Rachel A. Millstein

Impaired Stress-Coping and Fear Extinction and Abnormal Corticolimbic Morphology in Serotonin Transporter Knock-Out Mice

A lesser-expressing form of the human 5-HT transporter (5-HTT) gene has been associated with increased fear and anxiety and vulnerability to the effects of stress. These phenotypic abnormalities are linked to functional and anatomical disturbances in a neural pathway connecting the prefrontal cortex (PFC) and amygdala. Likewise, rodent and nonhuman primate studies indicate a major role for PFC and amygdala in the mediation of fear- and stress-related behaviors. We used a 5-HTT knock-out (KO) mouse to examine the effects of genetically driven loss of 5-HTT function for the following: (1) depression-related behavior in response to repeated stress, and pavlovian fear conditioning, extinction, and extinction recall; and (2) dendritic morphology and spine density of Golgi-stained pyramidal neurons in the infralimbic cortex (IL) and the basolateral amygdala (BLA). 5-HTT KO mice exhibited increased depressive-like immobility after repeated exposure to forced swim stress, compared with wild-type (WT) controls. Whereas fear conditioning and fear extinction was normal, 5-HTT KO mice exhibited a significant deficit in extinction recall. The apical dendritic branches of IL pyramidal neurons in 5-HTT KO mice were significantly increased in length relative to WT mice. Pyramidal neurons in BLA had normal dendritic morphology but significantly greater spine density in 5-HT KO mice compared with WT mice. Together, the present findings demonstrate a specific phenotypic profile of fear- and stress-related deficits in 5-HTT KO mice, accompanied by morphological abnormalities in two key neural loci. These data provide insight into the behavioral sequelae of loss of 5-HTT gene function and identify potential neural substrates underlying these phenotypes.

Early life genetic, epigenetic and environmental factors shaping emotionality in rodents

Childhood trauma is known to increase risk for emotional disorders and addiction. However, little is currently understood about the neurodevelopmental basis of these effects, or how genetic and epigenetic factors interact with the environment to shape the systems subserving emotionality. In this review, we discuss the use of rodent models of early life emotional experience to study these issues in the laboratory and present some of our pertinent findings. In rats, postnatal maternal separation can produce lasting increases in emotional behavior and stressor-reactivity, together with alterations in various brain neurotransmitter systems implicated in emotionality, including corticotropin-releasing factor, serotonin, norepinephrine, and glutamate. Genetic differences between inbred mouse strains have been exploited to further study how maternal behavior affects emotional development using techniques such as cross-fostering and generation of inter-strain hybrids. Together with our own recent data, the findings of these studies demonstrate the pervasive influence of maternal and social environments during sensitive developmental periods and reveal how genetic factors determine how these early life experiences can shape brain and behavior throughout life.

Genetic and dopaminergic modulation of reversal learning in a touchscreen-based operant procedure for mice.

Mice are uniquely suited as experimental subjects for various approaches to the study of the molecular and genetic basis of behavior, and there has been a corresponding explosion in the use of mice in behavioral neuroscience. Rats and monkeys, however, remain the preferred species for high-order cognitive models largely due to the unavailability of valid, reliable and translatable endpoint measures of behavior in the mouse. Here we present further development and validation of a touchscreen-based operant method for measuring cognition that is comparable to methods used in other species and human patients. C57BL/6J mice were found to show good performance on visual discrimination and reversal learning using this method. Demonstrating the sensitivity of the paradigm to genetic factors, C57BL/6J and DBA/2J mice exhibited marked differences in discrimination and reversal learning. Systemic treatment with the selective D1-like agonist, SKF81297, produced an impairment in the early phase of reversal learning, but did not alter visual discrimination, in C57BL/6J mice. The same treatment impaired spatial working memory on the T-maze delayed alternation task, but did not alter control measures of behavior including motivation and locomotor activity. These data demonstrate the sensitivity of visual discrimination and reversal learning measured by this method to genetic factors and pharmacological challenge, and thereby provide an extension and further validation of the method for measuring cognition in mice. When combined with emerging molecular techniques uniquely suited to this species such as genetic engineering and RNA modification this paradigm could provide a powerful new tool for behavioral neuroscience.

Effects of repeated maternal separation on prepulse inhibition of startle across inbred mouse strains

A growing body of research implicates genetic factors and childhood trauma in the etiology of neuropsychiatric diseases such as schizophrenia. However, there remains little understanding of how genetic variation influences early life stress to affect later disease susceptibility. Studies in rats have shown that postnatal maternal separation (MS) results in later deficits in prepulse inhibition of the acoustic startle response (PPI), an impairment in sensorimotor gating found in schizophrenic patients. In the present study, genetic differences in the effects of repeated MS on PPI were examined in eight inbred strains of mice (129S1/SvImJ, 129P3/J, A/J, BALB/cJ, BALB/cByJ C57BL/6J, DBA/2J and FVB/NJ). Mice were assigned to either MS (180 min/day on postnatal days P0–P13), ‘handling’ (15 min/day, P0–P13) or facility‐reared conditions and tested for PPI at 12 weeks of age. Results demonstrated major strain differences in the production of viable offspring irrespective of MS, leading to the exclusion of 129P3/J, A/J and BALB/cJ from the study. Pups from the five remaining strains exhibited marked differences in the acoustic startle response and PPI, confirming previous strain comparisons. However, MS produced no significant effects on PPI in any of the strains tested. A second form of postnatal stress (repeated footshock) also failed to alter PPI in the one strain studied, C57BL/6J. Present results demonstrate that the form of MS studied herein does not provide a robust model of early life stress effects on PPI in the mouse strains tested. The development and validation of a reliable mouse model of early life stress remains an important research goal.

Insertion mutation at the C-terminus of the serotonin transporter disrupts brain serotonin function and emotion-related behaviors in mice.

The 5-hydroxytryptamine transporter (5-HTT) regulates 5-hydroxytryptamine (5-HT) neurotransmission by removing 5-HT from the synaptic cleft. Emerging evidence from clinical and genetic studies implicates the 5-HTT in various neuropsychiatric conditions, including anxiety and depression. Here we report that a 5-HTT null mutant mouse line was generated by gene trapping that disrupted the sequence encoding the C-terminus of 5-HTT. This mutation resulted in significant reduction of 5-HTT mRNA and loss of 5-HTT protein. Brain levels of 5-HT and its major metabolite, 5-hydroxyindoleacetic acid, were markedly decreased in C-terminus 5-HTT -/- mice, while 5-HT uptake or 5-HT content in platelets was absent. Behavioral phenotyping showed that C-terminus 5-HTT -/- mice were normal on a screen for gross behavioral, neurological, and sensory functions. In the tail suspension test for depression-related behavior, C-terminus 5-HTT -/- mice showed increased immobility relative to their +/+ controls. By comparison, a previously generated line of 5-HTT -/- mice lacking exon 2, encoding the N-terminus of the 5-HTT, showed abnormally high immobility in response to repeated, but not acute, exposure to the tail suspension test. In a novel, brightly-lit open field, both C-terminus 5-HTT -/- mice and N-terminus 5-HTT -/- mice displayed decreased center time and reduced locomotor activity compared with their +/+ controls. Both mutant lines buried significantly fewer marbles than their +/+ controls in the marble burying test. These findings further demonstrate the neurobiological functions of the 5-HTT and add to a growing literature linking genetic variation in 5-HTT function with emotional abnormalities.

Ethanol Inhibits Clearance of Brain Serotonin by a Serotonin Transporter-Independent Mechanism

Brain serotonin (5-HT) modulates the neural and behavioral effects of ethanol in a manner that remains poorly understood. Here we show that treatment with physiologically relevant (i.e., moderately intoxicating) doses of ethanol inhibits clearance of 5-HT from extracellular fluid in the mouse hippocampus. This finding demonstrates, in vivo, a key molecular mechanism by which ethanol modulates serotonergic neurotransmission. The 5-HT transporter (5-HTT) is the principle means of 5-HT reuptake in the brain and an obvious candidate mechanism for the effect of ethanol to inhibit 5-HT clearance. However, our second major finding was that genetic inactivation of the 5-HTT in a knock-out mouse not only failed to prevent ethanol-induced inhibition of 5-HT clearance, but actually potentiated this effect. Ethanol-induced inhibition of 5-HT clearance was also potentiated in nonmutant mice by cotreatment with a 5-HTT antagonist. Providing a link with potential behavioral manifestations of this neural phenotype, 5-HTT knock-out mice also exhibited exaggerated sensitivity to behavioral intoxication, as assayed by the sedative/hypnotic effects of ethanol. This clearly demonstrates that the 5-HTT is not necessary for the neural and behavioral effects of ethanol observed herein and that genetic or pharmacological inactivation of the 5-HTT unmasks involvement of other principle mechanisms. These data are intriguing given growing evidence implicating the 5-HTT in the pathophysiology and treatment of alcoholism and neuropsychiatric conditions frequently comorbid with alcoholism, such as depression. The present findings provide new insights into the actions of ethanol on brain function and behavior.

Is the relationship between the built environment and physical activity moderated by perceptions of crime and safety

BackgroundDirect relationships between safety concerns and physical activity have been inconsistently patterned in the literature. To tease out these relationships, crime, pedestrian, and traffic safety were examined as moderators of built environment associations with physical activity.MethodsExploratory analyses used two cross-sectional studies of 2068 adults ages 20–65 and 718 seniors ages 66+ with similar designs and measures. The studies were conducted in the Baltimore, Maryland-Washington, DC and Seattle-King County, Washington regions during 2001–2005 (adults) and 2005–2008 (seniors). Participants were recruited from areas selected to sample high- and low- income and walkability. Independent variables perceived crime, traffic, and pedestrian safety were measured using scales from validated instruments. A GIS-based walkability index was calculated for a street-network buffer around each participant’s home address. Outcomes were total physical activity measured using accelerometers and transportation and leisure walking measured with validated self-reports (IPAQ-long). Mixed effects regression models were conducted separately for each sample.ResultsOf 36 interactions evaluated across both studies, only 5 were significant (p < .05). Significant interactions did not consistently support a pattern of highest physical activity when safety was rated high and environments were favorable. There was not consistent evidence that safety concerns reduced the beneficial effects of favorable environments on physical activity. Only pedestrian safety showed evidence of a consistent main effect with physical activity outcomes, possibly because pedestrian safety items (e.g., crosswalks, sidewalks) were not as subjective as those on the crime and traffic safety scales.ConclusionsClear relationships between crime, pedestrian, and traffic safety with physical activity levels remain elusive. The development of more precise safety variables and the use of neighborhood-specific physical activity outcomes may help to elucidate these relationships.

Association between anxiety and mortality in patients with coronary artery disease: A meta-analysis

BACKGROUND Depression and anxiety are common in patients with coronary artery disease (CAD). Although depression clearly has been associated with mortality in this population, the relationship between anxiety and mortality is less clear. Accordingly, we performed a series of meta-analyses to (1) examine the relationship between anxiety and mortality in patients with established CAD and (2) determine if this relationship differs in patients with stable CAD compared to those who have just had an acute coronary syndrome (ACS). METHODS AND RESULTS Systematic literature searches identified 44 articles (total N = 30,527) evaluating the prospective relationship between anxiety and mortality in individuals with established CAD. A series of 8 adjusted and unadjusted meta-analyses were performed to examine this relationship across all patients, with sensitivity analyses completed in post-ACS and stable CAD cohorts. In unadjusted analyses, anxiety was associated with a moderate increase in mortality risk (odds ratio 1.21 per SD increase in anxiety). However, when adjusting for covariates, nearly all associations became nonsignificant. In sensitivity analyses, anxiety was associated with an increased risk of poor outcomes in the stable CAD-but not post-ACS-cohort. CONCLUSIONS These analyses confirm that anxiety is associated with increased risk of mortality in patients with CAD; however, this relationship is not as strong as that of depression and may be explained partly by other clinical factors. If anxiety screening is performed, it should be performed during a period of clinical stability and should target anxiety disorders rather than anxiety symptoms alone.

The acute intoxicating effects of ethanol are not dependent on the vasopressin 1a or 1b receptors.

Studies of the role of vasopressin (Avp) in mediating the effects of ethanol have focused on Avps role in altering kidney function via its action through the vasopressin 2 receptor. However, alcohol consumption also has central effects that are poorly understood. There is evidence that Avp may mediate ethanol consumption as well as some of ethanols behavioral effects. Centrally only two Avp receptor subtypes are expressed: the 1a receptor (Avpr1a) and the 1b receptor (Avpr1b). To determine the extent to which these receptors mediate the behavioral effects of alcohol, we used mice with targeted disruptions of either their Avpr1a or Avpr1b gene. We examined the effects of genotype on the acute intoxicating effects of ethanol as well as on voluntary ethanol consumption. Surprisingly, our findings indicate that there is no interaction between either the Avpr1a or Avpr1b and ethanol on motor coordination, hypothermia, mood, or voluntary ethanol consumption.

Development, scoring, and reliability of the Microscale Audit of Pedestrian Streetscapes (MAPS)

BackgroundStreetscape (microscale) features of the built environment can influence people’s perceptions of their neighborhoods’ suitability for physical activity. Many microscale audit tools have been developed, but few have published systematic scoring methods. We present the development, scoring, and reliability of the Microscale Audit of Pedestrian Streetscapes (MAPS) tool and its theoretically-based subscales.MethodsMAPS was based on prior instruments and was developed to assess details of streetscapes considered relevant for physical activity. MAPS sections (route, segments, crossings, and cul-de-sacs) were scored by two independent raters for reliability analyses. There were 290 route pairs, 516 segment pairs, 319 crossing pairs, and 53 cul-de-sac pairs in the reliability sample. Individual inter-rater item reliability analyses were computed using Kappa, intra-class correlation coefficient (ICC), and percent agreement. A conceptual framework for subscale creation was developed using theory, expert consensus, and policy relevance. Items were grouped into subscales, and subscales were analyzed for inter-rater reliability at tiered levels of aggregation.ResultsThere were 160 items included in the subscales (out of 201 items total). Of those included in the subscales, 80 items (50.0%) had good/excellent reliability, 41 items (25.6%) had moderate reliability, and 18 items (11.3%) had low reliability, with limited variability in the remaining 21 items (13.1%). Seventeen of the 20 route section subscales, valence (positive/negative) scores, and overall scores (85.0%) demonstrated good/excellent reliability and 3 demonstrated moderate reliability. Of the 16 segment subscales, valence scores, and overall scores, 12 (75.0%) demonstrated good/excellent reliability, three demonstrated moderate reliability, and one demonstrated poor reliability. Of the 8 crossing subscales, valence scores, and overall scores, 6 (75.0%) demonstrated good/excellent reliability, and 2 demonstrated moderate reliability. The cul-de-sac subscale demonstrated good/excellent reliability.ConclusionsMAPS items and subscales predominantly demonstrated moderate to excellent reliability. The subscales and scoring system represent a theoretically based framework for using these complex microscale data and may be applicable to other similar instruments.