Abbreviations: Y, young; MA, middle-aged; A, aged; d, days; DBB, diagonal band of broca; EB: estradiol benzoate; 17^-E2, 17-beta estradiol; s.c., subcutaneously; OVLT, organum vasculosum of the lamina terminalis; P, progesterone; POA, preoptic area; SON, supraoptic nucleus; Rch, retrochiasmatic nucleus; IHC, immunohistochemistry.

hypothalamic mRNA. It is noteworthy that a study of mRNA expression in human subjects, both pre- and postmenopausal, also supported on overall increase in GnRH mRNA with age (Rance and Uswandi, 1996), in agreement with the findings of Gore et al. (2000a). However, it should be noted that the nuclei examined by Rubin et al. (1997) are thought to correspond to GnRH neurons involved in maintenance of the GnRH/ LH surge (Blake and Sawyer, 1974). Thus, localized decreases may occur in these nuclei, whereas an overall increase in GnRH gene expression occurs more generally in the aging hypothalamus, suggesting a mechanism of decline specific to the GnRH/LH surge generator.

Changes to GnRH mRNA expression are often, although not always, reflected by changes to protein expression. Immunohistochemistry can be used to detect the specific protein of interest, such as GnRH, or changes in the coexpression of GnRH protein with the immediate early genes c-jun and c-fos, thought to be markers of gene activation (discussed in the next paragraph). Several studies have compared GnRH cell numbers in young, middle-aged, and/or aged animals, the majority of which report no or very slight decreases in the aged hypothalamus (reviewed in Gore, (2001)). There have, however, been reports of age-associated changes in the distribution of GnRH neurons throughout the hypothalamus. In middle-aged animals undergoing the transition to irregularity, a decline of GnRH neurons in the medial septum was seen in irregularly cycling, middle-aged, proestrus females. No differences were seen in other nuclei such as OVLT, POA, or diagonal brand of Broca (Gore, 2001). Taken together with data suggesting specific roles of different subpopulations of GnRH neurons, with more caudal regions associated with LH pulsatility and more rostral with the GnRH/LH surge (Blake and Sawyer, 1974; Soper and Weick, 1980), researchers are beginning to examine these areas for specific age-related changes. This will enhance our understanding of the age-associated neuronal changes driving alterations of pituitary and gonadal output. Localized adjustments to GnRH neuronal populations would support a mechanism by which age-related changes to gonadotropin release and maintenance of pulsatility occur, and may explain the inconsistencies seen in studies reporting changes to hypothalamic GnRH neuronal expression.

Immediate early genes are transcription factors that are commonly used to measure the activity of a particular cell. Previously, c-fos activity has been correlated with GnRH neuronal activity during the preovulatory GnRH/ LH surge in young animals. Studies extending these findings to either intact, or OVX, steroid-treated middle-aged animals reported a decline in the coexpression of c-fos within GnRH neurons compared to young rats (see Table 43.1). Similar results were observed using the immediate early gene c-jun (Rubin, 2000). These findings support a decline in GnRH neuronal activity during the transition to anestrus.

In support of these observed molecular declines in GnRH activity, push-pull perfusion was used to sample the level of neuropeptide released. GnRH levels were lower in OVX, steroid-treated middle-aged versus young animal, and there was an overall decline in pulse frequency as well as amplitude (Rubin, 2000; Table 43.1). This result is in contrast to the report in the monkey showing an age-related increase in pulsatile GnRH release, measured by push-pull perfusion (Gore et al., 2004) as well as studies in humans showing age-related increases in gonadotropins (Gharib et al., 1990). This species difference highlights a shortcoming of the rat model, in which push-pull perfusion measurements may simply lack the sensitivity of that in monkeys. Nevertheless, taken together, these results show age-associated differences in GnRH gene expression, protein levels, activity, and release. Now we must focus on the mechanisms driving these changes.

GnRH neurons are regulated by a number of neurotransmitters, including (but not limited to) GABA, glutamate (GLU), catecholamines, and neuropeptide Y (NPY). Both protein and mRNA expression of receptors for these and other neurotransmitters have been coloca-lized to GnRH neurons (Smith and Jennes, 2001; Wise et al., 2002). Moreover, many of the neuromodulators synapsing onto GnRH neurons are colocalized with a large population of ERs (Smith and Jennes, 2001; Chakraborty et al., 2004). We propose that the modification of these inputs may be responsible for some of the age-associated differences in the HPG axis. Although there has been no indication of changes to synaptic input onto GnRH neurons in intact female aged rats, Romero et al. (1994) noted a decrease in the number of rough endoplasmic reticulum and Golgi apparati, suggestive of a decrease in GnRH biosynthesis. Additionally, long-term OVX (performed at 10 months of age and observed eight months following) resulted in a greater density of synaptic inputs. These data are suggestive of a role for aging, estrogen, and their possible interactions in GnRH synaptic input. These findings suggest that changes in activation of these estrogen sensitive cells may result in the observed changes to synaptic input onto GnRH neurons.

Following is a brief overview of some age-related changes in a few select neurotransmitters known to regulate GnRH neurons (GLU, GABA, catecholamines, and NPY, summarized in Table 43.2). Greater coverage of this subject can be found in (Gore, 2001; Smith and Jennes, 2001; Gore, 2002; Wise et al., 2002).

1. Glutamate: The most abundant excitatory neuro-transmitter in the brain and probably the best-studied neurotransmitter with respect to age-related changes in the regulation of GnRH neurons. Glutamate agonists potentiate, and antagonists attenuate, GnRH/LH release, and this is modulated by estrogen (Gore, 2001; Smith and Jennes, 2001;

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