Jean-Didier Vincent

Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study

An in vitro binding assay, using 125I-YLFQPQRFamide, a newly synthetized iodinated analog of FLFQPQRFamide, in which Phe1 (F) has been substituted by a Tyr (Y), was developed to demonstrate and characterize putative binding sites of this brain morphine modulating peptide. This radioligand bound in a time-dependent manner to rat spinal cord membrane preparation. This binding was dose-dependent, saturable and reversible. Both kinetic data and saturation measured at equilibrium lead to the existence of a homogenous population of high affinity binding sites with a Kd value of 0.09-0.1 nM and a maximal capacity Bmax of 14.5 +/- 2 fmol/mg protein. Results of competition experiments show that both FLFQPQRFamide and its analog YLFQPQRFamide had a similar capacity to inhibit the 125I-YLFQPQRFamide binding, suggesting that this radioiodinated analog is a good tool to study binding characteristics of FLFQPQRFamide receptors. The related octadecapeptide AGEGLSSPFWSLAAPQRFamide, another mammalian morphine modulating peptide competes for radioligand binding with similar potency. Our results also show that mu, delta and kappa opiate receptor agonists as well as the antagonist naloxone were not able to affect binding either in presence or in absence of 120 mM NaCl. Together, these data demonstrate that FLFQPQRFamide does not function as an endogenous opiate receptor antagonist and that is capacity to reduce opiate-induced analgesia is supported by specific binding sites.

Possible morphological bases for synchronisation of neuronal firing in the rat supraoptic nucleus during lactation

Abstract Oxytocin-containing neurones in the supraoptic and paraventricular nuclei of lactating rats display a periodic activation which results in a pulsatile release of hormone before each reflex milk ejection induced by suckling. This electrical activity occurs in an all-or-none fashion and is synchronised in the whole population of oxytocin neurones in both nuclei. The present report describes changes in the ultrastructure of the supraoptic nucleus of lactating animals which may serve as morphological bases for such a functional synchronisation. In the supraoptic nuclei of normal rats, neurosecretory neurones are usually separated by elements of the neuropil, particularly glial processes. At rare intervals, adjacent neurosecretory somata, and dendrites, are seen to be in direct apposition. The only specialisations apparent between the contiguous membranes are occasional attachment plates. In nuclei of lactating rats, quantitative analysis indicated that 34% of profiles of the sectioned neurosecretory cell bodies were in direct contact with each other and 22% with profiles of dendrites, a 5-fold increase over the corresponding frequencies observed in normal male and virgin female animals. Such contacts involved 10% of the total measured soma surface membrane (compared to 1.5% in the controls). The number of attachment plates supporting the apposing membranes also increased significantly as did the mean size of the individual appositions. There was also a higher incidence of presynaptic terminals contacting more than one post-synaptic element (soma or dendrite) in the same plane of section, a rare phenomenon in the normal nucleus. No further increases were evident in these appositional relations in virgin female and lactating rats deprived of water for one day, a stimulus which enhances vasopressin release. It is postulated that the structural reorganisation observed in the nuclei of lactating animals may lead to electrical interactions between the neurosecretory cells and may thus be one of the factors supporting the synchronisation of neuronal activity during the episodic release of oxytocin.

Dopamine D1 receptor modulates the voltage-gated sodium current in rat striatal neurones through a protein kinase A

1. Whole‐cell recordings were made from striatal neurones obtained from neonatal rats and maintained in primary cultures. The effects of dopamine D1 receptor activation were studied on the voltage‐gated sodium current. 2. Bath application of a specific D1 agonist, SKF38393 (1 microM), reduced the neuronal excitability recorded in current‐clamp by increasing the threshold for generation of action potentials. 3. In voltage‐clamp recordings, SKF38393 (1 microM) reversibly reduced the peak amplitude of the sodium current by 37.8 +/‐ 4.95%. This effect was reversed by the D1 antagonist SCH23390 and was blocked by the intracellular loading of GDP‐beta‐S (2 mM) suggesting GTP‐binding protein involvement. 4. The D1 agonist reduced the peak amplitude of the sodium current without significantly affecting (i) the voltage dependence of the current‐voltage relationship, (ii) the voltage dependence of the steady‐state activation and inactivation, (iii) the kinetics of the time‐dependent inactivation, and (iv) the kinetics of recovery from inactivation. 5. The peak amplitude of the sodium current was progressively reduced by intracellular loading of cyclic AMP‐dependent protein kinase (100 U ml‐1). 6. Diffusion of a specific peptide inhibitor of the cyclic AMP‐dependent protein kinase (PKI; 10 microM) into the cytosol of neurones blocked the effect of the D1 agonist on the sodium current amplitude. 7. These results demonstrate that dopamine acting at the D1 receptor reduces the amplitude of the sodium current without modifying its voltage‐ and time‐dependent properties. This effect involves activation of the cyclic AMP‐dependent protein kinase and results in a depression of the striatal neuronal excitability by increasing the threshold for generation of action potentials.

Activity of osmosensitive single cells in the hypothalamus of the behaving monkey during drinking

Summary The activity of cells in the diencephalon was analyzed during drinking; res to intracarotid hypertonic saline injections and to spontaneous or stimulus-associated arousal was also studied. All the osmosensitive cells studied exhibited a significant variation of their firing rate during drinking differing according to the type of cell. The neurosecretory cells in the nucleus supraopticus (NSO), identified by an antidromic stimulation from the posterior pituitary, were inhibited during drinking. The ‘specific’ osmosensitive cells located in the vicinity of the NSO, were either activated or inhibited by saline injection and showed the opposite response to drinking. Most of the ‘non-specific’ osmosensitive cells diffusely distributed in the diencephalon exhibited a change of firing rate during drinking relataed to sleep-walking variations. A group of cells located in the dorsal part of the lateral hypothalamus reacted differently to drinking, being activated by arousal but inhibited during drinking. These varying changes of firing rate observed during drinking suggest the existence of adaptive mechanisms which act in anticipation of the effects of the absorption of water on neuroendocrine and behavioral controls of water balance.

Septal release of vasopressin in response to osmotic, hypovolemic and electrical stimulation in rats

The central release of vasopressin was studied in anesthetized rats using push-pull perfusions and radioimmunoassay of the hormone. A basal release was observed in the lateral septum and in the lateral ventricle, whereas no vasopressin was detected in the perfusates from the caudate nucleus. Under osmotic stimulation, vasopressin release increased up to 12 and 60 times basal levels following i.p. injections of 5 ml and 10 ml/kg b.wt. of 2 M NaCl, respectively. This increase was blocked by using a calcium-free perfusion medium containing 0.1 mM EGTA. In the lateral ventricle, osmotic stimulation (5 ml/kg of 2 M NaCl i.p.) had the same effect as in the septum. In the caudate nucleus, no release was observed. Hemorrhage also increased the septal release of vasopressin in 5 out of 6 animals tested. Electrical stimulation of the pituitary stalk and of the supraoptic nucleus was used to evoke the release of vasopressin into the bloodstream. Septal release slightly decreased during pituitary stalk stimulation, whereas it did increase during stimulation of the supraoptic region. Our results show that systemic stimuli for vasopressin release evoke both a peripheral and a septal release of the hormone. The dissociation of the effects of electrical stimulation of the pituitary stalk and of the supraoptic nucleus suggests, however, that the vasopressinergic neurones responsible for septal release are distinct from those which project to the neurohypophysis.

Septal connections with identified oxytocin and vasopressin neurones in the supraoptic nucleus of the rat. An electrophysiological investigation

Abstract Neurosecretory cells in the supraoptic nucleus of the lactating rat were identified by antidromic stimulation and differentiated as oxytocin or vasopressin secreting by their electrical activity during suckling. Oxytocin-containing neurones show a slow irregular or fast continuous pattern of electrical activity which is interrupted every 5–15 min by a synchronous high-frequency discharge of action potentials, precipitating the reflex milk ejections. Vasopressin-containing neurones never react to suckling. They usually exhibit a slow irregular electrical activity, but under stimulation for vasopressin release, evolve a phasic pattern of neuronal discharge. In both types of neurone, electrical stimulation of the neurohypophysial tract elicited an antidromic activation followed by inhibition. Possible relations of these neurosecretory cells with the septum were investigated by studying their electrophysiological responses to electrical stimulation of the septum. Single pulse septal stimulation caused a short term inhibition in both vasopressin- and oxytocin-containing cells. Repetitive stimulation of the septum at low frequency decreased the background firing rates of oxytocin-containing neurones without affecting the high-frequency discharges at the time of milk ejections. In phasically firing vasopressin-containing neurones, such stimulation decreased the intraburst firing rates; at higher stimulus intensity, the phasic pattern was altogether disrupted. In slow firing vasopressin-containing neurones, stimulation produced a reduction of their firing rates. The septum appears, therefore, to exert an inhibitory action on both oxytocin and vasopressin systems. The observed effects of septal stimulation on vasopressin-containing neurones suggest that the septum could participate in the control of phasic activity, thus modulating the rate of hormone release. The role of the septum in oxytocin release remains unclear since, in our experiments, septal stimulation did not affect the reflex milk ejection induced by suckling. However, the inhibition of the background activity of oxytocin-containing neurones suggests that the septum could be involved in other situations where oxytocin might be released.

The effects of hypotension and hypovolaemia on the liberation of vasopressin during haemorrhage in the unanaesthetized monkey (Macaca Mulatta)

SummaryUsing unanaesthetized monkeys, experiments were performed to examine the effects of haemorrhage on the liberation of arginine vasopressin (AVP).Haemorrhages of 10%, 15% or 20% total blood volume were performed via a catheter with its tip in the abdominal vena cava. A catheter in the left internal jugular vein was used for blood sampling. Arterial blood pressure was monitored via a catheter whose tip rested in an iliac artery. The monkeys showed no signs of discomfort from this catheterisation. Blood samples for AVP assay were taken at different times from 0–90 min after the end of the haemorrhage. At the end of the experiment, blood removed was reinfused.Results show that haemorrhage resulted in liberation of AVP, but only if there was a fall in arterial blood pressure. AVP release occured more readily as the total volume of blood withdrawn increased, but the absolute rise in hormone concentration was not related to the total volume of blood withdrawn. However, comparing the area under the curve of mean arterial blood pressure with that for AVP concentration showed the two to have a significant exponential relationship.It is concluded that, as in other species, haemorrhage is a potent stimulus for AVP liberation in the monkey. However, in contrast to some other species, the fall in arterial pressure seems to be the prime stimulus rather than hypovolemia per se.

Synaptic and neuronal-glial plasticity in the adult oxytocinergic system in response to physiological stimuli.

Magnocellular oxytocinergic neurons in the hypothalamus offer a striking example of a mammalian neuronal system whose basic architecture and synaptic circuitry can be reversibly modified in adulthood. During parturition, lactation and prolonged osmotic stimulation, glial coverage of oxytocinergic neurons markedly diminishes and their surfaces are left in extensive juxtaposition; concurrently, there is formation of new synapses, which are predominantly GABAergic and which couple two or more oxytocinergic neurons simultaneously. These structural changes do not permanently modify the anatomy of the system since upon cessation of stimulation, neuronal juxtapositions and shared synapses disappear, to reappear upon new stimulation. At present, we can only speculate about the cellular mechanisms and factors responsible for these reversible neuroanatomical changes. However, oxytocin itself appears to be of primary importance since it can induce similar anatomical changes when chronically infused into the third ventricle.

Ion Channels and the Signal-Transduction Pathways in the Regulation of Growth-Hormone Secretion

The secretion of GH from pituitary somatotrophs is mainly regulated by alterations in the levels of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) that depend on the influx of Ca(2+) through voltage-gated Ca(2+) channels in the cell membrane. Hypothalamic stimulatory and inhibitory factors bind to specific receptors on the cell membrane to regulate membrane potential and activate second-messenger systems. The receptors are G-protein coupled, and activated G proteins directly influence membrane ion channels to regulate Ca(2+) influx. The function of cAMP-dependent protein kinase A is also modulated by receptor-coupled G proteins leading to the phosphorylation of Ca(2+) channel proteins and further alteration of Ca(2+) influx.

Effects of estrogen on the electrical activity of identified and unidentified hypothalamic units.

Experiments performed on unanesthetized ovariectomized female rabbits demonstrated the effects of estradiol benzoate (EB; 20 microng i.v.) on the electrical activity of hypothalamic units which send their axons to the median eminence. Of a total of 1,840 cells recorded in hypothalamic and preoptic areas, 46 (2.5%) were antidromically activated by stimulating the median eminence. Under the present experimental conditions, EB induced a progressive diminution in the mean firing rate of these cells observed throughout the recording period (30-120 min). In addition to cells projecting to the median eminence, neurons which could not be antidromically invaded using our techniques were observed to be sensitive to estrogen. Estrogen administration produced a long-lasting inhibition of antidromically activated cells and a depression of much shorter duration (15-20 min) of unidentified nonstimulated units. These data suggest the existence of two types of hypothalamic neurons sensitive to estrogen.