Yoko Momose-Sato

EVALUATION OF OPTIMAL VOLTAGE-SENSITIVE DYES FOR OPTICAL MONITORING OF EMBRYONIC NEURAL ACTIVITY

To evaluate the suitability of a variety of fast voltage-sensitive dyes for optical recording of rapid transmembrane potential activity in the embryonic nervous system, we screened over twenty dyes, including several newly synthesized probes, in three different embryonic neural preparations: cervical vagus nerve bundle, nodose ganglion, and brainstem from 7-day old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recordingsystem. Signal size, signal-to-noise ratio, photobleaching, and phototoxicity were examined. Several promising new merocyanine-rhodanine dyes for embryonic nervous systems were found.

A new simultaneous 1020-site optical recording system for monitoring neural activity using voltage-sensitive dyes

We have constructed a new 1020-site optical system for simultaneous recording of transmembrane electrical activity, using a 34 x 34-element photodiode array. This new apparatus permits analyses of the spatio-temporal pattern of neural activity, such as action potentials and postsynaptic potentials, in the central nervous system, at higher spatial and temporal resolutions.

Optical mapping of the early development of the response pattern to vagal stimulation in embryonic chick brain stem.

1. In both intact and slice preparations of vagus‐brain stem isolated from 3‐ to 8‐day‐old chick embryos, the spatial pattern of neural responses to vagal stimulation and its development were assessed by means of multiple‐site optical recording of electrical activity, using a voltage‐sensitive merocyanine‐rhodanine dye (NK2761) and a 12 x 12‐element photodiode array. 2. The first neural responses, viz. fast optical signals (related to the action potential), were recorded in the 4‐day‐old brain stem preparation, and slow optical signals (related to excitatory postsynaptic potentials) were detected from late 7‐ and 8‐day‐old brain stem preparations. 3. The evoked optical signals appeared to be concentrated longitudinally in the central region of the stimulated side of the intact brain stem preparation and in a limited dorsal area in the slice preparation. The signal size gradually increased and the response area expanded as development proceeded. 4. Based on the above results, we have constructed developmental maps of the spatial patterns of the fast and slow optical responses. In the maps, the positions of the peak‐size regions of the fast and slow signals were assessed and we have found that there were differences in the location of these areas for the fast vs. the slow signals in the late 7‐ and 8‐day‐old embryonic brain stem preparations. 5. In the maps for the late 7‐ and 8‐day‐old embryonic brain stems, the fast signal response area seems to correspond to the dorsal motor nucleus of the vagus nerve and the slow response area to the nucleus tractus solitarii.

Optical detection of postsynaptic potentials evoked by vagal stimulation in the early embryonic chick brain stem slice.

1. A voltage‐sensitive dye and multiple‐site optical recording of changes in membrane potential were used to reveal the postsynaptic potentials in the early embryonic chick brain stem slice preparation. 2. Vagus‐brain stem preparations were isolated from 8‐day‐old chick embryos and then transverse slice preparations were prepared with both the right and left vagus nerve fibres intact. The slice preparations were stained with a voltage‐sensitive merocyanine‐rhodanine dye (NK2761). 3. Voltage‐related optical (absorbance) changes evoked by vagus nerve stimulation with positive square current pulses using a suction electrode were recorded simultaneously from 127 contiguous loci in the preparation, using a 12 x 12‐element photodiode array. Optical responses appeared in a limited area near the dorsal surface of the stimulated side. 4. When relatively large stimulating currents were applied, optical changes having two (or sometimes three) components were recorded. One component was the fast spike‐like signal and another the delayed, long‐lasting slow signal. 5. The size of the slow signal was decreased by continuous stimulation, reduced by low external calcium ion concentrations and eliminated in the presence of manganese or cadmium ions. 6. The slow signals were eliminated in the presence of kynurenic acid, and they were reduced by 2‐APV (DL‐2‐amino‐5‐phosphono‐valeric acid) and by CNQX (6‐cyano‐7‐nitroquinoxaline‐2,3‐dione). We conclude that the slow signals correspond to excitatory postsynaptic potentials which are glutamate mediated.

Responses to glossopharyngeal stimulus in the early embryonic chick brainstem: spatiotemporal patterns in three dimensions from repeated multiple-site optical recording of electrical activity

In an effort to assess the spatial patterning of glossopharyngeal responses in the early embryonic chick brainstem, we used a multiple- site optical recording system with a 12 x 12 element photodiode array and a voltage-sensitive merocyanine-rhodanine dye (NK2761) to monitor neural transmembrane voltage activities. Seven and 8 d old embryonic chick brainstems were sliced into 1400–1600 microns thick sections with the glossopharyngeal and vagal nerves attached, and then stained with the dye. Neural voltage-related optical signals were evoked by a positive brief (depolarizing) square current pulse applied to the glossopharyngeal nerve with a microsuction electrode, and then recorded simultaneously from many loci in the objective two-dimensional image plane of a compound microscope. In addition to the multiple-site optical recording technique, we tried to introduce an optical sectioning method by changing the focal plane of the microscope to obtain three-dimensional information. Thus, we have been able to assess semiquantitatively the three-dimensional profiles of two glossopharyngeal response areas corresponding to the nucleus of the glossopharyngeal nerve (nucleus nervi glossopharyngei) and the nucleus of the tractus solitarius. Furthermore, glutaminergic excitatory postsynaptic potentials were determined within the response area corresponding to the nucleus of the tractus solitarius. In addition, we also compared the glossopharyngeal and vagal response areas and found that the cores of the related nuclei are separated in three dimensions.

Optical characterization of a novel GABA response in early embryonic chick brainstem

To examine the functional expression of embryonic GABA receptors, the inhibitory effects were studied of GABA (GABA responses) on the excitatory postsynaptic potentials evoked by vagal stimulus in seven- to 10-day-old embryonic chick brainstem slice preparations. A multiple-site optical recording technique was used, with a multiple element photodiode array system and a fast voltage-sensitive merocyanine-rhodanine dye (NK2761). First, in the GABA response, three components were pharmacologically identified: component 1, related to GABA(A) receptors; component 2, related to GABA(B) receptors; and component 3 which is insensitive to GABA(A) and GABA(B) antagonists, but is stimulated by both GABA(A) and GABA(B) agonists. Subsequently. the embryogenesis and early development of the three components were investigated, and early developmental maps of regional distribution patterns of the three components were constructed. Components 1 and 3 have already emerged in the seven-day-old embryonic brainstem preparation; component 2 appeared in the eight-day-old preparations. No component related to GABA(C) receptors was observed in the seven- to 10-day-old embryonic stages. From the pharmacological properties of component 3, we suggest that it is related to a new subtype, the GABA(D) receptor.

A novel γ-aminobutyric acid response in the embryonic brainstem as revealed by voltage-sensitive dye recording ☆

Using a multiple-site optical recording technique employing a fast voltage-sensitive dye, we found a novel type of gamma-aminobutyric acid (GABA) response, which is insensitive to GABAA and GABAB antagonists, but is stimulated by either GABAA or GABAB agonist. This evidence was identified in the early embryonic brainstem slice preparation.

Optical monitoring of early appearance of spontaneous membrane potential changes in the embryonic chick medulla oblongata using a voltage-sensitive dye

Using a voltage-sensitive merocyanine-rhodamine dye (NK2761) and a 12 x 12-element photodiode matrix array, we recorded optically spontaneous membrane potential changes in a slice preparation from the embryonic chick brain stem during early development. The spontaneous optical signals, related to membrane potential changes, showed a simple monophasic shape with a relatively long duration, and they were synchronized among the different regions in the medulla oblongata. The spontaneous signals were first detected from seven-day-old embryos, and were not present in six-day-old embryos. The spontaneous signals appeared sporadically, and their frequency was very low. Three modes of optical signals termed singlet-mode, doublet-mode, and triplet-mode were observed. In the doublet- and triplet-modes, the spatial pattern of the first signal was primarily similar to that of the singlet-mode signal, whereas the signal size and spatial extent of the second and third signals appeared to decay.

Optical determination of impulse conduction velocity during development of embryonic chick cervical vagus nerve bundles.

1. Employing an optical method for multiple‐site simultaneous recording of electrical activity, we have determined the conduction velocity in cervical vagus nerve bundles isolated from 5‐ to 21‐day‐old chick embryos, and investigated its developmental changes. 2. The preparations were stained with a voltage‐sensitive merocyanine‐rhodanine dye (NK2761), and action potential‐ (impulse‐) related optical signals were elicited by brief stimuli applied to the end of the vagus nerve bundle with a suction electrode. Optical signals were recorded simultaneously from many contiguous regions using a 12 x 12‐element photodiode array. 3. The optical signals spread with small delay from the site of stimulation. From the relationship between the delay and distance from the current‐applying electrode, conduction velocities were estimated in each tested preparation: the conduction velocity was very small and increased monotonically from about 0.1 m s‐1 at 5 days embryonic age to about 0.4 m s‐1 by hatching. The increase in the conduction velocity was closely related to a developmental increase in the diameter of the vagus nerve bundle. 4. In addition, we have examined the spread of electrotonic potentials. The space constant was very small (200‐450 microns) and increased as development proceeded. 5. Compound optical action signals having two distinct components were also recorded. They often appeared to be concentrated in the preparations from 8‐ to 12‐day‐old embryos. The conduction velocity of the second component was slower than that of the first. We suggest that appearance of the second component reflects degeneration of a subset of axons resulting from ‘neural cell death’ during the development of the vagus nerve.

Optical Monitoring of Postsynaptic Potential in the Early Embryonic Avian Brain Stem Using a Voltage-Sensitive Dye

The ontogenetic approach to generation of physiological events during natural development would be a useful and powerful strategy for studying central nervous systems: it would allow us to analyse progressively the complicated functional organization and architecture of nervous systems, in a manner reminiscent of the expansion of a complex function in a power series. However, the experimental analysis of early embryonic nervous systems is technically difficult because the cells are extremely inaccessible: the microelectrode examination of neural cells, which provides the most direct test of their electrophysiology, is often difficult because of the small size of the cells. For this reason, electrophysiological studies of very early developing embryonic nervous systems have been hampered.