Intersubnuclear connections within the rat trigeminal brainstem complex

Prior intracellular recording and labeling experiments have documented local-circuit and projection neurons in the spinal trigeminal (V) nucleus with axons that arborize in more rostral and caudal spinal trigeminal subnuclei and nucleus principalis. Anterograde tracing studies were therefore carried out to assess the origin, extent, distribution, and morphology of such intersubnuclear axons in the rat trigeminal brainstem nuclear complex (TBNC). Phaseolus vulgaris leucoagglutinin (PHA-L) was used as the anterograde marker because of its high sensitivity and the morphological detail provided. Injections restricted to TBNC subnucleus caudalis resulted in dense terminal labeling in each of the more rostral ipsilateral subnuclei. Subnucleus interpolaris projected ipsilaterally and heavily to magnocellular portions of subnucleus caudalis, as well as subnucleus oralis and nucleus principalis. Nucleus principalis, on the other hand, had only a sparse projection to each of the caudal ipsilateral subnuclei. Intersubnuclear axons most frequently traveled in the deep bundles within the TBNC, the V spinal tract, and the reticular formation. They gave rise to a number of circumscribed, highly branched arbors with many boutons of the terminal and en passant types. Retrograde single- or multiple-labeling experiments assessed the cells giving rise to TBNC intersubnuclear collaterals. Horseradish peroxidase (HRP) and/or fluorescent tracer injections into the thalamus, colliculus, cerebellum, nucleus principalis, and/or subnucleus caudalis revealed large numbers of neurons in subnuclei caudalis, interpolaris, and oralis projecting to the region of nucleus principalis. Cells projecting to more caudal spinal trigeminal regions were most numerous in subnuclei interpolaris and oralis. Some cells in lamina V of subnucleus caudalis and in subnuclei interpolaris and oralis projected to thalamus and/or colliculus, as well as other TBNC subnuclei. Such collateral projections were rare in nucleus principalis and more superficial laminae of subnucleus caudalis. TBNC cells labeled by cerebellar injections were not double-labeled by tracer injections into the thalamus, colliculus, or TBNC. These findings lend generality to currently available data obtained with intracellular recording and HRP labeling methods, and suggest that most intersubnuclear axons originate in TBNC local-circuit neurons, though some originate in cells that project to midbrain and/or diencephalon.     

Neonatal transection alters the percentage of substance-P-positive trigeminal ganglion cells that contribute axons to the regenerate infraorbital nerve

Neonatal transection results in a marked reduction of the number of trigeminal (V) ganglion cells that contribute axons to the regenerate infraorbital nerve (ION; Jacquin and Rhoades, 1985; Chiaia et al., 1987). Such lesions also produce a profound deafferentation of the V brain stem complex that appears to spare the innervation of layers I and II of subnucleus caudalis (SpC) by substance-P-positive (SP-positive) primary afferents (Jacquin and Rhoades, 1985; Rhoades et al., 1988). In the present study, we combined retrograde tracing with immunocytochemistry to determine whether neonatal transection of the ION alters the percentage of SP-positive V ganglion cells that contribute axons to this V branch upon regeneration. In V ganglia ipsilateral to the intact ION (n = 8), 11.6% +/- 3.2% of the cells labeled after application of true blue (TB) to the ION were also SP-positive. In ganglia ipsilateral to the neonatally damaged nerve (n = 8), 18.6% +/- 4.7% of the cells labeled after application of TB to the regenerate ION were also SP-positive (p less than 0.001). We also compared the SP content of intact ganglia (n = 10) with that of ganglia ipsilateral to the damaged nerve (n = 10) by means of radioimmunoassay. The normal V ganglia contained (mean +/- SD) 3496 +/- 774 pg SP/mg protein. The value for the ganglia ipsilateral to the damaged nerve was 5533 +/- 1746 pg SP/mg protein (p less than 0.01). There was no significant difference between SP levels on the control and partially deafferented sides of the brain stem in neonatally nerve-damaged adult rats. In one additional experiment, we injected TB into both vibrissa pads of seven rats on the day of birth prior to transection of the ION. After an 8-hr delay, the nerve on one side was then cut and allowed to regenerate, and both V ganglia were then processed for immunocytochemistry. On the nerve-damage side, 25.8% of the TB-labeled cells were SP-positive. The value for the intact side was 12.0% (p less than 0.000001). This result demonstrated that the lesion-induced change in the percentage of SP-positive ION cells was not the result of either late-growing axons from SP-positive ganglion cells that may have been missed by our nerve cuts or collateral sprouting into the regenerate ION by undamaged SP-positive ganglion cells.     

Differential expression of acetylcholinesterase in the brainstem, ventrobasal thalamus and primary somatosensory cortex of perinatal rats, mice, and hamsters

Acetylcholinesterase (AChE) is transiently expressed by thalamocortical axons in the rat, and staining for this enzyme has been used extensively to study the development of thalamocortical projections. In the present study, patterns of AChE staining were compared in the trigeminal brainstem, thalami and primary somatosensory cortices of perinatal rats, mice, and hamsters. As previously reported, the ventral posteromedial nucleus (VPM) of rats showed dense AChE staining from P-0 at least through P-8. The ventral posterolateral nucleus (VPL) contained heavy AChE staining at least through P-60. In the cortex, there was also dense AChE staining which was organized somatotopically in patches similar to those observed with other methods such as cytochrome oxidase (CO) staining. However, by adulthood, AChE staining revealed a negative image of the CO staining pattern in lamina IV. In the mouse and hamster, there was dense AChE staining inVPL from P-0 through adulthood, but VPM was much less heavily stained for this enzyme. Moreover, the staining in VPL of mice was markedly reduced after transection of axons that travel to the thalamus in the medial lemniscus, suggesting that much of it was contained in these afferent fibers. In the cortices of both perinatal and adult mice and hamsters, AChE staining yielded a negative image of the somatotopically organized patches demonstrable with CO staining. This negative image was apparent by P-2 in the mouse and P-4 in the hamster. These results document a dramatic species difference with respect to the expression of AChE in the thalami and cortices of developing rodents. The differences between the patterns observed in rats vs mice and hamsters probably reflect the fact that cortical AChE in the latter species is not contained in thalamocortical afferents arising from either VPM or VPL.     

Time course of expression and function of the serotonin transporter in the neonatal rat's primary somatosensory cortex

Immunocytochemical and autoradiographic techniques were employed to determine the time course of expression of the serotonin (5-HT) transporter (SERT) on thalamocortical afferents in the rat's primary somatosensory cortex (S-I), and to correlate this expression to the transient vibrissae-related patterning of 5-HT immunostaining previously described. In additional in vivo and in vitro experiments, 5-HT and 3H-5-HT were applied directly to the cortices of untreated and 5,7-dihydroxytryptamine-treated (5,7-DHT) rats in order to determine the period during which SERT functions on thalamocortical axons to take up 5-HT. In postnatal rats, SERT immunohistochemistry revealed a somatotopic patterning in S-I that persisted until P-15, which is 6 days after the disappearance of the vibrissae-related 5-HT immunostaining. 3H-citalopram autoradiography revealed a vibrissae-related pattern in layer IV of S-I until at least P-30. Following destruction of raphe-cortical afferents with 5,7-DHT on the day of birth, this binding pattern remained visible until at least P-25, indicating that SERT located on thalamocortical axons is responsible for the 3H-citalopram patterning observed in S-I. Tissue from 5,7-DHT-treated rats that had 5-HT applied directly to their cortices revealed a normal vibrissae-related pattern of 5-HT immunostaining in S-I at P-7 and P-11 but only a faint pattern at P-13 and none at P-14. In addition, 3H-5-HT injected directly into S-I labeled layer IV barrels at P-6 and P-12 but not at P-18. The results of these experiments demonstrate that SERT is expressed by thalamocortical afferents and remains functional long after the vibrissae-related 5-HT immunostaining in cortex disappears.     

Association study between copy number variation and beef fatty acid profile of Nellore cattle

The aim of this study was to analyze the association between the copy number variation regions (CNVRs) and fatty acid profile phenotypes for saturated (SFA), monosaturated (MUFA), polyunsaturated (PUFA), ω6 and ω3 fatty acids, PUFA/SFA and ω6/ω3 ratios, as well as for their sums, in Nellore cattle (Bos primigenius indicus). A total of 963 males were finished in feedlot and slaughtered with approximately 2 years of age. Animals were genotyped with the BovineHD BeadChip (Illumina Inc., San Diego, CA, USA). The copy number variation (CNV) detection was performed using the PennCNV algorithm. Log R ratio (LRR) and allele B frequency (BAF) were used to estimate the CNVs. The association analyses were done using the CNVRuler software and applying a logistic regression model. The phenotype was adjusted using a linear model considering the fixed effects of contemporary group and the animal age at slaughter. The fatty acid profile was analyzed on samples of longissimus thoracis muscle using gas chromatography with a 100-m capillary column. For the association analysis, the adjusted phenotypic values were considered for the traits, while the data was adjusted for the effects of the farm and year of birth, management groups at birth, weaning, and superannuation. A total of 186 CNVRs were significant for SFA (43), MUFA (42), PUFA (66), and omega fatty acid (35) groups, totaling 278 known genes. On the basis of the results, several genes were associated with several fatty acids of different saturations. Olfactory receptor genes were associated with C12:0, C14:0, and C18:0 fatty acids. The SAMD8 and BSCL2 genes, both related to lipid metabolic process, were associated with C12:0. The RAPGEF6 gene was found to be associated with C18:2 cis-9 cis-12 n-6, and its function is related to regulation of GTPase activity. Among the results, we highlighted the olfactory receptor activity (GO:0004984), G-protein-coupled receptor activity (GO:0004930), potassium:proton antiporter activity (GO:0015386), sodium:proton antiporter activity (GO:0015385), and odorant-binding (GO:0005549) molecular functions. A large number of genes associated with fatty acid profile within the CNVRs were identified in this study. These findings must contribute to better elucidate the genetic mechanism underlying the fatty acid profile of intramuscular fat in Nellore cattle.     

Effect of Formulation and Process Parameters on Chitosan Microparticles Prepared by an Emulsion Crosslinking Technique

There are many studies about the synthesis of chitosan microparticles; however, most of them have very low production rate, have wide size distribution, are difficult to reproduce, and use harsh crosslinking agents. Uniform microparticles are necessary to obtain repeatable drug release behavior. The main focus of this investigation was to study the effect of the process and formulation parameters during the preparation of chitosan microparticles in order to produce particles with narrow size distribution. The technique evaluated during this study was emulsion crosslinking technique. Chitosan is a biocompatible and biodegradable material but lacks good mechanical properties; for that reason, chitosan was ionically crosslinked with sodium tripolyphosphate (TPP) at three different ratios (32, 64, and 100%). The model drug used was acetylsalicylic acid (ASA). During the preparation of the microparticles, chitosan was first mixed with ASA and then dispersed in oil containing an emulsifier. The evaporation of the solvents hardened the hydrophilic droplets forming microparticles with spherical shape. The process and formulation parameters were varied, and the microparticles were characterized by their morphology, particle size, drug loading efficiency, and drug release behavior. The higher drug loading efficiency was achieved by using 32% mass ratio of TPP to chitosan. The average microparticle size was 18.7 μm. The optimum formulation conditions to prepare uniform spherical microparticles were determined and represented by a region in a triangular phase diagram. The drug release analyses were evaluated in phosphate buffer solution at pH 7.4 and were mainly completed at 24 h.     

Genomic prediction ability for beef fatty acid profile in Nelore cattle using different pseudo-phenotypes

The aim of the present study was to compare the predictive ability of SNP-BLUP model using different pseudo-phenotypes such as phenotype adjusted for fixed effects, estimated breeding value, and genomic estimated breeding value, using simulated and real data for beef FA profile of Nelore cattle finished in feedlot. A pedigree with phenotypes and genotypes of 10,000 animals were simulated, considering 50% of multiple sires in the pedigree. Regarding to phenotypes, two traits were simulated, one with high heritability (0.58), another with low heritability (0.13). Ten replicates were performed for each trait and results were averaged among replicates. A historical population was created from generation zero to 2020, with a constant size of 2000 animals (from generation zero to 1000) to produce different levels of linkage disequilibrium (LD). Therefore, there was a gradual reduction in the number of animals (from 2000 to 600), producing a “bottleneck effect” and consequently, genetic drift and LD starting in the generation 1001 to 2020. A total of 335,000 markers (with MAF greater or equal to 0.02) and 1000 QTL were randomly selected from the last generation of the historical population to generate genotypic data for the test population. The phenotypes were computed as the sum of the QTL effects and an error term sampled from a normal distribution with zero mean and variance equal to 0.88. For simulated data, 4000 animals of the generations 7, 8, and 9 (with genotype and phenotype) were used as training population, and 1000 animals of the last generation (10) were used as validation population. A total of 937 Nelore bulls with phenotype for fatty acid profiles (Sum of saturated, monounsaturated, omega 3, omega 6, ratio of polyunsaturated and saturated and polyunsaturated fatty acid profile) were genotyped using the Illumina BovineHD BeadChip (Illumina, San Diego, CA) with 777,962 SNP. To compare the accuracy and bias of direct genomic value (DGV) for different pseudo-phenotypes, the correlation between true breeding value (TBV) or DGV with pseudo-phenotypes and linear regression coefficient of the pseudo-phenotypes on TBV for simulated data or DGV for real data, respectively. For simulated data, the correlations between DGV and TBV for high heritability traits were higher than obtained with low heritability traits. For simulated and real data, the prediction ability was higher for GEBV than for Yc and EBV. For simulated data, the regression coefficient estimates (b_(Yc,DGV)), were on average lower than 1 for high and low heritability traits, being inflated. The results were more biased for Yc and EBV than for GEBV. For real data, the GEBV displayed less biased results compared to Yc and EBV for SFA, MUFA, n-3, n-6, and PUFA/SFA. Despite the less biased results for PUFA using the EBV as pseudo-phenotype, the b_(Yi,DGV estimates obtained for the different pseudo-phenotypes (Yc, EBV and GEBV) were very close. Genomic information can assist in improving beef fatty acid profile in Zebu cattle, since the use of genomic information yielded genomic values for fatty acid profile with accuracies ranging from low to moderate. Considering both simulated and real data, the ssGBLUP model is an appropriate alternative to obtain more reliable and less biased GEBVs as pseudo-phenotype in situations of missing pedigree, due to high proportion of multiple sires, being more adequate than EBV and Yc to predict direct genomic value for beef fatty acid profile.