"Simulations of voltage clamping poorly space-clamped voltage-dependent conductances in a uniform cylindrical neurite"

Hartline, D.K. and Castelfranco, A.M

. J. comput. Neuroscience (2003)
Abstract. Significant error is made by using a point voltage clamp to measure active ionic current properties in poorly space-clamped cells. This can even occur when there are no obvious signs of poor spatial control. We evaluated this error for experiments that employ an isochronal I(V) approach to analyzing clamp currents. Simulated voltage clamp experiments were run on a model neuron having a uniform distribution of a single voltage-gated inactivating ionic current channel along an elongate, but electrotonically compact, process. Isochronal Boltzmann I(V) and kinetic parameter values obtained by fitting the Hodgkin-Huxley equations to the clamp currents were compared with the values originally set in the model. Good fits were obtained for both inward and outward currents for moderate channel densities. Most parameter errors increased with conductance density. The activation rate parameters were more sensitive to poor space clamp than the I(V) parameters. Large errors can occur despite "normal"- looking clamp curves.

"Simulations of space-clamp errors in estimating parameters of voltage-gated conductances localized at different electrotonic distances"

A.M. Castelfranco and D.K. Hartline

Neurocomputing (2002: in press)
Abstract: Parameters of voltage-gated conductances measured in point voltage clamp experiments are in error if the membrane containing the conductance is under poor spatial control. The ability to correct such errors could rescue much flawed experimental data, but requires a detailed understanding of the errors. We evaluated such errors using simulated voltage-clamp experiments on a soma with a single (0.5 lambda) cylindrical process having a patch of voltage-dependent Hodgkin-Huxley channels placed various distances from the soma. Kinetic and steady-state parameters were obtained by curve-fitting the kinetics of soma clamp current flowing in response to step command depolarizations. Most errors in fitted parameters increased as the patch was located more distally on the neurite.

Multistable neurons: ionic mechanisms of bursting

Daniel K. Hartline

Sensory Systems of Arthropods eds Wiese, K., Gribakin, F., Popov, A.V. and Birkhauser, G.H. 557-566 (1993)

Increasingly, neurons are being found that have two quasi-stable states, high-frequency firing or silence, either of which may be present only transiently. Bursts of spikes can be triggered or terminated spontaneously, or by brief inputs, giving the output of the neuron some of the properties of an electronic monostable multivibrator. A voltage-dependent inward current giving rise to a regenerative "plateau potential" underlies this behavior in many neurons, arthropod and non-arthropod. Inactivation of inward current or slow activation of outward current may spontaneously terminate plateaus. Control of plateaus often involves a modulatory transmitter coupled to second messenger production. This mechanism can "reprogram" a neuron from a quasi-linear one into a bursty, bistable one.

Effects of soma isolation on outward currents measured under voltage clamp in spiny lobster stomatogastric motor neurons

Daniel K. Hartline, Donald V. Gassie, and Bradley R. Jones

Journal of Neurophysiology 69(6): 2056-2071 (1993)

  • 1. Outward currents in identified cell types from the pyloric system of the stomatogastric ganglion (STG) of the spiny lobster,Panulirus marginatus, were studied under two-microelectrode voltage clamp. A comparison was made between data from intact cells and somata isolated by ligation of the primary neurite of these monopolar neurons.

  • 2. Despite the elimination of current contributions from the extensive arborizations of STG neurons, few significant differences were found in the mean values of parameters for outward currents between populations of isolated somata and intact cells of a given type. Measurements that showed little difference included magnitude and activation threshold of a calcium-dependent outward current (IJ) and magnitude, activation threshold, voltage dependence, and inactivation time course of A current (IA). Although previous work has suggested that IJ, might reside predominantly in the soma, IA is known to be distributed in poorly space-clamped neurite processes. The absence of obvious effects of isolation was thus unexpected.

  • 3. To better understand the mechanisms involved, we used compartmental models derived from reconstructed neurons to simulate the effects of isolation. It was concluded that, for the particular conditions present in stomatogastric neurons, with a large, uniformly distributed outward current conductance activated, even though neurites and axon remain attached, most measured current flows through well-clamped soma membrane.

  • 4. Factors contributing to this result included the outward sign of the current, the large specific conductance activated in these neurons (among the larger reported in somata), and the presence of only a single major process leaving the soma. The potential for serious errors in voltage-clamp measurements from intact cells remains if these conditions are not met.

    Simulation of peptide processing, compartmentation and release in neurosecretory cells

    Daniel K. Hartline and Robert W. Newcomb

    Neurochem. Int. 19(3): 281-296 (1991)

    A theoretical framework is presented which mathematically describes the transport of secretory granules in neurons and the enzymatic processing of a prohormone and subsequent intermediates within such granules. The transport system represents the synthesis of individual granules, the migration of the granules among various ''diffusionally"-connected compartments, and the release of granules from designated compartmental release sites. The processing of prohormone is represented by a multiple-site cleavage reaction with separate rate constants for each cleavage site on each peptide fragment. The effects of neuron growth, processing enzyme activity decay, and changes in release probability with granule age are taken into account. Computer models were constructed based on these underlying assumptions. Simulations are used to illustrate how the distribution or peptide fragments contained in a neuron will depend on interactions of peptide processing kinetics and cellular transport and storage processes. The models provide a tool for testing how multiple cell-biological variables will interact to control the chemical mixture secreted from a peptidergic neuron.

    Quantitative analysis and computer simulation of oxytocin-neurophysin processing in the rat neurohypophysis

    Robert W. Newcomb, Daniel K. Hartline, Jean-Georges Lorentz, Antoine Depaulis and Jean J. Nordmann

    Neurochem. Int. 19(3): 297-312 (1991)

    A chromatographic procedure is used to obtain data on amounts of the oxytocin-neurophysin (OT-NP, or "A") and its more completely processed form (OT-NP', or "B"), which lacks the carboxyl terminal glutamic acid residue of the A form. Measurements of B +A and B/(B + A) are made for single neurohypophyses under varying physiological conditions, in release obtained by perfusion of isolated neurohypophysis with saline containing increased external potassium concentrations, and in isolated neural lobe nerve endings and nerve swellings. The chromatographic procedure is also combined with injection of [35S]cysteine over the supraoptic nucleus. This approach is used to obtain the in vivo A to B conversion rate, and to investigate the trafficking of newly synthesized secretory granules in different axonal compartments. The manner with which the conversion rate changes with secretory activity is investigated by comparing the conversion rate in control animals to that in animals subjected to osmotic stress. Generalized computer models of peptide processing, storage, and release in neurons are used to test various hypotheses of the control of secretory glanule movements in the neurohypophysis for sufficiency in explaining the changes in B + A and B/(B +A) with changing physiological state.

    Voltage clamp analysis of intact stomatogastric neurons

    Katherine Graubard and Daniel K. Hartline

    Brain Research 557: 241-254 (1991)

    Two-electrode voltage clamp of intact, identified pyloric neurons of the spiny lobster stomatogastric ganglion reveals two major outward currents. A rapidly inactivating, tetraethylammonium- (TEA) insensitive, 4-aminopyridine- (4AP) sensitive, outward current resembles IA of molluscan neurons; it activates rapidly on depolarizations above rest (e.g. -45 mV), delaying both thc axonal-sodium and the neuropil calcium spikes which escape voltage-clamp control. We infer that A-current is distributed both in a space clamped region (on or near the soma) and in a non-space clamped region with access to the generators for sodium and calcium spikes. A calcium-dependent outward current, Io(Ca), activates rapidly at clamp steps above -25 mV and inactivates at depolarizing holding voltages. Increasing depolarization results in an increase in both Io(Ca) and firing rate but a reduction in the amplitude of the sodium spike current. Blockage of Io(Ca) with Cd++ causes little change in spike firing pattern. These observations are consistent with Io(Ca) being activated primarily in the soma and nearby regions which are under good control with a soma voltage clamp (and distant from the Na+-spike trigger zone). While the lack of space clamp limits resolution of charging transients and tail currents, the identification of the major current subgroups can still be readily accomplished, and inferences about the location and function of currents can be made which would not be possible if the cells were space clamped or truncated.

    Simulation of restricted neural networks with reprogrmmable neurons

    Daniel K. Hartline

    IEEE Transactions on circuits and systems 36:(5) 653-660 (1989)

    This paper describes a network model composed of reprogrammable neurons, incorporating the following design features:
    1. Spikes can be generated by a model representing repetitive firing at axon (and dendritic) trigger zones.
    2. Active responses (plateau potentials; delaying mechanisms) are simulated with Hodgkin-Huxley type kinetics.
    3. Synaptic interactions, both spike-mediated and non-spiking chemical ("chemotonic"), simulate transmitter release and binding to postsynaptic receptors. Facilitation and antifacilitation of spike-mediated postsynaptic potentials (PSP's) are included.
    4. Chemical pools are used to simulate second messenger systems, trapping of ions in extracelluar spaces, and electrogenic pumps, as well as biochemical reaction chains of quite general character. Modulation of any of the parameters of any compartment can be effected through the pools.
    5. Intracellular messengers of three kinds are simulated explicitly: a) those produced by voltage-gated processes (e. g., Ca ); b) those dependent on transmitter (or hormone) binding; and c) those dependent on other internal messengers (e. g., internally released Ca; enzymatically activated pathways).

    Full-wave rectification from a mixed electrical chemical synapse

    Katherine Graubard and Daniel K. Hartline

    Science 237: 535-537(1987)

    Electrical and chemical synapses usually reinforce one another, but the pyloric late-to- lateral pyloric (PL-to-LP) neuronal connections in lobster stomatogastric ganglia create an inverted U-shaped transfer function between the two neurons: regardless of whether the PL membrane voltage swings positive or negative, the postsynaptic LP voltage will go negative. When the presynaptic cell voltage goes negative, the effect on the LP voltage is due to electrical coupling. During positive presynaptic voltages, the strong contribution of graded chemical inhibition from the PL to the LP neuron overrides the positive electrical coupling to produce net negativity.

    A microprocessor-controlled stimulator for generating voltage clamp command sequences

    Chien Chang, Ning Hsu and Daniel K. Hartline

    Journal of Neuroscience Methods 18: 361-370 (1986)

    This paper presents a design for a programmable stimulator which delivers a systematically varying sequence of command pulses to a voltage clamp or "current clamp" apparatus. The need for such stimulus sequencing is typical of many applications in neurophysiology. In voltage clamp studies, for example, the effects of "conditioning" pulses of varying magnitudes or durations will be examined on responses to subsequent "test" pulses. Such paradigms are used to characterize the activation and inactivation characteristics of ionic mechanisms. Although a labora- tory computer is easily programmed for such applications, its disadvantages in cost, size, ease of use, and competing demands make alternatives attractive. The micro- processor-controlled stimulator presented here has much of the flexibility of a laboratory computer in stimulus control, but it is compact, portable, convenient to use and economical.

    Synaptic regulation of cellular properties and burst oscillations of neurons in gastric mill system of spiny lobsters, Panulirus interruptus

    David F. Russell and Daniel K. Hartline

    Journal of Neurophysiology 52(1): 54-72 (1984)

    1. The properties of neurons in the stomatogastric ganglion (STG) participating in the pattern generator for the gastric mill rhythm were studied by intracellular current injection under several conditions: during on-going gastric rhythms, in the nonrhythmic isolated STG, after stimulation of the nerve carrying central nervous system (CNS) inputs to the STG, or under Ba2++ or Sr2++. 2. Slow regenerative depolarizations during ongoing rhythms were demonstrated in the interior median, cardiopyloric, lateral cardiac, gastropyloric, and continuous inhibitor (AM, CP, LC, GP, and Cl) neurons according to criteria such as voltage dependency, burst triggering, and termination by brief current pulses, etc. Experiments showed that regenerative like behavior was not due to synaptic network interactions. 3. The slow regenerative responses were abolished by isolating the stomatogastric ganglion but could be reestablished by stimulating the input nerve. This indicates that certain CNS inputs synaptically induce the regenerative property in specific gastric neurons. 4. Slow regenerative depolarizations were not demonstrable in gastric mill (GM) motor neurons. Their burst oscillations and firing rate were instead proportional to injected current. CNS inputs evoked a prolonged depolarization in GM motor neurons, apparently by a nonregenerative mechanism. 5. All the gastric cells showed prolonged regenerative potentials under 0.5 -1.5 mM Ba2++. 6. We conclude that the gastric neurons of the STG can be divided into three types according to their properties: Those with a regenerative capability, a repetitively firing type, and a nonregenerative "proportional" type. The cells are strongly influenced by several types of CNS inputs, including "gastric command fibers."

    Endogenous burst capability in a neuron of the gastric mill pattern generator of the spiny lobster Panulirus interruptus

    Daniel K. Hartline and David F. Russell

    Journal of Neurobiology 15(5): 345-364(1984)

    The gastric system of the lobster stomatogastric ganglion has previously been thought to include no neurons capable of endogenous bursting. We describe conditions under which one of the motorneurons, the CP cell, can burst endogenously in a free-running manner in the absence of other phasic network activity. Isolated preparations of the foregut nervous system were used, and the CP bursting was either spontaneous or was activated by continuous stimulation of an input nerve. Three criteria were applied to establish the endogenous nature of such burst generation in CP: absence of phasic input, reset of the bursting pattern by pulses of current in a characteristic phase-dependent manner, and modulation of burst rate by sustained injected current. (l) The firing of other cells which are known to be related synaptically to CP was monitored in nerve records. These other cells were either silent or fired only tonically. Cross-correlograms showed that CP bursting was not ascribable to phasic activity in these other network cells. (2) A depolarizing current pulse of sufficient strength injected intracellularly between bursts triggered a burst prematurely and reset the subsequent rhythm. A hyperpolarizing pulse during a burst terminated it and reset the subsequent rhythm. Reset behavior was similar to that described for other endogenous bursters. (3) Application of a positive-going ramp current initially caused an increase in burst rate, as described for other endogenous bursters. However, further depolarization caused a slower burst rate due to lengthening of the individual bursts, although mean firing frequency continued to increase throughout the range tested. Such free-running endogenous repetitive bursting appeared to result from the CP's ability to produce slow regenerative depolarizations ("plateau potentials"). When bursting was present, so was the plateau property, as determined by I-V analysis and by the ability of brief current pulses to trigger and terminate bursts. The previous inability to observe endogenous bursting in preparations with central input removed may be due to the usual absence of the plateau property in such preparations. CP bursting during normal gastric rhythms, while underlain by plateau potentials, is strongly controlled by network interactions. CP appears not to be well placed in the network to be considered a source of normal gastric rhythmicity. Nevertheless, endogenous bursting in CP may explain some of the partial gastric rhythms seen in behavioral studies, and illustrates one way that cellular properties might contribut to rhythmic behaviors.

    Graded synaptic transmission between identified spiking neurons

    Katherine Graubard, Jonathan A. Raper, and Daniel K. Hartline

    Journal of Neurophysiology 50(2): 508-521(1983)

    1. Graded synaptic transmission between spiking motoneurons of the pyloric group was studied in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. Intracellular microelectrodes were placed in the cell bodies of both pre- and postsynaptic neurons. 2. Graded synaptic transmission was found between all tested cell pairs that were known to display spike-evoked synaptic transmission, including PD to LP, PD to PE, PD to PL, PL to LP, and LP to PD. 3. Graded synaptic transmission was effective below the threshold for spikes. Thus, it was possible to study the influence of graded synaptic transmission in normally active ganglia without blockage of spikes by tetrodotoxin. PD and LP neurons that were known to produce spike-evoked inhibitory postsynaptic potentials (IPSPs) were also capable of producing inhibitory effects on postsynaptic cells below the threshold for spikes. 4. When tetrodotoxin (TTX) was used to eliminate both spikes and endogenous membrane oscillations, depolanzation of presynaptic neurons produeed hyperpolarization of postsynaptic cells. The presynaptic response to a current step usually showed a small early peak and a maintained, slightly lower plateau. The postsynaptic response had a delay,then a rise to a pronounced peak, and a roughly exponential decline to a maintained plateau. There was a presynaptic voltage threshold for any postsynaptic response; beyond the threshold, both pre- and postsynaptic peak and plateau responses increased with increasing current. PD neurons normally are depolarized beyond their release threshold in tetrodotoxin and, thus, released transmitter tonically for the many-hour duration of these experiments. 5. Chemical, tonic synaptic tranmission, here called graded synaptic transmission, was demonstrated by the presence of the following criteria: 1) reversal in sign of the postsynaptic response, 2) synaptic delay, 3) reversal potential, 4) postsynaptic conductance increase, 5) graded and reversible block by reduction of external Ca++, and 6)specific graded block of the LP-to-PD synapse without effect on the PD-to-LP synapse by less than 10 uM picrotoxin added to the bathing medium.