Microglia (tissue-resident macrophages) represent the main cell type of the innate immune system in the CNS; however, the mechanisms that control the activation of microglia are widely unknown. We systematically explored microglial activation and functional microglia-neuron interactions in organotypic hippocampal slice cultures, i.e., postnatal cortical tissue that lacks adaptive immunity. We applied electrophysiological recordings of local field potential and extracellular K + concentration, immunohistochemistry, design-based stereology, morphometry, Sholl analysis, and biochemical analyses. We show that chronic activation with either bacterial lipopolysaccharide through Toll-like receptor 4 (TLR4) or leukocyte cytokine IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, CD11b and CD68 up-regulation, and proinflammatory cytokine (IL-1β, TNF-α, IL-6) and nitric oxide (NO) release. Notably, these reactive phenotypes only moderately alter intrinsic neuronal excitability and gamma oscillations (30-100 Hz), which emerge from precise synaptic communication of glutamatergic pyramidal cells and fast-spiking, parvalbumin-positive GABAergic interneurons, in local hippocampal networks. Short-term synaptic plasticity and extracellular potassium homeostasis during neural excitation, also reflecting astrocyte function, are unaffected. In contrast, the coactivation of TLR4 and IFN-γ receptors results in neuronal dysfunction and death, caused mainly by enhanced microglial inducible nitric oxide synthase (iNOS) expression and NO release, because iNOS inhibition is neuroprotective. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-γ receptor signaling from peripheral immune cells, such as T helper type 1 and natural killer cells, to unleash neurotoxicity and inflammation-induced neurodegeneration. Our findings provide crucial mechanistic insight into the complex process of microglia activation, with relevance to several neurologic and psychiatric disorders.hippocampus | microglia | neuronal activity | slice culture | Toll-like receptor

Efficacy of postsynaptic inhibition through GABA A receptors in the mammalian brain depends on the maintenance of a Cl Ϫ gradient for hyperpolarizing Cl Ϫ currents. We have taken advantage of the reduced complexity under which Cl Ϫ regulation can be investigated in cultured neurons as opposed to neurons in other in vitro preparations of the mammalian brain. GABA is the main inhibitory transmitter in the mammalian brain. The dominant effect of GABA A receptor activation is a hyperpolarization caused by C l Ϫ flux into the cell (for review, see Sivilotti and Nistri, 1991;Kaila, 1994;Thompson, 1994). However, the direction of the C l Ϫ flux depends on the C l Ϫ gradient across the membrane. Indeed, GABA A receptor-mediated hyperpolarizing and /or depolarizing postsynaptic potentials have been observed (for review, see Kaila, 1994;Thompson, 1994). Some findings suggest variations in intracellular [C l Ϫ ] between different neurons and even a distinct C l Ϫ distribution in different compartments of a single neuron (Misgeld et al., 1986). Depolarizing GABA A responses, however, can be caused by bicarbonate efflux in combination with C l Ϫ influx or combined Cl Ϫ and HCO 3 Ϫ efflux (Grover et al., 1993;Kaila, 1994;Thompson, 1994;Staley et al., 1995;Perkins and Wong, 1996;Kaila et al., 1997). To be able to predict the direction of C l Ϫ currents flowing during GABA A receptor-mediated inhibition it is essential to understand the regulation of C l Ϫ homeostasis that provides the transmembrane gradient.A recently cloned K ϩ -Cl Ϫ cotransporter gene (KCC2) represents a perfect candidate for the regulation of neuronal Cl Ϫ homeostasis (Payne et al., 1996). In contrast to the ubiquitous presence of the K ϩ -Cl Ϫ cotransporter KCC1, expression of the K ϩ -Cl Ϫ cotransporter KCC2 is detected in C NS only and seems to be neuron specific. KCC2 is also distinct from KCC1 in that KCC2 is not involved in cell volume regulation and not activated by osmotic changes. Furthermore, KCC2 has a high affinity for extracellular K ϩ ions. The properties of KCC2 allow the regulation of [Cl Ϫ ] i to maintain Cl Ϫ gradients for hyperpolarizing GABAergic inhibition. Thermodynamic considerations predict that the electroneutral K ϩ -Cl Ϫ cotransporter KCC2 operates near equilibrium under physiological ionic conditions. Depending on [Cl Ϫ ] i and [K ϩ ] o (Payne, 1997), the transport will extrude or accumulate Cl Ϫ .The functional role of a particular Cl Ϫ transport system in neuronal Cl Ϫ regulation is difficult to establish in studies using integral preparations such as brain slices. One complicating factor is the presence of HCO 3 Ϫ anions. The HCO 3 Ϫ permeability of the GABA A channel (Bormann, 1988;Fatima-Shad and Barry, 1993) impedes conclusions toward actual [Cl Ϫ ] i if they are calculated from reversal potentials of GABA A receptor-mediated anion currents. Furthermore, a HCO 3 Ϫ /Cl Ϫ exchanger (RaleySusman et al., 1993) may well interfere (Chesler, 1990), and pH changes that result from manipulations of [HCO 3 Ϫ ] o strongly affect neur...

The development of hyperpolarizing inhibition is an important step in the maturation of neuronal networks. Hyperpolarizing inhibition requires Cl(-) outward transport that is accomplished by KCC2, a K(+)/Cl(-) cotransporter. We show that cultured hippocampal neurons initially contain an inactive form of the KCC2 protein, which becomes activated during subsequent maturation of the neurons. We also show that this process is accelerated by transient stimulation of IGF-1 receptors. Because the transporter can be rapidly activated by coapplication of IGF-1 and an Src kinase and can be deactivated by membrane-permeable protein tyrosine kinase inhibitors, we suggest that activation of K(+)/Cl(-) cotransporter function by endogenous protein tyrosine kinases mediates the developmental switch of GABAergic responses to hyperpolarizing inhibition.

Type II IFN (IFN-γ) is a proinflammatory T lymphocyte cytokine that serves in priming of microglia-resident CNS macrophagesduring the complex microglial activation process under pathological conditions. Priming generally permits an exaggerated microglial response to a secondary inflammatory stimulus. The impact of primed microglia on physiological neuronal function in intact cortical tissue (in situ) is widely unknown, however. We explored the effects of chronic IFN-γ exposure on microglia in hippocampal slice cultures, i.e., postnatal parenchyma lacking leukocyte infiltration (adaptive immunity). We focused on fast neuronal network waves in the gamma-band (30-70 Hz). Such gamma oscillations are fundamental to higher brain functions, such as perception, attention, and memory, and are exquisitely sensitive to metabolic and oxidative stress. IFN-γ induced substantial morphological changes and cell population expansion in microglia as well as moderate upregulation of activation markers, MHC-II, CD86, IL-6, and inducible nitric oxide synthase (iNOS), but not TNF-α. Cytoarchitecture and morphology of pyramidal neurons and parvalbumin-positive inhibitory interneurons were well-preserved. Notably, gamma oscillations showed a specific decline in frequency of up to 8 Hz, which was not mimicked by IFN-α or IL-17 exposure. The rhythm disturbance was caused by moderate microglial nitric oxide (NO) release demonstrated by pharmacological microglia depletion and iNOS inhibition. In conclusion, IFN-γ priming induces substantial proliferation and moderate activation of microglia that is capable of slowing neural information processing. This mechanism might contribute to cognitive impairment in chronic brain disease featuring elevated IFN-γ levels, blood-brain barrier leakage, and/or T cell infiltration, well before neurodegeneration occurs.interferon-gamma | microglia | neuroinflammation | neuronal electrical activity | nitric oxide synthase

Summary Lactate shuttled from blood, astrocytes, and/or oligodendrocytes may serve as the major glucose alternative in brain energy metabolism. However, its effectiveness in fueling neuronal information processing underlying complex cortex functions like perception and memory is unclear. We show that sole lactate disturbs electrical gamma and theta-gamma oscillations in hippocampal networks by either attenuation or neural bursts. Bursting is suppressed by elevating the glucose fraction in substrate supply. By contrast, lactate does not affect electrical sharp wave-ripple activity featuring lower energy use. Lactate increases the oxygen consumption during the network states, reflecting enhanced oxidative ATP synthesis in mitochondria. Finally, lactate attenuates synaptic transmission in excitatory pyramidal cells and fast-spiking, inhibitory interneurons by reduced neurotransmitter release from presynaptic terminals, whereas action potential generation in the axon is regular. In conclusion, sole lactate is less effective and potentially harmful during gamma-band rhythms by omitting obligatory ATP delivery through fast glycolysis at the synapse.