Brian M. Salzberg

Optical measurement of membrane potential

Optical measurement of membrane potential is a new tool for physiologists and has already found many applications. However, the number of possible pitfalls is alarming, particularly in situations where comparison with electrode measurements is impossible. Exhaustive and elaborate controls are clearly necessary; and yet they never provide complete assurance that an optical signal represents a change in membrane potential. In our opinion, the use of redistribution signals, which are slower, and thus more likely to represent to secondary effects of changes in membrane potential, and require permeant dyes with access to the internal millieu, may be more hazardous than the use of either fast or intrinsic signals. However, the larger size of the redistribution signals has endowed them with obvious appeal. If more sensitive fast signals can be found, the use of this kind of signal would be facilitated.

Optical recording of electrical activity from parallel fibres and other cell types in skate cerebellar slices in vitro.

1. A reliable and simple fish brain slice preparation was obtained from the cerebellum of the skate, and its properties were described. 2. A potentiometric oxonol dye, RH‐482, and multiple site optical recording of transmembrane voltage (MSORTV) were used to reveal the electrophysiological properties of the parallel fibre action potential and to measure its conduction (0.13 m/s). The parallel fibre action potential was blocked in the presence of tetrodotoxin (TTX) and prolonged by tetraethylammonium (TEA), suggesting that the upstroke depends upon sodium entry and the repolarization upon potassium efflux. An after‐hyperpolarization results from a calcium‐dependent potassium conductance. 3. A second potentiometric dye, RH‐155, differing only slightly from RH‐482, exhibited a high affinity for glial cell membrane, and could be used to monitor changes in extracellular potassium concentration by detecting changes in glial membrane potential. 4. Calcium channel blockers such as cadmium ions blocked the optical signal that reflected the extracellular accumulation of potassium. 5. Interventions that modified the extracellular volume, and thereby affected the accumulation of potassium, produced large changes in the optical signal that monitored glial depolarization. Hypertonic and hypotonic bathing solutions resulted in decreases and increases, respectively, in the magnitude of the extrinsic absorption change that tracked potassium accumulation. 6. Blocking sodium‐potassium pump activity by means of ouabain prolonged the time course of the optical signal that was related to potassium accumulation in the extracellular space. 7. Extracellular potassium accumulation was revealed to be critically dependent upon intracellular calcium ions.

Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale.

We have applied multiple site optical recording of transmembrane voltage (MSORTV) to patterned growth cultures of heart cells to analyze the effect of geometry per se on impulse propagation in excitable tissue, with cellular and subcellular resolution. Extensive dye screening led to the choice of di-8-ANEPPS as the most suitable voltage-sensitive dye for this application; it is internalized slowly and permits optical recording with signal-to-noise ratios as high as 40:1 (measured peak-to-peak) and average fractional fluorescence changes of 15% per 100 mV. Using a x 100 objective and a fast data acquisition system, we could resolve impulse propagation on a microscopic scale (15 microns) with high temporal resolution (uncertainty of +/- 5 microseconds). We could observe the decrease in conduction velocity of an impulse propagating along a narrow cell strand as it enters a region of abrupt expansion, and we could explain this phenomenon in terms of the micro-architecture of the tissue. In contrast with the elongated and aligned cells forming the narrow strands, the cells forming the expansions were aligned at random and presented 2.5 times as many cell-to-cell appositions per unit length. If the decrease in conduction velocity results entirely from this increased number of cell-to-cell boundaries per unit length, the mean activation delay introduced by each boundary can be estimated to be 70 microseconds. Using this novel experimental system, we could also demonstrate the electrical coupling of fibroblasts and endotheloid cells to myocytes in culture.

Novel naphthylstyryl-pyridinium potentiometric dyes offer advantages for neural network analysis

The submucous plexus of the guinea pig intestine is a quasi-two-dimensional mammalian neural network that is particularly amenable to study using multiple site optical recording of transmembrane voltage (MSORTV) [Biol. Bull. 183 (1992) 344; J. Neurosci. 19 (1999) 3073]. For several years the potentiometric dye of choice for monitoring the electrical activity of its individual neurons has been di-8-ANEPPS [Neuron 9 (1992) 393], a naphthylstyryl-pyridinium dye with a propylsulfonate headgroup that provides relatively large fluorescence changes during action potentials and synaptic potentials. Limitations to the use of this dye, however, have been its phototoxicity and its low water solubility which requires the presence of DMSO and Pluronic F-127 in the staining solution. In searching for less toxic and more soluble dyes exhibiting larger fluorescence signals, we first tried the dienylstyryl-pyridinium dye RH795 [J. Neurosci. 14 (1994) 2545] which is highly soluble in water. This dye yielded relatively large signals, but it was internalized quickly by the submucosal neurons resulting in rapid degradation of the signal-to-noise ratio. We decided to synthesize a series of naphthylstyryl-pyridinium dyes (di-n-ANEPPDHQ) having the same chromophore as di-8-ANEPPS and the quaternary ammonium headgroup (DHQ) of RH795 (resulting in two positive charges versus the neutral propylsulfonate-ring nitrogen combination), and we tested the di-methyl (JPW3039), di-ethyl (JPW2081), di-propyl (JPW3031), di-butyl (JPW5029), and di-octyl (JPW5037) analogues, all of them soluble in ethanol. We found that the di-propyl (di-3-ANEPPDHQ) and the di-butyl (di-4-ANEPPDHQ) forms yielded the best combination of signal-to-noise ratio, moderate phototoxicity and absence of dye internalization.

Charge-shift probes of membrane potential. Characterization of aminostyrylpyridinium dyes on the squid giant axon.

The characteristics of transmittance and fluorescence changes of 4-(p-aminostyryl)-1-pyridinium dyes in response to voltage-clamp pulses on the squid giant axon were examined. A zwitterionic styryl dye displays transmittance and excitation spectra on the voltage-clamped squid axon with shapes similar to those previously measured on a model membrane system and consistent with a postulated electrochromic mechanism. The speed of the transmittance response is faster than 1.2 microseconds. The size of the fluorescence change is a factor of 40 lower than on the model membrane; this diminution can be rationalized in terms of the background fluorescence from Schwann cells and the nonoptimal geometric arrangement of the axon membrane. When the emission spectrum is dissected from the excitation response, a nonelectrochromic component is found. This component might result from molecular motion during the excited state lifetime. A positively charged dye permeates the axon membrane and displays complex response waveforms dependent on the method of application and the axon holding potential. This contrasts markedly with model membrane results where the behavior of the cationic and zwitterionic dyes were indistinguishable.

A large change in dye absorption during the action potential

Recently, we began a search for easily detected changes in the optical properties of neurons that occur during excitation (1, 2). Until now, changes in extrinsic fluorescence were the most sensitive indicators of neuronal activity. More than 500 fluorescent dyes have been examined on giant axons from squid and a number were found (2) which gave fluorescence changes during single action potentials that were significantly larger than the noise in the measurements. Although it was subsequently demonstrated that one of these fluorescence signals was large enough to allow optical monitoring of action potentials in individual neurons of a leech segmental ganglion (3), larger signals would facilitate more complex experiments of this kind. Previous results (4-8) had suggested that there might be changes in light absorption of dyed axons during activity. We have now found absorption changes during the action potential using a number of dyes and several of these could be measured with a signal-to-noise ratio considerably larger than fluorescence changes obtained under the same conditions. Giant axons (diameters 320-500 ,m) from the squid, Loligo pealii, were cleaned of small fibers and placed horizontally in a chamber containing two pairs of platinum electrodes for stimulating and extracellular recording. The chamber was mounted on the stage of a Leitz Ortholux II microscope (E. Leitz, Inc., Rockleigh, N.J.). Light from a quartz-halogen tungsten-filament lamp was collimated, made quasimonochromatic with a bandpass interference filter, and focused on the axon by means of a bright-field condenser. Light collected by a 20 x (NA 0.4) objective was passed through a slit in the objective image plane which limited the light reaching a SGD-444 photodiode (E G & G, Inc., Salem, Mass.) to that originating from a 150 ,m length of axon. The axons were incubated with a solution of dye in seawater (2) for 20 min and then bathed in seawater bubbled with argon. In a few axons the intracellular potential was measured by impaling the axon with a glass microelectrode (filled with

Action Potentials and Frequency-Dependent Secretion in the Mouse Neurohypophysis

The frequency-dependence of secretion of arginine vasopressin (AVP) from the mouse neural lobe in vitro was studied and found to be comparable to that reported for the rat neural lobe in vitro. For a stimulus train of 600 pulses, the secretion of AVP per pulse (i.e., facilitation) increased to a maximum at 20 Hz. Compound intracellular action potentials were recorded from the mouse neural lobe using optical recording methods and potentiometric dyes. These extrinsic optical signals reflect the true time courses of transmembrane potential changes (e.g., action potentials), and the action potentials recorded from mouse neural lobes had a duration of 5 ms; at half-maximum peak height. Optical recordings during repetitive stimulation showed that significant spike broadening occurred in each subsequent spike at 10 and 16 Hz stimulation. These data are consistent with a spike broadening hypothesis of frequency-dependent facilitation in the neural lobe. However, 4-aminopyridine, a drug which causes spike broadening in neural tissues by blocking potassium channels, did not produce an increase in secretion of AVP per stimulus from the mouse neural lobe.

Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes

To record post synaptic potentials or electrical activity from processes of single cells in a central nervous system (CNS) preparation in situ, voltage sensitive dyes can be injected intracellularly, thereby staining only the cell under investigation. We report the structure, evaluation, and synthesis of 11 fluorescent styryl dyes developed for iontophoretic injection. The optical signals that represent small synaptic potentials from single processes of iontophoretically injected cells are expected to be very small and, therefore, such measurements are not easy. We report the methodology that permitted the optical recording of action potentials from a 3-micron axon and the recording of small synaptic potentials from the processes of single cells in the segmental ganglia of the leech. The same dyes also proved useful for optical recording of action potentials of anterogradely labeled axons, following local extracellular injection at a remote site in a mammalian CNS preparation.

Caffeine interaction with fluorescent calcium indicator dyes.

We report that caffeine, in millimolar concentrations, interacts strongly with four common calcium indicator dyes: mag-fura-2, magnesium green, fura-2, and fluo-3. Fluorescence intensities are either noticeably enhanced (mag-fura-2, fura-2) or diminished (magnesium green, fluo-3). The caffeine-induced changes in the fluorescence spectra are clearly distinct from those of metal ion binding at the indicator chelation sites. Binding affinities for calcium of either mag-fura-2 or magnesium green increased only slightly in the presence of caffeine. Caffeine also alters the fluorescence intensities of two other fluorescent dyes lacking a chelation site, fluorescein and sulforhodamine 101, implicating the fluorophore itself as the interaction site for caffeine. In the absence of caffeine, variation of solution hydrophobicity by means of water/dioxane mixtures yielded results similar to those for caffeine. These observations suggest that hydrophobic substances, in general, can alter dye fluorescence in a dye-specific manner. For the particular case of caffeine, and perhaps other commonly used pharmacological agents, the dye interactions can seriously distort fluorescence measurements of intracellular ion concentrations with metal indicator dyes.

The Challenge of Connecting the Dots in the B.R.A.I.N.

The Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative has focused scientific attention on the necessary tools to understand the human brain and mind. Here, we outline our collective vision for what we can achieve within a decade with properly targeted efforts and discuss likely technological deliverables and neuroscience progress.