K. P. Carlin

Dendritic L-type calcium currents in mouse spinal motoneurons: implications for bistability.

The intrinsic properties of mammalian spinal motoneurons provide them with the capability to produce high rates of sustained firing in response to transient inputs (bistability). Even though it has been suggested that a persistent dendritic calcium current is responsible for the depolarizing drive underlying this firing property, such a current has not been demonstrated in these cells. In this study, calcium currents are recorded from functionally mature mouse spinal motoneurons using somatic whole‐cell patch‐clamp techniques. Under these conditions a component of the current demonstrated kinetics consistent with a current originating at a site spatially segregated from the soma. In response to step commands this component was seen as a late‐onset, low amplitude persistent current whilst in response to depolarizing–repolarizing ramp commands a low voltage clockwise current hysteresis was recorded. Simulations using a neuromorphic motoneuron model could reproduce these currents only if a noninactivating calcium conductance was placed in the dendritic compartments. Pharmacological studies demonstrated that both the late‐onset and hysteretic currents demonstrated sensitivity to both dihydropyridines and the L‐channel activator FPL‐64176. Furthermore, the α1D subunits of L‐type calcium channels were immunohistochemically demonstrated on motoneuronal dendrites. It is concluded that there are dendritically located L‐type channels in mammalian motoneurons capable of mediating a persistent depolarizing drive to the soma and which probably mediate the bistable behaviour of these cells.

Characterization of calcium currents in functionally mature mouse spinal motoneurons.

Motoneurons integrate synaptic input and produce output in the form of trains of action potentials such that appropriate muscle contraction occurs. Motoneuronal calcium currents play an important role in the production of this repetitive firing. Because these currents change in the postnatal period, it is necessary to study them in animals in which the motor system is ‘functionally mature’, that is, animals that are able to weight‐bear and walk. In this study, calcium currents were recorded using whole‐cell patch‐clamp techniques from large (> 20 μm) ventral horn cells in lumbar spinal cord slices prepared from mature mice. Ninety percent (nine out of 10) of the recorded cells processed for choline acetyltransferase were found to be cholinergic, confirming their identity as motoneurons. A small number of motoneurons were found to have currents with low‐voltage‐activated (T‐type) characteristics. Pharmacological dissection of the high‐voltage‐activated current demonstrated ω‐agatoxin‐TK‐ (P/Q‐type), ω‐conotoxin GVIA‐ (N‐type), and dihydropyridine‐ and FPL‐64176‐sensitive (L‐type) components. A cadmium‐sensitive component of the current that was insensitive to these chemicals (R‐type) was also seen in these cells. These results indicate that the calcium current in lumbar spinal motoneurons from functionally mature mice is mediated by a number of different channel subtypes. The characterization of these calcium channels in mature mammalian motoneurons will allow for the future study of their modulation and their roles during behaviours such as locomotion.

Development of L-type calcium channels and a nifedipine-sensitive motor activity in the postnatal mouse spinal cord.

Intrinsic membrane properties are important in the regulation of motoneuronal output during such behaviours as locomotion. A conductance through L‐type calcium channels has been implicated as an essential component in the transduction of motoneuronal input to output during locomotion. Given the developmental changes in calcium currents occurring postnatally in some neurons, and the increasing interest in the study of spinal locomotor output in neonatal preparations, experiments were conducted to investigate the postnatal development of L‐type calcium channels in mouse motoneurons. This was assessed both physiologically, using a chemically induced rhythmic motor output, and anatomically, using immunohistochemical methods. The electrophysiological data were obtained during rhythmic bursting produced by application of N‐methyl‐d‐aspartate (NMDA) and strychnine to the isolated spinal cord at various postnatal ages. The L‐type calcium channel blocker nifedipine has no effect on this ventral root bursting in postnatal day (P) P2–P5 animals, but reversibly reduced the amplitude and/or burst duration of this activity in animals greater than P7. The immunohistochemical evidence demonstrates a dramatic change in the cellular profile of both the α1C and α1D subunits of L‐type calcium channels during postnatal development; the labelling of both subunits increases with age, approximating the adult pattern by P18. These results demonstrate that in the spinal cord, the L‐type calcium channel profile develops both physiologically and anatomically in the early postnatal period. This development parallels the development of the mature functional behaviours of weight bearing and walking, and may be necessary for the production of complex motor behaviour in the mature mammal.

Staircase currents in motoneurons: insight into the spatial arrangement of calcium channels in the dendritic tree.

In spinal motoneurons, activation of dendritically located depolarizing conductances can lead to amplification of synaptic inputs and the production of plateau potentials. Immunohistochemical and computational studies have implicated dendritic CaV1.3 channels in this amplification and suggest that CaV1.3 channels in spinal motoneurons may be organized in clusters in the dendritic tree. Our goal was to provide physiological evidence for the presence of multiple discrete clusters of voltage-gated calcium channels in spinal motoneurons and to explore the spatial arrangement of these clusters in the dendritic tree. We recorded voltage-gated calcium currents from spinal motoneurons in slices of mature mouse spinal cords. We demonstrate that single somatic voltage-clamp steps can elicit multiple inward currents with varying delays to onset, resulting in a current with a “staircase”-like appearance. Recordings from cultured dorsal root ganglion cells at different stages of neurite development provide evidence that these currents arise from the unclamped portions of the dendritic tree. Finally, both voltage- and current-clamp data were used to constrain computer models of a motoneuron. The resultant simulations impose two conditions on the spatial distribution of CaV channels in motoneuron dendrites: one of asymmetry relative to the soma and another of spatial separation between clusters of CaV channels. We propose that this compartmentalization would provide motoneurons with the ability to process multiple sources of input in parallel and integrate this processed information to produce appropriate trains of action potentials for the intended motor behavior.

Electrophysiological and Pharmacological Properties of Locomotor Activity-Related Neurons in cfos-EGFP Mice

Although locomotion is known to be generated by networks of spinal neurons, knowledge of the properties of these neurons is limited. Using neonatal transgenic mice that express enhanced green fluorescent protein (EGFP) driven by the c-fos promoter, we visualized EGFP-positive neurons in spinal cord slices from animals that were subjected to a locomotor task or drug cocktail [N-methyl-D-aspartate, serotonin (5-HT), dopamine, and acetylcholine (ACh)]. The activity-dependent expression of EGFP was also induced in dorsal root ganglion neurons with electrical stimulation of the neurons. Following 60-90 min of swimming, whole cell patch-clamp recordings were made from EGFP+ neurons in laminae VII, VIII, and X from slices of segments T(12) to L(4). The EGFP+ neurons (n = 55) could be classified into three types based on their responses to depolarizing step currents: single spike, phasic firing, and tonic firing. Membrane properties observed in these neurons include hyperpolarization-activated inward currents (29/55), postinhibitory rebound (11/55), and persistent-inward currents (31/55). Bath application of 10-40 microM 5-HT and/or ACh increased neuronal excitability or output with hyperpolarization of voltage threshold and changes in membrane potential. 5-HT also increased input resistance, reduced the afterhyperpolarization (AHP), and induced membrane oscillations, whereas ACh reduced the input resistance and increased the AHP. In this study, we demonstrate a new way of identifying neurons active in locomotion. Our results suggest that the EGFP+ neurons are a heterogeneous population of interneurons. The actions of 5-HT and ACh on these neurons provide insights into the neuronal properties modulated by these transmitters for generation of locomotion.

Postnatal Changes in the Inactivation Properties of Voltage-Gated Sodium Channels Contribute to the Mature Firing Pattern of Spinal Motoneurons

Most mammals are born with the necessary spinal circuitry to produce a locomotor-like pattern of neural activity. However, rodents seldom demonstrate weight-supported locomotor behavior until the second or third postnatal week, possibly due to the inability of the neuromuscular system to produce sufficient force during this early postnatal period. As spinal motoneurons mature they are seen to fire an increasing number of action potentials at an increasing rate, which is a necessary component of greater force production. The mechanisms responsible for this enhanced ability of motoneurons are not completely defined. In the present study we assessed the biophysical properties of the developing voltage-gated sodium current to determine their role in the maturing firing pattern. Using dissociated postnatal lumbar motoneurons in short-term culture (18-24 h) we demonstrate that currents recorded from the most mature postnatal age group (P10-P12) were significantly better able to maintain channels in an available state during repetitive stimulation than were the younger age groups (P1-P3, P4-P6, P7-P9). This ability correlated with the ability of channels to recover more quickly and more completely from an inactivated state. These age-related differences were seen in the absence of changes in the voltage dependence of channel gating. Differences in both closed-state inactivation and slow inactivation were also noted between the age groups. The results indicate that changes in the inactivation properties of voltage-gated sodium channels are important for the development of a mature firing pattern in spinal motoneurons.

Modulation of calcium currents in mouse ventral horn neurons by extracellular pH

Neuronal activity has been shown to modulate the pH of the extracellular environment. Since neuronal circuits in the ventral horn of the spinal cord are highly active during patterned movements, and voltage‐gated calcium channels play an important role in the production of spinal motoneuron output, the effects of changes in extracellular pH (pHe) on calcium currents in ventral horn neurons of the mouse spinal cord were examined. It is demonstrated that these channels are sensitive to modulation by pHe. The amplitude of the current mediated by these channels increased as the pHe was elevated. The elevated pHe also led to a hyperpolarizing shift in the voltage dependence of both activation and inactivation. The opposite effects were seen for a decrease in pHe. It was also noted that a decrease in pHe was associated with a faster inactivation of the current. It is concluded that voltage‐gated calcium currents in ventral horn neurons are modulated by changes in pHe, and that this modulation may play a physiologically important role in determining motoneuronal excitability during behaviors such as locomotion.

Simulation techniques for localising and identifying the kinetics of calcium channels in dendritic neurones

Abstract The present simulations were designed to determine whether the current–voltage (I–V) relationship obtained during voltage clamp was sufficient to characterise the kinetics and distribution of dendritic Ca2+ channels in spinal motoneurones. Two models were constructed, one based on a fully reconstructed adult cat motoneurone (neuromorphic model) the second a reduced two-compartment motoneurone model. The current–voltage (I–V) relationship in the neuromorphic model was used as a template to evaluate the I–V behaviour of the reduced model while simulating changes in location and kinetics of the calcium channels. The results show that the discriminative quality of the reduced model is low and the neuromorphic model remains the better qualitative match to the eletrophysiological results.

Rapid pH and PO2 changes in the tissue recording chamber during stoppage of a gas-equilibrated perfusate : effects on calcium currents in ventral horn neurons

In vitro studies often use bicarbonate‐buffered saline solutions to mimic the normal extracellular environment of tissues. These solutions are typically equilibrated with gaseous O2 and CO2, the latter interacting with bicarbonate ions to maintain a physiological pH. In vitro tissue chambers, like those used for electrophysiology, are usually continually perfused with the gassed buffer, but stopping the perfusion to add expensive chemicals or acquire imaging data is a common practice. The present study demonstrates that this procedure leads to rapid (< 30 s) increases in pH and decreases in PO2 of the detained solution in the tissue chamber. During the first 200 s, pH increased by 0.4 units and resulted in a 25% PO2 reduction of the detained solution. The rates of these changes were dependent on the volume of solution in the chamber. In experiments using acute transverse slices from the lumbar spinal cord of neonatal (postnatal day 0–10) mice, perfusion stoppage of the same duration was accompanied by a 34.7% enhancement of the peak voltage‐gated calcium current recorded from ventral horn neurons. In these cells both low voltage‐activated and high voltage‐activated currents were affected. These currents were unaffected by decreasing PO2 when a CO2‐independent buffer was used, suggesting that changes in pH were responsible for the observed effects. It is concluded that the procedure of stopping a bicarbonate/CO2‐buffered perfusate results in rapid changes in pH and PO2 of the solution detained in the tissue chamber, and that these changes have the potential to covertly influence experimental results.