Seizure-induced overexpression of NPY induces epileptic tolerance in a mouse model of spontaneous recurrent seizures.

Epileptic seizures result in pronounced over-expression of neuropeptide Y (NPY). In vivo and in vitro studies revealed that NPY exerts potent anticonvulsive actions through presynaptic Y2 receptors by suppressing glutamate release from principal neurons. We now investigated whether seizure-induced over-expression of NPY contributes to epileptic tolerance induced by preceding seizures. We used a previously established animal model based on selective inhibition of GABA release from parvalbumin (PV)-containing interneurons in the subiculum in mice. The animals present spontaneous recurrent seizures (SRS) and clusters of interictal spikes (IS). The frequency of SRS declined after five to six weeks, indicating development of seizure tolerance. In interneurons of the subiculum and sector CA1, SRS induced over-expression of NPY that persisted there for a prolonged time despite of a later decrease in SRS frequency. In contrast to NPY, somatostatin was not overexpressed in the respective axon terminals. Contrary to interneurons, NPY was only transiently expressed in mossy fibers. To demonstrate a protective function of endogenous, over-expressed NPY, we injected the selective NPY-Y2 receptor antagonist JNJ 5207787 simultaneously challenging the mice by a low dose of pentylenetetrazol (PTZ, 30 or 40 mg/kg, i.p.). In control mice, neither PTZ nor PTZ plus JNJ 5207787 induced convulsions. In mice with silenced GABA/PV neurons, PTZ alone only modestly enhanced EEG activity. When we injected JNJ 5207787 together with PTZ (either dose) the number of seizures, however, became significantly increased. In addition, in the epileptic mice CB1 receptor immunoreactivity was reduced in terminal areas of basket cells pointing to reduced presynaptic inhibition of GABA release from these neurons. Our experiments demonstrate that SRS result in overexpression of NPY in hippocampal interneurons. NPY overexpression persists for several weeks and may be related to later decreasing SRS frequency. Injection of the Y2 receptor antagonist JNJ 5207787 prevents this protective action of NPY only when release of the peptide is triggered by injection of PTZ and induces pronounced convulsions. Thus, over-expressed NPY released "on demand" by seizures may help terminating acute seizures and may prevent from recurrent epileptic activity.

Lipid mediator n-3 docosapentaenoic acid-derived protectin D1 enhances synaptic inhibition of hippocampal principal neurons by interaction with a G-protein-coupled receptor.

Epilepsy is a severe neurological disease manifested by spontaneous recurrent seizures due to abnormal hyper-synchronization of neuronal activity. Epilepsy affects about 1% of the population and up to 40% of patients experience seizures that are resistant to currently available drugs, thus highlighting an urgent need for novel treatments. In this regard, anti-inflammatory drugs emerged as potential therapeutic candidates. In particular, specific molecules apt to resolve the neuroinflammatory response occurring in acquired epilepsies have been proven to counteract seizures in experimental models, and humans. One candidate investigational molecule has been recently identified as the lipid mediator n-3 docosapentaenoic acid-derived protectin D1 (PD1n-3DPA ) which significantly reduced seizures, cell loss, and cognitive deficit in a mouse model of acquired epilepsy. However, the mechanisms that mediate the PD1n-3DPA effect remain elusive. We here addressed whether PD1n-3DPA has direct effects on neuronal activity independent of its anti-inflammatory action. We incubated, therefore, hippocampal slices with PD1n-3DPA and investigated its effect on excitatory and inhibitory synaptic inputs to the CA1 pyramidal neurons. We demonstrate that inhibitory drive onto the perisomatic region of the pyramidal neurons is increased by PD1n-3DPA , and this effect is mediated by pertussis toxin-sensitive G-protein coupled receptors. Our data indicate that PD1n-3DPA acts directly on inhibitory transmission, most likely at the presynaptic site of inhibitory synapses as also supported by Xenopus oocytes and immunohistochemical experiments. Thus, in addition to its anti-inflammatory effects, PD1n-3DPA anti-seizure and neuroprotective effects may be mediated by its direct action on neuronal excitability by modulating their synaptic inputs.

Silencing of Hippocampal Somatostatin Interneurons Induces Recurrent Spontaneous Limbic Seizures in Mice.

The hippocampus proper and the subiculum contain two major populations of somatostatin (SST)-containing interneurons, oriens-lacunosum moleculare (O-LM) cells projecting from the stratum oriens to the stratum lacunosum moleculare and bistratified cells with their cell bodies close to the pyramidal cell layer and axons terminating in the strata radiatum and oriens. Both types of interneurons innervate pyramidal cell dendrites and exert prominent feedback inhibition. We now investigated whether impairing this type of feed-back inhibition by selectively inhibiting GABA release from SST expressing interneurons in hippocampal sector CA1 and subiculum may be sufficient to induce spontaneous recurrent seizures. We injected transgenic mice expressing Cre-recombinase on the SST promoter unilaterally into the ventral CA1 sector and subiculum with an adeno-associated viral (AAV) vector expressing tetanus toxin light chain (TeLC) with its reading frame inverted in a flip-excision (FLEX) cassette. This treatment resulted in specific expression of TeLC and silencing of SST-containing interneurons. We continuously monitored the EEG and behavior of the mice for six weeks. Nine out of eleven mice within 10 days developed series of pre- or interictal spikes (IS, 21.4 ± 6.83 per week) and four mice exposed recurrent spontaneous seizures (SRS, 1.5 ± 0.29 per week). All 23 SRS observed were preceded by IS series. Our data demonstrate a critical role of feed-forward inhibition mediated by SST-containing interneurons suggesting that their sustained malfunctioning can be causatively involved in the development of TLE.

Increased expression of GABAA receptor subunits associated with tonic inhibition in patients with temporal lobe epilepsy.

Epilepsy animal models indicate pronounced changes in the expression and rearrangement of GABAA receptor subunits in the hippocampus and in para-hippocampal areas, including widespread downregulation of the subunits α5 and δ, and upregulation of α4, subunits that mediate tonic inhibition of GABA. In this case-control study, we investigated changes in the expression of subunits α4, α5 and δ in hippocampal specimens of drug resistant temporal lobe epilepsy patients who underwent epilepsy surgery. Using in situ hybridization, immunohistochemistry and α5-specific receptor autoradiography, we characterized expression of the receptor subunits in specimens from patients with and without Ammon's horn sclerosis compared to post-mortem controls. Expression of the α5-subunit was abundant throughout all subfields of the hippocampus, including the dentate gyrus, sectors CA1 and CA3, the subiculum and pre- and parasubiculum. Significant but weaker expression was detected for subunits α4 and δ notably in the granule cell/molecular layer of control specimens, but was faint in the other parts of the hippocampus. Expression of all three subunits was similarly altered in sclerotic and non-sclerotic specimens. Respective mRNA levels were increased by about 50-80% in the granule cell layer compared with post-mortem controls. Subunit α5 mRNA levels and immunoreactivities were also increased in the sector CA3 and in the subiculum. Autoradiography for α5-containing receptors using [3H]L-655,708 as ligand showed significantly increased binding in the molecular layer of the dentate gyrus in non-sclerotic specimens. Increased expression of the α5 and δ subunits is in contrast to the previously observed downregulation of these subunits in different epilepsy models, whereas increased expression of α4 in temporal lobe epilepsy patients is consistent with that in the rodent models. Our findings indicate increased tonic inhibition likely representing an endogenous anticonvulsive mechanism in temporal lobe epilepsy.

Regulation of Parvalbumin Interactome in the Perilesional Cortex after Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) causes 10-20% of structural epilepsy, with seizures typically originating in the cortex. Alterations in the neuronal microcircuits in the cortical epileptogenic zone, however, are poorly understood. Here, we assessed TBI-induced changes in perisomatic gamma aminobutyric acid (GABA)-ergic innervation in the perilesional cortex. We hypothesized that TBI will damage parvalbumin (PV)-immunoreactive inhibitory neurons and induce regulation of the associated GABAergic molecular interactome. TBI was induced in adult male Sprague-Dawley rats by lateral fluid-percussion injury. At 1-month post-TBI, the number of PV-positive somata was plotted on unfolded cortical maps and the distribution and density of immunopositive terminals analyzed. Qualitative analysis revealed either patchy microlesions of several hundred micrometers in diameter or diffuse neuronal loss. Quantitative analysis demonstrated a reduction in the number of PV-positive interneurons in patches down to 0% of that in sham-operated controls in the perilesional cortex. In the majority of patches, the cell numbers ranged from 71% to 90% that of the controls. The loss of PV-positive somata was accompanied by decreased axonal labeling. In situ hybridization revealed downregulated PV mRNA expression in the perilesional cortex. Gene Set Enrichment Analysis indicated a robustly downregulated expression profile of PV-related genes, which was confirmed by quantitative reverse transcriptase polymerase chain reaction. Specifically, we found that genes encoding postsynaptic GABA-A receptor genes, Gabrg2 and Gabrd, were downregulated in TBI animals compared with controls. Our data suggests that patchy reduction in PV-positive perisomatic inhibitory innervation contributes to the development of focal cortical inhibitory deficit after TBI.

Immunohistochemical distribution of 10 GABAA receptor subunits in the forebrain of the rhesus monkey Macaca mulatta.

GABAA receptors are composed of five subunits arranged around a central chloride channel. Their subunits originate from different genes or gene families. The majority of GABAA receptors in the mammalian brain consist of two α-, two ß- and one γ- or δ-subunit. This subunit organization crucially determines the physiological and pharmacological properties of the GABAA receptors. Using immunohistochemistry, we investigated the distribution of 10 GABAA receptor subunits (α1, α2, α3, α4, α5, ß1, ß2, ß3, γ2, and δ) in the fore brain of three female rhesus monkeys (Macaca mulatta). Within the cerebral cortex, subunits α1, α5, ß2, ß3, and γ2 were found in all layers, α2, α3, and ß1 were more concentrated in the inner and outer layers. The caudate/putamen was rich in α1, α2, α5, all three ß-subunits, γ2, and δ. Subunits α3 and α5 were more concentrated in the caudate than in the putamen. In contrast, α1, α2, ß1, ß2, γ2, and δ were highest in the pallidum. Most dorsal thalamic nuclei contained subunits α1, α2, α4, ß2, ß3, and γ2, whereas α1, α3, ß1, and γ2 were most abundant in the reticular nucleus. Within the amygdala, subunits α1, α2, α5, ß1, ß3, γ2, and δ were concentrated in the cortical nucleus, whereas in the lateral and basolateral amygdala α1, α2, α5, ß1, ß3, and δ, and in the central amygdala α1, α2, ß3, and γ2 were most abundant. Interestingly, subunit α3-IR outlined the intercalated nuclei of the amygdala. In the hippocampus, subunits α1, α2, α5, ß2, ß3, γ2, and δ were highly expressed in the dentate molecular layer, whereas α1, α2, α3, α5, ß1, ß2, ß3, and γ2 were concentrated in sector CA1 and the subiculum. The distribution of GABAA receptor subunits in the rhesus monkey was highly heterogeneous indicating a high number of differently assembled receptors. In most areas investigated, notably in the striatum/pallidum, amygdaloid nuclei and in the hippocampus it was more diverse than in the rat and mouse indicating a more heterogeneous and less defined receptor assembly in the monkey than in rodent brain.

Distinct gradients of various neurotransmitter markers in caudate nucleus and putamen of the human brain.

The caudate nucleus (CN) and the putamen (PUT) as parts of the human striatum are distinguished by a marked heterogeneity in functional, anatomical, and neurochemical patterns. Our study aimed to document in detail the regional diversity in the distribution of dopamine (DA), serotonin, γ-aminobuturic acid, and choline acetyltransferase within the CN and PUT. For this purpose we dissected the CN as well as the PUT of 12 post-mortem brains of human subjects with no evidence of neurological and psychiatric disorders (38-81 years old) into about 80 tissue parts. We then investigated rostro-caudal, dorso-ventral, and medio-lateral gradients of these neurotransmitter markers. All parameters revealed higher levels, turnover rates, or activities in the PUT than in the CN. Within the PUT, DA levels increased continuously from rostral to caudal. In contrast, the lowest molar ratio of homovanillic acid to DA, a marker of DA turnover, coincided with highest DA levels in the caudal PUT, the part of the striatum with the highest loss of DA in Parkinson's disease (N. Engl. J. Med., 318, 1988, 876). Highest DA concentrations were found in the most central areas both in the PUT and CN. We observed an age-dependent loss of DA in the PUT and CN that did not correspond to the loss described for Parkinson's disease indicating different mechanisms inducing the deficit of DA. Our data demonstrate a marked heterogeneity in the anatomical distribution of neurotransmitter markers in the human dorsal striatum indicating anatomical and functional diversity within this brain structure.

Structural and Functional Remodeling of Amygdala GABAergic Synapses in Associative Fear Learning.

Associative learning is thought to involve different forms of activity-dependent synaptic plasticity. Although previous studies have mostly focused on learning-related changes occurring at excitatory glutamatergic synapses, we found that associative learning, such as fear conditioning, also entails long-lasting functional and structural plasticity of GABAergic synapses onto pyramidal neurons of the murine basal amygdala. Fear conditioning-mediated structural remodeling of GABAergic synapses was associated with a change in mIPSC kinetics and an increase in the fraction of synaptic benzodiazepine-sensitive (BZD) GABAA receptors containing the α2 subunit without altering the intrasynaptic distribution and overall amount of BZD-GABAA receptors. These structural and functional synaptic changes were partly reversed by extinction training. These findings provide evidence that associative learning, such as Pavlovian fear conditioning and extinction, sculpts inhibitory synapses to regulate inhibition of active neuronal networks, a process that may tune amygdala circuit responses to threats.

Amygdala NPY Circuits Promote the Development of Accelerated Obesity under Chronic Stress Conditions.

Neuropeptide Y (NPY) exerts a powerful orexigenic effect in the hypothalamus. However, extra-hypothalamic nuclei also produce NPY, but its influence on energy homeostasis is unclear. Here we uncover a previously unknown feeding stimulatory pathway that is activated under conditions of stress in combination with calorie-dense food; NPY neurons in the central amygdala are responsible for an exacerbated response to a combined stress and high-fat-diet intervention. Central amygdala NPY neuron-specific Npy overexpression mimics the obese phenotype seen in a combined stress and high-fat-diet model, which is prevented by the selective ablation of Npy. Using food intake and energy expenditure as readouts, we demonstrate that selective activation of central amygdala NPY neurons results in increased food intake and decreased energy expenditure. Mechanistically, it is the diminished insulin signaling capacity on central amygdala NPY neurons under combined stress and high-fat-diet conditions that leads to the exaggerated development of obesity.

Effects of galanin receptor 2 and receptor 3 knockout in mouse models of acute seizures.

There exists solid evidence that endogenous galanin and galanin agonists exert anticonvulsive actions mediated both by galanin 1 receptor (GAL1-R) and galanin 2 receptor (GAL2-R). We have now investigated whether depletion of the recently identified third galanin receptor, GAL3-R, and that of GAL2-R, alters the threshold to the systemically applied γ-aminobutyric acid (GABA) antagonist pentylenetetrazole (PTZ) or to intrahippocampally administered kainic acid (KA). In neither model, GAL3-KO mice differed in their latency to the first seizure, mean seizure duration, total number of seizures, or time spent in seizures compared to wild-type controls. In addition, consistent with previous data, the response to PTZ was not altered in GAL2-KO mice. In contrast, intrahippocampal KA resulted in a significantly increased number of seizures and time spent in seizures in GAL2-KO mice, although the latency to the first seizure and the duration of individual seizures was not altered. These results are consistent with the previous data showing that GAL2-R knockdown does not affect the number of perforant path stimulations required for initiating status epilepticus but significantly increases the seizure severity during the ongoing status. In conclusion, our data support a specific role of GAL2-R but not of GAL3-R in mediating the anticonvulsive actions of endogenous galanin.

Hypothalamic CNTF volume transmission shapes cortical noradrenergic excitability upon acute stress.

Stress-induced cortical alertness is maintained by a heightened excitability of noradrenergic neurons innervating, notably, the prefrontal cortex. However, neither the signaling axis linking hypothalamic activation to delayed and lasting noradrenergic excitability nor the molecular cascade gating noradrenaline synthesis is defined. Here, we show that hypothalamic corticotropin-releasing hormone-releasing neurons innervate ependymal cells of the 3rd ventricle to induce ciliary neurotrophic factor (CNTF) release for transport through the brain's aqueductal system. CNTF binding to its cognate receptors on norepinephrinergic neurons in the locus coeruleus then initiates sequential phosphorylation of extracellular signal-regulated kinase 1 and tyrosine hydroxylase with the Ca2+-sensor secretagogin ensuring activity dependence in both rodent and human brains. Both CNTF and secretagogin ablation occlude stress-induced cortical norepinephrine synthesis, ensuing neuronal excitation and behavioral stereotypes. Cumulatively, we identify a multimodal pathway that is rate-limited by CNTF volume transmission and poised to directly convert hypothalamic activation into long-lasting cortical excitability following acute stress.

Arcuate nucleus and lateral hypothalamic CART neurons in the mouse brain exert opposing effects on energy expenditure.

Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the hypothalamus and an important regulator of energy homeostasis; however, the specific contributions of different CART neuronal populations to this process are not known. Here, we show that depolarization of mouse arcuate nucleus (Arc) CART neurons via DREADD technology decreases energy expenditure and physical activity, while it exerts the opposite effects in CART neurons in the lateral hypothalamus (LHA). Importantly, when stimulating these neuronal populations in the absence of CART, the effects were attenuated. In contrast, while activation of CART neurons in the LHA stimulated feeding in the presence of CART, endogenous CART inhibited food intake in response to Arc CART neuron activation. Taken together, these results demonstrate anorexigenic but anabolic effects of CART upon Arc neuron activation, and orexigenic but catabolic effects upon LHA-neuron activation, highlighting the complex and nuclei-specific functions of CART in controlling feeding and energy homeostasis.

Role of neuropeptide Y (NPY) in the differentiation of Trpm-5-positive olfactory microvillar cells.

The mouse olfactory neuroepithelium (ON) is comprised of anatomically distinct populations of cells in separate regions; apical (sustentacular and microvillar), neuronal (olfactory sensory neurons) and basal (horizontal and globose basal cells). The existence of microvillar cells (MVCs) is well documented but their nature and function remains unclear. An important transcription factor for the differentiation of MVCs is Skn-1a, with loss of function of Skn-1a in mice resulting in a complete loss of Trpm-5 expressing MVCs, while olfactory sensory neuron differentiation is normal. Our previous research has shown that neuropeptide Y (NPY) is expressed in MVCs and is important in the neuroproliferation of olfactory precursors. This study showed that following X-ray irradiation of the snout of wildtype mice, which decreases the proliferation of basal precursor cells, the numbers of Trpm-5-positive MVCs is increased at 2 and 5 weeks post-irradiation compared to controls. Skn-1a expression in the ON following X-ray irradiation also increases at 2 weeks post-irradiation in a regionally specific manner matching the expression pattern of Trpm-5-positive MVCs. In parallel, NPYCre knock-in mice were used to examine the expression of Skn-1a following activation of NPY unilaterally in the ON (unilateral nasal irrigation of AAV-NPY-FLEX). These experiments demonstrated that Skn-1a is only expressed when NPY is activated in MVCs. Therefore the expression of NPY is necessary for the transcription factor-mediated differentiation of olfactory MVCs.

Selective Silencing of Hippocampal Parvalbumin Interneurons Induces Development of Recurrent Spontaneous Limbic Seizures in Mice.

Temporal lobe epilepsy (TLE) is the most frequent form of focal epilepsies and is generally associated with malfunctioning of the hippocampal formation. Recently, a preferential loss of parvalbumin (PV) neurons has been observed in the subiculum of TLE patients and in animal models of TLE. To demonstrate a possible causative role of defunct PV neurons in the generation of TLE, we permanently inhibited GABA release selectively from PV neurons of the ventral subiculum by injecting a viral vector expressing tetanus toxin light chain in male mice. Subsequently, mice were subjected to telemetric EEG recording and video monitoring. Eighty-eight percent of the mice presented clusters of spike-wave discharges (C-SWDs; 40.0 ± 9.07/month), and 64% showed spontaneous recurrent seizures (SRSs; 5.3 ± 0.83/month). Mice injected with a control vector presented with neither C-SWDs nor SRSs. No neurodegeneration was observed due to vector injection or SRS. Interestingly, mice that presented with only C-SWDs but no SRSs, developed SRSs upon injection of a subconvulsive dose of pentylenetetrazole after 6 weeks. The initial frequency of SRSs declined by ∼30% after 5 weeks. In contrast to permanent silencing of PV neurons, transient inhibition of GABA release from PV neurons through the designer receptor hM4Di selectively expressed in PV-containing neurons transiently reduced the seizure threshold of the mice but induced neither acute nor recurrent seizures. Our data demonstrate a critical role for perisomatic inhibition mediated by PV-containing interneurons, suggesting that their sustained silencing could be causally involved in the development of TLE.SIGNIFICANCE STATEMENT Development of temporal lobe epilepsy (TLE) generally takes years after an initial insult during which maladaptation of hippocampal circuitries takes place. In human TLE and in animal models of TLE, parvalbumin neurons are selectively lost in the subiculum, the major output area of the hippocampus. The present experiments demonstrate that specific and sustained inhibition of GABA release from parvalbumin-expressing interneurons (mostly basket cells) in sector CA1/subiculum is sufficient to induce hyperexcitability and spontaneous recurrent seizures in mice. As in patients with nonlesional TLE, these mice developed epilepsy without signs of neurodegeneration. The experiments highlight the importance of the potent inhibitory action mediated by parvalbumin cells in the hippocampus and identify a potential mechanism in the development of TLE.

Effective G-protein coupling of Y2 receptors along axonal fiber tracts and its relevance for epilepsy.

Neuropeptide Y (NPY)-Y2 receptors are G-protein coupled receptors and, upon activation, induce opening of potassium channels or closing of calcium channels. They are generally presynaptically located. Depending on the neuron in which they are expressed they mediate inhibition of release of NPY and of the neuron's classical transmitter GABA, glutamate or noradrenaline, respectively. Here we provide evidence that Y2 receptor binding is inhibited dose-dependently by GTPγS along Schaffer collaterals, the stria terminalis and the fimbria indicating that Y2 receptors are functionally coupled to G-proteins along these fiber tracts. Double immune fluorescence revealed coexistence of Y2-immunoreactivity with ß-tubulin, a marker for axons in the stria terminalis, but not with synaptophysin labeling presynaptic terminals, supporting the localization of Y2 receptors along axonal tracts. After kainic acid-induced seizures in rats, GTPγS-induced inhibition of Y2 receptor binding is facilitated in the Schaffer collaterals but not in the stria terminalis. Our data indicate that Y2 receptors are not only located at nerve terminals but also along fiber tracts and are there functionally coupled to G-proteins.

Expression of class II histone deacetylases in two mouse models of temporal lobe epilepsy.

Epigenetic mechanisms like altered histone acetylation may have a crucial role in epileptogenesis. In two mouse models of temporal lobe epilepsy, we investigated changes in the expression of class II histone deacetylases (HDAC), a group of signal transducers that shuttle between nucleus and cytoplasm. Intrahippocampal injection of kainic acid (KA) induced a status epilepticus, development of spontaneous seizures (after 3 days), and finally chronic epilepsy and granule cell dispersion. Expression of class II HDAC mRNAs was investigated at different time intervals after KA injection in the granule cell layers and in sectors CA1 and CA3 contralateral to the site of KA injection lacking neurodegeneration. Increased expression of HDAC5 and 9 mRNAs coincided with pronounced granule cell dispersion in the KA-injected hippocampus at late intervals (14-28 days after KA) and equally affected both HDAC9 splice variants. In contrast, in the pilocarpine model (showing no granule cell dispersion), we observed decreases in the expression of HDAC5 and 9 at the same time intervals. Beyond this, striking similarities between both temporal lobe epilepsy models such as fast decreases in HDAC7 and 10 mRNAs during the acute status epilepticus were observed, notably also in the contralateral hippocampus not affected by neurodegeneration. The particular patterns of HDAC mRNA expression suggest a role in epileptogenesis and granule cell dispersion. Reduced expression of HDACs may result in increased expression of pro- and anticonvulsive proteins. On the other hand, export of HDACs from the nucleus into the cytoplasm could allow for deacetylation of cytoplasmatic proteins involved in axonal and dendritic remodeling, like granule cell dispersion. HDAC 5 and HDAC 9 expression is highly increased in granule cells of the KA-injected hippocampus and parallels granule cell dispersion. Both HDACs are thought to be targeted to the cytoplasm and to act there by deacetylating cytoplasmatic (e.g. cytosceleton-related) proteins.

Rapid changes in expression of class I and IV histone deacetylases during epileptogenesis in mouse models of temporal lobe epilepsy.

A prominent role of epigenetic mechanisms in manifestation of epilepsy has been proposed. Thus altered histone H3 and H4 acetylation has been demonstrated in experimental models of temporal lobe epilepsy (TLE). We now investigated changes in the expression of the class I and class IV histone deacetylases (HDAC) in two complementary mouse TLE models. Unilateral intrahippocampal injection of kainic acid (KA) induced a status epilepticus lasting 6 to 24h, development of spontaneous limbic seizures (2 to 3 days after KA injection) and chronic epilepsy, as revealed by telemetric recordings of the EEGs. Mice were killed at different intervals after KA injection and expression of HDAC mRNAs was investigated by in situ hybridization. We observed marked decreases in the expression of HDACs 1, 2 and 11 (by up to 75%) in the granule cell and pyramidal cell layers of the hippocampus during the acute status epilepticus (2 to 6h after KA injection). This was followed by increased expression of all class I HDAC mRNAs in all principal cell layers of the hippocampus after 12 to 48 h. In the chronic phase, 14 and 28 days after KA, only modest increases in the expression of HDAC1 mRNA were observed in granule and pyramidal cells. Immunohistochemistry using an antibody detecting HDAC2 revealed results consistent with the mRNA data and indicates also expression in glial cells on the injection side. Similar changes as seen in the KA model were observed after a pilocarpine-induced status epilepticus except that decreases in HDACs 2, 3 and 8 were also seen at the chronic 28 day interval. The prominent decreases in HDAC expression during status epilepticus are consistent with the previously demonstrated increased expression of numerous proteins and with the augmented acetylation of histone H4. It is suggested that respective putative gene products could facilitate proconvulsive as well as anticonvulsive mechanisms. The increased expression of all class I HDACs during the "silent phase", on the other hand, may be related to decreased histone acetylation, which could cause a decrease in expression of certain proteins, a mechanism that could also promote epileptogenesis. Thus, addressing HDAC expression may have a therapeutic potential in interfering with a status epilepticus and with the manifestation of TLE.

Calcium-binding proteins in focal cortical dysplasia.

OBJECTIVE: Alterations in γ-aminobutyric acid (GABA)-ergic cortical neurons have been reported in focal cortical dysplasia (FCD)Ia/IIIa, a malformation of cortical development associated with drug-resistant epilepsy. We compared numbers of neurons containing calcium-binding proteins parvalbumin (PV), calbindin (CB), and calretinin (CR) and densities of respective fibers in lateral temporal lobe surgical specimens of 17 patients with FCD with 19 patients who underwent anterior temporal lobe resection due to nonlesional temporal lobe epilepsy (non-FCD) as well as with 7 postmortem controls. METHODS: PV-, CB-, and CR-immunoreactive (IR) neurons were quantitatively investigated with use of two-dimensional cell counting and densitometry (reflecting mainly IR fibers) in cortical layers II, IV, and V. RESULTS: Numbers of PV-IR neurons, ratios of PV-containing to Nissl-stained neurons (correcting for eventual cell loss), and densities of PV-IR were higher in layer II of the cortex of FCD compared to non-FCD patients. Similarly, densities of CB-IR and CR-IR were also higher in layers II and V, respectively, of FCD than of non-FCD patients. Comparison with postmortem controls revealed significant higher cell numbers and fiber labeling for all three calcium-binding proteins in FCD cortex, whereas numbers of Nissl-stained neurons did not vary between FCD, non-FCD, and postmortem controls. In non-FCD versus postmortem controls, ratios of calcium-binding protein-IR cells to Nissl-stained neurons were unchanged in most instances except for increased CB/Nissl ratios and CB-IR densities in all cortical layers. SIGNIFICANCE: Increased numbers of PV neurons and fiber labeling in FCD compared to nondysplastic epileptic temporal neocortex and postmortem controls may be related to cortical malformation, whereas an increased number of CB-IR neurons and fiber labeling both in FCD and non-FCD specimens compared with postmortem controls may be associated with ongoing seizure activity. The observed changes may represent increased expression of calcium-binding proteins and thus compensatory mechanisms for seizures and neuronal loss in drug-resistant epilepsy.

Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury.

Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, ß2, ß3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, ß2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei.

GAL3 receptor KO mice exhibit an anxiety-like phenotype.

The neuropeptide galanin (GAL) is widely distributed in the central and peripheral nervous systems. It is a modulator of various physiological and pathological processes, and it mediates its effects via three G protein-coupled receptors (GAL1-3 receptors). A role for GAL as a modulator of mood and anxiety was suggested, because GAL and its receptors are highly expressed in limbic brain structures of rodents. In recent years, numerous studies of animal models have suggested an involvement of GAL and GAL1 and GAL2 receptors in anxiety- and depression-related behavior. However, to date, there is sparse literature implicating GAL3 receptors in behavioral functions. Therefore, we studied the behavior of GAL3 receptor-deficient (GAL3-KO) mice to elucidate whether GAL3 receptors are involved in mediating behavior-associated actions of GAL. The GAL3-KO mouse line exhibited normal breeding and physical development. In addition to behavioral tests, phenotypic characterization included analysis of hematology, amino acid profiles, metabolism, and sudomotor function. In contrast to WT littermates, male GAL3-KO mice exhibited an anxiety-like phenotype in the elevated plus maze, open field, and light/dark box tests, and they were less socially affiliated than WT animals to a stranger mouse in a social interaction test. In conclusion, our data suggest involvement of GAL3 receptors in GAL-mediated effects on mood, anxiety, and behavior, making it a possible target for alternative treatment strategies for mood disorders.