Reverse-translational identification of a cerebellar satiation network.

The brain is the seat of body weight homeostasis. However, our inability to control the increasing prevalence of obesity highlights a need to look beyond canonical feeding pathways to broaden our understanding of body weight control1-3. Here we used a reverse-translational approach to identify and anatomically, molecularly and functionally characterize a neural ensemble that promotes satiation. Unbiased, task-based functional magnetic resonance imaging revealed marked differences in cerebellar responses to food in people with a genetic disorder characterized by insatiable appetite. Transcriptomic analyses in mice revealed molecularly and topographically -distinct neurons in the anterior deep cerebellar nuclei (aDCN) that are activated by feeding or nutrient infusion in the gut. Selective activation of aDCN neurons substantially decreased food intake by reducing meal size without compensatory changes to metabolic rate. We found that aDCN activity terminates food intake by increasing striatal dopamine levels and attenuating the phasic dopamine response to subsequent food consumption. Our study defines a conserved satiation centre that may represent a novel therapeutic target for the management of excessive eating, and underscores the utility of a 'bedside-to-bench' approach for the identification of neural circuits that influence behaviour.

Cytokine enrichment in deep cerebellar nuclei is contributed by multiple glial populations and linked to reduced amyloid plaque pathology.

Alzheimer's disease (AD) pathology and amyloid-beta (Aß) plaque deposition progress slowly in the cerebellum compared to other brain regions, while the entorhinal cortex (EC) is one of the most vulnerable regions. Using a knock-in AD mouse model (App KI), we show that within the cerebellum, the deep cerebellar nuclei (DCN) has particularly low accumulation of Aß plaques. To identify factors that might underlie differences in the progression of AD-associated neuropathology across regions, we profiled gene expression in single nuclei (snRNAseq) across all cell types in the DCN and EC of wild-type (WT) and App KI male mice at age 7 months. We found differences in expression of genes associated with inflammatory activation, PI3K-AKT signalling, and neuron support functions between both regions and genotypes. In WT mice, the expression of interferon-response genes in microglia is higher in the DCN than the EC and this enrichment is confirmed by RNA in situ hybridisation, and measurement of inflammatory cytokines by protein array. Our analyses also revealed that multiple glial populations are responsible for establishing this cytokine-enriched niche. Furthermore, homogenates derived from the DCN induced inflammatory gene expression in BV2 microglia. We also assessed the relationship between the DCN microenvironment and Aß pathology by depleting microglia using a CSF1R inhibitor PLX5622 and saw that, surprisingly, the expression of a subset of inflammatory cytokines was increased while plaque abundance in the DCN was further reduced. Overall, our study revealed the presence of a cytokine-enriched microenvironment unique to the DCN that when modulated, can alter plaque deposition.

Postnatal TrkB ablation in corticolimbic interneurons induces social dominance in male mice.

The tight balance between synaptic excitation and inhibition (E/I) within neocortical circuits in the mammalian brain is important for complex behavior. Many loss-of-function studies have demonstrated that brain-derived neurotrophic factor (BDNF) and its cognate receptor tropomyosin receptor kinase B (TrkB) are essential for the development of inhibitory GABAergic neurons. However, behavioral consequences of impaired BDNF/TrkB signaling in GABAergic neurons remain unclear, largely due to confounding motor function deficits observed in previous animal models. In this study, we generated conditional knockout mice (TrkB cKO) in which TrkB was ablated from a majority of corticolimbic GABAergic interneurons postnatally. These mice showed intact motor coordination and movement, but exhibited enhanced dominance over other mice in a group-housed setting. In addition, immature fast-spiking GABAergic neurons of TrkB cKO mice resulted in an E/I imbalance in layer 5 microcircuits within the medial prefrontal cortex (mPFC), a key region regulating social dominance. Restoring the E/I imbalance via optogenetic modulation in the mPFC of TrkB cKO mice normalized their social dominance behavior. Taken together, our results provide strong evidence for a role of BDNF/TrkB signaling in inhibitory synaptic modulation and social dominance behavior in mice.

TrkB (tropomyosin-related kinase B) controls the assembly and maintenance of GABAergic synapses in the cerebellar cortex.

Inhibitory interneurons play a critical role in coordinating the activity of neural circuits. To explore the mechanisms that direct the organization of inhibitory circuits, we analyzed the involvement of tropomyosin-related kinase B (TrkB) in the assembly and maintenance of GABAergic inhibitory synapses between Golgi and granule cells in the mouse cerebellar cortex. We show that TrkB acts directly within each cell-type to regulate synaptic differentiation. TrkB is required not only for assembly, but also maintenance of these synapses and acts, primarily, by regulating the localization of synaptic constituents. Postsynaptically, TrkB controls the localization of a scaffolding protein, gephyrin, but acts at a step subsequent to the localization of a cell adhesion molecule, Neuroligin-2. Importantly, TrkB is required for the localization of an Ig superfamily cell adhesion molecule, Contactin-1, in Golgi and granule cells and the absence of Contactin-1 also results in deficits in inhibitory synaptic development. Thus, our findings demonstrate that TrkB controls the assembly and maintenance of GABAergic synapses and suggest that TrkB functions, in part, through promoting synaptic adhesion.

Graded activity of transcription factor Runx3 specifies the laminar termination pattern of sensory axons in the developing spinal cord.

Different functional classes of dorsal root ganglion sensory neurons project their axons to distinct target zones within the developing spinal cord. To explore the mechanisms that link sensory neuron subtype identity and axonal projection pattern, we analyzed the roles of Runx and ETS transcription factors in the laminar targeting of sensory afferents. Gain- and loss-of-function studies in chick embryos reveal that the status of Runx3 expression is a major determinant of the dorso-ventral position of termination of proprioceptive and cutaneous sensory axons. In addition, the level of expression and/or activity of Runx3 in individual proprioceptive sensory neurons appears to specify whether their axons terminate in intermediate or ventral regions. Our findings suggest that the selectivity of Runx3 expression, and its level of activity, control sensory afferent targeting in the developing spinal cord.

The Cerebellar Nuclei and Dexterous Limb Movements.

Dexterous forelimb movements like reaching, grasping, and manipulating objects are fundamental building blocks of the mammalian motor repertoire. These behaviors are essential to everyday activities, and their elaboration underlies incredible accomplishments by human beings in art and sport. Moreover, the susceptibility of these behaviors to damage and disease of the nervous system can lead to debilitating deficits, highlighting a need for a better understanding of function and dysfunction in sensorimotor control. The cerebellum is central to coordinating limb movements, as defined in large part by Joseph Babinski and Gordon Holmes describing motor impairment in patients with cerebellar lesions over 100 years ago (Babinski, 1902; Holmes, 1917), and supported by many important human and animal studies that have been conducted since. Here, with a focus on output pathways of the cerebellar nuclei across mammalian species, we describe forelimb movement deficits observed when cerebellar circuits are perturbed, the mechanisms through which these circuits influence motor output, and key challenges in defining how the cerebellum refines limb movement.

Myocyte Enhancer Factor 2c Regulates Dendritic Complexity and Connectivity of Cerebellar Purkinje Cells.

Mef2c haploinsufficiency is implicated in behavioral deficits related to autism, schizophrenia, and intellectual disability. Although perturbations in the cerebellum, notably Purkinje cells, have been linked to these neurological disorders, the underlying mechanisms remain poorly understood. In this study, we investigated the roles of Mef2c in cerebellar Purkinje cells during the first three weeks of postnatal development. Our analysis revealed that in comparison to other members of the Mef2 family, Mef2c expression is limited to postnatal Purkinje cells. Because the role of Mef2c has not been assessed in GABAergic neurons, we set out to determine the functional significance of Mef2c by knocking down the expression of Mef2c selectively in Purkinje cells. We found that the loss of Mef2c expression during the first and second postnatal week results in an increase in dendritic arborization without impact on the general growth and migration of Purkinje cells. The influence of Mef2c on dendritic arborization persists throughout the first three weeks, but is most prominent during the first postnatal week suggesting a critical period of Mef2c activity. Additionally, the loss of Mef2c expression results in an increase in the number of spines accompanied by an increase in Gad67 and vGluT1 puncta and decrease in vGluT2 puncta. Thus, our results reveal the specific expression and functional relevance of Mef2c in developing Purkinje cells and offer insight to how disruption of the expression of Mef2c in a GABAergic neuronal subtype may lead to pathogenesis of cerebellar-associated disorders.

Precision of Discrete and Rhythmic Forelimb Movements Requires a Distinct Neuronal Subpopulation in the Interposed Anterior Nucleus.

The deep cerebellar nuclei (DCN) represent output channels of the cerebellum, and they transmit integrated sensorimotor signals to modulate limb movements. But the functional relevance of identifiable neuronal subpopulations within the DCN remains unclear. Here, we examine a genetically tractable population of neurons in the mouse interposed anterior nucleus (IntA). We show that these neurons represent a subset of glutamatergic neurons in the IntA and constitute a specific element of an internal feedback circuit within the cerebellar cortex and cerebello-thalamo-cortical pathway associated with limb control. Ablation and optogenetic stimulation of these neurons disrupt efficacy of skilled reach and locomotor movement and reveal that they control positioning and timing of the forelimb and hindlimb. Together, our findings uncover the function of a distinct neuronal subpopulation in the deep cerebellum and delineate the anatomical substrates and kinematic parameters through which it modulates precision of discrete and rhythmic limb movements.

Distinct Activities of Tfap2A and Tfap2B in the Specification of GABAergic Interneurons in the Developing Cerebellum.

GABAergic inhibitory neurons in the cerebellum are subdivided into Purkinje cells and distinct subtypes of interneurons from the same pool of progenitors, but the determinants of this diversification process are not well defined. To explore the transcriptional regulation of the development of cerebellar inhibitory neurons, we examined the role of Tfap2A and Tfap2B in the specification of GABAergic neuronal subtypes in mice. We show that Tfap2A and Tfap2B are expressed in inhibitory precursors during embryonic development and that their expression persists into adulthood. The onset of their expression follows Ptf1a and Olig2, key determinants of GABAergic neuronal fate in the cerebellum; and, their expression precedes Pax2, an interneuron-specific factor. Tfap2A is expressed by all GABAergic neurons, whereas Tfap2B is selectively expressed by interneurons. Genetic manipulation via in utero electroporation (IUE) reveals that Tfap2B is necessary for interneuron specification and is capable of suppressing the generation of excitatory cells. Tfap2A, but not Tfap2B, is capable of inducing the generation of interneurons when misexpressed in the ventricular neuroepithelium. Together, our results demonstrate that the differential expression of Tfap2A and Tfap2B defines subtypes of GABAergic neurons and plays specific, but complementary roles in the specification of interneurons in the developing cerebellum.

An Optimization and Comparative Study of Air-Coupled CMUT Cells With Circular and Annular Geometries.

Air-coupled capacitive micromachined ultrasonic transducers (CMUTs) with annular cell geometry have recently been reported to have a promising transmit sensitivity. This paper reports three optimization schemes, which further improve the transmit sensitivity and also help achieve a reasonable comparison between the novel annular and conventional circular cells. Lumped element models of both cell types with laminate plate structures are presented. Based on these models, a design optimization flowchart was constructed to facilitate analytical optimization on the three schemes. Circular and annular CMUTs with a common 97-kHz natural resonance frequency were fabricated and characterized to verify the efficacy of the optimization principle. Using the optimization flowchart, annular and circular cells with frequencies ranging from 100 to 300 kHz were analytically optimized and then compared. The comparison results demonstrate that, given the same dc bias and ac excitation voltage, the output power density at the plate surface of the optimized annular cell is double that of the optimized circular cell. Additionally, when generating the same surface power density, an optimized annular cell requires either half the dc bias or half the ac excitation voltage of an optimized circular cell. This paper provides a practical optimization framework for CMUT cell design and demonstrates the superiority of annular cells for air-coupled applications.

Compensated Row-Column Ultrasound Imaging System Using Multilayered Edge Guided Stochastically Fully Connected Random Fields.

The row-column method received a lot of attention for 3-D ultrasound imaging. By reducing the number of connections required to address the 2-D array and therefore reducing the amount of data to handle, this addressing method allows for real time 3-D imaging. Row-column still has its limitations: the issues of sparsity, speckle noise inherent to ultrasound, the spatially varying point spread function, and the ghosting artifacts inherent to the row-column method must all be taken into account when building a reconstruction framework. In this research, we build on a previously published system and propose an edge-guided, compensated row-column ultrasound imaging system that incorporates multilayered edge-guided stochastically fully connected conditional random fields to address the limitations of the row-column method. Tests carried out on simulated and real row-column ultrasound images show the effectiveness of our proposed system over other published systems. Visual assessment show our proposed system's potential at preserving edges and reducing speckle. Quantitative analysis shows that our proposed system outperforms previously published systems when evaluated with metrics such as Peak Signal-to-Noise Ratio, Coefficient of Correlation, and Effective Number of Looks. These results show the potential of our proposed system as an effective tool for enhancing 3-D row-column imaging.

Lumped element modeling of air-coupled capacitive micromachined ultrasonic transducers with annular cell geometry.

Air-coupled capacitive micromachined ultrasonic transducers (CMUTs) based on annular cell geometry have recently been reported. Finite element analysis and experimental studies have demonstrated their significant improvement in transmit efficiency compared with the conventional circular-cell CMUTs. Extending the previous work, this paper proposed a lumped element model of annular-cell CMUTs. Explicit expressions of the resonance frequency, modal vector, and static displacement of a clamped annular plate under uniform pressure were first derived based on the plate theory and curve fitting method. The lumped model of an annular CMUT cell was then developed by adopting the average displacement as the spatial variable. Using the proposed model, the ratio of average-to-maximum displacement was derived to be 8/15. Experimental and simulation studies on a fabricated annular CMUT cell verified the effectiveness of the lumped model. The proposed model provides an effective and efficient way to analyze and design air-coupled annular-cell CMUTs.

Glutamatergic axon-derived BDNF controls GABAergic synaptic differentiation in the cerebellum.

To study mechanisms that regulate the construction of inhibitory circuits, we examined the role of brain-derived neurotrophic factor (BDNF) in the assembly of GABAergic inhibitory synapses in the mouse cerebellar cortex. We show that within the cerebellum, BDNF-expressing cells are restricted to the internal granular layer (IGL), but that the BDNF protein is present within mossy fibers which originate from cells located outside of the cerebellum. In contrast to deletion of TrkB, the cognate receptor for BDNF, deletion of Bdnf from cerebellar cell bodies alone did not perturb the localization of pre- or postsynaptic constituents at the GABAergic synapses formed by Golgi cell axons on granule cell dendrites within the IGL. Instead, we found that BDNF derived from excitatory mossy fiber endings controls their differentiation. Our findings thus indicate that cerebellar BDNF is derived primarily from excitatory neurons--precerebellar nuclei/spinal cord neurons that give rise to mossy fibers--and promotes GABAergic synapse formation as a result of release from axons. Thus, within the cerebellum the preferential localization of BDNF to axons enhances the specificity through which BDNF promotes GABAergic synaptic differentiation.

Capacitive micromachined ultrasonic transducers based on annular cell geometry for air-coupled applications.

A novel design of an air-coupled capacitive micromachined ultrasonic transducer (CMUT) with annular cell geometry (annular CMUT) is proposed. Finite element analysis shows that an annular cell has a ratio of average-to-maximum displacement (RAMD) of 0.52-0.58 which is 58-76% higher than that of a conventional circular cell. The increased RAMD leads to a larger volume displacement which results in a 48.4% improved transmit sensitivity and 127.3% improved power intensity. Single-cell annular CMUTs were fabricated with 20-µm silicon plates on 13.7-µm deep and 1.35-mm wide annular cavities using the wafer bonding technique. The measured RAMD of the fabricated CMUTs is 0.54. The resonance frequency was measured to be 94.5kHz at 170-V DC bias. The transmit sensitivity was measured to be 33.83Pa/V and 25.85Pa/V when the CMUT was excited by a continuous wave and a 20-cycle burst, respectively. The receive sensitivity at 170-V DC bias was measured to be 7.7mV/Pa for a 20-cycle burst, and 15.0mV/Pa for a continuous incident wave. The proposed annular CMUT design demonstrates a significant improvement in transmit efficiency, which is an important parameter for air-coupled ultrasonic transducers.

Compensated Row-Column Ultrasound Imaging System Using Fisher Tippett Multilayered Conditional Random Field Model.

3-D ultrasound imaging offers unique opportunities in the field of non destructive testing that cannot be easily found in A-mode and B-mode images. To acquire a 3-D ultrasound image without a mechanically moving transducer, a 2-D array can be used. The row column technique is preferred over a fully addressed 2-D array as it requires a significantly lower number of interconnections. Recent advances in 3-D row-column ultrasound imaging systems were largely focused on sensor design. However, these imaging systems face three intrinsic challenges that cannot be addressed by improving sensor design alone: speckle noise, sparsity of data in the imaged volume, and the spatially dependent point spread function of the imaging system. In this paper, we propose a compensated row-column ultrasound image reconstruction system using Fisher-Tippett multilayered conditional random field model. Tests carried out on both simulated and real row-column ultrasound images show the effectiveness of our proposed system as opposed to other published systems. Visual assessment of the results show our proposed system's potential at preserving detail and reducing speckle. Quantitative analysis shows that our proposed system outperforms previously published systems when evaluated with metrics such as Peak Signal to Noise Ratio, Coefficient of Correlation, and Effective Number of Looks. These results show the potential of our proposed system as an effective tool for enhancing 3-D row-column imaging.

A row-column addressed micromachined ultrasonic transducer array for surface scanning applications.

Row-column addressed arrays for ultrasonic non-destructive testing (NDT) applications are analyzed and demonstrated in this paper. Simulation and experimental results of a row-column addressed 32 by 32 capacitive micromachined ultrasonic transducer (CMUT) array are presented. The CMUT array, which was designed for medical imaging applications, has a center frequency of 5.3MHz. The CMUT array was used to perform C-scans on test objects with holes that have diameters of 1.0mm and 0.5mm. The array transducer has an aperture size of 4.8mm by 4.8mm, and it was used to scan an area of 4.0mm by 4.0mm. Compared to an N by N fully addressed 2-D array, a row-column addressed array of the same number of elements requires fewer (N instead of N(2)) pairs of interconnection and supporting electronic components such as pulsers and amplifiers. Even though the resulting field of view is limit by the aperture size, row-column addressed arrays and the row-column addressing scheme can be an alternative option of 2-D arrays for NDT applications.

An FPGA-based ultrasound imaging system using capacitive micromachined ultrasonic transducers.

We report the design and experimental results of a field-programmable gate array (FPGA)-based real-time ultrasound imaging system that uses a 16-element phased-array capacitive micromachined ultrasonic transducer fabricated using a fusion bonding process. The imaging system consists of the transducer, discrete analog components situated on a custom-made circuit board, the FPGA, and a monitor. The FPGA program consists of five functional blocks: a main counter, transmit and receive beamformer, receive signal pre-processing, envelope detection, and display. No dedicated digital signal processor or personal computer is required for the imaging system. An experiment is carried out to obtain the sector B-scan of a 4-wire target. The ultrasound imaging system demonstrates the possibility of an integrated system-in-a-package solution.

A 32 x 32 element row-column addressed capacitive micromachined ultrasonic transducer.

This paper presents characterization and initial imaging results of a 32 x 32 element two-dimensional capacitive micromachined ultrasonic transducer array. The devices are fabricated using a wafer bonding process in which both the insulation layer and the membrane are user-deposited silicon nitride. The transducers use a row-column addressing scheme to simplify the fabrication process and beamformer. By adjusting the number of rows and columns that are biased, the effective aperture of the transducer can be adjusted. This is significant because it permits imaging in the near-field of the transducer without the use of a lens. The effect on the transmit beam profile is demonstrated. The transducer has a center frequency of 5.9 MHz and a relative bandwidth of 110%. Images of horizontal and vertical wires are taken to demonstrate image resolution. A three-dimensional image of four pin heads is also demonstrated.