Thomas E. Fisher

Norepinephrine triggers release of glial ATP to increase postsynaptic efficacy

Glial cells actively participate in synaptic transmission. They clear molecules from the synaptic cleft, receive signals from neurons and, in turn, release molecules that can modulate signaling between neuronal elements. Whether glial-derived transmitters can contribute to enduring changes in postsynaptic efficacy, however, remains to be established. In rat hypothalamic paraventricular nucleus, we demonstrate an increase in the amplitude of miniature excitatory postsynaptic currents in response to norepinephrine that requires the release of ATP from glial cells. The increase in quantal efficacy, which likely results from an insertion of AMPA receptors, is secondary to the activation of P2X7 receptors, an increase in postsynaptic calcium and the activation of phosphatidylinositol 3-kinase. The gliotransmitter ATP, therefore, contributes directly to the regulation of postsynaptic efficacy at glutamatergic synapses in the CNS.

The function of Ca2+ channel subtypes in exocytotic secretion: new perspectives from synaptic and non-synaptic release

By mediating the Ca(2+) influx that triggers exocytotic fusion, Ca(2+) channels play a central role in a wide range of secretory processes. Ca(2+) channels consist of a complex of protein subunits, including an alpha(1) subunit that constitutes the voltage-dependent Ca(2+)-selective membrane pore, and a group of auxiliary subunits, including beta, gamma, and alpha(2)-delta subunits, which modulate channel properties such as inactivation and channel targeting. Subtypes of Ca(2+) channels are constituted by different combinations of alpha(1) subunits (of which 10 have been identified) and auxiliary subunits, particularly beta (of which 4 have been identified). Activity-secretion coupling is determined not only by the biophysical properties of the channels involved, but also by the relationship between channels and the exocytotic apparatus, which may differ between fast and slow types of secretion. Colocalization of Ca(2+) channels at sites of fast release may depend on biochemical interactions between channels and exocytotic proteins. The aim of this article is to review recent work on Ca(2+) channel structure and function in exocytotic secretion. We discuss Ca(2+) channel involvement in selected types of secretion, including central neurotransmission, endocrine and neuroendocrine secretion, and transmission at graded potential synapses. Several different Ca(2+) channel subtypes are involved in these types of secretion, and their function is likely to involve a variety of relationships with the exocytotic apparatus. Elucidating the relationship between Ca(2+) channel structure and function is central to our understanding of the fundamental process of exocytotic secretion.

The Expression of Voltage-Gated Ca2+ Channels in Pituicytes and the Up-Regulation of L-Type Ca2+ Channels During Water Deprivation

The primary components of the neurohypophysis are the neuroendocrine terminals that release vasopressin and oxytocin, and pituicytes, which are astrocytes that normally surround and envelop these terminals. Pituicytes regulate neurohormone release by secreting the inhibitory modulator taurine in an osmotically‐regulated fashion and undergo a marked structural reorganisation in response to dehydration as well as during lactation and parturition. Because of these unique functions, and the possibility that Ca2+ influx could regulate their activity, we tested for the expression of voltage‐gated Ca2+ channel α1 subunits in pituicytes both in situ and in primary culture. Colocalisation studies in neurohypophysial slices show that pituicytes (identified by their expression of the glial marker S100β), are immunoreactive for antibodies directed against Ca2+ channel α1 subunits CaV2.2 and CaV2.3, which mediate N‐ and R‐type Ca2+ currents, respectively. Pituicytes in primary culture express immunoreactivity for CaV1.2, CaV2.1, CaV2.2, CaV2.3 and CaV3.1 (which mediate L‐, P/Q‐, N‐, R‐ and T‐type currents, respectively) and immunoblotting studies confirmed the expression of these Ca2+ channel α1 subunits. This increase in Ca2+ channel expression may occur only in pituicytes in culture, or may reflect an inherent capability of pituicytes to initiate the expression of multiple types of Ca2+ channels when stimulated to do so. We therefore performed immunohistochemistry studies on pituitaries obtained from rats that had been deprived of water for 24 h. Pituicytes in these preparations showed a significantly increased immunoreactivity to CaV1.2, suggesting that expression of these channels is up‐regulated during the adaptation to long‐lasting dehydration. Our results suggest that Ca2+ channels may play important roles in pituicyte function, including a contribution to the adaptation that occurs in pituicytes when the need for hormone release is elevated.

Dehydration increases L‐type Ca2+ current in rat supraoptic neurons

The magnocellular neurosecretory cells of the hypothalamus (MNCs) regulate water balance by releasing vasopressin (VP) and oxytocin (OT) as a function of plasma osmolality. Release is determined largely by the rate and pattern of MNC firing, but sustained increases in osmolality also produce structural adaptations, such as cellular hypertrophy, that may be necessary for maintaining high levels of neuropeptide release. Since increases in Ca2+ current could enhance exocytotic secretion, influence MNC firing patterns, and activate gene transcription and translation, we tested whether Ca2+ currents in MNCs acutely isolated from the supraoptic nucleus (SON) of the hypothalamus are altered by 16–24 h of water deprivation. A comparison of whole‐cell patch‐clamp recordings demonstrated that dehydration causes a significant increase in the amplitude of current sensitive to the L‐type Ca2+ channel blocker nifedipine (from –56 ± 6 to –99 ± 10 pA; P < 0.001) with no apparent change in other components of Ca2+ current. Post‐recording immunocytochemical identification showed that this increase in current occurred in both OT‐ and VP‐releasing MNCs. Radioligand binding studies of tissue from the SON showed there is also an increase in the density of binding sites for an L‐type Ca2+ channel ligand (from 51.5 ± 4.8 to 68.1 ± 4.1 fmol (mg protein)−1; P < 0.05), suggesting that there was an increase in the number of L‐type channels on the plasma membrane of the MNCs or some other cell type in the SON. There were no changes in the measured number of binding sites for an N‐type Ca2+ channel ligand. Dehydration was not associated with changes in the levels of mRNA coding for Ca2+ channel α1 subunits. These data are consistent with the hypothesis that a selective increase of L‐type Ca2+ current may contribute to the adaptation that occurs in the MNCs during dehydration.

Novel Splice Variants of Rat CaV2.1 That Lack Much of the Synaptic Protein Interaction Site Are Expressed in Neuroendocrine Cells

Voltage-gated Ca2+ channels are responsible for the activation of the Ca2+ influx that triggers exocytotic secretion. The synaptic protein interaction (synprint) site found in the II–III loop of CaV2.1 and CaV2.2 mediates a physical association with synaptic proteins that may be crucial for fast neurotransmission and axonal targeting. We report here the use of nested PCR to identify two novel splice variants of rat CaV2.1 that lack much of the synprint site. Furthermore, we compare immunofluorescence data derived from antibodies directed against sequences in the CaV2.1 synprint site and carboxyl terminus to show that channel variants lacking a portion of the synprint site are expressed in two types of neuroendocrine cells. Immunofluorescence data also suggest that such variants are properly targeted to neuroendocrine terminals. When expressed in a mammalian cell line, both splice variants yielded Ca2+ currents, but the variant containing the larger of the two deletions displayed a reduced current density and a marked shift in the voltage dependence of inactivation. These results have important implications for CaV2.1 function and for the mechanisms of CaV2.1 targeting in neurons and neuroendocrine cells.

An osmosensitive voltage-gated K+ current in rat supraoptic neurons.

The magnocellular neurosecretory cells of the hypothalamus (MNCs) regulate their electrical behaviour as a function of external osmolality through changes in the activity of osmosensitive ion channels. We now present evidence that the MNCs express an osmosensitive voltage‐gated K+ current (the OKC). Whole‐cell patch‐clamp experiments on acutely isolated MNCs were used to show that increases in the external osmolality from 295 to 325 mosmol/kg cause an increase in a slow, tetraethylammonium‐insensitive outward current. The equilibrium potential for this current is close to the predicted EK in two different concentrations of external K+. The OKC is sensitive to block by Ba2+ (0.3 mm), and by the M‐type K+ current blockers linopirdine (150 μm) and XE991 (5 μm), and to enhancement by retigabine (10 μm), which increases opening of M‐type K+ channels. The OKC is suppressed by muscarine (30 μm) and is decreased by the L‐type Ca2+ channel blocker nifedipine (10 μm), but not by apamin (100 nm), which blocks SK‐type Ca2+‐dependent K+ currents. Reverse transcriptase‐polymerase chain reaction and immunocytochemical data suggest that MNCs express several members of the KV7 (KCNQ) family of K+ channels, including KV7.2, 7.3, 7.4 and 7.5. Extracellular recordings of individual MNCs in a hypothalamic explant preparation demonstrated that an XE991‐ and retigabine‐sensitive current contribute to the regulation of MNC firing. Our data suggest that the MNCs express an osmosensitive K+ current that could contribute to the regulation of MNC firing by external osmolality and that could be mediated by KV7/M‐type K+ channels.

A novel osmosensitive voltage gated cation current in rat supraoptic neurones.

The magnocellular neurosecretory cells of the hypothalamus (MNCs) regulate water balance by releasing vasopressin and oxytocin as a function of plasma osmolality. Release is determined largely by the rate and pattern of action potentials generated in the MNC somata. Changes in firing are mediated in part by a stretch‐inactivated non‐selective cation current that causes the cells to depolarize when increased osmolality leads to cell shrinkage. We have obtained evidence for a new current that may regulate MNC firing during changes in external osmolality, using whole‐cell patch clamp of acutely isolated rat MNC somata. In internal and external solutions lacking K+, with high concentrations of TEA, and with Na+ as the only likely permeant cation, the current appears as a slow inward current during depolarizations and yields a large tail current upon return to the holding potential of −80 mV. Approximately 60% of the MNCs tested (79 out of 134 cells) displayed a large increase in tail current density (from 5.2 ± 0.9 to 10.5 ± 1.4 pA pF−1; P < 0.001) following an increase in external osmolality from 295 to 325 mosmol kg−1. The current is activated by depolarization to potentials above −60 mV and does not appear to depend on changes in internal Ca2+. The current is carried by Na+ under these conditions, but is blocked by Cs+ and Ba2+ and by internal K+, which suggests that the current could be a K+ current under physiological conditions. This current could play an important role in regulating the response of MNCs to osmolality.

Osmotic activation of phospholipase C triggers structural adaptation in osmosensitive rat supraoptic neurons

Acutely isolated rat magnocellular neurosecretory cells (MNCs) display reversible hypertrophy when exposed to hypertonic saline (325 mosmol kg−1) for tens of minutes. Osmotically evoked hypertrophy is prevented by agents that block Na+ channels, TRPV1 channels (which mediate an osmotically evoked depolarization in these cells), L‐type Ca2+ channels, and exocytotic fusion and is associated with an increase in whole‐cell capacitance. Exposure to hypertonic saline activates phospholipase C leading to a decrease in plasma membrane phosphatidylinositol 4,5‐bisphosphate. Inhibition of phospholipase C or protein kinase C prevents osmotically evoked hypertrophy and activation of protein kinase C evokes hypertrophy in the absence of an increase in osmolality. These results suggest that osmotically evoked hypertrophy is triggered by an increase in MNC action potential firing, leading to Ca2+ influx, the activation of phospholipase C, an increase in diacylglycerol, and the activation of protein kinase C.

Expression of CaV2.2 and Splice Variants of CaV2.1 in Oxytocin‐ and Vasopressin‐Releasing Supraoptic Neurones

The magnocellular neurosecretory cells (MNCs) release vasopressin (VP) and oxytocin (OT) from their axon terminals into the circulation and from their somata and dendrites to exert paracrine effects on other MNCs. MNCs express several types of voltage‐gated Ca2+ channels, including CaV2.1 and CaV2.2. These two channels types are similar in structure and function in other cells, but although influx of Ca2+ through CaV2.2 triggers the release of both OT and VP into the circulation, CaV2.1 is involved in stimulating the release of VP but not OT. Release of OT from MNC somata is also triggered by CaV2.2 but not CaV2.1. These observations could be explained by differences in the level of expression of CaV2.1 in VP and OT MNCs or by differences in the way that the two channels interact with the exocytotic apparatus. We used immunohistochemistry to confirm earlier work suggesting that MNCs express variants of CaV2.1 lacking portions of an internal loop that enables the channels to interact with synaptic proteins. We used an antibody that would recognise both the full‐length CaV2.1 and the deletion variants to show that OT MNCs express fewer CaV2.1 channels than do VP MNCs in both somata and axon terminals. We used the reverse transcriptase‐polymerase chain reaction and immunocytochemistry to test whether MNCs express similar deletion variants of CaV2.2 and were unable to find any evidence to support this. Our data suggest that the different roles that CaV2.1 and CaV2.2 play in MNC secretion may be a result of the different levels of expression of CaV2.1 in VP and OT MNCs, as well as the expression in MNCs of deletion variants of CaV2.1 that do not interact with exocytotic proteins and therefore may be less likely to mediate exocytotic release.

Na(+) -Activated K(+) Channels in Rat Supraoptic Neurones.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus secrete the neurohormones vasopressin and oxytocin. The systemic release of these hormones depends on the rate and pattern of MNC firing and it is therefore important to identify the ion channels that contribute to the electrical behaviour of MNCs. In the present study, we report evidence for the presence of Na+‐activated K+ (KNa) channels in rat MNCs. KNa channels mediate outwardly rectifying K+ currents activated by the increases in intracellular Na+ that occur during electrical activity. Although the molecular identity of native KNa channels is unclear, their biophysical properties are consistent with those of expressed Slick (slo 2.1) and Slack (slo 2.2) proteins. Using immunocytochemistry and Western blot experiments, we found that both Slick and Slack proteins are expressed in rat MNCs. Using whole cell voltage clamp techniques on acutely isolated rat MNCs, we found that inhibiting Na+ influx by the addition of the Na+ channel blocker tetrodotoxin or the replacement of Na+ in the external solution with Li+ caused a significant decrease in sustained outward currents. Furthermore, the evoked outward current density was significantly higher in rat MNCs using patch pipettes containing 60 mm Na+ than it was when patch pipettes containing 0 mm Na+ were used. Our data show that functional KNa channels are expressed in rat MNCs. These channels could contribute to the activity‐dependent afterhyperpolarisations that have been identified in the MNCs and thereby play a role in the regulation of their electrical behaviour.