Diurnal and stress‐induced intra‐hippocampal corticosterone rise attenuated in 11β‐HSD1‐deficient mice: a microdialysis study in young and aged mice

11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) locally regenerates active glucocorticoids from their inert forms thereby amplifying intracellular levels within target tissues including the brain. We previously showed greater increases in intra‐hippocampal corticosterone (CORT) levels upon Y‐maze testing in aged wild‐type than in 11β‐HSD1^?/? mice coinciding with impaired and intact spatial memory, respectively. Here we examined whether ageing influences 11β‐HSD1 regulation of CORT in the dorsal hippocampus under basal conditions during the diurnal cycle and following stress. Intra‐hippocampal CORT levels measured by in vivo microdialysis in freely behaving wild‐type mice displayed a diurnal variation with peak levels in the evening that were significantly elevated with ageing. In contrast, the diurnal rise in intra‐hippocampal CORT levels was greatly diminished in 11β‐HSD1^?/? mice and there was no rise with ageing; basal intra‐hippocampal CORT levels were similar to wild‐type controls. Furthermore, a short (3 min) swim stress induced a longer lasting increase in intra‐hippocampal CORT levels in wild‐type mice than in 11β‐HSD1^?/? mice despite no genotypic differences in elevation of plasma CORT. These data indicate that 11β‐HSD1 activity contributes substantially to diurnal and stress‐induced increases in hippocampal CORT levels. This contribution is even greater with ageing. Thus, 11β‐HSD1 inhibition may be an attractive target for treating cognitive impairments associated with stress or ageing.     

Forebrain‐Specific Transgene Rescue of 11β‐HSD1 Associates with Impaired Spatial Memory and Reduced Hippocampal Brain‐Derived Neurotrophic Factor mRNA Levels in Aged 11β‐HSD1 Deficient Mice

Mice lacking the intracellular glucocorticoid‐regenerating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) are protected from age‐related spatial memory deficits. 11β‐HSD1 is expressed predominantly in the brain, liver and adipose tissue. Reduced glucocorticoid levels in the brain in the absence of 11β‐HSD1 may underlie the improved memory in aged 11β‐HSD1 deficient mice. However, the improved glucose tolerance, insulin sensitisation and cardioprotective lipid profile associated with reduced peripheral glucocorticoid regeneration may potentially contribute to the cognitive phenotype of aged 11β‐HSD1 deficient mice. In the present study, transgenic mice with forebrain‐specific overexpression of 11β‐HSD1 (Tg) were intercrossed with global 11β‐HSD1 knockout mice (HSD1KO) to examine the influence of forebrain and peripheral 11β‐HSD1 activity on spatial memory in aged mice. Transgene‐mediated delivery of 11β‐HSD1 to the hippocampus and cortex of aged HSD1KO mice reversed the improved spatial memory retention in the Y‐maze but not spatial learning in the watermaze. Brain‐derived neurotrophic factor (BDNF) mRNA levels in the hippocampus of aged HSD1KO mice were increased compared to aged wild‐type mice. Rescue of forebrain 11β‐HSD1 reduced BDNF mRNA in aged HSD1KO mice to levels comparable to aged wild‐type mice. These findings indicate that 11β‐HSD1 regenerated glucocorticoids in the forebrain and decreased levels of BDNF mRNA in the hippocampus play a role in spatial memory deficits in aged wild‐type mice, although 11β‐HSD1 activity in peripheral tissues may also contribute to spatial learning impairments in aged mice.     

Sex‐dependent modulation of age‐related cognitive decline by the L‐type calcium channel gene Cacna1c (Cav1.2)

Increased calcium influx through L‐type voltage‐gated calcium channels has been implicated in the neuronal dysfunction underlying age‐related memory declines. The present study aimed to test the specific role of Cacna1c (which encodes Ca_v1.2) in modulating age‐related memory dysfunction. Short‐term, spatial and contextual/emotional memory was evaluated in young and aged, wild‐type as well as mice with one functional copy of Cacna1c (haploinsufficient), using the novel object recognition, Y‐maze and passive avoidance tasks, respectively. Hippocampal expression of Cacna1c mRNA was measured by quantitative polymerase chain reaction. Ageing was associated with object recognition and contextual/emotional memory deficits, and a significant increase in hippocampal Cacna1c mRNA expression. Cacna1c haploinsufficiency was associated with decreased Cacna1c mRNA expression in both young and old animals. However, haploinsufficient mice did not manifest an age‐related increase in expression of this gene. Behaviourally, Cacna1c haploinsufficiency prevented object recognition deficits during ageing in both male and female mice. A significant correlation between higher Cacna1c levels and decreased object recognition performance was observed in both sexes. Also, a sex‐dependent protective role of decreased Cacna1c levels in contextual/emotional memory loss has been observed, specifically in male mice. These data provide evidence for an association between increased hippocampal Cacna1c expression and age‐related cognitive decline. Additionally, they indicate an interaction between the Cacna1c gene and sex in the modulation of age‐related contextual memory declines.     

Vitamin A deficiency impairs contextual fear memory in rats: Abnormalities in the glucocorticoid pathway

Vitamin A and its active metabolite, retinoic acid (RA), play a key role in the maintenance of cognitive functions in the adult brain. Depletion of RA using the vitamin A deficiency (VAD) model in Wistar rats leads to spatial memory deficits in relation to elevated intrahippocampal basal corticosterone (CORT) levels and increased hippocampal 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) activity. All of these effects are normalised by vitamin A supplementation. However, it is unknown whether vitamin A status also modulates contextual fear conditioning (CFC) in a glucocorticoid‐associated fear memory task dependent on the functional integrity of the hippocampus. In the present study, we investigated the impact of VAD and vitamin A supplementation in adult male rats on fear memory processing, plasma CORT levels, hippocampal retinoid receptors and 11β‐HSD1 expression following a novelty‐induced stress. We also examined whether vitamin A supplementation or a single injection of UE2316, a selective 11β‐HSD1 inhibitor, known to modulate local glucocorticoid levels, had any beneficial effects on contextual fear memory and biochemical parameters in VAD rats. We provide evidence that VAD rats exhibit a decreased fear conditioning response during training with a poor contextual fear memory 24 hours later. These VAD‐induced cognitive impairments are associated with elevated plasma CORT levels under basal conditions, as well as following a stressful event, with saturated CORT release, altered hippocampal retinoid receptors and 11β‐HSD1 expression. Vitamin A supplementation normalises VAD‐induced fear conditioning training deficits and all biochemical effects, although it cannot prevent fear memory deficits. Moreover, a single injection of UE2316 not only impairs contextual fear memory, but also reduces plasma CORT levels, regardless of the vitamin A status and decreases slightly hippocampal 11β‐HSD1 activity in VAD rats following stress. The present study highlights the importance of vitamin A status with respect to modulating fear memory conditioning in relation to plasma CORT levels and hippocampal 11β‐HSD1.     

Microglia express functional 11β‐hydroxysteroid dehydrogenase type 1

Glucocorticoids are potent regulators of inflammation exerting permissive, stimulatory, and suppressive effects. Glucocorticoid access to intracellular receptors is regulated by the activity of two distinct enzymes known as 11β‐hydroxysteroid dehydrogenase (11βHSD) Type 1 and Type 2, which catalyze the activation or deactivation of glucocorticoids. Although expression of these enzymes in major organ systems and their roles in the metabolic effects of glucocorticoids have been described, their role in the inflammatory response has only recently started to be addressed. In this report, we have studied the expression and activity of 11βHSD Type 1 and Type 2 in microglia cells. Microglia, the brain's resident macrophages, initiate and orchestrate CNS inflammatory responses. Importantly, activated microglia are implicated in most neurodegenerative conditions, making them key subjects of study. We found that microglia expressed 11βHSD‐1, but not 11βHSD‐2, both in ex vivo FACS‐sorted adult cells and in vitro primary cultures. 11βHSD‐1 expression was increased in LPS‐activated microglia. Moreover, 11βHSD‐1 catalyzed the metabolic conversion of 11‐dehydro‐corticosterone into corticosterone (CORT), which potently reduced cytokine production in activated microglia. We propose that 11βHSD‐1 may provide microglia with an intrinsic mechanism to autoregulate and inhibit proinflammatory mediator production through CORT formation. © 2010 Wiley‐Liss, Inc.     

Age‐associated changes in hippocampal‐dependent cognition in Diversity Outbred mice

Episodic memory impairment due to aging has been linked to hippocampal dysfunction. Evidence exists for alterations in specific circuits within the hippocampal system that are closely coupled to individual differences in the presence and severity of such memory loss. Here, we used the newly developed Diversity Outbred (DO) mouse that was designed to model the genetic diversity in human populations. Young and aged DO mice were tested in a hippocampal‐dependent water maze task. Young mice showed higher proficiency and more robust memory compared to the overall performance of aged mice. A substantial number of the older mice, however, performed on par with the normative performance of the younger mice. Stereological quantification of somatostatin‐immunoreactive neurons in the dentate hilus showed that high‐performing young and unimpaired aged mice had similar numbers of somatostatin‐positive interneurons, while aged mice that were impaired in the spatial task had significantly fewer such neurons. These data in the DO model tie loss of hilar inhibitory network integrity to age‐related memory impairment, paralleling data in other rodent models. © 2014 Wiley Periodicals, Inc.     

Hypothalamic-Pituitary-Adrenal Axis Abnormalities in Response to Deletion of 11β-HSD1 is Strain-Dependent

Inter‐individual differences in hypothalamic‐pituitary‐adrenal (HPA) axis activity underlie differential vulnerability to neuropsychiatric and metabolic disorders, although the basis of this variation is poorly understood. 11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) has previously been shown to influence HPA axis activity. 129/MF1 mice null for 11β‐HSD1 (129/MF1 HSD1^?/?) have greatly increased adrenal gland size and altered HPA activity, consistent with reduced glucocorticoid negative feedback. On this background, concentrations of plasma corticosterone and adrenocorticotrophic hormone (ACTH) were elevated in unstressed mice, and showed a delayed return to baseline after stress in HSD1‐null mice with reduced sensitivity to exogenous glucocorticoid feedback compared to same‐background genetic controls. In the present study, we report that the genetic background can dramatically alter this pattern. By contrast to HSD1^?/? mice on a 129/MF1 background, HSD1^?/? mice congenic on a C57Bl/6J background have normal basal plasma corticosterone and ACTH concentrations and exhibit normal return to baseline of plasma corticosterone and ACTH concentrations after stress. Furthermore, in contrast to 129/MF1 HSD1^?/? mice, C57Bl/6J HSD1^?/? mice have increased glucocorticoid receptor expression in areas of the brain involved in glucocorticoid negative feedback (hippocampus and paraventricular nucleus), suggesting this may be a compensatory response to normalise feedback control of the HPA axis. In support of this hypothesis, C57Bl/6J HSD1^?/? mice show increased sensitivity to dexamethasone‐mediated suppression of peak corticosterone. Thus, although 11β‐HSD1 appears to contribute to regulation of the HPA axis, the genetic background is crucial in governing the response to (and hence the consequences of) its loss. Similar variations in plasticity may underpin inter‐individual differences in vulnerability to disorders associated with HPA axis dysregulation. They also indicate that 11β‐HSD1 inhibition does not inevitably activate the HPA axis.     

Effect of Oestrogen Receptors on Brain NMDA Receptors of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Mice

Parkinson’s disease (PD) is characterised by the loss of nigrostriatal dopamine (DA) neurones and glutamate overactivity. There is substantial evidence to suggest that oestrogens prevent or delay the disease. 17β‐oestradiol has neuroprotective effects in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of PD and modulates brain NMDA receptors. In MPTP‐lesioned mice, oestrogen receptor (ER)α and ERβ are important in 17β‐oestradiol‐induced neuroprotection. To evaluate the role of ERs in the response of NMDA receptors to lesion, we compared wild‐type (WT) with ER knockout (KO) C57Bl/6 male mice that received 7, 9 or 11 mg/kg of MPTP. These mice were also treated with MPTP (9 mg/kg) and 17β‐oestradiol. [³H]Ro 25‐6981 specific binding autoradiography was used to label NMDA receptors containing NR2B subunits. In the frontal and cingulate cortex and striatum, vehicle‐treated WT mice had higher [³H]Ro 25‐6981 specific binding compared to ERKO mice. Cortical [³H]Ro 25‐6981 specific binding decreased with increasing doses of MPTP in WT and ERKOα but not ERKOβ mice, whereas a dose‐related decrease was only observed in the striatum of WT mice remaining low in ERKOα and ERKOβ mice. No effect of 17β‐oestradiol treatment in intact or MPTP‐lesioned mice of all three genotypes was observed in the cortex, whereas it increased striatal specific binding of intact ERKOβ and MPTP‐lesioned WT mice. Striatal [³H]Ro 25‐6981 specific binding positively correlated with striatal DA concentrations only in WT mice. MPTP and 17β‐oestradiol treatments had more limited effects in the hippocampus. Only in the CA3 and dentate gyrus did vehicle and 17β‐oestradiol‐treated ERKOα mice have higher [³H]Ro 25‐6981 specific binding than WT and ERKOβ mice, whereas MPTP decreased this specific binding only in the CA1, CA2 and CA3 of ERKOα mice. Hence, brain NMDA receptors were affected by the deletion of ERs, which affect the response to MPTP and 17β‐oestradiol treatments with brain region specificity.     

Genome‐wide gene expression profiling in GluR1 knockout mice: key role of the calcium signaling pathway in glutamatergically mediated hippocampal transmission

α‐Amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptors (AMPARs) convey fast synaptic transmission in the CNS and mediate various forms of hippocampal plasticity. Disruption of glutamate receptor type 1 (GluR1), a member of the AMPAR family, causes synaptic alterations and learning/memory deficits in mice. To gain mechanistic insight into the synaptic and behavioral changes associated with GluR1 deletion, hippocampal genome‐wide expression profiling was conducted using groups of GluR1 knockout (KO) mice and their wild‐type littermates. Regulation of 38 genes was found to be altered more than 30% (P < 0.01, n = 8), and seven of these genes were studied with additional quantitative experiments. A large portion of the altered genes encoded molecules involved in calcium signaling, including calcium channel components, calcium‐binding proteins and calcium‐calmodulin‐dependent protein kinase II subunits. At the protein level, we further evaluated some genes in the calcium pathway that were altered in GluR1 KO mice. Protein levels of two key molecules in the calcium pathway – GluR, ionotropic, N‐methyl‐d ‐aspartate‐1 and calcium/calmodulin‐dependent protein kinase II alpha – showed similar changes to those observed in mRNA levels. These findings raise the possibility that calcium signaling and other plasticity molecules may contribute to the hippocampal plasticity and behavioral deficits observed in GluR1 KO mice.     

Dysbindin‐1 loss compromises NMDAR‐dependent synaptic plasticity and contextual fear conditioning

Genetic variants in DTNBP1 encoding the protein dysbindin‐1 have often been associated with schizophrenia and with the cognitive deficits prominent in that disorder. Because impaired function of the hippocampus is thought to play a role in these memory deficits and because NMDAR‐dependent synaptic plasticity in this region is a proposed biological substrate for some hippocampal‐dependent memory functions in schizophrenia, we hypothesized that reduced dysbindin‐1 expression would lead to impairments in NMDAR‐dependent synaptic plasticity and in contextual fear conditioning. Acute slices from male mice carrying 0, 1, or 2 null mutant alleles of the Dtnbp1 gene were prepared, and field recordings from the CA1 striatum radiatum were obtained before and after tetanization of Schaffer collaterals of CA3 pyramidal cells. Mice homozygous for the null mutation in Dtnbp1 exhibited significantly reduced NMDAR‐dependent synaptic potentiation compared to wild type mice, an effect that could be rescued by bath application of the NMDA receptor coagonist glycine (10 μM). Behavioral testing in adult mice revealed deficits in hippocampal memory processes. Homozygous null mice exhibited lower conditional freezing, without a change in the response to shock itself, indicative of a learning and memory deficit. Taken together, these results indicate that a loss of dysbindin‐1 impairs hippocampal plasticity which may, in part, explain the role dysbindin‐1 plays in the cognitive impairments of schizophrenia. © 2013 Wiley Periodicals, Inc.     

Wfs1‐deficient animals have brain‐region‐specific changes of Na+, K+‐ATPase activity and mRNA expression of α1 and β1 subunits

Mutations in the WFS1 gene, which encodes the endoplasmic reticulum (ER) glycoprotein, cause Wolfram syndrome, a disease characterized by juvenile‐onset diabetes mellitus, optic atrophy, deafness, and different psychiatric abnormalities. Loss of neuronal cells and pancreatic β‐cells in Wolfram syndrome patients is probably related to the dysfunction of ER stress regulation, which leads to cell apoptosis. The present study shows that Wfs1‐deficient mice have brain‐region‐specific changes in Na⁺,K⁺‐ATPase activity and in the expression of the α₁ and β₁ subunits. We found a significant (1.6‐fold) increase of Na‐pump activity and β₁ subunit mRNA expression in mice lacking the Wfs1 gene in the temporal lobe compared with their wild‐type littermates. By contrast, exposure of mice to the elevated plus maze (EPM) model of anxiety decreased Na‐pump activity 1.3‐fold in the midbrain and dorsal striatum and 2.0‐fold in the ventral striatum of homozygous animals compared with the nonexposed group. Na‐pump α₁‐subunit mRNA was significantly decreased in the dorsal striatum and midbrain of Wfs1‐deficient homozygous animals compared with wild‐type littermates. In the temporal lobe, an increase in the activity of the Na‐pump is probably related to increased anxiety established in Wfs1‐deficient mice, whereas the blunted dopamine function in the forebrain of Wfs1‐deficient mice may be associated with a decrease of Na‐pump activity in the dorsal and ventral striatum and in the midbrain after exposure to the EPM. © 2014 Wiley Periodicals, Inc.     

α1‐Syntrophin–deficient mice exhibit impaired muscle force recovery after osmotic shock

Introduction: α1‐syntrophin, a member of the dystrophin complex, recruits membrane molecules, including aquaporin‐4, at the sarcolemma. The physiological functions of α1‐syntrophin are poorly understood. Methods: We examined the physiological characteristics of α1‐syntrophin–deficient muscles under osmotic stress conditions to test the possibility that mutant muscles are less tolerant of osmotic shock. Results: Isolated muscle bundles from mutant mice showed markedly reduced force production after hypo‐osmotic shock. In addition, the mutant muscle bundles showed delayed recovery of specific gravity after being exposed to hypo‐osmotic conditions. Two consecutive exercise tests on the treadmill revealed their performance in the second test was significantly lower than for wild‐type mice. Furthermore, mutant mice had higher serum lactate concentrations after treadmill exercise. Conclusions: Although the lack of α1‐syntrophin from the sarcolemma does not lead to muscle degeneration, our results suggest that it may be partly involved in the pathophysiology of dystrophin‐deficient Duchenne muscular dystrophy. Muscle Nerve 49 : 728–735, 2014     

In vivo labelling of hippocampal β‐amyloid in triple‐transgenic mice with a fluorescent acetylcholinesterase inhibitor released from nanoparticles

The drastic loss of cholinergic projection neurons in the basal forebrain is a hallmark of Alzheimer’s disease (AD), and drugs most frequently applied for the treatment of dementia include inhibitors of the acetylcholine‐degrading enzyme acetylcholinesterase (AChE). This protein is known to act as a ligand of β‐amyloid (Aβ) in senile plaques, a further neuropathological sign of AD. Recently, we have shown that the fluorescent, heterodimeric AChE inhibitor PE154 allows for the histochemical staining of cortical Aβ plaques in triple‐transgenic (TTG) mice with age‐dependent β‐amyloidosis and tau hyperphosphorylation, an established animal model for aspects of AD. In the present study, we have primarily demonstrated the targeting of Aβ‐immunopositive plaques with PE154 in vivo for 4 h up to 1 week after injection into the hippocampi of 13–20‐month‐old TTG mice. Numerous plaques, double‐stained for PE154 and Aβ‐immunoreactivity, were revealed by confocal laser‐scanning microscopy. Additionally, PE154 targeted hippocampal Aβ deposits in aged TTG mice after injection of carboxylated polyglycidylmethacrylate nanoparticles delivering the fluorescent marker in vivo. Furthermore, biodegradable core‐shell polystyrene/polybutylcyanoacrylate nanoparticles were found to be suitable, alternative vehicles for PE154 as a useful in vivo label of Aβ. Moreover, we were able to demonstrate that PE154 targeted Aβ, but neither phospho‐tau nor reactive astrocytes surrounding the plaques. In conclusion, nanoparticles appear as versatile carriers of AChE inhibitors and other promising drugs for the treatment of AD.     

The effects of C5aR1 on leukocyte infiltration following pilocarpine‐induced status epilepticus

This study aimed to determine the role C5aR1 plays in mediating immune responses acutely after pilocarpine‐induced status epilepticus (SE), specifically those of brain‐infiltrating leukocytes. Three days following pilocarpine SE, we determined by flow cytometry the brain immune cell phenotypes and measured key proinflammatory and antiinflammatory cytokine expression by infiltrating leukocytes and microglia in C5aR1‐deficient and wild‐type mice. Absence of C5aR1 reduced by 47% the numbers of Ly6G⁺ neutrophils in the brains of No‐SE mice and decreased neutrophil entry after SE to levels found in wild‐type brains that did not undergo SE (No‐SE). Moreover, C5aR1‐deficient mice showed increased interleukin (IL)‐4 expression in infiltrating leukocytes, but not in microglia. Increases in IL‐4 expression in infiltrating leukocytes coupled with decreased neutrophil invasion in C5aR1‐deficient mice after SE is likely to contribute to the reduced neuronal loss previously found in these mice compared to their wild‐type littermates. Although other SE models need to be investigated to substantiate our findings, this study provides further evidence that C5aR1 is an inflammatory mediator and may play a role in epileptogenesis.     

AMPA GluR‐A receptor subunit mediates hippocampal responsiveness in mice exposed to stress

Because stress represents a major precipitating event for psychiatric disorders, it is important to identify molecular mechanisms that may be altered in vulnerable individuals when exposed to stress. Here, we studied GluR‐A^?/? mice, animals with compromised AMPA receptor signaling, and characterized by a schizophrenic as well as depressive phenotype to investigate changes occurring in response to an acute stress. Wild‐type and GluR‐A^?/? mice were exposed to a single immobilization stress and sacrificed immediately after the end of the stress for the analysis of activity regulated genes and of glutamatergic synapse responsiveness. The acute stress produced a marked increase in the hippocampal expression of Arc (activity‐regulated cytoskeletal‐associated protein) in GluR‐A^?/?, but not in wild‐type mice, which was associated with a similar increase of phospho‐CaMKII, a partner in the action of Arc. When looking at the glutamatergic response to stress in wild‐type animals, we found that stress increased GluR‐A phosphorylation on serine831, an effect that was paralleled by a significant increase of the phosphorylation of the main NMDA receptor subunits, that is, NR‐1 and NR‐2B. Conversely, the stress‐induced modulation of NMDA receptor subunits was not observed in GluR‐A^?/? mice. We suggest that enhanced stress responsiveness in GluR‐A^?/? mice may be due, at least in part, to their inability to activate NMDA‐mediated glutamatergic neurotransmission, suggesting that the integrity of AMPA/NMDA receptor function may be important for successful coping under stressful conditions. © 2010 Wiley‐Liss, Inc.     

Neurones in the Preoptic Area of the Male Goldfish are Activated by a Sex Pheromone 17α,20β‐Dihydroxy‐4‐Pregnen‐3‐One

Pheromones are interesting molecules given their ability to evoke changes in the endocrine state and behaviours of animals. In goldfish, a sex pheromone, 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P), which is released by preovulatory females, is known to trigger the elevation of luteinising hormone (LH) levels, as well as reproductive behaviour in males. Interestingly, when 11‐ketotestosterone (11‐KT) is implanted into adult female fish, LH levels increase in response to the pheromone at any time of the day, which is normally a male‐specific response. However, the neural mechanisms underlying the male‐specific information processing of 17,20β‐P and its androgen dependence are yet unknown. In the present study, we focused on the preoptic area (POA), which plays important roles in the regulation of reproduction and reproductive behaviours. We mapped activity in the POA evoked by 17,20β‐P exposure using the immediate‐early gene c‐fos. We found that a population of ventral POA neurones close to kisspeptin2 (kiss2) neurones that appear to have important roles in reproduction was activated by 17,20β‐P exposure, suggesting that these activated neurones are important for the 17,20β‐P response. Next, we investigated the distribution of androgen receptor (ar) in the POA and its relationship with 17,20β‐P‐responsive and kiss2 neurones. We found that ar is widely expressed in the ventral POA, whereas it is only expressed in approximately 10% of 17,20β‐P‐activated neurones. On the other hand, it is expressed in almost 90% of the kiss2 neurones. Taken together, it is possible that ar expressing neurones in the ventral POA, most of which were not labelled by c‐fos in the present study, may at least partly account for androgen effects on responses to primer pheromones; the ar‐positive kiss2 neurones in the ventral POA may be a candidate. These results offer a novel insight into the mechanisms underlying male‐specific information processing of 17,20β‐P in goldfish.     

Investigation of neuromuscular abnormalities in neurotrophin‐3‐deficient mice

Neurotrophin‐3 (NT‐3) is a trophic factor that is essential for the normal development and maintenance of proprioceptive sensory neurons and is widely implicated as an important modulator of synaptic function and development. We have previously found that animals lacking NT‐3 have a number of structural abnormalities in peripheral nerves and skeletal muscles. Here we investigated whether haploinsufficiency‐induced reduction in NT‐3 resulted in impaired neuromuscular performance and synaptic function. Motor nerve terminal function was tested by monitoring the uptake/release of the fluorescent membrane dye FM1‐43 by the electrophysiological examination of synaptic transmission and electron microscopic determination of synaptic vesicle density at the presynaptic active zone. We investigated skeletal muscle form and function by measuring force in response to both nerve‐mediated and direct muscle stimulation and by quantification of fiber number and area from transverse sections. Synaptic transmission was not markedly different between the two groups, although the uptake and release of FM1‐43 were impaired in mature NT‐3‐deficient mice but not in immature mice. The electron microscopic examination of mature nerve terminals showed no genotype‐dependent variation in the number of synaptic vesicles near the active zone. NT‐3^+/? mice had normal soleus muscle fiber numbers but their fibers had smaller cross‐sectional areas and were more densely‐packed than wild‐type littermates. Moreover, the muscles of adult NT‐3‐deficient animals were weaker than those of wild‐type animals to both nerve and direct muscle stimulation. The results indicate that a reduction in NT‐3 availability during development impairs motor nerve terminal maturation and synaptic vesicle recycling and leads to a reduction in muscle fiber diameter.