Sparing of the dopaminergic neurons containing calbindin-D28k and of the dopaminergic mesocortical projections in weaver mutant mice.

In mice carrying the weaver mutation there is a spontaneous degeneration of dopaminergic neurons that is heterogeneous among cell groups: nigrostriatal neurons are more affected than mesolimbic neurons, while involvement of the mesocortical system is controversial. We questioned whether the pattern of cell loss in mesencephalon and fiber depletion in telencephalon could be related to the differential content of Calbindin-D28k in dopaminergic cells. The mesencephalon of seven-month-old mutants was serially sectioned and alternate series were immunostained with tyrosine hydroxylase and Calbindin-D28k. Cell counts indicated a 40% loss for the ensemble of dopamine mesencephalic neurons. However, double-immunostained preparations revealed that this cell loss was restricted to the neurons that lacked Calbindin-D28k, which were reduced by 72%, while the dopaminergic neurons containing Calbindin-D28k were completely spared. Calbindin-D28k was present in both the cytoplasm and nucleus of the dopaminergic cells. This nuclear localization was confirmed at the ultrastructural level. In the telencephalon of weaver mutants, areas receiving projections from the Calbindin-D28k-positive dopaminergic neurons, such as the cerebral cortex, contained normal densities of fibers, while areas harboring projections from the non-Calbindin-D28k dopaminergic neurons, such as the dorsal striatum, had reduced amounts of fibers. The vulnerability pattern in the mesencephalon of weaver mutants bears similarities to that described in idiopathic Parkinson's disease or in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism: Calbindin-D28k may thus delimit a group of dopaminergic neurons resistant to cell death in different conditions. On the other hand, the vulnerability pattern of dopaminergic fibers in weaver differs from that of Parkinson's disease, since there is a complete sparing of the dopaminergic mesocortical projection in weaver, contrasting with the damage of these projections in Parkinson's disease.

Serotonergic sprouting in primate MTP-induced hemiparkinsonism.

Immunocytochemistry to serotonin, dopamine beta hydroxylase (DBH), and tyrosine hydroxylase (TH) was studied in the brains of 2 cebus monkeys that had developed permanent hemiparkinsonism after intracarotid injection of 1.2 mg N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and were sacrificed after 10-12 months. A pronounced depletion of TH-immunoreactive neurons was found in the substantia nigra, caudate nucleus, and putamen, ipsilaterally to the injected side. In these dopamine-denervated structures, the number of serotonergic fibers was increased in the ipsilateral compared with the contralateral side, or with an untreated control monkey. Serotonergic neurons in the brainstem appeared to be unaffected. Topography and number of DBH-positive fibers in the control and the MPTP-injected sides were comparable.

Further indication that distinct dopaminergic subsets project to the rat cerebral cortex: lack of colocalization with neurotensin in the superficial dopaminergic fields of the anterior cingulate, motor, retrosplenial and visual cortices.

The extent of neurotensin (NT) colocalization in the different dopamine (DA) terminal fields of the rat cerebral cortex has been investigated and compared to previous data obtained in man (Gaspar et al., J. Comp. Neurol., 279 (1989) 249-271). Both innervations were revealed with single- or double-labeling immunocytochemical methods. Tyrosine hydroxylase (TH) was used as a specific marker of DA fibers after lesioning the noradrenergic system either with 6-hydroxydopamine (6-OHDA) at birth or DSP4 in adulthood. Three classes of afferents were observed which had a different regional and laminar distribution. First, a dense meshwork of finely dotted NT-positive varicosities occupied restricted areas of the limbic system: the granular retrosplenial and the deep entorhinal cortices and the subicular complex. These NT projections contained no double-labeled fibers and did not correspond to a mixed NT/TH pathway. Secondly, the mixed NT/DA projections identified previously in the prefrontal cortex (Studler et al., Neuropeptides, 11 (1988) 95-100), extended in fact rostrocaudally in layer VI of the whole cerebral cortex and formed small cluster-like groupings in layers II-III of the medial and lateral entorhinal cortex. In all these areas, the mixed NT/TH projections constituted approximately half of the DA terminals. Finally, the DA projections to the superficial layers of the anterior cingulate, motor, retrosplenial and visual cortices, were not colocalized with NT. The DA innervation of layers I-III of the rat anterior cingulate cortex displays striking similarities with that observed in the cingulate, primary motor, premotor and supplementary motor cortices in man: highest regional and laminar density of DA afferents and lack of colocalization with NT. It might thus represent a valuable model for understanding the pharmacology of the DA system besides the mixed DA/NT pathway which does not seem to have a counterpart in the human cerebral cortex. By contrast, that part of the NT innervation of the limbic system which is not colocalized with DA in rat, appears to represent the major fraction of the cortical NT innervation in man.

Neurotensin innervation of the human cerebral cortex: lack of colocalization with catecholamines.

We have localized neurotensin (NT) with immunocytochemical methods in the normal human cerebral cortex. Extensive areas of the frontal cortex, the hippocampal formation, and selected areas of the parietal, temporal and occipital lobes, were examined using post-mortem brain tissue. The peptidergic innervation was characteristically restricted to the limbic belt and to the dorsally contiguous regions. NT-labeled perikarya were found throughout the subiculum, including its dorsal supra-callosal continuation. NT terminal plexuses were particularly abundant in layers I-VI of the anterior cingulate cortex, in layer I of area 32 and of medical areas 9, 8, 6 and in layers II-III of area 29, of the presubiculum and entorhinal cortex. Elsewhere, NT fibers were scarce being more frequent in layer I. This regional and laminar pattern differed significantly from that of tyrosine hydroxylase (TH), which was used to label catecholaminergic axons, and preferentially the dopaminergic ones. Even in zones where TH and NT innervations were abundant, such as the anterior cingulate cortex or area 32, double-labeling procedures disclosed no colocalized fibers. The lack of NT-TH colocalization in human, contrasts with previous findings in the rodent cortex, where a contingent of the DA cortical afferents contains NT. The DA mesocortical neuronal population, labeled by TH antisera, thus seems to change its chemical phenotype, by losing the expression of an associated peptidergic neurotransmitter; this could be related to the predominant extension in the ascent of the phylogenetic scale of the non-colocalized, type of cortical DA innervation which is also found in rodents. The possible origins of the cortical, non-dopaminergic NT innervation in human are discussed: thalamo-cortical, subiculo-cortical or intrinsic. Such cortical NT innervation could be very important in limbic circuitry as a regulatory peptide in affective processes and could be involved in the physiology of pain and memory.

DARPP-32, a phosphoprotein enriched in dopaminoceptive neurons bearing dopamine D1 receptors: distribution in the cerebral cortex of the newborn and adult rhesus monkey.

DARPP-32, a dopamine (DA) and cAMP-regulated phosphoprotein, is associated with dopaminoceptive neurons bearing D-1 receptors in the basal ganglia. The present study addressed the distribution of DARPP-32 in the primate cerebral cortex and its putative association with D-1 receptor laden cells in this structure. DARPP-32-like immunoreactive (LIR) neurons were examined in the cerebral cortex of 3-day-old (P3), 6-week-old (P42), and adult rhesus monkeys. In the younger cases, a large number of DARPP-32 positive neurons, with the morphological characteristics of pyramidal cells, were observed throughout the cortex, in layers V-VI, and to a lesser extent in layer II and uppermost layer III. In the parietal, insular, temporal, and occipital cortices, DARPP-32 positive neurons were arranged in a monolayer in layer Va. They were often clustered in small groups with a bundling of their dendrites. In the primary motor cortex, Betz cells were among the labeled population. In the association and somatosensory areas, the basal dendrites of DARPP-32 positive neurons and the prominent tufting of their apical dendrites in layer I contributed to an essential bilaminar pattern resembling the distribution reported for DA afferents and D-1 receptors in these areas. The prominence and widespread distribution of DARPP-32 positive neurons in layer V may be a specialization of primate cortex since such cells are found only in restricted locations in rodents. The literature on the connections of the cerebral cortex suggests that a large number of the DARPP-32 positive neurons in layer VI and perhaps even in layer Va may be corticothalamic neurons. An important developmental observation was the presence of DARPP-32-LIR neurons in the white matter. They were prominent in the neonates but could not be seen in the adult. Their location as well as their type and shape were reminiscent of interstitial neurons. In the adult monkeys, the distribution of DARPP-32-LIR neurons was more circumscribed: they were numerous in the ventral temporal gyrus and in areas related to the limbic system: caudal orbitofrontal cortex, insula, temporal pole, entorhinal, and anterior cingulate cortex. Weak labeling was detected in layer Va of the superior temporal and parietal cortex, in some prefrontal areas (10, 13, and medial 9), and in the premotor and supplementary motor cortex; in adults, unlike neonates, few DARPP-32-LIR neurons were present in the dorsolateral prefrontal cortex, the primary motor or the primary visual or prestriate cortices.(ABSTRACT TRUNCATED AT 400 WORDS)

Subpopulations of somatostatin 28-immunoreactive neurons display different vulnerability in senile dementia of the Alzheimer type.

We tested whether the vulnerability of somatostatin (SST) neurons in senile dementia of the Alzheimer type (SDAT) depended upon their co-localization with neuropeptide Y (NPY). Density estimates of SST28- and NPY-immunoreactive neurons and percentage of double-labeled SST-NPY neurons were obtained in the cortex (areas 9 and 25) and the bed nucleus of stria terminalis (BST), in 6 SDAT and 5 control cases. Counts of senile plaques (SP) and neurofibrillary tangles (NFT) were done on thioflavin S stains. In both cortical areas, a decrease in the density of SST28-IR neurons was found in SDAT cases (-60% in area 25 and -80% in area 9), whereas density of NPY-IR neurons was unchanged. Accordingly, the proportion of single-labeled SST neurons decreased; this decrease was significantly correlated with SP (r = -0.89, P less than 0.001). We conclude that single SST-IR neurons, in cortical layers II-III, and V, are preferentially lost relative to co-localized SST-NPY neurons. In the BST, no significant reduction of SST-IR, NPY-IR neurons nor of the percentage of single labeled SST neurons was found, despite the presence of SP. Thus one subpopulation of SST neurons, defined by associated neurochemical characters (not co-localized with NPY nor with NADPH diaphorase) and by topography (cortical layers III and V) appears to be particularly vulnerable in SDAT. The potential importance of their position in neural circuitry is emphasized.

Catecholamine innervation of the human cerebral cortex as revealed by comparative immunohistochemistry of tyrosine hydroxylase and dopamine-beta-hydroxylase.

The organization of the cortical monoamine systems, dopamine (DA), and noradrenaline (NA), which have been studied extensively in the rat and more recently in the monkey, had not yet been investigated directly in the human brain. We report here the first systematic account of the regional and laminar distributions of the catecholamine fibers in the human cerebral cortex, using immunohistochemistry of the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH) in 13 cytoarchitectonic areas (4, 6, 9, 3b, 5, 40, 17, 18, 23, 24, 29, insula, and hippocampus) sampled postmortem. The noradrenergic (NA) innervation, mapped with DBH-immunoreactivity (DBH-IR), displayed a characteristic density gradient in the neocortex (highest in the primary sensorimotor areas, decreasing rostrally and caudally) that contrasted with the more uniform density in the limbic cortices (24, 23, 29, insula, hippocampus). NA axons were present in all cortical layers and were least numerous in layer I. The DBH-IR fibers were only partly TH-immunostained (10-50%, on double-labeled sections), suggesting a heterogeneity of the cortical NA axons. The putative dopaminergic (DA) fibers were identified by comparing alternate or double-immunolabeled (DBH-TH) sections, as the TH-IR fibers which contain no DBH-IR. A DA-like innervation was present in all cortical areas, with major regional differences in density and laminar distribution, which closely paralleled cytoarchitectural buildups: 1) the DA-like innervation was densest in the agranular areas, primary and secondary motor areas, anterior cingulate, and insula; it distributed throughout layers I-VI; 2) density was lower in the granular cortices, areas 9 (prefrontal cortex), 23, 3b, 5, 40, and 18, displaying a bilaminar pattern in layers I and V-VI. In all areas, DA-like fibers were most abundant in the molecular layer, with a predominant distribution in its deepest part. Convoluted and coily fibers represented a unique morphologic aspect of the CA innervation in the human cortex. These findings are in agreement with findings in nonhuman primates and demonstrate major evolutionary changes in the organization of the cortical aminergic input as compared with rodents. The most striking features are the expansion of the DA innervation to the whole cortex and the peak of highest density in the motor areas. The regional differentiation of NA innervation is also accentuated. Slight differences were found in the laminar distributions of the amines in humans and primates. These data seem quite promising and open new research fields in neurologic and psychiatric diseases.

Transient tyrosine hydroxylase-like immunoreactive neurons contain somatostatin and substance P in the developing amygdala and bed nucleus of the stria terminalis of the rat.

Tyrosine hydroxylase-like immunoreactive (TH-IR) neurons were observed from the embryonic day 17 (E17) to 6 weeks postnatally in two closely related nuclei of the limbic system, the bed nucleus of the stria terminalis (BNST) and the central nucleus of the amygdala (CNA) where they were restricted to circumscribed zones. These cells were scarce with an immature morphological aspect at E17. They progressively differentiated and increased in number until postnatal day 5 (P5), when their maximal density was reached. They were characterized as neurons by their ultrastructural appearance and the presence of both axo-somatic and axo-dendritic synaptic junctions. Moreover, TH-IR axons could be followed in the stria terminalis leaving the CNA, suggesting that part of TH-IR cells could be long projecting neurons rather than interneurons. A gradual decrease in the intensity of TH-IR and in density of labeled neurons was noted from P15 on, in both nuclei, (-50% at 4 weeks) until their total disappearance at 7 weeks. The significance of this TH-IR labeling regarding the catecholaminergic transmission remains unclear since these neurons did not contain the other catecholaminergic synthetic enzymes (DOPA-decarboxylase, dopamine-beta-hydroxylase, phenylethanolamine-N-methyl transferase) nor endogenous catecholamines. Double-labeling immunocytochemical methods, indicated that almost all the TH-IR neurons were colocalized with somatostatin 28 (SST) and with substance P (SP). Therefore these neurons expressed simultaneously 3 phenotypes, TH, SST and SP. This observation brings forth the notion of multiple neurotransmitter expression in transient neuronal populations and raises the question of neurotransmitter plasticity in the late postnatal development of the central nervous system (CNS). These neurons which were observed in two closely interconnected structures could be involved in early limbic circuits.

Tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex: a novel catecholaminergic group?

Tyrosine hydroxylase-like immunoreactive (TH-IR) neurons with morphological features of interneurons were found throughout the human cerebral cortex. Quantitative estimates in 14 different cytoarchitectonic areas revealed a specific regional distribution pattern, neurons being less dense in primary cortical areas and denser in higher order associative areas and some limbic related areas. A partial relationship was noted between the density of labeled neurons and that of the known dopaminergic innervation. The role of the cortical TH-IR neurons in catecholaminergic function, however, remains unclear since the presence of other catecholaminergic synthesizing enzymes, dopamine-beta-hydroxylase and DOPA decarboxylase, could not be demonstrated at their level. Similar neurons have been observed transiently in the rodent cortex during development; their persistence and topographical extension in the human brain warrants further study on their possible functional role.

Somatostatin 28 and neuropeptide Y innervation in the septal area and related cortical and subcortical structures of the human brain. Distribution, relationships and evidence for differential coexistence.

Somatostatin 28- and neuropeptide Y-containing innervations were mapped in the human medial forebrain (eight control brains) with immunohistochemistry, using the sensitive avidin-biotin-peroxidase method. Peptidergic perikarya and fibers had an extensive distribution: they were densest in the ventral striatum (nucleus accumbens, olfactory tubercle and bed nucleus of the stria terminalis) and infralimbic cortex, of intermediate density in the medial septal area and of lowest density in the dorsal and caudal lateral septal nucleus. Somatostatin-like immunoreactive perikarya and fibers were generally more numerous than the neuropeptide Y-like immunoreactive ones, but more faintly labeled. Their pattern of distribution was strikingly similar in some of the limbic structures studied but clearly distinct in others. Excellent overlap of neuropeptide Y and somatostatin-like immunoreactivity was detected in: (1) the medial septal area, where innervation occasionally formed perivascular clusters; (2) the nucleus accumbens and olfactory tubercle, characterized by dense patchy innervation; and (3) the laterodorsal septal nucleus, scarcely innervated. In the latter structures, most peptidergic neurons were double-labeled. On the other hand, both peptidergic innervations clearly differed in the lateroventral septal nucleus and the bed nucleus of the stria terminalis which contained distinct clusters of somatostatin-like immunoreactive neurons devoid of neuropeptide Y-like immunoreactivity. Also, the perineuronal and peridendritic axonal plexuses ('woolly fibers') present in these structures were only labeled with somatostatin. In the infralimbic cortex, the relation between the peptides varied according to the cortical laminae. Coexistence of somatostatin and neuropeptide Y frequently occurred in layer VI and in the subcortical white matter, whereas layer V and particularly layers II and III contained a contingent of neurons labeled only with somatostatin. Dense horizontal terminal networks in layers I and VI however were similar for both peptides. These findings support the existence of two different types of somatostatin-like immunoreactive perikarya as regards colocalization with neuropeptide Y. Their particular topographical segregation within the cortical and subcortical structures analysed suggest that they could have different connections and functional properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Transient expression of tyrosine hydroxylase immunoreactivity in some neurons of the rat neocortex during postnatal development.

Tyrosine hydroxylase-like immunoreactive neurons were observed in the dorsolateral and medial neocortex of the rat during postnatal development. They occurred from 8 up to 24 days of age and lacked other catecholamine synthetizing enzymes. They appeared to be insensitive to the suppression of cortical noradrenergic innervation induced by neonatal subcutaneous injections of 6-hydroxydopamine.

Postnatal ontogenesis of the dopaminergic innervation in the rat anterior cingulate cortex (area 24). Immunocytochemical and catecholamine fluorescence histochemical analysis.

The postnatal development of the dopaminergic input to the rat anterior cingulate cortex (area 24) was followed using anti-tyrosine hydroxylase immunocytochemistry and catecholamine fluorescence histochemistry in control and noradrenaline-depleted rats. Noradrenaline depletion in the cerebral cortex was obtained by peripheral injections of 6-hydroxydopamine (6-OHDA) at birth or N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP4) at various postnatal ages and controlled by the absence of dopamine-beta-hydroxylase-labelled axons. The superficial and deep components of the anterior cingulate dopaminergic field developed at a different rate in control as well as lesioned rats. The deep supragenual dopaminergic field was already present at birth like the dopaminergic innervation of the prefrontal cortex area 32. In the superficial field, the molecular layer was reached first from postnatal day 3 (P3) on by positive axons running through the anterior hippocampal continuation and from P5-P6 on by another dopaminergic contingent coming through the deep dopaminergic field and giving off collaterals for layer III. The adult distribution pattern and striking varicose aspect were not reached until P21-P30 and a further increased density was observed until P60. The superficial cingulate dopaminergic field extended into the pregenual part of area 24b. The innervation of the superficial and deep layers of the rat anterior cingulate cortex by two distinct dopaminergic subpopulations, one of them closely related to that of prefrontal cortex area 32, could be compared with other laminar differences. The important functional implications of these data are further discussed.