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.

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.

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.

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.

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.

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.

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.

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.

Glutamate decarboxylase 67 is expressed in hippocampal mossy fibers of temporal lobe epilepsy patients.

Recently, expression of glutamate decarboxylase-67 (GAD67), a key enzyme of GABA synthesis, was detected in the otherwise glutamatergic mossy fibers of the rat hippocampus. Synthesis of the enzyme was markedly enhanced after experimentally induced status epilepticus. Here, we investigated the expression of GAD67 protein and mRNA in 44 hippocampal specimens from patients with mesial temporal lobe epilepsy (TLE) using double immunofluorescence histochemistry, immunoblotting, and in situ hybridization. Both in specimens with (n = 37) and without (n = 7) hippocampal sclerosis, GAD67 was highly coexpressed with dynorphin in terminal areas of mossy fibers, including the dentate hilus and the stratum lucidum of sector CA3. In the cases with Ammon's horn sclerosis, also the inner molecular layer of the dentate gyrus contained strong staining for GAD67 immunoreactivity, indicating labeling of mossy fiber terminals that specifically sprout into this area. Double immunofluorescence revealed the colocalization of GAD67 immunoreactivity with the mossy fiber marker dynorphin. The extent of colabeling correlated with the number of seizures experienced by the patients. Furthermore, GAD67 mRNA was found in granule cells of the dentate gyrus. Levels, both of GAD67 mRNA and of GAD67 immunoreactivity were similar in sclerotic and nonsclerotic specimens and appeared to be increased compared to post mortem controls. Provided that the strong expression of GAD67 results in synthesis of GABA in hippocampal mossy fibers this may represent a self-protecting mechanism in TLE. In addition GAD67 expression also may result in conversion of excessive intracellular glutamate to nontoxic GABA within mossy fiber terminals.

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.

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.

The central and basolateral amygdala are critical sites of neuropeptide Y/Y2 receptor-mediated regulation of anxiety and depression.

Anxiety is integrated in the amygdaloid nuclei and involves the interplay of the amygdala and various other areas of the brain. Neuropeptides play a critical role in regulating this process. Neuropeptide Y (NPY), a 36 aa peptide, is highly expressed in the amygdala. It exerts potent anxiolytic effects through cognate postsynaptic Y1 receptors, but augments anxiety through presynaptic Y2 receptors. To identify the precise anatomical site(s) of Y2-mediated anxiogenic action, we investigated the effect of site-specific deletion of the Y2 gene in amygdaloid nuclei on anxiety and depression-related behaviors in mice. Ablating the Y2 gene in the basolateral and central amygdala resulted in an anxiolytic phenotype, whereas deletion in the medial amygdala or in the bed nucleus of the stria terminalis had no obvious effect on emotion-related behavior. Deleting the Y2 receptor gene in the central amygdala, but not in any other amygdaloid nucleus, resulted in an added antidepressant-like effect. It was associated with a reduction of presumably presynaptic Y2 receptors in the stria terminalis/bed nucleus of the stria terminalis, the nucleus accumbens, and the locus ceruleus. Our results are evidence of the highly site-specific nature of the Y2-mediated function of NPY in the modulation of anxiety- and depression-related behavior. The activity of NPY is likely mediated by the presynaptic inhibition of GABA and/or NPY release from interneurons and/or efferent projection neurons of the basolateral and central amygdala.

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.

Afamin is synthesized by cerebrovascular endothelial cells and mediates alpha-tocopherol transport across an in vitro model of the blood-brain barrier.

Alpha-tocopherol (alphaTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of alphaTocH transport into the CNS, transfer mechanisms across the blood-brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known alphaTocH-binding protein, contributes to alphaTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, alphaTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high-density lipoprotein-mediated transport, an essential and in vivo operating alphaTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to alphaTocH homeostasis at the BBB in vivo.

Dynamic up-regulation of prodynorphin transcription in temporal lobe epilepsy.

Dynorphin neuropeptides are believed to act as endogenous anticonvulsants, though direct evidence for such a role in humans is sparse. We now report pronounced increases of prodynorphin mRNA expression in the dentate gyrus of patients with temporal lobe epilepsy in comparison to controls. We detected a conspicuously right skewed, bimodal distribution of mRNA levels among patients, suggestive of a dynamic up-regulation of prodynorphin expression in epilepsy. Highest transcript levels were seen postictally. Our data argue for an essential role of dynorphin in the termination of seizures.

Effect of neuropeptide Y Y2 receptor deletion on emotional stress-induced neuronal activation in mice.

In different behavioral paradigms including the elevated plus maze (EPM), it was observed previously that deletion of the neuropeptide Y Y2 receptor subtype results in potent suppression of anxiety-related and stress-related behaviors. To identify neurobiological correlates underlying this behavioral reactivtiy, expression of c-Fos, an established early marker of neuronal activation, was examined in Y2 receptor knockout (Y2(-/-)) vs. wildtype (WT) mice. Mice were placed on the open arm (OA) or closed arm (CA) of the EPM for 10 min and the effect on regional c-Fos expression in the brain was investigated. The number of c-Fos positive neurons was significantly increased in both WT and Y2(-/-) lines after OA and CA exposure in 51 of 54 regions quantified. These regions included various cortical, limbic, thalamic, hypothalamic, and hindbrain regions. Genotype influenced c-Fos responses to arm exposures in 6 of the 51 activated regions: the cingulate cortex, barrel field of the primary somatosensory cortex, nucleus accumbens, dorsal lateral septum, amygdala and lateral periaqueductal gray. These differences in neuronal activity responses to the novel environments were more pronounced after OA than after CA exposure. Mice lacking Y2 receptors exhibited reduced neuronal activation when compared to WT animals in response to the emotional stressors. Reduced neuronal excitability in the identified brain areas relevant to the processing of motivated, explorative as well as anxiety-related behaviors is suggested to contribute to the reduced anxiety-related behavior observed in Y2(-/-) mice.

Neuropeptide Y gene therapy decreases chronic spontaneous seizures in a rat model of temporal lobe epilepsy.

Temporal lobe epilepsy remains amongst the most common and drug refractory of neurological disorders. Gene therapy may provide a realistic therapeutic approach alternative to surgery for intractable focal epilepsies. To test this hypothesis, we applied here a gene therapy approach, using a recombinant adeno-associated viral (rAAV) vector expressing the human neuropeptide Y (NPY) gene, to a progressive and spontaneous seizure model of temporal lobe epilepsy induced by electrical stimulation of the temporal pole of the hippocampus, which replicates many features of the human condition. rAAV-NPY or a control vector lacking the expression cassette (rAAV-Empty) was delivered into the epileptic rat hippocampi at an early progressive stage of the disease. Chronic epileptic rats were video-EEG monitored to establish pre-injection baseline recordings of spontaneous seizures and the effect of rAAV-NPY versus rAAV-Empty vector injection. Both non-injected stimulated controls and rAAV-empty injected rats showed a similar progressive increase of spontaneous seizure frequency consistent with epileptogenesis. The delivery of rAAV-NPY in epileptic rat brain leads to a remarkable decrease in the progression of seizures as compared to both control groups and this effect was correlated with the NPY over-expression in the hippocampus. Moreover, spontaneous seizure frequency was significantly reduced in 40% of treated animals as compared to their pre-injection baseline. Our data show that this gene therapy strategy decreases spontaneous seizures and suppresses their progression in chronic epileptic rats, thus representing a promising new therapeutic strategy.

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.