Osmoreception in magnocellular neurosecretory cells: from single channels to secretion

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Osmoreception/n magnocellularneurosecretorycells: from single channelsto secretion St~phane H. R. Oliet and Charles W. Bourque Stephane H. R. Oliet Recognizing that osmotic pressure is a principal factor and Charles W. controlling antidiuresis, Verney introduced the term Bourque areat the 'osmorec@toF to designate the mysterious cerebral Centre for Researchin structures that regulate vasopressin release from the Neuroscience, posterior pituitary. While hormone secretion from the

MontrealGeneral Hospitaland l)ept of neurohypophysis is influenced by synaptic inputs from Neurology and other osmoresponsive neurons, magnocdlular neuroNeurosurgery, McGil/ secretory cells currently provide our most comprehenUniversity, 1650 sive model of signal detection in an osmorec@tor.

CedarA ve, Montreal, P.O., Canada Vasopressin and oxytocin are hormones synthesized 1430 1,44.

by magnocelhlar neurosecretory cells (MNCs) of the supraoptic and paraventricular nuclei 1'2. While vasopressin and oxytocin are established for their role in antidiuresis and in the regulation of lactation and parturition respectively3, oxytocin has recently been shown also to display natriuretic activity at physiologically relevant concentrations 4. From their hypothalamic loci, the axon terminals of MNCs project to the posterior pituitary, where secretion into the blood occurs in proportion to the frequency of action potentials generated at MNC somata 3. In the rat, circulating concentrations of oxytocin and vasopressin both increase and decrease as plasma osmolality increases and decreases 3-7. By promoting Na + excretion and water retention respectively, these hormones lower the osmotic pressure of the plasma and, therefore, represent key homeostatic mechanisms for the regulation of fluid balance :~-6. Consistent with these observations, systemic hyperosmolality in the rat has been found to cause the excitation of both oxytocin- and vasopressin-releasing MNCs (Ref. 3).

Location of osmoreceptors regulating neurohypophysial hormone release Jewell and Verney, reporting the effects of brain lesions and arterial ligations on hormone release from the neurohypophysis, proposed that the osmoreceptors controlling vasopressin release were located in the anterior hypothalamus, perhaps even within the supraoptic nucleus 8. Consistent with the latter hypothesis, most MNCs recorded in the rat supraoptic nucleus were subsequently shown to respond electrically to the application of local osmotic stimuli in vivo 9 and during synaptic blockade in vitro 1°. While these results indicate that MNCs are osmosensitive intrinsically, neurohypophysial hormone-mediated responses to systemic hyperosmolality are attenuated by electrolytic lesions placed in a number of forebrain structures including: the subfornical organ 11 (SFO); the median preoptic nucleus 12 (MNPO); or the organum vasculosum lamina terrninalis 1:~ (OVLT). Interestingly, injections of hypertonic solutions into the area of the OVLT and MNPO have been found to excite MNCs (Refs 14-16), suggesting that osmosensitive forebrain regions contribute to the central regulation of vasopressin and oxytocin release. Anal340

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ogous experiments have revealed that osmosensitive afferents from the hepatic portal region can also modulate the electrical and secretory responses of MNCs (Ref. 17). In order to conciliate these findings, Leng and colleagues proposed that the release of neurohypophysial hormones is regulated by the concerted operation of an 'osmoreceptor complex 'ls'~v. According to this hypothesis, synaptic inputs from osmosensitive and non-osmosensitive neurons converge upon MNCs where their ability to influence electrical activity is modulated by the endogenous responsiveness of these cells to prevailing osmotic conditions. While neurons in the OVLT (Ref. 20) and SFO (Ref. 21) have recently been shown to be osmosensitive intrinsically, the results of electrophysiological studies on MNCs of the rat, at present, provide our most complete characterization of signal detection in an osmoreceptor.

Cationic conductance and osmosensitivity in supraoptic nucleus MNCs The endogenous osmosensitivity of MNCs was first demonstrated in 1980 when Mason reported that supraoptic neurons in hypothalamic slices were depolarized by hypertonic stimuli presented in solutions containing a low concentration of Ca 2+ and a high concentration of Mg ~÷ (Ref. 10). Voltage-clamp analysis showed that this response involved the activation of a non-selective cationic conductance 2~. More recently, experiments on neurons isolated acutely from the rat supraoptic nucleus have confirmed2:~and extended ~4'~5 these results, demonstrating that MNCs can also be hyperpolarized by hypotonic solutions (Fig. 1A). In contrast with the increased input conductance that occurs during exposure to hypertonic solutions~2'~:', hypotonic stimuli have been found to cause a reduction in membrane conductance ~4''~'~. Moreover, as shown in Fig. 1B, currentvoltage relationships measured under different osmotic conditions have been found to intersect at a common membrane potential ( - - 4 0 m V ) , suggesting that a single ionic mechanism might mediate the depolarizing and hyperpolarizing responses to increases or decreases in osmotic pressure. This hypothesis received additional support from the observation that changes in the extracellular concentration of Na ÷ elicit identical changes in the reversal potential of membrane current responses to both hypertonic and hypotonic stimuli~42"~. As shown by current responses to fixed voltage steps (Fig. 1C), modulation of cationic conductance can be evoked by osmolality changes of - 1 % , a sensitivity that is comparable to that reported for the osmotic regulation of hormone release from the neurohypophysis in vivo "~''~. These findings support a functional involvement of endogenous responses to the osmotic regulation of MNCs in situ ~' 1~ TINS, VoL 17, NO. 8, 1994

Effects of osmolality on A volume changes in mosmol kg-’ osmoreceptor cells In 1947, Vemey reported that NaCl was a more effective stimulus for antidiuresis than the same osmotic load of membrane per-meant ureaz‘j. These results suggest that changes in cell volume are a necess60mV ary feature of signal transduction in osmoreceptors. Confiing this micromorphometric hypothesis, analysis has revealed that acutely 6 mV isolated MNCs, exposed to osmosmol kg“ -80 -60 -40 -20 motic stimuli, undergo rapid and 4 I reversible changes in cell volume24. 298 295 305 295 In agreement with the absence I- Pi bd+rfof short-term accommodation in osmoreceptors controlling antidiuresis’“, osmotically evoked volume changes measured in MNCs 295 325 295 could be sustained during a 30 min stimulus period (S. H.R. 0. and C.W. B., unpublished observations), suggesting that they lack the rapid mechanisms volume-regulatory found in other cells27. The absence of volume regulation in MNCs has been proposed as a possible explanation for the apparent stability of an osmotic ‘set-point’ during Fig. 1. Effects of external osmolality changes mediated by the removal or addition of mannitol, on magnocellular neurosecretory cells (MNCs) isolated acutely from the rat supraoptic nucleus23. prolonged osmoreceptor stimu- (A) The firing activity of an isolated MNC (bottom) recorded in whole-cell current-clamp was lation in viv~P*~“. alternately enhanced and suppressed in response to osmotic stimuli (top). (6) Current-voltage As shown in Fig. 2, changes in relationships approximated from membrane current responses to voltage ramps (16 mV s-‘) were cell volume evoked by the pres- obtained during perfusion with solutions of varying osmolality. Note that the current traces entation of an osmotic stimulus intersect at a unique potential. (C) Effects of consecutive hypertonic stimuli (+3, +10 and +30 essentially mirror accompanying mosmolkg-‘) on input conductance measured from current responses to 30-mV hyperpolarizing changes in cationic conductancez4. steps (!I, = -70 mV). A and B are reproduced, with permission, from Ref. 25. This observation suggests that volume changes associated with osmotically evoked (Ref. 24). As shown in Fig. 3A, the activity of this water fluxes across the plasma membrane of MNCs channel can be modulated either by changing the are tightly coupled to the modulation of cationic osmolality of the extracellular fluid, or by applying a conductance. Interestingly, similar changes in cell small amount of pressure to the inside of the patch volume elicited by applying pressure to the inside of a pipette. While the mechanosensitivity of the channel whole-cell patch pipette can modulate the osmotically shown in Fig. 3 is established by its response to a gated conductance directly”‘. These findings indicate single pressure step, this procedure alone is inthat the cation channels underlying the osmosensitivity sufficient to determine whether the channel is actiof MNCs are directly sensitive to changes in cell vated or inactivated by membrane stretch”‘,“‘. Since volume rather than to changes in the concentration of an unknown amount of residual pressure occurs an intracellular solute. following seal formation”“, application of a single pressure pulse can either increase or, by opposing Mechanosensitive channels in supraoptic this residual value, decrease the local pressure on the neurons pipette side of the patch. Consequently, the characSpeculating on the possible basis for osmo- teristic activation profile of a mechanosensitive reception, Verney proposed that if ‘stretch receptors’ channel can only be determined from its response to were attached to the dendrites of supraoptic neurons, the application of a series of positive and negative osmotic pressure changes could be ‘transmuted into pressures. For example, the frequency of opening of electrical wave messages to the secretory endings of stretch-activated channels, increases upon application the supraoptico-hypophysial tracts”“. Almost 40 years of local positive or negative pressure since enhanced later Guharay and Sachs, providing the first charac- lateral tension is a necessary consequence of generterization of mechanosensitive channels, suggested ating either patch convexity or concavity”“-““. that ion channels of this type would be well suited to Consequently, such channels display a U-shaped ‘serve as osmoreceptors’“O. Recently, cell-attached activation curve, where minimal activity occurs near patch-clamp recordings from acutely isolated MNCs zero pressure. However, when examined over a have demonstrated the presence of a mechano- broad spectrum of pressures, the activity of sensitive cation channel displaying a slope conduc- mechanosensitive channels expressed in MNCs was tance of 32 pS and a reversal potential near -40 mV found to increase as a bell-shaped function of pipette

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control of spike discharge and hormone release from their axon terminals in the neurohypophysis. The 295 presence of cation channels, whose activity is inhibited directly by increases in membrane tension, provides a 140 possible explanation for this endogenous osmosensio~ tivity. As illustrated in Fig. 4A, swelling associated Q) 100 with hypotonic stimulation of MNCs will increase the 130 03 amount of tension experienced by the channel poput~o clation as a whole. As a result, an increasing proportion 03 c120 8 of these channels will be inhibited, thereby reducing cO 03 ~5 steady-state cationic inward current, and hyperpolarE 95 izing the cell. Conversely, neuronal shrinkage associ110 "~ cated with hypertonicity will attenuate membrane >0 O o tension, enhance channel activity, and result in o 100 • depolarization. One prediction of this model is that by increasing membrane tension progressively, solutions 90 i I I I I of decreasing tonicity should inhibit all channels 0 100 200 300 400 eventually, limiting the dynamic range of cationic responses at the lower end of external osmolalities. Time (s) The graph shown in Fig. 4B plots the regression fit of Fig. 2. Correlation between changes in input conductance (filled circles) and macroscopic cationic responses recorded as a function cell volume (open circles) during hyperosmotic stimulation. At each time point, of external osmolality that we have reported prethe percentage change of input conductance (relative to control) was obtained viouslyz~. Interestingly, the osmotic 'threshold' for from the membrane current response to a 2 0 m Y hyperpolarizing step (V~l = the appearance of cationic conductance approximates -70 mV). Changes in cell volume of magnocel/ular neurosecretory cells were plasma osmolality values at which circulating concenestimated from digitized images obtained using a scanning laser microscope. trations of neurohypophysial hormones become deData taken from Ref. 24. tectable by radioimmunoassay in the rat ~~. mosmol kg-1

pressure with maximal activity occurring at near zero Concluding r e m a r k s While synaptic inputs from additional cerebral or pressure ~1 (Fig. 3B). This finding indicates that the osmotically gated cation channels of rat MNCs are of peripheral osmoreceptors are known to be required to maintain the normal osmoresponsiveness of MNCs in the stretch-inactivated type :~:~. vivo, recent findings suggest that the intrinsic osmosensitivity of these cells might play an important role Stretch-inactivated channels and in the osmotic control of hormone release from the osmosensitivity Changes in external osmotic pressure modulate the neurohypophysis. Previous studies have shown that membrane potential of rat MNCs, contributing to the the resting membrane potential of MNCs recorded B

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Fig. 3. Properties of mechanosensitive cation channels in rat magnocellular neurosecretory cells (MNCs). (A) Recordings from a cell-attached patch (Vh = --100 mV) containing at least three channels. Note that overall channel activity can be enhanced either by raising the osmolality of the external solution, or by applying pressure to the membrane patch 24. (B) Effects of various pressures on channel activity expressed as the average total patch current recorded from experiments on seven MNCs. Zero pressure was defined arbitrarily as that corresponding to maximal patch current. Data taken from Ref. 24. 342

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ability that action potentials will be elicited by excitatory inputs 1°'19'22. Thus, the sensitivity of functional neurohypophysial responses to systemic hyperosmolality might arise from the concurrent activation of extrinsic and intrinsic mechanisms. Interestingly, hypoosmotic conditions have been found to reduce neurohypophysial responses to nonosmotic stimuli 'e9':~8':~9. While such effects could involve extrinsic inhibitory influences% it is interesting to note that changes in membrane potential modulate the effectiveness of all EPSPs, whether they originate from osmosensitive neurons or not ~~'zS. By analogy with the modulatory effects described above, the intrinsic hyperpolarization associated with hypotonic conditions will reduce the responsiveness of MNCs to synaptic excitation. Therefore, we hypothesize that by modulating the synaptic regulation of MNCs in a non-discriminatory manner, the intrinsic osmosensitivity of MNCs serves to adjust neurohypophysial secretory responses to the prevailing osmotic state of the animal.

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m o s m o l kg "1 Fig. 4. (A) Diagram illustrating the behaviour of stretchinactivated channels in response to changes in membrane tension and external osmolafity. (B) Cationic conductance (A G~,t) expressed as a function of external osmolafity. Solid line plots the regression fit of data reported in Ref. 25 expressed relative to that measured at 325 mosmol kg -1, the largest stimulus tested. The shaded area indicates that osmotically gated cationic conductance is absent at fluid osmolafities below ~275 mosmol kg -~, and increases as a steep function of osmolafity. A dashed line drawn through the normal resting extracellular fluid osmolality of rats indicates that a substantial amount of osmotically gated cationic conductance occurs near this 'set-point'.

under isotonic conditions usually lies below spike threshold ~9'ee':~r, and that the amplitude of many spontaneous EPSPs is insufficient to elicit firing 'e'e,:~r. While the intrinsic response of MNCs to osmotic stimuli is sensitive and dose-dependent (Fig. 4B), depolarizing responses to increases in osmolality of less than 2% are generally insufficient to stimulate firing 10'~2. Therefore, as proposed by Leng and colleagues ~9, it appears that by shifting the membrane potential toward spike threshold, depolarizing responses to hypertonicity serve to enhance the probTINS, Vol. 17, No. 8, 1994

1 Swaab, D. F., Pool, C. W. and Nijveldt, F. (1975) J. Neural Transm. 36, 195-215 2 Vandesande, F. and Dierickx, K. (1975) Cell Tiss. Res. 164, 153-162 3 Poulain, D. A. and Wakerley, J. B. (1982) Neuroscience 7, 772 - 808 4 Verbalis, J. G., Mangione, M. P. and Stricker, E. M. (1991) Endocrinology 128, 1317-1322 5 Dunn, F. L., Brennan, T. J., Nelson, A. E. and Robertson, G. L. (1973) J. C/in. Invest. 52, 3212-3219 6 Stricker, E. M. and Verbalis, J. G. (1986) Am. J. PhysioL 250, R267-R275 7 Johnson, A. K., Zardetto-Smith, A. M. and Edwards, G. L. (1992) Prog. Brain Res. 91, 381-393 8 Jewell, P. A. and Verney, E. B. (1957) Philos. Trans. R. Soc. London, Ser. B 240, 197-324 9 Leng, G. (1980) J. Physiol. 304, 405-414 10 Mason, W. T. (1980) Nature 287, 154-157 11 Mangiapane, M. L., Thrasher, T. N., Keil, L. C., Simpson, J. B. and Ganong, W. F. (1984) Brain Res. Bull. 13, 4 3 - 4 8 12 Mangiapane, M. L., Thrasher, T. N., Keil, L. C., Simpson, J. B. and Ganong, W. F. (1983) Neuroendocrinology 37, 73-77 13 Thrasher, T. N., Keil, L. C. and Ramsay, D. J. (1982) Endocrinology 110, 1837-1839 14 Honda, K., Negoro, H., Higuchi, T. and Tadokoro, Y. (1987) Am. J. Physiol. 252, R1039-R1045 15 Honda, K., Negoro, H., Higuchi, T. and Takano, S. (1990) Neurosci. Lett. 119, 167-170 16 Richard, D. and 8ourque, C. W. (1992) Neuroendocrinology 55, 609-611 17 8aertschi, A. J. and Vallet, P. G. (1981) J. Physiol. 315, 217-230 18 Leng, G., Mason, W. T. and Dyer, R. G. (1982) Neuroendocrinology 34, 75-82 19 Leng, G., Dyball, R. E. J. and Mason, W. T. (1985) in Vasopressin (Shrier, R. W,, ed.), pp. 333-342, Raven Press 20 Vivas, L., Chiaraviglio, E. and Carrer, H. F. (1990) Brain Res. 519, 294-300 21 Sibbald, J. R., Hubbard, J. I. and Sirett, N. E. (1988) Brain Res. 461,205-214 22 Bourque, C. W. (1989) J. Physiol. 417, 263-277 23 Oliet, S. H. R. and 8ourque, C. W. (1992) J. Physiot. 455, 291-306 24 Oliet, S. H. R. and Bourque, C. W. (1993) Nature 364, 341-343 25 Oliet, S. H. R. and Bourque, C. W. (1993)Am. J. PhysioL 265, R1475-R1479 26 Verney, E. B. (1947) Proc. R. Soc. London, Ser. B 135, 25-106 27 Hallows, K. R. and Knauf, P. A. (1993) in Cellular and Molecular Physiology of Cell Volume Regulation (Strange, K., ed.), pp. 3-29, CRC Press 28 Verbalis, J. G., Balwin, E. F. and Robinson, A. G. (1986) Am. J.

Acknowledgements Workin the authors' laboratory was supportedby the MedicalResearch Counci/of Canada. S.H.R.O.is a recipient of a studentshipfrom the Heart& Stroke Foundationof Canada.C W B, is an MRCScientist. 343

Physiol. 250, R444-R451 29 Verbalis, J. G. and Dohanics, J. (1991) Am. J. Physiol. 261, R1028-R1038 30 Guharay, F. and Sachs, F. (1984) J. PhysioL 352, 685-701 31 Morris, C. E. (1990)J. Membr. Biol. 113, 93-107 32 French, A. S. (1992) Annu. Rev. Physiol. 54, 135-152 33 Morris, C. E. and Sigurdson, W. J. (1989) Science 243, 807 - 809 34 Lecar, H. and Morris, C. E. (1993) in Mechanoreception by the Vascular Wa/l (Rubyani, G. M., ed.), pp. 1-11, Futura Publishing 35 Sokabe, M. and Sachs, F. (1992)in Advances in Comparative

and Enwronmental Physiology, Vol. 10 (Ito, F,, ed.), pp, 55-77, Springer 36 Sackin, H. (1993) in Cellular and Molecular Physiology of Cell Volume Regulation (Strange, K., ed.), pp. 215-240, CRC Press 37 Mason, W. T. (1983) Proc. R. Soc. London, Set. B 217, 141-161 38 Rosella-Dampman, L. M., Hartman, R. D. and Summy-Long, .L Y. (1987) Am. J. Physiol. 253, R31-R38 39 Dohanics, J., Hoffman, G. E. and Verbalis, J. G. (1991) Endocrinology 128, 331-340 40 Verbellis, J. G. (1993) Ann. N.Y. Acad. Sci. 689, 146-160

Neurotransmission:harnessingfusionmachineryat the synapse Thomas S611nerand James E. Rothman ThomasSdlnerand JamesE Rothmanare at the Cellular Biochemistryand BiophysicsProgram, MemoriaISIoan KettenngCancer Center, 1275 York Avenue, New York, NY 10021, USA.

Neurotransmission requires the docking of synaptic vesicles to the presynaptic plasma membrane, and their signal-dependent fusion. These processes use a general 'machinery' operating at several intracellular vesicular transport steps and, in addition, use a set of unique components that characterizes this specific form of regulated secretion. This review summarizes recent progress that has significantly increased our understanding of how intracellular transport vesicles dock and fuse with their target membrane, both in the synapse and elsewhere. Vesicles act as carriers in the secretory and endocytotic pathways, shuttling 'cargo' from one compartment to the next 1. Selective targeting of each vesicle to its correct destination is necessary to ensure and to propagate compartmental integrity of eukaryotic ceils. In some cases [for example, from endoplasmic reticulum (ER) to Golgi], the cargo is transported at

SynapUc vesicle

Presynaptic plasma membrane

Syntaxin SNAP-25

Fig. 1. The interaction of vesicle-associated membrane protein (VAMP) with syntaxin and synaptosome-associated protein 25 (SNAP-25) mediates the docking of synaptic vesicles with the presynaptic plasma membrane. These proteins form together with N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs) in the presence of nonhydrolyzable A TP, a 205 fusion particle. 344

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an almost constant rate. In other cases (for example, regulated exocytosis), it is accumulated in secretory vesicles whose release is tightly regulated, mainly at the fusion step, in response to an external stimulus. The exact delivery of cargo, including both biosynthetic and signaling molecules (subject to rigid spatial and temporal constraints), is prerequisite for the existence of higher organisms with their complex interactions between ceils, and their extensive communication with the environment. The release of neurotransmitters from the synapse is the best example of a secretory system operating under these conditions. In a resting nerve terminal, neurotransmitters are stored in synaptic vesicles. A subpopulation of these vesicles is docked to the 'active zone' at the presynaptic plasma membrane, and stimulation of the nerve terminal (usually by an action potential) leads to an influx of Ca2+, triggering fusion and release of the neurotransmitters. Recently, data from four different lines of study in different organisms, and by different methods, have come together to shed light on the underlying mechanisms of vesicle docking and fusion, and have revealed, to the surprise of many, that synaptic transmission does not use a unique mechanism. Rather, this crucial process makes use of a mechanism that is common to biology. First, functional assays using the vesicular transport system of the Golgi, representing constitutive secretion, led to the discovery and purification of several general components crucial for most, if not all, membrane-fusion processes and their membrane receptors in the synapse. Second, synaptic vesicles with their limited but welldefined protein composition enabled the identification and cloning of polypeptides that are potential components of regulated secretion. Third, identification of the targets of botulinum and tetanus neurotoxins, agents that block neurotransmission, confirmed the importance of these docking proteins in vivo. Fourth, the existence of yeast-gene products having structural and sequence homology to the components of the mammalian docking and fusion machinery emphasizes the generality of their function. This review summarizes recent developments that have significantly increased our insight into how intracellular transport vesicles dock and fuse with their appropriate target membrane, both in the TINS, VoL 17, NO. 8, 1994