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We developed genetically encoded voltage indicators using a transmembrane voltage-sensing domain and bright near-infrared fluorescent proteins derived from bacterial phytochromes. These new voltage indicators are excited by 640 nm light and emission is measured at 670 nm, allowing imaging in the near-infrared tissue transparency window. The spectral properties of our new indicators permit seamless voltage imaging with simultaneous blue-green light optogenetic actuator activation as well as simultaneous voltage-calcium imaging when paired with green calcium indicators. Iterative optimizations led to a fluorescent probe, here termed nirButterfly, which reliably reports neuronal activities including subthreshold membrane potential depolarization and hyperpolarization as well as spontaneous spiking or electrically- and optogenetically evoked action potentials. This enables largely improved all-optical causal interrogations of physiology.

Dendritic spikes in thin dendritic branches (basal and oblique dendrites) of pyramidal neurons are traditionally inferred from spikelets measured in the cell body. Here, we used laser-spot voltage-sensitive dye imaging in cortical pyramidal neurons (rat brain slices) to investigate the voltage waveforms of dendritic potentials occurring in response to spatially-restricted glutamatergic inputs. Local dendritic potentials lasted 200–500 ms and propagated to the cell body where they caused sustained 10-20 mV depolarizations. Plateau potentials propagating from dendrite to soma, and action potentials propagating from soma to dendrite, created complex voltage waveforms in the middle of the thin basal dendrite, comprised of local sodium spikelets, local plateau potentials, and back-propagating action potentials, superimposed on each other. Our model replicated these experimental observations and made the following predictions: (i) membrane time constant is shortened during the plateau; and (ii) synaptic responses are more effective drivers of neuronal action potentials during the plateau potential. Dendritic plateau potentials occurring in basal and oblique branches put pyramidal neurons into an activated neuronal state (“prepared state”), characterized by depolarized membrane potential and notably faster membrane responses. The prepared state provides a time window of 200-500 ms during which cortical neurons are particularly excitable and capable of following afferent inputs. At the network level, this predicts that sets of cells with simultaneous plateaus would provide cellular substrate for the formation of functional neuronal ensembles. Significance statement Strong and clustered glutamatergic inputs will have a major influence on activity at both neuronal and network scales. We recorded glutamate-mediated dendritic voltage plateaus using voltage imaging, and created a computer model that recreated experimental measures. Our model predicts the manner in which plateaus are triggered and the impact of inputs to an individual thin dendrite on the membrane response in the cell body. Plateau potentials profoundly change neuronal state -- a plateau potential triggered in one basal dendrite depolarizes the soma and shortens membrane time constant, making the cell more susceptible to firing triggered by other afferent inputs. We tested model predictions experimentally.

Subplate (SP) neurons exhibit spontaneous plateau depolarizations mediated by connexin hemichannels. Postnatal (P1–P6) mice show identical voltage pattern and drug-sensitivity as observed in slices from human fetal cortex; indicating that the mouse is a useful model for studying the cellular physiology of the developing neocortex. In mouse SP neurons, spontaneous plateau depolarizations were insensitive to blockers of: synaptic transmission (glutamatergic, GABAergic, or glycinergic), pannexins (probenecid), or calcium channels (mibefradil, verapamil, diltiazem); while highly sensitive to blockers of gap junctions (octanol), hemichannels (La3+, lindane, Gd3+), or glial metabolism (DLFC). Application of La3+ (100 μM) does not exert its effect on electrical activity by blocking calcium channels. Intracellular application of Gd3+ determined that Gd3+-sensitive pores (putative connexin hemichannels) reside on the membrane of SP neurons. Immunostaining of cortical sections (P1–P6) detected connexins 26, and 45 in neurons, but not connexins 32 and 36. Vimentin-positive glial cells were detected in the SP zone suggesting a potential physiological interaction between SP neurons and radial glia. SP spontaneous activity was reduced by blocking glial metabolism with DFLC or by blocking purinergic receptors by PPADS. Connexin hemichannels and ATP release from vimentin-positive glial cells may underlie spontaneous plateau depolarizations in the developing mammalian cortex.

The bAP-Ca2+ signal amplitudes in some apical tuft branches randomly vary from moment to moment. In repetitive measurements, successful AP invasions are followed by complete failures. Passive spread of voltage from the apical trunk into the tuft occasionally reaches the threshold for local Na+ spike, resulting in stronger Ca2+ influx. During a burst of three somatic APs, the peak of dendritic Ca2+ in the apical tuft occurs with a delay of 20-50 ms depending on AP frequency.

In response to food reward and other pertinent events, midbrain dopaminergic neurons fire short bursts of action potentials causing a phasic release of dopamine in the prefrontal cortex (rapid and transient increases in cortical dopamine concentration). Here we apply short (2s) iontophoretic pulses of glutamate, GABA, dopamine and dopaminergic agonists locally, onto layer 5 pyramidal neurons in brain slices of the rat medial prefrontal cortex (PFC). Unlike glutamate and GABA, brief dopaminergic pulses had negligible effects on the resting membrane potential. However, dopamine altered action potential firing in an extremely rapid (1s) and transient (5 min) manner, as every neuron returned to baseline in less than 5-min post-application. The physiological responses to dopamine differed markedly among individual neurons. Pyramidal neurons with a preponderance of D1-like receptor signaling respond to dopamine with a severe depression in action potential firing rate, while pyramidal neurons dominated by the D2 signaling pathway respond to dopamine with an instantaneous increase in spike production. Increasing levels of dopamine concentrations around the cell body resulted in a dose dependent response, which resembles an "inverted U curve" (Vijayraghavan S, Wang M, Birnbaum SG, Williams GV, Arnsten AF (2007) Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory. Nat Neurosci 10:376-384), but this effect can easily be caused by an iontophoresis current artifact. Our present data imply that one population of PFC pyramidal neurons receiving direct synaptic contacts from midbrain dopaminergic neurons would stall during the 0.5s of the phasic dopamine burst. The spillover dopamine, on the other hand, would act as a positive stimulator of cortical excitability (30% increase) to all D2-receptor carrying pyramidal cells, for the next 40s.

Basal dendrites of prefrontal cortical neurons receive strong synaptic drive from recurrent excitatory synaptic inputs. Synaptic integration within basal dendrites is therefore likely to play an important role in cortical information processing. Both synaptic integration and synaptic plasticity depend crucially on dendritic membrane excitability and the backpropagation of action potentials. We carried out multisite voltage-sensitive dye imaging of membrane potential transients from thin basal branches of prefrontal cortical pyramidal neurons before and after application of channel blockers. We found that backpropagating action potentials (bAPs) are predominantly controlled by voltage-gated sodium and A-type potassium channels. In contrast, pharmacologically blocking the delayed rectifier potassium, voltage-gated calcium, or Ih conductance had little effect on dendritic AP propagation. Optically recorded bAP waveforms were quantified and multicompartmental modeling was used to link the observed behavior with the underlying biophysical properties. The best-fit model included a nonuniform sodium channel distribution with decreasing conductance with distance from the soma, together with a nonuniform (increasing) A-type potassium conductance. AP amplitudes decline with distance in this model, but to a lesser extent than previously thought. We used this model to explore the mechanisms underlying two sets of published data involving high-frequency trains of APs and the local generation of sodium spikelets. We also explored the conditions under which IA down-regulation would produce branch strength potentiation in the proposed model. Finally, we discuss the hypothesis that a fraction of basal branches may have different membrane properties compared with sister branches in the same dendritic tree.

In CNS neurons most of synaptic integration takes place in thin dendritic branches that are difficult to study with conventional physiological recording techniques (electrodes). When cellular compartments are too small, or too many, for electrode recordings, optical methods bring considerable advantages. Here we focused our experimental effort on the development and utilization of new kinds of voltage-sensitive dyes (VSD). The new VSDs have bluish appearance in organic solvents, and hence are dubbed "blue dyes". They have preferred excitation windows for voltage recording that are shifted to longer wavelengths (approximately 660nm). Excitation in deep red light and emission in the near-infrared render "blue VSDs" potentially useful in measurements from fluorescent structures below the tissue surface because light scattering is minimized at longer wavelengths. Seven new molecules were systematically tested using intracellular injection. In comparison to the previously used red dye (JPW-3028) the blue dyes have better sensitivity (DeltaF/F) by approximately 40%. Blue dyes take little time to fill the dendritic tree, and in this aspect they are comparable with the fastest red dye JPW-3028. Based on our results, blue VSDs are well suited for experimental exploration of thin neuronal processes in semi intact preparations (brain slice). In some cases only six sweeps of temporal averaging were needed to acquire excellent records of individual action potentials in basal and oblique dendritic branches, or in axons and axon collaterals up to 200microm away from the cell body. Signal-to-noise ratio of these recordings was approximately 10. The combination of blue dyes and laser illumination approach imposed little photodynamic damage and allowed the total number of recording sweeps per cell to exceed 100. Using these dyes and a spot laser illumination technique, we demonstrate the first recording of action potentials in the oblique dendrite and distal axonal segment of the same pyramidal cell.

The origin of the action potential in the cochlea has been a long-standing puzzle. Because voltage-dependent Na+(Nav) channels are essential for action potential generation, we investigated the detailed distribution of Nav1.6 and Nav1.2 in the cochlear ganglion, cochlear nerve, and organ of Corti, including the type I and type II ganglion cells. In most type I ganglion cells, Nav1.6 was present at the first nodes flanking the myelinated bipolar cell body and at subsequent nodes of Ranvier. In the other ganglion cells, including type II, Nav1.6 clustered in the initial segments of both of the axons that flank the unmyelinated bipolar ganglion cell bodies. In the organ of Corti, Nav1.6 was localized in the short segments of the afferent axons and their sensory endings beneath each inner hair cell. Surprisingly, the outer spiral fibers and their sensory endings were well labeled beneath the outer hair cells over their entire trajectory. In contrast, Nav1.2 in the organ of Corti was localized to the unmyelinated efferent axons and their endings on the inner and outer hair cells. We present a computational model illustrating the potential role of the Nav channel distribution described here. In the deaf mutant quivering mouse, the localization of Nav1.6 was disrupted in the sensory epithelium and ganglion. Together, these results suggest that distinct Nav channels generate and regenerate action potentials at multiple sites along the cochlear ganglion cells and nerve fibers, including the afferent endings, ganglionic initial segments, and nodes of Ranvier.

Cortical information processing relies critically on the processing of electrical signals in pyramidal neurons. Electrical transients mainly arise when excitatory synaptic inputs impinge upon distal dendritic regions. To study the dendritic aspect of synaptic integration one must record electrical signals in distal dendrites. Since thin dendritic branches, such as oblique and basal dendrites, do not support routine glass electrode measurements, we turned our effort towards voltage-sensitive dye recordings. Using the optical imaging approach we found and reported previously that basal dendrites of neocortical pyramidal neurons show an elaborate repertoire of electrical signals, including backpropagating action potentials and glutamate-evoked plateau potentials. Here we report a novel form of electrical signal, qualitatively and quantitatively different from back-propagating action potentials and dendritic plateau potentials. Strong glutamatergic stimulation of an individual basal dendrite is capable of triggering a fast spike, which precedes the dendritic plateau potential. The amplitude of the fast initial spikelet was actually smaller that the amplitude of the backpropagating action potential in the same dendritic segment. Therefore, the fast initial spike was dubbed “spikelet”. Both the basal spikelet and plateau potential propagate decrementally towards the cell body, where they are reflected in the somatic whole-cell recordings. The low incidence of basal spikelets in the somatic intracellular recordings and the impact of basal spikelets on soma-axon action potential initiation are discussed.

To obtain a more complete description of individual neurons, it is necessary to complement the electrical patch pipette measurements with technologies that permit a massive parallel recording from many sites on neuronal processes. This can be achieved by using voltage imaging with intracellular dyes. With this approach, we investigated the functional structure of a mitral cell, the principal output neuron in the rat olfactory bulb. The most significant finding concerns the characteristics of EPSPs at the synaptic sites and surprisingly small attenuation along the trunk of the primary dendrite. Also, the experiments were performed to determine the number, location, and stability of spike trigger zones, the excitability of terminal dendritic branches, and the pattern and nature of spike initiation and propagation in the primary and secondary dendrites. The results show that optical data can be used to deduce the amplitude and shape of the EPSPs evoked by olfactory nerve stimulation at the site of origin (glomerular tuft) and to determine its attenuation along the entire length of the primary dendrite. This attenuation corresponds to an unusually large mean apparent “length constant” of the primary dendrite. Furthermore, the images of spike trigger zones showed that an action potential can be initiated in three different compartments of the mitral cell: the soma-axon region, the primary dendrite trunk, and the terminal dendritic tuft, which appears to be fully excitable. Finally, secondary dendrites clearly support the active propagation of action potentials.

Before the human cortex is able to process sensory information, young postmitotic neurons must maintain occasional bursts of action-potential firing to attract and keep synaptic contacts, to drive gene expression, and to transition to mature membrane properties. Before birth, human subplate (SP) neurons are spontaneously active, displaying bursts of electrical activity (plateau depolarizations with action potentials). Using whole-cell recordings in acute cortical slices, we investigated the source of this early activity. The spontaneous depolarizations in human SP neurons at midgestation (17-23 gestational weeks) were not completely eliminated by tetrodotoxin--a drug that blocks action potential firing and network activity--or by antagonists of glutamatergic, GABAergic, or glycinergic synaptic transmission. We then turned our focus away from standard chemical synapses to connexin-based gap junctions and hemichannels. PCR and immunohistochemical analysis identified the presence of connexins (Cx26/Cx32/Cx36) in the human fetal cortex. However, the connexin-positive cells were not found in clusters but, rather, were dispersed in the SP zone. Also, gap junction-permeable dyes did not diffuse to neighboring cells, suggesting that SP neurons were not strongly coupled to other cells at this age. Application of the gap junction and hemichannel inhibitors octanol, flufenamic acid, and carbenoxolone significantly blocked spontaneous activity. The putative hemichannel antagonist lanthanum alone was a potent inhibitor of the spontaneous activity. Together, these data suggest that connexin hemichannels contribute to spontaneous depolarizations in the human fetal cortex during the second trimester of gestation.

Understanding the biophysical properties of single neurons and how they process information is fundamental to understanding how the brain works. A technique that would allow recording of temporal and spatial dynamics of electrical activity in neuronal processes with adequate resolution would facilitate further research. Here, we report on the application of optical recording of membrane potential transients at many sites on neuronal processes of vertebrate neurons in brain slices using intracellular voltage-sensitive dyes. We obtained evidence that 1) loading the neurons with voltage-sensitive dye using patch electrodes is possible without contamination of the extracellular environment; 2) brain slices do not show any autofluorescence at the excitation/emission wavelengths used; 3) pharmacological effects of the dye were completely reversible; 4) the level of photodynamic damage already allows meaningful measurements and could be reduced further; 5) the sensitivity of the dye was comparable to that reported for invertebrate neurons; 6) the dye spread ∼500 μm into distal processes within 2 h incubation period. This distance should increase with longer incubation; 7) the optically recorded action potential signals from basolateral dendrites (that are difficult or impossible to approach by patch electrodes) and apical dendrites show that both direct soma stimulation and synaptic stimulation triggered action potentials that originated near the soma. The spikes backpropagated into both basolateral dendrites and apical processes; the propagation was somewhat faster in the apical dendrites.

Optimal dopamine tone is required for the normal cortical function; however it is still unclear how cortical-dopamine-release affects information processing in individual cortical neurons. Thousands of glutamatergic inputs impinge onto elaborate dendritic trees of neocortical pyramidal neurons. In the process of ensuing synaptic integration (information processing), a variety of calcium transients are generated in remote dendritic compartments. In order to understand the cellular mechanisms of dopaminergic modulation it is important to know whether and how dopaminergic signals affect dendritic calcium transients. In this chapter, we describe a relatively inexpensive method for monitoring dendritic calcium fluctuations at multiple loci across the pyramidal dendritic tree, at the same moment of time (simultaneously). The experiments have been designed to measure the amplitude, time course and spatial extent of action potential-associated dendritic calcium transients before and after application of dopaminergic drugs. In the examples provided here the dendritic calcium transients were evoked by triggering the somatic action potentials (backpropagation-evoked), and puffs of exogenous dopamine were applied locally onto selected dendritic branches.

The physiological responses of dendrites to dopaminergic inputs are poorly understood and controversial. We applied dopamine on one dendritic branch while simultaneously monitoring action potentials (APs) from multiple dendrites using either calcium-sensitive dye, voltage-sensitive dye or both. Dopaminergic suppression of dendritic calcium transients was rapid (

Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440–670 nm; the two-photon excitation range is 900–1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.

Our knowledge about the developing human cerebral cortex is based on the analysis of fixed postmortem material. Here we use electrical recordings from unfixed human postmortem tissue to characterize the synaptic physiology and spontaneous network activity of pioneer cortical neurons (“subplate neurons”). Our electrophysiological experiments show that functional glutamate or GABA ionotropic receptors are expressed on human subplate (SP) neurons as early as 20 gestational weeks. Extracellular (synaptic) stimulations evoked postsynaptic potentials in a very small fraction of SP neurons, suggesting that functional synaptic contacts are rare at midgestation. Although synaptic inputs were scarce, we regularly observed spontaneous (unprovoked) electrical activity among human SP neurons, comprised of sustained plateau depolarizations and bursts of action potential firing, which resembled cortical UP and DOWN states in the adult neocortex. Plateau depolarizations and bursts of action potential firing are thought to depend on the mature morphology and physiology of adult cortical network. However, our current data reveal that similar cortical rhythm is generated by a very immature ensemble of human fetal neurons. In the relative absence of sensory inputs, as in developmentin utero, or in slow-wave sleep (i.e., throughout the entire lifespan), the spontaneous slow oscillatory pattern (UP and DOWN states) is a fundamental aspect of human cortical physiology.

Neurons derived from human embryonic stem cells hold promise for the therapy of neurological diseases. Quality inspection of human embryonic stem cell-derived neurons has often been based on immunolabeling for neuronal markers. Here we put emphasis on their physiological properties. Electrophysiological measurements were carried out systematically at different stages of neuronal in vitro development, including the very early stage, neuroepithelial rosettes. Developing human neurons are able to generate action potentials (APs) as early as 10 days after the start of differentiation. Tyrosine hydroxylase (TH)-positive (putative dopaminergic, DA) neurons tend to aggregate into clumps, and their overall yield per coverslip is relatively low (8.3%) because of areas void of DA neurons. On the same in vitro day, neighboring neurons can be in very different stages of differentiation, including repetitive AP firing, single full-size AP, and abortive AP. Similarly, the basic electrophysiological parameters (resting membrane potential, input resistance, peak sodium, and peak potassium currents) are scattered in a wide range. Visual appearance of differentiating neurons, and number of primary and secondary dendrites cannot be used to predict the peak sodium current or AP firing properties of cultured neurons. Approximately 13% of neurons showed evidence of hyperpolarization-induced current (I(h)), a characteristic of DA neurons; however, no neurons with repetitive APs showed I(h). The electrophysiological measurements thus indicate that a standard DA differentiation (dibutyryl cyclic AMP-based) protocol, applied for 2-5 weeks, produces a heterogeneous ensemble of mostly immature neurons. The overall quality of human neurons under present conditions (survival factors were not used) begins to deteriorate after 12 days of differentiation.

In the field of cortical cellular physiology, much effort has been invested in understanding thick apical dendrites of pyramidal neurons and the regenerative sodium and calcium spikes that take place in the apical trunk. Here we focus on thin dendrites of pyramidal cells (basal, oblique, and tuft dendrites), and we discuss one relatively novel form of an electrical signal ("NMDA spike") that is specific for these branches. Basal, oblique, and apical tuft dendrites receive a high density of glutamatergic synaptic contacts. Synchronous activation of 10-50 neighboring glutamatergic synapses triggers a local dendritic regenerative potential, NMDA spike/plateau, which is characterized by significant local amplitude (40-50 mV) and an extraordinary duration (up to several hundred milliseconds). The NMDA plateau potential, when it is initiated in an apical tuft dendrite, is able to maintain a good portion of that tuft in a sustained depolarized state. However, if NMDA-dominated plateau potentials originate in proximal segments of basal dendrites, they regularly bring the neuronal cell body into a sustained depolarized state, which resembles a cortical Up state. At each dendritic initiation site (basal, oblique, and tuft) an NMDA spike creates favorable conditions for causal interactions of active synaptic inputs, including the spatial or temporal binding of information, as well as processes of short-term and long-term synaptic modifications (e.g., long-term potentiation or long-term depression). Because of their strong amplitudes and durations, local dendritic NMDA spikes make up the cellular substrate for multisite independent subunit computations that enrich the computational power and repertoire of cortical pyramidal cells. We propose that NMDA spikes are likely to play significant roles in cortical information processing in awake animals (spatiotemporal binding, working memory) and during slow-wave sleep (neuronal Up states, consolidation of memories).

Basal dendrites of neocortical pyramidal neurons are relatively short and directly attached to the cell body. This allows electrical signals arising in basal dendrites to strongly influence the neuronal output. Likewise, somatic action potentials (APs) should readily propagate back into the basilar dendritic tree to influence synaptic plasticity. Two recent studies, however, determined that sodium APs are severely attenuated in basal dendrites of cortical pyramidal cells, so that they completely fail in distal dendritic segments. Here we used the latest improvements in the voltage-sensitive dye imaging technique (Zhou et al., 2007) to study AP backpropagation in basal dendrites of layer 5 pyramidal neurons of the rat prefrontal cortex. With a signal-to-noise ratio of > 15 and minimal temporal averaging (only four sweeps) we were able to sample AP waveforms from the very last segments of individual dendritic branches (dendritic tips). We found that in short- (< 150 microm) and medium (150-200 microm in length)-range basal dendrites APs backpropagated with modest changes in AP half-width or AP rise-time. The lack of substantial changes in AP shape and dynamics of rise is inconsistent with the AP-failure model. The lack of substantial amplitude boosting of the third AP in the high-frequency burst also suggests that in short- and medium-range basal dendrites backpropagating APs were not severely attenuated. Our results show that the AP-failure concept does not apply in all basal dendrites of the rat prefrontal cortex. The majority of synaptic contacts in the basilar dendritic tree actually received significant AP-associated electrical and calcium transients.

Basal and oblique dendrites comprise ~2/3 of the total excitable membrane in the mammalian cerebral cortex, yet they have never been probed with glass electrodes, and therefore their electrical properties and overall impact on synaptic processing are unknown. In the present study, fast multi-site voltage-sensitive dye imaging combined with somatic recording was used to provide a detailed description of the membrane potential transients in basal and oblique dendrites of pyramidal neurons during single and trains of action potentials (APs). The optical method allowed simultaneous measurements from several dendrites in the visual field up to 200 microm from the soma, thus providing a unique report on how an AP invades the entire dendritic tree. In contrast to apical dendrites, basal and oblique branches: (1) impose very little amplitude and time course modulation on backpropagating APs; (2) are strongly invaded by the somatic spike even when somatic firing rates reach 40 Hz (activity-independent backpropagation); and (3) do not exhibit signs of a 'calcium shoulder' on the falling phase of the AP. A compartmental model incorporating AP peak latencies and half-widths obtained from the apical, oblique and basal dendrites indicates that the specific intracellular resistance (Ri) is less than 100 omicron cm. The combined experimental and modelling results also provide evidence that all synaptic locations along basal and oblique dendrites, situated within 200 microm from the soma, experience strong and near-simultaneous (latency1 ms) voltage transients during somatic firing. The cell body, axon hillock and basal dendritic compartments achieve unique synchronization during each AP. Therefore, with respect to a retrograde signal (AP), basal and proximal oblique dendrites should be considered as an integral part of the axo-somatic compartment.

Optical recording with a voltage-sensitive dye is advantageous where membrane potential must be recorded in many sites at once. This unit describes methods for making voltage-sensitive dye measurements on different preparations to study (1) how a neuron integrates its synaptic input into its action potential output by measuring membrane potential everywhere synaptic input occurs and where spikes are initiated; (2) how a nervous system generates a behavior in Aplysia abdominal ganglion; and (3) responses to sensory stimuli and generation of motor output in the vertebrate brain by simultaneous measurement of population signals from many areas. The approach is three-pronged: (1) find the dye with the largest signal-to-noise ratio; (2) reduce extraneous sources of noise; and (3) maximize the number of photons measured to reduce the relative shot noise. A discussion of optical recording methods including the choice of dyes, light sources, optics, cameras, and minimizing noise is also provided.

The present understanding of how individual nerve cells process information is incomplete. The classical model of the neuron (Rall, 1967) is one based on early studies of vertebrate spinal motoneurons (Coombs et al. 1957; Eccles, 1957; Fatt, 1957a,b) and the formal description developed by Rall using the simplifying assumption that dendrites behave as passive electrical elements. This model is currently being replaced by a more detailed description that involves complex, non-linear, active properties of the dendritic membrane (Wong et al. 1979; Llinas & Sugimori, 1980; Lasser-Ross & Ross, 1992; Stuart & Sakmann, 1994; De Schutter & Bower, 1994; Traub et al. 1994; Spruston et al. 1995; Chen et al. 1997; Cash & Yuste, 1999; Gongyu et al. 1999). An important consequence of active dendrites is that regional electrical properties of branching neuronal processes and the functional organization of individual neurons will be extraordinarily complex, dynamic and, in general, impossible to predict using a model derived in the absence of detailed measurements. To obtain such measurements, one would ideally like to be able to monitor, at multiple sites, subthreshold events as they propagate from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. It is important to be able to perform these measurements in at least partially intact neuronal structures (isolated invertebrate ganglia or tissue slices of vertebrate CNS) to ensure that highly specific regional electrical properties of individual neurons (Tauc & Hughes, 1963; Llinas & Sugimori, 1980; Stuart & Sakmann, 1994; Stuart & Hausser, 1994) and characteristic synaptic connections, largely lost in dissociated primary cultures, are preserved. The development of recording methods that would approximate these ideal requirements has been slow. Direct electrical measurements of the detailed spatial distribution and dynamics of voltage transients from neuronal processes are not possible due to size considerations. A true multi-site recording might, however, be achieved by using voltage-sensitive dyes (Cohen & Salzberg, 1978; Wu & Cohen, 1993). Recently, the sensitivity of intracellular voltage-sensitive dye techniques for monitoring voltage transients from neuronal processes in situ, introduced by Grinvald et al. (1987), has been improved 100-fold (Antic & Zecevic, 1995). We have used this approach previously to analyse the metacerebral cell from the terrestrial snail, Helix aspersa, and found multiple spike trigger zones for action potentials evoked by electrical stimulation of the soma. It was possible to determine the precise position of one of these sites (Zecevic, 1996). The metacerebral neuron is a bilaterally paired serotonergic modulatory interneuron whose activity accounts for some aspects of food-induced arousal in Helix, Aplysia and other molluscs (Kupferman & Weiss, 1982; Yeoman et al. 1996). The metacerebral neurons innervate the muscles controlling biting and serve to modulate muscle contractions resulting from the firing of motoneurons during feeding. It has been proposed that the metacerebral cell has gating/enabling function in feeding behaviour because a minimum level of tonic firing was required to support feeding in fine-wire recording experiments in the intact animal (Yeoman et al. 1994). The metacerebral cell fires action potentials in response to food stimuli applied to the lips of the animal. It has been postulated that excitatory inputs to metacerebral cells are mediated through interneurons (Horn et al. 1999). However, the exact nature of these synaptic inputs is unknown. Here, we analysed the initiation and propagation of action potentials evoked synaptically. First, the location of two trigger zones for synaptically evoked spikes was determined. The position of the trigger zones was stable and similar for both synaptically evoked action potentials and spikes elicited by electrical stimulation of the soma. Furthermore, we found that different sets of synaptic inputs activate different trigger zones. Also, we showed that a spike initiated at a remote axonal site did not always invade all parts of the neuron; the conduction of the axonal impulse was regularly blocked at particular locations and the failure of propagation was monitored directly. Finally, the propagating spikes in some axonal branches consistently reversed direction at certain branch points, a phenomenon known as reflection.

We carried out experiments to monitor optically the generation and spread of action potentials and subthreshold potentials in the processes of individual neurons in ganglia of the snail, Helix aspersa. The neurons were selectively stained by intracellular pressure injection of voltage-sensitive dyes. Optical signals were detected by a system for fast, multiple-site optical monitoring, utilizing a silicon photodiode array. After testing 30 voltage-sensitive dyes using absorption, we concluded that this mode was probably not sensitive enough to allow monitoring neuronal signals from distal processes. Satisfactory signals were obtained in fluorescence measurements using a newly synthesized styryl dye, JPW1114, specifically designed for intracellular application. There was an improvement in sensitivity (as defined by the signal-to-noise ratio) by a factor of about 50 over previously reported absorption and fluorescence signals from neuronal processes stained by either intra- or extracellular application of dyes. Recordings with good signal-to-noise ratio and adequate spatial and temporal resolution were obtained simultaneously from the cell body and long axonal branches. From this data, the site of action potential initiation was determined. Also, the propagation velocity of the action potential was calculated for different axonal segments; the results suggest that different regions have different velocities ranging from 0.53 m/sec to 0.07 m/sec. The present sensitivity was adequate to allow the recording of a 10 mV hyperpolarizing electrotonic response along axonal branches and to observe directly the decline of this passive response with distance from the site of stimulation. Relatively modest improvements in sensitivity will allow systematic analyses of the spread and summation of synaptic potentials in individual neurons.