Caramiphen edisylate as adjunct to standard therapy attenuates soman-induced seizures and cognitive deficits in rats.

The progression of epileptiform activity following soman (GD) exposure is characterized by a period of excessive cholinergic activity followed by excessive glutamatergic activity resulting in status epilepticus, which may lead to neuropathological damage and behavioral deficits. Caramiphen edisylate is an anticholinergic drug with antiglutamatergic properties, which conceptually may be a beneficial therapeutic approach to the treatment of nerve agent exposure. In the present study, rats were exposed to 1.2 LD50 GD or saline, treated with atropine sulfate (2mg/kg, im) and HI-6 (93.6mg/kg, im) 1min after GD exposure, and monitored for seizure activity. Rats were treated with diazepam (10mg/kg, sc) and caramiphen (0, 20 or 100mg/kg, im) 30min after seizure onset. Following GD exposure, performance was evaluated using a battery of behavioral tests to assess motor coordination and function, sensorimotor gating, and cognitive function. Caramiphen as adjunct to diazepam treatment attenuated GD-induced seizure activity, neuropathological damage, and cognitive deficits compared to diazepam alone, but did not attenuate the GD-induced sensorimotor gating impairment. These findings show that physiological, behavioral, and neuropathological effects of GD exposure can be attenuated by treatment with caramiphen as an adjunct to therapy, even if administration is delayed to 30min after seizure onset.

Olfactory cues increase avoidance behavior and induce Fos expression in the amygdala, hippocampus and prefrontal cortex of socially defeated mice.

Genes and proteins of the Fos family are used as markers of neuronal activity and can be modulated by stress. This study investigated whether social defeat (SD) or exposure to an olfactory cue associated with the SD experience activated Fos and FosB/DeltaFosB (ΔFosB) expression in brain regions implicated in the development of post-traumatic stress disorder. Mice exposed to acute SD showed more Fos positive cells in the basolateral amygdala (BLA), CA1 of the hippocampus and the medial prefrontal cortex (mPFC) 1h after SD, and had greater expression of the more persistent FosB/ΔFosB protein in the BLA 24 h after SD compared to controls. Mice exposed to an olfactory cue 24 h or 7 days after SD had higher levels of Fos expression in all three regions 1h after exposure to the cue, and displayed increased avoidance behavior compared to controls. While the avoidance response dissipated with time (less at 7 day vs 24 h after social defeat), Fos expression in the mPFC and CA1 in response to an olfactory cue was greater at 7 days relative to 24 h after social defeat. The results suggest additional processing of the cue-stress association and may provide further support for a role of the mPFC in fear inhibition. These findings may have implications for brain regions and circuitry involved in the avoidance of cues associated with a stressful event that may lead to context-dependent adaptive or maladaptive behavior.

The anticholinergic and antiglutamatergic drug caramiphen reduces seizure duration in soman-exposed rats: synergism with the benzodiazepine diazepam.

Therapy of seizure activity following exposure to the nerve agent soman (GD) includes treatment with the anticonvulsant diazepam (DZP), an allosteric modulator of γ-aminobutyric acid A (GABA(A)) receptors. However, seizure activity itself causes the endocytosis of GABA(A) receptors and diminishes the inhibitory effects of GABA, thereby reducing the efficacy of DZP. Treatment with an N-methyl-d-aspartic acid (NMDA) receptor antagonist prevents this reduction in GABAergic inhibition. We examined the efficacy of the NMDA receptor antagonist caramiphen edisylate (CED; 20mg/kg, im) and DZP (10mg/kg, sc), administered both separately and in combination, at 10, 20 or 30min following seizure onset for attenuation of the deleterious effects associated with GD exposure (1.2 LD(50); 132µg/kg, sc) in rats. Outcomes evaluated were seizure duration, neuropathology, acetylcholinesterase (AChE) activity, body weight, and temperature. We also examined the use of the reversible AChE inhibitor physostigmine (PHY; 0.2mg/kg, im) as a therapy for GD exposure. We found that the combination of CED and DZP yielded a synergistic effect, shortening seizure durations and reducing neuropathology compared to DZP alone, when treatment was delayed 20-30min after seizure onset. PHY reduced the number of animals that developed seizures, protected a fraction of AChE from GD inhibition, and attenuated post-exposure body weight and temperature loss independent of CED and/or DZP treatment. We conclude that: 1) CED and DZP treatment offers considerable protection against the effects of GD and 2) PHY is a potential therapeutic option following GD exposure, albeit with a limited window of opportunity.

WITHDRAWN: Effects of repeated sublethal VX exposure on operant time estimation in rats.

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Reduced isolation-induced aggressiveness in mice following NAALADase inhibition.

RATIONALE: Long-term individual housing increases aggressive behavior in mice, a condition termed isolation-induced aggression; this aggressiveness is reduced by some antidepressants and anxiolytics. NMDA antagonists also inhibit isolation-induced aggression in mice. The enzyme N-acetylated-alpha-linked acidic dipeptidase (NAALADase) hydrolyzes the neurotransmitter N-acetylaspartylglutamate (NAAG) to form glutamate and N-acetylaspartate; NAAG acts as a partial NMDA agonist as well as a full agonist at the presynaptic metabotropic glutamate receptor 3 (mGluR3), where it acts to reduce glutamate release. OBJECTIVE: We postulated that NAALADase inhibition would reduce isolation-induced aggression in mice. METHODS: We tested whether acute exposure to the NAALADase inhibitor 2-[[hydroxy[2,3,4,5,6-pentafluorophenyl)methyl]phosphinyl]methyl] pentanedioic acid (GPI-5232), administered 30 min prior to a social interaction test, would inhibit aggressive behavior in SJL mice that had been individually housed long term. RESULTS: Administration of GPI-5232 (30 mg/kg, IP) inhibited initiation of aggressive behavior, indicated by greater latencies to display tail-rattling, attack and biting, and by fewer mice initiating aggressive behavior, compared to mice that received vehicle. In addition, GPI-5232 treated mice had fewer tail-rattling responses to a non-aggressive conspecific. CONCLUSIONS: The effectiveness of GPI-5232 in this animal model suggests that NAALADase inhibition may be a novel therapeutic approach to reduce or inhibit heightened aggressiveness, and possibly to treat aggressive behavior associated with psychiatric disorders.

Accelerated Barnes maze test in mice for assessment of stress effects on memory.

Repeated restraint stress in rodents impairs spatial memory in a Y-maze test and induces hippocampal neuronal changes that last up to 5 d after the stressor ends. Our goal was to implement a Barnes maze spatial memory test in mice that could be used to validate our findings of social stress induced Y-maze impairment. We measured performance of mice in 5- and 9-day test paradigms previously used in rats and mice, respectively. Selecting features from each paradigm, we implemented a 5-d test (pre-training, training (4 trials/d/3 d) and probe testing for assessment of spatial memory in mice. Stress consisted of placing each test mouse in a stainless steel perforated box (25.5 cm x 21.5 cm x 16.5 cm) within an aggressor's home cage for 6 h/d for 21 d; direct agonistic encounters occurred randomly throughout stress periods. Barnes maze pre-training (habituation) was on day 21 of the stress exposures. In a preliminary experiment, mice that habituated following their last stressor performed poorly relative to unstressed and to those not habituated prior to the last stressor, as demonstrated by a greater latency to escape and more errors. We conclude that acute stress in a chronic stress paradigm may impair spatial memory acquisition.

A CRH1 antagonist into the amygdala of mice prevents defeat-induced defensive behavior.

Corticotropin-releasing hormone (CRH) is believed to play an important role in the regulation of behavioral responses to stress. CRH(1) receptor antagonists may reduce stress responsivity. Stress increases CRH in the amygdala, important in memory consolidation. We hypothesized that infusion of a CRH(1) antagonist into the amygdala following social defeat would prevent the development of generalized fear responses. Acute social defeat in mice increases defense towards intruders, even nonaggressive intruders, placed within their home cage. We infused the CRH(1) antagonist antalarmin (0.25 microg/125 nl) bilaterally into the amygdala of mice immediately after defeat and measured their response to a nonaggressive intruder stimulus mouse placed within their home cage 24 h after defeat. Defeated mice that received vehicle displayed high levels of crouch defensive posture and numerous flights from intruders, relative to nondefeated mice that received vehicle. Defeated mice that received antalarmin into the amygdala exhibited significantly less defensive posture than did vehicle-treated defeated mice. Display of defensive posture in antalarmin-treated mice approached that of vehicle-treated nondefeated mice. These findings support a role for CRH in the amygdala to promote consolidation of emotional memory and indicate that antagonism of CRH(1) receptors in the amygdala may prevent the development of exaggerated fear responses in stressed mice.

Vasopressin into the preoptic area increases grooming behavior in mice.

In mice, the neuropeptide arginine-8-vasopressin (AVP) induces excessive grooming, scratching, and hyperactivity when administered intracerebroventricularly. In hamsters, AVP infusion into the medial preoptic area/anterior hypothalamus (MPOA/AH) increases flank marking and flank mark grooming. We measured the behavioral effects of administration of AVP (0, 1, and 10 ng/250 nl) into the preoptic area (POA) of male C57BL/6 mice. Administration of AVP into the POA induced robust effects on grooming, including increased hindleg scratching and face washing. Rearing and olfactory investigation were inhibited by AVP into the POA. These findings indicate that the POA is one site in which AVP induces grooming behavior in mice.

Effects of social defeat and of diazepam on behavior in a resident-intruder test in male DBA/2 mice.

Social stress induces robust behavioral and physiological changes, some of which may alter the responsiveness to pharmacological agents, including diazepam (DZP). We used a resident-intruder paradigm to (1) develop a comprehensive ethogram of behavioral changes following social defeat (SD) in the socially reactive strain, DBA/2 male mice, (2) determine whether acute exposure of DBA/2 mice to low-dose DZP would induce flight or aggressive behavior, both of which have been observed in other rodent models and (3) to test whether prior social stress affects responses to DZP. Behavioral responses to a nonaggressive intruder (NAI) mouse 24 h post-SD were measured in resident subject mice exposed to DZP (0, 0.5, 2.0 mg/kg, ip) either prior to the resident-intruder test (Experiment 1) or immediately post-SD (Experiment 2); control mice were not defeated (NOSD). In general, SD mice displayed increased passive and active avoidance, defense, immobility, and risk assessment relative to NOSD mice. In Experiment 1, mice treated acutely with 0.5 mg/kg DZP had more approach and flight behavior, while those treated with 2.0 mg/kg DZP had more avoidance than vehicle-treated mice, independent of SD. In Experiment 2, acute DZP (2 mg/kg) induced effects 24 h later, possibly secondary to withdrawal. In a nonsocial context (Experiment 3), DZP increased exploratory activity.

Hormone-neurotransmitter interactions in the control of sexual behavior.

The stimuli from a receptive female and/or copulation itself leads to the release of dopamine (DA) in at least three integrative hubs. The nigrostriatal system promotes somatomotor activity; the mesolimbic system subserves numerous types of motivation; and the medial preoptic area (MPOA) focuses the motivation onto specifically sexual targets, increases copulatory rate and efficiency, and coordinates genital reflexes. The previous (but not necessarily concurrent) presence of testosterone is permissive for DA release in the MPOA, both during basal conditions and in response to a female. One means by which testosterone may increase DA release is by upregulating nitric oxide synthase, which produces nitric oxide, which in turn increases DA release. Hormonal priming in females may also increase DA release in the MPOA, and copulatory activity may further increase DA levels in females. One of the intracellular effects of stimulation of DA D1 receptors in the MPOA of male rats may be increased expression of the immediate-early gene c-fos, which may mediate longer term responses to copulation. Furthermore, increased sexual experience led to increased immunoreactivity to Fos, the protein product of c-fos, following copulation to one ejaculation. Another intracellular mediator of DA's effects, particularly in castrates, may be the phosphorylation of steroid receptors. Finally, while DA is facilitative to copulation, 5-HT is generally inhibitory. 5-HT is released in the LHA, but not in the MPOA, at the time of ejaculation. Increasing 5-HT in the LHA by microinjection of a selective serotonin reuptake inhibitor (SSRI) increased the latency to begin copulating and also the latency to the first ejaculation, measured from the time the male first intromitted. These data may at least partially explain the decrease in libido and the anorgasmia of people taking SSRI antidepressants. One means by which LHA 5-HT decreases sexual motivation (i.e. increases the latency to begin copulating) may be by decreasing DA release in the NAcc, a major terminal of the mesolimbic system. Thus, reciprocal changes in DA and 5-HT release in different areas of the brain may promote copulation and sexual satiety, respectively.

Social stress effects on territorial marking and ultrasonic vocalizations in mice.

Acute social defeat (SD) leads to transient and persistent physiological and behavioral changes. We examined the effects of acute SD on territorial urine marking and ultrasonic courtship vocalizations in DBA/2 male mice. Both behaviors are considered androgen dependent and are influenced by social status, with dominant mice displaying more of both behaviors. In Experiment 1, male mice that received SD displayed prolonged inhibition of territorial urine marking, relative to nondefeated control mice (NOSD). In addition, territorial marking increased with repeated tests. In Experiment 2, male mice that received 3 successive days of SD displayed fewer ultrasonic courtship vocalizations at 30 min. post-SD1 and 30 min. post-SD2, relative to NOSD mice. In Experiment 2, we also observed decreased territorial marking 4 weeks post-SD. In sum, SD induced prolonged inhibition of territorial marking, but had only transient effects on ultrasonic courtship vocalizations, suggesting that different mechanisms may mediate the maintenance of these behaviors.

Effects of a D1 antagonist and of sexual experience on copulation-induced Fos-like immunoreactivity in the medial preoptic nucleus.

The medial preoptic nucleus (MPN) of the medial preoptic area (MPOA) and the medial amygdala are two brain regions in which male rat sexual behavior increased Fos-like immunoreactivity (Fos-Li). Dopamine is released in the MPOA during male rat sexual behavior and facilitates copulation. Psychostimulants, which increase dopamine levels, induce Fos-Li in the striatum through D1 receptors. We examined whether copulation-induced Fos-Li in the MPN was also mediated through D1 receptors. In Experiment 1, sexually inexperienced male rats that received the D1 antagonist Schering 39166 prior to their first sexual experience had fewer Fos-Li cells in the MPN than did those that received vehicle. In Experiment 2, no significant effect of the D1 antagonist was observed on copulation-induced Fos-Li in male rats that had received repeated sexual experiences prior to the drug test day. Sexual experience increases copulatory efficiency; the mechanisms by which this improvement occurs are unclear. In Experiment 3, copulation by highly experienced male rats led to greater Fos-Li in the MPN than did copulation by sexually naive males. Although there were no differences between groups in amygdala Fos-Li in these studies, in several groups Fos-Li in the medial amygdala was positively correlated with the post-ejaculatory interval. These experiments indicate that (1) stimulation of D1 receptors may contribute to the transient copulation-induced increase in Fos-Li in the MPN, and (2) repeated sexual experiences enhanced copulation-induced Fos-Li in the MPN, which may represent a marker of altered responsiveness of neurons in the MPN to sexual or conditioned stimuli.

Possible acceleration of age effects on cognition following menopause.

Many cognitive functions have been shown to deteriorate with age. Because of the importance of the menopause as a milestone in the life cycles of women, we examined whether the aging-over-time process in some cognitive functions differs between women of reproductive age and postmenopausal women. It is demonstrated here that in some cognitive tests, including driving simulation, reaction time and some visuospatial tests, there is a significant acceleration in deterioration of functioning following menopause. It is suggested that this acceleration might be associated with the lack of gonadal hormones or other reproduction-related factors which may play a protective role against age-related deterioration in some cognitive functions in women.

The roles of nitric oxide in sexual function of male rats.

Nitric oxide (NO) may mediate penile erection by inhibiting smooth muscle of the corpora cavernosa, thereby allowing vasodilation of the corpora. In order to test the role of NO in the sexual function of intact male rats, either the precursor of NO (L-arginine, L-Arg) or an inhibitor of its synthesis (NG-nitro-L-arginine methyl ester, NAME) was administered systemically before tests of copulation, ex copula genital reflexes, or sexual motivation/motor activity. NAME impaired copulation in a dose dependent manner. It also decreased the number of ex copula erections, but it increased the number of ex copula seminal emissions and decreased the latency to the first seminal emission. L-Arg marginally increased the number of penile reflexes, but had no other effects. NAME had no effect on sexual motivation or motor activity. The results indicate that nitric oxide promotes erection in intact male rats, probably by mediating filling of the corpora cavernosa. The data also suggest that NO inhibits seminal emission, probably by decreasing sympathetic nervous system activity; this may help prevent premature ejaculation.

A D1 agonist in the MPOA facilitates copulation in male rats.

The classic dopamine agonist apomorphine, microinjected into the medial preoptic area (MPOA), enhances the copulatory behavior of male rats, while pharmacological blockade of endogenous dopamine inhibits sexual behavior. We now report that MPOA injections of 10 micrograms of the selective D1 agonist dihydroxyphenyl-tetrahydrothienopyridine (THP) significantly increased the number of ejaculations, while decreasing the latency to ejaculate in a 30-min test. These effects were not observed following coadministration of the selective D1 antagonist SCH-23390 with 10 micrograms THP. This enhancement may be related to a D1-stimulated facilitation of penile erections.

The multiple interactional biological processes that might lead to depression and gender differences in its appearance.

Several neurotransmitter systems have been implicated in the pathophysiology of depression. Gender differences have been demonstrated in some functions that involve these systems, mostly norepinephrine, serotonin, dopamine and acetylcholine. Several hormonal systems have been shown to be altered in depression and gender differences were demonstrated in their activity as well, notably in thyroid hormones and the hypothalamic-pituitary-adrenal system. Most gender differences in brain systems and their hormonal modulators might be attributed to developmental and state influences of gonadal hormones. It is suggested that gender differences in mechanisms that might underlie formation of depressive symptoms might be related mostly to interactional processes and destabilization of balance among multiple factors or circuitry in the central nervous system. Indeed, gender differences in specific neurotransmitters and hormonal systems can be demonstrated as well.

Opposite influence of medial preoptic D1 and D2 receptors on genital reflexes: implications for copulation.

Dopamine D1 and D2 receptors may synergize with or oppose each other's effects. We suggest that stimulation of D1 and D2 receptors in the medial preoptic area (MPOA) of male rats have opposing effects on genital reflexes. In Experiment 1 a D1 agonist injected into the MPOA increased the number of ex copula erections but decreased the number of seminal emissions. In Experiment 2 a D1 antagonist had the opposite effects (decreased erections and increased seminal emissions), as had a D2 agonist previously. We also suggest that D1 and D2 mechanisms in the MPOA have different thresholds of activation. In Experiment 3 a low dose of the mixed D1/D2 agonist apomorphine increased erections and anteroflexions, an effect blocked by the D1 antagonist. In Experiments 3 and 4 a high dose of apomorphine increased seminal emissions, an effect blocked by the D2 antagonist. Thus, low levels of dopaminergic stimulation may facilitate erections and anteroflexions (controlled by the parasympathetic system and striated muscles) via D1 receptors; higher or more prolonged stimulation may shift to seminal emission (controlled by the sympathetic system) via D2 receptors. This may explain the progression from erectile to ejaculatory mechanisms during copulation.

D2 receptors in the paraventricular nucleus regulate genital responses and copulation in male rats.

The D2 dopamine receptor agonist quinelorane (LY-163502), microinjected into the paraventricular nucleus (PVN), affected genital response of restrained supine male rats in a biphasic dose-dependent fashion. A moderate dose (1 microgram) facilitated penile responses (intense erections and penile movements), and decreased the latency to the first response. A high dose of quinelorane (10 micrograms) facilitated seminal emission while inhibiting penile responses. The addition of the D1 antagonist SCH-23390 to the 1 microgram dose of quinelorane potentiated quinelorane's increase in seminal emission. We suggest that D1 receptors in the PVN may be antagonistic to D2 receptor-mediated seminal emission, and possibly also penile responses. In copulation tests 1 microgram quinelorane decreased mount latency, whereas 10 micrograms quinelorane increased mount and intromission latencies and slowed copulatory rate. Both 1 and 10 micrograms quinelorane, and also 1 and 10 micrograms of the mixed D1 and D2 agonist apomorphine, decreased the number of intromissions preceding ejaculation.

Dose dependent D2 effects on genital reflexes after MPOA injections of quinelorane and apomorphine.

This study investigated the effects on genital reflexes of unilateral MPOA injections of 0.1, 1, 3, and 10 micrograms of the D2 agonist quinelorane (LY-163502), and of 3 micrograms quinelorane administered together with 3 micrograms of the D1 antagonist SCH-23390. In addition, the effects of an MPOA injection of 10 micrograms apomorphine were tested. All but the lowest dose of quinelorane significantly decreased the latency to the first reflex. The 3 and 10 micrograms doses of quinelorane, and the combination of quinelorane and SCH-23390, decreased the total number of reflexes. In addition, 10 micrograms quinelorane increased the number of seminal emissions. 10 micrograms apomorphine, like 10 micrograms quinelorane, decreased the latency to the first reflex and increased the number of seminal emissions, but did not decrease the numbers of erections or penile movements. The ratio of D1/D2 activity may influence the number of erections displayed during ex copula testing.