112
Methods for Setting Up Primary Cultures Specific to Animal Groups
(Tyndall effect), of the neurosecretory granules, which fill most of the
perikarya.
3. Dissect out the two groups of neurosecretory cells. Each group consists of approximately 200 neurons and an undetermined number of
glial cells. Each group of neurosecretory cells contains three cell types,
known as A, B, and C cells, which can be identified by their size, staining characteristics, and neurosecretory granule content. An average
of 70 type A cells is present in each group. The type B cells comprise
approximately one-third of the entire cell population. Approximately
10 type C cells are present in each group.
4. Transfer the neurosecretory cell groups into the culture medium in
small glass vessels (see Fig. 2).
Dissociation 1. Dissociate the explants in situ into cell clusters of various sizes or into
individual cells by using needles.
Culture 1. Gently press the cell clusters or individual cells onto the surface of a
set-u p
cover slip placed on the bottom of the culture vessel until the cell clusters or individual cells firmly adhered to it.
2. Place the culture vessels in desiccators and incubate at 29°C,
Results
According to Seshan and Levi-Montalcini (1973), no nerve fiber outgrowth is evident from neurosecretory explants up to the end of the first
week. When neurosecretory explants alone were cultured, all explants
had undergone disintegration within 1 month. However, if the explants
were co cultured with ovariole segments dissected out from late nymphal
or adult specimens, vigorous axonal outgrowth from cultured neurosecretory cells was observed after the second week. The number of nerve fibers increased progressively until the 2nd or 3rd month (Fig. 4). Electron micrographs of an axon of a neurosecretory cell cultured for 2
months showed the accumulation of a large number of electron-dense
granules.
Subprotocol 5
Corpora Cardiaca and Corpora Allata
The following method is based on the report of Seshan and LeviMontalcini (1971).
Materials
Equipment - Dissecting microscope
- Dissecting tray (see chapter I, section 3 "Tools")
- Scissors
Methods for Setting Up Primary Cultures Specific to Animal Groups
(Tyndall effect), of the neurosecretory granules, which fill most of the
perikarya.
3. Dissect out the two groups of neurosecretory cells. Each group consists of approximately 200 neurons and an undetermined number of
glial cells. Each group of neurosecretory cells contains three cell types,
known as A, B, and C cells, which can be identified by their size, staining characteristics, and neurosecretory granule content. An average
of 70 type A cells is present in each group. The type B cells comprise
approximately one-third of the entire cell population. Approximately
10 type C cells are present in each group.
4. Transfer the neurosecretory cell groups into the culture medium in
small glass vessels (see Fig. 2).
Dissociation 1. Dissociate the explants in situ into cell clusters of various sizes or into
individual cells by using needles.
Culture 1. Gently press the cell clusters or individual cells onto the surface of a
set-u p
cover slip placed on the bottom of the culture vessel until the cell clusters or individual cells firmly adhered to it.
2. Place the culture vessels in desiccators and incubate at 29°C,
Results
According to Seshan and Levi-Montalcini (1973), no nerve fiber outgrowth is evident from neurosecretory explants up to the end of the first
week. When neurosecretory explants alone were cultured, all explants
had undergone disintegration within 1 month. However, if the explants
were co cultured with ovariole segments dissected out from late nymphal
or adult specimens, vigorous axonal outgrowth from cultured neurosecretory cells was observed after the second week. The number of nerve fibers increased progressively until the 2nd or 3rd month (Fig. 4). Electron micrographs of an axon of a neurosecretory cell cultured for 2
months showed the accumulation of a large number of electron-dense
granules.
Subprotocol 5
Corpora Cardiaca and Corpora Allata
The following method is based on the report of Seshan and LeviMontalcini (1971).
Materials
Equipment - Dissecting microscope
- Dissecting tray (see chapter I, section 3 "Tools")
- Scissors