Optogenetic Interrogation of Functional Synapse Formation by Corticospinal Tract Axons in the Injured Spinal Cord

To restore function after injury to the CNS, axons must be stimulated to extend into denervated territory and, critically, must form functional synapses with appropriate targets. We showed previously that forced overexpression of the transcription factor Sox11 increases axon growth by corticospinal tract (CST) neurons after spinal injury. However, behavioral outcomes were not improved, raising the question of whether the newly sprouted axons are able to form functional synapses. Here we developed an optogenetic strategy, paired with single-unit extracellular recordings, to assess the ability of Sox11-stimulated CST axons to functionally integrate in the circuitry of the cervical spinal cord. Initial time course experiments established the expression and function of virally expressed Channelrhodopsin (ChR2) in CST cell bodies and in axon terminals in cervical spinal cord. Pyramidotomies were performed in adult mice to deprive the left side of the spinal cord of CST input, and the right CST was treated with adeno-associated virus (AAV)–Sox11 or AAV–EBFP control, along with AAV–ChR2. As expected, Sox11 treatment caused robust midline crossing of CST axons into previously denervated left spinal cord. Clear postsynaptic responses resulted from optogenetic activation of CST terminals, demonstrating the ability of Sox11-stimulated axons to form functional synapses. Mapping of the distribution of CST-evoked spinal activity revealed overall similarity between intact and newly innervated spinal tissue. These data demonstrate the formation of functional synapses by Sox11-stimulated CST axons without significant behavioral benefit, suggesting that new synapses may be mistargeted or otherwise impaired in the ability to coordinate functional output. SIGNIFICANCE STATEMENT As continued progress is made in promoting the regeneration of CNS axons, questions of synaptic integration are increasingly prominent. Demonstrating direct synaptic integration by regenerated axons and distinguishing its function from indirect relay circuits and target field plasticity have presented technical challenges. Here we force the overexpression of Sox11 to stimulate the growth of corticospinal tract axons in the cervical spinal cord and then use specific optogenetic activation to assess their ability to directly drive postsynaptic activity in spinal cord neurons. By confirming successful synaptic integration, these data illustrate a novel optogenetic-based strategy to monitor and optimize functional reconnection by newly sprouted axons in the injured CNS

Cryptococcal antigen testing in an integrated medical system: eastern Wisconsin

Context: Cryptococcosis is a serious environmentally-acquired endemic fungal infection causing meningitis, pneumonia and disseminated disease in usually immunocompromised hosts. Environmental associations of the pathogenic species include certain trees, soils, bird guano and sites such as parks. Little is known regarding the frequency or distribution of cryptococcosis in Wisconsin. Cryptococcal antigen detection tests (CrAg), more than 90% sensitive and specific, are frequently used to screen patients with likely disease. Objective: Explore the geodemographic and clinical features of Wisconsin patients tested with CrAg. Study Design: Retrospective review of CrAg-tested patients. Descriptive statistics were compared with chi-square or t-tests; binary logistic regression was used for multivariable analysis. Setting: Large Eastern Wisconsin medical system. Patients:All patients having CrAg performed 2013-April, 2017. Results: A total of 1465 CrAg (741 on serum, 723 on CSF, 1 other) were performed on 1211 unique patients (50.2% female, 73.9% White, mean age 53.7 +/-16.5) during this time. At least one CrAg was positive in 23/1211 patients (1.9%); 4 cases had pneumonia only; 21/23 were immunocompromised (6 transplant patients, 5 HIV, 4 malignancy, 3 steroid use, 2 diabetes, 1 combined deficiency). Positive patients were more likely to be male (82.6%) non-White (12/23 [3.8% of those tested] vs 11/23 [1.2% of those tested]), and these associations (both p Conclusion: Among ill CrAg-tested patients, male gender and non-White race/ethnicity, not natural environmental features, predicted positive tests

Cryptococcal Antigen Testing in an Integrated Medical System: Eastern Wisconsin

Cryptococcosis is a serious environmentally acquired endemic fungal infection commonly associated with immunocompromised hosts. Little is known regarding frequency or distribution in Wisconsin. We explored the geodemographic and clinical features of patients tested with cryptococcal antigen tests (CrAg) — previously shown to be \u3e90% sensitive and \u3e90% specific — within a large health care system located in eastern Wisconsin. To examine this, we retrospectively analyzed 1465 CrAg tests on 1211 unique patients (female: 50.2%; white race: 73.9%; mean age: 53.7 ± 16.5 years). At least one CrAg result was positive in 23 of 1211 patients (1.9%). From these, 21 of 23 were immunocompromised. Positive patients were disproportionately male (82.6%) and nonwhite (3.8% of those tested vs 1.2% of whites tested); P \u3c 0.01 for both. These associations remained in multivariable models. Positive patients were not significantly older (59.1 vs 53.6 years; P = 0.07). Overall, 17 separate zip codes had at least one positive case. Positive patients were more prevalent in the zip codes that included the city of Milwaukee (11 of 377 [2.9% of those tested] vs 12 of 834 [1.4% of all those tested in the remaining area of the state]), but this difference was not statistically significant. No other case clustering or close proximity to waterways was observed (41% were less than 162 m from green space, similar to historical controls). Overall, male sex, nonwhite race/ethnicity, and immunocompromised status, not zip code, were statistically associated with positive CrAg

Insulin sensitivity, leptin, adiponectin, resistin, and testosterone in adult male and female rats after maternal-neonatal separation and environmental stress

Care of premature infants often requires parental and caregiver separation, particularly during hypoxic and hypothermic episodes. We have established a neonatal rat model of human prematurity involving maternal-neonatal separation and hypoxia with spontaneous hypothermia prevented by external heat. Adults previously exposed to these neonatal stressors show a sex difference in the insulin and glucose response to arginine stimulation suggesting a state of insulin resistance. The current study used this cohort of adult rats to evaluate insulin resistance [homeostatic model assessment of insulin resistance (HOMA-IR)], plasma adipokines (reflecting insulin resistance states), and testosterone. The major findings were that daily maternal-neonatal separation led to an increase in body weight and HOMA-IR in adult male and female rats and increased plasma leptin in adult male rats only; neither prior neonatal hypoxia (without or with body temperature control) nor neonatal hypothermia altered subsequent adult HOMA-IR or plasma adiponectin. Adult male-female differences in plasma leptin were lost with prior exposure to neonatal hypoxia or hypothermia; male-female differences in resistin were lost in the adults that were exposed to hypoxia and spontaneous hypothermia as neonates. Exposure of neonates to daily hypoxia without spontaneous hypothermia led to a decrease in plasma testosterone in adult male rats. We conclude that neonatal stressors result in subsequent adult sex-dependent increases in insulin resistance and adipokines and that our rat model of prematurity with hypoxia without hypothermia alters adult testosterone dynamics

Programming of the adult HPA axis after neonatal separation and environmental stress in male and female rats

Maternal separation, hypoxia, and hypothermia are common stressors in the premature neonate. Using our rat model of human prematurity, we evaluated sexual dimorphisms in the long term effects of these neonatal stressors on behavior of the hypothalamic-pituitary-adrenal (HPA) axis in adult rats. Neonatal rats were exposed daily on postnatal days 2-6 to maternal separation with normoxia, with hypoxia allowing spontaneous hypothermia, with hypothermia per se, and with hypoxia while maintaining isothermia with external heat. The major findings were that (a) prior maternal-neonatal separation during the first week of postnatal life significantly attenuated the plasma ACTH and corticosterone response to restraint stress in adult male but not female rats, (b) prior neonatal hypothermia augmented the plasma ACTH and corticosterone response to restrain stress in adult male, but not female rats, and (c) changes in hypothalamic, pituitary, and adrenal mRNA expression did not account for most of these HPA axis effects. Most of the programming effects on adult HPA axis was attributed to prior maternal-neonatal separation alone (with normoxia) because the addition of hypoxia with spontaneous hypothermia, hypothermia per se, and hypoxia while preventing hypothermia during maternal-neonatal separation had minimal effects on the HPA axis. These results may inform strategies to prevent sexually dimorphic sequelae of neonatal stress including those due to medical interventions

Effect of a melanocortin type 2 Receptor (MC2R) antagonist on the corticosterone response to hypoxia and ACTH stimulation in the neonatal rat

The adrenal stress response in the neonatal rat shifts from ACTH-independent to ACTH-dependent between postnatal days 2 (PD2) and 8 (PD8). This may be due to an increase in an endogenous, bioactive, non-immunoreactive ligand to the MC2R. GPS1574 is a newly described MC2R antagonist that we have shown to be effective in vitro. Further experimentation with GPS1574 would allow better insight into this seemingly ACTH-independent steroidogenic response in neonates. We evaluated the acute corticosterone response to hypoxia or ACTH injection following pretreatment with GPS1574 (32 mg/kg) or Vehicle for GPS1574 in PD2, PD8, and PD15 rat pups. Pretreatment with GPS1574 decreased baseline corticosterone in PD2 pups, but increased baseline corticosterone in PD8 and PD15 pups. GPS1574 did not attenuate the corticosterone response to hypoxia in PD2 pups, and augmented the corticosterone response in PD8 and PD15 pups. GPS1574 augmented the corticosterone response to ACTH in PD2 and PD15 pups, but had no significant impact on the response in PD8 pups. Baseline adrenal Mrap and Star mRNA increased from PD2 to PD15, whereas Mrap2 mRNA expression was low and did not change with age. The data suggests that GPS1574 is not a pure MC2R antagonist, but rather acts as a bias agonist/antagonist. Its ability to attenuate or augment the adrenal response may depend on the ambient plasma ACTH concentration and/or developmental changes in early transduction steroidogenic pathway genes

Sex differences in adult rat insulin and glucose responses to arginine: programming effects of neonatal separation, hypoxia, and hypothermia

Acute neonatal hypoxia, a common stressor, causes a spontaneous decrease in body temperature which may be protective. There is consensus that hypothermia should be prevented during acute hypoxia in the human neonate; however, this may be an additional stress with negative consequences. We hypothesize that maintaining body temperature during hypoxia in the first week of postnatal life alters the subsequent insulin, glucose, and glucagon secretion in adult rats. Rat pups were separated from their dam daily from postnatal days (PD) 2-6 for the following 90 min experimental treatments: (1) normoxic separation (control), (2) hypoxia (8% O2) allowing spontaneous hypothermia, (3) normoxic hypothermia with external cold, and (4) exposure to 8% O2 while maintaining body temperature using external heat. An additional normoxic non-separated control group was performed to determine if separation per se changed the adult phenotype. Plasma insulin, glucose, and glucagon responses to arginine stimulation were evaluated from PD105 to PD133. Maternal separation (compared to non-separated neonates) had more pronounced effects on the adult response to arginine compared to the hypoxic, hypothermic, and hypoxic-isothermic neonatal treatments. Adult males exposed to neonatal maternal separation had augmented insulin and glucose responses to arginine compared to unseparated controls. Additionally, neonatal treatment had a significant effect on body weight gain; adults exposed to neonatal maternal separation were significantly heavier. Female adults had significantly smaller insulin and glucose responses to arginine regardless of neonatal treatment. Neonatal maternal separation during the first week of life significantly altered adult beta-cell function in a sexually dimorphic manner