transaortically first with saline containing 2 percent sodium nitrite and followed 
by 4 percent buffered paraformaldehyde (pH 6.8) containing 2 to 2.5 percent 
acrolein (EM grade). The brains were removed, sunk in 25 percent aqueous 
sucrose solution, and cut into 12 series of 25 urn sections, which were stored 
in cryoprotectant solution (Watson et al. 1986) until immunocytochemical 
procedures for c-Fos and LHRH were initiated. Immunocytochemical staining 
was accomplished with specific antisera for LHRH (LR-1) and antisera 
generated against the amino acids within the N-terminus of c-Fos (Cambridge 
Research Biochemicals sheep anti c-Fos, OA1 1-821 ; Dr. Tom Curran’s anti -alu- 
Fos). Sequential staining of first c-Fos followed by LHRH was performed with 
the ABC “elite” procedure allowing primary antisera concentrations to be 
1 :44, 000-50, 000 for the c-Fos antisera and 1 :1 00,000 for anti-LHRH (when 
double immunoperoxidase methods were used; if immunofluorescence was 
used instead, the concentration of anti-LHRH was increased to 1:30,000). 
The c-Fos staining following visualization of peroxidase activity with a nickel 
diaminobenzidine chromogen appeared blue black; LHRH reactivity was 
revealed with either diaminobenzidine (immunoperoxidase reactions), which 
stained golden brown, or Texas Red (immunofluorescence procedure), which 
fluoroesced red after excitation in the green range. An example of LHRH 
neurons activated during an LH surge stained with the immunofluorescent 
procedure is shown in figure 2. 
On analysis of plasma LH levels, the authors noted that rats whose plasma 
LH levels were high 30 to 60 minutes before the time of sacrifice had greater 
levels of c-Fos expression within their LHRH neurons than did rats whose 
LH levels were low prior to sacrifice (figure 3). Analysis of plasma LH and 
c-Fos immunoreactivity in LHRH neurons revealed a highly significant linear 
relationship (figure 4), indicating that the amplitude of LH secretion reflects the 
number of LHRH neurons activated. Further investigation of the relationship 
between LHRH stimulation and LH secretion was aimed at the use of a model in 
which the amplitude of an LH surge was attenuated by prevention of the actions 
of progesterone. Intact female rats were treated at 12:30 p.m. on the afternoon 
of proestrus with the progesterone antagonist RU 486 (5 mg, subcutaneously 
[SC]) and compared with untreated proestrus rats. In a separate series of 
experiments, castrated female rats treated with estradiol or with progesterone 
provided a second similar model. Rats were ovariectomized and 2 weeks 
later were administered 1 \iq estradiol benzoate subcutaneously (SC) at 9:00 
a.m. Twenty-four hours later a second injection of estradiol benzoate (50 \iq 
SC) was administered (also at 9:00 a.m.) followed by an SC injection of 
progesterone (5 mg) or vehicle at 12:30 p.m. Plasma LH was monitored as 
described above. In animals treated with RU 486 and in castrates receiving 
only estrogen, the blunting of the LH peak was accompanied by a decrease in 
the degree of LHRH c-Fos expression (figure 5) (Lee et al. 1990b). Close 
examination of the LHRH neurons revealed that, in addition to showing fewer 
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