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The hippocampal dentate gyrus is an important relay conveying sensory information from the entorhinal cortex to the hippocampus proper. During exploration, the dentate gyrus has been proposed to act as a pattern separator. However, the dentate gyrus also shows structured activity during immobility and sleep. The properties of these activity patterns at cellular resolution, and their role in hippocampal-dependent memory processes have remained unclear. Using dual-color in-vivo two-photon Ca2+ imaging, we show that in immobile mice dentate granule cells generate sparse, synchronized activity patterns associated with entorhinal cortex activation. These population events are structured and modified by changes in the environment; and they incorporate place- and speed cells. Importantly, they are more similar than expected by chance to population patterns evoked during self-motion. Using optogenetic inhibition, we show that granule cell activity is not only required during exploration, but also during immobility in order to form dentate gyrus-dependent spatial memories.

The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that, during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ third-order dendrites both in male and in female mice. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, whereas excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and inhibitory synapse maintenance. SIGNIFICANCE STATEMENT We show that dysmorphic neurons of human epileptogenic brain lesions have decreased levels of the Ste20-like kinase (SLK). Decreasing SLK expression in mouse neurons revealed that SLK has essential functions in forming the neuronal dendritic tree and in maintaining inhibitory connections with neighboring neurons.

SummaryThe size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ 3rd, order dendrites. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, while excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and of inhibitory synapse maintenance.

Focal cortical epilepsies are frequently refractory to available anticonvulsant drug therapies. One key factor contributing to this state is the limited availability of animal models that allow to reliably study focal cortical seizures and how they recruit surrounding brain areas in-vivo. In this study, we selectively expressed the inhibitory chemogenetic receptor, hM4D, in GABAergic neurons in focal cortical areas using viral gene transfer. Following focal silencing of GABAergic neurons by administration of Clozapine-N-Oxide (CNO), we demonstrated reliable induction of local epileptiform events in the electroencephalogram (EEG) signal of awake freely moving mice. Experiments in anesthetized mice showed consistent induction of focal seizures in two different brain regions – the barrel cortex (BC) and at the medial prefrontal cortex (mPFC). Seizures were accompanied by high frequency oscillations, a known characteristic of human focal seizures. Seizures propagated, but an analysis of seizure propagation revealed favored propagation pathways. CNO-induced epileptiform events propagated from the BC on one hemisphere to its counterpart and from the BC to the mPFC, but not vice-versa. Lastly, post-CNO epileptiform events in the BC could be triggered by sensory whisker-pad stimulation, indicating that this model, applied to sensory cortices, may be useful to study sensory-evoked seizures. Taken together, our results show that targeted chemogenetic inhibition of GABAergic neurons using hM4D can serve as a novel, versatile and reliable model of focal cortical epilepsy suitable to systematically study cortical ictogenesis in different cortical areas.Significance StatementFocal cortical epilepsies are often hard to alleviate using current anticonvulsant therapies while further drug discovery is impeded by the limited variety of suitable animal models. In this study, we established a novel model of focal cortical seizures induced by spatially-restricted chemogenetic silencing of cortical inhibitory neurons. We have shown this method to be effective at various cortical regions and reliably induce seizures that share key characteristics with known human epilepsy traits, including sensory triggering and seizure propagation. This model may thus be used to advance the discovery of new remedies for focal cortical epilepsies, as well as to improve our understanding of seizure spread along different cortical pathways.

Transgenic Cre-recombinase expressing mouse lines are widely used to express fluorescent proteins and opto-/chemogenetic actuators, making them a cornerstone of modern neuroscience. The investigation of interneurons in particular has benefitted from the ability to genetically target specific cell types. However, the specificity of some Cre driver lines has been called into question. Here, we show that nonspecific expression in a subset of hippocampal neurons can have substantial nonspecific functional effects in a somatostatin-Cre (SST-Cre) mouse line. Nonspecific targeting of CA3 pyramidal cells caused large optogenetically evoked excitatory currents in remote brain regions. Similar, but less severe patterns of nonspecific expression were observed in a widely used SST-IRES-Cre line, when crossed with a reporter mouse line. Viral transduction on the other hand yielded more specific expression but still resulted in nonspecific expression in a minority of pyramidal layer cells. These results suggest that a careful analysis of specificity is mandatory before the use of Cre driver lines for opto- or chemogenetic manipulation approaches.

The hippocampal dentate gyrus is an important relay conveying sensory information from the entorhinal cortex to the hippocampus proper. During exploration, the dentate gyrus has been proposed to act as a pattern separator. However, the dentate gyrus also shows structured activity during immobility and sleep. The properties of these activity patterns at cellular resolution, and their role in hippocampal-dependent memory processes have remained unclear. Using dual-color in-vivo two-photon Ca2+ imaging, we show that in immobile mice dentate granule cells generate sparse, synchronized activity patterns associated with entorhinal cortex activation. These population events are structured and modified by changes in the environment; and they incorporate place- and speed cells. Importantly, they recapitulate population patterns evoked during self-motion. Using optogenetic inhibition during immobility, we show that granule cell activity during immobility is required to form dentate gyrus-dependent spatial memories. These data suggest that memory formation is supported by dentate gyrus replay of population codes of the current environment.

1AbstractTransgenic Cre-recombinase expressing mouse lines are widely used to express fluorescent proteins and opto-/chemogenetic actuators, making them a cornerstone of modern neuroscience. Particularly, the investigation of interneurons has benefitted from the ability to target genetic constructs to defined cell types. However, the cell type specificity of some mouse lines has been called into questions. Here we show for the first time the functional consequences of unspecific expression in a somatostatin-Cre (SST-Cre) mouse line. We find large optogenetically evoked excitatory currents originating from unspecifically targeted CA3 pyramidal cells. We also used public Allen Brain Institute data to estimate expression specificity in other Cre lines. Another SST-Cre mouse lines shows comparable unspecificity, whereas a Parvalbumin-Cre mouse line shows much less unspecific expression. Finally, we make suggestions to ensure that the results from in-vivo use of Cre mouse lines are interpretable.

Focal cortical dysplasias (FCDs) represent common cortical malformations that are frequently associated with epilepsy. They have so far not been well understood in terms of their molecular pathogenesis, and with respect to mechanisms of seizure emergence.Several recent studies have succeeded in making significant advances in understanding the molecular genetics, in particular FCD type II. A second major advance has been the development of novel rodent models of FCDs that replicate a somatic mutation seen in humans, lead to a focal lesion, and recapitulate many phenotypic features of human FCDs. We will discuss these recent advances.These advances promise significant advances in understanding the heterogeneity of FCDs at the molecular genetic level. They also promise a much better understanding of cell-intrinsic and network mechanisms underlying increased seizure susceptibility and altered cognition. Systematic studies utilizing the approaches summarized here promise to lead to specific strategies regarding when and how to treat specific subgroups of FCDs.

Recent reports suggest that induced neurons (iNs), but not induced pluripotent stem cell (iPSC)-derived neurons, largely preserve age-associated traits. Here, we report on the extent of preserved epigenetic and transcriptional aging signatures in directly converted induced neural stem cells (iNSCs). Employing restricted and integration-free expression of SOX2 and c-MYC, we generated a fully functional, bona fide NSC population from adult blood cells that remains highly responsive to regional patterning cues. Upon conversion, low passage iNSCs display a profound loss of age-related DNA methylation signatures, which further erode across extended passaging, thereby approximating the DNA methylation age of isogenic iPSC-derived neural precursors. This epigenetic rejuvenation is accompanied by a lack of age-associated transcriptional signatures and absence of cellular aging hallmarks. We find iNSCs to be competent for modeling pathological protein aggregation and for neurotransplantation, depicting blood-to-NSC conversion as a rapid alternative route for both disease modeling and neuroregeneration., Induced neurons, but not induced pluripotent stem cell (iPSC)-derived neurons, preserve age-related traits. Here, the authors demonstrate that blood-derived induced neural stem cells (iNSCs), despite lacking a pluripotency transit, lose age-related signatures.

Cellular behavior is orchestrated by the complex interactions of a myriad of intracellular signal transduction pathways. To understand and investigate the role of individual components in such signaling networks, the availability of specific inhibitors is of paramount importance. We report the generation and validation of a novel variant of an RNA aptamer that selectively inhibits the mitogen‐activated kinase pathway in neurons. We demonstrate that the aptamer retains function under intracellular conditions and that application of the aptamer through the patch‐clamp pipette efficiently inhibits mitogen‐activated kinase‐dependent synaptic plasticity. This approach introduces synthetic aptamers as generic tools, readily applicable to inhibit different components of intraneuronal signaling networks with utmost specificity.

SummaryObjective The maturation of adult-born granule cells and their functional integration into the network is thought to play a key role in the proper functioning of the dentate gyrus. In temporal lobe epilepsy, adult-born granule cells in the dentate gyrus develop abnormally and possess a hilar basal dendrite (HBD). Although morphological studies have shown that these HBDs have synapses, little is known about the functional properties of these HBDs or the intrinsic and network properties of the granule cells that possess these aberrant dendrites. Methods We performed patch-clamp recordings of granule cells within the granule cell layer “normotopic” from sham-control and status epilepticus (SE) animals. Normotopic granule cells from SE animals possessed an HBD (SE+HBD+ cells) or not (SE+HBD− cells). Apical and basal dendrites were stimulated using multiphoton uncaging of glutamate. Two-photon Ca2+ imaging was used to measure Ca2+ transients associated with back-propagating action potentials (bAPs). Results Near-synchronous synaptic input integrated linearly in apical dendrites from sham-control animals and was not significantly different in apical dendrites of SE+HBD− cells. The majority of HBDs integrated input linearly, similar to apical dendrites. However, 2 of 11 HBDs were capable of supralinear integration mediated by a dendritic spike. Furthermore, the bAP-evoked Ca2+ transients were relatively well maintained along HBDs, compared with apical dendrites. This further suggests an enhanced electrogenesis in HBDs. In addition, the output of granule cells from epileptic tissue was enhanced, with both SE+HBD− and SE+HBD+ cells displaying increased high-frequency (>100 Hz) burst-firing. Finally, both SE+HBD− and SE+HBD+ cells received recurrent excitatory input that was capable of generating APs, especially in the absence of feedback inhibition. Significance Taken together, these data suggest that the enhanced excitability of HBDs combined with the altered intrinsic and network properties of granule cells collude to promote excitability and synchrony in the epileptic dentate gyrus.

SummaryNeuronal processing is classically conceptualized as dendritic input, somatic integration, and axonal output. The axon initial segment, the proposed site of action potential generation, usually emanates directly from the soma. However, we found that axons of hippocampal pyramidal cells frequently derive from a basal dendrite rather than from the soma. This morphology is particularly enriched in central CA1, the principal hippocampal output area. Multiphoton glutamate uncaging revealed that input onto the axon-carrying dendrites (AcDs) was more efficient in eliciting action potential output than input onto regular basal dendrites. First, synaptic input onto AcDs generates action potentials with lower activation thresholds compared with regular dendrites. Second, AcDs are intrinsically more excitable, generating dendritic spikes with higher probability and greater strength. Thus, axon-carrying dendrites constitute a privileged channel for excitatory synaptic input in a subset of cortical pyramidal cells.Video Abstract

The large isoforms of the Rab3 interacting molecule (RIM) family, RIM1α/β and RIM2α/β, have been shown to be centrally involved in mediating presynaptic active zone function. The RIM protein family contains two additional small isoforms, RIM3γ and RIM4γ, which are composed only of the RIM-specific C-terminal C2B domain and varying N-terminal sequences and whose function remains to be elucidated. Here, we report that both, RIM3γ and RIM4γ, play an essential role for the development of neuronal arborization and of dendritic spines independent of synaptic function. γ-RIM knock-down in rat primary neuronal cultures andin vivoresulted in a drastic reduction in the complexity of neuronal arborization, affecting both axonal and dendritic outgrowth, independent of the time point of γ-RIM downregulation during dendrite development. Rescue experiments revealed that the phenotype is caused by a function common to both γ-RIMs. These findings indicate that γ-RIMs are involved in cell biological functions distinct from the regulation of synaptic vesicle exocytosis and play a role in the molecular mechanisms controlling the establishment of dendritic complexity and axonal outgrowth.

In order to ensure operation of synaptic transmission within an appropriate dynamic range, neurons have evolved mechanisms of activity-dependent plasticity, including changes in presynaptic efficacy. The multi-domain protein RIM1α is an integral component of the cytomatrix at the presynaptic active[for full text, please go to the a.m. URL], 57th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN)

The pore-forming Ca(2+) channel subunit Ca(V)3.2 mediates a low voltage-activated (T-type) Ca(2+) current (I(CaT)) that contributes pivotally to neuronal and cardiac pacemaker activity. Despite the importance of tightly regulated Ca(V)3.2 levels, the mechanisms regulating its transcriptional dynamics are not well understood. Here, we have identified two key factors that up- and down-regulate the expression of the gene encoding Ca(V)3.2 (Cacna1h). First, we determined the promoter region and observed several stimulatory and inhibitory clusters. Furthermore, we found binding sites for the transcription factor early growth response 1 (Egr1/Zif268/Krox-24) to be highly overrepresented within the Ca(V)3.2 promoter region. mRNA expression analyses and dual-luciferase promoter assays revealed that the Ca(V)3.2 promoter was strongly activated by Egr1 overexpression in vitro and in vivo. Subsequent chromatin immunoprecipitation assays in NG108-15 cells and mouse hippocampi confirmed specific Egr1 binding to the Ca(V)3.2 promoter. Congruently, whole-cell I(CaT) values were significantly larger after Egr1 overexpression. Intriguingly, Egr1-induced activation of the Ca(V)3.2 promoter was effectively counteracted by the repressor element 1-silencing transcription factor (REST). Thus, Egr1 and REST can bi-directionally regulate Ca(V)3.2 promoter activity and mRNA expression and, hence, the size of I(CaT). This mechanism has critical implications for the regulation of neuronal and cardiac Ca(2+) homeostasis under physiological conditions and in episodic disorders such as arrhythmias and epilepsy.

SummaryHippocampal granule cells are important relay stations that transfer information from the entorhinal cortex into the hippocampus proper. This process is critically determined by the integrative properties of granule cell dendrites. However, their small diameter has so far hampered efforts to examine their properties directly. Using a combination of dual somatodendritic patch-clamp recordings and multiphoton glutamate uncaging, we now show that the integrative properties of granule cell dendrites differ substantially from other principal neurons. Due to a very strong dendritic voltage attenuation, the impact of individual synapses on granule cell output is low. At the same time, integration is linearized by voltage-dependent boosting mechanisms, only weakly affected by input synchrony, and independent of input location. These experiments establish that dentate granule cell dendritic properties are optimized for linear integration and strong attenuation of synaptic input from the entorhinal cortex, which may contribute to the sparse activity of granule cells in vivo.

Febrile seizures are a common childhood seizure disorder and a defining feature of genetic epilepsy with febrile seizures plus (GEFS+), a syndrome frequently associated with Na+ channel mutations. Here, we describe the creation of a knockin mouse heterozygous for the C121W mutation of the beta1 Na+ channel accessory subunit seen in patients with GEFS+. Heterozygous mice with increased core temperature displayed behavioral arrest and were more susceptible to thermal challenge than wild-type mice. Wild-type beta1 was most concentrated in the membrane of axon initial segments (AIS) of pyramidal neurons, while the beta1(C121W) mutant subunit was excluded from AIS membranes. In addition, AIS function, an indicator of neuronal excitability, was substantially enhanced in hippocampal pyramidal neurons of the heterozygous mouse specifically at higher temperatures. Computational modeling predicted that this enhanced excitability was caused by hyperpolarized voltage activation of AIS Na+ channels. This heat-sensitive increased neuronal excitability presumably contributed to the heightened thermal seizure susceptibility and epileptiform discharges seen in patients and mice with beta1(C121W) subunits. We therefore conclude that Na+ channel beta1 subunits modulate AIS excitability and that epilepsy can arise if this modulation is impaired.

SummaryNeurons possess elaborate dendritic arbors which receive and integrate excitatory synaptic signals. Individual dendritic subbranches exhibit local membrane potential supralinearities, termed dendritic spikes, which control transfer of local synaptic input to the soma. Here, we show that dendritic spikes in CA1 pyramidal cells are strongly regulated by specific types of prior input. While input in the linear range is without effect, supralinear input inhibits subsequent spikes, causing them to attenuate and ultimately fail due to dendritic Na+ channel inactivation. This mechanism acts locally within the boundaries of the input branch. If an input is sufficiently strong to trigger axonal action potentials, their back-propagation into the dendritic tree causes a widespread global reduction in dendritic excitability which is prominent after firing patterns occurring in vivo. Together, these mechanisms control the capability of individual dendritic branches to trigger somatic action potential output. They are invoked at frequencies encountered during learning, and impose limits on the storage and retrieval rates of information encoded as branch excitability.

Increasing evidence supports roles for the current mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels,Ih, in hippocampal maturation and specifically in the evolving changes of intrinsic properties as well as network responses of hippocampal neurons. Here, we describe a novel developmental plasticity of HCN channel expression in axonal and presynaptic compartments: HCN1 channels were localized to axon terminals of the perforant path (the major hippocampal afferent pathway) of immature rats, where they modulated synaptic efficacy. However, presynaptic expression and functions of the channels disappeared with maturation. This was a result of altered channel transport to the axons, because HCN1 mRNA and protein levels in entorhinal cortex neurons, where the perforant path axons originate, were stable through adulthood. Blocking action potential firingin vitroincreased presynaptic expression of HCN1 channels in the perforant path, suggesting that network activity contributed to regulating this expression. These findings support a novel developmentally regulated axonal transport of functional ion channels and suggest a role for HCN1 channel-mediated presynapticIhin hippocampal maturation.

Temporal lobe epilepsy (TLE) is the most common focal seizure disorder in adults. In many patients, transient brain insults, including status epilepticus (SE), are followed by a latent period of epileptogenesis, preceding the emergence of clinical seizures. In experimental animals, transcriptional upregulation of CaV3.2 T-type Ca2+-channels, resulting in an increased propensity for burst discharges of hippocampal neurons, is an important trigger for epileptogenesis. Here we provide evidence that the metal-regulatory transcription factor 1 (MTF1) mediates the increase of CaV3.2 mRNA and intrinsic excitability consequent to a rise in intracellular Zn2+ that is associated with SE. Adeno-associated viral (rAAV) transfer of MTF1 into murine hippocampi leads to increased CaV3.2 mRNA. Conversely, rAAV-mediated expression of a dominant-negative MTF1 abolishes SE-induced CaV3.2 mRNA upregulation and attenuates epileptogenesis. Finally, data from resected human hippocampi surgically treated for pharmacoresistant TLE support the Zn2+-MTF1-CaV3.2 cascade, thus providing new vistas for preventing and treating TLE., Temporal lobe epilepsy can cause ionic imbalance in the brain and alter transcriptional activities. Here, van Loo et al. show that the increase in neuronal zinc following status epilepticus can induce transcriptional change via metal-regulatory transcription factor 1, and alter voltage-gated calcium channel CaV3.2 and intrinsic neuronal excitability.

Dendritic voltage-gated ion channels profoundly shape the integrative properties of neuronal dendrites. In epilepsy, numerous changes in dendritic ion channels have been described, all of them due to either their altered transcription or phosphorylation. In pilocarpine-treated chronically epileptic rats, we describe a novel mechanism that causes an increased proximal dendritic persistent Na+current (INaP). We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of dendriticINaPis due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendriticINaPcauses augmented dendritic summation of excitatory inputs. These results establish a novel post-transcriptional modification of ion channels in chronic epilepsy and may provide a novel avenue for treatment of temporal lobe epilepsy.SIGNIFICANCE STATEMENTIn this paper, we describe a novel mechanism that causes increased dendritic persistent Na+current. We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of persistent Na+currents is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic persistent Na current causes augmented dendritic summation of excitatory inputs. We believe that these results establish a novel post-transcriptional modification of ion channels in chronic epilepsy.

The hyperpolarization-activated current (I(h)) contributes to intrinsic properties and network responses of neurons. Its biophysical properties depend on the expression profiles of the underlying hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and the presence of cyclic AMP (cAMP) that potently and differentially modulates I(h) conducted by HCN1, HCN2 and/or HCN4. Here, we studied the properties of I(h) in hippocampal CA1 pyramidal cells, the developmental evolution of the HCN-subunit isoforms that contribute to this current, and their interplay with age-dependent free cAMP concentrations, using electrophysiological, molecular and biochemical methods. I(h) amplitude increased progressively during the first four postnatal weeks, consistent with the observed overall increased expression of HCN channels. Activation kinetics of the current accelerated during this period, consonant with the quantitative reduction of mRNA and protein expression of the slow-kinetics HCN4 isoform and increased levels of HCN1. The sensitivity of I(h) to cAMP, and the contribution of the slow component to the overall I(h), decreased with age. These are likely a result of the developmentally regulated transition of the complement of HCN channel isoforms from cAMP sensitive to relatively cAMP insensitive. Thus, although hippocampal cAMP concentrations increased over twofold during the developmental period studied, the coordinated changes in expression of three HCN channel isoforms resulted in reduced effects of this signalling molecule on neuronal h currents.

T-type Ca2+ channels give rise to low-threshold inward currents that are central determinants of neuronal excitability. The availability of T-type Ca2+ channels is strongly influenced by voltage-dependent inactivation and recovery from inactivation. Here, we show that native and cloned T-type Ca2+ channel subunits selectively encode specific aspects of prior membrane potential changes via a powerful modulation of the rates with which these channels recover from inactivation. Increasing the duration of subthreshold (−70 to −55 mV) conditioning depolarizations caused a pronounced slowing of subsequent recovery from inactivation of both cloned (Cav3.1–3.3) and native T-type channels (thalamic neurones). The scaling of recovery rates with increasing duration of conditioning depolarizations could be well described by a power law function. Different T-type channel isoforms exhibited overlapping but complementary ranges of recovery rates. Intriguingly, scaling of recovery rates was dramatically reduced in Cav3.2 and Cav3.3, but not Cav3.1 subunits, when mock action potentials were superimposed on conditioning depolarizations. Our results suggest that different T-type channel subunits exhibit dramatic differences in scaling relationships, in addition to well-described differences in other biophysical properties. Furthermore, the availability of T-type channels is powerfully modulated over time, depending on the patterns of prior activity that these channels have encountered. These data provide a novel mechanism for cellular short-term plasticity on the millisecond to second time scale that relies on biophysical properties of specific T-type Ca2+ channel subunits.

Tuberous sclerosis complex (TSC) is a common hereditary disorder caused by mutations in either the TSC1 or TSC2 genes, and characterized by severe epilepsy, cerebral hamartomas and mental retardation. We have used rats that are heterozygous for an autosomal-dominant germline mutation in the TSC2 gene (TSC2+/- rats) to examine the consequences of TSC2 mutations for hippocampal synaptic plasticity. While basal synaptic transmission in the Schaffer collateral-CA1 synapse was not altered, paired-pulse plasticity was significantly enhanced in TSC2+/- rats (interpulse intervals 20-200 ms). Moreover, TSC2+/- rats exhibited a marked reduction of different forms of synaptic plasticity. Long-term potentiation (LTP) elicited following high-frequency tetanization of Schaffer collaterals was significantly decreased from 1.45 +/- 0.05-fold potentiation to 1.15 +/- 0.04 (measured after 60 min). This difference in LTP levels between TSC2+/- and wild-type rats also persisted in the presence of the gamma-aminobutyric acid (GABA)(A) receptor antagonist bicuculline. In addition to changed LTP, the level of long-term depression (LTD) elicited by different forms of low-frequency stimulation was significantly less in TSC2+/- rats. These results suggest that TSC2 mutations may cause hippocampal synapses to lose much of their potential for activity-dependent synaptic modification. An understanding of the underlying molecular pathways may suggest new therapeutic approaches aimed at inhibiting the development of the profound mental retardation in TSC.

Summary: Purpose: Embryonic stem (ES) cell–based therapy strategies are thought to bear considerable promise in chronic neurologic disorders. Nonetheless, studies addressing the functional properties of ES cell–derived progeny after transplantation into the adult, pathologically modified CNS are scarce. Methods: We therefore transplanted ES cell–derived neural precursors expressing enhanced green fluorescent protein only in neuronal progeny bilaterally into the hippocampi of pilocarpine-treated chronically epileptic and sham-control rats. Whole-cell patch-clamp recordings of identified ES cell–derived neurons (ESNs) in hippocampal slices were performed 13 to 34 days after transplantation. Results: Most ESNs were found in clusters at the transplant site and did not migrate into host tissue. However, they gave rise to a dense network of processes extending over large distances into the host tissue. All ESNs possessed the ability to generate action potentials and expressed voltage-gated Na+ and K+ currents, as well as hyperpolarization-activated currents. Likewise, most ESNs received non–N-methyl-d-aspartate (NMDA) and γ-aminobutyric acid (GABA)A receptor-mediated synaptic input. Both types of synapses displayed intact short-term plasticity. An unusual feature of the majority of ESNs was the occurrence of spontaneous pacemaking activity at frequencies ∼3 Hertz. No obvious differences were found between the functional properties of ESNs in sham-control and in pilocarpine-treated rats. Conclusions: After transplantation into adult control and epileptic rats, ESNs displayed intrinsic and synaptic properties characteristic of neurons. Even though ESNs remained close to the transplant site, the formation of extensive networks of graft-derived processes may be useful for ES cell–based substance delivery.

Specific agonists of metabotropic glutamate receptors (mGluRs) provide powerful tools to discriminate afferent fibers originating from different presynaptic neurons. The group II mGluR agonists L-CCG-I ((2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine) and DCG-IV ((2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine) are commonly used to distinguish between mossy fiber and associational-commissural (A/C) fiber input to the hippocampal CA3 region because only on the former group II mGluRs are expressed. Since previous reports indicated that L-CCG-I can activate group III mGluRs as well, we investigated whether L-CCG-I depresses A/C field potentials. L-CCG-I (10-300 microM) exhibited a significant dose-dependent and reversible reduction of A/C field potentials by 8 +/- 4% (10 microM), by 32 +/- 4% (100 microM, p0.001) and by 38 +/- 7% (300 microM, p0.05) that was accompanied by a concomitant increase in paired-pulse facilitation. Moreover, the selective group III antagonist (R,S)-alpha-methylserine-O-phosphate (MSOP; 100 microM) significantly reduced the field potential inhibition by L-CCG-I (100 microM) to 9 +/- 4% (p0.05). In contrast, DCG-IV did not affect A/C field potentials. In conclusion, the purported group II mGluR agonist L-CCG-I depresses A/C synaptic transmission by activation of group III mGluRs. For this reason, DCG-IV should be the drug of choice when aiming to discriminate between mossy fiber and A/C input to CA3 pyramidal cells.

Changes in the expression of ion channels, contributing to altered neuronal excitability, are emerging as possible mechanisms in the development of certain human epilepsies. In previous immature rodent studies of experimental prolonged febrile seizures, isoform-specific changes in the expression of hyperpolarization-activated cyclic nucleotide-gated cation channels (HCNs) correlated with long-lasting hippocampal hyperexcitability and enhanced seizure susceptibility. Prolonged early-life seizures commonly precede human temporal lobe epilepsy (TLE), suggesting that transcriptional dysregulation of HCNs might contribute to the epileptogenic process. Therefore, we determined whether HCN isoform expression was modified in hippocampi of individuals with TLE. HCN1 and HCN2 expression were measured usingin situhybridization and immunocytochemistry in hippocampi from three groups: TLE with hippocampal sclerosis (HS;n= 17), epileptic hippocampi without HS, or non-HS (NHS;n= 10), and autopsy material (n= 10). The results obtained in chronic human epilepsy were validated by examining hippocampi from the pilocarpine model of chronic TLE.In autopsy and most NHS hippocampi, HCN1 mRNA expression was substantial in pyramidal cell layers and lower in dentate gyrus granule cells (GCs). In contrast, HCN1 mRNA expression over the GC layer and in individual GCs from epileptic hippocampus was markedly increased once GC neuronal density was reduced by >50%. HCN1 mRNA changes were accompanied by enhanced immunoreactivity in the GC dendritic fields and more modest changes in HCN2 mRNA expression. Furthermore, similar robust and isoform-selective augmentation of HCN1 mRNA expression was evident also in the pilocarpine animal model of TLE. These findings indicate that the expression of HCN isoforms is dynamically regulated in human as well as in experimental hippocampal epilepsy. After experimental febrile seizures (i.e., early in the epileptogenic process), the preserved and augmented inhibition onto principal cells may lead to reduced HCN1 expression. In contrast, in chronic epileptic HS hippocampus studied here, the profound loss of interneuronal and principal cell populations and consequent reduced inhibition, coupled with increased dendritic excitation of surviving GCs, might provoke a “compensatory” enhancement of HCN1 mRNA and protein expression.

Different subtypes of voltage-dependent Ca(2+) currents in native neurones are essential in coupling action potential firing to Ca(2+) influx. For most of these currents, the underlying Ca(2+) channel subunits have been identified on the basis of pharmacological and biophysical similarities. In contrast, the molecular basis of R-type Ca(2+) currents remains controversial. We have therefore examined the contribution of the Ca(V)2.3 (alpha(1E)) subunits to R-type currents in different types of central neurones using wild-type mice and mice in which the Ca(V)2.3 subunit gene was deleted. In hippocampal CA1 pyramidal cells and dentate granule neurones, as well as neocortical neurones of wild-type mice, Ca(2+) current components resistant to the combined application of omega-conotoxin GVIA and MVIIC, omega-agatoxin IVa and nifedipine (I(Ca,R)) were detected that were composed of a large R-type and a smaller T-type component. In Ca(V)2.3-deficient mice, I(Ca,R) was considerably reduced in CA1 neurones (79 %) and cortical neurones (87 %), with less reduction occurring in dentate granule neurones (47 %). Analysis of tail currents revealed that the reduction of I(Ca,R) is due to a selective reduction of the rapidly deactivating R-type current component in CA1 and cortical neurones. In all cell types, I(Ca,R) was highly sensitive to Ni(2+) (100 microM: 71-86 % block). A selective antagonist of cloned Ca(V)2.3 channels, the spider toxin SNX-482, partially inhibited I(Ca,R) at concentrations up to 300 nM in dentate granule cells and cortical neurones (50 and 57 % block, EC(50) 30 and 47 nM, respectively). I(Ca,R) in CA1 neurones was significantly less sensitive to SNX-482 (27 % block, 300 nM SNX-482). Taken together, our results show clearly that Ca(V)2.3 subunits underlie a significant fraction of I(Ca,R) in different types of central neurones. They also indicate that Ca(V)2.3 subunits may give rise to Ca(2+) currents with differing pharmacological properties in native neurones.

Highly parallel expression monitoring by microarrays is a powerful tool to study human brain disorders. In contrast to various nonneuronal tissues, the CNS is composed of a multitude of different cell types. Changed mRNA levels in neuropathological conditions may simply reflect altered tissue composition, rather than specific gene transcription regulation. Therefore, it is crucial, to supplement expression array data of histologically heterogeneous brain samples with a detailed analysis at the cellular level. Here, we have used a two-step approach to identify specific changes in hippocampal gene expression in patients with a hippocampal seizure focus (TLE) and marked neuronal damage. Using comparative expression array hybridization, 21 genes appeared to be differentially regulated. Expression alterations of a subset of these genes, i.e. (up-regulation of ataxin-3 and glial fibrillary acid protein (GFAP) as well as down-regulation of calmodulin) was confirmed in an extended series of individuals by real-time quantitative RT-PCR (qRT-PCR). In order to determine the cellular localization of these mRNAs, we performed real-time qRT-PCR of individual laser-microdissected neurons and glial cells. While ataxin-3 was expressed only in hippocampal neurons, GFAP was detected in reactive astrocytes. The differential calmodulin expression found on the tissue level was not observed in mRNA analyses from single neurons, suggesting that lower calmodulin mRNA levels are a consequence of segmental cell loss and do not indicate reduced cellular expression. Ataxin-3 has been related to neuronal maintenance. Its functional role for TLE has to be further evaluated.

Mitochondrial function is a key determinant of both excitability and viability of neurons. Here, we demonstrate seizure-dependent changes in mitochondrial oxidative phosphorylation in the epileptic rat hippocampus. The intense pathological neuronal activity in pilocarpine-treated rats exhibiting spontaneous seizures resulted in a selective decline of the activities of NADH-CoQ oxidoreductase (complex I of the respiratory chain) and cytochrome c oxidase (complex IV of respiratory chain) in the CA3 and CA1 hippocampal pyramidal subfields. In line with these findings, high-resolution respirometry revealed an increased flux control of complex I on respiration in the CA1 and CA3 subfields and decreased maximal respiration rates in the more severely affected CA3 subfield. Imaging of mitochondrial membrane potential using rhodamine 123 showed a lowered mitochondrial membrane potential in both pyramidal subfields. In contrast to the CA1 and CA3 subfields, mitochondrial oxidative phosphorylation was unaltered in the dentate gyrus and the parahippocampal gyrus. The changes of oxidative phosphorylation in the epileptic rat hippocampus cannot be attributed to oxidative enzyme modifications but are very likely related to a decrease in mitochondrial DNA copy number as shown in the more severely affected CA3 subfield and in cultured PC12 cells partially depleted of mitochondrial DNA. Thus, our results demonstrate that seizure activity downregulates the expression of mitochondrial-encoded enzymes of oxidative phosphorylation. This mechanism could be invoked during diverse forms of pathological neuronal activity and could severely affect both excitability and viability of hippocampal pyramidal neurons.

Mutations inSCN1Aand other ion channel genes can cause different epileptic phenotypes, but the precise mechanisms underlying the development of hyperexcitable networks are largely unknown. Here, we present a multisystem analysis of anSCN1Amouse model carrying the NaV1.1-R1648H mutation, which causes febrile seizures and epilepsy in humans. We found a ubiquitous hypoexcitability of interneurons in thalamus, cortex, and hippocampus, without detectable changes in excitatory neurons. Interestingly, somatic Na+channels in interneurons and persistent Na+currents were not significantly changed. Instead, the key mechanism of interneuron dysfunction was a deficit of action potential initiation at the axon initial segment that was identified by analyzing action potential firing. This deficit increased with the duration of firing periods, suggesting that increased slow inactivation, as recorded for recombinant mutated channels, could play an important role. The deficit in interneuron firing caused reduced action potential-driven inhibition of excitatory neurons as revealed by less frequent spontaneous but not miniature IPSCs. Multiple approaches indicated increased spontaneous thalamocortical and hippocampal network activity in mutant mice, as follows: (1) more synchronous and higher-frequency firing was recorded in primary neuronal cultures plated on multielectrode arrays; (2) thalamocortical slices examined by field potential recordings revealed spontaneous activities and pathological high-frequency oscillations; and (3) multineuron Ca2+imaging in hippocampal slices showed increased spontaneous neuronal activity. Thus, an interneuron-specific generalized defect in action potential initiation causes multisystem disinhibition and network hyperexcitability, which can well explain the occurrence of seizures in the studied mouse model and in patients carrying this mutation.

T he quantitative study of gene expression in neurons under physiological and pathophysiological conditions has gained considerable importance. Consequently, a variety of different techniques to quantify gene expression have evolved. Generally, such approaches can be subdivided into strategies that provide absolute versus relative quantitation of a target transcript. Quantitative procedures such as quantitative Northern blots or conventional quantitative reverse transcription polymerase chain reaction (RT-PCR) methods have several disadvantages. For instance, Northern blots require substantial amounts of RNA, rendering investigation of mRNA levels from single cells impossible. Comparability of results is extremely dependent upon identical amounts of starting mRNA used in different samples, a condition that cannot usually be achieved with small cell numbers or minute amounts of tissue. To circumvent these obstacles, various techniques have been developed to quantify gene expression relative to a reference gene (Fink et al, 1998; Waha et al, 1998). This obviates the need to perform an accurate determination of initial mRNA concentrations. On the other hand, a suitable reference gene is essential as an internal control for relative mRNA quantitation. This internal control is crucial for a quantitative comparison of gene expression between different tissue types, at various developmental stages, and between control and disease tissue. Ideally, internal control transcripts should show constant, highlevel expression independent of experimental conditions. The genes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and b-actin have been commonly used as reference genes. In recent years, cyclophilin A and hypoxanthine phosphoribosyl-transferase (HPRT) have also been adopted for this purpose. However, several classical reference genes may exhibit considerable changes in expression in cell types or under disease conditions (Suzuki et al, 2000). Here, we have analyzed several of these genes as reference transcripts for activity-dependent chronic changes in central nervous system (CNS) gene expression. Pilocarpine-induced epilepsy in rats was employed as experimental paradigm. We find that GAPDH is not suitable for this purpose, whereas the neuron-specific gene synaptophysin appears remarkably stable following intense neuronal activity. To study activity-dependent alterations in reference gene expression, seizure activity was induced in vivo by applying a single dose of the convulsant pilocarpine (male Wistar rats 150–200 g, 300–380 mg/kg, ip) after prior administration of methyl-scopolamine (1 mg/kg, sc, 30 minutes before pilocarpine, ip). Seizures were terminated after 2 hours by application of diazepam (0.1 mg/kg, sc). Age-matched rats, injected with saline served only as controls. Thirty days after pilocarpine/saline injection, the brains of pilocarpinetreated and control animals (n 5 5 each) were removed. One hippocampus was immediately frozen in liquid nitrogen for in situ hybridization, whereas the contralateral hippocampus was used for preparation of 400-mm transverse vibratome slices. From these slices, single CA1 neurons were harvested with a glass pipette in less than 10 ml of extracellular solution (Beck et al, 1999). mRNA was isolated using the Dynabead mRNA Direct Micro Kit (Dynal, Hamburg, Germany) according to the manufacturer’s protocol. For real-time RT-PCR fluorescent hybridization, oligonucleotides and primers were chosen for the five putative reference genes: GAPDH, b-actin, cyclophilin A, HPRT, and synaptophysin (Eurogentec, Seraing, Received March 9, 2001. This work is supported by the Deutsche Forschungsgemeinschaft (SFB 400), the BONFOR program of the University of Bonn Medical Center, and the German-Israel Program of the Bundesministerium fur Bildung und Fors and Ministry of Science. Address reprint requests to: Dr. Albert J. Becker, Department of Neuropathology, University of Bonn Medical Center, Sigmund-Freud Str. 25, 53105 Bonn, Germany. E-mail: albert_becker@uni-bonn.de 0023-6837/01/8106-913$03.00/0 LABORATORY INVESTIGATION Vol. 81, No. 6, p. 913, 2001 Copyright © 2001 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A.

1. Fundamental to the understanding of CNS function is the question of how individual neurons integrate multiple synaptic inputs into an output consisting of a sequence of action potentials carrying information coded as spike frequency. The availability for activation of neuronal Na(+) channels is critical for this process and is regulated both by fast and slow inactivation processes. Here, we have investigated slow inactivation processes in detail in hippocampal neurons. 2. Slow inactivation was induced by prolonged (10-300 s) step depolarisations to -10 mV at room temperature. In isolated hippocampal dentate granule cells (DGCs), recovery from this inactivation was biexponential, with time constants for the two phases of slow inactivation tau(slow,1) and tau(slow,2) ranging from 1 to 10 s and 20 to 50 s, respectively. Both (slow,1) and tau(slow,2) were related to the duration of prior depolarisation by a power law function of the form tau(t) = a (t/a)b, where t is the duration of the depolarisation, a is a constant kinetic setpoint and b is a scaling power. This analysis yielded values of a = 0.034 s and b = 0.62 for tau(slow,1) and a = 24 s and b = 0.30 for tau(slow,2) in the rat. 3. When a train of action potential-like depolarisations of different frequencies (50, 100, 200 Hz) was used to induce inactivation, a similar relationship was found between the frequency of depolarisation and both tau(slow,1) and tau(slow,2) (a = 0.58 s, b = 0.39 for tau(slow,1) and a = 3.77 s and b = 0.42 for tau(slow,2)). 4. Using nucleated patches from rat hippocampal slices, we have addressed possible cell specific differences in slow inactivation. In fast-spiking basket cells a similar scaling relationship can be found (a = 3.54 s and b = 0.39) as in nucleated patches from DGCs (a = 2.3 s and b = 0.48) and non-fast-spiking hilar neurons (a = 2.57 s and b = 0.49). 5. Likewise, comparison of human and rat granule cells showed that properties of ultra-slow recovery from inactivation are conserved across species. In both species ultra-slow recovery was biexponential with both tau(slow,1) and tau(slow,2) being related to the duration of depolarisation t, with a = 0.63 s and b = 0.44 for tau(slow,1) and a = 25 s and b = 0.37 for tau(slow,2) for the human subject. 6. In summary, we describe in detail how the biophysical properties of Na(+) channels result in a complex interrelationship between availability of sodium channels and membrane potential or action potential frequency that may contribute to temporal integration on a time scale of seconds to minutes in different types of hippocampal neurons.

Mitochondria are cellular organelles crucial for energy supply and calcium homeostasis in neuronal cells, and their dysfunction causes seizure activity in some rare human epilepsies. To directly test whether mitochondrial respiratory chain enzymes are abnormal in the most common form of chronic epilepsy, temporal lobe epilepsy (TLE), living human brain specimens from 57 epileptic patients and 2 nonepileptic controls were investigated. In TLE patients with a hippocampal epileptic focus, we demonstrated a specific deficiency of complex I of the mitochondrial respiratory chain in the hippocampal CA3 region. In contrast, TLE patients with a parahippocampal epileptic focus showed reduced complex I activity only in parahippocampal tissue. Inhibitor titrations of the maximal respiration rate of intact human brain slices revealed that the observed reduction in complex I activity is sufficient to affect the adenosine triphosphate production rate. The abnormal complex I activity in the hippocampal CA3 region was paralleled by increased succinate dehydrogenase staining of neurons and marked ultrastructural abnormalities of mitochondria. Therefore, mitochondrial dysfunction is suggested to be specific for the epileptic focus and may constitute a pathomechanism contributing to altered excitability and selective neuronal vulnerability in TLE.

In mesial temporal lobe epilepsy (mTLE), the predominant form of epilepsy in adults, and in animal models of the disease, there is a conspicuous loss of the intracellular Ca2+-binding protein calbindin-D28k(CB) from granule cells (GCs) of the dentate gyrus. The role of this protein in nerve cell function is controversial, but here we provide evidence for its role in controlling Ca2+influx into human neurons. In patients with Ammon's horn sclerosis (AHS), the loss of CB from GCs markedly increased the Ca2+-dependent inactivation of voltage-dependent Ca2+currents (ICa), thereby diminishing Ca2+influx during repetitive neuronal firing. Introducing purified CB into GCs restored Ca2+current inactivation to levels observed in cells with normal CB content harvested from mTLE patients without AHS. Our data are consistent with the possibility of neuroprotection secondary to the CB loss. By limiting Ca2+influx through an enhanced Ca2+-dependent inactivation of voltage-dependent Ca2+channels during prolonged neuronal discharges, the loss of CB may contribute to the resistance of surviving human granule cells in AHS.

Selective hippocampal cell loss and altered neurotransmitter receptor expression have been proposed as pathogenic mechanisms in the development of chronic mesial temporal lobe epilepsy (TLE). Studies in animal models point to metabotropic glutamate receptors (mGluRs) as modulators of hippocampal epileptogenesis. In addition, mGluRs may constitute specific targets for the development of novel anticonvulsive drugs. As mGluR4 represents an inhibitory class III mGluR associated with the reduction of intracellular cyclic AMP levels and calcium influx, we have analyzed the regional and cellular expression of mGluR4 in surgical hippocampal specimens obtained from patients with TLE by using immunohistochemistry and in situ hybridization. Although the hippocampi of control specimens (n = 11) were almost devoid of mGluR4 immunolabeling, all TLE specimens (n = 35) showed a striking up-regulation of mGluR4 immunoreactivity, in particular within the dentate gyrus. Immunoelectron microscopy localized the receptor protein to the periphery of presynaptic and postsynaptic membranes. In situ hybridization revealed increased transcript levels of mGluR4 in dentate granule cells and residual CA4 neurons of TLE specimens compared with controls. Our results suggest a potential role of mGluR4 in counteracting excitatory hippocampal activity and in modulating seizure-associated vulnerability of hippocampal neurons. These data may also provide a basis for pharmacological studies of mGluR4 agonists as potential novel drugs in the treatment of TLE.

Aberrant axonal reorganization and altered distribution of neurotransmitter receptor subtypes have been proposed as major pathogenic mechanisms for hippocampal hyperexcitability in chronic temporal lobe epilepsies (TLE). Recent data point to excitatory class I metabotropic glutamate receptors (mGluR1 and mGluR5) as interesting candidates. Here, we have analyzed the hippocampal distribution and mRNA expression of mGluR1 and mGluR5 in two rat models of limbic seizures, i.e. electrical kindling and intraperitoneal kainate injections, as well as in human TLE. Quantitative RT-PCR analysis detected a significant increase of hippocampal mGluR1 gene transcript levels in kainate treated and kindled rats. In addition, microdissected hippocampal tissue samples localized this increase to the dentate gyrus. Using immunohistochemistry with mGluR1alpha subtype specific antibodies, increased labeling was observed within the dentate gyrus molecular layer (DG-ML). A similar pattern of increased mGluR1alpha neuropil staining was found within the DG-ML of epilepsy patients (n = 42) compared with peritumoral hippocampus specimens obtained from nonepileptic patients (biopsy controls, n = 3). This increase was detected in TLE patients with segmental hippocampal cell loss, as well as in TLE patients with focal lesions but no histopathological alterations of the hippocampus. In contrast, mGluR5 immunoreactivity and mRNA expression were not significantly altered in the DG-ML. Our data demonstrate a striking regional induction of mGluR1alpha in the hippocampal dentate gyrus of experimental animals with limbic seizures as well as in human patients with chronic, intractable TLE. This increase corresponds to functional alterations of class I mGluRs observed in seizure models and may significantly contribute to hippocampal hyperexcitability in focal human epilepsies.

Studies in rodents and nonhuman primates have linked the activity of N -methyl- d -aspartate (NMDA) receptors within the hippocampus to animals’ performance on memory-related tasks. However, whether these receptors are similarly essential for human memory is still an open question. Here we present evidence suggesting that hippocampal NMDA receptors, most likely within the CA1 region, do participate in human verbal memory processes. Words elicit a negative event-related potential (ERP) peaking around 400 ms within the anterior mesial temporal lobe (AMTL-N400). Ketamine, an NMDA-receptor antagonist, reduces the amplitude of the AMTL-N400 (in contrast to other hippocampal potentials) on initial presentation, eliminates the typical AMTL-N400 amplitude reduction with repetition, and leads to significant memory impairment. Of the various hippocampal subfields, only the density of CA1 neurons correlates with the word-related ERPs that are reduced by ketamine. Altogether, our behavioral, anatomical, and electrophysiological results indicate that hippocampal NMDA receptors are involved in human memory.

The localization and morphological assessment of plastic or newly formed synapses in the human brain remains difficult due to the lack of specific markers. The ectoenzyme 5'-nucleotidase may represent a useful marker of these structures, since in adult rodents synaptic 5'-nucleotidase activity is restricted to sites of spontaneous synaptic turnover and induced reactive synaptogenesis. However, it is unclear to what extent synaptic 5'-nucleotidase activity occurs in the normal human brain, and whether reactive synaptogenesis, as seen e.g. in temporal lobe epilepsy (TLE), is associated with this ectoenzyme. Therefore, we have investigated the histochemical distribution of 5'-nucleotidase in hippocampal control specimens (n = 3) and in the hippocampus of TLE patients (n = 13). In controls, 5'-nucleotidase activity was present in the dentate gyrus molecular layer (DG-ML) and the mossy fiber termination field within the CA4 and CA3 subfields. Compared with controls, TLE specimens revealed markedly increased 5'-nucleotidase labeling in the DG-ML, implying TLE-associated reactive synaptogenesis in this hippocampal region. In contrast to GAP-43, synaptophysin, and dynorphin A, synaptic 5'-nucleotidase activity may serve as a potential specific indicator of plastic synapses or newly formed terminals in the human brain and prove useful for the study of diseases involving aberrant sprouting or altered synaptic plasticity.

Mitochondria provide the main neuronal energy supply and are important organelles for the sequestration of intracellular Ca2+. This indicates a possible important role for mitochondria in modulating neuronal excitability in normal function as well as in disease. Therefore, we have investigated mitochondrial oxidative phosphorylation in the kainate model of epilepsy. We measured the oxygen consumption of single 400-micron rat hippocampal slices applying high resolution respirometry and determined mitochondrial NAD(P)H autofluorescence signal changes in single slices by laser-excited fluorescence spectroscopy. We observed an about 2-fold higher (p

Animals exposed to kainic acid (KA) induced status epilepticus display a striking pattern of selective neuronal vulnerability in the hippocampus. Neurons in the hilus/CA3 and CA1 subfields appear particularly sensitive whereas dentate gyrus (DG) granule cells are resistant. The molecular basis for this differential susceptibility remains largely unknown. Recently, an involvement of nitric oxide, c-Jun amino-terminal kinases (JNK) and interleukin-1 β converting enzyme (ICE)-related proteases has been proposed in KA induced neuronal cell death. In the present study, we have determined the regional expression of transcripts for two modulating genes operating in these pathways, i.e., the endogenous protein inhibitor of neuronal nitric oxide synthase (PIN), and a cytoplasmic inhibitor of the JNK signal transduction pathway, designated JNK interacting protein-1 (JIP-1) and of the gene for the apoptosis-executing protease Caspase-3 in KA-treated animals. The expression of PIN and JIP-1 was found significantly upregulated in granule cells of the resistant DG. In contrast, an induction of the ICE-related protease Caspase-3 was observed in vulnerable hippocampal regions, i.e. CA1, CA3 and hilus. These results point towards PIN and JIP-1 as antiapoptotic factors contributing to selective resistance of granule cells, whereas Caspase-3 may be involved in cell death of hippocampal CA1, CA3 and hilar neurons in the kainate epilepsy model.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and calcineurin represent neuronal Ca2+-dependent enzymes which dynamically modify several common substrates in the mammalian brain via phosphorylation/dephosphorylation cycles. Studies in animal models indicate that altered expression and activity of these enzymes may be involved in epilepsy. We have analyzed their immunohistochemical distribution in hippocampi of 28 temporal lobe epilepsy (TLE) patients and 13 controls. TLE specimens were classified as Ammon's horn sclerosis (AHS) or focal lesions without alteration of hippocampal cytoarchitecture. Compared to control and lesion-associated TLE specimens, striking changes in the distribution pattern of both enzymes were found in the dentate gyrus (DG) of AHS specimens: Whereas CaMKII labeling was significantly increased in the granule cell somata and their proximal dendrites, calcineurin immunoreactivity was significantly reduced in the granule cell somata. Furthermore, calcineurin staining in controls showed high levels in the inner molecular layer with a sharp demarcation towards the outer molecular layer. In AHS, calcineurin staining was reduced in the inner molecular layer, with partial loss of this demarcation. These findings raise the possibility, that an up-regulation of CaMKII with a concomitant down-regulation of calcineurin in the DG of AHS specimens may cause a pathogenetically relevant imbalance of neuronal Ca2+/calmodulin-dependent phosphorylation/dephosphorylation systems.

Ammon's horn sclerosis (AHS) is a common finding in patients with temporal lobe epilepsy (TLE). In addition to selective neuronal cell loss and axonal reorganization, AHS is also characterized by a striking astroglial reaction. However, the functional significance of reactive astrogliosis in the pathogenesis of TLE remains to be determined. Reactive astrocytes produce a variety of cell adhesion molecules and other extracellular matrix (ECM) components with potential effects on axonal growth, axonal branching, and neosynaptogenesis in the central nervous system (CNS). In the present study we describe the distribution of the ECM glycoprotein tenascin/cytotactin (TN-C) in 44 human hippocampal specimens from patients with TLE. The distribution of TN-C immunoreactivity was evaluated with the anti-human TN-C monoclonal antibody K8 by densitometrical analysis, and TN-C protein levels were detected by immunoblotting. In the normal human hippocampus, there were distinctive boundaries between areas of high and low TN-C expression. These border zones demarcated areas with major synaptic input, i.e., the dentate gyrus molecular layer (DG-ML) and the gray matter of the Ammon's horn. TN-C and the neurite growth-associated protein GAP-43 exhibited a complementary pattern of distribution. Densitometric and protein biochemical analysis showed a significant, 4.3-fold increase of TN-C in the hippocampus of TLE patients with AHS compared with normal hippocampus obtained at autopsy. This increase in TN-C immunoreactivity was accompanied by a loss of TN-C boundaries and closely correlated with the extent of reactive gliosis, as indicated by immunoreactivity for glial fibrillary acidic protein. Furthermore, a striking colocalization between TN-C and GAP-43 was observed in the DG-ML of patients with AHS. These observations raise the intriguing possibility of pathogenetically relevant glio-neuronal interactions in human TLE.

Genetic studies have implicated the evolutionary novel, anthropoid primate-specific gene locus G72/G30 in psychiatric diseases. This gene encodes the protein LG72 that has been discussed to function as a putative activator of the peroxisomal enzyme D-amino-acid-oxidase (DAO) and as a mitochondrial protein. We recently generated 'humanized' bacterial artificial chromosome transgenic mice (G72Tg) expressing G72 transcripts in cells throughout the brain. These mice exhibit several behavioral phenotypes related to psychiatric diseases. Here we show that G72Tg mice have a reduced activity of mitochondrial complex I, with a concomitantly increased production of reactive oxygen species. Affected neurons display deficits in short-term plasticity and an impaired capability to sustain synaptic activity. These deficits lead to an impairment in spatial memory, which can be rescued by pharmacological treatment with the glutathione precursor N-acetyl cysteine. Our results implicate LG72-induced mitochondrial and synaptic defects as a possible pathomechanism of psychiatric disorders.

The synapses formed by the mossy fiber (MF) axons of hippocampal dentate gyrus granule neurons onto CA3 pyramidal neurons exhibit an intriguing form of experience-dependent synaptic plasticity that is induced and expressed presynaptically. In contrast to most other CNS synapses, long-term potentiation (LTP) at the MF–CA3 synapse is readily induced even during blockade of postsynaptic glutamate receptors. Furthermore, blocking voltage-gated Ca2+channels prevents MF-LTP, supporting an involvement of presynaptic Ca2+signaling via voltage-gated Ca2+channels in MF-LTP induction. We examined the contribution of activity in both dentate granule cell somata and MF terminals to MF-LTP. We found that the induction of stable MF-LTP requires tetanization-induced action potentials not only at MF boutons, but also at dentate granule cell somata. Similarly, blocking Ca2+influx via voltage-gated Ca2+channels only at the granule cell soma was sufficient to disrupt MF-LTP. Finally, blocking protein synthesis or blocking fast axonal transport mechanisms via disruption of axonal tubulin filaments resulted in decremental MF-LTP.Collectively, these data suggest that—in addition to Ca2+influx at the MF terminals—induction of MF synaptic plasticity requires action potential-dependent Ca2+signaling at granule cell somata, protein synthesis, and fast axonal transport along MFs. A parsimonious interpretation of these results is that somatic activity triggers protein synthesis at the soma; newly synthesized proteins are then transported to MF terminals, where they contribute to the stabilization of MF-LTP. Finally, the present data imply that synaptic plasticity at the MF–CA3 synapse can be affected by local modulation of somatic and presynaptic Ca2+channel activity.

Genetic studies have implicated the evolutionary novel, primates-specific gene locus G72/G30 in schizophrenia, bipolar and panic disorders. It encodes for a protein LG72 whose function has been controversially discussed as putative regulator of the peroxisomal enzyme D-amino-acid-oxidase (DAO), or as a mitochondrial protein, which promotes robust mitochondrial fragmentation in mammalian cell lines including human and rat primary neurons. Because of this conserved function we here have generated "humanized" BAC transgenic mice (G72Tg) expressing alternatively spliced G72 and G30 transcripts, and the LG72 protein. G72 expression is prominent in granular cells of the cerebellum, the hippocampus, the cortex and the olfactory bulb. Most strikingly, G72Tg mice displayed deficits in sensorimotor gating which could be reversed with haloperidol, increased sensitivity to PCP, motor-coordination deficits, increased compulsive behaviors and deficits in smell identification. These results demonstrate that expression of the human G72/G30 gene locus in mice produces behavioral phenotypes that are relevant to psychiatric disorders.

We constructed balancer-chromosomes for the large autosomes ofDrosophila hydei and screened more than 16000 chromosomes for male sterile mutations in order to dissect spermatogenesis genetically. 365 mutants on the X chromosome and the autosomes 2, 3, and 4 were recovered and analysed cytologically in squash preparations under phase-contrast optics. The majority of the mutations allows a rather advanced differentiation of the spermatozoa. At the light-microscopical level, it is possible to classify these mutations with respect to individualization, coiling or motility of the mutant spermatozoa. In contrast, a small number of mutants exhibits conspicuous, pleiotropic phenotypes. Gonial divisions, the shaping of the spermatocyte nucleus and male meiotic divisions are controlled by X chromosomal or autosomal genes which can mutate to male sterile alleles. A number of nonallelic 3rd chromosome male sterile mutations interfere with the unfolding of the Y chromosomal lampbrush loops. Other autosomal male sterile mutations modify the morphology of these lampbrush loops. Another group of mutations inhibits the formation of the nebenkern while the development of the spermatid nucleus and the flagellum can proceed. Such male sterile mutations can decouple the development of nucleus, protein body, nebenkern, and flagellum of the spermatid. Thus, we can describe spermatogenesis inDrosophila as the coordinate execution of the individual developmental programs of the different components of the spermatozoon.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by deficiency in the sulfolipid degrading enzyme arylsulfatase A (ASA). In the absence of a functional ASA gene, 3-O-sulfogalactosylceramide (sulfatide; SGalCer) and other sulfolipids accumulate. The storage is associated with progressive demyelination and various finally lethal neurological symptoms. Lipid storage, however, is not restricted to myelin-producing cells but also occurs in neurons. It is unclear whether neuronal storage contributes to symptoms of the patients. Therefore, we have generated transgenic ASA-deficient [ASA(−/−)] mice overexpressing the sulfatide synthesizing enzymes UDP-galactose:ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST) in neurons to provoke neuronal lipid storage. CGT-transgenic ASA(−/−) [CGT/ASA(−/−)] mice showed an accumulation of C18:0 fatty acid-containing SGalCer in the brain. Histochemically, an increase in sulfolipid storage could be detected in central and peripheral neurons of both CGT/ASA(−/−) and CST/ASA(−/−) mice compared with ASA(−/−) mice. CGT/ASA(−/−) mice developed severe neuromotor coordination deficits and weakness of hindlimbs and forelimbs. Light and electron microscopic analyses demonstrated nerve fiber degeneration in the spinal cord of CGT/ASA(−/−) mice. CGT/ASA(−/−) and, to a lesser extent, young ASA(−/−) mice exhibited cortical hyperexcitability, with recurrent spontaneous cortical EEG discharges lasting 5–15 s. These observations suggest that SGalCer accumulation in neurons contributes to disease phenotype.