Optogenetics

Optogenetics in an important tool for probing neural circuitry and testing the influence of genetically targeted neurons on external subject behaviors. Optogenetics can be performed either independently or in the same site as a fiber photometry recording. Most users will be using either blue (465 nm) excitation for channelrhodopsin, or orange (590 nm) or red (635 nm) light for Chrimson, Jaws, and other opsins.

There is a choice of using an LED or a laser for the optogenetic light source. LEDs have broader light spectrums, but their peak powers are not as high as collimated lasers. You can see a list of Lux LED power outputs here. You should test the maximum power output of your intended optical setup with an external power meter (not the PM1 which does not have the range for opto stim powers) and consider the irradiation threshold of your opsin to see if you can deliver enough light power for optogenetic activation. You can find good predictions of irradiance values here

Optical Setup

Users will either be performing optogenetics in a dedicated optical implant (direct opto) or will be performing optogenetics at the same site as their fiber photometry recordings. The optical setups for these two scenarios are very different.

Direct Optostimulation The direct site optogenetic setup is easy. You will use a dedicated patch cord to go from your light source directly to the target.

Same Site Optostim + Fiber Photometry Performing optogenetics in the same site as your fiber photometry recordings is common, but the setup is more complicated. You will need to send your optogenetic light through an Optical Manifold to route into the same patch cord that is connected to your subject for photometry.

The filter ports in the Lux Manifold are bidirectional, which means that a port typically used for recording fluorescence of a specific color can pass that same color into the port as excitation. The dichroic mirrors are longpass, so anything above the wavelength cutoff will pass by without attenuation or reflection. In the above LxM6, red photometry (588 nm - 640 nm) is recorded in the F2 port. That same port can have red light of the same bandwidth input into it to then go through to the sample port and into the subject.

Important Considerations Fiber optic cables will generate red fluorescence when a blue or purple light pass through. This is a problem when trying to do blue optogenetic stimulation while simultaneously recording red photometry in the same site because the opto pulses will saturate the photosensor with red light. This can be mitigated by photobleaching your patch cord for a long time, either with the RZ10x or with an LxBB5. However, this is not guaranteed to work and you may still experience artifacts during optogenetics that make the fiber photometry recordings difficult to interpret during stimulation. A laser, which has a much narrower light bandwidth, may help.

Red LEDs are not as powerful as blue LEDs. While you can usually meet the required irradiation threshold needed to activate your opsin, the max power will still be fairly low. Consider using a red laser to make optostimulation easier.

Delivering High Powered LED Stimulation in the RZ10x

The RZ10x is capable of delivering high current (up to 1000 mA) to any of the LED driver slots to drive a bright LED output. Below are a series of images showing a typical opto stim setup using the Lower Bank of an RZ10x, Lux LEDs, and a Pulse Train gizmo.

Note

The RZ10x Lux banks are 'one application per bank', meaning that if you setup the LEDs for Legacy Control to perform opto stim, then you cannot access that same bank for fiber photometry recordings (you would need to use the Upper Bank for photometry in the listed example). If users want full access to both banks for photometry, then we strongly recommend the use of an external LED driver or laser that you can trigger with a digital IO pulse.

To perform opto with the Lux LEDs, you must first make the LEDs accessible on the analog outputs (DAC tab) to control with a voltage pulse. To do this, check 'Legacy Control' of whichever bank you want to drive, set the current to 1000 mA, and press 'Commit' at the bottom right. Once Synapse compiles, you will see the Drv-N ports available in the DAC tab.

The DAC tab is a place to route pulses generated in Synapse to an analog output. In the case of optogenetics, you will likely be using a pTrain gizmo to make voltage pulses from 0 V - 10 V.

Output: Float just means that the signal can vary beyond the typical 0 - 1 values that a logic pulse can produce. This is important for varying the light power of the LED. Please refer to the Pulse Train gizmo for more details about parameters and other configuration settings. We recommend you also set the Epoc ID to 'on Train1' to see your pulses at runtime.

Once the pTrain voltage pulse is routed to the DAC output of choice, you can then drive the LED on/ off with adjustable power at runtime using the pTrain controls.

Delivering TTL Pulses for External LEDs and Lasers

Some users may need to use an external laser for higher light powers, or they may want to trigger an external LED to keep both photometry banks functioning. Thorlabs has nice external LED drivers that can be triggered with TTL pulses.

TDT has a nice guide on digtial IO that you can read to learn more about generating TTL pulses and sending them out of the bit ports of the RZ10x.

Optogenetics with the iX6

We do not recommend using the iX6 for optogenetics. Instead, consider adding an iL24 to your iCon to perform opto with TTL pulses, as described earlier. Alternatively, you can consider adding or swapping for an iX7 which has simultaneous fiber photometry + integrated red optogenetic stimulation capabilities.