Behavioral comparison of the C57BL/6 inbred mouse strain and their CB6F1 siblings.

A large portion of basic biomedical research studies are conducted using genetically defined, inbred mouse strains. The C57BL/6 mouse strain is the most widely used genetic background in current rodent research. The rationale for using inbred strains is that all individuals are genetically identical with minimal phenotypic variation, allowing for more statistically powerful analyses. F1 hybrids between two inbred strains are also genetically identical to one another but are heterozygous at every locus at which the parental strains differ rather than homozygous. Both theoretical and empirical evidence suggests that this heterozygosity in F1 hybrids allow for potentially greater resilience in response to the inevitable stresses of laboratory environments. The purpose of this study was to characterize the differences in commonly used tests of physical performance (forelimb grip strength and rotarod) and anxiety-like behavior between the F1 hybrids created from BALB/c females mated to C57BL/6 males (called CB6F1 mice) and one of its parental strains, C57BL/6. We used a natural cross-fostering breeding scheme to minimize maternal care effects and emphasize the effects of genetic differences. We found significant correlations between anxiety-like behavioral measures and physical performance measures which are not traditionally associated with anxiety-like behavior, and which differ between strains. Findings from this study should be taken into consideration when designing behavioral studies and choosing model organisms.

Differences between adult and adolescent male mice in approach/avoidance and expression of hippocampal NPY in response to acute footshock.

Anxiety disorders are the most common neuropsychiatric disorders diagnosed in adolescence and adulthood. Stress can lead to an increase in anxiety-related behaviors, although the consequences of stress in rodents are typically investigated only in adults. The levels of Neuropeptide Y (NPY), a mediator of stress resilience, are reduced in adult patients with Post-Traumatic Stress Disorder. For rodents, footshock is a physical stressor that increases anxiety-like behavior and reduces NPY in adults, however, the effects in adolescents are unknown. Here we used a 30-min unpredictable footshock protocol to investigate the differences in behavior and stress-relevant molecules between adolescent (6 weeks) and adult (3 months) male C57Bl6/J mice. The protocol resulted in fear expression in both ages as observed by enhanced freezing during footshock and elevation in plasma corticosterone and NPY shortly after exposure. However, effects on approach/avoidance behavior were different between the two ages. One week after footshock exposure, adult mice showed reduced open arm time and entries on elevated plus maze (EPM), whereas adolescent mice showed no effect. Footshock mice in both age groups displayed reduced activity levels in EPM and open field. The hypolocomotion did not relate to motor deficits, as there were no differences between footshock and control groups using rotarod. Surprisingly, we found that the adolescent mice had elevated NPY peptide expression in hippocampus, whereas adults had reduced expression one week after footshock exposure. Together, these results demonstrate that stress differentially affects both behavior and the important stress resilience factor NPY in adolescents compared to adults.

A novel mouse model to evaluate neuropeptide Y-mediated melanocyte pathology.

Vitiligo is an autoimmune disease characterized by depigmented patches of skin due to loss of the pigment-producing melanocytes. No cure exists for vitiligo. The available treatments are inefficient for many patients, suggesting that universal treatment approaches may be inappropriate. Deeper understanding of the mechanistic basis for variability in vitiligo aetiologies is necessary. Genetic mutations in neuropeptide Y (NPY), a widely distributed protein, are associated with increased NPY expression and increased susceptibility for vitiligo. NPY is also upregulated in the circulation and lesional skin of some vitiligo patients. However, the contributions of NPY to melanocyte pathology are not understood, and presently there are no models with which to investigate this possibility. In this study, we employed NPY-overexpressing mice to explore the role of NPY in melanocyte dysfunction. Our results show that NPY overexpression induces progressive hair greying (depigmentation) due to premature depletion of follicular melanocyte stem cells. Additionally, NPY transcripts and protein are elevated in the skin and melanocytes of these mice, respectively, suggesting that these effects may be mediated locally. Together, these results suggest that supraphysiological levels of NPY in the skin can induce melanocyte dysfunction, thus identifying this mouse line as a novel model to study NPY-mediated melanocyte pathology.

Overexpression of neuropeptide Y decreases responsiveness to neuropeptide Y.

Neuropeptide Y (NPY) is an endogenous neuropeptide that is abundantly expressed in the central nervous system. NPY is involved in various neurological processes and neuropsychiatric disorders, including fear learning and anxiety disorders. Reduced levels of NPY are reported in Post-Traumatic Stress Disorder (PTSD) patients, and NPY has been proposed as a potential therapeutic target for PTSD. It is therefore important to understand the effects of chronic enhancement of NPY on anxiety and fear learning. Previous studies have shown that acute elevation of NPY reduces anxiety, fear learning and locomotor activity. Models of chronic NPY overexpression have produced mixed results, possibly caused by ectopic NPY expression. NPY is expressed primarily by a subset of GABAergic interneurons, providing specific spatiotemporal release patterns. Administration of exogenous NPY throughout the brain, or overexpression in cells that do not normally release NPY, can have detrimental side effects, including memory impairment. In order to determine the effects of boosting NPY only in the cells that normally release it, we utilized a transgenic mouse line that overexpresses NPY only in NPY+ cells. We tested for effects on anxiety related behaviors in adolescent mice, an age with high incidence of anxiety disorders in humans. Surprisingly, we did not observe the expected reduction in anxiety-like behavior in NPY overexpression mice. There was no change in fear learning behavior, although there was a deficit in nest building. The effect of exogenous NPY on synaptic transmission in acute hippocampal slices was also diminished, indicating that the function of NPY receptors is impaired. Reduced NPY receptor function could contribute to the unexpected behavioral outcomes. We conclude that overexpression of NPY, even in cells that normally express it, can lead to reduced responsiveness of NPY receptors, potentially affecting the ability of NPY to function as a long-term therapeutic.

Prefrontal cortex-dependent innate behaviors are altered by selective knockdown of Gad1 in neuropeptide Y interneurons.

GABAergic dysfunction has been implicated in a variety of neurological and psychiatric disorders, including anxiety disorders. Anxiety disorders are the most common type of psychiatric disorder during adolescence. There is a deficiency of GABAergic transmission in anxiety, and enhancement of GABA transmission through pharmacological means reduces anxiety behaviors. GAD67-the enzyme responsible for GABA production-has been linked to anxiety disorders. One class of GABAergic interneurons, Neuropeptide Y (NPY) expressing cells, is abundantly found in brain regions associated with anxiety and fear learning, including prefrontal cortex, hippocampus and amygdala. Additionally, NPY itself has been shown to have anxiolytic effects, and loss of NPY+ interneurons enhances anxiety behaviors. A previous study showed that knockdown of Gad1 from NPY+ cells led to reduced anxiety behaviors in adult mice. However, the role of GABA release from NPY+ interneurons in adolescent anxiety is unclear. Here we used a transgenic mouse that reduces GAD67 in NPY+ cells (NPYGAD1-TG) through Gad1 knockdown and tested for effects on behavior in adolescent mice. Adolescent NPYGAD1-TG mice showed enhanced anxiety-like behavior and sex-dependent changes in locomotor activity. We also found enhancement in two other innate behavioral tasks, nesting construction and social dominance. In contrast, fear learning was unchanged. Because we saw changes in behavioral tasks dependent upon prefrontal cortex and hippocampus, we investigated the extent of GAD67 knockdown in these regions. Immunohistochemistry revealed a 40% decrease in GAD67 in NPY+ cells in prefrontal cortex, indicating a significant but incomplete knockdown of GAD67. In contrast, there was no decrease in GAD67 in NPY+ cells in hippocampus. Consistent with this, there was no change in inhibitory synaptic transmission in hippocampus. Our results show the behavioral impact of cell-specific interneuron dysfunction and suggest that GABA release by NPY+ cells is important for regulating innate prefrontal cortex-dependent behavior in adolescents.