A. Calas

Efferents and afferents of the ventral tegmental-A10 region studied after local injection of [3H]leucine and horseradish peroxidase.

Abstract The efferents and the afferents of the VMT-A10 region were studied by using anterograde ([3H]leucine) and retrograde (HRP) tracing techniques. In order to produce very small injections in various parts of the VMT-A10 region, a slow diffusion technique for [3H]leucine labelling and a microiontophoretic injection for horseradish peroxidase labelling were developed. According to the histochemical and biochemical data, the [3H]leucine anterograde results were separated into three main types of projections. (1) Projections to regions rich in DA terminals. These projections certainly correspond to the efferents of the dopaminergic A10 neurones. According to various injection sites, we have been able to identify mesolimbic projections originating from the VMT-A10, pars medialis and mesostriatal-mesolimbic projections originating from the VMT-A10, pars lateralis. The mesolimbic projections include the prefrontal cortex, the medial part of the lateral septum, the interstitial nucleus of the stria terminalis, the accumbens nucleus and the olfactory tubercle. The mesostriatal-mesolimbic projections include the anteromedial part of the caudate nucleus, the cingular cortex, the entorhinal cortex, the amygdaloid complex, the accumbens nucleus, the olfactory tubercle and the piriform cortex to a lesser extent. (2) Projections to regions suspected of containing DA terminals. These ascending and descending projections which could represent the dopaminergic efferents of the VMT-A10 neurones have been demonstrated. Ascending projections originating either from the VMT-A10 pars medialis or pars lateralis region were found in the claustrum, the nucleus of the tractus diagonalis, the olfactory nuclei, the lateral habenula, the medial hypothalamus and the median eminence. The projections observed in the medial hypothalamus included the periventricular region, the arcuate nucleus, the ventral part of the ventromedial nucleus and the dorsomedial nucleus. The labelling of the anteromedial part of the dorsal hippocampus appeared to originate from the VMT-A10, pars posterior. The projections to the medial hypothalamus, median eminence and hippocampus may have a great functional significance, but further proof of their dopaminergic nature is needed. Descending projections were found ipsilateally to the dorsal raphe and to the cerebellum, and bilaterally to the locus coeruleus. The projections to the cerebellum are distributed to the nuclei interpositus and dentatus and to the Purkinje cell layer and granular layer of the cortex. These results raise the problem of descending dopaminergic projections from the A10 neurones. (3) Projections to regions not known to contain DA terminals. Anterior projections were found ipsilaterally to the supraoptic nucleus and bilaterally to the anterodorsal thalamic nucleus. Posterior projections were traced ipsilaterally to the limbic midbrain area, including the median raphe, the ventral and dorsal tegmental nucleus and the central gray. The horseradish peroxidase experiment supplied some clues as to the posterior afferents of the VMT-A10 region. Some labelled cells were found ipsilaterally in the substantia nigra, the medain raphe and the ventral tegmental nucleus. Numerous cells were labelled ipsilaterally in the dorsal raphe nucleus, and nuclei interpositus and dentatus of the cerebellum, and contralaterally in the locus coeruleus. These structures are likely to play an important role in the modulation of the activity of VMT-A10 neurones. The results of [3H]leucine and HRP experiments permitted us to demonstrate reciprocal connections between VMT-A10 region and anterior raphe nuclei, locus coeruleus and cerebellum.

Anatomical distribution and ultrastructural organization of the gabaergic system in the rat spinal cord. An immunocytochemical study using anti-GABA antibodies

gamma-Aminobutyric acid (GABA)-containing elements have been studied by light and electron microscopy in the rat spinal cord, using immunocytochemistry with anti-GABA antibodies. Light microscopy showed immunoreactive somata localized principally in laminae I-III, and occasionally in the deeper laminae of the dorsal horn and in the ventral horn. Small somata were also observed around the central canal. Punctate GABA-immunoreactive profiles were particularly concentrated in laminae I-III, and moderately abundant in the deeper laminae and in the ventral horn where they were observed surrounding the unlabelled motoneurons. At the ultrastructural level, the punctate profiles corresponded to GABA-containing axonal varicosities or small dendrites. GABA-immunoreactive varicosities were presynaptic to labelled or unlabelled dendrites and cell bodies. Some unlabelled terminals presynaptic to unlabelled dendrites received symmetrical synaptic contacts from GABA-immunoreactive terminals. These results confirm data obtained with L-glutamate decarboxylase immunocytochemistry, and support the role of GABA in pre- and postsynaptic inhibition in the spinal cord, respectively via axoaxonal and axosomatic or axodendritic synapses.

Ultrastructural localization of 5-hydroxytryptamine1A receptors in the rat brain.

5‐Hydroxytryptamine1A (5‐HT1A) receptors have been visualized at the electron microscopic level in selected areas (dorsal raphe nucleus, hippocampus, septum) of the rat brain using specific anti‐peptide antibodies.

Oxytocinergic innervation of autonomic nuclei controlling penile erection in the rat

In the rat, spinal autonomic neurons controlling penile erection receive descending pathways that modulate their activity. The paraventricular nucleus of the hypothalamus contributes oxytocinergic fibers to the dorsal horn and preganglionic sympathetic and parasympathetic cell columns. We used retrograde tracing techniques with pseudorabies virus combined with immunohistochemistry against oxytocin and radioligand binding detection of oxytocinergic receptors to evidence the oxytocinergic innervation of thoracolumbar and lumbosacral spinal neurons controlling penile erection. Spinal neurons labelled with pseudo-rabies virus transsynaptically transported from the corpus cavernosum were present in the intermediolateral cell column and the dorsal gray commissure of the thoracolumbar and lumbosacral spinal cord. Confocal laser scanning microscopic observation of the same preparations revealed close appositions between oxytocinergic varicosities and pseudorabies virus-infected neurons, suggesting strongly the presence of synaptic contacts. Electron microscopy confirmed this hypothesis. Oxytocin binding sites were present in the superficial layers of the dorsal horn, the dorsal gray commissure and the intermediolateral cell column in both the thoracolumbar and lumbosacral segments. In rats, stimulation of the paraventricular nucleus induces penile erection, but the link between the nucleus and penile innervation remains unknown. Our findings support the hypothesis that oxytocin, released by descending paraventriculo-spinal pathways, activates proerectile spinal neurons.

Anatomical relationships between the ventral mesencephalic tegmentum — A 10 region and the locus coeruleus as demonstrated by anterograde and retrograde tracing techniques

We have searched for anatomical connections between the ventral mesencephalic tegmentum (VMT), including the dopaminergic cell group A10 and the locus coeruleus (LC) region. Tritiated leucine (120–220 nl) and horseradish peroxidase delivered by electrophoresis were injected in the VMT-A10 region. We have demonstrated, on the one hand bilateral projections from the VMT-A10 region to LC, and on the other hand a possible contralateral projection from LC to VMT-A10 region. These relationships, reported for the first time may have some important functional significance.

Oxytocinergic and serotonergic innervation of identified lumbosacral nuclei controlling penile erection in the male rat

Penile erection is due to activation of proerectile neurons located in the sacral parasympathetic nucleus of the L6-S1 spinal cord in the rat. Contraction of the ischiocavernosus and bulbospongiosus striated muscles, controlled by motoneurons located in the ventral horn of the L5-L6 spinal cord, reinforces penile erection. Physiological and pharmacological arguments have been provided for a role of oxytocin and serotonin in the spinal regulation of penile erection. Immunohistochemistry of oxytocinergic and serotonergic fibres was performed at the lumbosacral level of the male rat spinal cord, and combined with retrograde tracing from the pelvic nerve or from the ischiocavernosus and bulbospongiosus muscles using wheat germ agglutinin-horseradish peroxidase. Sacral preganglionic neurons retrogradely labelled from the pelvic nerve formed a homogeneous population, predominant at the L6 level. Motoneurons retrogradely labelled from the ischiocavernosus and bulbospongiosus muscles were observed in the medial part of the dorsolateral and in the dorsomedial nuclei. Fibres immunoreactive for oxytocin were mainly distributed in the superficial layers of the dorsal horn, the dorsal gray commissure and the sacral parasympathetic nucleus. Some of these fibres were apposed to retrogradely-labelled sacral preganglionic neurons and at the ultrastructural level, some synapses were evidenced. Fibres immunoreactive for serotonin were largely and densely distributed in the dorsal horn, the dorsal gray commissure, the sacral parasympathetic nucleus and the ventral horn. Some serotonergic fibres occurred in close apposition with retrogradely-labelled sacral preganglionic neurons and motoneurons, and synapses were demonstrated at the ultrastructural level. This study provides morphological support for a role of oxytocin and serotonin on sacral preganglionic neurons innervating pelvic organs and motoneurons innervating the ischiocavernosus and bulbospongiosus muscles.

Distribution and characterization of neuropeptide Y-like immunoreactivity in the brain and pituitary of the goldfish

SummaryThe distribution of neuropeptide Y (NPY) immunoreactivity has been studied by means of immunocytochemistry and radioimmunoassay in the brain of the goldfish. It was found that NPY had a widespread distribution in the entire brain in particular in the telencephalon, diencephalon, optic tectum and rhombencephalon. In the pituitary gland, positive type-B fibers were observed in the various lobes frequently in direct contact with secretory cells, in particular the gonadotrophs, somatotrophs and MSH (melanocyte-stimulating hormone) secreting cells. When measured by radioimmunoassay, the highest NPY concentrations were found in the pituitary and telencephalon, confirming the results of immunocytochemistry. The displacement curves obtained with serial dilutions of brain extracts were parallel to that of synthetic porcine NPY. Following high performance liquid chromatography, the NPY-like material extracted from goldfish brain co-eluted as a single peak with synthetic porcine NPY. These data demonstrate the presence of an NPY-like substance widely distributed in the goldfish brain. The observation of NPY-immunoreactive fibers in the pituitary gland suggests that, among its other functions, NPY may play a role in the neuroendocrine regulation of pituitary function.

Gamma-aminobutyric acid-immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies

The distribution of GABA-immunoreactive neurons and axonal varicosities was investigated in the hippocampal region of the rat brain by means of an indirect peroxidase immunocytochemical method with recently developed anti-GABA antibodies. The immunolabeling was found to be restricted to nervous structures: neuronal cell bodies, dendrites and axon terminals. Myelinated axons showing GABA-immunoreactivity were also observed. GABA-immunoreactive neurons were found in great number in the stratum pyramidale, the superficial part of the stratum oriens and the deep part of the stratum radiatum in the Ammons horn. Less were found in the other regions; rare labeled cells were observed in the superficial part of the stratum radiatum and the middle part of the stratum oriens. The dentate gyrus exhibited numerous labeled cells in the granular layer, few in the hilus, rare in the molecular layer. A high density of GABA-immunoreactive terminals was found at the limit of the stratum oriens with the alveus, in the stratum pyramidale and in the stratum lacunosum. A lower density of labeled fibers was observed in the other areas. The somata and proximal dendrites of pyramidal and granular cells were encompassed by characteristic pericellular arrangements of GABA-immunoreactive varicosities. Ultrastructural observations revealed a diffuse immunoreaction product spread over the cytoplasm and the nucleus without specific relationship with the organelles, and immunoreactive aggregates in the cytoplasm. Labeled dendrites often showed enlargements displaying the immunoreaction whereas thinner segments were devoid of it. They received numerous asymmetrical synapses from unlabeled axon terminals. GABA-immunoreactive terminals were filled with small clear vesicles with immunopositive membranes and were observed in symmetrical contact with somata and dendrites.

Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain

Chronic pain states are characterized by long‐term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up‐regulation of Cav1.2‐comprising L‐type calcium channel (Cav1.2‐LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR‐103, simultaneously regulates the expression of the three subunits forming Cav1.2‐LTC in a novel integrative regulation. This regulation is bidirectional since knocking‐down or over‐expressing miR‐103, respectively, up‐ or down‐regulate the level of Cav1.2‐LTC translation. Functionally, we show that miR‐103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR‐103 is down‐regulated in neuropathic animals and that miR‐103 intrathecal applications successfully relieve pain, identifying miR‐103 as a novel possible therapeutic target in neuropathic chronic pain.

γ-Aminobutyric acid and 5-hydroxytryptamine interrelationship in the rat nucleus raphe dorsalis: Combination of radioautographic and immunocytochemical techniques at light and electron microscopy levels

Serotonin and gamma-aminobutyric acid (GABA) neurons in the nucleus raphe dorsalis were identified by immunocytochemistry using antibodies to 5-hydroxytryptamine or GABA. The pattern of the 5-hydroxytryptamine and GABA immunostaining presented similar features: 5-hydroxytryptamine or GABA immunoreactive somata were fusiform or ovoid (15-20 micron) and positive dendritic profiles were found either without any connection with other nerve elements or in contact with one or several terminals. In addition, some 5-hydroxytryptamine nerve endings were apposed to 5-hydroxytryptamine immunoreactive cell bodies or dendrites; also some GABA-immunopositive terminals were in contact with GABA-immunopositive nerve cell bodies. On the other hand, GABA and 5-hydroxytryptamine patterns may be differentiated in several respects: the 5-hydroxytryptamine-reactive nerve cell bodies were more numerous than the GABA ones. Some small, round (8-10 micron) nerve cell bodies were reactive with GABA antiserum, but no neurons of this type were reactive with a 5-hydroxytryptamine antiserum; finally, GABA nerve terminals were more numerous than 5-hydroxytryptamine ones. In order to understand the relationship between GABA and 5-hydroxytryptamine neurons, radioautographic and immunocytochemical procedures were combined: 5-hydroxytryptamine and GABA immunocytochemistry was combined with radioautography of [3H]GABA and [3H]5-hydroxytryptamine uptake, respectively. Some nerve cell bodies, dendrites or terminals, which were 5-hydroxytryptamine-immunopositive, were also capable of accumulating [3H]GABA and, conversely, some GABA-immunopositive elements were capable of accumulating [3H]5-hydroxytryptamine. Moreover, several nerve elements were reactive with both glutamate decarboxylase and 5-hydroxytryptamine antisera. These data confirm in electron microscopy previous studies suggesting the coexistence of both GABA and 5-hydroxytryptamine in the same neurons. The presence of uptake mechanisms for GABA and 5-hydroxytryptamine may indicate the action of both neurotransmitters in the same neuron. On the other hand, the [3H]GABA-labelled nerve endings in contact with 5-hydroxytryptamine-positive dendrites or nerve cell bodies indicate the possibility of a GABAergic control of the activity of some 5-hydroxytryptamine neurons; this corroborates biochemical and electrophysiological studies whereby a trans-synaptic control of the 5-hydroxytryptamine neurons by GABA may be envisaged.