Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination.

Significance: Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale ("mesoscopic") imaging of different aspects of neuronal activity with spectrally compatible fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. Aim: We aim to develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. Approach: Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm light-emitting diodes positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. Results: We demonstrate the performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake, head-fixed mice with a curved "crystal skull" window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRAB ACh 3.0 . Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. Conclusions: Our widefield microscope design with a single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination.

SIGNIFICANCE: Widefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale (mesoscopic) imaging of neuronal activity with fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. AIM: Develop a system for mesoscopic imaging of two fluorescent and two reflectance channels. APPROACH: Excitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625nm LEDs positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. RESULTS: We demonstrate performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake head-fixed mice with a curved crystal skull window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRABACh3.0 . Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. CONCLUSIONS: Our widefield microscope design with single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

Causal influence of brainstem response to transcutaneous vagus nerve stimulation on cardiovagal outflow.

BACKGROUND: The autonomic response to transcutaneous auricular vagus nerve stimulation (taVNS) has been linked to the engagement of brainstem circuitry modulating autonomic outflow. However, the physiological mechanisms supporting such efferent vagal responses are not well understood, particularly in humans. HYPOTHESIS: We present a paradigm for estimating directional brain-heart interactions in response to taVNS. We propose that our approach is able to identify causal links between the activity of brainstem nuclei involved in autonomic control and cardiovagal outflow. METHODS: We adopt an approach based on a recent reformulation of Granger causality that includes permutation-based, nonparametric statistics. The method is applied to ultrahigh field (7T) functional magnetic resonance imaging (fMRI) data collected on healthy subjects during taVNS. RESULTS: Our framework identified taVNS-evoked functional brainstem responses with superior sensitivity compared to prior conventional approaches, confirming causal links between taVNS stimulation and fMRI response in the nucleus tractus solitarii (NTS). Furthermore, our causal approach elucidated potential mechanisms by which information is relayed between brainstem nuclei and cardiovagal, i.e., high-frequency heart rate variability, in response to taVNS. Our findings revealed that key brainstem nuclei, known from animal models to be involved in cardiovascular control, exert a causal influence on taVNS-induced cardiovagal outflow in humans. CONCLUSION: Our causal approach allowed us to noninvasively evaluate directional interactions between fMRI BOLD signals from brainstem nuclei and cardiovagal outflow.

Cine gastric MRI reveals altered Gut-Brain Axis in Functional Dyspepsia: gastric motility is linked with brainstem-cortical fMRI connectivity.

BACKGROUND: Functional dyspepsia (FD) is a disorder of gut-brain interaction, and its putative pathophysiology involves dysregulation of gastric motility and central processing of gastric afference. The vagus nerve modulates gastric peristalsis and carries afferent sensory information to brainstem nuclei, specifically the nucleus tractus solitarii (NTS). Here, we combine MRI assessment of gastric kinematics with measures of NTS functional connectivity to the brain in patients with FD and healthy controls (HC), in order to elucidate how gut-brain axis communication is associated with FD pathophysiology. METHODS: Functional dyspepsia and HC subjects experienced serial gastric MRI and brain fMRI following ingestion of a food-based contrast meal. Gastric function indices estimated from 4D cine MRI data were compared between FD and HC groups using repeated measure ANOVA models, controlling for ingested volume. Brain connectivity of the NTS was contrasted between groups and associated with gastric function indices. KEY RESULTS: Propagation velocity of antral peristalsis was significantly lower in FD compared to HC. The brain network defined by NTS connectivity loaded most strongly onto the Default Mode Network, and more strongly onto the Frontoparietal Network in FD. FD also demonstrated higher NTS connectivity to insula, anterior cingulate and prefrontal cortices, and pre-supplementary motor area. NTS connectivity was linked to propagation velocity in HC, but not FD, whereas peristalsis frequency was linked with NTS connectivity in patients with FD. CONCLUSIONS & INFERENCES: Our multi-modal MRI approach revealed lower peristaltic propagation velocity linked to altered brainstem-cortical functional connectivity in patients suffering from FD suggesting specific plasticity in gut-brain communication.

S1 Brain Connectivity in Carpal Tunnel Syndrome Underlies Median Nerve and Functional Improvement Following Electro-Acupuncture.

Carpal Tunnel Syndrome (CTS) is a median nerve entrapment neuropathy that alters primary somatosensory cortex (S1) organization. While electro-acupuncture (EA), a form of peripheral neuromodulation, has been shown to improve clinical and neurophysiological CTS outcomes, the role of EA-evoked brain response during therapy (within and beyond S1) for improved outcomes is unknown. We investigated S1-associated whole brain fMRI connectivity during both a resting and sustained EA stimulation state in age-matched healthy controls (N = 28) and CTS patients (N = 64), at baseline and after 8 weeks of acupuncture therapy (local, distal, or sham EA). Compared to healthy controls, CTS patients at baseline showed decreased resting state functional connectivity between S1 and thalamic pulvinar nucleus. Increases in S1/pulvinar connectivity strength following verum EA therapy (combined local and distal) were correlated with improvements in median nerve velocity (r = 0.38, p = 0.035). During sustained local EA, compared to healthy controls, CTS patients demonstrated increased functional connectivity between S1 and anterior hippocampus (aHipp). Following 8 weeks of local EA therapy, S1/aHipp connectivity significantly decreased and greater decrease was associated with improvement in patients' functional status (r = 0.64, p = 0.01) and increased median nerve velocity (r = -0.62, p = 0.013). Thus, connectivity between S1 and other brain areas is also disrupted in CTS patients and may be improved following EA therapy. Furthermore, stimulus-evoked fMRI connectivity adds therapy-specific, mechanistic insight to more common resting state connectivity approaches. Specifically, local EA modulates S1 connectivity to sensory and affective processing regions, linked to patient function and median nerve health.

Non-uniform gastric wall kinematics revealed by 4D Cine magnetic resonance imaging in humans.

BACKGROUND: Assessment of gastric function in humans has relied on modalities with varying degrees of invasiveness, which are usually limited to the evaluation of single aspects of gastric function, thus requiring patients to undergo a number of often invasive tests for a full clinical understanding. Therefore, the development of a non-invasive tool able to concurrently assess multiple aspects of gastric function is highly desirable for both research and clinical assessments of gastrointestinal (GI) function. Recently, technological advances in magnetic resonance imaging (MRI) have provided new tools for dynamic (or "cine") body imaging. Such approaches can be extended to GI applications. METHODS: In the present work, we propose a non-invasive assessment of gastric function using a four-dimensional (4D, volumetric cine imaging), free-breathing MRI sequence with gadolinium-free contrast enhancement achieved through a food-based meal. In healthy subjects, we successfully estimated multiple parameters describing gastric emptying, motility, and peristalsis propagation patterns. KEY RESULTS: Our data demonstrated non-uniform kinematics of the gastric wall during peristaltic contraction, highlighting the importance of using volumetric data to derive motility measures. CONCLUSIONS & INFERENCES: MRI has the potential of becoming an important clinical and gastric physiology research tool, providing objective parameters for the evaluation of impaired gastric function.

Modulatory Effects of Respiratory-Gated Auricular Vagal Nerve Stimulation on Cardiovagal Activity in Hypertension.

The objective of this study was to determine potential effects of Respiratory-gated Auricular Vagal Afferent Nerve Stimulation (RAVANS) on cardiac autonomic activity in hypertensive patients.20 hypertensive subjects (57.3±6.2 years; 11 females, 9 males) were randomized to receive either active RAVANS at 25 Hz or sham stimulation for 5 consecutive days and were assessed 5 and 10 days later. Continuous electrocardiogram, pulse rate, and blood pressure signals were collected during 10-minute baseline, 30-minute stimulation, and 10-minute recovery periods for each session. LabChart was used to acquire and process heart rate variability and blood pressure indices. Percent changes of mean values during the recovery period were calculated comparing the final stimulation session and follow-up sessions to the first stimulation session. General linear models were applied to assess the effects of RAVANS on the variables evaluated, considering baseline values and sex as covariates in the models.We found that RAVANS increased high frequency (HF-HRV) power during recovery of the final stimulation session and both follow-up sessions in comparison to sham. RAVANS also lowered heart rate and increased average RR and root mean square of successive RR interval differences (RMSSD) during recovery on the final day of stimulation. No significant effects on blood pressure values were observed during these periods.These results suggest that RAVANS effectively stimulates cardiovagal activity in hypertension, with effects lasting up to 10 days. Future research incorporating larger sample sizes is needed to replicate the effects of RAVANS.Clinical Relevance- This research has implications for potential therapeutic effects of respiratory-gated tVNS on cardiovagal modulation in hypertensive patients.

Stimulus frequency modulates brainstem response to respiratory-gated transcutaneous auricular vagus nerve stimulation.

BACKGROUND: The therapeutic potential of transcutaneous auricular VNS (taVNS) is currently being explored for numerous clinical applications. However, optimized response for different clinical indications may depend on specific neuromodulation parameters, and systematic assessments of their influence are still needed to optimize this promising approach. HYPOTHESIS: We proposed that stimulation frequency would have a significant effect on nucleus tractus solitarii (NTS) functional MRI (fMRI) response to respiratory-gated taVNS (RAVANS). METHODS: Brainstem fMRI response to auricular RAVANS (cymba conchae) was assessed for four different stimulation frequencies (2, 10, 25, 100 Hz). Sham (no current) stimulation was used to control for respiration effects on fMRI signal. RESULTS: Our findings demonstrated that RAVANS delivered at 100 Hz evoked the strongest brainstem response, localized to a cluster in the left (ipsilateral) medulla and consistent with purported NTS. A co-localized, although weaker, response was found for 2 Hz RAVANS. Furthermore, RAVANS delivered at 100 Hz also evoked stronger fMRI responses for important monoamine neurotransmitter source nuclei (LC, noradrenergic; MR, DR, serotonergic) and pain/homeostatic regulation nuclei (i.e. PAG). CONCLUSION: Our fMRI results support previous localization of taVNS afference to pontomedullary aspect of NTS in the human brainstem, and demonstrate the significant influence of the stimulation frequency on brainstem fMRI response.

Characterization of plexinA and two distinct semaphorin1a transcripts in the developing and adult cricket Gryllus bimaculatus.

Guidance cues act during development to guide growth cones to their proper targets in both the central and peripheral nervous systems. Experiments in many species indicate that guidance molecules also play important roles after development, though less is understood about their functions in the adult. The Semaphorin family of guidance cues, signaling through Plexin receptors, influences the development of both axons and dendrites in invertebrates. Semaphorin functions have been extensively explored in Drosophila melanogaster and some other Dipteran species, but little is known about their function in hemimetabolous insects. Here, we characterize sema1a and plexA in the cricket Gryllus bimaculatus. In fact, we found two distinct predicted Sema1a proteins in this species, Sema1a.1 and Sema1a.2, which shared only 48% identity at the amino acid level. We include a phylogenetic analysis that predicted that many other insect species, both holometabolous and hemimetabolous, express two Sema1a proteins as well. Finally, we used in situ hybridization to show that sema1a.1 and sema1a.2 expression patterns were spatially distinct in the embryo, and both roughly overlap with plexA. All three transcripts were also expressed in the adult brain, mainly in the mushroom bodies, though sema1a.2 was expressed most robustly. sema1a.2 was also expressed strongly in the adult thoracic ganglia while sema1a.1 was only weakly expressed and plexA was undetectable.

The influence of respiration on brainstem and cardiovagal response to auricular vagus nerve stimulation: A multimodal ultrahigh-field (7T) fMRI study.

BACKGROUND: Brainstem-focused mechanisms supporting transcutaneous auricular VNS (taVNS) effects are not well understood, particularly in humans. We employed ultrahigh field (7T) fMRI and evaluated the influence of respiratory phase for optimal targeting, applying our respiratory-gated auricular vagal afferent nerve stimulation (RAVANS) technique. HYPOTHESIS: We proposed that targeting of nucleus tractus solitarii (NTS) and cardiovagal modulation in response to taVNS stimuli would be enhanced when stimulation is delivered during a more receptive state, i.e. exhalation. METHODS: Brainstem fMRI response to auricular taVNS (cymba conchae) was assessed for stimulation delivered during exhalation (eRAVANS) or inhalation (iRAVANS), while exhalation-gated stimulation over the greater auricular nerve (GANctrl, i.e. earlobe) was included as control. Furthermore, we evaluated cardiovagal response to stimulation by calculating instantaneous HF-HRV from cardiac data recorded during fMRI. RESULTS: Our findings demonstrated that eRAVANS evoked fMRI signal increase in ipsilateral pontomedullary junction in a cluster including purported NTS. Brainstem response to GANctrl localized a partially-overlapping cluster, more ventrolateral, consistent with spinal trigeminal nucleus. A region-of-interest analysis also found eRAVANS activation in monoaminergic source nuclei including locus coeruleus (LC, noradrenergic) and both dorsal and median raphe (serotonergic) nuclei. Response to eRAVANS was significantly greater than iRAVANS for all nuclei, and greater than GANctrl in LC and raphe nuclei. Furthermore, eRAVANS, but not iRAVANS, enhanced cardiovagal modulation, confirming enhanced eRAVANS response on both central and peripheral neurophysiological levels. CONCLUSION: 7T fMRI localized brainstem response to taVNS, linked such response with autonomic outflow, and demonstrated that taVNS applied during exhalation enhanced NTS targeting.

De novo assembly of a transcriptome for the cricket Gryllus bimaculatus prothoracic ganglion: An invertebrate model for investigating adult central nervous system compensatory plasticity.

The auditory system of the cricket, Gryllus bimaculatus, demonstrates an unusual amount of anatomical plasticity in response to injury, even in adults. Unilateral removal of the ear causes deafferented auditory neurons in the prothoracic ganglion to sprout dendrites across the midline, a boundary they typically respect, and become synaptically connected to the auditory afferents of the contralateral ear. The molecular basis of this sprouting and novel synaptogenesis in the adult is not understood. We hypothesize that well-conserved developmental guidance cues may recapitulate their guidance functions in the adult in order to facilitate this compensatory growth. As a first step in testing this hypothesis, we have generated a de novo assembly of a prothoracic ganglion transcriptome derived from control and deafferented adult individuals. We have mined this transcriptome for orthologues of guidance molecules from four well-conserved signaling families: Slit, Netrin, Ephrin, and Semaphorin. Here we report that transcripts encoding putative orthologues of most of the candidate developmental ligands and receptors from these signaling families were present in the assembly, indicating expression in the adult G. bimaculatus prothoracic ganglion.