Modulation of neurotransmitter release by dihydropyridine-sensitive calcium channels involves tyrosine phosphorylation

Cultured rat cerebellar granule cells depolarized by high KCl, display a large component of Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels as defined by a sensitivity to 1 μm nifedipine. This Ca²⁺ influx is not coupled to neurotransmitter exocytosis but has implications for neuronal development. KCl stimulation in the absence of external Ca²⁺ followed by the re-addition of Ca²⁺ allows the coupling of a class of L-type Ca²⁺ channels to neurotransmitter exocytosis as assessed by loading of glutamatergic pools with [³H]-d -aspartate. KCl stimulation in the absence of external Ca²⁺ (‘predepolarization’) enhances tyrosine phosphorylation of several cellular proteins, and inhibitors of tyrosine kinases block both phosphorylation and the neurotransmitter release coupled to the L-type Ca²⁺ channel. More specifically, an inhibitor of src family tyrosine kinases, PP1, blocks the effects of predepolarization suggesting a role for a src family kinase in the process. Furthermore, L-type Ca²⁺ channel recruitment and modulation of release could be activated with the tyrosine phosphatase inhibitor sodium orthovanadate. The phosphoproteins enhanced by predepolarization, which include the cytoskeletal proteins focal adhesion kinase (FAK) and vinculin, are also highly phosphorylated early on in culture when neurite outgrowth occurs. As the neurons develop a network of neurites, both tyrosine phosphorylation and L-type Ca²⁺ channel activity decrease. These results show a novel mechanism for the recruitment of L-type Ca²⁺ channels and their coupling to neurotransmitter release which involves tyrosine phosphorylation. This phenomenon has a role in cerebellar granule cell development.     

Endothelin-induced protein tyrosine phosphorylation of cultured astrocytes: its relationship to cytoskeletal actin organization.

Endothelins (ETs) promote cytoskeletal actin reorganization of cultured astrocytes (Koyama and Baba, Neuroscience 61:1007-1016, 1994; Koyama and Baba, Glia 16:342-350, 1996). In this study, we examined the signal transduction involved in that activity of ETs. Immunoblot analysis with an anti-phosphotyrosine antibody showed that ET-3 (1 nM) increased tyrosine phosphorylation of 120 Kda and 70 Kda astrocytic proteins. The tyrosine phosphorylations of both proteins reached a maximum at 1 nM ET-3. In morphological examinations, ET-3 (1 nM) induced stress fibers, an organized F-actin structure, and focal adhesions in 0.5 mM dibutyryl cAMP (DBcAMP)-treated astrocytes within 30 min. Immunochemical staining of phosphotyrosine revealed that the newly formed focal adhesions possessed phosphotyrosine immunoreactivity. Phorbol 12-myristate 13 acetate (PMA, 100 nM), bradykinin (1 microM), angiotensin II (100 nM), and A23187 (5 microM) did not induce astrocytic stress fibers and had no obvious effects on tyrosine phosphorylation of 120 Kda and 70 Kda proteins. Tyrosine phosphorylation of astrocytic 120 Kda and 70 Kda proteins was stimulated by 1 mM sodium orthovanadate (VO4(3-)), a protein tyrosine phosphatase inhibitor. VO4(3-) promoted reorganization of stress fibers and focal adhesions in DBcAMP-treated astrocytes. Neither chelation of intra- and extracellular Ca2+ nor pre-treatment with pertussis toxin (PTX) affected the ET-induced tyrosine phosphorylation and stress fiber formation in cultured astrocytes. These results suggest a relationship between cytoskeletal actin reorganization and the tyrosine phosphorylation of astrocytic proteins by ETs.     

Modification of Kv2.1 K+ currents by the silent Kv10 subunits

Human and rat Kv10.1a and b cDNAs encode silent K+ channel pore-forming subunits that modify the electrophysiological properties of Kv2.1. These alternatively spliced variants arise by the usage of an alternative site of splicing in exon 1 producing an 11-amino acid insertion in the linker between the first and second transmembrane domains in Kv10.1b. In human, the Kv10s mRNA were detected by Northern blot in brain kidney lung and pancreas. In brain, they were expressed in cortex, hippocampus, caudate, putamen, amygdala and weakly in substantia nigra. In rat, Kv10.1 products were detected in brain and weakly in testes. In situ hybridization in rat brain shows that Kv10.1 mRNAs are expressed in cortex, olfactory cortical structures, basal ganglia/striatal structures, hippocampus and in many nuclei of the amygdala complex. The CA3 and dentate gyrus of the hippocampus present a gradient that show a progression from high level of expression in the caudo-ventro-medial area to a weak level in the dorso-rostral area. The CA1 and CA2 areas had low levels throughout the hippocampus. Several small nuclei were also labeled in the thalamus, hypothalamus, pons, midbrain, and medulla oblongata. Co-injection of Kv2.1 and Kv10.1a or b mRNAs in Xenopus oocytes produced smaller currents that in the Kv2.1 injected oocytes and a moderate reduction of the inactivation rate without any appreciable change in recovery from inactivation or voltage dependence of activation or inactivation. At higher concentration, Kv10.1a also reduces the activation rate and a more important reduction in the inactivation rate. The gene that encodes for Kv10.1 mRNAs maps to chromosome 2p22.1 in human, 6q12 in rat and 17E4 in mouse, locations consistent with the known systeny for human, rat and mouse chromosomes.     

Modulation of baroreflex sensitivity by the state of protein tyrosine phosphorylation in the brainstem of the rat

Evidence accumulated recently suggests that protein tyrosine phosphorylation may play an important role in regulating neuronal functions. In the present study, we investigated if the state of protein tyrosine phosphorylation in the brainstem regulates baroreflex sensitivity. Anti-phosphotyrosine immunoblots of brainstem tissue revealed that several phosphotyrosine-containing proteins were present in the brainstem and their level of tyrosine phosphorylation was decreased by treatment of the slices with the protein tyrosine kinase (PTK) inhibitor genistein, and increased by treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate. In urethane-anaesthetized rats, we found that inhibiting PTK activity by topical application of genistein to the dorsal surface of the medulla reduced the phenylephrine-induced baroreflex bradycardiac response. Conversely, the baroreflex response was potentiated by activating endogenous PTK activity with insulin or by inhibiting PTP activity with pervanadate. Thus these results suggest that the state of cellular tyrosine phosphorylation within the dorsal medulla of the brainstem may regulate the baroreflex control of heart rate, thereby providing the first evidence for a role for protein tyrosine phosphorylation, a key process involved in diverse intracellular signalling pathways, in modulating baroreflex sensitivity.     

Modulation of Ca2+ currents in rat thalamocortical relay neurons by activity and phosphorylation

Rhythmic low and high frequency activity in thalamocortical networks depend critically on activation of low- and high-voltage-activated (LVA, HVA) Ca2+ currents. In order to test whether Ca2+ currents are modified during repetitive activation, acutely isolated thalamocortical relay neurons of rats, at postnatal days 12 (P12) to P20, were investigated using patch-clamp, Ca2+ imaging and Western blot techniques. High-voltage-activated, but not LVA Ca2+ currents were reduced significantly during 2 Hz stimulation. Ca2+ imaging experiments demonstrated a close correlation between the increase in intracellular Ca2+ levels and the decrease in HVA Ca2+ current amplitudes. Further examination of HVA Ca2+ currents revealed a 'U-shaped' inactivation curve and a time-dependent inactivation process that could be described by a two-exponential function. The 'U-shape' was significantly reduced, current amplitude was increased significantly and time-dependent inactivation revealed a one-exponential decline with Ba2+ as the charge carrier, following activation of the cAMP/PKA pathway, and following application of phosphatase inhibitors (ascomycin, calyculin A). Western blot analysis and the effect of ascomycin indicated an involvement of calcineurin in the inactivation process. Isolation of HVA Ca2+ current components by subtype-specific blockers revealed that changes in time-dependent inactivation, inactivation curve and current amplitude were carried mainly by L-type and N-type Ca2+ currents. Furthermore, Ca2+-dependent inactivation was operative during stimulation protocols mimicking tonic action potential firing. These data indicate a modulation of L- and N-type Ca2+ channels by phosphorylation, resulting jointly in an increased intracellular Ca2+ influx during activity of the ascending brainstem system, the latter occurring during states of wakefulness.     

Modulation of cytochrome P450 by inflammation in astrocytes.

Activation of systemic host defense mechanisms results in the down-regulation of cytochrome P450 enzymes in the liver. This occurs for various induced and constitutive isoforms of cytochrome P450 in response to cytokines such as IFNs, IL-1, IL-6, and TNF-alpha, which are produced during infection. Although the levels of cytochrome P450 in brain regions are low, the enzymes are regionally distributed and may play a critical role in the activation or degradation of drugs and chemicals in localized areas. If activation of the immune response in the CNS by LPS modulates the activity of cytochrome P450 forms in the brain, this may alter normal metabolic pathways or contribute to drug or chemical toxicity. This hypothesis was addressed by examining the effect of LPS on a major cytochrome P450 form in isolated astrocytes obtained from newborn rats. These cells were shown to express CYP1A1/2 when induced by dibenz[a, h]anthracene (DBA) as determined by enzyme activity, immunohistochemistry, and Western blotting. The treatment of these cells with LPS significantly attenuated the activity of these enzymes but had no effect on CYP1A1/2 protein levels as determined by Western blotting. The lack of effect by detoxified LPS indicated the requirement of the lipid A region on LPS to stimulate this response. Pentoxifylline (PNTX) prevented the LPS evoked decrease in CYP1A1/2 activity suggesting that cytokine release was a required component of this effect in astrocytes. These results indicate that stimulation of the immune response by LPS in isolated astrocytes decreases CYP1A1/2 activity. The release of cytokines is implicated in this effect and thought to participate in the functional inhibition of the enzyme as no effect on CYP1A1/2 protein levels was observed.     

Both cell-surface carbohydrates and protein tyrosine phosphatase are involved in the differentiation of astrocytes in vitro

Astrocytes are important in the development and maintenance of functions of the CNS, acting in cooperation with neurons and other glial cells. The glycans on astrocyte membrane are believed to play important roles in cell-cell communication. Plant lectins are useful probes, because the lectins can bind to certain cell surface receptors and elicit cellular responses that are normally activated by endogenous ligands for those receptors. In the present study, we investigated the effect of Datura stramonium agglutinin (DSA) on astrocytes and characterized several molecular events. The addition of DSA to a culture of flat, polygonal, immature astrocytes derived from the neonatal rat cerebellum caused the cells to become stellate in shape, similar to astrocytes observed in vivo, concomitant with an increase in expression of astrocyte-specific intermediate filament (glial fibrillary acidic protein [GFAP]) and inhibition of proliferation. These results indicate that DSA binds to astrocytes and triggers differentiation. We also found a decrease in the extent of tyrosine-phosphorylation of a 38-kDa protein. To elucidate the molecular events during astrocyte differentiation, we examined the effects of various signal transduction inhibitors on the transformation from the polygonal to stellate shape (stellation). Interestingly, only tyrosine phosphatase inhibitors, orthovanadate and phenylarsine oxide, showed an inhibitory effect. Our results suggest that DSA induced astrocyte differentiation acts via tyrosine dephosphorylation.     

Modulation of neuronal mitochondrial membrane potential by the NMDA receptor: role of arachidonic acid

Activation of NMDA receptors in dissociated cerebellar granule cells reduced mitochondrial membrane potential (MMP), as measured by rhodamine 123 fluorescence in a flow cytometer. This effect was inhibited by several NMDA-receptor antagonists with the following rank order of potency: MK-801>PCP>TCP>dextrorphan>dichlorokynurenic acid>

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-AP5>dextromethorphan. Neither spermine nor arcaine modified the NMDA-induced reduction in MMP, whereas ifenprodil and eliprodil inhibited this response in the micromolar range. The mechanism responsible for the alteration of MMP mediated by NMDA was studied. Mepacrine and dibucaine prevented the MMP reduction induced by NMDA, as did W₁₃ (calmodulin antagonist). In contrast, this effect was not blocked by cyclooxygenase or lipooxygenase inhibitors, H7 (a protein kinase C inhibitor) or nitroarginine (nitric oxide synthase inhibitor). These data suggest a direct interaction between NMDA-receptor activation and arachidonic acid formation, and indicate that NMDA receptor-mediated effect on MMP could involve arachidonic acid.     

Manipulation of the delayed rectifier Kv1.5 potassium channel in glial cells by antisense oligodeoxynucleotides

Glial cells have been shown to express several biophysically and pharmacology distinct potassium channel types. However, the molecular identity of most glial K⁺ channels is unknown. We have developed an antibody specific for the Shaker type potassium channel Kv1.5 protein, and demonstrate by immunohistochemistry the presence of this channel in glial cells of adult rat hippocampal and cerebellar slices, as well as in cultured spinal cord astrocytes. Immunoreactivity was particularly intense in the endfoot processes of astrocytes surrounding the microvasculature of the hippocampus. The specific contribution of this channel protein to the delayed rectifying K⁺ current of spinal cord astrocytes was determined by incubating these cells with antisense oligodeoxynucleotides complementary to the mRNA coding for Kv1.5 protein. Such treatment reduced delayed rectifier current density and shifted the potassium current steady-state inactivation, without altering current activation, cell capacitance, or cell resting potential. The tetraethylammonium acetate (TEA) sensitivity of astrocytic delayed rectifier current was enhanced following antisense oligodeoxynucleotide treatment, suggesting that Kv1.5 channel protein may provide a significant component of the TEA-insensitive current in this preparation. Our results suggest that Kv1.5 is widely expressed in glial cells of brain and spinal cord and that delayed rectifying K⁺ currents in astrocytes are largely mediated by Kv1.5 channel protein. © 1996 Wiley-Liss, Inc.     

Fibroblast growth factor-induced increases in calcium currents in the PC12 pheochromocytoma cell line are tyrosine phosphorylation dependent

The PC12 rat pheochromocytoma cell line is widely used to study neuronal differentiation by growth factors. In response to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), PC12 cells differentiate into sympathetic-like neurons and become electrically excitable. Using whole cell patch-clamp recording, with barium as a charge carrier, we looked at the effects of bFGF on calcium channel expression as reflected by changes in barium current amplitudes normalized to cell membrane area. Similar to the effect reported for NGF, we show that 7 day treatment with bFGF increased the barium current approximately 4-fold. The largest contributor to the increase in barium current with bFGF treatment is a 6-fold increase in the high threshold voltage activated Ω-conotoxin sensitive barium current. Smaller increases in current produced by bFGF treatment of PC12 cells are observed for the dihydropyridine sensitive and dihydropyridine/conotoxin insensitive currents. The bFGF-induced increases in barium currents are dependent on tyrosine phosphorylation, since the effects of bFGF are blocked by genistein, a tyrosine kinase inhibitor. This system will ultimately be useful in understanding the signaling pathways that control calcium channel expression in response to growth factors. © 1994 Wiley-Liss, Inc.     

HIV infection of fetal human astrocytes: the potential role of a receptor-mediated endocytic pathway

HIV infects microglia and astrocytes both in vivo and in vitro. Although there is a significant amount of information about microglial infection, data regarding astrocytes are more limited. For example, little is known about the initial membrane events occurring between HIV and astrocytes. Also, the mechanism by which HIV enters these cells remains to be determined. To address these questions, we exposed human astrocyte cultures to either HIV or to the HIV glycoprotein gp120. The cultures were analyzed for viral infection and gp120 binding to cultured cells by light and electron microscopy (EM) with and without immunocytochemistry, respectively; ligand-receptor biochemistry; and, Western, Northern and Southern blot analyses. The results of these studies showed that HIV binds to astrocytes via gp120 and a cell surface molecule weighing approximately 65 kDa that is neither CD4 nor galactocerebroside. Furthermore, binding of gp120 to astrocytes was concentration dependent and displayed a curve consistent with ligand-receptor binding. Additionally, radiolabeled gp120 binding was displaced by unlabeled gp120 but not by deglycosylated gp120, suggesting that the binding was specific. By EM, HIV virions were seen in clathrin-coated pits and in cytoplasmic vacuoles. This suggests linkage, in astrocytes, between a plasma membrane-associated protein that can act as a receptor for HIV and an endosomal pathway.     

GFAP phosphorylation studied in digitonin-permeabilized astrocytes: standardization of conditions

Cycles of assembly/disassembly of the intermediate filaments of astrocytes are modulated by the phosphorylation of glial fibrillary acidic protein (GFAP). The sites on GFAP are localized at the N-terminal where they are phosphorylated by cAMP-dependent and Ca(2+)-dependent protein kinases. Phosphorylation of GFAP has been investigated in brain slices, astrocyte cultures, cytoskeletal fractions and purified systems. Here we describe a different approach to study GFAP phosphorylation. We show that permeabilization of astrocytes in culture with digitonin allows direct access to the systems phosphorylating GFAP. Conditions for the permeabilization were established with an assay based on the exclusion of Trypan blue. Incubation of permeabilized cells with cAMP and Ca(2+) increased the phosphorylation state of GFAP. Immunocytochemistry with anti-GFAP showed that permeabilized astrocytes retained their typical flat, fibroblast morphology and exhibited well preserved glial filaments. On incubation with cAMP the filaments apparently condensed to form long processes. The results suggest the approach of studying structural changes in glial filaments in parallel to protein phosphorylation, in the presence of specific modulators of protein kinases and phosphatases has considerable potential.     

Membrane currents elicited by angiotensin II in astrocytes from the rat corpus callosum

The corpus callosum (CC) is the main white matter tract in the brain. It consists primarily of axons and glial cells. In the present work, membrane currents generated by angiotensin II (Ang II) in cultured astrocytes from the CC of newborn and 3-week-old rats were studied using the whole-cell voltage-clamp technique. After 4 days of culture, approximately 90% of cells were positive to glial fibrillary acidic protein (GFAP), indicating their astrocyte lineage. Ang II elicited inward currents in approximately 20% of cells and outward currents in approximately 4% of cells from the CC for newborn or 3-week-old rats. The main effect of Ang II on astrocytes from the newborn rat CC was a reduction of membrane conductance, by blocking of delayed rectifier K(+) currents in 96% of cells. However, no common action of Ang II was observed in cells from 3-week-old rat CC because the responses were quite variable, suggesting the participation of other ion currents. The partial agonist of AT(2) receptors, CGP-42112A, exerted effects on Ang II responses, whereas the AT(1) antagonist ZD7155 did not, suggesting that Ang II responses in CC astrocytes are predominantly mediated by activation of AT(2) receptors. This study is the first to show electrical responses generated by AT(2) receptors in glial cells from the rat central nervous system, and may help gain a better understanding of the functions of Ang II receptors in astrocytes from the rat CC in particular and of glial cells in general. (c) 2005 Wiley-Liss, Inc.     

Extracellular ATP-induced currents in astrocytes: Involvement of a cation channel

Whole-cell currents were measured with the perforated patch clamp technique in cultured rat astrocytes to analyze the underlying ionic mechanism for a P₂-purinoceptor-mediated depolarization. ATP (100 μM) induced an inward current with a mean amplitude of 130 pA and an EC₅₀ of 17 μM. The response desensitized during a 1 min application. Replacement of extracellular Na⁺ with NMDG or K⁺ abolished the ATP-evoked inward current. Replacement of Na⁺ with choline, however, resulted in an ATP-evoked response of one-third the amplitude in normal solution. This is indicative of a cation rather than Na⁺ channel. However, due to difficulties in voltage-clamping these gap junction-coupled cells at voltages different from the membrane resting potential, the current reversal potential could not be determined. Measurements with K⁺-sensitive microelectrodes showed that 100 μM ATP lowered the intracellular K⁺ concentration. Replacement of extracellular Ca²⁺ or Cl^? did not alter the ATP-induced inward currents. Fura-2 imaging experiments revealed a transient rise of the intracellular Ca²⁺ concentration during ATP application. Removal of extracellular Ca²⁺ did not influence the peak response; it did, however, shorten the time course. These results and previous observations that the permeability changes are caused by a P_2× receptor are indicative of an ATP-sensitive cation conductance. In addition, cytoplasmic Ca²⁺ is increased by mobilization from intracellular stores, and by additional influx across the cell membrane. Extracellular ATP released by neurons could evoke K⁺ release from astrocytes as well as be a mediator for cation changes that signal cell activation processes when released by damaged cells. © 1994 Wiley-Liss, Inc.     

Modulation of tyrosine hydroxylase gene expression in the rat adrenal gland by exercise: effects of age.

Both aging and exercise are associated with alterations in circulating levels of catecholamines. To determine the interactions of age and exercise on tyrosine hydroxylase (TH) activity and TH mRNA, Fischer-344 female rats aged 5 months (young) and 25 months (old) were trained by treadmill running for 10 weeks. The elevation in maximum oxygen consumption in both groups was equivalent following exercise, indicating that training had occurred. In control rats, both TH activity and TH mRNA were greater in the older groups when compared with the younger animals. In young rats, exercise decreased TH activity by 25% and TH mRNA by 27%. In older rats, exercise was not associated with a decrease in TH activity and TH mRNA. Choline acetyltransferase activity (ChAT) was decreased and glutamic acid decarboxylase activity (GAD) was increased by exercise in young rats. The decrease in ChAT activity and increase in GAD activity suggest that trans-synaptic mechanisms play a role in the exercise-induced alteration of TH gene expression. Neither ChAT nor GAD was altered by exercise in older groups. Our data suggest that the previously reported diminution in catecholamines associated with exercise may be due to a decrease in TH mRNA and a resulting decrease in TH activity. There was no effect of exercise in the old rats, supporting previous observations that the plasticity of the sympathoadrenal system diminishes with age.     

Modulation of sodium currents in rat sensory neurons by nucleotides

The effects of various nucleotides on the fast tetrodotoxin-sensitive (f-TTX-S) and the slow tetrodotoxin-resistant (s-TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were investigated using the patch-clamp technique. Nucleoside triphosphates (NTPs; ATP, GTP, UTP and CTP) and nucleoside diphosphates (NDPs; ADP, GDP, UDP and CDP) decreased f-TTX-S current, whereas they increased s-TTX-R current, when currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV. NTPs and NDPs shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction in both types of sodium currents. Most of them also increased the maximum conductance of s-TTX-R current. ITP, a derivative of ribonucleotide, and dTTP, a deoxyribonucleotide, modulated both types of sodium currents similarly to NTPs and NDPs. However, nucleoside monophosphates (NMPs; AMP, GMP, UMP and CMP) and adenosine had little or no effect on either type of sodium current. Therefore, it seems that nucleotides, regardless of the kind of base, should have two or more phosphates to be able to modulate sodium currents in DRG neurons. Extracellular nucleotides with di- or tri-phosphates would influence the perception by modulating sodium currents in sensory neurons. Particularly, the increase of the maximum conductance and the hyperpolarizing shift of the conductance-voltage relationship of s-TTX-R sodium current would result in an intensified nociception.     

Modulation of cytochrome P450 by inflammation in astrocytes

Activation of systemic host defense mechanisms results in the down-regulation of cytochrome P450 enzymes in the liver. This occurs for various induced and constitutive isoforms of cytochrome P450 in response to cytokines such as IFNs, IL-1, IL-6, and TNF-α, which are produced during infection. Although the levels of cytochrome P450 in brain regions are low, the enzymes are regionally distributed and may play a critical role in the activation or degradation of drugs and chemicals in localized areas. If activation of the immune response in the CNS by LPS modulates the activity of cytochrome P450 forms in the brain, this may alter normal metabolic pathways or contribute to drug or chemical toxicity. This hypothesis was addressed by examining the effect of LPS on a major cytochrome P450 form in isolated astrocytes obtained from newborn rats. These cells were shown to express CYP1A1/2 when induced by dibenz[a,h]anthracene (DBA) as determined by enzyme activity, immunohistochemistry, and Western blotting. The treatment of these cells with LPS significantly attenuated the activity of these enzymes but had no effect on CYP1A1/2 protein levels as determined by Western blotting. The lack of effect by detoxified LPS indicated the requirement of the lipid A region on LPS to stimulate this response. Pentoxifylline (PNTX) prevented the LPS evoked decrease in CYP1A1/2 activity suggesting that cytokine release was a required component of this effect in astrocytes. These results indicate that stimulation of the immune response by LPS in isolated astrocytes decreases CYP1A1/2 activity. The release of cytokines is implicated in this effect and thought to participate in the functional inhibition of the enzyme as no effect on CYP1A1/2 protein levels was observed.     

Modulation of connexin expression and gap junction communication in astrocytes by the gram-positive bacterium S. aureus

Gap junctions establish direct intercellular conduits between adjacent cells and are formed by the hexameric organization of protein subunits called connexins (Cx). It is unknown whether the proinflammatory milieu that ensues during CNS infection with S. aureus, one of the main etiologic agents of brain abscess in humans, is capable of eliciting regional changes in astrocyte homocellular gap junction communication (GJC) and, by extension, influencing neuron homeostasis at sites distant from the primary focus of infection. Here we investigated the effects of S. aureus and its cell wall product peptidoglycan (PGN) on Cx43, Cx30, and Cx26 expression, the main Cx isoforms found in astrocytes. Both bacterial stimuli led to a time-dependent decrease in Cx43 and Cx30 expression; however, Cx26 levels were elevated following bacterial exposure. Functional examination of dye coupling, as revealed by single-cell microinjections of Lucifer yellow, demonstrated that both S. aureus and PGN inhibited astrocyte GJC. Inhibition of protein synthesis with cyclohexamide (CHX) revealed that S. aureus directly modulates, in part, Cx43 and Cx30 expression, whereas Cx26 levels appear to be regulated by a factor(s) that requires de novo protein production; however, CHX did not alter the inhibitory effects of S. aureus on astrocyte GJC. The p38 MAPK inhibitor SB202190 was capable of partially restoring the S. aureus-mediated decrease in astrocyte GJC to that of unstimulated cells, suggesting the involvement of p38 MAPK-dependent pathway(s). These findings could have important implications for limiting the long-term detrimental effects of abscess formation in the brain which may include seizures and cognitive deficits.     

Modulation of dendritic synaptic processing in the lateral superior olive by hyperpolarization-activated currents

We have previously shown that mice lateral superior olive (LSO) neurons exhibit a large hyperpolarization-activated current (I(h) ), and that hyperpolarization-activated cyclic-nucleotide-gated type 1 channels are present in both the soma and dendrites of these cells. Here we show that the dendritic I(h) in LSO neurons modulates the integration of multiple synaptic inputs. We tested the LSO neuron's ability to integrate synaptic inputs by evoking excitatory post-synaptic potentials (EPSPs) in conjunction with brief depolarizing current pulses (to simulate a second excitatory input) at different time delays. We compared LSO neurons with the native I(h) present in both the soma and dendrites (control) with LSO neurons without I(h) (blocked with ZD7288) and with LSO neurons with I(h) only present peri-somatically (ZD7288+ computer-simulated I(h) using a dynamic clamp). LSO neurons without I(h) had a wider time window for firing in response to inputs with short time separations. Simulated somatic I(h) (dynamic clamp) could not reverse this effect. Blocking I(h) also increased the summation of EPSPs elicited at both proximal and distal dendritic regions, and dramatically altered the integration of EPSPs and inhibitory post-synaptic potentials. The addition of simulated peri-somatic I(h) could not abolish a ZD7288-induced increase of responsiveness to widely separated excitatory inputs. Using a compartmental LSO model, we show that dendritic I(h) can reduce EPSP integration by locally decreasing the input resistance. Our results suggest a significant role for dendritic I(h) in LSO neurons, where the activation/deactivation of I(h) can alter the LSO response to synaptic inputs.