Trigeminal mesencephalic (Mes V) neurons are critical components of the circuits controlling oral-motor activity. The possibility that they can function as interneurons necessitates a detailed understanding of the factors controlling their soma excitability. Using whole-cell patch-clamp recording, in vitro, we investigated the development of the ionic mechanisms responsible for the previously described subthreshold membrane oscillations and rhythmical burst discharge in Mes V neurons from rats ages postnatal day (P) 2-12. We found that the oscillation amplitude and frequency increased during development, whereas bursting emerged after P6. Furthermore, when bursting was initiated, the spike frequency was largely determined by the oscillation frequency. Frequency domain analysis indicated that these oscillations emerged from the voltage-dependent resonant properties of Mes V neurons. Low doses of 4-aminopyridine (<100 microm) reduced the oscillations and abolished resonance in most neurons, suggesting that the resonant current is a steady-state K(+) current (I(4-AP)). Sodium ion replacement or TTX reduced substantially the oscillations and peak amplitude of the resonance, suggesting the presence of a persistent Na(+) current (I(NaP)) that functions to amplify the resonance and facilitate the emergence of subthreshold oscillations and bursting.

Intracellular recordings and pharmacological manipulations were employed to investigate the ionic basis for serotonin-induced bistable membrane behaviors in guinea pig trigeminal motoneurons (TMNs). In voltage clamp, 10 microM serotonin (5-HT) induced a region of negative slope resistance (NSR) in the steady-state current-voltage (I-V) relationship at potentials less negative than -58 mV, creating the necessary conditions for membrane bistability. The contributions of sustained Na+ and Ca2+ currents to the generation of the NSR were investigated using specific ion channel antagonists and agonists. The NSR was eliminated by the L-type Ca2+ channel antagonist nifedipine (5-10 microM), indicating the contribution of L channels. In nifedipine, inward rectification was present in the I-V relationship in a similar voltage range (greater than -58 mV). This region was subsequently linearized by tetrodotoxin (TTX), indicating the presence of a persistent Na+ current. When the 5-HT-induced NSR was eliminated by perfusion in low Ca2+ solution (0.4 mM), it was restored by the Na+ channel agonist veratridine (10 microM). Commensurate with bistability, in current clamp during bath application of 5-HT, plateau potentials were elicited by transient depolarizing or hyperpolarizing stimuli. Plateau potentials evoked by depolarization were observed under control and TTX conditions, but were blocked by nifedipine, suggesting the participation of an L-type Ca2+ current. Plateau potentials initiated after release from hyperpolarization (anode break) were blocked by 300 microM Ni2+, suggesting the responses relied on deinactivation of a T-type Ca2+ current. Conditional bursting was also observed in 5-HT. Nifedipine or low Ca2+ solutions blocked bursting, and the L-channel agonist Bay K 8644 (10 microM) extended the duration of individual bursts, demonstrating the role of L-type Ca2+ currents. Interestingly, when bursting was blocked by nifedipine or low Ca2+, it could be restored by veratridine application via enhancement of the persistent Na+ current. We conclude that bistable membrane behaviors in TMNs are mediated by L-type Ca2+ and persistent Na+ currents. 5-HT is associated with enhancement of TMN activity during oral-motor activity; the induction of bistable membrane properties by 5-HT represents a cellular mechanism for this enhancement.

Intracellular recordings from guinea pig trigeminal motoneurons (TMNs) in brain stem slices were used to determine the underlying ionic mechanisms responsible for our previously demonstrated enhancement of TMN excitability during jaw movements by serotonin (5-HT). 5-HT (0.5-100 microM) depolarized motoneurons and increased input resistance in the majority of neurons tested. Additionally, 5-HT reduced the amplitude of the postspike medium-duration afterhyperpolarization, decreased the current threshold for maintained spike discharge, and increased the maximum slope of the steady-state spike frequency-current relationship. Under voltage clamp, from holding potentials close to resting potential, 5-HT produced an inward current and a decrease in instantaneous slope conductance, suggesting a reduction in a resting K+ leak conductance (I(leak)). The instantaneous current-voltage (I-V) relationship for the inward 5-HT current (I(5-HT)) was linear throughout most of the voltage range tested. However, the steady-state I-V relationship showed some degree of inward rectification at potentials starting around -70 mV. The mean reversal potential for the instantaneous I(5-HT) was -86.2 +/- 4.5 (SE) mV (n = 9), a value slightly negative to the predicted potassium equilibrium potential of -82 mV in these neurons. In the presence of 2 mM Ba2+, 5-HT application did not produce a further reduction in input conductance, but did expose a Ba2+-insensitive residual inward current that was resistant to Cs+ application. The instantaneous I-V relationship during 5-HT application in the presence of Ba2+ was shifted downward and parallel to control, suggesting that Ba2+ and 5-HT block the same resting I(leak). The residual Ba2+- and Cs+-insensitive component of the total inward I(5-HT) was voltage independent and was blocked when the extracellular Na+ was replaced by choline, suggesting that the predominant charge carrier for this residual current is Na+. 5-HT enhanced a hyperpolarization-activated cationic current, I(h). In the presence of Ba2+, the time course of I(5-HT) resembled that of I(h) and showed a similar voltage dependence that was blocked by extracellular Cs+ (1-3 mM). The effects of 5-HT on membrane potential, input resistance, and I(h) were partially mimicked by 5-HT2 agonists and suppressed by 5-HT2 antagonists. It is concluded that 5-HT enhances TMN membrane excitability through modulation of multiple intrinsic membrane conductances. This provides for a mechanism(s) to fine tune the input-output discharge properties of these neurons, thus providing them with greater flexibility in output in response to time-varying synaptic inputs during various movements of the jaw.

Subthreshold sodium currents are important in sculpting neuronal discharge and have been implicated in production and/or maintenance of subthreshold membrane oscillations and burst generation in mesencephalic trigeminal neurons (Mes V). Moreover, recent data suggest that, in some CNS neurons, resurgent sodium currents contribute to production of high-frequency burst discharge. In the present study, we sought to determine more directly the participation of these currents during Mes V electrogenesis using the action potentialclamp method. In postnatal day 8 -14 rats, the whole-cell patch-clamp method was used to record sodium currents by subtraction in response to application of TTX in voltage-clamp mode using the action potential waveform as the command protocol. We found that TTX-sensitive sodium current is the main inward current flowing during the interspike interval, compared with the h-current (I h ) and calcium currents. Furthermore, in addition to the transient sodium current that flows during the upstroke of action potential, we show that resurgent sodium current flows at the peak of afterhyperpolarization and persistent sodium current flows in the middle of the interspike interval to drive high-frequency firing. Additionally, transient, resurgent, and persistent sodium current components showed voltage-and time-dependent slow inactivation, suggesting that slow inactivation of these currents can contribute to burst termination. The data suggest an important role for these components of the sodium current in Mes V neuron electrogenesis.

Previous studies using pharmacological methods suggest that subthreshold sodium currents are critical for rhythmical burst generation in mesencephalic trigeminal neurons (Mes V). In this study, we characterized transient (I(NaT)), persistent (I(N)(aP)), and resurgent (I(res)) sodium currents in Na(v)1.6-null mice (med mouse, Na(v)1.6(-/-)) lacking expression of the sodium channel gene Scn8a. We found that peak transient, persistent, and resurgent sodium currents from med (Na(v)1.6(-/-)) mice were reduced by 18, 39, and 76% relative to their wild-type (Na(v)1.6(+/+)) littermates, respectively. Current clamp recordings indicated that, in response to sinusoidal constant amplitude current (ZAP function), all neurons exhibited membrane resonance. However, Mes V neurons from med mice had reduced peak amplitudes in the impedance-frequency relationship (resonant Q-value) and attenuated subthreshold oscillations despite the similar passive membrane properties compared with wild-type littermates. The spike frequency-current relationship exhibited reduced instantaneous discharge frequencies and spike block at low stimulus currents and seldom showed maintained spike discharge throughout the stimulus in the majority of med neurons compared with wild-type neurons. Importantly, med neurons never exhibited maintained stimulus-induced rhythmical burst discharge unlike those of wild-type littermates. The data showed that subthreshold sodium currents are critical determinants of Mes V electrogenesis and burst generation and suggest a role for resurgent sodium currents in control of spike discharge.

We investigated bursting behavior in rodent trigeminal neurons. The essential mechanisms operating in the biological systems were determined based on testable predictions of mathematical models. Bursting activity in trigeminal motoneurons is consistent with a traditional mechanism employing a region of negative slope resistance in the steady-state current-voltage relationship (Smith, T. G. 1975. Nature. 253:450-452). However, the bursting dynamics of trigeminal interneurons is inconsistent with the traditional mechanisms, and is far more effectively explained by a new model of bursting that exploits the unique stability properties associated with spike threshold (Baer, S. M., T. Erneux, and J. Rinzel. 1989. SIAM J. Appl. Math. 49:55-71).

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative motoneuron disease with presently no cure. Motoneuron (MN)hyperexcitability is commonly observed in ALS and is suggested to be a precursor for excitotoxic cell death. However, it is unknown whether hyperexcitability also occurs in MNs that are resistant to degeneration. Second, it is unclear whether all the MNs within homogeneous motor pools would present similar susceptibility to excitability changes since high-threshold MNs innervating fast fatigable muscle fibers selectively degenerate compared with low-threshold MNs innervating fatigue resistant slow muscle fibers. Therefore, we concurrently examined the excitability of ALS-vulnerable trigeminal motoneurons (TMNs) controlling jaw musculature and ALSresistant oculomotor neurons (OMNs) controlling eye musculature in a well studied SOD1 G93A ALS mouse model using in vitro patchclamp electrophysiology at presymptomatic ages P8 -P12. Our results show that hyperexcitability is not a global change among all the MNs, although mutant SOD1 is ubiquitously expressed. Instead, complex changes occur in ALS-vulnerable TMNs based on motor unit type and discharge characteristics. Firing threshold decreases among high-threshold TMNs and increases in a subpopulation of lowthreshold TMNs. The latter group was identified based on their linear frequency-current responses to triangular ramp current injections. Such complex changes in MN recruitment were absent in ALS-resistant OMNs. We simulated the observed complex changes in TMN excitability using a computer-based jaw closer motor pool model. Model results suggest that hypoexcitability may indeed represent emerging disease symptomology that causes resistance in muscle force initiation. Identifying the cellular and molecular properties of these hypoexcitable cells may guide effective therapeutic strategies in ALS.

To initiate and maintain bursts (and plateau potentials) in the presence of serotonin, guinea pig trigeminal motoneurons utilize L-type Ca2+ and persistent Na+ inward currents. However, the intrinsic currents that contribute to burst termination and determine the duration of the interburst interval are unknown. Therefore we investigated the roles of outward currents, whose slow activation is coupled to cytosolic cation (Ca2+ and Na+) accumulation. First we examined a Ca2+-dependent K+ current (IK-Ca) with apamin and Ba2+-substituted, low-Ca2+ solution. Blockade of IK-Ca lengthened burst duration and cycle time but did not abolish bursting. Next we studied the Na+/K+-ATPase pump current (Ip) with cardiac glycosides. In the presence of apamin or low-Ca2+/Ba2+ solution, blocking Ip (with ouabain or strophanthidin) decreased both burst duration and cycle time and ultimately transformed bursting into tonic spiking. We conclude that IK-Ca and Ip contribute to burst termination in trigeminal motoneurons. These currents influence temporal bursting properties such as burst duration and cycle time and may help determine the phasic activity of motoneurons during rhythmic oral-motor behaviors.