Embryos were obtained from Fertile White Leghorn chicken eggs incubated at 38®C in a forced-draft incubator. For the studies using purified neurons, the sensoiy
ganglia were dissected after 10, 12, and 14 days of incubation; the sympathetic chain ganglia after 10,12, 14, and 16 days; and the parasympathetic ciliary ganglia after 10, 12, and 16 days. For the studies using cultured neurons, sympathetic chain ganglia were dissected after 7 and 14 days of incubation, dorsal root ganglia (DRG) after 14 days, and dorsomedial trigeminal ganglia (DMTG) and trigeminal mesencephalic nuclei (TMN) after 10 days. For the study of trkB and BDNF mRNA expression, nodose and vestibular ganglia, heart, otic vesicle, and hindbrain were dissected from staged (Hamburger and Hamilton, 1951) chicken embryos early in development. Further details of the tissue dissection and cell culture techniques involved in this work are given in Chapter 5.
A detailed description of tissue dissecton is given in Davies (1988b) but a brief outline is shown below. All procedures were carried out using standard sterile technique in a laminar flow hood. Embryos were removed from the eggs by first holding the egg with the blunt end uppermost (where the airspace is located). The shell was wiped with 70% ethanol and allowed to dry, then cracked in a line around the airspace with forceps and the portion of shell removed along with the membrane lining the airspace. Embryos were removed using curved forceps placed beneath the neck, decapitated, and collected in a large petri dish.
The dissections were carried out in HEPES-buffered (20 mM) Hanks' balanced salt solution (Hanks, from Gibco BRL) under a stereomicroscope at x20 magnification. A fibre optic light source was used to avoid overheating the specimens. Electrolytically- sharpened tungsten needles were used to complete the dissections.
Sympathetic Ganglia and Dorsal Root Ganglia (DRG)
Abdominal and thoracic viscera were removed using watchmaker’s forceps and the posterior abdominal and thoracic walls washed with Hanks from a Pasteur pipette. Blunt forceps were used to slit the connective tissue sheath lying around the spinal column and the sheath carefully tom back. The sympathetic chain was carefully teased away from the spine using forceps, taking care not to separate the chain from the
underlying DRG. The chain was removed from mid-thoracic to sacral regions and separated from the connective tissue using sharpened tungsten needles. DRG (shown in figure 1, part A) were removed from the lumbosacral region by passing the pointed ends of forceps between the ganglia and spinal cord, so cutting the spinal roots. Each ganglion was removed using watchmaker's forceps, holding the nerve distal to the ganglion. The ganglia were collected in a petri dish of Hanks and associated nerves removed using tungsten needles. Between 20-30 of each ganglia were collected.
Cranial Sensory Ganglia
The locations of these ganglia in the chicken are shown in figure 1, part B. Nodose ganglia are located at the base of the neck, on either side of midline tissues in front of the vertebral column. They are glistening white and spindle-shaped in appearance with the attached vagus nerve passing rostrally. The ganglia were removed by taking hold of the vagus nerve with watchmaker's forceps. Associated nerve fibres were removed with tungsten needles.
The jugular, trigeminal, and vestibulo-acoustic (vestibular) ganglia were dissected from the cranial base after removal of the brain. A no. 15 scalpel blade was used to dissect the cranial base as shown in Davies (1988b). Tungsten needles were then used to complete the dissection of ganglia from the resultant tissue blocks, each ganglion being identified by its characteristic shape (see figure 1, part B). The trigeminal ganglion was then subdissected using tungsten needles into the regions containing dorsomedial neurons and ventrolateral neurons as described in Davies (1988b). 20-30 ganglia of each type were dissected out.
Trigeminal Mesencephalic Nucleus (TMN)
This was dissected from the midbrain. The cranial vault was removed with watchmaker's forceps and the brain removed by passing a small spatula between it and the cranial base. 20-40 brains were collected in Hanks and these were transferred to a fresh petri dish of Hanks. The dissection was completed using tungsten needles as
B
^ TMN
Figure 1 (A) Camera lucida drawing of the ventral aspect of the lumbosacral region of an ElO chick embryo after evisceration showing the location of the dorsal root ganglia. (From Davies, 1988b.) (B) Schematic illustration of an ElO chick embryo showing the locations of the cranial sensory ganglia. TMN, trigeminal mesencephalic nucleus; T, trigeminal ganglion; G, geniculate ganglion; VA, vestibulo-acoustic (vestibular) ganglion; J, jugular ganglion; P, petrosal ganglion. (From Davies and Lindsay, 1985.)
described in Davies (1988b). Briefly, the midbrain was isolated by two coronal incisions and the pia mater peeled off, initially from the ventral aspect. The TMN is located in the cerebral aqueduct. The roof of the cerebral aqueduct was then dissected from the midbrain and the median part of the TMN subdissected.
Ciliary Ganglia
The parasympathetic neurons of the ciliary ganglion were removed from embryos by simply scoring around the edge of the eye-ball with blunt forceps and then removing the eye from the socket. Care was taken to not damage the eye and the underlying tissues in the eye socket. The ciliary ganglia, situated near the stalk of the optic nerve, were then removed from the posterior part of the eye using blunt forceps. The ganglia were then cleaned from connective tissue using tungsten needles.