Srdjan D. Antic

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.

Where Is the Spike Generator of the Cochlear Nerve? Voltage-Gated Sodium Channels in the Mouse Cochlea

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.

Action potentials in basal and oblique dendrites of rat neocortical pyramidal neurons

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 μm 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 Ω cm. The combined experimental and modelling results also provide evidence that all synaptic locations along basal and oblique dendrites, situated within 200 μm from the soma, experience strong and near‐simultaneous (latency < 1 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.

Palette of fluorinated voltage-sensitive hemicyanine dyes

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.

Human Cortical Neurons Originate from Radial Glia and Neuron-Restricted Progenitors

Understanding the molecular and physiological determinants of cortical neuronal progenitor cells is essential for understanding the development of the human brain in health and in disease. We used surface marker fucose N-acetyl lactosamine (LeX) (also known as CD15) to isolate progenitor cells from the cortical ventricular/subventricular zone of human fetal brain at the second trimester of gestation and to study their progeny in vitro. LeX+ cells had typical bipolar morphology, radial orientation, and antigen profiles, characterizing them as a subtype of radial glia (RG) cells. Four complementary experimental techniques (clonal analysis, immunofluorescence, transfection experiments, and patch-clamp recordings) indicated that this subtype of RG generates mainly astrocytes but also a small number of cortical neurons. The neurogenic capabilities of RGs were both region and stage dependent. Present results provide the first direct evidence that RGs in the human cerebral cortex serve as neuronal progenitors. Simultaneously, another progenitor subtype was identified as proliferating cells labeled with neuronal (β-III-tubulin and doublecortin) but not RG markers [GFAP, vimentin, and BLBP (brain lipid-binding protein)]. Proliferative and antigenic characteristics of these cells suggested their neuron-restricted progenitor status. In summary, our in vitro study suggests that diverse populations of cortical progenitor cells, including multipotent RGs and neuron-restricted progenitors, contribute differentially to cortical neurogenesis at the second trimester of gestation in human cerebral cortex.

The Decade of the Dendritic NMDA Spike

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).

Electrical Excitability of Early Neurons in the Human Cerebral Cortex during the Second Trimester of Gestation

Information about development of the human cerebral cortex (proliferation, migration, and differentiation of neurons) is largely based on postmortem histology. Physiological properties of developing human cortical neurons are difficult to access experimentally and therefore remain largely unexplored. Animal studies have shown that information about the arousal of electrical activity in individual cells within fundamental cortical zones (subventricular zone [SVZ], intermediate zone, subplate [SP], and cortical plate [CP]) is necessary for understanding normal brain development. Here we ask where, in what cortical zone, and when, in what gestational week (gw), human neurons acquire the ability to generate nerve impulses (action potentials [APs]). We performed electrical recordings from individual cells in acute brain slices harvested postmortem from the human fetal cerebral cortex (16-22 gw). Tetrodotoxin-sensitive Na(+) current occurs more frequently among CP cells and with significantly greater peak amplitudes than in SVZ. As early as 16 gw, a relatively small population of CP neurons (27%) was able to generate sodium APs upon direct current injection. Neurons located in the SP exhibited the highest level of cellular differentiation, as judged by their ability to fire repetitive APs. At 19 gw, a fraction of human CP and SP neurons possess beta IV spectrin-positive axon initial segments populated with voltage-gated sodium channels (PanNav). These results yield the first physiological characterization of developing human fetal cortical neurons with preserved morphologies in intact surrounding brain tissue.

Voltage and calcium transients in basal dendrites of the rat prefrontal cortex.

Higher cortical functions (perception, cognition, learning and memory) are in large part based on the integration of electrical and calcium signals that takes place in thin dendritic branches of neocortical pyramidal cells (synaptic integration). The mechanisms underlying the synaptic integration in thin basal dendrites are largely unexplored. We use a recently developed technique, multisite voltage–calcium imaging, to compare voltage and calcium transients from multiple locations along individual dendritic branches. Our results reveal characteristic electrical transients (plateau potentials) that trigger and shape dendritic calcium dynamics and calcium distribution during suprathreshold glutamatergic synaptic input. We regularly observed three classes of voltage–calcium interactions occurring simultaneously in three different zones of the same dendritic branch: (1) proximal to the input site, (2) at the input site, and (3) distal to the input site. One hundred micrometers away from the synaptic input site, both proximally and distally, dendritic calcium transients are in tight temporal correlation with the dendritic plateau potential. However, on the same dendrite, at the location of excitatory input, calcium transients outlast local dendritic plateau potentials by severalfold. These Ca2+ plateaus (duration 0.5–2 s) are spatially restricted to the synaptic input site, where they cause a brief down‐regulation of dendritic excitability. Ca2+ plateaus are not mediated by Ca2+ release from intracellular stores, but rather by an NMDA‐dependent small‐amplitude depolarization, which persists after the collapse of the dendritic plateau potential. These unique features of dendritic voltage and calcium distributions may provide distinct zones for simultaneous long‐term (bidirectional) modulation of synaptic contacts along the same basal branch.

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.