Granule Cell Somata Research Articles

Pcdh11x controls target specification of mossy fiber sprouting.

Circuit formation is a defining characteristic of the developing brain. However, multiple lines of evidence suggest that circuit formation can also take place in adults, the mechanisms of which remain poorly understood. Here, we investigated the epilepsy-associated mossy fiber (MF) sprouting in the adult hippocampus and asked which cell surface molecules define its target specificity. Using single-cell RNAseq data, we found lack and expression of Pcdh11x in non-sprouting and sprouting neurons respectively. Subsequently, we used CRISPR/Cas9 genome editing to disrupt the Pcdh11x gene and characterized its consequences on sprouting. Although MF sprouting still developed, its target specificity was altered. New synapses were frequently formed on granule cell somata in addition to dendrites. Our findings shed light onto a key molecular determinant of target specificity in MF sprouting and contribute to understanding the molecular mechanism of adult brain rewiring.

Targeting of ribosomal protein S6 to dendritic spines by in vivo high frequency stimulation to induce long-term potentiation in the dentate gyrus.

ABSTRACTLate phase long-term potentiation (L-LTP) in the hippocampus is believed to be the cellular basis of long-term memory. Protein synthesis is required for persistent forms of synaptic plasticity, including L-LTP. Neural activity is thought to enhance local protein synthesis in dendrites, and one of the mechanisms required to induce or maintain the long-lasting synaptic plasticity is protein translation in the dendrites. One regulator of translational processes is ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. Although polyribosomes containing rpS6 are observed in dendritic spines, it remains unclear whether L-LTP induction triggers selective targeting of the translational machinery to activated synapses in vivo. Therefore, we investigated synaptic targeting of the translational machinery by observing rpS6 immunoreactivity during high frequency stimulation (HFS) for L-LTP induction in vivo. Immunoelectron microscopic analysis revealed a selective but transient increase in rpS6 immunoreactivity occurring as early as 15 min after the onset of HFS in dendritic spine heads at synaptic sites receiving HFS. Concurrently, levels of the rpS6 protein rapidly declined in somata of granule cells, as determined using immunofluorescence microscopy. These results suggest that the translational machinery is rapidly targeted to activated spines and that this targeting mechanism may contribute to the establishment of L-LTP.

Dendritic patch-clamp recordings from cerebellar granule cells demonstrate electrotonic compactness.

Cerebellar granule cells (GCs), the smallest neurons in the brain, have on average four short dendrites that receive high-frequency mossy fiber inputs conveying sensory information. The short length of the dendrites suggests that GCs are electrotonically compact allowing unfiltered integration of dendritic inputs. The small average diameter of the dendrites (~0.7 µm), however, argues for dendritic filtering. Previous studies based on somatic recordings and modeling indicated that GCs are electrotonically extremely compact. Here, we performed patch-clamp recordings from GC dendrites in acute brain slices of mice to directly analyze the electrotonic properties of GCs. Strikingly, the input resistance did not differ significantly between dendrites and somata of GCs. Furthermore, spontaneous excitatory postsynaptic potentials (EPSP) were similar in amplitude at dendritic and somatic recording sites. From the dendritic and somatic input resistances we determined parameters characterizing the electrotonic compactness of GCs. These data directly demonstrate that cerebellar GCs are electrotonically compact and thus ideally suited for efficient high-frequency information transfer.

KV 10.1 opposes activity-dependent increase in Ca²⁺ influx into the presynaptic terminal of the parallel fibre-Purkinje cell synapse.

Voltage-gated KV 10.1 potassium channels are widely expressed in the mammalian brain but their function remains poorly understood. We report that KV 10.1 is enriched in the presynaptic terminals and does not take part in somatic action potentials. In parallel fibre synapses in the cerebellar cortex, we find that KV 10.1 regulates Ca(2+) influx and neurotransmitter release during repetitive high-frequency activity. Our results describe the physiological role of mammalian KV 10.1 for the first time and help understand the fine-tuning of synaptic transmission. The voltage-gated potassium channel KV 10.1 (Eag1) is widely expressed in the mammalian brain, but its physiological function is not yet understood. Previous studies revealed highest expression levels in hippocampus and cerebellum and suggested a synaptic localization of the channel. The distinct activation kinetics of KV 10.1 indicate a role during repetitive activity of the cell. Here, we confirm the synaptic localization of KV 10.1 both biochemically and functionally and that the channel is sufficiently fast at physiological temperature to take part in repolarization of the action potential (AP). We studied the role of the channel in cerebellar physiology using patch clamp and two-photon Ca(2+) imaging in KV 10.1-deficient and wild-type mice. The excitability and action potential waveform recorded at granule cell somata was unchanged, while Ca(2+) influx into axonal boutons was enhanced in mutants in response to stimulation with three APs, but not after a single AP. Furthermore, mutants exhibited a frequency-dependent increase in facilitation at the parallel fibre-Purkinje cell synapse at high firing rates. We propose that KV 10.1 acts as a modulator of local AP shape specifically during high-frequency burst firing when other potassium channels suffer cumulative inactivation.

Inhibition and interneuron distribution in the dentate gyrus of p35 knockout mice

The p35 knockout (p35-/-) mouse is an animal model of temporal lobe epilepsy that recapitulates key neuroanatomic abnormalities-granule cell dispersion and mossy fiber sprouting-observed in the hippocampal formation of humans, as well as spontaneous seizure activity. It is a useful model in which to study the relationship between the abnormal neuronal structure and seizure activity to further our understanding of cortical dysplasia in epileptogenesis. Our previous work using this mouse model characterized the anatomic features of the dentate granule cells and the functional implications of these abnormalities on increased recurrent excitation. These data also suggested that there might be compromised inhibition in this animal model. We pursued this possibility, focusing our investigation on inhibitory circuitry. In preliminary investigations using neuroanatomic tools (immunocytochemistry, camera lucida reconstructions of individually labeled interneurons, and electron microscopy) combined with intracellular electrophysiology, we observed no significant reduction in the number of symmetric versus asymmetric synaptic contacts on dentate granule cell somata, and no statistically significant changes in evoked early or late inhibition. Although there were some abnormalities in the morphology/distribution of inhibitory interneurons (as well as a larger population of dentate granule cells) of the dentate gyrus, overall inhibition in the p35 knockout mouse appeared to be largely intact.

Rescue of neuronal migration deficits in a mouse model of fetal Minamata disease by increasing neuronal Ca2+spike frequency

In the brains of patients with fetal Minamata disease (FMD), which is caused by exposure to methylmercury (MeHg) during development, many neurons are hypoplastic, ectopic, and disoriented, indicating disrupted migration, maturation, and growth. MeHg affects a myriad of signaling molecules, but little is known about which signals are primary targets for MeHg-induced deficits in neuronal development. In this study, using a mouse model of FMD, we examined how MeHg affects the migration of cerebellar granule cells during early postnatal development. The cerebellum is one of the most susceptible brain regions to MeHg exposure, and profound loss of cerebellar granule cells is detected in the brains of patients with FMD. We show that MeHg inhibits granule cell migration by reducing the frequency of somal Ca(2+) spikes through alterations in Ca(2+), cAMP, and insulin-like growth factor 1 (IGF1) signaling. First, MeHg slows the speed of granule cell migration in a dose-dependent manner, independent of the mode of migration. Second, MeHg reduces the frequency of spontaneous Ca(2+) spikes in granule cell somata in a dose-dependent manner. Third, a unique in vivo live-imaging system for cell migration reveals that reducing the inhibitory effects of MeHg on somal Ca(2+) spike frequency by stimulating internal Ca(2+) release and Ca(2+) influxes, inhibiting cAMP activity, or activating IGF1 receptors ameliorates the inhibitory effects of MeHg on granule cell migration. These results suggest that alteration of Ca(2+) spike frequency and Ca(2+), cAMP, and IGF1 signaling could be potential therapeutic targets for infants with MeHg intoxication.

Rapid reversal of impaired inhibitory and excitatory transmission but not spine dysgenesis in a mouse model of mental retardation

Intellectual disability affects 2-3% of the population: those due to mutations of the X-chromosome are a major cause of moderate to severe cases (1.8/1000 males). Established theories ascribe the cellular aetiology of intellectual disability to malformations of dendritic spines. Recent work has identified changes in synaptic physiology in some experimental models. Here, we investigated the pathophysiology of a mouse model of intellectual disability using electrophysiological recordings combined with confocal imaging of dentate gyrus granule neurons. Lack of oligophrenin-1 resulted in reductions in dendritic tree complexity and mature dendritic spine density and in evoked and spontaneous EPSCs and IPSCs. In the case of inhibitory transmission, the physiological change was associated with a reduction in the readily releasable pool and vesicle recycling which impaired the efficiency of inhibitory synaptic transmission. Acute inhibition of the downstream signalling pathway of oligophrenin-1 fully reversed the functional changes in synaptic transmission but not the dendritic abnormalities. The impaired inhibitory (as well as excitatory) synaptic transmission at frequencies associated with cognitive function suggests a cellular mechanism for the intellectual disability, because cortical oscillations associated with cognition normally depend on inhibitory neurons firing on every cycle.

Clostridium perfringens epsilon toxin targets granule cells in the mouse cerebellum and stimulates glutamate release.

Epsilon toxin (ET) produced by C. perfringens types B and D is a highly potent pore-forming toxin. ET-intoxicated animals express severe neurological disorders that are thought to result from the formation of vasogenic brain edemas and indirect neuronal excitotoxicity. The cerebellum is a predilection site for ET damage. ET has been proposed to bind to glial cells such as astrocytes and oligodendrocytes. However, the possibility that ET binds and attacks the neurons remains an open question. Using specific anti-ET mouse polyclonal antibodies and mouse brain slices preincubated with ET, we found that several brain structures were labeled, the cerebellum being a prominent one. In cerebellar slices, we analyzed the co-staining of ET with specific cell markers, and found that ET binds to the cell body of granule cells, oligodendrocytes, but not astrocytes or nerve endings. Identification of granule cells as neuronal ET targets was confirmed by the observation that ET induced intracellular Ca2+ rises and glutamate release in primary cultures of granule cells. In cultured cerebellar slices, whole cell patch-clamp recordings of synaptic currents in Purkinje cells revealed that ET greatly stimulates both spontaneous excitatory and inhibitory activities. However, pharmacological dissection of these effects indicated that they were only a result of an increased granule cell firing activity and did not involve a direct action of the toxin on glutamatergic nerve terminals or inhibitory interneurons. Patch-clamp recordings of granule cell somata showed that ET causes a decrease in neuronal membrane resistance associated with pore-opening and depolarization of the neuronal membrane, which subsequently lead to the firing of the neuronal network and stimulation of glutamate release. This work demonstrates that a subset of neurons can be directly targeted by ET, suggesting that part of ET-induced neuronal damage observed in neuronal tissue is due to a direct effect of ET on neurons.

Stable Mossy Fiber Long-Term Potentiation Requires Calcium Influx at the Granule Cell Soma, Protein Synthesis, and Microtubule-Dependent Axonal Transport

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 Ca(2+) channels prevents MF-LTP, supporting an involvement of presynaptic Ca(2+) signaling via voltage-gated Ca(2+) 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 Ca(2+) influx via voltage-gated Ca(2+) 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 Ca(2+) influx at the MF terminals-induction of MF synaptic plasticity requires action potential-dependent Ca(2+) 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 Ca(2+) channel activity.

Improved Reversal Learning and Working Memory and Enhanced Reactivity to Novelty in Mice with Enhanced GABAergic Innervation in the Dentate Gyrus

The balance between excitation and inhibition controls fundamental aspects of the hippocampal function. Here, we report an increase in the ratio of inhibitory to excitatory neurons in the dentate gyrus, accompanied by γ-aminobutyric acid(A) (GABA(A)) receptor-dependent impairment of synaptic plasticity and enhancement of activity-dependent changes in excitability in anesthetized adult mice deficient for the extracellular matrix glycoprotein tenascin-R (TNR). TNR-deficient mice showed faster reversal learning, improved working memory, and enhanced reactivity to novelty than wild-type littermates. Remarkably, in wild-type and TNR-deficient mice, faster reversal learning rates correlated at the individual animal level with ratios of parvalbumin-positive interneurons to granule cells and densities of parvalbumin-positive terminals on somata of granule cells. Our data demonstrate that modification of the extracellular matrix by ablation of TNR leads to a new structural and functional design of the dentate gyrus, with enhanced GABAergic innervation, that is, enhanced ratio of inhibitory to excitatory cells, and altered plasticity, promoting working memory and reversal learning. In wild-type mice, the enhanced ratio of inhibitory to excitatory cells in the dentate gyrus also positively correlated with reversal learning, indicating that level of inhibition regulates specific aspects of learning independent of the TNR gene.

Differential regulation of mature and precursor microRNA expression by NMDA and metabotropic glutamate receptor activation during LTP in the adult dentate gyrus in vivo.

Regulation of microRNA (miRNA) expression and function in the context of activity-dependent synaptic plasticity in the adult brain is little understood. Here, we examined miRNA expression during long-term potentiation (LTP) in the dentate gyrus of adult anesthetized rats. Microarray expression profiling identified a subpopulation of regulated mature miRNAs 2 h after the induction of LTP by high-frequency stimulation (HFS) of the medial perforant pathway. Real-time polymerase chain reaction analysis confirmed modest upregulation of miR-132 and miR-212, and downregulation of miR-219, while no changes occurred at 10 min post-HFS. Surprisingly, pharmacological blockade of N-methyl-d-aspartate receptor (NMDAR)-dependent LTP enhanced expression of these mature miRNAs. This HFS-evoked expression was abolished by local infusion of the group 1 metabotropic glutamate receptor (mGluR) antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). AIDA had no effect on LTP induction or maintenance, but blocked activity-dependent depotentiation of LTP. Turning to the analysis of miRNA precursors, we show that HFS elicits 50-fold elevations of primary (pri) and precursor (pre) miR-132/212 that is transcription dependent and mGluR dependent, but insensitive to NMDAR blockade. Primary miR-219 expression was unchanged during LTP. In situ hybridization showed upregulation of the pri-miR-132/212 cluster restricted to dentate granule cell somata. Thus, HFS induces transcription miR-132/212 that is mGluR dependent and functionally correlated with depotentiation rather than LTP. In contrast, NMDAR activation selectively downregulates mature miR-132, -212 and -219 levels, indicating accelerated decay of these mature miRNAs. This study demonstrates differential regulation of primary and mature miRNA expression by mGluR and NMDAR signaling following LTP induction, the function of which remains to be defined.

Characterization of somatic Ca 2+ clearance mechanisms in young and mature hippocampal granule cells

Calcium is a key regulator for expression of genes relevant to survival and maturation of newborn neurons. Mammalian hippocampal dentate gyrus generates new granule cells (GCs) throughout adult life. We identified young and mature GCs in hippocampi of young adult mice according to their electrical properties, and investigated contributions of Na/Ca exchanger (NCX), sarco-endoplasmic reticulum Ca 2+-ATPase (SERCA), plasma membrane Ca 2+-ATPase (PMCA) and mitochondria to Ca 2+ clearance in somata of GCs. Somatic Ca 2+ clearance was increased by about 50% as GCs matured. NCX activity increased proportionally during maturation with its relative contribution kept about 40% both in young and mature GCs. On the other hand, the developmental increases in activities of mitochondria and SERCA resulted in higher contributions to Ca 2+ clearance in mature GCs than in young GCs. Especially mitochondrial function was most highly enhanced during maturation. PMCA activity, however, did not increase during maturation. Low Ca 2+ clearance in immature GCs might facilitate higher Ca 2+ accumulation during network activity, which in turn help survival of young GCs.

P2Y receptor mediated inhibitory modulation of noradrenaline release in response to electrical field stimulation and ischemic conditions in superfused rat hippocampus slices

In this study, the inhibitory regulation of the release of noradrenaline (NA) by P2 receptors was investigated in hippocampus slices pre-incubated with [(3)H]NA. Electrical field stimulation (EFS; 2 Hz, 240 shocks, and 1 ms) released NA in an outside [Ca(2+)]-dependent manner, and agonists of P2Y receptors inhibited the EFS-evoked [(3)H]NA release with pharmacological profile similar to that of the P2Y(1) and P2Y(13) receptor subtypes. This inhibitory modulation was counteracted by bicuculline and 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline + 2-amino-5-phosphonovalerate and 2-amino-4-phosphonobutyrate. In contrast, the excess release in response to 30 min combined oxygen and glucose deprivation was outside [Ca(2+)] independent, but still sensitive to the inhibition of both facilitatory P2X(1) and inhibitory P2Y(1) receptors. Whereas mRNA encoding P2Y(12) and P2Y(13) receptor subunits were expressed in the brainstem, P2Y(1) receptor immunoreactivity was localized to neuronal somata and dendrites innervated by the mossy fiber terminals in the CA3 region of the hippocampus, as well as somata of granule cells and interneurons in the dentate gyrus. In summary, in addition to the known facilitatory modulation via P2X receptors, EFS-evoked [(3)H]NA outflow in the hippocampus is subject to inhibitory modulation by P2Y(1)/P2Y(13) receptors. Furthermore, endogenous activation of both facilitatory and inhibitory P2 receptors may participate in the modulation of pathological NA release under ischemic-like conditions.

Nanodomain Coupling between Ca2+ Channels and Ca2+ Sensors Promotes Fast and Efficient Transmitter Release at a Cortical GABAergic Synapse

It is generally thought that transmitter release at mammalian central synapses is triggered by Ca2+ microdomains, implying loose coupling between presynaptic Ca2+ channels and Ca2+ sensors of exocytosis. Here we show that Ca2+ channel subunit immunoreactivity is highly concentrated in the active zone of GABAergic presynaptic terminals of putative parvalbumin-containing basket cells in the hippocampus. Paired recording combined with presynaptic patch pipette perfusion revealed that GABA release at basket cell-granule cell synapses is sensitive to millimolar concentrations of the fast Ca2+ chelator BAPTA but insensitive to the slow Ca2+ chelator EGTA. These results show that Ca2+ source and Ca2+ sensor are tightly coupled at this synapse, with distances in the range of 10-20 nm. Models of Ca2+ inflow-exocytosis coupling further reveal that the tightness of coupling increases efficacy, speed, and temporal precision of transmitter release. Thus, tight coupling contributes to fast feedforward and feedback inhibition in the hippocampal network.

Comparison of Changes in GAD65 and GAD67 Immunoreactivity and Levels in the Gerbil Main Olfactory Bulb Induced by Transient Ischemia

In the present study, we investigated changes in glutamate decarboxylase 65 (GAD65) and GAD67 immunoreactivity and protein levels in the main olfactory bulb (MOB) after 5 min of transient forebrain ischemia in gerbils. GAD65 immunoreactivity in the sham-operated group was shown in neurons and neuropil except for the somata of granule cells. GAD65 immunoreactivity was increased in neurons in the external plexiform layer 60 days after ischemia, and in mitral cells 30 and 60 days after ischemia. GAD67 immunoreactivity in the sham-operated group was shown in periglomerular cells, neuron in the external plexiform layer and granule cells with neuropil. GAD67 immunoreactivity in periglomerular cells was increased 10, 45 and 60 days after ischemia. GAD67 immunoreactivity in neurons in the external plexiform layer was increased 10 and 15 days after ischemia. Mitral cells showed strong GAD67 immunoreactivity 10 days after ischemia. However, GAD67 immunoreactivity in the granule cells was not changed with time after ischemia. In Western blot analysis for GAD65 and GAD67 protein levels in the ischemic gerbil MOB, GAD65 level was not changed after ischemia; GAD67 level was increased 10 days after ischemia. These results suggest that transient ischemia causes changes in GAD65 and GAD67 immunoreactivity in the gerbil MOB, and this change may induce a malfunction in olfaction after an ischemic insult.

Synaptic organization of the mouse cerebellar cortex in organotypic slice cultures

The cellular and synaptic organization of new born mouse cerebellum maintained in organotypic slice cultures was investigated using immunohistochemical and patch-clamp recording approaches. The histological organization of the cultures shared many features with that observed in situ. Purkinje cells were generally arranged in a monolayer surrounded by a molecular-like neuropil made of Purkinje cell dendritic arborizations. Purkinje cell axons ran between clusters of small round cells identified as granule cells by Kv3.1b potassium channel immunolabelling. The terminal varicosities of the Purkinje cells axons enwrapped presumptive neurons of the cerebellar nuclei whereas their recurrent collaterals were in contact with Purkinje cells and other neurons. Granule cell axons established contacts with Purkinje cell somata and dendrites. Parvalbumin and glutamine acid decarboxylase (GAD) immunohistochemistry revealed the presence of presumptive interneurons throughout the culture. The endings of granule cell axons were observed to be in contact with these interneurons. Similarly, interneurons endings were seen close to Purkinje cells and granule cells. Whole cell recordings from Purkinje cell somata showed AMPA receptor-mediated spontaneous excitatory post-synaptic currents (sEPSCs) and GABAA receptor-mediated spontaneous inhibitory post-synaptic currents (sIPSCs). Similar events were recorded from granule cell somata except that in this neuronal type EPSPs have both a NMDA component and an AMPA component. In addition, pharmacological experiments demonstrated a GABAergic control of granule cell activity and a glutamatergic control of GABAergic neurons by granule cells. This study shows that a functional neuronal network is established in such organotypic cultures even in the absence of the two normal excitatory afferents, the mossy fibers and the climbing fibers.

Developmental localization of potassium chloride co-transporter 2 in granule cells of the early postnatal mouse cerebellum with special reference to the synapse formation

In the adult CNS, GABA is the predominant inhibitory neurotransmitter, mediating the hyperpolarization of membrane potential and regulating the glutamatergic activity. In the immature CNS, on the other hand, GABA mediates depolarization and is involved in controlling morphogenesis. This developmental shift in GABA actions from depolarization to hyperpolarization occurs as a result of decreasing the intracellular chloride ion (Cl −) concentration ([Cl −] i) which is regulated by the potassium (K +)-Cl − co-transporter 2 (KCC2). To clarify the time-course of changes in the GABA actions during development, we examined the developmental localization of the KCC2 in the granule cells of the postnatal mouse cerebellum using specific antibodies against KCC2. The granule cell precursors and migrating granule cells were devoid of immunoreactivity against KCC2 antibodies. At postnatal day 3 (P3), the KCC2-immunolabeling was negative in the internal granular layer, although synaptophysin-positive mossy fiber terminals were detected. At P5, we first detected the KCC2-immunolabeling at the somata of granule cells and their dendrites before granule cells received inhibitory input from Golgi cells. Almost all KCC2-positive dendrites (more than 98%) attached to and formed synapses with mossy fiber terminals. As development proceeded, the number of KCC2-positive granule cells increased, and all granule cells became positive by P21. These results suggested that GABAergic transmission on granule cells might shift from excitation to inhibition after the synapse formation, and the excitatory synapse-formation and related factors might be the triggers for the expression and localization of the KCC2 in the granule cells. Furthermore, it was also suggested that formation of the GABAergic synapses and GABAergic transmission were not necessary for the KCC2-expression in the mouse cerebellar granule cells in vivo.

Main Determinants of Presynaptic Ca2+Dynamics at Individual Mossy Fiber–CA3 Pyramidal Cell Synapses

Synaptic transmission between hippocampal mossy fibers (MFs) and CA3 pyramidal cells exhibits remarkable use-dependent plasticity. The underlying presynaptic mechanisms, however, remain poorly understood. Here, we have used fluorescent Ca2+ indicators Fluo-4, Fluo-5F, and Oregon Green BAPTA-1 to investigate Ca2+ dynamics in individual giant MF boutons (MFBs) in area CA3 traced from the somata of granule cells held in whole-cell mode. In an individual MFB, a single action potential induces a brief peak of free Ca2+ (estimated in the range of 8-9 microm) followed by an elevation to approximately 320 nm, which slowly decays to its resting level of approximately 110 nm. Changes in the somatic membrane potential influence presynaptic Ca2+ entry at proximal MFBs in the hilus. This influence decays with distance along the axon, with a length constant of approximately 200 microm. In giant MFBs in CA3, progressive saturation of endogenous Ca2+ buffers during repetitive spiking amplifies rapid Ca2+ peaks and the residual Ca2+ severalfold, suggesting a causal link to synaptic facilitation. We find that internal Ca2+ stores contribute to maintaining the low resting Ca2+ providing approximately 22% of the buffering/extrusion capacity of giant MFBs. Rapid Ca2+ release from stores represents up to 20% of the presynaptic Ca2+ transient evoked by a brief train of action potentials. The results identify the main components of presynaptic Ca2+ dynamics at this important cortical synapse.

Identification of genes co‐upregulated with Arc during BDNF‐induced long‐term potentiation in adult rat dentate gyrus in vivo

Brain-derived neurotrophic factor (BDNF) is a critical regulator of transcription-dependent adaptive neuronal responses, such as long-term potentiation (LTP). Brief infusion of BDNF into the dentate gyrus of adult anesthetized rats triggers stable LTP at medial perforant path-granule synapses that is transcription-dependent and requires induction of the immediate early gene Arc. Rather than acting alone, Arc is likely to be part of a larger BDNF-induced transcriptional program. Here, we used cDNA microarray expression profiling to search for genes co-upregulated with Arc 3 h after BDNF-LTP induction. Of nine cDNAs encoding for known genes and up-regulated more than four-fold, we selected five genes, Narp, neuritin, ADP-ribosylation factor-like protein-4 (ARL4L), TGF-beta-induced immediate early gene-1 (TIEG1) and CARP, for further validation. Real-time PCR confirmed robust up-regulation of these genes in an independent set of BDNF-LTP experiments, whereas infusion of the control protein cytochrome C had no effect. In situ hybridization histochemistry further revealed up-regulation of all five genes in somata of post-synaptic granule cells following both BDNF-LTP and high-frequency stimulation-induced LTP. While Arc synthesis is critical for local actin polymerization and stable LTP formation, several of the co-upregulated genes have known functions in excitatory synaptogenesis, axon guidance and glutamate receptor clustering. These results provide novel insight into gene expression responses underlying BDNF-induced synaptic consolidation in the adult brain in vivo.

Metallothioneins and Zinc Dysregulation Contribute to Neurodevelopmental Damage in a Model of Perinatal Viral Infection

Neonatal Borna disease (NBD) virus infection in the Lewis rat results in life-long viral persistence and causes behavioral and neurodevelopmental abnormalities. A hallmark of the disorder is progressive loss of cerebellar Purkinje and dentate gyrus granule cells. Findings of increased brain metallothionein-I and -II (MT-I/-II) mRNA expression in cDNA microarray experiments led us to investigate MT isoforms and their relationship to brain zinc metabolism, cellular toxicity, and neurodevelopmental abnormalities in this model. Real-time PCR confirmed marked induction of MT-I/-II mRNA expression in the brains of NBD rats (40.5-fold increase in cerebellum, p<0.0001; 6.8-fold increase in hippocampus, p=0.003; and 9.5-fold increase in striatum, p=0.0012), whereas a trend toward decreased MT-III mRNA was found in hippocampus (1.25-fold decrease, p=0.0841). Double label immunofluorescence revealed prominent MT-I/-II expression in astrocytes throughout the brain; MT-III protein was decreased in granule cell neurons and increased in astrocytes, with differential subcellular distribution from cytoplasmic to nuclear compartments in NBD rat hippocampus. Modified Timm staining of hippocampus revealed reduced zinc in mossy fiber projections to the hilus and CA3, accumulation of zinc in glial cells and degenerating granule cell somata, and robust mossy fiber sprouting into the inner molecular layer of the dentate gyrus. Zinc Transporter 3 (ZnT-3) mRNA expression was decreased in hippocampus (2.3-fold decrease, p= 0.0065); staining for its correlate protein was reduced in hippocampal mossy fibers. Furthermore, 2 molecules implicated in axonal pathfinding and mossy fiber sprouting, the extracellular matrix glycoprotein, tenascin-R (TN-R), and the hyaluronan receptor CD44, were increased in NBD hippocampal neuropil. Abnormal zinc metabolism and mechanisms of neuroplasticity may contribute to the pathogenesis of disease in this model, raising more general implications for neurodevelopmental damage following viral infections in early life.