Stereotaxic injections were performed on P28-P36 mice in the prelimbic PFC, NAc, BLA and vHPC. Anterograde viruses included AAV2/1-hSyn-GFP, AAV2/1-CB7-mCherry and AAV2/1-hSyn-hChR2-eYFP (UPenn Vector Core). Retrograde tracers were 0.2% dilutions of Alexa-conjugated Cholera Toxin subunit B (CTB-Alexa 488 or CTB-Alexa 647; Invitrogen) or undiluted retrobeads (green or red; Lumafluor). After injections, animals were returned to their home cages for 2–3 weeks before experiments.
Alexa 647 ctb
Manufactured by Thermo Fisher Scientific
Alexa-647-CTB is a fluorescently labeled cholera toxin subunit B (CTB) conjugate. CTB binds to GM1 ganglioside receptors on the cell surface, and the Alexa-647 dye allows for fluorescent detection of CTB binding.
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5 protocols using alexa 647 ctb
1
Stereotaxic Viral Tracing in Mice
2
Mapping Collateralized MS Neuron Projections
3
Tracing Retinal Connections with CTB and AAV
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Intravitreal injection of cholera toxin subunit B (to trace retinal terminals) was performed as previously described (Monavarfeshani et al., 2018 (link); Su et al., 2011 (link)). Briefly, mice were anesthetized with isoflurane, and 1 μl of 1 mg/ml fluorescently conjugated Alexa‐647‐CTB (Invitrogen, C34778) was binocularly injected with a fine glass pipette using a picospritzer. The rapid, minor, and non‐invasive nature of these injections (which is not considered a surgery) precluded the need for additional analgesics. After 3 days, animals were killed and transcardially perfused with PBS followed by PFA. We opted for a binocular injection here because it is the best way to accurately delineate the border between vLGNe and vLGNi.
A similar intravitreal injection of AAV2/1‐hSyn‐Cre‐WPRE‐hGH (2.5 × 1013 GC/mL, here referred to as AAV1‐Cre) was used to monosynaptically label retinorecipient neurons in the vLGN. 1.2 μl of AAV‐Cre virus was binocularly injected at an approximate 45° angle relative to the optic axis. We opted for a binocular injection here to maximize the probability of AAV‐Cre infecting RGCs and the probability of trans‐synaptic infection in vLGN. AAV1‐Cre was a gift from James M. Wilson (Addgene viral prep #105553‐AAV1; RRID:Addgene_105553). Animals were killed and perfused with PFA as described above 6–10 weeks after injection.
A similar intravitreal injection of AAV2/1‐hSyn‐Cre‐WPRE‐hGH (2.5 × 1013 GC/mL, here referred to as AAV1‐Cre) was used to monosynaptically label retinorecipient neurons in the vLGN. 1.2 μl of AAV‐Cre virus was binocularly injected at an approximate 45° angle relative to the optic axis. We opted for a binocular injection here to maximize the probability of AAV‐Cre infecting RGCs and the probability of trans‐synaptic infection in vLGN. AAV1‐Cre was a gift from James M. Wilson (Addgene viral prep #105553‐AAV1; RRID:Addgene_105553). Animals were killed and perfused with PFA as described above 6–10 weeks after injection.
4
Tracing Retinal Connections with CTB and AAV
5
Retinal Tracing and Neuronal Labeling
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Intravitreal injection of cholera toxin subunit B (to trace retinal terminals) was performed as previously described (Monavarfeshani et al. 2018; Su et al. 2011) . Briefly, mice were anesthetized with isoflurane, and 1 μl of 1 mg/ml fluorescently conjugated Alexa-647-CTB (Invitrogen, C34778) was injected with a fine glass pipette using a picospritzer. After 3 days, animals were sacrificed and transcardially perfused with PBS followed by PFA.
A similar intravitreal injection of AAV2/1-hSyn-Cre-WPRE-hGH (2.5 × 10 13 GC/mL, here referred to as AAV-Cre) was used to monosynaptically label retinorecipient neurons in the vLGN. 1.2 μl of AAV-Cre virus was injected either monocularly or binocularly at an approximate 45° angle relative to the optic axis.
pENN.AAV.hSyn.Cre.WPRE.hGH was a gift from James M. Wilson (Addgene viral prep #105553-AAV1; RRID:Addgene_105553). Animals were sacrificed and perfused with PFA as described above 6-10 weeks after injection.
A similar intravitreal injection of AAV2/1-hSyn-Cre-WPRE-hGH (2.5 × 10 13 GC/mL, here referred to as AAV-Cre) was used to monosynaptically label retinorecipient neurons in the vLGN. 1.2 μl of AAV-Cre virus was injected either monocularly or binocularly at an approximate 45° angle relative to the optic axis.
pENN.AAV.hSyn.Cre.WPRE.hGH was a gift from James M. Wilson (Addgene viral prep #105553-AAV1; RRID:Addgene_105553). Animals were sacrificed and perfused with PFA as described above 6-10 weeks after injection.
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