Locus Coeruleus Noradrenergic Modulation of Diurnal Corticosterone, Stress Reactivity, and Cardiovascular Homeostasis in Male Rats.

INTRODUCTION: Activation of the locus coeruleus-noradrenergic (LC-NA) system during awakening is associated with an increase in plasma corticosterone and cardiovascular tone. These studies evaluate the role of the LC in this corticosterone and cardiovascular response. METHODS: Male rats, on day 0, were treated intraperitoneally with either DSP4 (50 mg/kg body weight) (DSP), an LC-NA specific neurotoxin, or normal saline (SAL). On day 10, animals were surgically prepared with jugular vein (hypothalamic-pituitary-adrenal [HPA] axis) or carotid artery (hemodynamics) catheters and experiments performed on day 14. HPA axis activity, diurnally (circadian) and after stress (transient hemorrhage [14 mL/kg body weight] or air puff-startle), and basal and post-hemorrhage hemodynamics were evaluated. On day 16, brain regions from a subset of rats were dissected for norepinephrine and corticotropin-releasing factor (CRF) assay. RESULTS: In DSP rats compared to SAL rats, (1) regional brain norepinephrine was decreased, but there was no change in median eminence or olfactory bulb CRF content; (2) during HPA axis acrophase, the plasma corticosterone response was blunted; (3) after hemorrhage and air puff-startle, the plasma adrenocorticotropic hormone response was attenuated, whereas the corticosterone response was dependent on stressor category; (4) under basal conditions, hemodynamic measures exhibited altered blood flow dynamics and systemic vasodilation; and (5) after hemorrhage, hemodynamics exhibited asynchronous responses. CONCLUSION: LC-NA modulation of diurnal and stress-induced HPA axis reactivity occurs via distinct neurocircuits. The integrity of the LC-NA system is important to maintain blood flow dynamics. The importance of increases in plasma corticosterone at acrophase to maintain short- and long-term cardiovascular homeostasis is discussed.

Early Life Stress Associated With Increased Striatal N-Acetyl-Aspartate: Cerebrospinal Fluid Corticotropin-Releasing Factor Concentrations, Hippocampal Volume, Body Mass and Behavioral Correlates.

INTRODUCTION: Using proton magnetic resonance spectroscopy imaging (1H-MRSI), the effects of early life stress (ELS) on nonhuman primate striatal neuronal integrity were examined as reflected by N-acetyl aspartate (NAA) concentrations. NAA measures were interrogated through examining their relationship to previously documented ELS markers -- cerebrospinal fluid (CSF) corticotropin-releasing factor (CRF) concentrations, hippocampal volume, body mass and behavioral timidity. Rodent models of depression exhibit increases in neurotrophic effects in the nucleus accumbens (NAcc). We hypothesized that rearing under conditions of ELS [Variable Foraging Demand: (VFD)] would produce persistent elevations of NAA concentrations (in absolute or ratio form) in ventral striatum/caudate nucleus (VS/CN) with altered correlation to ELS markers. METHODS: Eleven bonnet macaque males reared under VFD conditions and seven age-matched control subjects underwent 1H-MRSI during young adulthood. Voxels were placed over ventral striatum/caudate nucleus (VS/CN) to capture NAcc. Cisternal CSF CRF concentrations, hippocampal volume, body mass and response to a human intruder had been previously determined. RESULTS: VFD-reared monkeys exhibited significantly increased NAA/Cr concentrations in right VS/CN in comparison to normally-reared controls, controlling for multiple comparisons. In comparison to controls, VFD CSF CRF concentrations were directly associated with right VS/CN absolute NAA. Left hippocampal volume was inversely associated with left VS/CN N-acetyl aspartate/creatine (NAA/Cr) in VFD-reared but not in controls. Disruption of a normative inverse correlation between left VS/CN NAA and body mass was noted in VFD. Only non-VFD subjects exhibited a direct relationship between timidity response to an intruder and right VS/CN NAA. CONCLUSION: ELS produced persistent increases in VS/CN NAA, which demonstrated specific patterns of association (or lack thereof) to ELS markers in comparison to non-VFD subjects. The data are broadly consistent with a stable nonhuman primate phenotype of anxiety and mood disorder vulnerability whereby in vivo indicators of neuronal integrity, although reduced in hippocampus, are increased in striatum. The findings may provide a catalyst for further studies in humans and other species regarding a reciprocal hippocampal/NAcc relationship in affective disorders.

Reduced left ventricular dimension and function following early life stress: A thrifty phenotype hypothesis engendering risk for mood and anxiety disorders.

BACKGROUND: Early life stress (ELS) in macaques in the form of insecure maternal attachment putatively induces epigenetic adaptations resulting in a "thrifty phenotype" throughout the life cycle. For instance, ELS induces persistent increases in insulin resistance, hippocampal and corpus callosum atrophy and reduced "behavioral plasticity", which, taken together, engenders an increased risk for mood and anxiety disorders in humans but also a putative sparing of calories. Herein, we test the hypothesis whether a thrifty phenotype induced by ELS is peripherally evident as hypotrophy of cardiac structure and function, raising the possibility that certain mood disorders may represent maladaptive physiological and central thrift adaptations. METHODS: 14 adult bonnet macaques (6 males) exposed to the maternal variable foraging demand (VFD) model of ELS were compared to 20 non-VFD adult subjects (6 males). Left ventricle end-diastolic dimension (LVEDD), Left ventricle end-systolic dimension (LVESD) and stroke volume (SV) were calculated using echocardiography. Blood pressure and heart rate were measured only in females. Previously obtained neurobehavioral correlates available only in males were analyzed in the context of cardiac parameters. RESULTS: Reduced LVESD (p < 0.05) was observed when controlled for age, sex, body weight and crown-rump length whereas ejection fraction (EF) (p = 0.037) was greater in VFD-reared versus non-VFD subjects. Pulse pressure was lower in VFD versus non-VFD females (p < 0.05). Male timidity in response to a human intruder was associated with reduced LVEDD (p < 0.05). CONCLUSIONS: ELS is associated with both structural and functional reductions of left ventricular measures, potentially implying a body-wide thrifty phenotype. Parallel "thrift" adaptations may occur in key brain areas following ELS and may play an unexplored role in mood and anxiety disorder susceptibility.

Test-Retest Reliability of the SERT Imaging Agent 11C-HOMADAM in Healthy Humans.

The aim of this study was to measure the test-retest reliability of 11C-N,N-dimethyl-2-(2'-amino-4'-hydroxymethylphenylthio)benzylamine (11C-HOMADAM) imaging of serotonin transporter (SERT) density in healthy control subjects. Methods: Two female and 2 male volunteers participated in the study, with each undergoing three 90-min 11C-HOMADAM PET scans. Time-activity curves were derived from SERT-rich structures and fit to 2 models: a simplified reference tissue model and a multilinear graphical model. Binding potential, the ratio of specifically bound uptake to nondisplaceable uptake at equilibrium, was calculated from the model parameter estimates. Ninety-five percent confidence intervals and the intraclass correlation coefficient (ICC) were calculated and adjusted for repeated measures. Results: The ICC values ranged from -0.13 in the dorsal raphe to 0.88 in the caudate nucleus. The highest average ICC values were in the striatum, but other regions were sensitive to measurement outliers. Conclusion: Good-to-excellent test-retest reliability was observed for SERT binding in the striatum. The dorsal raphe ICC value was sensitive to a measurement outlier. 11C-HOMADAM binding potential calculated from the simplified reference tissue model and the multilinear graphical model were robust and in good agreement.

Adult glucocorticoid receptor mRNA expression volatility in response to an acute stressor and juvenile CSF corticotropin-releasing factor: A pilot neurodevelopmental study.

INTRODUCTION: Early life stress (ELS) has been shown to play a role in establishing persistent maladaptive HPA axis modifications that may contribute to the pathogenesis of mood and anxiety disorders. Central glucocorticoid receptor (GR) messenger RNA (mRNA) expression may facilitate (mal)adaptive responsivity to ELS. The role of adult monocytic GR mRNA expression, a putative CNS proxy, during acute stress exposure was explored as well as the ELS marker, juvenile cerebrospinal fluid (CSF) corticotropin-releasing factor. METHODS: Six adult macaques (three of which were exposed to variable foraging demand, a form of ELS) underwent acute restraint. Baseline GR expression and plasma cortisol concentrations were separately measured followed by subsequent measurements following stress completion (t=0min, 4h, 5 days and 7 days). Juvenile CSF CRF concentrations were available in five subjects to determine their developmental association with GR expression in response to stress. RESULTS: As expected acute restraint stress produced a significant increase in plasma cortisol concentrations most robustly observed at 4h post-stress time point. There was a significant juvenile CSF CRF concentration x time interaction in predicting adult GR mRNA expression in response to stress (partial η2=0.80). During acute stress juvenile CRF concentrations negatively predicted GR expression and during recovery, "flipped" to positively predict expression. Juvenile CSF CRF concentrations positively correlated with the volatility of adult GR mRNA expression. CONCLUSIONS: During acute stress, relatively high CSF CRF concentrations are associated with relatively rapid reductions in GR expression. Return to an ambient post-stress state was characterized by a direct relationship, consistent with increased HPA axis restraint in high CRF subjects. An ELS-associated allostatic adaptation suggests relative elevations of juvenile CSF CRF concentration set the stage for a relative hyper-volatility of adult GR mRNA expression in response to acute stress with potential long-term implications for HPA axis regulation.

Prenatal stress-induced increases in hippocampal von Willebrand factor expression are prevented by concurrent prenatal escitalopram.

Prenatal stress has been linked to deficits in neurological function including deficient social behavior, alterations in learning and memory, impaired stress regulation, and susceptibility to adult disease. In addition, prenatal environment is known to alter cardiovascular health; however, limited information is available regarding the cerebrovascular consequences of prenatal stress exposure. Vascular disturbances late in life may lead to cerebral hypoperfusion which is linked to a variety of neurodegenerative and psychiatric diseases. The known impact of cerebrovascular compromise on neuronal function and behavior highlights the importance of characterizing the impact of stress on not just neurons and glia, but also cerebrovasculature. Von Willebrand factor has previously been shown to be impacted by prenatal stress and is predictive of cerebrovascular health. Here we assess the impact of prenatal stress on von Willebrand factor and related angiogenic factors. Furthermore, we assess the potential protective effects of concurrent anti-depressant treatment during in utero stress exposure on the assessed cerebrovascular endpoints. Prenatal stress augmented expression of von Willebrand factor which was prevented by concurrent in utero escitalopram treatment. The functional implications of this increase in von Willebrand factor remain elusive, but the presented data demonstrate that although prenatal stress did not independently impact total vascularization, exposure to chronic stress in adulthood decreased blood vessel length. In addition, the current study demonstrates that production of reactive oxygen species in the hippocampus is decreased by prenatal exposure to escitalopram. Collectively, these findings demonstrate that the prenatal experience can cause complex changes in adult cerebral vascular structure and function.

In vivo investigation of escitalopram's allosteric site on the serotonin transporter.

Escitalopram is a commonly prescribed antidepressant of the selective serotonin reuptake inhibitor class. Clinical evidence and mapping of the serotonin transporter (SERT) identified that escitalopram, in addition to its binding to a primary uptake-blocking site, is capable of binding to the SERT via an allosteric site that is hypothesized to alter escitalopram's kinetics at the SERT. The studies reported here examined the in vivo role of the SERT allosteric site in escitalopram action. A knockin mouse model that possesses an allosteric-null SERT was developed. Autoradiographic studies indicated that the knockin protein was expressed at a lower density than endogenous mouse SERT (approximately 10-30% of endogenous mouse SERT), but the knockin mice are a viable tool to study the allosteric site. Microdialysis studies in the ventral hippocampus found no measurable decrease in extracellular serotonin response after local escitalopram challenge in mice without the allosteric site compared to mice with the site (p=0.297). In marble burying assays there was a modest effect of the absence of the allosteric site, with a larger systemic dose of escitalopram (10-fold) necessary for the same effect as in mice with intact SERT (p=0.023). However, there was no effect of the allosteric site in the tail suspension test. Together these data suggest that there may be a regional specificity in the role of the allosteric site. The lack of a robust effect overall suggests that the role of the allosteric site for escitalopram on the SERT may not produce meaningful in vivo effects.

Synthesis, binding affinity, radiolabeling, and microPET evaluation of 4-(2-substituted-4-substituted)-8-(dialkylamino)-6-methyl-1-substituted-3,4-dihydropyrido[2,3-b]pyrazin-2(1H)-ones as ligands for brain corticotropin-releasing factor type-1 (CRF1) receptors.

Compounds 1-14 were synthesized in a search for high-affinity CRF1 receptor ligands that could be radiolabeled with (11)C or (18)F for use as positron emission tomography (PET) radiotracers. Derivatives of 2 were developed which contained amide N-fluoroalkyl substituents. Compounds [(18)F]24 and [(18)F]25 were found to have appropriate lipophilicities of logP7.4=2.2 but microPET imaging with [(18)F]25 demonstrated limited brain uptake.

Synthesis, F-18 radiolabeling, and microPET evaluation of 3-(2,4-dichlorophenyl)-N-alkyl-N-fluoroalkyl-2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-amines as ligands of the corticotropin-releasing factor type-1 (CRF1) receptor.

A series of 3-(2,4-dichlorophenyl)-N-alkyl-N-fluoroalkyl-2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-amines were synthesized and evaluated as potential positron emission tomography (PET) tracers for the corticotropin-releasing factor type-1 (CRF1) receptor. Compounds 27, 28, 29, and 30 all displayed high binding affinity (⩽1.2 nM) to the CRF1 receptor when assessed by in vitro competition binding assays at 23 °C, whereas a decrease in affinity (⩾10-fold) was observed with compound 26. The logP7.4 values of [(18)F]26-[(18)F]29 were in the range of ∼2.2-2.8 and microPET evaluation of [(18)F]26-[(18)F]29 in an anesthetized male cynomolgus monkey demonstrated brain penetrance, but specific binding was not sufficient enough to differentiate regions of high CRF1 receptor density from regions of low CRF1 receptor density. Radioactivity uptake in the skull, and sphenoid bone and/or sphenoid sinus during studies with [(18)F]28, [(18)F]28-d8, and [(18)F]29 was attributed to a combination of [(18)F]fluoride generated by metabolic defluorination of the radiotracer and binding of intact radiotracer to CRF1 receptors expressed on mast cells in the bone marrow. Uptake of [(18)F]26 and [(18)F]27 in the skull and sphenoid region was rapid but then steadily washed out which suggests that this behavior was the result of binding to CRF1 receptors expressed on mast cells in the bone marrow with no contribution from [(18)F]fluoride.

Prenatal stress, regardless of concurrent escitalopram treatment, alters behavior and amygdala gene expression of adolescent female rats.

Depression during pregnancy has been linked to in utero stress and is associated with long-lasting symptoms in offspring, including anxiety, helplessness, attentional deficits, and social withdrawal. Depression is diagnosed in 10-20% of expectant mothers, but the impact of antidepressant treatment on offspring development is not well documented, particularly for females. Here, we used a prenatal stress model of maternal depression to test the hypothesis that in utero antidepressant treatment could mitigate the effects of prenatal stress. We also investigated the effects of prenatal stress and antidepressant treatment on gene expression related to GABAergic and serotonergic neurotransmission in the amygdala, which may underlie behavioral effects of prenatal stress. Nulliparous female rats were implanted with osmotic minipumps delivering clinically-relevant concentrations of escitalopram and mated. Pregnant dams were exposed to 12 days of mixed-modality stressors, and offspring were behaviorally assessed in adolescence (postnatal day 28) and adulthood (beyond day 90) to determine the extent of behavioral change. We found that in utero stress exposure, regardless of escitalopram treatment, increased anxiety-like behavior in adolescent females and profoundly influenced amygdala expression of the chloride transporters KCC2 and NKCC1, which regulate GABAergic function. In contrast, prenatal escitalopram exposure alone elevated amygdala expression of 5-HT1A receptors. In adulthood, anxiety-like behavior returned to baseline and gene expression effects in the amygdala abated, whereas deficits emerged in novel object recognition for rats exposed to stress during gestation. These findings suggest prenatal stress causes age-dependent deficits in anxiety-like behavior and amygdala function in female offspring, regardless of antidepressant exposure.

Early life stress and macaque amygdala hypertrophy: preliminary evidence for a role for the serotonin transporter gene.

BACKGROUND: Children exposed to early life stress (ELS) exhibit enlarged amygdala volume in comparison to controls. The primary goal of this study was to examine amygdala volumes in bonnet macaques subjected to maternal variable foraging demand (VFD) rearing, a well-established model of ELS. Preliminary analyses examined the interaction of ELS and the serotonin transporter gene on amygdala volume. Secondary analyses were conducted to examine the association between amygdala volume and other stress-related variables previously found to distinguish VFD and non-VFD reared animals. METHODS: Twelve VFD-reared and nine normally reared monkeys completed MRI scans on a 3T system (mean age = 5.2 years). RESULTS: Left amygdala volume was larger in VFD vs. control macaques. Larger amygdala volume was associated with: "high" cerebrospinal fluid concentrations of corticotropin releasing-factor (CRF) determined when the animals were in adolescence (mean age = 2.7 years); reduced fractional anisotropy (FA) of the anterior limb of the internal capsule (ALIC) during young adulthood (mean age = 5.2 years) and timid anxiety-like responses to an intruder during full adulthood (mean age = 8.4 years). Right amygdala volume varied inversely with left hippocampal neurogenesis assessed in late adulthood (mean age = 8.7 years). Exploratory analyses also showed a gene-by-environment effect, with VFD-reared macaques with a single short allele of the serotonin transporter gene exhibiting larger amygdala volume compared to VFD-reared subjects with only the long allele and normally reared controls. CONCLUSION: These data suggest that the left amygdala exhibits hypertrophy after ELS, particularly in association with the serotonin transporter gene, and that amygdala volume variation occurs in concert with other key stress-related behavioral and neurobiological parameters observed across the lifecycle. Future research is required to understand the mechanisms underlying these diverse and persistent changes associated with ELS and amygdala volume.

Prenatal antidepressant exposure: clinical and preclinical findings.

Pharmacological treatment of any maternal illness during pregnancy warrants consideration of the consequences of the illness and/or medication for both the mother and unborn child. In the case of major depressive disorder, which affects up to 10-20% of pregnant women, the deleterious effects of untreated depression on the offspring can be profound and long lasting. Progress has been made in our understanding of the mechanism(s) of action of antidepressants, fetal exposure to these medications, and serotonin's role in development. New technologies and careful study designs have enabled the accurate sampling of maternal serum, breast milk, umbilical cord serum, and infant serum psychotropic medication concentrations to characterize the magnitude of placental transfer and exposure through human breast milk. Despite this progress, the extant clinical literature is largely composed of case series, population-based patient registry data that are reliant on nonobjective means and retrospective recall to determine both medication and maternal depression exposure, and limited inclusion of suitable control groups for maternal depression. Conclusions drawn from such studies often fail to incorporate embryology/neurotransmitter ontogeny, appropriate gestational windows, or a critical discussion of statistically versus clinically significant. Similarly, preclinical studies have predominantly relied on dosing models, leading to exposures that may not be clinically relevant. The elucidation of a defined teratological effect or mechanism, if any, has yet to be conclusively demonstrated. The extant literature indicates that, in many cases, the benefits of antidepressant use during pregnancy for a depressed pregnant woman may outweigh potential risks.

Effects of duloxetine on norepinephrine and serotonin transporter activity in healthy subjects.

Duloxetine selectively inhibits the serotonin (5-HT) and norepinephrine (NE) transporters (5-HTT and NET, respectively), as demonstrated in vitro and in preclinical studies; however, transporter inhibition has not been fully assessed in vivo at the approved dose of 60 mg/d. Here, the in vivo effects of dosing with duloxetine 60 mg once daily for 11 days in healthy subjects were assessed in 2 studies: (1) centrally (n = 11), by measuring concentrations of 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylglycol (DHPG), and NE in cerebrospinal fluid, and (2) versus escitalopram 20 mg/d (n = 32) in a 2-period crossover study by assessing the ΔDHPG/ΔNE ratio in plasma during orthostatic testing and by pharmacokinetic/pharmacodynamic modeling of reuptake inhibition using subjects' serum in cell lines expressing cloned human 5-HTT or NET. At steady state, duloxetine significantly reduced concentrations of DHPG and 5-hydroxyindoleacetic acid (P < 0.05), but not NE, in cerebrospinal fluid; DHPG was also decreased in plasma and urine. The ΔDHPG/ΔNE ratio in plasma decreased significantly more with duloxetine than escitalopram (65% and 21%, respectively; P < 0.0001). Ex vivo reuptake inhibition of 5-HTT was comparable (EC50 = 44.5 nM) for duloxetine and escitalopram, but duloxetine inhibited NET more potently (EC50 = 116 nM and 1044 nM, respectively). Maximal predicted reuptake inhibition for 5-HTT was 84% for duloxetine and 80% for escitalopram, and that for NET was 67% and 14%, respectively. In summary, duloxetine significantly affected 5-HT and NE turnover in the central nervous system and periphery; these effects presumably occurred via inhibition of reuptake by the 5-HTT and NET, as indicated by effects on functional reuptake inhibition ex vivo.

Prenatal exposure to escitalopram and/or stress in rats produces limited effects on endocrine, behavioral, or gene expression measures in adult male rats.

Stress and/or antidepressants during pregnancy have been implicated in a wide range of long-term effects in the offspring. We investigated the long-term effects of prenatal stress and/or clinically relevant antidepressant exposure on male adult offspring in a model of the pharmacotherapy of maternal depression. Female Sprague-Dawley rats were implanted with osmotic minipumps that delivered clinically relevant exposure to the antidepressant escitalopram throughout gestation. Subsequently, pregnant females were exposed on gestational days 10-20 to a chronic unpredictable mild stress paradigm. The male offspring were analyzed in adulthood. Baseline physiological measurements were largely unaltered by prenatal manipulations. Behavioral characterization of the male offspring, with or without pre-exposure to an acute stressor, did not reveal any group differences. Prenatal stress exposure resulted in a faster return towards baseline following the peak response to an acute restraint stressor, but not an airpuff startle stressor, in adulthood. Microarray analysis of the hippocampus and hypothalamus comparing all treatment groups revealed no significantly-altered transcripts. Real time PCR of the hippocampus confirmed that several transcripts in the CRFergic, serotonergic, and neural plasticity pathways were unaffected by prenatal exposures. This stress model of maternal depression and its treatment indicate that escitalopram use and/or stress during pregnancy produced no alterations in our measures of male adult behavior or the transcriptome, however prenatal stress exposure resulted in some evidence for increased glucocorticoid negative feedback following an acute restraint stress. Study design should be carefully considered before implications for human health are ascribed to prenatal exposure to stress or antidepressant medication.

In vivo dialysis setup with a loop injection valve facilitates retrodialysis studies.

INTRODUCTION: Retrodialysis, as used in neuropharmacological research, is a technique for in vivo delivery of neuroactive agents with concurrent monitoring of their effects on cellular activity with a separation between certain degree of spatial and temporal resolution. Typically, this is accomplished either by the use of a liquid-switch requiring multiple pumps, or by exchange of flow tubing requiring stopping and restarting dialysis. In the present study, we describe the use of a medium pressure injection valve for retrodialysis that overcomes these problems. METHODS: The valve was configured with a loop to deliver 20µL of solution, and artificial CSF flow from the pump to the probe was established via this device. The application of this setup was evaluated in urethane anesthetized adult male C57BL/6J mice prepared with a CMA 11 probe implanted in the ventral hippocampus. By switching between the load and inject positions, the loop was filled with escitalopram solution (0.3µM) and delivered at a rate of 1µL/min at the probe for retrodialysis. Escitalopram (2mg/kg BW) was administered subcutaneously for microdialysis studies. During these treatments, dialysate fractions were collected for the determination of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). RESULTS: Irrespective of route of escitalopram administration, the pattern of dialysate 5-HT, and 5-HIAA response was comparable to that reported by other investigators. Accordingly, the in-line valve assembly did not compromise retrodialysis or microdialysis sampling. The manipulations to carry out retrodialysis using the valve setup are easy and simple. DISCUSSION: An in-line injection valve is a promising adaptation for retrodialysis studies and can be incorporated as a standard part of in vivo dialysis instrumentation.

Prenatal exposure to escitalopram and/or stress in rats: a prenatal stress model of maternal depression and its treatment.

RATIONALE: A rigorously investigated model of stress and antidepressant administration during pregnancy is needed to evaluate possible effects on the mother. OBJECTIVE: The objective of this study was to develop a model of clinically relevant prenatal exposure to an antidepressant and stress during pregnancy to evaluate the effects on maternal care behavior. RESULTS: Female rats implanted with 28-day osmotic minipumps delivering the SSRI escitalopram throughout pregnancy had serum escitalopram concentrations in a clinically observed range (17-65 ng/ml). A separate cohort of pregnant females exposed to a chronic unpredictable mild stress paradigm on gestational days 10-20 showed elevated baseline (305 ng/ml), and acute stress-induced (463 ng/ml), plasma corticosterone concentrations compared to unstressed controls (109 ng/ml). A final cohort of pregnant dams were exposed to saline (control), escitalopram, stress, or stress and escitalopram to determine the effects on maternal care. Maternal behavior was continuously monitored over the first 10 days after parturition. A reduction of 35 % in maternal contact and 11 % in nursing behavior was observed due to stress during the light cycle. Licking and grooming behavior was unaffected by stress or drug exposure in either the light or dark cycle. CONCLUSIONS: These data indicate that: (1) clinically relevant antidepressant treatment during human pregnancy can be modeled in rats using escitalopram; (2) chronic mild stress can be delivered in a manner that does not compromise fetal viability; and (3) neither of these prenatal treatments substantially altered maternal care post parturition.

Escitalopram alters gene expression and HPA axis reactivity in rats following chronic overexpression of corticotropin-releasing factor from the central amygdala.

We have previously demonstrated that viral-mediated overexpression of corticotropin-releasing factor (CRF) within the central nucleus of the amygdala (CeA) reproduces many of the behavioral and endocrine consequences of chronic stress. The present experiment sought to determine whether administration of the selective serotonin reuptake inhibitor (SSRI) escitalopram reverses the adverse effects of CeA CRF overexpression. In a 2×2 design, adult male rats received bilateral infusions of a control lentivirus or a lentivirus in which a portion of the CRF promoter is used to drive increased expression of CRF peptide. Four weeks later, rats were then implanted with an Alzet minipump to deliver vehicle or 10mg/kg/day escitalopram for a 4-week period of time. The defensive withdrawal (DW) test of anxiety and the sucrose-preference test (SPT) of anhedonia were performed both before and after pump implantation. Additional post-implant behavioral tests included the elevated plus maze (EPM) and social interaction (SI) test. Following completion of behavioral testing, the dexamethasone/CRF test was performed to assess HPA axis reactivity. Brains were collected and expression of HPA axis-relevant transcripts were measured using in situ hybridization. Amygdalar CRF overexpression increased anxiety-like behavior in the DW test at week eight, which was only partially prevented by escitalopram. In both CRF-overexpressing and control groups, escitalopram decreased hippocampal CRF expression while increasing hypothalamic and hippocampal expression of the glucocorticoid receptor (GR). These gene expression changes were associated with a significant decrease in HPA axis reactivity in rats treated with escitalopram. Interestingly, escitalopram increased the rate of weight gain only in rats overexpressing CRF. Overall these data support our hypothesis that amygdalar CRF is critical in anxiety-like behavior; because the antidepressant was unable to reverse behavioral manifestations of CeA CRF-OE. This may be a potential animal model to study treatment-resistant psychopathologies.

Neuroendocrine homeostasis after vagus nerve stimulation in rats.

BACKGROUND: The vagus nerve is important in maintaining HPA axis and sympatho-adrenal system (SAS) homeostasis, however little is known about the effect of vagus nerve stimulation (VNS), as used therapeutically, on these functions. Accordingly, the effect of VNS on plasma indices of HPA axis (ACTH, corticosterone), and SAS (norepinephrine, epinephrine) function were evaluated in rats. METHODS: Male rats, on day-0 (D0), underwent surgeries for implantation of catheters into the right jugular vein and programmable (VNP) or non-programmable (VND) neurocybernetic devices encircling the left cervical vagus. On D7, after a blood sample, the device in VNP rats was programmed to deliver 500 µs width, 0.25 mA current pulses at 20 Hz ('on' 30s, 'off' 5 min) followed by timed blood samples during the next 90 min. In acute studies, VNS was stopped at 60 min and the rats were perfused at 90 min to evaluate neuronal Fos immunoreactivity (Fos-IR). In chronic studies, the probe remained active. In these rats, the HPA axis response to airpuff-startle stressor (D17) and anterior pituitary CRF-receptor binding (D26) were evaluated. RESULTS: During acute VNS, plasma indices of HPA axis and SAS activity, as well as Fos-IR activation pattern in brain regions known to increase after stress, were not different between VND and VNP rats. During chronic VNS, stress-induced HPA axis responses exhibited a tendency toward faster recovery to baseline in VNP rats. CONCLUSIONS: Therapeutic VNS is not a stressor and does not compromise HPA axis or SAS homeostasis. Chronic VNS may facilitate development of efficient feedback mechanisms.

Modulation of vigilance in the primary hypersomnias by endogenous enhancement of GABAA receptors.

The biology underlying excessive daytime sleepiness (hypersomnolence) is incompletely understood. After excluding known causes of sleepiness in 32 hypersomnolent patients, we showed that, in the presence of 10 µM γ-aminobutyric acid (GABA), cerebrospinal fluid (CSF) from these subjects stimulated GABA(A) receptor function in vitro by 84.0 ± 40.7% (SD) relative to the 35.8 ± 7.5% (SD) stimulation obtained with CSF from control subjects (Student's t test, t = 6.47, P < 0.0001); CSF alone had no effect on GABA(A) signaling. The bioactive CSF component had a mass of 500 to 3000 daltons and was neutralized by trypsin. Enhancement was greater for α2 subunit- versus α1 subunit-containing GABA(A) receptors and negligible for α4 subunit-containing ones. CSF samples from hypersomnolent patients also modestly enhanced benzodiazepine (BZD)-insensitive GABA(A) receptors and did not competitively displace BZDs from human brain tissue. Flumazenil--a drug that is generally believed to antagonize the sedative-hypnotic actions of BZDs only at the classical BZD-binding domain in GABA(A) receptors and to lack intrinsic activity--nevertheless reversed enhancement of GABA(A) signaling by hypersomnolent CSF in vitro. Furthermore, flumazenil normalized vigilance in seven hypersomnolent patients. We conclude that a naturally occurring substance in CSF augments inhibitory GABA signaling, thus revealing a new pathophysiology associated with excessive daytime sleepiness.