The molecular signaling that underpins synapse loss in neuropathological conditions remains unknown. Concomitant upregulation of the neuronal nitric oxide (NO) synthase (nNOS) in neurodegenerative processes places NO at the center of attention. We found that de novo nNOS expression was sufficient to induce synapse loss from motoneurons at adult and neonatal stages. In brainstem slices obtained from neonatal animals, this effect required prolonged activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway and RhoA/Rho kinase (ROCK) signaling. Synapse elimination involved paracrine/retrograde action of NO. Furthermore, before bouton detachment, NO increased synapse myosin light chain phosphorylation (p-MLC), which is known to trigger actomyosin contraction and neurite retraction. NO-induced MLC phosphorylation was dependent on cGMP/PKG-ROCK signaling. In adulthood, motor nerve injury induced NO/cGMP-dependent synaptic stripping, strongly affecting ROCK-expressing synapses, and increased the percentage of p-MLCexpressing inputs before synapse destabilization. We propose that this molecular cascade could trigger synapse loss underlying early cognitive/motor deficits in several neuropathological states.
Rho-associated kinase (ROCK) regulates neural cell migration, proliferation and survival, dendritic spine morphology, and axon guidance and regeneration. There is, however, little information about whether ROCK modulates the electrical activity and information processing of neuronal circuits. At neonatal stage, ROCK␣ is expressed in hypoglossal motoneurons (HMNs) and in their afferent inputs, whereas ROCK is found in synaptic terminals on HMNs, but not in their somata. Inhibition of endogenous ROCK activity in neonatal rat brainstem slices failed to modulate intrinsic excitability of HMNs, but strongly attenuated the strength of their glutamatergic and GABAergic synaptic inputs. The mechanism acts presynaptically to reduce evoked neurotransmitter release. ROCK inhibition increased myosin light chain (MLC) phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons. Functional and ultrastructural changes induced by ROCK inhibition were fully prevented/reverted by MLC kinase (MLCK) inhibition. Furthermore, ROCK inhibition drastically reduced the phosphorylated form of p21-associated kinase (PAK), which directly inhibits MLCK. We conclude that endogenous ROCK activity is necessary for the normal performance of motor output commands, because it maintains afferent synaptic strength, by stabilizing the size of the readily releasable pool of synaptic vesicles. The mechanism of action involves a tonic inhibition of MLCK, presumably through PAK phosphorylation. This mechanism might be present in adults since unilateral microinjection of ROCK or MLCK inhibitors into the hypoglossal nucleus reduced or increased, respectively, whole XIIth nerve activity.
Excitotoxicity is a widely studied mechanism underlying motoneuron degeneration in amyotrophic lateral sclerosis (ALS). Synaptic alterations that produce an imbalance in the ratio of inhibitory/excitatory synapses are expected to promote or protect against motoneuron excitotoxicity. In ALS patients, motoneurons suffer a reduction in their synaptic coverage, as in the transition from the presymptomatic (2-month-old) to early-symptomatic (3-month-old) stage of the hSOD1(G93A) mouse model of familial ALS. Net synapse loss resulted from inhibitory bouton loss and excitatory synapse gain. Furthermore, in 3-month-old transgenic mice, remaining inhibitory but not excitatory boutons attached to motoneurons showed reduction in the active zone length and in the spatial density of synaptic vesicles in the releasable pool near the active zone. Bouton degeneration/loss seems to be mediated by bouton vacuolization and by mechanical displacement due to swelling vacuolated dendrites. In addition, chronic treatment with a nitric oxide (NO) synthase inhibitor avoided inhibitory loss but not excitatory gain. These results indicate that NO mediates inhibitory loss occurring from the pre- to early-symptomatic stage of hSOD1(G93A) mice. This work contributes new insights on ALS pathogenesis, recognizing synaptic re-arrangement onto motoneurons as a mechanism favoring disease progression rather than as a protective homeostatic response against excitotoxic events.
The main aim of this study was to assess the associations between the timing of lameness clinical case occurrence in lactation with productive and reproductive performances in grazing Holstein cows. A cohort study was carried out on a dataset with records from a commercial dairy herd (Buenos Aires, Argentina) for cows that calved and were dried off from January 2010 through June 2017. The first recorded event of lameness per lactation was considered for the study. Criteria for lactation inclusion included not having uterine diseases, mastitis, or anovulatory cysts during the studied risk period (i.e., up to 200 DIM). Therefore, a total of 7156 out of 20,086 lactations were included in the statistical analysis. The association between lameness case occurrence in lactation (cows not lame (LG0) vs. lame cows between parturition and first service (LG1) vs. lame cows between first service and first pregnancy (LG2)) with productive (i.e., accumulated milk yield to 150 DIM (MILK150) and 300 DIM (MILK305)) and reproductive performances (hazard of insemination and pregnancy) was analyzed with linear regression models and proportional hazard regression models, respectively. Lame cows produced 161 and 183 kg less MILK150 and MILK305 than non-lame herd mates, respectively. Moreover, LG1 cows produced 216 kg less MILK150 and 200 kg less MILK305 than LG0 cows, and LG2 cows also produced 58 kg less MILK150 and 158 kg less MILK305 than LG0 cows. The LG1 cows had a lower hazard of service than LG0 cows (HR = 0.43, 95%CI = 0.39–0.47). Furthermore, LG1 cows had a lower hazard of pregnancy than LG0 cows (HR = 0.52, 95%CI = 0.46–0.59) and took longer to get pregnant than LG0 cows (median [95%CI], 139 [132–144] vs. 101 [99–103]). Moreover, LG2 cows had a much lower hazard of pregnancy than LG0 cows (HR = 0.08, 95%CI = 0.05–0.12) and much longer calving to first pregnancy interval than LG0 cows (188 [183–196] vs. 101 [99–103]). In conclusion, cows that become lame in early lactation produce less milk and have lower hazards of insemination and pregnancy than herd mates that are healthy or become lame later in lactation. In addition, cows that become lame immediately after the voluntarily waiting period have the poorest reproductive performance (i.e., they have the lowest hazard of pregnancy and the longest calving to pregnancy interval).
The main objective of this study was to evaluate the risk factors for late embryonic loss (LEL) in supplemented grazing dairy cows. Additional objectives were to assess the incidence of LEL and its association with the reproductive performance of cows. A data set containing productive, reproductive, and health records of 13,551 lactations was used. A retrospective case-control study involving 631 cows with LEL (cases) and 2,524 controls (4 controls per case within each study year) was run. A case of LEL was defined when the embryo had no heartbeat or there was evidence of detached membranes or floating structures including embryo remnants by ultrasonography (US) at 28 to 42 d postartificial insemination (AI), whereas a non-case was defined as a cow diagnosed with positive pregnancy by US 28 to 42 d post-AI and reconfirmed as pregnant 90 ± 7 d post-AI. Four controls per case were randomly selected from the non-cases with a temporal matching criterion (±3 d around the date of the fecundating AI of the case). Multivariable logistic models were offered with the following predictors: year of LEL (2011 through 2015), season of LEL (summer vs. fall vs. winter vs. spring), parity (1 vs. 2 vs. ≥3), uterine disease (UD), non-uterine disease (NUD), body condition score at parturition, body condition score at 28 to 42 d post-AI (BCS-LEL), days in milk (DIM), and daily milk yield (MY). Statistical significance was set at P < 0.05 and a tendency was set at P ≤ 0.10. We found that 4.7, 22, and 23% of cows had LEL, UD, and NUD, respectively. Cases tended to have higher daily MY than controls (32.5 vs. 31.8 kg); also, cases had much longer calving to pregnancy interval (226 vs. 118 d), lower hazard of pregnancy [hazard ratio = 0.39, 95% confidence interval (CI) = 0.35-0.43], and higher odds for non-pregnancy [odds ratio (OR) = 2.89, 95% CI = 2.37-3.54] than controls. We found that the odds for LEL increased with parity number (OR = 2.48, 95% CI = 1.99-3.08 for parity ≥3) and with BCS-LEL <2.50 (OR = 1.81, 95% CI = 1.33-2.47). Conversely, the odds for LEL decreased with BCS-LEL >3.00 (OR = 0.70, 95% CI = 0.53-0.91). The odds for LEL increased with UD (OR = 1.23, 95% CI = 1.01-1.49), NUD (OR = 1.24, 95% CI = 1.01-1.54), DIM (OR = 1.03, 95% CI = 1.00-1.05), and daily MY (OR = 1.14, 95% CI = 1.04-1.25) in univariable models only. Finally, the odds for LEL were not associated with year, season, DIM, and body condition score at parturition. In conclusion, LEL is associated with extended calving to pregnancy interval, and among its risk factors are parity number and BCS-LEL.
Flow sorting cytometry has been shown to repeatedly produce viable sexed sperm at a level necessary for commercialization. Previous reports have shown that pregnancies rates were lower using sexed semen than commercial nonsexed semen. Sperm concentration, acrosome activation, and time to ovulation are some factors explaining the low conception. The objective of the present study was to compare pregnancy rates using sexed semen (3 × 106 sperm) deposited in different locations of the bovine uterus (body v. horn) and inseminated (AI) at 2 different times (52 h v. 58 h after progesterone intravaginal device removal). Holstein heifers, between 15 to 18 mo of age and a body condition score 3.05 ± 0.22 were used. All heifers were evaluated by transrectal ultrasonography to determine ovarian structures (284/357, 79.5% had a CL). On Day 0, all heifers received an intravaginal progesterone (P4) device (1.9 g of P4, CIDR, Pfizer, Buenos Aires, Argentina) plus 70 μg of D-cloprostenol (Bioprost, Biotay, Argentina) i.m. and 2 mg of estradiol benzoate (EB, Syntex SA, Buenos Aires, Argentina). On Day 8, CIDR devices were removed and heifers received 150 μg of D-cloprostenol i.m. at the same time. On Day 9, all heifers received 1 mg of EB i.m. and were randomly allocated in 4 different groups (2 × 2 factorial): group Body-52 h, AI in the body of the uterus at 52 h (n = 98); group Body-58 h: AI in the body of the uterus at 58 h (n = 104); group Horn-52 h: AI in the ipsilateral horn of the ovulatory follicle (detected by ultrasonography) at 52 h (n = 90) and group Horn-58 h: AI in the ipsilateral horn to the ovulatory follicle at 58 h (n = 65). All heifers were inseminated with only one dose of sexed semen. At time of AI an ultrasound examination was done to determine the size and the location of the preovulatory follicle. AI was done by a single inseminator using a traditional AI gun, and an embryo transfer gun (ET sheath, SBS Cryotec SA, Argentina) was used for deep AI. Pregnancy diagnosis was evaluated by transrectal ultrasonography using a 7.5-MHz transducer (Mindray 6600) 30 days after AI. Data were analyzed by logistic regression. Sixty percent of the preovulatory follicles were on the right ovary, and there were no differences between the sizes of the preovulatory follicle among groups (overall mean ± SEM: 15.5 ± 1.0 mm). Although pregnancy rates did not differ between horn (64/155, 41.2%) and body (70/202, 34.3%; P < 0.1) inseminations and between 52 h (67/188, 36.7% and 58 h (67/169, 39.6%), pregnancy rates were higher (P < 0.05) in heifers inseminated in the horn at 58 h (32/65, 49.2%) than those inseminated in the body at 58 h (35/104, 33.6%) and tended (P < 0.09) to be higher than heifers inseminated in the horn at 52 h (32/90, 35.5%) and in the body at 52 h (35/98, 35.7%). Fixed-time artificial insemination using ultrasonography and deep insemination could contribute to enhanced pregnancy rates using sexed semen in Holstein heifers. This research was done with the support of ADECO SA, SBS Cryo Tec SA.
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