Laurent Descarries

Cellular and subcellular distribution of the serotonin 5-HT2A receptor in the central nervous system of adult rat.

Light and electron microscope immunocytochemistry with a monoclonal antibody against the N‐terminal domain of the human protein was used to determine the cellular and subcellular localization of serotonin 5‐HT2A receptors in the central nervous system of adult rat. Following immunoperoxidase or silver‐intensified immunogold labeling, neuronal, somatodendritic, and/or axonal immunoreactivity was detected in numerous brain regions, including all those in which ligand binding sites and 5‐HT2A mRNA had previously been reported. The distribution of 5‐HT2A‐immunolabeled soma/dendrites was characterized in cerebral cortex, olfactory system, septum, hippocampal formation, basal ganglia, amygdala, diencephalon, cerebellum, brainstem, and spinal cord. Labeled axons were visible in every myelinated tract known to arise from immunoreactive cell body groups.

Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain

The 5‐HT1A and 5‐HT1B receptors of serotonin play important roles as auto‐ and heteroreceptors controlling the release of serotonin itself and of other neurotransmitters/modulators in the central nervous system (CNS). To determine the precise localization of these receptors, we examined their respective cellular and subcellular distributions in the nucleus raphe dorsalis and hippocampal formation (5‐HT1A) and in the globus pallidus and substantia nigra (5‐HT1B), using light and electron microscopic immunocytochemistry with specific antibodies. Both immunogold and immunoperoxidase preembedding labelings were achieved. In the nucleus raphe dorsalis, 5‐HT1A immunoreactivity was found exclusively on neuronal cell bodies and dendrites, and mostly along extrasynaptic portions of their plasma membrane. After immunogold labeling, the density of membrane‐associated 5‐HT1A receptors could be estimated to be at least 30–40 times that in the cytoplasm. In the hippocampal formation, the somata as well as dendrites of pyramidal and granule cells displayed 5‐HT1A immunoreactivity, which was also prominent on the dendritic spines of pyramidal cells. In both substantia nigra and globus pallidus, 5‐HT1B receptors were preferentially associated with the membrane of fine, unmyelinated, preterminal axons, and were not found on axon terminals. A selective localization to the cytoplasm of endothelial cells of microvessels was also observed. Because the 5‐HT1A receptors are somatodendritic, they are ideally situated to mediate serotonin effects on neuronal firing, both as auto‐ and as heteroreceptors. The localization of 5‐HT1B receptors to the membrane of preterminal axons suggests that they control transmitter release from nonserotonin as well as serotonin neurons by mediating serotonin effects on axonal conduction. The fact that these two receptor subtypes predominate at extrasynaptic and nonsynaptic sites provides further evidence for diffuse serotonin transmission in the CNS. J. Comp. Neurol. 417:181–194, 2000. ©2000 Wiley‐Liss, Inc.

Noradrenergic axon terminals in the cerebral cortex of rat. III. Topometric ultrastructural analysis

Summary Noradrenergic (NA) axons from the frontoparietal of an adult Sprague-Dawley rat were examined in 12 ribbons of 3 adjacent sections prepared for high resolution radioautography after specific labeling with [ 3 H]NA in vivo. During electron microscopic examination of the various cortical layers, care was taken to determine the location of 30–49 reactive sectional profiles, which were identified as and found to correspond to 2556 different axonal parts. These were subsequently analyzed in terms of general configuration, organelle content, pattern of labeling and intracellular relationships. The NA arborization in cortex may be described as widely dispersed, unmyelinated axons of very fine caliber (ca. 0.35 μm), bearing small spherical enlargements (ca. 1 μm in diameter) and spaced at short intervals (1–3 μm). When viewed in single thin sections, these axonal varicosities always show aggregates of small, pleomorphic, agranular ‘synaptic’ vesicles, often accompanied by one or more large granular vesicles (LGVs) and mitochondria. Within intervaricose segments, microtubules and smooth endoplasmic reticulum are visible, as well as scattered LGVs and mitochondria. [ 3 H]NA is highly concentrated inside the varicosities and to a lesser degree within intervaricose segments. It appears to be mainly associated with the small vesicles, some of the mitochondria, cisterns of smooth endoplasmic reticulum and LGVs. These preferential localizations probably reflect a strong binding capacity of the intra-axonal organelles toward endogenous norepinephrine. The figurative elements in cortical NA nerve endings are not sufficiently distinctive to be used as criteria for ultrastructural identification. Nevertheless, the examination of serial thin sections indicates that a small number of LGVs must be present in every NA varicosity. These data are compatible with a presumptive role of the LGVs in the transport of synthetic enzymes from the nerve cell bodies into the varicosities, wherein the storage of norepinephrine would be primarily ensured by the small vesicles. A very low proportion of cortical NA varicosities was found to be engaged in genuine synaptic relationships. Among 1835 reactive terminals, 341 of which were viewed in 2 or 3 adjacent sections, less than 5% exhibited typical junctional complexes, as opposed to 50% of unlabele boutons similarly sampled in the neighboring neuropil. The rare NA varicosities showing a synapse failed to reveal other structural differences with their congeners. Thus neither their intracortic repartition nor their cellular relationship support the existence of strategic contacts with restricted and/or specialized constituents of the parenchyma. It appears likely that in cortex endogenous norepinephrine may be liberated from all axonal varicosities where it is concentrated, and not only from the small number which form typical synapses. The fine structural characteristics of cortical NA fibers must therefore be considered in the light of recent demonstrations of their intricate and widespread distribution throughout the cerebral cortex, as well as distant, common origin in the locus coeruleus. In this context, it seems probable that the NA afferents might exert a diffuse, desynchronized and tonic influence on vast neuronal assemblies, and thus modulate integrative and/or specific cortical functions.

Modulatory role for biogenic amines in the cerebral cortex. Microiontophoretic studies

In order to investigate the mode of action of biogenic amines in rat cerebral cortex, the unitary activity of spontaneously firing neurons and their excitatory response to acetylcholine (ACh) were examined using microiontophoretic administration of dopamine (DA), noradrenaline (NA) and serotonin (5-HT). The predominant effect of these biogenic amines on the spontaneous activity was a profound and prolonged inhibition of firing (2-4 min), which attained its maximum within 15-120 sec. This response was generally more abrupt in onset and of greater magnitude with NA and 5-HT than with DA. Most units inhibited by DA, NA and 5-HT also showed marked depression of their excitatory response to ACh when pretreated with these biogenic amines. With repetitive administration of ACh, it could be shown that the total duration of inhibition of ACh responses by DA and NA was not as prolonged as the inhibition of the spontaneous firing of the same cells. With 5-HT, the initial ACh responses of many neurons could be completely blocked, and this inhibitory effect lasted as long as the inhibition of spontaneous firing. In view of the anatomical data demonstrating a relative sparsity of monoamine nerve terminals in cerebral cortex, the strong inhibition induced by DA, NA or 5-HT may have reflected slow inactivation of the biogenic amines. However, it could also be indicative of underlying mechanisms of action dependent on metabolic changes. Indeed, the interaction between biogenic amines and ACh might imply a balance between the intracellular pools of cAMP and cGMP is directly or indirectly influenced by the biogenic amines and ACh, respectively. This hypothesis would not exclude other modes of local interaction between DA, NA, 5-HT and ACh, and appears compatible with the modulatory role of biogenic amines in cerebral cortex.

Maladaptive plasticity of serotonin axon terminals in levodopa-induced dyskinesia.

Striatal serotonin projections have been implicated in levodopa‐induced dyskinesia by providing an unregulated source of dopamine release. We set out to determine whether these projections are affected by levodopa treatment in a way that would favor the occurrence of dyskinesia.

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 dead 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.

Noradrenaline axon terminals in adult rat neocortex: An immunocytochemical analysis in serial thin sections

Peroxidase-antiperoxidase electron microscope immunocytochemistry with an antiserum against noradrenaline-glutaraldehyde-protein conjugate was used to identify cortical noradrenaline terminals (axonal varicosities) from the upper layers of the frontal, parietal and occipital cortex in adult rat. A large number of immunostained varicosities were examined in serial thin sections, and compared with a control population of randomly chosen unlabeled terminals from the same sections. Both groups of varicosities were measured and scrutinized for the presence of a junctional complex indicative of synaptic specialization. Cellular elements juxtaposed to the membrane of both types of varicosities were also identified and counted. Noradrenaline varicosities in all three cortical regions averaged 0.65 microns in diameter. In contrast to their unlabeled counterparts, these profiles rarely showed a membrane differentiation characteristic of a synaptic contact (junctional complex). The rare junctional complexes formed by cortical noradrenaline varicosities were invariably symmetrical and almost always found on dendritic shafts. The microenvironment of noradrenaline varicosities also differed, exhibiting a greater number of apposed axonal varicosities and a smaller number of dendritic spines than that of the random population. The proportion of noradrenaline varicosities making a synaptic contact (synaptic incidence) was determined by plotting the incidence of visible junctions as a function of the number of thin sections available for examination. As extrapolated for whole varicosities after linear transformation (double reciprocal plot), this proportion was 17% or 26% depending on the stringency of the criteria used in identifying the junctional complex. The same analysis provided a figure of 98% for the control population. The present study largely confirmed our initial radioautographic characterization of the cortical noradrenaline innervation as a mostly non-junctional system, and also indicated that these varicosities are set in a particular microenvironment. These new data further support the eventuality of a diffuse release of cortical noradrenaline in the extracellular space, compatible with both its neuromodulatory role and multiplicity of actions on diverse cellular targets in the cerebral cortex. The functions assigned to the coeruleocortical noradrenaline system must therefore be viewed as the product of a widespread and ubiquitously distributed neuronal organization characterized by loose intercellular relationships. This system might be capable of selectivity and specificity of action, however, owing to the distribution of its receptors, and in view of intrinsically or extrinsically driven control mechanisms triggered by the release of its own or other transmitters and which may also involve target-initiated feedback mechanisms.

Serotonin nerve terminals in the locus coeruleus of adult rat: A radioautographic study

Serotonin (5-HT) nerve terminals in the locus coeruleus (LC) of adult rat were visualized by high-resolution radioautography, in order to examine their distribution, fine structural features and intimate relationships with norepinephrine neurons. In animals pretreated with a monoamine oxidase inhibitor, prolonged intraventricular perfusion of 10(-4) M [3H]5-HT resulted in a specific identification of most if not all 5-HT axonal varicosities in LC. These terminals were equally distributed between the dorsal and ventral divisions of the nucleus. Their density was approximated at 10(7) per cu.mm within the middle third of the LC. In electron microscope radioautographs, the labeled 5-HT varicosities averaged 0.9 micron in diameter. They all exhibited a distinctive storage organelle, in the form of microvesicles and microcanaliculi (15-25 nm in diameter) partly filled with electron-dense material and usually aggregated in association with several large dense-core vesicles. While this finding of intrinsic morphological characteristics appeared compatible with a special cellular origin or regional differentiation, it was also suggestive of particular functional properties and/or mode of action. In a sample comprised of some 500 sectional profiles from labeled 5-HT varicosities in LC, a small proportion only (less than 10%) exhibited morphologically defined synaptic junctions. These rare contacts were invariably made with dendritic processes and never observed on the noradrenergic perikarya. It is therefore concluded that, in the LC, non-synaptic as well as synaptic mechanisms might be involved in the modulation and transneuronal regulation of norepinephrine neurons by 5-HT afferents.

Ultrastructural evidence for diffuse transmission by monoamine and acetylcholine neurons of the central nervous system.

Publisher Summary Wealth of evidence has accumulated to confirm the reality and heuristic value of the volume transmission paradigm. In its largest acceptance, this diffuse mode of transmission may now be considered to apply not only to neuronal populations characterized by a paucity of synaptic junctions, but also to wholly junctional ones, such as the glycine-, GABA- and glutamate-containing neurons, which display spillover of their transmitter. Using immunoelectron microscopy, many of the receptors for these aminoacidergic transmitters, as well as for monoamines, ACh and neuropeptides, have been visualized in extrasynaptic membrane locations, on neuronal somata, dendrites, axons and axon terminals, as well as glia and endothelial cells, often remote from the corresponding release sites. The ambient level hypothesis has provided a framework for explaining some of the sustained and regulatory, as well as trophic-like effects of transmitters on a prolonged time scale.

Noradrenergic axon terminals in the cerebral cortex of rat. II. Quantitative data revealed by light and electron microscope radioautography of the frontal cortex.

Abstract Noradrenergic (NA) axons, labeled with dl -[ 3 H]norepinephrine ([ 3 H]NA), have been visualized in the frontal cortex of adult rats by means of light and electron microscope radioautography. Three hours after topical application of the tracer in the presence of a monoamine oxidase inhibitor, the radioactivity is mainly concentrated within axonal enlargements exhibiting synaptic vesicles. From electron microscope radioautographs, the average diameter of the labeled axon terminals may be calculated at 1.15 μm. Mapping of the reactive sites in light microscope radioautographs provides numerical data allowing estimates of the density and incidence of NA axon terminals in the frontal cortex. The total number of NA nerve endings is extrapolated to be 96.6 × 10 3 /cu. mm of cortex, representing a mean incidence of 1 NA terminal/8.8–14.5 × 10 3 cortical synapses. More than 35% of the labeled nerve endings occupy molecular layer I, where their incidence rises to 1/3.6–5.9 × 10 3 cortical synapses. In layers II–IV, there is a lower density of reactive terminals, while few are visible in the upper portion of layer V. Assuming that all NA axon terminals have been detected, the mean content of endogenous norepinephrine per NA nerve ending is approximately 2.33 × 10 −3 pg , indicating a concentration of 2900 μg/g wet weight or 0.3%.