Radial stretch reveals distinct populations of mechanosensitive mammalian somatosensory neurons.

Primary afferent somatosensory neurons mediate our sense of touch in response to changes in ambient pressure. Molecules that detect and transduce thermal stimuli have been recently identified, but mechanisms underlying mechanosensation, particularly in vertebrate organisms, remain enigmatic. Traditionally, mechanically evoked responses in somatosensory neurons have been assessed one cell at a time by recording membrane currents in response to application of focal pressure, suction, or osmotic challenge. Here, we used radial stretch in combination with live-cell calcium imaging to gain a broad overview of mechanosensitive neuronal subpopulations. We found that different stretch intensities activate distinct subsets of sensory neurons as defined by size, molecular markers, or pharmacological attributes. In all subsets, stretch-evoked responses required extracellular calcium, indicating that mechanical force triggers calcium influx. This approach extends the repertoire of stimulus paradigms that can be used to examine mechanotransduction in mammalian sensory neurons, facilitating future physiological and pharmacological studies.

High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics.

Understanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

Math1-driven GFP expression in the developing nervous system of transgenic mice.

Math1 is a bHLH transcription factor expressed in neural progenitor cells in multiple regions of the nervous system. Previously we identified a Math1 enhancer that directs expression of reporter genes in a Math1 specific pattern [Development 127 (2000) 1185]. We have used a portion of this enhancer to drive expression of a nuclear GFP reporter in the Math1 lineage in transgenic mice. In this transgenic mouse strain, GFP is expressed in Math1 domains in the (1). developing spinal cord in progenitors to dI1 dorsal interneurons, (2). granule-cell progenitors in the developing cerebellum, (3). Merkel cells in the skin, and (4). hair cells in the developing vestibular and auditory systems. Furthermore, non-Math1 related expression is detected that is likely due to the absence of inhibitory regulatory sequences from the transgene. These expression domains include (1). the apical ectodermal ridge in developing limbs, (2). post-mitotic cells in the developing cortex and spinal cord, (3). the dentate gyrus, (4). retina, and (5). olfactory epithelium. Because GFP marks specific neuronal cell types in living tissue, this transgenic strain is a powerful tool for future studies on the development and electrophysiological properties of distinct cell types in the central nervous system and in sensory systems.

Microscopic Delineation of Medulloblastoma Margins in a Transgenic Mouse Model Using a Topically Applied VEGFR-1 Probe.

The unambiguous demarcation of tumor margins is critical at the final stages in the surgical treatment of brain tumors because patient outcomes have been shown to correlate with the extent of resection. Real-time high-resolution imaging with the aid of a tumor-targeting fluorescent contrast agent has the potential to enable intraoperative differentiation of tumor versus normal tissues with accuracy approaching the current gold standard of histopathology. In this study, a monoclonal antibody targeting the vascular endothelial growth factor receptor 1 (VEGFR-1) was conjugated to fluorophores and evaluated as a tumor contrast agent in a transgenic mouse model of medulloblastoma. The probe was administered topically, and its efficacy as an imaging agent was evaluated in vitro using flow cytometry, as well as ex vivo on fixed and fresh tissues through immunohistochemistry and dual-axis confocal microscopy, respectively. Results show a preferential binding to tumor versus normal tissue, suggesting that a topically applied VEGFR-1 probe can potentially be used with real-time intraoperative optical sectioning microscopy to guide brain tumor resections.

Identification of cell surface targets through meta-analysis of microarray data.

High-resolution image guidance for resection of residual tumor cells would enable more precise and complete excision for more effective treatment of cancers, such as medulloblastoma, the most common pediatric brain cancer. Numerous studies have shown that brain tumor patient outcomes correlate with the precision of resection. To enable guided resection with molecular specificity and cellular resolution, molecular probes that effectively delineate brain tumor boundaries are essential. Therefore, we developed a bioinformatics approach to analyze micro-array datasets for the identification of transcripts that encode candidate cell surface biomarkers that are highly enriched in medulloblastoma. The results identified 380 genes with greater than a two-fold increase in the expression in the medulloblastoma compared with that in the normal cerebellum. To enrich for targets with accessibility for extracellular molecular probes, we further refined this list by filtering it with gene ontology to identify genes with protein localization on, or within, the plasma membrane. To validate this meta-analysis, the top 10 candidates were evaluated with immunohistochemistry. We identified two targets, fibrillin 2 and EphA3, which specifically stain medulloblastoma. These results demonstrate a novel bioinformatics approach that successfully identified cell surface and extracellular candidate markers enriched in medulloblastoma versus adjacent cerebellum. These two proteins are high-value targets for the development of tumor-specific probes in medulloblastoma. This bioinformatics method has broad utility for the identification of accessible molecular targets in a variety of cancers and will enable probe development for guided resection.

Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery.

A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500x500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections.

Merkel Cells in Somatosensation.

Merkel cells are rare epidermal cells whose function in the skin is still debated. These cells localize to highly touch-sensitive areas of vertebrate epithelia, including palatine ridges, touch domes and finger tips. In most cases, Merkel cells complex with somatosensory afferents to form slowly adapting touch receptors; it is unclear, however, whether mechanosensory transduction occurs in the Merkel cell, the somatosensory afferent or both. Classic anatomical results suggests that Merkel cells are sensory cells that transduce mechanical stimuli and then communicate with sensory afferents via neurotransmission. This model is supported by recent molecular, immunohistochemical and physiological studies of Merkel cells in vitro and in intact tissues. For example, Merkel cells express essential components of presynaptic machinery, including molecules required for release of the excitatory neurotransmitter glutamate. Moreover, Merkel cells in vitro and in vivo are activated by mechanical stimuli, including hypotonic-induced cell swelling. Although these findings support the hypothesis that Merkel cells are sensory receptor cells, a definitive demonstration that Merkel cells are necessary and sufficient to transduce touch awaits future studies.

Voltage-activated ion channels and Ca(2+)-induced Ca (2+) release shape Ca (2+) signaling in Merkel cells.

Ca(2+) signaling and neurotransmission modulate touch-evoked responses in Merkel cell-neurite complexes. To identify mechanisms governing these processes, we analyzed voltage-activated ion channels and Ca(2+) signaling in purified Merkel cells. Merkel cells in the intact skin were specifically labeled by antibodies against voltage-activated Ca(2+) channels (Ca(V)2.1) and voltage- and Ca(2+)-activated K(+) (BK(Ca)) channels. Voltage-clamp recordings revealed small Ca(2+) currents, which produced Ca(2+) transients that were amplified sevenfold by Ca(2+)-induced Ca(2+) release. Merkel cells' voltage-activated K(+) currents were carried predominantly by BK(Ca) channels with inactivating and non-inactivating components. Thus, Merkel cells, like hair cells, have functionally diverse BK(Ca) channels. Finally, blocking K(+) channels increased response magnitude and dramatically shortened Ca(2+) transients evoked by mechanical stimulation. Together, these results demonstrate that Ca(2+) signaling in Merkel cells is governed by the interplay of plasma membrane Ca(2+) channels, store release and K(+) channels, and they identify specific signaling mechanisms that may control touch sensitivity.

Swelling-activated Ca2+ channels trigger Ca2+ signals in Merkel cells.

Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception in the Merkel cell-neurite complex.

Molecular profiling reveals synaptic release machinery in Merkel cells.

Merkel cell-neurite complexes are somatosensory receptors that initiate the perception of gentle touch. The role of epidermal Merkel cells within these complexes is disputed. To ask whether Merkel cells are genetically programmed to be excitable cells that may participate in touch reception, we purified Merkel cells from touch domes and used DNA microarrays to compare gene expression in Merkel cells and other epidermal cells. We identified 362 Merkel-cell-enriched transcripts, including neuronal transcription factors, presynaptic molecules, and ion-channel subunits. Antibody staining of skin sections showed that Merkel cells are immunoreactive for presynaptic proteins, including piccolo, Rab3C, vesicular glutamate transporter 2, and cholecystokinin 26-33. These data indicate that Merkel cells are poised to release glutamate and neuropeptides. Finally, by using Ca(2+) imaging, we discovered that Merkel cells have L- and P/Q-type voltage-gated Ca(2+) channels, which have been shown to trigger vesicle release at synapses. These results demonstrate that Merkel cells are excitable cells and suggest that they release neurotransmitters to shape touch sensitivity.