Peripubertal GnRH and testosterone co-treatment leads to increased familiarity preferences in male sheep.

Chronic gonadotropin-releasing hormone agonist (GnRHa) treatment is effective for the medical suppression of the hypothalamic-pituitary-gonadal axis in situations like central precocious puberty and gender dysphoria. However, its administration during the peripubertal period could influence normal brain development and function because GnRH receptors are expressed in brain regions that regulate emotions, cognition, motivation and memory. This study used an ovine model to determine whether chronic peripubertal GnRHa-treatment affected the developmental shift from preference of familiarity to novelty. Experimental groups included Controls and GnRHa-treated rams. To differentiate between effects of altered GnRH signaling and those associated with the loss of sex steroids, a group was also included that received testosterone replacement as well as GnRHa (GnRHa + T). Preference for a novel versus familiar object was assessed during 5-min social isolation at 8, 28 and 46 weeks of age. Approach behavior was measured as interactions with and time spent near the objects, whereas avoidance behavior was measured by time spent in the entrance zone and attempts to escape the arena via the entry point. Emotional reactivity was measured by the number of vocalizations, escape attempts and urinations. As Control and GnRHa-treated rams aged, their approach behaviors showed a shift from preference for familiarity (8 weeks) to novelty (46 weeks). In contrast, relative to the Controls the GnRHa + T rams exhibited more approach behaviors towards both objects, at 28 and 46 weeks of age and preferred familiarity at 46 weeks of age. Vocalisation rate was increased in GnRHa treated rams in late puberty (28 weeks) compared to both Control and GnRHa + T rams but this effect was not seen in young adulthood (46 weeks). These results suggest that the specific suppression of testosterone during a developmental window in late puberty may reduce emotional reactivity and hamper learning a flexible adjustment to environmental change. The results also suggest that disruption of either endogenous testosterone signalling or a synergistic action between GnRH and testosterone signalling, may delay maturation of cognitive processes (e.g. information processing) that affects the motivation of rams to approach and avoid objects.

Spatial memory is impaired by peripubertal GnRH agonist treatment and testosterone replacement in sheep.

Chronic gonadotropin-releasing hormone agonist (GnRHa) is used therapeutically to block activity within the reproductive axis through down-regulation of GnRH receptors within the pituitary gland. GnRH receptors are also expressed in non-reproductive tissues, including areas of the brain such as the hippocampus and amygdala. The impact of long-term GnRHa-treatment on hippocampus-dependent cognitive functions, such as spatial orientation, learning and memory, is not well studied, particularly when treatment encompasses a critical window of development such as puberty. The current study used an ovine model to assess spatial maze performance and memory of rams that were untreated (Controls), had both GnRH and testosterone signaling blocked (GnRHa-treated), or specifically had GnRH signaling blocked (GnRHa-treated with testosterone replacement) during the peripubertal period (8, 27 and 41 weeks of age). The results demonstrate that emotional reactivity during spatial tasks was compromised by the blockade of gonadal steroid signaling, as seen by the restorative effects of testosterone replacement, while traverse times remained unchanged during assessment of spatial orientation and learning. The blockade of GnRH signaling alone was associated with impaired retention of long-term spatial memory and this effect was not restored with the replacement of testosterone signaling. These results indicate that GnRH signaling is involved in the retention and recollection of spatial information, potentially via alterations to spatial reference memory, and that therapeutic medical treatments using chronic GnRHa may have effects on this aspect of cognitive function.

A reduction in long-term spatial memory persists after discontinuation of peripubertal GnRH agonist treatment in sheep.

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.

Timing of Maternal Exposure and Foetal Sex Determine the Effects of Low-level Chemical Mixture Exposure on the Foetal Neuroendocrine System in Sheep.

We have shown that continuous maternal exposure to the complex mixture of environmental chemicals (ECs) found in human biosolids (sewage sludge), disrupts mRNA expression of genes crucial for development and long-term regulation of hypothalamic-pituitary gonadal (HPG) function in sheep. The present study investigated whether exposure to ECs only during preconceptional period or only during pregnancy perturbed key regulatory genes within the hypothalamus and pituitary gland and whether these effects were different from chronic (life-long) exposure to biosolid ECs. The findings demonstrate that the timing and duration of maternal EC exposure influences the subsequent effects on the foetal neuroendocrine system in a sex-specific manner. Maternal exposure prior to conception, or during pregnancy only, altered the expression of key foetal neuroendocrine regulatory systems such as gonadotrophin-releasing hormone and kisspeptin to a greater extent than when maternal exposure was 'life-long'. Furthermore, hypothalamic gene expression was affected to a greater extent in males than in females and, following EC exposure, male foetuses expressed more 'female-like' mRNA levels for some key neuroendocrine genes. This is the first study to show that 'real-life' maternal exposure to low levels of a complex cocktail of chemicals prior to conception can subsequently affect the developing foetal neuroendocrine system. These findings demonstrate that the developing neuroendocrine system is sensitive to EC mixtures in a sex-dimorphic manner likely to predispose to reproductive dysfunction in later life.

Reproduction Symposium: does grazing on biosolids-treated pasture pose a pathophysiological risk associated with increased exposure to endocrine disrupting compounds?

Biosolids (processed human sewage sludge), which contain low individual concentrations of an array of contaminants including heavy metals and organic pollutants such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), and polychlorinated dibenzodioxins/polychlorinated dibenzofurans known to cause physiological disturbances, are increasingly being used as an agricultural fertilizer. This could pose a health threat to both humans and domestic and wild animal species. This review summarizes results of a unique model, used to determine the effects of exposure to mixtures of environmentally relevant concentrations of pollutants, in sheep grazed on biosolids-treated pastures. Pasture treatment results in nonsignificant increases in environmental chemical (EC) concentrations in soil. Whereas EC concentrations were increased in some tissues of both ewes and their fetuses, concentrations were low and variable and deemed to pose little risk to consumer health. Investigation of the effects of gestational EC exposure on fetal development has highlighted a number of issues. The results indicate that gestational EC exposure can adversely affect gonadal development (males and females) and that these effects can impact testicular morphology, ovarian follicle numbers and health, and the transcriptome and proteome in adult animals. In addition, EC exposure can be associated with altered expression of GnRH, GnRH receptors, galanin receptors, and kisspeptin mRNA within the hypothalamus and pituitary gland, gonadotroph populations within the pituitary gland, and regional aberrations in thyroid morphology. In most cases, these anatomical and functional differences do not result in altered peripheral hormone concentrations or reproductive function (e.g., lambing rate), indicating physiological compensation under the conditions tested. Physiological compensation is also suggested from studies that indicate that EC effects may be greater when exposure occurs either before or during gestation compared with EC exposure throughout life. With regard to human and animal health, this body of work questions the concept of safe individual concentration of EC when EC exposure typically occurs as complex mixtures. It suggests that developmental EC exposure may affect many different physiological systems, with some sex-specific differences in EC sensitivity, and that EC effects may be masked under favorable physiological conditions.

Foetal hypothalamic and pituitary expression of gonadotrophin-releasing hormone and galanin systems is disturbed by exposure to sewage sludge chemicals via maternal ingestion.

Animals and humans are chronically exposed to endocrine disrupting chemicals (EDCs) that are ubiquitous in the environment. There are strong circumstantial links between environmental EDC exposure and both declining human/wildlife reproductive health and the increasing incidence of reproductive system abnormalities. The verification of such links, however, is difficult and requires animal models exposed to 'real life', environmentally relevant concentrations/mixtures of environmental contaminants (ECs), particularly in utero, when sensitivity to EC exposure is high. The present study aimed to determine whether the foetal sheep reproductive neuroendocrine axis, particularly gondotrophin-releasing hormone (GnRH) and galaninergic systems, were affected by maternal exposure to a complex mixture of chemicals, applied to pasture, in the form of sewage sludge. Sewage sludge contains high concentrations of a spectrum of EDCs and other pollutants, relative to environmental concentrations, but is frequently recycled to land as a fertiliser. We found that foetuses exposed to the EDC mixture in utero through their mothers had lower GnRH mRNA expression in the hypothalamus and lower GnRH receptor (GnRHR) and galanin receptor (GALR) mRNA expression in the hypothalamus and pituitary gland. Strikingly, this, treatment had no significant effect on maternal GnRH or GnRHR mRNA expression, although GALR mRNA expression within the maternal hypothalamus and pituitary gland was reduced. The present study clearly demonstrates that the developing foetal neuroendocrine axis is sensitive to real-world mixtures of environmental chemicals. Given the important role of GnRH and GnRHR in the regulation of reproductive function, its known role programming role in utero, and the role of galanin in the regulation of many physiological/neuroendocrine systems, in utero changes in the activity of these systems are likely to have long-term consequences in adulthood and represent a novel pathway through which EC mixtures could perturb normal reproductive function.

P2X7-like receptor subunits enhance excitatory synaptic transmission at central synapses by presynaptic mechanisms.

Recent studies demonstrate that P2X7 receptor subunits (P2X7RS) are present at central and peripheral synapses and suggest that P2X7RS can regulate transmitter release. In brainstem slices from 15 to 26 day old pentobarbitone-anesthetized mice, we examined the effect of P2X7RS activation on excitatory postsynaptic currents (EPSCs) recorded from hypoglossal motoneurons using whole-cell patch clamp techniques. After blockade of most P2X receptors with suramin (which is inactive at P2X7RS) and of adenosine receptors with 8-phenyltheophylline (8PT), bath application of the P2X receptor agonist 3'-0-(4-benzoyl)ATP (BzATP) elicited a 40.5+/-16.0% (mean+/-S.E.M., n = 8, P = 0.039) increase in evoked EPSC amplitude and significantly reduced paired pulse facilitation of evoked EPSCs. This response to BzATP (with suramin and 8PT present) was completely blocked by prior application of Brilliant Blue G (200 nM or 2 microM), a P2X7RS antagonist. In contrast, BzATP application with suramin and 8PT present did not alter miniature EPSC frequency or amplitude when action potentials were blocked with tetrodotoxin. These electrophysiological results suggest that P2X7RS activation increases central excitatory transmitter release via presynaptic mechanisms, confirming previous indirect measures of enhanced transmitter release. We suggest that possible presynaptic mechanisms underlying enhancement of evoked transmitter release by P2X7RS activation are modulation of action potential width or an increase in presynaptic terminal excitability, due to subthreshold membrane depolarization which increases the number of terminals releasing transmitter in response to stimulation.

Australian Neuroscience Society--20th Annual Meeting. 30 January-2 February 2000, Melbourne, Australia.

This meeting presented a large array of high quality neuroscience research, some of which held possible relevance for therapeutic development. The potential use of multipotent neural stem cells harvested from adult brain for transplantation and regeneration therapy was highlighted, as was the increasingly central role of the p75 neurotrophin receptor in regulating neuronal cell death. Of particular interest were strategies for the prevention of neuronal death by systemic administration of p75 receptor antisense oligonucleotides. Other significant data included a possible synergy between prostaglandin receptors and opioid receptors in cellular responses, thought to underlie pain perception and opioid analgesia. Groups in Melbourne, Brisbane and Bath, UK, have isolated novel alpha-conotoxins from Conus marine snails, and characterized their effects on neuronal nicotinic acetylcholine receptors (nAChRs), highlighting their subunit specificity and effects on synaptic transmission. While none of these findings are close to effective clinical use as yet, they hold great promise for the future, underlining the necessity for basic research as a starting point for novel therapies.

Adenosinergic modulation of respiratory neurones and hypoxic responses in the anaesthetized cat.

1. The modulatory effects of intracellularly injected adenosine on membrane potential, input resistance and spontaneous or evoked synaptic activity were determined in respiratory neurones of the ventral respiratory group. 2. The membrane potential hyperpolarized and sometimes reached values which were beyond the equilibrium potential of Cl(-)-dependent IPSPs. At the same time, neuronal input resistance decreased. 3. Spontaneous and stimulus-evoked postsynaptic activities were decreased, as were mean respiratory drive potentials. 4. Systemic injection of the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.01-0.05 mg kg-1) resulted in an increase in mean peak phrenic nerve activity when arterial chemoreceptors were denervated. In contrast, phrenic nerve activity decreased when arterial chemoreceptors were left intact. 5. The depressant effect of adenosine on synaptic activity was abolished after systemic DPCPX administration. DPCPX caused an increase in respiratory drive potentials, increased the amplitude of stimulus-evoked IPSPs, and hyperpolarized membrane potential. 6. Administration of DPCPX blocked the early hypoxic depression of stimulus-evoked IPSPs, doubled the delay of onset of hypoxic apnoea and shortened the time necessary for recovery of the respiratory rhythm. 7. The data indicate that adenosine acts on pre- and postsynaptic A1 receptors resulting in postsynaptic membrane hyperpolarization and depression of synaptic transmission. Blockade of A1 receptors increases respiratory activity, indicating that adenosine A1 receptors are tonically activated under control conditions. Further activation contributes to the hypoxic depression of synaptic transmission in the respiratory network.

Adenosine suppresses excitatory glutamatergic inputs to rat hypoglossal motoneurons in vitro.

Short-latency excitatory postsynaptic potentials (EPSPs), evoked by electrical stimulation lateral to the hypoglossal motor nucleus, were recorded from rat hypoglossal motoneurons (HMs) in brainstem slices. EPSPs were markedly suppressed or abolished by kynurenic acid (1 mM), showing that they were glutamatergic. The adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 100 nM) reduced EPSP amplitude to 42% of control, while the agonist 2-chloroadenosine (2-CA, 0.5-50 microM) caused a dose-dependent reduction of the EPSP. The adenosine receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 0.1-1 microM) increased the EPSP amplitude to 124% of control, and blocked EPSP reduction by CCPA or 2-CA. CCPA, 2-CA and DPCPX did not significantly alter HM input resistance or membrane potential. These data indicate that excitatory glutamatergic inputs to rat HMs are modulated by adenosine A1 receptors, most probably at a presynaptic site. This modulation may be especially significant in hypoxic responses of HMs.