D. Hough,a M. Bellingham,a I.R. Haraldsen,b M. McLaughlin,c J.E. Robinson,a A.K. Solbakk,d,e,f and N.P. Evansa,⁎
Psychoneuroendocrinology. 2017 Mar; 77: 1–8. doi: 10.1016/j.psyneuen.2016.11.029
Highlights
- •Peripubertal GnRHa impaired long-term spatial memory.
- •This impairment was not reversed after discontinuing GnRHa-treatment.
- •Spatial orientation and learning performance remained unaffected following GnRHa withdrawal.
- •Speed of progression through these spatial tasks was altered after discontinuing GnRHa.
- •GnRH irreversibly alters these cognitive functions during critical window of development.
Keywords: Spatial memory, Hippocampus, GnRH, Puberty, Precocious puberty, Gender identity disorder
Abstract
Chronic gonadotropin-releasing hormone agonist (GnRHa) administration is used where suppression of hypothalamic-pituitary-gonadal axis activity is beneficial, such as steroid-dependent cancers, early onset gender dysphoria, central precocious puberty and as a reversible contraceptive in veterinary medicine. GnRH receptors, however, are expressed outside the reproductive axis, e.g. brain areas such as the hippocampus which is crucial for learning and memory processes. Previous work, using an ovine model, has demonstrated that long-term spatial memory is reduced in adult rams (45 weeks of age), following peripubertal blockade of GnRH signaling (GnRHa: goserelin acetate), and this was independent of the associated loss of gonadal steroid signaling. The current study investigated whether this effect is reversed after discontinuation of GnRHa-treatment. The results demonstrate that peripubertal GnRHa-treatment suppressed reproductive function in rams, which was restored after cessation of GnRHa-treatment at 44 weeks of age, as indicated by similar testes size (relative to body weight) in both GnRHa-Recovery and Control rams at 81 weeks of age. Rams in which GnRHa-treatment was discontinued (GnRHa-Recovery) had comparable spatial maze traverse times to Controls, during spatial orientation and learning assessments at 85 and 99 weeks of age. Former GnRHa-treatment altered how quickly the rams progressed beyond a specific point in the spatial maze at 83 and 99 weeks of age, and the direction of this effect depended on gonadal steroid exposure, i.e. GnRHa-Recovery rams progressed quicker during breeding season and slower during non-breeding season, compared to Controls. The long-term spatial memory performance of GnRHa-Recovery rams remained reduced (P < 0.05, 1.5-fold slower) after discontinuation of GnRHa, compared to Controls. This result suggests that the time at which puberty normally occurs may represent a critical period of hippocampal plasticity. Perturbing normal hippocampal formation in this peripubertal period may also have long lasting effects on other brain areas and aspects of cognitive function.
Keywords: Spatial memory, Hippocampus, GnRH, Puberty, Precocious puberty, Gender identity disorder
1. Introduction
Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide that, following its release from axon terminals at the median eminence, stimulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the pituitary gland. GnRH can also reach the central nervous system (CNS), as GnRH neurones in the hypothalamus can have axons that extend into other regions of the CNS including the limbic system (Silverman et al., 1987). In addition, GnRH can cross the blood-brain barrier, from the median eminence, into the third ventricle cerebrospinal fluid, albeit with low efficiency (Caraty and Skinner, 2008). GnRH receptor expression has been demonstrated at sites within the CNS (Jennes et al., 1997, Albertson et al., 2009, Schang et al., 2011) and a range of peripheral tissues (Hapgood et al., 2005, Skinner et al., 2009). Thus, when GnRH analogs are used therapeutically in human and veterinary medicine, it is also important to consider the effects at these non-reproductive sites.
As GnRH agonists (GnRHa) result in continued receptor stimulation, as opposed to ultradian cyclic changes, theiradministration initially results in an increase in LH and FSH secretion (‘flare-effect’), followed by the down-regulation of GnRH receptor expression in the pituitary gland and suppression of reproductive axis function (Garner, 1994, Chen and Eugster, 2015). GnRHa is typically prescribed when the suppression of the reproductive axis is required, such as steroid-sensitive conditions like prostate cancer, uterine fibroids and endometriosis (Garner, 1994). In children and adolescents, GnRHa can be prescribed for treatment of central precocious puberty (CPP) (Chen and Eugster, 2015) and gender dysphoria (GD) (Hembree et al., 2009) to temporarily halt reproductive development.
Carel et al. (2009) emphasized the need for investigation of the potential psychological effects associated with peripubertal GnRHa-treatment in CPP. Similarly, the potential effects of GnRHa-treatment on cognition during this important developmental period are not well characterized. Wojniusz et al. (2016) recently demonstrated that peripubertal GnRHa increases emotional reactivity (i.e. emotional and behavioral responses to a fearful situation) in girls with CPP, whereas resting heart rate decreased and this effect was more pronounced with longer durations of GnRHa-treatment. Studies, using an ovine model, have also demonstrated that peripubertal GnRHa-treated rams display increased risk-taking behavior (Wojniusz et al., 2011), altered emotional reactivity (Evans et al., 2012) and reduced long-term spatial reference memory (Hough et al., 2016). Physiological changes within the limbic system have also been reported in this ovine model, as peripubertal GnRHa-treatment alters amygdala volume (Nuruddin et al., 2013a) and the expression of hippocampal genes that are involved in endocrine signaling and synaptic plasticity (Nuruddin et al., 2013b). With this growing body of evidence that peripubertal GnRHa-treatment may affect development of cognitive function, there is now a requirement to investigate whether these effects are reversible when GnRHa-treatment is discontinued.
In the current study, we investigated whether effects from peripubertal GnRHa-treatment, persisted in rams following the discontinuation of treatment. Specifically, we investigated whether the previously reported reduction in long-term spatial memory persists, or if effects on spatial orientation and learning emerge later in life, following the discontinuation of peripubertal GnRHa-treatment.
2. Materials and methods
2.1. Animals
All animal procedures were conducted at the University of Glasgow Cochno Farm and Research Centre (55° 55′N) under Home Office Regulations (Project License: 60/4422). The experimental animals used in this study were Scottish Mule, Texel cross males, born from same sex litters between 23 March and 12 April 2013. At birth, lambs were assigned to one of the treatment groups described below. Lambs from twin and triplet pregnancies were allocated to different groups, so that only 1 sibling was represented in each treatment group. Puberty was delayed in the GnRHa-Recovery (GnRHa-Rec, n = 25) lambs by subcutaneous implantation of the GnRHa, goserelin acetate (Zoladex 3.6 mg, kindly donated by Astra Zeneca, Macclesfield, UK), every four weeks from 8 to 44 weeks of age (average age of pubertal onset in male sheep is 10 weeks of age (Wood and Foster, 1998) and are expected to be sexually competent within the first year of life). Control (n = 30) and GnRHa-Rec rams were grazed on pasture, except during behavioral trials, when they were housed indoors with ad libitum access to hay or silage, with supplements as deemed necessary by standard management practices. All animals were euthanized at the end of the study period, when they were approximately 2 years of age.
2.2. Testes development
Approximately every four weeks, morphometric data were collected from all animals, including testes size, to monitor the effectiveness of GnRHa to suppress the reproductive axis. Scrotal length and circumference were measured with a tailor tape measure while sheep were held in a sitting-position. Testes size was calculated from the scrotal length × circumference and normalized to body weight. Testes size data at 28 (first breeding season), 44 (first anestrus), 81 (second breeding season) and 99 (second anestrus) weeks of age are presented, as these measurements were nearest to the dates of spatial maze performance assessment.
2.3. Assessment of spatial orientation and learning
This study used modifications of the assessment techniques and spatial mazes described previously (Hough et al., 2016), as it follows on from this study on the effects of chronic peripubertal GnRHa-treatment (with/without testosterone supplementation) on spatial orientation, learning and memory in rams from 8 to 45 weeks of age. The current study reports analyses of spatial maze performance data following the discontinuation of GnRHa-treatment at 44 weeks of age. Specifically, performance was evaluated at 83 and 95 weeks of age, and compared to that observed at 41 weeks of age (Hough et al., 2016) when the GnRH-Recovery group was still being treated with GnRHa.
2.3.1. Spatial orientation and learning
Sheep were individually assessed in spatial maze Layout 1 of Fig. 1 at 41 weeks of age, and in Layout 2 of Fig. 1 at 83 and 95 weeks of age. A change in maze layout was necessary, as some of the sheep were already familiar with the former layout (used in long-term spatial memory assessment at 45 weeks of age as reported by Hough et al., 2016). Each sheep was given three maze attempts within the same day (each attempt separated by ∼2 h) to traverse the maze and reunite with flock members in the audience pen. Approximately 30 sheep were assessed per day and kept in the audience pen throughout the day with ad libitum access to water and hay. During each maze attempt, a sheep was calmly ushered from the audience pen to the start of the maze. Sheep that failed to complete the maze within a 5 min time limit, were ushered back to the audience pen via the maze entrance so that the correct route remained unknown. On the last attempt of the day, unsuccessful sheep proceeded to the audience pen via the quickest route Spatial orientation was assessed as the performance of sheep in the first spatial maze attempt of the day, at each age. Spatial learning was assessed as the performance of sheep over three maze attempts within the same day, at each age….