Yoko Tominaga

Quantification of optical signals with electrophysiological signals in neural activities of Di-4-ANEPPS stained rat hippocampal slices

We have quantified the optical signals of synaptically induced neural activities in an in vitro brain slice preparation in terms of electrophysiological signals. The qualification was done using electrophysiologically well known neural activities in the CA1 area of rat hippocampal slices stained with externally applied fluorescent voltage-sensitive dye (VSD; Di-4-ANEPPS). Together with a newly designed CCD-based digital high-speed camera system and epi-fluorescent optics, our improvements were made on a protocol for staining using a newly designed chamber system. These improvements enabled us to make stable and reliable recordings of optical signals and electrophysiological measurements without affecting the physiological status and to make a quantitative comparison between them. The time course and amplitude of the optical signal showed fair agreement with intracellular and extracellular recordings, and was stable over 2 h. The optical signal followed synaptically induced long-term potentiation (LTP) as monitored by the electrophysiological signals. A regional difference in the amount of LTP was found in optical signals and was confirmed in the electrophysiological signals. These results demonstrate the capabilities of our improved method as an alternative but more potent tool to measure the neuronal activities of brain slice in addition to electrophysiological method.

A new nonscanning confocal microscopy module for functional voltage-sensitive dye and Ca2+ imaging of neuronal circuit activity

Recent advances in fluorescent confocal microscopy and voltage-sensitive and Ca(2+) dyes have vastly improved our ability to image neuronal circuits. However, existing confocal systems are not fast enough or too noisy for many live-cell functional imaging studies. Here, we describe and demonstrate the function of a novel, nonscanning confocal microscopy module. The optics, which are designed to fit the standard camera port of the Olympus BX51WI epifluorescent microscope, achieve a high signal-to-noise ratio (SNR) at high temporal resolution, making this configuration ideal for functional imaging of neuronal activities such as the voltage-sensitive dye (VSD) imaging. The optics employ fixed 100- × 100-pinhole arrays at the back focal plane (optical conjugation plane), above the tube lens of a usual upright microscope. The excitation light travels through these pinholes, and the fluorescence signal, emitted from subject, passes through corresponding pinholes before exciting the photodiodes of the imager: a 100- × 100-pixel metal-oxide semiconductor (MOS)-type pixel imager with each pixel corresponding to a single 100- × 100-μm photodiode. This design eliminated the need for a scanning device; therefore, acquisition rate of the imager (maximum rate of 10 kHz) is the only factor limiting acquisition speed. We tested the application of the system for VSD and Ca(2+) imaging of evoked neuronal responses on electrical stimuli in rat hippocampal slices. The results indicate that, at least for these applications, the new microscope maintains a high SNR at image acquisition rates of ≤0.3 ms per frame.

Paired Burst Stimulation Causes GABAA Receptor-Dependent Spike Firing Facilitation in CA1 of Rat Hippocampal Slices

The theta oscillation (4–8 Hz) is a pivotal form of oscillatory activity in the hippocampus that is intermittently concurrent with gamma (25–100 Hz) burst events. In in vitro preparation, a stimulation protocol that mimics the theta oscillation, theta burst stimulation (TBS), is used to induce long-term potentiation. Thus, TBS is thought to have a distinct role in the neural network of the hippocampal slice preparation. However, the specific mechanisms that make TBS induce such neural circuit modifications are still unknown. Using electrophysiology and voltage-sensitive dye imaging (VSDI), we have found that TBS induces augmentation of spike firing. The augmentation was apparent in the first couple of brief burst stimulation (100 Hz four pulses) on a TBS-train in a presence of NMDA receptor blocker (APV 50 μM). In this study, we focused on the characterizes of the NMDA independent augmentation caused by a pair of the brief burst stimulation (the first pair of the TBS; paired burst stimulation-PBS). We found that PBS enhanced membrane potential responses on VSDI signal and intracellular recordings while it was absent in the current recording under whole-cell clamp condition. The enhancement of the response accompanied the augmentation of excitatory postsynaptic potential (EPSP) to spike firing (E-S) coupling. The paired burst facilitation (PBF) reached a plateau when the number of the first burst stimulation (priming burst) exceeds three. The interval between the bursts of 150 ms resulted in the maximum PBF. Gabazine (a GABAA receptor antagonist) abolished PBF. The threshold for spike generation of the postsynaptic cells measured with a current injection to cells was not lowered by the priming burst of PBS. These results indicate that PBS activates the GABAergic system to cause short-term E-S augmentation without raising postsynaptic excitability. We propose that a GABAergic system of area CA1 of the hippocampus produce the short-term E-S plasticity that could cause exaggerated spike-firing upon a theta-gamma activity distinctively, thus making the neural circuit of the CA1 act as a specific amplifier of the oscillation signal.

Overall Assay of Neuronal Signal Propagation Pattern With Long-Term Potentiation (LTP) in Hippocampal Slices From the CA1 Area With Fast Voltage-Sensitive Dye Imaging

Activity-dependent changes in the input-output (I-O) relationship of a neural circuit are central in the learning and memory function of the brain. To understand circuit-wide adjustments, optical imaging techniques to probe the membrane potential at every component of neurons, such as dendrites, axons and somas, in the circuit are essential. We have been developing fast voltage-sensitive dye (VSD) imaging methods for quantitative measurements, especially for single-photon wide-field optical imaging. The long-term continuous measurements needed to evaluate circuit-wide modifications require stable and quantitative long-term recordings. Here, we show that VSD imaging (VSDI) can be used to record changes in circuit activity in association with theta-burst stimulation (TBS)-induced long-term potentiation (LTP) of synaptic strength in the CA1 area. Our optics, together with the fast imaging system, enabled us to measure neuronal signals from the entire CA1 area at a maximum frame speed of 0.1 ms/frame every 60 s for over 12 h. We also introduced a method to evaluate circuit activity changes by mapping the variation in recordings from the CA1 area to coordinates defined by the morphology of CA1 pyramidal cells. The results clearly showed two types of spatial heterogeneity in LTP induction. The first heterogeneity is that LTP increased with distance from the stimulation site. The second heterogeneity is that LTP is higher in the stratum pyramidale (SP)-oriens region than in the stratum radiatum (SR). We also showed that the pattern of the heterogeneity changed according to the induction protocol, such as induction by TBS or high-frequency stimulation (HFS). We further demonstrated that part of the heterogeneity depends on the I-O response of the circuit elements. The results show the usefulness of VSDI in probing the function of hippocampal circuits.

No-scanning type of confocal microscope for a fast functional imaging of neuronal circuit

In mammals, neural Hu proteins (HuB/C/D) are essential to induce neuronal development. Hu proteins are widely expressed in neurons and their expression persists from early developmental stage to adulthood. However the physiological functions of Hu in mature neuron are unclear. We found that HuC-deficient mice exhibited intentional tremor, gait abnormality and ataxia after 7 months of age. Prior to develop these symptoms, the morphology of Purkinje axons was gradually swollen and retracted at the deep cerebellar nuclei. None of these pathological changes were observed during cerebellum development. Intriguingly, Purkinje axons in HuC-deficient cerebellum were degenerated without Purkinje cells death. This is a rare case that axonal degeneration was occurred independently from cell death, so this would provide a good model to address the mechanism to sustain axonal functions in adult brains. In early stage of axonal degeneration, we found dysfunction of post-transcriptional mechanism-mediated axonal degeneration in HuC-deficient mice. And axonal transport system might be impaired which leading to that proteins and organelle are accumulated in the swollen Purkinje axons. We found that several motor proteins involving to an axonal transport as a candidate target mRNAs in adult cerebellum by RIP-CHIP assay. Among these mRNAs, HuC regulates Kinesin expression by a posttranscriptional mechanism in vitro and in vivo. The altered Kinesin expression could explain in part of the axonal degeneration in HuC-deficient cerebellum in vitro.

Modulation of feed-forward inhibition on CA1 pyramidal cells by patterned synaptic input probed with voltage-sensitive dye imaging in rat hippocampal slices

Diacylglycerol kinase (DGK) is involved in the intracellular signal transduction as a regulator of two second messengers, diacylglycerol and phosphatidic acid. Recently, we have reported that DGK is expressed in medium spiny neurons of the striatum and is highly concentrated at the perisynapse of dendritic spines. Furthermore, one of the transcripts derived from the human DGK locus is annotated in GenBank as being differentially expressed in bipolar disorder patients. However, it remains elusive how DGK is implicated in pathophysiological role in neurons at the cellular level. In the present study, we investigated a potential involvement of DGK in spine morphogenesis in transfected hippocampal neurons. We suggest that DGK is involved in the molecular machinery of dendrite outgrowth and spinogenesis through its kinase activity.