Carmela Rapisarda

Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes.

Cell-cell communication in astroglial syncytia is mediated by intracellular Ca(2+) ([Ca(2+)](i)) responses elicited by extracellular signaling molecules as well as by diverse physical and chemical stimuli. Despite the evidence that astrocytic swelling promotes [Ca(2+)](i) elevation through Ca(2+) influx, the molecular identity of the channel protein underlying this response is still elusive. Here we report that primary cultured cortical astrocytes express the transient receptor potential vanilloid-related channel 4 (TRPV 4), a Ca(2+)-permeable cation channel gated by a variety of stimuli, including cell swelling. Immunoblot and confocal microscopy analyses confirmed the presence of the channel protein and its localization in the plasma membrane. TRPV4 was functional because the selective TRPV4 agonist 4-alpha-phorbol 12,13-didecanoate (4alphaPDD) activated an outwardly rectifying cation current with biophysical and pharmacological properties that overlapped those of recombinant human TRPV4 expressed in COS cells. Moreover, 4alphaPDD and hypotonic challenge promoted [Ca(2+)](i) elevation mediated by influx of extracellular Ca(2+). This effect was abolished by low micromolar concentration of the TRPV4 inhibitor Ruthenium Red. Immunofluorescence and immunogold electron microscopy of rat brain revealed that TRPV4 was enriched in astrocytic processes of the superficial layers of the neocortex and in astrocyte end feet facing pia and blood vessels. Collectively, these data indicate that cultured cortical astroglia express functional TRPV4 channels. They also demonstrate that TRPV4 is particularly abundant in astrocytic membranes at the interface between brain and extracerebral liquid spaces. Consistent with its roles in other tissues, these results support the view that TRPV4 might participate in astroglial osmosensation and thus play a key role in brain volume homeostasis.

TWO DISTINCT INWARDLY RECTIFYING CONDUCTANCES ARE EXPRESSED IN LONG TERM DIBUTYRYL-CYCLIC-AMP TREATED RAT CULTURED CORTICAL ASTROCYTES

Long term incubation (1–3 weeks) with 250 μM dibutyryl‐cyclic‐AMP (dBcAMP) of pure cultured cortical astrocytes from newborn rats leads to the expression of voltage‐dependent, inward‐rectifying potassium (K+) and chloride (Cl−) currents which are lacking in shortly treated (4–24 h) and in control cultured astrocytes. Both conductances are already activated at the holding potential of −60 mV and are distinguishable for their gating kinetics and pharmacological sensitivity. K+ currents have a fast activation kinetic and show a time‐ and voltage‐dependent inactivation at potentials negative to −120 mV. The conductive property of the K+ currents increases upon elevation of the extracellular K+ concentration ([K+]0) and they are reversibly blocked by extracellular 0.1 mM barium ions (Ba2+). Cl− currents are activated only at negative membrane potentials; they display a slow activation kinetic, no time‐dependent inactivation and are not affected by 0.1 mM Ba2+. In individual astrocyte the K+ and Cl− conductances can be expressed singularly or in combination. The results indicate that the expression of these two conductances is controlled by a cAMP‐dependent molecular signalling, presumably by regulating a late gene activation. Thus, the strengthening of this signalling would contribute to promote the maturation of less differentiated astrocytes in culture, implicating the expression of K+ and Cl− membrane conductances which may operate together in the regulation of [K+]0 homeostasis via the mechanism of the local accumulation.

Characterization of an inwardly rectifying chloride conductance expressed by cultured rat cortical astrocytes.

The biophysical and pharmacological properties of the inwardly rectifying Cl− conductance (IClh), expressed in rat type‐1 neocortical cultured astrocytes upon a long‐term treatment (1–3 weeks) with dibutyryl‐cyclic‐AMP (dBcAMP), were investigated with the whole‐cell patch‐clamp technique. Using intra‐ and extra‐cellular solutions with symmetrical high Cl− content and with the monovalent cations replaced with N‐methyl‐D‐glucamine, time‐ and voltage‐dependent Cl− currents were elicited in response to hyperpolarizing voltage steps from a holding potential of 0 mV. The inward currents activated slowly and did not display any time‐dependent inactivation. The rising phase of the current traces was best fitted with two exponential components whose time constants decreased with larger hyperpolarization. The steady‐state activation of IClh was well described by a single Boltzmann function with a half‐maximal activation potential at −62 mV and a slope of 19 mV that yields to an apparent gating charge of 1.3. The anion selectivity sequence was Cl− = Br− = I− > F− > cyclamate ≥ gluconate. External application of the putative Cl− channel blockers 4,4 diisothiocyanatostilbene‐2,2 disulphonic acid or 4‐acetamido‐4‐isothiocyanatostilbene‐2,2‐disulphonic acid did not affect IClh. By contrast, anthracene‐9‐carboxylic acid, as well as Cd2+ and Zn2+, inhibited, albeit with different potencies, the Cl− current. Taken together, these results indicate that dBcAMP‐treated cultured rat cortical astrocytes express a Cl− inward rectifier, which exhibits similar but not identical features compared with those of the cloned and heterologously expressed hyperpolarization‐activated Cl− channel ClC‐2. GLIA 21:217–227, 1997.

Functional down-regulation of volume-regulated anion channels in AQP4 knockdown cultured rat cortical astrocytes

In the brain, the astroglial syncytium is crucially involved in the regulation of water homeostasis. Accumulating evidence indicates that a dysregulation of the astrocytic processes controlling water homeostasis has a pathogenetic role in several brain injuries. Here, we have analysed by RNA interference technology the functional interactions occurring between the most abundant water channel in the brain, aquaporin‐4 (AQP4), and the swelling‐activated Cl– current expressed by cultured rat cortical astrocytes. We show that in primary cultured rat cortical astrocytes transfected with control small interfering RNA (siRNA), hypotonic shock promotes an increase in cellular volume accompanied by augmented membrane conductance mediated by volume‐regulated anion channels (VRAC). Conversely, astroglia in which AQP4 was knocked down (AQP4 KD) by transfection with AQP4 siRNA changed their morphology from polygonal to process‐bearing, and displayed normal cell swelling but reduced VRAC activity. Pharmacological manipulations of actin cytoskeleton in rat astrocytes, and functional analysis in mouse astroglial cells, which retain their morphology upon knockdown of AQP4, suggest that stellation of AQP4 KD rat cortical astrocytes was not causally linked to reduction of VRAC current. Molecular analysis of possible candidates of swelling‐activated Cl– current provided evidence that in AQP4 KD astrocytes, there was a down‐regulation of chloride channel‐2 (CIC‐2), which, however, was not involved in VRAC conductance. Inclusion of ATP in the intracellular saline restored VRAC activity upon hypotonicity. Collectively, these results support the view that in cultured astroglial cells, plasma membrane proteins involved in cell volume homeostasis are assembled in a functional platform.

Hippocampal output and sensory mechanisms.

Abstract This study was performed with the aim of clarifying the mechanisms of the influence that the hippocampal output during theta rhythm exerts on sensory processes. The hippocampal output was simulated by low-frequency stimulation (single trains of 5 rectangular pulses: 5/sec, 0.5 msec, 3–9 V) of the dorsal part of the hippocampal formation. The changes induced by such output in the responsiveness of primary auditory areas to clicks were studied in curarized cats. Primary auditory responses ipsilateral and contralateral with respect to the hippocampus stimulated were modified when the interval between the last stimulus of the train and clicks ranged from 20 to 45 msec and from 30 to 55 msec respectively. The changes in the contralateral auditory response depended on the fact that stimulation of one hippocampus also involved the commissural activation of the other. Signs of hippocampal facilitation were not observed in medial geniculate responses to clicks. Coagulation of the fornix or of non-specific thalamic nuclei strongly depressed or suppressed the hippocampal influence on auditory responses. Laminar analysis of the small surface positive-negative responses elicited in primary auditory areas by train stimulation of the hippocampus alone showed that the hippocampal output activated the superficial layers of the neocortex.

Single-channel analysis of a ClC-2-like chloride conductance in cultured rat cortical astrocytes

The single‐channel behavior of the hyperpolarization‐activated, ClC‐2‐like inwardly rectifying Cl− current (I Clh), induced by long‐term dibutyryl‐cyclic‐AMP‐treated cultured cortical rat astrocytes, was analyzed with the patch‐clamp technique. In outside‐out patches in symmetrical 144 mM Cl− solutions, openings of hyperpolarization‐activated small‐conductance Cl− channels revealed burst activity of two equidistant conductance levels of 3 and 6 pS. The unitary openings displayed slow activation kinetics. The probabilities of the closed and conducting states were consistent with a double‐barrelled structure of the channel protein. These results suggest that the astrocytic ClC‐2‐like Cl− current I Clh is mediated by a small‐conductance Cl− channel, which has the same structural motif as the Cl− channel prototype ClC‐0.

Arachidonic acid activates an open rectifier potassium channel in cultured rat cortical astrocytes.

A pathophysiological increase in free arachidonic acid (AA) is thought to regulate the channel‐mediated astrocytic swelling occurring in several brain injuries. We report that in cultured rat type‐1 cortical astrocytes, exposure to 10 μM AA activates an open rectifier K+ channel, which exhibits many similarities with TREK/TRAAK members of the two‐pore‐domain K+ channel family KCNK. Patch‐clamp experiments showed that the current developed with a long latency and was preceded by a depression of the previously described outward rectifier K+ conductance. Pharmacologic studies indicate that the K+ open rectifier was differentially sensitive to classic K+‐channel blockers (quinine, quinidine, tetraethylammonium, and barium) and was inhibited potently by gadolinium ions. The activation of this K+ current occurred independently of the AA metabolism as pharmacologic inhibition of the lipoxygenase, cyclooxygenase, and cytochrome P450 epoxygenase signaling cascades did not alter the AA effect. Moreover, neither the neutralization of the NADPH‐oxidase pathway nor scavenging intracellular free radicals modified the AA response. Finally, the AA‐induced K+ current was unaffected by protein kinase C inhibitors. The activation mechanism of the K+ open rectifier was through an extracellular interaction of AA with the plasma membrane. RT‐PCR analysis revealed that the AA‐induced K+ conductance was mediated likely by TREK‐2 channels. Collectively, the results demonstrate that in cultured cortical astrocytes, pathological levels of AA directly activate an open rectifier K+ channel, which may play a role in the control of K+ homeostasis under pathophysiological conditions.

Guanosine promotes the up-regulation of inward rectifier potassium current mediated by Kir4.1 in cultured rat cortical astrocytes

Guanosine (Guo) is an endogenous neuroprotective molecule of the CNS, which has various acute and long‐term effects on both neurones and astroglial cells. Whether Guo also modulates the activity/expression of ion channels involved in homeostatic control of extracellular potassium by the astrocytic syncytium is still unknown. Here we provide electrophysiological evidence that chronic exposure (48 h) to Guo (500 μm) promotes the functional expression of an inward rectifier K+ (Kir) conductance in primary cultured rat cortical astrocytes. Molecular screening indicated that Guo promotes the up‐regulation of the Kir4.1 channel, the major component of the Kir current in astroglia in vivo. Furthermore, the properties of astrocytic Kir current overlapped those of the recombinant Kir4.1 channel expressed in a heterologous system, strongly suggesting that the Guo‐induced Kir conductance is mainly gated by Kir4.1. In contrast, the expression levels of two other Kir channel proteins were either unchanged (Kir2.1) or decreased (Kir5.1). Finally, we showed that inhibition of translational process, but not depression of transcription, prevents the Guo‐induced up‐regulation of Kir4.1, indicating that this nucleoside acts through de novo protein synthesis. Because accumulating data indicate that down‐regulation of astroglial Kir current contributes to the pathogenesis of neurodegenerative diseases associated with dysregulation of extracellular K+ homeostasis, these results support the notion that Guo might be a molecule of therapeutic interest for counteracting the detrimental effect of K+‐buffering impairment of the astroglial syncytium that occurs in pathological conditions.

Multiple Representations of the Body and Input-Output Relationships in the Agranular and Granular Cortex of the Chronic Awake Guinea Pig

The organization of somatosensory input and the input-output relationships in regions of the agranular frontal cortex (AGr) and granular parietal cortex (Gr) were examined in the chronic awake guinea pig, using the combined technique of single-unit recording and intracortical microstimulation (ICMS). AGr, which was cytoarchitectonically subdivided into medial (AGrm) and lateral (AGrl) parts, also can be characterized on a functional basis. AGrl contains the head, forelimb, and most hindlimb representations; only a small number of hindlimb neurons are confined in AGrm. Different distributions of submodalities exist in AGr and Gr: AGr receives predominantly deep input (with the exception of the vibrissa region, which receives cutaneous input), whereas neurons of Gr respond almost exclusively to cutaneous input. The cutaneous or deep receptive field (RF) of each neuron was determined by natural peripheral stimulation. All studied neurons were activated by small RFs, with the exception of lip, nose, pinna, and limb units of lateral Gr (Grl), for which the RFs were larger. Microelectrode mapping experiments revealed the existence of three spatially separate, incomplete body maps in which somatosensory and motor representations overlap. One body map, with limbs medially and head rostrolaterally, is contained in AGr. A second map, comparable to the first somatosensory cortex (SI) of other mammals, is found in Gr, with hindlimb, trunk, forelimb, and head representations in an orderly mediolateral sequence. An unresponsive zone separates the head area from the forelimb region. A third map, with the forelimb rostrally and the hindlimb caudally, lies adjacent and lateral to the SI head area. This limb representation, which is characterized by an upright and small size compared to that found in SI, can be considered to be part of the second somatosensory cortex (SII). A distinct head representation was not recognized as properly belonging to SII, but the evidence that neurons of the SI head region respond to stimulation of large RFs located in lips, nose, and pinna leads us to hypothesize that the SII face area overlaps that of SI to some extent, or, alternatively, that the two areas strictly contiguous and the limits are ambiguous, making them difficult to distinguish. The input-output relationships were based on the results of RF mapping and ICMS in the same electrode penetration. The intrinsic specific interconnections of cortical neurons whose afferent input and motor output is related to identical body regions show a considerable degree of refinement.(ABSTRACT TRUNCATED AT 400 WORDS)

pH modulation of an inward rectifier chloride current in cultured rat cortical astrocytes

The effects of changes in extra- and intracellular pH in the pathophysiological range (6.0-8.0) on astroglial plasma membrane ionic currents were investigated with the whole-cell patch-clamp technique. In cultured rat neocortical type-1 astrocytes differentiated by a long-term treatment with dibutyryl cyclic-AMP, exposure to an extracellular pH of 6.4 induced, as compared with the control extracellular pH at 7.3, a sustained and reversible increase in the holding current at -60mV. The rise in current was accompanied by a decrease in the apparent input resistance. Ion substitution experiments indicated that extracellular pH 6.4 upregulated the resting Cl(-) conductance, whereas an opposite effect could be observed at extracellular pH 8.0. Recordings of isolated Cl(-) currents showed that this modulation occurred on the previously identified hyperpolarization-activated, inwardly rectifying Cl(-) current, I(Clh). Extracellular acidification to pH 6.4 shifted the voltage dependence of I(Clh) activation by approximately 20mV towards more positive potentials, whereas a approximately 20mV opposite shift was observed upon exposure to extracellular pH 8.0. These effects were paralleled by an increase (extracellular pH 6.4) or decrease (extracellular pH 8.0) in the maximal conductance. Decreasing (6.0) or increasing (8.0) the intracellular pH shifted the steady-state activation of I(Clh) towards more negative or positive potentials, respectively, leaving unchanged the current sensitivity to extracellular pH modifications. The modulation of the inward rectifier Cl(-) current expressed by differentiated cultured neocortical astrocytes indicates that extra- and intracellular changes in pH occurring in a pathophysiological range may contribute to regulating Cl(-) accumulation in astroglial cells.