Dendrites Of Dentate Granule Cells Research Articles

Early detonation by sprouted mossy fibers enables aberrant dentate network activity

In temporal lobe epilepsy, sprouting of hippocampal mossy fiber axons onto dentate granule cell dendrites creates a recurrent excitatory network. However, unlike mossy fibers projecting to CA3, sprouted mossy fiber synapses depress upon repetitive activation. Thus, despite their proximal location, relatively large presynaptic terminals, and ability to excite target neurons, the impact of sprouted mossy fiber synapses on hippocampal hyperexcitability is unclear. We find that despite their short-term depression, single episodes of sprouted mossy fiber activation in hippocampal slices initiated bursts of recurrent polysynaptic excitation. Consistent with a contribution to network hyperexcitability, optogenetic activation of sprouted mossy fibers reliably triggered action potential firing in postsynaptic dentate granule cells after single light pulses. This pattern resulted in a shift in network recruitment dynamics to an "early detonation" mode and an increased probability of release compared with mossy fiber synapses in CA3. A lack of tonic adenosine-mediated inhibition contributed to the higher probability of glutamate release, thus facilitating reverberant circuit activity.

Inhibition of Protease-Activated Receptor 1 Does not Affect Dendritic Homeostasis of Cultured Mouse Dentate Granule Cells.

Protease-activated receptors (PARs) are widely expressed in the central nervous system (CNS). While a firm link between PAR1-activation and functional synaptic and intrinsic neuronal properties exists, studies on the role of PAR1 in neural structural plasticity are scarce. The physiological function of PAR1 in the brain remains not well understood. We here sought to determine whether prolonged pharmacologic PAR1-inhibition affects dendritic morphologies of hippocampal neurons. To address this question we employed live-cell microscopy of mouse dentate granule cell dendrites in 3-week old entorhino-hippocampal slice cultures prepared from Thy1-GFP mice. A subset of cultures were treated with the PAR1-inhibitor SCH79797 (1 μM; up to 3 weeks). No major effects of PAR1-inhibition on static and dynamic parameters of dentate granule cell dendrites were detected under control conditions. Granule cells of PAR1-deficient slice cultures showed unaltered dendritic morphologies, dendritic spine densities and excitatory synaptic strength. Furthermore, we report that PAR1-inhibition does not prevent dendritic retraction following partial deafferentation in vitro. Consistent with this finding, no major changes in PAR1-mRNA levels were detected in the denervated dentate gyrus (DG). We conclude that neural PAR1 is not involved in regulating the steady-state dynamics or deafferentation-induced adaptive changes of cultured dentate granule cell dendrites. These results indicate that drugs targeting neural PAR1-signals may not affect the stability and structural integrity of neuronal networks in healthy brain regions.

Blockade of excitatory synaptogenesis with proximal dendrites of dentate granule cells following rapamycin treatment in a mouse model of temporal lobe epilepsy.

Inhibiting the mammalian target of rapamycin (mTOR) signaling pathway with rapamycin blocks granule cell axon (mossy fiber) sprouting after epileptogenic injuries, including pilocarpine-induced status epilepticus. However, it remains unclear whether axons from other types of neurons sprout into the inner molecular layer and synapse with granule cell dendrites despite rapamycin treatment. If so, other aberrant positive-feedback networks might develop. To test this possibility stereological electron microscopy was used to estimate the numbers of excitatory synapses in the inner molecular layer per hippocampus in pilocarpine-treated control mice, in mice 5 days after pilocarpine-induced status epilepticus, and after status epilepticus and daily treatment beginning 24 hours later with rapamycin or vehicle for 2 months. The optical fractionator method was used to estimate numbers of granule cells in Nissl-stained sections so that numbers of excitatory synapses in the inner molecular layer per granule cell could be calculated. Control mice had an average of 2,280 asymmetric synapses in the inner molecular layer per granule cell, which was reduced to 63% of controls 5 days after status epilepticus, recovered to 93% of controls in vehicle-treated mice 2 months after status epilepticus, but remained at only 63% of controls in rapamycin-treated mice. These findings reveal that rapamycin prevented excitatory axons from synapsing with proximal dendrites of granule cells and raise questions about the recurrent excitation hypothesis of temporal lobe epilepsy.

Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation

We have previously reported that the surface expression of K+-dependent Na+/Ca2+ exchanger 2 (NCKX2) in the somatodendritic compartment is kept low by constitutive endocytosis, which results in the polarization of surface NCKX2 to the axon. Clathrin-mediated endocytosis is initiated by interaction of the μ subunit of adaptor protein complex 2 (AP-2) with the canonical tyrosine motif (YxxΦ) of a target molecule. We examined whether endocytosis of NCKX2 involves two putative tyrosine motifs (365YGKL and 371YDTM) in the cytoplasmic loop of NCKX2. Coimmunoprecipitation assay revealed that the 365YGKL motif is essential for the interaction with the μ subunit of AP-2 (AP2M1). Consistently, either overexpression of NCKX2-Y365A mutant or knockdown of AP2M1 in cultured hippocampal neurons significantly reduced the internalization of NCKX2 from the somatodendritic surface and thus abolished the axonal polarization of surface NCKX2. Next, we tested whether the interaction between the tyrosine motif and AP2M1 is regulated by phosphorylation of the 365th tyrosine residue (Tyr-365). Tyrosine phosphorylation of heterologously expressed NCKX2-WT, but not NCKX2-Y365A, was increased by carbachol (CCh) in PC-12 cells. The effect of CCh was inhibited by PP2, a Src family kinase (SFK) inhibitor. Moreover, PP2 facilitated the endocytosis of NCKX2 in both the somatodendritic and axonal compartments, suggesting that tyrosine phosphorylation of NCKX2 by SFK negatively regulates its endocytosis. Supporting this idea, activation of SFK enhanced the NCKX activity in the proximal dendrites of dentate granule cells (GCs). These results suggest that endocytosis of somatodendritic NCKX2 is regulated by SFK-dependent phosphorylation of Tyr-365.

Synaptic connections of hilar basal dendrites of dentate granule cells in a neonatal hypoxia model of epilepsy

Numerous animal models of epileptogenesis demonstrate neuroplastic changes in the hippocampus. These changes occur not only for the mature neurons and glia, but also for the newly generated granule cells in the dentate gyrus. One of these changes, the sprouting of mossy fiber axons, is derived predominantly from newborn granule cells in adult rats with pilocarpine-induced temporal lobe epilepsy. Newborn granule cells also mainly contribute to another neuroplastic change, hilar basal dendrites (HBDs), which are synaptically targeted by mossy fibers in the hilus. Both sprouted mossy fibers and HBDs contribute to recurrent excitatory circuitry that is hypothesized to be involved in increased seizure susceptibility and the development of spontaneous recurrent seizures (SRS) that occur following the initial pilocarpine-induced status epilepticus. Considering the putative role of these neuroplastic changes in epileptogenesis, a critical question is whether similar anatomic phenomena occur after epileptogenic insults to the immature brain, where the proportion of recently born granule cells is higher due to ongoing maturation. The current study aimed to determine if such neuroplastic changes could be observed in a standardized model of neonatal seizure-inducing hypoxia that results in development of SRS. We used immunoelectron microscopy for the immature neuronal marker doublecortin to label newborn neurons and their HBDs following neonatal hypoxia. Our goal was to determine whether synapses form on HBDs from neurons born after neonatal hypoxia. Our results show a robust synapse formation on HBDs from animals that experienced neonatal hypoxia, regardless of whether the animals experienced tonic-clonic seizures during the hypoxic event. In both cases, the axon terminals that synapse onto HBDs were identified as mossy fiber terminals, based on the appearance of dense core vesicles. No such synapses were observed on HBDs from newborn granule cells obtained from sham animals analyzed at the same time points. This aberrant circuit formation may provide an anatomic substrate for increased seizure susceptibility and the development of epilepsy.

Deafferentation enhances neurogenesis in the young and middle aged hippocampus but not in the aged hippocampus

Increased neurogenesis in the dentate gyrus (DG) after brain insults such as excitotoxic lesions, seizures, or stroke is a well known phenomenon in the young hippocampus. This plasticity reflects an innate compensatory response of neural stem cells (NSCs) in the young hippocampus to preserve function or minimize damage after injury. However, injuries to the middle-aged and aged hippocampi elicit either no or dampened neurogenesis response, which could be due to an altered plasticity of NSCs and/or the hippocampus with age. We examined whether the plasticity of NSCs to increase neurogenesis in response to a milder injury such as partial deafferentation is preserved during aging. We quantified DG neurogenesis in the hippocampus of young, middle-aged, and aged F344 rats after partial deafferentation. A partial deafferentation of the left hippocampus without any apparent cell loss was induced via administration of Kainic acid (0.5 μg in 1.0 μl) into the right lateral ventricle of the brain. In this model, degeneration of CA3 pyramidal neurons and dentate hilar neurons in the right hippocampus results in loss of commissural axons which leads to partial deafferentation of the dendrites of dentate granule cells and CA1-CA3 pyramidal neurons in the left hippocampus. Quantification of newly born cells that are added to the dentate granule cell layer at postdeafferentation days 4-15 using 5'-bromodeoxyuridine (BrdU) labeling revealed greatly increased addition of newly born cells (∼three fold increase) in the deafferented young and middle-aged hippocampi but not in the deafferented aged hippocampus. Measurement of newly born neurons using doublecortin (DCX) immunostaining also revealed similar findings. Analyses using BrdU-DCX dual immunofluorescence demonstrated no changes in neuronal fate-choice decision of newly born cells after deafferentation, in comparison to the age-matched naive hippocampus in all age groups. Thus, the plasticity of hippocampal NSCs to increase DG neurogenesis in response to a milder injury such as partial hippocampal deafferentation is preserved until middle age but lost at old age.

Differential distribution of KCC2 along the axo‐somato‐dendritic axis of hippocampal principal cells

The neuron-specific potassium-chloride cotransporter 2 (KCC2) plays a crucial role in adjusting intracellular Cl(-) concentrations. The lack of KCC2 in the plasma membrane of the axon initial segment (AIS) of pyramidal cells contributes to variable reversal potentials for perisomatic γ-aminobutyric acid (GABA)(A) receptor-mediated postsynaptic potentials, but the distribution of KCC2 in pyramidal dendrites remains to be established. We applied high-resolution pre-embedding immunolocalization to quantify KCC2 concentrations along dendritic, somatic and axonal regions of rat hippocampal principal cells. Confirming our results on neocortical pyramidal cells, membranes of AIS of CA1 pyramidal cells and dentate granule cells contained 6.4 ± 11.9% and 6.6 ± 14.1% of somatic KCC2 concentrations, respectively. Concentrations of KCC2 in basal dendritic shafts of stratum (str.) oriens were similar to somatic levels (109.2 ± 48.8%). Along apical dendritic shafts of CA1 pyramidal cells, the concentration of KCC2 showed a complex profile: normalized to somatic levels, the density of KCC2 was 124.5 ± 15.7%, 79 ± 12.4% and 98.2 ± 33.5% in the proximal and distal part of str. radiatum and in str. lacunosum moleculare, respectively. Dendritic spines of CA1 receiving excitatory inputs contained 39.9 ± 8.5% of KCC2 concentration measured in shafts of the same dendritic segments targeted by GABAergic inputs. Dendrites of dentate granule cells showed higher KCC2 concentration compared with the soma (148.9 ± 54%), but no concentration gradient was detected between proximal and distal dendrites. In conclusion, the density of KCC2 in hippocampal principal cells increases along the axo-somato-dendritic axis with cell type-specific distribution profiles within the dendritic tree.

The somato-dendritic A-type K+ channel complex: a ménage à trois

The somato-dendritic subthreshold-operating A-type K+ current (ISA) plays a crucial role in neuronal excitability. It regulates spike timing and frequency, dampens the back propagation of action potentials into dendrites and has an important role in dendritic signal processing (Hoffman et al., 1997). Hence, any alteration of ISA current density or gating kinetics could then have important consequences on dendritic integration and plasticity. Kv4 channel protein complexes represent the molecular correlate of the ISA current (Birnbaum et al., 2004; Maffie and Rudy, 2008). Studies in heterologous expression systems have shown that Kv4 pore-forming α subunits interact with two different auxiliary subunits: Kv-channel-interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins DPP6 and DPP10 (DPPX). Both auxiliary subunits affect channel gating and trafficking and their co-expression with Kv4 α subunits recapitulates the kinetics and gating properties of native ISA. The discovery of Nadal et al. (2003) showing that DPP6 proteins can be co-purified with Kv4.2 K+ channel proteins from rat cerebellum and suggesting that they might function as auxiliary subunits of the channel complex, opened new interesting perspectives for the role of this structurally-related dipeptidyl aminopeptidase. Indeed, recent studies showed that mutations in the DPP6 gene are associated with human neurological diseases, including amyotrophic lateral sclerosis (ALS), autism and progressive spinal muscular atrophy. DDP6 is a type II transmembrane protein, highly expressed in the nervous system and belonging to the prolyl oligopeptidase family of serine proteases. It consists of a large extracellular C-terminal domain, a single membrane-spanning domain and a short alternatively spliced intracellular N-terminus. DPP6 has a dimeric structure and has lost a catalytic serine residue, suggesting that it is unlikely to function as a dipeptidyl aminopeptidase (Strop et al., 2004). Though biochemical and functional data strongly suggest that DPP6 is an integral component of the Kv4.2 K+ channel complex and functions as an auxiliary subunit, the lack of good quality antibodies prevented high-resolution localization studies in native brain tissue. In the recent issue of Frontiers, Clark et al. (2008) provide a strong argument supporting the view that DPP6 is an integral part of native Kv4.2 channel complexes (ISA) in the brain. They raised antibodies against the whole extracellular domain of the protein, which was recombinantly expressed in baculovirus-infected Sf9 cells. Using this specific tool, they investigated DPP6 distribution in mouse brain and compared it with the distribution of Kv4 proteins (Clark et al., 2008). The three regions most prominently co-labeled with DPP6 and Kv4.2 antibodies were the striatum, the hippocampus and the cerebellar cortex. In the granule cells of the cerebellum, both Kv4.2 and DPP6 proteins were heavily stained in dendrites with a similar anterior–posterior gradient of expression. In line with the high density of ISA current found in dendrites of hippocampal pyramidal CA1 neurons (Hoffman et al., 1997), the dendrites of CA1–CA3 pyramidal cells were also prominently immunostained for both DPP6 and Kv4.2. In the molecular layer of the dentate gyrus, Kv4.2 and DPP6 staining co-localize in the dendrites of dentate granule cells. Thus, this study found remarkable similarities in the regional and cellular patterns of expression of Kv4.2 and DPP6 proteins. Similarities were also found in the dendritic subcellular pattern of expression of both proteins, notably in the CA1–CA3 hippocampal field and in the granule cells of the cerebellum. Such findings strengthen the notion that DPP6 is an integral part of the Kv4 channel complex that underlies the somato-dendritic ISA current in neurons. However, this study further provides intriguing findings showing that one brain structure, the hippocampal mossy fibers, which contain the axons of the granule cells of the dentate gyrus, do not share this pattern of Kv4 and DPP6 co-expression. The hippocampal mossy fiber axons lack Kv4 proteins but are heavily labeled by DPP6 antibodies, particularly in the terminal fields in the stratum lucidum of the CA3 region, thereby suggesting that DPP6 proteins may have additional, Kv4-unrelated functions. Altogether, these recent studies on DPP6(X) proteins raise important issues. Most likely, the Kv4 channel complex contains two dimers of DPP6 proteins. If so, does DPP6 assemble as homodimer or heterodimer and how the KChIP subunits are engaged into the Kv4 channel complex? Are KChIP and DPP6 exerting their effects on Kv4 channel surface expression via different mechanisms? The absence of Kv4 α subunits and the strong expression of DPP6 in the hippocampal mossy fibers indicate that DPP6(X) proteins may have presynaptic functions. It will be important to investigate the possible role of DPP6 in transmitter release as well as in motor neuron function and excitotoxicity, regarded as one of the main processes involved in the pathogenesis of ALS and progressive muscular spinal atrophy. Addressing these challenging issues will highlight the importance of DPP6 in Kv4-related and – unrelated functions.

Altered Localization of GABAAReceptor Subunits on Dentate Granule Cell Dendrites Influences Tonic and Phasic Inhibition in a Mouse Model of Epilepsy

Complex changes in GABA(A) receptors (GABA(A)Rs) in animal models of temporal lobe epilepsy during the chronic period include a decrease in the delta subunit and increases in the alpha4 and gamma2 subunits in the dentate gyrus. We used postembedding immunogold labeling to determine whether the subcellular locations of these subunits were also altered in pilocarpine-treated epileptic mice, and related functional changes were identified electrophysiologically. The ultrastructural studies confirmed a decrease in delta subunit labeling at perisynaptic locations in the molecular layer of the dentate gyrus where these subunits are critical for tonic inhibition. Unexpectedly, tonic inhibition in dentate granule cells was maintained in the epileptic mice, suggesting compensation by other GABA(A)Rs. An insensitivity of the tonic current to the neurosteroid tetrahydrodeoxy-corticosterone was consistent with decreased expression of the delta subunit. In the pilocarpine-treated mice, alpha4 subunit labeling remained at perisynaptic locations, but increased gamma2 subunit labeling was also found at many perisynaptic locations on granule cell dendrites, consistent with a shift of the gamma2 subunit from synaptic to perisynaptic locations and potential partnership of the alpha4 and gamma2 subunits in the epileptic animals. The decreased gamma2 labeling near the center of synaptic contacts was paralleled by a corresponding decrease in the dendritic phasic inhibition of granule cells in the pilocarpine-treated mice. These GABA(A)R subunit changes appear to impair both tonic and phasic inhibition, particularly at granule cell dendrites, and could reduce the adaptive responses of the GABA system in temporal lobe epilepsy.

CAMP Differentially Regulates Axonal and Dendritic Development of Dentate Granule Cells

Neurite polarity is a morphological characteristic of dentate gyrus granule cells, which extend axons to the hilar region and dendrites in the opposite direction, i.e. to the molecular layer. This remarkable polarity must require a differential system for axon and dendrite guidance. Here, we report that the axon and dendrites of a granule cell are differentially responsive to cAMP. In developing cultures of dispersed granule cells, dendritic growth cones were increased in number after pharmacological activation of cAMP signaling and decreased after blockade of cAMP signaling. Activation of cAMP signaling antagonized dendritic collapse induced by the potent repellents Sema3F and glutamate. In contrast to dendrites, axons were protected from Sema3F-induced collapse when cAMP signaling was inhibited. Axonal and dendritic growth cones both expressed type 1 adenylyl cyclase, but only axons showed a cAMP increase in response to Sema3F, and the elevated cAMP was sufficient to collapse axonal growth cones. Thus, the axons and dendrites of dentate granule cells differ in the regulation of cAMP levels as well as responsiveness to cAMP. cAMP may be crucial for shaping the information flow polarity in the dentate gyrus circuit.

Voluntary exercise alters the cytoarchitecture of the adult dentate gyrus by increasing cellular proliferation, dendritic complexity, and spine density

Voluntary exercise produces a dramatic increase in the number of bromodeoxyuridine (BrdU)-positive cells in the adult dentate gyrus (DG); however, it has never been determined whether this increase reflects neurogenic activity or some exercise-induced change in the metabolic processing of systemically injected BrdU. In these experiments, we show that 1) 200 mg/kg is a saturating dose for single injections of BrdU in both control and voluntary exercise animals; 2) there is significantly more cell labeling in animals that exercise when saturating doses of BrdU are employed; 3) high doses of BrdU do not affect the number, appearance, or distribution of labeled cells; 4) voluntary exercise leads to similar increases in the number of cells expressing Ki67, an intrinsic marker of cellular proliferation; 5) both dendritic length and complexity are significantly increased in the DG of animals that exercise; and 6) spine density is significantly greater on dendrites in the DG following voluntary exercise. This study demonstrates that exercise up-regulates neurogenic activity in the DG of adult rats, independently of any putative changes in altered BrdU metabolism, and that it also substantially alters the morphology of dentate granule cell dendrites. The dramatic changes in the cytoarchitecture of the DG induced by voluntary exercise might underlie the enhancement of hippocampal long-term potentiation and hippocampal-dependent memory that our group has previously described. These results suggest that exercise may be an effective component of therapeutic regimes aimed at improving the functioning of individuals with neuropathologies that involve the degradation of cells in the hippocampus.

Identification and characterization of a new Down syndrome model, Ts[Rb(12.1716)]2Cje, resulting from a spontaneous Robertsonian fusion between T(1716)65Dn and mouseChromosome 12

The segmental trisomy model, Ts65Dn, has been a valuable resource for the study of the molecular and developmental processes associated with the pathogenesis of Down syndrome. However, male infertility and poor transmission of the small marker chromosome, T(17(16))65Dn, carrying the distal end of mouse Chromosome 16 (MMU16) are limiting factors in the efficient production of these animals for experimental purposes. We describe here the identification and preliminary characterization of mice, designated Ts[Rb(12.17(16))]2Cje, carrying a chromosomal rearrangement of the Ts65Dn genome whereby the marker chromosome has been translocated to Chromosome 12 (MMU12) forming a Robertsonian chromosome. This stable rearrangement confers fertility in males and increases the frequency of transmitted segmental trisomy through the female germline. We confirm retention of a dosage imbalance of human Chromosome 21 (HSA21)-homologous genes from App to the telomere and expression levels similar to Ts65Dn within the triplicated region. In addition, we characterized the dendritic morphology of granule cells in the fascia dentata in Ts[Rb(12.17(16))2Cje and 2N control mice. Quantitative confocal microscopy revealed decreased spine density on the dendrites of dentate granule cells and significantly enlarged dendritic spines affecting the entire population in Ts[Rb(12.17(16))]2Cje as compared to 2N controls. These findings document that the structural dendritic spine abnormalities are similar to those previously observed in Ts65Dn mice. We conclude that this new model of Down syndrome offers reproductive advantages without sacrificing the integrity of the Ts65Dn model.

Synaptic structural abnormalities in the Ts65Dn mouse model of down syndrome

The Ts65Dn mouse is a genetic model for Down syndrome. Although this mouse shows abnormalities in cognitive function that implicate hippocampus as well as marked deficits in hippocampal long-term potentiation, the structure of the hippocampus has been little studied. We characterized synaptic structure in Ts65Dn and control (2N) mice, studying the hippocampus (fascia dentata, CA1) as well as the motor and somatosensory cortex, entorhinal cortex, and medial septum. Confocal microscopy was used to examine immunostained presynaptic boutons and to detail the structure of dendrites after Lucifer yellow microinjection. Both presynaptic and postsynaptic elements were significantly enlarged in Ts65Dn in all regions examined. The changes were detected at the youngest age examined (postnatal day 21) and in adults. In studies detailing the changes in fascia dentata and motor cortex, the enlargement of spines affected the entire population, resulting in the presence of spines whose volume was greatly increased. Electron microscopy confirmed that boutons and spines were enlarged and demonstrated abnormalities in the internal membranes of both. In addition, spine density was decreased on the dendrites of dentate granule cells, and there was reorganization of inhibitory inputs, with a relative decrease in inputs to dendrite shafts and an increase in inputs to the necks of spines. Taken together, the findings document widespread abnormalities of synaptic structure that recapitulate important features seen in Down syndrome. They establish the Ts65Dn mouse as a model for abnormal synapse structure and function in Down syndrome and point to the importance of studies to elucidate the mechanisms responsible for synapse enlargement.

Molecular mechanisms contributing to long-lasting synaptic plasticity at the temporoammonic-CA1 synapse.

The hippocampus and the nearby medial temporal lobe structures are required for the formation, consolidation, and retrieval of episodic memories. Sensory information enters the hippocampus via two inputs from entorhinal cortex (EC): One input (perforant path) makes synapses on the dendrites of dentate granule cells as the first set of synapses in the trisynaptic circuit, the other (temporoammonic; TA) makes synapses on the distal dendrites of CA1 neurons. Here we demonstrate that TA-CA1 synapses undergo both early- and late-phase long-term potentiation (LTP) in rat hippocampal slices. LTP at TA-CA1 synapses requires both NMDA receptor and voltage-gated Ca2+ channel activity. Furthermore, TA-CA1 LTP is insensitive to the blockade of fast inhibitory transmission (GABAA-mediated) and, interestingly, is dependent on GABAB-dependent slow inhibitory transmission. These findings indicate that the TA-CA1 synapses may rely on a refined modulation of inhibition to exhibit LTP.

Transient downregulation of sema3a mrna in a rat model for temporal lobe epilepsy: A novel molecular event potentially contributing to mossy fiber sprouting

Mossy fiber sprouting (MFS) in the hippocampal dentate gyrus is thought to play a critical role in the hyperexcitability of the hippocampus in temporal lobe epilepsy patients. The composition of molecular signals that is needed to direct this sprouting response has not yet been elucidated to a great extent. In the present study we investigated the expression profile of Sema3A mRNA and the axonal growth-associated protein GAP-43 mRNA during the process of electrically induced epileptogenesis in rats. Sema3A is an axon guidance molecule with repellent activity on dentate granule cell axons. It is produced by neurons in the entorhinal cortex, which synapse on the dendrites of dentate granule cells. Upregulation of GAP-43 expression in granule cells has often been reported in conjunction with MFS. After induction of status epilepticus, the expression of Sema3A mRNA was temporarily downregulated in the entorhinal cortex concomitantly with an upregulation of GAP-43 mRNA in dentate granule cells. In the following days, robust MFS into the dentate molecular layer was observed. When the induction of status epilepticus was incomplete the two responses appeared to dissociate, i.e., the downregulation of Sema3A mRNA did not occur, while upregulation of GAP-43 mRNA in dentate granule cells was still displayed. However, in these rats no significant MFS was observed. These findings indicate that Sema3A mRNA downregulation is temporarily correlated with MFS, while GAP-43 upregulation per se is not, and suggest that a loss of Sema3A in the molecular layer of the dentate gyrus could facilitate MFS into this area during epilepsy.

Plasticity in Hippocampal Peptidergic Systems Induced by Repeated Electroconvulsive Shock

The regulated secretion of bioactive peptides requires the coordinated actions of a variety of gene products ranging from peptide precursors to post-translational processing enzymes and the cytosolic machinery involved in vesicle exocytosis. To evaluate the role of plasticity of peptidergic processes in the clinical response to electroconvulsive treatment, we monitored expression of a peptide (neuropeptide Y, NPY), a post-translational processing enzyme (peptidylglycine α-amidating monooxygenase, PAM) and a cytosolic component involved in peptide secretion and neurite extension (kalirin) in the hippocampus. Adult male rats were subjected to single or repeated electroconvulsive shock. In general, levels of NPY, PAM and kalirin mRNA showed similar transient increases after acute and repeated electroconvulsive shock. In contrast, repeated, but not acute, electroconvulsive shock brought about widespread changes in protein expression. Increased amounts of NPY and PAM accumulated in mossy fibers, and dentate granule cell dendrites contained increased amounts of NPY, PAM and kalirin. CA1 pyramidal neurons expressed increased amounts of PAM and kalirin, with an accumulation of both proteins in their dendrites. Scattered interneurons contained increased levels of NPY and PAM after acute and repeated shocks. However, scattered interneurons contained increased levels of kalirin only after repeated shocks. The distinctly different effects of repeated vs. acute electroconvulsive shock support an important role for peptidergic plasticity in the therapeutic effects observed following electroconvulsive treatment.

Lamina-Specific Synaptic Activation Causes Domain-Specific Alterations in Dendritic Immunostaining for MAP2 and CAM Kinase II

Polyribosomal complexes are selectively localized beneath postsynaptic sites on neuronal dendrites; this localization suggests that the translation of the mRNAs that are present in dendrites may be regulated by synaptic activity. The present study tests this hypothesis by evaluating whether synaptic activation alters the immunostaining pattern for two proteins whose mRNAs are present in dendrites: the dendrite-specific cytoskeletal protein MAP2 and the alpha-subunit of CAMKII. High-frequency stimulation of the perforant path projections to the dentate gyrus, which terminate in a discrete band on the dendrites of dentate granule cells, produced a two-stage alteration in immunostaining for MAP2 in the dendritic laminae. Five minutes of stimulation (30 trains) caused a decrease in MAP2 immunostaining in the lamina in which the activated synapses terminate. After more prolonged periods of stimulation (1-2 hr), there was an increase in immunostaining in the sideband laminae just proximal and distal to the activated band of synapses. The same stimulation paradigm produced a modest increase in immunostaining for alpha-CAMKII in the activated laminae, with no detectable changes in the sideband laminae. The alterations in immunostaining for MAP2 were diminished, but not eliminated, by inhibiting protein synthesis; the increases in CAMKII were not. These findings reveal that patterned synaptic activity can produce domain-specific alterations in the molecular composition of dendrites; these alterations may be caused in part by local protein synthesis and in part by other mechanisms.

Occurrence of dentate granule cell LTP without proximal dendritic Ca2+ increase.

We investigated activity-dependent calcium increases in proximal dendrites of dentate granule cells in the rat hippocampus, and its relationship with induction of LTP at perforant path synapses (PP-synapses). LTP was induced at PP-synapses by high-frequency stimulation (HFS; 100 Hz for 0.4 s), and the same HFS evoked a dendritic calcium increase in the proximal dendrite. However, bath-application of the L-type voltage-dependent calcium channel (VDCC) blocker nimodipine noticeably reduced this calcium increase without abolishing induction of LTP. This calcium increase mediated by high-threshold VDCCs is likely to be evoked by action potentials. LTP induction at PP-synapses is hence suggested to be independent of action potential-induced calcium increases in the proximal dendrite.

Status epilepticus in epileptogenesis.

There has been direct evidence of gamma-aminobutyric acidA receptor modification during status epilepticus. Neuropeptides galanin and neuropeptide Y were demonstrated to play a role in terminating status epilepticus. Many of the CA3 pyramidal neurons destined to die as a consequence of status epilepticus were demonstrated to diminish expression of the GluR2 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors. It was demonstrated that the pattern of cell loss due to status epilepticus is distinct in immature pups compared with adult rats. The genetic basis for susceptibility to neuronal loss during status epilepticus was described. There was increasing evidence of unique receptors and ion channels in the epileptic brain. The molecular studies of epileptic gamma-aminobutyric acidA receptors present on dentate granule cells of rats with temporal lobe epilepsy revealed altered gene and receptor expression before onset of recurrent spontaneous seizures. They also revealed insertion of new gamma-aminobutyric acidA receptors in the inhibitory synapses present on soma and proximal dendrites of dentate granule cells.

Glutamate receptor subunits GluR1 and GluR2/3 distribution shows reorganization in the human epileptogenic hippocampus.

The AMPA-type glutamate receptor subunits GluR1 and GluR2/3 were localized by immunohistochemistry with subunit-specific antibodies in hippocampi removed surgically from patients with temporal lobe epilepsy for the control of seizures. The flip and flop splice variants of the subunits were localized by in situ hybridization histochemistry with specific oligoprobes. In patient hippocampi that were not the seizure focus, the GluR1 subunit proteins were diffusely expressed on the dendrites of neurons in all regions. In contrast, in these same hippocampi, the GluR2/3 subunit proteins were expressed strongly on the soma and proximal dendrites of principal neurons in all regions. The flip variant of these subunits was localized in the hilus and fields of Ammon's Horn (CA), while the flop variants were prominent on the dentate granule cells. In the epileptogenic hippocampus, while immunoreactivity was decreased in all fields that showed neuronal loss, there was an increased expression of GluR1 on the dendritic excrescences on the proximal dendrites of hilar neurons and CA3 pyramidal neurons, as well as expression of GluR2/3 in hilar neuron excrescences. Electron microscopic examination confirmed that the GluR1 immunoreactivity was only in dendritic processes, particularly dense at the postsynaptic membranes. Such expression of GluR1 may provide for an enhanced glutamatergic response by these neurons. GluR2/3 was also significantly increased on the dendrites of dentate granule cells in the epileptogenic hippocampus and may provide some protection against excitotoxic injury by reducing calcium flux into neurons.