A novel mouse model of Niemann-Pick type C disease carrying a D1005G-Npc1 mutation comparable to commonly observed human mutations.

We have identified a point mutation in Npc1 that creates a novel mouse model (Npc1(nmf164)) of Niemann-Pick type C1 (NPC) disease: a single nucleotide change (A to G at cDNA bp 3163) that results in an aspartate to glycine change at position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the NPC1 protein and is highly similar to commonly occurring human mutations. Genetic and molecular biological analyses, including sequencing the Npc1(spm) allele and identifying a truncating mutation, confirm that the mutation in Npc1(nmf164) mice is distinct from those in other existing mouse models of NPC disease (Npc1(nih), Npc1(spm)). Analyses of lifespan, body and spleen weight, gait and other motor activities, as well as acoustic startle responses all reveal a more slowly developing phenotype in Npc1(nmf164) mutant mice than in mice with the null mutations (Npc1(nih), Npc1(spm)). Although Npc1 mRNA levels appear relatively normal, Npc1(nmf164) brain and liver display dramatic reductions in Npc1 protein, as well as abnormal cholesterol metabolism and altered glycolipid expression. Furthermore, histological analyses of liver, spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol accumulation, glial activation and Purkinje cell loss at a slower rate than in the Npc1(nih) mouse model. Magnetic resonance imaging studies also reveal significantly less demyelination/dysmyelination than in the null alleles. Thus, although prior mouse models may correspond to the severe infantile onset forms of NPC disease, Npc1(nmf164) mice offer many advantages as a model for the late-onset, more slowly progressing forms of NPC disease that comprise the large majority of human cases.

Sex and exercise interact to alter the expression of anabolic androgenic steroid-induced anxiety-like behaviors in the mouse.

Anabolic androgenic steroids (AAS) are taken by both sexes to enhance athletic performance and body image, nearly always in conjunction with an exercise regime. Although taken to improve physical attributes, chronic AAS use can promote negative behavior, including anxiety. Few studies have directly compared the impact of AAS use in males versus females or assessed the interaction of exercise and AAS. We show that AAS increase anxiety-like behaviors in female but not male mice and that voluntary exercise accentuates these sex-specific differences. We also show that levels of the anxiogenic peptide corticotrophin releasing factor (CRF) are significantly greater in males, but that AAS selectively increase CRF levels in females, thus abrogating this sex-specific difference. Exercise did not ameliorate AAS-induced anxiety or alter CRF levels in females. Exercise was anxiolytic in males, but this behavioral outcome did not correlate with CRF levels. Brain-derived neurotrophic factor (BDNF) has also been implicated in the expression of anxiety. As with CRF, levels of hippocampal BDNF mRNA were significantly greater in males than females. AAS and exercise were without effect on BDNF mRNA in females. In males, anxiolytic effects of exercise correlated with increased BDNF mRNA, however AAS-induced changes in BDNF mRNA and anxiety did not. In sum, we find that AAS elicit sex-specific differences in anxiety and that voluntary exercise accentuates these differences. In addition, our data suggest that these behavioral outcomes may reflect convergent actions of AAS and exercise on a sexually differentiated CRF signaling system within the extended amygdala.

The Buzz about anabolic androgenic steroids: electrophysiological effects in excitable tissues.

Anabolic androgenic steroids (AAS) comprise a large and growing class of synthetic androgens used clinically to promote tissue-building in individuals suffering from genetic disorders, injuries, and diseases. Despite these beneficial therapeutic applications, the predominant use of AAS is illicit: these steroids are self-administered to promote athletic performance and body image. Hand in hand with the desired anabolic actions of the AAS are untoward effects on the brain and behavior. While the signaling routes by which the AAS impose both beneficial and harmful actions may be quite diverse, key endpoints are likely to include ligand-gated and voltage-dependent ion channels that govern the activity of electrically excitable tissues. Here, we review the known effects of AAS on molecular targets that play critical roles in controlling electrical activity, with a specific focus on the effects of AAS on neurotransmission mediated by GABA(A) receptors in the central nervous system.

Altered GABAA receptor-mediated synaptic transmission disrupts the firing of gonadotropin-releasing hormone neurons in male mice under conditions that mimic steroid abuse.

Gonadotropin-releasing hormone (GnRH) neurons are the central regulators of reproduction. GABAergic transmission plays a critical role in pubertal activation of pulsatile GnRH secretion. Self-administration of excessive doses of anabolic androgenic steroids (AAS) disrupts reproductive function and may have critical repercussions for pubertal onset in adolescent users. Here, we demonstrate that chronic treatment of adolescent male mice with the AAS 17alpha-methyltestosterone significantly decreased action potential frequency in GnRH neurons, reduced the serum gonadotropin levels, and decreased testes mass. AAS treatment did not induce significant changes in GABAA receptor subunit mRNA levels or alter the amplitude or decay kinetics of GABAA receptor-mediated spontaneous postsynaptic currents (sPSCs) or tonic currents in GnRH neurons. However, AAS treatment significantly increased action potential frequency in neighboring medial preoptic area (mPOA) neurons and GABAA receptor-mediated sPSC frequency in GnRH neurons. In addition, physical isolation of the more lateral aspects of the mPOA from the medially localized GnRH neurons abrogated the AAS-induced increase in GABAA receptor-mediated sPSC frequency and the decrease in action potential firing in the GnRH cells. Our results indicate that AAS act predominantly on steroid-sensitive presynaptic neurons within the mPOA to impart significant increases in GABAA receptor-mediated inhibitory tone onto downstream GnRH neurons, resulting in diminished activity of these pivotal mediators of reproductive function. These AAS-induced changes in central GABAergic circuits of the forebrain may significantly contribute to the disruptive actions of these drugs on pubertal maturation and the development of reproductive competence in male steroid abusers.

Chronic exposure to anabolic androgenic steroids alters neuronal function in the mammalian forebrain via androgen receptor- and estrogen receptor-mediated mechanisms.

Anabolic androgenic steroids (AAS) can promote detrimental effects on social behaviors for which GABA type A (GABA(A)) receptor-mediated circuits in the forebrain play a critical role. While all AAS bind to androgen receptors (AR), they may also be aromatized to estrogens and thus potentially impart effects via estrogen receptors (ER). Chronic exposure of wild-type male mice to a combination of chemically distinct AAS increased action potential (AP) frequency, selective GABA(A) receptor subunit mRNAs, and GABAergic synaptic current decay in the medial preoptic area (mPOA). Experiments performed with pharmacological agents and in AR-deficient Tfm mutant mice suggest that the AAS-dependent enhancement of GABAergic transmission in wild-type mice is AR-mediated. In AR-deficient mice, the AAS elicited dramatically different effects, decreasing AP frequency, spontaneous IPSC amplitude and frequency and the expression of selective GABA(A) receptor subunit mRNAs. Surprisingly, in the absence of AR signaling, the data indicate that the AAS do not act as ER agonists, but rather suggest a novel in vivo action in which the AAS inhibit aromatase and impair endogenous ER signaling. These results show that the AAS have the capacity to alter neuronal function in the forebrain via multiple steroid signaling mechanisms and suggest that effects of these steroids in the brain will depend not only on the balance of AR- versus ER-mediated regulation for different target genes, but also on the ability of these drugs to alter steroid metabolism and thus the endogenous steroid milieu.

The endocrine-disrupting compound, nonylphenol, inhibits neurotrophin-dependent neurite outgrowth.

Endocrine-disrupting compounds (EDCs) may interfere with neuronal development due to high levels of accumulation in biological tissue and potentially aberrant steroid signaling. Treatment of dissociated embryonic Xenopus spinal cord neurons with the EDC, nonylphenol (NP), did not alter cell survival or neurite outgrowth but inhibited neurotrophin-induced neurite outgrowth, effects that were recapitulated by treatment with comparable concentrations of 17 beta-estradiol (E2) and beta-estradiol 6-(O-carboxy-methyl)oxime: BSA (E2-BSA), but not a synthetic androgen. Effects of NP were not inhibited by the nuclear estrogen receptor antagonist, ICI 182,780, but were inhibited by the G protein antagonist, pertussis toxin. Nerve growth factor (NGF)-induced neurite outgrowth in Xenopus neurons was shown to require MAPK signaling. NP did not affect TrkA expression, MAPK signaling, or phosphatidylinositol 3' kinase-Akt-glycogen synthase kinase 3 beta (PI3K-Akt-GSK3 beta) signaling in Xenopus. The ability of NP to inhibit NGF-induced neurite outgrowth without altering survival was recapitulated in the rat pheochromocytoma (PC12) cell line. As with Xenopus neurons, the inhibitory actions of NP in PC12 cells were not antagonized by ICI 182,780 and did not involve alterations in signaling along either the MAPK or PI3K-Akt-GSK3 beta pathways. NP did significantly inhibit the ability of NGF to increase protein kinase A activity in this cell line. These data have important implications with respect to potentially deleterious effects of NP exposure during early neural development and highlight the fact that bioaccumulation of EDCs, such as NP, may elicit very disparate effects along divergent signaling pathways than those that arise from the actions of physiological levels of endogenous estrogens.

Sex- and age-specific effects of anabolic androgenic steroids on reproductive behaviors and on GABAergic transmission in neuroendocrine control regions.

Illicit use of anabolic androgenic steroids (AAS) has become a prevalent health concern not only among male professional athletes, but, disturbingly, among a growing number of women and adolescent girls. Despite the increasing use of AAS among women and adolescents, few studies have focused on the effects of these steroids in females, and female adolescent subjects are particularly underrepresented. Among the hallmarks of AAS abuse are changes in reproductive behaviors. Here, we discuss work from our laboratories on the actions of AAS on the onset of puberty and sexual behaviors in female rodents, AAS interactions and sex- and age-specific effects of these steroids on neural transmission mediated by gamma-aminobutyric acid receptors within forebrain neuroendocrine control regions that may underlie AAS-induced changes in these behaviors.

Environmental estrogens alter early development in Xenopus laevis.

A growing number of environmental toxicants found in pesticides, herbicides, and industrial solvents are believed to have deleterious effects on development by disrupting hormone-sensitive processes. We exposed Xenopus laevis embryos at early gastrula to the commonly encountered environmental estrogens nonylphenol, octylphenol, and methoxychlor, the antiandrogen, p,p-DDE, or the synthetic androgen, 17 alpha-methyltestosterone at concentrations ranging from 10 nM to 10 microM and examined them at tailbud stages (approximately 48 hr of treatment). Exposure to the three environmental estrogens, as well as to the natural estrogen 17 beta-estradiol, increased mortality, induced morphologic deformations, increased apoptosis, and altered the deposition and differentiation of neural crest-derived melanocytes in tailbud stage embryos. Although neural crest-derived melanocytes were markedly altered in embryos treated with estrogenic toxicants, expression of the early neural crest maker Xslug, a factor that regulates both the induction and subsequent migration of neural crest cells, was not affected, suggesting that the disruption induced by these compounds with respect to melanocyte development may occur at later stages of their differentiation. Co-incubation of embryos with the pure antiestrogen ICI 182,780 blocked the ability of nonylphenol to induce abnormalities in body shape and in melanocyte differentiation but did not block the effects of methoxychlor. Our data indicate not only that acute exposure to these environmental estrogens induces deleterious effects on early vertebrate development but also that different environmental estrogens may alter the fate of a specific cell type via different mechanisms. Finally, our data suggest that the differentiation of neural crest-derived melanocytes may be particularly sensitive to the disruptive actions of these ubiquitous chemical contaminants.

Chronic exposure to anabolic androgenic steroids alters activity and synaptic function in neuroendocrine control regions of the female mouse.

Disruption of reproductive function is a hallmark of abuse of anabolic androgenic steroids (AAS) in female subjects. To understand the central actions of AAS, patch clamp recordings were made in estrous, diestrous and AAS-treated mice from gonadotropin releasing hormone (GnRH) neurons, neurons in the medial preoptic area (mPOA) and neurons in the anteroventroperiventricular nucleus (AVPV); regions known to provide GABAergic and kisspeptin inputs to the GnRH cells. Action potential (AP) frequency was significantly higher in GnRH neurons of estrous mice than in AAS-treated or diestrous animals. No significant differences in AAS-treated, estrous or diestrous mice were evident in the amplitude or kinetics of spontaneous postsynaptic currents (sPSCs), miniature PSCs or tonic currents mediated by GABA(A) receptors or in GABA(A) receptor subunit expression in GnRH neurons. In contrast, the frequency of GABA(A) receptor-mediated sPSCs in GnRH neurons showed an inverse correlation with AP frequency across the three hormonal states. Surprisingly, AP activity in the medial preoptic area (mPOA), a likely source of GABAergic afferents to GnRH cells, did not vary in concert with the sPSCs in the GnRH neurons. Furthermore, pharmacological blockade of GABA(A) receptors did not alter the pattern in which there was lower AP frequency in GnRH neurons of AAS-treated and diestrous versus estrous mice. These data suggest that AAS do not impose their effects either directly on GnRH neurons or on putative GABAergic afferents in the mPOA. AP activity recorded from neurons in kisspeptin-rich regions of the AVPV and the expression of kisspeptin mRNA and peptide did vary coordinately with AP activity in GnRH neurons. Our data demonstrate that AAS treatment imposes a "diestrous-like" pattern of activity in GnRH neurons and suggest that this effect may arise from suppression of presynaptic kisspeptin-mediated excitatory drive arising from the AVPV. The actions of AAS on neuroendocrine regulatory circuits may contribute the disruption of reproductive function observed in steroid abuse.

Chronic anabolic androgenic steroid exposure alters corticotropin releasing factor expression and anxiety-like behaviors in the female mouse.

In the past several decades, the therapeutic use of anabolic androgenic steroids (AAS) has been overshadowed by illicit use of these drugs by elite athletes and a growing number of adolescents to enhance performance and body image. As with adults, AAS use by adolescents is associated with a range of behavioral effects, including increased anxiety and altered responses to stress. It has been suggested that adolescents, especially adolescent females, may be particularly susceptible to the effects of these steroids, but few experiments in animal models have been performed to test this assertion. Here we show that chronic exposure of adolescent female mice to a mixture of three commonly abused AAS (testosterone cypionate, nandrolone decanoate and methandrostenolone; 7.5 mg/kg/day for 5 days) significantly enhanced anxiety-like behavior as assessed by the acoustic startle response (ASR), but did not augment the fear-potentiated startle response (FPS) or alter sensorimotor gating as assessed by prepulse inhibition of the acoustic startle response (PPI). AAS treatment also significantly increased the levels of corticotropin releasing factor (CRF) mRNA and somal-associated CRF immunoreactivity in the central nucleus of the amygdala (CeA), as well as neuropil-associated immunoreactivity in the dorsal aspect of the anterolateral division of the bed nucleus of the stria terminalis (dBnST). AAS treatment did not alter CRF receptor 1 or 2 mRNA in either the CeA or the dBnST; CRF immunoreactivity in the ventral BnST, the paraventricular nucleus (PVN) or the median eminence (ME); or peripheral levels of corticosterone. These results suggest that chronic AAS treatment of adolescent female mice may enhance generalized anxiety, but not sensorimotor gating or learned fear, via a mechanism that involves increased CRF-mediated signaling from CeA neurons projecting to the dBnST.

Multiple endocrine neoplasia 2A due to a unique C609S RET mutation presents with pheochromocytoma and reduced penetrance of medullary thyroid carcinoma.

OBJECTIVE: We have identified a large kindred with multiple endocrine neoplasia 2A (MEN 2A) due to a mutation at RET codon 609 that results in a cysteine to serine substitution, a mutation previously identified in only one case in the literature. We characterized the clinical phenotype of the kindred and the biochemical mechanism of this new mutation. PATIENTS AND DESIGN: The index case, a 42-year-old woman, presented with pheochromocytoma. We screened 29 family members for the presence of the mutation. Of the 15 mutation-positive family members, 11 agreed to undergo further evaluation by physical examination, calcium and pentagastrin-stimulated calcitonin levels, measurement of urinary metanephrines, adrenal imaging and serum calcium levels. Biochemical characterization of the mutation was by transient transfection of human neuroblastoma cells and Western blot analysis. RESULTS: This kindred demonstrated an inheritance pattern consistent with autosomal dominant pheochromocytoma. Strikingly, no clinically evident case of medullary thyroid cancer (MTC) was observed among mutation-positive family members. Thyroidectomy in six cases revealed C-cell hyperplasia in all and microscopic MTC in two cases. Transfection experiments using human neuroblastoma cells showed that the mutant RET, unlike the wild-type receptor, is constitutively phosphorylated in the absence of ligand, and thus resembles other previously characterized MEN 2A mutations. CONCLUSIONS: The identification of a new mutation causing a MEN 2A phenotype that features pheochromocytoma and the surprising absence of clinically apparent MTC has significant implications for carriers of this mutation and provides further insights into the genotype-phenotype correlation in MEN 2A.

Adenoviral-mediated expression of functional Na+ channel beta1 subunits tagged with a yellow fluorescent protein.

Voltage-gated sodium (Na(+)) channels typically contain a pore-forming alpha subunit and one or two auxiliary beta subunits. Although initial characterization of known alpha and beta subunits has been facilitated by expression in heterologous cells, to understand fully the differences between individual subunits and the functional consequences of selective subunit expression, there is a need to acutely manipulate expression in cells that endogenously express Na(+) channels. To this end, we have constructed a recombinant adenovirus containing a cDNA for a mouse Na(+) channel beta1 subunit with a yellow fluorescent protein fused to its C-terminus (Ad-beta1-EYFP), and with fluorescence microscopy detected beta1-EYFP expression in primary cerebellar neurons and Chinese hamster ovary (CHO) cells upon transduction with this adenovirus, including expression in the plasma membrane. Consistent with this, patch clamp recordings confirmed that Na(+) currents in CHO cells expressing mouse Na(v)1.4 alpha subunits were appropriately modified by the viral-mediated expression of beta1-EYFP subunits. The results demonstrate that adenoviral-mediated gene delivery can be used effectively to express epitope-tagged Na(+) channel subunits with properties similar to wild-type subunits, and suggest that Ad-beta1-EYFP will be a useful reagent for investigating Na(+) channels in a variety of excitable cell types, including neurons.

The display of sexual behaviors by female rats administered ICI 182,780.

ICI 182,780 (ICI) is a pure antiestrogen that when administered systemically does not cross the blood-brain barrier, thus its actions are limited to the periphery. Four experiments were conducted to test the effects of ICI on the display of sexual behaviors in ovariectomized rats. Experiment 1 examined the effects of three doses of ICI (250, 500, and 750 micro g/rat) on sexual receptivity and paced mating behavior in rats primed with estradiol benzoate (EB) in combination with progesterone (P). Experiments 2 and 3 compared the display of sexual behaviors in rats primed with EB+P or EB alone and administered either 250 micro g ICI (Experiment 2) or 500 micro g ICI (Experiment 3). Experiment 4 tested the effects of ICI (250 and 500 micro g) on the expression of estrogen-induced progestin receptors in the uterus. ICI did not affect the display of sexual receptivity in any experiment. In rats primed with EB+P, paced mating behavior was altered by the 500 and 750 micro g, but not the 250 micro g, doses of ICI. The lowest (250 micro g) dose of ICI did alter paced mating behavior in rats primed with EB alone. The effects of ICI on paced mating behavior were manifested by a substantial lengthening of contact-return latencies following intromissions and ejaculations. The percentage of exits were not affected by ICI. Estrogen stimulation of uterine weight and induction of uterine progestin receptors was suppressed by ICI (250 and 500 micro g). ICI effects on paced mating behavior in hormone-primed female rats are likely to reflect antiestrogenic actions in the periphery, including interference with the estrogen induction of progestin receptors.

Single-cell analysis reveals cell-specific patterns of expression of a family of putative voltage-gated sodium channel genes in the leech.

To understand the molecular basis of nervous system function in the leech, Hirudo medicinalis, we have isolated four novel cDNAs encoding putative voltage-gated sodium (Na) channel alpha subunits, and have analyzed the expression of these genes in individual neurons of known function. To begin, degenerate oligonucleotide primers were used in combination with pre-existing cDNA libraries and reverse transcriptase-coupled polymerase chain reactions (RT-PCR). The putative leech Na channel cDNAs (LeNas) exhibit a higher degree of sequence homology to Na channel genes in other species than to voltage-gated calcium or potassium channel genes, including those expressed in leech. All LeNa cDNAs contain sequences corresponding to regions of functional importance in Na channel alpha subunits, including the "S4 region" involved in activation, the "pore loops" responsible for ion selectivity, and the "inactivation loop" between the third and fourth domains, though the latter lacks the highly conserved "IFM" motif critical for mammalian Na channel inactivation. Sequences corresponding to important determinants of tetrodotoxin sensitivity are found in some, but not all, LeNa cDNAs, consistent with prior electrophysiological evidence of Na channel heterogeneity in the leech with respect to this toxin. Subsequently, two different sets of isoform-specific primers and methods of RT-PCR, including a sensitive, fluorescence-based "real time" RT-PCR, were used to analyze LeNa isoform expression in functionally distinct neurons. The results from both approaches were consistent, and not only demonstrated that individual neurons often express more than one LeNa isoform, but also revealed cell-specific patterns of Na channel isoform expression in the leech nervous system.