Crucial role of astrocytes in temporal lobe epilepsy.

Astrocytes sense and respond to synaptic activity through activation of different neurotransmitter receptors and transporters. Astrocytes are also coupled by gap junctions, which allow these cells to redistribute through the glial network the K(+) ions excessively accumulated at sites of intense neuronal activity. Work over the past two decades has revealed important roles for astrocytes in brain physiology, and it is therefore not surprising that recent studies unveiled their involvement in the etiology of neurological disorders such as epilepsy. Investigation of specimens from patients with pharmacoresistant temporal lobe epilepsy and epilepsy models revealed alterations in expression, localization and function of astrocytic connexins, K(+) and water channels. In addition, disturbed gliotransmission as well as malfunction of glutamate transporters and of the astrocytic glutamate- and adenosine-converting enzymes - glutamine synthetase and adenosine kinase, respectively - have been observed in epileptic tissues. Accordingly, increasing evidence indicates that dysfunctional astrocytes are crucially involved in processes leading to epilepsy. These new insights might foster the search for new targets for the development of new, more efficient anti-epileptogenic therapies.

Dynamic signaling between astrocytes and neurons.

Astrocytes, a sub-type of glia in the central nervous system, are dynamic signaling elements that integrate neuronal inputs, exhibit calcium excitability, and can modulate neighboring neurons. Neuronal activity can lead to neurotransmitter-evoked activation of astrocytic receptors, which mobilizes their internal calcium. Elevations in astrocytic calcium in turn trigger the release of chemical transmitters from astrocytes, which can cause sustained modulatory actions on neighboring neurons. Astrocytes, and perisynaptic Schwann cells, by virtue of their intimate association with synapses, are strategically positioned to regulate synaptic transmission. This capability, that has now been demonstrated in several studies, raises the untested possibility that astrocytes are an integral element of the circuitry for synaptic plasticity. Because the highest ratio of glia-to-neurons is found at the top of the phylogenetic tree in the human brain, these recent demonstrations of dynamic bi-directional signaling between astrocytes and neurons leave us with the question as to whether astrocytes are key regulatory elements of higher cortical functions.

Cytosolic calcium oscillations in astrocytes may regulate exocytotic release of glutamate.

To obtain insights into the spatiotemporal characteristics and mechanism of Ca(2+)-dependent glutamate release from astrocytes, we developed a new experimental approach using human embryonic kidney (HEK) 293 cells transfected with the NMDA receptor (NMDAR), which act as glutamate biosensors, plated on cultured astrocytes. We here show that oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) in astrocytes trigger synchronous and repetitive [Ca(2+)](i) elevations in sensor HEK cells, and that these elevations are sensitive to NMDAR inhibition. By whole-cell patch-clamp recordings, we demonstrate that the activation of NMDARs in HEK cells results in inward currents that often have extremely fast kinetics, comparable with those of glutamate-mediated NMDAR currents in postsynaptic neurons. We also show that the release of glutamate from stimulated astrocytes is drastically reduced by agents that are known to reduce neuronal exocytosis, i.e., tetanus toxin and bafilomycin A(1). We conclude that [Ca(2+)](i) oscillations represent a frequency-encoded signaling system that controls a pulsatile release of glutamate from astrocytes. The fast activation of NMDARs in the sensor cells and the dependence of glutamate release on the functional integrity of both synaptobrevin and vacuolar H(+) ATPase suggest that astrocytes are endowed with an exocytotic mechanism of glutamate release that resembles that of neurons.

Reciprocal communication systems between astrocytes and neurones.

Over the past decade, a growing body of evidence has emerged on the existence in the brain of a close bidirectional communication system between neurones and astrocytes. This article reviews recent advances in understanding the rules governing these interactions and describes putative, novel functions attributable to astrocytes in neuronal transmission. Astrocytes can respond to the neurotransmitter released from active synaptic terminals, with cytosolic Ca(2+) oscillations whose frequency is under the dynamic control of neuronal activity. In response to these neuronal signals, astrocytes can signal back to neurones by releasing various neurone active compounds, such as the excitatory neurotransmitter glutamate. Interestingly, there is accumulating evidence that glutamate is released via a Ca(2+)-dependent mechanism which may share common properties with neurotransmitter exocytosis in neurones. This bidirectional communication system between neurones and astrocytes may lead to profound changes in neuronal excitability and synaptic transmission. While there clearly is an enormous amount of experimental and theoretical work yet to figure out, a coherent view is now emerging which incorporates the astrocyte, with the presynaptic terminal and the postsynaptic target neurone, as a possible third functional element of the synapse.

Cellular calcium handling in brain slices from calbindin D28k-deficient mice.

Cellular calcium handling was examined in brain slices from transgenic antisense mice with a regional deficiency in the neuronal calcium binding protein calbindin D28k and from their non transgenic wild type litter mate controls. Depolarization of brain slices with NMDA or potassium produced a prolonged elevation of neuronal calcium signal in neurons in brain slices from calbindin D28k-deficient transgenic mice. This effect was selective and was seen only in brain areas where the antisense construct produced a significant depletion of calbindin D28k protein. In other regions where calbindin D28k protein was not modified by the construct and in all glial cells whether from wild type or transgenic mice, cellular calcium handling was normal.

Nitric oxide-producing islet cells modulate the release of sensory neuropeptides in the rat substantia gelatinosa.

The substantia gelatinosa of the spinal cord (lamina II) is the major site of integration for nociceptive information. Activation of NMDA glutamate receptor, production of nitric oxide (NO), and enhanced release of substance P and calcitonin gene-related peptide (CGRP) from primary afferents are key events in pain perception and central hyperexcitability. By combining reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry for NO-producing neurons with immunogold labeling for substance P, CGRP, and glutamate, we show that (1) NO-producing neurons in lamina IIi are islet cells; (2) these neurons rarely form synapses onto peptide-immunoreactive profiles; and (3) NADPH diaphorase-positive dendrites are often in close spatial relationship with peptide-containing terminals and are observed at the periphery of type II glomeruli showing glutamate-immunoreactive central endings. By means of confocal fluorescent microscopy in acute spinal cord slices loaded with the Ca2+ indicator Indo-1, we also demonstrate that (1) NMDA evokes a substantial [Ca2+]i increase in a subpopulation of neurons in laminae I-II, with morphological features similar to those of islet cells; (2) a different neuronal population in laminae I-IIo, unresponsive to NMDA, displays a significant [Ca2+]i increase after slice perfusion with either substance P and the NO donor 3morpholinosydnonimine (SIN-1); and (3) the responses to both substance P and SIN-1 are either abolished or significantly inhibited by the NK1 receptor antagonist sendide. These results provide compelling evidence that glutamate released at type II glomeruli triggers the production of NO in islet cells within lamina IIi after NMDA receptor activation. The release of substance P from primary afferents triggered by newly synthesized NO may play a crucial role in the cellular mechanism leading to spinal hyperexcitability and increased pain perception.

On the role of voltage-dependent calcium channels in calcium signaling of astrocytes in situ.

Calcium ions play crucial roles in a large variety of cell functions. The recent proposal that changes in the intracellular calcium concentration ([Ca2+]i) in astrocytes underline a reciprocal communication system between neurons and astrocytes encourages the interest in the definition of the various components participating in this novel Ca2+ signaling system. We investigate here whether functional voltage-operated calcium channels (Ca2+ VOCs), which are clearly expressed in cultured astrocytes, participate in the regulation of [Ca2+]i also in astrocytes in situ. Depolarization with 40-60 mM K+ was used to analyze the activity of Ca2+ VOCs in Indo-1-loaded astrocytes in acute slices from the visual cortex and the CA1 hippocampal region of developing rats. We demonstrate here that the depolarization-induced [Ca2+]i increases in astrocytes are solely attributed to the activation of metabotropic receptors by neurotransmitters, such as glutamate, released by synaptic terminals on depolarization. In fact, (1) the K+-induced [Ca2+]i increases in astrocyte [Ca2+]i were potently reduced by alpha-methyl-4-carboxyphenylglycine, a metabotropic glutamate receptor competitive inhibitor; (2) after emptying intracellular Ca2+ stores with cyclopiazonic acid, none of the astrocytes displayed a [Ca2+]i increase on the depolarizing stimulus; and (3) after inhibiting neurotransmitter secretion in neurons by incubating the slices with tetanus neurotoxin, no [Ca2+]i increase on K+ stimulation was observed in astrocytes. Finally, patch-clamp whole-cell recordings from hippocampal astrocytes in acute brain slices failed to reveal any voltage-dependent calcium currents. On the basis of these results, the various roles proposed for astrocyte Ca2+ VOCs in the CNS should be reconsidered.

Prostaglandins stimulate calcium-dependent glutamate release in astrocytes.

Astrocytes in the brain form an intimately associated network with neurons. They respond to neuronal activity and synaptically released glutamate by raising intracellular calcium concentration ([Ca2+]i), which could represent the start of back-signalling to neurons. Here we show that coactivation of the AMPA/kainate and metabotropic glutamate receptors (mGluRs) on astrocytes stimulates these cells to release glutamate through a Ca2+-dependent process mediated by prostaglandins. Pharmacological inhibition of prostaglandin synthesis prevents glutamate release, whereas application of prostaglandins (in particular PGE2) mimics and occludes the releasing action of GluR agonists. PGE2 promotes Ca2+-dependent glutamate release from cultured astrocytes and also from acute brain slices under conditions that suppress neuronal exocytotic release. When applied to the CA1 hippocampal region, PGE2 induces increases in [Ca2+]i both in astrocytes and in neurons. The [Ca2+]i increase in neurons is mediated by glutamate released from astrocytes, because it is abolished by GluR antagonists. Our results reveal a new pathway of regulated transmitter release from astrocytes and outline the existence of an integrated glutamatergic cross-talk between neurons and astrocytes in situ that may play critical roles in synaptic plasticity and in neurotoxicity.

Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ.

The spatial-temporal characteristics of intracellular calcium ([Ca2+]i) changes elicited in neurons and astrocytes by various types of stimuli were investigated by means of confocal fluorescent microscopy in acute rat brain slices loaded with the Ca2+ indicator indo-1. Neurons and astrocytes from the visual cortex and CA1 hippocampal region were identified in situ on the basis of their morphological, electrophysiological, and pharmacological features. We show here that stimulation of neuronal afferents triggered periodic [Ca2+]i oscillations in astrocytes. The frequency of these oscillations was under a dynamic control by neuronal activity as it changed according to the pattern of stimulation. After repetitive episodes of neuronal stimulation as well as repetitive stimulation with a metabotropic glutamate receptor agonist, astrocytes displayed a long-lasting increase in [Ca2+]i oscillation frequency. Oscillating astrocytes were accompanied by repetitive [Ca2+]i elevations in adjacent neurons, most likely because of the release of glutamate via a tetanus toxin-resistant process. These results reveal that [Ca2+]i oscillations in astrocytes represent a highly plastic signaling system that underlies the reciprocal communication between neurons and astrocytes.

Brain-derived neurotrophic factor and nerve growth factor potentiate excitatory synaptic transmission in the rat visual cortex.

1. The effect of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) on excitatory synaptic transmission in the developing visual cortex was studied by whole-cell patch-clamp recordings from rat brain slices. 2. Both neurotrophins induced a rapid increase in the amplitude of impulse-evoked excitatory postsynaptic currents (EPSCs). BDNF also increased the frequency of spontaneous EPSCs. 3. Analysis of the currents revealed that alpha-amino-3-hydroxy-5-methyl-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated components contributing to the EPSC peak amplitude were equally potentiated by the neurotrophins. 4. When synaptic transmission was studied by minimal stimulation of intracortical afferents, neurotrophins induced a decrease in the occurrence of release failures. 5. A number of neurones were insensitive to the effects of the neurotrophins, possibly related to the considerable heterogeneity of neuronal types and to the uneven distribution of neurotrophin receptors in the visual cortex. 6. The probability of neurotransmitter release represents a rapidly modifiable synaptic feature by which neurotrophins can potentiate the efficacy of excitatory synaptic transmission in the visual cortex.

Long-lasting changes of calcium oscillations in astrocytes. A new form of glutamate-mediated plasticity.

Long-term changes of synaptic strength in the central nervous system are mediated by an increase of cytosolic calcium concentration ([Ca2+]i) following activation of excitatory neurotransmitter receptors. These phenomena, which represent a possible cellular basis for learning and memory processes in eukaryotes, are believed to be restricted to neurons. Here we provide evidence for a long-term change of the response elicited by the excitatory neurotransmitter glutamate in a non-neuronal cell population of the central nervous system, i.e. visual cortical astrocytes in culture. Stimulation with glutamate induces in astrocytes a regular pattern of [Ca2+]i oscillations. A second stimulation, after an interval ranging from 2 to 60 min, induces an oscillatory response characterized by an increased frequency. Induction of this change in the astrocyte response is abolished by a specific inhibitor of the nitric oxide synthase and recovers upon exogenous nitric oxide generation or addition of a permeant cGMP analogue. Local brief pulses of glutamate to individual astrocytes, at a rate of 1 Hz, also elicit [Ca2+]i oscillations whose frequency increases following a second series of pulses. The long-lasting modification in the [Ca2+]i oscillatory response induced by glutamate in astrocytes demonstrates that in the central nervous system cellular memory is not a unique feature of neurons.

Nerve growth factor promotes functional recovery of retinal ganglion cells after ischemia.

PURPOSE: To investigate the effect of a transient complete ischemia on the function of cat retina and to determine whether nerve growth factor (NGF), which was previously shown to enhance retinal ganglion cell (RGC) survival after optic nerve section in the adult rat, can promote recovery of retinal neurons after the ischemic insult. METHODS: Function of distal and proximal retina was assessed by recording the electroretinogram in response to both homogeneous flickering light (FERG) and contrast reversing gratings (PERG), respectively, 30 days after the induction of a 60-minute episode of ischemia. Visual evoked potentials in response to contrast reversing gratings were also recorded to evaluate visual acuity and contrast thresholds. Cell survival after ischemia was assessed in retinal whole-mounts stained with cresyl violet. Cats were intraocularly treated with NGF every other day, 3 times a week, for 30 days. Controls were treated with either phosphate buffered saline or cytochrome c. RESULTS: After ischemia, the FERG was not significantly affected. On the contrary, the PERG, visual acuity, and contrast thresholds were severely impaired. After NGF treatment, PERG response amplitudes were much less reduced compared to controls, and visual acuity and contrast thresholds were virtually normal. In addition, a larger number of presumed RGCs was present in the NGF-treated retinas compared to the cyt c-treated ones. CONCLUSIONS: The more proximally located retinal neurons, in particular RGCs, are highly vulnerable to ischemia. Intraocular NGF treatment was effective in enhancing the survival and functional recovery of these neurons. This suggests that NGF may represent a novel therapeutic agent for the treatment of ischemic ocular pathologies.

Developing rat retinal ganglion cells express the functional NGF receptor p140trkA.

The expression and cellular localization of NGF receptors in the developing rat retina were investigated immunocytochemically and biochemically. In in vitro preparations of retinal neurons from neonatal rats the functional NGF receptor p140trkA was immunocytochemically detected on retrogradely labeled retinal ganglion cells (RGCs). In transverse retinal sections p140trk-immunopositive cells were localized exclusively at the level of the RGC layer. Affinity labeling with 125I-NGF, chemical cross-linking, and immunoprecipitation with anti-NGF antibodies revealed the presence of three complexes which migrate on SDS-PAGE at approximately 90, 95, and 150 kDa. The bands at 90 and 95 kDa correspond to the so-called low affinity NGF receptor p75NGFR. Western blotting experiments using anti-TRK antibodies revealed that the slowest migrating band (150 kDa), which is not immunoprecipitated by monoclonal antibodies to p75NGFR, corresponds to p140trkA. The presence of the functional NGF receptor on RGCs provides the molecular explanation for the reported sensitivity of these cells to the biological action of NGF.

Effects of nerve growth factor on neuronal plasticity of the kitten visual cortex.

1. The effect of intraventricular administration of nerve growth factor (NGF) by means of a cannula-minipump system was studied in kittens monocularly deprived during the critical period. The ocular dominance of area 17 neurones of NGF-treated and control kittens was determined by conventional extracellular recordings. The soma size of cells in A and A1 laminae of the lateral geniculate nucleus (LGN) was also evaluated in Cresyl Violet preparations. 2. Binocularly responsive neurons were found to be significantly more numerous in NGF-treated than in control kittens. The shrinkage of cells from the deprived LGN laminae normally observed in control kittens was prevented by NGF administration. 3. Following an initial period of monocular deprivation (MD) kittens subsequently treated with NGF showed a substantial recovery of functional binocular connections. 4. These findings indicate that the administration of NGF during the period of deprivation reduces the amblyopic effects of MD, while its administration to kittens with both eyes open following the initial deprivation promotes recovery of the deprived eye. 5. Neurotrophic factors may contribute to the regulation of experience-dependent modifications of synaptic connectivity in the visual cortex.

Activity-dependent decrease in NMDA receptor responses during development of the visual cortex.

Plasticity of the developing visual system has been regarded as the best model for changes of neuronal connections under the influence of the environment. N-methyl-D-aspartate (NMDA) receptors are crucial for experience-dependent synaptic modifications that occur in the developing visual cortex. NMDA-mediated excitatory postsynaptic currents (EPSCs) in layer IV neurons of the visual cortex lasted longer in young rats than in adult rats, and the duration of the EPSCs became progressively shorter, in parallel with the developmental reduction in synaptic plasticity. This decrease in NMDA receptor-mediated EPSC duration is delayed when the animals are reared in the dark, a condition that prolongs developmental plasticity, and is prevented by treatment with tetrodotoxin, a procedure that inhibits neural activity. Application of L-glutamate to outside-out patches excised from layer IV neurons of young, but not of adult, rats activated prolonged bursts of NMDA channel openings. A modification of the NMDA receptor gating properties may therefore account for the age-dependent decline of visual cortical plasticity.

Distribution of protein gene product 9.5 (PGP 9.5) in the vertebrate retina: evidence that immunoreactivity is restricted to mammalian horizontal and ganglion cells.

Using light microscopic immunocytochemistry, we have studied the distribution of protein gene product 9.5 (PGP 9.5), a neuron-specific protein first extracted from human brain (Doran et al., '83:J. Neurochem. 40:1542-1547), in the vertebrate retina. Retinas were obtained from frog, chicken, rat, rabbit, cow, cat, dog, and human. No immunoreactivity was observed in frog and only a faint staining was present in chicken. In mammalian retinas, a strong positive reaction was restricted to horizontal and ganglion cells, with minor interspecies variations. Immunostaining was present throughout the cell body and the dendritic tree in horizontal cells. At the level of retinal ganglion cells, immunolabel was particularly abundant in cell bodies and axons forming the optic nerve. Only the main dendrites were stained, the remainder of the dendritic tree giving rise to a diffuse punctate reaction in the inner plexiform layer. In rats, displaced amacrine cells, which are known to contribute largely (40-50%) to the total neuronal population within the ganglion cell layer (Perry, '81: Neuroscience 6:931-944) were not immunoreactive, as demonstrated from (i) analysis of the morphology, cell size and cell density of immunoreactive neurons in wholemounts; (ii) colocalization of retrograde label and PGP 9.5 immunoreactivity in about 80% of ganglion cells after injection of peroxidase into the optic nerve; and (iii) reduction of immunoreactivity in the inner plexiform and ganglion cell layers following optic nerve transection. Western blot analysis of extracts from rabbit retinas indicated that the immunoreactive species is PGP 9.5 or a closely related molecule. Recent studies have demonstrated that PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase (Wilkinson et al., '89:Science 246:670-673). The present results, therefore, suggest that differences in the ubiquitination process exist between retinal neurons.

N-methyl-D-aspartate-induced neurotoxicity in the adult rat retina.

The present study provides evidence that the adult mammalian retina is highly sensitive to the excitotoxic action of NMDA. In particular, we have investigated the effects of a single intravitreal injection of different doses of N-methyl-D-aspartate (NMDA) (2-200 nmoles) on the adult rat retina. Morphological evaluation of transverse sections of retinae demonstrated a dose-dependent loss of cells in the ganglion cell layer (GCL) and a reduction in the thickness of the inner plexiform layer. No obvious alterations were noted in the more distal retinal layers. Quantitative analyses of Nissl-stained whole-mounted retinae revealed that administration of 20 nmoles of NMDA resulted in a 70% loss of cells with a soma diameter greater than 8 microns (presumed retinal ganglion cells); a 20% loss of cells with a soma diameter smaller than 8 microns (presumed displaced amacrine cells) was also observed. In addition, NMDA produced a dose-dependent decrease of retinal choline acetyltransferase (ChAT) activity, suggesting that NMDA affects cholinergic amacrine cells as well. MK-801, a non-competitive NMDA antagonist, completely prevented the NMDA-induced loss of cells in the GCL and blocked, in a dose-dependent manner, the NMDA-induced decrease of ChAT activity. The excitotoxic action of NMDA observed in these experiments is thus likely mediated through the NMDA receptor subtype. This "in vivo" model may be utilized to identify potential drugs that antagonize or limit the deleterious effects consequent to NMDA receptor overstimulation in the central nervous system.

Nerve growth factor prevents the amblyopic effects of monocular deprivation.

Monocular deprivation early in life causes dramatic changes in the functional organization of mammalian visual cortex and severe reduction in visual acuity and contrast sensitivity of the deprived eye. We tested whether or not these changes could be from competition between the afferents from the two eyes for a target-derived neurotrophic factor. Rats monocularly deprived during early postnatal development were treated with repetitive intraventricular injections or topical administration of nerve growth factor. The effects of monocular deprivation were then assessed electrophysiologically. In untreated animals visual acuity and contrast sensitivity of the deprived eye were strongly reduced, whereas in nerve growth factor-treated animals these parameters were normal.

Expression of NGF receptor and NGF receptor mRNA in the developing and adult rat retina.

Nerve growth factor (NGF) has been recently found to rescue axotomized retinal ganglion cells (RGCs) of the adult rat from degeneration. Because the trophic effect of NGF involves a receptor-coupling event, the characterization and cellular localization of the NGF receptor (NGFR) in the retina are essential to understanding the possible specific action of NGF in this district of the central nervous system. We report here that the NGFR mRNA is expressed in fetal, neonatal, and adult rat retina. Using monoclonal antibody 192-IgG to immunoprecipitate and immunohistochemically identify NGFR, we also found that the NGFR from the retina has a molecular weight identical to that of the NGFR from PC12 cells. The NGFR is localized on RGCs and Müller cells. Finally, following ligation of the optic nerve, NGFR-immunopositive material was found to accumulate both distal and proximal to the site of ligation, suggesting that RGC axons anterogradely and retrogradely transport the NGFR. These data raise the possibility that NGF may play a specific role in rat RGCs.