharmacological target for the safety use of morphine in pain management.

Pharmacological activation of dopamine D4 receptor modulates morphine-induced changes in the expression of GAD65/67 and GABAB receptors in the basal ganglia.

Dopamine D4 receptor (D4R) stimulation, in a putative D4R/µ opioid heteroreceptor (MOR) complex, counteracts the molecular, cellular and behavioural actions of morphine which are associated with morphine addiction, without any effect on its analgesic properties. In the present work, we have evaluated the role of D4R in modulating the effects of a continuous treatment with morphine on the GABAergic system in the basal ganglia. It has been demonstrated that the co-administration of a D4R agonist together with morphine leads to a restoration of GABA signaling by preventing drug-induced changes in GAD65/67 expression in the caudate putamen, globus palidus and substantia nigra. Results from GABABR1 and GABABR2 expression suggest a role of D4R in modulation of the GABAB heteroreceptor complexes along the basal ganglia, especially in the functional divisions of the caudate putamen. These results provide a new proof of the functional interaction between D4R and MOR and we postulate this putative heteroreceptor complex as a key target for the development of a new strategy to prevent the addictive effects of morphine in the treatment of pain. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.

Selective ablation of striatal striosomes produces the deregulation of dopamine nigrostriatal pathway.

The striatum is a complex structure in which the organization in two compartments (striosomes and matrix) have been defined by their neurochemical profile and their input-output connections. The striosomes receive afferences from the limbic brain areas and send projections to the dopamine neurons of the substantia nigra pars compacta. Thereby, it has been suggested that the striosomes exert a limbic control over the motor function mediated by the surrounding matrix. However, the functionality of the striosomes are not completely understood. To elucidate the role of the striosomes on the regulation of the nigral dopamine neurons, we have induced specific ablation of this compartment by striatal injections of the neurotoxin dermorphin-saporin (DS) and dopamine neurotransmission markers have been analyzed by immunohistochemistry. The degeneration of the striosomes resulted in a nigrostriatal projections imbalance between the two striatal compartments, with an increase of the dopamine neurotransmission in the striosomes and a decrease in the matrix. The present results highlight the key function of the striosomes for the maintenance of the striatal dopamine tone and would contribute to the understanding of their involvement in some neurological disorders such as Huntington's disease.

Males but not females show differences in calbindin immunoreactivity in the dorsal thalamus of the mouse model of fragile X syndrome.

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by the loss of the Fmr1 gene product, fragile X mental retardation protein. Here we analyze the immunohistochemical expression of calcium-binding proteins in the dorsal thalamus of Fmr1 knockout mice of both sexes and compare it with that of wildtype littermates. The spatial distribution pattern of calbindin-immunoreactive cells in the dorsal thalamus was similar in wildtype and knockout mice but there was a notable reduction in calbindin-immunoreactive cells in midline/intralaminar/posterior dorsal thalamic nuclei of male Fmr1 knockout mice. We counted the number of calbindin-immunoreactive cells in 18 distinct nuclei of the dorsal thalamus. Knockout male mice showed a significant reduction in calbindin-immunoreactive cells (range: 36-67% lower), whereas female knockout mice did not show significant differences (in any dorsal thalamic nucleus) when compared with their wildtype littermates. No variation in the calretinin expression pattern was observed throughout the dorsal thalamus. The number of calretinin-immunoreactive cells was similar for all experimental groups as well. Parvalbumin immunoreactivity was restricted to fibers and neuropil in the analyzed dorsal thalamic nuclei, and presented no differences between genotypes. Midline/intralaminar/posterior dorsal thalamic nuclei are involved in forebrain circuits related to memory, nociception, social fear, and auditory sensory integration; therefore, we suggest that downregulation of calbindin protein expression in the dorsal thalamus of male knockout mice should be taken into account when analyzing behavioral studies in the mouse model of FXS.

Phenotypic changes in calbindin D28K immunoreactivity in the hippocampus of Fmr1 knockout mice.

Fragile X syndrome (FXS), the most prevalent form of inherited mental retardation, is caused by the lack of FMRP (fragile mental retardation protein) as a result of the transcriptional silencing of the FMR1 gene. Here we analyze the immunohistochemical expression of the calbindin D28K protein in the hippocampus of Fmr1 knockout (KO) mice and compare it with that of their wildtype (WT) littermates. The spatial distribution pattern of calbindin-immunoreactive cells in the hippocampus was similar in WT and KO mice but for each age studied (ranging from 3.5-8 months) the dentate gyrus of Fmr1-KO mice showed a significant reduction in calbindin-immunoreactive granule cells. Also, the number of calbindin-immunoreactive cells was reduced in the CA1 pyramidal layer in KO mice compared to their WT littermates. In addition, Frm1-KO mice showed a group of calbindin-immunoreactive cells located only in the left CA3b subregion that was only sometimes observed in WT mice. Overall, the absence of FMRP results in a dysregulation of the calbindin protein expression in the hippocampus. This dysregulation is cell type- and time-dependent and as a consequence key elements of the hippocampal trisynaptic circuitry may lack calbindin in critical periods for normal memory/learning abilities to be achieved and may explain some of the FXS symptoms observed in the Fmr1-KO mouse model.

Distinct immunohistochemically defined areas in the medial amygdala in the developing and adult mouse.

The medial amygdala has been considered a subpallial structure, but various studies have shown that is a somewhat complex structure expressing both pallial and subpallial gene markers. In this regard, we analyzed the immunohistochemical expression of the neurotransmitter GABA, the vesicular glutamate transporter type 2 (VGLUT2), the neuronal nitric oxide synthase (nNOS), and the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the developing and adult mouse medial amygdala. From intermediate embryonic stages on, neurochemical data show a distinctive superficial region forming a band all along the medial amygdalar surface. This superficial band displays a strong VGLUT2-immunoreactive neuropil and numerous CR-immunoreactive fibers, as well as some nNOS-, CR- or CB-positive cells. In contrast, the superficial region of the posterior medial amygdala appears to be non-GABA immunoreactive. This band in the posterior medial amygdala matches a Tbr1-expressing territory. Our results also show differences between dorsal and ventral parts of the postnatal and adult posterior medial amygdala. Especially, a compact cell aggregate of nNOS immunoreactive cells was found in the ventral portion of the medial amygdala, whereas the dorsal part is occupied by scattered weakly stained cells. Comparison of our results with gene expression patterns and fiber-tracing studies, let us to propose that the superficial band is a pallial derivative, whereas the dorsal and ventral nuclei of the posterior medial amygdala receive each neurons from different subpallial compartments, and the latter one a subset of excitatory, pallial projection neurons.

Development and adult organization of the lateral part of the bed nucleus of the stria terminalis in the chicken.

The lateral part of the bed nucleus of the stria terminalis (BSTL) is a component of the subpallial amygdala located near the ventral sulcus of the lateral ventricle, but its limits have not been well defined in birds. In this study, we analyzed the expression patterns of a number of neurochemical markers: GABA, calbindin (CB), calretinin (CR), or neuronal nitric oxide synthase (nNOS), in the embryonic and adult chicken brain, to further characterize the organization of the avian BSTL. From embryonic day 16, it was possible to distinguish three different regions within BSTL on the basis of cytoarchitectonic and immunohistochemical features. A central region, referred to as lateral bed nucleus of the stria terminalis pars densocellularis (BSTLdc), is characterized by numerous tightly packed cell bodies, most of which are GABA-immunoreactive (ir), and two peripheral regions with lower cellular density displaying a moderate GABA expression, referred to as lateral bed nucleus of the stria terminalis, plexiform part 1 (BSTLp1) and plexiform part 2 (BSTLp2), respectively. In contrast to BSTLdc, both plexiform parts are characterized by the presence of many fibers and terminals immunoreactive for nNOS and CR, as well as some CR-ir scattered cells. A distinctive feature of BSTLp2 is a population of CB-ir cells embedded in a slightly CB-ir neuropil. Comparison of our immunohistochemical data with gene expression data suggests that BSTLdc and BSTLp1 are pallidal in nature, whereas BSTLp2 receives important contributions from the entopeduncular/preoptic area.

Calcium-binding proteins, neuronal nitric oxide synthase, and GABA help to distinguish different pallial areas in the developing and adult chicken. I. Hippocampal formation and hyperpallium.

To better understand the formation and adult organization of the avian pallium, we studied the expression patterns of gamma-aminobutyric acid (GABA), calbindin (CB), calretinin (CR), and neuronal nitric oxide synthase (nNOS) in the hippocampal formation and hyperpallium of developing and adult chicks. Each marker showed a specific spatiotemporal expression pattern and was expressed in a region (area)-specific but dynamic manner during development. The combinatorial expression of these markers was very useful for identifying and following the development of subdivisions of the chicken hippocampal formation and hyperpallium. In the hyperpallium, three separate radially arranged subdivisions were present since early development showing distinct expression patterns: the apical hyperpallium (CB-rich); the intercalated hyperpallium (nNOS-rich, CB-poor); the dorsal hyperpallium (nNOS-poor, CB-moderate). Furthermore, a novel division was identified (CB-rich, CR-rich), interposed between hyper- and mesopallium and related to the lamina separating both, termed laminar pallial nucleus. This gave rise at its surface to part of the lateral hyperpallium. Later in development, the interstitial nucleus of the apical hyperpallium became visible as a partition of the apical hyperpallium. In the hippocampal formation, at least five radial divisions were observed, and these were compared with the divisions proposed recently in adult pigeons. Of note, the corticoid dorsolateral area (sometimes referred as caudolateral part of the parahippocampal area) contained CB immunoreactivity patches coinciding with Nissl-stained cell aggregates, partially resembling the patches described in the mammalian entorhinal cortex. Each neurochemical marker was present in specific neuronal subpopulations and axonal networks, providing insights into the functional maturation of the chicken pallium.

Distribution of GABA, calbindin and nitric oxide synthase in the developing chick entopallium.

The distribution of GABA, calbindin and neuronal nitric oxide synthase (nNOS) was analyzed in the developing avian entopallium. The study was carried out in chick embryos from embryonic day (E)8 to hatching postnatal day (P)0, using immunohistochemical methods. At E8, GABA-positive cells were observed in pallial regions. Neither calbindin nor nNOS-immunoreactive cells were observed. At E10, the number of GABA neurons in the prospective entopallium increased and also nNOS cells were observed. Lightly stained nNOS neurons predominated over intensely stained ones. Calbindin immunoreactivity was not observed in the entopallium. At E12, the entopallial complex appeared as the pallial region displaying the highest density of GABA neurons. Also the whole entopallium displayed an intensely stained calbindin neuropil with many embedded stained cells. From E12 on, there was a decrease in the expression of nNOS. At E14-16, both GABA and calbindin-immunoreactive neurons were numerous and homogeneously distributed within the entopallium. The whole entopallium displayed a moderately stained neuropil. From E18 to P0, GABA and nNOS immunoreactivities remained similar to previous stages. At these stages, calbindin immunoreactivity within the entopallium consisted of a moderately stained central region bordered dorsally by a pale stained region. These two areas could correspond to the entopallial core and the perientopallial belt, respectively.

Distribution of nitric oxide-producing neurons in the developing and adult mouse amygdalar basolateral complex.

We analysed the expression of neuronal nitric oxide synthase (nNOS) in the mouse amygdalar basolateral complex (BLC) from embryonic day 15.5 to adult, using standard immunohistochemical methods. Our results indicate that each nucleus of the amygdalar basolateral complex displays a distinct nNOS expression pattern, which is established during the ontogenesis with minor changes in the adult. The basomedial nucleus (BM) exhibited the highest nNOS immunoreactivity in the basolateral complex, observable from early embryonic stages, whereas the lateral nucleus displayed the lowest level of immunoreactivity. The expression pattern for nNOS in the basolateral nucleus differed substantially from that of the lateral and basomedial nuclei, showing a slightly increase in the number of nNOS cells and neuropil staining from intermediate developmental until early postnatal stages. Two distinct types of nitrergic neurons, densely and lightly stained neurons, were observed in the developing basolateral complex. Both types of putative nitrergic neurons were unevenly distributed in the basolateral complex. On the basis of previous data regarding the colocalization between nNOS and GABA in the mouse claustrum, we suggest that nNOS expressing neurons in the basolateral amygdalar complex are both GABAergic and non-GABAergic.

Development of neurons and fibers containing calcium binding proteins in the pallial amygdala of mouse, with special emphasis on those of the basolateral amygdalar complex.

We studied the development of neurons and fibers containing calbindin, calretinin, and parvalbumin in the mouse pallial amygdala, with special emphasis on those of the basolateral amygdalar complex. Numerous calbindin-immunoreactive (CB+) cells were observed in the incipient basolateral amygdalar complex and cortical amygdalar area from E13.5. At E16.5, CB+ cells became more abundant in the lateral and basolateral nuclei than in the basomedial nucleus, showing a pattern very similar to that of gamma-aminobutyric acid (GABA)ergic neurons. Many CB+ cells observed in the pallial amygdala appeared to originate in the anterior entopeduncular area/ganglionic eminences of the subpallium. The density of CB+ cells gradually increased in the pallial amygdala until the first postnatal week and appeared to decrease later, coinciding with the postnatal appearance of parvalbumin cells and raising the possibility of a partial phenotypic shift. Calretinin (CR) immunoreactivity could be observed in a few cells and fibers in the pallial amygdala at E14.5, and by E16.5 it became a good marker of the different nuclei of the basolateral amygdalar complex. Numerous CB+ and CR+ varicosities, part of which have an intrinsic origin, were observed in the basolateral amygdalar complex from E16.5, and some surrounded unstained perikarya and/or processes before birth, indicating an early formation of inhibitory networks. Each calcium binding protein showed a distinct spatiotemporal expression pattern of development in the mouse pallial amygdala. Any alteration in the development of neurons and fibers containing calcium binding proteins of the pallial amygdala may result in important disorders of emotional and social behavior.

Embryonic and postnatal development of GABA, calbindin, calretinin, and parvalbumin in the mouse claustral complex.

We analyzed the development of immunoreactive expression patterns for the neurotransmitter gamma-aminobutyric acid (GABA) and the calcium-binding proteins calbindin, calretinin, and parvalbumin in the embryonic and postnatal mouse claustral complex. Each calcium-binding protein shows a different temporal and spatial pattern of development. Calbindin-positive cells start to be seen very early during embryogenesis and increase dramatically until birth, thus becoming the most abundant cell type during embryonic development, especially in the ventral pallial part of the claustrum. The distribution of calbindin neurons throughout the claustrum during embryonic development partly parallels that of GABA neurons, suggesting that at least part of the calbindin neurons of the claustral complex are GABAergic and originate in the subpallium. Parvalbumin cells, on the other hand, start to be seen only postnatally, and their number then increases while the density of calbindin neurons decreases. Based on calretinin expression in axons, the core/shell compartments of the dorsal claustrum start to be clearly seen at embryonic day 18.5 and may be related to the development of the thalamoclaustral input. Comparison with the expression of Cadherin 8, a marker of the developing dorsolateral claustrum, indicates that the core includes a central part of the dorsolateral claustrum, whereas the shell includes a peripheral area of the dorsolateral claustrum, plus the adjacent ventromedial claustrum. The present data on the spatiotemporal developmental patterns of several subtypes of GABAergic neurons in the claustral complex may help for future studies on temporal lobe epilepsies, which have been related to an alteration of the GABAergic activity.

Distinct types of nitric oxide-producing neurons in the developing and adult mouse claustrum.

We studied at the light and electron microscopic levels the nitric oxide-producing neurons in the mouse claustrum. Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemical staining were used to reveal putative nitrergic neurons. We also analyzed colocalization of nNOS with the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) as well as the ontogenesis of the nNOS-immunoreactive neurons, providing evidence for different populations of nitrergic neurons in the mouse claustrum. The general staining pattern was similar for the histochemical and the immunohistochemical methods, resulting in neuron and neuropil staining throughout the whole claustrum. We described two populations of nitric oxide-producing neurons in the mouse claustrum on the basis of a different level of nNOS expression. Densely nNOS-stained neurons were mostly GABA immunoreactive, displayed ultrastructural features typically seen in aspiny neurons, and may originate in the subpallium; they were first seen in the claustrum at embryonic stage 17.5 and probably represent local inhibitory interneurons. Densely stained cells were found from rostral to caudal levels throughout the dorsal claustrum and the endopiriform nucleus. Lightly nNOS-stained neurons, on the other hand, were more numerous than densely stained ones, especially in the dorsal claustrum. These claustral lightly stained cells, barely observed in the NADPH-diaphorase reacted sections, were mostly non-GABAergic, and appeared earlier during ontogenesis than densely stained cells (at embryonic stages 15.5-16.5). We suggest that these neurons are probably projection neurons.

Expression of calcium-binding proteins in the mouse claustrum.

The present paper describes the distribution of three calcium-binding proteins (calbindin D28k, calretinin, and parvalbumin) in the mouse dorsal claustrum and endopiriform nucleus. The three calcium-binding proteins were distinctly expressed in structures of both the claustrum and the endopiriform nucleus. Calbindin was the calcium-binding protein showing the highest expression in the claustrum and the endopiriform nucleus. In contrast, calretinin-immunoreactive structures, particularly cell bodies, were very scarce in these regions. Both calbindin-immunoreactive and parvalbumin-immunoreactive neurons were more abundant in the claustrum than in the endopiriform nucleus, and more in rostral than in caudal levels. Nevertheless, calcium-binding protein immunoreactive neurons constitute a minority population of claustral neurons. The colocalization study of calbindin and parvalbumin immunoreactivities has demonstrated that both calcium-binding proteins are mostly expressed by separate claustral neurons in the mouse. On the other hand, our results on parvalbumin and calretinin immunoreactivity match a novel subdivision of the mouse claustrum mostly based on the pattern of cadherin expression [Neuroscience 106 (2001) 505]. In this sense, we propose that a specific zone of the dorsal claustrum with cell bodies that strongly express Rcad and cadherin-8 would be the selective target for parvalbumin-expressing fibers, and that they would be mostly avoided by calretinin-expressing axons.

Mesencephalic and diencephalic afferent connections to the thalamic nucleus rotundus in the lizard, Psammodromus algirus.

The present work is an analysis of the afferent projections to the thalamic nucleus rotundus in a lizard, both at the light- and electron-microscopic level, using biotinylated dextran amine (BDA) as a neuroanatomical tracer. This study has confirmed previously reported afferent projections to nucleus rotundus in reptiles and has also identified a number of new cellular aggregates projecting to this dorsal thalamic nucleus. After BDA injections into nucleus rotundus, retrogradely labelled neurons were observed consistently within the following neuronal groups in the midbrain and the diencephalon: (i) the stratum griseum centrale of the optic tectum; (ii) the nucleus subpretectalis in the pretectum; (iii) the nucleus ansa lenticularis posterior, the posterior nucleus of the ventral supraoptic commissure, and the posteroventral nucleus, in the dorsal thalamus and (iv) the lateral suprachiasmatic nucleus and part of the reticular complex in the ventral thalamus. Tectal axons entering nucleus rotundus were fine and varicose and formed exclusively asymmetric synaptic contacts, mainly on small dendritic profiles. Rotundal neurons had symmetric synapses made by large boutons probably of nontectal origin. After comparing our results with those in other reptiles, birds and mammals, we propose that the sauropsidian nucleus rotundus forms part of a visual tectofugal pathway that conveys mesencephalic visual information to the striatum and dorsal ventricular ridge, and is similar to the mammalian colliculo-posterior/intralaminar-striatoamygdaloid pathway, the function of which may be to participate in visually guided behaviour.