Jorge Golowasch

Global Structure, Robustness, and Modulation of Neuronal Models

The electrical characteristics of many neurons are remarkably robust in the face of changing internal and external conditions. At the same time, neurons can be highly sensitive to neuromodulators. We find correlates of this dual robustness and sensitivity in a global analysis of the structure of a conductance-based model neuron. We vary the maximal conductance parameters of the model neuron and, for each set of parameters tested, characterize the activity pattern generated by the cell as silent, tonically firing, or bursting. Within the parameter space of the five maximal conductances of the model, we find directions, representing concerted changes in multiple conductances, along which the basic pattern of neural activity does not change. In other directions, relatively small concurrent changes in a few conductances can induce transitions between these activity patterns. The global structure of the conductance-space maps implies that neuromodulators that alter a sensitive set of conductances will have powerful, and possibly state-dependent, effects. Other modulators that may have no direct impact on the activity of the neuron may nevertheless change the effects of such direct modulators via this state dependence. Some of the results and predictions arising from the model studies are replicated and verified in recordings of stomatogastric ganglion neurons using the dynamic clamp.

A Model Neuron with Activity-Dependent Conductances Regulated by Multiple Calcium Sensors

Membrane channels are subject to a wide variety of regulatory mechanisms that can be affected by activity. We present a model of a stomatogastric ganglion (STG) neuron in which several Ca2+-dependent pathways are used to regulate the maximal conductances of membrane currents in an activity-dependent manner. Unlike previous models of this type, the regulation and modification of maximal conductances by electrical activity is unconstrained. The model has seven voltage-dependent membrane currents and uses three Ca2+ sensors acting on different time scales. Starting from random initial conditions over a given range, the model sets the maximal conductances for its active membrane currents to values that produce a predefined target pattern of activity ∼90% of the time. In these models, the same pattern of electrical activity can be produced by a range of maximal conductances, and this range is compared with voltage-clamp data from the lateral pyloric neuron of the STG. If the electrical activity of the model neuron is perturbed, the maximal conductances adjust to restore the original pattern of activity. When the perturbation is removed, the activity pattern is again restored after a transient adjustment period, but the conductances may not return to their initial values. The model suggests that neurons may regulate their conductances to maintain fixed patterns of electrical activity, rather than fixed maximal conductances, and that the regulation process requires feedback systems capable of reacting to changes of electrical activity on a number of different time scales.

Network stability from activity-dependent regulation of neuronal conductances

Activity-dependent plasticity appears to play an important role in the modification of neurons and neural circuits that occurs during development and learning. Plasticity is also essential for the maintenance of stable patterns of activity in the face of variable environmental and internal conditions. Previous theoretical and experimental results suggest that neurons stabilize their activity by altering the number or characteristics of ion channels to regulate their intrinsic electrical properties. We present both experimental and modeling evidence to show that activity-dependent regulation of conductances, operating at the level of individual neurons, can also stabilize network activity. These results indicate that the stomatogastric ganglion of the crab can generate a characteristic rhythmic pattern of activity in two fundamentally different modes of operation. In one mode, the rhythm is strictly conditional on the presence of neuromodulatory afferents from adjacent ganglia. In the other, it is independent of neuromodulatory input but relies on newly developed intrinsic properties of the component neurons.

Neuromodulators, Not Activity, Control Coordinated Expression of Ionic Currents

Electrical activity in identical neurons across individuals is often remarkably similar and stable over long periods. However, the ionic currents that determine the electrical activity of these neurons show wide animal-to-animal amplitude variability. This seemingly random variability of individual current amplitudes may obscure mechanisms that globally reduce variability and that contribute to the generation of similar neuronal output. One such mechanism could be the coordinated regulation of ionic current expression. Studying identified neurons of the Cancer borealis pyloric network, we discovered that the removal of neuromodulatory input to this network (decentralization) was accompanied by the loss of the coordinated regulation of ionic current levels. Additionally, decentralization induced large changes in the levels of several ionic currents. The loss of coregulation and the changes in current levels were prevented by continuous exogenous application of proctolin, an endogenous neuromodulatory peptide, to the pyloric network. This peptide does not exert fast regulatory actions on any of the currents affected by decentralization. We conclude that neuromodulatory inputs to the pyloric network have a novel role in the regulation of ionic current expression. They can control, over the long term, the coordinated expression of multiple voltage-gated ionic currents that they do not acutely modulate. Our results suggest that current coregulation places constraints on neuronal intrinsic plasticity and the ability of a network to respond to perturbations. The loss of conductance coregulation may be a mechanism to facilitate the recovery of function.

Neuromodulation independently determines correlated channel expression and conductance levels in motor neurons of the stomatogastric ganglion

Neuronal identity depends on the regulated expression of numerous molecular components, especially ionic channels, which determine the electrical signature of a neuron. Such regulation depends on at least two key factors, activity itself and neuromodulatory input. Neuronal electrical activity can modify the expression of ionic currents in homeostatic or nonhomeostatic fashion. Neuromodulators typically modify activity by regulating the properties or expression levels of subsets of ionic channels. In the stomatogastric system of crustaceans, both types of regulation have been demonstrated. Furthermore, the regulation of the coordinated expression of ionic currents and the channels that carry these currents has been recently reported in diverse neuronal systems, with neuromodulators not only controlling the absolute levels of ionic current expression but also, over long periods of time, appearing to modify their correlated expression. We hypothesize that neuromodulators may regulate the correlated expression of ion channels at multiple levels and in a cell-type-dependent fashion. We report that in two identified neuronal types, three ionic currents are linearly correlated in a pairwise manner, suggesting their coexpression or direct interactions, under normal neuromodulatory conditions. In each cell, some currents remain correlated after neuromodulatory input is removed, whereas the correlations between the other pairs are either lost or altered. Interestingly, in each cell, a different suite of currents change their correlation. At the transcript level we observe distinct alterations in correlations between channel mRNA amounts, including one of the cell types lacking a correlation under normal neuromodulatory conditions and then gaining the correlation when neuromodulators are removed. Synaptic activity does not appear to contribute, with one possible exception, to the correlated expression of either ionic currents or of the transcripts that code for the respective channels. We conclude that neuromodulators regulate the correlated expression of ion channels at both the transcript and the protein levels.

Membrane Capacitance Measurements Revisited: Dependence of Capacitance Value on Measurement Method in Nonisopotential Neurons

During growth or degeneration neuronal surface area can change dramatically. Measurements of membrane protein concentration, as in ion channel or ionic conductance density, are often normalized by membrane capacitance, which is proportional to the surface area, to express changes independently from cell surface variations. Several electrophysiological protocols are used to measure cell capacitance, all based on the assumption of membrane isopotentiality. Yet, most neurons violate this assumption because of their complex anatomical structure, raising the question of which protocol yields measurements that are closest to the actual total membrane capacitance. We measured the capacitance of identified neurons from crab stomatogastric ganglia using three different protocols: the current-clamp step, the voltage-clamp step, and the voltage-clamp ramp protocols. We observed that the current-clamp protocol produced significantly higher capacitance values than those of either voltage-clamp protocol. Computational models of various anatomical complexities suggest that the current-clamp protocol can yield accurate capacitance estimates. In contrast, the voltage-clamp protocol estimates rapidly deteriorate as isopotentiality is reduced. We provide a mathematical description of these results by analyzing a simple two-compartment model neuron to facilitate an intuitive understanding of these methods. Together, the experiments, modeling, and mathematical analysis indicate that accurate total membrane capacitance measurements cannot be obtained with voltage-clamp protocols in nonisopotential neurons. Furthermore, although current-clamp steps can theoretically yield accurate measurements, experimentalists should be aware of limitations imposed by step duration and numerical errors during fitting procedures to obtain the membrane time constant.

The innervation of the pyloric region of the crab, Cancer borealis: homologous muscles in decapod species are differently innervated.

SummaryThe muscles of the pyloric region of the stomach of the crab,Cancer borealis, are innervated by motorneurons found in the stomatogastric ganglion (STG). Electrophysiological recording and stimulating techniques were used to study the detailed pattern of innervation of the pyloric region muscles. Although there are two Pyloric Dilator (PD) motorneurons in lobsters, previous work reported four PD motorneurons in the crab STG (Dando et al. 1974; Hermann 1979a, b). We now find that only two of the crab PD neurons innervate muscles homologous to those innervated by the PD neurons in the lobster,Panulirus interrruptus. The remaining two PD neurons innervate muscles that are innervated by pyloric (PY) neurons inP. interruptus. The innervation patterns of the Lateral Pyloric (LP), Ventricular Dilator (VD), Inferior Cardiac (IC), and PY neurons were also determined and compared with those previously reported in lobsters. Responses of the muscles of the pyloric region to the neurotransmitters, acetylcholine (ACh) and glutamate, were determined by application of exogenous cholinergic agonists and glutamate. The effect of the cholinergic antagonist, curare, on the amplitude of the excitatory junctional potentials (EJPs) evoked by stimulation of the pyloric motor nerves was measured. These experiments suggest that the differences in innervation pattern of the pyloric muscles seen in crab and lobsters are also associated with a change in the neurotransmitter active on these muscles. Possible implications of these findings for phylogenetic relations of decapod crustaceans and for the evolution of neural circuits are discussed.

Adult mouse basal forebrain harbors two distinct cholinergic populations defined by their electrophysiology

We performed whole-cell recordings from basal forebrain (BF) cholinergic neurons in transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the choline acetyltransferase promoter. BF cholinergic neurons can be differentiated into two electrophysiologically identifiable subtypes: early and late firing neurons. Early firing neurons (∼70%) are more excitable, show prominent spike frequency adaptation and are more susceptible to depolarization blockade, a phenomenon characterized by complete silencing of the neuron following initial action potentials. Late firing neurons (∼30%), albeit being less excitable, could maintain a tonic discharge at low frequencies. In voltage clamp analysis, we have shown that early firing neurons have a higher density of low voltage activated (LVA) calcium currents. These two cholinergic cell populations might be involved in distinct functions: the early firing group being more suitable for phasic changes in cortical acetylcholine release associated with attention while the late firing neurons could support general arousal by maintaining tonic acetylcholine levels.

Pacemaker neuron and network oscillations depend on a neuromodulator-regulated linear current

Linear leak currents have been implicated in the regulation of neuronal excitability, generation of neuronal and network oscillations, and network state transitions. Yet, few studies have directly tested the dependence of network oscillations on leak currents or explored the role of leak currents on network activity. In the oscillatory pyloric network of decapod crustaceans neuromodulatory inputs are necessary for pacemaker activity. A large subset of neuromodulators is known to activate a single voltage-gated inward current IMI, which has been shown to regulate the rhythmic activity of the network and its pacemaker neurons. Using the dynamic clamp technique, we show that the crucial component of IMI for the generation of oscillatory activity is only a close-to-linear portion of the current-voltage relationship. The nature of this conductance is such that the presence or the absence of neuromodulators effectively regulates the amount of leak current and the input resistance in the pacemaker neurons. When deprived of neuromodulatory inputs, pyloric oscillations are disrupted; yet, a linear reduction of the total conductance in a single neuron within the pacemaker group recovers not only the pacemaker activity in that neuron, but also leads to a recovery of oscillations in the entire pyloric network. The recovered activity produces proper frequency and phasing that is similar to that induced by neuromodulators. These results show that the passive properties of pacemaker neurons can significantly affect their capacity to generate and regulate the oscillatory activity of an entire network, and that this feature is exploited by neuromodulatory inputs.

Ionic current correlations underlie the global tuning of large numbers of neuronal activity attributes.

Ionic conductances in identified neurons are highly variable. This poses the crucial question of how such neurons can produce stable activity. Coexpression of ionic currents has been observed in an increasing number of neurons in different systems, suggesting that the coregulation of ionic channel expression, by thus linking their variability, may enable neurons to maintain relatively constant neuronal activity as suggested by a number of recent theoretical studies. We examine this hypothesis experimentally using the voltage- and dynamic-clamp techniques to first measure and then modify the ionic conductance levels of three currents in identified neurons of the crab pyloric network. We quantify activity by measuring 10 different attributes (oscillation period, spiking frequency, etc.), and find linear, positive and negative relationships between conductance pairs and triplets that can enable pyloric neurons to maintain activity attributes invariant. Consistent with experimental observations, some of the features most tightly regulated appear to be phase relationships of bursting activity. We conclude that covariation (and probably a tightly controlled coregulation) of ionic conductances can help neurons maintain certain attributes of neuronal activity invariant while at the same time allowing conductances to change over wide ranges in response to internal or environmental inputs and perturbations. Our results also show that neurons can tune neuronal activity globally via coordinate expression of ion currents.