CRISPR-based genome editing of a diurnal rodent, Nile grass rat (Arvicanthis niloticus).

BACKGROUND: Diurnal and nocturnal mammals have evolved distinct pathways to optimize survival for their chronotype-specific lifestyles. Conventional rodent models, being nocturnal, may not sufficiently recapitulate the biology of diurnal humans in health and disease. Although diurnal rodents are potentially advantageous for translational research, until recently, they have not been genetically tractable. The present study aims to address this major limitation by developing experimental procedures necessary for genome editing in a well-established diurnal rodent model, the Nile grass rat (Arvicanthis niloticus). RESULTS: A superovulation protocol was established, which yielded nearly 30 eggs per female grass rat. Fertilized eggs were cultured in a modified rat 1-cell embryo culture medium (mR1ECM), in which grass rat embryos developed from the 1-cell stage into blastocysts. A CRISPR-based approach was then used for gene editing in vivo and in vitro, targeting Retinoic acid-induced 1 (Rai1), the causal gene for Smith-Magenis Syndrome, a neurodevelopmental disorder. The CRISPR reagents were delivered in vivo by electroporation using an improved Genome-editing via Oviductal Nucleic Acids Delivery (i-GONAD) method. The in vivo approach produced several edited founder grass rats with Rai1 null mutations, which showed stable transmission of the targeted allele to the next generation. CRISPR reagents were also microinjected into 2-cell embryos in vitro. Large deletion of the Rai1 gene was confirmed in 70% of the embryos injected, demonstrating high-efficiency genome editing in vitro. CONCLUSION: We have established a set of methods that enabled the first successful CRISPR-based genome editing in Nile grass rats. The methods developed will guide future genome editing of this and other diurnal rodent species, which will promote greater utility of these models in basic and translational research.

CRISPR-based Genome Editing of a Diurnal Rodent, Nile Grass Rat ( Arvicanthis niloticus).

Diurnal and nocturnal mammals have evolved distinct pathways to optimize survival for their chronotype-specific lifestyles. Conventional rodent models, being nocturnal, may not sufficiently recapitulate the biology of diurnal humans in health and disease. Although diurnal rodents are potentially advantageous for translational research, until recently, they have not been genetically tractable. Here, we address this major limitation by demonstrating the first successful CRISPR genome editing of the Nile grass rat ( Arvicanthis niloticus ), a valuable diurnal rodent. We establish methods for superovulation; embryo development, manipulation, and culture; and pregnancy maintenance to guide future genome editing of this and other diurnal rodent species.

Effects of bright light therapy on neuroinflammatory and neuroplasticity markers in a diurnal rodent model of Seasonal Affective Disorder.

BACKGROUND: Bright light therapy (BLT) is widely used for treating Seasonal Affective Disorder (SAD). However, the neural mechanisms underlying the therapeutic effects of BLT remain largely unexplored. The present study used a diurnal rodent (Nile grass rats; Arvicanthis niloticus) to test the hypothesis that the therapeutic effects of BLT could be, in part, due to reduced neuroinflammation and/or enhanced neuroplasticity. Our previous research has demonstrated that compared to grass rats housed in a summer-like daytime bright light condition (1000 lux), those housed in a winter-like daytime dim light condition (50 lux) showed increased depression- and anxiety-like behaviours, as well as impaired sociosexual behaviours and spatial memory, similar to what is observed in patients suffering from SAD. MATERIALS AND METHODS: In the present study, male and female grass rats were housed under the winter-like dim daytime light condition (lights on 600-1800 hr, 50 lux). The experimental groups received daily 1-h early morning BLT from 0600-0700 using full-spectrum light (10,000 lux), while the control groups received narrowband red light (λmax, 780 nm). Following 4 weeks of treatment, the expression of several neuroinflammatory or plasticity markers was examined in the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), and the CA1 of the dorsal hippocampus. RESULTS: For the neuroinflammatory markers, BLT reduced TNF-α in the BLA of females, and upregulated CD11b in the mPFC and IL6 in the BLA in males. For the neuroplasticity markers, BLT downregulated BDNF in the CA1 and TrkB in all three brain regions in females but upregulated BDNF in the BLA and CA1 in males. CONCLUSIONS: These results indicate that the therapeutic effects of BLT on sleep, mood, and cognition may be attributed in part to mechanisms involving neuroinflammation and neuroplasticity in corticolimbic brain regions. Moreover, these effects appear to vary between sexes.

Effects of light therapy on sleep/wakefulness, daily rhythms, and the central orexin system in a diurnal rodent model of seasonal affective disorder.

BACKGROUND: Bright light therapy (BLT) is the first-line treatment for seasonal affective disorder. However, the neural mechanisms underlying BLT are unclear. To begin filling this gap, the present study examined the impact of BLT on sleep/wakefulness, daily rhythms, and the wakefulness-promoting orexin/hypocretin system in a diurnal rodent, Nile grass rats (Arvicanthis niloticus). METHODS: Male and female grass rats were housed under a 12:12 h light/dark cycle with dim light (50 lx) during the day. The experimental group received daily 1-h early morning BLT (full-spectrum white light, 10,000 lx), while the control group received narrowband red light for 4 weeks. Sleep/wakefulness and in-cage locomotor activity were monitored, followed by examination of hypothalamic prepro-orexin and orexin receptors OX1R and OX2R expression in corticolimbic brain regions. RESULTS: The BLT group had higher wakefulness during light treatment, better nighttime sleep quality, and improved daily rhythm entrainment compared to controls. The impact of BLT on the orexin system was sex- and brain region-specific, with males showing higher OX1R and OX2R in the CA1, while females showed higher prepro-orexin but lower OX1R and OX2R in the BLA, compared to same-sex controls. LIMITATIONS: The present study focused on the orexin system in a limited number of brain regions at a single time point. Sex wasn't a statistical factor, as male and female cohorts were run independently. CONCLUSIONS: The diurnal grass rats show similar behavioral responses to BLT as humans, thus could be a good model for further elucidating the neural mechanisms underlying the therapeutic effects of BLT.

Daytime Light Deficiency Leads to Sex- and Brain Region-Specific Neuroinflammatory Responses in a Diurnal Rodent.

Seasonal changes in peripheral inflammation are well documented in both humans and animal models, but seasonal changes in neuroinflammation, especially the impact of seasonal lighting environment on neuroinflammation remain unclear. To address this question, the present study examined the effects of environmental lighting conditions on neuroinflammation in a diurnal rodent model, Nile grass rats (Arvicanthis niloticus). Male and female grass rats were housed in either bright (brLD) or dim (dimLD) light during the day to simulate a summer or winter light condition, respectively. After 4 weeks, microglia markers Iba-1 and CD11b, as well as pro-inflammatory cytokines TNF-α and IL-6, were examined in the anterior cingulate cortex (ACC), basolateral amygdala (BLA), and dorsal hippocampus (dHipp). The results revealed that winter-like dim light during the day leads to indicators of increased neuroinflammation in a brain site- and sex-specific manner. Specifically, relatively few changes in the neuroinflammatory markers were observed in the ACC, while numerous changes were found in the BLA and dHipp. In the BLA, winter-like dimLD resulted in hyper-ramified microglia morphology and increased expression of the pro-inflammatory cytokine IL-6, but only in males. In the dHipp, dimLD led to a higher number and hyper-ramified morphology of microglia as well as increased expression of CD11b and TNF-α, but only in females. Neuroinflammatory state is thus influenced by environmental light, differently in males and females, and could play a role in sex differences in the prevalence and symptoms of psychiatric or neurological disorders that are influenced by season or other environmental light conditions. Diurnal Nile grass rats were housed under bright or dim light during the day for 4 weeks, simulating seasonal fluctuations in daytime lighting environment. Dim light housing resulted in hyper-ramified morphology of microglia (scale bar, 15 µm) and altered expression of pro-inflammatory cytokines (TNF-α) in a sex- and brain region-specific manner.

Impact of daytime light intensity on the central orexin (hypocretin) system of a diurnal rodent (Arvicanthis niloticus).

The neuropeptide orexin/hypocretin is implicated in sleep and arousal, energy expenditure, reward, affective state and cognition. Our previous work using diurnal Nile grass rats (Arvicanthis niloticus) found that orexin mediates the effects of environmental light, particularly daytime light intensity, on affective and cognitive behaviours. The present study further investigated how daytime light intensity affects the central orexin system in male and female grass rats. Subjects were housed for 4 weeks in 12:12 hr dim light:dark (50 lux, dimLD) or in 12:12 hr bright light:dark cycle (1000 lux, brightLD). Day/night fluctuations in some orexin measures were also assessed. Despite similar hypothalamic prepro-orexin mRNA expression across all conditions, there were significantly more orexin-immunoreactive neurons, larger somata, greater optical density or higher orexin A content at night (ZT14) than during the day (ZT2), and/or in animals housed in brightLD compared to dimLD. Grass rats in brightLD also had higher cisternal CSF levels of orexin A. Furthermore, orexin receptor OX1R and OX2R proteins in the medial prefrontal cortex were higher in brightLD than dimLD males, but lower in brightLD than dimLD females. In the CA1 and dorsal raphe nucleus, females had higher OX1R than males without any significant effects of light condition, and OX2R levels were unaffected by sex or light. These results reveal that daytime light intensity alters the central orexin system of both male and female diurnal grass rats, sometimes sex-specifically, and provides insight into the mechanisms underlying how daytime light intensity impacts orexin-regulated functions.

Postpartum State, but Not Maternal Caregiving or Level of Anxiety, Increases Medial Prefrontal Cortex GAD65 and vGAT in Female Rats.

Upregulation of the inhibitory neurotransmitter, GABA, is involved in many of the behavioral differences between postpartum and nulliparous female rodents. This is evidenced by studies showing that pharmacological blockade of GABAergic activity impairs maternal caregiving and postpartum affective behaviors. However, the influence of motherhood on the capacity for GABA synthesis or release in the medial prefrontal cortex (mPFC; brain region involved in many social and affective behaviors) is not well-understood. Western blotting was used to compare postpartum and nulliparous rats in protein levels of the 65-kD isoform of glutamic acid decarboxylase (GAD65; synthesizes most GABA released from terminals) and vesicular GABA transporter (vGAT; accumulates GABA into synaptic vesicles for release) in the mPFC. We found that postpartum mothers had higher GAD65 and vGAT compared to virgins, but such differences were not found between maternally sensitized and non-sensitized virgins, indicating that reproduction rather than just the display of maternal caregiving is required. To test whether GAD65 and vGAT levels in the mPFC were more specifically related to anxiety-related behavior within postpartum mothers, we selected 8 low-anxiety and 8 high-anxiety dams based on their time spent in the open arms of an elevated plus maze on postpartum day 7. There were no significant differences between the anxiety groups in either GAD65 or vGAT levels. These data further indicate that frontal cortical GABA is affected by female reproduction and more likely contributes to differences in the display of socioemotional behaviors across, but not within, female reproductive state.

Low Daytime Light Intensity Disrupts Male Copulatory Behavior, and Upregulates Medial Preoptic Area Steroid Hormone and Dopamine Receptor Expression, in a Diurnal Rodent Model of Seasonal Affective Disorder.

Seasonal affective disorder (SAD) involves a number of psychological and behavioral impairments that emerge during the low daytime light intensity associated with winter, but which remit during the high daytime light intensity associated with summer. One symptom frequently reported by SAD patients is reduced sexual interest and activity, but the endocrine and neural bases of this particular impairment during low daylight intensity is unknown. Using a diurnal laboratory rodent, the Nile grass rat (Arvicanthis niloticus), we determined how chronic housing under a 12:12 h day/night cycle involving dim low-intensity daylight (50 lux) or bright high-intensity daylight (1,000 lux) affects males' copulatory behavior, reproductive organ weight, and circulating testosterone. We also examined the expression of mRNAs for the aromatase enzyme, estrogen receptor 1 (ESR1), and androgen receptor (AR) in the medial preoptic area (mPOA; brain site involved in the sensory and hormonal control of copulation), and mRNAs for the dopamine (DA) D1 and D2 receptors in both the mPOA and nucleus accumbens (NAC; brain site involved in stimulus salience and motivation to respond to reward). Compared to male grass rats housed in high-intensity daylight, males in low-intensity daylight displayed fewer mounts and intromissions when interacting with females, but the groups did not differ in their testes or seminal vesicle weights, or in their circulating levels of testosterone. Males in low-intensity daylight unexpectedly had higher ESR1, AR and D1 receptor mRNA in the mPOA, but did not differ from high-intensity daylight males in D1 or D2 mRNA expression in the NAC. Reminiscent of humans with SAD, dim winter-like daylight intensity impairs aspects of sexual behavior in a male diurnal rodent. This effect is not due to reduced circulating testosterone and is associated with upregulation of mPOA steroid and DA receptors that may help maintain some sexual motivation and behavior under winter-like lighting conditions.