A novel micro-ECoG recording method for recording multisensory neural activity from the parietal to temporal cortices in mice.

Characterization of inter-regional interactions in brain is essential for understanding the mechanism relevant to normal brain function and neurological disease. The recently developed flexible micro (µ)-electrocorticography (µECoG) device is one prominent method used to examine large-scale cortical activity across multiple regions. The sheet-shaped µECoG electrodes arrays can be placed on a relatively wide area of cortical surface beneath the skull by inserting the device into the space between skull and brain. Although rats and mice are useful tools for neuroscience, current µECoG recording methods in these animals are limited to the parietal region of cerebral cortex. Recording cortical activity from the temporal region of cortex in mice has proven difficult because of surgical barriers created by the skull and surrounding temporalis muscle anatomy. Here, we developed a sheet-shaped 64-channel µECoG device that allows access to the mouse temporal cortex, and we determined the factor determining the appropriate bending stiffness for the µECoG electrode array. We also established a surgical technique to implant the electrode arrays into the epidural space over a wide area of cerebral cortex covering from the barrel field to olfactory (piriform) cortex, which is the deepest region of the cerebral cortex. Using histology and computed tomography (CT) images, we confirmed that the tip of the µECoG device reached to the most ventral part of cerebral cortex without causing noticeable damage to the brain surface. Moreover, the device simultaneously recorded somatosensory and odor stimulus-evoked neural activity from dorsal and ventral parts of cerebral cortex in awake and anesthetized mice. These data indicate that our µECoG device and surgical techniques enable the recording of large-scale cortical activity from the parietal to temporal cortex in mice, including somatosensory and olfactory cortices. This system will provide more opportunities for the investigation of physiological functions from wider areas of the mouse cerebral cortex than those currently available with existing ECoG techniques.

Synaptotagmins 1 and 7 Play Complementary Roles in Somatodendritic Dopamine Release.

The molecular mechanisms underlying somatodendritic dopamine (DA) release remain unresolved, despite the passing of decades since its discovery. Our previous work showed robust release of somatodendritic DA in submillimolar extracellular Ca2+ concentration ([Ca2+]o). Here we tested the hypothesis that the high-affinity Ca2+ sensor synaptotagmin 7 (Syt7), is a key determinant of somatodendritic DA release and its Ca2+ dependence. Somatodendritic DA release from SNc DA neurons was assessed using whole-cell recording in midbrain slices from male and female mice to monitor evoked DA-dependent D2 receptor-mediated inhibitory currents (D2ICs). Single-cell application of an antibody to Syt7 (Syt7 Ab) decreased pulse train-evoked D2ICs, revealing a functional role for Syt7. The assessment of the Ca2+ dependence of pulse train-evoked D2ICs confirmed robust DA release in submillimolar [Ca2+]o in wild-type (WT) neurons, but loss of this sensitivity with intracellular Syt7 Ab or in Syt7 knock-out (KO) mice. In millimolar [Ca2+]o, pulse train-evoked D2ICs in Syt7 KOs showed a greater reduction in decreased [Ca2+]o than seen in WT mice; the effect on single pulse-evoked DA release, however, did not differ between genotypes. Single-cell application of a Syt1 Ab had no effect on train-evoked D2ICs in WT SNc DA neurons, but did cause a decrease in D2IC amplitude in Syt7 KOs, indicating a functional substitution of Syt1 for Syt7. In addition, Syt1 Ab decreased single pulse-evoked D2ICs in WT cells, indicating the involvement of Syt1 in tonic DA release. Thus, Syt7 and Syt1 play complementary roles in somatodendritic DA release from SNc DA neurons.SIGNIFICANCE STATEMENT The respective Ca2+ dependence of somatodendritic and axonal dopamine (DA) release differs, resulting in the persistence of somatodendritic DA release in submillimolar Ca2+ concentrations too low to support axonal release. We demonstrate that synaptotagmin7 (Syt7), a high-affinity Ca2+ sensor, underlies phasic somatodendritic DA release and its Ca2+ sensitivity in the substantia nigra pars compacta. In contrast, we found that synaptotagmin 1 (Syt1), the Ca2+ sensor underlying axonal DA release, plays a role in tonic, but not phasic, somatodendritic DA release in wild-type mice. However, Syt1 can facilitate phasic DA release after Syt7 deletion. Thus, we show that both Syt1 and Syt7 act as Ca2+ sensors subserving different aspects of somatodendritic DA release processes.

Activity-dependent somatodendritic dopamine release in the substantia nigra autoinhibits the releasing neuron.

Somatodendritic dopamine (DA) release from midbrain DA neurons activates D2 autoreceptors on these cells to regulate their activity. However, the source of autoregulatory DA remains controversial. Here, we test the hypothesis that D2 autoreceptors on a given DA neuron in the substantia nigra pars compacta (SNc) are activated primarily by DA released from that same cell, rather than from its neighbors. Voltage-clamp recording allows monitoring of evoked D2-receptor-mediated inhibitory currents (D2ICs) in SNc DA neurons as an index of DA release. Single-cell application of antibodies to Na+ channels via the recording pipette decreases spontaneous activity of recorded neurons and attenuates evoked D2ICs; antibodies to SNAP-25, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, also decrease D2IC amplitude. Evoked D2ICs are nearly abolished by the light chain of botulinum neurotoxin A, which cleaves SNAP-25, whereas synaptically activated GABAB-receptor-mediated currents are unaffected. Thus, somatodendritic DA release in the SNc autoinhibits the neuron that releases it.

Lithium increases synaptic GluA2 in hippocampal neurons by elevating the δ-catenin protein.

Lithium (Li+) is a drug widely employed for treating bipolar disorder, however the mechanism of action is not known. Here we study the effects of Li+ in cultured hippocampal neurons on a synaptic complex consisting of δ-catenin, a protein associated with cadherins whose mutation is linked to autism, and GRIP, an AMPA receptor (AMPAR) scaffolding protein, and the AMPAR subunit, GluA2. We show that Li+ elevates the level of δ-catenin in cultured neurons. δ-catenin binds to the ABP and GRIP proteins, which are synaptic scaffolds for GluA2. We show that Li+ increases the levels of GRIP and GluA2, consistent with Li+-induced elevation of δ-catenin. Using GluA2 mutants, we show that the increase in surface level of GluA2 requires GluA2 interaction with GRIP. The amplitude but not the frequency of mEPSCs was also increased by Li+ in cultured hippocampal neurons, confirming a functional effect and consistent with AMPAR stabilization at synapses. Furthermore, animals fed with Li+ show elevated synaptic levels of δ-catenin, GRIP, and GluA2 in the hippocampus, also consistent with the findings in cultured neurons. This work supports a model in which Li+ stabilizes δ-catenin, thus elevating a complex consisting of δ-catenin, GRIP and AMPARs in synapses of hippocampal neurons. Thus, the work suggests a mechanism by which Li+ can alter brain synaptic function that may be relevant to its pharmacologic action in treatment of neurological disease.

Presynaptic D1 heteroreceptors and mGlu autoreceptors act at individual cortical release sites to modify glutamate release.

The aim of this work was to study release of glutamic acid (GLU) from one-axon terminal or bouton at-a-time using cortical neurons grown in vitro to study the effect of presynaptic auto- and heteroreceptor stimulation. Neurons were infected with release reporters SypHx2 or iGluSnFR at 7 or 3 days-in-vitro (DIV) respectively. At 13-15 DIV single synaptic boutons were identified from images obtained from a confocal scanning microscope before and after field electrical stimulation. We further stimulated release by raising intracellular levels of cAMP with forskolin (10µM). Forskolin-mediated effects were dependent on protein kinase A (PKA) and did not result from an increase in endocytosis, but rather from an increase in the size of the vesicle readily releasable pool. Once iGluSnFR was confirmed as more sensitive than SypHx2, it was used to study the participation of presynaptic auto- and heteroreceptors on GLU release. Although most receptor agonizts (carbamylcholine, nicotine, dopamine D2, BDNF) did not affect electrically stimulated GLU release, a significant increase was observed in the presence of metabotropic D1/D5 heteroreceptor agonist (SKF38393 10µM) that was reversed by PKA inhibitors. Interestingly, stimulation of group II metabotropic mGLU2/3 autoreceptors (LY379268 50nM) induced a decrease in GLU release that was reversed by the specific mGLU2/3 receptor antagonist (LY341495 1µM) and also by PKA inhibitors (KT5720 200nM and PKI14-22 400nM). These changes in release probability at individual release sites suggest another level of control of the distribution of transmitter substances in cortical tissue.

beta-Phorbol ester-induced enhancement of exocytosis in large mossy fiber boutons of mouse hippocampus.

beta-Phorbol esters (BPE), synthetic analogues of diacylglycerol (DAG), induce the potentiation of transmission in many kinds of synapses through activating the C(1) domain-containing receptors. However, their effects on synaptic vesicle exocytosis have not yet been investigated. Here, we evaluated the vesicular exocytosis directly from individual large mossy fiber boutons (LMFBs) in hippocampal slices from transgenic mice that selectively express synaptopHluorin (SpH). We found that the activity-dependent increment of SpH fluorescence (DeltaSpH) was enhanced by 4beta-phorbol 12,13-diacetate (PDAc), one of the BPEs, without influencing the recycled component of SpH. These PDAc effects on DeltaSpH were almost completely inhibited by staurosporine, a non-selective antagonist of protein kinases. However, intermittent synaptic transmission was still potentiated through a staurosporine-resistant mechanism. The staurosporine-sensitive cascade may facilitate the vesicle replenishment, thus maintaining the fidelity of transmission at a high level during repetitive firing of the presynaptic neuron.

Photocurrent attenuation by a single polar-to-nonpolar point mutation of channelrhodopsin-2.

Channelrhodopsin-2 (ChR2), one of the algal light-gated cation channel rhodopsins, contains five peculiar glutamic acid residues in the N-terminal region corresponding to the second to third transmembrane helices. Here we made systematic mutations of these polar amino acid residues of ChR2 into nonpolar alanine, and evaluated their photocurrent properties. Amongst them, the photocurrent generated by the E97A mutation, ChR2(E97A), was much smaller than expected from its expression. The ChR2(E97A) photocurrent was similar to wild-type ChR2 in the kinetic profiles, the reversal potential and the dependency to the light power density. Our results suggest that the residue E97 is one of the molecular determinants involved in the ion flux regulation.

Molecular determinants differentiating photocurrent properties of two channelrhodopsins from chlamydomonas.

A light signal is converted into an electrical one in a single molecule named channelrhodopsin, one of the archaea-type rhodopsins in unicellular green algae. Although highly homologous, two molecules of this family, channelrhodopsin-1 (ChR1) and -2 (ChR2), are distinct in photocurrent properties such as the wavelength sensitivity, desensitization, and turning-on and -off kinetics. However, the structures regulating these properties have not been completely identified. Photocurrents were analyzed for several chimera molecules made by replacing N-terminal segments of ChR2 with the homologous counterparts of ChR1. We found that the wavelength sensitivity of the photocurrent was red-shifted with negligible desensitization and slowed turning-on and -off kinetics when replacement was made with the segment containing the fifth transmembrane helix of ChR1. Therefore, this segment is involved in the determination of photocurrent properties, the wavelength sensitivity, and the kinetics characterizing ChR1 and ChR2. Eight amino acid residues differentiating this segment were exchanged one-by-one, and the photocurrent properties of each targeted mutant ChR2 were further analyzed. Among them, position Tyr(226)(ChR1)/Asn(187)(ChR2) is one of the molecular determinants involved in the wavelength sensitivity, desensitization, and turning-on and -off kinetics. It is suggested that these amino acid residues directly or indirectly interact with the chromophore as well as with the protein structure determining the photocurrent kinetics. Some of the chimera channelrhodopsins are suggested to have several advantages over the wild-type ChR2 in the introduction of light-induced membrane depolarization for the purpose of artificial stimulation of neurons in vivo and visual prosthesis for photoreceptor degeneration.

Synaptic vesicle dynamics in the mossy fiber-CA3 presynaptic terminals of mouse hippocampus.

The mossy fiber (MF)-CA3 synapse in the hippocampus is unique in the CNS because of its wide dynamic range of transmitter release during short- and long-term plasticity. The presynaptic mechanisms underlying the fidelity of transmission were investigated for the MF-CA3 synapses. The relative size of readily releasable pool (RRP) of vesicles was estimated by counting the number of docked vesicles at an active zone (AZ) on the transmission electron microscopy (TEM) image. The size of the releasable pool and the exo-endocytosis kinetics were directly measured from individual large MF boutons in hippocampal slices of transgenic mice that selectively express synaptopHluorin (SpH), a pH-sensitive GFP fused to the lumenal aspect of one of the vesicular membrane proteins, VAMP-2, in these boutons. Here we found (1) there are distinct two vesicle pools, the resting pool which is resistant to exocytosis, and the releasable pool, (2) the initially docked vesicles are easily depleted and the RRP is maintained by refilling from the reserve subpopulation of releasable pool ("reserve" releasable pool), and (3) the contribution of rapid reuse of recycled vesicles is relatively small. Therefore, the fidelity of transmission is suggested to be ensured by the rapid refilling rate of RRP.

Transgenic mouse lines expressing synaptopHluorin in hippocampus and cerebellar cortex.

We generated six transgenic mouse lines in which synaptopHluorin (SpH), one of green fluorescent protein-based sensors of vesicular exocytosis, was expressed under the control of neuron-specific Thy-1.2 promoter. In situ hybridization study revealed that SpH mRNA was expressed in a broad spectrum of brain regions in four of them, whereas in others it was expressed in the specific regions of the hippocampus. In one particular line, SpH immunoreactivity was specifically observed in the mossy fiber presynaptic terminals of both hippocampus and cerebellar cortex. The fluorescence intensity of these presynaptic terminals was somewhat decreased by acidic buffer superfusion and greatly increased by vesicular neutralization of pH, indicating that the SpH molecules are mainly distributed in the synaptic vesicles. The exocytosis-dependent fluorescence increment was measured upon activation of a single presynaptic terminal. These transgenic lines are expected to facilitate morphological and physiological studies of presynaptic terminals in a variety of regions of the brain.