Charles W. Bourque

Central mechanisms of osmosensation and systemic osmoregulation

Systemic osmoregulation is a vital process whereby changes in plasma osmolality, detected by osmoreceptors, modulate ingestive behaviour, sympathetic outflow and renal function to stabilize the tonicity and volume of the extracellular fluid. Furthermore, changes in the central processing of osmosensory signals are likely to affect the hydro-mineral balance and other related aspects of homeostasis, including thermoregulation and cardiovascular balance. Surprisingly little is known about how the brain orchestrates these responses. Here, recent advances in our understanding of the molecular, cellular and network mechanisms that mediate the central control of osmotic homeostasis in mammals are reviewed.

Transient Receptor Potential Vanilloid 1 Is Required for Intrinsic Osmoreception in Organum Vasculosum Lamina Terminalis Neurons and for Normal Thirst Responses to Systemic Hyperosmolality

Recent studies have indicated that members of the transient receptor potential vanilloid (TRPV) family of cation channels are required for the generation of normal osmoregulatory responses, yet the mechanism of osmosensory transduction in primary osmoreceptor neurons of the CNS remains to be defined. Indeed, despite ample evidence suggesting that the organum vasculosum lamina terminalis (OVLT) serves as the primary locus of the brain for the detection of osmotic stimuli, evidence that neurons in the OVLT are intrinsically osmosensitive has remained elusive. Here we show that murine OVLT neurons are intrinsically sensitive to increases in the osmolality of the extracellular fluid. Hypertonic conditions provoked increases in membrane cation conductance that resulted in the generation of an inward current, depolarizing osmoreceptor potentials, and enhanced action potential discharge. Moreover, we found that this osmosensory signal transduction cascade was absent in OVLT neurons from TRPV1 knock-out (TRPV1−/− ) mice and that responses of wild type (WT) OVLT neurons could be blocked by ruthenium red, an inhibitor of TRPV channels. Finally, TRPV1−/− mice showed significantly attenuated water intake in response to systemic hypertonicity compared with WT controls. These findings indicate that OVLT neurons act as primary osmoreceptors and that a product of the trpv1 gene is required for osmosensory transduction.

Synaptic control of rat supraoptic neurones during osmotic stimulation of the organum vasculosum lamina terminalis in vitro.

1. The effects of osmotic or electrical stimulation of the organum vasculosum lamina terminalis (OVLT) were examined during intracellular recordings (32 degrees C) obtained from ninety‐five supraoptic nucleus magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus. 2. Brief (10‐20 s) applications of hypertonic and hypotonic solutions to the area of the OVLT caused prolonged (> 1 min) increases and decreases, respectively, in electrical activity in seventy of seventy‐four trials performed on neurones with membrane potentials near spike threshold (approximately ‐55 mV). Changes in firing frequency were related to changes in external osmolality in a dose‐dependent manner between 275 and 355 mosmol kg‐1. 3. When 30 s periods recorded immediately before, and 30 s following, the application of an osmotic stimulus were examined, the frequency of spontaneous EPSPs (sEPSPs) was related in a dose‐dependent manner to the osmolality of the solution superfusing the OVLT region. The increased EPSP frequency was maintained and did not adapt if the osmolality of the medium was raised for periods of > 10 min. In contrast, the frequency of spontaneous IPSPs (sIPSPs) was virtually unaffected by changes in external osmotic pressure. 4. Osmotically evoked changes in MNC firing were strongly correlated with accompanying changes in the frequency of sEPSPs (slope, 0.9; correlation coefficient (r) = 0.7), but not sIPSPs (r = 0.2), suggesting that changes in firing rate following osmotic stimulation of the OVLT are selectively mediated by changes in synaptic excitation. 5. In the presence of bicuculline (5‐10 microM), electrical stimulation of the OVLT evoked fast EPSPs in forty‐seven of forty‐eight MNCs tested. These responses were reversibly reduced by application of 20‐40 microM kynurenic acid (n = 3) or 20‐40 microM 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; n = 11). Similarly, bath application of CNQX (n = 3) or kynurenic acid (n = 4) reversibly abolished the excitatory response of supraoptic neurones following hypertonic stimulation of the OVLT. 6. Brief (10‐15 s) applications of gamma‐aminobutyric acid (GABA) over the OVLT reversibly abolished increases in sEPSP frequency and action potential firing rate evoked by hyperosmotic stimulation of the OVLT. In the presence of GABA, the rates of sEPSP and sIPSP frequency were reduced to 37 +/‐ 10 and 44 +/‐ 13% (means +/‐ S.E.M.), respectively, of those observed under isotonic conditions (295 mosmol kg‐1). 7. These results suggest that inhibitory and excitatory pathways originating from neurones located within the OVLT are tonically active under resting osmotic conditions in rat hypothalamic explants. Osmotically evoked changes in MNC firing, however, are selectively mediated through increases or decreases in the intensity of the excitatory component of OVLT‐derived inputs.

NMDA receptor-mediated rhythmic bursting activity in rat supraoptic nucleus neurones in vitro.

1. Intracellular recordings were obtained from 112 supraoptic nucleus magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus maintained in vitro. The effects of glutamate receptor agonists and antagonists were examined at 32‐34 degrees C. 2. In control solutions, spontaneously active (> 5 Hz) phasic or continuous neurones showed interspike interval distributions slightly skewed toward short intervals, but did not feature pauses in the 0.4‐2 s range. Current injection to alter the rate of cell discharge shifted the histograms according to the mean firing rate, but failed to induce intermittent pauses in the 0.4‐2 s range. 3. Application of N‐methyl‐D‐aspartate (NMDA) induced a mode of firing in which bimodal interspike interval distributions reflected a high incidence of clusters of short interspike intervals (0.5‐1.5 s) recurring every 1‐3 s. In contrast, firing evoked by application of D,L‐alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxalone propionic acid (AMPA) was not associated with a clustering of impulse discharge. 4. The putative endogenous excitatory amino acid transmitters L‐glutamate, L‐aspartate and quinolinate all mimicked the effects of NMDA. Clustered spiking responses to these agents were reversibly blocked by D,L‐2‐amino‐5‐phosphono‐valerate (APV), but not by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). In contrast, the non‐NMDA receptor ligands kainate and quisqualate caused CNQX‐sensitive increases in firing rate, but these responses were not associated with the appearance of clustered activity. 5. When applied to cells showing negative resting potentials (< ‐70 mV), or to neurones hyperpolarized by current injection, responses to NMDA consisted of rhythmic (approximately 1 Hz) voltage oscillations associated with bursts of spike discharge. In the presence of TTX, NMDA could induce subthreshold voltage oscillations in the absence of action potentials. 6. Application of a voltage clamp to potentials between ‐75 and ‐55 mV during rhythmic bursting responses failed to reveal any rhythmic oscillation of the membrane current. In all cases, rhythmic bursting activity resumed upon returning to the current‐clamp mode. 7. Rhythmic bursting responses to NMDA application were abolished in Mg(2+)‐free solutions, suggesting that the voltage dependence of NMDA channels served to promote regenerative voltage changes throughout the cycle. The NMDA‐induced current itself, however, did not appear to decrease with time, suggesting that a distinct, outward current, was necessary to initiate the repolarizing phase of each cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage‐gated calcium currents in the magnocellular neurosecretory cells of the rat supraoptic nucleus.

1. Whole‐cell patch‐clamp techniques were used to analyse voltage‐dependent calcium currents in acutely isolated somata of magnocellular neurosecretory cells (MNCs) from the supraoptic nucleus of the hypothalamus of adult rats. Currents were characterized on the basis of their rates of inactivation and their sensitivity to a series of calcium channel blocking agents. 2. Curve fitting analysis of series of long lasting depolarizing voltage steps from a holding potential of ‐80 mV revealed three current components with different voltage dependences and rates of inactivation (n = 36). These include a low threshold (‐60 mV), rapidly inactivating (tau = 42 +/‐ 3 ms at ‐10 mV) component, a high threshold (‐30 mV), slowly inactivating (tau = 1790 +/‐ 70 ms) component and a component with an intermediate threshold (‐50 mV) and rate of inactivation (tau = 187 +/‐ 15 ms). There is also a non‐inactivating portion of evoked calcium current with a threshold of ‐50 mV. 3. Based on its voltage dependence, rate of inactivation, greater sensitivity to the divalent cation nickel than to cadmium and insensitivity to omega‐conotoxin GVIA (omega‐CgTX), the low threshold current appears to be a T‐type calcium current. The rate of inactivation, voltage dependence, and sensitivity to omega‐CgTX of the slowly inactivating component suggests that it is an N‐type current. The characteristics of the intermediate component do not correspond to any identified calcium current type. 4. Portions of the non‐inactivating calcium current are sensitive to nifedipine (23 +/‐ 2% of the total non‐inactivating current at ‐10 mV; n = 10), suggesting the presence of L‐type currents, omega‐agatoxin‐IVA (omega‐Aga‐IVA; 20 +/‐ 6% of total; n = 11), suggesting the presence of P‐type channels, and omega‐CgTX (39 +/‐ 3% of total; n = 19), suggesting the presence of a non‐inactivating N‐type current. The non‐inactivating component at low potentials (> or = ‐50 mV) was selectively blocked by nifedipine, suggesting the presence of a novel, low threshold L‐type current. 5. We conclude that MNC soma express T‐, N‐, L‐, and P‐type calcium currents, as well as a novel low threshold nifedipine‐sensitive current and an unidentified inactivating component. This complement of currents is different from that seen in the terminals of these cells, suggesting a spatial and functional segregation of calcium current types in MNCs.

Properties of supraoptic magnocellular neurones isolated from the adult rat.

1. Magnocellular neurosecretory cells (MNCs) were isolated from the supraoptic nucleus of adult Long‐Evans rats using an enzymatic procedure. Immunocytochemical staining with antibodies against vasopressin and oxytocin revealed that MNCs can be identified by size. The membrane properties of these cells were examined at 32‐34 degrees C using intracellular recording methods. 2. Isolated MNCs displayed a mean (+/‐ S.E.M.; n = 109) resting membrane potential of ‐64.1 +/‐ 1.0 mV, an input resistance of 571 +/‐ 34 M omega, and a time constant of 8.7 +/‐ 0.4 ms. Measurements of specific resistivity and input capacitance revealed that the soma of these cells accounts for a mere 20% of their total somato‐dendritic membrane in situ. 3. Voltage‐current relations measured near ‐60 mV were linear negative to spike threshold. From more hyperpolarized membrane potentials, voltage responses to depolarizing current steps displayed transient outward rectification and delayed impulse discharge. 4. Action potentials (76.6 +/‐ 0.9 mV) triggered from an apparent threshold of ‐59.3 +/‐ 0.1 mV broadened progressively at the onset of spontaneous or current‐evoked spike trains. Steady‐state spike duration increased as a logarithmic function of firing frequency with a maximum near 25 Hz. These effects were abolished in Ca(2+)‐free solutions. 5. In all cells, evoked spike trains were followed by a prolonged Ca(2+)‐sensitive after‐hyperpolarization. In contrast, only a small proportion (16%) of MNCs displayed spontaneous bursting activity or depolarizing after‐potentials following brief current‐evoked bursts. 6. Isolated MNCs responded to amino acids (glutamate and GABA) and to the neuropeptide cholecystokinin, indicating that receptors for these neurotransmitters are expressed postsynaptically by MNCs and are retained following dissociation. 7. Increasing the osmolality of the superfusing solution by 5‐30 mosmol kg‐1 caused a membrane depolarization associated with a decrease of input resistance and accelerated spontaneous spike discharge in each of thirty‐six MNCs tested. Current‐clamp analysis suggested that these responses resulted from the activation of a cationic conductance. Excitatory effects of hyperosmolality were not observed in non‐magnocellular neurones (n = 6).

Intraterminal recordings from the rat neurohypophysis in vitro

1. Intracellular recordings were obtained from neurosecretory terminals (endings of Herring bodies), axons and pars intermedia cells in the isolated neuro‐intermediate lobe of the rat. Responses to current injection and stimulation of the neural stalk (NS) were examined at 34 degrees C. Cellular identity was verified following injection of Lucifer Yellow. 2. Neurosecretory terminals were identified by a constant‐latency action potential response to NS stimulation and an appropriate collision test. This response was not blocked by Ca2(+)‐free solutions. Hyperpolarization of the terminals could block the generation of a local spike. Injection of Lucifer Yellow into eleven units confirmed that such responses were recorded from neurosecretory terminals. 3. Neurohypophysial nerve terminals had a resting potential of ‐60.4 +/‐ 1.1 mV and displayed a spike amplitude of 72.4 +/‐ 1.9 mV. The local spike threshold was ‐41.6 +/‐ 1.9 mV. Voltage‐current relations were linear near resting potential, but displayed strong outward rectification positive to ‐55 mV. While terminals could fire at high frequencies during NS stimulation, repetitive activity could not be evoked by prolonged depolarizing current pulses. 4. During the initial 1‐3 s of a train of brief depolarizing pulses or NS stimuli, nerve terminals showed a progressive broadening of their action potentials. At steady state, the duration of these impulses increased logarithmically with firing rate, showing a maximum near 25 Hz. Spike broadening in nerve terminals was reversibly abolished by superfusion of the neural lobe with Ca2(+)‐free, Mn2(+)‐containing solutions. In the absence of external Ca2+, action potentials were smaller, and lacked a prominent shoulder on their repolarizing phase. 5. Sustained (greater than 5 s) repetitive stimulation at 10‐20 Hz led to a gradual increase in the latency for invasion and eventual failure of the spike‐generating mechanism within the terminal. This effect required several seconds to recover. In contrast, action potentials recorded within the axon followed continuous repetitive stimulation and did not show any frequency‐dependent changes in duration (0.7 +/‐ 0.1 ms). 6. Cells of the pars intermedia (PI) displayed an input resistance of 215.7 +/‐ 47.4 M omega and fired a single action potential in response to current injection. The amplitude of this current‐evoked spike decreased during repetitive stimulation, but its duration was not affected. In 87% of the PI cells tested, stimulation of the NS evoked a Ca2(+)‐sensitive synaptic response which reversed near ‐40 mV, but no cell was directly activated by the stimulus.

Ionic basis for the intrinsic activation of rat supraoptic neurones by hyperosmotic stimuli.

1. Magnocellular neurosecretory cells (MNCs) were impaled in the supraoptic nucleus of rat hypothalamic explants maintained in vitro. Current‐ and voltage‐clamp analysis of the osmotically induced response was performed at 34 degrees C. 2. Addition of mannitol or NaCl to cause a rise in fluid osmolarity (greater than +6 mosM) caused a membrane depolarization whose amplitude increased when elicited from more hyperpolarized levels. Changes in temperature (34‐28 degrees C), addition of TTX, or superfusion with Na(+)‐free or Ca2(+)‐free solutions did not block the osmotically induced depolarization. In control solutions the response was consistently accompanied by an increase in the frequency of spontaneous postsynaptic potentials. Thus, osmotic stimuli have a direct effect on MNCs, and they also apparently activate other neurones which are presynaptic to these cells. 3. Under voltage‐clamp, hyperosmotic stimuli induced an inward current (Io) accompanied by an increase in membrane conductance. The current was unaffected or slightly enhanced by doubling the external K+ concentration. Io was also characterized by a linear I‐V relation (between ‐100 and ‐50mV) and an extrapolated reversal potential near ‐10 mV. Io presumably results from the activation of a voltage‐independent and non‐selective cationic conductance. 4. Hyperosmotic stimuli did not affect the depolarizing after‐current (IDAP) responsible for the production of phasic bursts. However, the inward shift of the post‐spike I‐V curve caused by Io could reduce or eliminate the region of net outward current which lies negative to spike threshold in silent neurones. Thus in MNCs displaying IDAP, activation of Io by a rise in osmotic pressure can induce or enhance phasic bursting activity. 5. Application of hyperosmotic stimuli sufficient to excite most MNCs (+20 to +80 mosM) did not elicit a response from any of seventeen neurones impaled in areas lateral and caudal to the supraoptic nucleus. Recordings obtained from three CA1 neurones in slices of rat hippocampus revealed that stimuli in excess of +100 mosM are required to evoke appreciable non‐specific depolarizations. 6. These studies indicate that the specific endogenous osmosensitivity of MNCs results from the activation of the intrinsic current Io. Furthermore, interactions between Io and IDAP explain how osmotic stimuli can lead to the induction of phasic bursting activity, a response which is known to potentiate the secretion of vasopressin from the neural lobe.

Chapter 2.1.1 Osmoregulation of vasopressin neurons: A synergy of intrinsic and synaptic processes

The release of vasopressin into the general circulation varies as a function of plasma osmolality and therefore plays a major role in systemic osmoregulation. In vivo, the secretion of this hormone in the neurohypophysis is primarily determined by the rate of action potential discharge of the magnocellular neurosecretory cells (MNCs) in the hypothalamus. Experiments done over the past 20 years have clarified much of the neurophysiological basis underlying this important osmoregulatory reflex. As discussed here, recent findings indicate that the regulation of the firing rate of MNCs during changes in systemic osmolality involves the concerted modulation of mechanosensitive ion channels in MNCs, as well as excitatory glutamatergic inputs derived from forebrain regions such as the organum vasculosum of the lamina terminalis.

Integration of sodium and osmosensory signals in vasopressin neurons.

Vasopressin (antidiuretic hormone) release has been thought to be controlled by interacting osmoreceptors and Na(+)-detectors for > 20 years. Only recently, however, have molecular and cellular advances revealed how changes in the external concentration of Na+ and osmolality are detected during acute and chronic osmotic perturbations. In rat vasopressin-containing neurons, local osmosensitivity is conferred by intrinsic stretch-inactivated cation channels and by taurine release from surrounding glia. Na+ detection is accomplished by acute regulation of the permeability of stretch-inactivated channels and by changes in Na+ channel gene expression. These features provide a first glimpse of the integrative processes at work in a central osmoregulatory reflex.