Astrocytic glutamate is not necessary for the generation of epileptiform neuronal activity in hippocampal slices.

The release of glutamate from astrocytes activates synchronous slow inward currents (SICs) in hippocampal pyramidal neurons, which are mediated by the NMDA receptor and represent a nonsynaptic mechanism to promote the synchronization of neuronal activity. Two recent studies demonstrate that SICs generate neuronal paroxysmal depolarizations resembling those typical of interictal epileptiform activity and proposed that there could be an astrocytic basis of epilepsy (Kang et al., 2005; Tian et al., 2005). We tested this hypothesis using two in vitro models of epileptiform activity in hippocampal slices. Removal of extracellular Mg2+ and application of picrotoxin or perfusion with 0.5 mM Mg2+ and 8.5 mM K+-containing saline result mainly in neuronal ictal- and interictal-like epileptiform activity, respectively. Although both models trigger epileptiform activity, astrocytic Ca2+ oscillations were increased only after slice perfusion with 0 mM Mg2+ and picrotoxin. The activation of astrocytic Ca2+ signaling correlates with an increased frequency of SICs, and, when paired neurons were within 100 microm of one another with synchronous neuronal Ca2+ elevations, the generation of synchronous neuronal depolarizations and action potential discharges. TTX blocked both ictal- and interictal-like epileptiform activity without affecting SICs or SIC-mediated neuronal synchronization. In contrast, NMDA receptor antagonists, which block SICs, did not prevent the generation of either ictal- or interictal-like events. Based on this clear-cut pharmacology, our data demonstrate that nonsynaptic glutamate release from astrocytes is not necessary for the generation of epileptiform activity in vitro, although we cannot exclude the possibility that it may modulate the strength of the ictal (seizure)-like event.

Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors.

Fast excitatory neurotransmission is mediated by activation of synaptic ionotropic glutamate receptors. In hippocampal slices, we report that stimulation of Schaffer collaterals evokes in CA1 neurons delayed inward currents with slow kinetics, in addition to fast excitatory postsynaptic currents. Similar slow events also occur spontaneously, can still be observed when neuronal activity and synaptic glutamate release are blocked, and are found to be mediated by glutamate released from astrocytes acting preferentially on extrasynaptic NMDA receptors. The slow currents can be triggered by stimuli that evoke Ca2+ oscillations in astrocytes, including photolysis of caged Ca2+ in single astrocytes. As revealed by paired recording and Ca2+ imaging, a striking feature of this NMDA receptor response is that it occurs synchronously in multiple CA1 neurons. Our results reveal a distinct mechanism for neuronal excitation and synchrony and highlight a functional link between astrocytic glutamate and extrasynaptic NMDA receptors.

Neurotrophins in spinal cord nociceptive pathways.

Neurotrophins are a well-known family of growth factors for the central and peripheral nervous systems. In the course of the last years, several lines of evidence converged to indicate that some members of the family, particularly NGF and BDNF, also participate in structural and functional plasticity of nociceptive pathways within the dorsal root ganglia and spinal cord. A subpopulation of small-sized dorsal root ganglion neurons is sensitive to NGF and responds to peripheral NGF stimulation with upregulation of BDNF synthesis and increased anterograde transport to the dorsal horn. In the latter, release of BDNF appears to modulate or even mediate nociceptive sensory inputs and pain hypersensitivity. We summarize here the status of the art on the role of neurotrophins in nociceptive pathways, with special emphasis on short-term synaptic and intracellular events that are mediated by this novel class of neuromessengers in the dorsal horn. Under this perspective we review the findings obtained through an array of techniques in naïve and transgenic animals that provide insight into the modulatory mechanisms of BDNF at central synapses. We also report on the results obtained after immunocytochemistry, in situ hybridization, and monitoring intracellular calcium levels by confocal microscopy, that led to hypothesize that also NGF might have a direct central effect in pain modulation. Although it is unclear whether or not NGF may be released at dorsal horn endings of certain nociceptors in vivo, we believe that these findings offer a clue for further studies aiming to elucidate the putative central effects of NGF and other neurotrophins in nociceptive pathways.

Glutamate-mediated cytosolic calcium oscillations regulate a pulsatile prostaglandin release from cultured rat astrocytes.

The synaptic release of glutamate evokes in astrocytes periodic increases in [Ca2+]i, due to the activation of metabotropic glutamate receptors (mGluRs). The frequency of these [Ca2+]i oscillations is controlled by the level of neuronal activity, indicating that they represent a specific, frequency-coded signalling system of neuron-to-astrocyte communication. We recently found that neuronal activity-dependent [Ca2+]i oscillations in astrocytes are the main signal that regulates the coupling between neuronal activity and blood flow, the so-called functional hyperaemia. Prostaglandins play a major role in this fundamental phenomenon in brain function, but little is known about a possible link between [Ca2+]i oscillations and prostaglandin release from astrocytes. To investigate whether [Ca2+]i oscillations regulate the release of vasoactive prostaglandins, such as the potent vasodilator prostaglandin E2 (PGE2), from astrocytes, we plated wild-type human embryonic kidney (HEK)293 cells, which respond constitutively to PGE2 with [Ca2+]i elevations, onto cultured astrocytes, and used them as biosensors of prostaglandin release. After loading the astrocyte-HEK cell co-cultures with the calcium indicator Indo-1, confocal microscopy revealed that mGluR-mediated [Ca2+]i oscillations triggered spatially and temporally coordinated [Ca2+]i increases in the sensor cells. This response was absent in a clone of HEK cells that are unresponsive to PGE2, and recovered after transfection with the InsP3-linked prostanoid receptor EP1. We conclude that [Ca2+]i oscillations in astrocytes regulate prostaglandin releases that retain the oscillatory behaviour of the [Ca2+]i changes. This finely tuned release of PGE2 from astrocytes provides a coherent mechanistic background for the role of these glial cells in functional hyperaemia.

Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation.

The cellular mechanisms underlying functional hyperemia--the coupling of neuronal activation to cerebral blood vessel responses--are not yet known. Here we show in rat cortical slices that the dilation of arterioles triggered by neuronal activity is dependent on glutamate-mediated [Ca(2+)](i) oscillations in astrocytes. Inhibition of these Ca(2+) responses resulted in the impairment of activity-dependent vasodilation, whereas selective activation--by patch pipette--of single astrocytes that were in contact with arterioles triggered vessel relaxation. We also found that a cyclooxygenase product is centrally involved in this astrocyte-mediated control of arterioles. In vivo blockade of glutamate-mediated [Ca(2+)](i) elevations in astrocytes reduced the blood flow increase in the somatosensory cortex during contralateral forepaw stimulation. Taken together, our findings show that neuron-to-astrocyte signaling is a key mechanism in functional hyperemia.