Combining Optogenetics with Electrophysiology Measurements

Transcription

1 Combining Optogenetics with Electrophysiology Measurements This research was supported by the National Institute of Aging of the National Institutes of Health under award number R44AG The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

2 Pinnacle s Optogenetic System A turn-key optogenetics system with integrated biopotential/neurotransmitter recordings for mice and rats. LED fiber probes deliver > 100 mw/mm 2 for most wavelengths 445, 515, 590, 640 & 660 nm. No optical commutator is required. The system has precision timing, TTL inputs/ outputs and multiple head stage configurations (opto only, sleep + opto, seizure + opto, biosensor + opto, sleep or seizure + biosensor + opto, etc.) The next few slides will illustrate in vivo data available from this system for EEG and biosensors

3 Methods Transgenic mice [(Thy1-COP4/EYFP) 9Gfng - Jackson Laboratory] (n=4) expressing channelrhodopsin (activated by blue light) in the hippocampus and cortex and wild-type C57 mice (n=4) were used. All mice were implanted with a cannula and depth electrode targeting the hippocampus, and two cortical electrodes. In a subset of mice (n=2), a second cannula was placed in the anterior cortex. All procedures were approved by the University of Kansas IACUC. After 1 week, a blue 445 nm LED fiber probe was inserted in the hippocampal guide cannula and optical stimulations were performed. Next, the probe was replaced with a deep red 660 nm probe and the experiment repeated. In mice with a second cannula, a glutamate biosensor was inserted ~12 h prior to optogenetic stimulation. Light was delivered for either 20 seconds (EEG) or 40 seconds (EEG + biosensor) at an intensity > 100 mw/mm2 at 20 Hz, 10% duty cycle. Five to ten trials were conducted on each animal with 10 minutes between stimulation trials.

4 Stimulation Induces Seizure Activity In Transgenic Mice EEG activity during and after stimulation with blue (445 nm) and deep red (660 nm) light in the same animal. Stimulation parameters: 20 Hz, 5 ms 20 s (10% duty cycle) 600 ma LED probe intensity Blue: 130 mw/mm 2 Deep red : 155 mw/mm Seconds

5 Stimulation Does Not Induce Seizure in Wild-Type Mice 20 Seconds EEG activity during and after stimulation with blue (445 nm) light in a wild-type C57 mouse. Light pulses were delivered at 20 Hz, 5 ms duration (10% duty cycle) for 20 seconds with a current of 600mA. Blue LED intensity was measured prior to implantation at 100 mw/mm 2. No response or artifact was seen in any of the control mice (n=4).

6 Stimulation Induces Glutamate Change in Transgenic Mice Average change in extracellular glutamate concentration in the frontal cortex during a single 40 s stimulation (20 Hz, 10% duty cycle, 600 ma) with blue (445 nm) and deep red (660 nm) light in the same animal (n=5). Representative EEG depth electrode activity is plotted during time of stimulation. Bars represent S.E.M.

7 Considerations when Integrating Optogenetics and Recording Systems Merge Optogenetics with other measurements (EEG, EMG, FSCV, Biosensors, Microdialysis ). Add very tight timing and control for light delivery. Ensure that the systems are synchronized. Possibly merge multiple software packages. Provide a method for feedback (i.e. when a seizure is detected, trigger optical stimulation). And put this all on the head of a freely moving mouse/rat in a way that does not alter behavior, neurochemistry or physiology.

8 Pinnacle s Solution A turn-key system that can be readily used by any researcher. Move the LED to the head of the animal, so no optical commutator is needed.

9 Core System The key component of the Pinnacle system is the Optogenetics Interface Module which provides precise timing and illumination control when used with our existing 3- and 4-channel data acquisition systems. This unit connects to an electrical commutator and headstage. The system uses LED fiber probes which are compatible with standard cannula placement techniques and do not require an optical commutator. Nine headstages are available for mice or rats.

10 Pinnacle s LED Fiber Probes

11 Opsins and LEDs

12 Heating Impact of LED Probes

13 One More Tool to Make Your Research Easier An LED Probe with a Depth Electrode Tip detail with depth electrode next to optical fiber