Presynaptic inhibition of diverse afferents to the locus ceruleus by kappa-opiate receptors: a novel mechanism for regulating the central norepinephrine system.

The norepinephrine nucleus, locus ceruleus (LC), is activated by diverse stimuli and modulates arousal and behavioral strategies in response to these stimuli through its divergent efferent system. Afferents communicating information to the LC include excitatory amino acids (EAAs), corticotropin-releasing factor (CRF), and endogenous opioids acting at mu-opiate receptors. Because the LC is also innervated by the endogenous kappa-opiate receptor (kappa-OR) ligand dynorphin and expresses kappa-ORs, this study investigated kappa-OR regulation of LC neuronal activity in rat. Immunoelectron microscopy revealed a prominent localization of kappa-ORs in axon terminals in the LC that also contained either the vesicular glutamate transporter or CRF. Microinfusion of the kappa-OR agonist (trans)-3,4-dichloro-N-methyl-N-[2-1-pyrrolidinyl)-cyclo-hexyl] benzeneacetamide (U50488) into the LC did not alter LC spontaneous discharge but attenuated phasic discharge evoked by stimuli that engage EAA afferents to the LC, including sciatic nerve stimulation and auditory stimuli and the tonic activation associated with opiate withdrawal. Inhibitory effects of the kappa-OR agonist were not restricted to EAA afferents, as U50488 also attenuated tonic LC activation by hypotensive stress, an effect mediated by CRF afferents. Together, these results indicate that kappa-ORs are poised to presynaptically inhibit diverse afferent signaling to the LC. This is a novel and potentially powerful means of regulating the LC-norepinephrine system that can impact on forebrain processing of stimuli and the organization of behavioral strategies in response to environmental stimuli. The results implicate kappa-ORs as a novel target for alleviating symptoms of opiate withdrawal, stress-related disorders, or disorders characterized by abnormal sensory responses, such as autism.

"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies.

Sociability Deficits and Altered Amygdala Circuits in Mice Lacking Pcdh10, an Autism Associated Gene.

BACKGROUND: Behavioral symptoms in individuals with autism spectrum disorder (ASD) have been attributed to abnormal neuronal connectivity, but the molecular bases of these behavioral and brain phenotypes are largely unknown. Human genetic studies have implicated PCDH10, a member of the δ2 subfamily of nonclustered protocadherin genes, in ASD. PCDH10 expression is enriched in the basolateral amygdala, a brain region implicated in the social deficits of ASD. Previous reports indicate that Pcdh10 plays a role in axon outgrowth and glutamatergic synapse elimination, but its roles in social behaviors and amygdala neuronal connectivity are unknown. We hypothesized that haploinsufficiency of Pcdh10 would reduce social approach behavior and alter the structure and function of amygdala circuits. METHODS: Mice lacking one copy of Pcdh10 (Pcdh10+/-) and wild-type littermates were assessed for social approach and other behaviors. The lateral/basolateral amygdala was assessed for dendritic spine number and morphology, and amygdala circuit function was studied using voltage-sensitive dye imaging. Expression of Pcdh10 and N-methyl-D-aspartate receptor (NMDAR) subunits was assessed in postsynaptic density fractions of the amygdala. RESULTS: Male Pcdh10+/- mice have reduced social approach behavior, as well as impaired gamma synchronization, abnormal spine morphology, and reduced levels of NMDAR subunits in the amygdala. Social approach deficits in Pcdh10+/- male mice were rescued with acute treatment with the NMDAR partial agonist d-cycloserine. CONCLUSIONS: Our studies reveal that male Pcdh10+/- mice have synaptic and behavioral deficits, and establish Pcdh10+/- mice as a novel genetic model for investigating neural circuitry and behavioral changes relevant to ASD.

Activation of basolateral amygdala in juvenile C57BL/6J mice during social approach behavior.

There is a strong need to better understand the neurobiology of juvenile sociability (tendency to seek social interaction), a phenotype of central relevance to autism spectrum disorders (ASD). Although numerous genetic mouse models of ASD showing reduced sociability have been reported, and certain brain regions, such as the amygdala, have been implicated in sociability, there has been little emphasis on delineating brain structures and circuits activated during social interactions in the critical juvenile period of the mouse strain that serves as the most common genetic background for these models-the highly sociable C57BL/6J (B6) strain. We measured expression of the immediate early genes Fos and Egr-1 to map activation of brain regions following the Social Approach Test (SAT) in juvenile male B6 mice. We hypothesized that juvenile B6 mice would show activation of the amygdala during social interactions. The basolateral amygdala (BLA) was activated by social exposure in highly sociable, 4-week-old B6 mice. In light of these data, and the many lines of evidence indicating alteration of amygdala circuits in human ASD, future studies are warranted to assess structural and functional alterations in the BLA, particularly at BLA synapses, in various mouse models of ASD.

cAMP response element-binding protein is required for stress but not cocaine-induced reinstatement.

Reinstatement of previously extinguished conditioned place preference (CPP) is precipitated by stress or drug exposure. Here, we show that acute exposure to forced swim stress (FS), in a context distinct from conditioning, induces reinstatement of cocaine CPP in wild-type mice. This behavior is accompanied by a pattern of phosphorylated cAMP response element-binding protein (pCREB) activation in discrete brain regions that is distinct from the pattern observed after cocaine-induced reinstatement. For example, previous cocaine conditioning increases pCREB levels in the amygdala, and acute exposure to FS, but not to cocaine, further augments these changes. In contrast, previous cocaine conditioning does not alter levels of pCREB in the nucleus accumbens, but acute exposure to FS increases pCREB levels in this region on reinstatement day. Furthermore, to determine whether these alterations of CREB are necessary in FS or cocaine-induced reinstatement, we examined the effect of these stimuli on reinstatement behavior in mice deficient in alpha and Delta isoforms of CREB. The CREB(alphaDelta) mutant mice show deficits in FS-induced reinstatement of conditioned place preference. In contrast, they show robust cocaine-induced reinstatement. This deficit in stress but not drug-induced reinstatement indicates a specific requirement for CREB in stress-induced behavioral responses to drugs of abuse.

Development of home cage social behaviors in BALB/cJ vs. C57BL/6J mice.

BALB/cJ and C57BL/6J inbred mouse strains have been proposed as useful models of low and high levels of sociability (tendency to seek social interaction), respectively, based primarily on behaviors of ∼30-day-old mice in the Social Approach Test (SAT). In the SAT, approach and sniffing behaviors of a test mouse toward an unfamiliar stimulus mouse are measured in a novel environment. However, it is unclear whether such results generalize to a familiar environment with a familiar social partner, such as with a littermate in a home cage environment. We hypothesized that C57BL/6J mice would show higher levels of social behaviors than BALB/cJ mice in the home cage environment, particularly at 30 days-of-age. We measured active and passive social behaviors in home cages by pairs of BALB/cJ or C57BL/6J littermates at ages 30, 41, and 69 days. The strains did not differ robustly in their active social behaviors. C57BL/6J mice were more passively social than BALB/cJ mice at 30 days, and C57BL/6J levels of passive social behaviors declined to BALB/cJ levels by 69 days. The differences in passive social behaviors at 30 days-of-age were primarily attributable to differences in huddling. These results indicate that different test conditions (SAT conditions vs. home cage conditions) elicit strain differences in distinct types of behaviors (approach/sniffing vs. huddling behaviors, respectively). Assessment of the more naturalistic social interactions in the familiar home cage environment with a familiar littermate will provide a useful component of a comprehensive assessment of social behaviors in mouse models relevant to autism.

Sociability and brain development in BALB/cJ and C57BL/6J mice.

Sociability--the tendency to seek social interaction--propels the development of social cognition and social skills, but is disrupted in autism spectrum disorders (ASD). BALB/cJ and C57BL/6J inbred mouse strains are useful models of low and high levels of juvenile sociability, respectively, but the neurobiological and developmental factors that account for the strains' contrasting sociability levels are largely unknown. We hypothesized that BALB/cJ mice would show increasing sociability with age but that C57BL/6J mice would show high sociability throughout development. We also hypothesized that littermates would resemble one another in sociability more than non-littermates. Finally, we hypothesized that low sociability would be associated with low corpus callosum size and increased brain size in BALB/cJ mice. Separate cohorts of C57BL/6J and BALB/cJ mice were tested for sociability at 19-, 23-, 31-, 42-, or 70-days-of-age, and brain weights and mid-sagittal corpus callosum area were measured. BALB/cJ sociability increased with age, and a strain by age interaction in sociability between 31 and 42 days of age suggested strong effects of puberty on sociability development. Sociability scores clustered according to litter membership in both strains, and perinatal litter size and sex ratio were identified as factors that contributed to this clustering in C57BL/6J, but not BALB/cJ, litters. There was no association between corpus callosum size and sociability, but smaller brains were associated with lower sociability in BALB/cJ mice. The associations reported here will provide directions for future mechanistic studies of sociability development.

Stress-induced potentiation of cocaine reward: a role for CRF R1 and CREB.

Both clinical and preclinical research have shown that stress can potentiate drug use; however, the underlying mechanisms of this interaction are unknown. Previously, we have shown that a single exposure to forced swim (FS) reinstates extinguished conditioned place preference (CPP) to cocaine and that cAMP response element binding protein (CREB) is necessary for this response. CREB can be activated by corticotropin releasing factor (CRF) receptor type 1 (CRF(R1)) binding, which mediates neuroendocrine and behavioral responses to stress as well as to drugs of abuse. The present experiments investigate whether changes in cocaine reward elicited by previous exposure to stress are mediated by CREB and/or CRF(R1). Chronic exposure to FS in advance of conditioning enhances cocaine CPP in wild-type mice, but this is blocked in CREB-deficient mice. In addition, pretreatment with the CRF(R1) antagonist, antalarmin, before FS exposure blocks this stress-induced enhancement of cocaine CPP. Furthermore, FS-induced increase in phosphorylated CREB (pCREB), specifically in the lateral septum (LS) and nucleus accumbens (NAc) is also blocked by antalarmin. Taken together, these studies suggest that both CREB and CRF(R1) activation are necessary for stress-induced potentiation of drug reward.

Neuregulin 1 transgenic mice display reduced mismatch negativity, contextual fear conditioning and social interactions.

INTRODUCTION: Neuregulin-1 (NRG1) is one of susceptibility genes for schizophrenia and plays critical roles in glutamatergic, dopaminergic and GABAergic signaling. Using mutant mice heterozygous for Nrg1 (Nrg1(+/-)) we studied the effects of Nrg1 signaling on behavioral and electrophysiological measures relevant to schizophrenia. EXPERIMENTAL PROCEDURE: Behavior of Nrg1(+/-) mice and their wild type littermates was evaluated using pre-pulse inhibition, contextual fear conditioning, novel object recognition, locomotor, and social choice paradigms. Event-related potentials (ERPs) were recorded to assess auditory gating and novel stimulus detection. RESULTS: Gating of ERPs was unaffected in Nrg1(+/-) mice, but mismatch negativity in response to novel stimuli was attenuated. The Nrg1(+/-) mice exhibited behavioral deficits in contextual fear conditioning and social interactions, while locomotor activity, pre-pulse inhibition and novel object recognition were not impaired. SUMMARY: Nrg1(+/-) mice had impairments in a subset of behavioral and electrophysiological tasks relevant to the negative/cognitive symptom domains of schizophrenia that are thought to be influenced by glutamatergic and dopaminergic neurotransmission. These mice are a valuable tool for studying endophenotypes of schizophrenia, but highlight that single genes cannot account for the complex pathophysiology of the disorder.

Chronically increased Gsalpha signaling disrupts associative and spatial learning.

The cAMP/PKA pathway plays a critical role in learning and memory systems in animals ranging from mice to Drosophila to Aplysia. Studies of olfactory learning in Drosophila suggest that altered expression of either positive or negative regulators of the cAMP/PKA signaling pathway beyond a certain optimum range may be deleterious. Here we provide genetic evidence of the behavioral and physiological effects of increased signaling through the cAMP/PKA pathway in mice. We have generated transgenic mice in which the expression of a constitutively active form of Gsalpha (Gsalpha* Q227L), the G protein that stimulates adenylyl cyclase activity, is driven in neurons within the forebrain by the promoter from the CaMKIIalpha gene. Despite significantly increased adenylyl cyclase activity, Gsalpha* transgenic mice exhibit PKA-dependent decreases in levels of cAMP due to a compensatory up-regulation in phosphodiesterase activity. Interestingly, Gsalpha* transgenic mice also exhibit enhanced basal synaptic transmission. Consistent with a role for the cAMP/PKA pathway in learning and memory, Gsalpha* transgenic mice show impairments in spatial learning in the Morris water maze and in contextual and cued fear conditioning tasks. The learning deficits observed in these transgenic mice suggest that associative and spatial learning requires regulated Gsalpha protein signaling, much as does olfactory learning in Drosophila.

CREB gene transcription factors: role in molecular mechanisms of alcohol and drug addiction.

This article presents the proceedings of a symposium presented at the meeting of the Research Society on Alcoholism, held in Vancouver, British Columbia, Canada, in June 2004. The organizers and chairpersons were Subhash C. Pandey and Fulton Crews. The presentations were (1) Ethanol Modulation of CREB: Role in Dependence and Withdrawal, by Fulton Crews; (2) Effects of D1 Dopamine Receptor Activation During Withdrawal From Chronic Morphine: Enhanced CREB Activation and Decreased Conditioned Place Aversion, by Elena H. Chartoff; (3) CREB-Haplodeficient Mice: Role in Anxiety and Alcohol-Drinking Behaviors, by Subhash C. Pandey; and (4) A Role for CREB in Stress and Drug Addiction, by Julie A. Blendy.