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.

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.

Ultrastructural and morphometric features of the acetylcholine innervation in adult rat parietal cortex: an electron microscopic study in serial sections.

This study was aimed at characterizing the ultrastructural morphology of the normal acetylcholine (ACh) innervation in adult rat parietal cortex. After immunostaining with a monoclonal antibody against purified rat brain choline acetyltransferase (ChAT), more than 100 immunoreactive axonal varicosities (terminals) from each layer of the Par 1 area were photographed and examined in serial thin sections across their entire volume. These varicosities were relatively small, averaging 0.6 micron in diameter, 1.6 microns 2 in surface, and 0.12 micron 3 in volume. In every layer, a relatively low proportion exhibited a synaptic membrane differentiation (10% in layer I, 14% in II-III, 11% in IV, 21% in V, 14% in VI), for a I-VI average of 14%. These synaptic junctions were usually single, symmetrical (> 99%), and occupied a small portion of the surface of varicosities (< 3%). A majority were found on dendritic branches (76%), some on spines (24%), and none on cell bodies. On the whole, the ACh junctional varicosities were significantly larger than their nonjunctional counterparts, and both synaptic and nonsynaptic varicosities could be observed on the same fiber. A subsample of randomized single thin sections from these whole varicosities yielded similar values for size and synaptic frequency as the result of a stereological extrapolation. Also analyzed in single sections, the microenvironment of the ChAT-immunostained varicosities appeared markedly different from that of unlabeled varicosity profiles randomly selected from their vicinity, mainly due to a lower incidence of synaptically targeted dendritic spines. Thus, the normal ACh innervation of adult rat parietal cortex is predominantly nonjunctional (> 85% of its varicosities), and the composition of the microenvironment of its varicosities suggests some randomness in their distribution at the microscopic level. It is unlikely that these ultrastructural characteristics are exclusive to the parietal region. Among other functional implications, they suggest that this system depends predominantly on volume transmission to exert its modulatory effects on cortical activity.

Ultrastructural characterization of the acetylcholine innervation in adult rat neostriatum.

The ultrastructural features of acetylcholine axon terminals (varicosities) in adult rat neostriatum were characterized by electron microscopy after immunostaining with a sensitive monoclonal antibody against rat choline acetyltransferase. Several hundred single sections from these varicosities were analysed for shape, size and content, presence of a synaptic membrane specialization, and composition of the microenvironment. An equivalent number of unlabeled varicosities selected at random from the same micrographs were similarly examined. The immunostained varicosity profiles were relatively small and seldom showed a junctional membrane specialization. Stereological extrapolation to the whole volume of these varicosities indicated that less than 10% were synaptic. Far fewer dendritic spines were juxtaposed to these predominantly asynaptic profiles than to their unlabeled counterparts. This difference seemed imputable to the low synaptic incidence of the acetylcholine varicosities and was consistent with the view that these are randomly distributed in relation to surrounding elements. The bulk of the data was suggestive of volume transmission. This raised the possibility that, in such a densely innervated area, a basal level of acetylcholine is permanently maintained around all cellular elements, contributing to the modulatory properties of this transmitter. This basal level of acetylcholine could also serve as a regulatory signal controlling the expression of different receptor subtypes in neurons, glia and blood vessels.

Quantitative and morphometric data indicate precise cellular interactions between serotonin terminals and postsynaptic targets in rat substantia nigra.

We have quantified the density of serotonin axonal varicosities, their synaptic incidence and their distribution among potential targets in the pars reticulata and pars compacta of the rat substantia nigra. Serotonin axonal varicosities, counted at the light microscopic level following in vitro [3H]serotonin uptake and autoradiography, amounted to 9 x 10(6)/mm3 in the pars reticulata and 6 x 10(6)/mm3 in the pars compacta, among the densest serotonin innervations in brain. As determined at the electron microscopic level following immunolabelling for serotonin, virtually all serotonin varicosities in the pars reticulata and 50% of those in the pars compacta formed a synapse, essentially with dendrites. The combination of serotonin immunocytochemistry with tyrosine hydroxylase immunolabelling of dopamine neurons reveals that 20% of the serotonin synaptic contacts in the pars reticulata are on dopamine dendrites and 6% are on a type of unlabelled dendrite characterized by its peculiarly high cytoplasmic content of microtubules. The comparison of the diameter of the dendritic profiles that were in synaptic contact with serotonin-immunoreactive varicosities with the diameter of all other dendritic profiles of the same type suggests that serotoninergic varicosities innervate dopamine dendrites uniformly along their length, whereas they tend to contact microtubule-filled dendrites in more proximal regions and the other, unidentified dendrites in more distal regions. Furthermore, the size of the serotonin-immunoreactive varicosities and of their synaptic junctions is significantly smaller on dopamine dendrites and larger on microtubule-filled dendrites than on other, unidentified dendrites, indicating that the nature of the postsynaptic target is an important determinant of synaptic dimensions. These data should help to clarify the role of serotonin in the nigral control of motor functions. They indicate that this dense serotonin input to the substantia nigra is very precisely organized, acting through both "non-junctional" and "junctional" modes of neurotransmission in the pars compacta, which projects to the neostriatum and the limbic system, whereas the predominant mode of serotonin transmission appears to be of the "junctional" type in the pars reticulata, where serotonin can finely control the motor output of the basal ganglia by acting on the GABA projection neurons either directly or through the local release of dopamine by dopaminergic dendrites. The data also raise the possibility that the postsynaptic targets have trophic retrograde influences on serotoninergic terminals.

Acetylcholine innervation of sensory and motor neocortical areas in adult cat: a choline acetyltransferase immunohistochemical study.

Light microscopic choline acetyltransferase (ChAT) immunocytochemistry was used to examine the distribution of the acetylcholine innervation in primary motor (4 gamma) and sensory (3a, 3b, 41 and 17) cortical areas of adult cat. In every area, scattered immuno-reactive cell bodies were present and a relatively dense meshwork of ChAT immunoreactive axons pervaded the whole cortical thickness. These axons were generally thin and bore innumerable varicosities of different sizes. A few thicker and smoother fibers and occasional clusters of unusually large varicosities were also visible. Overall, area 17 was less densely innervated than the other areas. In each area, layer I showed the densest innervation. Innervation of underlying layers was rather uniform in area 17, but patterned in other areas. In areas 4 gamma and 3a, layers II, upper III and V showed preferential innervation. Innervation of layer IV was the strongest in areas 3b and 41. Area 3a was transitional between 4 gamma and 3b. Except in area 17, the laminar pattern of acetylcholinesterase staining was consistent with that of ChAT. In the light of current data on the distribution of this cortical innervation in different species, and of its presumed ultrastructural features, it appears likely that such regional and laminar features subtend widespread, modulatory roles of ACh.

Recovery of choline acetyltransferase activity without sprouting of the residual acetylcholine innervation in adult rat cerebral cortex after lesion of the nucleus basalis.

In view of the divergent literature concerning the long-term effects of ibotenic acid lesions of the nucleus basalis of Meynert (NBM) on the choline acetyltransferase (ChAT) activity in adult rat cerebral cortex, we have critically reassessed the issue of an eventual recovery of this enzymatic activity by sprouting of the residual acetylcholine (ACh) innervation. At short (1 week) and long survival time (3 months) after unilateral ibotenic acid lesion, ChAT activity was biochemically measured in the ipsi and contralateral fronto-parietal cortex of several rats in which the extent of ACh neuronal loss in NBM was also estimated by counts of ChAT-immunostained cell bodies on the lesioned vs. non-lesioned side. In other lesioned rats, particular attention was paid to the distribution of the residual cortical ACh (ChAT-immunostained) innervation, and that of immunostained vasoactive intestinal polypeptide (VIP) axon terminals known to belong in part to intrinsic cortical ACh neurons which co-localize this peptide. One week after NBM lesion, profound decreases of ipsilateral cortical ChAT activity were tightly correlated with the extent of ACh cell body loss in the nucleus. A significant recovery of cortical ChAT activity could be documented after 3 months, despite persistence of NBM cell body losses as severe as after 1 week. At both survival times, the number of ChAT-immunostained axons was markedly reduced throughout the ipsilateral fronto-parietal cortex, demonstrating that most ACh fibers of extrinsic origin had been permanently removed. This result also indicated that the long-term recovery of ChAT activity had occurred without sprouting of the residual ACh innervation. The laminar distribution and number of VIP-immunostained terminals remained the same on the lesioned and intact side and comparable to normal, ruling out an extensive sprouting of intrinsic ACh/VIP or VIP alone fibers. The return to a near normal cortical ChAT activity in severely ACh-denervated cortex suggested that the intrinsic ACh innervation was primarily responsible for this recovery.

Relational features of acetylcholine, noradrenaline, serotonin and GABA axon terminals in the stratum radiatum of adult rat hippocampus (CA1).

In a well-defined sector of adult rat hippocampus (CA1, stratum radiatum), the ultrastructural features of acetylcholine (ACh), noradrenaline (NA), serotonin (5-HT) and GABA axon terminals (varicosities) were compared by electron microscopy after immunostaining with antibodies against choline acetyltransferase, NA, 5-HT and glutamic acid decarboxylase. Approximately 100 sectional profiles of each type were analyzed for size, presence of a synaptic membrane specialization (synaptic incidence) and composition of the microenvironment. An equivalent number of immunonegative varicosity profiles selected at random from the same micrographs were similarly examined. ACh, NA and 5-HT varicosity profiles were of comparable size, and significantly smaller than GABA profiles. They exhibited a low frequency of junctional specialization, amounting to 7%, 15% and 21%, respectively, when extrapolated to the whole volume of these terminals. In contrast, GABA varicosities appeared entirely synaptic. The ACh, NA and 5-HT varicosities also differed from their GABA counterparts in being juxtaposed to a greater number of unlabeled axonal varicosities and a lower number of dendritic branches. In addition, the microenvironment of immunostained terminals showed a much lower number of dendritic spines than that of immunonegative varicosities. This latter finding was viewed as another indication that predominantly asynaptic varicosities do not maintain particular relationships with their immediate surround. It was also concluded that volume transmission represents a major mode of transmission for ACh, NA and 5-HT in adult rat hippocampus, thus contributing to the properties and functions assigned to these transmitters in this part of brain.