High voltage-activated Ca2+ currents in rat supraoptic neurones: biophysical properties and expression of the various channel alpha1 subunits.

The diversity of Ca2+ currents was studied in voltage-clamped acutely dissociated neurones from the rat supraoptic nucleus (SON), and the expression of the various corresponding pore-forming alpha1 subunits determined by immunohistochemistry. We observed the presence of all high voltage-activated L-, N-, P/Q- and R-type currents. We did not observe low-voltage-activated T-type current. The multimodal current/voltage relationships of L- and R-type currents indicated further heterogeneity within these current types, each exhibiting two components that differed by a high (-20 mV) and a lower (-40 mV) threshold potential of activation. L- and R-type currents were fast activating and showed time-dependent inactivation, conversely to N- and P/Q-type currents, which activated more slowly and did not inactivate. The immunocytochemical staining indicated that the soma and proximal dendrites of SON neurones were immunoreactive for Cav1.2, Cav1.3 (forming L-type channels), Cav2.1 (P/Q-type), Cav2.2 (N-type) and Cav2.3 subunits (R-type). Each subunit exhibited further specificity in its distribution throughout the nucleus, and we particularly observed strong immunostaining of Cav1.3 and Cav2.3 subunits within the dendritic zone of the SON. These data show a high heterogeneity of Ca2+ channels in SON. neurones, both in their functional properties and cellular distribution. The lower threshold and rapidly activating L- and R-type currents should underlie major Ca2+ entry during action potentials, while the slower and higher threshold N- and P/Q-type currents should be preferentially recruited during burst activity. It will be of key interest to determine their respective role in the numerous Ca2+-dependent events that control the activity and physiology of SON neurones

The V1a and V1b, but not V2, vasopressin receptor genes are expressed in the supraoptic nucleus of the rat hypothalamus, and the transcripts are essentially colocalized in the vasopressinergic magnocellular neurons.

We have identified and visualized the vasopressin (VP) receptors expressed by hypothalamic magnocellular neurons in supraoptic and paraventricular nuclei. To do this, we used RT-PCR on total RNA extracts from supraoptic nuclei or on single freshly dissociated supraoptic neurons, and in situ hybridization on frontal sections of hypothalamus of Wistar rats. The RT-PCR on supraoptic RNA extracts revealed that mainly V1a, but also V1b, subtypes of VP receptors are expressed from birth to adulthood. No V2 receptor messenger RNA (mRNA) was detected. Furthermore, the single-cell RT-nested PCR indicated that the V1a receptor mRNA is present in vasopressinergic magnocellular neurons. In light of these results, in situ hybridization was performed to visualize the V1a and V1b receptor mRNAs in supraoptic and paraventricular nuclei. Simultaneously, we coupled this approach to: 1) in situ hybridization detection of oxytocin or VP mRNAs; or 2) immunocytochemistry to detect the neuropeptides. This provided a way of identifying the neurons expressing perceptible amounts of V1a or V1b receptor mRNAs as vasopressinergic neurons. Here, we suggest that the autocontrol exerted specifically by VP on vasopressinergic neurons is mediated through, at least, V1a and V1b subtype receptors.

A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to a retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons.

A peptide nucleic acid (PNA) antisense for the AUG translation initiation region of prepro-oxytocin mRNA was synthesized and coupled to a r etro-inverso peptide that is rapidly taken up by cells. This bioconjugate was internalized by cultured cerebral cortex neurons within minutes, according to the specific property of the vector peptide. The PNA alone also entered the cells, but more slowly. Cell viability was unaffected when the PNA concentrations were lower than 10 microM and incubation times less than for 24 h. Magnocellular neurons from the hypothalamic supraoptic nucleus, which produce oxytocin and vasopressin, were cultured in chemically defined medium. Both PNA and vector peptide-PNA depressed the amounts of the mRNA coding for prepro-oxytocin in these neurons. A scrambled PNA had no effect and the very cognate prepro-vasopressin mRNA was not affected. The antisense PNA also depressed the immunocytochemical signal for prepro-oxytocin in this culture in a dose- and time-dependent manner. These results show that PNAs driven by the retro-inverso vector peptide are powerful antisense reagents for use on cells in culture.

NMDA receptor properties in rat supraoptic magnocellular neurons: characterization and postnatal development.

Hypothalamo-neurohypophysial magnocellular neurons display specific electrical activities in relation to the mode of release of their hormonal content (vasopressin or oxytocin). These activities are under strong glutamatergic excitatory control. The implication of NMDA receptors in the control of vasopressinergic and oxytocinergic neurons is still a matter of debate. We here report the first detailed characterization of functional properties of NMDA receptors in voltage-clamped magnocellular neurons acutely dissociated from the supraoptic nucleus. All cells responded to NMDA with currents that reversed polarity around 0 mV and were inhibited by D-2-amino-5-phosphonovalerate (D-APV) and by 100 microM extracellular Mg2+ (at -80 mV). Sensitivity to the co-agonist glycine (EC50, 2 microM) was low compared with most other neuronal preparations. The receptors displayed low sensitivity to ifenprodil, were insensitive to glycine-independent potentiation by spermine, and had a unitary conductance of 50 pS. No evidence was found for two distinct cell populations, suggesting that oxytocinergic and vasopressinergic neurons express similar NMDA receptors. Characterization of NMDA receptors at different postnatal ages revealed a transient increase in density of NMDA currents during the second postnatal week. This was accompanied by a specific decrease in sensitivity to D-APV, with no change in NMDA sensitivity or any other properties studied. Supraoptic NMDA receptors thus present characteristics that strikingly resemble those of reconstituted receptors composed of NR1 and NR2A subunits. Understanding the functional significance of the development of NMDA receptors in the supraoptic nucleus will require further knowledge about the maturation of neuronal excitability, synaptic connections and neurohormone release mechanisms.

Differential distribution of a potassium current in immunocytochemically identified supraoptic magnocellular neurones of the rat.

Magnocellular hypothalamo-neurohypophysial neurones display characteristic firing patterns, related to the hormone they release. To identify the membrane currents that may underlie these firing patterns, we performed whole-cell recording of freshly dissociated magnocellular neurones from the supraoptic nucleus. After recording, cells were immunocytochemically identified by using highly selective monoclonal antibodies raised respectively against vasopressin (AVP) and oxytocin (OT) neurophysin. In 64 out of 131 neurones (48.8%), we detected the presence of a transient potassium current whose kinetic properties were characteristic of an A-current. The A-current was activated by depolarisation over -40 mV, and inactivated rapidly with a monoexponential decay (tau = 28 +/- 2.7 ms; n = 33 at 0 mV). Using conditioning prepulses of 50 ms, the voltage dependence of the inactivation was determined, and the data were adequately fit with a Boltzman equation (half-maximal inactivation: -42.5 mV). The steady-state time-dependent inactivation curve was determined using a prepulse potential at -40 mV, and data were best described with a mono-exponential equation (tau = 89.7 ms). The sensitivity to 1 mM 4-amino-pyridine (63 +/- 9% inhibition, n = 6), and a reversal potential close to the theoretical Nernst equilibrium for potassium (-56.3 +/- 1 mV, n = 6, vs. -58 mV) confirmed that the transient current studied was indeed an A-type potassium current. Immunocytochemical identification revealed that the A-current was selectively expressed in OT-neurophysin-positive cells. As previous work in hypothalamic slice preparations suggests that the A-current is expressed by both AVP cells and OT cells, the present data suggest that whereas the A-current is expressed in the soma of OT cells, it may be expressed only on the dendritic tree of AVP cells, which is truncated in the dispersed cell preparation used here. This distribution may play a role in the specific firing characteristics of magnocellular neurones.

Photic regulation of c-fos gene expression in the suprachiasmatic nucleus and the circadian rhythm of photosensitivity in the mink.

The relationship between the photic stimulation of the c-fos gene product in cells of the suprachiasmatic nuclei and the photoperiodic control of testicular activity were examined in mink. Mink were kept in a short photoperiod (control, LD4:20), or in 'asymmetric skeleton photoperiods' (groups A and B). The light period for groups A and B was divided into two fractions (3 h 30 min and 30 min); the shorter fraction occurred in the night, 4 h (group A) or 8 h (group B) after the end of the main light period. There was no photic activation of the proto-oncogene c-fos on the control or group A, and 4 weeks on this photoperiodic treatment produced marked testicular development. In contrast, in group B, c-fos mRNA was induced 30 min after the end of the secondary photofraction, was maximal 30 min later and then decreased. Fos-like immunoreactivity was detected 2 h after the end of the secondary photofraction, with activity peaking 1 h later. The animals of this group remained sexually quiescent. These results suggest that photo-induction of the proto-oncogene c-fos is implicated in the gonadal inhibition induced in this species when the light period, extends into the photosensitive phase of the circadian rhythm of photosensitivity.

Seasonal changes in the hypothalamic vasopressinergic system of a wild Sahelian rodent, Taterillus petteri.

Seasonal variations in the immunoreactivity of vasopressinergic perikarya in the paraventricular (PVN), supraoptic (SON) and suprachiasmatic nuclei (SCN), and in the labelling of vasopressinergic fibres in the internal zone of the median eminence were studied in Taterillus petteri, a rodent that is found in the north Burkina Faso (formerly Upper Volta). In this region, there are four seasonal climatic combinations: the humid and hot, humid and cold, dry and cold, and dry and hot seasons. In the dry hot season, the rodents experience phases of torpor (adaptation to dryness). Immunoreactivity of the PVN and SON is highest during the dry cold season. Labelling is intense during the dry hot and humid hot seasons, and is at its lowest during the humid cold season. In the SCN, labelling of the perikarya is only dense during the dry hot season, whereas for the rest of the year, the immunoreactivity is weak or undetectable. The pattern of immunoreactive variations of vasopressin-positive fibres located in the internal zone of the median eminence is similar to those of vasopressinergic perikarya in the PVN and SON. These results suggest that there is an association between: (1) seasonal modifications in the immunoreactivity of PVN and SON vasopressinergic perikarya and vasopressinergic fibres of the internal median eminence, and (2) climatic conditions, water metabolism, behavioural activity and diet. It is not possible to establish a correlation between seasonal variations in water availability and fluctuations in the labelling of vasopressinergic perikarya in the SCN. However, labelling is intense when the animals are in torpor during the dry hot season.

Localization of melatonin binding sites in the pars tuberalis of the mink at three times during the seasonal testicular cycle.

The strict photoperiodic dependence of gonadotropic function observed in mink provides an excellent physiological model for studying the activity of GnRH hypothalamic neurons. In mink, exposure to more than 10 h light inhibits the activity of this neurohormonal system. Melatonin plays an essential role in this type of regulation of gonadotropic function. In mink, contrary to results in many other photosensitive species, melatonin was observed to mediate the gonadostimulating effect of short days. However, the binding sites and action of this substance have not yet been defined. We thus attempted to identify melatonin binding sites in the mink brain. The study was carried out at three times during the seasonal testicular cycle in male minks maintained under natural environmental conditions. Coronal sections were taken from the whole brain and pituitary gland. Using 2-[125I]iodomelatonin, we observed a strong concentration of binding sites in the pars tuberalis and not in the part of the median eminence surrounded by the pars tuberalis. This concentrated localization of melatonin binding sites brings up the problem of defining the action of this substance in photoregulating the activity of the GnRH neurohormonal system.

In situ hybridization of GnRH mRNA in the rat and the mink hypothalamus using biotinylated synthetic oligonucleotide probes.

The cellular localization of GnRH messenger RNA (mRNA) in the rat and the mink hypothalamus has been examined using a newly developed highly sensitive non-radioactive in situ hybridization procedure. Synthetic oligonucleotides labeled by addition of a biotin-21-dUTP tail at their 3' end can be used to detect GnRH mRNA in both species. Streptavidin-alkaline phosphatase revealed with nitroblue tetra-zolium-bromo-chloro-indolyl-phosphate as substrate makes possible detection of the biotinylated oligonucleotides. In the rat, our findings confirm results previously obtained using synthetic radioactive probes, and demonstrate the potency of and interest in using biotinylated oligonucleotides to identify related sequences of bases in tissues. The principle advantages include rapid signal detection, excellent spatial resolution, and low background. In the mink, the in situ hybridization method clearly confirms the characterization of GnRH-producing cells and also allows detection of GnRH cell bodies in conditions in which they are not detected by immunohistochemistry. Adaptation of the in situ hybridization to the detection of GnRH mRNA in species like the mink which shows seasonal reproductive activity is a crucial step. This method offers a new approach to problems as fundamental as changes in gene expression depending on photoperiod or under a variety of experimental conditions.

Short-day stimulation of testicular activity and immunoreactivity of the hypothalamic GnRH system in mink following deafferentation of the pineal body by bilateral superior cervical ganglionectomy and melatonin replacement.

The effects of superior cervical ganglionectomy on testicular function (testis volume and plasma testosterone levels) and the immunocytochemical activity of the GnRH hypothalamic system were studied in the mink, a short-day breeder. Animals reared in a natural photoperiod were (i) ganglionectomized at four different times during the period extending from the end of summer to the end of autumn (September 15, October 20, October 28, and December 1), and (ii) reared for 50 days in a short gonadostimulatory photoperiod (4L:20D). Lastly, an attempt was made to overcome the effects of superior cervical ganglion removal by administering melatonin to mink reared in a natural photoperiod. In mink reared in a natural photoperiod, deafferentation of the pineal on September 15 (L = 12.5 h) or October 20 (L = 10.5 h) resulted in consistently low values of testicular volume and plasma testosterone until the end of the experiment (February). When the operation was performed on October 28 (L = 10 h) testicular activity was initiated but only lasted a short time and did not allow maximal gonadal development. When superior cervical ganglionectomy was carried out on December 1 (L = 8.5 h), during the phase of renewed testicular activity, the increases in testicular volume and testosterone levels were not affected by the operation and the subsequent variation of these parameters was identical to that observed in intact animals. Similarly, in mink reared for 50 days in a photoperiod of 4L:20D before superior cervical ganglionectomy, deafferentation of the pineal did not prevent gonadostimulation induced by short days.(ABSTRACT TRUNCATED AT 250 WORDS)

Suprachiasmatic nucleus lesions abolish photoperiod-induced changes in the testis function and GnRH immunoreactivity in the mink, a short-day breeder.

Testicular activity (testis volume and plasma testosterone) and immunoreactive GnRH hypothalamic system were examined after suprachiasmatic nucleus (SCN) lesion in the mink, a short-day breeding mammal, whose sexual activity is inhibited by day lengths exceeding 10 h. In animals maintained under a natural photoperiod, SCN destruction performed during the period of maximum sexual activity (February) was shown to have no effect on onset of the testicular inactive period which begins at the end of winter and continues through spring. On the other hand, while gonadal activity began again at the end of autumn in intact animals, minks that had undergone SCN destruction remained sexually inactive until the end of the experiment period (February). The SCN could thus be crucial to the onset of sexual activity triggered by the reduction of day length, whereas onset of sexual inactivity is a spontaneous phenomenon. This was confirmed in a second experiment demonstrating that a short photoperiod (4 L:20 D), highly gonadostimulatory in intact animals, had no effect on testicular activity after SCN destruction. An immunocytochemical study of the hypothalamic GnRH system (staining intensity and number of labeled perikarya and immunoreactive endings in the external layer of the median eminence) also showed consistent by very low rates of immunoreactivity and number of labeled perikarya and endings in operated animals.

Morphofunctional evidence for the involvement of hypothalamic dopaminergic and GABAergic neurons in the mechanisms of photoperiod-dependent prolactin release in the mink.

This study was designed to examine possible relationships between the photoperiodic regulation of prolactin secretion and the activity of dopaminergic and GABAergic neurons projecting to the external layer of the median eminence. The study was carried out on the mink whose remarkable photosensitivity has been clearly demonstrated. The animals were reared in short (4L:20D) or long (20L:4D) photoperiods. The experiment began in November when day length is short (9.5 h). Dopaminergic and GABAergic neurons were studied using immunocytochemical methods allowing evaluation of the immunoreactivities of tyrosine hydroxylase (TH) and glutamate decarboxylase (GAD), which are respective markers of these neurons. The results were quantified by image analysis. The plasma prolactin level of animals maintained in 4L:20D decreased after 60 days and TH and GAD immunoreactivity were strongly stimulated. After 110 days, the prolactin concentration and TH and GAD immunoreactivity recovered their starting levels. In animals maintained in 20L:4D, the prolactin level was 3 times higher than at the beginning of the photoperiodic treatment but only dopaminergic neurons showed a change, i.e. a decrease in immunoreactivity. At the end of the experiment, prolactin secretion was no longer affected by the stimulatory effect of long-day treatment, and TH immunoreactivity remained low. These results confirm the generally accepted concept that dopaminergic neurons are potent PIF-producing components. GABAergic hypothalamic system appears to be implicated in photoperiodic PRL regulation, but this remains to be clearly demonstrated.

Long-term effects of pinealectomy on testicular function, luteinizing hormone-releasing hormone hypothalamic system, and plasma prolactin levels in the mink, a short-day breeder.

Experiments in minks, as in a number of other seasonal breeders, clearly demonstrate that the pineal gland is essential for the photoperiodic control of reproduction. While maintenance of pineal-intact minks under natural photoperiods results in a set of seasonally appropriate changes in testicular activity, pinealectomized minks undergo none of these changes but rather remain sexually inactive as under long-day conditions. Thus, the consequences of pinealectomy differ from one photoperiodic species to another, but the unifying feature is the organism's need for the pineal gland to respond appropriately to changes in day length. Although the precise mechanism by which the pineal regulates hypothalamic-pituitary gonadal function remains unknown, the results of the present study indicate that, in the mink, luteinizing hormone-releasing hormone axonal transport is affected by pinealectomy. Furthermore, our results suggest that the pineal does not act exclusively upon the neuroendocrine-gonadal system but also acts on other functions that are influenced by photoperiod. Pinealectomized minks left in natural conditions cannot adjust their prolactin secretion in response to either long or short photoperiods. Operated animals continued to have plasma prolactin variations but at irregular intervals and with no apparent relation to the time of the year. The data strengthen the hypothesis that melatonin may act at some point on the hypothalamic neuroendocrine systems, which regulate the two functions differently, and that melatonin is not an anti- or progonadal substance but rather a seasonal transducer.

Peptidergic neurohormonal systems in the basal hypothalamus of the ferret and the mink: immunocytochemical study of variations during the annual reproductive cycle.

The hypothalamic systems secreting corticotropin-releasing hormone (CRF), somatostatin, oxytocin, vasopressin and luteinizing hormone-releasing hormone (LHRH) were characterized using immunochemistry, and variations were studied in relation to the recrudescence of testicular activity in the ferret and the mink, two species with opposite photoregulation of their annual reproductive cycles. Under the present conditions of study, the immunoreactivity of the CRF, somatostatin, and oxytocin systems showed no significant variation in either species. In contrast, in these two species, the immunoreactivity of the LHRH system varied considerably depending on the date of observation. The increase in the number and immunoreactivity of the LHRH-secreting neurons that occurred in November in the mink and in January in the ferret, is in agreement with previous results showing that the photoperiod plays an essential role in regulating the annual activity of the testis and that the photoperiodic environmental conditions required for the activation of the LHRH system differ between the species. Similarly, correlations could be found between an increase in immunoreactivity of the vasopressinergic axons projecting to the external median eminence and the recrudescence of testicular activity.

Circadian participation in the photoregulation of testis activity and prolactin secretion in the mink, a short-day breeder.

It has been demonstrated that an endogenous mechanism is involved in photoperiodic time measurement in the mink, a short-day-breeding mannal. A study of testicular activity (testicular volume, plasma testosterone concentration) and plasma prolactin level was carried out in sexually resting minks (the experiment began in November). Groups of minks were kept in the natural photoperiod or subjected to different resonance light-dark (LD) cycles (LD 4:8, LD 4:20, LD 4:32, LD 4:44); an additional group of animals was reared in an ahemeral photoperiod (LD 4:16). A rapid increase of testicular activity was observed in control animals or those kept in LD 4:20 (T 24) and LD 4:44 (T 48). In the other groups of animals, those kept in LD 4:8 (T 12), LD 4:32 (T 36), and LD 4:16 (T 20), testicular function remained at rest. Prolactin secretion was, in contrast, stimulated in the groups kept in LD 4:8 (T 12). LD 4:32 (T 36), and LD 4:16 (T 20), and remained low in the groups kept in LD 4:20 (T 24) and LD 4:44 (T 48). These results show that the effects of the different photoperiodic regimens do not depend on the duration of the photophase, but rather on the period of the LD cycles. The LD cycles that allow an increase of testicular function are those that are inhibitory to reproduction in birds and long-day-breeding mammals. To explain these results, it is suggested that in the mink exposure to light during the circadian photosensitive phase induces inhibition of testicular activity and stimulation of prolactin secretion. To explain the opposite effects of a single photoperiod on testicular function and secretion of prolactin, the hypothesis has been advanced that, in the mink, long days might simultaneously inhibit hypothalamic luteinizing-hormone-releasing hormone (LH-RH) activity and prolactin-inhibiting factor (PIF) activity.

[Effect of thyroidectomy on variations during the spring and summer of the testicular activity and blood prolactin in the mink]. / Influence de la thyroïdectomie sur les variations, au cours du printemps et de l'été, de l'activité testiculaire et de la prolactinémie chez le vison.

The possible role of thyroid hormones in the setting of sexual quiescence was investigated in the mink, since levels of thyroid hormones were earlier shown to rise while testicular activity decreased. When performed at the beginning of the sexual period, thyroidectomy transiently stimulated testosterone production, and significantly prolonged the duration of maximal testicular development. These results indicate that mink conforms to a pattern of inhibitory thyroid-testis interactions similar to that previously described in several species of birds and mammals. Thyroidectomy was unable, however, to prevent ultimately the installation of sexual quiescence which also appears independent of the photoperiod. On the other hand, thyroidectomy did not modify, from February to October, the general pattern of prolactin secretion, even though the vernal stimulation of prolactin secretion, induced by increasing daylength, was significantly enhanced in the absence of thyroid hormones.

Incidence of a circadian cycle of photosensitivity in the regulation of the annual testis cycle in the mink: a short-day mammal.

Like the birds or long-day mammals studied up until now the photoregulation of the annual testicular cycle in the mink, a short-day mammal, depends on phase relationships existing between the daily cycle of alternating period of light-dark and the circadian cycle of photosensitivity. Our results show, however, that the characteristics of photoresponse in the mink are exactly the opposite of those of long-day animal species. For long-day species light has a stimulating effect on the central machinery of gonadotropic control (LH-RH) whereas in the mink, the secretion of gonadotropins is induced by short days. Interpreted according to this hypothesis, the sexual cycle of the mink under natural photoperiodic conditions is also explained by seasonal gonadotropic stimulation beginning after the autumn equinox when in our latitudes daily light duration is less than 12 hr. However, the end of the reproduction period which, in nature, seems to be the result of the inhibition of the gonadotropic function by long days could, in fact, be the result of a more complex mechanism. Different inhibiting mechanisms already shown in long-day animals could also be involved during this phase of the sexual cycle in the mink.

[Testicular activity of the ferret kept for over 4 years under permanent light and constant temperature]. / Activité testiculaire du furet maintenu pendant plus de 4 ans en lumiére permanente et température constante.

Five male ferrets, aged 2 months, were kept in constant light (4-5 lx) and temperature (15 +/- 1 degree C) from July 1979 to September 1983. Under these experimental conditions, the testicular size displayed spontaneous variations. Four cycles, characterized by a marked testicular enlargement followed by an equivalent decrease, were observed in three ferrets. The two others exhibited only three spontaneous cycles. The mean interval between two successive maximums equalled 340 +/- 28 days.

[Circadian photosensitivity and photoperiodic control of the annual cycle of blood prolactin in the mink]. / Photosensibilité circadienne et contrôle photopériodique du cycle annuel de la prolactinémie chez le vison.

An annual cycle in plasma prolactin was evidenced in male Minks with a minimum in fall and a maximum in June. In two series of night interruption experiments, it was found that the photoperiodic control of the prolactin cycle involved a circadian rhythm in sensitivity to light whose photosensitive phase culminated 16 hrs. after dawn. The same photosensitivity rhythm also induced the annual testis cycle, although, in the latter case "long days" had an inhibitory effect instead of the stimulatory control for prolactin.