Neuronal population activity in the olivocerebellum encodes the frequency of essential tremor in mice and patients.

Essential tremor (ET) is the most prevalent movement disorder, characterized primarily by action tremor, an involuntary rhythmic movement with a specific frequency. However, the neuronal mechanism underlying the coding of tremor frequency remains unexplored. Here, we used in vivo electrophysiology, optogenetics, and simultaneous motion tracking in the Grid2dupE3 mouse model to investigate whether and how neuronal activity in the olivocerebellum determines the frequency of essential tremor. We report that tremor frequency was encoded by the temporal coherence of population neuronal firing within the olivocerebellums of these mice, leading to frequency-dependent cerebellar oscillations and tremors. This mechanism was precise and generalizable, enabling us to use optogenetic stimulation of the deep cerebellar nuclei to induce frequency-specific tremors in wild-type mice or alter tremor frequencies in tremor mice. In patients with ET, we showed that deep brain stimulation of the thalamus suppressed tremor symptoms but did not eliminate cerebellar oscillations measured by electroencephalgraphy, indicating that tremor-related oscillations in the cerebellum do not require the reciprocal interactions with the thalamus. Frequency-disrupting transcranial alternating current stimulation of the cerebellum could suppress tremor amplitudes, confirming the frequency modulatory role of the cerebellum in patients with ET. These findings offer a neurodynamic basis for the frequency-dependent stimulation of the cerebellum to treat essential tremor.

Nyquist-exceeding high voxel rate acquisition in mesoscopic multiphoton microscopy for full-field submicron resolution resolvability.

The Nyquist-Shannon criterion has never been realized in a laser-scanning mesoscopic multiphoton microscope (MPM) with a large field-of-view (FOV)-resolution ratio, especially when employing a high-frequency resonant-raster-scanning. With a high optical resolution nature, a current mesoscopic-MPM either neglects the criterion and degrades the digital resolution to twice the pixel size, or reduces the FOV and/or the raster-scanning speed to avoid aliasing. We introduce a Nyquist figure-of-merit (NFOM) parameter to characterize a laser-scanning MPM in terms of its optical-resolution retrieving ability. Based on NFOM, we define the maximum aliasing-free FOV, and subsequently, a cross-over excitation wavelength, below which the FOV becomes NFOM-constrained irrespective of an optimized optical design. We validate our idea in a custom-built mesoscopic-MPM with millimeter-scale FOV yielding an ultra-high FOV-resolution ratio of >3,000, while securing up-to a 1.6 mm Nyquist-satisfied aliasing-free FOV, a ∼400 nm lateral resolution, and a 70 M/s effective voxel-sampling rate, all at the same time.

Longitudinal intravital imaging nerve degeneration and sprouting in the toes of spared nerve injured mice.

Neuropathic pain is pain caused by damage to the somatosensory nervous system. Both degenerating injured nerves and neighboring sprouting nerves can contribute to neuropathic pain. However, the mesoscale changes in cutaneous nerve fibers over time after the loss of the parent nerve has not been investigated in detail. In this study, we followed the changes in nerve fibers longitudinally in the toe tips of mice that had undergone spared nerve injury (SNI). Nav1.8-tdTomato, Thy1-GFP and MrgD-GFP mice were used to observe the small and large cutaneous nerve fibers. We found that peripheral nerve plexuses degenerated within 3 days of nerve injury, and free nerve endings in the epidermis degenerated within 2 days. The timing of degeneration paralleled the initiation of mechanical hypersensitivity. We also found that some of the Nav1.8-positive nerve plexuses and free nerve endings in the fifth toe survived, and sprouting occurred mostly from 7 to 28 days. The timing of the sprouting of nerve fibers in the fifth toe paralleled the maintenance phase of mechanical hypersensitivity. Our results support the hypotheses that both injured and intact nerve fibers participate in neuropathic pain, and that, specifically, nerve degeneration is related to the initiation of evoked pain and nerve sprouting is related to the maintenance of evoked pain.

An Index Combining Lost and Remaining Nerve Fibers Correlates with Pain Hypersensitivity in Mice.

Multiple peripheral nerves are known to degenerate after nerve compression injury but the correlation between the extent of nerve alteration and pain severity remains unclear. Here, we used intravital two-photon fluorescence microscopy to longitudinally observe changes in cutaneous fibers in the hind paw of Nav1.8-Cre-tdTomato mice after chronic constriction injury (CCI). Results showed that the CCI led to variable loss of the skin nerve plexus and intraepidermal nerve fibers. The timing of Nav1.8 nerve fiber loss correlated with the development of mechanical hypersensitivity. We compared a scoring approach that assessed whole-paw nerve degeneration with an index that quantified changes in the nerve plexus and terminals in multiple small regions of interest (ROI) from intravital images of the third and fifth toe tips. We found that the number of surviving nerve fibers was not linearly correlated with mechanical hypersensitivity. On the contrary, at 14 days after CCI, the moderately injured mice showed greater mechanical hypersensitivity than the mildly or severely injured mice. This indicates that both surviving and injured nerves are required for evoked neuropathic pain. In addition, these two methods may have the estimative effect as diagnostic and prognostic biomarkers for the assessment of neuropathic pain.

Cerebellar oscillations driven by synaptic pruning deficits of cerebellar climbing fibers contribute to tremor pathophysiology.

Essential tremor (ET) is one of the most common movement disorders and the prototypical disorder for abnormal rhythmic movements. However, the pathophysiology of tremor generation in ET remains unclear. Here, we used autoptic cerebral tissue from patients with ET, clinical data, and mouse models to report that synaptic pruning deficits of climbing fiber (CF)-to-Purkinje cell (PC) synapses, which are related to glutamate receptor delta 2 (GluRδ2) protein insufficiency, cause excessive cerebellar oscillations and might be responsible for tremor. The CF-PC synaptic pruning deficits were correlated with the reduction in GluRδ2 expression in the postmortem ET cerebellum. Mice with GluRδ2 insufficiency and CF-PC synaptic pruning deficits develop ET-like tremor that can be suppressed with viral rescue of GluRδ2 protein. Step-by-step optogenetic or pharmacological inhibition of neuronal firing, axonal activity, or synaptic vesicle release confirmed that the activity of the excessive CF-to-PC synapses is required for tremor generation. In vivo electrophysiology in mice showed that excessive cerebellar oscillatory activity is CF dependent and necessary for tremor and optogenetic-driven PC synchronization was sufficient to generate tremor in wild-type animals. Human validation by cerebellar electroencephalography confirmed that excessive cerebellar oscillations also exist in patients with ET. Our findings identify a pathophysiologic contribution to tremor at molecular (GluRδ2), structural (CF-to-PC synapses), physiological (cerebellar oscillations), and behavioral levels (kinetic tremor) that might have clinical applications for treating ET.

Saturated two-photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth.

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near-infrared spectrum can improve the present situation. Here, the capability of saturated two-photon saturated excitation (TP-SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering-enlarged point spread function (PSF), we find that TP-SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single-photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering-enlarged PSF, TP-SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.

Spatiotemporal Changes of Neuronal Responses in the Primary Somatosensory Cortex to Noxious Tail Stimulation in Awake and Pentobarbital-Anesthetized Rats.

Primary somatosensory cortex (SI) is a key area in the processing of nociceptor inputs to our consciousness. To clarify the columnar and laminar organization of SI for pain processing, we compared spatiotemporal changes in neuronal activities of the primary sensorimotor cortex (SmI) of the rat in response to noxious laser heat stimulation applied to the mid-tail. Longitudinal and vertical array microelectrodes were chronically implanted in the cerebral cortex. Evoked neuronal activities, including intracortical local field potentials (LFP) and ensemble single-unit activity (SU) around SmI were simultaneously recorded. The effect of pentobarbital on the neuronal responses was evaluated in comparison with the neuronal responses in conscious animals to explore the potential substrate of nociceptive processing in the conscious state. The results from the experiment with longitudinal microelectrode arrays indicated that noxious stimulation induced a neuronal response which was spread widely around the SmI of the conscious rat, and the range of neuronal responses was limited to the tail region of the SmI under anesthesia. The results from the experiment with vertical microelectrode arrays showed the universal neuronal responses through all cortical layers of the SmI in conscious rats, and sodium pentobarbital suppressed these neuronal responses in the supragranular layers significantly relative to the deeper layers and basal activity. These results imply that a wider range of cortical activation, both in the horizontal or vertical dimension, might be important for nociceptive processing in the conscious state.

Long-term neuroplasticity of the face primary motor cortex and adjacent somatosensory cortex induced by tooth loss can be reversed following dental implant replacement in rats.

Tooth loss is common, and exploring the neuroplastic capacity of the face primary motor cortex (face-M1) and adjacent primary somatosensory cortex (face-S1) is crucial for understanding how subjects adapt to tooth loss and their prosthetic replacement. The aim was to test if functional reorganization of jaw and tongue motor representations in the rat face-M1 and face-S1 occurs following tooth extraction, and if subsequent dental implant placement can reverse this neuroplasticity. Rats (n = 22) had the right maxillary molar teeth extracted under local and general anesthesia. One month later, seven rats had dental implant placement into healed extraction sites. Naive rats (n = 8) received no surgical treatment. Intracortical microstimulation (ICMS) and recording of evoked jaw and tongue electromyographic responses were used to define jaw and tongue motor representations at 1 month (n = 8) or 2 months (n = 7) postextraction, 1 month postimplant placement, and at 1-2 months in naive rats. There were no significant differences across study groups in the onset latencies of the ICMS-evoked responses (P > 0.05), but in comparison with naive rats, tooth extraction caused a significant (P < 0.05) and sustained (1-2 months) decreased number of ICMS-defined jaw and tongue sites within face-M1 and -S1, and increased thresholds of ICMS-evoked responses in these sites. Furthermore, dental implant placement reversed the extraction-induced changes in face-S1, and in face-M1 the number of jaw sites even increased as compared to naive rats. These novel findings suggest that face-M1 and adjacent face-S1 may play a role in adaptive mechanisms related to tooth loss and their replacement with dental implants.

Neuroplastic changes in the sensorimotor cortex associated with orthodontic tooth movement in rats.

Orthodontic tooth movement (OTM) causes transient pain and changes in the dental occlusion that may lead to altered somatosensory inputs and patterns of mastication. This study used intracortical microstimulation (ICMS) and electromyographic (EMG) recordings to test whether neuroplastic changes occur in the ICMS-defined motor representations of left and right anterior digastric (LAD, RAD), masseter, buccinator, and genioglossus (GG) muscles within the rat's face primary motor cortex (face-M1) and adjacent face primary somatosensory cortex (face-S1) during OTM. Analyses included any changes in the number of ICMS sites representing these muscles and in the onset latencies of ICMS-evoked responses in the muscles. Sprague-Dawley rats were divided into experimental (E), sham (S), and naive (N) groups; OTM was induced in the E group. Statistical analyses involved a mixed model repeated-measures analysis of variance (MMRM ANOVA). OTM resulted in significant neuroplastic changes in the number of positive sites in the E group for LAD, RAD, and GG muscles in face-M1 and face-S1 at days 1, 7, and 28 of continuous orthodontic force application, and in the number of sites in face-M1 from which ICMS could simultaneously evoke EMG responses in different combinations of LAD, RAD, and GG muscles. However, the onset latencies of ICMS-evoked responses were not significantly different between groups or between face-M1 and face-S1. The neuroplastic changes documented in this study may reflect adaptive sensorimotor changes in response to the altered environment in the oral cavity induced by OTM.

Modulation dynamics in the orofacial sensorimotor cortex during motor skill acquisition.

The orofacial sensorimotor cortex is known to play a role in motor learning. However, how motor learning changes the dynamics of neuronal activity and whether these changes differ between orofacial primary motor (MIo) and somatosensory (SIo) cortices remain unknown. To address these questions, we used chronically implanted microelectrode arrays to track learning-induced changes in the activity of simultaneously recorded neurons in MIo and SIo as two naive monkeys (Macaca mulatta) were trained in a novel tongue-protrusion task. Over a period of 8-12 d, the monkeys showed behavioral improvements in task performance that were accompanied by rapid and long-lasting changes in neuronal responses in MIo and SIo occurring in parallel: (1) increases in the proportion of task-modulated neurons, (2) increases in the mutual information between tongue-protrusive force and spiking activity, (3) reductions in the across-trial firing rate variability, and (4) transient increases in coherent firing of neuronal pairs. More importantly, the time-resolved mutual information in MIo and SIo exhibited temporal alignment. While showing parallel changes, MIo neurons exhibited a bimodal distribution of peak correlation lag times between spiking activity and force, whereas SIo neurons showed a unimodal distribution. Moreover, coherent activity between pairs of MIo neurons was higher and centered around force onset compared with pairwise coherence of SIo neurons. Overall, the results suggest that the neuroplasticity in MIo and SIo occurring in parallel serves as a substrate for linking sensation and movement during sensorimotor learning, whereas the differing dynamic organizations reflect specific ways to control movement parameters as learning progresses.

Characterization of esophageal defects in the Crouzon mouse model.

BACKGROUND: Mutations in Fibroblastic Growth Factor Receptors (FGFR) have been associated with human craniosynostotic birth defects like Crouzon syndrome. Several anecdotes and case reports have indicated higher incidence of gastrointestinal tract disorders in FGFR-associated craniosynostotic birth defects. Our objective was to characterize esophageal defects in a mouse model of human Crouzon syndrome, with a mutation in codon 290 of FGFR2. METHODS: Dissected esophagi of Fgfr2(W290R) postnatal heterozygous (HET) and wild-type mice were analyzed by histological staining, immunohistochemically with cell proliferation marker, and functionally by strain gauge measures of electrically evoked contractile force. RESULTS: The esophagi of HETs were noticeably smaller but with wider lumen than those of wild-type littermates. The HET esophagi showed a decrease in proliferation and an increase in expression of Sonic Hedgehog as compared to wild-type esophagi. Histological investigations revealed reduced amounts and disorganization of collagen in muscle layers. Functional analysis revealed altered contractile properties in HET with reduced peak amplitude and prolonged duration of evoked contractile force response and lower stimulation threshold. CONCLUSION: The defects observed in the esophagus of the mutant may explain some of the clinical symptoms observed in humans, for example, recurrent vomiting, gastroesophageal reflux, and esophageal strictures. Taken together, our results provide evidence for the importance of Fibroblastic Growth Factor signaling in the growth and patterning of the esophagus, providing a possible scientific basis for the gastrointestinal tract clinical findings in craniosynostotic patients. Furthermore, the findings also provide a sound scientific rationale for any changes in the clinical management of gastrointestinal tract problems in patients with craniosynostotic defects.

Jaw-opening reflex and corticobulbar motor excitability changes during quiet sleep in non-human primates.

STUDY OBJECTIVE: To test the hypothesis that the reflex and corticobulbar motor excitability of jaw muscles is reduced during sleep. DESIGN: Polysomnographic recordings in the electrophysiological study. SETTING: University sleep research laboratories. PARTICIPANTS AND INTERVENTIONS: The reflex and corticobulbar motor excitability of jaw muscles was determined during the quiet awake state (QW) and quiet sleep (QS) in monkeys (n = 4). MEASUREMENTS AND RESULTS: During QS sleep, compared to QW periods, both tongue stimulation-evoked jaw-opening reflex peak and root mean square amplitudes were significantly decreased with stimulations at 2-3.5 × thresholds (P < 0.001). The jaw-opening reflex latency during sleep was also significantly longer than during QW. Intracortical microstimulation (ICMS) within the cortical masticatory area induced rhythmic jaw movements at a stable threshold (≤ 60 µA) during QW; but during QS, ICMS failed to induce any rhythmic jaw movements at the maximum ICMS intensity used, although sustained jaw-opening movements were evoked at significantly increased threshold (P < 0.001) in one of the monkeys. Similarly, during QW, ICMS within face primary motor cortex induced orofacial twitches at a stable threshold (≤ 35 µA), but the ICMS thresholds were elevated during QS. Soon after the animal awoke, rhythmic jaw movements and orofacial twitches could be evoked at thresholds similar to those before QS. CONCLUSIONS: The results suggest that the excitability of reflex and corticobulbar-evoked activity in the jaw motor system is depressed during QS.

Face sensorimotor cortex and its neuroplasticity related to orofacial sensorimotor functions.

This review describes evidence in subprimates and primates that the face primary somatosensory cortex (face SI) and primary motor cortex (face MI) are involved in sensorimotor integration and control of orofacial motor functions that include semiautomatic movements (e.g., chewing, swallowing) and voluntary movements (e.g., jaw-opening). The review also notes that the neuroplastic capabilities of the face SI and face MI have recently been documented, and may reflect or allow for functional adaptation (or maladaptation) of the orofacial sensorimotor system to an altered oral state or oral motor behaviour. They may contribute to the processes whereby patients undergoing oral rehabilitation can (or cannot) restore the lost orofacial sensorimotor functions. Such understanding is important since pain, injuries to the oral tissues, and alterations to the dental occlusion induced by tooth loss or attrition are common occurrences in humans that may sometimes be accompanied by impaired oral sensorimotor functions. Furthermore, impaired oral sensorimotor functions are common in many neurological disorders, sometimes making the most vital functions of eating, swallowing and speaking difficult and thereby reducing the patient's quality of life. It has also been well documented that such negative consequences can be improved following oral rehabilitation as patients adapt, for example, to a new dental prosthesis aimed at restoring function. Therefore, understanding the mechanisms and cortical neuroplastic processes underlying orofacial sensorimotor functions and adaptation is also important for the development of new therapeutic strategies to facilitate recovery of patients suffering from orofacial pain and sensorimotor disorders and improve their quality of life.

Chapter 9--face sensorimotor cortex neuroplasticity associated with intraoral alterations.

Loss of teeth or dental attrition is a common clinical occurrence associated with altered somatosensation and impaired oral motor behavior (e.g., mastication, deglutition, phonation). Oral rehabilitation aims at restoring these sensorimotor functions to improve patients' quality of life. Recent studies have implicated neuroplastic changes within the primary motor cortex (M1) in the control of limb motor behaviors following manipulations of sensory inputs to or motor outputs from the central nervous system as well as in learning and adaptation processes. However, limited data are available of the neuroplastic capabilities of face-M1 in relation to orofacial motor functions. The overall objective of our series of studies was to use intracortical microstimulation (ICMS) and recordings of evoked muscle electromyographic activity to test if neuroplastic changes occur in the ICMS-defined motor representations of the tongue-protrusive (genioglossus, GG) and jaw-opening (anterior digastric, AD) muscles within the rat face-M1 and adjacent face primary somatosensory cortex (face-S1) following several different types of intraoral manipulations. We found that a change in diet consistency was not associated with statistically significant changes in AD and GG motor representations. However, incisor extraction resulted, one week later, in a significantly increased AD representation within the contralateral face-M1 and face-S1, and incisor trimming produced time-dependent changes in the AD motor representation. These novel findings underscore the neuroplastic capabilities of the face sensorimotor cortex and point to its possible role in adaptation to an altered peripheral state or altered sensorimotor behavior. Further insights into the neuroplastic capabilities of the face sensorimotor cortex promise to improve therapeutic strategies aimed at the restoration of oral functions, particularly in patients suffering from orofacial sensorimotor deficits or pain.

Effects of incisor extraction on jaw and tongue motor representations within face sensorimotor cortex of adult rats.

Loss of teeth is associated with changes in somatosensory inputs and altered patterns of mastication, but it is unclear whether tooth loss is associated with changes in motor representations within face sensorimotor cortex of rats. We used intracortical microstimulation (ICMS) and recordings of cortically evoked muscle electromyographic (EMG) activities to test whether changes occur in the ICMS-defined motor representations of the left and right jaw muscles [masseter, anterior digastric (LAD, RAD)] and tongue muscle [genioglossus (GG)] within the cytoarchitectonically defined face primary motor cortex (face-M1) and adjacent face primary somatosensory cortex (face-S1) 1 week following extraction of the right mandibular incisor in anesthetized (ketamine-HCl) adult male Sprague-Dawley rats. Under local and general anesthesia, an "extraction" group (n = 8) received mucoalveolar bone surgery and extraction of the mandibular right incisor. A "sham-extraction" group (n = 6) received surgery with no extraction. A "naive" group (n = 6) had neither surgery nor extraction. Data were compared by using mixed-model repeated-measures ANOVA. Dental extraction was associated with a significantly increased number of sites within face-M1 and face-S1 from which ICMS evoked RAD EMG activities, a lateral shift of the RAD and LAD centers of gravity within face-M1, shorter onset latencies of ICMS-evoked GG activities within face-M1 and face-S1, and an increased number of sites within face-M1 from which ICMS simultaneously evoked RAD and GG activities. Our novel findings suggest that dental extraction may be associated with significant neuroplastic changes within the rat's face-M1 and adjacent face-S1 that may be related to the animal's ability to adapt to the altered oral state.

Motor cortex neuroplasticity associated with lingual nerve injury in rats.

The aim of this study was to determine if lingual nerve trauma affects the features of face primary motor cortex (MI) defined by intracortical microstimulation (ICMS). The left lingual nerve was transected in adult male rats by an oral surgical procedure; sham rats (oral surgery but no nerve transection) as well as naive intact rats served as control groups. ICMS was applied at post-operative days 0, 7, 14, 21, and 28 to map the jaw and tongue motor representations in face MI by analyzing ICMS-evoked movements and electromyographic activity recorded in the genioglossus (GG) and anterior digastric (AD) muscles. There were no statistically significant effects of acute (day 0) nerve transection or sham procedure (p > 0.05). The surgery in the sham animals was associated with limited post-operative change; this was reflected in a significant (p < 0.05) increase in the number of GG sites in left MI at post-operative day 14 compared to day 0. However, nerve transection was associated with significant increases in the total number of AD and GG sites in left or right MI or specifically the number of GG sites in rats at post-operative days 21 or 28 compared to earlier time periods. There were also significant differences between nerve-transected and sham groups at post-operative days 7, 14, or 21. These findings suggest that lingual nerve transection is associated with significant time-dependent neuroplastic changes in the tongue motor representations in face MI.

Properties and plasticity of the primate somatosensory and motor cortex related to orofacial sensorimotor function.

1. The lateral pericentral region of the cerebral cortex has been well documented in primates to be important in sensorimotor integration and control and in the learning of new motor skills. 2. The present article provides, first, an overview of limb sensorimotor cortical mechanisms and, second, outlines recent evidence pointing to an important role for the face sensorimotor cortex in semi-automatic, as well as trained, orofacial motor behaviour and to its propensity for neuroplastic changes in association with orofacial motor skill acquisition or an altered oral environment.