Intraspinal injections to double-infect L2 spinal interneurons that project to L5 in the adult rat

doi:10.1038/protex.2017.125

Method Article

Intraspinal injections to double-infect L2 spinal interneurons that project to L5 in the adult rat

https://doi.org/10.1038/protex.2017.125

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Neural circuitry in the lumbar spinal cord governs two principal features of locomotion, rhythm and pattern, which reflect intra- and interlimb movement. These features are functionally organized into a hierarchy that precisely controls stepping in a stereotypic, speed-dependent fashion. Here, we show that a specific component of the locomotor pattern can be independently manipulated. Silencing spinal L2 interneurons that project to L5 selectively disrupts hindlimb alternation allowing a continuum of walking-to-hopping to emerge from the otherwise intact network. This perturbation, which was is independent of speed and occurred occurs spontaneously with each step, did does not disrupt multi-joint movements or forelimb alternation, nor did does it translate to a non-weight bearing locomotor activity. Both the underlying rhythm and the usual relationship between speed and spatiotemporal characteristics of stepping persisted. These data illustrate that hindlimb alternation can be manipulated independently from other core features of stepping, revealing a striking freedom in an otherwise precisely-controlled system.

Neuroscience

Intraspinal injections

lumbar interneurons

viral vectors

HiRet-TRE-eTeNT.EGFP (Kinoshita et al 2012)

AAV2-CMV-rtTAV16 (Kinoshita et al 2012)

Pneumatic picopump

Stereotaxic frame

Spinal stabilizers

Pulled, beveled micropipettes (30 um outside diameter)

Anesthetize the rats with an i.p. injection of sodium pentobarbital (Oak Pharmaceuticals Inc.; IL, USA).
After confirming animals are fully anesthetized (e.g. no pedal-paw reflex following toe pinch), perform a midline incision to expose the underlying musculature.
After careful dissection of the underling musculature to expose the vertebral column, place the animals into a spinal stabilization unit and perform a T13 laminectomy (rostral half) to expose spinal L5. Perform a duratomy by gently scraping the surface of the cord with the bent tip of a 30G x 1/2" needle. Be careful not to damage the surface of the cord.
Using a stereotaxic device (with a pulled and beveled micropipette, outside diameter of 0.30 µm) connected to a pneumatic picopump (World Precision Instruments), perform microinjections of HiRet-TRE-EGFP.eTeNT into the intermediate gray matter of spinal L5 (0.4 mm mediolateral, 1.4 mm dorsoventral). Four injections should be given, two on each side of the spinal cord, each separated by 1.5 mm rostrocaudal. Injections should be given as two, 0.25 µl boluses with three minute incubations to allow for viral uptake.
Following the injections, close the incision site in layers with sutures and apply surgical stables to seal the wound.
Provide post-operative care, including gentamicin (20 mg/kg, s.c.; Boehringer Ingelheim; 7 days), glycopyrrolate (0.02 mg/kg, s.c.; Butler Schein), and lactated Ringer’s solution (10 c.c, s.c.; Hospira). Provide pain management care as needed (buprenorphine [10mg/kg, s.c.; Par Pharmaceuticals] every 12 hours for the first 48 hours post-surgery). Animals should recovery voluntary bladder control within 24 hours of surgery.
One week following the L5 intraspinal injections, re-anesthetized the animals (ketamine:xylazine; 80 mg/kg:4 mg/kg, i.p.; Hospira; IL, USA; Akorn Animal Health).
Perform L2 intraspinal injections with the AAV2-CMV-rtTAV16 viral vector. To do this, repeat steps 2-4 described above with the following modifications: (a) perform a rostral T12 laminectomy to expose spinal L2, and (b) use the mediolateral and dorsoventral coordinates of 0.6 mm and 1.5 mm, respectively.
Repeat steps 5-6 for post-operative care, with the addition of providing the reversal agent Yohimbine at 0.1 mg/kg, i.m. (Lloyd Laboratories) to counteract the effects of xylazine.
Animals should recover for at least one week prior to behavioral testing.

The authors declare no competing financial interests.

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Intraspinal injections to double-infect L2 spinal interneurons that project to L5 in the adult rat

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