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PubMed1985

Optical mapping of electrical activity in rat somatosensory and visual cortex.

We have investigated the use of optical methods for monitoring neuron activity in mammalian cortex. The cortex was stained with a voltage-sensitive dye and fluorescence was simultaneously measured from 124 areas using a photodiode array. Optical signals were detected in rat somatosensory cortex in response to small whisker movements and in visual cortex in response to light flashes to the eye. Relatively large signals were obtained during focal interictal epileptiform discharges induced by bicuculline. The measuring system had a time resolution of milliseconds and a spatial resolution of a few hundred micrometers. Simultaneous, multi-site optical recordings of activity may provide a new and potentially powerful method for studying function and dysfunction in mammalian cortex.

Animals↗
PubMed1997

Optical mapping of conduction patterns of normal and tachycardia-like excitations in the rat atrium.

Multiple-site optical recording of transmembrane voltage activity (MSORTV), using a voltage-sensitive dye and a 12 x 12-element photodiode array, was employed to monitor action potentials in the rat atrium. Atrial preparations including the sinus node area, caval area, and atrial septum were dissected from adult rat hearts and stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). For suppression of optical artefacts due to contractile movements, a bathing medium containing 2,3-butanedione monoxime (BDM: 10-20 mM) was used. The spread of spontaneous excitation from the pacemaker was assessed optically by timing the initiation of the action potential-related extrinsic absorption changes. The optical signals were recorded from more than 300 contiguous loci in the atrium by sliding the photodiode array over the image of the preparation; in this way, the intra-atrial conduction pattern of spontaneous excitation was mapped. The obtained maps revealed a non-radial spread of excitatory waves originating in the pacemaking area over the atrium. Furthermore, we also mapped the conduction pattern of long-lasting tachycardia-like excitation evoked by a short train of electrical stimulation (5-10 Hz, 1-2s) applied with a bipolar electrode. These maps suggest that excitatory waves are propagated in a circular pathway which often surrounds the ostium of the superior or inferior vena cava. Various patterns for the pathway were also demonstrated optically.

Action Potentials↗
PubMed1991

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.

Action Potentials↗
PubMed1998

Optical mapping of neural responses in the embryonic rat brainstem with reference to the early functional organization of vagal nuclei.

We examined the functional organization of the vagal nuclei of the rat embryo during morphogenesis, using multiple-site optical recording with a voltage-sensitive dye. Slice preparations with vagus nerve fibers were dissected from 13- to 16-d-old embryonic (E13-E16) rat brainstems, and they were stained with the dye. Electrical activity in response to vagal stimulation was recorded optically from many sites. In the E13-E14 preparations, two types of spike-like optical signals were recorded: one was a narrow signal (type I), and the other was a broader signal (type II). Comparison with the morphology revealed by DiI labeling suggests that the type I signal response area corresponds to the nucleus of the tractus solitarius, and the type II signal response area corresponds to the dorsal motor nucleus of the vagus nerve. In the E15-E16 preparations, type I signals were followed by a slow signal related to glutamate-mediated excitatory postsynaptic potentials, suggesting that synaptic function is organized in the nucleus of the tractus solitarius by the 15-d-old embryonic stage. In the E14 preparation, a small, slow signal was evoked only in Mg2+-free solution, implying that postsynaptic function related to NMDA receptors emerges, in latent form, at the 14-d-old embryonic stage. In the E15 and E16 preparations, although the nucleus ambiguus is identified morphologically, no neural response-related optical signal was observed there, indicating that the embryonic organization of morphology and physiological function is not necessarily temporally coincident. We have mapped the dynamic spatiotemporal patterns of the evoked optical signals and have outlined the early phase of the functional organization of the cranial nuclei related to the vagus.

Animals↗
PubMed1994

Optical mapping of early embryonic expressions of Mg(2+)-/APV-sensitive components of vagal glutaminergic EPSPs in the chick brainstem.

Multiple-site optical recording of transmembrane voltage activity, using a voltage-sensitive dye (NK2761) and a 12 x 12 element photodiode array, was employed to monitor neural responses in the slice preparations that were dissected from 6-9-d-old embryonic brainstems. Transmembrane voltage-related optical responses evoked by a brief square current pulse applied with a microsuction electrode to the vagus nerve were recorded simultaneously from many sites in the preparation. In preparations from 6-d-old embryos, only action potential-related spike-like optical signals were often detected in the normal bathing solution containing 0.5 mM Mg2+. However, in several loci of some preparations, excitatory postsynaptic potential-related slow signals were elicited in a Mg(2+)-free bathing medium, and these elicited slow signals were blocked by DL-2-amino-5-phosphonovaleric acid (2-APV). These results show a possible embryonic expression of NMDA receptor-mediated excitatory postsynaptic potentials. The slow signals were usually detected from tested preparations from 7-9-d-old embryos in normal bathing solution. The later phase of the slow signals was enhanced in a Mg(2+)-free bathing medium, and the enhanced component was also sensitive to 2-APV and insensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). There were regional differences in the degree of the enhancement of the slow signals in the Mg(2+)-free bathing medium, and of the 2-APV-induced reduction of the slow signals. We have constructed early developmental maps of the spatial patterns of the slow signals related to Mg(2+)- and 2-APV-sensitive postsynaptic potentials. These maps reflect the spatial distribution of functional expression of the NMDA receptors during early development of the chick brainstem. The present findings are new data that have not been reported previously, and they have been obtained for the first time using the multiple-site optical recording technique with a voltage-sensitive dye.

2-Amino-5-phosphonovalerate↗
PubMed1999

Optical mapping of neural network activity in chick spinal cord at an intermediate stage of embryonic development.

We have applied multiple-site optical recording of transmembrane potential changes to recording of neuronal pathway/network activity from embryonic chick spinal cord slice preparations. Spinal cord preparations were dissected from 8-day-old chick embryos at Hamburger-Hamilton stage 33, and transverse slice preparations were prepared with the 13th cervical spinal nerve or with the 2nd or 5th lumbosacral spinal nerve intact. The slice preparations were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). Transmembrane voltage-related optical (dye-absorbance) changes evoked by spinal nerve stimulation with positive square-current pulses using a suction electrode were recorded simultaneously from many loci in the preparation, using a 128- or 1,020-element photodiode array. Optical responses were detected from dorsal and ventral regions corresponding to the posterior (dorsal) and anterior (ventral) gray horns. The optical signals were composed of two components, fast spike-like and slow signals. In the dorsal region, the fast spike-like signal was identified as the presynaptic action potential in the sensory nerve and the slow signal as the postsynaptic potential. In the ventral region, the fast spike-like signal reflects the antidromic action potential in motoneurons, and the slow signal is related to the postsynaptic potential evoked in the motoneuron. In preparations in which the ventral root was cut microsurgically, the antidromic action potential-related optical signals were eliminated. The areas of the maximal amplitude of the evoked signals in the dorsal and ventral regions were located near the dorsal root entry zone and the ventral root outlet zone, respectively. Quasiconcentric contour-line maps were obtained in the dorsal and ventral regions, suggesting the functional arrangement of the dorsal and ventral synaptic connections. Synaptic fatigue induced by repetitive stimuli in the ventral synapses was more rapid than in the dorsal synapses. The distribution patterns of the signals were essentially similar among C13, LS2, and LS5 preparations, suggesting that there is no difference in the spatiotemporal pattern of the neural responses along the rostrocaudal axis of the spinal cord at this developmental stage. In the ventral root-cut preparations, comparing the delay times between the ventral slow optical signals, we have been able to demonstrate that neural network-related synaptic connections are generated functionally in the embryonic spinal cord at Hamburger-Hamilton stage 33.

Animals↗
PubMed1999

Optical mapping of DNA polymerase I action and products.

Single molecule approaches to the characterization of biochemical systems offer an intrinsically simple and direct approach to address difficult, previously unyielding problems. Optically based approaches have recently been used to construct high resolution, ordered restriction maps from a variety of clone types. Advancements in surface technologies have enabled the reliable elongation and fixation of large DNA molecules onto specially derivatized substrates with retention of biochemical accessibility. In this study, the addition of fluorescently labeled nucleotides to surface-mounted DNA molecules by the action of DNA polymerase I is investigated using fluorescence microscopy to image individual template molecules. Molecules undergoing nick translation and containing only a few fluorochromes are readily imaged. These novel results suggest that surface-bound molecules may serve as a substrate for a broad range of enzymatic actions, and may offer new routes to analysis when coupled to advanced imaging techniques.

Bacteriophage lambda↗
PubMed1999

Optical mapping reveals the functional organization of the trigeminal nuclei in the chick embryo.

The functional organization of the trigeminal nuclei during embryogenesis was investigated using multiple-site optical recording with a fast voltage-sensitive dye. Brainstem preparations with three classified trigeminal nerve afferents, the ophthalmic, maxillary and mandibular nerves, together with motor nerve fibers, were dissected from five- to eight-day-old chick embryos. Electrical responses evoked by trigeminal nerve stimulations were optically recorded simultaneously from many loci of the stained preparations. We identified three response areas related to the trigeminal nerve: area I, located cephalic to the level of the trigeminal ganglion; area II, located caudal to the level of the trigeminal ganglion; and area III, located at the level of the trigeminal root. The neural responses in areas I and II were evoked by ophthalmic, maxillary or mandibular nerve stimulation, while the responses in area III were detected when the stimulation was applied to the trigeminal motor nerve. In comparison with the morphology indicated by DiI labeling, the results suggest that areas I, II and III correspond to the principal sensory nucleus of the trigeminal nerve, the spinal sensory nucleus of the trigeminal nerve and the trigeminal motor nucleus, respectively. We identified two components of the optical response: a fast and a slow signal. In five-day-old preparations, fast spike-like signals related to action potentials were recorded from the three response areas. In six-day-old preparations, slow optical signals which reflect glutamate-mediated excitatory postsynaptic potentials were detected from area II only when the ophthalmic nerve was stimulated: no slow signal was evoked by maxillary or mandibular nerve stimulation. In seven- and eight-day-old preparations, slow signals were detected from both areas I and II with every nerve stimulation. These results suggest that synaptic function is first generated in the spinal trigeminal nucleus by the six-day embryonic stage, and the developmental organization of synaptic function is not the same in the three trigeminal nerves or in the two sensory nuclei. Contour line maps of the signal amplitude revealed that the size and the area of the neural responses within the trigeminal nuclei changed dramatically with development. We compared the spatial distribution and temporal dynamics of the optical signals between the ophthalmic, maxillary and mandibular nerve stimulations, and we found that somatotopic organization is less clear in a rostrocaudal/mediolateral X-Y plane, although the areas of the maxillary and mandibular nerves appeared to separate in the lateral direction.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1994

Optical mapping of repolarization and refractoriness from intact hearts.

BACKGROUND: Heterogeneities of repolarization (R) across the myocardium have been invoked to explain most reentrant arrhythmias. The measurement of refractory periods (RPs) has been widely used to assess R, but conventional electrode and extrastimulus mapping techniques have not provided reliable maps of RPs. METHODS AND RESULTS: Guinea pig hearts were stained with a voltage-sensitive dye to measure fluorescence (F) action potentials (APs) from 124 sites with a photodiode array. AP duration (APD) was defined as the time between depolarization (dF/dt)max and R time points (ie, the time when AP returns to baseline or some percent thereof). However, R time points are difficult to determine because AP downstrokes are often encumbered by drifting baselines and motion artifacts, which make this definition ambiguous. In optical and microelectrode recordings, the second derivative of AP downstrokes is shown to contain an easily detected, unique local maximum. The correlation between the position of this maximum (d2F/dt2)max and R has been tested during altered AP characteristics induced by changes in cycle length, ischemia, and hypoxia. Under these various modifications of the AP, the time points of (d2F/dt2)max fell at 97.0 +/- 2.1% of recovery to baseline. Extrastimulus techniques applied to (1) isolated myocytes, (2) intact hearts, and (3) mathematical simulations indicated that (d2V/dt2)max coincided with the effective RPs of APs. The coincidence of RPs and (d2V/dt2)max was valid within 5 milliseconds, for resting potentials of -75 to -90 mV and extrastimuli three times threshold voltage. CONCLUSIONS: Thus, optical APs and (d2F/dt2)max can be used to map activation, R, and RPs with AP recordings from a single heartbeat.

Animals↗
PubMed2000

Simultaneous optical mapping of transmembrane potential and intracellular calcium in myocyte cultures.

INTRODUCTION: Fast spatially resolved measurements of transmembrane potential (Vm) and intracellular calcium (Ca(i)2+) are important for studying mechanisms of arrhythmias and defibrillation. The goals of this work were (1) to develop an optical technique for simultaneous multisite optical recordings of Vm and Ca(i)2+, and (2) to determine the relationship between Vm and Ca(i)2+ during normal impulse propagation in myocyte cultures. METHODS AND RESULTS: Monolayers of neonatal rat myocytes were stained with fluorescent dye RH-237 (Vm) and Fluo-3AM (Ca(i)2+). Both dyes were excited at the same wavelength range. The emitted fluorescence was optically separated into components corresponding to changes in Vm and Ca(i)2+ and measured using two 16 x 16 photodiode arrays at a spatial resolution of up to 27.5 microm per diode and sampling rate of 2.5 kHz. The optical setup was adjusted so that there was no optical cross-talk between the two types of measurements, which was validated in experiments involving staining with either RH-237 or Fluo-3. The amplitude of Fluo-3 signals rapidly decreased during experiments due to dye leakage. Dye leakage was substantially reduced by application of 1 mM probenecid, a blocker of organic anion transport, which had no effect on action potential duration and only minor effect on conduction velocity. In double-stained preparations, during regular pacing Ca(i)2+ transients had a rise time of 14.2 +/- 2 msec, and they followed Vm upstrokes with a delay of 5.3 +/- 1 msec (n = 9). Durations of Vm and Ca(i)2+ transients determined at 50% level of signal recovery were 54.6 +/- 10 msec and 136 +/- 8 msec, respectively. Application of 2 microM nifedipine reduced the amplitude and duration of Ca(i)2+ transients without significantly affecting conduction velocity. CONCLUSION: The results demonstrate feasibility of simultaneous optical recordings of Vm and Ca(i)2+ transients with high spatial and temporal resolution.

Aniline Compounds↗
PubMed1996

Anisotropic activation spread in heart cell monolayers assessed by high-resolution optical mapping. Role of tissue discontinuities.

The role of tissue discontinuities in anisotropic impulse propagation was assessed in two-dimensional anisotropic monolayers of neonatal rat myocytes cultured on a growth-directing substrate of collagen. Activation spread and distribution of maximal upstroke rate of rise (Vmax) of the action potential were measured with an optical system using a voltage-sensitive fluorescent dye (RH-327) and a 10x10 photodiode array with a spatial resolution ranging from 7 to 15 microns. Activation maps were compared with the cellular architecture and the distribution of gap junctions obtained from immunostaining the gap junction protein connexin43 (Cx43). Four types of structures were studied: (1) dense cell cultures, (2) cultures with anisotropic intercellular clefts of variable size, (3) discontinuities created by inclusion of nonmyocyte cells, and (4) discontinuities resulting from nonuniform expression of gap junctions. In dense monolayers, activation spread was continuous with microinhomogeneities in both longitudinal and transverse directions. The average cell dimensions in such monolayers were smaller than in adult canine myocardium. However, the degree of cellular anisotropy (length-to-width ratio of 5.3 +/- 1.4) and connectivity were similar. The presence of small intercellular clefts (less than one cell in length) did not disturb the general pattern of transverse or longitudinal activation spread, but it was associated with wave front microcollisions during transverse propagation and a concomitant increase of Vmax beyond the cleft. Long intercellular clefts caused discontinuous transverse propagation. Conduction velocity and Vmax decreased significantly at narrow isthmuses formed by closely apposed clefts, rendering such sites susceptible for conduction block. In contrast Vmax increased when the wave front faced the borders of the clefts. Nonmyocyte cells were electrically connected to myocytes and served as sinks for electrotonic currents, thereby producing localized conduction slowing and a decrease in Vmax. Localized inhomogeneity in Cx43 distribution correlated accurately with circumscribed conduction block and changes in Vmax. Our results provide direct experimental evidence that the cellular structure and gap junction distribution correlate with action potential propagation and distribution of Vmax. We suggest that in tissue with a nonuniform anisotropy, connective tissue separating fiber bundles or sites of inhomogeneous connexin distribution may represent predilective sites for block in transverse direction.

Animals↗
PubMed2001

Optical mapping of activation patterns in an animal model of congenital heart block.

Congenital heart block (CHB) is associated with high mortality and affects children of mothers with autoantibodies (IgG) to ribonucleoproteins SSB/La and SSA/Ro. IgG from mothers of children with CHB (positive IgG) was used to assess activation patterns in both the right atrium (RA) and right ventricle (RV) of Langendorff-perfused young rabbit hearts. Optical action potentials (AP) were obtained by using a 124-site photodiode array with 4-[-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium. Optical APs were recorded to simultaneously image activation patterns from the RA and RV. Perfusion of positive IgG (800--1,200 micro resulted in sinus bradycardia and varying degrees of heart block. Activation maps revealed marked conduction delay at the sinoatrial junction but only minor changes in overall atrial and ventricular activation patterns. No conduction disturbances were seen in the presence of IgG from mothers with healthy children. In conclusion, besides atrioventricular (AV) block, positive IgG induces sinus bradycardia. These results establish that the sequelae of CHB are associated with impaired intrasinus and/or sinoatrial conduction. The findings raise the possibility that sinus bradycardia in the developing heart may indicate the potential for AV conduction disturbances.

Action Potentials↗
PubMed1996

New polyacrylamide gel-based methods of sample preparation for optical microscopy: immobilization of DNA molecules for optical mapping.

New methods have been developed for rapid immobilization of biological macromolecules and other microscopic objects from aqueous solution at gel/gel, gel/solid and gel/solution interfaces using thin polyacrylamide gels covalently bound to glass surfaces. When quickly spread over a dry gel, an aqueous sample loses most of its water and low-molecular-weight solutes due to migration of these components into the gel. All optically observable objects thus become concentrated at the gel surface and may be easily located by light microscopy. Based on this, a procedure for binding DNA at a positively charged gel/solution interface was developed. A mild immobilization of the DNA molecules was obtained, allowing 'all in focus' observations of DNA digestion by restriction endonucleases with an apparent rate close to that in solution.

Acrylamides↗
PubMed1995

Optical mapping of translucence changes in rat hippocampal slices during hypoxia.

We have evaluated the effects of hypoxia on changes in light transmittance (delta T/T) in rat hippocampal interface slices at 36 degrees C, using a digital imaging system. Slice translucence increased only slightly (delta T/T = 4.65% in CA1; control 2.27%) during brief hypoxia in which hypoxic spreading depression-like depolarization was not induced. If hypoxia duration was sufficient to trigger spreading depression in CA1, slice translucence increased rapidly in CA1 (delta T/T = 27.5%) and smaller, delayed changes were noted in other regions. These spreading depression-induced changes reversed slowly to control levels upon reoxygenation. Measurement of intrinsic optical signals revealed both spatial and temporal patterns of cell volume changes associated with hypoxia and spreading depression in hippocampus.

Animals↗
PubMed1983

Fiber optic mapping of the Xenopus visual system: shift in the retinotectal projection during development.

Two new techniques for assaying the retina to tectum connections in the lower vertebrate visual system are presented. These techniques allow defined regions of the retina to be stimulated, thus circumventing some of the difficulties of the more conventional retinotectal mapping techniques. Applying these techniques to the Xenopus visual system demonstrates that the retina-to-tectum projection shifts during development. The central part of the retinotectal projection moves medially and caudally about 150 microns (10% of the size of the tectum) in two weeks. The presence of such plasticity in a normal developing animal indicates that the plasticity previously observed in experimentally altered animals probably reflects a normal developmental process.

Animals↗