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

Correlation between electrophysiological and morphological characteristics during maturation of rat supraoptic neurons.

The neurohypophysial peptides oxytocin (OT) and vasopressin (AVP) are well known for their role in reproductive functions and fluid balance regulation, respectively. During development, these peptides are thought to act as trophic factors on both peripheral and central structures. However, despite this early developmental function, the maturation of their secreting neurons remains poorly investigated. In this study, we have characterized the electrical and morphological characteristics displayed by OT and AVP supraoptic (SO) neurons between embryonic day 21 and postnatal day 20. Transient changes in passive membrane properties, correlated with a transient increase in the dendritic arborization, were observed at the beginning of the second postnatal week (PW2). The action potential matured mostly during PW1 and its threshold progressively hyperpolarized in parallel with the resting membrane potential. During PW1, SO neurons displayed unique characteristics with a low-threshold Ca(2+)-dependent depolarizing potential and a prominent hyperpolarization-activated current (I(h) ). This latter is involved in a depolarizing sag during hyperpolarization and an after hyperpolarizing potential following a depolarization. During this period, maintaining E(Cl) unchanged by the use of gramicidin-perforated patch recordings revealed excitatory GABAergic potentials, that became inhibitory during PW2, whilst glutamatergic potential appeared. The electrical activity was very erratic in young neurons and progressively differentiated in the typical firing observed in mature neurons (tonic and phasic for OT and AVP neurons, respectively) during PW2--3. These results show that the development of electrical properties of SO neurons is correlated with the maturation of their dendritic arborization.

Developmental regulation of a local positive autocontrol of supraoptic neurons.

Mature oxytocin (OT) and vasopressin (AVP) magnocellular neurons of the hypothalamic supraoptic nuclei (SON) autocontrol their electrical activity via somatodendritic release of their respective peptides. Because OT and AVP are synthesized early in development and could play an important role in the maturation of these neurons, we checked whether the peptides are released within the SON and act on their secreting neurons during 3 weeks of postnatal development. We used patch-clamp recordings from SON neurons in rat hypothalamic horizontal slices to show that the spontaneous electrical activity of immature SON neurons is blocked by OT or AVP receptor antagonists, demonstrating a basal somatodendritic release of the peptides. Application of OT or AVP depolarizes SON neurons and stimulates activity typical of the corresponding mature neurons. This effect is directly on SON neurons because it is recorded in dissociated neurons. Radioimmunoassays from isolated SON were used to show that each peptide facilitates its own release at a somatodendritic level, exhibiting a self-sustaining positive feedback loop. This autocontrol is not uniform during development because the proportion of neurons depolarized by the peptides, the amplitude of the depolarization, and the propensity of the peptides to facilitate their own release are maximal during the second postnatal week and decrease thereafter. These data are consistent with a role of autocontrol in the maturation of SON neurons because it is maximal during the delimited period of postnatal development that corresponds to the period of major synapse formation.

Osmotic regulation of neuronal activity: a new role for taurine and glial cells in a hypothalamic neuroendocrine structure.

Maintenance of osmotic pressure is a primary regulatory process essential for normal cell function. The osmolarity of extracellular fluids is regulated by modifying the intake and excretion of salts and water. A major component of this regulatory process is the neuroendocrine hypothalamo-neurohypophysial system, which consists of neurons located in the paraventricular and supraoptic nuclei. These neurons synthesize the neurohormones vasopressin and oxytocin and release them in the blood circulation. We here review the mechanisms responsible for the osmoregulation of the activity of these neurons. Notably, the osmosensitivity of the supraoptic nucleus is described including the recent data that suggests an important participation of taurine in the transmission of the osmotic information. Taurine is an amino acid mainly known for its involvement in cell volume regulation, as it is one of the major inorganic osmolytes used by cells to compensate for changes in extracellular osmolarity. In the supraoptic nucleus, taurine is highly concentrated in astrocytes, and released in an osmodependent manner through volume-sensitive anion channels. Via its agonist action on neuronal glycine receptors, taurine is likely to contribute to the inhibition of neuronal activity induced by hypotonic stimuli. This inhibitory influence would complement the intrinsic osmosensitivity of supraoptic neurons, mediated by excitatory mechanoreceptors activated under hypertonic conditions. These observations extend the role of taurine from the regulation of cell volume to that of the whole body fluid balance. They also point to a new role of supraoptic glial cells as active components in a neuroendocrine regulatory loop.

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.

Vasopressin regularizes the phasic firing pattern of rat hypothalamic magnocellular vasopressin neurons.

Vasopressin (AVP) magnocellular neurons of hypothalamic nuclei express specific phasic firing (successive periods of activity and silence), which conditions the mode of neurohypophyseal vasopression release. In situations favoring plasmatic secretion of AVP, the hormone is also released at the somatodendritic level, at which it is believed to modulate the activity of AVP neurons. We investigated the nature of this autocontrol by testing the effects of juxtamembrane applications of AVP on the extracellular activity of presumed AVP neurons in paraventricular and supraoptic nuclei of anesthetized rats. AVP had three effects depending on the initial firing pattern: (1) excitation of faintly active neurons (periods of activity of <10 sec), which acquired or reinforced their phasic pattern; (2) inhibition of quasi-continuously active neurons (periods of silences of <10 sec), which became clearly phasic; and (3) no effect on neurons already showing an intermediate phasic pattern (active and silent periods of 10-30 sec). Consequently, AVP application resulted in a narrower range of activity patterns of the population of AVP neurons, with a Gaussian distribution centered around a mode of 57% of time in activity, indicating a homogenization of the firing pattern. The resulting phasic pattern had characteristics close to those established previously for optimal release of AVP from neurohypophyseal endings. These results suggest a new role for AVP as an optimizing factor that would foster the population of AVP neurons to discharge with a phasic pattern known to be most efficient for hormone release.

Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurones: possible role in osmoregulation.

1. To evaluate the implication of taurine in the physiology of supraoptic neurones, we (i) investigated the agonist properties of taurine on glycine and GABAA receptors of supraoptic magnocellular neurones acutely dissociated from adult rats, using whole-cell voltage clamp, (ii) studied the effects of taurine and strychnine in vivo by extracellular recordings of supraoptic vasopressin neurones in anaesthetized rats, and (iii) measured the osmolarity-dependent release of endogenous taurine from isolated supraoptic nuclei by HPLC. 2. GABA, glycine and taurine evoked rapidly activating currents that all reversed close to the equilibrium potential for Cl-, indicating activation of Cl(-)-selective channels. Glycine-activated currents were reversibly blocked by strychnine (IC50 of 35 nM with 100 microM glycine), but were unaffected by the GABAA antagonist gabazine (1-3 microM). GABA-activated currents were reversibly antagonized by 3 microM gabazine, but not by strychnine (up to 1 microM). 3. Responses to 1 mM taurine were blocked by strychnine but not by gabazine and showed no additivity with glycine-induced currents, indicating selective activation of glycine receptors. Responses to 10 mM taurine were partially antagonized by gabazine, the residual current being blocked by strychnine. Thus, taurine is also a weak agonist of GABAA receptors. 4. In the presence of gabazine, taurine activated glycine receptors with an EC50 of 406 microM. Taurine activated at most 70% of maximal glycine currents, suggesting that it is a partial agonist of glycine receptors. 5. In vivo, locally applied strychnine (300 nM) increased and taurine (1 mM) decreased the basal electrical activity of vasopressin neurones in normally hydrated rats. The effect of strychnine was markedly more pronounced in water-loaded rats. 6. Taurine, which is concentrated in supraoptic glial cells, could be released from isolated supraoptic nuclei upon hyposmotic stimulation. Decreases in osmolarity of 15 and 30% specifically enhanced basal release of taurine by 42 and 124%, respectively. 7. We conclude that supraoptic neurones express high amounts of glycine receptors, of which taurine may be regarded as a major natural agonist. We postulate that taurine, which can be released in hyposmotic situations, acts on glycine receptors to exert an inhibitory control on magnocellular neurones during alterations of body fluid homeostasis, implicating an active participation of glial cells in this neuroendocrine regulatory loop.

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.

Postnatal maturation of rat hypothalamoneurohypophysial neurons: evidence for a developmental decrease in calcium entry during action potentials.

Action potentials and voltage-gated currents were studied in acutely dissociated neurosecretory cells from the rat supraoptic nucleus during the first three postnatal weeks (PW1-PW3), a period corresponding to the final establishment of neuroendocrine relationships. Action potential duration (at half maximum) decreased from 2.7 to 1.8 ms; this was attributable to a decrease in decay time. Application of cadmium (250 microM) reduced the decay time by 43% at PW1 and 21% at PW3, indicating that the contribution of calcium currents to action potentials decreased during postnatal development. The density of high-voltage-activated calcium currents increased from 4.4 to 10.1 pA/pF at postnatal days 1-5 and 11-14, respectively. The conductance density of sustained potassium current, measured at +20 mV, increased from 0.35 (PW1) to 0.53 (PW3) nS/pF. The time to half-maximal amplitude did not change. Conductance density and time- and voltage-dependent inactivation of the transient potassium current were stable from birth. At PW1, the density and time constant of decay (measured at 0 mV) were 0.29 nS/pF (n = 12) and 17.9 ms (n = 10), respectively. Voltage-dependent properties and density (1.1 nS/pF) of the sodium current did not change postnatally. During PW1, fitting the mean activation data with a Boltzmann function gave a half-activation potential of -25 mV. A double Boltzman equation was necessary to adequately fit the inactivation data, suggesting the presence of two populations of sodium channels. One population accounted for approximately 14% of the channels, with a half-inactivation potential of -86 mV; the remaining population showed a half-inactivation potential of -51 mV. A mathematical model, based on Hodgkin-Huxley equations, was used to assess the respective contributions of individual currents to the action potential. When the densities of calcium and sustained potassium currents were changed from immature to mature values, the decay time of the action potentials generated with the model decreased from 2.85 to 1.95 ms. A similar reduction was obtained when only the density of the potassium current was increased. Integration of the calcium currents generated during mature and immature action potentials demonstrated a significant decrease in calcium entry during development. We conclude that the developmental reduction of the action potential duration 1) is a consequence of the developmentally regulated increase in a sustained potassium current and 2) leads to a reduction of the participation of calcium currents in the action potential, resulting in a decreased amount of calcium entering the cell during each action potential.

Developmental autoregulation of calcium currents in mammalian central neurones.

The influence of calcium currents expressed at early stages on the subsequent development of calcium currents was studied in embryonic rat hypothalamic neurones in culture. Voltage-activated calcium currents and spontaneous fluctuations of intracellular free calcium concentration ([Ca2+]i) were monitored. Acute application of nickel chloride (0.1 mM) to 6- to 7-day-old cultures strongly reduced calcium currents and [Ca2+]i fluctuations. When cultures were maintained for 6-7 days in the presence of NiCl2 (0.05-0.1 mM), expression of the low voltage-activated current was strongly inhibited; this treatment did not affect high voltage-activated currents. Our results suggest that spontaneous activation of calcium currents early during development promotes calcium influx that regulates expression of calcium current in mature neurones.

Synchronous development of spontaneous and evoked calcium-dependent properties in hypothalamic neurons.

The development of various related parameters was compared in hypothalamic neurons grown in primary culture. We measured: (i) low- and high-voltage-activated calcium currents; (ii) spontaneous and N-methyl-D-aspartate (NMDA)-induced fluctuations of intracellular calcium concentration; (iii) basal and NMDA- or potassium-evoked somatostatin release. Spontaneous calcium fluctuations appeared after 5 days in culture and increased progressively in amplitude and frequency over the next 8 days studied. Basal release of somatostatin was not detectable in 3 day-old cultures and reached a plateau at day 5. Responses evoked by exogenous stimulations (voltage-activated calcium currents, agonist-induced intracellular calcium rise and somatostatin release) appeared early in culture, increased in amplitude during 7-10 days and then stabilized. We conclude that, in hypothalamic neurons, the main neuronal functions develop in synchrony over a limited period of time.

In vivo development of voltage-dependent ionic currents in embryonic Xenopus spinal neurons.

Initial evidence that electrical excitability is both an early aspect of neuronal differentiation and a developmentally regulated property was obtained from recordings of action potentials in vivo. Subsequently, the analysis of the underlying voltage-dependent currents during early stages of embryogenesis was facilitated by investigation of dissociated neurons and muscle cells differentiating in culture. Calcium and potassium currents play a major role in the differentiation of the action potential of Xenopus spinal neurons, and calcium influx triggers specific features of neuronal differentiation. However, the extent to which differentiation of currents in vitro parallels that in vivo is uncertain. We have undertaken a study of in vivo differentiation of these macroscopic currents in Xenopus embryos. Spinal cords were isolated from embryos at several early stages of neurogenesis. Neurons in these isolated spinal cords were accessible to patch-clamp electrodes. Neuronal currents were recorded within 1 hr to assure that the characteristics of the currents resulted from developmental events occurring in vivo prior to the experiment. Whole-cell voltage-clamp recordings from neurons in these acutely isolated and intact embryonic spinal cords demonstrate that both the delayed-rectifier and inactivating potassium current and a low-voltage-activated calcium current mature in a manner closely parallel to that observed in culture. The results validate those from the culture system and indicate that the spinal cord is another region of the CNS accessible to cellular analysis in an intact preparation.

Role of calcium and protein kinase C in development of the delayed rectifier potassium current in Xenopus spinal neurons.

The delayed rectifier current of embryonic Xenopus spinal neurons plays the central role in developmental conversion of calcium-dependent action potentials to sodium-dependent spikes. During its maturation, this potassium current undergoes a pronounced increase in rate of activation. The mechanism underlying the change in kinetics was analyzed with whole-cell voltage clamp of neurons cultured under various conditions. Calcium is necessary at an early stage of development, to permit influx that triggers subsequent release of calcium from intracellular stores. Its action is prevented by depletion of protein kinase C and mimicked by stimulation of the kinase. Calcium influx through voltage-dependent channels at early stages of development regulates the differentiation of potassium current kinetics and modulation of the ionic dependence of action potentials.