Neuropeptide Y, calcitonin gene-related peptide, and neurokinin A in brain regions of HAB rats correlate with anxiety-like behaviours.

Anxiety disorders are pervasive psychiatric disorders causing great suffering. The high (HAB) and low (LAB) anxiety-related behaviour rats were selectively bred to investigate neurobiological correlates of anxiety. We compared the level of neuropeptides relevant for anxiety- and depression-related behaviours in selected brain regions of HAB and LAB rats. Increased anxiety and depression-like behaviours of male and female HAB rats in the elevated plus-maze and forced swim tests were accompanied by elevated levels of neuropeptide Y (NPY) in the prefrontal (PFC), frontal (FC) and cingulate cortex (CCx), the striatum, and periaqueductal grey (PAG). Moreover, HAB rats displayed sex-dependent, elevated levels of calcitonin gene-related peptide (CGRP) in PFC, FC, CCx, hippocampus, and PAG. Higher neurokinin A (NKA) levels were detected in CCx, striatum, and PAG in HAB males and in CCx and hypothalamus in HAB females. Increased neurotensin was detected in CCx and PAG in HAB males and in hypothalamus in HAB females. Elevated corticotropin-releasing hormone (CRH) levels appeared in female HAB hypothalamus. Significant correlations were found between anxiety-like behaviour and NPY, CGRP, NKA, and neurotensin, particularly with NPY in CCx and striatum, CGRP in FC and hippocampus, and NKA in entorhinal cortex. This is the first report of NPY, CGRP, NKA, Neurotensin, and CRH measurements in brain regions of HAB and LAB rats, which showed widespread NPY and CGRP alterations in cortical regions, with NKA and neurotensin changes localised in sub-cortical areas. The results may contribute to elucidate pathophysiological mechanisms underlying anxiety and depression and should facilitate identifying novel therapeutic targets.

CGRP in a gene-environment interaction model for depression: effects of antidepressant treatment.

OBJECTIVE: Genetic and environmental factors interact in the development of major depressive disorder (MDD). While neurobiological correlates have only partially been elucidated, altered levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) in animal models and in the cerebrospinal fluid of depressed patients were reported, suggesting that CGRP may be involved in the pathophysiology and/or be a trait marker of MDD. However, changes in CGRP brain levels resulting from interactions between genetic and environmental risk factors and the response to antidepressant treatment have not been explored. METHODS: We therefore superimposed maternal separation (MS) onto a genetic rat model (Flinders-sensitive and -resistant lines, FSL/FRL) of depression, treated these rats with antidepressants (escitalopram and nortriptyline) and measured CGRP-LI in selected brain regions. RESULTS: CGRP was elevated in the frontal cortex, hippocampus and amygdala (but not in the hypothalamus) of FSL rats. However, MS did not significantly alter levels of this peptide. Likewise, there were no significant interactions between the genetic and environmental factors. Most importantly, neither escitalopram nor nortriptyline significantly altered brain CGRP levels. CONCLUSION: Our data demonstrate that increased brain levels of CGRP are present in a well-established rat model of depression. Given that antidepressants have virtually no effect on the brain level of this peptide, our study indicates that further research is needed to evaluate the functional role of CGRP in the FSL model for depression.

Sex-dependent behavior, neuropeptide profile and antidepressant response in rat model of depression.

A plethora of animal models of depression is described in the literature, aiming at mimicking different aspects of depression. Understanding the link between depression and stress has been and remains a major focus area for development of animal models, but lines of research with a more mechanistic focus targeting deficiencies in neurotransmitter systems or dysfunctional neuronal circuitries and neuroinflammation are also pursued vigorously. The main objectives of the present study were systematically to evaluate strain and sex characteristics of a genetic animal model, the Flinders Sensitive Line (FSL)/ Flinders Resistant Line (FRL), by applying behavioral, molecular and pharmacological measures relevant to depression, and compare it with the outbred Sprague Dawley rat. In addition, we aimed at comparing across strains and sex the expression of NPY, CRF, CGRP in brain regions critically involved in mood regulation, and investigating the responses to escitalopram. In line with the comparisons of FSL and FRL rats, the FSL rats weighed significantly less than SD rats. Overall, escitalopram treatment for 5-6 weeks did not have a major impact on weight, but displayed a significant antidepressant-like effect, however without any changes in NPY, CRH and CGRP expression. Our comparative study of FSL and SD rat with respect to behavioral characteristic, neuropeptide levels in various brain regions (protein and mRNA levels), and response to long-term antidepressant treatment revealed that female FSL rats showed the most pronounced depressive-like phenotype and response to SSRI treatment. However, these findings were not paralleled by changes in measures of NPY, CRH and CGRP function.

Nortriptyline influences protein pathways involved in carbohydrate metabolism and actin-related processes in a rat gene-environment model of depression.

Although most available antidepressants increase monoaminergic neurotransmission, their therapeutic efficacy is likely mediated by longer-term molecular adaptations. To investigate the molecular changes induced by chronic antidepressant treatment we analysed proteomic changes in rat pre-frontal/frontal cortex and hippocampus after nortriptyline (NT) administration. A wide-scale analysis of protein expression was performed on the Flinders Sensitive Line (FSL), a genetically-selected rat model of depression, and the control Flinders Resistant Line (FRL). The effect of NT treatment was examined in a gene-environment interaction model, applying maternal separation (MS) to both strains. In the forced swim test, FSL rats were significantly more immobile than FRL animals, whereas NT treatment reduced immobility time. MS alone did not modify immobility time, but it impaired the response to NT in the FSL strain. In the proteomic analysis, in FSL rats NT treatment chiefly modulated cytoskeleton proteins and carbohydrate metabolism. In the FRL strain, changes influenced protein polymerization and intracellular transport. After MS, NT treatment mainly affected proteins in nucleotide metabolism in FSL rats and synaptic transmission and neurite morphogenesis pathways in FRL rats. When the effects of NT treatment and MS were compared between strains, carbohydrate metabolic pathways were predominantly modulated.

CSF neurochemicals during tryptophan depletion in individuals with remitted depression and healthy controls.

The purpose of this study was to examine the differential effects of acute tryptophan (TRP) depletion vs. sham condition on plasma, cerebrospinal fluid (CSF) biochemical parameters, and mood in the following three subject groups: (1) nine antidepressant-free individuals with remitted depression, (2) eight paroxetine-treated individuals with recently remitted depression, and (3) seven healthy controls. Plasma TRP decreased during TRP depletion and increased during sham condition (p<.01). CSF TRP and 5-hydroxyindoleacetic acid were lower during TRP depletion than sham condition (p<.01 each). During TRP depletion, CSF TRP correlated significantly with the plasma sum of large neutral amino acids (SigmaLNAA) (R=-.52, p=.01), but did not significantly correlate with plasma TRP (R=.15, p=.52). The correlation between CSF TRP and ratio of TRP to SigmaLNAA was R=.41 and p=.06 during TRP depletion, and R=-.44 and p=.04 during sham condition. A negative correlation trend was observed between CSF-TRP levels and peak Hamilton Depression Rating Scale scores during TRP depletion in patients recovered from depression (R=-.45, p=.07), but not in healthy controls (R=-.01, p=.98). CSF neuropeptide Y was higher during TRP depletion than sham condition (t=1.75, p<.10). These results illustrate the importance of assessing plasma SigmaLNAA when using the TRP depletion paradigm. The use of a single CSF sampling technique although practical may result in data acquisition limitations.

Early-life stress and antidepressant treatment involve synaptic signaling and Erk kinases in a gene-environment model of depression.

Stress has been shown to interact with genetic vulnerability in pathogenesis of psychiatric disorders. Here we investigated the outcome of interaction between genetic vulnerability and early-life stress, by employing a rodent model that combines an inherited trait of vulnerability in Flinders Sensitive Line (FSL) rats, with early-life stress (maternal separation). Basal differences in synaptic signaling between FSL rats and their controls were studied, as well as the consequences of early-life stress in adulthood, and their response to chronic antidepressant treatment (escitalopram). FSL rats showed basal differences in the activation of synapsin I and Erk1/2, as well as in alpha CaM kinase II/syntaxin-1 and alpha CaM kinase II/NMDA-receptor interactions in purified hippocampal synaptosomes. In addition, FSL rats displayed a blunted response of Erk-MAP kinases and other differences in the outcome of early-life stress in adulthood. Escitalopram treatment restored some but not all alterations observed in FSL rats after early-life stress. The marked alterations found in key regulators of presynaptic release/neurotransmission in the basal FSL rats, and as a result of early-life stress, suggest synaptic dysfunction. These results show that early gene-environment interaction may cause life-long synaptic changes affecting the course of depressive-like behavior and response to drugs.

Increased levels of cocaine and amphetamine regulated transcript in two animal models of depression and anxiety.

The neurobiological bases of mood disorders remain elusive but both monoamines and neuropeptides may play important roles. The neuropeptide cocaine and amphetamine regulated transcript (CART) was shown to induce anxiety-like behavior in rodents, and mutations in the human CART gene are associated with depression and anxiety. We measured CART-like immunoreactivity (-LI) in genetic rat models of depression and anxiety, i.e. the Flinders Sensitive Line (FSL) and rats selected for High Anxiety-related Behavior (HAB) using a radioimmunoassay. CART-LI was significantly increased in the periaqueductal grey in FSL rats, whereas in the HAB strain it was increased in the hypothalamus, both compared with their respective controls. No line-dependent changes were found in the hippocampus, striatum or frontal cortex. Our results confirm human genetic studies indicating CART as a neurobiological correlate of depression and anxiety, and suggest that its differential regulation in specific brain regions may play a role for the behavioral phenotypes.

Nortriptyline mediates behavioral effects without affecting hippocampal cytogenesis in a genetic rat depression model.

A prevailing hypothesis is that neurogenesis is reduced in depression and that the common mechanism for antidepressant treatments is to increase it in adult hippocampus. Reduced neurogenesis has been shown in healthy rats exposed to stress, but it has not yet been demonstrated in depressed patients. Emerging studies now indicate that selective serotonin reuptake inhibitors can, exert behavioral effects without affecting neurogenesis in mice. Here we extend our previous findings demonstrating that the number of BrdU positive cells in hippocampus was significantly higher in a rat model of depression, the Flinders Sensitive Line (FSL) compared to the control strain the Flinders Resistant Line (FRL). We also show that chronic treatment with the tricyclic antidepressant nortriptyline exerts behavioral effects in the Porsolt forced swim test without affecting hippocampal cell proliferation in the FSL model. These results strengthen the arguments against hypothesis of neurogenesis being necessary in etiology of depression and as requisite for effects of antidepressants, and illustrate the importance of using a disease model and not healthy animals to assess effects of potential therapies for major depressive disorder.

Remodelling by early-life stress of NMDA receptor-dependent synaptic plasticity in a gene-environment rat model of depression.

An animal model of depression combining genetic vulnerability and early-life stress (ELS) was prepared by submitting the Flinders Sensitive Line (FSL) rats to a standard paradigm of maternal separation. We analysed hippocampal synaptic transmission and plasticity in vivo and ionotropic receptors for glutamate in FSL rats, in their controls Flinders Resistant Line (FRL) rats, and in both lines subjected to ELS. A strong inhibition of long-term potentiation (LTP) and lower synaptic expression of NR1 subunit of the NMDA receptor were found in FSL rats. Remarkably, ELS induced a remodelling of synaptic plasticity only in FSL rats, reducing inhibition of LTP; this was accompanied by marked increase of synaptic NR1 subunit and GluR2/3 subunits of AMPA receptors. Chronic treatment with escitalopram inhibited LTP in FRL rats, but this effect was attenuated by prior ELS. The present results suggest that early gene-environment interactions cause lifelong synaptic changes affecting functional and molecular aspects of plasticity, partly reversed by antidepressant treatments.

Acute stress differentially affects corticotropin-releasing hormone mRNA expression in the central amygdala of the "depressed" flinders sensitive line and the control flinders resistant line rats.

Preclinical and clinical evidence suggests that neuropeptides play a role in the pathophysiology of mood disorders. In the present study, we investigated the involvement of the peptides corticotropin-releasing hormone (CRH), neuropeptide Y (NPY) and nociceptin/orphanin FQ (N/OFQ) and of their receptors in the regulation of emotional behaviours. In situ hybridization experiments were performed in order to evaluate the mRNA expression levels of these neuropeptidergic systems in limbic and limbic-related brain regions of the Flinders Sensitive Line (FSL) rats, a putative genetic animal model of depression. The FSL and their controls, the Flinders Resistant Line (FRL) rats, were subjected to one hour acute restraint and the effects of the stress exposure, including possible strain specific changes on these neuropeptidergic systems, were studied. In basal conditions, no significant differences between FSL and FRL rats in the CRH mRNA expression were found, however an upregulation of the CRH mRNA hybridization signal was detected in the central amygdala of the stressed FRL, compared to the non stressed FRL rats, but not in the FSL, suggesting a hypoactive mechanism of response to stressful stimuli in the "depressed" FSL rats. Baseline levels of NPY and N/OFQ mRNA were lower in the FSL rats compared to the FRL in the dentate gyrus of hippocampus and in the medial amygdala, respectively. However, the exposure to stress induced a significant upregulation of the N/OFQ mRNA levels in the paraventricular thalamic nucleus, while in the same nucleus the N/OFQ receptor mRNA expression was higher in the FSL rats. In conclusion, selective alterations of the NPY and N/OFQ mRNA in limbic and limbic-related regions of the FSL rats, a putative animal model of depression, provide further support for the involvement of these neuropeptides in depressive disorders. Moreover, the lack of CRH activation following stress in the "depressed" FSL rats suggests a form of allostatic load, that could alter their interpretation of environmental stimuli and influence their behavioural response to stressful situations.

Search for biological correlates of depression and mechanisms of action of antidepressant treatment modalities. Do neuropeptides play a role?

Dysregulation of the monoaminergic systems is likely a sufficient but not a necessary cause of depression. A wealth of data indicates that neuropeptides, e.g., NPY, CRH, somatostatin, tachykinins and CGRP play a role in affective disorders and alcohol use/abuse. This paper focuses on NPY in etiology and pathophysiology of depression. Decreased peptide and mRNA NPY were found in hippocampus of both the genetic, e.g., the FSL strain, and environmental rat models of depression, e.g., chronic mild stress and early life maternal separation paradigms. Rat models of alcoholism also show altered NPY. Furthermore, NPY is also reduced in CSF of depressed patients. Antidepressive treatments tested so far (lithium, topiramate, SSRIs, ECT and ECS, wheel running) increase NPY selectively in rat hippocampus and in human CSF. Moreover, NPY given icv to rat has antidepressive effects which are antagonized by NPY-Y1 blockers. The data support our hypothesis that the NPY system dysregulation constitutes one of the biological underpinnings of depression and that one common mechanism of action of antidepressive treatment modalities may be effects on NPY and its receptors. In a novel paradigm, early life maternal separation was superimposed on "depressed" FSL and control rats and behavioral and brain neurochemistry changes observed in adulthood. The consequences were more deleterious in genetically vulnerable FSL. Early antidepressive treatment modulated the adult sequelae. Consequently, if these data are confirmed, the ethical and medical question that will be asked is whether it is permissible and advisable to consider prophylactically treating persons at risk.

Chronic amphetamine treatment reduces NGF and BDNF in the rat brain.

Amphetamines (methamphetamine and d-amphetamine) are dopaminergic and noradrenergic agonists and are highly addictive drugs with neurotoxic effect on the brain. In human subjects, it has also been observed that amphetamine causes psychosis resembling positive symptoms of schizophrenia. Neurotrophins are molecules involved in neuronal survival and plasticity and protect neurons against (BDNF) are the most abundant neurotrophins in the central nervous system (CNS) and are important survival factors for cholinergic and dopaminergic neurons. Interestingly, it has been proposed that deficits in the production or utilization of neurotrophins participate in the pathogenesis of schizophrenia. In this study in order to investigate the mechanism of amphetamine-induced neurotoxicity and further elucidate the role of neurotrophins in the pathogenesis of schizophrenia we administered intraperitoneally d-amphetamine for 8 days to rats and measured the levels of neurotrophins NGF and BDNF in selected brain regions by ELISA. Amphetamine reduced NGF levels in the hippocampus, occipital cortex and hypothalamus and of BDNF in the occipital cortex and hypothalamus. Thus the present data indicate that chronic amphetamine can reduce the levels of NGF and BDNF in selected brain regions. This reduction may account for some of the effects of amphetamine in the CNS neurons and provides evidences for the role of neurotrophins in schizophrenia.

The effects of social isolation on neuropeptide Y levels, exploratory and anxiety-related behaviors in rats.

NPY is one of the most abundantly expressed peptides within the CNS, and has been previously demonstrated to be altered in several animal models of depression, as well as to be differentially regulated by acute and repeated stress. The effect of social deprivation, through isolation housing, on brain NPY concentrations in adult rats has not been previously investigated. The effects of 12 weeks of social isolation, in adult rats, on anxiety-related behaviors and central concentrations of NPY in: hypothalamus, amygdala, caudate-putamen, hippocampus, and frontal cortex were evaluated. Single housed animals spent significantly more time on the open arms of the elevated plus maze and in the central area of the open field as compared to pair housed controls. These data are most likely indicative of enhanced exploration and novelty seeking. Concentrations of neuropeptide Y were increased in the caudate-putamen of the single housed subjects. NPY levels in caudate/putamen and hypothalamus were also significantly correlated with time spent in the open arms of the elevated plus maze. These data suggest that chronic social isolation, in these adult Wistar rats, did not increase anxiety but produced enhanced exploration in tests of anxiety, an effect that was associated with NPY concentrations in the striatum and hypothalamus.

Adult life behavioral consequences of early maternal separation are alleviated by escitalopram treatment in a rat model of depression.

In order to study the gene-environment interaction as well as investigate prophylactic/ameliorative effects of early intervention on development of adult life psychopathology, we superimposed maternal separation on an animal model of depression the Flinders Sensitive Line (FSL) rats and their controls the Flinders Resistant Line (FRL) rats and studied behavior following treatment with escitalopram. Animals were maternally separated for 180 min/day from postnatal day 2 (PND 2) to 14. The control groups were left undisturbed. Treatment with escitalopram or vehicle admixed to food pellets was commenced on PND 43 and continued until PND 73. The Porsolt swim test was carried out on PND 65. Baseline FRL/FSL differences in body weight and swim duration, considered to be an inverse index of depression, were found (p's<0.001). In the FSL, maternal separation further decreased swim duration (p<0.001) while the FRL strain was unaffected. Escitalopram had no effect in FRL, but increased swim duration in both maternally non-separated and separated FSL (p<0.05 and p=0.001; respectively). These results confirm the strain differences between the FSL and FRL and demonstrate that the long-term effects of early life adverse experience will to a significant degree depend on the genetic make-up of an individual. Finally, antidepressant treatment reversed behavioral abnormalities caused by genetic and environmental factors. This study highlights the importance of genetic factors in susceptibility to early life adverse events, and demonstrates the efficiency of early antidepressant treatment in reversing behavioral abnormalities, both those caused by genetic factors and by environmental factors.

Increased CRF-like and NPY-like immunoreactivity in adult rats exposed to nicotine during adolescence: relation to anxiety-like and depressive-like behavior.

OBJECTIVE: Recently, animal models have been developed that demonstrate that adolescent nicotine exposure produces neurobehavioral changes which persist into adulthood. This study further examined the impact of adolescent nicotine exposure on anxiety-like and depressive-like behavior, as well as on levels of corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) in this model. METHODS: Male adolescent rats (35-40 days old) were administered nicotine using Nicoderm CQ patches (Smith-Kline Beecham). Behavior in the elevated plus maze (EPM) and forced swim test (FST) was assessed 2-3 weeks after exposure ended. Brain levels of CRF and NPY were then assessed 5-6 weeks after behavioral tests were completed. In addition, blood and brain levels of nicotine resulting from nicotine treatment were examined. RESULTS: After 5 days of exposure to 5 mg/kg/day nicotine, blood levels of nicotine averaged 66+/-5 ng/ml and brain nicotine levels averaged 52+/-4 ng/g. Rats exposed to nicotine displayed an anxiety-like profile in the EPM (i.e., decreased time spent in the open arms) and an antidepressant-like profile in the FST (i.e., less time spent immobile). Rats exposed to nicotine also had increased hypothalamic and frontal cortical CRF, increased hypothalamic and hippocampal NPY, and a decreased ratio of NPY to CRF in the amygdala. CONCLUSIONS: This study demonstrates that adolescent nicotine exposure produces lasting increases in anxiety-like behavior and may reduce depressive-like behavior. These behavioral changes also occurred in concert with alterations in CRF and NPY systems. Thus, lasting neurobehavioral changes associated with adolescent nicotine exposure may be related to allostatic changes in stress peptide systems.

Neuropeptide y in euthymic lithium-treated women with bipolar disorder.

Plasma neuropeptide Y-like immunoreactivity (NPY-LI) and platelet cyclic AMP (cAMP) activity were determined in 13 women with bipolar disorder stabilized on lithium (Li) and 12 matched healthy controls. No differences in plasma NPY-LI were found between the two groups. In euthymic Li-treated bipolar patients, there was an inverse correlation between plasma NPY-LI levels and intracellular cAMP in prostaglandin E1-stimulated platelets. A positive correlation was found between plasma NPY-LI levels and age in both the patient and the control group. Our findings support earlier findings that NPY is capable of inhibiting adenylyl cyclase and that aging is a physiological factor in regulating NPY-LI levels.

Decreased plasma prolactin release in euthymic lithium-treated women with bipolar disorder.

In order to evaluate the effect of treatment with citalopram (CIT) and lithium (Li) on hormone levels in women with bipolar disorder, morning plasma prolactin (PRL) and cortisol (CORT) were measured in 14 nonmedicated depressed patients, 13 depressed patients responding to CIT treatment, 17 euthymic patients on long-term Li treatment, and 11 healthy controls. Plasma PRL values in the Li group were significantly lower than those of the three other groups, suggesting a net inhibitory impact of augmentative effects of Li on dopaminergic activity and serotonergic neurotransmission in the central nervous system. Plasma CORT values in nonmedicated depressed patients were significantly higher than those of healthy controls, indicating hyperactivity of the hypothalamic-pituitary-adrenal system in depression, which appears to be a state-dependent phenomenon, and is normalized upon successful treatment with Li and CIT.

Calcium homeostasis in long-term lithium-treated women with bipolar affective disorder.

The authors investigated the effect of long-term lithium administration on intracellular calcium mobilization. The subjects were 13 women with bipolar affective disorder stabilized on lithium and 12 matched healthy controls. Total and ionized serum calcium, intracellular calcium ion concentration, plasma parathyroid hormone (PTH) and tyrotropin (TSH), serum electrolytes and cyclic AMP (cAMP) activity in platelets were measured. The serum electrolytes sodium, potassium and creatinine and plasma PTH and TSH were all within normal ranges in patients and controls and no differences were found between the two groups. No difference was found in basal and prostaglandin E1 (PGE1)-stimulated cAMP generation in platelets between patients and controls. However, total serum calcium and ionized serum calcium levels were higher in patients than in controls and there was a significant correlation between these two measures. In the patient group, serum lithium concentration correlated positively with stimulated levels of intracellular calcium in platelets. In the present study, no distinct hyperparathyroidism was found in lithium-treated patients. However, our findings indicate that lithium administration affects calcium metabolism in patients with bipolar affective disorder inducing mild hypercalcemia and a dose-dependent normalized calcium mobilization. Furthermore, our results did not support the hypothesis that lithium's primary site of action in bipolar illness may be on signal transduction mechanisms.