Dejan Zecevic

Imaging membrane potential with voltage-sensitive dyes

Membrane potential can be measured optically using a variety of molecular probes. These measurements can be useful in studying function at the level of an individual cell, for determining how groups of neurons generate a behavior, and for studying the correlated behavior of populations of neurons. Examples of the three kinds of measurements are presented. The signals obtained from these measurements are generally small. Methodological considerations necessary to optimize the resulting signal-to-noise ratio are discussed.

Voltage Imaging from Dendrites of Mitral Cells: EPSP Attenuation and Spike Trigger Zones

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.

Action Potentials Initiate in the Axon Initial Segment and Propagate through Axon Collaterals Reliably in Cerebellar Purkinje Neurons

Purkinje neurons are the output cells of the cerebellar cortex and generate spikes in two distinct modes, known as simple and complex spikes. Revealing the point of origin of these action potentials, and how they conduct into local axon collaterals, is important for understanding local and distal neuronal processing and communication. By using a recent improvement in voltage-sensitive dye imaging technique that provided exceptional spatial and temporal resolution, we were able to resolve the region of spike initiation as well as follow spike propagation into axon collaterals for each action potential initiated on single trials. All fast action potentials, for both simple and complex spikes, whether occurring spontaneously or in response to a somatic current pulse or synaptic input, initiated in the axon initial segment. At discharge frequencies of less than ∼250 Hz, spikes propagated faithfully through the axon and axon collaterals, in a saltatory manner. Propagation failures were only observed for very high frequencies or for the spikelets associated with complex spikes. These results demonstrate that the axon initial segment is a critical decision point in Purkinje cell processing and that the properties of axon branch points are adjusted to maintain faithful transmission.

Hundreds of neurons in the Aplysia abdominal ganglion are active during the gill-withdrawal reflex

A combination of optical and electrode recording methods was used to obtain an overview of the neuron activity in the Aplysia abdominal ganglion in response to a light touch to the siphon skin. Spike activity was detected in up to 150 different neurons. Habituation and sensitization of the gill-withdrawal reflex was accompanied by large changes in the number of activated neurons. It is likely that these recordings are incomplete; the actual number of activated neurons is estimated to be about 300 in the acutely sensitized preparation. While we presume that not all 300 of these neurons are involved in the gill- withdrawal reflex, the number of neurons is so large that it may be difficult to determine the role of each activated neuron with presently available experimental tools.

Imaging brain activity with voltage- and calcium-sensitive dyes

This paper presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes and then discusses the methodological aspects of the measurements that are needed to achieve an optimal signal-to-noise ratio.Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations.Both invertebrate and vertebrate ganglia can be bathed in voltage-sensitive dyes to stain all of the cell bodies in the preparation. These dyes can then be used to follow the spike activity of many neurons simultaneously while the preparations are generating behaviors.Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb. There they can be used to measure the input from the nose to the bulb.Three kinds of noise are discussed. a. Shot noise from the random emission of photons from the preparation. b. Vibrational noise from external sources. c. Noise that occurs in the absence of light, the dark noise.Three different parts of the light measuring apparatus are discussed: the light sources, the optics, and the cameras.The major effort presently underway to improve the usefulness of optical recordings of brain activity are to find methods for staining individual cell types in the brain. Most of these efforts center around fluorescent protein sensors of activity.

The spatio‐temporal characteristics of action potential initiation in layer 5 pyramidal neurons: a voltage imaging study

Non‐technical summary  Axons are long process of nerve cells that mediate communication and control within the organism. Mammalian neurons have developed a complex ion channel clustering mechanism in axons to optimize rapid signalling. It has been discovered recently that intricate details of the spatial pattern of channel clustering play a critical role in signal processing in the axon. The electrical properties of axons, however, have been difficult to study using electrodes because axons are very small in diameter. We took advantage of a critical methodological improvement in the high sensitivity membrane potential imaging (Vm imaging) technique to study electrical correlates of channel clustering in the axon of cortical neurons. Voltage imaging revealed the location and length of the axonal site for nerve impulse initiation as well as the pattern of saltatory conduction in myelinated axons in the form of dynamic spatial maps of transmembrane potential.

Dendritic signals from rat hippocampal CA1 pyramidal neurons during coincident pre‐ and post‐synaptic activity: a combined voltage‐ and calcium‐imaging study

The non‐linear and spatially inhomogeneous interactions of dendritic membrane potential signals that represent the first step in the induction of activity‐dependent long‐term synaptic plasticity are not fully understood, particularly in dendritic regions which are beyond the reach of electrode measurements. We combined voltage‐sensitive‐dye recordings and Ca2+ imaging of hippocampal CA1 pyramidal neurons to study large regions of the dendritic arbor, including branches of small diameter (distal apical and oblique dendrites). Dendritic membrane potential transients were monitored at high spatial resolution and correlated with supra‐linear [Ca2+]i changes during one cycle of a repetitive patterned stimulation protocol that typically results in the induction of long‐term potentiation (LTP). While the increase in the peak membrane depolarization during coincident pre‐ and post‐synaptic activity was required for the induction of supra‐linear [Ca2+]i signals shown to be necessary for LTP, the change in the baseline‐to‐peak amplitude of the backpropagating dendritic action potential (bAP) was not critical in this process. At different dendritic locations, the baseline‐to‐peak amplitude of the bAP could be either increased, decreased or unaltered at sites where EPSP–AP pairing evoked supra‐linear summation of [Ca2+]i transients. We suggest that modulations in the bAP baseline‐to‐peak amplitude by local EPSPs act as a mechanism that brings the membrane potential into the optimal range for Ca2+ influx through NMDA receptors (0 to −15 mV); this may require either boosting or the reduction of the bAP, depending on the initial size of both signals.

Rapid time course of action potentials in spines and remote dendrites of mouse visual cortex neurons

Axonally initiated action potentials back‐propagate into spiny dendrites of central mammalian neurons and thereby regulate plasticity at excitatory synapses on individual spines as well as linear and supralinear integration of synaptic inputs along dendritic branches. Thus, the electrical behaviour of individual dendritic spines and terminal dendritic branches is critical for the integrative function of nerve cells. The actual dynamics of action potentials in spines and terminal branches, however, are not entirely clear, mostly because electrode recording from such small structures is not feasible. Additionally, the available membrane potential imaging techniques are limited in their sensitivity and require substantial signal averaging for the detection of electrical events at the spatial scale of individual spines. We made a critical improvement in the voltage‐sensitive dye imaging technique to achieve multisite recordings of backpropagating action potentials from individual dendritic spines at a high frame rate. With this approach, we obtained direct evidence that in layer 5 pyramidal neurons from the visual cortex of juvenile mice, the rapid time course of somatic action potentials is preserved throughout all cellular compartments, including dendritic spines and terminal branches of basal and apical dendrites. The rapid time course of the action potential in spines may be a critical determinant for the precise regulation of spike timing‐dependent synaptic plasticity within a narrow time window.

Functional profile of the giant metacerebral neuron of Helix aspersa: temporal and spatial dynamics of electrical activity in situ

1 Understanding the biophysical properties of single neurons and how they process information is fundamental to understanding how the brain works. However, action potential initiation and the preceding integration of the synaptic signals in neuronal processes of individual cells are complex and difficult to understand in the absence of detailed, spatially resolved measurements. Multi‐site optical recording with voltage‐sensitive dyes from individual neurons in situ was used to provide these kinds of measurements. We analysed in detail the pattern of initiation and propagation of spikes evoked synaptically in an identified snail (Helix aspersa) neuron in situ. 2 Two main spike trigger zones were identified. The trigger zones were activated selectively by different sets of synaptic inputs which also produced different spike propagation patterns. 3 Synaptically evoked action potentials did not always invade all parts of the neuron. The conduction of the axonal spike was regularly blocked at particular locations on neuronal processes. 4 The propagating spikes in some axonal branches consistently reversed direction at certain branch points, a phenomenon known as reflection. 5 These experimental results, when linked to a computer model, could allow a new level of analysis of the electrical structure of single neurons.

Imaging Inhibitory Synaptic Potentials Using Voltage Sensitive Dyes

Studies of the spatio-temporal distribution of inhibitory postsynaptic potentials (IPSPs) in a neuron have been limited by the spatial information that can be obtained by electrode recordings. We describe a method that overcomes these limitations by imaging IPSPs with voltage-sensitive dyes. CA1 hippocampal pyramidal neurons from brain slices were loaded with the voltage-sensitive dye JPW-1114 from a somatic patch electrode in whole-cell configuration. After removal of the patch electrode, we found that neurons recover their physiological intracellular chloride concentration. Using an improved voltage-imaging technique, dendritic GABAergic IPSPs as small as 1 mV could be resolved optically from multiple sites with spatial averaging. We analyzed the sensitivity of the technique, in relation to its spatial resolution. We monitored the origin and the spread of IPSPs originating in different areas of the apical dendrite and reconstructed their spatial distribution. We achieved a clear discrimination of IPSPs from the dendrites and from the axon. This study indicates that voltage imaging is a uniquely suited approach for the investigation of several fundamental aspects of inhibitory synaptic transmission that require spatial information.