Journal: The Journal of Neuroscience

Article Title: Dynamic Network Activation of Hypothalamic MCH Neurons in REM Sleep and Exploratory Behavior

doi: 10.1523/JNEUROSCI.0305-19.2019

Figure Lengend Snippet: Deep-brain imaging of MCH neurons. A, Schematic of transfection of MCH neurons in MCH-Cre mice with AAV-DIO-GCaMP6 followed by placement of the GRIN lens in region transfected with GCaMP6 (slow or medium). The miniscope is attached to the GRIN lens via a baseplate on the skull. B, Photomicrograph depicts the location of the GRIN lens (outlined in dashed lines) atop the body of GCaMP6s containing neurons in the hypothalamus in a representative MCH-Cre mouse. The brain region containing the GRIN lens was sectioned along the coronal axis of the brain, and tissue containing the GCaMP6s neurons were identified. f, Fornix. Scale bar, 300 μm. C, Immunohistochemistry revealed that GCaMP6s-infected neurons (green) were also immunopositive for MCH. The coronal sections were incubated with the MCH antibody and visualized using a Leica confocal microscope. Scale bar, 80 μm. D, The field of view of the GRIN lens with fluorescence (ΔF/F0) in somata and processes during REM sleep in neurons extracted automatically by PCA-ICA analysis. We have labeled the three neurons (labeled 1, 2, and 3) whose Ca2+ fluorescence is plotted in E. E, GCaMP6s fluorescence (ΔF/F0) in MCH neurons is associated with REM sleep. Ca2+ imaging was performed simultaneously with recording of cortical EEG and EMG activity in the nuchal muscles. Behavioral video recordings were obtained and examined to identify behaviors such as walking, eating, grooming, or eating. Activity in the EEG (depicted as power spectra, 0.3–15 Hz) and the EMG is used to identify wake, NREM, and REM sleep states (labeled as hypnogram). The traces depict the change in fluorescence (ΔF/F) during wake–sleep bouts of the three neurons identified in D. In each neuron, the ΔF/F0 (expressed as a z-score) varies with the wake–sleep state of the animal, with peak fluorescence associated with REM sleep. The hypnogram categorizes the sleep–wake states in the following colors: purple, active wake; blue, quiet wake; green, NREM; yellow, pre-REM sleep; red, REM sleep. F, The same field of view as in D, but this image shows the PCA-ICA extracted neurons (ΔF/F0) while the mouse was engaged in exploring novel objects placed in its home cage. This image shows that some neurons that were evident in REM sleep (D) were also activated during exploratory behavior. However, some neurons in D were not evident during exploratory behavior, indicating selective activation of these neurons during REM sleep (D). Thirty percent of the neurons were activated during REM sleep but not during exploratory behavior, indicating that a subset of MCH neurons is selectively active in REM sleep. G, GCaMP6s fluorescence in MCH neurons while exploring novel objects. The traces are from the same neurons represented in REM sleep (E). Note that the GCaMP6s has a rapid response and a slow rate of decay, which makes it difficult to infer whether the imaged neuron fired as single spikes or in clusters.

Article Snippet: The sleep–wake states were identified based on EEG, EMG and video data (Neuroexplorer; Plexon).

Techniques: Imaging, Transfection, Immunohistochemistry, Infection, Incubation, Microscopy, Fluorescence, Labeling, Activity Assay, Muscles, Activation Assay