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. 2019 May 23:(147):10.3791/59232.
doi: 10.3791/59232.

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

Karam Khateeb  1 , Devon J Griggs  2 , Philip N Sabes  3 , Azadeh Yazdan-Shahmorad  4
Affiliations

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

Karam Khateeb et al. J Vis Exp. .

Abstract

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (>130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.

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Figures

Figure 1.
Figure 1.
MR-compatible cylinder and cannula injection grid. A,B) custom-designed nylon injection grid. C) MR-compatible fixed cylinder. D, E) Rotating MR-compatible cylinder. F) MR-compatible infusion cylinder with fixed grid position. The arrows point to the cavities that are designed to be filled with wet sterile absorbable gelatin keeping the surface of the brain moist for the duration of injection. G) Cannula inserted in the grid. This figure has been modified from Yazdan-Shahmorad et al. 2016.
Figure 2.
Figure 2.
A) T1-weighted image of vitamin E that was attached to the top of the injection grid that enables us to measure the distance to the surface of the brain (white arrow). B) T2-weighted image of the brain helps to plan the location of injection from the cannula grid filled with saline. C) MR image of the infusion chamber and the saline-filled cannula grid. The orthogonal lines represent the sagittal (yellow) and coronal (purple) planes. D) Photo of the reflux resistant injection cannula tip with the reflux resistant step (black arrow). E) Infusion lines. This figure has been modified from Yazdan-Shahmorad et al. 2016.
Figure 3.
Figure 3.
A) Spread of 50 μL of the viral vector in coronal sections of Monkey G for one injection site in S1 (shown with an arrow in B). B) MRI surface rendering of the cortical surface below the cylinder for Monkeys G and J, respectively. The S1 infusion locations are shown in blue, M1 locations in red. C) MR Volume reconstruction of the spread of viral vector during CED infusion. Brain is shown in light gray; S1 and M1 infusion volumes are shown in blue and red, respectively. No MR volume reconstruction is available for the M1 infusions for monkey G since they were not done in the MR scanner. This figure has been modified from Yazdan-Shahmorad et al. 2016.
Figure 4.
Figure 4.
Electrophysiological recordings of light-evoked activity. Micro-Electrocorticography (μECoG) recordings occurred during pulsed optical stimulation. Recording traces were from the closest electrode to the site of stimulation for examples of M1 (red) and S1 (green) stimulation locations. Shaded areas around the traces represent standard error across 25 trials. The blue squares on the traces show the timing of stimulation (1 ms). The full set of stimulus-triggered waveforms for both sample pairs of stimulation and recording sites are superimposed on the mean waveform as shown on the left side of the panel. This figure has been modified from Yazdan-Shahmorad et al. 2016.
Figure 5.
Figure 5.
Histological analysis. A) Baseline coronal MR image in monkey G. B) Spread of contrast agent after the infusion for the same MR coronal slice as in A. C) A coronal tissue section from approximately the same site as in A and B; peroxidase staining reflects expression of the EYFP-reporter. D) Good alignment is observed between the area of EYFP expression measured with surface epiflourescence (dark green areas) and with histological staining (light green lines). These include the region of vector spread estimated from MR images (white line); white dots indicate injection sites, and the entire black region represents the area exposed by the craniotomy. The two left most injection sites are located in M1, and the two right most sites are located in S1. E) Low magnification image of the coronal section stained with anti-GFP antibody showing the medio-lateral aspect of YFP expression in the somatosensory cortex of monkey J (areas 1, 2, 3). The black arrowhead indicates the location of the cannula track; the adjacent tissue (black frame) is shown in F, at greater magnification to show laminar distribution of the YFP-positive cells. F) Densely populated regions of YFP-positive cells are located predominantly in layers II-III and V-VI, and also show typical pyramidal morphology (cells in white frames are further enlarged in panels G-I). White arrowheads on bottom panels G-I point to typical pyramidal cells in layers II-III (G); layer V (H); and layer VI (I). Scale bars: A, 2 mm; B, 200 μm; C-E, 100 μm. This figure has been modified from Yazdan-Shahmorad et al. 2016 and Yazdan-Shahmorad et al. 2018.
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