Neuropeptide content and connectivity of the rat claustrum.

The rat claustrum has a homogeneous distribution of the neuropeptides somatostatin (SOM), cholecystokinin (CCK) and vasoactive intestinal polypeptide (VIP) along its rostrocaudal axis. In general, neuropeptide levels are comparable to those of overlying pyriform cortex. Visualization of mRNA encoding SOM, CCK and VIP in cell bodies of the claustrum by in situ hybridization histochemistry demonstrates that all 3 neuropeptides are contained in intrinsic claustral neurons. Mid-coronal section of the claustrum itself, or interruption of potential rostral, caudal or medial connections between the claustrum and the rest of the brain did not significantly alter levels of VIP, SOM or CCK in claustrum, cerebral cortex, or basal ganglia. Isolation of the claustrum from the cerebral cortex immediately dorsal to it along its rostrocaudal aspect caused no change in peptide levels in claustrum indicating that VIP, SOM and CCK projections to claustrum do not arrive from dorsal cortical areas. Transection of the external capsule above the claustrum caused a 50-100% elevation of all 3 peptides on the contralateral side of the lesion, suggesting that VIP, SOM and CCK synthesis and/or release within the claustrum may be regulated by projections from the contralateral side. VIP, SOM and CCK are candidates for neurotransmitters contained in neurons whose cell bodies are within the claustrum and possibly also immediately overlying lateral neocortex, and have their terminals mainly within the claustrum itself.

Purification and cDNA cloning of ARPP-16, a cAMP-regulated phosphoprotein enriched in basal ganglia, and of a related phosphoprotein, ARPP-19.

ARPP-16 (cAMP-regulated phosphoprotein of Mr = 16,000) is a substrate for cAMP-dependent protein kinase and is enriched in the basal ganglia. ARPP-16 has been purified to homogeneity from the soluble fraction of bovine caudate nuclei. An additional substrate for cAMP-dependent protein kinase of Mr = 19,000 (ARPP-19) was found to cross-react with rabbit anti-serum prepared against purified ARPP-16. Immunological analysis indicated that ARPP-16 was enriched in the basal ganglia while ARPP-19 was present in similar levels in all brain regions studied and was also present in non-neuronal tissues. ARPP-19 was also purified to homogeneity from bovine caudate nucleus cytosol. Using oligonucleotide probes based on the partial amino acid sequence of purified ARPP-16, cDNA clones for ARPP-16 and ARPP-19 were isolated from a bovine caudate nucleus cDNA library and sequenced. The predicted amino acid sequences of the two proteins were identical except that ARPP-19 had an additional 16 amino acids at the NH2-terminal. The two cDNA clones share an identical 3'-untranslated region of 756 nucleotides. The cDNA clone for ARPP-16 contained 806 additional nucleotides located 3' to this common sequence. The 5'-untranslated regions of the two clones were entirely different. The results suggest the possibility that ARPP-16 and ARPP-19 are produced by alternative transcription and splicing.

Distribution of DARPP-32 in the basal ganglia: an electron microscopic study.

DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein, has been studied by light and electron microscopical immunocytochemistry in the rat caudatoputamen, globus pallidus and substantia nigra. In the caudatoputamen, DARPP-32 was present in neurons of the medium-sized spiny type. Immunoreactivity for DARPP-32 was present in dendritic spines, dendrites, perikaryal cytoplasm, most but not all nuclei, axons and a small number of axon terminals. Immunoreactive axon terminals in the caudatoputamen formed symmetrical synapses with immunolabeled dendritic shafts or somata. Neurons having indented nuclei were never immunoreactive. In the globus pallidus and substantia nigra pars reticulata, DARPP-32 was present in myelinated and unmyelinated axons and in axon terminals. The labelled axon terminals in these regions formed symmetrical synaptic contacts on unlabelled dendritic shafts or on unlabelled somata. These data suggest that DARPP-32 is present in striatal neurons of the medium-sized spiny type and that these DARPP-32-immunoreactive neurons form symmetrical synapses on target neurons in the globus pallidus and substantia nigra. The presence of DARPP-32 in these striatal neurons and in their axon terminals suggests that DARPP-32 mediates part of the response of medium-size spiny neurons in the striatum to dopamine D-1 receptor activation.

Sexually dimorphic level of CCK-8-like immunoreactive neuronal somata within several basal forebrain nuclei of the rat.

Using indirect immunocytochemical technique CCK-8-like immunoreactive somata could be demonstrated in different basal forebrain nuclei of adult rat (olfactory tubercle, nucleus accumbens septi, nucleus tractus diagonalis BROCA, nucleus septi medialis et lateralis, caudate-putamen-complex). The comparison of these findings with those of rapid Golgi technique and AChE histochemistry suggested that all CCK-8 immunostaining nerve cells should be aspiny neurons and, partly at least, a coexistence of CCK-8 and acetylcholine is assumed. In the number of neuronal somata with CCK-8-like immunoreactivity in all nuclei, especially in the caudate-putamen-complex, a distinct difference between male and female rats exist.

The distribution of cholecystokinin receptors in the vertebrate brain: species differences studied by receptor autoradiography.

The regional distribution of cholecystokinin receptors in the brain of several vertebrate species including pigeon, rat, mouse, guinea-pig, cat, monkey and human was studied by in vitro auto-radiography using [125I] Bolton-Hunter cholecystokinin-octapeptide as a ligand. Cholecystokinin receptors presented marked species differences in several brain regions particularly in the laminar distribution of the cerebellar and cortical layers, in the hippocampal formation and in the basal ganglia. No binding was observed in the cerebellum of pigeon and rat, while intermediate to high densities of binding was observed in the cerebellum of the other species studied. In the pigeon external striatum, corresponding to the mammalian neocortex, low densities of cholecystokinin binding sites were observed. In contrast, mammalian neocortex was rich in cholecystokinin binding sites. However, the lamination was different from species to species, with high densities predominating in lamina IV in rat, mouse and guinea-pig cortex, in lamina II and VI in cat and monkey cortex and in lamina V in human cortex. Another area presenting important species differences was the hippocampal formation. The pigeon hippocampus showed intermediate densities of cholecystokinin receptors. In the mammals studied, very different patterns and densities of cholecystokinin binding sites were observed in the hippocampal formation. The basal ganglia were labelled in all species examined except the mouse. While biochemical and pharmacoganglia were labelled in all species examined except the mouse. While biochemical and pharmacological studies have shown that cholecystokinin receptors in different species present comparable characteristics, our autoradiographic investigations indicate that cholecystokinin binding sites are expressed differentially in several brain regions of different vertebrate species. The present autoradiographic study provides a morphological basis for further analysis on the expression and functionality of cholecystokinin receptors.

[A rapid radioenzyme method for determining dopamine and 3,4-dihydroxyphenylacetic acid in the basal ganglia of the rat brain]. / Bystryi radiofermentnyi metod opredeleniia dofamina i 3,4-dioksifeniluksusnoi kisloty v bazal'nykh gangliiakh mozga krys.

A rapid and high sensitive radioenzymatic method of determination in the rat brain basal ganglia of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DHPA) whose methylated derivatives were divided by extraction with organic solvents is proposed. The method sensitivity for DA is 0.25 ng in a sample and for DHPA 0.1 ng in a sample. The yield of the internal standard is 80-85%. The method is high specific, the cross reaction during determination of DA and DHPA does not exceed 5%. The study of the effects of some dopaminergic agents (apomorphine, haloperidol. L-DOPA and pargyline on the contents of DA and DHPA in the rat brain basal ganglia in vivo confirms a high specificity of the method. The method may be used for screening of pharmacological compounds possessing the suggested dopaminergic and antimonoamine oxidase activity.

Autoradiographic analysis of tritiated imipramine binding in the human brain post mortem: effects of suicide.

In vitro quantitative autoradiography of high-affinity tritiated imipramine binding sites was performed on brains of 12 suicide victims and 12 matched controls. Region-specific differences in imipramine binding were found between the two groups. Thus, the pyramidal and molecular layers of the cornu ammoni hippocampal fields and the hilus of the dentate gyrus exhibited 80%, 60%, and 90% increases in binding in the suicide group, respectively. The postcentral cortical gyrus, insular cortex, and claustrum had 45%, 28%, and 75% decreases in binding in the suicide group, respectively. No difference in imipramine binding was observed in prefrontal cortical regions, in the basal ganglia, and in mesencephalic nuclei. No sex and postmortem delay effects on imipramine binding were found. Imipramine binding was positively correlated with age, the effect of age being most pronounced in portions of the basal ganglia and temporal cortex.

Changes within the basal nucleus in Parkinson's disease.

The magnocellular nuclei in the basal forebrain--the medial septal nucleus, the nucleus of the diagonal band, and the basal nucleus within the substantia innominata--are tightly connected with each other. Large multipolar neurons clearly predominating in number can be differentiated from a spindle-shaped and a small globular type of neuron on account of their typical pattern of lipofuscin pigmentation. As an additional feature some of the multipolar neurons exhibit intracytoplasmic acidophilic granules. At the ultrastructural level, these granules reveal a homogeneous substructure, and they are occasionally located within mitochondria. The percentage of large multipolar neurons displaying acidophilic granules varies in a topographical manner (medial septal nucleus and vertical limb nucleus of the diagonal band: 10%, horizontal limb nucleus of the diagonal band and anteromedial subnucleus of the basal nucleus: 36%, posterolateral subnucleus of the basal nucleus: 54%). In parkinsonian cases a significant decrease of granule-bearing neurons within the posterolateral subnucleus of the basal nucleus is noted. This decrease might be associated with the appearance of neurons containing Lewy-bodies. It is also conceivable that the granule-bearing neurons show a higher vulnerability and underlie neuronal death. Analyzing the lipofuscin pigmentation within the two subnuclei of the basal nucleus in parkinsonian cases, one notices that the number of large multipolar neurons displaying pigment granules in a dendritic stem is significantly higher than in controls. Moreover, it is apparent that the degree of this pigment penetration is more pronounced in cases of Parkinson's disease. These alterations may be interpreted as a sign of plasticity of this neuronal population.

Nerve growth factor receptor and choline acetyltransferase remain colocalized in the nucleus basalis (Ch4) of Alzheimer's patients.

Previous investigations have demonstrated an almost exclusive "coupling" between the receptor for nerve growth factor and cholinergic neurons within the basal forebrain. The present series of experiments were carried out to address two questions. First, what is the status of nerve growth factor receptor-containing neurons within the basal forebrain of patients with histopathologically confirmed diagnoses of Alzheimer's disease (AD)? More importantly, the second experiment assesses the degree to which the receptor for nerve growth factor and choline acetyltransferase remain colocalized within AD basal forebrain. A "decoupling" of this relationship, in which nerve growth factor receptors are no longer present upon magnocellular cholinergic neurons, would suggest that a loss of trophic support is functionally antecedent to the neuronal shrinkage and neuronal death seen in the basal forebrain in AD. Data obtained from six AD cases and four normal controls demonstrated an extensive reduction in number and shrinkage in size of nerve growth factor receptor containing neurons within the Ch4 region of the basal forebrain. Double label studies using either immunofluorescence or immunoperoxidase techniques demonstrated that the receptor for nerve growth factor and choline acetyltransferase remain colocalized in AD patients. This was true for neurons exhibiting either healthy or dystrophic morphological profiles. These data confirm previous studies, demonstrating that both a loss and shrinkage of cholinergic neurons occurs within the AD basal forebrain. The results of the present immunohistochemical investigation suggest that the degenerative changes associated with these neurons do not result from impaired trophic support related to a loss of NGF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Basal forebrain efferents to the medial dorsal thalamic nucleus in the rhesus monkey.

Thalamic efferent connections of the basal forebrain (BF); medial septal nucleus (MS), vertical limb of the diagonal band (VDB), horizontal limb of the diagonal band (HDB), nucleus basalis (NB), and ventral pallidum (VP) were investigated in twelve rhesus monkeys. In five animals, injections of radioactively labeled amino acids were placed in the BF. In four animals, the injections involved different divisions of the NB, HDB, and the most ventral part of the VDB. In those four cases, labeled fibers in the medial forebrain bundle were observed traveling caudally towards the hypothalamus where some turned dorsally to enter the inferior thalamic peduncle. These fibers terminated in the ventral half of the magnocellular part of the medial dorsal thalamic nucleus (MDmc). In a fifth case, the amino acid injection involved most of the MS and the VDB. Labeled fibers traveled caudally from the injection site and entered the stria medullaris. These fibers then traveled caudally before turning ventrally to terminate in the dorsal half of MDmc. To determine which of the diverse neuronal types in the BF gives rise to these thalamic projections, in two monkeys injections of horseradish peroxidase (HRP) were placed into MDmc. Labeled neurons were observed throughout the full extent of the NB, the VDB, the MS, and part of the VP. In order to determine the extent of the cholinergic input to MDmc from the BF, one of the HRP cases was processed for the simultaneous visualization of HRP, and acetylcholinesterase (AChE), the hydrolytic enzyme for acetylcholine, and a second case was processed for simultaneous visualization of HRP, and choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine. We observed that 30-50% of the HRP-labeled neurons were putatively cholinergic. In order to determine if the NB projection to MD is a collateral of the NB projection to orbital frontal cortex, one fluorescent retrograde tracer was injected into the orbital frontal cortex and one into MD. This case showed that approximately 5% of the BF neurons that project to MDmc also project to the orbital frontal cortex. These results confirm a significant subcortical projection by which the cholinergic system of the basal forebrain may influence higher cortical functions through the thalamus.

Distribution of enkephalin-immunoreactive nerve fibres and terminals in the region of the nucleus basalis magnocellularis of the rat: a light and electron microscopic study.

This investigation was carried out on the distribution of enkephalin-containing nerve fibres and terminals in the region of the nucleus basalis magnocellularis (NBM) of the rat. At the light microscope (LM) level, enkephalin-immunoreactive sites and endogenous choline acetyltransferase (ChAT) were demonstrated by employing the two-colour immunoperoxidase staining technique, using highly specific monoclonal antibodies against enkephalin and ChAT. A pharmacohistochemical procedure to reveal acetylcholinesterase (AChE)-synthesizing neurons combined with the peroxidase-antiperoxidase (PAP) immunocytochemical technique to detect endogenous enkephalins, provided ultrastructural data on the relationships of neuronal elements containing AChE and enkephalins in the region of the NBM. At the LM level, cholinergic neurons of the NBM were surrounded by a dense network of enkephalin-immunoreactive nerve fibres. Electron microscopic (EM) observations of histochemically characterized structures, that were first identified in the LM, revealed that intensely AChE-stained structures in the region of the NBM received sparse synaptic inputs from enkephalin immunoreactive terminals. Synaptic inputs of immunoreactive terminals onto intensely AChE-stained neuron cell bodies were not detected. Synaptic contacts onto proximal AChE-positive dendrites were sparse, but the density increased on more distal regions of the dendrites. All immunoreactive boutons studied established symmetrical synaptic contacts with AChE-positive post-synaptic structures. The pattern of the synaptic input to these cells differs strikingly from that onto typical globus pallidus neurons. The perikarya and dendrites of the latter neurons were characteristically ensheathed in immunoreactive synaptic boutons. Results are consistent with the view that enkephalin-like substances in the rat might be synaptic transmitters or neuromodulators in the area of the NBM and that cholinergic neurons of the NBM (Ch4) are integrated into the circuitry of the basal ganglia. Enkephalins may play an important role regulating the extrinsic cholinergic innervation of the neocortex.

Calcium binding protein in the basal ganglia system of a non-mammalian vertebrate: an immunohistochemical study in the reptile Caiman crocodilus.

In Caiman, calbindin D28K immunoreactivity (CaBP) was observed within many neurons of the substantia nigra (SN) but only in the caudal portion of the area ventralis of Tsai (AVT). A dense CaBP fiber plexus showing some regional inhomogeneity was observed in the dorsolateral portion of the telencephalic ventrolateral area (VLA). These results are consistent with previous reports that the SN and AVT project to the dorsolateral and medial portions of the VLA, and strongly support the theory that the caiman VLA contains cell populations homologous to those found in the mammalian corpus striatum.

Imaging of the aging brain. Part I. Normal findings.

A thorough knowledge of the normal changes that occur in the brain with age is critical before abnormal findings are analyzed. Magnetic resonance (MR) imaging improves the ability to distinguish normal and abnormal findings in the brain. The major changes that may occur in elderly individuals without neurologic deficits include enlargement of the ventricles, cortical sulci, and vermian subarachnoid spaces; multifocal areas of hyperintensity in the white matter and basal ganglia; a progressive prominence of hypointensity on T2-weighted images of the putamen, almost equal to that of the globus pallidus; an increase in the oxygen extraction ratio with normal or mildly decreased neuron metabolism; arteriosclerosis in large and small arteries and amyloid angiopathy in leptomeningeal cortical vessels; and decreased dopamine receptor binding in the corpus striatum. Since approximately half of the elderly population exhibits only negligible brain alterations, MR imaging may facilitate the distinction between usual (no neurologic dysfunction) and successful (no brain or vascular changes) aging.

A detailed examination of substance P in pathologically graded cases of Huntington's disease.

Substance P concentrations have been found to be reduced in the basal ganglia in Huntington's disease (HD). In order to further examine this finding in the present study we measured substance P-like immunoreactivity (SPLI) in cases of HD which had been graded as to the severity of pathological changes in the striatum. Marked significant reductions of SPLI were found in all striatal nuclei which were significantly correlated with the percentage of neuronal loss in the varying pathologic grades. Similar changes were found in the projection sites of striatal substance P neurons, the globus pallidus interna and the substantia nigra. These changes are consistent with a loss of striatal substance P containing projection neurons in HD. Significant reductions in SPLI were also found in the external pallidum, bed nucleus of the stria terminalis and the subthalamic nucleus. Small significant increases in SPLI (20-30%) were found in 3 frontal cortical regions (Brodmann areas 6, 8 and 9). The finding of neurochemical changes in the subthalamic nucleus is of particular interest since lesions in this nucleus are known to result in chorea and therefore might contribute to the chorea which is a cardinal symptom of HD.

In situ hybridization of nucleus basalis neurons shows increased beta-amyloid mRNA in Alzheimer disease.

To determine which cells within the brain produce beta-amyloid mRNA and to assess expression of the beta-amyloid gene in Alzheimer disease, we analyzed brain tissue from Alzheimer and control patients by in situ hybridization. Our results demonstrate that beta-amyloid mRNA is produced by neurons in the nucleus basalis of Meynert and cerebral cortex and that nucleus basalis perikarya from Alzheimer patients consistently hybridize more beta-amyloid probe than those from controls. These observations support the hypothesis that increased expression of the beta-amyloid gene plays an important role in the deposition of amyloid in the brains of patients with Alzheimer disease.

Subcellular distribution of mammalian tachykinins in rat basal ganglia.

A combined differential and density gradient centrifugation procedure was used to study the subcellular localisation of the mammalian tachykinins in rat caudateputamen and substantia nigra. Substance P, neurokinin A, neuropeptide K, and neurokinin B were found to be concentrated in the synaptosomal fractions and in fractions containing heavy synaptic vesicles in both regions studied. In contrast, the catecholamines dopamine and noradrenaline had a more widespread distribution throughout the gradient. HPLC analysis of the immunoreactivity recovered showed that the tachykinin immunoreactivity coeluted with the relevant synthetic tachykinins, except in the soluble gradient fraction where neurokinin A immunoreactivity eluted in position consistent with neurokinin A3-10. These results suggest that, in the basal ganglia, the mammalian tachykinins are localised in fractions containing large dense cored synaptic vesicles. This vesicular localisation would be consistent with the proposed role of the tachykinins as neurotransmitters and neuromodulators.

DARPP-32 and phosphatase inhibitor-1, two structurally related inhibitors of protein phosphatase-1, are both present in striatonigral neurons.

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of Mr = 32,000) and phosphatase inhibitor-1, two previously characterized inhibitors of protein phosphatase-1, were identified in both the neostriatum and the substantia nigra. Phosphatase inhibitor-1 was partially purified from bovine caudate nucleus and found to be distinct from DARPP-32 in some of its biochemical properties. The neuronal localization of DARPP-32 and phosphatase inhibitor-1 within the rat neostriatum and substantia nigra was investigated by studying the effects of kainic acid. Injection into the neostriatum of kainic acid, which destroys striatonigral neurons and striatonigral fibers, decreased the amounts of DARPP-32 and phosphatase inhibitor-1 to the same extent, both in the lesioned neostriatum and in the ipsilateral substantia nigra. The specific activity of protein phosphatase-1 in the neostriatum was unaffected by kainic acid. The results indicate that, in rat brain, DARPP-32 and phosphatase inhibitor-1 are both present in striatal neurons and in striatonigral fibers, and that they probably coexist in at least a subpopulation of striatonigral neurons. In contrast, protein phosphatase-1 does not appear to be enriched in any specific neuronal subpopulation in the neostriatum.

Receptor autoradiography as a tool for the study of the phylogeny of the basal ganglia.

Recent studies on the neurotransmitter organization of the basal ganglia and forebrain in lower vertebrates suggest that, in contrast to the old concepts of the phylogeny of the brain, there are many similarities between the chemical organization of the brain throughout evolution. By examining neurotransmitter receptors using in vitro autoradiography we have attempted to further our understanding of the evolution of the brain. Receptors enriched in different parts of the basal ganglia in mammals appear to be also enriched in the homologous areas in lower vertebrates. Thus, for example, dopamine and muscarinic receptors, but not serotonin-1A, are enriched in the paleostriatum augmentatum while GABA/benzodiazepine receptors are enriched in the paleostriatum primitivum corresponding with their localization to the caudate-putamen and globus pallidus respectively. Our results support the concept of a more conservative evolution of the vertebrate brain and demonstrate the usefulness of receptor autoradiography in the understanding of brain evolution.