Brandon M. Stell

Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by δ subunit-containing GABAA receptors

Neuroactive steroids are potent modulators of γ-aminobutyric acid type A receptors (GABAARs), and their behavioral effects are generally viewed in terms of altered inhibitory synaptic transmission. Here we report that, at concentrations known to occur in vivo, neuroactive steroids specifically enhance a tonic inhibitory conductance in central neurons that is mediated by extrasynaptic δ subunit-containing GABAARs. The neurosteroid-induced augmentation of this tonic conductance decreases neuronal excitability. Fluctuations in the circulating concentrations of endogenous neuroactive steroids have been implicated in the genesis of premenstrual syndrome, postpartum depression, and other anxiety disorders. Recognition that δ subunit-containing GABAARs responsible for a tonic conductance are a preferential target for neuroactive steroids may lead to novel pharmacological approaches for the treatment of these common conditions.

Ovarian cycle-linked changes in GABA A receptors mediating tonic inhibition alter seizure susceptibility and anxiety

Disturbances of neuronal excitability changes during the ovarian cycle may elevate seizure frequency in women with catamenial epilepsy and enhance anxiety in premenstrual dysphoric disorder (PMDD). The mechanisms underlying these changes are unknown, but they could result from the effects of fluctuations in progesterone-derived neurosteroids on the brain. Neurosteroids and some anxiolytics share an important site of action: tonic inhibition mediated by δ subunit–containing GABAA receptors (δGABAARs). Here we demonstrate periodic alterations in specific GABAAR subunits during the estrous cycle in mice, causing cyclic changes of tonic inhibition in hippocampal neurons. In late diestrus (high-progesterone phase), enhanced expression of δGABAARs increases tonic inhibition, and a reduced neuronal excitability is reflected by diminished seizure susceptibility and anxiety. Eliminating cycling of δGABAARs by antisense RNA treatment or gene knockout prevents the lowering of excitability during diestrus. Our findings are consistent with possible deficiencies in regulatory mechanisms controlling normal cycling of δGABAARs in individuals with catamenial epilepsy or PMDD.

Altered Expression of the δ Subunit of the GABAA Receptor in a Mouse Model of Temporal Lobe Epilepsy

δ Subunit-containing GABAA receptors are located predominantly at nonsynaptic sites in the dentate gyrus where they may play important roles in controlling neuronal excitability through tonic inhibition and responses to GABA spillover. Immunohistochemical methods were used to determine whether δ subunit expression was altered after pilocarpine-induced status epilepticus in C57BL/6 mice in ways that could increase excitability of the dentate gyrus. In pilocarpine-treated animals, the normal diffuse labeling of the δ subunit in the dentate molecular layer was decreased by 4 d after status epilepticus (latent period) and remained low throughout the period of chronic seizures. In contrast, diffuse labeling of α4 and γ2 subunits, potentially interrelated GABAA receptor subunits, was increased during the chronic period. Interestingly, δ subunit labeling of many interneurons progressively increased after pilocarpine treatment. Consistent with the observed changes in δ subunit labeling, physiological studies revealed increased excitability in the dentate gyrus of slices obtained from the pilocarpine-treated mice and demonstrated that physiological concentrations of the neurosteroid tetrahydrodeoxycorticosterone were less effective in reducing excitability in the pilocarpine-treated animals than in controls. The findings support the idea that alterations in nonsynaptic δ subunit-containing GABAA receptors in both principal cells and interneurons could contribute to increased seizure susceptibility in the hippocampal formation in a temporal lobe epilepsy model.

Axonal GABAA receptors

Type A GABA receptors (GABAARs) are well established as the main inhibitory receptors in the mature mammalian forebrain. In recent years, evidence has accumulated showing that GABAARs are prevalent not only in the somatodendritic compartment of CNS neurons, but also in their axonal compartment. Evidence for axonal GABAARs includes new immunohistochemical and immunogold data: direct recording from single axonal terminals; and effects of local applications of GABAAR modulators on action potential generation, on axonal calcium signalling, and on neurotransmitter release. Strikingly, whereas presynaptic GABAARs have long been considered inhibitory, the new studies in the mammalian brain mostly indicate an excitatory action. Depending on the neuron that is under study, axonal GABAARs can be activated by ambient GABA, by GABA spillover, or by an autocrine action, to increase either action potential firing and/or transmitter release. In certain neurons, the excitatory effects of axonal GABAARs persist into adulthood. Altogether, axonal GABAARs appear as potent neuronal modulators of the mammalian CNS.

Activation of Presynaptic GABAA Receptors Induces Glutamate Release from Parallel Fiber Synapses

The parallel fibers relay information coming into the cerebellar cortex from the mossy fibers, and they form synapses with molecular layer interneurons (MLIs) and Purkinje cells. Here we show that activation of ionotropic GABA receptors (GABAARs) induces glutamate release from parallel fibers onto both MLIs and Purkinje cells. These GABA-induced EPSCs have kinetics and amplitudes identical to random spontaneous currents (sEPSCs), but, unlike sEPSCs, they occur in bursts of between one and five successive events. The variation in amplitude of events within bursts is significantly less than the variation of all sEPSC amplitudes, suggesting that the bursts result from repetitive activation of single presynaptic fibers. Electron microscopy of immunogold-labeled α-1 subunits revealed GABAARs on parallel fiber terminals. We suggest that the activation of these receptors underlies the increased amplitude of parallel fiber-evoked Purkinje cell EPSCs seen with application of exogenous GABA or after the release of GABA from local interneurons. These results occur only when molecular layer GABAARs are activated, and the effects are abolished when the receptors are blocked by the GABAAR antagonist gabazine (5 μm). From these data, we conclude that GABAARs located on parallel fibers depolarize parallel fiber terminals beyond the threshold for Na+ channel activation and thereby induce glutamate release onto MLIs and Purkinje cells.

Interneurons of the cerebellar cortex toggle Purkinje cells between up and down states

We demonstrate that single interneurons can toggle the output neurons of the cerebellar cortex (the Purkinje cells) between their two states. The firing of Purkinje cells has previously been shown to alternate between an “up” state in which the cell fires spontaneous action potentials and a silent “down” state. We show here that small hyperpolarizing currents in Purkinje cells can bidirectionally toggle Purkinje cells between down and up states and that blockade of the hyperpolarization-activated cation channels (H channels) with the specific antagonist ZD7288 (10 μM) blocks the transitions from down to up states. Likewise, hyperpolarizing inhibitory postsnyaptic potentials (IPSPs) produced by small bursts of action potentials (10 action potentials at 50 Hz) in molecular-layer interneurons induce these bidirectional transitions in Purkinje cells. Furthermore, single interneurons in paired interneuron → Purkinje cell recordings, produce bidirectional switches between the two states of Purkinje cells. The ability of molecular-layer interneurons to toggle Purkinje cells occurs when Purkinje cells are recorded under whole-cell patch-clamp conditions as well as when action potentials are recorded in an extracellular loose cell-attached configuration. The mode switch demonstrated here indicates that a single presynaptic interneuron can have opposite effects on the output of a given Purkinje cell, which introduces a unique type of synaptic interaction that may play an important role in cerebellar signaling.

Biphasic action of axonal GABA-A receptors on presynaptic calcium influx.

Although ionotropic γ-aminobutyric acid A receptors (GABA(A)Rs) have long been known to exist on the axons of many different cells, their effect on axon excitability and synaptic transmission remains controversial. Here, using high-speed Ca(2+) imaging, it is shown that they induce a biphasic effect in parallel fibers of the cerebellar cortex. Multicellular measurements indicate a facilitation of action potential (AP)-evoked Ca(2+) transients, which is subsequently followed by depression. However, the receptor activation does not increase influx of Ca(2+) into individual fibers but instead, increases the probability of AP generation. These results provide a description of the effect of presynaptic GABA(A)R activation and explain why reports of the effect of their activation have been so varied.

A Tale of Timing and Transport

Excitatory and inhibitory synapses show long-term plasticity, but spike timing-dependent plasticity was seen only at excitatory connections. No longer. In this issue of Neuron, Woodin et al. demonstrate that coincident pre- and postsynaptic activity acts on the neuronal K+/Cl- cotransporter KCC2 to shift the reversal potential for Cl- and thus alters the effectiveness of GABAergic synapses.