Brain infiltration of leukocytes contributes to the pathophysiology of temporal lobe epilepsy.

Clinical and experimental evidence indicates that inflammatory processes contribute to the pathophysiology of epilepsy, but underlying mechanisms remain mostly unknown. Using immunohistochemistry for CD45 (common leukocyte antigen) and CD3 (T-lymphocytes), we show here microglial activation and infiltration of leukocytes in sclerotic tissue from patients with mesial temporal lobe epilepsy (TLE), as well as in a model of TLE (intrahippocampal kainic acid injection), characterized by spontaneous, nonconvulsive focal seizures. Using specific markers of lymphocytes, microglia, macrophages, and neutrophils in kainate-treated mice, we investigated with pharmacological and genetic approaches the contribution of innate and adaptive immunity to kainate-induced inflammation and neurodegeneration. Furthermore, we used EEG analysis in mutant mice lacking specific subsets of lymphocytes to explore the significance of inflammatory processes for epileptogenesis. Blood-brain barrier disruption and neurodegeneration in the kainate-lesioned hippocampus were accompanied by sustained ICAM-1 upregulation, microglial cell activation, and infiltration of CD3(+) T-cells. Moreover, macrophage infiltration was observed, selectively in the dentate gyrus where prominent granule cell dispersion was evident. Unexpectedly, depletion of peripheral macrophages by systemic clodronate liposome administration affected granule cell survival. Neurodegeneration was aggravated in kainate-lesioned mice lacking T- and B-cells (RAG1-knock-out), because of delayed invasion by Gr-1(+) neutrophils. Most strikingly, these mutant mice exhibited early onset of spontaneous recurrent seizures, suggesting a strong impact of immune-mediated responses on network excitability. Together, the concerted action of adaptive and innate immunity triggered locally by intrahippocampal kainate injection contributes seizure-suppressant and neuroprotective effects, shedding new light on neuroimmune interactions in temporal lobe epilepsy.

Adoptive transfer of T lymphocytes in immunodeficient mice influences epileptogenesis and neurodegeneration in a model of temporal lobe epilepsy.

Intra-hippocampal injection of kainic acid (KA) in adult mice causes a focal lesion in the CA1 area and hilus of the dentate gyrus, as well as pronounced granule cell hypertrophy and dispersion. The lesion results in chronic focal seizures, with a two-week delay following KA-induced status epilepticus. Furthermore, seizures are preceded by infiltration of T lymphocytes into the lesioned tissue and of macrophage-like cells, strongly immunopositive for the monocyte marker F4/80, into the dentate gyrus, where they regulate granule cell dispersion and survival. Unexpectedly, depletion of CD4(+) and/or CD8(+) T lymphocytes by targeted gene deletion results in a marked shortening of the delay prior to seizure onset, suggesting a role of adaptive immunity in epileptogenesis (Zattoni et al. 2011, J. Neurosci. 31, 4037). Here, we investigated the specific role of adaptive immunity in this TLE model by adoptive i.v. transfer of immunopurified T cells in mutant mice lacking either CD4(+) T cells (MHCII-knockout), CD8(+) T cells (ß2-microglobulin-knockout), or both populations (RAG1-knockout mice). EEG analysis in mutants mice injected with KA two days after the T cell transfer revealed that grafting of the missing T cell population had no influence on seizure onset, but strongly influenced F4/80(+) macrophage-like cell infiltration in the dentate gyrus. Specifically, CD8(+) T cells in ß2-microgloblin-knockout mice enhanced macrophage recruitment, whereas CD4(+) T cells transferred in MHCII-knockout and in RAG1-knockout mice blocked macrophage infiltration, leading to reduced granule cell dispersion and survival, thereby worsening the KA-induced lesion. These results suggest that intact adaptive immunity is required for delayed seizure onset in this mouse model of TLE and unravel complex interactions between T cells and mononuclear phagocytes for the control of neuronal integrity and survival in the lesioned brain.

Changes in expression and function of extrasynaptic GABAA receptors in the rat hippocampus during pregnancy and after delivery.

Pregnancy is associated with changes in mood and anxiety level as well as with marked hormonal fluctuations. Increases in the brain concentrations of neuroactive steroids during pregnancy in rats are accompanied by changes in expression of subunits of the GABA type A receptor (GABA(A)-R) in the brain. Granule cells of the dentate gyrus (DGGCs) exhibit two components of inhibitory GABAergic transmission: a phasic component mediated by synaptic GABA(A)-Rs, and a tonic component mediated by extrasynaptic GABA(A)-Rs. Recordings of GABAergic currents were obtained from hippocampal slices prepared from rats in estrus, at pregnancy day 15 (P15) or P19, or at 2 d after delivery. Exogenous GABA or 3alpha,5alpha-THP induced an increase in tonic current in DGGCs that was significantly greater at P19 than in estrus. Neither tonic nor phasic currents were affected by pregnancy in CA1 pyramidal cells. Immunohistochemical analysis revealed a marked increase in the abundance of the delta subunit of the GABA(A)-R and a concomitant decrease in that of the gamma(2) subunit in the hippocampus at P19. Expression of the alpha(4) subunit did not change during pregnancy but was increased 2 d after delivery. Treatment of rats from P12 to P18 with the 5alpha-reductase inhibitor finasteride prevented the changes in tonic current and in delta and gamma(2) subunit expression normally apparent at P19. These data suggest that the number of extrasynaptic GABA(A)-Rs is increased in DGGCs during late pregnancy as a consequence of the associated marked fluctuations in the brain levels of neuroactive steroids.

Neuroactive steroids and GABAA receptor plasticity in the brain of the WAG/Rij rat, a model of absence epilepsy.

The role of neuroactive steroids and GABA(A) receptors in the generation of spontaneous spike-and-wave discharges (SWDs) was investigated in the WAG/Rij rat model of absence epilepsy. The plasma, cerebrocortical, and thalamic concentrations of the progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) were increased in the WAG/Rij rat at 2 months of age compared with those in control (Wistar) rats. In contrast, the brain and peripheral levels of 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC) did not differ between the two rat strains at this age. At 6 months of age, when absence epilepsy worsens in WAG/Rij rats, the plasma concentration of 3alpha,5alpha-TH PROG remained high whereas that of 3alpha,5alpha-TH DOC had increased, the cerebrocortical levels of both 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC had increased, and the thalamic concentrations of these metabolites had decreased. At 6 months of age the expression of the alpha(4) and delta subunits of the GABA(A) receptor in relay nuclei was increased. Finally, chronic stress induced by social isolation elicited a reduction in the amount of 3alpha,5alpha-TH PROG in the thalamus of 2-month-old WAG/Rij rats that was associated with a reduction in the number and overall duration of SWDs at 6 months of age. Absence epilepsy in the WAG/Rij rat is thus associated with changes in the abundance of neuroactive steroids and in the expression of specific GABA(A) receptor subunits in the thalamus, a brain area key to the pathophysiology of this condition.

Low tolerance and dependence liabilities of etizolam: molecular, functional, and pharmacological correlates.

The effects of prolonged exposure to and subsequent withdrawal of the thienotriazolobenzodiazepine etizolam on gamma-aminobutyric acid (GABA) type A receptor gene expression and function were compared with those of the benzodiazepine lorazepam. Exposure of rat hippocampal neurons in culture to 10 microM etizolam for 5 days reduced the amounts of alpha5 and gamma2S receptor subunit mRNAs, whereas etizolam withdrawal was associated with a persistent reduction in gamma2S mRNA and an increase in alpha2 and alpha3 mRNAs. Neither chronic exposure to nor withdrawal of etizolam affected the acute modulatory effects of etizolam or lorazepam on GABA-evoked Cl- current. Treatment with 10 microM lorazepam for 5 days reduced the amounts of alpha1 and gamma2S subunit mRNAs and increased that of alpha3 mRNA, whereas lorazepam withdrawal was associated with persistence of the changes in alpha3 and gamma2S mRNAs and an increase in alpha2 and alpha4 mRNAs. Parallel changes in the abundance of alpha1 and alpha4 subunit proteins induced by chronic exposure to and withdrawal of lorazepam, but not etizolam, were detected by immunocytofluorescence analysis. Chronic lorazepam treatment resulted in a reversible reduction in the modulatory efficacy of this drug and conferred on flumazenil the ability to potentiate GABA-evoked Cl- current. The anticonvulsant action of etizolam was not altered in mice chronically treated with this drug, whereas lorazepam-treated animals became tolerant to the acute anticonvulsant effect of this benzodiazepine. These data suggest that etizolam is endowed with a reduced liability to induce tolerance and dependence compared with classical benzodiazepines.

Changes in expression of the delta subunit of the GABA (A) receptor and in receptor function induced by progesterone exposure and withdrawal.

Type A receptors for GABA (GABA(A) receptors) that contain the delta subunit are located predominantly at extrasynaptic sites and are implicated in modulation of neuronal excitability through tonic inhibition. We have examined the effects of chronic exposure to and subsequent withdrawal of progesterone or the progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THPROG) on expression of the delta subunit of GABA(A) receptors and on receptor function in cultured rat hippocampal neurons. Progesterone treatment for 1 day increased the amounts of both delta subunit mRNA and protein, whereas such treatment for 6 days induced marked decreases in the abundance of both the mRNA and protein. Subsequent progesterone withdrawal up-regulated expression of the delta subunit, which was significantly increased at 9-12 h after withdrawal. These effects of progesterone were mimicked by 3alpha,5alpha-THPROG and blocked by the 5alpha-reductase inhibitor finasteride. They were also accompanied by parallel changes in the function of GABA(A) receptors in hippocampal neurons. These results show that chronic exposure to and withdrawal of progesterone induce differential effects on both expression of the delta subunit of GABA(A) receptors and receptor function that are mediated by 3alpha,5alpha-THPROG. They are consistent with the notion that this progesterone metabolite plays a key physiological role in modulation of GABAergic synapses.

Social isolation-induced increase in alpha and delta subunit gene expression is associated with a greater efficacy of ethanol on steroidogenesis and GABA receptor function.

Previously we have demonstrated that social isolation of rats reduces both the cerebrocortical and plasma concentrations of 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and potentiates the positive effects of acute ethanol administration on the concentrations of this neurosteroid. We now show that the ethanol-induced increase in 3alpha,5alpha-TH PROG is more pronounced in the brain than in the plasma of isolated rats. The ability of ethanol to inhibit isoniazid-induced convulsions is greater in isolated rats than in group-housed animals and this effect is prevented by treatment with finasteride. Social isolation modified the effects of ethanol on the amounts of steroidogenic regulatory protein mRNA and protein in the brain. Moreover, ethanol increased the amplitude of GABA(A) receptor-mediated miniature inhibitory postsynaptic currents recorded from CA1 pyramidal neurones with greater potency in hippocampal slices prepared from socially isolated rats than in those from group-housed rats, an effect inhibited by finasteride. The amounts of the alpha(4) and delta subunits of the GABA(A) receptor in the hippocampus were increased in isolated rats as were GABA(A) receptor-mediated tonic inhibitory currents in granule cells of the dentate gyrus. These results suggest that social isolation results in changes in GABA(A) receptor expression in the brain, and in an enhancement of the stimulatory effect of ethanol on brain steroidogenesis, GABA(A) receptor function and associated behaviour.

Distinct patterns of expression and regulation of GABA receptors containing the delta subunit in cerebellar granule and hippocampal neurons.

Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABA(A)R) for gamma-aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABA(A)R plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the delta subunit of GABA(A)Rs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of delta subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down-regulated delta subunit expression in cerebellar granule neurons and gradually normalized that in hippocampal neurons. These effects of ethanol exposure and withdrawal were accompanied by corresponding functional changes in GABA(A)Rs. GABA(A)Rs containing the delta subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABA(A)Rs that contain the delta subunit in different neuronal types.