Vijayalakshmi Santhakumar

Long-term hyperexcitability in the hippocampus after experimental head trauma

Head injury is a causative factor in the development of temporal lobe epilepsy. However, whether a single episode of concussive head trauma causes a persistent increase in neuronal excitability in the limbic system has not been unequivocally determined. This study used the rodent fluid percussion injury (FPI) model, in combination with electrophysiological and histochemical techniques, to investigate the early (1 week) and long‐term (1 month or longer) changes in the hippocampus after head trauma. Low‐frequency, single‐shock stimulation of the perforant path revealed an early granule cell hyperexcitability in head‐injured animals that returned to control levels by 1 month. However, there was a persistent decrease in threshold to induction of seizure‐like electrical activity in response to high‐frequency tetanic stimulation in the hippocampus after head injury. Timm staining revealed both early‐ and long‐term mossy fiber sprouting at low to moderate levels in the dentate gyrus of animals that experienced FPI. There was a long‐lasting increase in the frequency of spontaneous inhibitory postsynaptic currents in dentate granule cells after FPI, and ionotropic glutamate receptor antagonists selectively decreased the spontaneous inhibitory postsynaptic current frequency in the head‐injured animals. These results demonstrate that a single episode of experimental closed head trauma induces long‐lasting alterations in the hippocampus. These persistent structural and functional alterations in inhibitory and excitatory circuits are likely to influence the development of hyperexcitable foci in posttraumatic limbic circuits.

Granule cell hyperexcitability in the early post-traumatic rat dentate gyrus: the 'irritable mossy cell' hypothesis.

1 Cytochemical and in vitro whole‐cell patch clamp techniques were used to investigate granule cell hyperexcitability in the dentate gyrus 1 week after fluid percussion head trauma. 2 The percentage decrease in the number of hilar interneurones labelled with either GAD67 or parvalbumin mRNA probes following trauma was not different from the decrease in the total population of hilar cells, indicating no preferential survival of interneurones with respect to the non‐GABAergic hilar cells, i.e. the mossy cells. 2 Dentate granule cells following trauma showed enhanced action potential discharges, and longer‐lasting depolarizations, in response to perforant path stimulation, in the presence of the GABAA receptor antagonist bicuculline. 3 There was no post‐traumatic alteration in the perforant path‐evoked monosynaptic excitatory postsynaptic currents (EPSCs), or in the intrinsic properties of granule cells. However, after trauma, the monosynaptic EPSC was followed by late, polysynaptic EPSCs, which were not present in controls. 4 The late EPSCs in granule cells from fluid percussion‐injured rats were not blocked by the NMDA receptor antagonist 2‐amino‐5‐phosphonovaleric acid (APV), but were eliminated by both the non‐NMDA glutamate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and the AMPA receptor antagonist GYKI 53655. 5 In addition, the late EPSCs were not present in low (0·5 mM) extracellular calcium, and they were also eliminated by the removal of the dentate hilus from the slice. 6 Mossy hilar cells in the traumatic dentate gyrus responded with significantly enhanced, prolonged trains of action potential discharges to perforant path stimulation. 7 These data indicate that surviving mossy cells play a crucial role in the hyperexcitable responses of the post‐traumatic dentate gyrus.

Rapid Deletion of Mossy Cells Does Not Result in a Hyperexcitable Dentate Gyrus: Implications for Epileptogenesis

Loss of cells from the hilus of the dentate gyrus is a major histological hallmark of human temporal lobe epilepsy. Hilar mossy cells, in particular, are thought to show dramatic numerical reductions in pathological conditions, and one prominent theory of epileptogenesis is based on the assumption that mossy cell loss directly results in granule cell hyperexcitability. However, whether it is the disappearance of hilar mossy cells from the dentate gyrus circuitry after various insults or the subsequent synaptic-cellular alterations (e.g., reactive axonal sprouting) that lead to dentate hyperexcitability has not been rigorously tested, because of the lack of available techniques to rapidly remove specific classes of nonprincipal cells from neuronal networks. We developed a fast, cell-specific ablation technique that allowed the targeted lesioning of either mossy cells or GABAergic interneurons in horizontal as well as axial (longitudinal) slices of the hippocampus. The results demonstrate that mossy cell deletion consistently decreased the excitability of granule cells to perforant path stimulation both within and outside of the lamella where the mossy cell ablation took place. In contrast, ablation of interneurons caused the expected increase in excitability, and control aspirations of the hilar neuropil or of interneurons in the presence of GABA receptor blockers caused no alteration in granule cell excitability. These data do not support the hypothesis that loss of mossy cells from the dentate hilus after seizures or traumatic brain injury directly results in hyperexcitability.

Contributions of the GABAA Receptor α6 Subunit to Phasic and Tonic Inhibition Revealed by a Naturally Occurring Polymorphism in the α6 Gene

GABAA receptors (GABARs) are heteromultimeric proteins composed of five subunits. The specific subunit composition determines critical properties of a GABAR such as pharmacological sensitivities and whether the receptor contributes to synaptic or extrasynaptic forms of inhibition. Classically, synaptic but not extrasynaptic GABARs are thought to respond to benzodiazepines, whereas the reverse has been suggested for ethanol. To examine the effects of subunit composition on GABAR function in situ, we took advantage of two naturally occurring alleles of the rat gene for GABAR subunit α6 (Gabra6100R and Gabra6100Q). Depending on their subunit partners, these two variants of α6 can lead to differential sensitivities to benzodiazepines and ethanol. An examination of synaptic and extrasynaptic GABA-mediated currents in cerebellar granule cells from Gabra6100R/100R and Gabra6100Q/100Q rats uncovered marked allele-dependent differences in benzodiazepine sensitivity. Unexpectedly, we found that the benzodiazepines flunitrazepam and diazepam enhanced extrasynaptic inhibition mediated by δ subunit-containing GABARs in Gabra6100Q/100Q rats. Complementary experiments on recombinant GABARs confirmed that, at subsaturating [GABA], flunitrazepam potentiates α6/δ subunit-containing GABARs. Based on data and a simple theoretical analysis, we estimate that the average extrasynaptic [GABA] is ∼160 nm in perfused slices. These results (1) demonstrate contributions of α6 subunits to both synaptic and extrasynaptic GABA responses, (2) establish that δ subunit-containing GABARs are benzodiazepine sensitive at subsaturating [GABA] and, (3) provide an empirical estimate of extrasynaptic [GABA] in slices.

DEVELOPMENTAL REGULATION AND NEUROPROTECTIVE EFFECTS OF STRIATAL TONIC GABAA CURRENTS

Striatal neurons are known to express GABA(A) receptor subunits that underlie both phasic and tonic inhibition. Striatal projection neurons, or medium spiny neurons (MSNs), are divided into two classes: MSNs containing the dopamine D1 receptor (D1-MSNs) form the direct pathway to the substantia nigra and facilitate movement while MSNs expressing the dopamine D2 receptor (D2-MSNs) form the pallidal pathway that inhibits movement. Consequently, modulating inhibition in distinct classes of MSNs will differentially impact downstream network activity and motor behavior. Given the powerful role of extrasynaptic inhibition in controlling neuronal excitability, we examined the nature of striatal tonic inhibition and its potential role in preventing excitotoxicity. Consistent with earlier studies in young (P16-P25) mice, tonic GABA currents in D2-MSNs were larger than in D1-MSNs. However, with age (>P30 mice) the tonic GABA currents increased in D1-MSNs but decreased in D2-MSNs. These data demonstrate a developmental switch in the MSN subtype expressing larger tonic GABA currents. Compared to wild-type, MSNs from adult mice lacking the GABA(A)R delta subunit (Gabrd(-/-) mice) had both decreased tonic GABA currents and reduced survival following an in vitro excitotoxic challenge with quinolinic acid. Furthermore, muscimol-induced tonic GABA currents were accompanied by reduced acute swelling of striatal neurons after exposure to NMDA in WT mice but not in Gabrd(-/-) mice. Our data are consistent with a role for tonic inhibition mediated by GABA(A)R delta subunits in neuroprotection against excitotoxic insults in the adult striatum.

Decrease in Tonic Inhibition Contributes to Increase in Dentate Semilunar Granule Cell Excitability after Brain Injury

Brain injury is an etiological factor for temporal lobe epilepsy and can lead to memory and cognitive impairments. A recently characterized excitatory neuronal class in the dentate molecular layer, semilunar granule cell (SGC), has been proposed to regulate dentate network activity patterns and working memory formation. Although SGCs, like granule cells, project to CA3, their typical sustained firing and associational axon collaterals suggest that they are functionally distinct from granule cells. We find that brain injury results in an enhancement of SGC excitability associated with an increase in input resistance 1 week after trauma. In addition to prolonging miniature and spontaneous IPSC interevent intervals, brain injury significantly reduces the amplitude of tonic GABA currents in SGCs. The postinjury decrease in SGC tonic GABA currents is in direct contrast to the increase observed in granule cells after trauma. Although our observation that SGCs express Prox1 indicates a shared lineage with granule cells, data from control rats show that SGC tonic GABA currents are larger and sIPSC interevent intervals shorter than in granule cells, demonstrating inherent differences in inhibition between these cell types. GABAA receptor antagonists selectively augmented SGC input resistance in controls but not in head-injured rats. Moreover, post-traumatic differences in SGC firing were abolished in GABAA receptor blockers. Our data show that cell-type-specific post-traumatic decreases in tonic GABA currents boost SGC excitability after brain injury. Hyperexcitable SGCs could augment dentate throughput to CA3 and contribute substantively to the enhanced risk for epilepsy and memory dysfunction after traumatic brain injury.

H-channels in epilepsy: new targets for seizure control?

Hyperpolarization-activated cation channels (h-channels) are key regulators of neuronal excitation and inhibition, and have a rich diversity of subunit composition, distribution, modulation and function. Recent results indicate that the behavior of h-channels can be altered significantly by seizures. The activity-dependent, short-term and long-term plasticity of h-channels can, in turn, modulate neuronal excitability. The reciprocal interactions between neuronal activity and h-channels indicate that these ion channels could be promising novel targets for anti-epileptic therapies.

Status epilepticus enhances tonic GABA currents and depolarizes GABA reversal potential in dentate fast-spiking basket cells

Temporal lobe epilepsy is associated with loss of interneurons and inhibitory dysfunction in the dentate gyrus. While status epilepticus (SE) leads to changes in granule cell inhibition, whether dentate basket cells critical for regulating granule cell feedforward and feedback inhibition express tonic GABA currents (I(GABA)) and undergo changes in inhibition after SE is not known. We find that interneurons immunoreactive for parvalbumin in the hilar-subgranular region express GABAA receptor (GABA(A)R) δ-subunits, which are known to underlie tonic I(GABA). Dentate fast-spiking basket cells (FS-BCs) demonstrate baseline tonic I(GABA) blocked by GABA(A)R antagonists. In morphologically and physiologically identified FS-BCs, tonic I(GABA) is enhanced 1 wk after pilocarpine-induced SE, despite simultaneous reduction in spontaneous inhibitory postsynaptic current (sIPSC) frequency. Amplitude of tonic I(GABA) in control and post-SE FS-BCs is enhanced by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), demonstrating the contribution of GABA(A)R δ-subunits. Whereas FS-BC resting membrane potential is unchanged after SE, perforated-patch recordings from FS-BCs show that the reversal potential for GABA currents (E(GABA)) is depolarized after SE. In model FS-BCs, increasing tonic GABA conductance decreased excitability when E(GABA) was shunting and increased excitability when E(GABA) was depolarizing. Although simulated focal afferent activation evoked seizurelike activity in model dentate networks with FS-BC tonic GABA conductance and shunting E(GABA), excitability of identical networks with depolarizing FS-BC E(GABA) showed lower activity levels. Thus, together, post-SE changes in tonic I(GABA) and E(GABA) maintain homeostasis of FS-BC activity and limit increases in dentate excitability. These findings have implications for normal FS-BC function and can inform studies examining comorbidities and therapeutics following SE.

Postsynaptic effects of GABAergic synaptic diversity: regulation of neuronal excitability by changes in IPSC variance

GABAergic synaptic inputs to principal cells are heterogeneous in terms of their anatomical, molecular and physiological properties. Whether diversity in GABAergic synaptic inputs affects the efficacy of GABAergic inhibition is not understood. Here we show that alterations in the heterogeneity of IPSC populations arriving at single cells can significantly modify the effects of GABAergic inputs on neuronal excitability. The effects of IPSC diversity were examined in a computational model that incorporated experimentally measured values for spontaneous IPSCs and CA1 pyramidal cell electrophysiological properties. The simulations showed that increased variance in the conductance or decay of IPSCs could potently modulate the firing rate of the postsynaptic cells. The actual direction of the IPSC variance-induced modulation in postsynaptic cell discharges depended on the mean IPSC conductance and mean decay time constant around which the variance was introduced, as well as on the degree of depolarization and firing of the postsynaptic cell. Further analysis of the underlying mechanisms determined that these effects of IPSC variance on neuronal excitability were entirely predicted from the non-linear actions of IPSCs on action potential generation. The variance effects on neuronal excitability could be strong enough to overcome even large changes in mean IPSC conductance, demonstrating that increased mean synaptic conductance (or increased mean IPSC or IPSP) alone does not necessarily imply a more effective inhibition, a finding which has important implications for epilepsy research. These data show that the degree of heterogeneity of the GABAergic synaptic inputs to principal cells can powerfully modulate the efficacy of GABAergic inhibition. The results indicate the functional importance of the diversity of interneurons in cortical and hippocampal circuits, and suggest that plastic changes in GABAergic synaptic diversity may modulate neuronal excitability under both normal and pathological conditions.

Plasticity of interneuronal species diversity and parameter variance in neurological diseases

Interneuronal diversity reflects the division of labor between numerous highly specialized interneuronal species, each performing a set of specific functions in neuronal networks. The rich diversity of interneurons found in the normal healthy brain is often significantly altered in neurological and psychiatric diseases. In genetic and developmental disorders, the diversity of interneuronal networks is compromised because of disturbances in the generation, specification or migration of specific interneuronal subtypes. Following insults related to trauma and seizures, the relative abundance of interneuronal subtypes might change, and entire interneuronal species can be lost from the network. In addition to the complete or partial loss of interneuronal subgroups, heterogeneity can also be altered in more subtle ways, as a result of changes in cell-to-cell variance of a particular parameter within specific interneuronal populations. Computational and experimental studies show that alterations in cellular and synaptic GABAergic heterogeneity can significantly modulate both firing rates and network coherence, indicating that plasticity of interneuronal diversity is likely to be an important mechanistic component of malfunctioning cortical networks in many pathological states.