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Neuron-microglia communication through the Cx3cr1-Cx3cl1 axis is essential for the development and refinement of neural circuits, which determine their function into adulthood. In the present work we set out to extend the behavioral characterization of Cx3cr1

The diathesis–stress model of schizophrenia posits that a constitutive factor increases the vulnerability to secondary stressors. Alterations in neuron–microglia communication through the fractalkine pathway is a potential predisposing factor. Wild-type (WT) and Cx3cr1−/− (KO) mice of both sexes randomly received either a low (0.5 mg/kg) or high dose (1 mg/kg) of MK-801 or saline during early postnatal development. Neuronal apoptosis was assessed at a midpoint of the pharmacological protocol. Survival and growth rates were determined up to adulthood when innate behaviors, unconditioned anxiety, contextual memory and seizure susceptibility were evaluated, as well as hippocampal local field potential and sensory gating. Fractalkine receptor (CX3CR1) depletion and MK-801 treatment had a synergistic effect, increasing neuronal apoptosis and overall mortality. Both factors independently induced long-lasting cognitive impairments in the wide array of tasks assessed. Low MK-801 dose treatment greatly augmented the mortality of pentylenetetrazol-induced seizures in WT mice, an effect prevented by CX3CR1 depletion. MK-801 treatment induced a shift in the power spectrum of the hippocampal local field potential towards higher frequencies that was averted in Cx3cr1−/− mice by an opposite shift. CX3CR1 depletion severely increases the vulnerability to neonatal NMDA antagonism with additional complex interactions regarding cognitive and neurophysiological effects.

Background: Deficits in odor detection and discrimination are premature symptoms of Alzheimer’s disease (AD) that correlate with pathological signs in the olfactory bulb (OB) and piriform cortex (PCx). Similar olfactory dysfunction has been characterized in AD transgenic mice that overproduce amyloid-β peptide (Aβ), which can be prevented by reducing Aβ levels by immunological and pharmacological means, suggesting that olfactory dysfunction depends on Aβ accumulation and Aβ-driven alterations in the OB and/or PCx, as well as on their activation. However, this possibility needs further exploration. Objective: To characterize the effects of Aβ on OB and PCx excitability/coupling and on olfaction. Methods: Aβ oligomerized solution (containing oligomers, monomers, and protofibrils) or its vehicle were intracerebroventricularlly injected two weeks before OB and PCx excitability and synchrony were evaluated through field recordings in vivo and in brain slices. Synaptic transmission from the OB to the PCx was also evaluated in slices. Olfaction was assessed through the habituation/dishabituation test. Results: Aβ did not affect lateral olfactory tract transmission into the PCx but reduced odor habituation and cross-habituation. This olfactory dysfunction was related to a reduction of PCx and OB network activity power in vivo. Moreover, the coherence between PCx-OB activities was also reduced by Aβ. Finally, Aβ treatment exacerbated the 4-aminopyridine-induced excitation in the PCx in slices. Conclusion: Our results show that Aβ-induced olfactory dysfunction involves a complex set of pathological changes at different levels of the olfactory pathway including alterations in PCx excitability and its coupling with the OB. These pathological changes might contribute to hyposmia in AD.

Transgenic mice have been used to make valuable contributions to the field of neuroscience and model neurological diseases. The simultaneous functional analysis of hippocampal cell activity combined with hippocampal dependent innate task evaluations provides a reliable experimental approach to detect fine changes during early phases of neurodegeneration. To this aim, we used a merge of patch-clamp with two hippocampal innate behavior tasks. With this experimental approach, whole-cell recordings of CA1 pyramidal cells, combined with hippocampal-dependent innate behaviors, have been crucial for evaluating the early mechanism of neurodegeneration and its consequences. Here, we present our protocol for ex vivo whole-cell recordings of CA1 pyramidal cells and hippocampal dependent innate behaviors in an adolescent (p30) mice.

Hydrogen peroxide (H2O2) is a messenger involved in both damaging neuroinflammatory responses and physiological cell communication. The ventrolateral medulla, which regulates several vital functions including breathing and blood pressure, is highly influenced by hydrogen peroxide, whose extracellular levels could be determined by hypoxia and microglial activity, both of which modulate ventrolateral medulla function. Therefore, in this study we aimed to test whether different patterns of hypoxia and/or putative microglial modulators change extracellular hydrogen peroxide in the ventrolateral medulla by using an enzymatic reactor online sensing procedure specifically designed for this purpose. With this new technique, we detected extracellular levels of hydrogen peroxide in the ventrolateral medulla in vitro, which spontaneously fluctuated. These fluctuations are reduced by minocycline, a putative microglial inhibitor, and by the microglial toxin liposomal clodronate. Suitably, lipopolysaccharide increases extracellular hydrogen peroxide, while minocycline and liposomal clodronate reduce this increase. Application of blue light to slices with microglia expressing channelrhodopsin-2 also increases extracellular hydrogen peroxide. Moreover, long-lasting and intermittent hypoxia (as well as subsequent reoxygenation) increase extracellular hydrogen peroxide to similar levels, which is partially prevented by minocycline. The effect of long-lasting hypoxia was reproduced in vivo. Overall, our data show that changes in oxygen concentration, and possibly microglial function, modulate extracellular H2O2 levels in the ventrolateral medulla, which could influence the function of this neural circuit under normal and pathological conditions related to inflammation and/or hypoxia.

Tau hyperphosphorylation at several sites, including those close to the microtubule domain region (MDr), is considered a key pathological event in the development of Alzheimer's disease (AD). Recent studies indicate that at the very early stage of this disease, increased phosphorylation in Tau's MDr domain correlates with reduced levels of neuronal excitability. Mechanistically, we show that pyramidal neurons and some parvalbumin-positive interneurons in 1-month-old triple-transgenic AD mice accumulate hyperphosphorylated Tau protein and that this accumulation correlates with changes in theta oscillations in hippocampal neurons. Pyramidal neurons from young triple-transgenic AD mice exhibited less spike accommodation and power increase in subthreshold membrane oscillations. Furthermore, triple-transgenic AD mice challenged with the potassium channel blocker 4-aminopyridine had reduced theta amplitude compared with 4-aminopyridine–treated control mice and, unlike these controls, displayed no seizure-like activity after this challenge. Collectively, our results provide new insights into AD pathogenesis and suggest that increases in Tau phosphorylation at the initial stages of the disease represent neuronal responses that compensate for brain circuit overexcitation.

Chronic intermittent hypoxia (CIH) is the most distinct feature of obstructive sleep apnea (OSA), a common breathing and sleep disorder that leads to several neuropathological consequences, including alterations in the hippocampal network and in seizure susceptibility. However, it is currently unknown whether these alterations are permanent or remit upon normal oxygenation. Here, we investigated the effects of CIH on hippocampal spontaneous network activity and hyperexcitability in vitro and explored whether these alterations endure or fade after normal oxygenation. Results showed that applying CIH for 21 days to adult rats increases gamma-band hippocampal network activity and aggravates 4-Aminopyridine-induced epileptiform activity in vitro. Interestingly, these CIH-induced alterations remit after 30 days of normal oxygenation. Our findings indicate that hippocampal network alterations and increased seizure susceptibility induced by CIH are not permanent and can be spontaneously reverted, suggesting that therapeutic interventions against OSA in patients with epilepsy, such as surgery or continuous positive airway pressure (CPAP), could be favorable for seizure control.

Soluble amyloid beta peptide (Aβ) is responsible for the early cognitive dysfunction observed in Alzheimer's disease. Both cholinergically and glutamatergically induced hippocampal theta rhythms are related to learning and memory, spatial navigation, and spatial memory. However, these two types of theta rhythms are not identical; they are associated with different behaviors and can be differentially modulated by diverse experimental conditions. Therefore, in this study, we aimed to investigate whether or not application of soluble Aβ alters the two types of theta frequency oscillatory network activity generated in rat hippocampal slices by application of the cholinergic and glutamatergic agonists carbachol or DHPG, respectively. Due to previous evidence that oscillatory activity can be differentially affected by different Aβ peptides, we also compared Aβ25−35 and Aβ1−42 for their effects on theta rhythms in vitro at similar concentrations (0.5 to 1.0 μM). We found that Aβ25−35 reduces, with less potency than Aβ1−42, carbachol-induced population theta oscillatory activity. In contrast, DHPG-induced oscillatory activity was not affected by a high concentration of Aβ25−35 but was reduced by Aβ1−42. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of exerting a generalized inhibitory effect on neuronal network function.

It is accepted that the main- and accessory- olfactory systems exhibit overlapping responses to pheromones and odorants. We performed whole-cell patch-clamp recordings in adult rat olfactory bulb slices to define a possible interaction between the first central relay of these systems: the accessory olfactory bulb (AOB) and the main olfactory bulb (MOB). This was tested by applying electrical field stimulation in the dorsal part of the MOB while recording large principal cells (LPCs) of the anterior AOB (aAOB). Additional recordings of LPCs were performed at either side of the plane of intersection between the aAOB and posterior-AOB (pAOB) halves, or linea alba, while applying field stimulation to the opposite half. A total of 92 recorded neurons were filled during whole-cell recordings with biocytin and studied at the light microscope. Neurons located in the aAOB (n = 6, 8%) send axon collaterals to the MOB since they were antidromically activated in the presence of glutamate receptor antagonists (APV and CNQX). Recorded LPCs evoked orthodromic excitatory post-synaptic responses (n = 6, aAOB; n = 1, pAOB) or antidromic action potentials (n = 8, aAOB; n = 7, pAOB) when applying field stimulation to the opposite half of the recording site (e.g., recording in aAOB; stimulating in pAOB, and vice-versa). Observation of the filled neurons revealed that indeed, LPCs send axon branches that cross the linea alba to resolve in the internal cellular layer. Additionally, LPCs of the aAOB send axon collaterals to dorsal-MOB territory. Notably, while performing AOB recordings we found a sub-population of neurons (24% of the total) that exhibited voltage-dependent bursts of action potentials. Our findings support the existence of: 1. a direct projection from aAOB LPCs to dorsal-MOB, 2. physiologically active synapses linking aAOB and pAOB, and 3. pacemaker-like neurons in both AOB halves. This work was presented in the form of an Abstract on SfN 2014 (719.14/EE17).

Oscillatory activity in the entorhinal cortex has been associated with several cognitive functions. Accordingly, Alzheimer Disease-associated cognitive decline has been related to amyloid beta-induced disturbances in several of these oscillatory patterns. We have previously shown that acute application of amyloid beta inhibits the generation of slow frequency oscillations (7-20 Hz). In contrast, alterations in faster oscillations recorded in Alzheimer Disease-transgenic mice that over-express amyloid beta have been controversial. Since transgenic mice may produce complex responses due to compensatory mechanisms, we tested the effect of acute application of amyloid beta on fast oscillations (beta-gamma bursts) generated by entorhinal cortex slices in vitro in a Mg2+ -ree solution. We also explored the participation of the enzyme glycogen synthase kinase 3 (GSK-3) in this effect. Our results show that bath application of a clinically relevant concentration of amyloid beta (10 nM) activates GSK-3 and reduces the power of beta-gamma bursts in the entorhinal cortex. The reduction of beta-gamma bursts by amyloid beta is blocked by inhibiting GSK-3 either with lithium or with SB 216763. Our results suggest that amyloid beta-induced inhibition of entorhinal cortex beta-gamma activity involves GSK-3 activation, which may provide a molecular mechanism for amyloid beta-induced neural network disruption and support the use of GSK-3 inhibitors to treat Alzheimer Disease.

a b s t r a c t Breathing and the activity of its generator (the pre-Botzinger complex; pre-BotC) are highly regulated functions. Among neuromodulators of breathing, somatostatin (SST) is unique: it is synthesized by a sub- set of glutamatergic pre-BotC neurons, but acts as an inhibitory neuromodulator. Moreover, SST regulates breathing both in normoxic and in hypoxic conditions. Although it has been implicated in the neuromod- ulation of breathing, neither the locus of SST modulation, nor the receptor subtypes involved have been identified. In this study, we aimed to fill in these blanks by characterizing the SST-induced regulation of inspiratory rhythm generation in vitro and in vivo. We found that both endogenous and exogenous SST depress all preBotC-generated rhythms. While SST abolishes sighs, it also decreases the frequency and increases the regularity of eupnea and gasping. Pharmacological experiments showed that SST modulates inspiratory rhythm generation by activating SST receptor type-2, whose mRNA is abundantly expressed in the pre-Botzinger complex. In vivo, blockade of SST receptor type-2 reduces gasping amplitude and consequently, it precludes auto-resuscitation after asphyxia. Based on our findings, we suggest that SST functions as an inhibitory neuromodulator released by excitatory respiratory neurons when they become overactivated in order to stabilize breathing rhythmicity in normoxic and hypoxic conditions.

Alzheimer disease (AD) patients show alterations in both neuronal network oscillations and the cognitive processes associated to them. Related to this clinical observation, it has been found that amyloid beta protein (Abeta) differentially affects some hippocampal network activities, reducing theta and gamma oscillations, without affecting sharp waves and ripples. Beta-like oscillations is another cognitive-related network activity that can be evoked in hippocampal slices by several experimental manipulations, including bath application of kainate and increasing extracellular potassium. Here, we tested whether or not different Abeta peptides differentially affect beta-like oscillatory patterns. We specifically tested the effects of fresh dissolved Abeta(25-35) and oligomerized Abeta(1-42) and found that kainate-induced oscillatory network activity was affected, in a slightly concentration dependent-manner, by both fresh dissolved (mostly monomeric) Abeta(25-35) and oligomeric Abeta(1-42). In contrast, potassium-induced oscillatory activity, which is reduced by oligomeric Abeta(1-42), is not affected by monomeric Abeta(25-35) at any of the concentrations tested. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of a generalized inhibitory effect of Abeta peptides on neuronal network function.

Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (-1.8 mOsm/min) maintained constant volume up to -50% external osmolarity (pi(o)), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl-, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, D-[3H]aspartate (as marker for glutamate) of pi(o) -2% and -19%, respectively, and more delayed thresholds of -30% for [3H]glycine and -25% and -29%, respectively, for Cl- (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five-fold over other amino acids) and the largest cell content decrease. Taurine and Cl- efflux were abolished by niflumic acid and 86Rb by 15 mM Ba2+. Niflumic acid essentially prevented IVR in all ranges of pi(o). Cl--free medium impaired IVR when pi(o) decreased to -24% and Ba2+ blocked it only at a late phase of -30% pi(o). These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of pi(o) is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl- and amino acids, particularly taurine; (iii) Cl- contribution to IVR is delayed with respect to other niflumic acid-sensitive osmolyte fluxes (osmolarity threshold of -25% pi(o)); and (iv), K+ fluxes do not contribute to IVR until a late phase (< -30% pi(o)).

Non-selective cationic channels (NSCC) are a heterogeneous family of channels, widely expressed in non-excitable and excitable cells, that share several functional characteristics but have diverse molecular origin. NSCC can be formed by transient receptor potential (TRP) channels, calcium activated non-selective channels, hyperpolarization activated cation currents, acid-sensitive cationic channels (ASIC), etc. As a result of its wide expression, as well as to the fact that the activation of such currents produce a persistent membrane depolarization, NSCC have been involved in a variety of neuronal processes such as signal transduction, firing pattern (including plateau potentials and bursting mechanisms) as well as synaptic transmission. Due to the relevance of such channels, alterations in their normal function have been associated with the pathophysiology of several nervous system diseases. Over the last years several blockers of such channels have been discovered. Here we review the pharmacology of NSCC blockers including trivalent cations, verapamil derivates, flufenamic acid, the "typical" TRP blockers 2-APB, ACA and SKF 96365 as well as ASIC blockers. This review focuses on the pharmacological properties of such drugs and their potential use for the understanding of the nervous system as well as for the treatment of neurological diseases.

TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 μm CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 ± 0.4 to 1.56 ± 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 μm) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.

Exposure of cultured cerebellar granule neurons (24 h serum-starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non-receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl-inositol-3-kinase (PI3K). The hyposmotic-induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85-95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co-immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84-91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4-Src-PI3K signaling cascade is indicated by the 48-66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.

Exposure of cultured Swiss 3T3 fibroblasts to 35% hyposmotic solution activated epidermal growth factor receptor (EGFR) phosphorylation to a greater extent than the ligand, EGF. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) had the same effect. EGFR phosphorylation seems to be involved in the transduction signalling for hyposmotically induced taurine release, as suggested by the latter's reduction when EGFR phosphorylation was blocked by 50 microM AG213 or AG112 and, conversely, its potentiation by EGF (200 ng/ml). The relationship between hyposmotically induced taurine efflux and reduced osmolarity showed saturable kinetics, following a sigmoidal function. EGF shifted the relationship to the left, implying an increase in sensitivity to hyposmolarity. EGF increased taurine efflux only marginally under isosmotic conditions. EGF and agglutinins also potentiated the hyposmotically induced release of 86Rb but, in contrast to taurine, the efflux was unaffected by EGFR inhibition. EGF and agglutinins markedly increased 86Rb release under isosmotic conditions. The EGF-evoked isosmotic 86Rb release, together with the hyposmotic efflux, accounted fully for the observed potentiation by EGF, raising the possibility of an overlapping of these two effects, rather than a true potentiation. A link between EGFR, phosphatidylinositide-3-kinase (PI3K) and hyposmotically induced taurine (but not 86Rb) release is suggested by the increase in PI3K activity elicited by hyposmolarity, which was fully prevented by EGFR inhibition, and by a marked reduction of hyposmotically induced taurine (but not 86Rb) release, by wortmannin. The present findings, together with results showing EGF activation of osmosensitive Cl- fluxes implicate EGFR as an important modulator of osmolyte efflux pathways.

A decrease in external osmolarity results in cell swelling and the immediate activation of a mechanism to restore cell volume, known as regulatory volume decrease (RVD). When exposed to a gradual osmolarity decrease (GODE), some cells do not swell. This reflects the operation of an active regulatory process known as isovolumetric regulation (IVR). The mechanisms underlying IVR appear similar to those activated during RVD, namely the extrusion of K+, Cl-, amino acids, and other organic molecules. A previous study has documented IVR in cerebellar granule neurons, parallel to an early efflux of taurine and Cl-, whereas K+ efflux is delayed. In this work we briefly review the importance of amino acids in the mechanisms of cell volume control in the brain, with emphasis on IVR. We also present experiments showing the response to GODE in cerebellar astrocytes. The currents activated during GODE, recorded in the whole-cell configuration of the patch clamp technique, indicate the early activation of an anion current, followed by a more delayed cation current. A correlation between the time course of amino acid efflux during GODE and the occurrence or not of IVR in various cell types, suggest the importance of these osmolytes in the volume regulatory process in this model.

G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results.

Hyposmolarity activates amino acid efflux as part of the corrective volume process in a variety of cells. This review discusses the mechanism of amino acid release in brain cells preparations. Results present evidence of substantial differences between the efflux of taurine and that of GABA and glutamate, which besides a possible role as osmolytes, have a main function as synaptic transmitters. The differences found concern the efflux time course, the sensitivity to Cl− channel blockers, the modulation by tyrosine kinases, the influence of PKC and the effect of cytoskeleton disruptive agents. While taurine efflux features fit well with the mechanisms so far described in most cell types, the efflux of GABA and glutamate does not. Alternate mechanisms for the release of these two amino acids are discussed, including a PKC-modulated, actin-dependent exocytosis.

The calcium (Ca2+) dependence of potassium (K+) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments 86Rb release and changes in cytosolic Ca2+ ([Ca2+]i). Hyposmotic (50%) medium increased [Ca2+]i from 117 to 386 nM, with contributions of extracellular Ca2+ and Ca2+ from the endoplasmic reticulum. Hyposmotic medium increased 86Rb efflux rate from 0.015 min−1 to a maximal of 0.049 min−1 and a net release of 30%. This osmosensitive efflux was inhibited by Ba2+ (0.028 min−1), quinidine (0.024 min−1), and charybdotoxin (0.040 min−1), but was unaffected by TEA, 4-AP, or apamin. Removal of external Ca2+ from the hyposmotic medium increased 86Rb efflux to a maximal rate constant of 0.056 min−1 and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca2+ removal in isosmotic medium. This isosmotic 86Rb efflux was unaffected by TEA or 4-AP, reduced by verapamil, and abolished by Ba2+, nitrendipine, and Mg2+. With the swelling-induced [Ca2+]i rise suppressed by ethyleneglycoltetraacetic acid-acetoxy-methyl ester (EGTA-AM), hyposmotic 86Rb was 30% reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect 86Rb efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca2+, because these agents did not affect the [Ca2+]i rise and the inhibition persisted in the absence of external Ca2+. The phospholipase C blocker U-73122 did not affect [Ca2+]i nor 86Rb efflux. Blockers of Ca2+/calmodulin W7 and KN-93 decreased 86Rb efflux to the same extent as EGTA-AM. Ionomycin markedly potentiated 86Rb release in hyposmotic conditions only when [Ca2+]i was raised to about 1 μM, suggesting the implication of maxi-K+ channels at this [Ca2+]i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that 86Rb efflux in cerebellar astrocytes is largely (70%) Ca2+-independent and the Ca2+-dependent fraction is sustained essentially by Ca2+ released from the endoplasmic reticulum and mediated by a mechanism involving Ca2+/calmodulin. J. Neurosci. Res. 57:350–358, 1999. © 1999 Wiley-Liss, Inc.

Hyposmotic swelling increased 86Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. 86Rb release was partially blocked by quinidine, Ba2+, and Cs+ but not by TEA, 4-AP, or Gd3+. 86Rb efflux decreased in Cl−-depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl− and K+ fluxes. Swelling induced a substantial increase in [Ca2+]i to which both external and internal sources contribute. However, 86Rb efflux was independent of [Ca2+]o, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling-activated 86Rb efflux in differentiated granule neurons after 8 DIV, which express Ca2+-sensitive K+ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca2+-dependence, or pharmacological sensitivity. We conclude that the swelling-activated K+ efflux in cerebellar granule neurons is not mediated by Ca2+-sensitive large conductance K+ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage-gated K+ channels. J. Neurosci. Res. 53:626–635, 1998. © 1998 Wiley-Liss, Inc.

Early cognitive deficit characteristic of early Alzheimer's disease seems to be produced by the soluble forms of beta-amyloid protein. Such cognitive deficit correlates with neuronal network dysfunction that is reflected as alterations in the electroencephalogram of both Alzheimer patients and transgenic murine models of such disease. Correspondingly, recent studies have demonstrated that chronic exposure to betaAP affects hippocampal oscillatory properties. However, it is still unclear if such neuronal network dysfunction results from a direct action of betaAP on the hippocampal circuit or it is secondary to the chronic presence of the protein in the brain. Therefore, we aimed to explore the effect of acute exposure to betaAP(25-35) on hippocampal network activity both in vitro and in vivo, as well as on intrinsic and synaptic properties of hippocampal neurons. We found that betaAP(25-35), reversibly, affects spontaneous hippocampal population activity in vitro. Such effect is not produced by the inverse sequence betaAP(35-25) and is reproduced by the full-length peptide betaAP(1-42). Correspondingly betaAP(25-35), but not the inverse sequence betaAP(35-25), reduces theta-like activity recorded from the hippocampus in vivo. The betaAP(25-35)-induced disruption in hippocampal network activity correlates with a reduction in spontaneous neuronal activity and synaptic transmission, as well as with an inhibition in the subthreshold oscillations produced by pyramidal neurons in vitro. Finally, we studied the involvement of Fyn-kinase on the betaAP(25-35)-induced disruption in hippocampal network activity in vitro. Interestingly, we found that such phenomenon is not observed in slices obtained from Fyn-knockout mice. In conclusion, our data suggest that betaAP acutely affects proper hippocampal function through a Fyn-dependent mechanism. We propose that such alteration might be related to the cognitive impairment observed, at least, during the early phases of Alzheimer's disease.

TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.

Exposure of cultured cerebellar granule neurons (24 h serum-starved) during 3 min to 30% hyposmotic medium activated the tyrosine kinase receptor ErbB4 in the absence of its ligand. Hyposmolarity also activated the non-receptor tyrosine kinases, Src, focal adhesion kinase (FAK), extracellular signal-regulated protein kinase (ERK)1/2, and the tyrosine kinase target phosphatidyl-inositol-3-kinase (PI3K). The hyposmotic-induced activation of these kinases required the prior phosphorylation of ErbB4 as shown by the effect of ErbB4 blockade with AG213 reducing by 85-95% the phosphorylation of FAK and ERK1/2, by 74% and 36% that of PI3K and Src, respectively. These results suggest a key role of ErbB4 as a signal integrator of events associated with hyposmolarity. PI3K seems to be an important connecting element in the signaling network evoked by the hyposmolarity/ErbB4 activation as: (i) the p85 regulatory subunit of PI3K co-immunoprecipitates with ErbB4 and with FAK; (ii) PI3K blockade with wortmannin reduced the hyposmotic activation of FAK (90%) and ERK1/2 (84-91%). Inhibition of Src with PP2 reduced ErbB4 phosphorylation and inhibited the subsequent cytosolic kinase activation with the same potency as ErbB4 blockade. These results point to Src and ErbB4 and as early targets of the hyposmotic stimulus and osmosignaling. The functional significance for cell volume regulation of the ErbB4-Src-PI3K signaling cascade is indicated by the 48-66% decrease of the hyposmotic taurine efflux observed by inhibition of these kinases.

Volume changes and whole cell ionic currents activated by gradual osmolarity reductions (GOR) of 1.8 mosM/min were characterized in C6 glioma cells. Cells swell less in GOR than after sudden osmolarity reductions (SOR), the extent of swelling being partly Ca2+ dependent. In nominally Ca2+-free conditions, GOR activated predominantly whole cell outward currents. Cells depolarized from the initial −79 mV to a steady state of −54 mV reached at 18% osmolarity reduction [hyposmolarity of −18% (H-18%)]. Recordings of Cl− and K+ currents showed activation at H-3% of an outwardly rectifying Cl− current, with conductance of 1.6 nS, sensitive to niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, followed at H-18% by an outwardly rectifying K+ current with conductance of 4.1 nS, inhibited by clofilium but insensitive to the typical K+ channel blockers. With 200 nM Ca2+ in the patch pipette, whole cell currents activated at H-3% and at H-13% cells depolarized from −77 to −63 mV. A K+ current activated at H-1%, showing a rapid increase in conductance, suppressed by charybdotoxin and insensitive to clofilium. These results show the operation of two different K+ channels in response to GOR in the same cell type, activated by Ca2+ and osmolarity and with different osmolarity activation thresholds. Taurine and glutamate efflux, monitored by labeled tracers, showed delayed osmolarity thresholds of H-39 and H-33%, respectively. This observation clearly separates the Cl− and amino acid osmosensitive pathways. The delayed amino acid efflux may contribute to counteract swelling at more stringent osmolarity reductions.

The ability to regulate cell volume is an ancient conserved trait present in essentially all species through evolution. The maintenance of a constant cell volume is a homeostatic imperative in animal cells. Changes in cell water content affecting the concentration of intracellular messenger molecules impair the complex signaling network, crucial for cell functioning and intercellular communication. Although the renal homeostatic mechanisms exert a precise control of extracellular fluid osmolarity, this is challenged in a variety of pathological situations. The intracellular volume constancy is continuously compromised by the generation of local and transient osmotic microgradients, associated with nutrients uptake, secretion, cytoskeleton remodeling and transynaptic ionic gradients1.

Water gain in the brain consequent to hyponatremia is counteracted by mechanisms that initially include a compensatory displacement of liquid from the interstitial space to cerebrospinal fluid and systemic circulation and subsequently an active reduction in cell water accomplished by extrusion of intracellular osmolytes to reach osmotic equilibrium. Potassium (K + ), chloride (Cl − ), amino acids, polyalcohols, and methylamines all contribute to volume regulation, with a major contribution of ions at the early phase and of organic osmolytes at the late phase of the regulatory process. Experimental models in vitro show that osmolyte fluxes occur via leak pathways for organic osmolytes and separate channels for Cl − and K + . Osmotransduction signaling cascades for Cl − and taurine efflux pathways involve tyrosine kinases and phosphoinositide kinases, while Ca 2+ and serine-threonine kinases modulate K + pathways. In-depth knowledge of the cellular and molecular adaptive mechanisms of brain cells during hyponatremia contributes to a better understanding of the associated complications, including the risks of inappropriate correction of the hyponatremic condition.

Hyposmolarity activates amino acid efflux as part of the corrective volume process in a variety of cells. This review discusses the mechanism of amino acid release in brain cells preparations. Results present evidence of substantial differences between the efflux of taurine and that of GABA and glutamate, which besides a possible role as osmolytes, have a main function as synaptic transmitters. The differences found concern the efflux time course, the sensitivity to C1- channel blockers, the modulation by tyrosine kinases, the influence of PKC and the effect of cytoskeleton disruptive agents. While taurine efflux features fit well with the mechanisms so far described in most cell types, the efflux of GABA and glutamate does not. Alternate mechanisms for the release of these two amino acids are discussed, including a PKC-modulated, actin-dependent exocytosis.

Following hyposmotic swelling induced, cell volume regulation is achieved by the efflux of osmolytes and osmotically obligated water. The role of amino acids as organic osmolytes is widely reported in brain preparations. Results on mechanisms of the osmosensitive taurine release have been often extrapolated to other amino acids. In the present work we present results showing that amino acids such as glutamate, which serve the dual role of osmolytes and neurotransmitters deviate from the taurine release features in several brain preparations, including cultured astrocytes and neurons, and brain slices. Taurine efflux as previously reported is inhibited by chloride channel blockers and by tyrosine kinase and PI3K inhibitors, and was insensitive to manipulation of PKC activity. Glutamate efflux (followed as 3H-d-aspartate) was reduced by chloride channel blockers in cultured cell preparations, while it was unaffected in brain slices. PKC activation markedly enhanced hyposmotic-induced glutamate release in all preparations, pointing to a role of this kinase in the osmosensitive glutamate release. These results suggest that in cell cultures glutamate may share a common efflux pathway with taurine, but in more integrated preparation, the hyposmotic challenge activates a different pathway modulated by PKC. This mechanism may be identical to the stimulation-secretion vesicular release typical of neurotransmitter amino acids. However, no difference in the release mechanism was observed between neuroblasts and differentiated neurons. Moreover, the PKC-sensitive pathway is also present in fibroblasts in which such vesicular release does not operate. However, this does not exclude the activation by hyposmolarity of exocytotic mechanisms dependent on PKC activity.