Neuronal plasticity and seasonal reproduction in sheep

Abstract

Seasonal reproduction represents a naturally occurring example of functional plasticity in the adult brain as it reflects changes in neuroendocrine pathways controlling gonadotropin-releasing hormone (GnRH) secretion and, in particular, the responsiveness of GnRH neurons to estradiol negative feedback. Structural plasticity within this neural circuitry may, in part, be responsible for seasonal switches in the negative feedback control of GnRH secretion that underlie annual reproductive transitions. We review evidence for structural changes in the circuitry responsible for seasonal inhibition of GnRH secretion in sheep. These include changes in synaptic inputs onto GnRH neurons, as well as onto dopamine neurons in the A15 cell group, a nucleus that plays a key role in estradiol negative feedback. We also present preliminary data suggesting a role for neurotrophins and neurotrophin receptors as an early mechanistic step in the plasticity that accompanies seasonal reproductive transitions in sheep. Finally, we review recent evidence suggesting that kisspeptin cells of the arcuate nucleus constitute a critical intermediary in the control of seasonal reproduction. Although a majority of the data for a role of neuronal plasticity in seasonal reproduction has come from the sheep model, the players and principles are likely to have relevance for reproduction in a wide variety of vertebrates, including humans, and in both health and disease.

Figure 1

Seasonal plasticity in synaptic inputs onto GnRH neurons in the sheep. A: GnRH neurons in the preoptic area receive about half the density of synaptic inputs (synapses/10 µm plasma membrane) during anestrus compared to the breeding season. This change was seen in both ovary-intact (intact) and ovariectomized ewes bearing 1 cm sc E₂ implants (OVX+E). B: Adjacent non-identified neurons in the preoptic area showed no significant changes in their synapses; n=8/group for A and B. C: Thyroidectomy (THX) failed to block the decrease in synaptic density onto GnRH neurons observed in thyroid (Thy)-intact ewes during anestrus; n=8/group. D: Glial-neuronal interactions are important for plasticity of GnRH inputs. Electron micrograph of a GnRH cell from an anestrous ewe demonstrating an axon terminal (at) almost entirely surrounded by a GnRH cell but separated from it by extensions of a thin glial process (gl). Bar = 2 µm. (A, B modified from (Xiong et al., 1997); C: modified from (Adams et al., 2006)).

Figure 2

A: Confocal image (1µm thick) of synapsin-positive contacts (e.g., white arrows) onto a tyrosine hydroxylase (TH)-positive cell in the A15 of an anestrous ewe. Bar = 20 µm. B: Electron micrograph demonstrating synapsin-positive axon terminals (asterisks) contacting an unidentified dendrite (d) in the A15 of an anestrus ewe. Bar = 2 µm. C: Seasonal change in mean number (±SEM) of synapsin-positive terminals onto TH-immunoreactive dendrite and cell bodies, TH-immunoreactive dendrite length, and mean number of dendritic bifurcations of A15 neurons. n=6/anestrous group, n=8/breeding season group. * p<0.05. (modified from (Adams et al., 2006)).

Figure 3

Representative confocal images (1µm thick) of vGlut1 (red, top row) and vGlut2 (red, bottom row) close contacts (e.g., arrows) onto TH-immunoreactive cells (green) in thyroid-intact ewes sacrificed during the breeding season (Thy-intact/BrS) or anestrus (Thy-intact/An), and thyroidectomized ewes sacrificed during anestrus (THX/An). Bar = 20 µm.

Figure 4

Number of close contacts/100µm cell surface (mean ± SEM) of vGlut1-positive (A) and vGlut2-positive (B) terminals onto TH-immunoreactive A15 cells (soma, dendrite, combined total) in thyroid-intact ewes sacrificed during the breeding season (Thy-intact/BrS; black bars; n=6) or anestrus (Thy-intact/An; white bars; n=6), and in thyroidectomized ewes sacrificed during anestrus (THX/An; grey bars, n=6). Mean density of inputs were compared between groups using a one-way ANOVA, followed by post hoc analyses using the Fisher LSD Method. *indicates significant difference from Thy-intact/BrS (p<0.05); # indicates significant difference from THX/An (p<0.05).

Figure 5

Percentage fold change ( mean ± SEM) of neurotrophin and receptor mRNA expression by real-time qPCR in the preoptic area (POA), A15, and arcuate nucleus (ARC) of the hypothalamus for NGF (A), TrkA (B), BDNF (C), and TrkB (D) in thyroid-intact (Thy-intact/BrS, black bars, n=5) and thyroidectomized (THX/BrS, white bars, n=6) ewes; * indicate significant difference from Thy-intact/BrS, p<0.05.

Figure 6

Seasonal changes in kisspeptin cells (A) and their inputs to GnRH neurons (B). A: The number of kisspeptin-immunoreactive neurons in the middle and caudal ARC is decreased during anestrous (* p<.001). Images show examples of sections through the middle ARC immunostained for kisspeptin in OVX+E ewes perfused during either the breeding season or anestrus. Bar = 100 µm. B: The percentage of GnRH cells in the MBH that receive one or more kisspeptin close contact was less in anestrous than in breeding season ewes (* p<.001); GnRH cells in the POA or anterior hypothalamic area (AHA) showed no significant seasonal differences. Images show examples of dual immunoperoxidase stained sections in which close contacts (e.g., arrows) are seen between kisspeptin terminals (blue-black) and GnRH somas (brown). Bar = 15 µm. (A and B modified from (Smith et al., 2008b))

Figure 7

A: Schematic depiction of the neural circuitry in the hypothalamus regulating seasonal control of estradiol (E₂) negative feedback, and sites at which morphological plasticity may contribute to seasonal reproductive transitions. E₂ acts upon ER-a (ER) containing cells in the ventromedial preoptic area (vmPOA) and retrochiasmatic area (RCh); these neurons, in turn, contact A15 dopamine cells, which project caudally to the mediobasal hypothalamus (MBH), containing GnRH cells directly at the level of their terminals in the median eminence, and/or via kisspeptin (Kiss) cells in the arcuate nucleus. Thyroid hormone-dependent seasonal plasticity in this circuit has been demonstrated at the level of (1) synaptic contacts onto A15 neurons, specifically those which are glutamatergic, and (2) the dendritic morphology of dopaminergic A15 neurons. In addition, there are seasonal changes in the number of kisspeptin inputs to GnRH neurons in the MBH (3), and plasticity may exist at the level of kisspeptin cell bodies and their inputs as well. B, C: An example of how seasonal plasticity may underlie reproductive transitions in the ewe. Changes in the length of A15 dendrites (red) and the number of glutamatergic synaptic contacts (blue) onto them, that occur between the breeding season and anestrus, may result in a functional “reconnection” of this circuitry, and underlie the ability of E₂ to inhibit GnRH secretion at this time of year. The latter may be mediated either directly by A15 projections to GnRH neurons or indirectly via arcuate kisspeptin cells. OCh = optic chiasm.