Identification of Active Membrane Areas in the Giant Neuron of Aplysia

Intracellular and extracellular potentials were simultaneously recorded from the soma and different parts of the axon of the giant cell of Aplysia. Evidence was obtained that for all modes of stimulation the spike originates in the axon at some distance from the cell body. The conduction of the spike is blocked at a distance of 200 to 300 µ from the soma for the antidromic spike, closer to the soma for an orthodromic spike. This event is recorded in the soma as a small or A spike. After some delay, a spike is initiated in the resting part of the axon and in the axon hillock; the soma is invaded only afterwards. The response of these three parts of the neuron is recorded in the soma as the big or S spike.     

Electrical excitability of the soma of sensory neurons is required for spike invasion of the soma,but not for through-conduction

The cell soma of primary sensory neurons is electrically excitable, and is invaded by action potentials as they pass from the peripheral nerve, past the dorsal root ganglion (DRG) and toward the spinal cord. However, there are virtually no synapses in the DRG, and no signal processing is known to occur there. Why, then, are DRG cell somata excitable? We have constructed and validated an explicit model of the primary sensory neuron and used it to explore the role of electrical excitability of the cell soma in afferent signaling. Reduction and even elimination of soma excitability proved to have no detectable effect on the reliability of spike conduction past the DRG and into the spinal cord. Through-conduction is affected, however, by major changes in neuronal geometry in the region of the t-junction. In contrast to through-conduction, excitability of the soma and initial segment is essential for the invasion of afferent spikes into the cell soma. This implies that soma invasion has a previously unrecognized role in the physiology of afferent neurons, perhaps in the realm of metabolic coupling of the biosynthesis of signaling molecules required at the axon ends to functional demand, or in cell-cell interaction within sensory ganglia. Spike invasion of the soma in central nervous system neurons may play similar roles.     

Electrophysiology of Supramedullary Neurons in Spheroides maculatus : I. Orthodromic and antidromic responses

This series of three papers presents data on a system of neurons, the large supramedullary cells (SMC) of the puffer, Spheroides maculatus, in terms of the physiological properties of the individual cells, of their afferent and efferent connections, and of their interconnections. Some of these findings are verified by available anatomical data, but others suggest structures that must be sought for in the light of the demonstration that these cells are not sensory neurons. Analysis on so broad a scale was made possible by the accessibility of the cells in a compact cluster on the dorsal surface of the spinal cord. Simultaneous recordings were made intracellularly and extracellularly from individual cells or from several, frequently with registration of the afferent or efferent activity as well. The passive and active electrical properties of the SMC are essentially similar to those of other neurons, but various response characteristics have been observed which are related to different excitabilities of different parts of the neuron, and to specific anatomical features. The SMC produce spikes to direct stimuli by intracellular depolarization, or by indirect synaptic excitation from many afferent paths, including tactile stimulation of the skin. Responses that were evoked by intracellular stimulation of a single cell cause an efferent discharge bilaterally in many dorsal roots, but not in the ventral. Sometimes several distinct spikes occurred in the same root, and behaved independently. Thus, a number of axons are efferent from each neuron. They are large unmyelinated fibers which give rise to the elevation of slowest conduction in the compound action potential of the dorsal root. A similar component is absent in the ventral root action potential. Antidromic stimulation of the axons causes small potentials in the cell body, indicating that the antidromic spikes are blocked distantly to the soma, probably in the axon branches. The failure of antidromic invasion is correlated with differences in excitability of the axons and the neurite from which they arise. As recorded in the cell body, the postsynaptic potentials associated with stimulation of afferent fibers in the dorsal roots or cranial nerves are too small to discharge the soma spike. The indirect spike has two components, the first of which is due to the synaptically initiated activity of the neurite and which invades the cell body. The second component is then produced when the soma is fired. The neurite impulse arises at some distance from the cell body and propagates centrifugally as well as centripetally. An indirect stimulus frequently produces repetitive spikes which are observed to occur synchronously in all the cells examined at one time. Each discharge gives rise to a large efferent volley in each of the dorsal roots and cranial nerves examined. The synchronized responses of all the SMC to indirect stimulation occur with slightly different latencies. They are due to a combination of excitation by synaptic bombardment from the afferent pathways and by excitatory interconnections among the SMC. Direct stimulation of a cell may also excite all the others. This spread of activity is facilitated by repetitive direct excitation of the cell as well as by indirect stimulation.     

Extra spike formation in sensory neurons and the disruption of afferent spike patterning

The peculiar pseudounipolar geometry of primary sensory neurons can lead to ectopic generation of "extra spikes" in the region of the dorsal root ganglion potentially disrupting the fidelity of afferent signaling. We have used an explicit model of myelinated vertebrate sensory neurons to investigate the location and mechanism of extra spike formation, and its consequences for distortion of afferent impulse patterning. Extra spikes originate in the initial segment axon under conditions in which the soma spike becomes delayed and broadened. The broadened soma spike then re-excites membrane it has just passed over, initiating an extra spike which propagates outwards into the main conducting axon. Extra spike formation depends on cell geometry, electrical excitability, and the recent history of impulse activity. Extra spikes add to the impulse barrage traveling toward the spinal cord, but they also travel antidromically in the peripheral nerve colliding with and occluding normal orthodromic spikes. As a result there is no net increase in afferent spike number. However, extra spikes render firing more staccato by increasing the number of short and long interspike intervals in the train at the expense of intermediate intervals. There may also be more complex changes in the pattern of afferent spike trains, and hence in afferent signaling.     

The Spread of Excitation among Neurons in the Heart Ganglion of the Stomatopod, Squilla oratoria

Neurons in the heart ganglion of the mantis shrimp (a stomatopod crustacean) are functionally tightly linked together. The extracellular action potential from the whole trunk very often shows a complex form, but the response is all-or-none to the applied stimulus, indicating that the excitation in one neuron spreads very rapidly to all others. Application of isotonic MgCl₂ solution or repetitive stimulation sometimes separates the spike into its components. The resting potential of the soma membrane is 50 to 60 mv. External stimulation elicits a spike of 60 to 80 mv amplitude with a step on its rising phase. Hyperpolarization reveals one more inflection on the rising phase. These inflections divide the soma action potential into three parts, A₁, A₂, and B spikes in that order from the foot. The B spike disappears on increasing the hyperpolarization, but A₁ and A₂ remain, indicating that B originates from the soma membrane, whereas A₁ and A₂ originate from the two axons of the bipolar cell. Thus the impulse invades the soma from two directions, one from the stimulated side, the other from the other side via the "parallel axons" and the "side-connections;" the latter are presumed to interconnect the axons. When the parallel axons are cut, conduction takes place across the soma with a greatly reduced safety factor and a prolonged conduction time. Neuron-to-neuron transmission takes place in either direction.     

Electrophysiological effects of a neurotoxin extracted from the skin of the Australian frog Pseudophryne coriacea

1. The electrophysiological effects of a pumiliotoxin-B-like alkaloid extracted from the skin of the Australian frog Pseudophryine coriacea (PsC) have been studied in rat superior cervical ganglia at 37°C.2. PsC (50 mg/ml) elicits a broadening of the evoked compound action potential and, at rest, the appearance of spontaneous spike discharge at 10–20 Hz. Action potentials presumably originate far away from the soma, which is invaded in a typical ISSD sequence.3. The toxin effect is not related to any direct action on the preganglionic fibers of the sympathetic trunk, and does not involve synaptic mechanisms.4. Two-electrode voltage-clamp experiments showed that the main properties of the major voltage-dependent ionic currents are apparently unaffected by the toxin, while the cell input resistance is considerably reduced.5. The data are consistent with the hypothesis that PsC elicits a cationic permeability increase generating a pacemaker current in a region close to the cell soma.     

Modes of Initiation and Propagation of Spikes in the Branching Axons of Molluscan Central Neurons

A study has been made of Aplysia nerve cells, mainly in the pleural ganglia, in which the main axon divides into at least two branches in the neighbourhood of the soma. Conduction between these branches was investigated by intracellular recordings from the soma following antidromic stimulation via the nerves containing the axonal branches. It has been shown that transmission between separate branches need not involve discharge of the soma but only of the axonal region between the soma and the origin of the branches. In some cells, the spike may fail to invade the other axonal branch, whereas transmission in the opposite direction is readily achieved. Often spikes in none of the branches are transmitted to the others, unless facilitated. Indications about the geometry of the neuron in the vicinity of the soma may be obtained from the study of the relative size of the A spikes originated in different branches. These observations, together with the presence of different sizes of A spikes, produced by orthodromic stimulation, provide evidence that spikes initiated at separate axonal "trigger zones" of Aplysia neurons may be conducted selectively to the effectors or other neurons innervated by the particular branch.     

Asymmetry in Signal Propagation between the Soma and Dendrites Plays a Key Role in Determining Dendritic Excitability in Motoneurons

It is widely recognized that propagation of electrophysiological signals between the soma and dendrites of neurons differs depending on direction, i.e. it is asymmetric. How this asymmetry influences the activation of voltage-gated dendritic channels, and consequent neuronal behavior, remains unclear. Based on the analysis of asymmetry in several types of motoneurons, we extended our previous methodology for reducing a fully reconstructed motoneuron model to a two-compartment representation that preserved asymmetric signal propagation. The reduced models accurately replicated the dendritic excitability and the dynamics of the anatomical model involving a persistent inward current (PIC) dispersed over the dendrites. The relationship between asymmetric signal propagation and dendritic excitability was investigated using the reduced models while varying the asymmetry in signal propagation between the soma and the dendrite with PIC density constant. We found that increases in signal attenuation from soma to dendrites increased the activation threshold of a PIC (hypo-excitability), whereas increases in signal attenuation from dendrites to soma decreased the activation threshold of a PIC (hyper-excitability). These effects were so strong that reversing the asymmetry in the soma-to-dendrite vs. dendrite-to-soma attenuation, reversed the correlation between PIC threshold and distance of this current source from the soma. We propose the tight relation of the asymmetric signal propagation to the input resistance in the dendrites as a mechanism underlying the influence of the asymmetric signal propagation on the dendritic excitability. All these results emphasize the importance of maintaining the physiological asymmetry in dendritic signaling not only for normal function of the cells but also for biophysically realistic simulations of dendritic excitability.     

Dendritic I h Selectively Blocks Temporal Summation of Unsynchronized Distal Inputs in CA1 Pyramidal Neurons

The active dendritic conductances shape the input-output properties of many principal neurons in different brain regions, and the various ways in which they regulate neuronal excitability need to be investigated to better understand their functional consequences. Using a realistic model of a hippocampal CA1 pyramidal neuron, we show a major role for the hyperpolarization-activated current, Ih, in regulating the spike probability of a neuron when independent synaptic inputs are activated with different degrees of synchronization and at different distances from the soma. The results allowed us to make the experimentally testable prediction that the Ih in these neurons is needed to reduce neuronal excitability selectively for distal unsynchronized, but not for synchronized, inputs.     

Cholinergic modulation of neuronal spike reactions to dendritic and somatic application of excitatory acids

Acetylcholine effects on neuronal firing responses evoked by somatic or dendritic applications of excitatory amino acids were studied in slices of guinea-pig parietal cortex. Excitatory reactions initiated by dendritic activation were enhanced by acetylcholine wherever it was iontophoretically applied: either to soma or dendrites. The effect consisted in shortening spike response latencies and increasing response intensity and duration. The modified responses were recorded within 1-min interval after acetylcholine microinjections at a distance within 300 microns of the soma. Parameters of responses to somatic applications of excitatory amino acids were not significantly changed by acetylcholine. The results suggest that acetylcholine improves dendritic propagation rather than membrane excitability.     

Amplitude variability and extracellular low-pass filtering of neuronal spikes

The influence of neural morphology and passive electrical parameters on the width and amplitude of extracellular spikes is investigated by combined analytical and numerical investigations of idealized and anatomically reconstructed pyramidal and stellate neuron models. The main results are: 1), All models yield a low-pass filtering effect, that is, a spike-width increase with increasing distance from soma. 2), A neuron's extracellular spike amplitude is seen to be approximately proportional to the sum of the dendritic cross-sectional areas of all dendritic branches connected to the soma. Thus, neurons with many, thick dendrites connected to soma will produce large amplitude spikes, and therefore have the largest radius of visibility. 3), The spike shape and amplitude are found to be dependent on the membrane capacitance and axial resistivity, but not on the membrane resistivity. 4), The spike-amplitude decay with distance r is found to depend on dendritic morphology, and is decaying as 1/rⁿ with 1 ≤ n ≤ 2 close to soma and n ≥ 2 far away.     

Impulse Origin and Propagation in a Bipolar Sensory Neuron

Intracellular recording techniques were used to study electrical activity in bipolar sensory cells associated with crayfish tactile receptors. Several lines of evidence indicate that spikes evoked by natural stimulation of the receptor originate at a dendritic locus. Although overshooting spikes are recorded in the soma in response to both natural and antidromic stimulation receptor potentials are observed only rarely, and, when present, their amplitude is less than 5 mv. Impulses propagating centrifugally into the soma following antidromic stimulation always exhibit an inflection in the rising phase of the spike; however, orthodromic spikes are usually uninflected. Occasionally, orthodromic responses (in the soma) exhibit rather unusual wave forms. Such spikes evoked by natural stimuli are indistinguishable from those elicited electrically in the dendrite, but they do not resemble antidromic impulses. Because the axonal and dendritic boundaries of the soma have a low safety factor for spike transmission, at high frequencies invasion of the soma by dendritic spikes is impeded and often blocked. The soma region can thus act as a low-pass filter. The significance of this self-limiting mechanism for the behavior of the animal is not known; it is suggested, however, that this impediment is a potentially critical one, and may, in other situations, have encouraged the evolution of alternative arrangements.     

GM1 ganglioside contributes to retain the neuronal conduction and neuronal excitability in visceral and baroreceptor afferents

GM1 ganglioside has a great impact on the function of nodes of Ranvier on myelinated fiber, suggesting its potential role to maintain the electrical and neuronal excitability of neurons. Here we first demonstrate that visceral afferent conduction velocity of myelinated and unmyelinated fibers are reduced significantly by tetrodotoxin (TTX) or cholera toxin-B subunits (CTX-B), and only the effects mediated by CTX-B are prevented by GM1 pre-treatment. At soma of myelinated A and unmyelinated C-type nodose ganglion neurons (NGNs), the action potential spike frequency reduced by CTX-B is also prevented by GM1. Additionally, the current density of both TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na⁺ channels were significantly decreased by CTX-B without changing the voltage-dependent property. These data confirm that endogenous GM1 may play a dominant role in maintaining the electrical and neuronal excitability via modulation of sodium (Na⁺) channel around nodes and soma as well, especially TTX-S Na⁺ channel, which is also confirmed by the reduction of spike amplitude and depolarization. Similar data are also extended to fluorescently identified and electrophysiologically characterized aortic baroreceptor neurons. These findings suggest that GM1 plays an important role in the neural modulation of electric and neuronal excitability in visceral afferent system.     

Steps in the production of motoneuron spikes

1. Spikes evoked in spinal motoneurons by antidromic stimulation normally present an inflection in their rising phase. A similar inflection is present in spikes evoked by direct stimulation with short pulses. 2. In either case the inflection becomes less prominent if the motoneuron membrane is depolarized and more prominent when it is hyperpolarized. Both antidromic and direct spikes may fall from the level of the inflection thus evoking a "small spike" only if sufficient hyperpolarization is applied. Similar events occur when antidromic or direct spikes are evoked in the aftermath of a preceding spike. 3. Spikes evoked by direct stimuli applied shortly after firing of a "small spike" may also become partially blocked at a critical stimulus interval. At shorter intervals, however, spike size again increases and no inflection can be detected in the rising phase. 4. When a weak direct stimulus evokes a small spike only, a stronger stimulus may evoke a full spike. Curves of the strength of the stimuli required for eliciting small or full spikes have been constructed in a number of conditions. 5. To explain the results it is assumed that threshold of the major portions of the soma membrane is higher than the threshold of the axon, the transition occurring over a finite area near the axon hillock. Following antidromic or direct stimulation, soma excitation is then initiated in the region of the axon hillock. Spread of activity towards the soma occurs at first slowly and with low safety factor. At this stage block may be easily evoked. Safety factor for propagation increases rapidly as the growing impulse involves larger and larger areas of the soma membrane so that, once the critical areas are excited, activation of the remaining portions of the soma membrane will suddenly occur.     

Advantages and Limitations of the Use of Optogenetic Approach in Studying Fast-Scale Spike Encoding

Understanding single-neuron computations and encoding performed by spike-generation mechanisms of cortical neurons is one of the central challenges for cell electrophysiology and computational neuroscience. An established paradigm to study spike encoding in controlled conditions in vitro uses intracellular injection of a mixture of signals with fluctuating currents that mimic in vivo-like background activity. However this technique has two serious limitations: it uses current injection, while synaptic activation leads to changes of conductance, and current injection is technically most feasible in the soma, while the vast majority of synaptic inputs are located on the dendrites. Recent progress in optogenetics provides an opportunity to circumvent these limitations. Transgenic expression of light-activated ionic channels, such as Channelrhodopsin2 (ChR2), allows induction of controlled conductance changes even in thin distant dendrites. Here we show that photostimulation provides a useful extension of the tools to study neuronal encoding, but it has its own limitations. Optically induced fluctuating currents have a low cutoff (~70Hz), thus limiting the dynamic range of frequency response of cortical neurons. This leads to severe underestimation of the ability of neurons to phase-lock their firing to high frequency components of the input. This limitation could be worked around by using short (2 ms) light stimuli which produce membrane potential responses resembling EPSPs by their fast onset and prolonged decay kinetics. We show that combining application of short light stimuli to different parts of dendritic tree for mimicking distant EPSCs with somatic injection of fluctuating current that mimics fluctuations of membrane potential in vivo, allowed us to study fast encoding of artificial EPSPs photoinduced at different distances from the soma. We conclude that dendritic photostimulation of ChR2 with short light pulses provides a powerful tool to investigate population encoding of simulated synaptic potentials generated in dendrites at different distances from the soma.     

A re-excitation mechanism for strychnine-induced doublets in molluscan neurons

The effects of strychnine on Aplysia R2 neurons were evaluated using simultaneous intracellular recordings of the soma and axon potentials. 1 mM strychnine produced a slight enlargement of the somatic spike and a large increase of the axon spike duration. Following direct stimulation, the soma displayed depolarizing afterpotentials ( DAPs ) which might trigger extra-spikes, both produced electronically by long-lasting axon spikes. Cobalt suppressed both the axon spike lengthening and the somatic extra-spikes or DAPs , and induced large depolarizing shifts in the soma. The region of largest spike lengthening (proximal axon) had a large density of Ca channels. The different effects of strychnine on the soma and on the axon were assumed to result from a selective blockage of the V-dependent K channels which would predominate in the axon whereas Ca-activated K channels would predominate in the soma.     

Regulation of somatic firing dynamics by backpropagating dendritic spikes

Pyramidal cells of the apteronotid ELL have been shown to display a characteristic mechanism of burst discharge, which has been shown to play an important role in sensory coding. This form of bursting depends on a reciprocal dendro-somatic interaction, in which discharge of a somatic spike causes a dendritic spike, which in turn contributes a dendro-somatic current flow to create a depolarizing afterpotential (DAP) in the soma. We review here our recent work showing how the timing of this DAP influences the somatic firing dynamics, and how the degree of inactivation of dendritic Na⁺ currents can cause an increased delay between somatic and dendritic spikes. This ultimately allows the DAP to become more effective at increasing the excitability of the somatic spike generating mechanism. Further, this delay between dendritic and somatic spiking can be regulated by strongly hyperpolarizing GABA_B mediated dendritic inhibition, allowing the burst dynamics to fall under synaptic regulation. In contrast, a weaker, shunting inhibition due to GABA_A mediated dendritic inhibition can regulate the dendritic spike waveform to decrease the dendro-somatic current flow and the resulting DAP. We therefore show that the qualitative behaviour of an individual cell can depend on the degree of synaptic input, and the exact timing of events across the spatial extent of the neuron. Thus, our results serve to illustrate the complex dynamics that can be observed in cells with significant dendritic arborisation, a nearly ubiquitous adaptation amongst principal neurons.     

Effects of Lithium on Different Membrane Components of Crayfish Stretch Receptor Neurons

Unlike several other varieties of input membrane, that of the crayfish stretch receptor develops a generator potential in response to stretch when all the Na of the medium is replaced with Li. However, Li depolarizes the receptor neuron, the soma membrane becoming more depolarized than that of the axon. During exposure to Li the cell usually fires spontaneously for a period, and when it becomes quiescent spike electrogenesis fails in the soma but persists in the axon. These effects are seen in the rapidly adapting as well as the slowly adapting cells. The block of spike electrogenesis of the soma membrane is only partly due to the Li-induced depolarization and a significant role must be ascribed to a specific effect of Li.     

Multiple spike initiation zones in a neuron implicated in learning in the leech: a computational model

Sensitization of the defensive shortening reflex in the leech has been linked to a segmentally repeated tri-synaptic positive feedback loop. Serotonin from the R-cell enhances S-cell excitability, S-cell impulses cross an electrical synapse into the C-interneuron, and the C-interneuron excites the R-cell via a glutamatergic synapse. The C-interneuron has two unusual characteristics. First, impulses take longer to propagate from the S soma to the C soma than in the reverse direction. Second, impulses recorded from the electrically unexcitable C soma vary in amplitude when extracellular divalent cation concentrations are elevated, with smaller impulses failing to induce synaptic potentials in the R-cell. A compartmental, computational model was developed to test the sufficiency of multiple, independent spike initiation zones in the C-interneuron to explain these observations. The model displays asymmetric delays in impulse propagation across the S–C electrical synapse and graded impulse amplitudes in the C-interneuron in simulated high divalent cation concentrations.