Priming of microglia by type II interferon is lasting and resistant to modulation by interleukin-10 in situ.

Immunological priming by type II interferon (IFN-γ) is crucial for evoking neurotoxic phenotypes of microglia (tissue-resident macrophages). We report that serial exposure of hippocampal slice cultures to IFN-γ and lipopolysaccharide (Toll-like receptor 4 ligand) induces high release of IL-6, TNF-α and nitric oxide, concomitant loss of electrical network activity (neuronal gamma oscillations) and neurodegeneration. Notably, these effects are still present after 3 days of IFN-γ removal but neither mimicked by IFN-α nor attenuated by anti-inflammatory cytokine, IL-10. Our findings might be relevant for brain diseases featuring elevated IFN-γ levels, such as viral and bacterial infections, multiple sclerosis and Alzheimer's disease.

TLR2- and TLR3-activated microglia induce different levels of neuronal network dysfunction in a context-dependent manner.

Recognition of pathogen- or damage-associated molecular patterns (PAMPs, DAMPs) by innate Toll-like receptors (TLRs) is central to the activation of microglia (brain macrophages) in many CNS diseases. Notably, TLR-mediated microglial activation is complex and modulated by additional exogenous and endogenous immunological signals. The impact of different microglial reactive phenotypes on electrical activity and neurotransmission is widely unknown, however. We explored the effects of TLR ligands on microglia and neuronal network function in rat organotypic hippocampal slice cultures (in situ), i.e., postnatal cortical tissue lacking adaptive immunity. Single exposure of slice cultures to TLR2 or TLR3 ligands [PGN, poly(I:C)] for 2-3 days induced moderate microglial activation featuring IL-6 and TNF-α release and only mild alterations of fast neuronal gamma band oscillations (30-70 Hz) that are fundamental to higher cognitive functions, such as perception, memory and behavior. Paired exposure to TLR3/TLR2 or TLR3/TLR4 ligands (LPS) induced nitric oxide (NO) release, enhanced TNF-α release, and associated with advanced network dysfunction, including slowing to the beta frequency band (12-30 Hz) and neural bursts (hyperexcitability). Paired exposure to a TLR ligand and the leukocyte cytokine IFN-γ enhanced NO release and associated with severe network dysfunction, albeit sensitive parvalbumin- and somatostatin-positive inhibitory interneurons were preserved. Notably, the neuronal disturbance was prevented by either microglial depletion or pharmacological inhibition of oxidant-producing enzymes, inducible NO synthase (iNOS) and NADPH oxidase. In conclusion, TLR-activated microglia can induce different levels of neuronal network dysfunction, in which severe dysfunction is mainly caused by reactive oxygen and nitrogen species rather than proinflammatory cytokines. Our findings provide a mechanistic insight into microglial activation and functional neuronal network impairment, with relevance to neuroinflammation and neurodegeneration observed in, e.g., meningoencephalitis, multiple sclerosis and Alzheimer's disease.

GM-CSF induces noninflammatory proliferation of microglia and disturbs electrical neuronal network rhythms in situ.

BACKGROUND: The granulocyte-macrophage colony-stimulating factor (GM-CSF) (or CSF-2) is involved in myeloid cell growth and differentiation, and, possibly, a major mediator of inflammation in body tissues. The role of GM-CSF in the activation of microglia (CNS resident macrophages) and the consequent impacts on neuronal survival, excitability, and synaptic transmission are widely unknown, however. Here, we focused on electrical neuronal network rhythms in the gamma frequency band (30-70 Hz). Gamma oscillations are fundamental to higher brain functions, such as perception, attention, and memory, and they are exquisitely sensitive to metabolic and oxidative stress. METHODS: We explored the effects of chronic GM-CSF exposure (72 h) on microglia in male rat organotypic hippocampal slice cultures (in situ), i.e., postnatal cortex tissue lacking leukocyte invasion (adaptive immunity). We applied extracellular electrophysiological recordings of local field potential, immunohistochemistry, design-based stereology, biochemical analysis, and pharmacological ablation of microglia. RESULTS: GM-CSF triggered substantial proliferation of microglia (microgliosis). By contrast, the release of proinflammatory cytokines (IL-6, TNF-α) and nitric oxide, the hippocampal cytoarchitecture as well as the morphology of parvalbumin-positive inhibitory interneurons were unaffected. Notably, GM-CSF induced concentration-dependent, long-lasting disturbances of gamma oscillations, such as slowing (beta frequency band) and neural burst firing (hyperexcitability), which were not mimicked by the T lymphocyte cytokine IL-17. These disturbances were attenuated by depletion of the microglial cell population with liposome-encapsulated clodronate. In contrast to priming with the cytokine IFN-γ (type II interferon), GM-CSF did not cause inflammatory neurodegeneration when paired with the TLR4 ligand LPS. CONCLUSIONS: GM-CSF has a unique role in the activation of microglia, including the potential to induce neuronal network dysfunction. These immunomodulatory properties might contribute to cognitive impairment and/or epileptic seizure development in disease featuring elevated GM-CSF levels, blood-brain barrier leakage, and/or T cell infiltration.

Neuronal gamma oscillations and activity-dependent potassium transients remain regular after depletion of microglia in postnatal cortex tissue.

Microglial cells (resident macrophages) feature rapid activation in CNS disease and can acquire multiple phenotypes exerting neuroprotection or neurotoxicity. The functional impact of surveying ("resting") microglia on neural excitability and neurotransmission in physiology is widely unknown, however. We addressed this issue in male rat hippocampal slice cultures (in situ) by pharmacological microglial ablation within days and by characterizing neuronal gamma-band oscillations (30-70 Hz) that are highly sensitive to neuromodulators and disturbances in ion and energy regulation. Gamma oscillations support action potential timing and synaptic plasticity, associate with higher brain functions like perception and memory, and require precise communication between excitatory pyramidal cells and inhibitory (GABAergic) interneurons. The slice cultures featured well-preserved hippocampal cytoarchitecture and parvalbumin-positive interneuron networks, microglia with ramified morphology, and low basal levels of IL-6, TNF-α, and nitric oxide (NO). Stimulation of slice cultures with the pro-inflammatory cytokine IFN-γ or bacterial LPS serving as positive controls for microglial reactivity induced MHC-II expression and increased cytokine and NO release. Chronic exposure of slice cultures to liposome-encapsulated clodronate reduced the microglial cell population by about 96%, whereas neuronal structures, astrocyte GFAP expression, and basal levels of cytokines and NO were unchanged. Notably, the properties of gamma oscillations reflecting frequency, number and synchronization of synapse activity were regular after microglial depletion. Also, electrical stimulus-induced transients of the extracellular potassium concentration ([K+ ]o ) reflecting cellular K+ efflux, clearance and buffering were unchanged. This suggests that nonreactive microglia are dispensable for neuronal homeostasis and neuromodulation underlying network signaling and rhythm generation in cortical tissue.

Lactate Attenuates Synaptic Transmission and Affects Brain Rhythms Featuring High Energy Expenditure.

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.

Selective inhibition of mitochondrial respiratory complexes controls the transition of microglia into a neurotoxic phenotype in situ.

Microglia are tissue resident macrophages (innate immunity) and universal sensors of alterations in CNS physiology. In response to pathogen or damage signals, microglia feature rapid activation and can acquire different phenotypes exerting neuroprotection or neurotoxicity. Although transcriptional aspects of microglial phenotypic transitions have been described, the underlying metabolic reprogramming is widely unknown. Employing postnatal organotypic hippocampal slice cultures, we describe that microglia transformed into a mild reactive phenotype by single TLR4 stimulation with lipopolysaccharide (LPS), which was boosted into a severe neurotoxic phenotype by IFN-γ (LPS + INF-γ). The two reactive phenotypes associated with reduction of microglial homeostatic "surveillance" markers, increase of cytokine release (IL-6, TNF-α) as well as enhancement of tissue energy demand and lactate production. These reactive phenotypes differed in the pattern of inhibition of the respiratory chain in mitochondria, however. TLR4 stimulation induced succinate dehydrogenase (complex II) inhibition by the metabolite itaconate. By contrast, TLR4 + IFN-γ receptor stimulation mainly resulted in complex IV inhibition by nitric oxide (NO) that also associated with severe oxidative stress, neuronal dysfunction and death. Notably, pharmacological depletion of microglia or treatment with itaconate resulted in effective neuroprotection reflected by well-preserved cytoarchitecture and electrical network activity, i.e., neuronal gamma oscillations (30-70 Hz) that underlie higher cognitive functions in vivo. Our findings provide in situ evidence that (i) proinflammatory microglia can substantially alter brain energy metabolism and (ii) fine-tuning of itaconate and NO metabolism determines microglial reactivity, impairment of neural network function and neurodegeneration. These data add mechanistic insights into microglial activation, with relevance to disorders featuring neuroinflammation and to drug discovery.

Mild metabolic stress is sufficient to disturb the formation of pyramidal cell ensembles during gamma oscillations.

Disturbances of cognitive functions occur rapidly during acute metabolic stress. However, the underlying mechanisms are not fully understood. Cortical gamma oscillations (30-100 Hz) emerging from precise synaptic transmission between excitatory principal neurons and inhibitory interneurons, such as fast-spiking GABAergic basket cells, are associated with higher brain functions, like sensory perception, selective attention and memory formation. We investigated the alterations of cholinergic gamma oscillations at the level of neuronal ensembles in the CA3 region of rat hippocampal slice cultures. We combined electrophysiology, calcium imaging (CamKII.GCaMP6f) and mild metabolic stress that was induced by rotenone, a lipophilic and highly selective inhibitor of complex I in the respiratory chain of mitochondria. The detected pyramidal cell ensembles showing repetitive patterns of activity were highly sensitive to mild metabolic stress. Whereas such synchronised multicellular activity diminished, the overall activity of individual pyramidal cells was unaffected. Additionally, mild metabolic stress had no effect on the rate of action potential generation in fast-spiking neural units. However, the partial disinhibition of slow-spiking neural units suggests that disturbances of ensemble formation likely result from alterations in synaptic inhibition. Our study bridges disturbances on the (multi-)cellular and network level to putative cognitive impairment on the system level.

The mitochondrial calcium uniporter is crucial for the generation of fast cortical network rhythms.

The role of the mitochondrial calcium uniporter (MCU) gene (Mcu) in cellular energy homeostasis and generation of electrical brain rhythms is widely unknown. We investigated this issue in mice and rats using Mcu-knockout and -knockdown strategies in vivo and in situ and determined the effects of these genetic manipulations on hippocampal gamma oscillations (30-70 Hz) and sharp wave-ripples. These physiological network states require precise neurotransmission between pyramidal cells and inhibitory interneurons, support spike-timing and synaptic plasticity and are associated with perception, attention and memory. Absence of the MCU resulted in (i) gamma oscillations with decreased power (by >40%) and lower synchrony, including less precise neural action potential generation ('spiking'), (ii) sharp waves with decreased incidence (by about 22%) and decreased fast ripple frequency (by about 3%) and (iii) lack of activity-dependent pyruvate dehydrogenase dephosphorylation. However, compensatory adaptation in gene expression related to mitochondrial function and glucose metabolism was not detected. These data suggest that the neuronal MCU is crucial for the generation of network rhythms, most likely by influences on oxidative phosphorylation and perhaps by controlling cytoplasmic Ca2+ homeostasis. This work contributes to an increased understanding of mitochondrial Ca2+ uptake in cortical information processing underlying cognition and behaviour.

Priming of microglia with IFN-γ slows neuronal gamma oscillations in situ.

Type II IFN (IFN-γ) is a proinflammatory T lymphocyte cytokine that serves in priming of microglia-resident CNS macrophages-during 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 up-regulation 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.

Metabolic modulation of neuronal gamma-band oscillations.

Gamma oscillations (30-100 Hz) represent a physiological fast brain rhythm that occurs in many cortex areas in awake mammals, including humans. They associate with sensory perception, voluntary movement, and memory formation and require precise synaptic transmission between excitatory glutamatergic neurons and inhibitory GABAergic interneurons such as parvalbumin-positive basket cells. Notably, gamma oscillations are exquisitely sensitive to shortage in glucose and oxygen supply (metabolic stress), with devastating consequences for higher cognitive functions. Herein, we explored the robustness of gamma oscillations against changes in the availability of alternative energy substrates and amino acids, which is partially regulated by glial cells such as astrocytes. We used organotypic slice cultures of the rat hippocampus expressing acetylcholine-induced persistent gamma oscillations under normoxic recording conditions (20% oxygen fraction). Our main findings are (1) partial substitution of glucose with pyruvate and the ketone body ß-hydroxybutyrate increases the frequency of gamma oscillations, even at different stages of neuronal tissue development. (2) Supplementation with the astrocytic neurotransmitter precursor glutamine has no effect on the properties of gamma oscillations. (3) Supplementation with glycine increases power, frequency, and inner coherence of gamma oscillations in a dose-dependent manner. (4) During these treatments switches to other frequency bands or pathological network states such as neural burst firing or synchronized epileptic activity are absent. Our study indicates that cholinergic gamma oscillations show general robustness against these changes in nutrient and amino acid composition of the cerebrospinal fluid; however, modulation of their properties may impact on cortical information processing under physiological and pathophysiological conditions.

TLR4-activated microglia require IFN-γ to induce severe neuronal dysfunction and death in situ.

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-1ß, 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.

A reliable model for gamma oscillations in hippocampal tissue.

Gamma oscillations (30-100 Hz) reflect a fast brain rhythm that provides a fundamental mechanism of complex neuronal information processing in the hippocampus and in the neocortex in vivo. Gamma oscillations have been implicated in higher brain functions, such as sensory perception, motor activity, and memory formation. Experimental studies on synaptic transmission and bioenergetics underlying gamma oscillations have primarily used acute slices of the hippocampus. This study tests whether organotypic hippocampal slice cultures of the rat provide an alternative model for cortical gamma oscillations in vitro. Our findings are that 1) slice cultures feature well-preserved laminated architecture and neuronal morphology; 2) slice cultures of different maturation stages (7-28 days in vitro) reliably express gamma oscillations at about 40 Hz as induced by cholinergic (acetylcholine) or glutamatergic (kainate) receptor agonists; 3) the peak frequency of gamma oscillations depends on the temperature, with an increase of ∼ 3.5 Hz per degree Celsius for the range of 28-36 °C; 4) most slice cultures show persistent gamma oscillations for ∼ 1 hr during electrophysiological local field potential recordings, and later alterations may occur; and 5) in slice cultures, glucose at a concentration of 5 mM in the recording solution is sufficient to power gamma oscillations, and additional energy substrate supply with monocarboxylate metabolite lactate (2 mM) exclusively increases the peak frequency by ∼ 4 Hz. This study shows that organotypic hippocampal slice cultures provide a reliable model to study agonist-induced gamma oscillations at glucose levels near the physiological range.

Widespread activation of microglial cells in the hippocampus of chronic epileptic rats correlates only partially with neurodegeneration.

Activation of microglial cells (brain macrophages) soon after status epilepticus has been suggested to be critical for the pathogenesis of mesial temporal lobe epilepsy (MTLE). However, microglial activation in the chronic phase of experimental MTLE has been scarcely addressed. In this study, we questioned whether microglial activation persists in the hippocampus of pilocarpine-treated, epileptic Wistar rats and to which extent it is associated with segmental neurodegeneration. Microglial cells were immunostained for the universal microglial marker, ionized calcium-binding adapter molecule-1 and the activation marker, CD11b (also known as OX42, Mac-1). Using quantitative morphology, i.e., stereology and Neurolucida-based reconstructions, we investigated morphological correlates of microglial activation such as cell number, ramification, somatic size and shape. We find that microglial cells in epileptic rats feature widespread, activation-related morphological changes such as increase in cell number density, massive up-regulation of CD11b and de-ramification. The parameters show heterogeneity in different hippocampal subregions. For instance, de-ramification is most prominent in the outer molecular layer of the dentate gyrus, whereas CD11b expression dominates in hilus. Interestingly, microglial activation only partially correlates with segmental neurodegeneration. Major neuronal death in the hilus, CA3 and CA1 coincides with strong up-regulation of CD11b. However, microglial activation is also observed in subregions that do not feature neurodegeneration, such as the molecular and granular layer of the dentate gyrus. This in vivo study provides solid experimental evidence that microglial cells feature widespread heterogeneous activation that only partially correlates with hippocampal segmental neuronal loss in experimental MTLE.

Energy substrates that fuel fast neuronal network oscillations.

Fast neuronal network oscillations in the gamma-frequency band (30--100 Hz) provide a fundamental mechanism of complex neuronal information processing in the hippocampus and neocortex of mammals. Gamma oscillations have been implicated in higher brain functions such as sensory perception, motor activity, and memory formation. The oscillations emerge from precise synapse interactions between excitatory principal neurons such as pyramidal cells and inhibitory GABAergic interneurons, and they are associated with high energy expenditure. However, both energy substrates and metabolic pathways that are capable to power cortical gamma oscillations have been less defined. Here, we investigated the energy sources fueling persistent gamma oscillations in the CA3 subfield of organotypic hippocampal slice cultures of the rat. This preparation permits superior oxygen supply as well as fast application of glucose, glycolytic metabolites or drugs such as glycogen phosphorylase inhibitor during extracellular recordings of the local field potential. Our findings are: (i) gamma oscillations persist in the presence of glucose (10 mmol/L) for greater than 60 min in slice cultures while (ii) lowering glucose levels (2.5 mmol/L) significantly reduces the amplitude of the oscillation. (iii) Gamma oscillations are absent at low concentration of lactate (2 mmol/L). (iv) Gamma oscillations persist at high concentration (20 mmol/L) of either lactate or pyruvate, albeit showing significant reductions in the amplitude. (v) The breakdown of glycogen significantly delays the decay of gamma oscillations during glucose deprivation. However, when glucose is present, the turnover of glycogen is not essential to sustain gamma oscillations. Our study shows that fast neuronal network oscillations can be fueled by different energy-rich substrates, with glucose being most effective.

Intracellular acidification in neurons induced by ammonium depends on KCC2 function.

The Cl(-)-extruding neuron-specific K(+)-Cl(-) cotransporter KCC2, which establishes hyperpolarizing inhibition, can transport NH(4) (+) instead of K(+). It is, however, not clear whether KCC2 provides the only pathway for neuronal NH(4) (+) uptake. We therefore investigated NH(4) (+) uptake in cultured rat brain neurons. In neurons cultured for > 4 weeks, the response to NH(4)Cl applications (5 mM) consisted of an alkaline shift which reversed to an acid shift within seconds. Rebound acid shifts which followed brief applications of NH(4)Cl were blocked by furosemide (100 microM). They were rather insensitive to bumetanide (1 and 100 microM), in contrast to those induced in cultured glial cells. Rebound acid shifts persisted in the presence of 1 mM Ba(2+) and in Na(+)-free solution but were inhibited by extracellular K(+). In neurons with depolarizing GABA responses, indicating the absence of functional KCC2, applications of NH(4)Cl barely induced an acidosis. However, large rebound acid shifts occurred in neurons that had changed their GABA response from Ca(2+) increases to Ca(2+) decreases. Rebound acid shifts continued to increase even after the change in the GABA response had occurred and could be induced earlier in neurons transfected with KCC2 cDNA. We conclude that KCC2 provides the main pathway for fast neuronal NH(4) (+) uptake. Therefore, NH(4)Cl-induced rebound acid shifts can be used to indicate the development of KCC2 function. Further, the well known up-regulation of KCC2 function during development has the inevitable consequence of opening a major pathway for NH(4) (+) influx, which can be relevant under pathophysiological conditions.

Hyperpolarizing inhibition develops without trophic support by GABA in cultured rat midbrain neurons.

During a limited period of early neuronal development, GABA is depolarizing and elevates [Ca2+]i, which mediates the trophic action of GABA in neuronal maturation. We tested the attractive hypothesis that GABA itself promotes the developmental change of its response from depolarizing to hyperpolarizing (Ganguly et al. 2001). In cultured midbrain neurons we found that the GABA response changed from depolarizing to hyperpolarizing, although GABAA receptors had been blocked throughout development. In immature neurons prolonged exposure of the cells to nanomolar concentrations of GABA or brief repetitive applications of GABA strongly diminished the elevation of [Ca+]i by GABA. As revealed by gramicidin perforated-patch recording, reduced [Ca2+]i responses were due to a diminished driving force for Cl-. This suggests that immature neurons do not have an efficient inward transport that can compensate the loss of cytosolic Cl-resulting from sustained GABAA receptor activation by ambient GABA. Transient increases in external K+, which can induce voltage-dependent Cl- entry, restored GABA-induced [Ca2+]i elevations. In mature neurons, GABA reduced [Ca2+]i provided that background [Ca2+]i was elevated by the application of an L-type Ca2+ channel agonist. This was probably due to a hyperpolarization of the membrane by Cl- currents. K(+)-Cl- cotransport maintained the gradient for hyperpolarizing Cl-currents. We conclude that in immature midbrain neurons an inward Cl- transport is not effective although the GABA response is depolarizing. Further, GABA itself is not required for the developmental switch of GABAergic responses from depolarizing to hyperpolarizing in cultured midbrain neurons.

KCC2 mediates NH4+ uptake in cultured rat brain neurons.

Elevated levels of NH4+ in the brain impair neuronal function. We studied the effects of NH4+ on postsynaptic inhibition of cultured rat brain neurons using whole cell recording under nominally HCO3- -free conditions. Application of NH4+ shifted the reversal potentials for spontaneous inhibitory postsynaptic currents and currents elicited by dendritic GABA applications in a positive direction because [Cl-]i increased. The positive shift of the reversal potentials of GABA-induced Cl- currents was equal on equimolar elevation of [NH4+]o or [K+]o, respectively. The NH4+-induced increase in [Cl-]i was reversed by an inhibitor of cation-anion cotransport, furosemide (0.1 mM), but not by bumetanide (0.01 mM) or by replacement of [Na+]o by Li+. We conclude that neuron-specific K-Cl cotransporter (KCC2) transports NH4+ similar to K+. Despite this fact, the small increase of [NH4+]o during metabolic encephalopathies will barely elevate [Cl-]i. However, an impairment of neuronal function may result because KCC2 provides a pathway to accumulate NH4+, and thereby, a continuous acid load to neurons.