Ameliorating schizophrenia-like symptoms in vasopressin deficient male Brattleboro rat by chronic antipsychotic treatment.

Due to its various function vasopressin has been associated with many psychiatric disorders, including schizophrenia. Our previous study confirmed that vasopressin-deficient (di/di) Brattleboro rat can be a good genetic model for schizophrenia. Our present aim was to confirm whether the treatment effects of marketed antipsychotics are similar in di/di rats to those seen in human schizophrenic patients. Chronic subcutaneous administration of aripiprazole (5 mg/kg), clozapine (1 mg/kg), haloperidol (0.1 mg/kg), olanzapine (0.3 mg/kg) or risperidone (0.25 mg/kg) was used for 15 days in control (+/+ Brattleboro) and di/di rats. Social discrimination, social avoidance and prepulse inhibition tests were conducted on day 1, 8 and 15 of the treatment. Vasopressin-deficient rats showed social memory- and sensorimotor gating deficit. All used antipsychotics successfully normalized the reduced prepulse inhibition of di/di animals. However, most were effective only after prolonged treatment. Aripiprazole, clozapine, and olanzapine normalized the social memory deficit, while the effects of haloperidol and risperidone were not unequivocal. All drugs reduced social interest to some extent both in control and in di/di animals, aripiprazole being the less implicated in this regard during the social avoidance test. The restoration of schizophrenia-like behavior by antipsychotic treatment further support the utility of the vasopressin-deficient Brattleboro rat as a good preclinical model. Reduced social interest might be a general side-effect of antipsychotics, and aripiprazole has the most favorable profile in this regard.

Depressive- and anxiety-like behaviors and stress-related neuronal activation in vasopressin-deficient female Brattleboro rats.

Vasopressin can contribute to the development of stress-related psychiatric disorders, anxiety and depression. Although these disturbances are more common in females, most of the preclinical studies have been done in males. We compared female vasopressin-deficient and +/+ Brattleboro rats. To test anxiety we used open-field, elevated plus maze (EPM), marble burying, novelty-induced hypophagia, and social avoidance tests. Object and social recognition were used to assess short term memory. To test depression-like behavior consumption of sweet solutions (sucrose and saccharin) and forced swim test (FST) were studied. The stress-hormone levels were followed by radioimmunoassay and underlying brain areas were studied by c-Fos immunohistochemistry. In the EPM the vasopressin-deficient females showed more entries towards the open arms and less stretch attend posture, drank more sweet fluids and struggled more (in FST) than the +/+ rats. The EPM-induced stress-hormone elevations were smaller in vasopressin-deficient females without basal as well as open-field and FST-induced genotype-differences. On most studied brain areas the resting c-Fos levels were higher in vasopressin-deficient rats, but the FST-induced elevations were smaller than in the +/+ ones. Similarly to males, female vasopressin-deficient animals presented diminished depression- and partly anxiety-like behavior with significant contribution of stress-hormones. In contrast to males, vasopressin deficiency in females had no effect on object and social memory, and stressor-induced c-Fos elevations were diminished only in females. Thus, vasopressin has similar effect on anxiety- and depression-like behavior in males and females, while only in females behavioral alterations are associated with reduced neuronal reactivity in several brain areas.

Enduring abolishment of remote but not recent expression of conditioned fear by the blockade of calcium-permeable AMPA receptors before extinction training.

RATIONALE: Calcium-permeable (GluA2 subunit-free) AMPA receptors (CP-AMPAR) play prominent roles in fear extinction; however, no blockers of these receptors were studied in tests relevant to extinction learning so far. METHODS: The CP-AMPAR antagonist IEM-1460 was administered once before extinction trainings, which were started either 1 or 28 days after fear conditioning (FC). We used a mild extinction protocol that durably decreased but did not abolish conditioned fear. The messenger RNA (mRNA) expression of GluA1 and GluA2 subunits were investigated at both time points in the ventromedial prefrontal cortex (vmPFC) and amygdala. RESULTS: IEM-1460 transiently facilitated extinction 1 day after conditioning, but learned fear spontaneously recovered 4 weeks later. When the extinction protocol was applied 28 days after training, IEM-1460 enhanced extinction memory, moreover abolished conditioned fear for at least a month. The expression of GluA1 and GluA2 mRNAs was increased at both time points in the vmPFC. In the basolateral and central amygdala, the GluA1/GluA2 mRNA ratio increased, suggesting a shift towards the preponderance of GluA1 over GluA2 expression. CONCLUSIONS: AMPAR blockade lastingly enhanced the extinction of remote but not recent fear memories. Time-dependent changes in AMPA receptor subunit mRNA expression may explain the differential effects of CP-AMPAR blockade on recent and remote conditioned fear, further supporting the notion that the mechanisms maintaining learned fear change over time. Our findings suggest clinical implications for CP-AMPAR blockers, particularly for acquired anxieties (e.g., post-traumatic stress disorder) which have a slow onset and are durable.

Anxiogenic role of vasopressin during the early postnatal period: maternal separation-induced ultrasound vocalization in vasopressin-deficient Brattleboro rats.

Both animal and human studies suggest that in adulthood, plasma vasopressin level correlates well with anxiety. Little is known about the mood regulation during the perinatal period. Here, we aim to investigate the influence of vasopressin on anxiety during the early postnatal age. As a sign of distress, rat pups emit ultrasonic vocalizations (USVs) when they are separated from their mother. This USV was detected in 7- to 8-day-old vasopressin-deficient Brattleboro pups, and they were compared to their heterozygote littermates and wild-type pups. The results were confirmed by V1b antagonist treatment (SSR149415 10 mg/kg ip 30 min before test) in wild-types. Chlordiazepoxide (3 mg/kg ip 30 min before test)-an anxiolytic-was used to test the interaction with the GABAergic system. At the end of the test, stress-hormone levels were measured by radioimmunoassay. Vasopressin-deficient pups vocalized substantially less than non-deficient counterparts. Treatment with V1b antagonist resulted in similar effect. Chlordiazepoxide reduced the frequency and duration of the vocalization only in wild-types. Reduced vocalization was accompanied by smaller adrenocorticotropin levels but the level of corticosterone was variable. Our results indicate that the anxiolytic effect of vasopressin deficiency (both genetic and pharmacological) exists already during the early postnatal age. Vasopressin interacts with the GABAergic system. As mood regulation does not go parallel with glucocorticoid levels, we suggest that vasopressin might have a direct effect on special brain areas.

Restoration of peripheral V2 receptor vasopressin signaling fails to correct behavioral changes in Brattleboro rats.

Beside its hormonal function in salt and water homeostasis, vasopressin released into distinct brain areas plays a crucial role in stress-related behavior resulting in the enhancement of an anxious/depressive-like state. We aimed to investigate whether correction of the peripheral symptoms of congenital absence of AVP also corrects the behavioral alterations in AVP-deficient Brattleboro rats. Wild type (WT) and vasopressin-deficient (KO) male Brattleboro rats were tested. Half of the KO animals were treated by desmopressin (V2-receptor agonist) via osmotic minipump (subcutaneous) to eliminate the peripheral symptoms of vasopressin-deficiency. Anxiety was studied by elevated plus maze (EPM), defensive withdrawal (DW) and marble burying (MB) tests, while depressive-like changes were monitored in forced swimming (FS) and anhedonia by sucrose preference test. Cell activity was examined in septum and amygdala by c-Fos immunohistochemistry after 10 min FS. KO rats spent more time in the open arm of the EPM, spent less time at the periphery of DW and showed less burying behavior in MB suggesting a reduced anxiety state. KO animals showed less floating behavior during FS revealing a less depressive phenotype. Desmopressin treatment compensated the peripheral effects of vasopressin-deficiency without a significant influence on the behavior. The FS-induced c-Fos immunoreactivity in the medial amygdala was different in WT and KO rats, with almost identical levels in KO and desmopressin treated animals. There were no differences in central and basolateral amygdala as well as in lateral septum. Our data confirmed the role of vasopressin in the development of affective disorders through central mechanisms. The involvement of the medial amygdala in the behavioral alterations of vasopressin deficient animals deserves further attention.

Increase in Alzheimer's related markers preceeds memory disturbances: studies in vasopressin-deficient Brattleboro rat.

Alzheimer's disease (AD) is the most common form of dementia in the elderly. For more effective therapy early diagnostic markers could be beneficial. Therefore we compared one year old rats with adults and examined if changes in possible brain markers of AD preceeded memory decline. We also tested if vasopressin-deficient animals were useful model of AD as vasopressin has well known positive effect on memory and AD patient has decreased vasopressin production. We compared adult (3 month) and old (12 month), normal and vasopressin-deficient Brattleboro rats. To receive a comprehensive picture about their memory we examined their social discrimination, object discrimination and conditioned learning abilities (shuttle box). Amyloid precursor protein (APP), mitogen-activated protein kinase 1 (MAPK1), ß-actin and tryptophan 2,3-dioxygenase 2 (TDO2) mRNA levels was measured by quantitative PCR. There was no difference between the memory of adult and aged groups. The vasopressin-deficient rats at both ages showed a weaker performance in the course of social and object discrimination tests and a higher escape failure during the shuttle box experiment. The brain marker mRNAs of the elder animals were higher than the levels of the adults, but the absence of vasopressin had no influence on them. Thus, the one year old rats showed elevated levels of AD-related markers, but memory deficits were observable only in vasopressin deficient animals. Vasopressin does not seem to have pathogenic role in AD. Changes in the studied markers might predict later symptoms, although further studies are required for confirmation.

A quick assessment tool for human-directed aggression in pet dogs.

Many test series have been developed to assess dog temperament and aggressive behavior, but most of them have been criticized for their relatively low predictive validity or being too long, stressful, and/or problematic to carry out. We aimed to develop a short and effective series of tests that corresponds with (a) the dog's bite history, and (b) owner evaluation of the dog's aggressive tendencies. Seventy-three pet dogs were divided into three groups by their biting history; non-biter, bit once, and multiple biter. All dogs were exposed to a short test series modeling five real-life situations: friendly greeting, take away bone, threatening approach, tug-of-war, and roll over. We found strong correlations between the in-test behavior and owner reports of dogs' aggressive tendencies towards strangers; however, the test results did not mirror the reported owner-directed aggressive tendencies. Three test situations (friendly greeting, take-away bone, threatening approach) proved to be effective in evoking specific behavioral differences according to dog biting history. Non-biters differed from biters, and there were also specific differences related to aggression and fear between the two biter groups. When a subsample of dogs was retested, the test revealed consistent results over time. We suggest that our test is adequate for a quick, general assessment of human-directed aggression in dogs, particularly to evaluate their tendency for aggressive behaviors towards strangers. Identifying important behavioral indicators of aggressive tendencies, this test can serve as a useful tool to study the genetic or neural correlates of human-directed aggression in dogs.

Maternal neglect with reduced depressive-like behavior and blunted c-fos activation in Brattleboro mothers, the role of central vasopressin.

Early mother-infant relationships exert important long-term effects in offspring and are disturbed by factors such as postpartum depression. We aimed to clarify if lack of vasopressin influences maternal behavior paralleled by the development of a depressive-like phenotype. We compared vasopressin-deficient Brattleboro mothers with heterozygous and homozygous normal ones. The following parameters were measured: maternal behavior (undisturbed and separation-induced); anxiety by the elevated plus maze; sucrose and saccharin preference and forced swim behavior. Underlying brain areas were examined by c-fos immunocytochemistry among rest and after swim-stress. In another group of rats, vasopressin 2 receptor agonist was used peripherally to exclude secondary changes due to diabetes insipidus. Results showed that vasopressin-deficient rats spend less time licking-grooming their pups through a centrally driven mechanism. There was no difference between genotypes during the pup retrieval test. Vasopressin-deficient mothers tended to explore more the open arms of the plus maze, showed more preference for sucrose and saccharin and struggled more in the forced swim test, suggesting that they act as less depressive. Under basal conditions, vasopressin-deficient mothers had more c-fos expression in the medial preoptic area, shell of nucleus accumbens, paraventricular nucleus of the hypothalamus and amygdala, but not in other structures. In these areas the swim-stress-induced activation was smaller. In conclusion, vasopressin-deficiency resulted in maternal neglect due to a central effect and was protective against depressive-like behavior probably as a consequence of reduced activation of some stress-related brain structures. The conflicting behavioral data underscores the need for more sex specific studies.

The vasopressin-deficient Brattleboro rat: lessons for the hypothalamo-pituitary-adrenal axis regulation.

Adaptation to stress is indispensable to life and the hypothalamo-pituitary-adrenocortical axis is one of the major components of the adaptation. The hypothalamic component consists of corticotropin-releasing hormone and arginine vasopressin, with a questionable contribution of the latter. Vasopressin was more important in the regulation of the adrenocorticotropin secretion in the perinatal vasopressin-deficient Brattleboro rats than in adulthood, where its role depended on the nature of the stressor encountered. In adults, the vasopressin deficiency did not influence the development of chronic stress response. In the neonatal rats, the role of vasopressin was supported by the inhibitory action of a V1b antagonist and vasopressin antiserum. As the corticosterone response to stress did not follow the adrenocorticotropin levels, we assume the presence of an adrenocorticotropin independent adrenal gland regulation in the neonates. We have shown that the apparent dissociation of the corticosterone and adrenocorticotropin responses is not due to the different time course of the two hormone responses, to different level of the corticosterone binding globulin or to changes in the adrenal gland sensitivity. In vitro experiments point to the contribution of beta-adrenoceptors in the process. It was also confirmed by in vivo tests using the vasopressin-deficient Brattleboro pup as a model organism, where corticosterone levels may rise without adrenocorticotropin level changes. Another important question is the role of adrenocorticotropin beyond the corticosterone secretion regulation, which could be supposed, e.g., in cardiovascular events, immunological processes, and metabolism. We can conclude that Brattleboro rats gave us much information about the stress-axis regulation far beyond the role of vasopressin itself.

Temporal changes in c-Fos activation patterns induced by conditioned fear.

Mechanisms underlying shock-induced conditioned fear - a paradigm frequently used to model posttraumatic stress disorder, PTSD - are usually studied shortly after shocks. Some of the brain regions relevant to conditioned fear were activated in all the c-Fos studies published so far, but the overlap between the activated regions was small across studies. We hypothesized that discrepant findings were due to dynamic neural changes that followed shocks, and a more consistent picture would emerge if consequences were studied after a longer interval. Therefore, we exposed rats to a single session of footshocks and studied their behavioral and neural responses one and 28 days later. The neuronal activation marker c-Fos was studied in 24 brain regions relevant for conditioned fear, e.g. in subdivisions of the prefrontal cortex, hippocampus, amygdala, hypothalamic defensive system, brainstem monoaminergic nuclei and periaqueductal gray. The intensity of conditioned fear (as shown by the duration of contextual freezing) was similar at the two time-points, but the associated neuronal changes were qualitatively different. Surprisingly, however, Multiple Regression Analyses suggested that conditioned fear-induced changes in neuronal activation patterns predicted the duration of freezing with high accuracy at both time points. We suggest that exposure to electric shocks is followed by a period of plasticity where the mechanisms that sustain conditioned fear undergo qualitative changes. Neuronal changes observed 28 days but not 1 day after shocks were consistent with those observed in human studies performed in PTSD patients.

Changes in adaptability following perinatal morphine exposure in juvenile and adult rats.

The problem of drug abuse among pregnant women causes a major concern. The aim of the present study was to examine the adaptive consequences of long term maternal morphine exposure in offspring at different postnatal ages, and to see the possibility of compensation, as well. Pregnant rats were treated daily with morphine from the day of mating (on the first two days 5mg/kgs.c. than 10mg/kg) until weaning. Male offspring of dams treated with physiological saline served as control. Behavior in the elevated plus maze (EPM; anxiety) and forced swimming test (FST; depression) as well as adrenocorticotropin and corticosterone hormone levels were measured at postpartum days 23-25 and at adult age. There was only a tendency of spending less time in the open arms of the EPM in morphine treated rats at both ages, thus, the supposed anxiogenic impact of perinatal exposure with morphine needs more focused examination. In response to 5min FST morphine exposed animals spent considerable longer time with floating and shorter time with climbing at both ages which is an expressing sign of depression-like behavior. Perinatal morphine exposure induced a hypoactivity of the stress axis (adrenocorticotropin and corticosterone elevations) to strong stimulus (FST). Our results show that perinatal morphine exposure induces long term depression-like changes. At the same time the reactivity to the stress is failed. These findings on rodents presume that the progenies of morphine users could have lifelong problems in adaptive capability and might be prone to develop psychiatric disorders.

Circadian expression of Bmal1 and serotonin-N-acetyltransferase mRNAs in chicken retina cells and pinealocytes in vivo and in vitro.

Unlike mammals, rhythmic changes in serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) transcripts in chicken pineal cells are controlled by an oscillator located in the pinealocytes themselves, which is comprised of clock genes. Asimilar clock-dependent pathway has been postulated to regulate the retinal melatonin rhythm. In chicken retinal photoreceptor cells and pinealocytes, the chicken AANAT gene (cAANAT) is coexpressed with clock genes, including cBmal1 and cClock, which might regulate cAANAT transcription. Here, we have studied the temporal profile of cBmal1, cClock, and cAANAT mRNAexpressions in retinal cells in vivo with chickens housed in a 14/10-h light/dark (LD) cycle for 2 wk and in vitro cultured in a superfusion system for 4 LD cycles. mRNA levels of these genes were analyzed by RT-PCR and compared with their corresponding pineal transcripts. cBmal1 mRNA showed a peak during the light phase between Zeitgeber time (ZT) 8 and 10, preceding the amplitude of the nocturnal increase in cAANAT expression at ZT 16-17. Retinal cBmal1 and cAANAT mRNAs exhibited less robust cycling than their corresponding pineal transcripts in the same animal. cClock mRNAlevels failed to exhibit a well-detectable rhythm. The phase of the rhythms of retinal cBmal1 and cAANAT mRNAs suggests a link between retinal cBmal1 and cAANAT expressions similar to the regulation of pineal cAANAT transcription. Based on the highly conserved nature of the clockwork, it is reasonable to consider that chicken retina and pineal gland might serve as a useful tool for the development of drugs that could influence clock function in man.

Suppression of serotonin N-acetyltransferase transcription and melatonin secretion from chicken pinealocytes transfected with Bmal1 antisense oligonucleotides containing locked nucleic acid in superfusion system.

In birds, rhythmic changes in pineal serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, Aanat) transcripts are controlled by an oscillator located in the pinealocytes themselves which is comprised by clock genes. Our previous data indicated a temporal association between the expressions of chicken Bmal1 clock gene and Aanat suggesting a functional molecular link between them. Here, we studied the effect of cBmal1 antisense oligonucleotides containing locked nucleic acid on cAanat transcripts and melatonin production in cultured chicken pinealocytes transfected in superfusion system. These oligonucleotides synthesized for activating RNase H or blocking the binding of the translation machinery were able to reduce significantly cAanat transcription and melatonin secretion, whereas control inverted oligonucleotides were ineffective. These results indicate the key role of cBmal1 in the regulation of indole metabolism. The superfusion cell culture with reduced transfection toxicity may provide a useful tool for antisense drug design to influence the highly conserved clockwork also in man.