GRINjector

GRINjector is a device for injecting gradient-refractive index (GRIN) lens probes into animals. The advantages are:

1. The injection needle displaces brain tissue, improving targetting and minimizing tissue compression.
2. The GRIN is held in place during retraction of the placement needle from the GRIN and the GRINJector device.
3. The guide needle can be easily swapped for other guide needles depending on the diameter and length of the GRIN lens probe.

This repository contains CAD files for printing GRINjector version 3.0 components and assembling them into a usable GRINjector.

For those conducting calcium imaging experiments and analysis, we have a software package called CIAtah: https://github.com/bahanonu/ciatah.

Questions? Contact: Biafra Ahanonu (github [at] bahanonu [dot] com).

Components

The below figure illustrates several of the main components:

Below is a list of components needed to assemble a GRINjector:

• Vernier micrometer. SM-13. Newport.
• Bearing stage. 9066-COM. Newport.
• 4x lock screws that come with 9066-COM.
• Model 1770, Kopf Instruments. For attaching GRINjector to stereotaxic arm.
• GRINjector bottom connector (grinjector_bottom_connector)
• GRINjector main needle connector top (grinjector_mainneedle_connector_top)
• GRINjector main needle connector bottom (grinjector_mainneedle_connector_bottom)
• Stereotaxic holder attachment (stereotaxic_holder_attachment)
• 2x - 8-32 x 1'' cap screw.
• 1x - 4-40 x 3/16'' cap screw.
• 1x - 4-40 x 1/4'' cap screw.
• 4x - 4-40 x 5/16'' cap screw.
• Luer lock needle (Holder needle) matched to the size of the GRIN lens probe.
  • For 1-mm GRIN lens probe, recommend 17G needle that is 0.04-0.042'' inner diameter (e.g. https://www.mcmaster.com/6710A24/).
  • For 0.5-mm GRIN lens probe, recommend 21G needle (e.g. https://www.mcmaster.com/6710A28/).
• Thin needle (Stability needle) that is ~80-mm long and between 0.25-0.5-mm diameter (smaller version for smaller GRIN lens probes).
  • 0.25-mm diameter: https://www.mcmaster.com/6710A48/.
  • 0.5-mm diameter: https://www.mcmaster.com/6710A28/.
  • Used to hold the GRIN probe in place as outer sheath is removed. For the above pieces, may need to cut the metal piece from the Luer lock portion.

Printing

The main parts are provided as STEP and STL files to allow printing on nearly all commonly used 3D printers (Straysys, uPrint, ). We recommend printing with a high-resolution printer to ensure optimal fit and one with easy to remove support material—we have used VeroBlackPlus on a Stratasys Objet30 and found that to work quite well.

Assembly

Below are step-by-step by step instructions for assembling the GRINjector after building the necessary parts.

1. Connect the SM-13 and 9066-COM to form a functional setup. Remove the locking plate from 9066-COM as below (see https://www.newport.com/medias/sys_master/images/images/he5/h5b/9366526427166/906806B-9066-9067-User-Manual.pdf).

2. Using 8-32 cap screws, connect the Stereotaxic holder attachment to the back of the 9066-COM, connecting to the top-most holes near the micrometer.

3. Next, pull down the bearing stage so that the two 4-40 holes in the back are exposed, place 4-40 x 5/16'' cap screws in each hole.

4. Tap the grinjector_mainneedle_connector_bottom holes with a 4-40 tap then place it onto 9066-COM as below and screw in the 4-40 x 5/16'' cap screws (red below).

5. Align the lock screws that comes with 9066-COM to each of the four holes on each side of the grinjector_bottom_connector.

6. Next, place a blunt or syringe tip Holder needle that matches the size of your GRIN lens probe into the groove of the grinjector_mainneedle_connector_bottom piece then place the grinjector_mainneedle_connector_top piece on top. Screw the grinjector_mainneedle_connector_top in place with 4-40 x 5/16'' cap screws then secure the Holder needle with a 4-40 x 1/4'' cap screw.

• Syringe tip Luer-lock needles allow easier parting of the underlying tissue as the GRINjector is lowered into the brain or spinal cord tissue.

7. Next, obtain a long, Stability needle (e.g. ~80-mm long, 0.5-mm diameter) and thread through the small opening at the top of grinjector_bottom_connector. The Stability needle will then go through the Holder needle. Lock the needle in place with a 4-40 x 3/16'' cap screw.

• Note that the end of the Stability needle can be sanded or polished to avoid the chance that it will scratch the GRIN lens probe during implantation and retraction.

8. Lastly, verify that the chosen Holder needle fits the GRIN lens probe tightly and that you can push the GRIN lens probe out of the Holder needle smoothly by twisting the SM-13 micrometer.

You are now done assembled the GRINjector! Happy imaging.

Animal experiments

To use the GRINjector in an animal:

• Attach the GRINjector onto a stereotaxic arm then perform your normal GRIN lens probe crainotomy surgery.
• Lower the GRIN into the brain using the GRINjector (below diagram, left).
• After placement at desired coordinates in tissue, raise the blunt tip needle by twisting the micrometer until the needle is clear of the GRIN lens probe. The red needle secures the GRIN in place during retraction of the device.
• Secure the GRIN in place with dental cement, UV glue, or your adhesive of choice then after it as set, slowly raise the GRINjector using the stereotaxic arm.
• If your stereotaxic arm allows angles, the GRINjector can be used to target the same site at different angles to avoid vasculature or vital brain regions (below diagram, right).

Notes

Holder needle:

• SAI Infusion works well: https://www.sai-infusion.com/products/blunt-needles?variant=333055723. The plastic has some give that works well with the 3D printed parts.
• Check that the needle inner diameter is as close to the GRIN lens size as possible, e.g. for 1-mm that would be 17G needles that are ~0.04-0.042'' (https://www.mcmaster.com/6710A24/).
• Sometimes users have to check a couple needles within the same batch since there will be slight size variations that will optimally fit their GRIN lenses.

Stability needle:

• We have found that a metal needle (e.g. could go with https://www.mcmaster.com/6710A48/ or similar then cut the needle to the desired length) does not cause significant scratching.
• If you have a grinder (handheld or bench), polisher (e.g. https://www.ultratecusa.com/product/ultrapol-end-edge-polisher/), or similar surface, then you can make the needle surface smoother and flat.
• Alternatively can place a small drop of optical glue on the end of the needle and cure it or look for plastic needles.

Acknowledgements

I would like to thank Prof. Benjamin Grewe for helping inspire and provide feedback on the initial GRINjector design.

References

We have a book chapter pending describing this device. In the meantime, if you used this device in your research please cite our publication Corder*, Ahanonu*, et al. Science 2019 :

@article{corderahanonu2019amygdalar,
  title={An amygdalar neural ensemble that encodes the unpleasantness of pain},
  author={Corder, Gregory and Ahanonu, Biafra and Grewe, Benjamin F and Wang, Dong and Schnitzer, Mark J and Scherrer, Gr{\'e}gory},
  journal={Science},
  volume={363},
  number={6424},
  pages={276--281},
  year={2019},
  publisher={American Association for the Advancement of Science}
}

License

Copyright (C) 2015-2021 Biafra Ahanonu

This project is licensed under the terms of the MIT license. See LICENSE file for details.

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