Scott H. Chandler

NMDA receptor function in mouse models of Huntington disease

Huntington disease (HD) is an autosomal dominant disorder in which degeneration of medium‐sized spiny striatal neurons occurs. The HD gene and the protein it encodes, huntingtin, have been identified but their functions remain unknown. Transgenic mouse models for HD have been developed and we examined responses of medium‐sized striatal neurons recorded in vitro to application of N‐methyl‐D‐aspartate (NMDA) in two of these. The first model (R6/2) expresses exon 1 of the human HD gene with approximately 150 CAG repeats. In the R6/2 an enhancement of currents induced by selective activation of NMDA receptors as well as an enhancement of intracellular Ca2+ flux occurred in both presymptomatic and symptomatic mice. These alterations appeared specific for the NMDA receptor because α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptor‐mediated currents were reduced in symptomatic R6/2s. In R6/2 animals there were parallel increases in NMDA‐R1 and decreases in NMDA‐R2A/B subunit proteins as established by immunohistochemistry. The second model (YAC72) contains human genomic DNA spanning the full‐length gene and all its regulatory elements with 72 CAG repeats. The phenotypical expression of the disorder develops more gradually than in the R6/2. In YAC72 mice we found similar but less marked increases in responses of medium‐sized striatal neurons to NMDA. These findings indicate that alterations in NMDA receptor function may predispose striatal neurons to excitotoxic damage, leading to subsequent neuronal degeneration and underscore the functional importance of NMDA receptors in HD. J. Neurosci. Res. 66:525–539, 2001.

Alterations in N-methyl-D-aspartate receptor sensitivity and magnesium blockade occur early in development in the R6/2 mouse model of Huntington's disease.

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disorder that affects primarily the striatum and cerebral cortex. A search for the factors that increase the vulnerability of striatal neurons will lead to a better understanding of the pathological cascades of this disease. A current hypothesis for neurodegeneration of striatal medium‐sized spiny neurons in HD is an alteration in N‐methyl‐D‐aspartate (NMDA) receptor function. In the present study we examined electrophysiological properties of NMDA receptors in the R6/2 transgenic mouse model. These animals express exon 1 of the human HD gene and present an overt behavioral phenotype at about 5 weeks of age. Whole‐cell voltage clamp recordings from acutely dissociated striatal neurons were obtained from three different age groups of transgenic mice (15, 21, and 40 days old) and their littermate controls (WT). In transgenic animals, two groups of neurons were found with respect to NMDA and Mg2+ sensitivity. One group of R6/2 cells displayed responses similar to those of WT, whereas the other showed increased responses to NMDA and decreased Mg2+ sensitivity. These cells were encountered in all age groups. The abnormal sensitivity to NMDA and Mg2+ indicates that NMDA receptor alterations occur very early in development and suggest the presence of constitutively abnormal NMDA receptors. These alterations may contribute to an enhancement of NMDA responses at hyperpolarized membrane potentials that may be a key factor in striatal neuronal dysfunction.

Immunohistochemical localization of glutamate and glutaminase in guinea pig trigeminal premotoneurons.

Previous electrophysiological experiments in guinea pigs from our laboratory [11,36,37] have suggested that synaptic transmission between last-order interneurons (premotoneurons) and trigeminal motoneurons during reflex activation or cortically induced rhythmical jaw movements is mediated by excitatory amino acids (EAAs). In the present study, we performed a series of double-labeling experiments in guinea pigs to determine the location of neurons which contain glutamate or glutaminase and project to the trigeminal motor nucleus (Mo5). This was accomplished by combining immunohistochemical staining and standard retrograde tract-tracing techniques. Injections of a retrograde tracer, colloidal-gold bound to inactivated WGA-HRP (gWGA-HRP), into the trigeminal motor nucleus labeled a column of neurons originating adjacent to Mo5, including the supratrigeminal nucleus, intertrigeminal nucleus and the mesencephalic nucleus of V. The column extended caudally into the parvocellular reticular formation and adjacent trigeminal sensory nucleus oralis and oralis gamma subdivision. In all of these regions, immunoreactivity to glutamate or glutaminase was observed co-localized with gWGA-HRP.

Rhythmical Oral-Motor Activity Recorded in an In Vitro Brainstem Preparation

The present study employed the neonatal rat isolated brainstem preparation to determine whether oral-motor rhythmical activity, a substrate for the complex behaviors of suckling and chewing, could be elicited in vitro by path application of excitatory amino acids (EAAs). Bath application of EAA agonists (kainate [KA], [+/-]-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid [AMPA], N-methyl-D, L-aspartate [NMA]), in conjunction with the gamma-aminobutyric acid antagonist bicuculline, either failed to induce rhythmic activity (n = 17 preparations) or induced a low-amplitude, low-frequency burst discharge (< 1 Hz, n = 10 preparations) from the motor branches of the trigeminal nerves when the brainstem was contiguous from the spinomedullary junction to the superior colliculus. Burst activity was in most cases bilaterally synchronous. However, when a discrete coronal transection was made at the level of the facial colliculus, between the trigeminal and facial motor nuclei, the rhythmic bursts produced by the resultant 3- to 5-mm block of tissue following bath application of EAA agonists increased in amplitude and frequency (4-8 Hz, n = 35). Application of 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), a non-N-methyl-D-aspartate (non-NMDA) receptor antagonist, blocked the rhythm induced by non-NMDA receptor agonist (n = 4) but was less effective in suppressing NMA-induced rhythmicity. In contrast, D, L-2-amino-5-phosphonovaleric acid (APV) blocked by both NMA-induced (n = 5) and, in most cases, KA-induced (n = 5) rhythmicity, suggesting an essential role for NMA receptors in production of EAA-induced rhythmical oral-motor activity in the neonatal rat. The present data demonstrate that a narrow, bilaterally distributed region of brainstem surrounding the trigeminal motor nucleus contains sufficient neuronal circuitry for the production of oral-motor rhythmogenesis.

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.

Localization of oral-motor rhythmogenic circuits in the isolated rat brainstem preparation

Using an in vitro isolated brainstem preparation from neonatal rat (0-2 days), the minimal circuitry for production of rhythmical oral-motor activity was determined. In the presence of the excitatory amino acid agonist, N-methyl-D,L-aspartate (NMA), and the GABAA antagonist, bicuculline (BIC), rhythmical oral-motor activity was recorded from the motor branch of the trigeminal nerve. In preparations where the brainstem was isolated in continuity between the rostral inferior colliculus and the obex, oral-motor activity was not observed. However, when the brainstem was serially transected in the coronal plane starting at the obex and proceeding rostrally, rhythmogenic activity emerged and became more stable until the level of the rostral facial nucleus (facial colliculus, FC) was approached. Transections more rostral than the FC produced rhythms that progressively deteriorated until the trigeminal motor nucleus (MoV) was reached, at which point all activities ceased. Surgical isolation of an ipsilateral quadrant of the brainstem encompassing the tissue between the FC and inferior colliculus, rostro-caudally, and the midline to lateral brainstem, medio-laterally, exhibited oral-motor activity as well. The remaining contralateral side of brainstem was devoid of rhythmical trigeminal activity. However, further coronal transection of the remaining brainstem at the level of the FC induced rhythmical oral-motor activity in the trigeminal nerve. The data suggest the existence of bilaterally coordinated rhythmogenic circuits in each half of brainstem between the rostral trigeminal nucleus and the rostral facial nucleus, which are tonically inhibited by brainstem circuits caudal to the facial nucleus.

Evidence for excitatory amino acid transmission between mesencephalic nucleus of V afferents and jaw-closer motoneurons in the guinea pig

Previous studies have suggested that monosynaptic transmission between spinal primary afferent fibers and motoneurons is mediated by an excitatory amino acid, most likely glutamate or aspartate. No such comparable studies have been carried out in the trigeminal system. In an attempt to elucidate the neurotransmitter(s) mediating monosynaptic transmission between mesencephalic of V nucleus afferents (Mes V) and trigeminal jaw-closer motoneurons, the effect of iontophoretic application of excitatory amino acid antagonists on the Mes V-induced field potential, recorded in the trigeminal motor nucleus (Mot V), was examined. Application of DL-2-amino-4-phosphonobutyrate (APB) and the broad spectrum amino acid antagonists, kynurenic acid (KYN) and gamma-D-glutamylglycine (DGG), for 3-4 min reversibly reduced the amplitude of the Mes V induced field potential. The effect of APB was much greater than any of the other compounds tested. On the other hand, the specific N-methyl-D-aspartate (NMDA) receptor blocker DL-2-amino-5-phosphonovaleric acid (APV), was without effect on the field potential. Based on current-response curves for each antagonist tested, the order of potency was determined to be APB greater than KYN greater than DGG greater than APV. These antagonists were also compared with respect to their efficacy in blocking individual jaw-closer motoneuron activity induced by iontophoretic application of amino acid receptor excitants glutamate (Glut), aspartate (Asp), kainate (K), and quisqualate (Q). NMDA application was without effects on these motoneurons. The profile of activity of these antagonists on these amino acid excitants was similar to that found in other areas of the CNS by other investigators. KYN and DGG both significantly reduced responses induced by all excitants tested, whereas APB had more modest effects on K and Q excitation and was without effect on Glut and Asp excitations in most cells tested. The data suggest that an excitatory amino acid, activating non-NMDA receptors, mediates some component of synaptic transmission between Mes V afferents and jaw-closer motoneurons. The data is also consistent with the proposal made in other systems that APB blocks synaptic transmission by a mechanism other than postsynaptic receptor blockade.

The effects of orofacial sensory input on spontaneously occurring and apomorphine-induced rhythmical jaw movements in the anesthetized guinea pig

The effects of tonic mandibular loading (jaw depression) on spontaneously occurring and apomorphine (APO)-induced rhythmical jaw movements (RJMs) were examined in the anesthetized guinea pig. It was found that this type of perturbation significantly increased only the amplitude and burst duration of the masseter (jaw closer) EMG activity, whereas the frequency of RJMs was not changed. The data suggest that jaw closer muscle spindle or temporomandibular joint feedback does not strongly influence the activity of the neural networks responsible for determining the frequency of RJMs in the anesthetized guinea pig.

NMDA receptor NR1 and NR2A/B subunit expression in trigeminal neurons during early postnatal development

Trigeminal motoneurons (Mo5), mesencephalic trigeminal neurons (Me5), and supratrigeminal (Su5) and intertrigeminal (I5) neurons are important constituents of the neural circuitry responsible for jaw movements observed during ingestive behaviors. In addition, in adult animals, N‐methyl‐D‐aspartate (NMDA) receptors are a critical component of the brainstem circuitry responsible for reflex‐ and centrally activated jaw movements. However, little is known about the expression of this receptor in circuitry used to produce neonatal jaw movements. Receptor immunohistochemistry was used to describe changes in the expression of NMDA NR1 and NR2A/B receptor subunits in Mo5, Me5, Su5, and I5 neurons during postnatal development. Rats at postnatal days (P) 1, 3, 8, 15–16, 21–24, and 28–35 were used. An affinity‐purified polyclonal antibody against the NR1 subunit and an affinity‐purified polyclonal antibody that recognizes both NR2A and 2B subunits were used to depict the expression of these subunits. In Mo5, immunoreactivity was noted for both antibodies throughout the time frame sampled. NR1 expression in Me5 neurons emerged at P1. NR2A/B expression emerged at P3 in caudal and middle regions of Me5 and at P8 for rostral regions of the nucleus. NR1 immunoreactivity was present at P1 for neurons in I5 and at P3 for neurons in the Su5 region. NR2A/B subunit expression in Su5 and I5 neurons emerged at P8. These results provide evidence for NMDA receptor subunits in neonatal trigeminal neurons used in oral–motor circuitry and suggest a role for the NMDA receptor in synaptogenesis associated with these neurons during postnatal development. J. Comp. Neurol. 409:237–249, 1999.

The effects of nanoliter ejections of lidocaine into the pontomedullary reticular formation on cortically induced rhythmical jaw movements in the guinea pig

In the ketamine/urethane anesthetized guinea pig, electromyographic (EMG) responses of the anterior digastric muscle were studied when loci within the lower brainstem were microejected with lidocaine (2%) during rhythmical jaw movements (RJMs) evoked by repetitive electrical stimulation of the masticatory area of the cortex. The area investigated was between the trigeminal motor nucleus (Mot V) and the rostral pole of the inferior olive. Microejections of lidocaine, contralateral to the cortical stimulus site, into the ventral-medial portion of Mot V where digastric motoneurons are known to be located, resulted in reduction or complete abolishment of the digastric EMG activity ipsilateral to the ejection with no effective change in mean cycle duration (CD) or mean percent normalized integrated amplitude of the contralateral digastric EMG. Microejections of lidocaine, contralateral to the cortical stimulus site, into the ponto-medullary reticular formation in areas that included portions of the caudal nucleus pontis caudalis (PnC), nucleus gigantocellularis (GC), medial nucleus parvocellularis (PCRt), and dorsal paragigantocellularis (dPGC), in most cases produced a bilateral reduction in the mean normalized integrated amplitude and a bilateral increase in the mean cycle duration. In these sites, the bilateral increase in mean cycle duration of digastric EMG bursts was also associated with a significant increase of coefficient of variation in CD. In many cases, microejection of lidocaine completely abolished rhythmical digastric activity, bilaterally. HRP injections into Mot V were performed to determine the locations of trigeminal premotoneurons and their relationship to effective lidocaine sites for rhythmical jaw movement suppression. Retrogradely labeled cells were found mainly in the mesencephalic nucleus of V; trigeminal principal and spinal V sensory nuclei, bilaterally; and within the intermediate and lateral regions of reticular formation, bilaterally. No labeling was found in the medial reticular formation, including the nucleus gigantocellularis and dorsal paragigantocellularis.(ABSTRACT TRUNCATED AT 400 WORDS)