Visual stimulation drives retinotopic acetylcholine release in the mouse visual cortex.

Cholinergic signaling is involved with a variety of brain functions including learning and memory, attention, and behavioral state modulation. The spatiotemporal characteristics of neocortical acetylcholine (ACh) release in response to sensory inputs are poorly understood, but a lack of intra-region topographic organization of cholinergic projections from the basal forebrain has suggested diffuse release patterns and volume transmission. Here, we use mesoscopic imaging of fluorescent ACh sensors to show that visual stimulation results in ACh release patterns that conform to a retinotopic map of visual space in the mouse primary visual cortex, suggesting new modes of functional cholinergic signaling in cortical circuits.x.

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.

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.

Chemogenetic modulation of sensory afferents induces locomotor changes and plasticity after spinal cord injury.

Neuromodulatory therapies for spinal cord injury (SCI) such as electrical epidural stimulation (EES) are increasingly effective at improving patient outcomes. These improvements are thought to be due, at least in part, to plasticity in neuronal circuits. Precisely which circuits are influenced and which afferent classes are most effective in stimulating change remain important open questions. Genetic tools, such as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), support targeted and reversible neuromodulation as well as histological characterization of manipulated neurons. We therefore transduced and activated lumbar large diameter peripheral afferents with excitatory (hM3Dq) DREADDs, in a manner analogous to EES, in a rat hemisection model, to begin to trace plasticity and observe concomitant locomotor changes. Chronic DREADDs activation, coupled with thrice weekly treadmill training, was observed to increase afferent fluorescent labeling within motor pools and Clarke's column when compared to control animals. This plasticity may underlie kinematic differences that we observed across stages of recovery, including an increased and less variable hindquarters height in DREADDs animals, shorter step durations, a more flexed ankle joint early in recovery, a less variable ankle joint angle in swing phase, but a more variable hip joint angle. Withdrawal of DREADDs agonist, clozapine-N-oxide (CNO) left these kinematic differences largely unaffected; suggesting that DREADDs activation is not necessary for them later in recovery. However, we observed an intermittent "buckling" phenomenon in DREADDs animals without CNO activation, that did not occur with CNO re-administration. Future studies could use more refined genetic targeted of specific afferent classes, and utilize muscle recordings to find where afferent modulation is most influential in altering motor output.