transsynaptic tract tracing  (Addgene inc)

Journal: The Journal of Neuroscience

Article Title: Superior Colliculus Controls the Activity of the Rostromedial Tegmental Nuclei in an Asymmetrical Manner

doi: 10.1523/JNEUROSCI.1556-20.2021

Figure Lengend Snippet: RMTg neurons monosynaptically innervated by the contralateral SC project to the midbrain dopaminergic system. A , Exemplary brain image showing the site of unilateral injection of anterograde transsynaptic AAV (AAV1-hSyn-Cre-hGH) containing the gene for Cre recombinase into the SC. Immunohistochemical staining was performed to visualize Cre-recombinase (Alexa Fluor 647; yellow color). B , Transsynaptically labeled Cre-positive neurons within RMTg. The orange dashed line indicates the RMTg boundaries based on the anti-FoxP1 immunostaining. C , Representative image of RMTg after the follow-up injection of AAV (AAV2-EF1α-DIO-mCherry) carrying Cre-dependent gene for fluorescent protein (mCherry; red color). The orange dashed line indicates the RMTg boundaries based on the anti-FoxP1 immunostaining. D , Top, Representative image of VTA and SNc after the injections of anterograde transsynaptic AAV (containing Cre recombinase gene) into the SC and AAV with Cre-dependent gene for mCherry into the contralateral (right in this case) RMTg. Immunohistochemical staining was performed to visualize TH-positive neurons (Alexa Fluor 488; green color). Bottom panels, Magnified regions of VTA and SNc with mCherry-expressing axons originating in the RMTg neurons innervated by the contralateral SC. E , Top, Exemplary image of binarized mCherry-positive axons within the VTA and SNc used for area fraction calculation. Bottom, Mediolateral distribution of normalized area fraction of mCherry-expressing axons originating from the RMTg neurons innervated by the contralateral SC. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In the transsynaptic tract-tracing experiments, 80–200 nl of AAV1-hSyn-Cre-hGH (AAV; viral titer: 1.8 × 10 13 vg/ml; Addgene) was injected into the SC unilaterally (−6.4 to −6.6 mm caudally, ±1.8 to ±2.0 mm laterally, and −4.4 to −4.6 mm ventrally from the bregma point), and, in some of these rats, 300 nl of AAV2-EF1α-DIO-mCherry (viral titer: 5.3 × 10 12 vg/ml; UNC Vector Core) was injected into the contralateral RMTg (10° lateral angle, −6.8 to −7.0 mm caudally, +1.7 mm laterally, and −7.4 mm ventrally from the bregma point).

Techniques: Injection, Immunohistochemical staining, Staining, Labeling, Immunostaining, Expressing

Journal: The Journal of Neuroscience

Article Title: Superior Colliculus Controls the Activity of the Rostromedial Tegmental Nuclei in an Asymmetrical Manner

doi: 10.1523/JNEUROSCI.1556-20.2021

Figure Lengend Snippet: Stimulation of RMTg neurons innervated by the contralateral SC inhibit DA-like neurons in the VTA and SNc. A , Scheme of the experiment. SC was unilaterally injected with the transsynaptic viral vector (AAV1-hSyn-Cre-hGH) carrying the gene for Cre recombinase and contralateral RMTg was injected with AAV2-EF1α-DIO-ChR2(H134R)-mCherry carrying Cre-dependent genes for ChR2 and mCherry fluorescent protein. At least 2 weeks later, extracellular recordings of the activity of midbrain DA-like neurons was conducted while RMTg neurons, monosynaptically innervated by the contralateral SC, were optogenetically stimulated. The shape of a typical action potential of a DA-like neuron is shown in the inset on the left side. B , C , Top panels, Heatmaps of peristimulus firing rates (normalized to baseline) showing the responses of all recorded DA-like neurons to RMTg stimulation with trains of laser light pulses (473 nm, <20 mW, 5 ms pulses at 40 Hz over 1 s; B ) or single laser light pulses (473 nm, 100 ms, <20 mW; C ). Responses of individual neurons are shown in rows and are sorted by the amplitude of the response during the stimulation time (marked by vertical dashed line). Bottom panels, Peristimulus mean firing rate (normalized to baseline; ±SEM marked with gray color) and peristimulus mean change in firing rate (±SEM marked with gray color). The stimulation time is marked with a blue vertical bar. D , Localization of all recorded DA-like neurons with color-coded type of response to the optogenetic stimulation of RMTg neurons monosynaptically innervated by the contralateral SC. E , G , Pie charts showing the proportions of response types (inhibition, no effect, excitation) of VTA (marked by green outer ring) and SNc (marked by purple outer ring) DA-like neurons elicited by train (5 ms at 40 Hz; E ) or single-pulse (100 ms; G ) optogenetic stimulation of RMTg neurons innervated by the contralateral SC. F , H , Mean firing rates before, during, and after the train (5 ms at 40 Hz; F ) or single-pulse (100 ms; H ) optogenetic stimulation of RMTg neurons innervated by the contralateral SC (100 ms bins). I , J , From left to right: percentage change in firing rate, duration, and latency to the minimum of the response of DA-like neurons both in VTA (green bars) and SNc (purple bars) caused by train (5 ms at 40 Hz; I ) or single-pulse (100 ms; J ) optogenetic stimulation of RMTg neurons innervated by the contralateral SC. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, Nonsignificant.

Article Snippet: In the transsynaptic tract-tracing experiments, 80–200 nl of AAV1-hSyn-Cre-hGH (AAV; viral titer: 1.8 × 10 13 vg/ml; Addgene) was injected into the SC unilaterally (−6.4 to −6.6 mm caudally, ±1.8 to ±2.0 mm laterally, and −4.4 to −4.6 mm ventrally from the bregma point), and, in some of these rats, 300 nl of AAV2-EF1α-DIO-mCherry (viral titer: 5.3 × 10 12 vg/ml; UNC Vector Core) was injected into the contralateral RMTg (10° lateral angle, −6.8 to −7.0 mm caudally, +1.7 mm laterally, and −7.4 mm ventrally from the bregma point).

Techniques: Injection, Plasmid Preparation, Activity Assay, Inhibition