In situ hybridization in wholemounted lamprey spinal cord: localization of netrin mRNA expression

The lamprey has been used as a model for the study of vertebrate neuronal circuitry and spinal cord regeneration. One of the advantages of this preparation is the ability to view the entire CNS in wholemount, including several identified neurons and neuron groups. However, because of difficulties in penetration of molecular reagents past the dense meninx primitiva and glia limitans, there has been no reliable method for in situ hybridization in spinal cord wholemounts. We now describe a protocol that accomplishes this while preserving the structural integrity of the cord. In order to enhance penetration of probes and antibodies, the m. primitiva was surgically stripped from the spinal cord after incubation of the fresh tissue in 0.1% collagenase I. Additional modifications that enhanced hybridization signal include (a) increasing the amount of Tween-20 in the hybridization mix to 2%, instead of the typical 0.2%; (b) carrying out the hybridization for 36 h and applying the anti-digoxigenin antibody to the tissue for 48 h. Using this protocol, we showed that netrin mRNA is expressed in dorsal cells, in medium sized neurons of the lateral gray matter and in the glial/ependymal cells of the spinal cords of lampreys. This method will help to study the expression of molecules of interest in identified neurons and neuronal groups without the need for serial section reconstruction.