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.     

Depletion type floating gate p-channel MOS transistor for recording action potentials generated by cultured neurons

We report the realization of electrical coupling between neurons and depletion type floating gate (FG) p-channel MOS transistors. The devices were realized in a shortened 0.5 μm CMOS technology. Increased boron implant dose was used to form the depletion type devices. Post-CMOS processing steps were added to expose the devices sensing area. The neurons are coupled to the polycrystalline silicon (PS) FG through 420 Å thermal oxide in an area which is located over the thick field oxide away from the transistor. The combination of coupling area pad having a diameter of 10 or 15 μm and sensing transistor with W/L of 50/0.5 μm results in capacitive coupling ratio of the neuron signal of about 0.5 together with relatively large transistor transconductance. The combination of the FG structure with a depletion type device, leads to the following advantages. (a) No need for dc bias between the solution in which the neurons are cultured and the transistor with expected consequences to the neuron as well as the silicon die durability. (b) The sensing area of the neuron activity is separated from the active area of the transistor. Thus, it is possible to design the sensing area and the channel area separately. (c) The channel area, which is the most sensitive part of the transistor, can be insulated and shielded from the ionic solution in which the neurons are cultured. (d) There is an option to add a switching transistor to the FG and use the FG also for the neuron stimulation.     

The action potential in mammalian central neurons

The action potential of the squid giant axon is formed by just two voltage-dependent conductances in the cell membrane, yet mammalian central neurons typically express more than a dozen different types of voltage-dependent ion channels. This rich repertoire of channels allows neurons to encode information by generating action potentials with a wide range of shapes, frequencies and patterns. Recent work offers an increasingly detailed understanding of how the expression of particular channel types underlies the remarkably diverse firing behaviour of various types of neurons.     

In vivo recording of monophasic action potentials in awake dogs

Assessment of cardiac repolarization in dogs in vivo is of interest in numerous experimental canine models. Previous studies have used monophasic action potentials (MAPs) to investigate repolarization in vitro and in vivo in anesthetized animal models. Therefore, an approach for recording MAPs in awake dogs without the interference of anesthesia is desirable. We describe an experimental technique to record MAPs in conscious dogs by means of conventional rubber introducers which are implanted into the internal jugular vein. Atrial as well as ventricular MAPs can be simultaneously measured without complications. Pacing thresholds are low and stable over time. Continuous MAP recordings of stable amplitude can be achieved from the same endocardial site for periods up to 1h. Antegrade and retrograde atrioventricular nodal conduction properties can be assessed. Programmed stimulation can be performed to simultaneously determine local refractory periods and MAP duration at cycle lengths from 500 to 200ms. In awake, unsedated dogs measuring MAPs via rubber introducers permits safe, long-term recording of MAPs. Such recordings may be useful for safety pharmacologic studies in evaluating cardiovascular and noncardiovascular drugs with respect to their effects on repolarization. In various canine in vivo models including in vivo models of long QT syndrome, heart failures or sudden cardiac death, the present technique permits electrophysiologic measurements without the interference of anesthesia.     

Voltage-sensitive dye recording of action potentials and synaptic potentials from sympathetic microcultures.

Given the appropriate multicell electrophysiological techniques, small networks of cultured neurons (microcultures) are well suited to long-term studies of synaptic plasticity. To this end, we have developed an apparatus for optical recording from cultured vertebrate neurons using voltage-sensitive fluorescent dyes (Chien, C.-B., and J. Pine. 1991. J. Neurosci. Methods. 38:93-105). We evaluate here the usefulness of this technique for recording action potentials and synaptic potentials in microcultures of neurons from the rat superior cervical ganglion (SCG). After extensive dye screening and optimization of conditions, we chose the styryl dye RH423, which gave fast linear fluorescence changes of approximately 1%/100 mV for typical recordings. The root mean square noise of the apparatus (limited by shot noise) was typically 0.03%, equivalent to 3 mV of membrane potential. Illumination for at least 100 flashes of 100 ms each caused no noticeable photodynamic damage. Our results show that voltage-sensitive dyes can be used to record from microcultures of vertebrate neurons with high sensitivity. Dye signals were detected from both cell bodies and neurites. Signals from presumptive dendrites showed hyperpolarizations and action potentials simultaneous with those in the cell body, while those from presumptive axons showed delayed propagating action potentials. Subthreshold synaptic potentials in the cell body were occasionally detectable optically; however, they were usually masked by signals from axons passing through the same pixel. This is due to the complex anatomy of SCG microcultures, which have many crisscrossing neurites that often pass over cell bodies. Given a simpler microculture system with fewer neurites, it should be possible to use dye recording to routinely measure subthreshold synaptic strengths.     

Regulation of back-propagating action potentials in hippocampal neurons.

Protein kinase C has recently been shown to modulate the slow recovery from inactivation of Na+ channels in apical dendrites of hippocampal CA1 pyramidal neurons. Moreover, dendritic, A-type K+ channels have been found to be modulated by protein kinases A and C and by mitogen-activated protein kinase. The electrical signalling ability of these dendrites is thus highly regulated by a number of neurotransmitters and second-messenger systems.     

Optical recording of the electrical activity of synaptically interacting Aplysia neurons in culture using potentiometric probes.

We used multiple-site optical recording methods, in conjunction with impermeant molecular probes of the cell membrane potential, to record the electrical activity of model neural circuits in vitro. Our system consisted of co-cultured pairs of left upper quadrant neurons from the abdominal ganglion of the marine gastropod Aplysia. These neurons interact via inhibitory synapses in vitro. Photodynamic damage to the neurons was essentially eliminated over the time course of the measurements, approximately less than 30 s, by removing oxygen from the recording solution and replacing it with argon. This procedure did not affect the synaptic interactions. We observed repetitive spiking activity in single-trace optical recordings with a maximum signal-to-noise ratio per detector of approximately 50. Individual optical signals that corresponded to either the activity of the presynaptic neuron or that of the postsynaptic neuron were clearly identified. This allowed us to monitor the activity of synaptically interacting neurons, observed as a reduction of the firing rate of the postsynaptic cell after activity of the presynaptic cell. Our results demonstrate that optical methods are appropriate for recording prolonged, asynchronous activity from synaptically interacting neurons in culture.     

Optical recording of action potential propagation in demyelinated frog nerve.

Conduction in focally demyelinated frog nerves has been measured optically using potential-sensitive dyes. Absorption changes were recorded with an array of photodiodes positioned in the image plane of a microscope. Both the amplitude and conduction velocity of the optical signals decreased in the demyelinated region. Conduction was improved after exposure to the potassium channel blocking agent 4-aminopyridine.     

Stepwise action potentials of the soma membrane of molluscan neurons

Experiments were carried out on neurons of the visceral complex of ganglia ofHelix pomatia. Application of strong hyperpolarizing stimuli ("electro-convulsive shock") throughan intracellular microelectrode led to dissociation of the original action potential into small components. Repetition of the "electro-convulsive shock" intensified these phenomena. Regular hyperpolarizing stimuli led to the formation of action potentials whose amplitude depended on the intensity and duration of the hyperpolarizing stimuli. The possibility that trigger zones are located on the soma membrane of molluscan neurons is discussed on the basis of the results.     

Dipole characterization of single neurons from their extracellular action potentials

The spatial variation of the extracellular action potentials (EAP) of a single neuron contains information about the size and location of the dominant current source of its action potential generator, which is typically in the vicinity of the soma. Using this dependence in reverse in a three-component realistic probe + brain + source model, we solved the inverse problem of characterizing the equivalent current source of an isolated neuron from the EAP data sampled by an extracellular probe at multiple independent recording locations. We used a dipole for the model source because there is extensive evidence it accurately captures the spatial roll-off of the EAP amplitude, and because, as we show, dipole localization, beyond a minimum cell-probe distance, is a more accurate alternative to approaches based on monopole source models. Dipole characterization is separable into a linear dipole moment optimization where the dipole location is fixed, and a second, nonlinear, global optimization of the source location. We solved the linear optimization on a discrete grid via the lead fields of the probe, which can be calculated for any realistic probe + brain model by the finite element method. The global source location was optimized by means of Tikhonov regularization that jointly minimizes model error and dipole size. The particular strategy chosen reflects the fact that the dipole model is used in the near field, in contrast to the typical prior applications of dipole models to EKG and EEG source analysis. We applied dipole localization to data collected with stepped tetrodes whose detailed geometry was measured via scanning electron microscopy. The optimal dipole could account for 96% of the power in the spatial variation of the EAP amplitude. Among various model error contributions to the residual, we address especially the error in probe geometry, and the extent to which it biases estimates of dipole parameters. This dipole characterization method can be applied to any recording technique that has the capabilities of taking multiple independent measurements of the same single units.     

Dendritic action potentials of pyramidal tract neurons in the cat sensorimotor cortex

The intracellular activity of pyramidal tract neurons was studied during electrical stimulation of ventrolateral and ventroposterolateral thalamic nuclei in acute experiments on cats immobilized by myorelaxants. Somatic action potentials were observed and spontaneous spikes were also produced by single and rhythmic stimulation of the thalamic nuclei at the rate of 8–14 Hz, by iontophoretic application of strychnine, and by intracellular depolarizing current pulses. These potentials had a relatively low and variable amplitude of 5–60 mV and are presumed to be dendritic action potentials. It is postulated that these variable potentials arise in the dendrites of pyramidal neurons with multiple zones generating such activity. No interaction was observed where somatic and dendritic action potentials occur simultaneously. The possible functional role of dendritic action potentials is discussed.I. S. Beritashvili Institute of Physiology, Academy of Sciences of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 18, No. 4, pp. 435–443, July–August, 1986.     

Properties of action potentials carried by divalent cations in identified leech neurons

Properties of divalent cation potentials carried by either Sr2+ or Ca2+ ions in Na+-free, TEA-Ringer solution were characterized in identified neurons of two species of leeches (Macrobdella and Haementeria). In Macrobdella, the overshoot of the potentials varied logarithmically with [Sr2+]0 (28.5 mV per 10-fold change). The overshoot, Vmax, and duration of the potentials increased with increasing divalent cation concentration and saturated at about 20 to 30 mM [Sr2+]0. The Vmax, amplitude, and duration of the potentials were reversibly blocked by Co2+ and Mn2+. The block by Mn2+ could be well-fitted by a reverse Langmuir-curve with an apparent KI of 100 micromolar. The local anesthetic procaine also reversibly inhibited the Vmax and duration of the potentials. The inhibition was greater at alkaline pH suggesting that procaine blocks the calcium channel from inside the membrane. The identified leech neurons examined in Macrobdella varied considerably in their ability to sustain somatic divalent cation potentials. Stimulation of T cells and most motoneurons produced no or only weak potentials, whereas stimulation of Retzius, N, Nut, and AP cells evoked overshooting potentials of several seconds' duration. Stimulation of the ALG cell of Haementeria in normal Ringer solution evoked a slowly-rising, purely Ca2+-dependent potential of approximately 100 ms duration. This response was TTX-resistant, unaffected by complete removal of Na+ from the Ringer solution, and abolished by 1 mM Mn2+. The overshoot varied logarithmically with a slope of 28 mV/decade change in [Ca2+]0.     

Zinc-dependent action potentials in giant neurons of the snail,Euhadra quaestia

Summary In giant neurons of subesophageal ganglion of the Japanese land snail,Euhadra quaestia Deshayes, permeation of Zn ions through Ca channels were investigated with a conventional current clamp method.All-or-none action potentials of long duration (90 to 120 sec) were evoked in 24mm Zn containing salines. The overshoots were about +10 mV and the maximum rate of rises (MRRs) was about 2.9 V/sec. The amplitudes and the MRRs of the action potentials depended on external Zn ion concentrations.The action potentials were suppressed by specific Ca-channel inhibitors such as Co²⁺, La³⁺ and Verapamil, but they were resistant to Na-channel inhibitor, tetrodotoxin, even at 30 m.It is concluded that these action potentials are generated by Zn ions permeating Ca channels in snail neuronal membrane.On the basis of Hagiwara and Takahashi's (S. Hagiwara & K. Takahashi, 1967,J. Gen. Physiol. 50:583) model of Ca channels, it is inferred that Zn ions are 5 to 10 times stronger in affinity to Ca channels than Ca ions, but 10 to 20 times less permeable.     

Complex action potentials of secondary neurons of the rat olfactory bulb

Complex action potentials arising spontaneously or evoked by stimulation of the lateral olfactory tract in secondary neurons of the rat olfactory bulb were recorded. The amplitude and duration of the complex potentials varied depending on synchronization of onset of the individual components (of which more than four were distinguished) and their combination. It is suggested that complex potentials were recorded in cases when the electrode was located in the region of the junction between spike-generating zones (the branching node of the dendrite, the junction of the soma with the dendrites and axon). It is concluded that there are numerous generating zones in the dendrites of the secondary olfactory neurons. Evoked action potentials appeared after the following latent periods: first, about 1 msec; second, about 2 msec; and third, about 3 msec. The results of the analysis showed that the antidromic response appeared after the shortest latent period. These results are evidence of the existence of considerable and varied representation of excitatory synapses in secondary neurons (besides the excitatory input in the olfactory glomeruli).M. B. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 8, No. 6, pp. 575–582, November–December, 1976.