Behavioral and Transcriptomic Changes Following Brain-Specific Loss of Noradrenergic Transmission.

Noradrenaline (NE) plays an integral role in shaping behavioral outcomes including anxiety/depression, fear, learning and memory, attention and shifting behavior, sleep-wake state, pain, and addiction. However, it is unclear whether dysregulation of NE release is a cause or a consequence of maladaptive orientations of these behaviors, many of which associated with psychiatric disorders. To address this question, we used a unique genetic model in which the brain-specific vesicular monoamine transporter-2 (VMAT2) gene expression was removed in NE-positive neurons disabling NE release in the entire brain. We engineered VMAT2 gene splicing and NE depletion by crossing floxed VMAT2 mice with mice expressing the Cre-recombinase under the dopamine ß-hydroxylase (DBH) gene promotor. In this study, we performed a comprehensive behavioral and transcriptomic characterization of the VMAT2DBHcre KO mice to evaluate the role of central NE in behavioral modulations. We demonstrated that NE depletion induces anxiolytic and antidepressant-like effects, improves contextual fear memory, alters shifting behavior, decreases the locomotor response to amphetamine, and induces deeper sleep during the non-rapid eye movement (NREM) phase. In contrast, NE depletion did not affect spatial learning and memory, working memory, response to cocaine, and the architecture of the sleep-wake cycle. Finally, we used this model to identify genes that could be up- or down-regulated in the absence of NE release. We found an up-regulation of the synaptic vesicle glycoprotein 2c (SV2c) gene expression in several brain regions, including the locus coeruleus (LC), and were able to validate this up-regulation as a marker of vulnerability to chronic social defeat. The NE system is a complex and challenging system involved in many behavioral orientations given it brain wide distribution. In our study, we unraveled specific role of NE neurotransmission in multiple behavior and link it to molecular underpinning, opening future direction to understand NE role in health and disease.

Regulation of GluA1 phosphorylation by d-amphetamine and methylphenidate in the cerebellum.

Prescription stimulants, such as d-amphetamine or methylphenidate are used to treat suffering from attention-deficit hyperactivity disorder (ADHD). They potently release dopamine (DA) and norepinephrine (NE) and cause phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 in the striatum. Whether other brain regions are also affected remains elusive. Here, we demonstrate that d-amphetamine and methylphenidate increase phosphorylation at Ser845 (pS845-GluA1) in the membrane fraction of mouse cerebellum homogenate. We identify Bergmann glial cells as the source of pS845-GluA1 and demonstrate a requirement for intact NE release. Consequently, d-amphetamine-induced pS845-GluA1 was prevented by ß1-adenoreceptor antagonist, whereas the blockade of DA D1 receptor had no effect. Together, these results indicate that NE regulates GluA1 phosphorylation in Bergmann glial cells in response to prescription stimulants.

Antidepressive effects of targeting ELK-1 signal transduction.

Depression, a devastating psychiatric disorder, is a leading cause of disability worldwide. Current antidepressants address specific symptoms of the disease, but there is vast room for improvement 1 . In this respect, new compounds that act beyond classical antidepressants to target signal transduction pathways governing synaptic plasticity and cellular resilience are highly warranted2-4. The extracellular signal-regulated kinase (ERK) pathway is implicated in mood regulation5-7, but its pleiotropic functions and lack of target specificity prohibit optimal drug development. Here, we identified the transcription factor ELK-1, an ERK downstream partner 8 , as a specific signaling module in the pathophysiology and treatment of depression that can be targeted independently of ERK. ELK1 mRNA was upregulated in postmortem hippocampal tissues from depressed suicides; in blood samples from depressed individuals, failure to reduce ELK1 expression was associated with resistance to treatment. In mice, hippocampal ELK-1 overexpression per se produced depressive behaviors; conversely, the selective inhibition of ELK-1 activation prevented depression-like molecular, plasticity and behavioral states induced by stress. Our work stresses the importance of target selectivity for a successful approach for signal-transduction-based antidepressants, singles out ELK-1 as a depression-relevant transducer downstream of ERK and brings proof-of-concept evidence for the druggability of ELK-1.

Genetic elimination of dopamine vesicular stocks in the nigrostriatal pathway replicates Parkinson's disease motor symptoms without neuronal degeneration in adult mice.

The type 2 vesicular monoamine transporter (VMAT2), by regulating the storage of monoamines transmitters into synaptic vesicles, has a protective role against their cytoplasmic toxicity. Increasing evidence suggests that impairment of VMAT2 neuroprotection contributes to the pathogenesis of Parkinson's disease (PD). Several transgenic VMAT2 mice models have been developed, however these models lack specificity regarding the monoaminergic system targeting. To circumvent this limitation, we created VMAT2-KO mice specific to the dopamine (DA) nigrostriatal pathway to analyze VMAT2's involvement in DA depletion-induced motor features associated to PD and examine the relevance of DA toxicity in the pathogenesis of neurodegeneration. Adult VMAT2 floxed mice were injected in the substancia nigra (SN) with an adeno-associated virus (AAV) expressing the Cre-recombinase allowing VMAT2 removal in DA neurons of the nigrostriatal pathway solely. VMAT2 deletion in the SN induced both DA depletion exclusively in the dorsal striatum and motor dysfunction. At 16 weeks post-injection, motor symptoms were accompanied with a decreased in food and water consumption and weight loss. However, despite an accelerating death, degeneration of nigrostriatal neurons was not observed in this model during this time frame. This study highlights a non-cytotoxic role of DA in our genetic model of VMAT2 deletion exclusively in nigrostriatal neurons.

VMAT2-Mediated Neurotransmission from Midbrain Leptin Receptor Neurons in Feeding Regulation.

Leptin receptors (LepRs) expressed in the midbrain contribute to the action of leptin on feeding regulation. The midbrain neurons release a variety of neurotransmitters including dopamine (DA), glutamate and GABA. However, which neurotransmitter mediates midbrain leptin action on feeding remains unclear. Here, we showed that midbrain LepR neurons overlap with a subset of dopaminergic, GABAergic and glutamatergic neurons. Specific removal of vesicular monoamine transporter 2 (VMAT2) in midbrain LepR neurons (KO mice) disrupted DA accumulation in vesicles, but failed to cause a significant change in the evoked release of either glutamate or GABA to downstream neurons. While KO mice showed no differences on chow, they presented a reduced high-fat diet (HFD) intake and resisted to HFD-induced obesity. Specific activation of midbrain LepR neurons promoted VMAT2-dependent feeding on chow and HFD. When tested with an intermittent access to HFD where first 2.5-h HFD eating (binge-like) and 24-h HFD feeding were measured, KO mice exhibited more binge-like, but less 24-h HFD feeding. Interestingly, leptin inhibited 24-h HFD feeding in controls but not in KO mice. Thus, VMAT2-mediated neurotransmission from midbrain LepR neurons contributes to both binge-like eating and HFD feeding regulation.

Resilience to chronic stress is mediated by noradrenergic regulation of dopamine neurons.

Dopamine (DA) neurons in the ventral tegmental area (VTA) help mediate stress susceptibility and resilience. However, upstream mechanisms controlling these neurons remain unknown. Noradrenergic (NE) neurons in the locus coeruleus, implicated in the pathophysiology of depression, have direct connections within the VTA. Here we demonstrate that NE neurons regulate vulnerability to social defeat through inhibitory control of VTA DA neurons.

Selective genetic disruption of dopaminergic, serotonergic and noradrenergic neurotransmission: insights into motor, emotional and addictive behaviour.

BACKGROUND: The monoaminergic transmitters dopamine (DA), noradrenaline (NE) and serotonin (5-HT) modulate cerebral functions via their extensive effects in the brain. Investigating their roles has led to the creation of vesicular monoaminergic transporter-2 (VMAT2) knockout (KO) mice. While this mutation results in postnatal death, VMAT2-heterozygous (HET) mice are viable and show a complex behavioural phenotype. However, the simultaneous alteration of the 3 systems prevents investigations into their individual functions. METHODS: To assess the specific role of NE, 5-HT and DA, we genetically disrupted their neurotransmission by creating conditional VMAT2-KO mice with targeted recombination. These specific recombinations were obtained by breeding VMAT2(lox/lox) mice with DBHcre, SERTcre and DATcre mice, respectively. We conducted a complete neurochemical and behavioural characterization of VMAT2-HET animals in each system. RESULTS: Conditional VMAT2-KO mice revealed an absence of VMAT2 expression, and a specific decrease in the whole brain levels of each monoamine. Although NE- and 5-HT-depleted mice are viable into adulthood, DA depletion results in postnatal death before weaning. Interestingly, alteration of the DA transmission fully accounted for the increased amphetamine response formerly observed in the VMAT2-HET mice, whereas alteration of the 5-HT system was solely responsible for the increase in cocaine response. LIMITATIONS: We used VMAT2-HET mice that displayed a mild phenotype. Because the VMAT2-KO in DA neurons is lethal, it precluded a straightforward comparison of the full KOs in the 3 systems. CONCLUSION: Given the intermingled functions of NE, 5-HT and DA in regulating cognitive and affective functions, this model will enhance understanding of their respective roles in the pathophysiology of psychiatric disorders.

Presynaptic D2 dopamine receptors control long-term depression expression and memory processes in the temporal hippocampus.

BACKGROUND: Dysfunctional mesocorticolimbic dopamine signaling has been linked to alterations in motor and reward-based functions associated with psychiatric disorders. Converging evidence from patients with psychiatric disorders and use of antipsychotics suggests that imbalance of dopamine signaling deeply alters hippocampal functions. However, given the lack of full characterization of a functional mesohippocampal pathway, the precise role of dopamine transmission in memory deficits associated with these disorders and their dedicated therapies is unknown. In particular, the positive outcome of antipsychotic treatments, commonly antagonizing D2 dopamine receptors (D2Rs), on cognitive deficits and memory impairments remains questionable. METHODS: Following pharmacologic and genetic manipulation of dopamine transmission, we performed anatomic, neurochemical, electrophysiologic, and behavioral investigations to uncover the role of D2Rs in hippocampal-dependent plasticity and learning. Naïve mice (n = 4-21) were used in the different procedures. RESULTS: Dopamine modulated both long-term potentiation and long-term depression in the temporal hippocampus as well as spatial and recognition learning and memory in mice through D2Rs. Although genetic deletion or pharmacologic blockade of D2Rs led to the loss of long-term potentiation expression, the specific genetic removal of presynaptic D2Rs impaired long-term depression and performances on spatial memory tasks. CONCLUSIONS: Presynaptic D2Rs in dopamine fibers of the temporal hippocampus tightly modulate long-term depression expression and play a major role in the regulation of hippocampal learning and memory. This direct role of mesohippocampal dopamine input as uncovered here adds a new dimension to dopamine involvement in the physiology underlying deficits associated with neuropsychiatric disorders.

Is unpredictable chronic mild stress (UCMS) a reliable model to study depression-induced neuroinflammation?

Unipolar depression is one of the leading causes of disability. The pathophysiology of depression is poorly understood. Evidence suggests that inflammation is associated with depression. For instance, pro-inflammatory cytokines are found to be elevated in the peripheral blood of depressed subjects. Cytokine immunotherapy itself is known to induce depressive symptoms. While the epidemiological and biochemical relationship between inflammation and depression is strong, little is known about the possible existence of neuroinflammation in depression. The use of animal models of depression such as the Unpredictable Chronic Mild Stress (UCMS) has already contributed to the elucidation of the pathophysiological mechanisms of depression such as decreased neurogenesis and HPA axis alterations. We used this model to explore the association of depressive-like behavior in mice with changes in peripheral pro-inflammatory cytokines IL-1ß, TNFα and IL-6 level as well as the neuroinflammation by quantifying CD11b expression in brain areas known to be involved in the pathophysiology of depression. These areas include the cerebral cortex, the nucleus accumbens, the bed nucleus of the stria terminalis, the caudate putamen, the amygdala and the hippocampus. The results indicate that microglial activation is significantly increased in the infralimbic, cingulate and medial orbital cortices, nucleus accumbens, caudate putamen, amygdala and hippocampus of the mouse brain as a function of UCMS, while levels of pro-inflammatory cytokines did not differ among the groups. This finding suggests that neuroinflammation occurs in depression and may be implicated in the subject's behavioral response. They also suggest that UCMS could be a potentially reliable model to study depression-induced neuroinflammation.

Fluoxetine effect on aortic nitric oxide-dependent vasorelaxation in the unpredictable chronic mild stress model of depression in mice.

OBJECTIVE: Major depression is an independent risk factor for the development of cardiovascular diseases. However, the exact mechanism by which depression may induce cardiovascular events is unclear. Endothelial dysfunction has been reported as a possible link between depression and subsequent cardiovascular events as described in depressed subjects. The purpose of this study was to investigate endothelial dysfunction and atherosclerosis formation in the aorta of mice exposed to the unpredictable chronic mild stress (UCMS) procedure. METHODS: BALB/c mice were exposed to two 7-week UCMS procedures separated by 6 weeks. Treatments (fluoxetine 10 mg/kg; NaCl 0.9%) started at the third week until the end of the seventh week of each procedure. Endothelial function was evaluated by in vitro assessment of acetylcholine-induced vasorelaxation in aortic rings. By using specific inhibitors for nitric oxide (NO)- and prostacyclin-dependent relaxation, we assessed the part played by NO, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF)-like mediators in endothelium-dependent relaxation. Atherosclerosis was evaluated by histological examination. RESULTS: Depression-like behavior was increased in the UCMS versus unstressed group and was reversed by chronic fluoxetine treatment. Vascular reactivity study indicated that UCMS induced a decrease in the NO-dependent relaxation that was partially compensated by an EDHF-like dependent relaxation. Because fluoxetine per se increased the NO-dependent relaxation, fluoxetine was able to reverse UCMS effect on the NO component and abolished the EDHF-like component. Atherosclerotic lesion was found in aorta of UCMS and nonstressed animals. CONCLUSIONS: As an independent risk factor, UCMS reproduced the endothelial alterations observed in depression but was not sufficient to provoke morphological alterations.

Altered aortic vascular reactivity in the unpredictable chronic mild stress model of depression in mice: UCMS causes relaxation impairment to ACh.

Major depression is an independent risk factor for the development of cardiovascular disease. This impact of depression on vascular function seems to be mediated by the endothelial dysfunction, defined as an impairment of endothelium-dependent vasorelaxation, which represents a reliable predictor of atherosclerosis and has been regularly found to be associated with depression. This study aimed at investigating aortic vascular reactivity in mice submitted to the unpredictable chronic mild stress (UCMS) procedure, a reliable model of depression. The results confirm the effectiveness of the UCMS procedure to induce neuroendocrine, physical and behavioral depression-like alterations as well as a significant decrease of acetylcholine-induced vasorelaxation without any effect on phenylephrine-induced vasoconstriction. In this study, we reveal an altered vascular reactivity in an animal model of depression, demonstrating an endothelial dysfunction reminiscent to the one found in depressed patients.

Early and late-onset effect of chronic stress on vascular function in mice: a possible model of the impact of depression on vascular disease in aging.

Depression is recognized as a predictor of increased cardiac morbidity and mortality. In addition, depressed patients exhibit an increase in the serum markers of endothelial dysfunction and platelet activation involved in the cascade of events leading to atherosclerosis. The purpose of this study was to determine the early and late-onset expression of various vascular markers in a rodent model of depression. Male DBA (an inbred strain of mice)/2J mice were exposed to either 7 weeks of controlled living conditions or unpredictable chronic mild stress (UCMS), and subsequently given daily fluoxetine (10 mg/kg) or NaCl (9%) during the last 5 weeks of the experiment. Depressive-like behavior was evaluated by using motivational and self-care behavior, including the assessment of the animal's coat state and grooming behavior. Enzyme-linked immunoassay was used to quantify matrix metalloproteinase-9 (MMP-9), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and plasminogen activator inhibitor-1 (PAI-1) expression either immediately after the end of the UCMS procedure (short term condition) or 10 months later (long-term condition). Results indicate that 1) UCMS procedure induces a short-term depressive-like behavior in mice, defined as coat state deterioration, an effect that is prevented by fluoxetine treatment; 2) UCMS procedure has no effect on the short-term expression of the studied markers; however, UCMS increases expression of plasminogen activator inhibitor-1 only in the long-term group; 3) fluoxetine treatment is unable to counteract this UCMS-induced change; 4) aging induces behavioral perturbation, defined as a decrease in grooming motivation, and an increase of all the vascular markers in both control and UCMS groups and 5) pretreatment with fluoxetine has no protective effects on aging-induced behavioral and vascular alterations. Thus, in this model of depression-like behavior, UCMS appears to induce late-onset physiological changes, which are consistent with human studies indicating that depression is a risk factor for the development of heart disease.

Association between repeated unpredictable chronic mild stress (UCMS) procedures with a high fat diet: a model of fluoxetine resistance in mice.

Major depressive disorder is a debilitating disease. Unfortunately, treatment with antidepressants (ADs) has limited therapeutic efficacy since resistance to AD is common. Research in this field is hampered by the lack of a reliable natural animal model of AD resistance. Depression resistance is related to various factors, including the attendance of cardiovascular risk factors and past depressive episodes. We aimed to design a rodent model of depression resistance to ADs, associating cardiovascular risk factors with repeated unpredicted chronic mild stress (UCMS). Male BALB/c mice were given either a regular (4% fat) or a high fat diet (45% fat) and subjected to two 7-week periods of UCMS separated by 6 weeks. From the second week of each UCMS procedure, vehicle or fluoxetine (10 mg/kg, i.p.) was administrated daily. The effects of the UCMS and fluoxetine in both diet conditions were assessed using physical (coat state and body weight) and behavioural tests (the reward maze test and the splash test). The results demonstrate that during the second procedure, UCMS induced behavioural changes, including coat state degradation, disturbances in self-care behaviour (splash test) and anhedonia (reward maze test) and these were reversed by fluoxetine in the regular diet condition. In contrast, the high-fat diet regimen prevented the AD fluoxetine from abolishing the UCMS-induced changes. In conclusion, by associating UCMS-an already validated animal model of depression-with high-fat diet regimen, we designed a naturalistic animal model of AD resistance related to a sub-nosographic clinical entity of depression.

Depressive behavior and vascular dysfunction: a link between clinical depression and vascular disease?

As chronic stress and depression have become recognized as significant risk factors for peripheral vascular disease in patients with no prior history of vasculopathy, we interrogated this relationship utilizing an established mouse model of chronic stress/depressive symptoms from behavioral research. Male mice were exposed to 8 wk of unpredictable chronic mild stress (UCMS; e.g., wet bedding, predator sound/smell, random disruption of light/dark cycle), with indexes of depressive behavior (coat status, grooming, and mobility) becoming exacerbated vs. controls. In vascular rings, constrictor (phenylephrine) and endothelium-independent dilator (sodium nitroprusside) responses were not different between groups, although endothelium-dependent dilation (methacholine) was attenuated with UCMS. Nitric oxide synthase (NOS) inhibition was without effect in UCMS but nearly abolished reactivity in controls, while cyclooxygenase inhibition blunted dilation in both. Combined blockade abolished reactivity in controls, although a significant dilation remained in UCMS that was abolished by catalase. Arterial NO production was attenuated by UCMS, although H2O2 production was increased. UCMS mice demonstrated an increased, although variable, insulin resistance and inflammation. However, while UCMS-induced vascular impairments were consistent, the predictive power of aggregate plasma levels of insulin, TNF-alpha, IL-1beta, and C-reactive peptide were limited. However, when separated into tertiles with regard to vascular outcomes, insulin resistance and hypertension were predictive of the most severe vascular impairments. Taken together, these data suggest that aggregate insulin resistance, inflammation, and hypertension in UCMS mice are not robust predictors of vascular dysfunction, suggesting that unidentified mechanisms may be superior predictors of poor vascular outcomes in this model.

Endothelial dysfunction: A potential therapeutic target for geriatric depression and brain amyloid deposition in Alzheimer's disease?

Depression and Alzheimer's disease (AD) are among the most prevalent mental disorders in the elderly. Strong evidence suggests that vascular diseases and vascular risk factors are associated with both depression and AD, and could partially explain the coexistence or the concurrent onset of these two diseases. In particular, endothelial dysfunction appears to play a critical role in the neurobiology of depression and amyloid deposition in the brains of patients with AD. Antidepressants have a significant impact on endothelial function. In addition, several drugs used to treat vascular disease or vascular risk factors, such as calcium-channel blockers, angiotensin-converting enzyme inhibitors and statins, have, to variable extents, significant clinical effects on depressive symptomatology or amyloid deposition in AD. Furthermore, preclinical and clinical data suggest that the nitric oxide and VEGF signaling pathways may be of value for the treatment of depression and AD.

Cocaine-induced reinstatement in rats: evidence for a critical role of cocaine stimulus properties.

RATIONALE: The exact behavioral nature of drug-induced reinstatement of drug seeking is still debated. As an incentive, the drug can have general facilitatory influences on appetitive behaviors. As an interoceptive stimulus, the drug can acquire discriminative properties and control behavior. OBJECTIVE: This study assessed the relative contribution of the incentive versus discriminative properties of cocaine in food-seeking reinstatement. METHODS: In Experiment 1, eight groups of rats were trained to press a lever for food pellets and experienced cocaine (0, 5, 10, or 15 mg/kg; i.p.), either during the operant conditioning sessions or 4 h after, in another environment without food access. In Experiment 2, to dissociate the role of the operant response per se from the consummatory response, two groups of rats experienced food consumption under cocaine (10 mg/kg; i.p.) either during operant conditioning sessions or during alternate sessions of free access to the food. Then, for both experiments, food pellets were withheld and cocaine injections ceased (extinction). The reinstating effects of noncontingent cocaine (10 mg/kg; i.p.) and food pellet delivery were assessed. Locomotor activity was recorded to probe expression of behavioral sensitization. RESULTS: Cocaine reinstated lever pressing only in rats having previously performed the operant responses under cocaine. In contrast, food pellet delivery reinstated lever pressing independently of rats' history with cocaine. Locomotor sensitization was evidenced for all cocaine-pre-exposed rats, dissociating sensitization from reinstatement. CONCLUSIONS: When present during operant conditioning, the stimulus "cocaine" acquires conditioned properties which can then promote reinstatement of the extinguished behavior.