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. 2020 Jun 1:339:108730.
doi: 10.1016/j.jneumeth.2020.108730. Epub 2020 Apr 14.

Methods for mechanical delivery of viral vectors into rhesus monkey brain

J Megan Fredericks  1 , Kiana E Dash  2 , Emilia M Jaskot  2 , Thomas W Bennett  3 , Walter Lerchner  2 , George Dold  3 , David Ide  3 , Alexander C Cummins  2 , Violette H Der Minassian  2 , Janita N Turchi  2 , Barry J Richmond  2 , Mark A G Eldridge  4
Affiliations

Methods for mechanical delivery of viral vectors into rhesus monkey brain

J Megan Fredericks et al. J Neurosci Methods. .

Abstract

Background: Modern molecular tools make it possible to manipulate neural activity in a reversible and cell-type specific manner. For rhesus monkey research, molecular tools are generally introduced via viral vectors. New instruments designed specifically for use in monkey research are needed to enhance the efficiency and reliability of vector delivery.

New method: A suite of multi-channel injection devices was developed to permit efficient and uniform vector delivery to cortical regions of the monkey brain. Manganese was co-infused with virus to allow rapid post-surgical confirmation of targeting accuracy using MRI. A needle guide was designed to increase the accuracy of sub-cortical targeting using stereotaxic co-ordinates.

Results: The multi-channel injection devices produced dense, uniform coverage of dorsal surface cortex, ventral surface cortex, and intra-sulcal cortex, respectively. Co-infusion of manganese with the viral vector allowed for immediate verification of injection accuracy. The needle guide improved accuracy of targeting sub-cortical structures by preventing needle deflection.

Comparison with existing method(s): The current methods, hand-held injections or single slow mechanical injection, for surface cortex transduction do not, in our hands, produce the density and uniformity of coverage provided by the injector arrays and associated infusion protocol.

Conclusions: The efficiency and reliability of vector delivery has been considerably improved by the development of new methods and instruments. This development should facilitate the translation of chemo- and optogenetic studies performed in smaller animals to larger animals such as rhesus monkeys.

Keywords: Chemogenetic; DREADD; Lentivirus; Non-human primates; Optogenetic; Rhesus monkey.

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Conflict of interest statement

Declaration of Competing Interest No competing interests declared

Figures

Figure 1:
Figure 1:
Multi-channel array for surface injections into ventral cortex. (A) Components: four needles inserted into the dorsal surface holes, 3D printed base, hypodermic tubing is inserted into the lateral surface (see Supplemental Figure 2 for additional detail). (B) Computer Aid Design (CAD) model of the assembled ventral array. (C) Photo of assembled ventral array. The array is connected to the infusion apparatus (see Supplemental Figure 1) by 10 cm silicone tubing for each channel, which is secured to the array using Krazy glue.
Figure 2:
Figure 2:
Multi-channel array for surface injections of dorsal cortex. (A) Components: 4 hypodermic tubing inserted from above, 3D printed base, 4 needles inserted from below (see Supplemental Figure 2 for additional detail). (B) CAD model of assembled array. (C) Photo of assembled array. The array is connected to the infusion apparatus (Supplemental Figure 1) by 5 cm silicone tubing. The center hole (not shown) in the 3D printed base allows for the insertion of 23-gauge wire to stabilize the connection to the infusion apparatus.
Figure 3:
Figure 3:
Needle array for injections into sulci. (A) Components: hypodermic tubing inserted from above, 3D printed base, needles inserted from below (see Supplemental Figure 2 for additional detail). (B) CAD model of assembled array. (C) Photo of assembled array. The array is connected to the infusion apparatus (Supplemental Figure 1) by 5 cm silicone tubing. The center hole (not shown) in the 3D printed base allows for the insertion of 23-gauge wire to stabilize the connection to the infusion apparatus.
Figure 4:
Figure 4:
Needle guide for precise sub-cortical targeting. (A) The pump is mounted on a stereotaxic arm. (B) The needle guide is attached to the arm with a ‘C’-bracket. (C & D) The needle guide aperture allows the needle to pass through. Component dimensions of the foot can be found in Supplemental Figure 3.
Figure 5:
Figure 5:
Visualization of cortical injections using MR contrast reagent. MR images acquired immediately after surgery of single injections in which virus was co-infused with 0.5 μL of (A) no Mn2+, (B) 0.1 mM Mn2+, or (C) 5 mM Mn2+.
Figure 6:
Figure 6:
In vivo and ex vivo visualization of cortical viral injection/expression using injector arrays. (AI) Schematic representation of injector type used. (AII) Skull-stripped MR image of Monkey A acquired after virus co-infused with 5 mM Mn2+ into area 12 of ventrolateral prefrontal cortex (vlPFC). (AIII) Bright field visualization of CFP expression. (AIV) Fluorescent imaging of CFP expression. (BI – BIV) Same conventions as ‘A’, but illustrating the results of injections into primary somatosensory cortex (S1) using the needle array for surface injections of dorsal cortex of Monkey B using 0.1 mM Mn2+. (CI – CIV) Same conventions as ‘A’, but illustrating the results of two injections, one into central sulcus and one into intraparietal sulcus of Monkey B using the needle array for sulcal injections with 0.1 mM Mn2+.
Figure 7:
Figure 7:
In vivo and ex vivo visualization of subcortical viral injection/expression using a needle guide for enhanced accuracy. Targeting Tail of Caudate: the pre-op scan of Monkey C (A) used to calculate the coordinates (B) for targeting of injections. A post-op scan (C) was acquired 8 hours after the injection of 10 μL of virus co-infused with 0.5 mM Mn2+. All MR images are skull stripped (D) Bright field visualization of CFP expression in Monkey D. (E) Confocal imaging of CFP expression in Monkey D.
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