[PhD Thesis Presentation] ‐ Mr. Neil Dalphin -"Functional Voltage Imaging of Mouse Super-Granular Layers, in Vivo"

Presenter: Mr. Neil Dalphin

Title: Functional Voltage Imaging of Mouse Super-Granular Layers, in Vivo

Supervisor: Professor Bernd Kuhn

Unit: Optical Neuroimaging Unit

Abstract:

Layer 1 of cortex is theorized to be responsible for gain control of cortical output or attentional focusing. However, layer 1 is often neglected in research, due to the difficulty of recording from it. Here, I describe two voltage imaging methods which allow recording neural activity, response to sensory stimuli and oscillations, in the supra-granular cortical layers (including layer 1) under anesthetized and awake conditions, and show first results.
I developed a new method to load a newly synthesized, water soluble voltage dye (DiMethyl-ANNINE-6plus) into layer 1. This involved creating a space between the dura and brain, by applying dilute hydrogen peroxide to the open craniotomy, and then injecting the dye into this space. This allowed the dye to diffuse through layer 1 without damaging the brain tissue. Two photon microscopy allowed optical sectioning, so neural activity could be investigated within these upper cortical areas. This protocol is promising for its ease of use in investigating sensory stimuli in layer 1. Additionally all major neural oscillations up to 40Hz can be detected with this method, so we could observe how they change with brain state, and cortical depth.
Alternatively I made recordings in layers 1 and 2, with bulk-loaded voltage dye (ANNINE-6). Voltage responses showed a fast and slow response to whisker stimulation, with a 10-20ms initial response, followed by a prolonged depolarization (peak around 200ms following stimulus), which was larger (~1.03% Δf/f) when the mouse was anesthetized than when it was awake (~0.61% Δf/f). Looking further into voltage responses, gamma frequency oscillations were found through layers 1 and 2, which also showed a power increase following whisker stimulation, but mainly in awake conditions.
I combined ANNINE-6 voltage imaging with GCaMP-6f calcium imaging, to compare the average membrane potential collected by the voltage dye with intracellular calcium responses. Calcium responses showed almost no activity during anesthesia, which indicates a lack of thalamic inputs into layer 1, during anesthesia, but showed a very large response when the mouse was awake. In comparison, voltage changes were stronger in anesthetized than awake animals, although the fast initial response may get larger during wakefulness.
Together these data show fundamental changes in cortical processing from anesthesia to wakefulness, suggesting a thalamic role in cortical integration and local brain oscillations. Additionally the presence of gamma in layer 1 sets an interesting question into its origin.