Alain Beaudet

Serotonin axon terminals in the ventral tegmental area of the rat: fine structure and synaptic input to dopaminergic neurons.

The serotoninergic (5-hydroxytryptamine, 5-HT) innervation of the rat ventral tegmental area (VTA) was examined by light and electron microscopic radioautography following intraventricular infusion of [3H]5-HT. The [3H]5-HT labeled processes were characterized with respect to their regional distribution, ultrastructure and relationships with all neurons, including dopaminergic neurons, identified in the same sections using immunocytochemistry for the localization of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). By light microscopy, [3H]5-HT labeled axons and axonal varicosities were detected throughout the interfascicular nucleus and ventral portion of the VTA. By electron microscopy, [3H]5-HT-labeled axons were found to be mainly small and unmyelinated, although a few showed several lamellae of myelin. The labeled varicosities measured 0.6 micron in mean diameter and contained many small, round or flattened agranular vesicles and a few large granular vesicles. More than 18% showed synaptic specializations in single thin sections. Most of these synapses were asymmetric and established on dendritic shafts. Based on the probability of seeing such synaptic specializations in single thin sections, it was estimated that as many as 50% of the labeled 5-HT terminals formed synaptic contacts in the VTA. In dually labeled light microscopic sections, [3H]5-HT-accumulating processes often appeared adjacent to TH-immunoreactive perikarya and proximal dendrites. Electron microscopy demonstrated that terminals with radioautographic labeling for 5-HT formed conventional synapses both with TH-labeled and unlabeled dendrites in the VTA. Many additional 5-HT terminals lacking recognizable synaptic densities were directly apposed to TH-labeled dendrites and were isolated from the rest of the neuropil by thin glial leaflets. These results suggest that 5-HT neurons innervate both dopaminergic and non-dopaminergic neurons in the VTA and may influence mesocortical and mesolimbic efferent systems through synaptic as well as non-synaptic mechanisms.

Ultrastructural features of six types of neurons in rat dorsal root ganglia

SummarySections of glutaraldehyde-fixed lumbar dorsal root ganglia of the rat were examined in the electron microscope following impregnation with the uranyl-lead-copper technique or postfixation in potassium ferrocyanide-reduced osmium. Three types of ganglion cells (A, B, C) were identified on the basis of their size and the distribution of their organelles. They were further subdivided into six subtypes according to the arrangement and three-dimensional organization of the Nissl bodies and Golgi apparatus in the perikarya. Type A1 cells were large, clear neurons in which Nissl bodies, separated from each other by pale narrow strands of cytoplasm containing small stacks of Golgi saccules and rod-like mitochondria, were evenly distributed throughout the perikaryon. In type A2, the Nissl bodies assumed a similar distribution but were separated by much wider strands of cytoplasm. Type A3, the smallest of the type A category, displayed densely packed Nissl bodies and long stacks of Golgi saccules which formed a perinuclear ring in the mid-portion of the perikaryon.Type B cells were smaller and showed a concentric zonation of their organelles. In type B1, large Nissl bodies located in an outer cytoplasmic zone were made of long piles of parallel cisternae interrupted by curved Golgi stacks. Type B2 was characterized by a ring-like Golgi apparatus separating the perikaryon in a cortical zone composed mainly of Nissl substance and a juxtanuclear zone containing mitochondria and smooth endoplasmic reticulum. Type C cells were the smallest of the ganglion cells and contained small, poorly demarcated Nissl bodies and a juxtanuclear Golgi apparatus.

Up-regulation and trafficking of δ opioid receptor in a model of chronic inflammation: implications for pain control

&NA; Pharmacological and physiological evidence supports a role for delta (&dgr;) opioid receptors in the nociceptive mechanisms of inflammation. However, few data exist regarding &dgr; opioid receptor expression and localization in such conditions. In this study, we have assessed the distribution and function of &dgr; opioid receptors in the rat spinal cord following induction of chronic inflammation by intraplantar injection of complete Freunds adjuvant (CFA). Intrathecal administration of the selective &dgr; opioid receptor agonist, d‐[Ala2, Glu4] deltorphin, dose‐dependently reversed thermal hyperalgesia induced by CFA. In situ hybridization and Western blotting experiments revealed an increase in &dgr; opioid receptor mRNA and protein levels, respectively, in the dorsal lumbar spinal cord ipsilateral to the CFA injection site compared to the contralateral side and sham‐injected controls. By electron microscopy, immunopositive &dgr; opioid receptors were evident in neuronal perikarya, dendrites, unmyelinated axons and axon terminals. Quantification of immunopositive signal in dendrites revealed a twofold increase in the number of immunogold particles in the ipsilateral dorsal spinal cord of CFA‐injected rats compared to the contralateral side and to sham‐injected rats. Moreover, the relative frequency of immunogold particles associated with or in close proximity to the plasma membrane was increased in the ipsilateral dorsal spinal cord, indicating a more efficient targeting of &dgr; opioid receptors to neuronal plasma membranes. These data demonstrate that CFA induces an up‐regulation and increased membrane targeting of &dgr; opioid receptors in the dorsal spinal cord which may account for the enhanced antinociceptive effects of &dgr; opioid receptor agonists in chronic inflammatory pain models.

CELLULAR DISTRIBUTION OF NEUROTENSIN RECEPTORS IN RAT BRAIN : IMMUNOHISTOCHEMICAL STUDY USING AN ANTIPEPTIDE ANTIBODY AGAINST THE CLONED HIGH AFFINITY RECEPTOR

Receptors for the neuropeptide, neurotensin, were localized by immunohistochemistry in the rat brain by using an antibody raised against a sequence of the third intracellular loop of the cloned high affinity receptor. Selective receptor immunostaining was observed throughout the brain and brainstem. This immunostaining was totally prevented by preadsorbing the antibody with the immunogenic peptide. The regional distribution of the immunoreactivity conformed for the most part to that of [3H]‐ or [125I]‐neurotensin binding sites previously identified by autoradiography. Thus, the highest levels of immunostaining were observed in the islands of Calleja, diagonal band of Broca, magnocellular preoptic nucleus, pre‐ and parasubiculum, suprachiasmatic nucleus, anterodorsal nucleus of the thalamus, substantia nigra, ventral tegmental area, pontine nuclei and dorsal motor nucleus of the vagus, all of which had previously been documented to contain high densities of neurotensin binding sites. There were, however, a number of regions reportedly endowed with neurotensin binding sites, including the central amygdaloid nucleus, periaqueductal gray, outer layer of the superior colliculus and dorsal tegmental nucleus, which showed no or divergent patterns of immunostaining, suggesting that they might be expressing a molecularly distinct form of the receptor. At the cellular level, neurotensin receptor immunoreactivity was predominantly associated with perikarya and dendrites in some regions (e.g., in the basal forebrain, ventral midbrain, pons and rostral medulla) and with axons and axon terminals in others (e.g., in the lateral septum, bed nucleus of the stria terminalis, neostriatum, paraventricular nucleus of the thalamus and nucleus of the solitary tract). These data indicate that neurotensin may act both post‐ and presynaptically in the central nervous system and confirm that some of its effects are exerted on projection neurons. There were also areas, such as the cerebral cortex, nucleus accumbens and para‐ and periventricular nucleus of the hypothalamus, which contained both immunoreactive perikarya/dendrites and axon terminals, consistent with either a joint association of the receptor with afferent and efferent elements or its presence on interneurons. Taken together, these results also suggest that the neurotensin high affinity receptor protein is associated with a neuronal population that is more extensive than originally surmised from in situ hybridization studies.

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%.

Sensory presynaptic and widespread somatodendritic immunolocalization of central ionotropic P2X ATP receptors.

Recent evidence suggests that extracellular ATP plays a neurotransmitter role in the central nervous system. Its fast ionotropic effects are exerted through a family of P2X ATP-gated channels expressed in brain and spinal cord. To determine the physiological significance of central ATP receptors, we have investigated the localization of a major neuronal P2X receptor at the cellular and subcellular levels using affinity-purified antibodies directed against the C-terminal domain of P2X4 subunit. Subunit-specific anti-P2X4 antibodies detected a single band of 57,000 +/- 3000 mol. wt in transfected HEK-293 cells and in homogenates from adult rat brain. The strongest expression of central P2X receptors was observed in the olfactory bulb, lateral septum, cerebellum and spinal cord. P2X4 immunoreactivity was also evident in widespread areas including the cerebral cortex, hippocampus, thalamus and brainstem. In all regions examined, P2X receptors were associated with perikarya and dendrites where they were concentrated at the level of afferent synaptic junctions, confirming a direct involvement of postsynaptic ATP-gated channels in fast excitatory purinergic transmission. Moreover, P2X4-containing purinoceptors were localized in axon terminals in the olfactory bulb and in the substantia gelatinosa of nucleus caudalis of the medulla and dorsal horn of the spinal cord, demonstrating an important selective presynaptic role of ATP in the modulation of neurotransmitter release in central sensory systems.

VIP neurons as prime synaptic targets for serotonin afferents in rat suprachiasmatic nucleus: a combined radioautographic and immunocytochemical study

SummaryCellular relationships between serotonin (5-HT) axon terminals and neurons containing vasoactive intestinal peptide (VIP) were characterized by combined radioautography and immunocytochemistry in rat suprachiasmatic nucleus (SCN). Light microscopic immunoradioautographs showed significant overlap between (3H)5-HT uptake sites and VIP-immunoreactive elements in the ventral half of the SCN. Of the 255 (3H)5-HT-labelled axonal profiles detected in a systematic electron microscopic survey of single thin sections from this area, 75 (30%) were directly apposed to VIP-immunoreactive nerve cell bodies and/or dendrites. Radioautographically labelled 5-HT varicosities often showed well-differentiated, symmetrical or asymmetrical synaptic junctions, 60% of which were established on VIP-immunoreactive nerve cell bodies or dendrites. In a separate sampling of 198 (3H)5-HT-labelled terminals seen in apposition with VIP-immunoreactive elements, 50 showed a junctional complex at the site of contact. Postsynaptic immunoreactive elements were mostly dendrites but also included nerve cell bodies. Despite the methodological limitations inherent to the present double labelling approach, these data strongly support the view that VIP neurons are prime synaptic targets for 5-HT afferents in the SCN. VIP/5-HT interactions are thus likely to play an important functional role in this nucleus and may in particular subserve the 5-HT mediated regulation of certain circadian rhythms, including that of pituitary hormone secretion.

Morphine and Pain-Related Stimuli Enhance Cell Surface Availability of Somatic δ-Opioid Receptors in Rat Dorsal Root Ganglia

The present study demonstrates that perikaryalδ-opioid receptors (δORs) in rat dorsal root ganglion (DRG) neurons bind and internalize opioid ligands circulating in the CSF. Using confocal and electron microscopy, we found that prolonged morphine treatment increased the cell surface density of these perikaryal δORs and, by way of consequence, receptor-mediated internalization of the fluorescent deltorphin (DLT) analog ω-Bodipy 576/589 deltorphin-I 5-aminopentylamide (Fluo-DLT) in all three types of DRG neurons (small, medium, and large). In contrast, chronic inflammatory pain induced by the injection of complete Freunds adjuvant (CFA) into one hindpaw selectively increased Fluo-DLT internalization in small and medium-sized DRG neurons ipsilateral to the inflammation. Based on our previous studies in the spinal cord of μ-opioid receptor (μOR) knock-out mice, it may be assumed that the enhanced membrane recruitment of δORs observed after sustained morphine is attributable to stimulation of μORs. However, the selectivity of the effect induced by inflammatory pain suggests that it involves a different mechanism, namely a modality-specific and pain-related activation of C and Aδ fibers. Indeed, stimulation by capsaicin of transient receptor potential vanilloid 1 receptors, which are selectively expressed by small diameter (< 600 μm2) DRG neurons, increased Fluo-DLT internalization exclusively in this cell population. The present results, therefore, demonstrate that DRG neurons express perikaryal δORs accessible to CSF-circulating ligands and that the density and, hence, presumably also the responsiveness, of these receptors may be modulated by both pain-related stimuli and sustained exposure to μOR agonists.

Radioautographic characterization of a serotonin-accumulating nerve cell group in adult rat hypothalamus.

Intensely labeled nerve cell bodies were identified by radioautography within the pars ventralis of nucleus dorsomedialis hypothalami (hdv), following intraventricular perfusion with 10(-5) or 10(-4) M tritiated serotonin [3H]5-HT in adult rats pretreated with a monoamine oxidase inhibitor. This selective reaction, which involved approximately 1000 neurons on each side of the third ventricle, was unaltered by concomitant administration of 10(-3) M non-radioactive norepinephrine, and was absent after intraventricular injection of 10(-5) or 10(-4) M tritiated norepinephrine. The 3H-labeled 5-HT nerve cell bodies were loosely grouped within the inner and caudal half of the hdv, and appeared morphologically similar to the unreactive neurons among which they were interspersed. Within the same region, numerous labeled axonal varicosities were also detected, which were never found in synaptic contact with the reactive cells. If the 3H-labeled 5-HT neurons contain endogenous 5-HT, they might constitute an intrinsic source of 5-HT innervation in the adult rat hypothalamus.

Distribution of NTS3 receptor/sortilin mRNA and protein in the rat central nervous system

The neurotensin (NT) receptor, NTS3, originally identified as the intracellular sorting protein sortilin, is a member of a recently discovered family of receptors characterized by a single transmembrane domain. The present study provides the first comprehensive description of the distribution of NTS3/sortilin mRNA and protein in adult rat brain using in situ hybridization and immunocytochemistry. Both NTS3/sortilin mRNA and immunoreactivity displayed a widespread distribution throughout the brain. High levels of NTS3/sortilin expression and immunoreactivity were found in neuronal cell bodies and dendrites of allocortical areas such as the piriform cortex and hippocampus. Regions expressing both high levels of NTS3/sortilin mRNA and protein also included several neocortical areas, the islands of Calleja, medial and lateral septal nuclei, amygdaloid nuclei, thalamic nuclei, the supraoptic nucleus, the substantia nigra, and the Purkinje cell layer of the cerebellar cortex. In the brainstem, all cranial nerve motor nuclei were strongly labeled. NTS3/sortilin mRNA and immunoreactivity were also detected over oligodendrocytes in major fiber tracts. Subcellularly, NTS3/sortilin was predominantly concentrated over intracytoplasmic membrane‐bound organelles. Many of the areas exhibiting high levels of NTS3/sortilin (e.g., olfactory cortex, medial septum, and periaqueductal gray) have been documented to contain high concentrations of NT nerve cell bodies and axons, supporting the concept that NTS3/sortilin may play a role in NT sorting and/or signaling. Other areas (e.g., hippocampal CA fields, cerebellar cortex, and cranial nerve motor nuclei), however, are NT‐negative, suggesting that NTS3/sortilin also exerts functions unrelated to NT signaling. J. Comp. Neurol. 461:483–505, 2003.