AAV Vector Mediated Delivery of NG2 Function Neutralizing Antibody and Neurotrophin NT-3 Improves Synaptic Transmission, Locomotion, and Urinary Tract Function after Spinal Cord Contusion Injury in Adult Rats.

NG2 is a structurally unique transmembrane chondroitin sulfate proteoglycan (CSPG). Its role in damaged spinal cord is dual. NG2 is considered one of key inhibitory factors restricting axonal growth following spinal injury. Additionally, we have recently detected its novel function as a blocker of axonal conduction. Some studies, however, indicate the importance of NG2 presence in the formation of synaptic contacts. We hypothesized that the optimal treatment would be neutralization of inhibitory functions of NG2 without its physical removal. Acute intraspinal injections of anti-NG2 monoclonal antibodies reportedly prevented an acute block of axonal conduction by exogenous NG2. For prolonged delivery of NG2 function neutralizing antibody, we have developed a novel gene therapy: adeno-associated vector (AAV) construct expressing recombinant single-chain variable fragment anti-NG2 antibody (AAV-NG2Ab). We examined effects of AAV-NG2Ab alone or in combination with neurotrophin NT-3 in adult female rats with thoracic T10 contusion injuries. A battery of behavioral tests was used to evaluate locomotor function. In vivo single-cell electrophysiology was used to evaluate synaptic transmission. Lower urinary tract function was assessed during the survival period using metabolic chambers. Terminal cystometry, with acquisition of external urethral sphincter activity and bladder pressure, was used to evaluate bladder function. Both the AAV-NG2Ab and AAV-NG2Ab combined with AAV-NT3 treatment groups demonstrated significant improvements in transmission, locomotion, and bladder function compared with the control (AAV-GFP) group. These functional improvements associated with improved remyelination and plasticity of 5-HT fibers. The best results were observed in the group that received combinational AAV-NG2Ab+AAV-NT3 treatment.SIGNIFICANCE STATEMENT We recently demonstrated beneficial, but transient, effects of neutralization of the NG2 proteoglycan using monoclonal antibodies delivered intrathecally via osmotic mini-pumps after spinal cord injury. Currently, we have developed a novel gene therapy tool for prolonged and clinically relevant delivery of a recombinant single-chain variable fragment anti-NG2 antibody: AAV-rh10 serotype expressing scFv-NG2 (AAV-NG2Ab). Here, we examined effects of AAV-NG2Ab combined with transgene delivery of Neurotrophin-3 (AAV-NT3) in adult rats with thoracic contusion injuries. The AAV-NG2Ab and AAV-NG2Ab+AAV-NT3 treatment groups demonstrated significant improvements of locomotor function and lower urinary tract function. Beneficial effects of this novel gene therapy on locomotion and bladder function associated with improved transmission to motoneurons and plasticity of axons in damaged spinal cord.

Gonadectomy but not biological sex affects burst-firing in dopamine neurons of the ventral tegmental area and in prefrontal cortical neurons projecting to the ventral tegmentum in adult rats.

The mesocortical and mesolimbic dopamine systems regulate cognitive and motivational processes and are strongly implicated in neuropsychiatric disorders in which these processes are disturbed. Sex differences and sex hormone modulation are also known for these dopamine-sensitive behaviours in health and disease. One relevant mechanism of hormone impact appears to be regulation of cortical and subcortical dopamine levels. This study asked whether this regulation of dopamine tone is a consequence of sex or sex hormone impact on the firing modes of ventral midbrain dopamine neurons. To address this, single unit extracellular recordings made in the ventral tegmental area and substantia nigra were compared among urethane-anaesthetized adult male, female, gonadectomized male rats. These comparisons showed that gonadectomy had no effect on nigral cells and no effects on pacemaker, bursty, single-spiking or random modes of dopamine activity in the ventral tegmental area. However, it did significantly and selectively increase burst firing in these cells in a testosterone-sensitive, estradiol-insensitive manner. Given the roles of prefrontal cortex (PFC) in modulating midbrain dopamine cell firing, we next asked whether gonadectomy's effects on dopamine cell bursting had correlated effects on the activity of ventral tegmentally projecting prefrontal cortical neurons. We found that gonadectomy indeed significantly and selectively increased burst firing in ventral tegmentally projecting but not neighbouring prefrontal cells. These effects were also androgen-sensitive. Together, these findings suggest a working model wherein androgen influence over the activity of PFC neurons regulates its top-down modulation of mesocortical and mesolimbic dopamine systems and related dopamine-sensitive behaviours.

The transformation of synaptic to system plasticity in motor output from the sacral cord of the adult mouse.

Synaptic plasticity is fundamental in shaping the output of neural networks. The transformation of synaptic plasticity at the cellular level into plasticity at the system level involves multiple factors, including behavior of local networks of interneurons. Here we investigate the synaptic to system transformation for plasticity in motor output in an in vitro preparation of the adult mouse spinal cord. System plasticity was assessed from compound action potentials (APs) in spinal ventral roots, which were generated simultaneously by the axons of many motoneurons (MNs). Synaptic plasticity was assessed from intracellular recordings of MNs. A computer model of the MN pool was used to identify the middle steps in the transformation from synaptic to system behavior. Two input systems that converge on the same MN pool were studied: one sensory and one descending. The two synaptic input systems generated very different motor outputs, with sensory stimulation consistently evoking short-term depression (STD) whereas descending stimulation had bimodal plasticity: STD at low frequencies but short-term facilitation (STF) at high frequencies. Intracellular and pharmacological studies revealed contributions from monosynaptic excitation and stimulus time-locked inhibition but also considerable asynchronous excitation sustained from local network activity. The computer simulations showed that STD in the monosynaptic excitatory input was the primary driver of the system STD in the sensory input whereas network excitation underlies the bimodal plasticity in the descending system. These results provide insight on the roles of plasticity in the monosynaptic and polysynaptic inputs converging on the same MN pool to overall motor plasticity.

Initiating daily acute intermittent hypoxia (dAIH) therapy at 1-week after contusion spinal cord injury (SCI) improves lower urinary tract function in rat.

Spinal cord injury (SCI) above the level of the lumbosacral spinal cord produces lower urinary tract (LUT) dysfunction, resulting in impairment of urine storage and elimination (voiding). While spontaneous functional recovery occurs due to remodeling of spinal reflex micturition pathways, it is incomplete, indicating that additional strategies to further augment neural plasticity following SCI are essential. To this end, acute intermittent hypoxia (AIH) exposure has been proposed as a therapeutic strategy for improving recovery of respiratory and other somatic motor function following SCI; however, the impact of AIH as a therapeutic intervention to improve LUT dysfunction remains to be determined. Therefore, we examined the effects of daily AIH (dAIH) on both spontaneous micturition patterns and reflex micturition event (rME) behaviors in adult female Sprague-Dawley rats with mid-thoracic moderate contusion SCI. For these experiments, dAIH gas exposures (five alternating 3 min 12% O2 and 21% O2 episodes) were delivered for 7 consecutive days beginning at 1-week after SCI, with awake micturition patterns being evaluated weekly for 2-3 sessions before and for 4 weeks after SCI and rME behaviors elicited by continuous infusion of saline into the bladder being evaluated under urethane anesthesia at 4-weeks after SCI; daily normoxia (dNx; 21% O2 episodes) served as a control. At 1-week post-SCI, both an areflexic phenotype (i.e., no effective voiding events) and a functional voiding phenotype (i.e., infrequent voiding events with large volumes) were observed in spontaneous micturition patterns (as expected), and subsequent dAIH, but not dNx, treatment led to recovery of spontaneous void frequency pattern to pre-SCI levels; both dAIH- and dNx-treated rats exhibited slightly increased void volumes. At 4-weeks post-SCI, rME behaviors showed increased effectiveness in voiding in dAIH-treated (compared to dNx-treated) rats that included an increase in both bladder contraction pressure (delta BP; P = 0.014) and dynamic voiding efficiency (P = 0.018). Based on the voiding and non-voiding bladder contraction behaviors (VC and NVC, respectively) observed in the BP records, bladder dysfunction severity was classified into mild, moderate, and severe phenotypes, and while rats in both treatment groups included each severity phenotype, the primary phenotype observed in dAIH-treated rats was mild and that in dNx-treated rats was moderate (P = 0.044). Taken together, these findings suggest that 7-day dAIH treatment produces beneficial improvements in LUT function that include recovery of micturition pattern, more efficient voiding, and decreased NVCs, and extend support to the use of dAIH therapy to treat SCI-induced LUT dysfunction.

External urethral sphincter motor unit recruitment patterns during micturition in the spinally intact and transected adult rat.

In the rat, external urethral sphincter (EUS) activation during micturition consists of three sequential phases: 1) an increase in tonic EUS activity during passive filling and active contraction of the bladder (guarding reflex), 2) synchronized phasic activity (EUS bursting) associated with voiding, and 3) sustained tonic EUS activity that persists after bladder contraction. These phases are perturbed following spinal cord injury. The purpose of the present study was to characterize individual EUS motor unit (MU) patterns during micturition in the spinally intact and transected adult rat. EUS MU activity was recorded from either the L5 or L6 ventral root (intact) or EUS muscle (transected) during continuous flow cystometry in urethane-anesthetized adult female Sprague-Dawley rats. With the use of bladder pressure threshold and timing of activation, four distinct patterns of EUS MU activity were identified in the intact rat: low threshold sustained, medium/high threshold sustained, medium/high threshold not sustained, and burst only. In general, these MUs displayed little frequency modulation during active contraction, generated high-frequency bursts of action potentials during EUS bursting, and varied in terms of the duration of sustained tonic activity. In contrast, three general patterns of EUS MU activity were identified in the transected rat: low threshold, medium threshold, and high threshold. These MUs exhibited considerable frequency modulation during active contraction of the bladder, no bursting behavior and little to no sustained firing. The prominent frequency modulation of EUS MUs is likely due to the enhanced guarding reflex seen in EUS whole muscle electromyogram recordings in transected rats (D'Amico SC, Schuster IP, Collins WF 3rd. Exp Neurol 228: 59-68, 2011). In addition, EUS MU recruitment in transected rats more closely followed predictions by the size principle than in intact rats. This may reflect the influence of local synaptic circuits or intrinsic properties of EUS motoneurons that are active in intact rats but attenuated or absent in transected rats.

Serial transurethral cystometry: A novel method for longitudinal evaluation of reflex lower urinary tract function in adult female rats.

AIMS: The aim of the study is to develop a minimally invasive method for longitudinal evaluation of lower urinary tract function that allows for simultaneous measurements of bladder pressure and external urethral sphincter (EUS) electromyographic (EMG) activity. METHODS: To evaluate the reliability of serial transurethral cystometry (STUC), rats (n = 12) underwent three sessions of STUC, one session a week for 3 weeks. During each session, rats were anesthetized with ketamine-xylazine (90 mg/kg and 10 mg/kg), and micturition reflex data were acquired using transurethral cystometry and percutaneous recording of EUS (EMG) activity during continuous infusion of saline into the bladder. The reliability and consistency of the STUC method were assessed using intra-class correlation (ICC) analysis and repeated measures ANOVA. RESULTS: ICC values calculated from five successive events during the first micturition session indicate good to excellent reliability for measurements of peak bladder pressure, threshold bladder pressure, minimum bladder pressure, volume threshold, duration of EUS bursting, and number of EUS burst events. Across the three recording sessions no significant difference was observed in peak bladder pressure, threshold bladder pressure, minimum bladder pressure, volume threshold, number of EUS burst events, and duration of EUS bursting using repeated measures ANOVA. CONCLUSION: Serial transurethral cystometry under ketamine-xylazine anesthesia with simultaneous percutaneous EUS EMG recording is a novel, reliable, accurate, and minimally invasive method for quantitative assessment of lower urinary tract (LUT) function in adult female rats over extended periods of time.

No inbreeding depression in sperm storage ability or offspring viability in Drosophila melanogaster females.

Mating between relatives usually decreases genetic quality of progeny as deleterious recessive alleles are expressed in inbred individuals. Inbreeding degrades sperm traits but its effects on sperm storage and fate within females are currently unknown. We quantified the relationship between the degrees of inbreeding relevant to natural populations (f=0, 0.25 and 0.50) and the number of sperm inseminated and stored, sperm swimming speed, long-term sperm viability while in storage, pattern of sperm precedence, mating latency, and offspring viability of female Drosophila melanogaster. The use of transgenic flies that have either red or green fluorescent sperm heads allowed us to distinguish two ejaculates in the female reproductive tract and facilitated quantification of sperm storage and use traits. We found no inbreeding depression in either long- or short-term sperm storage ability. The most inbred females exhibited significantly longer mating latency, which could be explained by males preferring to mate with outbred females. On the other hand, as no evidence for cryptic male choice in the form of ejaculate tailoring of sperm number was found, the most inbred females might just be less eager to mate. We also found no evidence that the degree of maternal inbreeding influenced offspring viability. Comparison with a contemporaneous study of male inbreeding consequences for ejaculate quality suggests that inbreeding depression is more severe in males than in females in our study population.

Rapid diversification of sperm precedence traits and processes among three sibling Drosophila species.

Postcopulatory sexual selection is credited with driving rapid evolutionary diversification of reproductive traits and the formation of reproductive isolating barriers between species. This judgment, however, has largely been inferred rather than demonstrated due to general lack of knowledge about processes and traits underlying variation in competitive fertilization success. Here, we resolved processes determining sperm fate in twice-mated females, using transgenic Drosophila simulans and Drosophila mauritiana populations with fluorescently labeled sperm heads. Comparisons among these two species and Drosophila melanogaster revealed a shared motif in the mechanisms of sperm precedence, with postcopulatory sexual selection potentially occurring during any of the three discrete stages: (1) insemination; (2) sperm storage; and (3) sperm use for fertilization, and involving four distinct phenomena: (1) sperm transfer; (2) sperm displacement; (3) sperm ejection; and (4) sperm selection for fertilizations. Yet, underlying the qualitative similarities were significant quantitative differences in nearly every relevant character and process. We evaluate these species differences in light of concurrent investigations of within-population variation in competitive fertilization success and postmating/prezygotic reproductive isolation in hybrid matings between species to forge an understanding of the relationship between microevolutionary processes and macroevolutionary patterns as pertains to postcopulatory sexual selection in this group.

Postcopulatory sexual selection generates speciation phenotypes in Drosophila.

BACKGROUND: Identifying traits that reproductively isolate species, and the selective forces underlying their divergence, is a central goal of evolutionary biology and speciation research. There is growing recognition that postcopulatory sexual selection, which can drive rapid diversification of interacting ejaculate and female reproductive tract traits that mediate sperm competition, may be an engine of speciation. Conspecific sperm precedence (CSP) is a taxonomically widespread form of reproductive isolation, but the selective causes and divergent traits responsible for CSP are poorly understood. RESULTS: To test the hypothesis that postcopulatory sexual selection can generate reproductive isolation, we expressed GFP or RFP in sperm heads of recently diverged sister species, Drosophila simulans and D. mauritiana, to enable detailed resolution of species-specific sperm precedence mechanisms. Between-species divergence in sperm competition traits and mechanisms prompted six a priori predictions regarding mechanisms of CSP and degree of cross asymmetry in reproductive isolation. We resolved four distinct mechanisms of CSP that were highly consistent with predictions. These comprise interactions between multiple sex-specific traits, including two independent mechanisms by which females exert sophisticated control over sperm fate to favor the conspecific male. CONCLUSIONS: Our results confirm that reproductive isolation can quickly arise from diversifying (allopatric) postcopulatory sexual selection. This experimental approach to "speciation phenotypes" illustrates how knowledge of sperm precedence mechanisms can be used to predict the mechanisms and extent of reproductive isolation between populations and species.

Inbreeding reveals mode of past selection on male reproductive characters in Drosophila melanogaster.

Directional dominance is a prerequisite of inbreeding depression. Directionality arises when selection drives alleles that increase fitness to fixation and eliminates dominant deleterious alleles, while deleterious recessives are hidden from it and maintained at low frequencies. Traits under directional selection (i.e., fitness traits) are expected to show directional dominance and therefore an increased susceptibility to inbreeding depression. In contrast, traits under stabilizing selection or weakly linked to fitness are predicted to exhibit little-to-no inbreeding depression. Here, we quantify the extent of inbreeding depression in a range of male reproductive characters and then infer the mode of past selection on them. The use of transgenic populations of Drosophila melanogaster with red or green fluorescent-tagged sperm heads permitted in vivo discrimination of sperm from competing males and quantification of characteristics of ejaculate composition, performance, and fate. We found that male attractiveness (mating latency) and competitive fertilization success (P2) both show some inbreeding depression, suggesting they may have been under directional selection, whereas sperm length showed no inbreeding depression suggesting a history of stabilizing selection. However, despite having measured several sperm quality and quantity traits, our data did not allow us to discern the mechanism underlying the lowered competitive fertilization success of inbred (f = 0.50) males.

Quantification of external urethral sphincter and bladder activity during micturition in the intact and spinally transected adult rat.

The goal of this study was to determine the effect of chronic mid-thoracic spinal cord transection on the time course of external urethral sphincter (EUS) and bladder activity associated with micturition events in the rat. Adult female Sprague-Dawley rats, either spinally intact or transected (T(9)-T(10)), were anesthetized with urethane and set up for continuous flow urodynamic recording of bladder intravesical pressure (BP) and EUS electromyography (EMG). Spinal transections were performed under isoflurane anesthesia 1-8 weeks prior to the terminal experiment. Four major differences between intact and transected rats were observed: 1) While the frequency of micturition events in the intact rat was dependent upon the rate of bladder filling, the bladder contraction and associated EUS activation in transected rats exhibited an intrinsic rhythm that was independent of the rate of bladder filling and post-transection survival time. 2) EUS activation was augmented at the beginning of active bladder contraction in the transected rat, indicating an amplified guarding reflex. 3) Phasic EUS activity at the peak of bladder contraction (EUS bursting) in the intact rat was markedly reduced or absent in the transected rat. 4) The sustained tonic EUS activity following bladder relaxation in the intact rat was absent in the transected rat. These data are discussed in the context of understanding the pathophysiology of spinal cord injury (SCI) induced destrusor-sphincter dyssynergia (DSD).