Excitatory amino acids such as glutamate are critical to the regulation of the hypothalamic-pituitary-gonadal axis. Glutamatergic N
-aspartate (NMDA) and non-NMDA receptors are located throughout the hypothalamus and affect gonadotrophin-releasing hormone (GnRH) release directly, via expression on GnRH neurones, and/or indirectly via expression on neurones regulating GnRH release (1
). The focus of this study is the NMDA receptor, which plays a physiological role in the regulation of the reproductive axis. Administration of NMDA receptor analogues excite, and antagonists inhibit, GnRH release in vitro
and luteinising hormone (LH) release in vivo
). Furthermore, these actions of glutamate on GnRH neurones are diminished with the onset of reproductive ageing (2
), although the mechanisms behind these changes are yet to be identified.
The NMDA receptor is a heteromeric receptor composed of various combinations of the seven identified subunits (NR1, NR2a-d, NR3a-b). The NR1, NR2a and NR2b subunits are abundantly expressed in the hypothalamus in general and on GnRH cell bodies in particular, with evidence suggesting that these three subunits underlie many of the functional responses of GnRH neurones to NMDA receptor modulators (1
). The NR1 subunit is obligatory for a functional channel, and the NR2 subunits contribute to the binding of glutamate and the channel properties of the receptor (8
). In the hypothalamus, the more likely subunit compositions are thought to be NR1/NR2a, NR1/NR2b or NR1/NR2a/NR2b (12
), which differ in their physiological channel properties (13
During ageing, studies in rodents show that there is a loss of GnRH responsiveness to NMDA receptor analogues/antagonists, and this finding may relate to the decrements in GnRH/LH pulsatility and surge generation. Considering the importance of the NMDA receptor composition in determining channel kinetics, it is likely that age-associated changes to the subunit population of these receptors in the hypothalamus are involved in the onset of reproductive ageing in rats. Indeed, measurements of mRNA levels of NMDA receptor subunits in large macro-dissections of the hypothalamus have demonstrated both regional differences and steroid hormone modulation, effects that vary between the subunits (1
). A further study focusing on protein expression of NMDA receptor subunits in the anteroventral periventricular nucleus (AVPV) showed an age-related decline in NR1 levels (14
). The AVPV is an integration centre for somatosensory pathways that converge onto GnRH neurones (15
) and plays a key regulatory role in the proper functioning of the GnRH/LH surge and oestrous cyclicity (16
). Additionally, the AVPV shows high co-expression of oestrogen receptor alpha and NMDA receptors (specifically NR1), suggesting that it is a likely region for the interaction of oestradiol and glutamate signalling (7
). Finally, the AVPV, together with associated periventricular regions referred to collectively as the rostral periventricular area of the third ventricle (RP3V), is strongly implicated as having direct inputs to GnRH neurones that play key roles in the mediation of oestradiol feedback (17
The expression of the NR2b subunit appears to be key to the functionality of the population of hypothalamic NMDA receptors. Previous and ongoing studies in our laboratory demonstrate that the administration of the NR2b selective antagonist ifenprodil results in the upregulation of various parameters of LH pulsatile release in both young and middle-aged rats (19
); it causes a disruption of the steroid-induced LH surge in middle-aged rats (J. A. Maffucci and A. C. Gore, unpublished data); and it differentially affects GnRH mRNA levels in young and middle-aged rats (19
). These data suggest that the NR2b subunit, specifically, plays an important regulatory role in two very important physiological aspects of the female reproductive system (i.e. GnRH/LH pulsatile release and GnRH/LH surge) and is subject to age-associated changes in expression levels.
The present study examined age-associated changes in the expression of the NR2b subunit in the AVPV of young, middle-aged and aged rats. In addition, the effects of steroid hormone treatment were evaluated. Hormones modulate the effects of NMDA receptor analogues/antagonists on the hypothalamic-pituitary-gonadal axis, a response that may differ with ageing, and which in turn may be the result of differences in the NMDA receptor subunit composition. First, changes in NR2b protein expression were quantified by stereology and brightfield microscopy. Second, age- and hormone-associated alterations in those NMDA receptors expressing NR1 and NR2b either singly- or doubly-labelled, were analysed using confocal microscopy.