It is possible that the NMDA receptor plays an important role in c-/bs induction 
in cortical neurons via a variety of stimuli. For example, section of the 
infraorbital nerve induced Fos in brain stem but decreased Fos in cortex (Sharp 
et al. 1989c). The explanation for the decreases of Fos expression in cortex 
was unclear. It is hypothesized that cortical induction of Fos in most rats 
handled in the laboratory is related to stress of handling, injections, and surgery 
(Sharp et al. 1991). If true, then the decreases in Fos expression in cortex 
following infraorbital section could be explained by the fact that concurrent 
activation of the following two receptors is required: (1) activation of stress 
receptors via unknown pathways and transmitters and (2) concurrent activation 
of NMDA receptors by thalamocortical fibers. To test the idea that induction of 
c -fos in cortical neurons might require the concurrent action of two receptors, 
studies of cultured cortical neurons were undertaken (Hisanaga et al., in press). 
METHODS 
Dissociated neocortical cells were prepared from 16- or 17-day-old Sprague- 
Dawley rats using methods previously described by Hisanaga and Sharp 
(1990). Brains were removed and neocortex was dissected in LI 5 medium. 
Following incubation for 40 minutes at 37 °C in 10 mL phosphate-buffered 
saline (PBS) containing 100 U papain, 0.02 percent DNAase, 25 mM glucose, 
and 1 mM cysteine, the tissue was rinsed in 30 percent heat-inactivated horse 
serum and triturated with a plastic pipette in 8 mL of medium. Cells were 
centrifuged at 600 g for 5 minutes. Following aspiration of medium, cells were 
suspended in minimal essential medium (MEM) containing 0.5 mM glutamine, 
20 mM glucose, 100 U/mL penicillin, 100 ng/mL streptomycin, and 10 percent 
fetal calf serum. The cells were passed through an 80 urn mesh, counted, and 
plated on poly-D-lysine (20 ng/mL)-coated plates at a density of 1x10 s cells per 
cm 2 . Cells were maintained at 37 °C in 5 percent COg/95 percent air. The 
medium was changed to serum-free MEM or serum- and magnesium-free MEM 
4 to 5 hours following seeding. Cultured neurons were studied 3 days after 
seeding. 
Cultured cortical neurons were treated with (1) glutamate (10 nM), (2) zinc 
chloride (100 ^iM), (3) carbachol (1,10, 50 mM), (4) dibutyryl-cyclic AMP (db- 
cAMP) (1 mM), (5) 12-O-tetra-decanoyiphorbol- 13-acetate (TPA) (50 nM), (6) 
potassium chloride (46 mM), (7) vasoactive intestinal polypeptide (V IP) (1 piM), 
(8) somatostatin (1 ^M), (9) neuropeptide Y (NPY) (1 ^M), (10) basic fibroblast 
growth factor (bFGF) (.1 ng/mL), (11) nerve growth factor (NGF) (5 ng/mL), (12) 
EGF (20 ng/mL), and (13) platelet-derived growth factor (PDGF) (1 U/mL). 
NMDA receptor blockers (MK801, 0.1 |xM; CPP, 10 ^M; and APH, 30 to 100 
nM) were added 15 minutes prior to each stimulant. 
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