Axon regeneration in the central nervous system is limited both by inhibitory extracellular cues and by an intrinsically low capacity for axon growth in some CNS populations. Chondroitin sulfate proteoglycans (CSPGs) are well-studied inhibitors of axon growth in the CNS, and degradation of CSPGs by chondroitinase has been shown to improve the extension of injured axons. Alternatively, axon growth can be improved by targeting the neuron-intrinsic growth capacity through forced expression of regeneration-associated transcription factors. For example, a transcriptionally active chimera of Krüppel-like Factor 7 (KLF7) and a VP16 domain improves axon growth when expressed in corticospinal tract neurons. Here we tested the hypothesis that combined expression of chondroitinase and VP16-KLF7 would lead to further improvements in axon growth after spinal injury. Chondroitinase was expressed by viral transduction of cells in the spinal cord, while VP16-KLF7 was virally expressed in sensory neurons of the dorsal root ganglia or corticospinal tract (CST) neurons. After transection of the dorsal columns, both chondroitinase and VP16-KLF7 increased the proximity of severed sensory axons to the injury site. Similarly, after complete crush injuries, VP16-KLF7 expression increased the approach of CST axons to the injury site. In neither paradigm however, did single or combined treatment with chondroitinase or VP16-KLF7 enable regenerative growth distal to the injury. These results substantiate a role for CSPG inhibition and low KLF7 activity in determining the net retraction of axons from sites of spinal injury, while suggesting that additional factors act to limit a full regenerative response.

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wordsMain Text: 4252 words Abstract Background: Imbalance in prefrontal cortical (PFC) pyramidal neuron excitation:inhibition is thought to underlie symptomologies shared across stress-related disorders and neuropsychiatric disease, including dysregulation of emotion and cognitive function. G protein-gated inwardly rectifying K + (GIRK/Kir3) channels mediate excitability of medial PFC pyramidal neurons, however the functional role of these channels in mPFC-dependent regulation of affect, cognition, and cortical dynamics is unknown. Methods:In mice harboring a 'floxed' version of the kcnj3 (Girk1) gene, we used a viral-cre approach to disrupt GIRK1-containing channel expression in pyramidal neurons within the prelimbic (PL) or infralimbic (IL) cortices. Additional studies used a novel model of chronic unpredictable stress (CUS) to determine the impact on PL GIRK-dependent signaling and cognitive function. Results:In males, loss of pyramidal GIRK-dependent signaling in the PL, but not IL, differentially impacted measures of affect and motivation, and impaired working memory and cognitive flexibility. CUS produced similar deficits in affect and cognition that paralleled a reduction in PL pyramidal GIRK-dependent signaling akin to viral approaches. Viral-and stress-induced behavioral deficits were rescued by systemic injection of a novel, GIRK1-selective agonist, ML-297. Unexpectedly, neither ablation of PL GIRK-dependent signaling or exposure to the CUS regimen impacted affect or cognition in female mice.Conclusions: GIRK-dependent signaling in male mice, but not females, is critical for maintaining optimal PL function and behavioral control. Disruption of this inhibition may underlie stress-related dysfunction of the PL and represent a therapeutic target for treating stress-induced deficits in affect regulation and impaired cognition that reduce quality of life.

Leptin signaling pathways, stemming primarily from the hypothalamus, are necessary for maintaining normal energy homeostasis and body weight. In both rodents and humans, dysregulation of leptin signaling leads to morbid obesity and diabetes. Since leptin resistance is considered a primary factor underlying obesity, understanding the regulation of leptin signaling could lead to therapeutic tools and provide insights into the causality of obesity. While leptin actions in some hypothalamic regions such as the arcuate nuclei have been characterized, less is known about leptin activity in the hypothalamic ventromedial nuclei (VMN). Recently, pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to reduce feeding behavior and alter metabolism when administered into the VMN in a pattern similar to that of leptin. In the current study, we examined whether leptin and PACAP actions in the VMN share overlapping pathways in the regulation of energy balance. Interestingly, PACAP administration into the VMN increased STAT3 phosphorylation and SOCS3 mRNA expression, both of which are hallmarks of leptin receptor activation. In addition, BDNF mRNA expression in the VMN was increased by both leptin and PACAP administration. Moreover, antagonizing PACAP receptors fully reversed the behavioral and cellular effects of leptin injections into the VMN. Electrophysiological studies further illustrated that leptin-induced effects on VMN neurons were blocked by antagonizing PACAP receptors. We conclude that leptin dependency on PACAP signaling in the VMN suggests a potential common signaling cascade, allowing a tonically and systemically secreted neuropeptide to be more precisely regulated by central neuropeptides.

The impact of CNS disorders is exacerbated by the difficulty in developing safe, effective glutamatergic therapeutics. Synaptic glutamate transmission is vital for neural physiology throughout the brain, which contributes to the vast therapeutic potential and safety risk of glutamatergic therapeutics. Here, we created a genetically modified rat (MSxc) to survey the range of brain functions impacted by the loss of glutamate release from astrocytes involving system xc- (Sxc). Eliminating Sxc activity was not lethal and did not alter growth patterns, activity states, novel object recognition or performance of other simple tasks. In contrast, MSxc rats differed from WT in Pavlovian Conditioned Approach and cocaine self-administration/reinstatement paradigms. Both WT and MSxc rats readily learned that a cue predicted food delivery during Pavlovian Conditioned Approach training. However, WT rats were more likely to approach the food tray (i.e., goal tracking) whereas MSxc rats were more likely to approach the food-predicted cue (i.e., sign tracking) even when this behavior was punished. In the self-administration/reinstatement paradigm, MSxc rats had higher levels of cocaine-primed drug seeking in the absence of altered extinction or cocaine self-administration. These data demonstrate that Sxc-mediated glutamate release from astrocytes regulates non-reinforced and negative-outcome behaviors without altering simple learning or other forms of basic brain function.

OBJECTIVES/GOALS: We aim to determine whether non-neuronal, non-synaptic glutamate signaling mechanisms can be targeted to produce highly specific, narrow changes in brain function that would benefit CNS disorders. To do this, we investigated cognitive changes produced through manipulating the activity of the astrocytic glutamate release mechanism system xc-. METHODS/STUDY POPULATION: System xc- (Sxc) activity was eliminated by mutating the gene Slc7a11 through pronuclear injection of zinc-finger nucleases into Sprague Dawley rat embryos to create a line of rats lacking Sxc (MSxc rats). To confirm a lack of Sxc activity, we verified that tissue from MSxc rats had a complete lack of xCT, which is the regulatory subunit of Sxc that is encoded by Slc7a11. We also verified that astrocyte cultures generated from MSxc tissue lacked cystine-evoked glutamate release. Next, we measured development (body weight), CNS regulation of metabolism, and other indicators of generalized, non-specific brain function as well as behaviors that are reliant on executive function, such as cognitive flexibility, impulse control, decision-making, and response inhibition. RESULTS/ANTICIPATED RESULTS: Eliminating Sxc was not lethal and did not impair development or produce widespread changes in brain function as is commonly observed when deleting other glutamate mechanisms. MSxc rats did not differ from wildtype in growth rate, central regulation of metabolism as reflected by absolute or diurnal changes in core body temperature, locomotor activity in a familiar or novel environment, or simple forms of cognition such as novel object recognition, or operant responding (food and cocaine-reinforced). In contrast, behaviors that rely on executive function were impaired. MSxc rats displayed deficits in cocaine reinstatement and attentional set-shifting. We anticipate MSxc rats to also show impairments in decision-making in the rat gambling task and response inhibition in the stop-signal reaction time task. DISCUSSION/SIGNIFICANCE: Eliminating Sxc activity in rats produced deficits in behaviors reliant on executive function without impacting development or simple brain function. These results highlight the potential of targeting Sxc to enhance cognition without generating therapeutically limiting adverse effects resulting from non-specific changes in brain function.

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