Neuronal intracellular chloride concentration [Cl(-)](i) is an important determinant of γ-aminobutyric acid type A (GABA(A)) receptor (GABA(A)R)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCCs) move Cl(-) across the membrane, but accumulating evidence suggests factors other than the bulk concentrations of transported ions determine [Cl(-)](i). Measurement of [Cl(-)](i) in murine brain slice preparations expressing the transgenic fluorophore Clomeleon demonstrated that cytoplasmic impermeant anions ([A](i)) and polyanionic extracellular matrix glycoproteins ([A](o)) constrain the local [Cl(-)]. CCC inhibition had modest effects on [Cl(-)](i) and neuronal volume, but substantial changes were produced by alterations of the balance between [A](i) and [A](o). Therefore, CCCs are important elements of Cl(-) homeostasis, but local impermeant anions determine the homeostatic set point for [Cl(-)], and hence, neuronal volume and the polarity of local GABA(A)R signaling.
In in vitro models of acute brain injury, neuronal death may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. Neurons undergoing programmed cell death are in this queue, and are the most visible and frequently quantified measure of neuronal death after injury. However, the size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for 2 weeks. Altering phagocytosis rates (e.g., by changing the number of microglia) dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for stains that label dying neurons. Canonically neuroprotective interventions, such as seizure blockade, and neurotoxic maneuvers, such as perinatal ethanol exposure, were mediated by effects on microglial activity and the membrane permeability of neurons undergoing programmed cell death. These canonically neuroprotective and neurotoxic interventions had either no or opposing effects on healthy surviving neurons identified by the ongoing expression of transgenic fluorescent proteins. SIGNIFICANCE STATEMENT In in vitro models of acute brain injury, microglial phagocytosis is overwhelmed by the number of dying cells. Under these conditions, the assumptions on which assays for neuroprotective and neurotoxic effects are based are no longer valid. Thus, longitudinal assays of healthy cells, such as serial assessment of the fluorescence emission of transgenically expressed proteins, provide more accurate estimates of cell death than do single-time point anatomic or biochemical assays of the number of dying neurons. More accurate estimates of death rates in vitro will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.
Coincident pre- and postsynaptic activity of hippocampal neurons alters the strength of gamma-aminobutyric acid (GABA(A))-mediated inhibition through a Ca(2+)-dependent regulation of cation-chloride cotransporters. This long-term synaptic modulation is termed GABAergic spike-timing dependent plasticity (STDP). In the present study, we examined whether the properties of the GABAergic synapses themselves modulate the required postsynaptic Ca(2+) influx during GABAergic STDP induction. To do this we first identified GABAergic synapses between cultured hippocampal neurons based on their relatively long decay time constants and their reversal potentials which lay close to the resting membrane potential. GABAergic STDP was then induced by coincidentally (±1 ms) firing the pre- and postsynaptic neurons at 5 Hz for 30 s, while postsynaptic Ca(2+) was imaged with the Ca(2+)-sensitive fluorescent dye Fluo4-AM. In all cases, the induction of GABAergic STDP increased postsynaptic Ca(2+) above resting levels. We further found that the magnitude of this increase correlated with the amplitude and polarity of the GABAergic postsynaptic current (GPSC); hyperpolarizing GPSCs reduced the Ca(2+) influx in comparison to both depolarizing GPSCs, and postsynaptic neurons spiked alone. This relationship was influenced by both the driving force for Cl(-) and GABA(A) conductance (which had positive correlations with the Ca(2+) influx). The spike-timing order during STDP induction did not influence the correlation between GPSC amplitude and Ca(2+) influx, which is likely accounted for by the symmetrical GABAergic STDP window.
After acute brain injury, neuronal apoptosis may overwhelm the capacity for microglial phagocytosis, creating a queue of dying neurons awaiting clearance. The size of this queue should be equally sensitive to changes in neuronal death and the rate of phagocytosis. Using rodent organotypic hippocampal slice cultures as a model of acute perinatal brain injury, serial imaging demonstrated that the capacity for microglial phagocytosis of dying neurons was overwhelmed for two weeks. Altering phagocytosis rates, e.g. by changing the number of microglia, dramatically changed the number of visibly dying neurons. Similar effects were generated when the visibility of dying neurons was altered by changing the membrane permeability for vital stains. Canonically neuroprotective interventions such as seizure blockade and neurotoxic maneuvers such as perinatal ethanol exposure were mediated by effects on microglial activity and the membrane permeability of apoptotic neurons, and had either no or opposing effects on healthy surviving neurons.After acute brain injury, microglial phagocytosis is overwhelmed by the number of dying cells. Under these conditions, the assumptions on which assays for neuroprotective and neurotoxic effects are based are no longer valid. Thus longitudinal assays of healthy cells, such as assessment of the fluorescence emission of transgenically-expressed proteins, provide more accurate estimates of cell death than do single-time-point anatomical or biochemical assays. More accurate estimates of death rates will increase the translatability of preclinical studies of neuroprotection and neurotoxicity.
Fatally injured neurons may necrose and rupture immediately, or they may initiate a programmed cell death pathway and then wait for microglial phagocytosis. Biochemical and histopathologic assays of neuronal death assess the numbers of neurons awaiting phagocytosis at a particular time point after injury. This number varies with the fraction of neurons that have necrosed vs initiated programmed cell death, the time elapsed since injury, the rate of phagocytosis, and the assay’s ability to detect neurons at different stages of programmed cell death. Many of these variables can be altered by putatively neurotoxic and neuroprotective interventions independent of the effects on neuronal death. This complicates analyses of neurotoxicity and neuroprotection and has likely contributed to difficulties with clinical translation of neuroprotective strategies after brain injury. Time-resolved assays of neuronal health, such as ongoing expression of transgenic fluorescent proteins, are a useful means of avoiding these problems.
We appreciate the interest in our paper and the opportunity to clarify theoretical and technical aspects describing the influence of Donnan equilibria on neuronal chloride ion (Cl(-)) distributions.
Abstract In the mature CNS, coincident pre‐ and postsynaptic activity decreases the strength of γ‐aminobutyric acid (GABA) A ‐mediated inhibition through a Ca 2+ ‐dependent decrease in the activity of the neuron‐specific K + ‐Cl – cotransporter KCC2. In the present study we examined whether coincident pre‐ and postsynaptic activity can also modulate immature GABAergic synapses, where the Na + ‐K + ‐2Cl – (NKCC1) cotransporter maintains a relatively high level of intracellular chloride ([Cl – ] i ). Dual perforated patch‐clamp recordings were made from cultured hippocampal neurons prepared from embryonic Sprague–Dawley rats. These recordings were used to identify GABAergic synapses where the reversal potential for Cl – (E Cl ) was hyperpolarized with respect to the action potential threshold but depolarized with respect to the resting membrane potential. At these synapses, repetitive postsynaptic spiking within ± 5 ms of GABAergic synaptic transmission resulted in a hyperpolarizing shift of E Cl by 10.03 ± 1.64 mV, increasing the strength of synaptic inhibition. Blocking the inward transport of Cl – by NKCC1 with bumetanide (10 µ m ) hyperpolarized E Cl by 16.14 ± 4.8 mV, and prevented this coincident activity‐induced shift of E Cl . The bumetanide‐induced hyperpolarization of E Cl occluded furosemide, a K + ‐Cl – cotransporter antagonist, from producing further shifts in E Cl . Together, this indicates that brief coincident pre‐ and postsynaptic activity strengthens inhibition through a regulation of NKCC1. This study further demonstrates ionic plasticity as a mechanism underlying inhibitory synaptic plasticity.
Abstract Intraventricular haemorrhage is a common complication of premature birth. Survivors are often left with cerebral palsy, intellectual disability and/or hydrocephalus. Animal models suggest that brain tissue shrinkage, with subsequent vascular stretch and tear, is an important step in the pathophysiology, but the cause of this shrinkage is unknown. Clinical risk factors for intraventricular haemorrhage are biomarkers of hypoxic–ischaemic stress, which causes mature neurons to swell. However, immature neuronal volume might shift in the opposite direction in these conditions. This is because immature neurons express the chloride, salt and water transporter NKCC1, which subserves regulatory volume increases in non-neural cells, whereas mature neurons express KCC2, which subserves regulatory volume decreases. When hypoxic–ischaemic conditions reduce active ion transport and increase the cytoplasmic membrane permeability, the effects of these transporters are diminished. Consequentially, mature neurons swell (cytotoxic oedema), whereas immature neurons might shrink. After hypoxic–ischaemic stress, in vivo and in vitro multi-photon imaging of perinatal transgenic mice demonstrated shrinkage of viable immature neurons, bulk tissue shrinkage and blood vessel displacement. Neuronal shrinkage was correlated with age-dependent membrane salt and water transporter expression using immunohistochemistry. Shrinkage of immature neurons was prevented by prior genetic or pharmacological inhibition of NKCC1 transport. These findings open new avenues of investigation for the detection of acute brain injury by neuroimaging, in addition to prevention of neuronal shrinkage and the ensuing intraventricular haemorrhage, in premature infants.
