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 Ca_V2.2 and Ca_V2.3, which mediate N‐ and R‐type Ca²⁺ currents, respectively. Pituicytes in primary culture express immunoreactivity for Ca_V1.2, Ca_V2.1, Ca_V2.2, Ca_V2.3 and Ca_V3.1 (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 Ca_V1.2, 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.     

Electrophysiological properties of reactive glial cells in the kainate-lesioned hippocampal slice

Kainate-lesioned hippocampal slices provide an excellent system for examining the electrophysiological properties of non-cultured glial cells without interfering signals from surrounding neurons. Intracellular recordings in the intensely gliotic CA3 region indicated that the resting membrane potential and input resistance of these reactive glial cells were similar to those reported for non-reactive astrocytes. Dye-coupling typical of astrocytes was also demonstrated amongst these cells, and was considerably reduced by cytoplasmic acidification. Occasionally these cells demonstrated spontaneous, rhythmic oscillations of membrane potential associated with large changes in whole cell input resistance. The action potentials reported in cultured astrocytes were not observed in reactive glial cells, even under conditions that maximize the observation of Ca2(+)-dependent responses. This suggests that reactive glial cells in this preparation have either no voltage-activated Ca2+ channels or a very low density of such channels. These cells also lack a bicarbonate conductance, but they do appear to have an apamin-sensitive conductance, possibly a Ca2(+)-activated gK.     

Dendritic excitability during increased synaptic activity in rat neocortical L5 pyramidal neurons

Recent years have seen increased study of dendritic integration, mostly in acute brain slices. However, due to the low background activity in brain slices the integration of synaptic input in slice preparations may not truly reflect conditions in vivo. To investigate dendritic integration, back‐propagation of the action potential (AP) and initiation of the dendritic Ca²⁺ spike we simultaneously recorded membrane potential at the soma and apical dendrite of layer 5 (L5) pyramidal neurons in quiescent and excited acute brain slices. After excitation of the brain slice the somatic input resistance decreased and the apparent passive space constant shortened. However, the back‐propagating AP and dendritic Ca²⁺ spike were robust during increased synaptic activity. The dendritic Ca²⁺ spike was suppressed by the ionic composition of the bath solution required for slice excitation, suggesting that Ca²⁺ spikes may be smaller in vivo than in the acute slice preparation. The results presented here suggest that, under the conditions of slice excitation examined in this study, the increased membrane conductance induced by activation of voltage‐gated channels during back‐propagation of the AP and dendritic Ca²⁺ spike initiation is sufficiently larger than the membrane conductance at subthreshold potentials to allow these two regenerative dendritic events to remain robust over several levels of synaptic activity in the apical dendrite of L5 pyramidal neurons.     

Periaqueductal gray neurons' activity in a mesencephalic slice preparation

The electrophysiological characteristics of periaqueductal gray (PAG) neurons were studied using intracellular techniques in guinea pig brainstem slices maintained ‘in vitro’. Input resistance and time constant ranged between 60–110 MΩ and 11–20 ms respectively. Direct activation elicited action potentials generated by a Na⁺-conductance and characterized by a remarkable Ca²⁺-dependent plateau in the falling phase and a long-lasting afterhyperpolarization which is probably due to a Ca²⁺-dependent K⁺-conductance. These cells also showed a slow return to the resting membrane potential after hyperpolarizing pulses which was Ca²⁺-dependent. All PAG neurons had a low resting membrane potential and displayed a tonic spontaneous firing at a frequency around 7 impulses/s.     

Excitatory action of opioid peptides and opiates on cultured hippocampal pyramidal cells

Bath application of low concentrations of opioid peptides and higher concentrations of opiates increased the amplitude and duration of excitatory postsynaptic potentials of pyramidal cells and induced long-lasting depolarization shifts. These actions were reversible and blocked by the opiate antagonist naloxone. Synaptic isolation of the cells by exposure of the cultures to 8 mM Mg2+ not only abolished all spiking and synaptic activity, but also obliterated the peptide effects on pyramidal cells, although these cells were still excited by bath-applied glutamate. The opioid peptides had no detectable effect on resting membrane potential and on the input resistance of the penetrated cells. Experiments in which pyramidal cells were synaptically activated by field stimulation provided direct evidence for a disinhibitory action of the peptides.     

Membrane Properties of Identified Guinea-Pig Paraventricular Neurons and their Response to an Opioid μ-Receptor Agonist: Evidence for an Increase in K+ Conductance

Intracellular recordings were obtained from neurons in the paraventricular nucleus (PVN) of guinea-pig hypothalamic slices. Passive and active properties of the neurons were determined, and when possible, recorded neurons were injected with biocytin. The effects of a μ-receptor opioid agonist [D-Ala², Nme-Phe⁴, Gly⁵-ol]enkephalin (DAGO) were studied in order to determine which types of cells have μ receptors and to test the hypothesis that an increase in K⁺ conductance causes μ-receptor-mediated inhibition in the PVN. The recorded cells inside the PVN were divided into two groups, primarily on the basis of the presence or absence of a low threshold Ca²⁺ spike (LTS). In one group of neurons, LTS potentials could not be evoked (non-LTS cells, n = 42). In another group of neurons (n = 35), LTS potentials with one or two Na⁺ spikes could be initiated with depolarizing pulses superimposed on steady hyperpolarizing currents; however, these neurons did not fire robust bursts (i.e. non-bursting LTS cells). The mean time constant of non-LTS cells (19.9±1.6ms; mean ± SEM) was significantly shorter than that of non-bursting LTS cells (26.7 ± 2.1 ms). There were no differences in the mean resting membrane potential, spike amplitude, spike duration, input resistance, spike threshold and pattern of synaptic inputs between the two groups. Intracellular labeling with biocytin combined with cresyl violet counter-staining demonstrated that the two types of cells were located in the PVN. The mean diameters of non-LTS cells along their long axis (25.4 ± 1.7 μm) and short axis (17.5 ±2.2 μm, n = 7) were larger than those of non-bursting LTS cells (22.1 ±1.0 μm, 15.2 ± 1.1 μm, respectively, n = 12); this suggests that non-LTS cells are magnocellular neurons and non-bursting LTS cells are parvocellular neurons, as previously described in the rat. Bath application of DAGO at a concentration of 1 μM inhibited 7 of 25 (28%) non-LTS cells tested, and 4 of 21 (19%) non-bursting LTS cells. The main effect of DAGO on PVN cells was a hyperpolarization of membrane potential (4 to 15 mV) with a decrease of input resistance (20% to 38%). Quinine (100 μM to 1 mM), a K⁺ channel blocker, eliminated or reduced inhibitions mediated by DAGO. The apparent reversal potential of the hyperpolarization induced by the μ-receptor agonist was about ?85 mV. These data in the guinea-pig PVN suggest that non-LTS cells are putative magnocellular neurons, and non-bursting LTS cells are parvocellular neurons. The μ-receptor agonist, DAGO, directly hyperpolarizes approximately equal numbers of each of these cell types. Experiments with quinine and injected currents support the hypothesis that DAGO activation of μ receptors on neurons in the region of the PVN leads to an increase in K⁺ conductance.     

Augmented astrocyte microdomain Ca2+ dynamics and parenchymal arteriole tone in angiotensin II-infused hypertensive mice

Hypertension is an important contributor to cognitive decline but the underlying mechanisms are unknown. Although much focus has been placed on the effect of hypertension on vascular function, less is understood of its effects on nonvascular cells. Because astrocytes and parenchymal arterioles (PA) form a functional unit (neurovascular unit), we tested the hypothesis that hypertension-induced changes in PA tone concomitantly increases astrocyte Ca²⁺. We used cortical brain slices from 8-week-old mice to measure myogenic responses from pressurized and perfused PA. Chronic hypertension was induced in mice by 28-day angiotensin II (Ang II) infusion; PA resting tone and myogenic responses increased significantly. In addition, chronic hypertension significantly increased spontaneous Ca²⁺ events within astrocyte microdomains (MD). Similarly, a significant increase in astrocyte Ca²⁺ was observed during PA myogenic responses supporting enhanced vessel-to-astrocyte signaling. The transient potential receptor vanilloid 4 (TRPV4) channel, expressed in astrocyte processes in contact with blood vessels, namely endfeet, respond to hemodynamic stimuli such as increased pressure/flow. Supporting a role for TRPV4 channels in aberrant astrocyte Ca²⁺ dynamics in hypertension, cortical astrocytes from hypertensive mice showed augmented TRPV4 channel expression, currents and Ca²⁺ responses to the selective channel agonist GSK1016790A. In addition, pharmacological TRPV4 channel blockade or genetic deletion abrogated enhanced hypertension-induced increases in PA tone. Together, these data suggest chronic hypertension increases PA tone and Ca²⁺ events within astrocytes MD. We conclude that aberrant Ca²⁺ events in astrocyte constitute an early event toward the progression of cognitive decline.     

Calcium spikes and calcium currents in neurons from the medial preoptic nucleus of rat

Ca²⁺ spikes, their contribution to firing patterns, and the underlying Ca²⁺ currents in neurons of the medial preoptic nucleus of rat were investigated by tight-seal whole-cell recordings in a slice preparation. Two different types of spikes were recorded: Low-threshold spikes were generated from membrane potentials <−75 mV. High-threshold spikes were recorded when K⁺ currents were reduced, and were readily evoked from membrane potentials near −40 mV. Both types of spikes were blocked by substitution of Co²⁺ for Ca²⁺ in the external medium, but were insensitive to 2.0 μM TTX. Under voltage-clamp conditions, two main types of Ca²⁺ currents were characterized: low-threshold currents that activated at membrane potentials >−60 mV, and high-threshold currents that activated at potentials >−30 mV. The low-threshold current and the low-threshold spike were more sensitive to block by external Ni²⁺ than to block by Cd²⁺, whereas the high-threshold current and the high-threshold spike were more sensitive to block by external Cd²⁺ than to block by Ni²⁺. Significant fractions of the high-threshold currents were blocked by 10 μM nifedipine, 1.0 μM ω-conotoxin GVIA, 50 nM ω-agatoxin IVA and 1.0 μM ω-conotoxin MVIIC, suggesting the presence of L-, N-, P- and Q-type Ca²⁺ channels. There were also a high-threshold current component insensitive to the above mentioned toxins. It is proposed that the low-threshold current serves as a trigger for short bursts of fast spikes from hyperpolarized levels, whereas the high-threshold current is involved in the Cd²⁺-sensitive burst firing seen in relatively depolarized neurons.     

Effect of D890 on membrane properties of neocortical neurons

D890, a derivative of the Ca²⁺ channel antagonist D600, was intracellularly applied from conventional microelectrodes into pyramidal neurons of neocortical slices. The effects of D890 were ascertained by evaluating alterations in membrane properties following drug administration and by comparing these neurons to untreated controls. The amplitude of action potentials (APs) evoked by depolarizing current pulses was attenuated by up to 30% within about 15 min after impalement with D890-containing electrodes. AP rate of rise was reduced by up to 80% and duration was increased. These effects were dependent upon the rate of stimulation. When depolarizing pulses were delivered at low rates of stimulation (e.g. 0.1 Hz), the overshoot of evoked APs declined by about 10%. At higher frequencies (2Hz) the AP overshoot decreased by up to 90%. These effects were mostly reversible on decreasing the frequency of stimulation. A half maximal effect was attained at about 1 Hz, when APs of control neurons were unaltered. In neurons impaled with D890-containing electrodes, depolarizing current pulses delivered in the presence of tetraethylammonium (TEA) and tetrodotoxin elicited high threshold calcium spikes which had a duration between 20 and 200 ms. In the early phase of D890 application, the duration of Ca²⁺ spikes decreased in a reversible frequency-dependent manner; after prolonged application, however, the recovery was incomplete. On the average, Ca²⁺ spike amplitude and duration decreased by 20% and 50%, respectively, suggesting that D890 usually produces an incomplete blockade of the underlying CA current. The duration of the slow envelope of orthodromically evoked epileptiform paroxysmal depolarizing shifts (DSs), induced by bath application of 10⁻⁵ M bicuculline, was frequency dependent and consistently increased from about 20 ms to 150 ms (half amplitude width) at frequencies above 0.5 Hz. In the presence of D890, the action potentials superimposed on the slow envelope of the DS were attenuated, but neither the amplitude nor the frequency-dependent progressive prolongation of the DS was altered. Application of TEA in the presence of bicuculline (10⁻⁵ M) increased the amplitude and duration of the DS in neurons impaled with D890-containing electrodes. Under these conditions, the durations of DSs evoked by low frequency orthodromic stimulation (0.5Hz) were still progressively prolonged, while, in the same neuron, directly evoked Ca²⁺ spikes progressively decreased in amplitude and duration. During DSs, the membrane potential depolarized to levels beyond those required for directly eliciting high threshold Ca²⁺ spikes, however, a Ca²⁺-spike component was not obvious during the DS. A high conductance state of the membrane at the peak of the DS may limit Ca²⁺ spike electrogenesis. The results suggest that D890 affects fast Na⁺ currents and the conventional Ca²⁺ conductance underlying the Ca²⁺ spike. The absence of effects of D890 on the DS slow envelope suggests that Ca²⁺ conductances do not make a large contribution to the latter in the neurons studied, or that Ca²⁺ flows through channels with different pharmacological properties during the DS and the Ca²⁺ spike.     

From excitation to intracellular Ca2+ movements in skeletal muscle: Basic aspects and related clinical disorders

In skeletal muscle fiber, excitation-contraction coupling corresponds to the sequence of events occurring from action potential firing to initiation of contraction by an increase in cytosolic Ca²⁺. These events are elicited in response to excitation of the motor neuron which induces trains of action potentials in the muscle cell that spread along the sarcolemma and in depth along the T-tubule membrane. Depolarization of the T-tubule membrane induces a conformational change in a protein complex, called the dihydropyridine receptor, which opens a calcium channel anchored in the membrane of the sarcoplasmic reticulum, called the ryanodine receptor, in charge of release of Ca²⁺ ions that activate contractile proteins. Ryanodine receptors shut upon return of the T-tubule membrane potential to its resting value and muscle cell relaxation results from the removal of cytosolic Ca²⁺ that is pumped back into the SR lumen through the sarcoplasmic reticulum Ca²⁺ ATPase. Mutations in genes encoding either plasma membrane ion channels, the main subunit of the dihydropyridine receptor, ryanodine receptor, sarcoplasmic reticulum Ca²⁺ ATPase or proteins interfering with trans-sarcolemmal Ca²⁺ influx or sarcoplasmic reticulum Ca²⁺ efflux lead to clinical disorders that manifest as myotonia, muscle weakness, paralysis or muscle wasting.     

Receptor-activated Ca2+ increases in vibrodissociated cortical astrocytes: A nonenzymatic method for acute isolation of astrocytes

A new nonenzymatic method for the acute isolation of astrocytes from rat cerebral cortex is described. A vibratory device was used to dissociate the cells from thin brain slices, and the method yielded fresh and relatively well-preserved astrocytes without previous enzyme incubation. These cells were examined in a microspectrofluorometric system for measurement of changes in intracellular free calcium concentrations ([Ca²⁺]_i), and their expression of various neurotransmitter receptors was determined. Acutely isolated glial fibrillary acidic protein (GFAP)-positive astrocytes (p7–p18) were seen to respond to the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 10^-4 M) with increases in [Ca²⁺]_i, and this response was blocked by (RS)-1-aminoindan-1,5 dicarboxylic acid (AIDA, 10^-3 M), an antagonist to group 1 metabotropic glutamate receptors. The δ-opioid receptor agonist D-Pen², D-Pen⁵-enkehalin (DPDPE, 10^-6 M) evoked [Ca²⁺]_i increases that were blocked by the δ-opioid antagonist ICI 174.388 (10^-5 M). The astrocytes failed to respond to 5-hydroxytryptamine (5-HT, 10^-5 M), although the same cells subsequently were found to respond to other agonists. Furthermore, [Ca²⁺]_i responses evoked by phenylephrine (10^-5 M) were blocked by prazosin (0.2⋅10^-6 M), suggesting the expression of α₁-adrenergic receptors on the acutely isolated astrocytes. The cells were also shown to react with [Ca²⁺]_i increases in response to depolarization with high extracellular potassium concentrations (50⋅10^-3 M). The signals induced by depolarization were not seen in Ca²⁺-free buffer, indicating the presence of voltage-activated calcium channels in these cells. Thus, the present study confirms some of the results earlier obtained in cell cultures, suggesting that cortical astrocytes in vivo express glutamate, opiate, and adrenergic receptors, coupled to increases in [Ca²⁺]_i, whereas no receptors for 5-HT could be detected. J. Neurosci. Res. 54:390–401, 1998. © 1998 Wiley-Liss, Inc.     

Abnormal calcium homeostasis in astrocytes from the trisomy 16 mouse

The regulation of intracellular Ca²⁺ was investigated in cultured astrocytes from the trisomy 16 (Ts16) mouse, an animal model for Down syndrome and Alzheimer's disease (AD). The cytoplasmic ionized Ca²⁺ concentration ([Ca²⁺]_cyt) was determined using digital imaging of fura-2-loaded cells. The relative Ca²⁺ content of internal endoplasmic reticulum (ER) stores was estimated from the magnitude of the transient increase in [Ca²⁺]_cyt induced by cyclopiazonic acid (CPA), an inhibitor of Ca²⁺ sequestration into ER stores. At rest, the average [Ca²⁺]_cyt was 140 nM in euploid (normal) astrocytes, but over twice as high, 320 nM, in Ts16 cells. In the absence of extracellular Ca²⁺, CPA induced a transient increase in [Ca²⁺]_cyt to over 1200 nM in Ts16 astrocytes as compared to only 500 nM in euploid cells, indicating an increased amount of Ca²⁺ in the Ts16 astrocyte ER. In contrast to euploid astrocytes, both resting [Ca²⁺]_cyt and the amount of Ca²⁺ in the ER stores varied widely among individual Ts16 astrocytes. These results show that Ts16 produces a dysregulation of Ca²⁺ homeostasis leading to increased cytoplasmic and stored Ca²⁺. Since increases in [Ca²⁺]_cyt have been implicated in the etiology of neurodegenerative diseases, including AD, this finding of abnormal Ca²⁺ homeostasis in a genetic model of human neurological disorders suggests that Ca²⁺ dysregulation may be a common feature underlying neurodegenerative processes. GLIA 19:352–368, 1997. © 1997 Wiley-Liss, Inc.     

Penicillin- and barium-induced epileptiform bursting in hippocampal neurons: Actions on Ca++ and K+ potentials

Both barium (Ba⁺⁺) and penicillin produce spontaneous epileptiform burst generation in hippocampal neurons in vitro. Recent investigations suggest that Ba⁺⁺ acts by both adding to a calcium (Ca⁺⁺)-mediated depolarization and reducing potassium (K⁺) conductance. In contrast, it has been proposed that pencillin produces burst generation by attenuating inhibitory postsynaptic potentials. However, some evidence suggests that penicillin may also directly alter intrinsic membrane properties. We therefore compared the actions of penicillin and Ba⁺⁺ on three intrinsic Ca⁺⁺- or K⁺-mediated membrane events, namely, Ca⁺⁺ spikes, Ca⁺⁺-dependent anomalous rectification, and K⁺-dependent afterhyperpolarization. Ba⁺⁺ augmented the Ca⁺⁺ potentials and attenuated the K⁺-dependent afterhyperpolarization: penicillin had no demonstrable effect on these events. Ba⁺⁺ produced rhythmical burst firing and oscillations of the membrane potentials, while penicillin caused sporadic burst generation followed by a longlasting afterhyperpolarization. Synchronized, orthodromically evoked burst firing occurred after exposure to penicillin but not to Ba⁺⁺. Ba⁺⁺ and penicillin are prototypes of agents which induce epileptogenesis in mammalian cortical neurons by two different but probably interrelated mechanisms. Ba⁺⁺ causes burst generation by disrupting a delicate balance between depolarizing Ca⁺⁺ potentials and repolarizing, hyperpolarizing K⁺ potentials. Pencillin does not affect Ca⁺⁺- or K⁺-mediated membrane events; other data suggest that it produces burst generation in hippocampal pyramidal neurons by attenuating γ-aminobutyric acid-mediated synaptic inhibition, which in turn ordinarily limits intrinsic bursting.     

Dendritic electrogenesis in rat hippocampal CA1 pyramidal neurons: functional aspects of Na+ and Ca2+ currents in apical dendrites

The regenerative properties of CA1 pyramidal neurons were studied through differential polarization with external electrical fields. Recordings were obtained from somata and apical dendrites in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino-5-phosphonovaleric acid (APV), and bicuculline. S+ fields hyperpolarized the distal apical dendrites and depolarized the rest of the cell, whereas S÷ fields reversed the polarization. During intradendritic recordings, S+ fields evoked either fast spikes or compound spiking. The threshold response consisted of a low-amplitude fast spike and a slow depolarizing potential. At higher field intensities the slow depolarizing potential increased in amplitude, and additional spikes of high amplitude appeared. During intrasomatic recordings, S+ field evoked repetitive firing of fast spikes, whereas S÷ fields evoked a slow depolarizing, potential on top of which high- and low-amplitude spikes were evoked. Tetrodotoxin (TTX) blocked all types of responses in both dendrites and somata. Perfusion with Ca²⁺-free, Co²⁺-containing medium increased the frequency and amplitude of fast spikes evoked by S+ field and substantially reduced the slow depolarizing potential evoked by S÷ fields. Antidromic stimulation revealed that an all-or-none dendritic component was activated in the distal apical dendrites by back-propagating somatic spikes. The dendritic component had an absolute refractory period of about 4 ms and a relative refractory period of 10–12 ms. Ca²⁺-dependent spikes in the dendrites were followed by a long-lasting afterhyperpolarization (AHP) and a decrease in membrane input resistance, during which dendritic excitability was selectively reduced. The data suggest that generation of fast Na⁺ currents and slow Ca²⁺ currents in the distal part of apical dendrites is highly sensitive to the dynamic state of the dendritic membrane. Depending on the mode and frequency of activation these currents can exert a substantial influence on the input-output behavior of the pyramidal neurons. © 1996 Wiley-Liss, Inc.     

Effects of trifluoperazine on synaptically evoked potentials and membrane properties of CA1 pyramidal neurons of rat hippocampus in situ and in vitro

The effects of trifluoperazine (TFP), a phenothiazine antipsychotic, on hippocampal activity were studied in the CA1 subfield, both in situ and in slices. In the extracellular studies in situ and in vitro, both somatic population spikes and dendritic excitatory postsynaptic potentials (EPSP) fields were depressed reversibly by TFP, applied by microiontophoresis or in the bath (50-100 μM). Similar effects were also seen during iontophoretic applications of sphingosine in situ. Like TFP (at micromolar concentrations) sphingosine is a dual Ca²⁺/calmodulin-dependent kinase and protein kinase C (PKC) inhibitor. In intracellular recordings from slices, 50-100 μM TFP induced a slow depolarization and a decrease in input resistance (R_N), probably through a β-aminobutyric acid (GABA)-mediated increase in Cl^? conductance (G_Cl). TFP also reduced the slow afterhyperpolarization (AHP) as well as electrically evoked inhibitory postsynaptic potentials (IPSPs), but EPSPs were augmented in both amplitude and duration. When CA1 neurons were voltage clamped, TFP elicited a corresponding inward current (consistent with depolarization), increased the leak conductance, and enhanced excitatory synaptic currents; whereas inhibitory synaptic currents and high-threshold Ca²⁺ currents were reduced. In conclusion, these effects of TFP–which cannot be readily explained by its potent antidopamine action–are in keeping with other evidence that both Ca²⁺/calmodulin-dependent kinase and PKC can modulate G_Cl-conductance and high-threshold Ca²⁺ -conductance, as well as inhibitory and excitatory postsynaptic currents. © 1993 Wiley-Liss, Inc.     

Glucocorticoids—potent modulators of astrocytic calcium signaling

Glucocorticoids are the first line of choice in the treatment of cerebral edema associated with brain tumors. High‐dose glucocorticoids reduce the extent of edema within hours, often relieving critical increases in intracranial pressure, but the mechanisms by which glucocorticoids modulate brain water content are not well‐understood. A possible target of action may be glucocorticoid receptor‐expressing astrocytes, which are the primary regulators of interstitial ion homeostasis in brain. In this study, we demonstrate that two glucocorticoids, methylprednisolone and dexamethasone, potentiate astrocytic signaling, via long‐range calcium waves. Glucocorticoid treatment increased both resting cytosolic calcium (Ca²⁺_i) level and the extent and amplitude of Ca²⁺ wave propagation two‐fold, compared to matched controls. RU‐486, a potent steroid receptor antagonist, inhibited the effects of methylprednisolone. The glucocorticoid‐associated potentiation of Ca²⁺ signaling may result from upregulation of the cellular ability to mobilize Ca²⁺ and release ATP, because both agonist‐induced Ca²⁺_i increments (via ATP and bradykinin) and ATP release were proportionally enhanced by glucocorticoids. In contrast, neither gap junction expression (as manifested connexin 43 immunoreactivity) nor functional coupling was significantly affected by methylprednisolone. Confocal microscopy revealed both the expression of glucocorticoid receptors and nuclear translocation of these receptors when exposed to methylprednisolone. We postulate that the edemolytic effects of glucocorticoids may result from enhanced astrocytic calcium signaling. GLIA 28:1–12, 1999. © 1999 Wiley‐Liss, Inc.     

Semaphorin 3A induces acute changes in membrane excitability in spiral ganglion neurons in vitro

The development and survival of spiral ganglion neurons (SGNs) are dependent on multiple trophic factors as well as membrane electrical activity. Semaphorins (Sema) constitute a family of membrane‐associated and secreted proteins that have garnered significant attention as a potential SGN “navigator” during cochlea development. Previous studies using mutant mice demonstrated that Sema3A plays a role in the SGN pathfinding. The mechanisms, however, by which Sema3A shapes SGNs firing behavior are not known. In these studies, we found that Sema3A plays a novel role in regulating SGN resting membrane potential and excitability. Using dissociated SGN from pre‐hearing (P3–P5) and post‐hearing mice (P12–P15), we recorded membrane potentials using whole‐cell patch clamp recording techniques in apical and basal SGN populations. Recombinant Sema3A was applied to examine the effects on intrinsic membrane properties and action potentials evoked by current injections. Apical and basal SGNs from newborn mice treated with recombinant Sema3A (100 ng/ml) displayed a higher resting membrane potential, higher threshold, decreased amplitude, and prolonged latency and duration of spikes. Although a similar phenomenon was observed in SGNs from post‐hearing mice, the resting membrane potential was essentially indistinguishable before and after Sema3A exposure. Sema3A‐mediated changes in membrane excitability were associated with a significant decrease in K⁺ and Ca²⁺ currents. Sema3A acts through linopirdine‐sensitive K⁺ channels in apical, but not in the basal SGNs. Therefore, Sema3A induces differential effects in SGN membrane excitability that are dependent on age and location, and constitutes an additional early and novel effect of Sema3A SGNs in vitro.     

Calcium electrogenesis in neocortical pyramidal neurons in vivo

Much of what is known about Ca²⁺ electrogenesis in neocortical cells has been derived from in vitro studies. Since Ca²⁺ currents are controlled by various modulators, comparing these findings to in vivo data is essential. Here, we analysed tetrodotoxin (TTX)-resistant, presumably Ca²⁺-mediated potentials in intracellularly recorded neocortical neurons in vivo. TTX was applied locally to block Na⁺ channels. Its effectiveness was demonstrated by the elimination of fast spikes and orthodromic responses. In response to depolarizing current pulses bringing the membrane potential beyond ≈–33 mV, 71% of neurons generated high-threshold Ca²⁺ spikes averaging 17 mV. This is in contrast with in vitro findings, where high-threshold spikes could only be elicited following the blockade of K⁺ conductances. Consistent with this, neurons dialysed with K⁺ channel blockers in vivo generated high-threshold spikes that had a lower threshold (≈–40 mV) and, with intracellular Cs⁺, a larger amplitude, indicating the presence of K⁺ currents opposing the activation of Ca²⁺ channels. Only 15% of cortical cells displayed low-threshold Ca²⁺ spikes. To compare high-threshold Ca²⁺ spikes evoked by synaptic stimuli or current injection, another group of cortical neurons was dialysed with QX-314 and Cs⁺, in the absence of extracellular TTX. Synaptic stimuli applied on a background of membrane depolarization elicited presumed Ca²⁺ spikes whose amplitude varied in a stepwise fashion. Thus, although there are numerous similarities between in vivo and in vitro data, some significant differences were found, which suggest that the high-voltage activated Ca²⁺ currents and/or the K⁺ conductances that oppose them are subjected to different modulatory influences in vivo than in vitro.     

Mechanosensory Signaling in Astrocytes

Mechanosensitivity is a well-known feature of astrocytes, however, its underlying mechanisms and functional significance remain unclear. There is evidence that astrocytes are acutely sensitive to decreases in cerebral perfusion pressure and may function as intracranial baroreceptors, tuned to monitor brain blood flow. This study investigated the mechanosensory signaling in brainstem astrocytes, as these cells reside alongside the cardiovascular control circuits and mediate increases in blood pressure and heart rate induced by falls in brain perfusion. It was found that mechanical stimulation-evoked Ca²⁺ responses in astrocytes of the rat brainstem were blocked by (1) antagonists of connexin channels, connexin 43 (Cx43) blocking peptide Gap26, or Cx43 gene knock-down; (2) antagonists of TRPV4 channels; (3) antagonist of P2Y₁ receptors for ATP; and (4) inhibitors of phospholipase C or IP3 receptors. Proximity ligation assay demonstrated interaction between TRPV4 and Cx43 channels in astrocytes. Dye loading experiments showed that mechanical stimulation increased open probability of carboxyfluorescein-permeable membrane channels. These data suggest that mechanosensory Ca²⁺ responses in astrocytes are mediated by interaction between TRPV4 and Cx43 channels, leading to Cx43-mediated release of ATP which propagates/amplifies Ca²⁺ signals via P2Y₁ receptors and Ca²⁺ recruitment from the intracellular stores. In astrocyte-specific Cx43 knock-out mice the magnitude of heart rate responses to acute increases in intracranial pressure was not affected by Cx43 deficiency. However, these animals displayed lower heart rates at different levels of cerebral perfusion, supporting the hypothesis of connexin hemichannel-mediated release of signaling molecules by astrocytes having an excitatory action on the CNS sympathetic control circuits.SIGNIFICANCE STATEMENT There is evidence suggesting that astrocytes may function as intracranial baroreceptors that play an important role in the control of systemic and cerebral circulation. To function as intracranial baroreceptors, astrocytes must possess a specialized membrane mechanism that makes them exquisitely sensitive to mechanical stimuli. This study shows that opening of connexin 43 (Cx43) hemichannels leading to the release of ATP is the key central event underlying mechanosensory Ca²⁺ responses in astrocytes. This astroglial mechanism plays an important role in the autonomic control of heart rate. These data add to the growing body of evidence suggesting that astrocytes function as versatile surveyors of the CNS metabolic milieu, tuned to detect conditions of potential metabolic threat, such as hypoxia, hypercapnia, and reduced perfusion.     

Spontaneous and Repetitive Calcium Transients in C2C12 Mouse Myotubes during In Vitro Myogenesis

Fluorescence videomicroscopy was used to monitor changes in the cytosolic free Ca²⁺ concentration ([Ca²⁺]i in the mouse muscle cell line C2C12 during in vitro myogenesis. Three different patterns of changes in [Ca²⁺]i were observed: (i) [Ca²⁺]i oscillations; (ii) faster Ca²⁺ events confined to subcellular regions (localized [Ca²⁺]i spikes) and (iii) [Ca²⁺]i spikes detectable in the entire myotube (global [Ca²⁺]i spikes). [Ca²⁺]i oscillations and localized [Ca²⁺]i spikes were detectable following the appearance of caffeine-sensitivity in differentiating C2C12 cells. Global [Ca²⁺]i spikes appeared later in the process of myogenesis in cells exhibiting coupling between voltage-operated Ca²⁺ channels and ryanodine receptors. In contrast to [Ca²⁺]i oscillations and localized [Ca²⁺]i spikes, the global events immediately stopped when cells were perfused either with a Ca²⁺-free solution, or a solution with TTX, TEA and verapamil. To explore further the mechanism of the global [Ca²⁺]i spikes, membrane currents and fluorescence signals were measured simultaneously. These experiments revealed that global [Ca²⁺]i spikes were correlated with an inward current. Moreover, while the depletion of the Ca²⁺ stores blocked [Ca²⁺]i oscillations and localized [Ca²]i spikes, it only reduced the amplitude of global [Ca²⁺]i spikes. It is suggested that, during the earlier stages of the myogenesis, spontaneous and repetitive [Ca²⁺]i changes may be based on cytosolic oscillatory mechanisms. The coupling between voltage-operated Ca²⁺ channels and ryanodine receptors seems to be the prerequisite for the appearance of global [Ca²⁺]i spikes triggered by a membrane oscillatory mechanism, which characterizes the later phases of the myogenic process.