Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Abstract

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Dorsal Horn Gastrin-Releasing Peptide Expressing Neurons Transmit Spinal Itch But Not Pain Signals

“…Intraspinal virus injections were made in isoflurane-anesthetized mice using a motorized stereotaxic frame (David Kopf Instruments and Neurostar). Three separate unilateral injections of 300 nl each were performed at a depth of 300 m using glass micropipettes connected to a PHD ultra nanomite syringe pump (Harvard Apparatus) as described previously (Albisetti et al, 2017;Haenraets et al, 2018). The following viruses were used: for chemogenetic activation, AAV1.hSyn.flex.…”

The Neurokinin-1 Receptor is Expressed with Gastrin-Releasing Peptide Receptor in Spinal Interneurons and Modulates Itch

“…Using a scalpel, a skin incision was made over the T12-L3 vertebrae. Small scissors were used to cut through fascia, and #5 forceps were used to separate muscle from the T12-T13 vertebrae, exposing the intervertebral space above the left L3/L4 spinal segments [10,23]. Laminectomies were avoided to minimize spinal cord trauma.…”

Targeted up-regulation of Drp1 in dorsal horn attenuates neuropathic pain hypersensitivity by increasing mitochondrial fission

“…The AAV vector was injected into the SDH of mice as described previously [ 90 ]. Mice were anaesthetized with pentobarbital and the target vertebral column position was located using palpation.…”