Michinori Ichikawa

Entorhinal-Hippocampal Interactions Revealed by Real-Time Imaging

The entorhinal cortex provides the major cortical input to the hippocampus, and both structures have been implicated in memory processes. The dynamics of neuronal circuits in the entorhinal-hippocampal system were studied in slices by optical imaging with high spatial and temporal resolution. Reverberation of neural activity was detected in the entorhinal cortex and was more prominent when the inhibition due to γ-aminobutyric acid was slightly suppressed. Neural activity was transferred in a frequency-dependent way from the entorhinal cortex to the hippocampus. The entorhinal neuronal circuit could contribute to memory processes by holding information and selectively gating the entry of information into the hippocampus.

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.

Axonal microtubules necessary for generation of sodium current in squid giant axons. I: Pharmacological study on sodium current and restoration of sodium current by microtubule proteins and 260K protein

SummaryEffects of the reagents suppressing or supporting axoplasmic microtubule assembly were studied on the Na ionic current of squid giant axons by perfusing the axon internally with the solution containing the reagent. Among the reagents suppressing the assembly, colchicine, vinblastine, podophyllotoxin, sulfhydryl reagents such as DTNB and NEM, and chaotropic anions such as iodide and bromide, were examined. These reagents reduced maximum Na conductance and shifted the voltage dependence of steady-state Na activation in a depolarizing direction along the voltage axis. They also made the voltage dependence less steep, but did not affect sodium inactivation appreciably. Effects on Na ionic current of reagents which support microtubule assembly (Taxol, DMSO, D2O and temperature) were opposite the effects of those agents suppressing assembly. At the same time, we demonstrated that after Na currents were partially reduced, they could be restored by internally perfusing the axon with a solution containing microtubule proteins, 260K proteins and cAMP under conditions favorable for microtubule assembly. For full restoration, it was found that the following conditions were necessary: (1) The microenvironment within the axon is suitable for microtubule assembly. (2) Tubulins incorporated into microtubules are fully tyrosinated at their C-termini. (3) A peripheral protein having a molecular weight of 260,000 daltons (260K protein) is indispensable. These results suggest that axoplasmic microtubules and 260K proteins in the structure underlying the axolemma play a role in generating Na currents in squid giant axons.

High-speed CCD imaging system for monitoring neural activity in vivo and in vitro, using a voltage-sensitive dye

We have designed and constructed a high-speed CCD imaging system for optically detecting neural activity from preparations stained externally with a voltage-sensitive dye, and have used this system to image evoked and epileptiform neural activity in the rat somatosensory cortex. The imaging system uses a commercially available 1/3-in. CCD chip, and it can continuously capture images for more than 8 s, at 1000 frames/s, with a spatial resolution of 128 x 62 pixels. The spatial/temporal resolution of the CCD sensor is variable by changing the geometry of on-chip binning pixels, which can be controlled by a PC/AT computer. Dye bleaching correction was not necessary for long-term imaging of epileptiform neural events, since the sensitivity of the CCD sensor was increased by combining the signal from adjacent pixels.

An improved cryofixation method: cryoquenching of small tissue blocks during microwave irradiation

The metal contact method of rapid freezing is greatly improved by irradiating the specimen with microwaves at 2.45 GHz for a short period of time (50 ms), while pushing the specimen onto the surface of the copper block cooled by liquid N2. The microwave irradiation, together with two technical improvements (a light‐mass plunger and a recently developed β‐gel shock absorber) for preventing bounce, produces a good freezing zone for squid retina, with high reproducibility for each experimental trial, extending from the contact surface to a depth of about 15 μm, which is comparable to the depth obtained by the metal contact method using liquid He in the absence of microwave irradiation. A good freezing zone was also experimentally demonstrated in specimens of rat liver and heart muscle. Microwave irradiation does not have appreciable effects on the ultrastructure of squid retina. The mechanism underlying the improvement in the rapid freezing under the microwave irradiation is discussed.

Differential alteration of hippocampal synaptic strength induced by pituitary adenylate cyclase activating polypeptide-38 (PACAP-38)

We investigated the effect of pituitary adenylate cyclase activating polypeptide-38 (PACAP-38) on synaptic transmission in rat hippocampal slices using extracellular recordings. Brief bath application of PACAP-38 induced a long-lasting depression of transmission at the Schaffer collateral-CA1 synapse while at the same time causing enhancement of the perforant path-granule cell synapse in the dentate gyrus. Depression at the CA1 synapse was not occluded by low frequency-induced long-term depression (LTD), nor was enhancement at the granule cell synapse blocked by previous high frequency-induced long-term potentiation (LTP). Both actions of PACAP-38 could be elicited in the presence of an N-methyl-D-aspartate (NMDA) receptor antagonist, or in the presence of inhibitors of cyclic AMP- or Ca2+-dependent protein kinases, suggesting a novel mechanism of synaptic modulation.

Axonal microtubules necessary for generation of sodium current in squid giant axons: II. Effect of colchicine upon asymmetrical displacement current

SummaryEffect of internal colchicine on asymmetrical displacement currents was studied by internally perfusing squid giant axons with a solution containing colchicine. It was found that (1) asymmetrical displacement currents were composed of two parts; colchicine-sensitive and colchicine-resistant; that (2) the colchicine-sensitive part had a definite rising phase while the colchicine-resistant one showed an instantaneous jump, followed by exponential decay; and that (3) the colchicine-sensitive part related to normal Na channels.

Anticalmodulin drugs block the sodium gating current of squid giant axons.

SummaryThe effects of calmodulin (CaM) antagonists (W-7, W-5, trifluoperazine, chlorpromazine, quinacrine, diazepam, propericyazine and carmidazolium) on the sodium and potassium channels were studied on the intracellularly perfused and voltage-clamped giant axon of the squid. It was found that the drugs are more potent blockers of the sodium current than of the potassium current. The drugs also reduce the sodium gating current. The blockage of the sodium and gating current can be explained by assuming that the drugs interact with the sodium gating subunit in one of its closed states. The site of action is probably the intracellular surface of the axolemma where presumably a Ca2+-calmodulin complex can be formed.

Massively parallel classification of single-trial EEG signals using a min-max Modular neural network

This paper presents a method for classifying single-trial electroencephalogram (EEG) signals using min-max modular neural networks implemented in a massively parallel way. The method has three main steps. First, a large-scale, complex EEG classification problem is simply divided into a reasonable number of two-class subproblems, as small as needed. Second, the two-class subproblems are simply learned by individual smaller network modules in parallel. Finally, all the individual trained network modules are integrated into a hierarchical, parallel, and modular classifier according to two module combination laws. To demonstrate the effectiveness of the method, we perform simulations on fifteen different four-class EEG classification tasks, each of which consists of 1491 training and 636 test data. These EEG classification tasks were created using a set of non-averaged, single-trial hippocampal EEG signals recorded from rats; the features of the EEG signals are extracted using wavelet transform techniques. The experimental results indicate that the proposed method has several attractive features. 1) The method is appreciably faster than the existing approach that is based on conventional multilayer perceptrons. 2) Complete learning of complex EEG classification problems can be easily realized, and better generalization performance can be achieved. 3) The method scales up to large-scale, complex EEG classification problems.

Simultaneous multi-site recordings of neural activity with an inline multi-electrode array and optical measurement in rat hippocampal slices

Understanding neuronal network function requires multi-site recording. An optical imaging method using voltage-sensitive dyes (VSD) is one such suitable method. It is also important to complement this technique with methods that measure other physiological parameters of neuronal network activity. We aimed to develop a multi-channel electrode method that would easily permit the simultaneous optical imaging of neural activity in vitro. There are several multi-electrode systems; some of these are commercially available, but they are not easy to use and are also expensive. We developed a novel, less expensive, stainless steel inline electrode array that utilizes the standard etching method of circuit board manufacturing. This method, along with widely available computer drawing software, allowed us to produce electrode arrays with uniform quality in electrical characteristics and shapes. The electrode array was designed so that it could easily substitute for single electrode systems without modifying the conventional set-up used for electrophysiological measurements and optical recordings. Simultaneous measurement of evoked neural activities in area CA1 of the rat hippocampal slice with our optical imaging method using the VSD Di-4-ANEPPS is demonstrated in this paper. The electrode array was also used as a stimulating electrode, and the evoked response was measured by the optical recording method. The results demonstrate the advantages of simultaneous recordings obtained by these complementary multi-site recording methods in vitro.