Activity-dependent neuronal control of gap-junctional communication in astrocytes.

A typical feature of astrocytes is their high degree of intercellular communication through gap junction channels. Using different models of astrocyte cultures and astrocyte/neuron cocultures, we have demonstrated that neurons upregulate gap-junctional communication and the expression of connexin 43 (Cx43) in astrocytes. The propagation of intercellular calcium waves triggered in astrocytes by mechanical stimulation was also increased in cocultures. This facilitation depends on the age and number of neurons, indicating that the state of neuronal differentiation and neuron density constitute two crucial factors of this interaction. The effects of neurons on astrocytic communication and Cx43 expression were reversed completely after neurotoxic treatments. Moreover, the neuronal facilitation of glial coupling was suppressed, without change in Cx43 expression, after prolonged pharmacological treatments that prevented spontaneous synaptic activity. Altogether, these results demonstrate that neurons exert multiple and differential controls on astrocytic gap-junctional communication. Since astrocytes have been shown to facilitate synaptic efficacy, our findings suggest that neuronal and astrocytic networks interact actively through mutual setting of their respective modes of communication.

Bidirectional transmission at the rectifying electrotonic synapse: a voltage-dependent process.

Rectifying properties of electrotonic synapses established by the crayfish giant motor fiber are associated with a more negative resting membrane potential in the presynaptic than in the postsynaptic side of the junction. An increased junctional conductance and bidirectional transmission are produced, with almost no delay, by inverting this polarization.

Gap junctions in cultured astrocytes: single-channel currents and characterization of channel-forming protein.

Currents from gap junction channels were recorded from pairs of astrocytes in primary culture using the double whole-cell recording technique. In weakly coupled pairs, single-channel events could be resolved without pharmacological uncoupling treatment. Under these conditions, unitary conductance was 56 +/- 7 pS, and except for multiples of this value, no other level of conductance was observed consistently. To characterize the type of junctional protein constituting astrocyte gap junction channels, immunological and biochemical experiments were carried out on the same material. Specific cDNA probes for three connexins identified in mammals (Cx26, Cx32, and Cx43) showed that only Cx43 mRNA was expressed in cultured astrocytes. The presence of Cx43 protein in cultured astrocytes was demonstrated by immunoblotting, immunofluorescence, and immunogold labeling using anti-peptide antibodies specific to Cx43. These results strongly suggest that gap junctions in astrocytes have a 50-60 pS unitary conductance associated with channels composed of Cx43 protein.

Glia: the fulcrum of brain diseases.

Neuroglia represented by astrocytes, oligodendrocytes and microglial cells provide for numerous vital functions. Glial cells shape the micro-architecture of the brain matter; they are involved in information transfer by virtue of numerous plasmalemmal receptors and channels; they receive synaptic inputs; they are able to release 'glio'transmitters and produce long-range information exchange; finally they act as pluripotent neural precursors and some of them can even act as stem cells, which provide for adult neurogenesis. Recent advances in gliology emphasised the role of glia in the progression and handling of the insults to the nervous system. The brain pathology, is, to a very great extent, a pathology of glia, which, when falling to function properly, determines the degree of neuronal death, the outcome and the scale of neurological deficit. Glial cells are central in providing for brain homeostasis. As a result glia appears as a brain warden, and as such it is intrinsically endowed with two opposite features: it protects the nervous tissue as long as it can, but it also can rapidly assume the guise of a natural killer, trying to eliminate and seal the damaged area, to save the whole at the expense of the part.

Control of gap-junctional communication in astrocytic networks.

Astrocytes, which constitute the most abundant cell type in mammalian brain, are extensively coupled to one another through gap junctions composed mainly of connexin43. In regions exhibiting high levels of connexin43 expression, tens of astrocytes are labeled following single-cell intracellular injection. Importantly, both the expression and the permeability of gap junctions are tightly regulated. Such long- and short-term regulations indicate that astrocytic networks might be subject to remodeling and to some plasticity. Since evidence for neuro-glial interaction exists, the degree of coupling between astrocytes could participate to set the tone of neuronal activity and to determine the sphere of influenced neurons. Research in this area is still at its early stages and significant progress requires a transition from the understanding of basic properties to the study of function.

Astroglial connexin43 contributes to neuronal suffering in a mouse model of Alzheimer's disease.

In Alzheimer's disease (AD), astrocyte properties are modified but their involvement in this pathology is only beginning to be appreciated. The expression of connexins, proteins forming gap junction channels and hemichannels, is increased in astrocytes contacting amyloid plaques in brains of AD patients and APP/PS1 mice. The consequences on their channel functions was investigated in a murine model of familial AD, the APPswe/PS1dE9 mice. Whereas gap junctional communication was not affected, we revealed that hemichannels were activated in astrocytes of acute hippocampal slices containing Aß plaques. Such hemichannel activity was detected in all astrocytes, whatever their distance from amyloid plaques, but with an enhanced activity in the reactive astrocytes contacting amyloid plaques. Connexin43 was the main hemichannel contributor, however, a minor pannexin1 component was also identified in the subpopulation of reactive astrocytes in direct contact with plaques. Distinct regulatory pathways are involved in connexin and pannexin hemichannel activation. Inflammation triggered pannexin hemichannel activity, whereas connexin43 hemichannels were activated by the increase in resting calcium level of astrocytes. Importantly, hemichannel activation led to the release of ATP and glutamate that contributed to maintain a high calcium level in astrocytes placing them in the center of a vicious circle. The astroglial targeted connexin43 gene knocking-out in APPswe/PS1dE9 mice allowed to diminish gliotransmitter release and to alleviate neuronal damages, reducing oxidative stress and neuritic dystrophies in hippocampal neurons associated to plaques. Altogether, these data highlight the importance of astroglial hemichannels in AD and suggest that blocking astroglial hemichannel activity in astrocytes could represent an alternative therapeutic strategy in AD.

Relearning of Activities of Daily Living: A Comparison of the Effectiveness of Three Learning Methods in Patients with Dementia of the Alzheimer Type.

This study examined the effectiveness of three different learning methods: trial and error learning (TE), errorless learning (EL) and learning by modeling with spaced retrieval (MR) on the relearning process of IADL in mild-to-moderately severe Alzheimer's Dementia (AD) patients (n=52), using a 6-weeks randomized controlled trial design. The participants had to relearn three IADLs. Repeated-measure analyses during pre-intervention, post-intervention and 1-month delayed sessions were performed. All three learning methods were found to have similar efficiency. However, the intervention produced greater improvements in the actual performance of the IADL tasks than on their explicit knowledge. This study confirms that the relearning of IADL is possible with AD patients through individualized interventions, and that the improvements can be maintained even after the intervention.

Application of antimony microelectrodes to intracellular pH monitoring.

Some novel studies of the properties of the antimony microelectrode used for intracellular pH measurements are described. First, it is shown that currents in the picoampere range, such as those encountered as leakage in some electrometers, induce important changes in pH sensitivity. The response time of the electrode has also been measured and indicates that the electrode exhibits a rapid time course which would be very useful for dynamic cytoplasmic pH investigations. An example of internal pH recording during cellular acidification in Xenopus laevis oocyte is also presented.

Postnatal development of GFAP, connexin43 and connexin30 in cat visual cortex.

In cat visual cortex, neurons acquire progressively mature functional properties during the first postnatal months. The aim of this study was to analyze the development of astrocytes during this period. The patterns of expression of the glial fibrillary acidic protein (GFAP) as well as of two gap junction proteins expressed in astrocytes, connexin43 (Cx43) and connexin30 (Cx30), were investigated by immunohistochemistry and optical density measurements, in visual cortical areas 17 and 18 at four different ages: 2 weeks (postnatal days 12 to 15, P12-15), 1 month (P27-31), 2 months (P60-62) and beyond 1 year. Since visual experience is a key factor for neural development, the patterns of expression of these three proteins were studied both in normally-reared and monocularly deprived animals. Interestingly, the distribution of GFAP, Cx43 and Cx30 was found to change dramatically but independently of visual experience, during postnatal development, even beyond P60. During the first postnatal month, GFAP and Cx43 were mainly localized in the white matter underlying the visual cortical areas 17 and 18. Then, their distributions evolved similarly with a progressive decrease of their density in the white matter associated with an increase in the cortex. Connexin30 expression appeared only from the second postnatal month, strictly in the cortex and with a laminar distribution which was similar to that of Cx43 at the same age. In adults, a specific laminar distribution was observed, that was identical for GFAP, Cx43 and Cx30: their density was higher in layers II/III and V than in the other cortical layers.

[Gap junction-mediated intercellular communication in astrocytes and neuroprotection]. / Communication jonctionnelle entre astrocytes et neuroprotection.

Neuroglial interaction represents a concept that is now more and more integrated in the attempts to understand who does what and how in neuronal processing and survival, in normal as well as in pathological situations. The purpose of the review is to provide an overlook about the role of glial cells, mainly astrocytes, in neuroprotection. Since a typical feature of glia is to be connected by gap junctions that allow them to be organized as a communicating network(s), we will focus this review on what is known about the contribution of astrocyte gap junctions (AGJ) in neuronal survival. As neuroglial interaction and AGJ are both affected during neurodegenerative diseases, we will also consider the above mentioned glial properties in a pathological context with a special interest in Alzheimer's disease.

Proximity of excitatory synapses and astroglial gap junctions in layer IV of the mouse barrel cortex.

Neurons and astrocytes, the two major cell populations in the adult brain, are characterized by their own mode of intercellular communication--the synapses and the gap junctions (GJ), respectively. In addition, there is increasing evidence for dynamic and metabolic neuroglial interactions resulting in the modulation of synaptic transmission at the so-called "tripartite synapse". Based on this, we have investigated at the ultrastructural level how excitatory synapses (ES) and astroglial GJ are spatially distributed in layer IV of the barrel cortex of the adult mouse. We used specific antibodies for connexin (Cx) 30 and 43 to identify astroglial GJ, these two proteins are known to be present in the majority of astroglial GJ in the cerebral cortex. In electron-microscopic images, we measured the distance between two ES, between two GJ and between a GJ and its nearest ES. We found a ratio of two GJ per three ES in the hollow and septal areas. Taking into account the size of an astrocyte domain, the high density of GJ suggests the occurrence of reflexive type, i.e. GJ between processes of the same astrocyte. Interestingly, the distance between an ES and an astroglial GJ was found to be significantly lower than that between either two synapses or between two GJ. These observations indicate that the two modes of cell-to-cell communication are not randomly distributed in layer IV of the barrel cortex. Consequently, this feature may provide the morphological support for the recently reported functional interactions between neuronal circuits and astroglial networks.

Proliferation of C6 glioma cells is blunted by the increase in gap junction communication caused by tolbutamide.

We have previously reported that tolbutamide prevents the inhibition of gap junction communication in astrocytes. Here, we show that tolbutamide increases gap junction communication and connexin 43 expression in poorly coupled C6 glioma cells. The increase in communication is concurrent with the inhibition of the rate of proliferation due to a block of the progression of C6 glioma cells through the S phase of the cell cycle. The effects of tolbutamide were quantitatively similar to that found after the elevation of intracellular cAMP. Furthermore, the effects of tolbutamide and cAMP were additive. The possible beneficial effect of tolbutamide on gene therapy for gliomas is discussed.

Ontogeny of electrophysiological properties and dendritic pattern in second-order chick vestibular neurons.

The pattern of development of several subpopulations of second-order vestibular neurons was investigated by using intracellular recordings from chicken brain slices to define the timing of morphological and electrophysiological changes occurring at 3 critical ages. Two embryonic stages, embryonic day 13 (E13) and E15-16, and also newborn chicks were selected according to previous anatomical findings showing the differentiation of primary vestibular afferents and their synapses within a distinctive brainstem vestibular nucleus, the tangential nucleus. The responses of these cells to depolarizing and hyperpolarizing current pulses and their postsynaptic responses to vestibular nerve stimulation were recorded, while simultaneously biocytin was injected for subsequent morphogenetic analysis. From this study, developmental schedules of membrane properties, synaptic responses, and dendritic differentiation were established for the 2 cell populations of the tangential nucleus and other neurons located in the surrounding vestibular nuclei. Compared with all other second-order vestibular neurons, the principal cells of the tangential nucleus exhibited a distinctive schedule. Mainly, this includes their pattern of firing on depolarization, the shape and duration of the vestibular-evoked excitatory postsynaptic potential, and the time of onset of dendritic outgrowth. In regard to these observations, E15-16 appears to be a turning point in principal cell ontogeny, whereas these features occur earlier in development for other second-order vestibular neurons. These findings, which indicate that the principal cells may have distinct membrane properties at specific ages, are discussed in light of their unique vestibular innervation and the possible consequences for vestibular signal processing.

Ammonium sulfate induced uncouplings of crayfish septate axons with and without increased junctional resistance.

Cytoplasmic pH (pH1) of crayfish lateral giant axons was monitored using antimony microelectrodes placed near septate junctions and variations of internal pH was induced by short applications of ammonium sulfate in the perfusing bath of the preparation. This treatment produced a rapid cell alkalinization followed, after wash, by acidification rebound. Simultaneously, two successive phases of uncoupling of the septate junction occurred; they had the same time course as those of their associated pH1 movements. Calculation of the electronic coupling parameters indicated that, during alkalinization, coupling was accompanied by an increased axonal membrane conductance (the intimate origin of which was beyond the scope of this study) and resulted from a shunt of the gap junctions; the resistance proper of the latter was unaffected; thus involvement of Ca2+ was ruled out and uncoupling was only an indirect consequence of the electrotonic junction's network configuration. In contrast, and as expected from previous investigations, the junctional membrane resistance was increased during the second phase of cytoplasmic acidification. Evidence that uncoupling can be brought about by a non-junctional membrane increased permeability raises questions about some of the criteria commonly used during investigations of electrotonic transmission.

The first developing "mixed" synapses between vestibular sensory neurons mediate glutamate chemical transmission.

In the present study, the nature of the synaptic transmission responsible for a monophasic potential generated by vestibular nerve stimulation of the principal cells in the chick tangential nucleus was established. This work was performed in slice preparations at the critical embryonic age of 15-16 days, the time of first observation of morphologically mixed (chemical and electrical) synapses at the axosomatic endings called spoon endings. The spoon endings are formed by the primary vestibular fibers with the largest diameters, the colossal vestibular fibers. This monophasic potential fits the criteria for chemical rather than electrical transmission due to the following responses in most cases: (i) the absence of collision between a direct spike initiated by depolarization in the principal cell and a vestibular-evoked action potential; (ii) failure to follow high frequency stimulation (up to 50 Hz); (iii) sensitivity to low calcium solution (0.1 mM). These tests indicate that strong electrical coupling between spoon endings and principal cells does not prevail at this stage. The recordings were obtained from principal cells injected intracellularly with biocytin, allowing their identification by morphological criteria. The lack of tracer coupling between the stained principal cells and their innervating vestibular fibers (n = 17) is consistent with the absence of electrical coupling. Identification of the neurotransmitter involved in this vestibular response was achieved by bath application of glutamate receptor antagonists, DL-2-amino-5-phosphonovaleric acid (40 microM) and 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM), which blocked transmission reversibly. These results suggest that at the onset of formation of these "mixed" vestibular synapses, the gap junctions identified morphologically are likely not functional, and that the main response of the principal cells to vestibular nerve stimulation is mediated by glutamate.

ATP-induced inhibition of gap junctional communication is enhanced by interleukin-1 beta treatment in cultured astrocytes.

Nucleotides are signaling molecules involved in variety of interactions between neurons, between glial cells as well as between neurons and glial cells. In addition, ATP and other nucleotides are massively released following brain insults, including inflammation, and may thereby be involved in mechanisms of cerebral injury. Recent concepts have shown that in astrocytes intercellular communication through gap junctions may play an important role in neuroprotection. Therefore, we have studied the effects of nucleotides on gap junction communication in astrocytes. Based on measurement of intercellular dye coupling and recording of junctional currents, the present study shows that ATP (10-100 microM) induces a rapid and a concentration-dependent inhibition of gap junction communication in cultured cortical astrocytes from newborn mice. Effects of agonists and antagonists of purinergic receptors indicate that the inhibition of gap junctional communication by ATP mainly involves the stimulation of metabotropic purinergic 1 (P2Y(1)) receptors. Pretreatment with the pro-inflammatory cytokine interleukin-1beta (10 ng/ml, 24 h), which has no effect by itself on gap junctional communication, increases the inhibitory effect of ATP and astrocytes become sensitive to uridine 5'-triphosphate (UTP). As indicated by the enhanced expression of P2Y(2) receptor mRNA, P2Y(2) receptors are responsible for the increased responses evoked by ATP and UTP in interleukin-1beta-treated cells. In addition, the effect of endothelin-1, a well-known inhibitor of gap junctional communication in astrocytes was also exacerbated following interleukin-1beta treatment. We conclude that ATP decreases intercellular communication through gap junctions in astrocytes and that the increased sensitivity of gap junction channels to nucleotides and endothelin-1 is a characteristic feature of astrocytes exposed to pro-inflammatory treatments.

Spontaneous synaptic activity in chick vestibular nucleus neurons during the perinatal period.

The principal cells of the chick tangential nucleus are second-order vestibular neurons involved in the vestibuloocular and vestibulocollic reflexes. The spontaneous synaptic activity of morphologically identified principal cells was characterized in brain slices from 1-day-old hatchlings (H1) using whole-cell voltage-clamp recordings and Cs-gluconate pipet solution. The frequency was 1.45 Hz for spontaneous excitatory postsynaptic currents (sEPSCs) and 1.47 Hz for spontaneous inhibitory postsynaptic currents (sIPSCs). Using specific neurotransmitter receptor antagonists, all of the sEPSCs were identified as AMPA receptor-mediated events, whereas 56% of the sIPSCs were glycine and 44% were GABA(A) receptor-mediated events. On exposure to TTX, the frequency of EPSCs decreased by 68%, while the frequency of IPSCs decreased by 33%, indicating greater EPSC dependency on presynaptic action potentials. These data on spontaneous synaptic activity at H1 were compared with those obtained in previous studies of 16-day old embryos (E16). After birth, the spontaneous synaptic activity exhibited increased EPSC frequency, increased ratio for excitatory to inhibitory events, increased percentage of TTX-dependent EPSCs, and faster kinetics. In addition, the ratio for glycine/GABA receptor-mediated events increased significantly. Altogether, these data indicate that at hatching spontaneous synaptic activity of vestibular nucleus neurons in brain slices of the chick tangential nucleus undergoes appreciable changes, with increased frequency of EPSCs and glycinergic activity playing more important roles compared with the late-term chick embryo when GABAergic activity prevailed. The definition of this developmental pattern of synaptic activity in vestibular nucleus neurons should contribute to understanding how vestibular reflex activity is established in the hatchling chick.

Microglia and astrocytes may participate in the shaping of visual callosal projections during postnatal development.

In the adult cat, axons running through the corpus callosum interconnect the border between the visual cortical areas 17 and 18 (A17 and A18) of both hemispheres. This specific pattern emerges during postnatal development, under normal viewing conditions (NR), from the elimination of initially exuberant callosal projections. In contrast, if the postnatal visual experience is monocular from birth (MD), juvenile callosal projections are stabilised throughout A17 and A18. The present study aimed at using such a model in vivo to find indications of a contribution of glial cells in the shaping of projections in the developing CNS through interactions with neurones, both in normal and pathological conditions. As a first stage, the distribution and the morphology of microglial cells and astrocytes were investigated from 2 weeks to adulthood. Microglial cells, stained with isolectin-B4, were clustered in the white matter below A17 and A18. Until one month, these clustered cells displayed an ameboid morphology in NR group, while they were more ramified in MD animals. Their phenotype thus depends on the postnatal visual experience, which indicates that microglial cells may interact with axons of visual neurones. It also suggests that they may differentially contribute to the elimination and the stabilisation of juvenile exuberant callosal fibres in NR and MD animals respectively. Beyond one month, microglial cells were very ramified in both experimental groups. Astrocytes were labelled with a GFAP-antibody. The distributions of connexins 43 (Cx43) and 30 (Cx30), the main proteic components of gap junction channels in astrocytes, were also investigated using specific antibodies. Both in NR and MD groups, until 1 month, GFAP-positive astrocytes and Cx43 were mainly localised within the subcortical white matter. Then GFAP, Cx43 and Cx30 stainings progressively appeared within the cortex, throughout A17 and A18 but with a differential laminar expression according to the age. Thus, the distributions of both astrocytes and connexins changed with age; however, the monocular occlusion had no visible effect. This suggests that astrocytes may contribute to the postnatal development of neuronal projections to the primary visual cortex, including visual callosal projections.

Lack of biocytin transfer at gap junctions in the chicken vestibular nuclei.

In vivo experiments were designed to test for functional gap junctions at 'mixed' synapses that were morphologically characterized between the large-diameter, primary vestibular fibers and second-order vestibular neurons in the chicken, Gallus gallus. In previous intracellular recordings and dye injections into these neurons from brain slice preparations of chick embryos (E15/16) and also newborn hatchlings (HI-2), no evidence was obtained for functional gap junctions. Therefore, biocytin, a low molecular weight tracer that permeates gap junction channels, was extracellularly applied to either the ampullary nerves or to the vestibular ganglion of 3-6 day old hatchlings and adult chickens (9 months). This procedure resulted in the uptake of the dye and heavy staining of both the thick and thin fibers composing the vestibular nerve and in loading of vestibular efferent neurons. However, no dye transfer was observed between the large-diameter, primary vestibular fibers and second-order vestibular neurons. This observation, which was performed using a relatively non-invasive approach on intact animals, suggests that the gap junctions at these mixed synapses are probably not functional under the conditions of these experiments.

Uncoupling of invertebrate electrotonic synapses by carbon dioxide.

Cell acidification was obtained by exposing to carbon dioxide the lateral giant axon of the crayfish and the buccal ganglion of Navanax inermis; electrophysiological measurements show that the resulting intracellular pH uncouples neurons and that this uncoupling is the consequence of increased junctional resistance at the level of the gap junctions. Conditions under which the uncoupling is reversible are also described.