Stellate and pyramidal neurons in goldfish telencephalon respond differently to anoxia and GABA receptor inhibition.

With oxygen deprivation, the mammalian brain undergoes hyper-activity and neuronal death while this does not occur in the anoxia-tolerant goldfish (Carassius auratus). Anoxic survival of the goldfish may rely on neuromodulatory mechanisms to suppress neuronal hyper-excitability. As γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, we decided to investigate its potential role in suppressing the electrical activity of goldfish telencephalic neurons. Utilizing whole-cell patch-clamp recording, we recorded the electrical activities of both excitatory (pyramidal) and inhibitory (stellate) neurons. With anoxia, membrane potential (Vm) depolarized in both cell types from -72.2 mV to -57.7 mV and from -64.5 mV to -46.8 mV in pyramidal and stellate neurons, respectively. While pyramidal cells remained mostly quiescent, action potential frequency (APf) of the stellate neurons increased 68-fold. Furthermore, the GABAA receptor reversal potential (E-GABA) was determined using the gramicidin perforated-patch-clamp method and found to be depolarizing in pyramidal (-53.8 mV) and stellate neurons (-42.1 mV). Although GABA was depolarizing, pyramidal neurons remained quiescent as EGABA was below the action potential threshold (-36 mV pyramidal and -38 mV stellate neurons). Inhibition of GABAA receptors with gabazine reversed the anoxia-mediated response. While GABAB receptor inhibition alone did not affect the anoxic response, co-antagonism of GABAA and GABAB receptors (gabazine and CGP-55848) led to the generation of seizure-like activities in both neuron types. We conclude that with anoxia, Vm depolarizes towards EGABA which increases APf in stellate neurons and decreases APf in pyramidal neurons, and that GABA plays an important role in the anoxia tolerance of goldfish brain.

Ovulation but not milt production is inhibited in fathead minnows (Pimephales promelas) exposed to a reproductively inhibitory pulp mill effluent.

BACKGROUND: A 5-day fathead minnow (FHM) spawning assay is used by industry to monitor pulp mill effluent quality, with some mill effluents capable of completely inhibiting spawning. The purpose of this report is to characterize the effect of an inhibitory effluent on egg and milt production in FHM. METHODS: Eight tanks were treated with an inhibitory effluent while eight were kept with clean water. Each tank contained two males and four females as per the 5-day FHM spawning assay used by industry. Females were stripped of ovulated eggs and males of milt in four effluent-exposed and four control tanks. Eggs oviposited in every tank were also counted and checked for fertilization and data analyzed with 2-way ANOVA. RESULTS: We show that female, but not male, fathead minnow reproductive function is impaired in the 5-day fathead minnow spawning assay used by industry to evaluate pulp mill effluent quality in Canada. Milt production was not changed in the control or exposed males mid-way and at the end of the five day exposure (p > 0.05; n = 8). Total egg production (stripped + oviposited) was impaired (p < 0.05) in fathead minnows exposed to effluent (288 eggs/tank, n = 4 tanks) compared to those in control tanks (753 eggs/tank, n = 4 tanks). CONCLUSIONS: Our results indicate that males are able to detect female signals and prepare appropriately for spawning while in females inhibition of ovulation is occurring somewhere along the hypothalamus-pituitary-gonad reproductive axis. These results suggest female-specific neuroendocrine disruption and provide mechanistic insight into an assay used by industry to assess pulp mill effluent quality.

Estrogen-like effects in male goldfish co-exposed to fluoxetine and 17 alpha-ethinylestradiol.

The antidepressant fluoxetine (FLX) and the synthetic estrogen, 17 alpha-ethinylestradiol (EE2), are present in municipal sewage discharges. To better understand possible interactions between them, male goldfish were exposed to an ethanol control or to nominal concentrations of FLX (0.54 µg/L) and EE2 (5 ng/L) alone and in combination for 14 days. Real-time reverse-transcription polymerase chain reaction was used to assess effects on hepatic gene expression and liquid chromatography tandem mass spectrometry to analyze the plasma proteome. The results showed an increase in estrogen receptor alpha (esr1) and vitellogenin (vtg) gene expression by 1.9-2.4-fold in the FLX and EE2 groups, but this did not reach statistical significance. In contrast, co-exposure up regulated esr1 and vtg gene expression by 5.5- and 5.3-fold, respectively. Fluoxetine and EE2 alone did not affect estrogen receptor beta (esr2), but the co-exposure down regulated esr2 expression by 50%. There was a significant increase in the number of plasma proteins that were related to endocrine system disorders in the FLX and FLX plus EE2 groups. The level of VTG protein was increased in the plasma from goldfish exposed to EE2, FLX, and FLX plus EE2. Our study demonstrates that low concentrations of FLX and EE2 in a simple mixture produce strong estrogen-like effects in the male goldfish.

Rapid modulation of gene expression profiles in the telencephalon of male goldfish following exposure to waterborne sex pheromones.

Sex pheromones rapidly affect endocrine physiology and behaviour, but little is known about their effects on gene expression in the neural tissues that mediate olfactory processing. In this study, we exposed male goldfish for 6h to waterborne 17,20ßP (4.3 nM) and PGF2α (3 nM), the main pre-ovulatory and post-ovulatory pheromones, respectively. Both treatments elevated milt volume (P=0.001). Microarray analysis of male telencephalon following PGF2α treatment identified 71 unique transcripts that were differentially expressed (q<5%; 67 up, 4 down). Functional annotation of these regulated genes indicates that PGF2α pheromone exposure affects diverse biological processes including nervous system functions, energy metabolism, cholesterol/lipoprotein transport, translational regulation, transcription and chromatin remodelling, protein processing, cytoskeletal organization, and signalling. By using real-time RT-PCR, we further validated three candidate genes, ependymin-II, calmodulin-A and aldolase C, which exhibited 3-5-fold increase in expression following PGF2α exposure. Expression levels of some other genes that are thought to be important for reproduction were also determined using real-time RT-PCR. Expression of sGnRH was increased by PGF2α, but not 17,20ßP, whereas cGnRH expression was increased by 17,20ßP but not PGF2α. In contrast, both pheromones increase the expression of glutamate (GluR2a, NR2A) and γ-aminobutyric acid (GABAA γ2) receptor subunit mRNAs. Milt release and rapid modulation of neuronal transcription are part of the response of males to female sex pheromones.

Modulation of epileptiform activity by three subgroups of GABAergic interneurons in mouse somatosensory cortex.

4-Aminopyridine (4-AP) induces ictal-like epileptiform discharges in a variety of brain regions. These events are associated with enhanced inhibitory and excitatory synaptic neurotransmission. The relative contribution of specific subclasses of GABAergic interneurons (INs) to epileptiform activity in the 4-AP model is not well characterized. We have used genetically encoded channelrhodopsin (ChR) and Archaerhodopsin (Arch) expression in parvalbumin (PV), somatostatin (SST) and vasoactive intestinal polypeptide (VIP) INs to investigate the role of interneuron subclasses in 4-AP-induced epileptiform discharges. Whole-cell patch-clamp recordings were obtained from L5 pyramidal cells (PYRs) in somatosensory cortex of 30-to-70-day old mice. In the presence of 100 µM 4-AP, photostimulation of ChR in PV and SST, but not VIP INs, evoked epileptiform discharges similar to spontaneous and electrically evoked events. Light activation of Arch in PV INs was more effective in reducing epileptiform activity compared to SST and VIP INs. Epileptiform discharges were evoked at offset of Arch induced hyperpolarizations in PV and SST interneurons but not VIP INs. PV and SST INs could both initiate and inhibit 4-AP-induced epileptiform activity in L5 PYRs. VIP INs did not contribute significantly to eliciting or inhibiting epileptiform discharges. These results suggest that subclasses of INs contribute differently to the initiation and modulation of epileptiform discharges in cortical networks.

Electrophysiological characterization of male goldfish (Carassius auratus) ventral preoptic area neurons receiving olfactory inputs.

Chemical communication via sex pheromones is critical for successful reproduction but the underlying neural mechanisms are not well-understood. The goldfish is a tractable model because sex pheromones have been well-characterized in this species. We used male goldfish forebrain explants in vitro and performed whole-cell current clamp recordings from single neurons in the ventral preoptic area (vPOA) to characterize their membrane properties and synaptic inputs from the olfactory bulbs (OB). Principle component and cluster analyses based on intrinsic membrane properties of vPOA neurons (N = 107) revealed five (I-V) distinct cell groups. These cells displayed differences in their input resistance (Rinput: I < II < IV < III = V), time constant (TC: I = II < IV < III = V), and threshold current (Ithreshold: I > II = IV > III = V). Evidence from electrical stimulation of the OB and application of receptor antagonists suggests that vPOA neurons receive monosynaptic glutamatergic inputs via the medial olfactory tract, with connectivity varying among neuronal groups [I (24%), II (40%), III (0%), IV (34%), and V (2%)].

Waterborne fluoxetine disrupts the reproductive axis in sexually mature male goldfish, Carassius auratus.

Fluoxetine (FLX) is a pharmaceutical acting as a selective serotonin reuptake inhibitor and is used to treat depression in humans. Fluoxetine and the major active metabolite norfluoxetine (NFLX) are released to aquatic systems via sewage-treatment effluents. They have been found to bioconcentrate in wild fish, raising concerns over potential endocrine disrupting effects. The objective of this study was to determine effects of waterborne FLX, including environmental concentrations, on the reproductive axis in sexually mature male goldfish. We initially cloned the goldfish serotonin transporter to investigate tissue and temporal expression of the serotonin transporter, the FLX target, in order to determine target tissues and sensitive exposure windows. Sexually mature male goldfish, which showed the highest levels of serotonin transporter expression in the neuroendocrine brain, were exposed to FLX at 0.54µg/L and 54µg/L in a 14-d exposure before receiving vehicle or sex pheromone stimulus consisting of either 4.3nM 17,20ß-dihydroxy-4-pregnene-3-one (17,20P) or 3nM prostaglandin F2(α) (PGF2(α)). Reproductive endpoints assessed included gonadosomatic index, milt volume, and blood levels of the sex steroids testosterone and estradiol. Neuroendocrine function was investigated by measuring blood levels of luteinizing hormone, growth hormone, pituitary gene expression of luteinizing hormone, growth hormone and follicle-stimulating hormone and neuroendocrine brain expression of isotocin and vasotocin. To investigate changes at the gonadal level of the reproductive axis, testicular gene expression of the gonadotropin receptors, both the luteinizing hormone receptor and the follicle-stimulating hormone receptor, were measured as well as expression of the growth hormone receptor. To investigate potential impacts on spermatogenesis, testicular gene expression of the spermatogenesis marker vasa was measured and histological samples of testis were analyzed qualitatively. Estrogen indices were measured by expression and activity analysis of gonadal aromatase, as well as liver expression analysis of the estrogenic marker, esr1. After 14d, basal milt volume significantly decreased at 54µg/L FLX while pheromone-stimulated milt volume decreased at 0.54µg/L and 54µg/L FLX. Fluoxetine (54µg/L) inhibited both basal and pheromone-stimulated testosterone levels. Significant concentration-dependent reductions in follicle-stimulating hormone and isotocin expression were observed with FLX in the 17,20P- and PGF2(α)-stimulated groups, respectively. Estradiol levels and expression of esr1 concentration-dependently increased with FLX. This study demonstrates that FLX disrupts reproductive physiology of male fish at environmentally relevant concentrations, and potential mechanisms are discussed.