Burst firing is required for induction of Hebbian LTP at lateral perforant path to hippocampal granule cell synapses.

High frequency burst firing is critical in summation of back-propagating action potentials (APs) in dendrites, which may greatly depolarize dendritic membrane potential. The physiological significance of burst firings of hippocampal dentate GCs in synaptic plasticity remains unknown. We found that GCs with low input resistance could be categorized into regular-spiking (RS) and burst-spiking (BS) cells based on their initial firing frequency (Finit) upon somatic rheobase current injection, and investigated how two types of GCs differ in long-term potentiation (LTP) induced by high-frequency lateral perforant pathway (LPP) inputs. Induction of Hebbian LTP at LPP synapses required at least three postsynaptic APs at Finit higher than 100 Hz, which was met in BS but not in RS cells. The synaptically evoked burst firing was critically dependent on persistent Na+ current, which was larger in BS than RS cells. The Ca2+ source for Hebbian LTP at LPP synapses was primarily provided by L-type calcium channels. In contrast, Hebbian LTP at medial PP synapses was mediated by T-type calcium channels, and could be induced regardless of cell types or Finit of postsynaptic APs. These results suggest that intrinsic firing properties affect synaptically driven firing patterns, and that bursting behavior differentially affects Hebbian LTP mechanisms depending on the synaptic input pathway.

L-type Ca2+ channels mediate regulation of glutamate release by subthreshold potential changes.

Subthreshold depolarization enhances neurotransmitter release evoked by action potentials and plays a key role in modulating synaptic transmission by combining analog and digital signals. This process is known to be Ca2+ dependent. However, the underlying mechanism of how small changes in basal Ca2+ caused by subthreshold depolarization can regulate transmitter release triggered by a large increase in local Ca2+ is not well understood. This study aimed to investigate the source and signaling mechanisms of Ca2+ that couple subthreshold depolarization with the enhancement of glutamate release in hippocampal cultures and CA3 pyramidal neurons. Subthreshold depolarization increased presynaptic Ca2+ levels, the frequency of spontaneous release, and the amplitude of evoked release, all of which were abolished by blocking L-type Ca2+ channels. A high concentration of intracellular Ca2+ buffer or blockade of calmodulin abolished depolarization-induced increases in transmitter release. Estimation of the readily releasable pool size using hypertonic sucrose showed depolarization-induced increases in readily releasable pool size, and this increase was abolished by the blockade of calmodulin. Our results provide mechanistic insights into the modulation of transmitter release by subthreshold potential change and highlight the role of L-type Ca2+ channels in coupling subthreshold depolarization to the activation of Ca2+-dependent signaling molecules that regulate transmitter release.

Rewiring of Prelimbic Inputs to the Nucleus Accumbens Core Underlies Cocaine-Induced Behavioral Sensitization.

BACKGROUND: Unbalanced activity of medium spiny neurons (MSNs) of the direct and indirect pathways mediates reward-related behaviors induced by addictive drugs. Prelimbic (PL) input to MSNs in the nucleus accumbens core (NAcC) plays a key role in cocaine-induced early locomotor sensitization (LS). However, the adaptive plastic changes at PL-to-NAcC synapses underlying early LS remain unclear. METHODS: Using transgenic mice and retrograde tracing, we identified NAcC-projecting pyramidal neurons (PNs) in the PL cortex based on the expression of dopamine receptor types (D1R or D2R). To examine cocaine-induced alterations in PL-to-NAcC synapses, we measured excitatory postsynaptic current amplitudes evoked by optostimulation of PL afferents to MSNs. Riluzole was chosen to test the effects of PL excitability on cocaine-induced changes of PL-to-NAcC synapses. RESULTS: NAcC-projecting PNs were segregated into D1R- and D2R-expressing PNs (D1- and D2-PNs, respectively), and their excitability was opposingly regulated by respective dopamine agonists. Both D1- and D2-PNs exhibited balanced innervation of direct MSNs and indirect MSNs in naïve animals. Repeated cocaine injections resulted in biased synaptic strength toward direct MSNs through presynaptic mechanisms in both D1- and D2-PNs, although D2R activation reduced the D2-PN excitability. Under group 1 metabotropic glutamate receptors coactivation, however, D2R activation enhanced the D2-PN excitability. The cocaine-induced rewiring accompanied LS, and both rewiring and LS were precluded by PL infusion of riluzole, which reduced the intrinsic excitability of PL neurons. CONCLUSIONS: These findings indicate that cocaine-induced rewiring of PL-to-NAcC synapses correlates well with early behavioral sensitization and that rewiring and LS can be prevented by riluzole-induced reduction of excitability of PL neurons.

Involvement of Ca2+ in Signaling Mechanisms Mediating Muscarinic Inhibition of M Currents in Sympathetic Neurons.

Acetylcholine can excite neurons by suppressing M-type (KCNQ) potassium channels. This effect is mediated by M1 muscarinic receptors coupled to the Gq protein. Although PIP2 depletion and PKC activation have been strongly suggested to contribute to muscarinic inhibition of M currents (IM), direct evidence is lacking. We investigated the mechanism involved in muscarinic inhibition of IM with Ca2+ measurement and electrophysiological studies in both neuronal (rat sympathetic neurons) and heterologous (HEK cells expressing KCNQ2/KCNQ3) preparations. We found that muscarinic inhibition of IM was not blocked either by PIP2 or by calphostin C, a PKC inhibitor. We then examined whether muscarinic inhibition of IM uses multiple signaling pathways by blocking both PIP2 depletion and PKC activation. This maneuver, however, did not block muscarinic inhibition of IM. Additionally, muscarinic inhibition of IM was not prevented either by sequestering of G-protein ßγ subunits from Gα-transducin or anti-Gßγ antibody or by preventing intracellular trafficking of channel proteins with blebbistatin, a class-II myosin inhibitor. Finally, we re-examined the role of Ca2+ signals in muscarinic inhibition of IM. Ca2+ measurements showed that muscarinic stimulation increased intracellular Ca2+ and was comparable to the Ca2+ mobilizing effect of bradykinin. Accordingly, 20-mM of BAPTA significantly suppressed muscarinic inhibition of IM. In contrast, muscarinic inhibition of IM was completely insensitive to 20-mM EGTA. Taken together, these data suggest a role of Ca2+ signaling in muscarinic modulation of IM. The differential effects of EGTA and BAPTA imply that Ca2+ microdomains or spatially local Ca2+ signals contribute to inhibition of IM.

Voltage-gated calcium channels contribute to spontaneous glutamate release directly via nanodomain coupling or indirectly via calmodulin.

Neurotransmitter release occurs either synchronously with action potentials (evoked release) or spontaneously (spontaneous release). Whether the molecular mechanisms underlying evoked and spontaneous release are identical, especially whether voltage-gated calcium channels (VGCCs) can trigger spontaneous events, is still a matter of debate in glutamatergic synapses. To elucidate this issue, we characterized the VGCC dependence of miniature excitatory postsynaptic currents (mEPSCs) in various synapses with different coupling distances between VGCCs and synaptic vesicles, known as a critical factor in evoked release. We found that most of the extracellular calcium-dependent mEPSCs were attributable to VGCCs in cultured autaptic hippocampal neurons and the mature calyx of Held where VGCCs and vesicles were tightly coupled. Among loosely coupled synapses, mEPSCs were not VGCC-dependent at immature calyx of Held and CA1 pyramidal neuron synapses, whereas VGCCs contribution was significant at CA3 pyramidal neuron synapses. Interestingly, the contribution of VGCCs to spontaneous glutamate release in CA3 pyramidal neurons was abolished by a calmodulin antagonist, calmidazolium. These data suggest that coupling distance between VGCCs and vesicles determines VGCC dependence of spontaneous release at tightly coupled synapses, yet VGCC contribution can be achieved indirectly at loosely coupled synapses.

Gradual decorrelation of CA3 ensembles associated with contextual discrimination learning is impaired by Kv1.2 insufficiency.

The associative network of hippocampal CA3 is thought to contribute to rapid formation of contextual memory from one-trial learning, but the network mechanisms underlying decorrelation of neuronal ensembles in CA3 is largely unknown. Kv1.2 expressions in rodent CA3 pyramidal cells (CA3-PCs) are polarized to distal apical dendrites, and its downregulation specifically enhances dendritic responses to perforant pathway (PP) synaptic inputs. We found that haploinsufficiency of Kv1.2 (Kcna2+/-) in CA3-PCs, but not Kv1.1 (Kcna1+/-), lowers the threshold for long-term potentiation (LTP) at PP-CA3 synapses, and that the Kcna2+/- mice are normal in discrimination of distinct contexts but impaired in discrimination of similar but slightly distinct contexts. We further examined the neuronal ensembles in CA3 and dentate gyrus (DG), which represent the two similar contexts using in situ hybridization of immediate early genes, Homer1a and Arc. The size and overlap of CA3 ensembles activated by the first visit to the similar contexts were not different between wild type and Kcna2+/- mice, but these ensemble parameters diverged over training days between genotypes, suggesting that abnormal plastic changes at PP-CA3 synapses of Kcna2+/- mice is responsible for the impaired pattern separation. Unlike CA3, DG ensembles were not different between two genotype mice. The DG ensembles were already separated on the first day, and their overlap did not further evolve. Eventually, the Kcna2+/- mice exhibited larger CA3 ensemble size and overlap upon retrieval of two contexts, compared to wild type or Kcna1+/- mice. These results suggest that sparse LTP at PP-CA3 synapse probably supervised by mossy fiber inputs is essential for gradual decorrelation of CA3 ensembles.

Dopamine Receptor Supports the Potentiation of Intrinsic Excitability and Synaptic LTD in Temporoammonic-CA1 Synapse.

Dopaminergic projection to the hippocampus from the ventral tegmental area or locus ceruleus has been considered to play an essential role in the acquisition of novel information. Hence, the dopaminergic modulation of synaptic plasticity in the hippocampus has been widely studied. We examined how the D1 and D2 receptors influenced the mGluR5-mediated synaptic plasticity of the temporoammonic-CA1 synapses and showed that the dopaminergic modulation of the temporoammonic-CA1 synapses was expressed in various ways. Our findings suggest that the dopaminergic system in the hippocampal CA1 region regulates the long-term synaptic plasticity and processing of the novel information.

Calbindin regulates Kv4.1 trafficking and excitability in dentate granule cells via CaMKII-dependent phosphorylation.

Calbindin, a major Ca2+ buffer in dentate granule cells (GCs), plays a critical role in shaping Ca2+ signals, yet how it regulates neuronal function remains largely unknown. Here, we found that calbindin knockout (CBKO) mice exhibited dentate GC hyperexcitability and impaired pattern separation, which co-occurred with reduced K+ current due to downregulated surface expression of Kv4.1. Relatedly, manipulation of calbindin expression in HT22 cells led to changes in CaMKII activation and the level of surface localization of Kv4.1 through phosphorylation at serine 555, confirming the mechanism underlying neuronal hyperexcitability in CBKO mice. We also discovered that Ca2+ buffering capacity was significantly reduced in the GCs of Tg2576 mice to the level of CBKO GCs, and this reduction was restored to normal levels by antioxidants, suggesting that calbindin is a target of oxidative stress. Our data suggest that the regulation of CaMKII signaling by Ca2+ buffering is crucial for neuronal excitability regulation.

Postnatal maturation of glutamate clearance and release kinetics at the rat and mouse calyx of Held synapses.

Although calyx of Held synapses undergo dramatic changes around the hearing onset, previous in vivo studies suggest that the calyx synapses undergo further post-hearing maturation process. While developmental changes over the hearing onset have been extensively studied, this post-hearing maturation process remained relatively little investigated. Because of post-hearing maturation, previous results from studies around hearing onset and studies of post-hearing calyx synapses are somewhat inconsistent. Here, we characterized the post-hearing maturation of calyx synapses with regard to in vitro electrophysiological properties in rats and mice. We found that parameters for residual glutamate in the cleft during a train, EPSC kinetics, and vesicle pool size became close to a full mature level by P14, but they further matured until P16 in the rats. Consistently, the phasic and slow EPSCs evoked by action potential trains at P16 calyx synapses were not different from those at P18 or P25 under physiological extracellular [Ca2+ ]o (1.2 mM). In contrast, the parameters for residual current and EPSC kinetics displayed drastic changes until P16 in mice, and slow EPSCs during the train further decreased between P16 and P18, suggesting that maturation of calyx synapses progresses at least up to P16 in rats and P18 in mice.

Impaired pattern separation in Tg2576 mice is associated with hyperexcitable dentate gyrus caused by Kv4.1 downregulation.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that causes memory loss. Most AD researches have focused on neurodegeneration mechanisms. Considering that neurodegenerative changes are not reversible, understanding early functional changes before neurodegeneration is critical to develop new strategies for early detection and treatment of AD. We found that Tg2576 mice exhibited impaired pattern separation at the early preclinical stage. Based on previous studies suggesting a critical role of dentate gyrus (DG) in pattern separation, we investigated functional changes in DG of Tg2576 mice. We found that granule cells in DG (DG-GCs) in Tg2576 mice showed increased action potential firing in response to long depolarizations and reduced 4-AP sensitive K+-currents compared to DG-GCs in wild-type (WT) mice. Among Kv4 family channels, Kv4.1 mRNA expression in DG was significantly lower in Tg2576 mice. We confirmed that Kv4.1 protein expression was reduced in Tg2576, and this reduction was restored by antioxidant treatment. Hyperexcitable DG and impaired pattern separation in Tg2576 mice were also recovered by antioxidant treatment. These results highlight the hyperexcitability of DG-GCs as a pathophysiologic mechanism underlying early cognitive deficits in AD and Kv4.1 as a new target for AD pathogenesis in relation to increased oxidative stress.

CaV α2δ Autoimmune Encephalitis: A Novel Antibody and its Characteristics.

OBJECTIVE: Discovery of a novel antibody would enable diagnosis and early treatment of autoimmune encephalitis. The aim was to discover a novel antibody targeting a synaptic receptor and characterize the pathogenic mechanism. METHOD: We screened for unknown antibodies in serum and cerebrospinal fluid samples from autoimmune encephalitis patients. Samples with reactivity to rat brain sections and no reactivity to conventional antibody tests underwent further processing for antibody discovery, using immunoprecipitation to primary neuronal cells, mass-spectrometry analysis, an antigen-binding assay on an antigen-overexpressing cell line, and an electrophysiological assay with cultured hippocampal neurons. RESULTS: Two patients had a novel antibody against CaV α2δ (voltage-gated calcium channel alpha-2/delta subunit). The patient samples stained neuropils of the hippocampus, basal ganglia, and cortex in rat brain sections and bound to a CaV α2δ-overexpressing cell line. Knockdown of CaV α2δ expression in cultured neurons turned off the immunoreactivity of the antibody from the patients to the neurons. The patients were associated with preceding meningitis or neuroendocrine carcinoma and responded to immunotherapy. In cultured neurons, the antibody reduced neurotransmitter release from presynaptic nerve terminals by interfering with tight coupling of calcium channels and exocytosis. INTERPRETATION: Here, we discovered a novel autoimmune encephalitis associated with anti-CaV α2δ antibody. Further analysis of the antibody in autoimmune encephalitis might promote early diagnosis and treatment. ANN NEUROL 2021;89:740-752.

Inter-spike mitochondrial Ca2+ release enhances high frequency synaptic transmission.

KEY POINTS: Presynaptic mitochondria not only absorb but also release Ca2+ during high frequency stimulation (HFS) when presynaptic [Ca2+ ] is kept low (<500 nm) by high cytosolic Ca2+ buffer or strong plasma membrane calcium clearance mechanisms under physiological external [Ca2+ ]. Mitochondrial Ca2+ release (MCR) does not alter the global presynaptic Ca2+ transients. MCR during HFS enhances short-term facilitation and steady state excitatory postsynaptic currents by increasing vesicular release probability. The intra-train MCR may provide residual calcium at interspike intervals, and thus support high frequency neurotransmission at central glutamatergic synapses. ABSTRACT: Emerging evidence indicates that mitochondrial Ca2+ buffering contributes to local regulation of synaptic transmission. It is unknown, however, whether mitochondrial Ca2+ release (MCR) occurs during high frequency synaptic transmission. Confirming the previous notion that 2 µm tetraphenylphosphonium (TPP+ ) is a specific inhibitor of the mitochondrial Na+ /Ca2+ exchanger (mNCX), we studied the role of MCR via mNCX in short-term plasticity during high frequency stimulation (HFS) at the calyx of Held synapse of the rat. TPP+ reduced short-term facilitation (STF) and steady state excitatory postsynaptic currents during HFS at mature calyx synapses under physiological extracellular [Ca2+ ] ([Ca2+ ]o  = 1.2 mm), but not at immature calyx or at 2 mm [Ca2+ ]o . The inhibitory effects of TPP+ were stronger at synapses with morphologically complex calyces harbouring many swellings and at 32°C than at simple calyx synapses and at room temperature. These effects of TPP+ on STF were well correlated with those on the presynaptic mitochondrial [Ca2+ ] build-up during HFS. Mitochondrial [Ca2+ ] during HFS was increased by TPP+ at mature calyces under 1.2 mm [Ca2+ ]o , and further enhanced at 32°C, but not under 2 mm [Ca2+ ]o or at immature calyces. The close correlation of the effects of TPP+ on mitochondrial [Ca2+ ] with those on STF suggests that mNCX contributes to STF at the calyx of Held synapses. The intra-train MCR enhanced vesicular release probability without altering global presynaptic [Ca2+ ]. Our results suggest that MCR during HFS elevates local [Ca2+ ] near synaptic sites at interspike intervals to enhance STF and to support stable synaptic transmission under physiological [Ca2+ ]o .

Cellular and systemic mechanisms for glucose sensing and homeostasis.

Glucose is a major source of energy in animals. Maintaining blood glucose levels within a physiological range is important for facilitating glucose uptake by cells, as required for optimal functioning. Glucose homeostasis relies on multiple glucose-sensing cells in the body that constantly monitor blood glucose levels and respond accordingly to adjust its glycemia. These include not only pancreatic ß-cells and α-cells that secrete insulin and glucagon, but also central and peripheral neurons regulating pancreatic endocrine function. Different types of cells respond distinctively to changes in blood glucose levels, and the mechanisms involved in glucose sensing are diverse. Notably, recent studies have challenged the currently held views regarding glucose-sensing mechanisms. Furthermore, peripheral and central glucose-sensing cells appear to work in concert to control blood glucose level and maintain glucose and energy homeostasis in organisms. In this review, we summarize the established concepts and recent advances in the understanding of cellular and systemic mechanisms that regulate glucose sensing and its homeostasis.

Kv4.1, a Key Ion Channel For Low Frequency Firing of Dentate Granule Cells, Is Crucial for Pattern Separation.

The dentate gyrus (DG) in the hippocampus may play key roles in remembering distinct episodes through pattern separation, which may be subserved by the sparse firing properties of granule cells (GCs) in the DG. Low intrinsic excitability is characteristic of mature GCs, but ion channel mechanisms are not fully understood. Here, we investigated ionic channel mechanisms for firing frequency regulation in hippocampal GCs using male and female mice, and identified Kv4.1 as a key player. Immunofluorescence analysis showed that Kv4.1 was preferentially expressed in the DG, and its expression level determined by Western blot analysis was higher at 8-week than 3-week-old mice, suggesting a developmental regulation of Kv4.1 expression. With respect to firing frequency, GCs are categorized into two distinctive groups: low-frequency (LF) and high-frequency (HF) firing GCs. Input resistance (Rin) of most LF-GCs is lower than 200 MΩ, suggesting that LF-GCs are fully mature GCs. Kv4.1 channel inhibition by intracellular perfusion of Kv4.1 antibody increased firing rates and gain of the input-output relationship selectively in LF-GCs with no significant effect on resting membrane potential and Rin, but had no effect in HF-GCs. Importantly, mature GCs from mice depleted of Kv4.1 transcripts in the DG showed increased firing frequency, and these mice showed an impairment in contextual discrimination task. Our findings suggest that Kv4.1 expression occurring at late stage of GC maturation is essential for low excitability of DG networks and thereby contributes to pattern separation.SIGNIFICANCE STATEMENT The sparse activity of dentate granule cells (GCs), which is essential for pattern separation, is supported by high inhibitory inputs and low intrinsic excitability of GCs. Low excitability of GCs is thought to be attributable to a high K+ conductance at resting membrane potentials, but this study identifies Kv4.1, a depolarization-activated K+ channel, as a key ion channel that regulates firing of GCs without affecting resting membrane potentials. Kv4.1 expression is developmentally regulated and Kv4.1 currents are detected only in mature GCs that show low-frequency firing, but not in less mature high-frequency firing GCs. Furthermore, mice depleted of Kv4.1 transcripts in the dentate gyrus show impaired pattern separation, suggesting that Kv4.1 is crucial for sparse coding and pattern separation.

Disparities in Short-Term Depression Among Prefrontal Cortex Synapses Sustain Persistent Activity in a Balanced Network.

Persistent activity of cue-representing neurons in the prefrontal cortex (PFC) is regarded as a neural basis for working memory. The contribution of short-term synaptic plasticity (STP) at different types of synapses comprising the cortical network to persistent activity, however, remains unclear. Characterizing STP at synapses of the rat PFC layer 5 network, we found that PFC synapses exhibit distinct STP patterns according to presynaptic and postsynaptic identities. Excitatory postsynaptic currents (EPSCs) from corticopontine (Cpn) neurons were well sustained throughout continued activity, with stronger depression at synapses onto fast-spiking interneurons than those onto pyramidal cells. Inhibitory postsynaptic currents (IPSCs) were sustained at a weaker level compared with EPSC from Cpn synapses. Computational modeling of a balanced network incorporating empirically observed STP revealed that little depression at recurrent excitatory synapses, combined with stronger depression at other synapses, could provide the PFC with a unique synaptic mechanism for the generation and maintenance of persistent activity.

Prenatal selective serotonin reuptake inhibitor (SSRI) exposure induces working memory and social recognition deficits by disrupting inhibitory synaptic networks in male mice.

Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed antidepressant drugs in pregnant women. Infants born following prenatal exposure to SSRIs have a higher risk for behavioral abnormalities, however, the underlying mechanisms remains unknown. Therefore, we examined the effects of prenatal fluoxetine, the most commonly prescribed SSRI, in mice. Intriguingly, chronic in utero fluoxetine treatment impaired working memory and social novelty recognition in adult males. In the medial prefrontal cortex (mPFC), a key region regulating these behaviors, we found augmented spontaneous inhibitory synaptic transmission onto the layer 5 pyramidal neurons. Fast-spiking interneurons in mPFC exhibited enhanced intrinsic excitability and serotonin-induced excitability due to upregulated serotonin (5-HT) 2A receptor (5-HT2AR) signaling. More importantly, the behavioral deficits in prenatal fluoxetine treated mice were reversed by the application of a 5-HT2AR antagonist. Taken together, our findings suggest that alterations in inhibitory neuronal modulation are responsible for the behavioral alterations following prenatal exposure to SSRIs.

Intracellular Zn2+ Signaling Facilitates Mossy Fiber Input-Induced Heterosynaptic Potentiation of Direct Cortical Inputs in Hippocampal CA3 Pyramidal Cells.

Repetitive action potentials (APs) in hippocampal CA3 pyramidal cells (CA3-PCs) backpropagate to distal apical dendrites, and induce calcium and protein tyrosine kinase (PTK)-dependent downregulation of Kv1.2, resulting in long-term potentiation of direct cortical inputs and intrinsic excitability (LTP-IE). When APs were elicited by direct somatic stimulation of CA3-PCs from rodents of either sex, only a narrow window of distal dendritic [Ca2+] allowed LTP-IE because of Ca2+-dependent coactivation of PTK and protein tyrosine phosphatase (PTP), which renders non-mossy fiber (MF) inputs incompetent in LTP-IE induction. High-frequency MF inputs, however, could induce LTP-IE at high dendritic [Ca2+] of the window. We show that MF input-induced Zn2+ signaling inhibits postsynaptic PTP, and thus enables MF inputs to induce LTP-IE at a wide range of [Ca2+]i values. Extracellular chelation of Zn2+ or genetic deletion of vesicular zinc transporter abrogated the privilege of MF inputs for LTP-IE induction. Moreover, the incompetence of somatic stimulation was rescued by the inhibition of PTP or a supplement of extracellular zinc, indicating that MF input-induced increase in dendritic [Zn2+] facilitates the induction of LTP-IE by inhibiting PTP. Consistently, high-frequency MF stimulation induced immediate and delayed elevations of [Zn2+] at proximal and distal dendrites, respectively. These results indicate that MF inputs are uniquely linked to the regulation of direct cortical inputs owing to synaptic Zn2+ signaling.SIGNIFICANCE STATEMENT Zn2+ has been mostly implicated in pathological processes, and the physiological roles of synaptically released Zn2+ in intracellular signaling are little known. We show here that Zn2+ released from hippocampal mossy fiber (MF) terminals enters postsynaptic CA3 pyramidal cells, and plays a facilitating role in MF input-induced heterosynaptic potentiation of perforant path (PP) synaptic inputs through long-term potentiation of intrinsic excitability (LTP-IE). We show that the window of cytosolic [Ca2+] that induces LTP-IE is normally very narrow because of the Ca2+-dependent coactivation of antagonistic signaling pairs, whereby non-MF inputs become ineffective in inducing excitability change. The MF-induced Zn2+ signaling, however, biases toward facilitating the induction of LTP-IE. The present study elucidates why MF inputs are more privileged for the regulation of PP synapses.

Association of mGluR-Dependent LTD of Excitatory Synapses with Endocannabinoid-Dependent LTD of Inhibitory Synapses Leads to EPSP to Spike Potentiation in CA1 Pyramidal Neurons.

The input-output relationships in neural circuits are determined not only by synaptic efficacy but also by neuronal excitability. Activity-dependent alterations of synaptic efficacy have been extensively investigated, but relatively less is known about how the neuronal output is modulated when synaptic efficacy changes are associated with neuronal excitability changes. In this study, we demonstrate that paired pulses of low-frequency stimulation (PP-LFS) induced metabotropic glutamate receptor (mGluR)-dependent LTD at Schaffer collateral (SC)-CA1 synapses in Sprague Dawley rats (both sexes), and this LTD was associated with EPSP to spike (E-S) potentiation, leading to the increase in action potential (AP) outputs. Threshold voltage (Vth) for APs evoked by synaptic stimulation and that by somatic current injection were hyperpolarized significantly after PP-LFS. Blockers of GABA receptors mimicked and occluded PP-LFS effects on E-S potentiation and Vth hyperpolarization, suggesting that suppression of GABAergic mechanisms is involved in E-S potentiation after PP-LFS. Indeed, IPSCs and tonic inhibitory currents were reduced after PP-LFS. The IPSC reduction was accompanied by increased paired-pulse ratio, and abolished by AM251, a blocker for Type 1 cannabinoid receptors, suggesting that PP-LFS suppresses presynaptic GABA release by mGluR-dependent endocannabinoids signaling. By contrast, a Group 1 mGluR agonist, 3, 5-dihydroxyphenylglycine, induced LTD at SC-CA1 synapses but failed to induce significant IPSC reduction and AP output increase. We propose that mGluR signaling that induces LTD coexpression at excitatory and inhibitory synapses regulates an excitation-inhibition balance to increase neuronal output in CA1 neurons.SIGNIFICANCE STATEMENT Long-lasting forms of synaptic plasticity are usually associated with excitability changes, the ability to fire action potentials. However, excitability changes have been regarded to play subsidiary roles to synaptic plasticity in modifying neuronal output. We demonstrate that, when metabotropic glutamate receptor-dependent LTD is induced by paired pulses of low-frequency stimulation, the action potential output in response to a given input paradoxically increases, indicating that increased excitability is more powerful than synaptic depression. This increase is mediated by the suppression of a presynaptic GABA release via metabotropic glutamate receptor-dependent endocannabinoid signaling. Our study shows that neuronal output changes do not always follow the direction of synaptic plasticity at excitatory synapses, highlighting the importance of regulating inhibitory tone via endocannabinoid signaling.

Reliable autapse formation using the single-cell patterning method.

Auto neuronal synapses, or autapses, are aberrant structures where the synaptic contact of a neuron forms onto its own branch. The functions of autapses, however, remain unknown. Here, we introduce a simple patterning method for capturing a single-cell, in which we maintained the isolated cell until it reached maturity, and developed arrays of autapses for electrophysiological analysis using multi-electrode arrays (MEA). The pattern arrays were formed by selective patterning of poly-L-lysine and various cell repellent materials. We tested the efficiency of single neuron pattern formed according to materials and pattern dimensions. Autapse formation was verified by immunostaining synaptic markers and physiological measurements via recordings from MEA. The results demonstrated that our multiscale patterning method increased the number of autapses consisting of a single neuron, which matured to connect onto themselves. The proposed patterning method (4.06 ± 0.33 isolated single-cells mm-2) is at least twelve times more efficient and productive than the spray method (0.31 ± 0.10 isolated single-cells mm-2). The spontaneous activity of a single neuron on the patterned MEA occured after 11 d in vitro. The single neuron activity consisted of bursts followed by spike trains (the burst rate was 2.56 min-1). This indicates that our method could be used for electrophysiological analysis, including MEA.

Enhancement of dendritic persistent Na+ currents by mGluR5 leads to an advancement of spike timing with an increase in temporal precision.

Timing and temporal precision of action potential generation are thought to be important for encoding of information in the brain. The ability of single neurons to transform their input into output action potential is primarily determined by intrinsic excitability. Particularly, plastic changes in intrinsic excitability represent the cellular substrate for spatial memory formation in CA1 pyramidal neurons (CA1-PNs). Here, we report that synaptically activated mGluR5-signaling can modulate the intrinsic excitability of CA1-PNs. Specifically, high-frequency stimulation at CA3-CA1 synapses increased firing rate and advanced spike onset with an improvement of temporal precision. These changes are mediated by mGluR5 activation that induces cADPR/RyR-dependent Ca2+ release in the dendrites of CA1-PNs, which in turn causes an increase in persistent Na+ currents (INa,P) in the dendrites. When group I mGluRs in CA1-PNs are globally activated pharmacologically, afterdepolarization (ADP) generation as well as increased firing rate are observed. These effects are abolished by inhibiting mGluR5/cADPR/RyR-dependent Ca2+ release. However, the increase in firing rate, but not the generation of ADP is affected by inhibiting INa,P. The differences between local and global activation of mGluR5-signaling in CA1-PNs indicates that mGluR5-dependent modulation of intrinsic excitability is highly compartmentalized and a variety of ion channels are recruited upon their differential subcellular localizations. As mGluR5 activation is induced by physiologically plausible brief high-frequency stimulation at CA3-CA1 synapses, our results suggest that mGluR5-induced enhancement of dendritic INa,P in CA1-PNs may provide important implications for our understanding about place field formation in the hippocampus.