Distribution of G-protein-coupled receptor kinase (GRK) isoforms 2, 3, 5 and 6 mRNA in the rat brain.

There is limited knowledge about the distribution of the different G-protein-coupled receptor kinases (GRKs) in the rat brain, especially for the recently cloned isoforms GRK5 and GRK6. In this work an overview will be given of the mRNA expression patterns of four G-protein-coupled receptor kinases, GRK2 (betaARK1), GRK3 (betaARK2), GRK5 and GRK6 in the rat brain. As now shown by us and recently by others GRK2 and GRK3 are widely distributed in rat brain with nearly the same expression pattern. But GRK3, in general, appeared to be weaker expressed than GRK2 in most brain areas. Exceptions were the islands of Calleja, the compact part of the substantia nigra and the locus coeruleus. GRK3 mRNA was very low expressed or absent in the striatum and in some hypothalamic and thalamic nuclei. The expression pattern of GRK6 was also similar to GRK2. In the caudate putamen GRK6 yielded the strongest hybridization signal of all GRK types. GRK5 took a special position. The message for this form was not expressed ubiquitously in the brain but was mainly localized in limbic brain regions with a very prominent expression in the lateral septal area. GRK5 may therefore be involved in reward and addiction. Accordingly, a higher expression level of GRK5 mRNA was found in the lateral septum of cocaine-sensitized rats as compared to controls.     

D3 dopamine receptor mRNA is widely expressed in the human brain

Considerable attention has been given to the association of the D₃ dopamine receptor subtype and limbic function based on the abundant localization of D₃ receptor sites and mRNA expression in the islands of Calleja and nucleus accumbens in experimental animals. Though most human anatomical studies have focused on the role of D₃ receptors in limited brain structures, detailed information about the overall anatomical organization of the D₃ receptor in the human brain is still, however, not available. In the current study, we examined the anatomical distribution of D₃ receptor mRNA expression at different levels of the human brain in whole hemisphere horizontal cryosections using in situ hybridization. This approach made it possible to establish for the first time the wide and heterogenous expression of the D₃ receptor gene throughout the human brain. As expected, the most abundant D₃ mRNA expression levels were found in the islands of Calleja and discrete cell cluster populations within the ventral striatum/nucleus accumbens region. High levels were also evident within the dentate gyrus and striate cortex. Low to moderate D₃ mRNA expression levels were apparent in most brain areas including all other cortical regions (highest in the anterior cingulate/subcallosal gyrus), caudate nucleus, putamen, anterior and medial thalamic nucleus, mammillary body, amygdala, hippocampal CA region, lateral geniculate body, substantia nigra pars compacta, locus coeruleus, and raphe nuclei. While the current anatomical map of D₃ receptor mRNA expression in the human brain does confirm previous reports that D₃ receptors may play important roles in limbic-related functions such as emotion and cognition, the findings also suggest other non-limbic functions for D₃ mRNA-expressing cell populations such as processing of motor and sensory information.     

Language gene]

The human capacity for acquiring speech and language must derive, at least in part, from the genome. Recent advance in the field of molecular genetics finally discovered 'Language Gene'. Disruption of FOXP2 gene, the firstly identified 'language gene' causes severe speech and language disorder. To elucidate the anatomical basis of language processing in the brain, we examined the expression pattern of FOXP2/Foxp2 genes in the monkey and rat brains through development. We found the preferential expression of FOXP2/Foxp2 in the striosomal compartment of the developing striatum. Thus, we suggest the striatum, particularly striosomal system may participate in neural information processing for language and speech. Our suggestion is consistent with the declarative/ procedural model of language proposed by Ullman (1997, 2001), which the procedural memory-dependent mental grammar is rooted in the basal ganglia and the frontal cortex, and the declarative memory-dependent mental lexicon is rooted in the temporal lobe.     

Autoradiographic distribution of binding sites of [3H]SKF 38393, a selective dopamine D1 receptor agonist, in the mouse forebrain

Distribution of binding sites of [3H]SKF 38393, a selective dopamine D1 receptor agonist, was studied on forebrain coronal sections of CXBI/ByJ mice by radioligand binding and digital subtraction autoradiography. Highest levels of [3H]SKF 38393 binding were detected in olfactory tubercle and caudate nucleus/putamen. Intermediate to low concentrations of receptors were indicated in cortex, amygdala, hypothalamus, claustrum, and septal area, whereas the lowest binding was found in white matter tracts (commissura anterior, corpus callosum). Analysis of data on caudate nucleus/putamen indicates a striosomal pattern of binding with a gradient of binding sites from medial to lateral caudate/putamen. The distribution of [3H]SKF 38393 binding sites corresponds to that of dopaminergic projection fields in the studied areas.     

Distribution of the histamine H(2) receptor in monkey brain and its mRNA localization in monkey and human brain

The distribution of histamine H(2) receptor mRNA was determined by in situ hybridization histochemistry in human and monkey brain. In the case of monkey brain, we combined this technique with receptor ligand autoradiography to compare the distribution of mRNA and receptor binding sites. [(125)I]Iodoaminopotentidine ([(125)I]-APT), a reversible, high specific activity antagonist with high affinity and selectivity for the H(2) receptor, was used for receptor autoradiography. Radiolabeled oligonucleotides derived from the human mRNA sequence encoding this receptor were used as hybridization probes. The highest density of the H(2) receptor mRNA in human and monkey brain was found in caudate and putamen nuclei and external layers of cerebral cortex. Moderate levels were seen in the hippocampal formation and lower densities in the dentate nucleus of cerebellum. Areas such as globus pallidus, amygdaloid complex, cerebellar cortex, and substantia nigra were devoid of hybridization signal. The distribution of H(2) receptor mRNA in monkey brain is generally in good agreement with that of the corresponding binding sites: prominent in caudate, putamen, accumbens nuclei, and cortical areas. The hippocampus showed lower densities of receptors and low levels were detected in the globus pallidus pars lateralis. No binding sites were seen in amygdaloid complex and substantia nigra. The distribution of histaminergic innervation is in good correlation with the areas of high density for H(2) receptors: caudate, putamen, and external layers of cerebral cortex in monkey and human brain. The presence of mRNA in caudate and putamen nuclei, together with its absence from substantia nigra, suggests that the H(2) receptors found in the striatum are synthesized by intrinsic cells and not by nigral dopaminergic cells. These striatal H(2) receptors may be located on short circuit striatal interneurons or somatodendritically on striatal projection neurons which project to the globus pallidus pars lateralis. In conclusion, the present results, which constitute, to our knowledge, the first report of the regional distribution of mRNA encoding H(2) receptors detected by in situ hybridization, define the sites of synthesis of H(2) receptors and are the basis for future, more detailed studies that should result in a better understanding of H(2) receptor function.     

Cholinergic innervation of the human striatum, globus pallidus, subthalamic nucleus, substantia nigra, and red nucleus.

The anatomical organization of cholinergic markers such as acetylcholinesterase, choline acetyltransferase, and nerve growth factor receptors was investigated in the basal ganglia of the human brain. The distribution of choline acetyltransferase-immunoreactive axons and varicosities and their relationship to regional perikarya showed that the caudate, putamen, nucleus accumbens, olfactory tubercle, globus pallidus, substantia nigra, red nucleus, and subthalamic nucleus of the human brain receive widespread cholinergic innervation. Components of the striatum (i.e., the putamen, caudate, olfactory tubercle, and nucleus accumbens) displayed the highest density of cholinergic varicosities. The next highest density of cholinergic innervation was detected in the red nucleus and subthalamic nucleus. The level of cholinergic innervation was of intermediate density in the globus pallidus and the ventral tegmental area and low in the pars compacta of the substantia nigra. Immunoreactivity for nerve growth factor receptors (NGFr) was confined to the cholinergic neurons of the basal forebrain and their processes. Axonal immunoreactivity for NGFr was therefore used as a marker for cholinergic projections originating from the basal forebrain (Woolf et al., '89: Neuroscience 30:143-152). Although the vast majority of striatal cholinergic innervation was NGFr-negative and, therefore, intrinsic, the striatum also contained NGFr-positive axons, indicating the existence of an additional cholinergic input from the basal forebrain. This basal forebrain cholinergic innervation was more pronounced in the putamen than in the caudate. The distribution of NGFr-positive axons suggested that the basal forebrain may also project to the globus pallidus but probably not to the subthalamic nucleus, substantia nigra, or red nucleus. The great majority of cholinergic innervation to these latter three structures and to parts of the globus pallidus appeared to come from cholinergic neurons outside the basal forebrain, most of which are probably located in the upper brainstem. These observations indicate that cholinergic neurotransmission originating from multiple sources is likely to play an important role in the diverse motor and behavioral affiliations that have been attributed to the human basal ganglia.     

Preprotachykinin-A mRNA expression in the human and monkey brain: An in situ hybridization study.

The mRNA expression for preprotachykinin-A (PPT-A) was studied throughout the human and cynomolgus monkey brain to assess the neuroanatomical expression pattern of the PPT-A gene in primates. In situ hybridization showed that the PPT-A mRNA is expressed highly in specific regions of the postmortem human brain, including the striatum, islands of Calleja, hypothalamus (posterior, premammillary, medial mammillary, and ventromedial nuclei), superior and inferior colliculi, periaqueductal gray, and oculomotor nuclear complex. PPT-A mRNA-expressing neurons also were present in the paranigralis (ventral tegmental area) and were scattered in the bed nucleus stria terminalis throughout the sublenticular substantia innominata region, including the diagonal band of Broca and the nucleus basalis of Meynert. In the hippocampus, high PPT-A mRNA expression was localized predominantly to the polymorphic layer of the dentate gyrus; no labeled cells were present in the granular layer. Positively labeled cells also were found scattered in the CA regions as well as in the amygdaloid complex. Neocortical expression of PPT-A mRNA was localized mainly to the deep laminae (layers V/VI), except for the striate cortex (labeling was seen also in superficial layers). The subiculum, thalamus, globus pallidus, ventral pallidum, substantia nigra pars compacta, red nucleus, pontine nuclei, and cerebellum were characterized by very weak to undetectable expression of PPT-A mRNA. An expression pattern was evident in the monkey forebrain similar to that observed in the human, except for the absence of PPT mRNA-expressing cells in the medial mammillary nucleus despite intense expression in supramammillary, lateral mammillary, and premammillary nuclei. Overall, more similarities than differences are apparent between primate species in the expression pattern of the PPT-A gene. J. Comp. Neurol. 411;56-72, 1999.     

Immunohistochemical demonstration of differential substance P-, met-enkephalin-, and glutamic-acid-decarboxylase-containing cell body and axon distributions in the corpus striatum of the cat

The immunohistochemical localization of neuronal cell bodies and axons reactive for substance P (SP) and methionine-enkephalin (ME) was investigated in the corpus striatum of the adult cat brain and compared with that of glutamate decarboxylase (GAD), synthetic enzyme for gamma-aminobutyric acid. Striatal cell bodies reactive for ME could be identified only in colchicine treated cats, are medium size, ovoid striatal cells, and are found in large numbers in a more or less even distribution throughout the caudate nucleus, putamen, and nucleus accumbens. The striatal region most densely occupied by ME-immunoreactive cells is the ventral and central part of the caudate head. Modest numbers of larger ME-reactive neurons are dispersed throughout the entopeduncular nucleus and the pars reticulata of the substantia nigra. Striatal cells of medium size reactive for SP could be identified, with or without colchicine, in largest numbers in the medial half of the caudal three-fourths of the putamen and in clusters of irregular size and shape in the head of the caudate nucleus. Cells reactive for SP are also common in layer II and the islands of Calleja of the olfactory tubercle. We could not reliably visualize GAD-positive cell bodies in the striatum, even with colchicine treatment; however, they could be seen readily in all pallidal structures such as the globus pallidus, ventral pallidum, entopeduncular nucleus, and substantia nigra. Axons reactive for ME are found mainly in the globus pallidus where they form a dense and even network throughout the nucleus. The globus pallidus is almost devoid of SP reactivity except near its extreme caudal pole. Conversely, SP-immunoreactive axons form dense meshworks in the entopeduncular nucleus and substantia nigra where ME immunoreactivity is minimal. Fewer, but still ample numbers, of SP-reactive axons are present also in the ventral tegmental and retrorubral areas of the midbrain tegmentum and in the ventral pallidum of the basal forebrain, but only sparse ME-reactive axons are present in these areas. This differential distribution of SP- and ME-containing axons in the pallidal and nigral structures stands in contrast to the relatively homogeneous and dense distribution of GAD-containing axons throughout the dorsal and ventral pallidum, entopeduncular nucleus, and substantia nigra.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of 125I-neurotensin binding sites in human forebrain: comparison with the localization of acetylcholinesterase

The distribution of 125I-neurotensin binding sites was compared with that of acetylcholinesterase reactivity in the human basal forebrain by using combined light microscopic radioautography/histochemistry. High 125I-neurotensin binding densities were observed in the bed nucleus of the stria terminalis, islands of Calleja, claustrum, olfactory tubercle, and central nucleus of the amygdala; lower levels were seen in the caudate, putamen, medial septum, diagonal band nucleus, and nucleus basalis of Meynert. Adjacent sections processed for cholinesterase histochemistry demonstrated a regional overlap between the distribution of labeled neurotensin binding sites and that of intense acetylcholinesterase staining in all of the above regions, except in the bed nucleus of the stria terminalis, claustrum, and central amygdaloid nucleus, where dense 125I-neurotensin labeling was detected over areas containing only weak to moderate cholinesterase staining. At higher magnification, 125I-neurotensin-labeled binding sites in the islands of Calleja, supraoptic nucleus of the hypothalamus, medial septum, diagonal band nucleus, and nucleus basalis of Meynert were selectively associated with neuronal perikarya found to be cholinesterase-positive in adjacent sections. Moderate 125I-neurotensin binding was also apparent over the cholinesterase-reactive neuropil of these latter three regions. These data suggest that neurotensin (NT) may directly influence the activity of magnocellular cholinergic neurons in the human basal forebrain, and may be involved in the physiopathology of dementing disorders such as Alzheimer's disease, in which these neurons have been shown to be affected.     

Hemiparkinsonism in a monkey after unilateral internal carotid artery infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is associated with regional ipsilateral changes in striatal dopamine D-2 receptor density

Infusion of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into the right internal carotid artery of a cynomologus monkey (Macaca fascicularis) induced an almost complete loss of the dopaminergic innervation of the right caudate-putamen and hemiparkinsonism. Digital subtraction autoradiography revealed that at 8 weeks postinjection, a major increase in [3H]spiroperidol binding to D-2 sites in the lateral regions of the right caudate nucleus and putamen occurred, without a significant change in the medial caudate nucleus and putamen. The 92-96% decrease in specific [3H]mazindol binding observed in the right striatum extended into the medial caudate nucleus and putamen and confirmed the extensive loss of dopamine inputs to this structure. The region of the increase in D-2 receptor density is innervated by somatosensory, motor and parietal cortex. This indicates that the increase in D-2 receptor density in this region of the striatum may play a particularly important role in the L-dihydroxyphenylanine-induced motoric recovery observed in such animals.     

Distribution of D2 dopamine receptor mRNA in the primate brain.

1. The distribution of the messenger RNA (mRNA) encoding the D₂dopamine receptor has been mapped in the monkey brain by hybridization.

2. Using [35s]-labelled riboprobes corresponding to the region of the D2 dopamine receptor spanning the third cytosolic loop and the sixth and seventh transmembrane domains, specific hybridization was observed in a number of neural structures.

3. High levels of mRNA expression were observed in the caudate, putamen, and claustrum. Significant amounts were also identified in the hippocampus, lateral geniculate nucleus, much of the cortex, amygdala, pons, and thalamus. High levels of this mRNA were also visualized in the substantia nigra, likely reflecting autoreceptor synthesis.

4. While the distribution of D₂ dopamine receptor mRNA was similar between the monkey and previously published maps in the rat, several differences were noted.

5. These results demonstrate the feasibility of visualizing this mRNA in the primate brain, and suggest that a similar analysis of human postmortem brain material may be possible.     

Effects of haloperidol and clozapine on Fos expression in the primate striatum

In cynomolgus monkeys, the typical neuroleptic haloperidol induced strong expression of the immediate early gene product Fos in both the nucleus accumbens shell and the dorsal striatum. In the caudate nucleus, haloperidol induced staining was more marked in the striosomes than the matrix. The atypical neuroleptic clozapine also induced Fos expression in the nucleus accumbens, but, in contrast to haloperidol, had only a small effect in the dorsal striatum. Additionally, clozapine was more potent than haloperidol at inducing Fos-like immunoreactivity in the islands of Calleja. These results are similar to those typically obtained in rodents, and suggest that the basic mechanisms underlying the regional specificity of the effects of atypical neuroleptics are likely be conserved between these two mammalian orders.     

Differential regulation of the dopamine D1, D2 and D3 receptor gene expression and changes in the phenotype of the striatal neurons in mice lacking the dopamine transporter

Mice with a genetic disruption of the dopamine transporter (DAT-/-) exhibit locomotor hyperactivity and profound alterations in the homeostasis of the nigrostriatal system, e.g. a dramatic increase in the extracellular dopamine level. Here, we investigated the adaptive changes in dopamine D1, D2 and D3 receptor gene expression in the caudate putamen and nucleus accumbens of DAT-/- mice. We used quantitative in situ hybridization and found that the constitutive hyperdopaminergia results in opposite regulations in the gene expression for the dopamine receptors. In DAT-/- mice, we observed increased mRNA levels encoding the D3 receptor (caudate putamen, +60-85%; nucleus accumbens, +40-107%), and decreased mRNA levels for both D1 (caudate putamen, -34%; nucleus accumbens, -45%) and D2 receptors (caudate putamen, -36%; nucleus accumbens, -33%). Furthermore, we assessed the phenotypical organization of the striatal efferent neurons by using double in situ hybridization. Our results show that in DAT+/+ mice, D1 and D2 receptor mRNAs are segregated in two different main populations corresponding to substance P and preproenkephalin A mRNA-containing neurons, respectively. The phenotype of D1 or D2 mRNA-containing neurons was unchanged in both the caudate putamen and nucleus accumbens of DAT-/- mice. Interestingly, we found an increased density of preproenkephalin A-negative neurons that express the D3 receptor mRNA in the nucleus accumbens (core, +35%; shell, +46%) of DAT-/- mice. Our data further support the critical role for the D3 receptor in the regulation of D1-D2 interactions, an action being restricted to neurons coexpressing D1 and D3 receptors in the nucleus accumbens.     

Mapping of c-fos gene expression in the brain during morphine dependence and precipitated withdrawal, and phenotypic identification of the striatal neurons involved

The c-fos gene is expressed in the central nervous system in response to various neuronal stimuli. Using in situ hybridization, we examined the effects of chronic morphine treatment and withdrawal on c-fos mRNA in the rat brain, and particularly within identified striatal neurons. Morphine dependence was induced by subcutaneous implantation of two pellets of morphine for 6 days and withdrawal was precipitated by administration of naltrexone. Placebo animals and morphine-dependent rats showed a very weak c-fos mRNA expression in all the structures studied. Our study emphasized the spatial variations in c-fos mRNA expression, and also revealed a peak expression of c-fos mRNA at 1 h after naltrexone-precipitated withdrawal in the projection areas of dopaminergic neurons, noradrenergic neurons and in several regions expressing opiate receptors. Interestingly, morphine withdrawal induces c-fos mRNA expression in the two efferent populations of the striatum (i.e. striatonigral and striatopallidal neurons) both in the caudate putamen and nucleus accumbens. Moreover, the proportions of activated neurons during morphine withdrawal are different in the caudate putamen (mostly in striatopallidal neurons) and in the shell and core parts of the nucleus accumbens (mostly in striatonigral neurons). The activation of striatopallidal neurons suggests a predominant dopaminergic regulation on c-fos gene expression in the striatum during withdrawal. On the contrary, c-fos induction in striatonigral neurons during withdrawal seems to involve a more complex regulation like opioid-dopamine interactions via the mu opioid receptor and the D1 dopamine receptor coexpressed on this neuronal population or the implication of other neurotransmitter systems.     

Cell death atlas of the postnatal mouse ventral forebrain and hypothalamus: Effects of age and sex

Naturally occurring cell death is essential to the development of the mammalian nervous system. Although the importance of developmental cell death has been appreciated for decades, there is no comprehensive account of cell death across brain areas in the mouse. Moreover, several regional sex differences in cell death have been described for the ventral forebrain and hypothalamus, but it is not known how widespread the phenomenon is. We used immunohistochemical detection of activated caspase‐3 to identify dying cells in the brains of male and female mice from postnatal day (P) 1 to P11. Cell death density, total number of dying cells, and regional volume were determined in 16 regions of the hypothalamus and ventral forebrain (the anterior hypothalamus, arcuate nucleus, anteroventral periventricular nucleus, medial preoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus, and ventromedial nucleus of the hypothalamus; the basolateral, central, and medial amygdala; the lateral and principal nuclei of the bed nuclei of the stria terminalis; the caudate‐putamen; the globus pallidus; the lateral septum; and the islands of Calleja). All regions showed a significant effect of age on cell death. The timing of peak cell death varied between P1 to P7, and the average rate of cell death varied tenfold among regions. Several significant sex differences in cell death and/or regional volume were detected. These data address large gaps in the developmental literature and suggest interesting region‐specific differences in the prevalence and timing of cell death in the hypothalamus and ventral forebrain. J. Comp. Neurol. 521:2551–2569, 2013. © 2013 Wiley Periodicals, Inc.