Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation.

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive performance. These effects may be due to widespread connections of the trigeminal system to the ascending reticular activating system (ARAS), to which noradrenergic neurons of the locus coeruleus (LC) belongs. LC neurons contain projections to the whole brain, and it is known that their discharge co-varies with pupil size. LC activation is necessary for eliciting task-related mydriasis. If chewing effects on cognitive performance are mediated by the LC, it is reasonable to expect that changes in cognitive performance are correlated to changes in task-related mydriasis. Two novel protocols are presented here to verify this hypothesis and document that chewing effects are not attributable to aspecific motor activation. In both protocols, performance and pupil size changes observed during specific tasks are recorded before, soon after, and half an hour following a 2 min period of either: a) no activity, b) rhythmic, bilateral handgrip, c) bilateral chewing of soft pellet, and d) bilateral chewing of hard pellet. The first protocol measures level of performance in spotting target numbers displayed within numeric matrices. Since pupil size recordings are recorded by an appropriate pupillometer that impedes vision to ensure constant illumination levels, task-related mydriasis is evaluated during a haptic task. Results from this protocol reveal that 1) chewing-induced changes in performance and task-related mydriasis are correlated and 2) neither performance nor mydriasis are enhanced by handgrip. In the second protocol, use of a wearable pupillometer allows measurement of pupil size changes and performance during the same task, thus allowing even stronger evidence to be obtained regarding LC involvement in the trigeminal effects on cognitive activity. Both protocols have been run in the historical office of Prof. Giuseppe Moruzzi, the discoverer of ARAS, at the University of Pisa.

Improving sensitivity, specificity, and reproducibility of individual brainstem activation.

Functional imaging of the brainstem may open new avenues for clinical diagnostics. However, for reliable assessments of brainstem activation, further efforts improving signal quality are needed. Six healthy subjects performed four repeated functional magnetic resonance imaging (fMRI) sessions on different days with jaw clenching as a motor task to elicit activation in the trigeminal motor nucleus. Functional images were acquired with a 7 T MR scanner using an optimized multiband EPI sequence. Activation measures in the trigeminal nucleus and a control region were assessed using different physiological noise correction methods (aCompCor and RETROICOR-based approaches with variable numbers of regressors) combined with cerebrospinal fluid or brainstem masking. Receiver-operating characteristic analyses accounting for sensitivity and specificity, activation overlap analyses to estimate the reproducibility between sessions, and intraclass correlation analyses (ICC) for testing reliability between subjects and sessions were used to systematically compare the physiological noise correction approaches. Masking the brainstem led to increased activation in the target ROI and resulted in higher values for the area under the curve (AUC) as a combined measure for sensitivity and specificity. With the highest values for AUC, activation overlap, and ICC, the most favorable physiological noise correction method was to control for the cerebrospinal fluid time series (aCompCor with one regressor). Brainstem motor nuclei activation can be reliably identified using high-field fMRI with optimized acquisition and processing strategies-even on single-subject level. Applying specific physiological noise correction methods improves reproducibility and reliability of brainstem activation encouraging future clinical applications.

[Functional plasticity of trigeminal motor nucleus in unilateral mastication model rats].

To observe the plasticity changes of trigeminal motor nucleus (Mo5) and masseter H-reflex in unilateral mastication model rats and explore the possible mechanism of functional plasticity in motor center involved in unilateral mastication, 54 adult male Wistar rats were randomly divided into 1-month (n = 10), 3-month (n = 10), and 16-month (n = 7) model groups and their corresponding control groups, respectively. Unilateral mastication model rats were prepared by intermittent removal of clinical crowns of left teeth (model side). Rats were anesthetized (20% urethane, i.p.), and bilateral Mo5 were chosen to conduct extracellular recordings, while bilateral electromyography (EMG) of masseter muscle and its H-reflex were simultaneously recorded by a polygraph. It was observed that the firing rate of Mo5 neurons in model sides was significantly lower than that of right sides in 3 model groups, and that of left sides in their control groups. The response latency of Mo5, which was evoked by electrical stimulation of masseter nerve in model sides of 1-month and 3-month model groups, was significantly longer than that of left sides in their control groups. Moreover, the amplitude of H-wave in model sides of 3-month and 16-month model groups was lower than that of left sides in their control groups when H-reflex was evoked by electrical stimulation of left masseter nerve. These results suggest that unilateral mastication in model rats decreases the Mo5 neuron excitability, and this may be one of the functional plasticity mechanisms in motor center involved in unilateral mastication.

Reevaluation of reflex responses of the human masseter muscle to electrical lip stimulation.

We examined the reflex response of the human masseter muscle to electrical stimulation of the lip using both single motor unit and surface electromyogram based methods. Using the classical analysis methods, reflex response to mild electrical stimuli generated two distinct short-lasting inhibitions. This pattern may reflect the development of combinations of short- and long-latency inhibitory postsynaptic potentials as a result of the mildly painful electrical lip stimulation. However, this pattern appearing in the classical analysis methods may have developed as a consequence of earlier responses and may not be genuine. This study examined the genuineness of these responses using both the classical analysis methods and the discharge rate method to uncover the realistic postsynaptic potentials in human trigeminal motor nucleus. Using the discharge rate method, we found that the electrical lip stimulation only generated a long-lasting single or compound inhibitory response that is followed by late, long-lasting excitation. These findings have important implications on the redrawing of the neuronal pathways of the trigeminal nerve that are frequently used to judge neuromuscular disorders of the trigeminal region.NEW & NOTEWORTHY We examined the human masseter reflex response to electrical stimulation of lower lip to uncover realistic postsynaptic potentials in the trigeminal motor nucleus. We found that the stimulation generates a long-lasting single or compound inhibitory response that is followed by a late, long-lasting excitation. These findings have important implications on the redrawing of the neuronal pathways of the trigeminal nerve that are frequently used to judge neuromuscular disorders of the trigeminal region.

ISL1-based LIM complexes control Slit2 transcription in developing cranial motor neurons.

LIM-homeodomain (HD) transcription factors form a multimeric complex and assign neuronal subtype identities, as demonstrated by the hexameric ISL1-LHX3 complex which gives rise to somatic motor (SM) neurons. However, the roles of combinatorial LIM code in motor neuron diversification and their subsequent differentiation is much less well understood. In the present study, we demonstrate that the ISL1 controls postmitotic cranial branchiomotor (BM) neurons including the positioning of the cell bodies and peripheral axon pathfinding. Unlike SM neurons, which transform into interneurons, BM neurons are normal in number and in marker expression in Isl1 mutant mice. Nevertheless, the movement of trigeminal and facial BM somata is stalled, and their peripheral axons are fewer or misrouted, with ectopic branches. Among genes whose expression level changes in previous ChIP-seq and microarray analyses in Isl1-deficient cell lines, we found that Slit2 transcript was almost absent from BM neurons of Isl1 mutants. Both ISL1-LHX3 and ISL1-LHX4 bound to the Slit2 enhancer and drove endogenous Slit2 expression in SM and BM neurons. Our findings suggest that combinations of ISL1 and LHX factors establish cell-type specificity and functional diversity in terms of motor neuron identities and/or axon development.

Corticofugal projection patterns of whisker sensorimotor cortex to the sensory trigeminal nuclei.

The primary (S1) and secondary (S2) somatosensory cortices project to several trigeminal sensory nuclei. One putative function of these corticofugal projections is the gating of sensory transmission through the trigeminal principal nucleus (Pr5), and some have proposed that S1 and S2 project differentially to the spinal trigeminal subnuclei, which have inhibitory circuits that could inhibit or disinhibit the output projections of Pr5. Very little, however, is known about the origin of sensorimotor corticofugal projections and their patterns of termination in the various trigeminal nuclei. We addressed this issue by injecting anterograde tracers in S1, S2 and primary motor (M1) cortices, and quantitatively characterizing the distribution of labeled terminals within the entire rostro-caudal chain of trigeminal sub-nuclei. We confirmed our anterograde tracing results by injecting retrograde tracers at various rostro-caudal levels within the trigeminal sensory nuclei to determine the position of retrogradely labeled cortical cells with respect to S1 barrel cortex. Our results demonstrate that S1 and S2 projections terminate in largely overlapping regions but show some significant differences. Whereas S1 projection terminals tend to cluster within the principal trigeminal (Pr5), caudal spinal trigeminal interpolaris (Sp5ic), and the dorsal spinal trigeminal caudalis (Sp5c), S2 projection terminals are distributed in a continuum across all trigeminal nuclei. Contrary to the view that sensory gating could be mediated by differential activation of inhibitory interconnections between the spinal trigeminal subnuclei, we observed that projections from S1 and S2 are largely overlapping in these subnuclei despite the differences noted earlier.

Electrophysiological and morphological properties of rat supratrigeminal premotor neurons targeting the trigeminal motor nucleus.

The electrophysiological and morphological characteristics of premotor neurons in the supratrigeminal region (SupV) targeting the trigeminal motor nucleus (MoV) were examined in neonatal rat brain stem slice preparations with Ca(2+) imaging, whole cell recordings, and intracellular biocytin labeling. First, we screened SupV neurons that showed a rapid rise in intracellular free Ca(2+) concentration ([Ca(2+)]i) after single-pulse electrical stimulation of the ipsilateral MoV. Subsequent whole cell recordings were generated from the screened SupV neurons, and their antidromic responses to MoV stimulation were confirmed. We divided the antidromically activated premotor neurons into two groups according to their discharge patterns during the steady state in response to 1-s depolarizing current pulses: those firing at a frequency higher (HF neurons, n = 19) or lower (LF neurons, n = 17) than 33 Hz. In addition, HF neurons had a narrower action potential and a larger afterhyperpolarization than LF neurons. Intracellular labeling revealed that the axons of all HF neurons (6/6) and half of the LF neurons (4/9) entered the MoV from its dorsomedial aspect, whereas the axons of the remaining LF neurons (5/9) entered the MoV from its dorsolateral aspect. Furthermore, the dendrites of three HF neurons penetrated into the principal sensory trigeminal nucleus (Vp), whereas the dendrites of all LF neurons were confined within the SupV. These results suggest that the types of SupV premotor neurons targeting the MoV with different firing properties have different dendritic and axonal morphologies, and these SupV neuron classes may play unique roles in diverse oral motor behaviors, such as suckling and mastication.