Intraneural distribution of exogenous norepinephrine in the central nervous system of the rat.

Catecholaminergic neurons, which take up and retain exogenous norepinephrine labeled with tritium, were studied by means of high resolution radioautography, in the substantia nigra, the substantia grisea periventricularis, and the locus coeruleus of the rat. Under the conditions required for the radioautographic detection of exogenous norepinephrine-(3)H, it was established that (1) glutaraldehyde was the most suitable fixative for preserving the labeled amine in situ; (2) norepinephrine-(3)H itself, rather than metabolites, accounted for most of the reactions detected in catecholaminergic neurons. At various time intervals after an intraventricular injection of norepinephrine-(3)H, the tracer reached a concentration 15-100 times higher, and disappeared at a slower rate, in presynaptic axons (t(1/2):4 hr) than in nerve cell bodies (t(1/2):0.8-1.3 hr). After pretreatment with a monoamine oxidase inhibitor, the radioautographic reactions increased and persisted longer, especially in the preterminal axons. Within neurons, the labeled amine was ubiquitously distributed in the nerve cell body and concentrated in presynaptic axons and synaptic terminals of various morphological types. Although large granular vesicles were usually present in the labeled axonal bulbs, no structural characteristic could be specifically ascribed to catecholaminergic neurons. It is suggested that exogenous norepinephrine bound to macromolecular complexes is present in all parts of catecholaminergic neurons and mainly concentrated within presynaptic axons.

Unchanged density of 5-HT(1A) autoreceptors on the plasma membrane of nucleus raphe dorsalis neurons in rats chronically treated with fluoxetine.

5-HT(1A) autoreceptors regulate the firing of 5-HT neurons and their release of 5-HT. In previous immuno-electron microscopic studies, we have demonstrated an internalization of 5-HT(1A) autoreceptors in the nucleus raphe dorsalis (NRD) of rats, after the acute administration of a single dose of the specific agonist 8-hydroxy-2-(di-n-propylamine)tetralin (8-OH-DPAT) or of the selective 5-HT reuptake inhibitor, fluoxetine. Twenty-four hours after either treatment, the receptors were back in normal density on the plasma membrane of NRD neurons. Here, we examined the subcellular localization of these receptors and the in vivo binding of the 5-HT(1A) radioligand 4,2-(methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-fluorobenzamido]ethylpiperazine labeled with [(18)F]fluorine ([(18)F]MPPF) after chronic fluoxetine treatment (10 mg/kg daily for 3 weeks, by minipump). Unexpectedly, after such a treatment, there were no more differences between treated and control rats in either the density of plasma membrane labeling of NRD dendrites, or in the in vivo binding of [(18)F]MPPF, as measured with beta-microprobes. This was in keeping with earlier reports of an unchanged density of 5-HT(1A) receptor binding sites after chronic fluoxetine treatment, but quite unexpected from the strong electrophysiological and biochemical evidence for a desensitization of 5-HT(1A) autoreceptors under such conditions. Indeed, when the fluoxetine-treated rats were challenged with a single dose of 8-OH-DPAT, there was no internalization of the 5-HT(1A) autoreceptors, at variance with the controls. Interestingly, several laboratories have reported an uncoupling of 5-HT(1A) autoreceptors from their G protein in the NRD of rats chronically treated with fluoxetine. Therefore, the best explanation for our results is that, after repeated internalization and retargeting, functional 5-HT(1A) autoreceptors are replaced by receptors uncoupled from their G protein on the plasma membrane of NRD 5-HT neurons. Thus, the regulatory function of these autoreceptors may depend on a dynamic balance among their production, activation, internalization and recycling to the plasma membrane in inactivated (desensitized) form.

Enhanced glutamatergic phenotype of mesencephalic dopamine neurons after neonatal 6-hydroxydopamine lesion.

There is increasing evidence that a subset of midbrain dopamine (DA) neurons uses glutamate as a co-transmitter and expresses vesicular glutamate transporter (VGLUT) 2, one of the three vesicular glutamate transporters. In the present study, double in situ hybridization was used to examine tyrosine hydroxylase (TH) and VGLUT2 mRNA expression during the embryonic development of these neurons, and postnatally, in normal rats and rats injected with 6-hydroxydopamine (6-OHDA) at P4 to destroy partially DA neurons. At embryonic days 15 and 16, there was a regional overlap in the labeling of TH and VGLUT2 mRNA in the ventral mesencephalon, which was no longer found at late embryonic stages (E18-E21) and postnatally. In normal pups from P5 to P15, only 1-2% of neurons containing TH mRNA in the ventral tegmental area (VTA) and substantia nigra, pars compacta, also displayed VGLUT2 mRNA. In contrast, after the cerebroventricular administration of 6-OHDA at P4, 26% of surviving DA neurons in the VTA of P15 rats expressed VGLUT2. To search for a colocalization of TH and VGLUT2 protein in axon terminals of these neurons, the nucleus accumbens of normal and 6-OHDA-lesioned P15 rats was examined by electron microscopy after dual immunocytochemical labeling. In normal rats, VGLUT2 protein was found in 28% of TH positive axon terminals in the core of nucleus accumbens. In 6-OHDA-lesioned rats, the total number of TH positive terminals was considerably reduced, and yet the proportion also displaying VGLUT2 immunoreactivity was modestly but significantly increased (37%). These results lead to the suggestion that the glutamatergic phenotype of a VTA DA neurons is highly plastic, repressed toward the end of normal embryonic development, and derepressed postnatally following injury. They also support the hypothesis of co-release of glutamate and DA by mesencephalic neurons in vivo, at least in the developing brain.

Regionally selective changes in neurotransmitter receptors in the brain of the 5-HT1B knockout mouse.

The serotonin1B receptor knockout (5-HT1B KO) mouse is a valuable animal model of addiction to psychostimulants. We previously found selective increases in dopamine (DA) turnover in the nucleus accumbens of these mice, in addition to several changes in their central serotonin system. Here, we searched for further DA adaptations by measuring D1 and D2 receptor as well DA plasma membrane transporter (DAT) sites by ligand binding autoradiography, and G-protein coupling to D1 and D2 receptors by [35S]GTP gamma S autoradiography. Except for a slight increase in the lateral septum, D1 receptor binding did not differ from wild-type in twenty-one other neocortical, limbic or basal ganglia regions examined in the KO. Nor were there changes in D1 agonist-stimulated G-protein coupling in any of these regions, including the lateral septum. Increases in D2 binding sites, presumably involving GABAergic projection neurons, were measured in the nucleus accumbens, olfactory tubercle and ventral tegmental area of the 5-HT1B KO. However, no activation of the efficacy of D2 receptor coupling to G-protein could be measured in these and other brain regions. Binding to DAT was unchanged throughout brain. Because of their implication in cocaine addiction, the functionality of mu-opioid and GABAB receptors was also assessed by [35S]GTP gamma S autoradiography. 5-HT1B KO showed selective decreases in G-protein coupling to mu-opioid receptors in the paraventricular thalamic nucleus, and to GABAB receptors in the basolateral nucleus of amygdala. It is likely that these latter changes underlie some aspects of the addictive behavior of the 5-HT1B KO mouse.

Catecholaminergic activation of G-protein coupling in rat spinal cord: further evidence for the existence of dopamine and noradrenaline receptors in spinal grey and white matter.

[35S]GTPgammaS autoradiography of slide-mounted tissue sections was used to examine G-protein coupling in the rat spinal cord, as stimulated by dopamine, the D1 receptor agonist SKF 38393, noradrenaline, and noradrenaline in the presence of the alpha adrenoceptor antagonist, phentolamine. Measurements were obtained from the different laminae of spinal grey and from the dorsal, lateral, and ventral columns of white matter, at cervical, thoracic, and lumbar levels. At every level, there was a relatively strong basal incorporation of GTPgammaS in laminae II-III>lamina IV-X of spinal grey, even in presence of DPCPX to block endogenous activation by adenosine A1 receptors. Dopamine, and to a lesser degree SKF 38393, but not the D2 receptor agonist quinpirole, stimulated G-protein coupling in laminae IV-X. Both dopamine and SKF 38393 also induced a weak but significant activation throughout the white matter. In both grey and white matter, the activation by dopamine was markedly reduced in presence of a selective D1 receptor antagonist. Noradrenaline strongly stimulated coupling throughout the spinal grey at all levels, an effect that was uniformly reduced in the presence of phentolamine. With or without phentolamine, there was also significant stimulation by noradrenaline in the white matter. Under the same experimental conditions, alpha 1, alpha 2, and beta adrenergic receptor agonists failed to activate GTPgammaS incorporation in either grey or white matter. However, in the presence of selective alpha 1 or alpha 2 receptor antagonist, significant reductions of noradrenaline-stimulated GTPgammaS incorporation were observed in both grey and white matter. The beta antagonist propanolol reduced GTPgammaS incorporation in grey matter only. Thus, the results confirmed the existence of D1 dopamine receptors and of alpha 1, alpha 2, and beta adrenergic receptors in the grey matter of rat spinal cord. In white matter, they strongly suggested the presence of dopamine D1, and of alpha 1 and alpha 2 adrenergic receptors on glia and/or microvessels, that might be activated by diffuse transmission in vivo.

Diffuse transmission by acetylcholine in the CNS.

Recent immunoelectron microscopic studies have revealed a low frequency of synaptic membrane differentiations on ACh (ChAT-immunostained) axon terminals (boutons or varicosities) in adult rat cerebral cortex, hippocampus and neostriatum, suggesting that, besides synaptic transmission, diffuse transmission by ACh prevails in many regions of the CNS. Cytological analysis of the immediate micro-environment of these ACh terminals, as well as currently available immunocytochemical data on the cellular and subcellular distribution of ACh receptors, is congruent with this view. At least in brain regions densely innervated by ACh neurons, a further aspect of the diffuse transmission paradigm is envisaged: the existence of an ambient level of ACh in the extracellular space, to which all tissue elements would be permanently exposed. Recent experimental data on the various molecular forms of AChE and their presumptive role at the neuromuscular junction support this hypothesis. As in the peripheral nervous system, degradation of ACh by the prevalent G4 form of AChE in the CNS would primarily serve to keep the extrasynaptic, ambient level of ACh within physiological limits, rather than totally eliminate ACh from synaptic clefts. Long-lasting and widespread electrophysiological effects imputable to ACh in the CNS might be explained in this manner. The notions of diffuse transmission and of an ambient level of ACh in the CNS could also be of clinical relevance, in accounting for the production and nature of certain cholinergic deficits and the efficacy of substitution therapies.

Agonist-induced internalization of serotonin-1a receptors in the dorsal raphe nucleus (autoreceptors) but not hippocampus (heteroreceptors).

Serotonin-1A (5-HT(1A)) receptors in the CNS are a major target for psychotropic drugs. In nucleus raphe dorsalis (NRD) and hippocampus (CA3), the selective 5-HT(1A) agonist (+)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT) reduces the firing activity of serotoninergic (5-HT) and pyramidal neurons, respectively. When located on 5-HT (autoreceptors), but not on non-5-HT (heteroreceptors) neurons, 5-HT(1A) receptors are known to be subject to desensitization. Using quantitative electron microscopy after pre-embedding immunogold labeling with specific antibodies, we examined the subcellular distribution of these receptors after acute administration of 8-OH-DPAT (0.5 mg/kg, i.v.). Silver-intensified immunogold particles associated with the plasma membrane or the cytoplasm were counted in somata and dendrites within the NRD, 15 min, 1 hr and 24 hr after 8-OH-DPAT injection, and in hippocampal dendrites 1 hr after the same treatment. Significant decrease in the density of membrane labeling and concomitant increase of cytoplasmic labeling were demonstrated in the NRD, 15 min and 1 hr after 8-OH-DPAT administration, with a return to baseline level at 24 hr. Internalization was blocked by previous administration of the 5-HT(1A) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide (WAY 100635), which, by itself, was without apparent effect. In hippocampus (CA3), there were no apparent changes in the distribution of the receptor after 8-OH-DPAT administration. These findings are in line with earlier results showing a desensitization of 5-HT(1A) autoreceptors but not heteroreceptors after treatment with 5-HT(1A) receptor agonist. They suggest that this desensitization is the result of autoreceptor internalization.

Selective cholinergic denervation, independent from oxidative stress, in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by increases in amyloid-beta (Abeta) peptides, neurofibrillary tangles, oxidative stress and cholinergic deficits. However, the selectivity of these deficits and their relation with the Abeta pathology or oxidative stress remain unclear. We therefore investigated amyloidosis-related changes in acetylcholine (ACh) and serotonin (5-HT) innervations of hippocampus and parietal cortex by quantitative choline acetyltransferase (ChAT) and 5-HT immunocytochemistry, in 6, 12/14 and 18 month-old transgenic mice carrying familial AD-linked mutations (hAPP(Sw,Ind)). Further, using manganese superoxide dismutase (MnSOD) and nitrotyrosine immunoreactivity as markers, we evaluated the relationship between oxidative stress and the ACh deficit in 18 month-old mice. Thioflavin-positive Abeta plaques were seen in both regions at all ages; they were more numerous in hippocampus and increased in number (>15-fold) and size as a function of age. A majority of plaques exhibited or were surrounded by increased MnSOD immunoreactivity, and dystrophic ACh or 5-HT axons were seen in their immediate vicinity. Counts of immunoreactive axon varicosities revealed significant decreases in ACh innervation, with a sparing of the 5-HT, even in aged mice. First apparent in hippocampus, the loss of ACh terminals was in the order of 20% at 12/14 months, and not significantly greater (26%) at 18 months. In parietal cortex, the ACh denervation was significant at 18 months only, averaging 24% across the different layers. Despite increased perivascular MnSOD immunoreactivity, there was no evidence of dystrophic ACh varicosities or their accentuated loss in the perivascular area. Moreover, there was virtually no sign of tyrosine nitration in ChAT nerve terminals or neuronal cell bodies. These data suggest that aggregated Abeta exerts an early, non-selective and focal neurotoxic effect on both ACh and 5-HT axons, but that a selective, plaque- and oxidative stress-independent diffuse cholinotoxicity, most likely caused by soluble Abeta assemblies, is responsible for the hippocampal and cortical ACh denervation.

Effect of Prior Dopamine Denervation on Survival and Fiber Outgrowth from Intrastriatal Fetal Mesencephalic Grafts.

[3H]Dopamine (DA) uptake radioautography and tyrosine hydroxylase (TH) immunocytochemistry were used to assess quantitatively the effects of the presence or absence of host mesostriatal DA afferents on the survival and fiber outgrowth from fetal ventral mesencephalic DA neurons grafted into the neostriatum of adult recipient rats. Rats received bilateral intrastriatal transplants of fetal ventral mesencephalic tissue 1 month after a unilateral injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle (denervated side). Five to six months later, some of the grafted rats received a second 6-OHDA injection in the left nigrostriatal bundle (acutely denervated or 'intact' side). After a further 7 days, slices of each hemisphere from the latter rats were incubated with [3H]DA and processed for film and high resolution radioautography. The density of the film radioautographs was measured with a computerized image analysis system and calibrated by silver grain cluster (i.e. DA terminal) counting over selected areas of the same sections in light microscope radioautographs. The brains of the remaining grafted rats were processed for TH immunoreactivity 6 - 12 months after graft surgery. Neither the size of the grafts, nor the number of surviving TH-positive graft neurons showed any significant difference between the nondenervated and the denervated sides. However, the size of the TH-positive cell bodies was significantly greater in the grafts on the denervated side. In the [3H]DA uptake radioautographs, considerable outgrowth of DA fibers was evident in the neostriatum on the 'intact' side in spite of the presence of an intact host DA innervation until 7 days before sacrifice. The overall DA fiber outgrowth was nevertheless almost two-fold greater on the denervated side, and extended deeper into the host neostriatum than on the 'intact' side; only 7% of the total neostriatal area, on average, was at background level compared to 30% on the 'intact' side, and the overall density of neostriatal DA innervation amounted to 36% of normal as compared to 20% on the 'intact' side. The correlation between the overall density of graft-derived DA innervation and the size of the grafts was linear on the 'intact' side, but reached a plateau with relatively small grafts on the denervated side. However, the ventral striatum on both sides was very poorly innervated by these grafts. These findings demonstrate that the mature neostriatal tissue can support axonal growth and innervation from grafted fetal DA neurons even in the presence of a normal complement of endogenous DA fibers. Prior removal of the host striatal DA innervation does not influence the overall size of the grafts nor the number of surviving DA neurons, but induces an increase in the cell body size and fiber outgrowth of the grafted DA neurons.

Monoamine innervation of the organum vasculosum laminae terminalis (OVLT): a high resolution radioautographic study in the rat.

The monoamine innervation of the organum vasculosum laminae terminalis (OVLT) was examined in the adult rat by light and electron microscope radioautography after intraventricular administration of tritiated serotonin [( 3H]5-HT) or dopamine [( 3H]DA). Radioautographic and biochemical controls after 5,7-dihydroxytryptamine or 6-hydroxydopamine lesioning established the respective serotonin (5-HT) and catecholamine (CA) identities of the axonal varicosities labeled under the conditions of the present experiments. For descriptive purposes, the OVLT was subdivided in three parts: two parenchymal zones, one juxtaventricular, the other juxtavascular, and the vascular core. Almost 10% of all axonal varicosities in the OVLT were found to be labeled with [3H]5-HT. This 5-HT innervation was most prominent in the rostrocaudal and ventrodorsal portions of the juxtaventricular zone and the dorsal aspect of the juxtavascular zone; there was none in the vascular core. [3H]DA-labeled varicosities were much less abundant and yet more numerous than earlier histofluorescent and immunohistochemical studies would have predicted. They predominated in the juxtavascular zone, where a majority presumably had a dopamine (DA) rather than a noradrenaline identity. Some were also found in the vascular core, where they most likely corresponded to peripheral autonomic noradrenaline endings. In the juxtaventricular zone of the OVLT, a significant proportion of the [3H]5-HT-labeled varicosity profiles could be observed to form axodendritic synapses, but in the juxtavascular zone no 5-HT or any [3H]DA-labeled ones were ever seen in synaptic junction. In the juxtavascular zone, the 5-HT and the presumed DA endings established close relationships with neurosecretory axons, and with astrocytic or tanycytic processes on which they occasionally formed "synaptoid contacts." A few endings of either type were also seen to about directly on the outer basement membrane of the perivascular space. It therefore appears probable that in OVLT monoamines influence neural and nonneural elements. At a proximal level of regulation (juxtaventricular zone), 5-HT could act both synaptically and nonsynaptically as an interneuronal transmitter or modulator. In contrast, distally (juxtavascular zone), both DA and 5-HT could be released as neurohormones in addition to modulating neurosecretion. 5-HT and DA varicosities in the OVLT could also behave as sensors for circulating factors that do not cross the blood-brain barrier.

Serotonin-immunoreactive neurons in the cnidarian Renilla koellikeri.

The cellular localization of 5-hydroxytryptamine (5-HT) was investigated in the pennatulid anthozoan Renilla koellikeri by means of peroxidase-antiperoxidase-immunohistochemistry with an antiserum against 5-HT-formaldehyde-protein conjugate. In many colonies, strong 5-HT-immunoreactivity was displayed by the cell bodies and beaded processes of relatively small neuronlike elements predominating in the outer ectoderm or scattered in the underlying mesoglea. The immunostained neurons of the mesoglea were generally bipolar and their relatively short processes extended toward myoepithelial cells. In the ectoderm, most immunostained neurons appeared pseudounipolar. These cell bodies were endowed with a small, superficially directed, conical appendage reaching the outer surface of the epithelium. Their neurites emerged from the inner pole of the perikarya and branched toward other immunopositive ectodermal and mesogleal nerve cells, or nematocytes in the tentacles. The networklike distribution of the presumed 5-HT ectodermal neurons varied between the different regions of colonies and along the autozooid column. In the context of earlier observations in cnidarians, these cytological features suggest a sensory as well as a modulatory function for 5-HT in Renilla koellikeri.

Cholinergic innervation in adult rat cerebral cortex: a quantitative immunocytochemical description.

A method for determining the length of acetylcholine (ACh) axons and number of ACh axon varicosities (terminals) in brain sections immunostained for choline acetyltransferase (ChAT) was used to estimate the areal and laminar densities of this innervation in the frontal (motor), parietal (somatosensory), and occipital (visual) cortex of adult rat. The number of ACh varicosities per length of axon (4 per 10 microm) appeared constant in the different layers and areas. The mean density of ACh axons was the highest in the frontal cortex (13.0 m/mm(3) vs. 9.9 and 11.0 m/mm(3) in the somatosensory and visual cortex, respectively), as was the mean density of ACh varicosities (5.4 x 10(6)/mm(3) vs. 3.8 and 4.6 x 10(6)/mm(3)). In all three areas, layer I displayed the highest laminar densities of ACh axons and varicosities (e.g., 13.5 m/mm(3) and 5.4 x 10(6)/mm(3) in frontal cortex). The lowest were those of layer IV in the parietal cortex (7.3 m/mm(3) and 2.9 x 10(6)/mm(3)). The lengths of ACh axons under a 1 mm(2) surface of cortex were 26.7, 19.7, and 15.3 m in the frontal, parietal, and occipital areas, respectively, for corresponding numbers of 11.1, 7.7, and 6.4 x 10(6) ACh varicosities. In the parietal cortex, this meant a total of 1.2 x 10(6) synaptic ACh varicosities under a 1 mm(2) surface, 48% of which in layer V alone, according to previous electron microscopic estimates of synaptic incidence. In keeping with the notion that the synaptic component of ACh transmission in cerebral cortex is preponderant in layer V, these quantitative data suggest a role for this innervation in the processing of cortical output as well as input. Extrapolation of particular features of this system in terms of total axon length and number of varicosities in whole cortex, length of axons and number of varicosities per cortically projecting neuron, and concentration of ACh per axon varicosity, should also help in arriving at a better definition of its roles and functional properties in cerebral cortex.

Dopaminergic projection from nucleus raphe dorsalis to neostriatum in the rat.

The existence of a dopamine (DA) projection from nucleus raphe dorsalis (RD) to neostriatum was demonstrated in the rat by combined tyrosine hydroxylase (TH) immunohistochemistry and radioautography after retrograde axonal transport of [3H]noradrenaline ([3H]NA). Intrastriatal injections of [3H]NA were carried out in normal rats or after ipsilateral destruction of the nigrostriatal DA system by injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra. Some 1,000 TH-positive nerve cell bodies were counted within the confines of RD as defined by its content in serotonin (5-HT) neurons. These DA neurons occupied the upper third of the RD and they were part of its small cell population. In all cases, a small proportion of the TH-immunoreactive nerve cell bodies in RD were retrogradely radiolabeled. Radiolabeled but immunonegative cells were exceedingly rare. The double-labeled neurons were generally more numerous after elimination of the nigrostriatal DA innervation than in normal rats. They mostly lay within the ventral portion of the medial subdivision of RD and always predominated on the [3H]NA- injected side. Some were also present in nucleus linearis caudalis. It was concluded that [3H]NA had been taken up and retrogradely transported exclusively by catecholamine neurons; part of the DA cell group in RD projects to the neostriatum; and that most if not all non-5-HT neurons projecting from RD to neostriatum are likely to be dopaminergic.

Distribution of GABA-immunoreactive neurons in the basal ganglia of the squirrel monkey (Saimiri sciureus).

The distribution of GABA-immunoreactive neurons was visualized in the basal ganglia of the squirrel monkey (Saimiri sciureus), by using a highly specific antiserum raised against GABA-glutaraldehyde-lysyl-protein conjugate and revealed by the indirect peroxidase-antiperoxidase immunohistochemical method. In the dorsal striatum, GABA-immunoreactive nerve cell bodies were small to medium in size (sectional area ranging from 90 to 125 microns2), but some larger ones (500-600 microns2) were also found. These cells displayed no obvious clustering but were significantly more numerous in the caudate nucleus than in the putamen; their number was also markedly greater at caudal than at rostral striatal levels. A moderate number of evenly distributed positive axon terminals were visible in both the caudate nucleus and the putamen. In the ventral striatum, GABA-immunoreactive nerve cell bodies and axon terminals were seen in fair number within the nucleus accumbens and in the deep layers of the olfactory tubercle. Many positive terminals but no somata were found in the islands of Calleja. In the globus pallidus, virtually all nerve cell bodies were GABA-immunoreactive and the neuropil exhibited a multitude of positive terminals. In the substantia innominata, clusters of small, globular GABA-immunoreactive somata were scattered among aggregates of larger, nonimmunoreactive neurons belonging to the nucleus basalis, and the whole region showed a low to moderate number of evenly spread GABA-positive terminals. In the subthalamic nucleus, nerve cell bodies were generally surrounded by several GABA-positive terminals but were not themselves immunoreactive. The substantia nigra showed many GABA-immunoreactive somata, which predominated in the pars lateralis and diminished progressively in number along the lateromedial axis of the pars reticulata. These cells formed a rather pleomorphic group comprising round, fusiform, or polygonal elements of relatively large size (sectional area ranging from 200 to 800 microns2). In the pars compacta and ventral tegmental area, a few GABA-immunoreactive neurons of small size were dispersed among larger, unreactive neurons. In both pars lateralis and pars reticulata of the substantia nigra, the number of GABA-positive terminals was high and their distribution was rather uniform; a smaller number were visible in the pars compacta of the substantia nigra and in the ventral tegmental area. The present results demonstrate that GABA-containing neurons are widely and heterogeneously distributed in the various components of the squirrel monkey's basal ganglia.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural relationships of serotonin axon terminals in the cerebral cortex of the adult rat.

PAP immunocytochemistry with an antiserum against serotonin (5-HT)-glutaraldehyde-protein conjugate (kindly donated by M. Geffard) was used to analyze the ultrastructural relationships of 5-HT axon terminals (varicosities) in the frontal (Fr1-Fr2), parietal (Par1), and occipital (Oc1M-Oc2) cortex of adult rats. One hundred-forty-five immunostained varicosities from Fr1-Fr2 (54 from layers I-II; 91 from layer VI) and 97 each from the upper layers (I-II) of Par1 and OcM1-Oc2 were examined in groups of serial thin sections (mean number of sections in series: 3.2 to 7.3). These terminals were of comparable shape and size in the 4 cortical sectors examined, and averaged 0.66 +/- 0.2 microns in mean diameter. The proportion of varicosities engaged in synaptic contact was evaluated by linear transformation of the relationship between the frequency of observed synaptic junctions and the number of thin sections available for examination. Reliability of the sampling was evidenced by a high coefficient of correlation (r greater than 0.95) in each cortical sector. The synaptic incidence extrapolated for whole varicosities ranged from 28% (layer VI of Fr1-Fr2) to 46% (Par1), without statistically significant differences between the 4 sectors examined. The interregional mean could thus be evaluated at 38%. The synaptic 5-HT terminals always made asymmetrical junctions, which were exclusively found on dendritic spines and shafts, and appeared more frequent on spines than shafts in the deep frontal and the upper occipital cortex. In all 4 sectors, dendritic shafts and spines and other axonal varicosities were frequently encountered in the immediate microenvironment of the immunostained varicosities. It is concluded that the cortical 5-HT innervation is predominantly nonjunctional throughout the neocortex of the adult rat, which reinforces earlier views of a highly divergent afferent system with particular functional properties and perhaps capable of widespread, global and/or sustained influences in this part of the brain.

Light and electron microscopic immunocytochemical analysis of the neurovascular relationships of choline acetyltransferase and vasoactive intestinal polypeptide nerve terminals in the rat cerebral cortex.

Acetylcholine or vasoactive intestinal peptide (VIP) nerve terminals closely related to intracortical blood vessels have previously been reported. Recent physiological evidence indicates that these central neuronal systems are involved in the fine control of local cerebral blood flow. In the present study, the intimate associations between choline acetyltransferase (ChAT) and VIP axon terminals and intracortical microvessels were characterized by light (LM) and electron microscopic (EM) immunocytochemistry. In semithin sections, LM analysis of the distribution of ChAT- and VIP-immunostained puncta juxtaposed to small intraparenchymal blood vessels demonstrated that neither type of terminal was enriched or impoverished around microvessels within the cerebral cortex. At the EM level, most ChAT- or VIP-immunolabelled elements located within a 3 microns perimeter around vessel walls were axon terminals. These perivascular terminals were associated primarily with capillaries but also, to a lesser extent, with microarterioles. Even though ChAT and VIP terminals were frequently found in the immediate vicinity (< or = 0.25 microns) of microvessels, they almost never contacted the outer basal lamina, usually abutting onto perivascular astroglial leaflets. There were no membrane specializations at the site of contact between ChAT or VIP terminals and perivascular astroglia. In all cortical areas examined, the average size of VIP-immunolabelled varicosities (0.56 +/- 0.04 microns 2) was significantly larger than that of their ChAT counterparts (0.32 +/- 0.02 microns 2; P < 0.001). Perivascular VIP terminals were more frequently engaged in synaptic contact than those immunostained for ChAT, which rarely exhibited a synaptic junction even in serial thin sections. Neither VIP nor ChAT immunostaining was ever observed in endothelial cells. These results suggest that both acetylcholine and VIP exert their effects on intracortical microvessels through indirect, paracrine mechanisms. The marked difference in synaptic incidence and average size between both types of perivascular terminals indicates that these two vasoactive agents are primarily located in distinct neuronal populations. Further, our results show that the astrocytic glia is the major direct target for both ChAT and VIP perivascular terminals and suggest that neuronal/glial/vascular interactions are a key element in the neurogenic control of the intracortical microcirculation.

Decrease and long-term recovery of choline acetyltransferase immunoreactivity in adult cat somatosensory cortex after peripheral nerve transections.

The functional reorganization of cerebral cortex following peripheral deafferentation is associated with changes in a number of neurotransmitters and related molecules. Acetylcholine (ACh) enhances neuronal responsiveness and could play a role in activity-dependent cortical plasticity. In this study, choline acetyltransferase (ChAT) immunohistochemistry was used to investigate ACh innervation of the primary somatosensory cortex in cats sustaining complete unilateral forearm and paw denervations. Survival times of 2-52 weeks were examined. The deafferented contralateral cortex was defined electrophysiologically, and quantitative estimates of ChAT-immunoreactive fiber density were obtained from the forelimb and hindlimb sectors of area 3b in both hemispheres. In the 3b forelimb sector contralateral to the deafferentation, a decrease in density of ChAT-positive fibers relative to the ipsilateral hemisphere was apparent at 2 weeks and most pronounced at 13 weeks, involving all cortical layers except layer I. There was no such decrease in the hindlimb sector, but the loss of ChAT immunoreactivity extended to sectors representing proximal forelimb and trunk. Changes in ChAT immunoreactivity were no longer found after 1 year of survival. This long-lasting but reversible lowering of ChAT immunoreactivity could result from a loss of afferent activity in basalis neurons and/or trophic influences retrogradely exerted by cortex on these cells. Reduced ACh transmission might then contribute to the loss of gamma aminobutyric acid (GABA) inhibition in the deafferented cortex by decreasing the activation of inhibitory interneurons. The long-term recovery of a normal ChAT immunoreactivity in cortex could be a consequence of its functional reorganization.

Dual character, asynaptic and synaptic, of the dopamine innervation in adult rat neostriatum: a quantitative autoradiographic and immunocytochemical analysis.

Dopamine (DA) axon terminals (varicosities) in the neostriatum of adult rats were examined for shape, size, content, synaptic incidence, type of junction, synaptic targets, and microenvironment after electron microscopic identification either by [3H]DA uptake autoradiography or by immunocytochemistry with monoclonal antibodies against DA-glutaraldehyde-protein conjugate. Both approaches yielded comparable results. Whether they were from the paraventricular or the mediodorsal neostriatum, respectively, the [3H]DA-labeled and DA-immunostained varicosities were generally oblong and relatively small; more than 60% contained one or more mitochondria. Sixty to seventy percent were asynaptic, and 30-40% were endowed with a synaptic membrane differentiation (junctional complex), as inferred by stereological extrapolation from single thin sections (both approaches) or observed directly in long, uninterrupted series of thin sections (immunocytochemistry). The synaptic DA varicosities always displayed symmetrical junctions: 67% with dendritic branches, 30% with dendritic spines, and 2-3% with neuronal cell bodies. DA varicosities juxtaposed to one another were frequent. Other axonal varicosities were more numerous in the immediate vicinity of DA varicosities than around randomly selected, unlabeled terminals. The respective microenvironments of DA and unlabeled varicosities also showed enrichment in the preferred synaptic targets of both groups of varicosities, with dendritic branches for DA and dendritic spines for the unlabeled ones. These data suggest a dual mode of operation that is diffuse as well as synaptic for the nigrostriatal DA system. In such a densely DA-innervated brain region, they also lead to the hypothesis that a basal level of extracellular DA might be maintained permanently around every tissue constituent and, thus, contribute to the mechanisms of action, properties, and functions (or dysfunctions) of DA within the neostriatum itself and as part of the basal ganglia circuitry.

The serotonin neurons in nucleus raphe dorsalis of adult rat: a light and electron microscope radioautographic study.

The serotoninergic nerve cell body population of nucleus raphe dorsalis (RD) was identified by radioautography following cerebroventricular instillation of tritiated serotonin ([3H]5-HT) in adult rats pretreated with a monoamine oxidase inhibitor. Series of histological sections taken throughout the midbrain and upper pons exhibited a similar distribution and number of labeled nerve cell bodies in RD after prolonged administration of either 10-5 or 10-4M [3H]5-HT or 10-4M [3H]5-HT and 10-3M nonradioactive noradrenaline. This allowed systematic mapping and quantification of serotoninergic nerve cell bodies at various levels of the RD. Their extrapolated total number averaged 11,500. Twice as many unreactive (nonserotoninergic) neurons were present within the same region. In electron microscope radioautographs, the labeled cells were usually larger (17.9 micrometer mean diameter) than their unlabeled congeners (13.1 micrometer), but stereological sampling of their perikarial organelle content failed to reveal any difference in cytoplasmic composition. Few [3H]5-HT-labeled axonal varicosities were observed in RD and none were found in close apposition or in synaptic junction with labeled nerve cell bodies, dendrites, or unreactive perikarya. A detailed statistical analysis of silver grain distribution in both labeled and "unlabeled" nerve cell bodies, indicated that in the former, but not in the latter, dense bodies had a relatively high affinity for [3H]5-HT. Mitochondria and the cytoplasmic membrane were the only other organelles to show higher labeling indices in labeled than in unlabeled cells. Other sites of [3H]5-HT localization could be ascribed to artefactitious cross-linkage of the tracer by the fixative, since they had the same relative affinity in the two cell populations. These results provide new insights into the morphology and cytofunctional properties of the 5-HT neurons of rat RD.

Quantified regional and laminar distribution of the noradrenaline innervation in the anterior half of the adult rat cerebral cortex.

The regional and laminar distribution of the noradrenaline (NA) innervation in the adult rat cerebral cortex was quantified in radioautographs of semithin sections from whole hemisphere slices incubated with tritiated catecholamines and a monoamine oxidase inhibitor. Uptake-labeled axonal varicosities (aggregates of silver grains) were counted with the help of a computerized image analyzer in seven cytoarchitectonic areas of the rostral half of the cortex: Cg3, rostral AID, Cg2, Fr1, Par1, caudal AID, and Pir (prepiriform) according to Zilles's nomenclature. Both dopamine (DA) and NA terminals were detected after incubation with [3H]DA and citalopram or with [3H]NA alone. In the presence of desipramine (DMI), DA terminals alone were demonstrated; the number of NA terminals was then obtained by subtraction from counts in adjacent slices incubated with or without DMI. These counts suggested that DA and NA varicosities were fully visualized only after labeling with their respective tritiated amine. Similar numbers of labeled NA varicosities as inferred after [3H]NA incubation with or without DMI were observed after [3H]NA incubation in the presence of benztropine (BZ). This indicated that NA terminals were then maximally detected to the exclusion of the DA ones, and the latter approach was adopted for the acquisition of normative data. Since the average diameter of the labeled NA varicosities was known from earlier measurements in electron microscope radioautographs, the initial counts of labeled sites/mm2 of histological section could be expressed as numbers of varicosities/mm3 of tissue following a double correction for incomplete detection at the chosen duration of radioautographic exposure and section thickness. The overall density of NA innervation was thus estimated at 1.2 million varicosities/mm3 of tissue, with no statistically significant differences between the seven cortical areas examined. In every region, the number of NA terminals was the greatest in the molecular layer (1.5-2 times the density in the rest of cortex) and then progressively decreased in the underlying cortex, with a two- to threefold difference between upper and lower layers. These numerical data allowed an estimation to be made of the possible number of cortical NA varicosities per locus coeruleus nerve cell body of origin (at least 300,000), of their average number per cortical neuron (30-50), their actual incidence among all terminals in the cortex (1/1,000), their mean endogenous amine content per varicosity (0.22 fg), and the mean number of recognition sites for the uptake blocker DMI (4,500/varicosity).(ABSTRACT TRUNCATED AT 400 WORDS)