Chie-Fang Hsiao

Participation of Sodium Currents in Burst Generation and Control of Membrane Excitability in Mesencephalic Trigeminal Neurons

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 potential-clamp 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 (Ih) 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.

Evidence for a Novel Bursting Mechanism in Rodent Trigeminal Neurons

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).

Intrinsic membrane properties and morphological characteristics of interneurons in the rat supratrigeminal region

The membrane properties and morphological features of interneurons in the supratrigeminal area (SupV) were studied in rat brain slices using whole‐cell patch clamp recording techniques. We classified three morphological types of neurons as fusiform, pyramidal, and multipolar and four physiological types of neurons according to their discharge pattern in response to a 1‐sec depolarizing current pulse from −80 mV. Single‐spike neurons responded with a single spike, phasic neurons showed an initial burst of spikes and were silent during the remainder of the stimulus, delayed‐firing (DF) neurons exhibited a slow depolarization and delay to initial spike onset, and tonic (T) neurons showed maintained a discharge throughout the stimulus pulse. In a subpopulation of neurons (10%), membrane depolarization to around −44 mV produced a rhythmic burst discharge (RB) that was associated with voltage‐dependent subthreshold membrane oscillations. Both these phenomena were blocked by the sodium channel blocker riluzole at a concentration that did not affect the fast transient spike. Low doses of 4‐AP, which blocks low‐threshold K+ currents, transformed bursting into low‐frequency tonic discharge. In contrast, bursting occurred with exposure to cadium, a calcium‐channel blocker. This suggests that persistent sodium currents and low‐threshold K+ currents have a role in intrinsic burst generation. Importantly, RB cells were most often associated with multipolar neurons that exhibited either a DF or a T discharge. Thus, the SupV contains a variety of physiological cell types with unique morphologies and discharge characteristics. Intrinsic bursting neurons form a unique group in this region.

Characteristics of a fast transient outward current in guinea pig trigeminal motoneurons

A fast transient voltage dependent outward current (TOC) in trigeminal motoneurons (TMNs) was studied in guinea pig brainstem slices by use of sharp electrodes in combination with single electrode voltage clamp techniques. In solutions containing TTX, low Ca2+/Mn2+ and 20 mM TEA this current activated around -55 to -60 mV from holding potentials negative to resting potential, obtained its peak amplitude within 5 ms and decayed as a single exponential with a time constant of 6-8 ms. Half maximal values for inactivation and activation were -72 and -37 mV, respectively. Bath application of 5 mM 4-AP suppressed this current by approximately 90% and eliminated the early depolarizing transient membrane rectification observed in response to a constant depolarizing current pulse, prolonged the action potential duration, and reduced the threshold voltage and delay to onset of the action potential. It is suggested that this current resembles the typical A-current observed in many CNS neurons and, as a result of its voltage and time dependent properties, could contribute to control of motoneuronal discharge and timing of burst onset during rhythmical jaw movements. Therefore, any cellular models of masticatory activity should include the properties of this current.

Participation of a persistent sodium current and calcium-activated nonspecific cationic current to burst generation in trigeminal principal sensory neurons

The properties of neurons participating in masticatory rhythmogenesis are not clearly understood. Neurons within the dorsal trigeminal principal sensory nucleus (dPrV) are potential candidates as components of the masticatory central pattern generator (CPG). The present study examines in detail the ionic mechanisms controlling burst generation in dPrV neurons in rat (postnatal day 8-12) brain stem slices using whole cell and perforated patch-clamp methods. Nominal extracellular Ca(2+) concentration transformed tonic discharge in response to a maintained step pulse of current into rhythmical bursting in 38% of nonbursting neurons. This change in discharge mode was suppressed by riluzole, a persistent Na(+) current (INaP) antagonist. Veratridine, which suppresses the Na(+) channel inactivation mechanism, induced rhythmical bursting in nonbursting neurons in normal artificial cerebrospinal fluid, suggesting that INaP contributes to burst generation. Nominal extracellular Ca(2+) exposed a prominent afterdepolarizing potential (ADP) following a single spike induced by a 3-ms current pulse, which was suppressed, but not completely blocked, by riluzole. Application of BAPTA, a Ca(2+) chelator, intracellularly, or flufenamic acid, a Ca(2+)-activated nonspecific cationic channel (ICAN) antagonist, extracellularly to the bath, suppressed rhythmical bursting and the postspike ADP. Application of drugs to alter Ca(2+) release from endoplasmic reticulum also suppressed bursting. Finally, voltage-clamp methods demonstrated that nominal Ca(2+) facilitated INaP and induced ICAN. These data demonstrate for the first time that the previously observed induction in dPrV neurons of rhythmical bursting in nominal Ca(2+) is mediated by enhancement of INaP and onset of ICAN, which are dependent on intracellular Ca(2+).

Homeostatic Dysregulation in Membrane Properties of Masticatory Motoneurons Compared with Oculomotor Neurons in a Mouse Model for Amyotrophic Lateral Sclerosis

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 ALS-resistant oculomotor neurons (OMNs) controlling eye musculature in a well studied SOD1G93A ALS mouse model using in vitro patch-clamp 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 low-threshold 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.