Historadioautographic localization of oxytocin and V1a vasopressin binding sites in the kidney of developing and adult rabbit, mouse and merione and of adult human.

The localization of oxytocin (OT) binding sites and vasopressin (VP) binding sites of the V1a subtype was investigated by radioautography in kidneys of rabbits, mice and meriones during postnatal development and in the adult, and in the human kidney. Kidney sections were incubated in the presence of selective radioiodinated OT and V1a antagonists, respectively. The localizations were compared with those previously described in the rat. The main finding of the study was the almost constant presence in the cortex of V1a binding sites in the connecting tubule, the cortical collecting duct and in the juxtaglomerular apparatus (on the intra- and extraglomerular mesangium and the afferent arteriole). This distribution suggests an interaction of VP via V1a receptors and the kallikrein-kinin system in the kidney. OT binding sites, in comparison with V1a binding sites, were fewer and less constantly detectable in the kidney of the different species. In the mouse, their presence on the limbs of Henle's loop in the medulla points to the possibility of their involvement in the medullary concentrating process. In the kidneys of the various species, OT and V1a binding sites occurred always in differential structures. In contrast, in the human kidney cortex, a colocalization of OT and V1a binding sites was almost constantly observed. This raises the question as to the specificity of the neurohypophysial hormone receptors in the human kidney.

Tenascin and laminin function in target recognition and central synaptic differentiation.

The influence of the target cell-issued extracellular molecules tenascin-C and laminin on synaptogenesis was studied in mixed primary cultures of pituitary melanotrophs and hypothalamic neurons. We could demonstrate in this neuron-target co-culture system a new role for tenascin-C, which appeared to be expressed as an early and transitory signal of target recognition for selective afferent fibers. Tenascin-C expression disappeared from the melanotrophs soon after the establishment of neural contacts. Concomitantly, the melanotrophs became immunoreactive for laminins, and more specifically for the synaptic isoform beta2 chain-containing laminin. The laminin signal appeared to be involved in the induction of synaptic differentiation, selectively with fibers containing both dopamine and GABA, like those innervating the melanotrophs in situ.

The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein.

MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The steroidogenic acute regulatory protein is located in the inner membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain. The present work was undertaken to determine which step of the intracellular cholesterol pathway MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the amino-terminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signals, we show that a dileucine motif (Leu(66)-Leu(67)) and a tyrosine residue (Tyr(89)) are critical for the targeting or the proper folding of the molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s).

Adventitia-derived nitric oxide in rat aortas exposed to endotoxin: cell origin and functional consequences.

The role of adventitial cells in bacterial lipopolysaccharide (LPS)-induced vascular nitric oxide (NO) overproduction has been largely ignored. In rat aortas exposed to LPS in vitro or in vivo, it was found that adventitia contained the major part of NO synthase (NOS)-2 protein (Western blot and immunohistochemistry) and generated the largest amount of NO (electron paramagnetic resonance spin trapping). NOS-2 immunoreactive cells were mainly resident macrophages at an early stage (5 h, in vitro or in vivo) and fibroblasts at a later stage (20 h, in vitro). Adventitial NOS-2 activity largely accounted for 1) the relaxing effect of L-arginine in rings exposed to LPS in vivo, 2) generation of an "NO store" revealed by N-acetylcysteine-induced relaxation, and 3) formation of protein-bound dinitrosyl iron complexes in the medial layer of aortic rings exposed to LPS in vitro. In conclusion, the adventitia is a powerful source of NO triggered by LPS in the rat aorta. This novel source of NO has an important impact on smooth muscle function and might be implicated in various inflammatory diseases.

[Pulmonary toxicity of oxygen]. / A propos de la toxicité pulmonaire de l'oxygène.

Recent progress in artificial ventilation procedures sheds doubt on the toxicity of oxygen in the lung. Histological studies of the lung by light and electron microscopy were made in rats exposed to 100% oxygen at 1 ATA in spontaneous ventilation. The results are compared with the lesions observed in 19 human patients suffering from acute respiratory distress syndrome and with the data on retinopathy of prematurity. This study suggests to keep in mind that oxygen may, as well as infections or baro-traumatic factors, be involved in causing or aggravating lung fibrosis. The complexity of the mechanisms of this action is emphasized. The production of highly reactive free radicals is one of the essential factors of this aggression. These elements illustrate the value of further studies on ways of preventing these changes, especially using appropriate antioxidant molecules.

Synaptically released 5-HT modulates the activity of tonically discharging neuronal populations in the rostral ventral medulla (RVM).

There is substantial evidence for an important modulating role of monoamines (catecholamines and serotonin, 5-HT) in the rostral ventral medulla (RVM), a region which plays an important role in cardiovascular and nociceptive functions. We investigated in slices the role of endogenous monoamines in the synaptic control of the activity of rat RVM neuronal populations using intracellular recordings in the lateral RVM plus lateral aspect of nucleus paragigantocellularis lateralis. A triple-labelling protocol allowed us to identify the location of impaled neurons and their eventual monoaminergic phenotype within the serotonergic and catecholaminergic populations of the RVM. Focal electrical stimulation revealed the existence of a functional monoaminergic input onto RVM neurons which was mediated by endogenous 5-HT acting at inhibitory 5-HT1A receptors but did not involve noradrenergic neurotransmission. The slow 5-HT-mediated inhibitory postsynaptic potential (IPSP) was only observed in the regularly discharging neurons, which were found to be neither catecholaminergic nor serotonergic. The synaptic release of 5-HT was, itself, under an inhibitory control involving GABAA (gamma-aminobutyric acid) receptors. Moreover, we characterized the effect of the 5-HT-releasing agent fenfluramine on this functional 5-HT-mediated synaptic transmission. Our results show that the effect of fenfluramine is biphasic consisting of an initial prolongation of the serotonergic IPSP followed by a decrease in amplitude. Our data provide a basis for the previously reported inhibitory effects of exogenously applied serotonin agonists/antagonists on the autonomic functions controlled by the RVM. This 5-HT pathway, which functionally links the serotonergic and catecholaminergic regions, might play an important role in cardiovascular and nociceptive functions.

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.

Historadioautographic localization, pharmacology and ontogeny of V(1a) vasopressin binding sites in the rat kidney.

The localization and pharmacological characteristics of vasopressin (VP) binding sites of the V(1a) subtype in developing and adult rat kidney were investigated by radioautography on kidney sections incubated in the presence of a radioiodinated selective V(1a) antagonist. Their localization after in vivo systemic infusion of the radioligand was also investigated. V(1a) binding sites first appear at embryonic day 16 on vascular elements. In the adult, they were localized in the cortex (vascular and tubular structures, juxtaglomerular apparatus), the outer medulla outer stripe (vasa recta) and inner stripe (thin descending limbs of short looped nephrons) and the inner medulla (collecting ducts). Data obtained in vitro were confirmed by in vivo binding at postnatal day 30 (PN30). Whatever their localizations, the V(1a) binding sites exhibited full V(1a) pharmacological profile in postnatal stages rats and in adult rats: a high affinity (nM range) for VP and for the V(1a) agonist, a lower affinity (microM range) for oxytocin and no affinity for the oxytocin agonist. The presence of V(1a) binding sites in these different structures raises the question of the putative roles of VP in modulating renal functions. A striking finding is the presence of V(1a) binding sites in the outer medullary thin descending limbs of short looped nephrons suggesting their colocalization with urea transporters.

The pituitary intermediate lobe of the hydrated and dehydrated gerbil. An immunohistochemical and electron-microscopic study.

The pituitary intermediate lobe was studied by immunocytochemistry on semithin sections and by electron microscopy in gerbils (Gerbillus pyramidum) caught in summer and winter in the natural biotope or experimentally submitted to chronic hydration or dehydration. In the gerbil, the intermediate lobe was formed by a predominating population of tightly packed melanotrophic cells the immunocytochemical and morphological features of which were comparable to those described in other mammals. A few typical corticotrophic cells were scattered in the contact zone with the neural lobe. Folliculostellate cells labelled with antibodies against glial fibrillary acid protein and vimentin were interspersed between the glandular cells; they formed small follicles in the vicinity of which the apical cytoplasm contained conspicuous dense granules. Both glandular cell types were innervated by axons most probably colocalizing dopamine and gamma-aminobutyric acid. In the pituitaries from the gerbils caught in winter, as from those having free access to hydrated food, the melanotrophic cells exhibited morphological characteristics of high functional activity. In the gerbils caught in summer or receiving exclusively dry food, the secretory activity of the cells was obviously depressed. The corticotrophic cells were unaffected. These observations raise the question of the role of the intermediate lobe in the adaptation to desert life.

Historadioautographic localisation of oxytocin and vasopressin binding sites in the central nervous system of the merione (Meriones shawi).

The distribution of vasopressin and oxytocin binding sites in the central nervous system of the merione (Meriones shawi), a rodent adapted to desert life, was studied by means of conventional film radioautography at macroscopic scale and historadioautography at cellular level using radioiodinated ligands highly selective for either oxytocin or type V1 a vasopressin receptors. Both types of binding sites exhibited the same selectivity for endogenous peptides as in the rat. Distribution of oxytocin binding sites was similar in some structures (limbic system, spinal cord) to that described in the rat and in other rodents. Vasopressin binding sites were much more widely distributed in the merione than in the rat brain. In addition to locations common to most rodents (lateral septum and suprachiasmatic nucleus), in merione vasopressin binding sites occurred in several areas known to express oxytocin binding sites in the rat (olfactory system, hypothalamus). Colocalisation of vasopressin and oxytocin binding sites, which occurred in the CA1 and CA2 fields of Ammon's horns of the hippocampus, the caudate-putamen and the fundus striati of the merione, has so far not been reported in any other rodent.

L1 and laminin: their expression during rat hypophysis ontogenesis and in adult neurohemal areas.

L1 is a murine multidomain glycoprotein implicated in cell aggregation, fasciculation. neurite outgrowth and synaptogenesis. Laminin, a trimeric polypeptide, is implicated in neuronal survival, growth cone guidance, neurite outgrowth and cell differentiation. Laminin can also interact with the cell adhesion molecule L1. Their expressions were investigated from embryonic day 15 (E15) to adult in the rat hypophysis, and in adult neurohemal zones. Detected in the neural lobe from E17, the L1 immunoreactivity increased during prenatal development and persisted in adulthood mainly related to the neuropeptidergic fibers. Pituicytes were only labelled on the plasmalemma apposed to axons. In the intermediate lobe, L1 appeared at birth on folliculo-stellate cells extensions, constituting a network which densified during postnatal development. L1 is also expressed in all neurohemal areas on neuronal profiles. Laminin was clearly detectable in the hypophysis at E15 before the first blood vessels penetrate the Rathke pouch. At E20, all the basal membranes of the blood vessels were stained. In the intermediate lobe, a spotted laminin immunoreactivity was detected at E21. At this stage, we observed the staining of intercellular spaces and the intracellular labelling of melanotrophs, concerning reticulum or vesicles. The staining of melanotrophs seemed to maintain during adulthood. In contrast with blood vessels of the adult cerebral tissue, adult capillaries of the neural lobe and the others neuro-hemal zones were intensely labelled with the anti-laminin antibody. These results suggest that neurite outgrowth and neurite guidance could be promoted by L1 and laminin in the neurointermediate lobe. The "intercellular tunnels" could also be an important guidance cue for migrating cells in the intermediate lobe. These data also demonstrate that melanotrophic cells. secreting the laminin, have a role in the ontogenesis of the gland. Finally, we suggest that L1 and laminin can collaborate to reinforce "neurons-capillaries" interactions in neurohemal zones.

Dystrophins in neurohypophysial lobe of normal and dehydrated rats: immunolocalization and biochemical characterization.

Dystrophin, utrophin and dystroglycan are present not only in muscle but also in brain. In muscle, they link the extracellular matrix to the cytoskeleton. Their function in brain is not understood. Here we show their presence in the hypothalamo-neurohypophysial system which secretes the neurohormones oxytocin and vasopressin. Using immunocytochemistry, we showed that dystrophins are present in the neurohypophysis of control rats. After water deprivation, immunoreactivity dramatically decreased and appeared in axonal swellings in the hypothalamic tract. Dystrophin immunostaining can be ascribed to dystrophin and/or utrophin as well as the DMD (Duchenne Muscular Dystrophy) gene short products Dp140 and Dp71 as revealed by Western immunoblots of synaptosomes isolated from neurohypophyses of control rats. In synaptosomes isolated from rats under water deprivation, the immunoreactivity entirely disappeared. Further biochemichal characterization of isolated neurosecretory granules (NSG) showed that Dp140 and Dp71 are enriched in the NSG stored in the swellings of the neurohypophysis whereas the NSG of the nerve endings are devoid of these proteins. In addition we observed that the presence of beta-dystroglycan and actin correlates with the presence of dystrophins. Our data favor a direct implication of the dystrophins and/or utrophin, dystroglycan and actin in the neurosecretory processes of the hypothalamo-neurohypophysial system.

Brain inflammatory response induced by intracerebroventricular infusion of lipopolysaccharide: an immunohistochemical study.

Inflammatory processes may play a critical role in the pathogenesis of the degenerative changes associated with Alzheimer's disease (AD). In the present study, we used an animal model of brain inflammation in order to study a possible mechanism involved in AD. Lipopolysaccharide (LPS) was used to produce global microglial reactivity within the brain of young rats. Time-dependent changes in the inflammatory reaction and the participation of glial cells after acute injection of LPS (50 or 100 microg) into the lateral ventricle or the fourth ventricle were compared with the chronic infusion of LPS (0.15, 0.5, 1.5 or 5.0 microg/h) into the fourth ventricle (14 days). Several immunohistochemical markers were used to characterize the microglial response. Acute and chronic exposure to LPS induced major histocompatibility complex class II (MHC II) antigen expression, detected with OX-6 antibody, in a sub-population of microglial cells in defined brain areas. The morphological features and distribution of OX-6 positive cells observed in the proximity of the cannula track after LPS injection into the lateral ventricle suggested the recruitment of monocytes/macrophages from the periphery. The activation of the resident microglial cells was delayed and mainly concentrated within the temporal lobe regions and the limbic system. Chronic infusion to LPS into the fourth ventricle induced a comparable activation of microglial cells. Quantitative analysis of OX-6 positive cells showed a dose-dependent response to LPS exposure.

Electrophysiological properties of cultured neonatal rat dorsal horn neurons containing GABA and met-enkephalin-like immunoreactivity.

We have developed a culture of neurons dissociated from the most superficial laminae of the neonatal rat spinal cord dorsal horn (DH). By using the perforated patch-clamp technique, we distinguished four types of neurons based on their firing properties in response to intracellular injection of 900 ms lasting current pulses. Type 1 neurons were characterized by a tonic firing. Type 2 neurons displayed marked spike accommodation and fired brief (<500 ms) bursts of action potentials, whereas type 3 neurons fired a single spike. Type 4 neurons exhibited different types of firing patterns, but all of them possessed a time-dependent inwardly rectifying current activated by membrane hyperpolarization. Met-enkephalin-like immunoreactivity (met-ENK-LI) and glutamic acid decarboxylase-like immunoreactivity (GAD-LI) were colocalized in 42% of the neurons (n = 59), which were previously identified electrophysiologically. Type 1-4 neurons represented respectively 4, 64, 20, and 12% of the population of neurons colocalizing met-ENK-LI and GAD-LI. We conclude that the electrophysiological properties of DH neurons present in our cultures are similar to those described in acute slice or hemisected spinal cord preparations and that met-ENK-LI and GABA-LI are preferentially colocalized in type 2 neurons.

Oxytocin modulates glutamatergic synaptic transmission between cultured neonatal spinal cord dorsal horn neurons.

The functional characteristics of binding sites for the neuropeptide oxytocin (OT) detected by radioautography in laminae I and II of the dorsal horn (DH) and on cultured neonatal DH neurons were studied on the latter using perforated patch-clamp recordings. The neurons were identified by their spike discharge properties and on the basis of the presence of met-enkephalin-like and glutamate decarboxylase-like immunoreactivities. OT (100 nM) never induced any membrane current at a holding potential of -60 mV but increased the frequency of spontaneously occurring AMPA receptor-mediated EPSCs or the mean amplitude of electrically evoked EPSCs in a subset (35%) of neurons. The frequency of miniature EPSCs (m-EPSCs) recorded in the presence of 0.5 microM tetrodotoxin was also increased by OT (100 nM) without any change in their mean amplitude, indicating an action at a site close to the presynaptic terminal. The decay kinetics of any type of EPSC were never modified by OT. The effect of OT was reproduced by [Thr4, Gly7]-OT (100 nM), a selective OT receptor agonist, and blocked by d(CH2)5-[Tyr(Me)2,Thr4,Tyr-NH29]-ornithine vasotocin (100 nM), a specific OT receptor antagonist. Reducing the extracellular Ca2+ concentration from 2.5 to 0.3 mM in the presence of Cd2+ (100 microM) reversibly blocked the effect of OT on m-EPSCs. The OT receptors described here may represent the substrate for modulatory actions of descending hypothalamo-spinal OT-containing pathways on the nociceptive system.

GABA- and peptide-immunoreactivities co-localize in the rat central extended amygdala.

The central amygdaloid nucleus and the lateral bed nucleus of the stria terminalis are two similar telencephalic structures belonging to the central extended amygdala. These regions contain numerous peptidergic and GABAergic neurones which maintain the neurones projecting to the brain stem under tight intrinsic control. Using immunocytochemistry in colchicine-treated rats, we showed that, in the lateral subdivision of the central amygdaloid nucleus and in the dorsal part of the lateral bed nucleus of the stria terminalis, a population of GABAergic neurones is able to co-synthesize either corticotropin-releasing factor or methionine-enkephalin, but never both peptides. These results suggest that, in the GABAergic intrinsic circuits of the central extended amygdala, co-liberated peptides can have a modulatory role on GABAergic actions.

[Implication of type II glucocorticoid receptors in aldosterone induced apoptosis of rat thymocytes]. / Implication des récepteurs glucocorticoïdes de type II dans l'action apoptotique de l'aldostérone sur les thymocytes de rat.

The thymolytic action of dexamethasone (DEXA) and aldosterone (ALDO) has been studied in vitro and in vivo. In vitro, in the presence of DEXA, the number of apoptotic cells increased with time. After 6 hours of incubation, 55 and 86% of thymocytes are dead with 10(-7) and 10(-5) M of DEXA, respectively. Whereas, in the presence of equivalent concentrations of ALDO, the rate of mortality of cells was only 30-40%. In vivo study confirmed these results and showed that apoptotic action of ALDO remained less potent than that of DEXA. On the other hand, addition of the potent glucocorticoid antagonist, RU 38486 prevents not only the dexamethasone but also the aldosterone-stimulated cell death. We conclude that the thymolytic action of the endogenous mineralocorticoid hormone is not mediated by its specific receptor but paradoxically by type II glucocorticoid receptor.

Two oxytocin-binding site subtypes in rat kidney: pharmacological characterization, ontogeny and localization by in vitro and in vivo autoradiography.

The localization of oxytocin (OT)-binding sites in the developing rat kidney and their pharmacological characterization were investigated by means of autoradiographic techniques. The cellular localization was studied by application of the histoautoradiographic technique to (1) frozen sections and semithin sections from kidney slices incubated in vitro in the presence of a 125I-labelled OT antagonist and (2) frozen and semithin sections from kidneys after in vivo systemic infusion of the radioligand. Pharmacological characteristics were determined in competition experiments by using quantitative film autoradiography. Specific OT-binding sites were first detected at embryonic day 17 (E17) in the cortex. At early stages up to postnatal days (PN30), the cortical OT-binding sites were highly concentrated on the juxta- and paraglomerular portion of the distal tubule; in the adult they were restricted to the macula densa. In the medulla, OT-binding sites were first detected at E19 when this region is forming; they were localized on the thin limb of Henle's loop. These data obtained by in vivo binding were confirmed by in vivo binding at PN30 which showed, in addition, the presence in one rat of OT-binding sites in the inner stripe of the outer medulla. At all stages examined (PN15 to PN90), cortical OT-binding sites had a higher selectivity for OT versus vasopressin (IC50 = 0.78 +/- 0.04 nM and 8 +/- 0.5 nM respectively at PN90) than medullary sites (IC50 = 1.9 +/- 0.27 nM and 2 +/- 1.13 nM respectively at PN90). These data suggest that the OT-binding sites of the macula densa and thin Henle's loop, detected in the rat kidney, represent two subtypes of OT receptors which could mediate distinct effects of OT on kidney function.

[A physico-chemical analysis of glucocorticoid receptors in corticosensitive and corticoresistant thymocytes: decrease in nuclear transfer]. / Analyse physico-chimique des récepteurs glucocorticoïdes des thymocytes cortico-résistants et cortico-sensibles: diminution du transfert nucléaire.

We have demonstrated in prior work that the density of cytoplasm receptors was comparable in DEXA-resistant and DEXA-sensible thymocyte populations (1). The present study was aimed at examining whether the resistance of thymocytes to the lytic action of glucocorticoids was linked or not to variation in the physico-chemical properties of the receptor itself. Our results show that the cytoplasmic receptors of DEXA-resistant thymocytes were, in terms of specificity, stability and elution profile on DEAE Sephacel columns, similar to those of DEXA-sensible cells. However, under condition of identical concentrations of DNA and receptors, we revealed a decrease in the nuclear transfer of glucocorticoid receptor in the DEXA-resistant thymocytes. This deficiency may account in part for the glucocorticoid resistance of these cells which may be related to intrathymic cell differentiation phenomenon.

Target-dependent synaptogenesis: inductive effect of pituitary melanotrophs on their central innervation.

The glandular activity of the vertebrate pituitary intermediate lobe (IL) is regulated by direct cellular innervation, in contrast with the purely humoral regulation of adjacent pituitary anterior lobe (AL). Thus in the rat IL, melanotrophs receive a dopaminergic and GABAergic innervation from the basal hypothalamus, which tonically inhibit their glandular activity. We studied this model of neuron-target interactions in cocultures in defined medium of fetal hypothalamic neurons with neonate pituitary glandular cells. In the cocultures with IL cells, neuroglandular contacts occurred after 4 days in vitro (DIV) but required another 8 DIV to exhibit ultrastructural and immunocytochemical features of fully differentiated functional synapses; by contrast, neuroneuronal synapses developed much faster and could already be detected after 4 DIV. In the cocultures with AL cells, neuroglandular contacts never mature in differentiated synapses. Confocal microscope observation revealed that dopaminergic neurons, which represented less than 1% of total neurons in the cocultures, established 50% of the synapses detected on the melanotrophs. These cells are thus able, contrary to the AL cells, to promote the establishment of functional synapses and, to some extent, to select their specific innervation.