Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse.

The coupling between Ca2+ channels and release sensors is a key factor defining the signaling properties of a synapse. However, the coupling nanotopography at many synapses remains unknown, and it is unclear how it changes during development. To address these questions, we examined coupling at the cerebellar inhibitory basket cell (BC)-Purkinje cell (PC) synapse. Biophysical analysis of transmission by paired recording and intracellular pipette perfusion revealed that the effects of exogenous Ca2+ chelators decreased during development, despite constant reliance of release on P/Q-type Ca2+ channels. Structural analysis by freeze-fracture replica labeling (FRL) and transmission electron microscopy (EM) indicated that presynaptic P/Q-type Ca2+ channels formed nanoclusters throughout development, whereas docked vesicles were only clustered at later developmental stages. Modeling suggested a developmental transformation from a more random to a more clustered coupling nanotopography. Thus, presynaptic signaling developmentally approaches a point-to-point configuration, optimizing speed, reliability, and energy efficiency of synaptic transmission.

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains.

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

In vivo nanoscopic landscape of neurexin ligands underlying anterograde synapse specification.

Excitatory synapses are formed and matured by the cooperative actions of synaptic organizers, such as neurexins (Nrxns), neuroligins (Nlgns), LRRTMs, and Cbln1. Recent super-resolution nanoscopy developments have revealed that many synaptic organizers, as well as glutamate receptors and glutamate release machinery, exist as nanoclusters within synapses. However, it is unclear how such nanodomains interact with each other to organize excitatory synapses in vivo. By applying X10 expansion microscopy to epitope tag knockin mice, we found that Cbln1, Nlgn1, and LRRTM1, which share Nrxn as a common presynaptic receptor, form overlapping or separate nanodomains depending on Nrxn with or without a sequence encoded by splice site 4. The size and position of glutamate receptor nanodomains of GluD1, NMDA, and AMPA receptors were regulated by Cbln1, Nlgn1, and LRRTM1 nanodomains, respectively. These findings indicate that Nrxns anterogradely regulate the postsynaptic nanoscopic architecture of glutamate receptors through competition and coordination of Nrxn ligands.

Dendritic Homeostasis Disruption in a Novel Frontotemporal Dementia Mouse Model Expressing Cytoplasmic Fused in Sarcoma.

Cytoplasmic aggregation of fused in sarcoma (FUS) is detected in brain regions affected by amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which compose the disease spectrum, FUS proteinopathy. To understand the pathomechanism of ALS-FTD-associated FUS, we examined the behavior and cellular properties of an ALS mouse model overexpressing FUS with nuclear localization signal deletion. Mutant FUS transgenic mice showed hyperactivity, social interactional deficits, and impaired fear memory retrieval, all of which are compatible with FTD phenotypes. Histological analyses showed decreased dendritic spine and synaptic density in the frontal cortex before neuronal loss. Examination of cultured cells confirmed that mutant but not wild-type FUS was associated with decreased dendritic growth, mRNA levels, and protein synthesis in dendrites. These data suggest that cytoplasmic FUS aggregates impair dendritic mRNA trafficking and translation, in turn leading to dendritic homeostasis disruption and the development of FTD phenotypes.

Neural differentiation of human embryonic stem cells induced by the transgene-mediated overexpression of single transcription factors.

Pluripotent human embryonic stem cells (hESCs) can differentiate into multiple cell lineages, thus, providing one of the best platforms to study molecular mechanisms during cell differentiation. Recently, we have reported rapid and efficient differentiation of hESCs into functional neurons by introducing a cocktail of synthetic mRNAs encoding five transcription factors (TFs): NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. Here we further tested a possibility that even single transcription factors, when expressed ectopically, can differentiate hESCs into neurons. To this end, we established hESC lines in which each of these TFs can be overexpressed by the doxycycline-inducible piggyBac vector. The overexpression of any of these five TFs indeed caused a rapid and rather uniform differentiation of hESCs, which were identified as neurons based on their morphologies, qRT-PCR, and immunohistochemistry. Furthermore, calcium-imaging analyses and patch clamp recordings demonstrated that these differentiated cells are electrophysiologically functional. Interestingly, neural differentiations occurred despite the cell culture conditions that rather promote the maintenance of the undifferentiated state. These results indicate that over-expression of each of these five TFs can override the pluripotency-specific gene network and force hESCs to differentiate into neurons.

Chemical labelling for visualizing native AMPA receptors in live neurons.

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

Synaptotagmin 2 Is the Fast Ca2+ Sensor at a Central Inhibitory Synapse.

GABAergic synapses in brain circuits generate inhibitory output signals with submillisecond latency and temporal precision. Whether the molecular identity of the release sensor contributes to these signaling properties remains unclear. Here, we examined the Ca2+ sensor of exocytosis at GABAergic basket cell (BC) to Purkinje cell (PC) synapses in cerebellum. Immunolabeling suggested that BC terminals selectively expressed synaptotagmin 2 (Syt2), whereas synaptotagmin 1 (Syt1) was enriched in excitatory terminals. Genetic elimination of Syt2 reduced action potential-evoked release to ∼10%, identifying Syt2 as the major Ca2+ sensor at BC-PC synapses. Differential adenovirus-mediated rescue revealed that Syt2 triggered release with shorter latency and higher temporal precision and mediated faster vesicle pool replenishment than Syt1. Furthermore, deletion of Syt2 severely reduced and delayed disynaptic inhibition following parallel fiber stimulation. Thus, the selective use of Syt2 as release sensor at BC-PC synapses ensures fast and efficient feedforward inhibition in cerebellar microcircuits.

Nanodomain coupling explains Ca²âº independence of transmitter release time course at a fast central synapse.

A puzzling property of synaptic transmission, originally established at the neuromuscular junction, is that the time course of transmitter release is independent of the extracellular Ca(2+) concentration ([Ca(2+)]o), whereas the rate of release is highly [Ca(2+)]o-dependent. Here, we examine the time course of release at inhibitory basket cell-Purkinje cell synapses and show that it is independent of [Ca(2+)]o. Modeling of Ca(2+)-dependent transmitter release suggests that the invariant time course of release critically depends on tight coupling between Ca(2+) channels and release sensors. Experiments with exogenous Ca(2+) chelators reveal that channel-sensor coupling at basket cell-Purkinje cell synapses is very tight, with a mean distance of 10-20 nm. Thus, tight channel-sensor coupling provides a mechanistic explanation for the apparent [Ca(2+)]o independence of the time course of release.

One-year follow-up for the therapeutic efficacy of pregabalin in patients with leg symptoms caused by lumbar spinal stenosis.

BACKGROUND: Pregabalin is a well-accepted treatment option for patients with neuropathic pain. However, the therapeutic efficacy of pregabalin for reducing the incidence of spinal surgery to treat leg symptoms in patients with lumbar spinal stenosis remains unknown. The purpose of this study was to analyze the therapeutic efficacy of pregabalin for reducing the incidence of spinal surgery for leg symptoms in patients with lumbar spinal stenosis during the first year of treatment. METHODS: Consecutive patients diagnosed with lumbar spinal stenosis at our hospital from January to June 2009 were treated with nonsteroidal anti-inflammatory drug monotherapy and formed the control group (n = 47; 22 males, 25 females). Patients diagnosed with lumbar spinal stenosis at our hospital between August 2010 and October 2011 were treated with a nonsteroidal anti-inflammatory drug and pregabalin combination therapy and formed the pregabalin group (n = 49; 27 males, 22 females). The proportions of patients who underwent spinal surgery during the first year of treatment were assessed and compared between the two groups using the Mann-Whitney U test. In addition, the periods in which patients decided to undergo spinal surgery were compared using the Kaplan-Meier method. RESULTS: Six patients (12.2%) in the pregabalin group and 22 patients (46.8%) in the control group underwent spinal surgery during the first year of treatment (P = 0.0035). The period in which patients decided to undergo spinal surgery was significantly delayed in the pregabalin group compared with the control group in those for whom spinal surgery was necessary (P = 0.0128). CONCLUSIONS: Nonsteroidal anti-inflammatory drug and pregabalin combination therapy may result in a lower incidence of spinal surgery during the first year of treatment or a delayed period before undergoing spinal surgery if necessary compared with nonsteroidal anti-inflammatory drug monotherapy in patients with leg symptoms caused by lumbar spinal stenosis.

Four-year follow-up of pregnancy-associated osteoporosis: a case report.

A 22-year-old woman presented with complaints of severe pain in a wide region of the thoracolumbar spine. She developed severe pain in the thoracolumbar spine region 2 months after her first delivery and was referred 1 month later. A lateral thoracic X-ray showed depressed degenerative vertebrae (T7, T9). One month after the initial examination, thoracic sagittal magnetic resonance imaging showed low intensity areas on T1-weighted imaging and iso-high intensity areas on T2-weighted imaging at T5, 7, 8, 9 and 11. Bone mineral density measured by ultrasound was low (%YAM 76%). The bone metabolic markers were high, suggesting accelerated osteoclast activity. These findings prompted a diagnosis of pregnancy-associated osteoporosis. She was asked to stop breastfeeding and to wear a lumbar brace, and treatment with nutritional calcium, activated vitamin D3, and risedronate sodium was started. Her low back pain almost disappeared after treatment. Bone metabolic markers showed normalization 8 months after the initial examination. Risedronate sodium was stopped 2 years and 2 months after the initial examination. Teriparatide treatment was started because her bone mineral density remained low; however, the osteoblast marker P1NP was not increased 5 months after the start of teriparatide treatment.

Therapeutic efficacy of pregabalin in patients with leg symptoms due to lumbar spinal stenosis.

The purpose of this study was to evaluate the therapeutic efficacy of pregabalin in patients with leg symptoms due to lumbar spinal stenosis. Study subjects were classified into two groups according to their pharmacotherapy: the pregabalin group, treated with nonsteroidal anti-inflammatory drug and pregabalin combination therapy, and the control group, treated with nonsteroidal anti-inflammatory drug monotherapy. The two groups were compared in terms of the duration of pain after the onset of leg symptoms and the type of neurogenic intermittent claudication, whether radicular-, caudal-, or mixed-type. Numerical rating scale and Roland-Morris Disability Questionnaire scores were evaluated before and 3 months after treatment. After 3 months of treatment, there were significant differences in the numerical rating scale for radicular- and mixed-types, but not for caudal-type, between the two groups in the subjects with leg symptoms for greater than 3 months. There were significant differences between the two groups in Roland-Morris Disability Questionnaire scores for mixed-type, but not for radicular- and caudal-types, in the subjects with leg symptoms for less than 3 months and for radicular- and mixed-types, but not for caudal-type, in the subjects with leg symptoms for greater than 3 months. Nonsteroidal anti-inflammatory drug and pregabalin combination therapy may be more effective than nonsteroidal anti-inflammatory drug monotherapy for the relief of leg symptoms due to lumbar spinal stenosis, preventing aggravation of subjective symptoms and improving quality of life for patients with radicular- and mixed-types in subjects with leg symptoms for greater than 3 months, although it may be necessary to consider alternative therapy for patients with caudal-type.

Active roles of electrically coupled bipolar cell network in the adult retina.

Gap junctions are frequently observed in the adult vertebrate retina. It has been shown that gap junctions function as passive electrotonic pathways and play various roles, such as noise reduction, synchronization of electrical activities, regulation of the receptive field size, and transmission of rod signals to cone pathways. The presence of gap junctions between bipolar cells has been reported in various species but their functions are not known. In the present study, we applied dual whole-cell clamp techniques to the adult goldfish retina to elucidate the functions of gap junctions between ON-type bipolar cells with a giant axon terminal (Mb1-BCs). Electrophysiological and immunohistochemical experiments revealed that Mb1-BCs were coupled with each other through gap junctions that were located at the distal dendrites. The coupling conductance between Mb1-BCs under light-adapted conditions was larger than that under dark-adapted conditions. The gap junctions showed neither rectification nor voltage dependence, and behaved as a low-pass filter. Mb1-BCs could generate Ca(2+) spikes in response to depolarization, especially under dark-adapted conditions. The Ca(2+) spike evoked electrotonic depolarization through gap junctions in neighboring Mb1-BCs, and the depolarization in turn could trigger Ca(2+) spikes with a time lag. A brief depolarizing pulse applied to an Mb1-BC evoked a long-lasting EPSC in the postsynaptic ganglion cell. The EPSC was shortened in duration when gap junctions were pharmacologically or mechanically impaired. These results suggest that the spread of Ca(2+) spikes through gap junctions between bipolar cells may play a key role in lateral interactions in the adult retina.

Changes of low back pain after vascular reconstruction for abdominal aortic aneurysm and high aortic occlusion: a retrospective study.

BACKGROUND: The purpose of the present study is to clarify the influence of acute and chronic interruption of blood flow from lumbar arteries as well as the influence of vascular reconstruction on low back pain, back muscles, and lumbar discs. METHODS: Subjects were 34 patients with AAA in whom vascular reconstruction was performed. A second group was comprised of 9 patients with HAO. The presence of low back pain before surgery and at follow-up examination was retrospectively examined in the AAA group and the HAO group to investigate postoperative changes. The CSA and degeneration of the multifidus muscle and the lumbar discs on magnetic resonance imaging were assessed in the AAA group and control group. RESULTS: Low back pain, significant atrophy, or degeneration of the multifidus muscle or degeneration of the lumbar disc did not newly develop after surgery in the AAA group. These results indicated that acute interruption of lumbar arteries did not induce the development or deterioration of low back pain and organic changes in the back muscles or lumbar discs. The frequency of low back pain before surgery was significantly higher in the HAO group than that in the AAA group. However, the frequency of low back pain after surgery did not differ significantly between the 2 groups because low back pain in the HAO group was improved after surgery. CONCLUSION: The finding that low back pain was improved by merely performing treatment for the vascular system might provide support for the presence of vascular backache.

Group III metabotropic glutamate receptors and exocytosed protons inhibit L-type calcium currents in cones but not in rods.

Light responses of photoreceptors (rods and cones) are transmitted to the second-order neurons (bipolar cells and horizontal cells) via glutamatergic synapses located in the outer plexiform layer of the retina. Although it has been well established that postsynaptic group III metabotropic glutamate receptors (mGluRs) of ON bipolar cells contribute to generating the ON signal, presynaptic roles of group III mGluRs remain to be elucidated at this synaptic connection. We addressed this issue by applying the slice patch-clamp technique to the newt retina. OFF bipolar cells and horizontal cells generate a steady inward current in the dark and a transient inward current at light offset, both of which are mediated via postsynaptic non-NMDA receptors. A group III mGluR-specific agonist, L-2-amino-4-phosphonobutyric acid (L-AP-4), inhibited both the steady and off-transient inward currents but did not affect the glutamate-induced current in these postsynaptic neurons. L-AP-4 inhibited the presynaptic L-type calcium current (ICa) in cones by shifting the voltage dependence of activation to more positive membrane potentials. The inhibition of ICa was most prominent around the physiological range of cone membrane potentials. In contrast, L-AP-4 did not affect L-type ICa in rods. Paired recordings from photoreceptors and the synaptically connected second-order neurons confirmed that L-AP-4 inhibited both ICa and glutamate release in cones but not in rods. Furthermore, we found that exocytosed protons also inhibited ICa in cones but not in rods. Selective modulation of ICa in cones may help broaden the dynamic range of synaptic transfer by controlling the amount of transmitter release from cones.

Indomethacin blocks the nucleus pulposus-induced effects on nerve root function. An experimental study in dogs with assessment of nerve conduction and blood flow following experimental disc herniation.

Inflammatory mechanisms have been suggested to be involved in the basic pathophysiologic events leading to nerve root injury after local application of nucleus pulposus. To assess if these nucleus pulposus-induced effects could be blocked by anti-inflammatory treatment, 41 dogs were exposed to either incision of the L6-7 disc to induce experimental disc herniation with (n=12) or without (n=14) indomethacin treatment per os (5 mg/kg per day), and no incision with (n=5) or without (n=10) indomethacin. Intraneural blood flow and nerve conduction velocity were assessed after 7 days to evaluate the degree of nerve injury. Disc incision induced a reduction in nerve root and dorsal ganglion blood flow as well as nerve function, similarly to previous studies. However, simultaneous treatment with indomethacin efficiently blocked the negative effects on both blood flow and nerve conduction but had no effects per se. The present study thus indicates that inflammatory mechanisms may be of relevance in the pathophysiology of nucleus pulposus-induced nerve root injury and thereby also for sciatica.

Light-evoked oscillatory discharges in retinal ganglion cells are generated by rhythmic synaptic inputs.

In the visual system, optimal light stimulation sometimes generates gamma-range (ca. 20 approximately 80 Hz) synchronous oscillatory spike discharges. This phenomenon is assumed to be related to perceptual integration. Applying a planar multi-electrode array to the isolated frog retina, Ishikane et al. demonstrated that dimming detectors, off-sustained type ganglion cells, generate synchronous oscillatory spike discharges in response to diffuse dimming illumination. In the present study, applying the whole cell current-clamp technique to the isolated frog retina, we examined how light-evoked oscillatory spike discharges were generated in dimming detectors. Light-evoked oscillatory ( approximately 30 Hz) spike discharges were triggered by rhythmic ( approximately 30 Hz) fluctuations superimposed on a depolarizing plateau potential. When a suprathreshold steady depolarizing current was injected into a dimming detector, only a few spikes were evoked at the stimulus onset. However, repetitive spikes were triggered by a gamma-range sinusoidal current superimposed on the steady depolarizing current. Thus the light-evoked rhythmic fluctuations are likely to be generated presynaptically. The light-evoked rhythmic fluctuations were suppressed not by intracellular application of N-(2,6-dimethyl-phenylcarbamoylmethyl)triethylammonium bromide (QX-314), a Na(+) channel blocker, to the whole cell clamped dimming detector but by bath-application of tetrodotoxin to the retina. The light-evoked rhythmic fluctuations were suppressed by a GABA(A) receptor antagonist but potentiated by a GABA(C) receptor antagonist, whereas these fluctuations were little affected by a glycine receptor antagonist. Because amacrine cells are spiking neurons and because GABA is one of the main transmitters released from amacrine cells, amacrine cells may participate in generating rhythmically fluctuated synaptic input to dimming detectors.