Behavioral flexibility is the ability of an animal to adapt to changes in its environment. One example is altering a strategy to achieve a goal when the previously chosen strategy no longer works. A hallmark of many psychiatric diseases, such as depression, is a distinct decrease in behavioral flexibility (Uddin, 2021). This contributes to individuals feeling “stuck” and not being able to adapt to dynamic environments. One structure that plays a crucial role in negative emotion and depression is an epithalamic structure called the lateral habenula (LHb). In fact, LHb hyperactivity is a consistent neural marker of depression (Caldecott-Hazard et al., 1988). Interestingly, the LHb is implicated in a variety of contexts, including memory formation and spatial navigation (Mathis et al., 2017, Goutagny et al., 2013, Baker et al., 2019). We sought to discover precisely how the LHb contributes to behavioral flexibility with the use of a complex strategy switching task. This task is performed on an elevated plus maze, requiring rats to recognize changes in reward contingency and adapt their strategy accordingly. Animals must either alternate between reward locations (e.g. east or west maze arms) or go the same reward arm (e.g. only east or only west), regardless of start location. After animals were fully trained on the task, we inactivated the LHb using muscimol, a GABA-A agonist. Our preliminary results (n=6 rats) show that strategy switching and other behavioral metrics are impaired when the LHb undergoes inactivation, indicating that the LHb and its downstream circuitry play an important role in behavioral flexibility.

The mechanisms guiding the sensory detection of pain and the subsequent sensitization of damaged tissue to mechanical and thermal stimuli are relatively well understood. However, mechanisms guiding the transformation of nociception into the negative feelings associated with pain remains largely unknown. This affective component, notably in chronic pain, translates into an intense emotional impact on patients and can contribute to the development of comorbid psychiatric disorders. The elderly population has a propensity to be socially isolated and face exacerbated effects of chronic pain. In 2021, an estimated 20.9% of U.S adults suffer from chronic pain with persons over 65 years of age having the greatest propensity of acquiring the disease. Due to this, clinical intervention models call for a more holistic approach to pain intervention that incorporates lifestyle and nutritional factors, extending beyond pharmacological treatments. One of these promising non-pharmacological interventions is positive social interaction, which has been shown to alleviate pain and suffering. Several studies show that humans who maintain strong social bonds recover from injuries faster than people without them. However, it has not yet been evaluated the extent to which this phenomenon occurs in geriatric animals and its relative efficacy as a social intervention to alleviate chronic pain in injured mice. My project seeks to gauge whether social intervention can alleviate chronic pain symptoms in aged mice and to unveil the underlying mechanisms guiding these successful non-pharmacological treatments. I will achieve this through two aims: an evaluation of social self administration as an intervention for chronic pain, and histological analysis to identify gene expression changes as a result of social interaction. Future research will include mini-scope endomicroscopy recordings to visualize communication among major brain regions, and comparison of cell ensemble activity between groups of mice will lead to the identification of relevant neural ensembles and molecules.

In mammals, circadian rhythms are regulated by a hierarchy of oscillators governed by a central circadian pacemaker in the suprachiasmatic nucleus (SCN), which is principally entrained by the light-dark (LD) cycle. Recent experiments in our lab have revealed that cyclic 24-h fearful stimuli can act as a potent nonphotic zeitgeber, entraining circadian rhythms of behavior in mice and rats. This discovery utilized a naturalistic rodent cage with a safe nesting area separated from a foraging area where feeding and drinking occur. While foraging behaviors naturally occur at night, when the foraging area is rendered dangerous by nocturnal aversive stimuli (footshocks), animals entrain behaviors to the shock schedule by shifting activity to the daytime. Under conditions of fear-entrainment, SCN clock gene expression remains loyal to the LD cycle and the SCN is necessary but not sufficient for sustaining diurnal activity. Therefore, we propose the existence of extra-SCN fear-entrained oscillators capable of overriding SCN output and influencing behavioral timing. Here, we subjected 16 mice to either diurnal shocks (DS; control) or nocturnal shocks (NS) under a 12:12 LD cycle. Following confirmation of fear-entrainment, animals were released into constant conditions and sacrificed between 24-36h after the last presentation of footshocks, either CT 1 or CT13. Brains were dissected, sliced, prepared for immunohistochemistry processing, and c-Fos protein quantification is currently underway in the SCN, basolateral amygdala, paraventricular nucleus of the thalamus, and dentate gyrus. We hypothesize that c-Fos expression within the SCN will align with the LD cycle, while centers involved in fear processing and memory will exhibit altered levels of c-Fos expression in response to time-specific fear. Results from this study may be useful for identifying putative brain regions containing fear-entrainable oscillator(s).

Leigh syndrome (LS) is the most common form of mitochondrial disease in children. It affects 1 in every 40,000 births and its clinical manifestations include ataxia, seizures, failure to thrive and premature death. Genetic mutations in more than 75 different genes have been associated with LS. Among them is NDUFS4, the gene that codes for a subunit of the protein complex I of the mitochondria. Mice carrying a whole-body knockout (KO) of this gene greatly model this illness; they recapitulate multiple phenotypes of LS in patients. Prior studies in the lab have shown that the KO of Ndufs4 in GABAergic neurons, not in excitatory neurons, across all brain regions, reproduce the epilepsy phenotype seen in the global KO mice. Surprisingly, new KO mice with Ndufs4 inactivation restricted to GABAergic neurons of the brainstem and cerebellum interneurons, mediated by GlycineT2Cre, also have epilepsy. In this study, we sought to uncover the brain regions that house neurons involved in seizure activity in these mice. Brain regions experiencing neuronal hyperactivity during seizures in this new model of LS were examined. A thermal seizure was induced in the Ndufs4 GlycineT2Cre KO mice. For control condition, mice were exposed to a sham experiment. Forty-five minutes after the seizures or sham procedure, the mice were anaesthetized, and their brains were fixed and harvested. Brain slices were prepared and stained with a c-Fos antibody and finally imaged on the confocal microscope. Interestingly, high c-Fos immunoactivity was observed in the cerebellum alone and not in forebrain brain regions generally known to be involved in seizure generation. These findings indicate the participation of the cerebellum in seizure generation in Leigh syndrome epilepsy. In future studies we plan to increase the sample size and confirm the results with statistical methods.

Photoreceptor cells in the retina use several opsin proteins to detect light and confer visual information. Mutations to genes encoding opsins are associated with varying degrees of color blindness and retinal degeneration. In a previous mouse model for color blindness the retina degenerated, with a significant reduction of healthy cones by 3 months (Ma, et al., 2022, Human Gene Therapy). I am characterizing a new mouse line that was gene edited to lack both blue and green sensitive cone opsins, making it a double knock out. We fix and embed the eyes before freezing them, and then I stain frozen eye sections with fluorescent antibodies.  Using a high-resolution microscope, I am able to examine retina health and cone populations. I am comparing knockout animals with a wild type strain of normal mice and a retinal degeneration strain processed in the same way. I confirmed the absence of both blue and green cone opsin in the knockout mice, and despite being a model for color blindness, the mice maintain a healthy population of ospin-less cones for at least one year. However, at one year the knockout mice have approximately 30% fewer cones in their retinas than normal mice. The populations of dying cells and immune response cells in the knockout retinas match those seen in the wild type retinas, and are significantly lower than the populations seen in the degeneration model. This suggests that the retina is not in an active state of degeneration for at least one year. This model will be useful for future development of cone opsin gene therapies, and can serve as a model for color blindness. It also has implications for the health of cones without the cone opsin protein.