Maja Djurisic

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.

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.

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.

Functional Structure of the Mitral Cell Dendritic Tuft in the Rat Olfactory Bulb

The input–output transform performed by mitral cells, the principal projection neurons of the olfactory bulb, is one of the key factors in understanding olfaction. We used combined calcium and voltage imaging from the same neuron and computer modeling to investigate signal processing in the mitral cells, focusing on the glomerular dendritic tuft. The main finding was that the dendritic tuft functions as a single electrical compartment for subthreshold signals within the range of amplitudes detectable by voltage-sensitive dye recording. These evoked EPSPs had uniform characteristics throughout the glomerular tuft. The Ca2+ transients associated with spatially uniform subthreshold synaptic potentials were comparable but not equal in amplitude in all regions. The average range of normalized amplitudes of the EPSP-driven Ca2+ signals from different locations on dendritic branches in the glomerular tuft was relatively narrow and appeared to be independent of the dendritic surface-to-volume ratio. The computer simulations constrained by the imaging data indicated that a synchronized activation of ∼100 synapses randomly distributed on tuft branches was sufficient to generate spatially homogenous EPSPs. This number of activated synapses is consistent with the data from anatomical studies. Furthermore, voltage attenuation of the EPSP along the primary dendrite at physiological temperature was weak compared with other cell types. In the model, weak attenuation of the EPSP along the primary dendrite could be accounted for by passive electrical properties of the mitral cell.

Optical monitoring of neural activity using voltage-sensitive dyes.

Publisher Summary This chapter discusses the optical monitoring of neural activity using voltage-sensitive dyes. An optical measurement of membrane potential using a molecular probe can be beneficial in a variety of circumstances. One advantage is the possibility of simultaneous measurements from many locations. Several different optical properties of membrane-bound dyes are sensitive to membrane potential, including fluorescence, absorption, dichroism, birefringence, fluorescence resonance energy transfer (FRET), nonlinear second harmonic generation, and resonance Raman absorption. Different kinds of staining are used in the two experiments described in the chapter: (1) For studying the membrane potential in individual dendrites of a neuron, the dye was released from an intracellular electrode in the soma and then allowed to spread into the dendritic tree, and (2) for the population signals, the olfactory bulb was superfused for 60 min in a solution of the dye. Neuron-type-specific staining can make it possible to determine the role of specific neuron types in generating the input-output function of a brain region.

Wide-field and two-photon imaging of brain activity with voltage- and calcium-sensitive dyes.

This chapter presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes. Because experimental measurements are limited by low sensitivity, the chapter then discusses the methodological aspects that are critical for optimal signal-to-noise ratio. Two of the examples use wide-field (1-photon) imaging and the third uses two-photon scanning microscopy. These methods have relatively high temporal resolution ranging from 10 to 10,000 Hz. The three examples are the following: (1) Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations. These experiments are directed at understanding how individual neurons convert the complex input synaptic activity into the output spike train. (2) Recently developed methods for staining many individual cells in the mammalian brain with calcium-sensitive dyes together with two-photon microscopy made it possible to follow the spike activity of many neurons simultaneously while in vivo preparations are responding to stimulation. (3) 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 glomeruli. There, the population signals can be used as a measure of the input from the nose to the bulb. Three kinds of noise in measuring light intensity are discussed: (1) Shot noise from the random emission of photons from the preparation. (2) Extraneous (technical) noise from external sources. (3) Noise that occurs in the absence of light, the dark noise. In addition, we briefly discuss the light sources, the optics, and the detectors and cameras. The commonly used organic voltage and ion sensitive dyes stain all of the cell types in the preparation indiscriminately. A major effort is underway to find methods for staining individual cell types in the brain selectively. Most of these efforts center around fluorescent protein activity sensors because transgenic methods can be used to express them in individual cell types.

Imaging Nervous System Activity with Voltage‐Sensitive Dyes

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.

Imaging of Spiking and Subthreshold Activity of Mitral Cells with Voltage‐Sensitive Dyes

Abstract: To obtain a more complete description of individual neurons, it is necessary to complement electrical measurements with technologies such as voltage imaging with intracellular dyes, which permit massive parallel recording from many sites on neuronal processes. Utilizing such an approach, we investigate the functional structure of the mitral cell, the principal output neuron in the rat olfactory bulb. These experiments were designed to determine the number, location, and the stability of spike trigger zones, the excitability of terminal dendritic branches, the pattern and nature of spike initiation and propagation in the primary dendrite, the basic characteristics of the evoked EPSPs at the site of origin (the glomerular tuft), and its attenuation along the primary dendrite. 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. The amplitude of the EPSPs evoked by olfactory nerve stimulation was determined at the site of origin (glomerular tuft) and its attenuation was monitored optically along the entire length of the primary dendrite.

Determinants of Low EPSP Attenuation in Primary Dendrites of Mitral Cells Modeling Study

Abstract: Efficiency of synaptic potential propagation through neurons depends mainly on their membrane properties and intracellular resistivity. We use a morphologically realistic compartmental model of a mitral cell and data obtained from whole‐cell patch‐clamp and voltage‐imaging experiments to explore passive parameter space in which reported low EPSP attenuation is observed.