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.

Differential effects of olanzapine, haloperidol and risperidone on calcitonin gene-related peptide in the rat brain.

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide which acts on central nervous system (CNS) neurons and is involved in activities related to dopamine. These effects of CGRP suggest that the peptide may have a role in pathophysiology and treatment of schizophrenia where dopaminergic system hypoactivity in the frontal cortex and hyperactivity in the subcortical structures have been demonstrated. In this study we measured by radioimmunoassay (RIA) the brain levels of CGRP-like immunoreactivity (CGRP-LI) in rats treated with either classical (haloperidol) or atypical (risperidone and olanzapine) antipsychotic drugs. Both haloperidol and risperidone decreased CGRP-LI in the striatum. Risperidone also decreased CGRP-LI in the occipital cortex. On the other hand, olanzapine increased CGRP-LI in the striatum, the frontal cortex and hypothalamus. The differential effects on CGRP could reflect a different profile of side effects and further suggest that CGRP is involved in CNS functions related to psychiatric disorders.

Escitalopram reduces increased hippocampal cytogenesis in a genetic rat depression model.

Hippocampal neurogenesis is potentially implicated in etiology of depression and as the final common mechanism underlying antidepressant treatments. However, decreased neurogenesis has not been demonstrated in depressed patients and, in animals, reduced cytogenesis was shown in healthy rats exposed to stressors, but, so far, not in models of depression. Here we report that the number of BrdU positive cells in hippocampus was (1) significantly higher in a rat model of depression, the Flinders Sensitive Line (FSL) compared to control FRL, (2) increased in both FSL and FRL following maternal separation, (3) reduced by escitalopram treatment in maternally separated animals to the level found in non-separated animals. These results argue against the prevailing hypothesis that adult cytogenesis is reduced in depression and that the common mechanism underlying antidepressant treatments is to increase adult cytogenesis. The results also point to the importance of using a disease model and not healthy animals for testing effects of potential treatments for human depression and suggest other cellular mechanisms of action than those that had previously been proposed for escitalopram.

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.

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.

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.

Effects of acute and subchronic d-amphetamine on ventral striatal concentrations of neurotensin and neuropeptide Y in rats treated with antipsychotic drugs.

We have reported that acute d-amphetamine increases extracellular concentrations (efflux) of neurotensin-like immunoreactivity (NT-LI) and neuropeptide Y-LI (NPY-LI) in the ventral striatum (VSTR) of freely moving rats, effects that are abolished by chronic administration of haloperidol and risperidone admixed to food pellets. In this study we further investigated the d-amphetamine effects on NT-LI and NPY-LI efflux in VSTR and their content in selected brain regions. Rats received haloperidol, risperidone or vehicle for 30days and saline or d-amphetamine either on days 22-29 and/or day 30. Seven day d-amphetamine administration decreased basal NT-LI and NPY-LI efflux in vehicle-treated rats; pretreatment with haloperidol counteracted these effects, while pretreatment with risperidone had effect only on NT-LI. Acute d-amphetamine after the seven day d-amphetamine increased NT-LI only. Pretreatment with haloperidol or risperidone abolished the effects of acute d-amphetamine on NT-LI and NPY-LI. Acute and seven day d-amphetamine increased NT-LI and NPY-LI contents in striatum; seven day d-amphetamine also increased NT-LI in frontal and occipital cortex and both NT-LI and NPY-LI in hippocampus. Our results suggest that NT and NPY are involved in both the pathophysiology and the therapeutics of schizophrenia.

Effect of chronic olanzapine treatment on nerve growth factor and brain-derived neurotrophic factor in the rat brain.

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are proteins involved in neuronal survival, neurite outgrowth and synapse formation. Recent observations suggest that treatment with typical and atypical antipsychotic drugs affect NGF and BDNF levels in the rat brain. The atypical antipsychotic olanzapine has a low incidence of side effects, such as extrapyramidal and anticholinergic symptoms. Since NGF and BDNF are involved in the regulation of cholinergic, dopaminergic and serotonergic neurons in the central nervous system (CNS) we hypothesized that chronic olanzapine treatment will influence the distribution of NGF and BDNF in the rat brain. To test this hypothesis we administered olanzapine for 29 days in the drinking water at the doses of 3 and 15 mg/kg body weight and measured the levels of NGF and BDNF in the brain of Wistar rats. Olanzapine increased NGF in the hippocampus, occipital cortex and hypothalamus. In contrast, olanzapine decreased BDNF in the hippocampus and frontal cortex. Although the significance of these findings is not clear, a heuristic hypothesis is that olanzapine's clinical effects and a favorable side effect profile are in part mediated by neurotrophins.

Effects of haloperidol and risperidone on neurotensin levels in brain regions and neurotensin efflux in the ventral striatum of the rat.

Neurotensin (NT) may play a role in the pathophysiology of schizophrenia and in the mechanism of action of antipsychotic drugs. Here we studied the effects of a 30-day regimen of haloperidol (1.15 mg/100 g food) and risperidone (1.15 and 2.3 mg/100 g food) on NT-like immunoreactivity (-LI) levels in brain tissue and NT-LI efflux in the ventral striatum (VSTR) of the rat. Haloperidol, but not risperidone, increased NT-LI levels in the striatum. In the occipital cortex, risperidone, but not haloperidol, decreased levels of NT-LI. In the hippocampus and the frontal cortex both haloperidol and risperidone (the higher dose) increased NT-LI levels. In the VSTR, haloperidol and risperidone (the higher dose) decreased NT-LI efflux and abolished the stimulatory effect of d-amphetamine (1.5 mg/kg, s.c.). Thus, changes in NT occur in response to antipsychotic drugs and psychostimulants that may be relevant for the pathophysiology and treatment of schizophrenia.

Chronic antipsychotic treatment selectively alters nerve growth factor and neuropeptide Y immunoreactivity and the distribution of choline acetyl transferase in rat brain regions.

Neuropeptides and neurotrophins play a number of roles in the central nervous system (CNS). Nerve growth factor (NGF), the first characterized member of the family of neurotrophins, influences the synthesis of some neuropeptides, including neuropeptide Y (NPY), a peptide amply expressed in the CNS, interacting with catecholamines and modifying behaviour. In this study we investigated whether antipsychotic treatment affects the constitutive levels of NGF-, NPY- and choline acetyl transferase (ChAT)-like immunoreactivities (-LI) in the CNS. Rats were fed food supplemented with haloperidol (1.15 mg/100 g food), risperidone (1.15 or 2.3 mg/100 g food), or vehicle. After 29 d treatment animals were sacrificed with focused high-energy microwave irradiation for radioimmunoassay (RIA) of NPY-LI, by decapitation for analysis of NGF, and by perfusion for immunocytochemistry. Haloperidol and risperidone elevated NGF-LI concentrations in the hypothalamus but decreased NGF-LI in the striatum and hippocampus. In contrast, antipsychotics did not alter NPY-LI in the striatum. Haloperidol increased NPY-LI concentration in the occipital cortex, while risperidone increased NPY-LI in the occipital cortex, hippocampus, and hypothalamus. Significant decreases in ChAT immunoreactivity in large-size neurons following both haloperidol and risperidone treatments in the septum as well as Meynert's nucleus were observed. Our findings demonstrate that antipsychotic drugs alter the regional brain levels of NGF-LI, NPY-LI and ChAT-LI and raise the possibility that these effects are implicated in their pharmacological and therapeutic properties.

Extracellular levels of NPY in the dorsal hippocampus of freely moving rats are markedly elevated following a single electroconvulsive stimulation, irrespective of anticonvulsive Y1 receptor blockade.

Neuropeptide Y (NPY) has been proposed to play a role in the pathophysiology of depression and also to act as an endogenous anticonvulsant. Repeated administration of electroconvulsive stimulations (ECS) has been shown to induce a long-term increase in hippocampal NPY neurotransmission, while the effects of single ECS are largely unexplored. In this study, we assessed extracellular levels of NPY in the dorsal hippocampus of freely moving rats following a single ECS. We also studied the effect of locally administered BIBP3226, a selective NPY Y1 receptor antagonist with reported anticonvulsant properties, on the duration of the ECS-induced seizure and NPY release in freely moving animals. Our data demonstrate that a single ECS increases extracellular NPY-like immunoreactivity (LI) levels in the dorsal hippocampus, reaching statistical significance 2h following the treatment. KCl transiently and calcium-dependently increased extracellular levels of NPY, suggesting that the measured NPY-LI is derived from functional neurons. Local BIBP3226 perfusion essentially abolished the ECS-induced seizure but had no effect on the basal NPY-LI outflow or on the ECS-induced rise in extracellular NPY levels. Our data are in line with the hypothesis that one mechanism of action of ECS is to release NPY in the hippocampus and suggest that the increase is in itself not associated with anticonvulsant activity but may represent other properties of NPY.

Effects of olanzapine on extracellular concentrations and tissue content of neurotensin in rat brain regions.

We have previously shown that both the psychostimulant d-amphetamine and the antipsychotics haloperidol and risperidone affect extracellular concentrations and tissue content of neurotensin (NT) in distinct brain regions. This study investigated the effects of acute olanzapine (1, 5mg/kg, s.c.) on extracellular NT-like immunoreactivity (-LI) concentrations in the ventral striatum (vSTR) and the medial prefrontal cortex (mPFC), and the effects of acute d-amphetamine (1.5mg/kg, s.c.) on extracellular NT-LI in these brain regions after a 30-day olanzapine (15mg/kg, p.o.) administration in rats. The effects of a 30-day olanzapine (3, 15mg/kg, p.o.) administration and d-amphetamine (1.5mg/kg, s.c.) coadministration during either the last day (acute) or the last 8days (chronic) on NT-LI tissue content in distinct rat brain regions were also studied. Acute olanzapine increased extracellular NT-LI, in both the vSTR and the mPFC. Chronic olanzapine increased and decreased basal extracellular NT-LI in the vSTR and the mPFC, respectively, and abolished the stimulatory effects of acute d-amphetamine on extracellular NT-LI in these brain regions. Chronic olanzapine as well as acute and chronic d-amphetamine affected NT-LI tissue content in a brain region-dependent manner. Chronic olanzapine prevented the effects of acute and chronic d-amphetamine on NT-LI tissue content in certain brain regions. The fact that olanzapine and d-amphetamine affected extracellular NT-LI in the vSTR and mPFC as well as NT-LI tissue content in distinct brain regions further supports the notion that NT plays a role in the therapeutic actions of antipsychotic drugs and possibly also in the pathophysiology of schizophrenia.

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.

Housing conditions modulate escitalopram effects on antidepressive-like behaviour and brain neurochemistry.

Despite limited understanding of the pathophysiology of depression and the underlying mechanisms mediating antidepressant effects, there are several efficient treatments. The anhedonia symptoms of depression are characterized by decreased motivation and drive and imply possible malfunctioning of the mesolimbic dopamine system, whereas cognitive deficits might reflect decreased plasticity in hippocampus. In female Flinders Sensitive Line (FSL) rats, a model of depression, we compared the effects of three long-term antidepressant treatments: voluntary running, escitalopram and the combination of both on antidepressant-like behaviour in the Porsolt swim test (PST), and on regulation of mRNA for dopamine and neuropeptides in striatal dopamine pathways and brain-derived neurotrophic factor (BDNF) in hippocampus. Escitalopram diet attenuated running behaviour in FSL rats but not in non-depressed controls rats. In the PST the running group had increased climbing activity (noradrenergic/dopaminergic response), whereas the combination of escitalopram and running-wheel access increased swimming (serotonergic response). Running elevated mRNA for dynorphin in caudate putamen and BDNF in hippocampus. The combined treatment down-regulated D1 receptor and enkephalin mRNA in accumbens. Escitalopram alone did not affect behaviour or mRNA levels. We demonstrate a novel behavioural effect of escitalopram, i.e. attenuation of running in 'depressed' rats. The antidepressant-like effect of escitalopram was dependent on the presence of a running wheel, but not actual running indicating that the environment influenced the antidepressant effect of escitalopram. Different patterns of mRNA changes in hippocampus and brain reward pathways and responses in the PST by running and escitalopram suggest that antidepressant-like responses by running and escitalopram are achieved by different mechanisms.

Social isolation increases number of newly proliferated cells in hippocampus in female flinders sensitive line rats.

Genetic background influences the responsiveness to stress and plays a crucial role in the pathophysiology of depression. In an animal model of depression, Flinders Sensitive Line rats, and Sprague Dawley controls we analyzed if 7 weeks of social isolation of adult animals affect the number of newly proliferated cells in the dentate gyrus or mRNAs of Neuropeptide Y (NPY), the NPY-Y1 receptor, nociceptin, BDNF, and the serotonin 5HT1A and 5HT2A receptors, which are molecules involved in hippocampal plasticity. Since depressive illness more frequently affects women than men, and females seem to respond differently to stressful experiences than males, female rats were used in this study. Bromodeoxyuridine, which is a thymidin analogue that is incorporated into the DNA of newly formed cells, was administered during 9 days to even out the effects of hormonal fluctuations. Social isolation increased the number of newly proliferated Bromodeoxyuridine-immunoreactive cells in the Flinders Sensitive Line rats, whereas it had no impact on the number of cells in the Sprague Dawley strain. Group housed Sprague Dawley rats had a higher expression of BDNF, NPY, and the serotonin 5HT2A receptor mRNA than "depressed" Flinders Sensitive Line. Social isolation downregulated these molecules in Sprague Dawley but not in Flinders Sensitive Line rats thereby eliminating the differences between the two strains. We demonstrate strain and gender specific responses to stress induced regulation of factors important for hippocampal plasticity.

d-Amphetamine-induced increase in neurotensin and neuropeptide Y outflow in the ventral striatum is mediated via stimulation of dopamine D1 and D2/3 receptors.

The neuroanatomical and functional relationships between dopamine (DA) and neurotensin (NT) and DA and neuropeptide Y (NPY) suggest a role for these neuropeptides in DA-related neuropsychiatric disorders. By employing a microdialysis technique in conjunction with radioimmunoassay (RIA), the effects of d-amphetamine per se or after pretreatment with DA receptor antagonists on NT and NPY outflow were determined in the ventral striatum (VSTR) of the rat. One hour after a subcutaneous (s.c.) injection of saline, the DA-D(1) receptor antagonist SCH 23390 (0.3 mg/kg), or the DA-D(2/3) receptor antagonist raclopride (1.0 mg/kg), animals were injected s.c. with either saline or d-amphetamine (1.5 mg/kg). d-Amphetamine significantly increased extracellular NT- and NPY-like immunoreactivity (LI) concentrations compared with control animals. Administration of SCH 23390 or raclopride did not significantly affect NT-LI or NPY-LI concentrations. However, pretreatment with either SCH 23390 or raclopride abolished the stimulatory effect of d-amphetamine on NT-LI and NPY-LI. These findings demonstrate that d-amphetamine increases extracellular concentrations of NT-LI and NPY-LI in the VSTR through a mechanism that initially involves stimulation of either DA-D(1) or DA-D(2/3) receptors but appears to require both. In conclusion, changes in dopaminergic neurotransmission via DA-D(1) and DA-D(2/3) receptors affect the outflow of both NT and NPY in the VSTR.