Abstract The anatomical and physiological properties of GABAergic inhibitory neurotransmission were investigated in organotypic slice cultures of rat hippocampus. Interneurons and terminal‐like elements containing GABA‐like immunoreactivity were numerous in tissue kept for 13–26 days in culture and showed a similar morphology and distribution to those known from investigations on the hippocampal formation in situ. Furthermore, after 8–30 days in culture, spontaneous and evoked IPSPs were observed in all CA3 pyramidal cells tested, resulting from an increase in chloride conductance, and were shown to be mediated by activation of GABA receptors. No functional decrement in the efficacy of GABAergic inhibitory synaptic transmission following chronic isolation and long‐term maintenance in vitro was noticed. In particular, neither the magnitude of the synaptic conductance underlying the inhibitory postsynaptic currents nor its reversal potential varied with time in culture. Taken together, the present physiological and immunohistochemical data show that GABAergic inhibition is well expressed in organotypic hippocampal slice cultures and is maintained over periods of at least 4 weeks in vitro.
Abstract Expression of neurotrophins (NTs) and their receptors is elevated in the adult CNS under several neuropathological conditions. We have investigated the anatomical and electrophysiological consequences of chronic NT‐3 or NT‐4/5 treatment on established organotypic hippocampal slice cultures maintained in vitro for > 14 days. Both NT‐3 and NT‐4/5 increased spontaneous, action potential‐dependent excitatory synaptic activity (sEPSCs), but only NT‐3 increased inhibitory synaptic activity (sIPSCs) in CA3 pyramidal cells. Both NTs strongly promoted spontaneous synaptic bursting activity. Spontaneous bursts of EPSCs were observed after either NT treatment but only NT‐3‐treated cultures exhibited an increase in spontaneous bursts of IPSCs. In addition, sIPSC bursts were eliminated by blocking glutamatergic excitation. The frequency of miniature inhibitory postsynaptic currents, but not miniature excitatory postsynaptic currents, was also increased by both NT‐3 and NT‐4/5. Furthermore, NT‐3 and NT‐4/5 induced an up‐regulation of the growth‐associated protein GAP‐43, suggesting that neurotrophins may be able to induce axonal reorganization in established neuronal networks. CA1 pyramidal cells exhibited slight alterations in dendritic branching after NT‐4/5, but not NT‐3 treatment. We conclude that chronic treatment with NT‐3 or NT‐4/5 can affect an established hippocampal network by elevating spontaneous inhibitory and excitatory synaptic activity and inducing coordinated pre‐ and postsynaptic structural changes.
An excitatory action of l-2-amino-4-phosphonobutanoate (l-AP4), a glutamate analogue, is observed following pre-exposure of tissue to quisqualate. We have studied the mechanism of sensitization of l-AP4 responses by quisqualate in voltage-clamped CA3 pyramidal cells in rat hippocampal slice cultures in the presence of tetrodotoxin. Prior to quisqualate addition, CA3 cells did not respond to l-AP4 (50 - 1000 microM). Following brief application of quisqualate (500 nM for 30 s), l-AP4 (50 - 200 microM) induced a complex excitatory response which could be obtained for >1 h. l-AP4 caused an ionotropic inward current associated with a conductance increase. This response was in part sensitive to 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and in part sensitive to d-2-amino-5-phosphonovalerate (d-AP5) and Mg2+ ions. At depolarizing potentials, in the presence of CNQX and d-AP5, l-AP4 caused excitation by depressing K+ currents, mimicking the metabotropic action of glutamate. This indicates that the action of l-AP4 is mediated by three different receptor types: N-methyl-d-aspartate (NMDA) receptors, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors, and glutamatergic metabotropic receptors. The l-AP4 response persisted in solutions containing low Ca2+ and high Mg2+ concentrations or 100 - 200 microM Cd2+, suggesting that it is independent of extracellular Ca2+. We were unable to identify any substance other than quisqualate capable of sensitizing the l-AP4 action. This effect also occurred when quisqualate was applied in Ca2+-free solution or in solutions containing low concentrations of Na+ or Cl-. Sensitization of l-AP4 responses by quisqualate was not observed in acutely dissociated pyramidal cells recorded by means of the whole-cell recording mode, although ionotropic quisqualate responses were present. Sensitization was readily reversed by short applications of the endogenous excitatory amino acids glutamate, aspartate and homocysteate at concentrations of 10 - 100 microM. Our data are consistent with the hypothesis that the excitatory action of l-AP4 results from a Ca2+-independent release of endogenous excitatory amino acids from some presynaptic neuronal or glial site.
Abstract: Cocultures of septal and hippocampal tissues taken from 6‐ to 7‐day‐old rats were maintained in culture for up to 30 days in the presence and absence of nerve growth factor (NGF). and their Chol‐1 contents determined at varying time intervals by a modified enzyme‐linked immunosorption assay (ELISA). The major brain gangliosides were determined densitometrically after spraying chromatograms with resorcinol reagent. There was little change in the contribution of the major gangliosides to the total ganglioside content of the explants either with time or the presence or absence of NGF, the only exception being an NGF‐insensitive fall in the contribution of GM1 to about 60% of its initial value at 20 and 30 days. By contrast, the concentration of Chol‐1 expressed either per unit weight of ganglioside sialic acid or protein increased considerably in culture and this increase was enhanced by NGF. The effect of NGF resembles that on other cholinergic markers, choline acetyltransferase and acetylcholinesterase, and may be attributed to an NGF‐stimulated hippocampal ingrowth of cholinergic fibres and enhanced survival of cholinergic septal neurons. The Chol‐1 concentration finally attained in the presence of NGF and the time course of its increase parallel those previously found in vivo and indicate the potential usefulness of scptal‐hippocampal cocultures for investigating the function of Chol‐1.
