Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

This method facilitates the reproducible dissection and slicing of rodent brainstems for the captured analysis of the Mirian Medullary Respiratory Network. This technique offers great experimental flexibility, making it possible to prepare either an in-vitro unblock or a slice preparation for recording. This method may be used for electrophysiology experiments to explore the neural control circuit that generates breathing rhythms and to understand the pathology and disregulation of breathing in genetically modified rodents.

After dissection of the neuraxis, quickly transfer the isolated trunk of the animal to an aerated dissection chamber under a dissection microscope and place the tissue dorsal-side up, with the rostral end facing the front of the chamber. Pin the tissue at the shoulders and the most caudal end of the spinal cord and make a midsagittal incision through the skull following the parietal suture to avoid damaging the cortex and the brainstem underlying the skull. Beginning at the sagittal suture and working laterally, snip the occipital sutures of the skull to create flaps of bone that may be reflected and pinned to anchor and stabilize the rostral part of the skull.

After reflecting both skull flaps, excise the remainder of the cerebral cortex, leaving the caudal portion of the cerebellum relatively intact to perform a dorsal laminectomy. Use micro spring scissors and forceps to remove the musculature surrounding the skull and vertebral column. Remove the tissue along the dorsal side of the ribcage, leaving the ribs intact and carefully snip away the lateral processes of the vertebral lamina.

After cutting away any tissue overlying the pons and medulla, the vermis, cerebellum, pons and beginning of the spinal cord, will be clearly visible. To perform a ventral laminectomy, place the tissue dorsal-side down and pin at the ribcage and the most caudal end of the spine. Use the skull flaps to pin down the rostral-side of the tissue and remove the ventral half of the ribcage, including the sternum and all of the abdominal organs.

Dissect away the soft tissue attached to the ribcage to expose the ribs and spinal cord and remove the tongue, esophagus, trachea, larynx and all of the other soft tissue and musculature overlying the base of the skull and the spinal column. In order to identify the hard pallet, dissect the tissue overlying this rectangular plate of bone at the base of the skull, that includes a v-shape indentation. Then cut along the midline of the pallet, carefully lifting the tissue upwards and perform a transverse cut to remove it.

To begin a ventral laminectomy, remove the lamina, exposing the ventral surface of the brainstem and the spinal cord from the first cervical vertebra to approximately the thoracic vertebra seven and snip five to 10 millimeters along both sides of the spinal column at the lamina. When the spinal cord has been exposed, snip the rootlets approximately 20 to 25 millimeters bilaterally, along the spinal column to approximately T7 and carefully lift the rostral edge of C1, C2 and C3 with hooked or bent forceps to allow snipping beneath the bone for removal of the three vertebra. When the desired length of spinal cord has been isolated from the vertebral column, make a transverse cut to remove the spinal cord tissue.

After removing the dura, place the brainstem in the center of the paraffin platform on the plastic cutting block and use fine insect pins, trimmed to no more than one centimeter in length to pin the caudal end of the brainstem through the distal spinal cord. Then align the paraffin covered cutting block with the pinned brainstem in the Vibratone block holder, so that the blade will cut perpendicular to the rostral face of the brainstem. Next, make an initial slice to remove 200 to 300 micrometers of uneven extraneous tissue on the rostral-most end of the tissue, making small adjustments as necessary to ensure PARP linearity and small cuts to remove any uneven tissue.

The glossopharyngeal rootlets will become visible at the lateral edge of the brainstem, cut a 300 to 500 micrometer slice of brainstem from these rostral neuroanatomical markers to capture the pre-Botzinger complex and the associated transmission circuitry. All of the slicing procedure steps are critical for creating a reproducible, viable slice, with all of the necessary neuronal circuitry in the correct orientation to produce robust rhythm. All of the minimally necessary neural circuit elements for generating and transmitting inspiratory rhythm can be captured in a thin slice using this method.

Including the pre-Botzinger complex, pre-motor neurons projecting to the hypoglossal motor neurons and the hypoglossal nerve rootlets. The pre-Botzinger complex, the hypoglossal or 12th cranial nerve rootlets and C4 nerve rootlets can then be used for inspiratory recording. The key aspect of this procedure is the careful isolation of the rootlets and confirmation that the brainstem is not damaged before cutting the final slice.

Following this procedure, electrophysiology recordings can be made using patch clamp methods, extra cellular recordings or suction electrodes to record the electrical activity produced by the network. Previously published protocols have provided little step-by-step detail, making it difficult for new researchers to use this method without traveling to another laboratory and spending time with a seasoned investigator.