Endocrine disruption and health effects of caged mussels, Elliptio complanata, placed downstream from a primary-treated municipal effluent plume for 1 year

Freshwater mussels, Elliptio complanata, were caged in special benthic pens and were immersed at one upstream (Ups) site and two downstream sites (8 and 11 km) of a primary-treated municipal effluent plume for 1 year. The levels of metallothionein-like proteins (MT), lipid peroxidation, protein-free DNA strands and glutathione S-transferase (GST) activity were assayed in digestive gland, gill and gonad tissues to evaluate biological effects and damage. The levels of monoamines (serotonin and dopamine) in nerve ganglia, ATP-dependent transport activity and monoamine oxidase (MAO) activity were also investigated in the homogenates, synaptosomes and mitochondria, respectively. Results showed that significant amounts of sediment accumulated in cages and 82% of mussels survived the yearlong exposure period at the downstream sites. MT-like proteins were induced in all tissues with the following response intensity: gill (3-fold), digestive gland (1.4-fold) and gonad tissues (1.3-fold). Lipid peroxidation decreased (2.5-fold) in digestive gland but increased in gill (1.6-fold) and in gonad tissues (1.5-fold). GST activity was readily increased in digestive gland (2.5-fold), suggesting the presence of organic contaminants in the plume. Levels of protein-free DNA strands did not vary significantly in digestive gland and gill tissues but were significantly reduced in gonad tissues (2.5-fold) relative to the upstream site. In visceral nerve ganglia, both serotonin and ATP-dependent serotonin transport decreased 1.7-fold with a 4-fold increase of 5-hydroxyindole acetate (5-HIAA, a serotonin metabolite) level relative to the upstream site. However, MAO activity was somewhat reduced at downstream sites (0.7- to 0.9-fold of the activity at the upstream site). Dopamine levels were found to be decreased (1.5-fold), but dopamine ATP-dependent transport activity was increased 1.8-fold, suggesting reduced dopaminergic activity. These results indicate that estrogenic chemicals are likely at play, and the increased dopamine and decreased serotonin ATP-dependent transport suggest that the municipal plume was serotonergic for mussels located at the downstream sites. Mussels exposed for 1 year display a complex but characteristic pattern of responses that could lead to harmful health effects including neuroendocrine disruption of reproduction.     

Inflammatory properties of municipal effluents to Elliptio complanata mussels--lack of effects from anti-inflammatory drugs

The purpose of this study was to identify the pharmacological effects of anti-inflammatory drugs in freshwater mussels (Elliptio complanata) exposed to a primary-treated municipal effluent. Mussel specimens were injected with either increasing concentrations of ibuprofen or with a municipal effluent extract, and then left to stand for 24 h at 15 degrees C. They were also exposed to dilutions of a primary-treated effluent for 30 days at 15 degrees C under semi-static conditions. Gill and gonad cylcooxygenase (COX) were then determined after the incubation period. The influence of various drugs found in municipal effluents on serotonin and dopamine synaptosome transport was determined in visceral ganglia. The results show that injections of ibuprofen reduced COX activity nearly 4-fold in gills and 1.4-fold in gonads. However, COX activity was induced in both tissues after 24 h in mussels injected with a municipal effluent extract and after 30 days in those exposed to dilutions of the effluent. Moreover, synaptosomal dopamine transport activity was increased by ibuprofen, aspirin, caffeine and estradiol-17beta (E2), and decreased by loperamide and carbamazepine, suggesting increased and decreased turnover rates of this catecholamine, respectively. Serotonin transport activity was much less affected, decreasing with high doses of loperamide and increasing with ibuprofen, but with less intensity than with dopamine. The results suggest that although ibuprofen can effectively reduce COX activity in gill and gonadal tissues, exposure to both the municipal effluent and its organic extract increased COX activities, indicating the absence of NSAID (non-steroidal anti-inflammatory drugs)-related effects. Besides their known estrogenic and serotonergic properties, municipal effluents appear to elicit a state similar to inflammation in freshwater mussels.     

Neurotoxicological effects of a primary and ozonated treated wastewater on freshwater mussels exposed to an experimental flow-through system

The neurotoxic potential of a primary-treated and ozonated municipal effluent was examined using feral freshwater Elliptio complanata mussels. Specimens were exposed to increasing concentrations (0, 1, 3, 10 and 20% v/v) of a primary-treated effluent before and after treatment with 10 mg/L of ozone in a mesocosm-type experiment for 30 days. A suite of biomarkers was used to assess the potential neurotoxic stress of the wastewaters on these benthic invertebrates: opiate binding sites, gamma-aminobutyric acid (GABA) metabolism, monoamines levels (serotonin, dopamine), monoamine oxidase, acetylcholinesterase and lipid peroxidation. Gametogenic activity was also determined by the gonado-somatic index and by vitellogenin-like proteins. The results show that the number of opiate binding sites increased slightly, especially after ozonation. GABA metabolism was generally reduced, suggesting higher glutamate stimulation than GABA dampening effects in mussel ganglia. This excitatory state was further confirmed by decreased acetylcholinesterase activity in gonadal tissues. The turnover of dopamine was enhanced with increased serotonin levels, but accompanied by reduced catabolism, as evidenced by decreased monoamine oxidase activity. Moreover, oxidative stress was increased, as determined by lipid peroxidation in the gonad (containing ganglia), which was significantly correlated with acetylcholinesterase activity and dopamine metabolism. The gonado-somatic index was significantly reduced with increased levels of vitellogenin-like proteins, again confirming the estrogenic action of these wastewaters. The data suggest that exposure to a primary-treated municipal effluent before and after ozonation leads to an excitotoxic syndrome implicating perturbations in GABA, dopamine and acetylcholine signaling. The increase in dopamine metabolism may be associated with the occurrence of opiate-like compounds (i.e. morphine) in the effluent. In general, ozonation reduced the severity of the responses, indicating that this disinfection strategy does not increase neurotoxicity to mussels.     

Neurochemical effects of benzodiazepine and morphine on freshwater mussels

The purpose of this study was to examine the neurochemical effects of morphine, diazepam, a common benzodiazepine, and an effluent concentrate on the endemic freshwater mussel Elliptio complanata. Mussels were exposed to the drugs and to the solid-phase concentrate of a municipal effluent and left to stand at 15 °C for 48 h. Neurochemical effects were determined by monitoring changes in dopamine, serotonin, glutamate and γ-aminobutyric acid (GABA) levels in the visceral mass (containing the nerve ganglia) of mussels. The activities of acetylcholinesterase (AChE), dopamine and serotonin-dependent adenylyl cyclase (ADC) were also determined in the mussels. Oxidative stress was determined by tracking changes in lipid peroxidation (LPO) in the mitochondrial and post-mitochondrial fractions. The results revealed that the drugs and the effluent extract were biologically active in mussels. Morphine reduced serotonin and increased dopamine in mussel tissues while reducing AChE activity and increasing GABA levels. This suggests the induction of a relaxation state in mussels. Diazepam also reduced serotonin levels but produced no change in dopamine levels. However, dopamine-sensitive ADC activity was readily activated, indicating the potential effect on opiate signaling. Diazepam increased glutamate levels slightly, but AChE remained stable. The increase in both dopamine ADC activity and glutamate concentrations was also associated with greater oxidative stress on the mitochondrial and post-mitochondrial fractions in cells. A comparison of the global response pattern of these drugs with those of the effluent extract revealed only a relative proximity to morphine. In conclusion, the data warrant more studies on the analysis of opiates and benzodiazepines in municipal effluents to better address the potential environmental hazard of these neuroactive drug classes to aquatic organisms.     

Evidence of feminization in wild Elliptio complanata mussels in the receiving waters downstream of a municipal effluent outfall

The endocrine-disrupting activity of municipal effluents has the potential to alter the reproductive system and induce feminization to aquatic organisms. The purpose of this study was to examine the sex ratio, vitellogenin (Vtg)-like proteins, serotonin, arachidonate cyclooxygenase (COX) activity and dopamine status in wild mussels living at sites upstream and downstream of two municipal effluent outfalls in the Mille-Îles River (Quebec, Canada). Gonad integrity was also studied by monitoring the gonado-somatic index (GSI), the activity of the rate-limiting enzyme aspartate transcarbamoylase (ATC) for purine synthesis, and changes in lipid peroxidation (LPO). The results showed that the proportion of females was dramatically increased from 30% at the upstream sites to 80% at the downstream sites. The levels of Vtg-like proteins were significantly elevated in the male mussels only. Male mussels downstream of the municipal effluent plumes expressed female-specific protein bands (Vtg-like), as determined by high-resolution gel electrophoresis and silver staining. The serotonin/dopamine ratio was significantly decreased in the downstream mussels, indicating that the gonad was in a state of early vitellogenesis. However, this change was not accompanied by changes in ATC, suggesting no significant egg production was underway; this was confirmed by the observation that the downstream mussels displayed significantly low GSIs. GSIs were rather dependent on the serotonin/dopamine ratio (r = 0.44; p < 0.001), while Vtg-like proteins were dependent on dopamine levels (r = 0.50; p < 0.001). The increase in COX activity at the downstream sites and its close relationship with increased serotonin levels suggest a concomitant serotonergic signalling in addition to VTG production. The production of Vtg-like proteins combined with the serotonergic effects of the municipal effluents was associated with oxidative damage (LPO) in the gonad. This study provides the first evidence of feminization in wild mussel populations and the disruption in gonad physiology by exposure to municipal effluents.     

Evaluation of estrogenic effects of municipal effluents to the freshwater mussel Elliptio complanata

Municipal effluents are an important source of estrogens to the aquatic environment. The purpose of this study was to examine the estrogenicity of municipal effluents to the indigenous freshwater mussel, Elliptio complanata. First, estradiol-binding sites in gonad homogenates were characterized to determine the binding affinity and specificity of estrogens. Mussels were exposed to increasing concentrations of a municipal effluent for 96 h at 15 degrees C. In another experiment, mussels were placed in cages and submerged for 62 days at 1.5 km upstream and 5 km downstream of a municipal effluent plume in the St. Lawrence River. Mussels were harvested for assessment of vitellogenin-like proteins in the hemolymph and determination of total lipid, carbohydrate and protein in the gonad. The presence of specific estrogen-binding sites was found in both male and female gonads. Binding of estradiol to cytosol proteins reached saturation, yielding a dissociation constant of 0.4 nM. Vitellogenin (Vg) levels increased significantly in both the hemolymph and the gonad after exposure to the effluent. Moreover, females appeared to be more sensitive than males to producing Vg. Mussels exposed in situ to contaminated surface waters had higher levels of Vg at the downstream site, again, females had higher levels of Vg than did males. On the other hand, lipid and sugar levels in male gonads were significantly increased at the downstream site. Moreover, mussels at the downstream site had decreased shell growth length and increased total and soft tissue weights. We conclude that municipal effluents contain bio-available xenoestrogens at levels sufficient to elicit effects in freshwater mussels.     

Alterations in DNA metabolism in Elliptio complanata mussels after exposure to municipal effluents

This study sought to examine the genotoxic potential in Elliptio complanata freshwater mussels exposed to a physically and chemically treated municipal effluent before and after ozone treatment. Mussels were continuously exposed to increasing concentrations of the effluents for 14 days. Genotoxicity was determined by tracking changes in key enzymes for purine and pyrimidine synthesis (dehydrofolate reductase and aspartate transcarbamoylase), catabolism of purines (xanthine oxido-reductase) and DNA strand-break levels as determined by the alkaline precipitation assay. Other biomarkers related to xenobiotic biotransformation (cytochrome P4503A and glutathione S-transferase activities), metal metabolism (labile zinc and redox state of metathioneins) and oxidative stress (superoxide dismutase activity) were also determined in the mussels. The data revealed that dehydrofolate reductase activity was reduced by the initial effluent and increased by the ozonated effluent. Aspartate transcarbamoylase activity was significantly induced only with the ozonated effluent. The levels of DNA strand breaks responded in a biphasic manner in mussels exposed to the physically and chemically treated effluent where an initial decrease was observed at a low effluent concentration (3% v/v) followed by an increase in DNA strand breaks at a higher effluent concentration (20%). This response pattern was lost in the ozonated effluent, where only a decrease in DNA breaks was found. Xanthine oxidoreductase activity was not significantly affected but did correlate significantly with dehydrofolate reductase activity. Multivariate factorial and canonical analyses revealed that oxidative stress and metal/xenobiotic metabolism markers were strongly correlated with DNA strand breaks in mussels, suggesting that the presence of metals (zinc) and planar hydroxylated hydrocarbons present in these effluents were strong contributors to the observed response. We conclude that municipal effluents contain a complex mixture of pollutants that could modulate DNA synthesis and repair mechanisms in mussels.     

Neuromediator mechanisms of the effect of the antihistamine agent dimebone on the brain

Dimebone was shown to inhibit monoamine oxidase (MAO) deaminating dopamine and serotonin, decrease dopamine metabolism in the basal ganglia of the rat brain, increase noradrenaline level and depress dopamine deamination in the hypothalamus. Dimebone first increased and then diminished the release of dopamine in the cortex, with the concomitant MAO activation and the increase in dopamine and noradrenaline levels. The in vitro experiments have demonstrated that dimebone (10(-4)) preferentially inhibited MAO activity, type B and dopamine deamination in homogenates of different rat brain structures. The role of MAO inhibition in the mechanism of dimebone action on the catecholamine metabolism in the brain structures and its stimulating effect on CNS are discussed.     

Survival,Growth and Condition of Freshwater Mussels: Effects of Municipal Wastewater Effluent

Freshwater mussels (Family Unionidae) are among the most imperiled group of organisms in the world, with nearly 65% of North American species considered endangered. Anthropogenic disturbances, including altered flow regimes, habitat alteration, and pollution, are the major driver of this group''s decline. We investigated the effects of tertiary treated municipal wastewater effluent on survivorship, growth, and condition of freshwater mussels in experimental cages in a small Central Texas stream. We tested the effluent effects by measuring basic physical parameters of native three ridge mussels (Amblema plicata) and of non-native Asian clams (Corbicula fluminea), before and after 72-day exposure at four sites above and below a municipal wastewater treatment plant outfall. Survivorship and growth of the non-native Asian clams and growth and condition indices of the native three ridge mussels were significantly higher at the reference site above the outfall than in downstream sites. We attribute this reduction in fitness below the outfall to elevated nutrient and heavy metal concentrations, and the potential presence of other untested-for compounds commonly found in municipal effluent. These results, along with an absence of native mussels below the discharge, indicate a significant negative impact of wastewater effluent on both native and non-native mussels in the stream.     

Disorders of the dynamics of postnatal development of monoamine oxidase activity in the brain as affected by the antenatal action of alcohol

The activity of different types of monoamine oxidase (MAO)-MAO, type A (substrate serotonin) and two types of mixed MAO forms using tyramine or dopamine as substrates, in different brain regions of the rat offspring exposed prenatally to ethanol was investigated on the 30th and 60th day postnatally. The present study has revealed differences in the development of brain MAO activity during ontogenesis. Disturbances in the activity of all MAO types investigated as well as the distortion of their postnatal development have been observed in the brain of the rat offspring exposed prenatally to ethanol. The possible teratogenic effect of ethanol on the developing fetal brain is discussed.     

Decrease of core body temperature in mice by dehydroepiandrosterone

Dietary dehydroepiandrosterone (DHEA) reduces food intake in mice, and this response is under genetic control. Moreover, both food restriction and DHEA can prevent or ameliorate certain diseases and mediate other biological effects. Mice fed DHEA (0.45% w/w of food) and mice pair-fed to these mice (food restricted) for 8 weeks were tested for changes in body temperature. DHEA was more efficient than food restriction alone in causing hypothermia. DHEA injected intraperitoneally also induced hypothermia that reached a nadir at 1 to 2 hr, and slowly recovered by 20 to 24 hr. This effect was dose dependent (0.5-50 mg). Each mouse strain tested (four) was susceptible to this effect, suggesting that the genetics differ for induction of hypophagia and induction of hypothermia. Because serotonin and dopamine can regulate (decrease) body temperature, we treated mice with haloperidol (dopamine receptor antagonist), 5,7-dihydroxytryptamine (serotonin production inhibitor), or ritanserin (serotonin receptor antagonist) prior to injection of DHEA. All of these agents increased rather than decreased the hypothermic effects of DHEA. DHEA metabolites that are proximate (5-androstene-3beta, 17beta-diol and androstenedione) or further downstream (estradiol-17beta) were much less effective than DHEA in inducing hypothermia. However, the DHEA analog, 16alpha-chloroepiandrosterone, was as active as DHEA. Thus, DHEA administered parentally seems to act directly on temperature-regulating sites in the body. These results suggest that DHEA induces hypothermia independent of its ability to cause food restriction, to affect serotonin or dopamine functions, or to act via its downstream steroid metabolites.     

Toxicological effects of primary-treated urban wastewaters, before and after ozone treatment, on freshwater mussels (Elliptio complanata)

This study examined the toxic potential of a primary-treated municipal effluent, before and after ozonation, in freshwater mussels. Animals were exposed to various concentrations (0, 1, 3, 10 and 20% v/v) of a primary-treated effluent and also after a treatment with ozone at 10 mg/L in continuous flow-through mode for seven weeks. A suite of biomarkers was used to assess the potential toxic effects of various contaminants typically present in municipal wastewaters: heavy metal metabolism (metallothioneins and labile zinc), cytochrome P4501A1 and 3A4, glutathione S-transferase activities (biotransformation of organic compounds), lipid peroxidation and xanthine oxidoreductase (oxygen radical scavenging), DNA damage, mitochondrial electron transport activity at various temperatures and gonad lipid levels (cellular energy allocation) and aspartate transcarbamoylase and dihydrofolate reductase (gonad activity). On the one hand, some biomarkers, including metallothioneins, labile zinc, glutathione S-transferase, cytochrome P4503A4 activity, dehydrofolate reductase and aspartate transcarbamoylase, were readily decreased. In contrast, these biomarkers, cytochrome P4501A1, gill lipid peroxidation, DNA strand breaks in gills and digestive gland, mitochondrial electron transport at high and low temperatures (temperature-dependent activity) and total gonad lipids, were readily increased. In general, ozone treatment reduced adverse effects by either decreasing the intensity of the toxic responses or increasing the threshold concentration. For gill lipid peroxidation, however, intensity was greater at a higher threshold concentration. Ozone treatment eliminated the temperature sensitivity of the mitochondrial electron transport system, indicating a loss of interaction between temperature and urban pollution in terms of energy expenditure in mussels. Ozone treatment could significantly decrease either the toxic potency or intensity of urban pollutants at the expense of increased oxidative stress in gills of freshwater mussels.     

Changes in monoamine transmitter concentration in freshwater mussel tissues.

Freshwater mussels were analyzed for biogenic amine transmitter substances in gill tissue, suprabranchial nerve and blood. Gill tissue from normal pondwater-acclimated mussels contained significant amounts of monoamine neurotransmitter substances. In comparison with the suprabranchial nerve the gill tissue contained 42% of the dopamine, 7% of the serotonin and 490% of norepinephrine. Exposing the animals to deionized water (salt-depleted) resulted in a loss of transmitter substances from gill tissue, but serotonin reduction was modest. The mussel gill tissue content of serotonin and the precursor tryptophan was regulated at nearly constant levels. Serotonin is an important transmitter substance in mussels and the many functions it controls, including sodium transport regulation, would depend on its continued presence.     

Methyl parathion induced alterations in monoaminergic system of developing rat pups

Methyl parathion induced alterations in the level of monoamines, viz. norepinephrine, dopamine and serotonin were studied in discrete regions of developing central nervous system of rat pups. A significant decrease in the level of monoamines noticed in methyl parathion toxicosis may be related to the altered neuronal activity and inefficiency, leading to depression and impairment in various behavioural activities. In contrast to AChE inhibition, monoamine oxidase (MAO) activity showed an increasing trend and it could cause deamination of catecholamines and accumulation of its metabolites. This suggests that an increased AChE inhibition may indirectly stimulate MAO activity in developing rat pups exposed to methyl parathion.     

Hormone mediated changes in monoamine stores and regulation of enzymes of biosynthesis and metabolism in the rat adrenal gland. Influence of progesterone, estradiol, ACTH and testosterone administration

The influence of daily injections, for 7 days, of 17-hydroxyprogesterone caproate (8 mg/100 g body weight pro die), of estradiol-17 beta 17-undecylate (1.2 mg/100 g body weight pro die) of testosterone enanthate (2 mg/100 g body weight pro die) and of ACTH (4 IU/100 g body weight pro die) to male Wistar rats, weighing 200 g, were investigated. Monamine storage and regulation of enzymes phenylethanolamine N-methyl transferase (PNMT), monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) were studied in the adrenal glands. Results from testosterone treated rats were especially significant : the hormone administration decreased the stores of dopamine and norepinephrine (epinephrine precursors), stimulated PNMT and decreased metabolism of epinephrine by MAO and COMT. However, the adrenal content of epinephrine remained in the normal range. These findings suggest that uptake of epinephrine is accelerated in peripheral tissues by testosterone.     

On the role of brain serotonin system in the pathway from gene to behaviour

This paper concentrates on involvement of protein elements in the brain neurotransmitter serotonin system (key enzymes in serotonin metabolism and 5-HT(1A) receptors) in the genetic control of behaviour. The data were obtained using Norway rats selected for more that 50 generations for lack of aggressive response and for aggressive behaviour towards humans (fear-induced aggression), inbred mouse strains, and MAO A knockout mice. The review provides converging line of evidence that: 1) brain serotonin contributes to critical mechanism underlying genetically defined individual differences in aggressiveness, and 2) genes encoding pivotal enzymes in serotonin metabolism (tryptophan hydroxylase, MAO A) and 5-HT(1A) receptors belong to a group of genes that modulate aggressive behaviour.     

Comparative neurochemical profile of 3,4-methylenedioxymethamphetamine and its metabolite alpha-methyldopamine on key targets of MDMA neurotoxicity

The neurotoxicity of MDMA or "Ecstasy" in rats is selectively serotonergic, while in mice it is both dopaminergic and serotonergic. MDMA metabolism may play a key role in this neurotoxicity. The function of serotonin and dopamine transporter and the effect of MDMA and its metabolites on them are essential to understand MDMA neurotoxicity. The aim of the present study was to investigate and compare the effects of MDMA and its metabolite alpha-methyldopamine (MeDA) on several molecular targets, mainly the dopamine and serotonin transporter functionality, to provide evidence for the role of this metabolite in the neurotoxicity of MDMA in rodents. MeDA had no affinity for the serotonin transporter but competed with serotonin for its uptake. It had no persistent effects on the functionalism of the serotonin transporter, in contrast to the effect of MDMA. Moreover, MeDA inhibited the uptake of dopamine into the serotonergic terminal and also MAO(B) activity. MeDA inhibited dopamine uptake with a lower IC(50) value than MDMA. After drug washout, the inhibition by MeDA persisted while that of MDMA was significantly reduced. The effect of MDMA on the dopamine transporter is related with dopamine release from vesicular stores, as this inhibition disappeared in reserpine-treated animals. However, the effect of MeDA seems to be a persistent conformational change of this transporter. Moreover, in contrast with MDMA, MeDA did not show affinity for nicotinic receptors, so no effects of MeDA derived from these interactions can be expected. The metabolite reduced cell viability at lower concentrations than MDMA. Apoptosis plays a key role in MDMA induced cellular toxicity but necrosis is the major process involved in MeDA cytotoxicity. We conclude that MeDA could protect against the serotonergic lesion induced by MDMA but potentiate the dopaminergic lesion as a result of the persistent blockade of the dopamine transporter induced this metabolite.     

Cholinergic receptors, ion channels, neurotransmitter synthesis, and neurite outgrowth are independently regulated during the in vitro differentiation of a human neuroblastoma cell line

The differentiation of human nerve cells was investigated using a cell model comprising human neuroblastoma (IMR32) cells that were induced to differentiate by the addition of 5-bromo-2'-deoxyuridine (BrdU) or N6-O2-dibutyryl cyclic adenosine 3'-5' monophosphate (Bt2cAMP). As parameters of differentiation, we studied neurite outgrowth, cholinergic receptors, voltage-activated ion channels, tyrosine hydroxylase activity, and neurotransmitter content. BrdU induced marked morphological differentiation, as indicated by the number and length of neurites, as well as an increase in the number of alpha-bungarotoxin binding sites, muscarinic receptors, and voltage-dependent Na channels. In addition, BrdU induced an increase in tyrosine hydroxylase activity as well as in serotonin, dopamine, and noradrenaline content. Bt2cAMP had a less dramatic effect on the morphological appearance of the cells, induced the expression of alpha-bungarotoxin binding sites (but not of muscarinic receptors), and produced a marked increase in the serotonin and noradrenaline content. Not only the number but also the functional properties of nicotinic and muscarinic receptors were differently affected by the two drugs. We conclude that Bt2cAMP and BrdU induce a different pattern of differentiation in the same cells, and that the expression of specific neuronal markers can be modulated to yield functionally different neurons.     

The distribution of neurones immunoreactive for β-tyrosine hydroxylase,dopamine and serotonin in the ventral nerve cord of the cricket,Gryllus bimaculatus

The cellular localization of the biogenic amines dopamine and serotonin was investigated in the ventral nerve cord of the cricket, Gryllus bimaculatus, using antisera raised against dopamine, -tyrosine hydroxylase and serotonin. Dopamine-(n<-70) and serotonin-immunoreactive (n<-120) neurones showed a segmental arrangement in the ventral nerve cord. Some neuromeres, however, did not contain dopamine-immunoreactive cell bodies. The small number of stained cells allowed complete identification of brain and thoracic cells, including intersegmentally projecting axons and terminal arborizations. Dopamine-like immunostaining was found primarily in plurisegmental interneurones with axons descending to the soma-ipsilateral hemispheres of the thoracic and abdominal ganglia. In contrast, serotonin-immunostaining occurred predominantly in interneurones projecting via soma-contralaterally ascending axons to the thorax and brain. In addition, serotonin-immunoreactivity was also present in efferent cells and afferent elements. Serotonin-immunoreactive, but no dopamine-immunoreactive, varicose fibres were observed on the surface of some peripheral nerves. Varicose endings of both dopamine-and serotonin-immunoreactive neurones occurred in each neuromere and showed overlapping neuropilar projections in dorsal and medial regions of the thoracic ganglia. Ventral associative neuropiles lacked dopamine-like immunostaining but were innervated by serotonin-immunoreactive elements. A colocalization of the two amines was not observed. The topographic representation of neurone types immunoreactive for serotonin and dopamine is discussed with respect to possible modulatory functions of these biogenic amines in the central nervous system of the cricket.     

Adaptive Changes in Postsynaptic Dopamine Receptors Despite Unaltered Dopamine Dynamics in Mice Lacking Monoamine Oxidase B

Monoamine oxidase (MAO) B is considered a key enzyme in dopamine metabolism. The present studies, conducted in MAO B knockout mice, show that lack of MAO B does not alter extracellular levels of dopamine in striatum. Similarly, the synthesis, storage, uptake, and release of dopamine are also unaltered. However, autoradiography revealed a significant up-regulation of the D2-like dopamine receptors in the striatum of MAO B knockout mice. Mutant mice also exhibit a functional supersensitivity of D1-dopamine receptors in the nucleus accumbens. Thus, the agonist SKF 38,393-induced c-Fos immunoreactivity was significantly increased in knockout mice as compared with wild-type controls. In view of the apparently normal basal dopamine dynamics observed in MAO B knockout mice, we hypothesize that a dopamine-independent mechanism underlies adaptations in dopamine receptor function that occur as a consequence of MAO B depletion. Finally, these findings suggest that chronic administration of MAO inhibitors, as occurs in the treatment of Parkinson's disease and depression, may be associated with an increased responsiveness of CNS neurons to dopamine receptor ligands.