Protocol to visualize ion channel trafficking in acutely isolated rodent neurons using live-cell immunocytochemistry.

Here, we present a protocol for live-cell immunocytochemistry to demonstrate reversible translocation of ion channels to the neuronal cell surface. We describe steps for cell preparation and isolation, experimental treatment, antibody binding prior to fixation, specific pipetting techniques, troubleshooting, and expected outcomes of correct use of the protocol. This protocol will be useful to study regulated translocation of ion channels and other membrane proteins. For complete details on the use and execution of this protocol, please refer to Haan et al.1.

Osmotically evoked PLCδ1-dependent translocation of ΔN-TRPV1 channels in rat supraoptic neurons.

Osmoregulation is an essential homeostatic process that requires constant release of vasopressin during sustained increases in plasma osmolality. The magnocellular neurosecretory cells (MNCs) respond to increases in external osmolality through increases in the activity of ΔN-TRPV1 channels, which leads to increased action potential firing and vasopressin release. We show that sustained exposure of acutely isolated rat and mouse MNCs to hypertonic solutions (90 min) causes a reversible translocation of ΔN-TRPV1 channels from internal stores to the plasma membrane that depends on the activation of phospholipase C and on SNARE-dependent exocytosis. ΔN-TRPV1 channel translocation is absent in MNCs isolated from transgenic mice lacking the PLCδ1 isoform, suggesting that PLCδ1 is essential for triggering this process. The translocation of ΔN-TRPV1 channels to the cell surface could contribute to the maintenance of MNC excitability during sustained increases in osmolality. Our data therefore have important implications for the mechanisms underlying mammalian osmoregulation.

PLCδ1 Plays Central Roles in the Osmotic Activation of ΔN-TRPV1 Channels in Mouse Supraoptic Neurons and in Murine Osmoregulation.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus play a vital role in osmoregulation, but the mechanisms underlying MNC osmosensitivity are not fully understood. We showed previously that high osmolality activates phospholipase C (PLC) in rat MNCs in a Ca2+-dependent manner and that PLC activation is necessary for full osmotic activation of an N-terminal variant of the TRPV1 (ΔN-TRPV1) channel. We therefore hypothesized that the Ca2+-dependent δ1 isoform of PLC contributes to ΔN-TRPV1 activation and tested whether MNC function is defective in a transgenic PLCδ1 KO mouse. Water deprivation for 24 h caused greater increases in serum osmolality and losses in body weight in PLCδ1 KO mice than it did in control mice. Action potentials and ΔN-TRPV1 currents were measured in acutely isolated mouse MNCs using whole-cell patch clamp before and after exposure to hypertonic solutions. This treatment elicited a significant activation of ΔN-TRPV1 currents and an increase in firing rate in MNCs isolated from control mice, but not from PLCδ1 KO mice. Submembranous filamentous actin was measured in isolated MNCs before and after treatment with angiotensin II and hypertonic solution. Both treatments caused an increase in filamentous actin fluorescence in MNCs isolated from control mice, but both responses were significantly attenuated in MNCs from PLCδ1 KO mice. Our data demonstrate that the PLCδ1 isoform plays a key role in the activation of ΔN-TRPV1 channels and in osmosensory transduction in MNCs. This study advances our understanding of the molecular mechanisms underlying mammalian osmoregulation.SIGNIFICANCE STATEMENT Magnocellular neurosecretory cells (MNCs) of the hypothalamus play a central role in osmoregulation. We have identified a key role for the PLCδ1 isoform in the activation of ΔN-TRPV1 channels and osmosensory transduction in MNCs. The data indicate that the PLCδ1 isoform is activated by the Ca2+ influx occurring during MNC action potentials and exerts a positive feedback on ΔN-TRPV1 channels to enhance MNC excitability. This study provides evidence that PLCδ1 is a key molecule underlying osmosensory transduction, the regulation of VP release, and osmoregulation.

Osmotic activation of a Ca2+-dependent phospholipase C pathway that regulates ∆N TRPV1-mediated currents in rat supraoptic neurons.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus regulate body fluid balance by releasing the hormones vasopressin (VP) and oxytocin (OT) in an osmolality-dependent manner. Elevations of external osmolality increase MNC firing and hormone release. MNC osmosensitivity is largely due to activation of a mechanosensitive non-selective cation current that responds to osmotically-evoked changes in MNC volume and is mediated by an N-terminal variant of the TRPV1 channel (∆N TRPV1). We report a novel mechanism by which increases in osmolality may modulate ∆N TRPV1-mediated currents and thus influence MNC electrical behaviour. We showed previously that acute elevations of external osmolality activate the enzyme phospholipase C (PLC) in isolated MNCs. We now show that the osmotic activation of PLC has a time course and dose-dependence that is consistent with a role in MNC osmosensitivity and that it contributes to the osmotically-evoked increase in non-selective cation current in MNCs through a protein kinase C-dependent pathway. We furthermore show that the mechanism of osmotic activation of PLC requires an increase in internal Ca2+ that depends on influx through L-type Ca2+ channels. Our data therefore suggest that MNCs possess an osmotically-activated Ca2+-dependent PLC that contributes to the osmotic activation of ∆N TRPV1 and may therefore be important in MNC osmosensitivity and in central osmoregulation.

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

The magnocellular neurosecretory cells of the hypothalamus (MNCs) synthesize and secrete vasopressin or oxytocin. A stretch-inactivated cation current mediated by TRPV1 channels rapidly transduces increases in external osmolality into a depolarization of the MNCs leading to an increase in action potential firing and thus hormone release. Prolonged increases in external osmolality, however, trigger a reversible structural and functional adaptation that may enable the MNCs to sustain high levels of hormone release. One poorly understood aspect of this adaptation is somatic hypertrophy. We demonstrate that hypertrophy can be evoked in acutely isolated rat MNCs by exposure to hypertonic solutions lasting tens of minutes. Osmotically evoked hypertrophy requires activation of the stretch-inactivated cation channel, action potential firing, and the influx of Ca(2+). Hypertrophy is prevented by pretreatment with a cell-permeant inhibitor of exocytotic fusion and is associated with an increase in total membrane capacitance. Recovery is disrupted by an inhibitor of dynamin function, suggesting that it requires endocytosis. We also demonstrate that hypertonic solutions cause a decrease in phosphatidylinositol 4,5-bisphosphate in the plasma membranes of MNCs that is prevented by an inhibitor of phospholipase C (PLC). Inhibitors of PLC or protein kinase C (PKC) prevent osmotically evoked hypertrophy, and treatment with a PKC-activating phorbol ester can elicit hypertrophy in the absence of changes in osmolality. These studies suggest that increases in osmolality cause fusion of internal membranes with the plasma membrane of the MNCs and that this process is mediated by activity-dependent activation of PLC and PKC.

The Ca2+ channel ß2 subunit is selectively targeted to the axon terminals of supraoptic neurons.

The assembly of high voltage-activated Ca(2+) channels with different ß subunits influences channel properties and possibly subcellular targeting. We studied ß subunit expression in the somata and axon terminals of the magnocellular neurosecretory cells, which are located in the supraoptic nucleus (SON) and neurohypophysis, respectively. Antibodies directed against the 4 Ca(V)ß subunits (Ca(V)ß(1)-Ca(V)ß(4)) were used for immunoblots and for immunostaining of slices of these two tissues. We found that all 4 ß subunits are expressed in both locations, but that Ca(V)ß(2) had the highest relative expression in the neurohypophysis. These data suggest that the Ca(V)ß(2) subunit is selectively targeted to axon terminals and may play a role in targeting and/or regulating the properties of Ca(2+) channels.

Primate follicular development and oocyte maturation in vitro.

The factors and processes involved in primate follicular development are complex and not fully understood. An encapsulated three-dimensional (3D) follicle culture system could be a valuable in vitro model to study the dynamics and regulation of folliculogenesis in intact individual follicles in primates. Besides the research relevance, in vitro follicle maturation (IFM) is emerging as a promising approach to offer options for fertility preservation in female patients with cancer. This review summarizes the current published data on in vitro follicular development from the preantral to small antral stage in nonhuman primates, including follicle survival and growth, endocrine (ovarian steroid hormone) and paracrine/autocrine (local factor) function, as well as oocyte maturation and fertilization. Future directions include major challenges and strategies to further improve follicular growth and differentiation with oocytes competent for in vitro fertilization and subsequent embryonic development, as well as opportunities to investigate primate folliculogenesis by utilizing this 3D culture system. The information may be valuable in identifying optimal conditions for human follicle culture, with the ultimate goal of translating the experimental results and products to patients, thereby facilitating diagnostic and therapeutic approaches for female fertility.

Lifestyle alterations for the amelioration of hot flashes.

Hot flashes are a common complaint among women as they transition through menopause. This article reviews the evidence of lifestyle alterations for the amelioration of hot flashes including obesity, exercise, smoking, relaxation techniques, and acupuncture. Further randomized controlled trials regarding these lifestyle alterations are needed to determine their full potential benefits regarding hot flashes.

Survival, growth, and maturation of secondary follicles from prepubertal, young, and older adult rhesus monkeys during encapsulated three-dimensional culture: effects of gonadotropins and insulin.

A three-dimensional culture system supports the development of primate preantral follicles to the antral stage with appreciable steroid production. This study assessed i) whether in vitro developmental competence of follicles is age dependent, ii) the role of gonadotropins and insulin in supporting folliculogenesis, and iii) anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) production by growing follicles. Ovaries were obtained from prepubertal, young, and older adult rhesus macaques. Secondary follicles were encapsulated into alginate beads and cultured individually for 40 days in media containing 0.05 or 5  µg/ml insulin, with or without recombinant human (rh) FSH (500  mIU/ml). No follicles survived in the culture without rhFSH. In the presence of rhFSH, survival was lower for follicles from older animals, whereas growth, i.e. follicle diameter, was less by day 40 for follicles from prepubertal animals. The surviving follicles were categorized as no-grow (NG; ≤ 250 µm), slow-grow (SG; 250-500 µm), and fast-grow (FG; ≥ 500  µm) according to their diameters. SG follicles cultured with 5 µg/ml insulin produced more ovarian steroids than those cultured with 0.05  µg/ml insulin by week 5. SG and FG follicles produced more AMH and VEGF than the NG, and levels peaked at weeks 2 and 5 respectively. After 100  ng/ml rh chorionic gonadotropin treatment for 34 h, more healthy oocytes were retrieved from young adults whose follicles were cultured with 5  µg/ml insulin. This culture system offers an opportunity to characterize the endocrine and paracrine function of primate follicles that influence follicle growth and oocyte maturation.

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 E(K) in two different concentrations of external K(+). The OKC is sensitive to block by Ba(2+) (0.3 mm), and by the M-type K(+) current blockers linopirdine (150 microm) and XE991 (5 microm), and to enhancement by retigabine (10 microm), which increases opening of M-type K(+) channels. The OKC is suppressed by muscarine (30 microm) and is decreased by the L-type Ca(2+) channel blocker nifedipine (10 microm), but not by apamin (100 nm), which blocks SK-type Ca(2+)-dependent K(+) currents. Reverse transcriptase-polymerase chain reaction and immunocytochemical data suggest that MNCs express several members of the K(V)7 (KCNQ) family of K(+) channels, including K(V)7.2, 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 K(V)7/M-type K(+) channels.

Novel splice variants of rat CaV2.1 that lack much of the synaptic protein interaction site are expressed in neuroendocrine cells.

Voltage-gated Ca(2+) channels are responsible for the activation of the Ca(2+) influx that triggers exocytotic secretion. The synaptic protein interaction (synprint) site found in the II-III loop of Ca(V)2.1 and Ca(V)2.2 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 Ca(V)2.1 that lack much of the synprint site. Furthermore, we compare immunofluorescence data derived from antibodies directed against sequences in the Ca(V)2.1 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 Ca(2+) 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 Ca(V)2.1 function and for the mechanisms of Ca(V)2.1 targeting in neurons and neuroendocrine cells.

Dehydration increases L-type Ca(2+) 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 Ca(2+) current could enhance exocytotic secretion, influence MNC firing patterns, and activate gene transcription and translation, we tested whether Ca(2+) 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 Ca(2+) channel blocker nifedipine (from -56 +/- 6 to -99 +/- 10 pA; P < 0.001) with no apparent change in other components of Ca(2+) 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 Ca(2+) 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 Ca(2+) channel ligand. Dehydration was not associated with changes in the levels of mRNA coding for Ca(2+) channel alpha(1) subunits. These data are consistent with the hypothesis that a selective increase of L-type Ca(2+) current may contribute to the adaptation that occurs in the MNCs during dehydration.

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.

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 P2X(7) 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.