mRNAs or one mRNA and an immunocytochemically identified marker are 
not yet sufficiently reliable to be easily applied; generally, there is great 
compromise in the quality of either the mRNA label, the immunocytochemical 
marker, or both. Another advantage to localizing the proteins is that their 
appearance is delayed by at least 30 to 45 minutes after the stimulus is 
delivered, unlike the mRNA, which increases within 5 to 10 minutes after 
stimulation. This delay allows the investigator to move or manipulate the 
animal prior to sacrifice without concern that such handling will result in I EG 
expression. The case is well illustrated for studies of light activation. In this 
instance, for study of mRNA changes, animals must be sacrificed under 
safelight conditions, whereas immunocytochemical analysis would allow 
an animal to be anesthetized and brought into the light for perfusion 
without allowing sufficient time for the light stimulus to be translated into 
new IEG proteins. A disadvantage in using IEG products localized by 
immunocytochemistry rather than by in situ hybridization of their mRNA is 
the lack of precise quantitation inherent in the immunocytochemical methods. 
In this chapter, the authors present evidence that, in spite of this problem, 
localization of IEG products with standard immunohistochemical techniques 
permits assessment of relative changes in gene expression. The studies 
presented in this chapter use natural stimuli and document that the expression 
of the lEGs is not only induced by drug treatment or other experimental 
manipulation but also is involved in normal homeostatic function. 
C-FOS AS A MARKER FOR NEURON ACTIVATION: LUTEINIZING 
HORMONE-RELEASING HORMONE NEURONS AS THE MODEL 
Most of the data presented here focus on the authors’ studies of neuroendocrine 
regulation. In particular, study of the regulation of reproductive function has 
been most revealing. The study of neuroendocrine-adenohypophyseal function 
has presented challenges to physiologists in that conventional means of 
assessing neuronal activity are impractical owing to the small size and scattered 
distribution of the neuroendocrine neurons regulating the anterior pituitary. The 
case in point is well illustrated for one neuroendocrine system: the luteinizing 
hormone-releasing hormone (LHRH) (also known as gonadotropin-releasing 
hormone or GnRH) neurons, which stimulate the release of both luteinizing 
hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary. 
LHRH neurons are small, few in number, and widely scattered. The rat brain 
contains only 1,200 LHRH neurons (Wray and Hoffman 1986a, 1986b), 70 
percent of which send their axons to the median eminence to effect LH and FSH 
release (Merchanthaler et al. 1989). The LHRH cells are scattered over the full 
rostral-to-caudal extent of the forebrain and generally do not reside within classic 
cytoarchitectonic boundaries (Hoffman and Gibbs 1982; Silverman et al. 1982). 
To make matters worse, the LHRH somata typically measure only 8 to 10 urn in 
diameter; even in sites where the LHRH neurons are most numerous, they are 
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