Intranasal neuropeptide Y reverses anxiety and depressive-like behavior impaired by single prolonged stress PTSD model.

PTSD is a debilitating neuropsychiatric disorder and many patients do not respond sufficiently to current treatments. Neuropeptide Y (NPY) is suggested to provide resilience to the development of PTSD and co-morbid depression. Injections of NPY to the rodent brain are anxiolytic. Recently we showed that intranasal delivery of NPY to rats before or immediately after exposure to single prolonged stress (SPS) animal model of PTSD prevented development of many biochemical and behavioral symptoms of PTSD, indicating its prophylactic potential. Here, we investigated whether intranasal NPY might provide benefits once symptoms have already developed. One week after exposure to SPS stressors, animals were given intranasal NPY or vehicle and tested on elevated plus maze 2h or 2 days later. The NPY treated rats had lower anxiety-like behavior than vehicle treated rats as indicated by more entries into open arms and fewer into closed arms, lower anxiety index, higher risk assessment and unprotected head dips and reduced grooming time. Their anxiety index was similar to that of unstressed controls. On most of these variables there was no effect of time interval and rats displayed similar overall changes 2h or 2 days after the infusion. Moreover, intranasal NPY led to reduced depressive-like behavior, assessed by forced swim test. Thus, intranasal NPY reversed several behavioral impairments triggered by the traumatic stress of SPS and has potential for non-invasive PTSD therapeutic intervention.

Single intranasal neuropeptide Y infusion attenuates development of PTSD-like symptoms to traumatic stress in rats.

Exposure to severe stress leads to development of neuropsychiatric disorders, including depression and Post-Traumatic Stress Disorder (PTSD) in at-risk individuals. Neuropeptide Y (NPY) is associated with resilience or improved recovery. Therefore exogenous administration to the brain has therapeutic potential although peripheral administration can trigger undesirable side effects. Here, we established conditions with intranasal (IN) NPY infusion to rats to obtain CSF concentrations in the proposed anxiolytic range without significant change in plasma NPY. Rats were pretreated with IN NPY or vehicle before exposure to single prolonged stress (SPS) animal model of PTSD and compared to untreated controls. The IN NPY appeared to lessen the perceived severity of stress, as these animals displayed less time immobile in forced swim part of the SPS. Thirty minutes after SPS the elevation of plasma adrenocorticotropic hormone (ACTH) and corticosterone was not as pronounced in NPY-infused rats and the induction of tyrosine hydroxylase (TH) in locus coeruleus (LC) was attenuated. Seven days after SPS, they displayed lower depressive-like behavior on Forced Swim Test and reduced anxiety-like behavior on Elevated Plus Maze. The prolonged effect of SPS on Acoustic Startle Response was also lower in NPY-infused rats. Plasma ACTH, corticosterone, and hippocampal glucocorticoid receptor levels were significantly above controls only in the vehicle - but not IN NPY-treated group 1week after SPS. Baseline TH mRNA levels in LC did not differ among groups, but increased with forced swim in the vehicle - but not NPY-pretreated animals. Administration of IN NPY after exposure to SPS led to similar, but not identical, reduction in development of anxiety, depressive-like behavior and hyperarousal. The results show that single IN NPY can alter stress-triggered dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and activation of central noradrenergic activity. These findings provide proof of concept for potential of IN NPY for non-invasive prophylactic treatment or early intervention in response to traumatic stress.

Varied mechanisms of oestradiol-mediated regulation of dopamine ß-hydroxylase transcription.

Experiments performed in vivo and in cell culture have demonstrated that oestradiol induces dopamine ß-hydroxylase (DBH) gene transcription. In the present study, we examined oestrogen-responsive elements of the rat DBH gene promoter aiming to characterise the mechanisms of oestradiol-induced DBH transcription. Various mutations and deletions of DBH promoter reporter constructs were tested for responsiveness to 17ß-oestradiol (E(2) ). Mutation of the half palindromic oestrogen response element (ERE) at position -759 reduced the response to E(2) in PC12 cells co-transfected with oestrogen receptor (ER) α, indicating a functional role for this motif. In cells co-transfected with ERß, mutations at the -759 site were unresponsive to E(2) . To characterise the additional E(2) responsive elements, mediated by ERα, the DBH promoter was truncated to the proximal 249 or 200 nucleotides upstream of the transcription start site. Despite either truncation, 10 nm E(2) still elicited an approximately two-fold induction of DBH promoter activity. Mutation of a possible ERE-like sequence at -59 had no effect. The lack of a functional ERE in the proximal region of the rat DBH promoter despite E(2) -mediated DBH promoter activity, suggests regulation by a nonclassical mechanism, such as a membrane-initiated signalling pathway. Moreover, the induction of DBH promoter activity and the rise in DBH mRNA levels were observed within hours. To determine whether membrane-initiated E(2) signalling is involved in rat DBH gene transcription, a membrane impermeable E(2) conjugate, ß-oestradiol-6-(O-carboxy-methyl) oxime-bovine serum albumin (E(2) BSA), was used. Incubation with E(2) -BSA induced luciferase activity and elicited a significant rise in DBH mRNA levels in the ERα transfected cells. The findings indicate two different mechanisms whereby DBH transcription is regulated by E(2) in the presence of ERα. The results implicate both genomic and membrane-initiated mechanisms, mediated by ERα, in E(2) -induced DBH gene transcription.

Adrenocorticotropic hormone elevates gene expression for catecholamine biosynthesis in rat superior cervical ganglia and locus coeruleus by an adrenal independent mechanism.

Classically, upon hypothalamic stimulation, adrenocorticotropic hormone (ACTH) is released from the pituitary and acts on melanocortin 2 receptors (MC2R) in the adrenal cortex, stimulating glucocorticoid synthesis and release. Our earlier studies suggested that ACTH might have a direct effect on sympathetic ganglia. To analyze further the involvement of ACTH in regulation of gene expression of norepinephrine (NE) biosynthetic enzymes, we examined the effect of bilateral adrenalectomy (ADX) of Sprague-Dawley male rats. Fourteen days post-ADX, as expected, plasma ACTH was elevated, and levels of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and MC2R mRNAs in superior cervical ganglia (SCG), and TH mRNA in locus coeruleus (LC) were increased compared with sham-operated animals. To determine effect of pulsatile elevation of ACTH, corticosterone pellets were implanted to ADX rats. Similar to immobilization (IMO) stress ACTH injections to these animals caused a rise in ACTH in plasma and triggered elevation of TH and DBH mRNAs in SCG and in LC with single and repeated daily injections, and MC2R mRNA in SCG with single injections. To study the effect of ACTH in isolated cells, primary cultures of rat SCG were transfected with TH and DBH promoter constructs and treated with ACTH. In agreement with the in vivo data, ACTH elevated their promoter activities similar to levels triggered by cyclic AMP analog. ACTH in the human SK-N-SH neuroblastoma cells increased TH and DBH promoter activity and endogenous DBH mRNA levels. The results show that ACTH can have a direct effect on transcription and gene expression of NE biosynthetic enzymes even without contribution of adrenal hormones.

Estrogen-triggered activation of GTP cyclohydrolase 1 gene expression: role of estrogen receptor subtypes and interaction with cyclic AMP.

Guanosinetriphosphate cyclohydrolase I (GTPCH) catalyzes the initial step in the de novo biosynthesis of (6R)-5,6,7,8-tetrahydrobiopterin, an important determinant of the rate of catecholamine and nitric oxide biosynthesis. Administration of estrogen in vivo was found to elevate GTPCH mRNA levels in several catecholaminergic locations. To examine the mechanism, PC12 cells were co-transfected with a reporter construct containing 2988 bp of rat GTPCH promoter fused to luciferase gene, and expression vectors for estrogen receptors. Addition of 2.5-20 nM of 17 beta-estradiol increased GTPCH promoter-driven luciferase activity in the presence of either estrogen receptor alpha or estrogen receptor beta indicating, for the first time, that 17 beta-estradiol can regulate GTPCH gene expression via transcriptional mechanisms. However, there were differences in dose dependence and time course with estrogen receptor alpha or estrogen receptor beta. With estrogen receptor alpha, the effect was greater with lower doses of 17 beta-estradiol. At the same dose, the response with estrogen receptor beta was observed somewhat earlier than with estrogen receptor alpha and with 20 nM 17 beta-estradiol was effective even after 6 h. These responses to 17 beta-estradiol required estrogen receptors and specific agonists for estrogen receptor alpha and estrogen receptor beta, 4,4,4,-(4-propil-[1H-pyrazole-1,3,5-triyl)tris-phenol and 2,3-bis[4-hydroxyphenyl]propionitrile respectively, triggered increased GTPCH promoter activity. In addition, neither estradiol, nor the selective agonists activated GTPCH promoter without transfection of appropriate estrogen receptor expression vectors. Addition of 17 beta-estradiol, or the selective agonists, also elevated endogenous GTPCH mRNA levels. The results demonstrate that estrogen can have a direct effect on GTPCH gene expression. Although estradiol increased GTPCH promoter activity in the presence of estrogen receptors, it attenuated the response of the promoter and endogenous gene to cyclic AMP, suggesting the crosstalk between estrogen and cyclic AMP pathways in the regulation of GTPCH gene expression. These findings reveal the significance of estrogen in modulating regulation of rate limiting enzyme in the (6R)-5,6,7,8-tetrahydrobiopterin biosynthesis, which may have implications for sex-related differences in vulnerability in related disorders.

Estrogen modifies stress response of catecholamine biosynthetic enzyme genes and cardiovascular system in ovariectomized female rats.

Estrogen is likely involved in the gender specific differences in coping with stress. Activation of catecholamine (CA) biosynthetic enzyme gene expression in central and peripheral CA systems plays a key role in response to stress and in regulation of the cardiovascular system. Here we examined whether estradiol can modulate response of hypothalamic-pituitary-adrenal axis (HPA), gene expression of enzymes related to CA biosynthesis in several noradrenergic locations, tetrahydrobiopterin (BH4) concentration and blood pressure (BP) in response to immobilization stress (IMO) of ovariectomized female rats. Rats were injected with 25 mug/kg estradiol benzoate (EB) or sesame oil once daily for 16 days and subsequently exposed to two hours of IMO. The IMO triggered elevation in plasma ACTH was lessened in EB-pretreated animals. However, estradiol did not alter the IMO-elicited rise of tyrosine hydroxylase mRNA levels in adrenal medulla (AM) and in the nucleus of solitary track (NTS) compared with controls. The response of GTP cyclohydrolase I (GTPCH) mRNA in AM to IMO was also similar in both groups. Several responses to IMO in EB-treated rats were reversed. Instead of IMO-elicited elevation in dopamine beta-hydroxylase mRNA levels in the locus coeruleus, GTPCH mRNA and BH4 levels in the NTS, they were reduced by IMO. In a parallel experiment, BP was monitored during restraint stress. The elevation of BP in response to single or repeated restraint stress was sustained during 2 h in controls and reduced after 70 min stress in EB treated rats. One month after withdrawal of EB treatment, the BP response to restraint was similar to that of rats which never received EB. The results demonstrate that estrogen can modulate responses to stress affecting HPA axis, CA biosynthesis, in central and peripheral noradrenergic systems, and BP.

Heightened transcription for enzymes involved in norepinephrine biosynthesis in the rat locus coeruleus by immobilization stress.

BACKGROUND: The locus coeruleus (LC), a target for CRH neurons, is critically involved in responses to stress. Various physiological stresses increase norepinephrine turnover, tyrosine hydroxylase (TH) enzymatic activity, protein and mRNA levels in LC cell bodies and terminals; however, the effect of stress on other enzymes involved in norepinephrine biosynthesis in the LC is unknown. METHODS: Rats were exposed to single (2 hour) or repeated (2 hour daily) immobilization stress (IMO). Recombinant rat dopamine b-hydroxylase (DBH) cDNA was expressed in E. coli and used to generate antisera for immunohistochemistry and immunoblots in LC. Northern blots were used to assess changes in mRNA levels for TH, DBH, and GTP cyclohydrolase I (GTPCH) in the LC in response to the stress. Conditions were found to isolate nuclei from LC and to use them for run-on assays of transcription. RESULTS: Repeated stress elevated the DBH immunoreactive protein levels in LC. Parallel increases in TH, DBH and GTPCH mRNA levels of about 300% to 400% over control levels were observed with single IMO, and remained at similar levels after repeated IMO. This effect was transcriptionally mediated, and even 30 min of a single IMO significantly increased the relative rate of transcription. CONCLUSIONS: This study is the first to reveal transcriptional activation of the genes encoding catecholamine biosynthetic enzymes in the LC by stress. In addition to TH, changes in DBH and GTPCH gene expression may also contribute to the development of stress-triggered affective disorders.

[Interaction central GABA-B receptors with serotoninergic brain system during regulation of the hypothalamo-hypophyseal-testicular axis by the negative feedback mechanism in rats]. / Vzaimodeistvie tsentral'nylh GAMK-B retseptorov s serotoninergicheskoi sistemoi golovnogo mozga krys v reguliatsii gipotalamo-gipofizarno-semennikovogo kompleksa mekhanizmom otritsatel'noi obratnoi sviazi.

The findings suggest that the GABA inhibits testosterone compensation after hemicastration, due to activation of the mediobasal hypothalamus serotoninergic system in Wistar rats. The GABA seems also to exert an activating effect via the GABA-receptors upon the hypothalamo-pituitary-testicular feedback regulation.

c-Fos deficiency inhibits induction of mRNA for some, but not all, neurotransmitter biosynthetic enzymes by immobilization stress.

Recent studies indicated that c-Fos protein may be mediating stress-elicited transcriptional activation of genes involved in neurotransmitter biosynthesis. However, direct evidence for c-Fos mediating these changes in gene expression has been lacking. Mice with disrupted c-fos gene (+/- or -/- genotypes) were used to examine the effect of immobilization stress on a group of stress-responsive genes. In male adrenals, c-Fos was found not essential for stress-elicited activation of expression of tyrosine hydroxylase, dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase, or neuropeptide Y. In females, immobilization failed to induce adrenal DBH in the c-Fos-deficient mice. In brainstem, c-Fos was indispensable for elevation of DBH mRNA in both genders. The gene, gender, and tissue specificity in the requirement for c-Fos points to diversity in adaptation mechanisms to stress.

Selective in vivo stimulation of stress-activated protein kinase in different rat tissues by immobilization stress.

Stress activated protein kinases (SAPK) are key enzymes mediating the cellular response to stressful stimuli. While they are intensively studied in cultured cells, little is known about their physiological role in vivo, or relevance to pathological conditions. Therefore we examined the effect of various times of immobilization on c-Jun N-terminal protein kinase (JNK) activity in several rat stress responsive tissues and in a number of other locations. The abundance and relative distribution of JNK isoforms, the basal levels, time course and relative magnitude of stress induced JNK activity differed among tissues and regions of the brain of the same animal. JNK immunoreactive proteins were most abundant in the brain, especially in the hippocampus, hypothalamus and frontal cortex. Marked activation in response to immobilization stress was observed in adrenal medulla, adrenal cortex, aorta and hippocampus, less pronounced in locus coeruleus. JNK was not affected in superior cervical ganglia, pituitary, hypothalamus, frontal cortex and cerebellum. In adrenal medulla, the activation of JNK by single immobilization stress is correlated with increased transcription of stress-responsive genes, tyrosine hydroxylase and dopamine beta-hydroxylase. These data suggest a potential role of JNK signal transduction pathway in mediating the long term adaptation to stressful stimuli in vivo.

[Selection vector and ontogenetic development of behavior under domestication of wild Norway rats]. / Vektor otbora i ontogeneticheskie zakonomernosti formirovaniia povedeniia pri deomestikatsii serykh krys.

Hereditary variation in rates of ontogenetic formation of exploratory behavior, glucocorticoid functions, and neurotransmitter systems in wild Norway rats during selection for absence of defensive response towards humans (domestication) is demonstrated. Interrelated shifts in the development of behavior and neurohormonal systems are shown. A comparison of the data obtained with the results of domestication of another species-silver fox-suggests that equally vectorized selection of animals belonging to different taxa produces equally directed changes of their regulatory systems.

[The circadian dynamics of the biochemical activity of the epiphysis in silver foxes]. / Sutochnaia dinamika biokhimicheskoi aktivnosti épifiza serebristo-chernykh lisits.

Specific features of light-dependent biosynthesis of melatonin in the epiphysis are analyzed in female silver-black foxes. Just as in most previously studied animals, the serotonin and 5-hydroxyindolylacetic acid content of epiphysis decreased during the dark time of the 24 h period, while the activity of the melatonin-synthesizing enzyme N-acetyltransferase and melatonin content of epiphysis and plasma, on the contrary, markedly and reliably increased. However, the pattern of changes in the neurotransmitters dopamine and noradrenaline shows specific features.

[Monoamine oxidase activity in the brain of male laboratory mice differing in the capacity for social dominance]. / Aktivnost' monoaminoksidaz v golovnom mozge samtsov laboratornykh myshei, otlichaiushchikhsia po sposobnosti k dominirovaniiu.

The activity of type A and B monoamine oxidases (MAO-A and MAO-B) was studied in the brain stem and cerebral hemispheres of male mice from the PT and CBA inbred laboratory lines with opposite dominance capabilities and in their direct and reciprocal F1 hybrids. MAO-B activity was shown to be lower in the hemispheres of intact PT males and F1 hybrid males, which are genetically predisposed to social domination, than in CBA males. One-hour social stress imposed by grouping mice into a micropopulation resulted in a decrease in MAO-A and MAO-B activity in the hemispheres of dominant PT and F1 hybrid males and an increase in MAO-B activity in the brain stem of subordinant CBA and dominant F1 males.

[Catecholamine levels and tyrosine hydroxylase activity in the brain structures of male mice with a determined behavioral type in a population]. / Urovni katekholaminov i aktivnost' tirozingidroksilazy v strukturakh golovnogo mozga samtsov myshei s determinirovannym tipom povedeniia v populiatsii.

Biosynthetic activity in brain catecholaminergic neurons was studied in male mice with genetically determined dominant (PT strain) and subordinate (CBA) behaviour. The dominant PT mice were characterized by higher levels of catecholamines and tyrosine hydroxylase activity in most of the brain regions in comparison with subordinate CBA mice. Percent of animals with dominant type of behaviour was high among the hybrid F1 males derived from PT and CBA mice. These offsprings inherited also the high levels of noradrenaline and dopamine as well as the high tyrosine hydroxylase activity in the striatum and the brain stem. The obtained data suggest that the level of biosynthetic activity in the brain catecholaminergic neurons is essential for coordination of the brain neurochemical systems in expression of the dominant male mice behaviour in a population.

Participation of gamma-aminobutyric acid in the negative feedback mechanisms of the hypothalamohypophyseotesticular complex.

It is known that gamma-aminobutyric acid (GABA) participates in the regulation of the secretion of many adenohypophyseal hormones [2, 9]. In addition, GABAergic mechanisms may be involved in the regulation of testosterone-dependent aggressive [5] and sexual [8] behavior. A substantial number of studies suggest that GABA and its receptors are capable of participating in the regulation of the secretions of luteinizing hormone (LH). However, its role in this process is not entirely clear, since according to the data of various authors, GABA exerts both inhibitory and activating influences on the secretion of LH [3, 12]. Still less is known of the role of GABA and its receptors in the regulation of the secretion of LH by the negative feedback mechanism, which has been studied in bilaterally castrated rats [3]. However, such a model offers the possibility of investigating only a separate link of this mechanism which regulates the hypothalamohypophyseotesticular complex (HHTC). With regard to the study of the neurochemical regulation of the integral negative feedback system, the use of unilaterally castrated rats is more appropriate for this purpose. In such animals the compensation of the insufficiency of androgens is determined by negative feedback stimulation, and not by the administration of exogenous steroids. In addition, the level of testosterone in the peripheral blood more adequately reflects the state of this mechanism than the level of the gonadotropins [7]. However, the role of GABA and its receptors in the regulation of the integral negative feedback mechanism of the HHTC, so far as we know, remains entirely unstudied. This was in fact the purpose of the present study.

Involvement of the brain catecholaminergic system in the regulation of dominant behavior.

The role of the brain catecholaminergic system in establishing dominant-subordinate relationships in mice of different genotypes was studied using inhibitors of tyrosine hydroxylase (alpha-methyl-p-tyrosine) or of dopamine-beta-hydroxylase (FLA-57) or FLA-57 plus the dopamine precursor, DOPA. Demotion in all dominant and subdominant animals was associated with decreased noradrenaline levels, but the aggressive behavior of dominant male mice depended on the noradrenaline/dopamine ratio. Alterations in this relationship seem to have specific effects on social dominance in animals in the micropopulation, as drug-treated mice do not exhibit changes in their general activity. It can be concluded that brain catecholamines are of prime importance in maintenance of dominance.