Claudia Schmauss

Heterosynaptic Dopamine Neurotransmission Selects Sets of Corticostriatal Terminals

Dopamine input to the striatum is required for voluntary motor movement, behavioral reinforcement, and responses to drugs of abuse. It is speculated that these functions are dependent on either excitatory or inhibitory modulation of corticostriatal synapses onto medium spiny neurons (MSNs). While dopamine modulates MSN excitability, a direct presynaptic effect on the corticostriatal input has not been clearly demonstrated. We combined optical monitoring of synaptic vesicle exocytosis from motor area corticostriatal afferents and electrochemical recordings of striatal dopamine release to directly measure effects of dopamine at the level of individual presynaptic terminals. Dopamine released by either electrical stimulation or amphetamine acted via D2 receptors to inhibit the activity of subsets of corticostriatal terminals. Optical and electrophysiological data suggest that heterosynaptic inhibition was enhanced by higher frequency stimulation and was selective for the least active terminals. Thus, dopamine, by filtering less active inputs, appears to reinforce specific sets of corticostriatal synaptic connections.

Physiological Modulation of Intestinal Motility by Enteric Dopaminergic Neurons and the D2 Receptor: Analysis of Dopamine Receptor Expression, Location, Development, and Function in Wild-Type and Knock-Out Mice

Dopaminergic neurons are present in both plexuses of the murine bowel and are upregulated after extrinsic denervation but play unknown roles in enteric nervous system (ENS) physiology. Transcripts encoding dopamine (DA) receptors D1–D5 were analyzed by reverse transcription-PCR in stomach ≈ duodenum ≈ ileum ≈ proximal ≫ distal colon. Dissected muscle and myenteric plexus contained transcripts encoding D1–D3 and D5, whereas mucosa contained D1 and D3–D5. D1–D5 expression began in fetal gut [embryonic day 10 (E10)], before the appearance of neurons (E12), and was sustained without developmental regulation through postnatal day 1. In situ hybridization revealed that subsets of submucosal and myenteric neurons contained mRNA encoding D2 or D3. Immunoblots confirmed that D1, D2, and D5 receptor proteins were present from stomach through distal colon. Subsets of submucosal and myenteric neurons were also D1, D2, or D3 immunoreactive. When double labeled by in situ hybridization, these neurons contained mRNA encoding the respective receptors. Total gastrointestinal transit time (TGTT) and colonic transit time (CTT) were measured in mice lacking D2, D3, or D2 plus D3. Both TGTT and CTT were decreased significantly (motility increased) in D2 and D2 plus D3, but not D3, knock-out animals. Mice lacking D2 and D2 plus D3 but not D3 were smaller than wild-type littermates, yet ate significantly more and had greater stool frequency, water content, and mass. Because motility is abnormal when D2 is absent, the net inhibitory DA effect on motility is physiologically significant. The early expression of DA receptors is also consistent with the possibility that DA affects ENS development.

How Stress and Fluoxetine Modulate Serotonin 2C Receptor Pre-mRNA Editing

In two inbred strains of mice, C57BL/6 and 129Sv, the majority of forebrain neocortical pre-mRNA encoding the serotonin 2C (5-HT2C) receptor is altered by adenosine-to-inosine editing. As a result, >60% of all mRNAs encode receptors with reduced constitutive and agonist-stimulated activity. However, in the BALB/c strain, a genetically distinct inbred strain with lower forebrain serotonin levels, spontaneously elevated anxiety, and increased stress reactivity, the majority of 5-HT2C mRNA is nonedited and encodes receptors with the highest constitutive activity and the highest agonist affinity and potency. Neither acute stress (the forced swim test) nor chronic treatment with the serotonin-selective reuptake inhibitor fluoxetine elicit significant changes in 5-HT2C pre-mRNA editing in C57BL/6 mice. In contrast, exposure of BALB/c mice to acute stress and chronic treatment of nonstressed BALB/c mice with fluoxetine elicit significant, site-specific increases in 5-HT2C pre-mRNA editing that increase the pool of mRNA encoding receptors with reduced function. These changes in 5-HT2C pre-mRNA editing resemble those detected previously in the prefrontal cortex of subjects with major depression. However, when chronic fluoxetine treatment is combined with stress exposure of BALB/c mice, these changes in 5-HT2C pre-mRNA editing are no longer detected. These findings illustrate that 5-HT2C pre-mRNA editing responses to stress and chronic fluoxetine are modulated by the genetic background, as well as the behavioral state of the animal. They suggest further that the changes in 5-HT2C pre-mRNA editing found in major depression reflect a previously unrecognized molecular response to stress that can be prevented by chronic antidepressant treatment.

Early Life Stress Alters Adult Serotonin 2C Receptor Pre-mRNA Editing and Expression of the α Subunit of the Heterotrimeric G-Protein Gq

Infant maternal separation, a paradigm of early life stress in rodents, elicits long-lasting changes in gene expression that persist into adulthood. In BALB/c mice, an inbred strain with spontaneously elevated anxiety and stress reactivity, infant maternal separation led to increased depression-like behavioral responses to adult stress and robustly increased editing of serotonin 2C receptor pre-mRNA. Chronic fluoxetine treatment of adult BALB/c mice exposed to early life stress affected neither their behavioral responses to stress nor their basal 5-HT2C pre-mRNA editing phenotype. However, when fluoxetine was administered during adolescence, depression-like behavioral responses to stress were significantly diminished in these mice, and their basal and stress-induced 5-HT2C pre-mRNA editing phenotypes were significantly lower. Moreover, when BALB/c mice exposed to early life stress were raised in an enriched postweaning environment, their depression-like behavioral responses to adult stress were also significantly diminished. However, their 5-HT2C pre-mRNA editing phenotype remained unaltered. Hence, the similar behavioral effects of enrichment and fluoxetine treatment during adolescence were not accompanied by similar changes in 5-HT2C pre-mRNA editing. Enriched and nonenriched BALB/c mice exposed to early life stress also exhibited significantly increased expression of mRNA and protein encoding the Gαq subunit of G-protein that couples to 5-HT2A/2C receptors. In contrast, Gαq expression levels were significantly lower in fluoxetine-treated mice. These findings suggest that compensatory changes in Gαq expression occur in mice with persistently altered 5-HT2C pre-mRNA editing and provide an explanation for the dissociation between 5-HT2C receptor editing phenotypes and behavioral stress responses.

Antidepressant drug-induced stimulation of mouse hippocampal neurogenesis is age-dependent and altered by early life stress

The continuous generation of new neurons in the adult hippocampus exhibits remarkable plasticity. Decreased neurogenesis is thought to underlie depression‐like behaviors, and increased neurogenesis is thought to occur following antidepressant drug treatment. Studies on different strains of mice, however, yielded contrasting results with regard to the link between behavioral modifications induced by antidepressant drugs or environmental enrichment and changes in adult hippocampal neurogenesis. Therefore, we conducted a comparative study on the inbred strains Balb/c and C57Bl/6 that differ substantially in emotionality, stress reactivity, and behavioral responses to chronic antidepressant drugs. Quantitative assessments of progenitor cell proliferation and immature neuronal differentiation in the dentate gyrus revealed that, despite significantly different basal proliferation rates between both strains, neither strain exhibited changes in adult neurogenesis after exposure to early life stress or adult chronic fluoxetine treatment. A stimulatory effect of fluoxetine on adult hippocampal neurogenesis was only detected when treatment was initiated during adolescence, and this effect was abolished in mice exposed to early life stress, a prominent risk factor for developing adult‐onset depression‐like behaviors. Thus, in both strains of mice neither adult fluoxetine treatment nor adolescent fluoxetine treatment following early life stress exposure increased the proliferation and early differentiation of adult neural progenitor cells. J. Comp. Neurol. 509:372–381, 2008.

Early life stress triggers sustained changes in histone deacetylase expression and histone H4 modifications that alter responsiveness to adolescent antidepressant treatment.

Early life stress can elicit long-lasting changes in gene expression and behavior. Recent studies on rodents suggest that these lasting effects depend on the genetic background. Whether epigenetic factors also play a role remains to be investigated. Here we exposed the stress-susceptible mouse strain Balb/c and the more resilient strain C57Bl/6 to a powerful early life stress paradigm, infant maternal separation. In Balb/c mice, infant maternal separation led to decreased expression of mRNA encoding the histone deacetylases (HDACs) 1, 3, 7, 8, and 10 in the forebrain neocortex in adulthood, an effect accompanied by increased expression of acetylated histone H4 proteins, especially acetylated H4K12 protein. These changes in HDAC expression and histone modifications were not detected in C57Bl/6 mice exposed to early life stress. Moreover, a reversal of the H4K12 hyperacetylation detected in infant maternally separated Balb/c mice (achieved with chronic adolescent treatment with a low dose of theophylline that only activates HDACs) worsened the abnormal emotional phenotype resulting from this early life stress exposure. In contrast, fluoxetine, a drug with potent antidepressant efficacy in infant maternally separated Balb/c mice, potentiated all histone modifications triggered by early life stress. Moreover, in non-stressed Balb/c mice, co-administration of an HDAC inhibitor and fluoxetine, but not fluoxetine alone, elicited antidepressant effects and also triggered changes in histone H4 expression that were similar to those provoked by fluoxetine treatment of mice exposed to early life stress. These results suggest that Balb/c mice develop epigenetic modifications after early life stress exposure that, in terms of the emotive phenotype, are of adaptive nature, and that enhance the efficacy of antidepressant drugs.

Dopamine receptor functions: lessions from knockout mice

In the past few years, a number of laboratories have used gene targeting via homologous recombination to generate mice deficient for key molecules involved in dopaminergic (DAergic) transmission. This tremendous effort has resulted in the successful generation and characterization of mice deficient for the neurotransmitter DA, the main terminator of DAergic neurotransmission (the DA transporter), and all five subtypes of DA receptors. This review summarizes the results from studies of the various DA receptor knockout mice and of mice deficient in proteins that mediate DA receptor signaling. It focuses on a comparison of the locomotor phenotypes and responses to drugs of abuse (psychostimulants), and reviews the results of anatomic studies examining the morphological and neurochemical differentiation of the striatum in these mutants. Moreover, an overview of recently published results highlighting the physiological relevance of the interaction between different DA receptors and between DA receptors and other neurotransmitter receptors in the modulation of behavioral and molecular responses to DAergic stimulation is presented. Finally, in view of the recently discovered heteroligomeric assemblies of neurotransmitter receptors that involve DA receptor subtypes, the potential value of knockout mice as a tool for testing the in vivo significance of these heteroligomeric receptors is discussed.

A role for dopamine D2 receptors in reversal learning.

Reversal learning has been shown to require intact serotonergic innervation of the forebrain neocortex. Whether dopamine acting through D2 receptors plays a complementary role in this anatomic area is still unclear. Here we show that mice lacking dopamine D2 receptors exhibited significantly impaired performance in the reversal learning phase of an attention-set-shifting task (ASST) and that wild type mice treated chronically with the D2-like receptor antagonist haloperidol exhibited the same cognitive deficit. The test-phase-specific deficits of D2 mutants and haloperidol-treated mice were also accompanied by deficits in the induction of expression of early growth response gene 2 (egr-2), a regulatory transcription factor previously shown to be selectively induced in the ventrolateral orbital frontal cortex and the pre- and infralimbic medial prefrontal cortex of ASST-tested mice. D2-receptor knockout mice and haloperidol-treated wild type, however, exhibited lower egr-2 expression in these anatomic regions after completion of an ASST-test phase that required reversal learning but not after completion of set-shifting phases without rule reversals. In contrast, mice treated chronically with clozapine, an atypical neuroleptic drug with lower D2-receptor affinity and broader pharmacological effects, had deficits in compound discrimination phases of the ASST, but also these deficits were accompanied by lower egr-2 expression in the same anatomic subregions. Thus, the findings indicate that egr-2 expression is a sensitive indicator of test-phase-specific performance in the ASST and that normal function of D2 receptors in subregions of the orbital frontal and the medial prefrontal cortex is required for cognitive flexibility in tests involving rule reversals.

Strain-specific cognitive deficits in adult mice exposed to early life stress.

Early life stress is a prominent risk factor for the development of adult psychopathology. Numerous studies have shown that early life stress leads to persistent changes in behavioral and endocrine responses to stress. However, despite recent findings of gene expression changes and structural abnormalities in neurons of the forebrain neocortex, little is known about specific cognitive deficits that can result from early life stress. Here we examined five cognitive functions in two inbred strains of mice, the stress-resilient strain C57Bl/6 and the stress-susceptible strain Balb/c, which were exposed to an infant maternal separation paradigm and raised to adulthood. Between postnatal ages P60 to P90, mice underwent a series of tests examining five cognitive functions: Recognition memory, spatial working memory, associative learning, shifts of attentional sets, and reversal learning. None of these functions were impaired in IMS C57Bl/6 mice. In contrast, IMS Balb/c mice exhibited deficits in spatial working memory and extradimensional shifts of attention, that is, functions governed primarily by the medial prefrontal cortex. Thus, like recently discovered changes in frontocortical gene expression, the emergence of specific cognitive deficits associated with the medial prefrontal cortex is also strain-specific. These findings illustrate that early life stress can indeed affect specific cognitive functions in adulthood, and they support the hypothesis that the genetic background and environmental factors are critical determinants in the development of adult cognitive deficits in subjects with a history of early life stress.

Induction of early growth response gene 2 expression in the forebrain of mice performing an attention-set-shifting task.

Early growth response (egr) genes encode transcription factors that are induced by stimuli that cause synaptic plasticity. Here we show that the expression of one member of this family, egr-2, is induced in the orbital frontal cortex (OFC) and medial prefrontal cortex (mPFC) of mice performing an attention-set-shifting task (ASST). The ASST is a series of two-choice perceptual discriminations between different odors and textures. Within the OFC and mPFC, different subregions exhibited egr-2 induction in response to different test-related features. In the medial OFC and the anterior cingulate subregion of the mPFC, egr-2 induction occurred in response to exposure to the novel odor stimulus. In the ventrolateral OFC and the pre- and infralimbic mPFC, additional egr-2 induction occurred during the associative learning phase of the ASST. In the infralimbic mPFC, further egr-2 induction occurred when mice performed set-shifting and reversal learning phases of the ASST. Mice with enhanced set-shifting performance exhibited decreased egr-2 induction in the mPFC indicating that the magnitude of egr-2 induction correlates with the magnitude of attentional demand. This decrease was largest in the infralimbic mPFC suggesting further that egr-2 induction in this region plays a role in the attentional control during set-shifting. In contrast to egr-2, neither egr-1 nor egr-3 expression was altered in ASST-tested mice, and no egr-2 induction occurred in mice that performed a spatial working memory task. These findings suggest a specific role of egr-2-mediated transcriptional activation in cognitive functions associated with attention.