A one-photon endoscope for simultaneous patterned optogenetic stimulation and calcium imaging in freely behaving mice.

Optogenetics and calcium imaging can be combined to simultaneously stimulate and record neural activity in vivo. However, this usually requires two-photon microscopes, which are not portable nor affordable. Here we report the design and implementation of a miniaturized one-photon endoscope for performing simultaneous optogenetic stimulation and calcium imaging. By integrating digital micromirrors, the endoscope makes it possible to activate any neuron of choice within the field of view, and to apply arbitrary spatiotemporal patterns of photostimulation while imaging calcium activity. We used the endoscope to image striatal neurons from either the direct pathway or the indirect pathway in freely moving mice while activating any chosen neuron in the field of view. The endoscope also allows for the selection of neurons based on their relationship with specific animal behaviour, and to recreate the behaviour by mimicking the natural neural activity with photostimulation. The miniaturized endoscope may facilitate the study of how neural activity gives rise to behaviour in freely moving animals.

Thalamic projections to the subthalamic nucleus contribute to movement initiation and rescue of parkinsonian symptoms.

The parafascicular nucleus (Pf) of the thalamus provides major projections to the basal ganglia, a set of subcortical nuclei involved in action initiation. Here, we show that Pf projections to the subthalamic nucleus (STN), but not to the striatum, are responsible for movement initiation. Because the STN is a major target of deep brain stimulation treatments for Parkinson's disease, we tested the effect of selective stimulation of Pf-STN projections in a mouse model of PD. Bilateral dopamine depletion with 6-OHDA created complete akinesia in mice, but Pf-STN stimulation immediately and markedly restored a variety of natural behaviors. Our results therefore revealed a functionally novel neural pathway for the initiation of movements that can be recruited to rescue movement deficits after dopamine depletion. They not only shed light on the clinical efficacy of conventional STN DBS but also suggest more selective and improved stimulation strategies for the treatment of parkinsonian symptoms.

Ultrasound Stimulations Induce Prolonged Depolarization and Fast Action Potentials in Leech Neurons.

Objective: Ultrasound (US) stimulation carries the promise of a selective, reversible, and non-invasive modulation of neural activity without the need for genetic manipulation of neural structures. However, the mechanisms of US-induced generation of action potentials (APs) are still unclear. Methods: Here we address this issue by analyzing intracellularly recorded responses of leech nociceptive neurons to controlled delivery of US. Results: US induced a depolarization linearly accumulating in time and outlasting the duration of the stimulation. Spiking activity was reliably induced for an optimal US intensity range. Moreover, we found that APs induced by US differ in smaller amplitude and faster repolarization from those induced by electrical stimulation in the same cell but display the same repolarization rate. Conclusions: These results shed light on the mechanism by which spikes are induced by US and pave the way for designing more efficient US stimulation patterns.

Improved neuron culture using scaffolds made of three-dimensional PDMS micro-lattices.

Tissue engineering strives to create functional components of organs with different cell types in vitro. One of the challenges is to fabricate scaffolds for three-dimensional (3D) cell culture under physiological conditions. Of particular interest is the investigation of the morphology and function of the central nervous system cultured using such scaffolds. Here, we used an elastomer-polydimethylsiloxane (PDMS)-to produce lattice-type scaffolds from a photolithography-defined template. The photomask with antidot arrays was spin-coated by a thick layer of resist, and was downward mounted on a rotating stage at an angle of 45°. After the exposure was repeated three or more times, maintaining the same exposure plan but rotated by the same angle, a photoresist was developed to produce a 3D porous template. Afterwards, a pre-polymer mixture of PDMS was poured in and cured, followed by a resist etch, resulting in lattice-type PDMS features. Before cell culture, the PDMS lattices were surface functionalized. A culture test was conducted using NIH-3T3 cells and primary hippocampal cells from rats, showing homogenous cell infiltration and 3D attachment. As expected, a much higher cell number was found in the 3D PDMS lattices compared to the 2D culture. We also found a higher neuron-to-astrocyte ratio and a higher degree of cell ramification in the 3D culture compared to the 2D culture due to the change of scaffold topography and the elastic properties of the PDMS micro-lattices. Our results demonstrate that the 3D PDMS micro-lattices improve the survival and growth of cells, as well as the network formation of neurons. We believe that such an enabling technology is useful for research and clinical applications, including disease modeling, regenerative medicine, and drug discovery/drug cytotoxicity studies.

Effective motor neuron differentiation of hiPSCs on a patch made of crosslinked monolayer gelatin nanofibers.

Human induced pluripotent stem cells (hiPSCs) are differentiated into mature motor neurons by using a culture patch made of crosslinked monolayer gelatin nanofibers. Compared to the conventional culture dish method, the patch method is more effective for culture and differentiation of stem cells, because cells are supported by a net-like structure made of crosslinked monolayer nanofibers instead of a planar substrate. The pores of the net-like structure have sizes smaller than those of cells but large enough to minimize the exogenous cell-material contact and to increase the permeability as well as the efficiency of cell-cell interactions. As expected, the differentiated hiPSCs showed the up-regulation of the expression of neuron specific proteins and the signature of matured motor neurons, allowing plug-and-play with a commercial multi-electrode array for neuron spike recording.