Survival, re-expansion, and pregnancy outcome following vitrification of dromedary camel cloned blastocysts: A possible role of vitrification in improving clone pregnancy rate by weeding out poor competent embryos.

There is a clinical demand for efficient cryopreservation of cloned camel embryos with considerable logistic and economic advantage. Vitrification of in vivo derived embryos has been reported in camels, but there is no study on vitrification of cloned embryos. Moreover, whether characteristic differences between cloned and in vivo derived embryos imply different vitrification requirement is unresolved. Here, we compared survival, re-expansion and pregnancy rates of cloned embryos vitrified using two commercial vitrification kits (Cryotec and Kitazato), developed basically for human embryos, and a vitrification protocol developed for in vivo camel embryos (CVP). Cloned embryos responded dynamically to vitrification-warming steps in commercial kits, with a flat shrinkage in the final vitrification solution and a quick re-expansion to the original volume immediately after transferring to the isotonic warming solution. Contrarily, full shrinkage was not observed in CVP method, and majority of embryos were still collapsed post-warming. The immediate re-expansion was highly associated and predictive of higher survival and total cell number, and also better redox state of embryos vitrified by Cryotec and Kitazato kits compared to CVP method. Importantly, while 30% blastomere loss, verified by differential dye exclusion test, was tolerated in vitrified embryos, >50% blastomeres loss in non-expanded blastocysts implied the minimal essential cell survival rate for blastocoelic cavity re-expansion in vitrified cloned camel blastocysts, irrespective of vitrification method. A protocol-based exposure of embryos to cryoprotectants indicated that cryoprotectant toxicity, per se, may not be involved in lower cryosurvival of embryos in CVP vs. Cryotec and Kitazato. The initial pregnancy rates were numerically higher in Cryotec and Kitazato frozen transfers compared to fresh transfer (56.3, 60 and 33.3%, respectively), and importantly, a higher percentage of established pregnancies in vitrified groups passed the critical 3 months period of early embryonic loss compared to sibling fresh clone pregnancies (50, 40, and 10%, respectively). Results confirmed the suitability of Cryotec and Kitazato kits for vitrification of cloned camel embryos and that vitrification may improve pregnancy outcome by weeding out poor competent embryos.

Unexpected global impact of VTA dopamine neuron activation as measured by opto-fMRI.

Dopamine neurons in the ventral tegmental area (VTA) are strongly implicated in cognitive and affective processing as well as in psychiatric disorders, including schizophrenia, depression, attention-deficit hyperactivity disorder and substance abuse disorders. In human studies, dopamine-related functions are routinely assessed using functional magnetic resonance imaging (fMRI) measures of blood oxygenation-level-dependent (BOLD) signals during the performance of dopamine-dependent tasks. There is, however, a critical void in our knowledge about whether and how activation of VTA dopamine neurons specifically influences regional or global fMRI signals. Here, we used optogenetics in Th::Cre rats to selectively stimulate VTA dopamine neurons while simultaneously measuring global hemodynamic changes using BOLD and cerebral blood volume-weighted (CBVw) fMRI. Phasic activation of VTA dopamine neurons increased BOLD and CBVw fMRI signals in VTA-innervated limbic regions, including the ventral striatum (nucleus accumbens). Surprisingly, basal ganglia regions that receive sparse or no VTA dopaminergic innervation, including the dorsal striatum and the globus pallidus, were also activated. In fact, the most prominent fMRI signal increase in the forebrain was observed in the dorsal striatum that is not traditionally associated with VTA dopamine neurotransmission. These data establish causation between phasic activation of VTA dopamine neurons and global fMRI signals. They further suggest that mesolimbic and non-limbic basal ganglia dopamine circuits are functionally connected and thus provide a potential novel framework for understanding dopamine-dependent functions and interpreting data obtained from human fMRI studies.

A randomized, double-blind, placebo-controlled trial on the efficacy and tolerability of sertraline in Iranian veterans with post-traumatic stress disorder.

BACKGROUND: Unlike civilian post-traumatic stress disorder (PTSD), the efficacy of sertraline for the treatment of combat-related PTSD has not yet been proven. The present study aimed to evaluate the clinical efficacy of sertraline against combat-related PTSD in a randomized, double-blind, placebo-controlled trial. METHOD: Seventy Iranian veterans of the Iran-Iraq war who met the DSM-IV criteria for diagnosis of PTSD were randomized to receive either flexibly dosed sertraline (50-200 mg/day) (n=35, completers=32) or placebo (n=35, completers=30) for 10 weeks. Efficacy was evaluated by the Impact of Event Scale--Revised (IES-R) and the Clinical Global Impression scale--Severity (CGI-S) and Improvement (CGI-I) ratings. Responder criteria were defined as a ≥30% reduction in the IES-R total score plus a CGI-I rating of 'much' or 'very much' improved. RESULTS: On both intention-to-treat (ITT) and per protocol (completer) methods of analysis, the mean reductions in the IES-R total and subscale (re-experiencing/intrusion, avoidance/numbing and hyperarousal) scores (p<0.001) and also in the CGI-S score (p<0.01) were significantly greater in the sertraline group than in the placebo group. For the CGI-I, the mean endpoint score was significantly lower in the sertraline group than in the placebo group (p≤0.001). The number of responders in the sertraline group was significantly higher than in the placebo group (44% v. 3%, p≤0.001). Sertraline was well tolerated, with a 6% discontinuation rate as a result of adverse reactions. CONCLUSIONS: The results of this study suggest that sertraline can be an effective, safe and tolerable treatment for combat-related PTSD in Iranian veterans.

Microdeletion 15q13.3: a locus with incomplete penetrance for autism, mental retardation, and psychiatric disorders.

BACKGROUND: Microdeletions within chromosome 15q13.3 are associated both with a recently recognised syndrome of mental retardation, seizures, and dysmorphic features, and with schizophrenia. METHODS AND RESULTS: Based on routine diagnostic testing of approximately 8200 samples using array comparative genomic hybridisation, we identified 20 individuals (14 children and six parents in 12 families) with microdeletions of 15q13.3. Phenotypes in the children included developmental delay, mental retardation, or borderline IQ in most and autistic spectrum disorder (6/14), speech delay, aggressiveness, attention deficit hyperactivity disorder, and other behavioural problems. Both parents were available in seven families, and the deletion was de novo in one, inherited from an apparently normal parent in four, and inherited from a parent with learning disability and bipolar disorder in two families. Of the 14 children, six in five families were adopted, and DNA was available for only one of these 10 biological parents; the deletion was very likely inherited for one of these families with two affected children. Among the unavailable parents, two mothers were described as having mental retardation, another mother as having "mental illness", and one father as having schizophrenia. We hypothesise that some of the unavailable parents have the deletion. CONCLUSIONS: The occurrence of increased adoption, frequent autism, bipolar disorder, and lack of penetrance are noteworthy findings in individuals with deletion 15q13.3. A high rate of adoption may be related to the presence of the deletion in biological parents. Unconfirmed histories of antisocial behaviours in unavailable biological parents raise the concern that future research may show that deletion 15q13.3 is associated with such behaviours.

Reversal of phencyclidine effects by a group II metabotropic glutamate receptor agonist in rats.

Glutamatergic abnormalities have been associated with several psychiatric disorders, including schizophrenia and addiction. Group II metabotropic glutamate receptors were targeted to normalize glutamatergic disruptions associated with an animal model of schizophrenia, the phencyclidine model. An agonist of this group of receptors, at a dose that was without effects on spontaneous activity and corticolimbic dopamine neurotransmission, attenuated the disruptive effects of phencyclidine on working memory, stereotypy, locomotion, and cortical glutamate efflux. This behavioral reversal occurred in spite of sustained dopamine hyperactivity. Thus, targeting this group of receptors may present a nondopaminergic therapeutic strategy for treatment of psychiatric disorders.

Serotonergic and noradrenergic lesions suppress the enhancing effect of maternal exercise during pregnancy on learning and memory in rat pups.

The beneficial effects of exercise on learning and memory are well documented but the effects of prenatal exposure to maternal exercise on offspring are not clear yet. Using a two-trial-per-day Morris water maze for five consecutive days, succeeded by a probe trial 2 days later we showed that maternal voluntary exercise (wheel running) by pregnant rats increased the acquisition phase of the pups' learning. Maternal forced swimming by pregnant rats increased both acquisition and retention phases of the pups' learning. Also we found that the rat pups whose mother was submitted to forced-swimming during pregnancy had significantly higher brain, liver, heart and kidney weights compared with their sedentary counterparts. On the other hand we estimated the cell number of different regions of the hippocampus in the rat pups. We found that both exercise models during pregnancy increased the cell number in cornus ammonis subregion 1 (CA1) and dentate gyrus of the hippocampus in rat pups. To determine the role that noradrenergic and serotonergic neurotransmission and N-methyl-D-aspartate (NMDA) receptors hold in mediation of the maternal exercise in offspring, we used N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), p-chloroamphetamine (PCA) and MK-801 to eliminate or block the above systems, respectively. Blocking the NMDA receptors, significantly abolished learning and memory in rat pups from all three experimental groups. Elimination of noradrenergic or serotonergic input did not significantly attenuate the learning and memory in rat pups whose mothers were sedentary, while it significantly reversed the positive effects of maternal exercise during pregnancy on rat pups' learning and memory. The presented results suggest that noradrenergic and serotonergic systems in offspring brain seem to have a crucial specific role in mediating the effects of maternal physical activity during pregnancy on rat pups' cognitive function in both models of voluntary and forced exercise.

Amygdala regulation of nucleus accumbens dopamine output is governed by the prefrontal cortex.

A dynamic interaction between the prefrontal cortex (PFC), amygdala, and nucleus accumbens (NAc) may be fundamental to regulation of goal-directed behavior by affective and cognitive processes. This study demonstrates that a mechanism for this triadic relationship is an inhibitory control by prefrontal cortex on accumbal dopamine release during amygdala activation. In freely moving rats, microstimulation of basolateral amygdala at intensities that produced mild behavioral activation produced an expected rapid increase in glutamate efflux in the prefrontal cortex and the nucleus accumbens shell region of the ventral striatum. However, during the stimulation, dopamine release increased only in the prefrontal cortex, not in the nucleus accumbens. An increase in accumbal dopamine release was observed during the stimulation if glutamate activation in the prefrontal cortex was inhibited at either presynaptic or postsynaptic levels. Some behaviors expressed during the stimulation were intensified in animals in which prefrontal cortex glutamate activation was blocked. In addition, these animals continued to express stimulus-induced behaviors after the termination of stimulation, whereas normal poststimulus behaviors such as ambulation and grooming were not displayed as frequently. Considering that dopamine neurotransmission in the nucleus accumbens is thought to play an integral role in goal-directed motor behavior, these findings suggest that the prefrontal cortex influences the behavioral impact of amygdala activation via a concomitant active suppression of accumbal dopamine release. Absence of this cortical influence appears to result in an aberrant pattern of behavioral expression in response to amygdala activation, including behavioral perseveration after stimulus termination.

Flow regime in a restored wetland determines trophic links and species composition in the aquatic macroinvertebrate community.

In a restored wetland (South of Spain), where different flow regimes control water exchange with the adjacent Guadalquivir estuary, the native Palaemon varians coexists with an exotic counterpart species Palaemon macrodactylus. This controlled m\acrocosm offers an excellent opportunity to investigate how the effects of water management, through different flow regimes, and the presence of a non-native species affect the aquatic community and the trophic niche (by gut contents and C-N isotopic composition) of the native shrimp Palaemon varians. We found that increased water exchange rate (5% day(-1) in mixed ponds vs. 0.1% day(-1) in extensive ponds) modified the aquatic community of this wetland; while extensive ponds are dominated by isopods and amphipods with low presence of P. macrodactylus, mixed ponds presented high biomass of mysids, corixids, copepods and both shrimp species. An estuarine origin of nutrients and primary production might explain seasonal and spatial differences found among ponds of this wetland. A combined analysis of gut contents and isotopic composition of the native and the exotic species showed that: (1) native P. varians is mainly omnivorous (2) while the non-native P. macrodactylus is more zooplanktivorous and (3) a dietary overlap occurred when both species coexist at mixed ponds where a higher water exchange and high abundance of mysids and copepods diversifies the native species' diet. Thus differences in the trophic ecology of both species are clearly explained by water management. This experimental study is a valuable tool for integrated management between river basin and wetlands since it allows quantification of wetland community changes in response to the flow regime.

Effect of clozapine, haloperidol, or M100907 on phencyclidine-activated glutamate efflux in the prefrontal cortex.

BACKGROUND: The increase in glutamate efflux in the prefrontal cortex by the psychotomimetic drugs phencyclidine (PCP) and ketamine may produce the dopaminergic and some of the behavioral effects of these drugs. Here, we examined whether antipsychotic drugs influence this increase. METHODS: The effect of haloperidol, clozapine or the 5-HT(2A) antagonist, M100907, on PCP-induced increase in cortical glutamate efflux was examined by microdialysis. Because previous studies had suggested that M100907 attenuates some behavioral effects of PCP, we also examined the effect of M100907 on PCP-induced cortical and accumbal dopamine activation while making concomitant measures of locomotion and stereotypy. RESULTS: Haloperidol, clozapine or M100907 did not significantly block hyperglutamatergic effects of PCP. M100907 was ineffective in inhibiting the dopaminergic and motoric effects of PCP. CONCLUSIONS: These results contrast previous findings with glutamatergic drugs, such as AMPA antagonists or group II metabotropic glutamate agonists, that blocked glutamatergic and motoric effects of PCP. Thus, the PCP glutamate activation model lacks predictive validity for conventional antipsychotics; however, this model may be useful for design of novel classes of drugs that target those symptoms of schizophrenia that are not generally treated with monoamine-based antipsychotics.

Clozapine preferentially increases dopamine release in the rhesus monkey prefrontal cortex compared with the caudate nucleus.

Despite substantial differences between species in the organization and elaboration of the cortical dopamine innervation, little is known about the pharmacological response of cortical or striatal sites to antipsychotic medications in nonhuman primates. To examine this issue, rhesus monkeys were chronically implanted with guide cannulae directed at the principal sulcus, medial prefrontal cortex, premotor cortex, and caudate nucleus. Alterations in dopamine release in these discrete brain regions were measured in response to administration of clozapine or haloperidol. Clozapine produced significant and long-lasting increases in dopamine release in the principal sulcus, and to a lesser extent, in the caudate nucleus. Haloperidol did not produce a consistent effect on dopamine release in the principal sulcus, although it increased dopamine release in the caudate. Clozapine's preferential augmentation of dopamine release in the dorsolateral prefrontal cortex supports the idea that clozapine exerts its therapeutic effects in part by increasing cortical dopamine neurotransmission.

Temporal dynamics of glutamate efflux in the prefrontal cortex and in the hippocampus following repeated stress: effects of pretreatment with saline or diazepam.

Acute stress has been associated with activation of glutamate efflux in forebrain structures. The present study sought to characterize the extracellular dynamics of glutamate in response to acute and repeated stress in the prefrontal cortex and hippocampus in rats. One-minute sampling of extracellular glutamate levels was performed during repeated tail-pinch stress. Animals were stressed three times, beginning at approximately 10.30 a.m. and continuing at 2.5-h intervals. In the prefrontal cortex, the initial 10-min tail pinch produced a robust increase in extracellular levels of glutamate. This increase was apparent immediately (i.e. 1 min) after the start of the stress procedure. The second tail pinch produced a smaller increase in glutamate levels while the third tail pinch did not significantly increase these levels. In the hippocampus, the initial stress response was smaller in magnitude than that observed in the prefrontal cortex. Furthermore, responses to subsequent tail pinches were similar to that seen following the first tail pinch. Treatment with diazepam (3 mg/kg/i.p.) 30 min before the first stress session abolished the stress response in the prefrontal cortex and hippocampus. However, in the prefrontal cortex, the second tail pinch (performed approximately 3 h after diazepam administration) produced a robust increase in glutamate efflux. In contrast, in the hippocampus of diazepam-treated rats, the second tail pinch produced a small delayed response. Pretreatment with saline resulted in non-significant responses to all three tail pinches in the prefrontal cortex. The present study suggests that: (i) stress produces a rapid increase in glutamate efflux in the prefrontal cortex and hippocampus, (ii) repeated stress reveals tolerance in the glutamatergic response in the prefrontal cortex, (iii) saline and diazepam pretreatment reduce the stress-induced efflux of glutamate in the prefrontal cortex, and (iv) exposure to diazepam may prevent the prefrontal cortex from adapting its response to the subsequent stressor. These finding are consistent with the role of the prefrontal cortex as a region which may regulate reactions to aversive stimuli.

Characterization of dopamine release in the rat medial prefrontal cortex as assessed by in vivo microdialysis: comparison to the striatum.

Using the technique of perfusion microdialysis combined with a small-bore liquid chromatography system we have measured the basal and drug-induced fluxes of extracellular dopamine in the medial prefrontal cortex of chloral hydrate-anesthetized rats and have compared our findings in the cortex to that observed in the striatum. The results were as follows. (1) At a flow rate of 2 microliter/min, the basal level of dopamine in the medial prefrontal cortex was 0.28 +/- 0.1 (n = 32) fmol/microliter perfusate, which was nearly an order of magnitude less than that obtained from the striatum. (2) alpha-Methyl-para-tyrosine (150 mg/kg i.v.) significantly decreased the extracellular levels of striatal and cortical dopamine. The magnitude and duration of the responses were similar in both regions. (3) Local perfusion with 30 mM K+ had a more profound effect on dopamine release in the striatum than in the medial prefrontal cortex. The K(+)-induced release in both regions was significantly attenuated in the absence of Ca2+. (4) The anxiogenic beta carboline FG 7142 (15 mg/kg, i.p.) enhanced the release of cortical dopamine by about 50% while it was without an effect in the striatum. (5) Amphetamine (1 mg/kg, i.v.) significantly elevated, while reserpine (5 mg/kg, i.p.) rapidly attenuated, the dopamine level in the medial prefrontal cortex. These studies demonstrate that perfusion microdialysis, in conjunction with small-bore liquid chromatography with electrochemical detection, can be used to measure the basal release of dopamine in the rat medial prefrontal cortex and that the dopamine release process in this region, as has been shown in the striatum, is sensitive to stimulation conditions and pharmacological manipulations.(ABSTRACT TRUNCATED AT 250 WORDS)

Preferential activation of cortical dopamine neurotransmission by clozapine: functional significance.

Dopamine projections to the prefrontal cortex are thought to be essential for the proper functioning of this region and are proposed to be involved in negative (deficit) symptomatology of schizophrenia. Our studies in the rodent indicate that clozapine, the most effective antipsychotic drug for the treatment of negative symptoms, causes an increase in the basal output of dopamine neurons projecting to the prefrontal cortex. This finding is in contrast to the effect of clozapine in the basal ganglia and the effect of typical antipsychotic drugs such as haloperidol in the prefrontal cortex. The ability of clozapine to increase dopamine release in the prefrontal cortex and its relatively weak affinity for some types of dopamine receptors suggest that this drug may exert its therapeutic influence in part by increasing dopaminergic function in the prefrontal cortex.

Distinct contributions of glutamate and dopamine receptors to temporal aspects of rodent working memory using a clinically relevant task.

RATIONALE: Understanding the mechanistic basis of working memory, the capacity to hold representation "on line," is important for delineating the processes involved in higher cognitive functions and the pathophysiology of thought disorders. OBJECTIVES: We compared the contribution of glutamate and dopamine receptor subtypes to temporal aspects of working memory using a modified rodent spatial working memory task that incorporates important elements of clinical working memory tasks. METHODS: A discrete paired-trial variable-delay T-maze task was used. Initial characterization studies indicated that performance on this task is stable at seconds-long retention intervals, is sensitive to retention interval and proactive interference, and is dependent on the integrity of the medial prefrontal cortex. RESULTS: Consistent with clinical findings, low dose amphetamine (0.25 mg/kg) produced a delay-dependent improvement in performance, while higher doses impaired performance at all retention intervals. D1 receptor blockade produced the predicted dose- and delay-dependent impairment. D2 receptor blockade had no effect. Activation of metabotropic glutamate 2/3 (mGluR2/3) receptors, which in the prefrontal cortex inhibits the slow asynchronous phase of glutamate release, also produced a delay-dependent impairment. Low doses of an AMPA/kainate antagonist had effects similar to the mGluR2/3 agonist. In contrast, NMDA receptor antagonist-induced impairment was memory load-insensitive, resulting in chance-level performance at all retention intervals. CONCLUSIONS: These findings suggest that activation of NMDA receptors is necessary for the formation of mnemonic encoding while modulatory components involving slow asynchronous release of glutamate and phasic release of dopamine contribute to the active maintenance of information during the delay period.

Mechanisms of action of atypical antipsychotic drugs. Implications for novel therapeutic strategies for schizophrenia.

The mechanisms which contribute to the actions of atypical antipsychotic drugs, such as clozapine and the putative atypical agents remoxipride and raclopride, are reviewed. Examination of available preclinical and clinical data leads to two hypotheses concerning the mode of action of atypical antipsychotic drugs. The first hypothesis is that antagonism of the dopamine D2 receptor is both necessary and sufficient for the atypical profile, but that interaction with subtypes of the D2 receptor differentiates typical from atypical antipsychotic drugs. The second hypothesis has been previously advanced, and suggests that a relatively high ratio of serotonin 5-HT2:dopamine D2 receptor antagonism may subserve the atypical profile. It seems likely that the atypical antipsychotic drug profile may be achieved in more than one way.

Electrocoating carbon fiber microelectrodes with Nafion improves selectivity for electroactive neurotransmitters.

A method which improves carbon fiber microelectrode selectivity for cationic amines by electrocoating the fiber with a thin film of the ionic polymer, Nafion, is described. The selectivity and response speed of these electrodes for the detection of electroactive cationic and anionic species found in brain extracellular fluid was evaluated using differential pulse voltammetry and chronoamperometry and compared to uncoated fibers. Carbon fiber microelectrodes electrocoated with Nafion are highly sensitive to cationic amines such as dopamine and serotonin and have minimal sensitivity to anions such as ascorbic acid and uric acid at physiological concentrations.

Effect of a pharmacological stressor on glutamate efflux in the prefrontal cortex.

The anxiogenic beta-carboline, FG 7142 (20 mg/kg) significantly increased glutamate efflux in the prefrontal cortex of conscious rats as assessed by microdialysis. Pretreatment with the benzodiazepine receptor agonist, diazepam (5 mg/kg), abolished this effect. These findings indicate that anxiogenic compounds produce an effect similar to physical stressors on the outflow of glutamate, and implicate the GABA/benzodiazepine receptor complex in the stress-induced activation of glutamate systems in the prefrontal cortex.

Do endogenous excitatory amino acids influence striatal dopamine release?

In vivo microdialysis techniques were used to examine whether endogenous excitatory amino acids exert a tonic facilitatory influence on striatal dopamine release. Local application of NMDA and non-NMDA antagonists at 10 microM was without an effect on basal dopamine release while 100 microM and 1 mM of these drugs significantly enhanced the release. Our findings do not support the idea that excitatory amino acids have a tonic excitatory effect on striatal dopamine release.

Regional differences in resting extracellular potassium levels of rat brain.

The extracellular potassium concentration in the cerebral cortex of the mammalian brain has been repeatedly reported to be approximately 3.0 mM. We have made detailed measurements with potassium-selective microelectrodes and have found significantly lower extracellular potassium concentrations in unstimulated rat brain caudate and thalamus (1.9-2.5 mM) when compared to cortex and cerebral spinal fluid (3.0-3.5 mM). These regional differences may be caused by variations in spontaneous activity of neurons, regional permeability differences in endothelial cells of brain capillaries to potassium, or caused by variations in uptake by glia.