The modern concept of neurogenesis in the adult brain is predicated on the premise that multipotent glial cells give rise to new neurons throughout life. Although extensive evidence exists indicating that this is the case, the transition from glial to neuronal phenotype remains poorly understood. A unique monolayer cellculture system was developed to induce, expose, and recapitulate the entire developmental series of events of subventricular zone (SVZ) neurogenesis. We show here, using immunophentoypic, ultrastructural, electrophysiological, and time-lapse analyses, that SVZ-derived glial fibrillary acidic protein low ͞A2B5 ؉ ͞nestin ؉ candidate founder cells undergo metamorphosis to eventually generate large numbers of fully differentiated interneuron phenotypes. A model of postnatal neurogenesis is considered in light of known embryonic events and reveals a limited developmental potential of SVZ stem͞progenitor cells, whereby ancestral cells in both embryonic and postnatal͞adult settings give rise to glia and GABAergic interneurons.adult stem cells ͉ electrophysiology ͉ in vitro ͉ neurogenesis ͉ subventricular zone A ttempts to trace the cellular source of neurogenesis in the adult CNS have recently led to the surprising conclusion that dedicated glial cells give rise to new neurons throughout life (1-5). Even though neurons and glia are both derived from the embryonic neuroepithelium, sharing common signaling pathways and downstream transcription factors during development (6), it is difficult to imagine how one major cell class in the adult brain can transpose into the other. Postnatal neurogenesis in the subventricular zone (SVZ) of rodents proceeds as a characteristic series of events, where multipotent glial cells (referred to as type-B cells) can divide to form colonies of neuroblasts (type-A cells) through a transit-amplifying cell population (type-C cells) (7). Newborn neuroblasts migrate from the SVZ through the rostral migratory stream and mature to GABAergic granule cells and periglomerular cells, which, 3-4 weeks after generation, integrate as inhibitory interneurons into the olfactory bulb of rodents (8-10). Certain features, such as nestinand glial-fibrillary acidic protein (GFAP) expression, are ascribed to the founder cells of postnatal neurogenesis (3,(11)(12)(13)(14), but their distinctive antigenic and functional profiles remain elusive. Traditional approaches for the isolation and characterization of persistent neurogenesis have relied on the in vitro neurosphere (NS) assay (15, 16) or on post hoc identification, depending on the incorporation of BrdUrd and͞or retroviral constructs to label precursors during cell division. However, neither of these methods affords the recognition of the dynamic processes involved in the maturation of individual cells en route from stem cells to fully differentiated neural phenotypes.Here, we present an alternative culture model that closely recapitulates in vivo postnatal͞adult SVZ neurogenesis, allowing us to monitor the entire sequence of hierarchical eve...

The dentate gyrus (DG) normally functions as a filter, preventing propagation of synchronized activity into the seizure-prone hippocampus. This filter or 'gatekeeper' attribute of the DG is compromised in various pathological states, including temporal lobe epilepsy (TLE). This study examines the role that altered inhibition may play in the deterioration of this crucial DG function. Using the pilocarpine animal model of TLE, we demonstrate that inhibitory synaptic function is altered in principal cells of the DG. Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in dentate granule cells (DGCs) from epileptic animals were larger, more sensitive to blockade by zinc and less sensitive to augmentation by the benzodiazepine type site 1 modulator zolpidem. Furthermore, mIPSCs examined during a quiescent period following injury but preceding onset of epilepsy were significantly smaller than those present either in control or in TLE DGCs, and had already acquired sensitivity to blockade by zinc prior to the onset of spontaneous seizures. Rapid agonist application experiments demonstrated that prolonged (>35 ms) exposure to zinc is required to block GABA A receptors (GABA A Rs) in patches pulled from epileptic DGCs. Therefore, zinc must be tonically present to block DGC GABA A Rs and alter DG function. This would occur only during repetitive activation of mossy fibres. Thus, in the pilocarpine animal model of TLE, an early, de novo, expression of zinc-sensitive GABA A Rs is coupled with delayed, epilepsy-induced development of a zinc delivery system provided by aberrant sprouting of zinc-containing mossy fibre recurrent collaterals. The temporal and spatial juxtaposition of these pathophysiological alterations may compromise normal 'gatekeeper' function of the DG through dynamic zinc-induced failure of inhibition, predisposing the hippocampal circuit to generate seizures.

In the CNS, inhibitory synaptic function undergoes profound transformation during early postnatal development. This is due to variations in the subunit composition of subsynaptic GABA(A) receptors (GABA(A)Rs) at differing developmental stages as well as other factors. These include changes in the driving force for chloride-mediated conductances as well as the quantity and/or cleft lifetime of released neurotransmitter. The present study was undertaken to investigate the nature and time course of developmental maturation of GABAergic synaptic function in hippocampal CA1 pyramidal neurons. In neonatal [postnatal day (P) 1-7] and immature (P8-14) CA1 neurons, miniature inhibitory postsynaptic currents (mIPSCs) were significantly larger, were less frequent, and had slower kinetics compared with mIPSCs recorded in more mature neurons. Adult mIPSC kinetics were achieved by the third postnatal week in CA1 neurons. However, despite this apparent maturation of mIPSC kinetics, significant differences in modulation of mIPSCs by allosteric agonists in adolescent (P15-21) neurons were still evident. Diazepam (1-300 nM) and zolpidem (200 nM) increased the amplitude of mIPSCs in adolescent but not adult neurons. Both drugs increased mIPSC decay times equally at both ages. These differential agonist effects on mIPSC amplitude suggest that in adolescent CA1 neurons, inhibitory synapses operate differently than adult synapses and function as if subsynaptic receptors are not fully occupied by quantal release of GABA. Rapid agonist application experiments on perisomatic patches pulled from adolescent neurons provided additional support for this hypothesis. In GABA(A)R currents recorded in these patches, benzodiazepine amplitude augmentation effects were evident only when nonsaturating GABA concentrations were applied. Furthermore nonstationary noise analysis of mIPSCs in P15-21 neurons revealed that zolpidem-induced mIPSC augmentation was not due to an increase in single-channel conductance of subsynaptic GABA(A)Rs but rather to an increase in the number of open channels responding to a single GABA quantum, further supporting the hypothesis that synaptic receptors may not be saturated during synaptic function in adolescent neurons. These data demonstrate that inhibitory synaptic transmission undergoes a markedly protracted postnatal maturation in rat CA1 pyramidal neurons. In the first two postnatal weeks, mIPSCs are large in amplitude, are slow, and occur infrequently. By the third postnatal week, mIPSCs have matured kinetically but retain distinct responses to modulatory drugs, possibly reflecting continued immaturity in synaptic structure and function persisting through adolescence.

γ-Aminobutyric acidAreceptors (GABARs) are heteromeric proteins composed of multiple subunits. Numerous subunit subtypes are expressed in individual neurons, which assemble in specific preferred GABAR configurations. Little is known, however, about the coordination of subunit expression within individual neurons or the impact this may have on GABAR function. To investigate this, it is necessary to profile quantitatively the expression of multiple subunit mRNAs within individual cells. In this study, single-cell antisense RNA amplification was used to examine the expression of 14 different GABAR subunit mRNAs simultaneously in individual human dentate granule cells (DGCs) harvested during hippocampectomy for intractable epilepsy. α4, β2, and δ-mRNA levels were tightly correlated within individual DGCs, indicating that these subunits are expressed coordinately. Levels of α3- and β2-mRNAs, as well as ε- and β1-mRNAs, also were strongly correlated. No other subunit correlations were identified. Coordinated expression could not be explained by the chromosomal clustering of GABAR genes and was observed in control and epileptic rats as well as in humans, suggesting that it was not species-specific or secondary to epileptogenesis. Benzodiazepine augmentation of GABA-evoked currents also was examined to determine whether levels of subunit mRNA expression correlated with receptor pharmacology. This analysis delineated two distinct cell populations that differed in clonazepam modulation and patterns of α-subunit expression. Clonazepam augmentation correlated positively with the relative expression of α1- and γ2-mRNAs and negatively with α4- and δ-mRNAs. These data demonstrate that specific GABAR subunit mRNAs exhibit coordinated control of expression in individual DGCs, which has significant impact on inhibitory function.

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Zinc is found throughout the CNS at synapses co-localized with glutamate in presynaptic terminals. In particular, dentate granule cells' (DGC) mossy fiber (MF) axons contain especially high concentrations of zinc co-localized with glutamate within vesicles. To study possible physiological roles of zinc, visualized slice-patch techniques were used to voltage-clamp rat CA3 pyramidal neurons, and miniature excitatory postsynaptic currents (mEPSCs) were isolated. Bath-applied zinc (200 microM) enhanced median mEPSC peak amplitudes to 153.0% of controls, without affecting mEPSC kinetics. To characterize this augmentation further, rapid agonist application was performed on perisomatic outside-out patches to coapply zinc with glutamate extremely rapidly for brief (1 ms) durations, thereby emulating release kinetics of these substances at excitatory synapses. When zinc was coapplied with glutamate, zinc augmented peak glutamate currents (mean +/- SE, 116.6 +/- 2.8% and 143.8 +/- 9.8% of controls at 50 and 200 microM zinc, respectively). This zinc-induced potentiation was concentration dependent, and pharmacological isolation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated currents (AMPAR currents) gave results similar to those observed with glutamate application (mean, 115.0 +/- 5.4% and 132.5 +/- 9.1% of controls at 50 and 200 microM zinc, respectively). Inclusion of the AMPAR desensitization blocker cyclothiazide in the control solution, however, abolished zinc-induced augmentation of glutamate-evoked currents, suggesting that zinc may potentiate AMPAR currents by inhibiting AMPAR desensitization. Based on the results of the present study, we hypothesize that zinc is a powerful modulator of both excitatory synaptic transmission and glutamate-evoked currents at physiologically relevant concentrations. This modulatory role played by zinc may be a significant factor in enhancing excitatory neurotransmission and could significantly regulate function at the mossy fiber-CA3 synapse.

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