In situ activation of antigen-specific CD8+ T cells in the presence of antigen in organotypic brain slices.

The activation of Ag-specific T cells locally in the CNS could potentially contribute to the development of immune-mediated brain diseases. We addressed whether Ag-specific T cells could be stimulated in the CNS in the absence of peripheral lymphoid tissues by analyzing Ag-specific T cell responses in organotypic brain slice cultures. Organotypic brain slice cultures were established 1 h after intracerebral OVA Ag microinjection. We showed that when OVA-specific CD8(+) T cells were added to Ag-containing brain slices, these cells became activated and migrated into the brain to the sites of their specific Ags. This activation of OVA-specific T cells was abrogated by the deletion of CD11c(+) cells from the brain slices of the donor mice. These data suggest that brain-resident CD11c(+) cells stimulate Ag-specific naive CD8(+) T cells locally in the CNS and may contribute to immune responses in the brain.

Properties of a population of GABAergic cells in murine auditory cortex weakly excited by thalamic stimulation.

Feedforward inhibition triggered by thalamocortical (TC) afferents sharpens onset responses and shapes receptive fields of pyramidal cells in auditory cortex (ACx). Previous studies focused only on interneurons located in and around layer IV in primary ACx, target of the dense thalamic projections from ventral medial geniculate. We investigated a population of feedforward interneurons located throughout layers I-V and activated by both afferents from primary and nonprimary thalamus using recordings from auditory TC brain slices obtained from mice expressing green fluorescent protein under control of the glutamic acid decarboxylase (GAD65) promoter in a subpopulation of cortical GABAergic cells. We studied the responses of these interneurons and of pyramidal cells in ACx to thalamic stimulation and to hyper- and depolarizing current pulses. Most interneurons exhibited monosynaptic responses to thalamic stimulation, but this excitation was weak and subthreshold. Interneurons had multipolar dendritic morphology with widespread and dense axonal projections extending several hundred micrometers from the soma. In pyramidal cells from layers II-IV, thalamic excitatory postsynaptic potentials were significantly larger than in interneurons and were superthreshold in 40% of cells, but in these cells, there was no evidence of feedforward inhibition. By contrast, feedforward inhibition was observed in 12 of 18 layer V pyramidal cells. Thus feedforward inhibition in supragranular layers of ACx is weak, and these interneurons require coincident excitation to be activated by thalamic inputs.

Modulation of gamma-aminobutyric acid type A receptor-mediated spontaneous inhibitory postsynaptic currents in auditory cortex by midazolam and isoflurane.

BACKGROUND: Anesthetic agents that target gamma-aminobutyric acid type A (GABA(A)) receptors modulate cortical auditory evoked responses in vivo, but the cellular targets involved are unidentified. Also, for agents with multiple protein targets, the relative contribution of modulation of GABA(A) receptors to effects on cortical physiology is unclear. The authors compared effects of the GABA(A) receptor-specific drug midazolam with the volatile anesthetic isoflurane on spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal cells of auditory cortex. METHODS: Whole cell recordings were obtained in murine brain slices at 34 degrees C. GABA(A) sIPSCs were isolated by blocking ionotropic glutamate receptors. Effects of midazolam and isoflurane on time course, amplitude, and frequency of sIPSCs were measured. RESULTS: The authors detected no effect of midazolam at 0.01 microM on sIPSCs, whereas midazolam at 0.1 and 1 microM prolonged the decay of sIPSCs by approximately 25 and 70%, respectively. Isoflurane at 0.1, 0.25, and 0.5 mm prolonged sIPSCs by approximately 45, 150, and 240%, respectively. No drug-specific effects were observed on rise time or frequency of sIPSCs. Isoflurane at 0.5 mm caused a significant decrease in sIPSC amplitude. CONCLUSIONS: The dose dependence of isoflurane effects on GABA(A) sIPSCs in pyramidal cells is consistent with effects on auditory evoked response in vivo. By contrast, comparable effects of midazolam on GABA(A) sIPSCs arise at concentrations exceeding those currently thought to be achieved in vivo, suggesting that the cellular targets of midazolam reside elsewhere in the thalamocortical circuit or that the concentration of midazolam reached in the brain is higher than currently believed.