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.

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.

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.

Neuropeptide Y overexpression using recombinant adeno-associated viral vectors.

Gene therapy may represent a promising alternative treatment of epileptic patients who are resistant to conventional anti-epileptic drugs. Among the various approaches for the application of gene therapy in the treatment of CNS disorders, recombinant adeno-associated viral (AAV) vectors have been most widely used. Preclinical studies using a selection of "therapeutic" genes injected into the rodent brain to correct the compromised balance between inhibitory and excitatory transmission in epilepsy, showed significant reduction of seizures and inhibition of epileptogenesis. In particular, transduction of neuropeptide genes, such as galanin and neuropeptide Y (NPY) in specific brain areas in experimental models of seizures resulted in significant anticonvulsant effects. Recent findings showed a long-lasting NPY over-expression in the rat hippocampus by local application of recombinant AAV vectors associated with reduced generalization of seizures, delayed kindling epileptogenesis, and strong reduction of chronic spontaneous seizures. These results establish a proof-of-principle evidence of the efficacy of gene therapy as anticonvulsant treatment. Additional investigations are required to address safety concerns and possible side effects in more detail.

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.

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.

Gene therapy in epilepsy: the focus on NPY.

Gene therapy represents an innovative and promising alternative for the treatment of epileptic patients who are resistant to conventional antiepileptic drugs. Among the various approaches for the application of gene therapy in the treatment of CNS disorders, recombinant viral vectors have been most widely used so far. Several gene targets could be used to correct the compromized balance between inhibitory and excitatory transmission in epilepsy. Transduction of neuropeptide genes such as galanin and neuropeptide Y (NPY) in specific brain areas in experimental models of seizures resulted in significant anticonvulsant effects. In particular, the long-lasting NPY over-expression obtained in the rat hippocampus using intracerebral application of recombinant adeno-associated viral (AAV) vectors reduced the generalization of seizures from their site of onset, delayed acquisition of fully kindled seizures and afforded neuroprotection. These results establish a proof-of-principle for the applicability of AAV-NPY vectors for the inhibition of seizures in epilepsy. Additional investigations are required to demonstrate a therapeutic role of gene therapy in chronic models of seizures and to address in more detail safety concerns and possible side-effects.

Increased expression of Nogo-A in hippocampal neurons of patients with temporal lobe epilepsy.

Mesial temporal lobe epilepsy (TLE) is associated with pronounced anatomical and biochemical changes in the hippocampal formation including extensive neurodegeneration, reorganization of mossy fibres and sprouting of interneurons. Although the anatomical features and some of the physiological consequences of hippocampal remodeling have been well documented, the molecular mechanisms underlying the profound and orientated outgrowth of hippocampal neurons in TLE are not yet understood. The reticulon protein Nogo-A has been associated with an inhibitory action on axon growth and plasticity. Using immunohistochemistry and in situ hybridization, we investigated the expression of Nogo-A in specimens obtained at surgery from patients with TLE compared with those obtained from autopsy controls. In control specimens, Nogo-A immunoreactivity and mRNA were mainly confined to oligodendrocytes. Only approximately 40% of the specimens revealed low expression of Nogo-A mRNA in neurons. In contrast, in TLE patients with and without Ammon's horn sclerosis, Nogo-A mRNA and immunoreactivity were markedly up-regulated in most neurons (3.6- and 4.4-fold increases in Nogo-A mRNA in granule cells of sclerotic and nonsclerotic specimens) and their processes throughout the hippocampal formation. Similar elevations in Nogo-A mRNA and protein levels were determined by quantitative RT-PCR and Western blotting. Since Nogo-A expression was also up-regulated in specimens without hippocampal sclerosis, it may be induced by seizures prior to progressing neurodegeneration.

Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus.

Neuropeptide Y (NPY) inhibits seizures in experimental models and reduces excitability in human epileptic tissue. We studied the effect of long-lasting NPY overexpression in the rat hippocampus with local application of recombinant adeno-associated viral (AAV) vectors on acute kainate seizures and kindling epileptogenesis. Transgene expression was significantly increased by 7 d, reached maximal expression by 2 weeks, and persisted for at least 3 months. Serotype 2 AAV vector increased NPY expression in hilar interneurons, whereas the chimeric serotype 1/2 vector caused far more widespread expression, also including mossy fibers, pyramidal cells, and the subiculum. EEG seizures induced by intrahippocampal kainate were reduced by 50-75%, depending on the vector serotype, and seizure onset was markedly delayed. In rats injected with the chimeric serotype 1/2 vector, status epilepticus was abolished, and kindling acquisition was significantly delayed. Thus, targeted NPY gene transfer provides a potential therapeutic principle for the treatment of drug-resistant partial epilepsies.

Expression of plasma membrane GABA transporters but not of the vesicular GABA transporter in dentate granule cells after kainic acid seizures.

Kainic acid-induced seizures cause a marked increase in the expression of glutamate decarboxylase 67 (GAD67) in granule cells of the dentate gyrus. To determine the possible modes of sequestration of newly formed gamma-aminobutyric acid (GABA), we used in situ hybridization and immunocytochemistry to investigate the expression of several proteins related to GABA in dentate granule cells of rats 4 h to 60 days after kainic acid-induced status epilepticus and in controls. GAD67 and GAD65 mRNA levels were increased by up to 300% and 800%, respectively, in the granule cell layer 6-24 h after kainate injection. Subsequently, increased GAD and GABA immunoreactivity was observed in the terminal field of mossy fibers and in presumed dendrites of granule cells. mRNA of both known plasma membrane GABA transporters (GAT-1 and GAT-3) was expressed in granule cells of control rats. GAT-1 mRNA levels increased (by 30%) 9 h after kainate injection but were reduced by about 25% at later intervals. GAT-3 mRNA was reduced (by 35-75%) in granule cells 4 h to 30 days after kainic acid injection. In contrast, no expression of the mRNA or immunoreactivity of the vesicular GABA transporter was detected in granule cells or in mossy fibers, respectively. GABA transaminase mRNA was only faintly expressed in granule cells, and its levels were reduced (by 60-65%) 12 h to 30 days after kainate treatment. The results indicate that GABA can be taken up and synthesized in granule cells. No evidence for the expression of the vesicular GABA transporter (VGAT) in granule cells was obtained. After sustained epileptic seizures, the markedly increased expression of glutamate decarboxylase and the reduced expression of GABA transaminase may result in increased cytoplasmic GABA concentrations in granule cells. It is suggested that, during epileptic seizures, elevated intracellular GABA and sodium concentration could then result in nonvesicular release of GABA from granule cell dendrites. GABA could then act on GABA-A receptors, protecting granule cells from overexcitation.

Increased expression of GABA(A) receptor beta-subunits in the hippocampus of patients with temporal lobe epilepsy.

It has been postulated that dysfunction of the GABA-ergic transmission is causatively related to the development of epilepsy. Animal models of temporal lobe epilepsy (TLE) revealed considerable changes in the expression of GABA(A) receptor subunits in the hippocampus. Using immunocytochemistry, we investigated the expression of GABA(A) receptor subunits alpha1, alpha3, beta1-3, and gamma2 in hippocampal specimens obtained at surgery from TLE patients with and without hippocampal sclerosis and in autopsy controls. Consistent with the severe neurodegeneration in the CA1 sector, significant decreases in alpha1-, alpha3-, beta3-, and gamma2-subunit immunoreactivity (IR) were detected in sclerotic but not in nonsclerosic specimens. In contrast, pronounced increases in IR of all 3 beta-subunits were observed in most sectors of the hippocampal formation both in sclerotic and nonsclerotic specimens, being especially pronounced in the dentate molecular layer and in the subiculum where subunit alpha3- and gamma2-IR were also elevated. Using in situ hybridization for subunits beta2 and beta3, increased expression of the respective mRNAs was detected in dentate granule cells of patients with and without hippocampal sclerosis. Beta-subunits are important constituents of the GABA(A) receptor and contribute to the binding site of GABA. Our data indicate pronounced adaptive changes in the expression of these GABA(A) receptor subunits related to seizure activity and indicate altered assembly of GABA(A) receptors in TLE.

Important role of hypothalamic Y2 receptors in body weight regulation revealed in conditional knockout mice.

Neuropeptide Y is implicated in energy homeostasis, and contributes to obesity when hypothalamic levels remain chronically elevated. To investigate the specific role of hypothalamic Y2 receptors in this process, we used a conditional Y2 knockout model, using the Cre-lox system and adenoviral delivery of Cre-recombinase. Hypothalamus-specific Y2-deleted mice showed a significant decrease in body weight and a significant increase in food intake that was associated with increased mRNA levels for the orexigenic NPY and AgRP, as well as the anorexic proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) in the arcuate nucleus. These hypothalamic changes persisted until at least 34 days after Y2 deletion, yet the effect on body weight and food intake subsided within this time. Plasma concentrations of pancreatic polypeptide and corticosterone were 3- to 5-fold increased in hypothalamus-specific Y2 knockout mice. Germ-line Y2 receptor knockout also produced a significant increase in plasma levels of pancreatic polypeptide. However, these mice differed from conditional knockout mice in that they showed a sustained reduction in body weight and adiposity associated with increased NPY and AgRP but decreased POMC and CART mRNA levels in the arcuate nucleus. The transience of the observed effects on food intake and body weight in the hypothalamus-specific Y2 knockout mice, and the difference of this model from germ-line Y2 knockout mice, underline the importance of conditional models of gene deletion, because developmental, secondary, or extrahypothalamic mechanisms may mask such effects in germ-line knockouts.

Neuropeptide Levels after Pentylenetetrazol Kindling in the Rat.

Levels of several neuropeptides were measured in the frontal cortex, dorsal hippocampus, striatum, and amygdala/pyriform cortex in rats kindled for 5 weeks by daily injection of pentylenetetrazol (30 mg/kg, i.p.). Significantly increased concentrations (by 30 - 140%) were found in all examined brain areas for neuropeptide Y, somatostatin (except hippocampus) and neurokinin-like immunoreactivity 10 days after the last kindling session. Similar but less pronounced changes were also found 24 h after the last seizure. The increase in total neurokinin-like immunoreactivity was due to a marked increase in neurokinin B as revealed by HPLC analysis. Increases in peptide levels, however, were restricted to fully kindled animals. At the same time no changes in levels of substance P, vasoactive intestinal polypeptide and calcitonin gene-regulated peptide were observed. Cholecystokinin octapeptide was enhanced only in the hippocampus (by 46%). The increases in neuropeptide Y, somatostatin, and neurokinin-like immunoreactivity subsided after 3 months. A markedly decreased seizure threshold was observed 10 days and 2 months after the final kindling session. No nerve cell degeneration was observed in kindled rats 24 h or 10 days after the last pentylenetetrazol injection. Some animals (2 of 4), however, exhibited signs of blood - brain barrier damage when examined 24 h after the last kindling session which may reflect the preceding convulsions. No such changes were detected after 10 days. The increases in peptide levels may suggest increased activity of respective neurons which, at least to some degree, may be associated with gamma-aminobutyric acid. The changes in peptide levels may be more closely related to the kindling procedure itself than to the decreased seizure threshold of the animals.