Shufflebrain

The larva's small size necessitates the use of a stereoscopic dissecting microscope for operations. With experience, the surgery becomes routine and requires only a few inexpensive tools. An anesthetic (MS 222), dissolved in the water, renders the animal unconscious.[2] A cream-colored Vermont marble clay lines the bottom of the operating dish, the clay molded to complement the contours of a salamander's belly. Ordinary straight pins, plunged into the clay and then crossed, truss the animal into the desired position. An ordinary needle with a honed point becomes the scalpel. But I use iridectomy (iris) scissors for most of the cutting, and "Genuine du Mont et fils" Swiss watchmakers' forceps for grasping, manipulating and blunt-dissecting.

The larva's skull cap consists of two microscopically thin translucent membranes, the forerunners of bony plates. The membranes meet at a midline seam, or raphé and a quick, upward, longitudinal stroke with the tips of the iridectomy scissors quickly separates them, exposing the brain. Even after the skull ossifies into bone, the opening up of the cranium requires no more force than does clipping a fingernail. Hemorrhage is not serious, and no problem at all with larvae. Working under the dissecting microscope, the operator can see and avoid the tiniest blood vessels. When the experiment makes cutting a vessel necessary, clots rapidly form and quickly plug the leaks. Unlike the organs of higher animals, those of the salamander can survive for days without a blood supply, particularly in a cool environment. In fact, the animal may be bled entirely and yet remain alive for a time. (In the early 1950s, the embryologist, Meryl Rose actually raised a colony of bloodless tadpoles.) The chilled animal's slowed metabolism reduces its demand for oxygen. After surgery, the thin skin lets sufficient oxygen pass in from the water to keep a transplanted organ alive until the host's vasculature hooks into it.

For operations on larvae, sutures are unnecessary. (Adult skin may require stitches, however.) But even with larvae, it is essential to cover a wound with skin. When gently pressed together, the cut edges of the skin adhere and send battalions of epithelial cells over the rift. Within days, the line of incision has disappeared completely.

The salamander larva was an excellent candidate for shufflebrain experiments on an additional count. After the healing period, messages would relay freely between the spinal cord and the shuffled brain. How did I know this? Some of my confidence grew out of an extensive series of preliminary experiments I will mention briefly in a moment. But as a student trying to teach myself the art of transplanting tiny organs, I assigned myself exercises involving the larval salamander's brain. First, I would make a tunnel in the Jell-O-like connective tissue of the dorsal fin. Then I would remove the brain, and store it in the tunnel. The animal, of course, went into a stupor. How did I know whether the brain had survived (whether I had passed or flunked my test)? After some days, I would return the brain to the cranium. Most animals survived. And in eight to twenty days, they recovered consciousness.

Preliminary to shufflebrain experiments, I conducted a systematic investigation of the salamander larva's medulla, the transition zone between the rest of the brain and the spinal cord. When I destroyed the medulla, the animals became unconscious and died within two weeks. If I left the medulla intact and amputated the brain immediately in front of it, the animals went into permanent stupor but remained alive for many months.

Here's a section through the head of an essentially brainless salamander larva, an example of "blanking." The large circular objects toward the edges of the photo are eyes. The zone between the eyes would ordinarily be filled with brain. Here we see it packed with connective tissue cells that had migrated in from elsewhere. This animal lived for many months after its brain had been amputated; it was behaviorally inert during the entire period. (Brainless animals do not undergo metamorphosis, incidentally, because they don't have the pituitary gland, one lobe of which makes the hormones that drive the process.)

To interpret my experimental results, I had to control variables associated with brain regeneration. Other investigators had reported that a larval salamander's cerebral hemispheres regenerate but that lower regions of its brain do not.

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