Group B7 Research Articles

Histopathological changes in extraocular muscles of rabbits following injection of Bupivacaine 5mg/ml versus 7.5mg/ml.

: Purpose To compare the histopathological effects of injecting 2 concentrations of Bupivacaine (5mg/ml and 7.5mg/ml) in the superior rectus muscle of rabbits, and to compare these to conventional extraocular muscle surgery in previous studies. Methods Eighteen albino rabbits’ eyes were used. The superior rectus muscles were injected with Bupivacaine 5mg/ml (Group B5, 10 eyes) or 7.5mg/ml (Group B7, 8 eyes). The rabbits were sacrificed and eyes enucleated 6 weeks later for histopathological evaluation. Results were compared to the average of those obtained, by 3 previous studies, after conventional superior rectus resection in rabbits. Results Foreign body reaction was absent in all specimens. Conjunctival and scleral inflammation, perimuscular adhesions, intramuscular fibrosis, conjunctival and scleral oedema and muscle atrophy were higher in group B7, while conjunctival hyperaemia and muscle hypertrophy were higher in group B5 (p > .05). On comparison to conventional surgery, conjunctival inflammation and hyperaemia, foreign body reaction, and adhesions were less after bupivacaine injection (p >.05 for all except for intensity of conjunctival inflammation in B5 versus conventional surgery). Scleral inflammation was more frequent after bupivacaine injection (p <.05). Muscle fibrosis was more frequent in group B7 and conventional surgery than in group B5 (p >.05). Conclusions Both Bupivacaine concentrations effectively produced the desired muscle hypertrophy and fibrosis, so the lower concentration may be used for muscle strengthening to correct strabismus. Bupivacaine injection, although produced no foreign body reaction, did not significantly lower the development of undesired postoperative adhesions and caused more scleral inflammation.

Effects of immunohistochemical conditions on the results of PD-L1 (22C3) staining

Objective: To investigate the optimal experimental conditions (including antigen retrieval time, antibody titers and antibody incubation time) for reliable detection of programmed death-ligand 1 (PD-L1) expression using PD-L1 (22C3) antibody concentrate, and to establish a laboratory developed test for PD-L1 detection. Methods: Using Dako PD-L1 IHC 22C3 pharmDX staining procedure and scoring guidelines as the standard reference (group A), the PD-L1 expression in 25 tissue specimens (including 15 lung cancer tissues, 5 tonsil tissues and 5 placenta tissues) was detected with Flex+/HRP detection kit (EnVision) under 8 different experimental conditions (groups B1 to B8). The staining results were then compared to those in group A. Results: In group B1, 3 tissue samples showed the percentages of PD-L1 positive tumor cells were similar to those in group A, while the percentages of PD-L1 positive tumor cells were lower than those in group A in the other samples. In group B7, two case showed a positive rate higher than that in group A that was also above the positive cut-off value, and the rest of the samples had a percentage of PD-L1 positive tumor cells slightly higher than that in group A, but still below the positive cut-off value. The staining results of group B8 were the closest to those of group A compared with the other groups. Although the percentages of PD-L1 positive tumor cells in the B2 to B6 groups were decreased in various degrees as compared with group A, they were still concordant with group A's classification (positive vs. negative) and would not change the choice of clinical treatments. Conclusions: The experimental conditions are associated with detection rate of PD-L1 expression using 22C3 antibody. In the present study, the most-suitable alterative conditions in the PD-L1 detection using 22C3 antibody concentrate are those applied in the group B8 (including antigen retrieval in Dako PT Link tank at 97 ℃, pH 6.0 for 40 min and incubation with 22C3 antibodies (1∶100 dilution) at room temperature for 60 min, incubation with EnVision Flex+Linker at room temperature for 30 min, incubation with EnVision/HRP at room temperature for 30 min and DAB staining for 5 min), which could provide reliable results at minimum costs.

Potential effect of one-alpha-hydroxy vitamin D3 (alfacalcidol) on experimentally induced allergic asthma

Background: Alfacalcidol is a vitamin D analog mainly used in the management of osteoporosis. Apart from the well-known role of vitamin D in bone and calcium metabolism, an immune-modulator role of vitamin D in allergic diseases was suggested. Aim: To evaluate the potential effect of alfacalcidol either alone or in combination with salbutamol or/and prednisolone and its role in the protection and management of experimentally induced allergic asthma. Materials and methods: Male guinea pigs were divided into two groups: group A constituted normal guinea pigs (received saline) and group B constituted ovalbumin-sensitized guinea pigs, which were divided into eight subgroups: group B1 (control): groups from B2 to B4 was treated with salbutamol, prednisolone, alfacalcidol, respectively, for 1 week, while group B5 was treated with salbutamol+prednisolone; group B6 was treated with salbutamol+alfacalcidol; group B7 was treated with prednisolone+alfacalcidol; and group B8 was treated with salbutamol, prednisolone, and alfacalcidol for 1 week. Twenty-four hours after the last dose, the animals were subjected to both: (a)pharmacological studies, (b) histopathological studies (group A, group B1, B3, and B4). Results: (a) Pharmacological studies: sensitization of guinea pigs caused a significant increase in amplitude of histamine-induced contractions of isolated tracheal smooth muscles in comparison to that of the normal one. Treatment of sensitized guinea pigs with salbutamol, prednisolone, or alfacalcidol produced a significant reduction in the amplitude of histamine-induced contractions in comparison to sensitized nontreated guinea pigs. Addition of alfacalcidol to salbutamol or prednisolone in the treatment of sensitized guinea pigs caused more decrease in the amplitude of histamine-induced contractions. However, the highest reduction in the amplitude of contractions of isolated tracheal strips was produced by the treatment of guinea pigs with a combination of salbutamol, prednisolone, and alfacalcidol. (b) Histopathological examinations: treatment of sensitized guinea pigs with alfacalcidol resulted in an anti-remodeling effect, while prednisolone showed better anti-inflammatory effect, when it was compared with that of the control group. Conclusion: Alfacalcidol was shown to possess anti-inflammatory and anti-remodeling effects in allergic asthma. It was superior to prednisolone regarding the anti-remodeling effect. So, alfacalcidol could be beneficial in the management of allergic asthma.

Expression change and significance of homebox gene-A10 in rat cryptorchidism

To explore the effects of HOXA10 gene expression in undescended and descended testis. Twenty-four male Sprague Dawley rats were surgically prepared for unilateral cryptorchidism model. Their undescended and descended testes were removed at days 7 (infancy period, group B7) and 60 (sexual maturity period, group B60) post-operation. And contralateral descended testis served as controls (group A7, group A60). The expression of HOXA10 with histologic changes in experimental cryptorchidism were detected with one-step real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Compared with the control group, the levels of HOXA10 gene for groups B7 and B60 decreased markedly to 0.78 ± 0.11 (P < 0.01) and 0.56 ± 0.03 (P < 0.01) respectively.However the levels of HOXA10 gene and protein in group B60 were significantly lower than those in group B7 (P < 0.01). There is a lower expression of HOXA10 in undescended testes than in normally developed counterparts. And the expression of HOXA10 decreases with the elapsing time of cryptorchidism.

Epidural Volume Extension In Combined Spinal Epidural Anaesthesia For Rapid Motor Recovery After Elective Caesarean SectionA Comparative Study

Background: Epidural volume extension with saline solution can contribute to enhancement of a small dose intrathecal local anaesthetic. The resultant substantial reduction in local anaesthetic dose produces a sufficient effective block, decreased side effect and less motor blockade. The aim of this study was to evaluate the sensory and motor block characteristics of 10ml epidural saline after intrathecal small dose local anaesthetic and opioid. Setting and Design: This prospective, randomized, double-blind study was conducted in a operation room setting of a university hospital. One hundred and five women (25-40 years) of ASA 1 and 11 physical status electively undergoing caesarean section under regional anaesthesia were investigated.Method and Material: After hospital Ethics Committee approval and written informed consent, 105 women were allocated randomly, into three groups (n=35), received 7mg of spinal hyperbaric 0.5% Bupivacaine (groupB7), 7mg of spinal hyperbaric 0.5% Bupivacaine followed by 10ml of Normal Saline through the epidural catheter, 5 minutes thereafter (group B7EVE) and 10mg of spinal hyperbaric 0.5% Bupivacaine without epidural volume extension (groupB10). All women also received 25mcg of Fentanyl intrathecally. The patients were assessed at 2.5 minutes interval for sensory block level to loss of pain from pin prick and for motor block using modified Bromage scale. We also recorded visual analogue scale, peak sensory block height, highest Modified Bromage scale, motor block recovery, incidences of side effects and ephedrine dose requirement.Result: Demographic data, duration of surgery, VAS, incidence of maternal side effects and ephedrine dose requirement were similar in all the groups. There was no difference in the peak sensory block heights between the groups during the study. The motor score was significantly lower in the group B7-EVE (grade-1 vs grade-3 in groupB7and groupB10, P=0.014). This was associated with a significantly faster motor recovery to Modified Bromage 0 in group B7-EVE (61-80mts vs 121-140mts in group B7 and 141-160mts in groupB10, P=0.0001).Conclusion: This study demonstrates a benefit in using epidural volume extension with 10ml normal saline, as a part of a combined spinal epidural technique by providing a more rapid motor recovery of the lower limbs after elective caesarean section.

Epidural volume extension in combined spinal epidural anaesthesia for elective caesarean section: a randomised controlled trial

We investigated the effect of epidural volume extension on spinal blockade in pregnant women undergoing elective caesarean section with a combined spinal-epidural technique. We randomly allocated 90 healthy subjects to three groups to receive spinal hyperbaric bupivacaine 7.5 mg (group B7.5), spinal hyperbaric bupivacaine 7.5 mg immediately followed by epidural volume extension with saline 5 ml (group B7.5-EVE) or spinal hyperbaric bupivacaine 10 mg without epidural volume extension (group B10). We evaluated the height of the block every 5 min for 15 min following the spinal injection. The overall sensory block level increased with time (p < 0.001), regardless of the group studied, and there were significantly fewer failures of block in the group B10 compared with both B7.5 and B7.5-EVE groups (p = 0.001). In conclusion, we could not demonstrate a benefit in using epidural volume extension with 5 ml saline as part of a combined spinal epidural technique in term parturients undergoing elective caesarean section.

Inheritance of seed condensed tannins and their relationship with seed-coat color and pattern genes in common bean (Phaseolus vulgaris L.)

Condensed tannins are major flavonoid end products that affect the nutritional quality of many legume seeds. They chelate minerals and interact with proteins, thus reducing their bioavailability. Tannins also contribute to seed coat color and pigment distribution or intensity. The objective of this study was to analyze the relationship between quantitative trait loci (QTL) for seed tannin concentration in common bean and Mendelian genes for seed coat color and pattern. Three populations of recombinant inbred lines, derived from crosses between the Andean and Mesoamerican genepools were used for QTL identification and for mapping STS markers associated with seed color loci. Seed coat condensed tannins were determined with a butanol-HCl method and a total of 12 QTL were identified on separate linkage groups (LGs) in each of the populations with individual QTL explaining from 10 to 64% of the phenotypic variation for this trait. Loci on linkage groups B3 and B10 were associated with the Mendelian genes Z and Bip for partly colored seed coat pattern, while a QTL on linkage group B7 was associated with the P gene which is the primary locus for the control of color expression in beans. In conclusion, this study found that the inheritance of tannin concentration fits an oligogenic model and identifies novel putative alleles at seed coat color and pattern genes that control tannin accumulation. The results will be important for the genetic improvement of nutritionally enhanced or biofortified beans that have health promoting effects from higher polyphenolics or better iron bioavailability.

Genetic dissection of the resistance to nine anthracnose races in the common bean differential cultivars MDRK and TU

Resistance to nine races of the pathogenic fungus Colletotrichum lindemuthianum, causal agent of anthracnose, was evaluated in F(3) families derived from the cross between the anthracnose differential bean cultivars TU (resistant to races, 3, 6, 7, 31, 38, 39, 102, and 449) and MDRK (resistant to races, 449, and 1545). Molecular marker analyses were carried out in the F(2) individuals in order to map and characterize the anthracnose resistance genes or gene clusters present in these two differential cultivars. The results of the combined segregation indicate that at least three independent loci conferring resistance to anthracnose are present in TU. One of them, corresponding to the previously described anthracnose resistance locus Co-5, is located in linkage group B7, and is formed by a cluster of different genes conferring specific resistance to races, 3, 6, 7, 31, 38, 39, 102, and 449. Evidence of intra-cluster recombination between these specific resistance genes was found. The second locus present in TU confers specific resistance to races 31 and 102, and the third locus confers specific resistance to race 102, the location of these two loci remains unknown. The resistance to race 1545 present in MDRK is due to two independent dominant genes. The results of the combined segregation of two F(4) families showing monogenic segregation for resistance to race 1545 indicates that one of these two genes is linked to marker OF10(530), located in linkage group B1, and corresponds to the previously described anthracnose resistance locus Co-1. The second gene conferring resistance to race 1545 in MDRK is linked to marker Pv-ctt001, located in linkage group B4, and corresponds to the Co-3/Co-9 cluster. The resistance to race 449 present in MDRK is conferred by a single gene, located in linkage group B4, probably included in the same Co-3/Co-9 cluster.

Quantitative Trait Loci Linked to White Mold Resistance in Common Bean

White mold disease (WM) of common bean (Phaseolus vulgaris L.), caused by Sclerotinia sclerotiorum (Lib.) de Bary, reduces crop yield and quality throughout the United States. The development of cultivars with resistance to WM would be facilitated by the identification and use of molecular markers linked to resistance genes. The objectives of this research were (i) to characterize WM reaction in a recombinant inbred line (RIL) population derived from a cross between resistant and susceptible germplasm, (ii) to validate the effect of a previously reported quantitative trait locus (QTL) for WM resistance, and (iii) to locate additional QTL associated with WM resistance. A RIL population that consisted of 94 lines was derived from a cross between G122 (resistant) and CO72548 (susceptible). The population was evaluated for WM reaction in three greenhouse tests and one field environment, and for molecular markers throughout the genome. Two RIL were identified with higher resistance levels (P &lt; 0.05) than the resistant parent G122. A previously reported QTL on linkage group B7 was significant (P &lt; 0.01) in single‐factor analysis of variance, but not with composite interval mapping. Five QTL for resistance to WM were found (likelihood odds ratio [LOD] &gt; 2.7) on linkage groups B1, B2b, B8, and B9. The QTL were contributed from both parents and together accounted for 48% of the phenotypic variation (R2). For field resistance, one QTL (R2 = 12%) on linkage group B8 was detected. These results confirm polygenic resistance to WM in common bean.

Marker-Assisted Backcrossing QTL for Partial Resistance to Sclerotinia White Mold in Dry Bean

White mold caused by Sclerotinia sclerotiorum (Lib.) de Bary is a major disease limiting dry bean (Phaseolus vulgaris L.) production. Genetic resistance provides some control but is difficult to breed for because of low heritability. We sought to determine if marker-assisted selection for quantitative trait loci (QTL) conferring partial resistance could facilitate breeding for resistance to white mold in dry bean. The Phs marker linked with a QTL derived from landrace G122 on linkage group B7 was backcrossed into ‘Winchester’ pinto, forming two BC3F4:6 inbred line populations. The AW9.1200 and SS18.1650 markers linked with a QTL from snap bean breeding line NY6020-4 on B8 were backcrossed into ‘Maverick’ pinto and ‘Matterhorn’ great northern, forming BC2F4:6 inbred line populations. The B7 QTL in the BC3F4:6 populations on average explained 52% of the phenotypic variation for disease reaction in the greenhouse test and 10% across four field tests. The B8 QTL explained 30% in the greenhouse and 10% in the field. Averaged across tests and populations the B7 and B8 QTL conditioned 15 and 17% reduction in disease severity score, respectively. Linkage drag from selection of the B7 QTL was observed for yield. MAS for the B7 and B8 QTL was an effective breeding tool for introgressing partial resistance to white mold into susceptible pinto and great northern dry bean market classes, but further selection for agronomic performance may be required to obtain lines worthy of commercial production.

Generation and Molecular Mapping of a Sequence Characterized Amplified Region Marker Linked with theBctGene for Resistance toBeet curly top virusin Common Bean

ABSTRACT A random amplified polymorphic DNA (RAPD) marker directly linked (0.0 cM) with a resistance gene was identified in a snap bean recombinant inbred population (Moncayo x Primo) consisting of 94 F(5:7) recombinant inbred lines that had uniform segregation for disease reaction to Beet curly top virus (BCTV) across three field locations. Resistance was conditioned by a single dominant allele tentatively designated Bct. Seven hundred and fifty decamer primers were screened to obtain the linked RAPD marker that was then converted to a sequence characterized amplified region (SCAR) marker SAS8.1550. The SCAR mapped within a cluster of resistance genes on linkage group B7 of the core map. A survey of 103 BCTV-resistant and -susceptible snap and dry bean genotypes was conducted using SAS8.1550. Results showed that the SCAR would be highly useful for marker-assisted selection of Bct in snap and dry bean originating from the Andean gene pool. Marker-assisted selection for Bct will expedite the development of BCTV-resistant cultivars and minimize the need for cumbersome pathogen tests.

A molecular marker tightly linked to P, a gene required for flower and seedcoat color in common bean (Phaseolus vulgaris L.), contains the Ty3-gypsy retrotransposon Tpv3g.

In common bean (Phaseolus vulgaris L.), the expression of color in flower and seedcoat tissues requires the dominant allele of the P gene. The fully recessive p allele completely suppresses color expression in these tissues, whereas in specific genetic backgrounds, the p(gri) allele potentiates a grayish-white seedcoat and pale violet (nearly white) flowers with two violet dots on the banner petals. As a first step to gaining a better understanding of this important gene, we phenotypically scored an F2 population segregating for P and p(gri) and subsequently screened contrasting bulk DNA samples with oligonucleotide primers to uncover random amplified polymorphic DNA (RAPD) fragments. OU3(2300), an RAPD marker linked in coupling phase to the dominant allele, mapped 1.3 cM from P. The core 'BAT93' x 'Jalo EEP558' recombinant inbred population was scored, and the marker mapped to linkage group B7. The segregating fragment was cloned, sequenced, and shown to possess significant homology to the Ty3-gypsy class of retrotransposons. We have named the element Tpv3g. It is estimated that about 100 copies of the element are present in the common bean genome. Phylogenetic analysis placed Tpv3g in the class A group of plant retrotransposons.

QTL Conditioning Physiological Resistance and Avoidance to White Mold in Dry Bean

Physiological resistance is an important component of integrated strategies used to control white mold [caused by Sclerotinia sclerotiorum (Lib.) de Bary], a major disease of common bean (Phaseolus vulgaris L.) in North America. Information pertaining to inheritance of physiological resistance, as detected by the greenhouse straw test, and its relationship with field resistance is lacking. The objectives of this study were to compare physiological resistance as detected by two separate straw tests with field resistance, evaluate heritability of physiological resistance, and to characterize the disease reaction of G 122 by quantitative trait locus (QTL) analysis. This was done in a recombinant inbred population (A 55/G 122) consisting of 67 F8‐derived lines. The greenhouse tests with five and six replications, respectively, and the field test with three replications were conducted in randomized complete block designs. Moderate heritability for disease reaction (scored from 1 = no symptoms to 9 = severe disease) was observed across the straw tests (0.65) and in the field (0.78). Inheritance of disease reaction was further investigated with a framework linkage map composed of 74 markers. Interval mapping detected a QTL on linkage group B7 near the phaseolin seed protein (Phs) locus that explained 38% of the phenotypic variation for disease score across the straw tests. The same B7 QTL (26%), and an additional QTL (18%) on B1 near the fin gene for determinate growth habit, conditioned field resistance. A QTL (34%) for canopy porosity, a measure of potential disease avoidance, also mapped to the fin locus. Results confirmed that physiological resistance as detected by the straw test was an integral component of field resistance, and that both physiological and avoidance mechanisms contributed to field resistance in the A 55/G 122 population. The landrace G 122 clearly provides breeders with a heritable source of physiological resistance to combat white mold disease.

Bacterial, Fungal, and Viral Disease Resistance Loci Mapped in a Recombinant Inbred Common Bean Population (`Dorado'/XAN 176)

Understanding the genomic associations among disease resistance loci will facilitate breeding of multiple disease resistant cultivars. We constructed a genetic linkage map in common bean (Phaseolus vulgaris L.) containing six genes and nine quantitative trait loci (QTL) comprising resistance to one bacterial, three fungal, and two viral pathogens of bean. The mapping population consisted of 79 F5:7 recombinant inbred lines (RILs) derived from a `Dorado'/XAN 176 hybridization. There were 147 randomly amplified polymorphic DNA (RAPD) markers, two sequence characterized amplified region (SCAR) markers, one intersimple sequence repeat (ISSR) marker, two seedcoat color genes R and V, the Asp gene conditioning seed brilliance, and two rust [Uromyces appendiculatus var. appendiculatus (Pers.:Pers) Unger] resistance genes: one conditioning resistance to Races 53 and 54 and the other conditioning resistance to Race 108. These markers mapped across eleven linkage groups, one linked triad, and seven linked pairs for an overall map length of 930 cM (Kosambi). Genes conditioning resistance to anthracnose (Co-2) [Colletotrichum lindemuthianum (Sacc. and Magnus) Lams.-Scrib.], bean rust (Ur-5), and bean common mosaic virus (I and bc-3) (BCMV) did not segregate in this population, but were mapped by inference using linked RAPD and SCAR markers identified in other populations. Nine previously reported quantitative trait loci (QTL) conditioning resistance to a variety of pathogens including common bacterial blight [Xanthomonas campestris pv. phaseoli (Smith) Dye], ashy stem blight [Macrophomina phaseolina (Tassi) Goid.], and bean golden mosaic virus (BGMV), were located across four linkage groups. Linkage among QTL for resistance to ashy stem blight, BGMV, and common bacterial blight on linkage group B7 and ashy stem blight, BGMV, and rust resistance loci on B4 will complicate breeding for combined resistance to all four pathogens in this population.

Effects of acute and chronic treatment with imipramine on 5-hydroxytryptamine nerve cell groups and on bulbospinal 5-hydroxytryptamine/substance P/thyrotropin releasing hormone immunoreactive neurons in the rat. A morphometric and microdensitometric analysis

Groups of male rats were treated for a period of 14 days with imipramine (10 mumol/kg) given twice daily. Separate groups of rats received a single dose treatment using the same dose and experimental design as for the repeated treatment. Employing the avidin-biotin immunoperoxidase technique for immunohistochemistry 5-hydroxytryptamine (5-HT)-, substance P(SP)- and thyrotropin releasing hormone (TRH)-like immunoreactivities (IRs) were visualized in consecutive coronal sections of the brain stem and of the spinal cord. The IRs were studied by means of morphometric and microdensitometric procedures using automatic image analysis on profiles representing nerve terminal networks of the ventral horn of the cervical and lumbar enlargements of the spinal cord as well as their coexistence (5-HT/SP and 5-HT/TRH). With the same technique 5-HT IR was measured in the 5-HT nerve cell groups of the medulla oblongata (B1, B2, B3) and of the nucleus raphe dorsalis (B7) of the midbrain. In addition 5-HT and 5-hydroxyindolacetic acid (5-HIAA) levels were measured in the ventral and dorsal horns of the cervical and lumbar enlargements of the spinal cord using high performance liquid chromatography (HPLC). In the same parts of the spinal cord SP IR was studied by means of radioimmunoassay (RIA). The microdensitometric studies showed that chronic, but not acute, imipramine treatment selectively increased SP IR in the 5-HT/SP/TRH costoring nerve terminals of the medial part of the ventral horn in both the cervical and the lumbar enlargements. Furthermore, quantitative analysis of the entity of coexistence in the 5-HT nerve terminals networks of these areas showed that all the 5-HT nerve terminals contained SP and TRH IRs and that this phenomenon remained after acute and chronic imipramine treatment. The microdensitometric studies on the 5-HT nerve cell groups of the medulla oblongata and of the nucleus raphe dorsalis demonstrated that chronic, but not acute, imipramine treatment selectively increased 5-HT IR in the nerve cell bodies of the lateral part of group B3 as evaluated from the median grey values. Acute, but not chronic, imipramine treatment significantly increased the field area of 5-HT IR of nerve cell bodies in group B7, reflecting an increase in the mean profile area of the 5-HT IR nerve cell body profiles. Instead, the mean profile area of 5-HT IR cell bodies of group B1 was acutely reduced by imipramine.(ABSTRACT TRUNCATED AT 400 WORDS)

The nuclei of origin of brainstem serotonergic projections to the rodent spinal trigeminal nucleus

The origins of the serotonergic projections to the spinal trigeminal nucleus in the rat were determined utilizing the combined HRP retrograde transport-immunohistochemical technique. The areas providing the greatest number of serotonergic afferent projections to the spinal trigeminal nucleus are the raphe magnus, raphe obscurus, nucleus reticularis gigantocellularis pars α, and the nucleus reticularis paragigantocellularis. A smaller serotonergic input to the spinal trigeminal nucleus was found to arise from the raphe pallidus, periaqueductal gray, raphe dorsalis, raphe pontis and mesencephalic reticular formation. These results indicate that the spinal trigeminal nucleus receives serotonergic input from all levels of the brainstem including the midbrain cell group B7.

Morphometric characterization of transmitter-identified nerve cell groups: Analysis of mesencephalic 5-HT nerve cell bodies

Morphometric techniques have been used to introduce criteria to objectively define transmitter-identified nerve cell groups and subpopulations of nerve cells within such groups. The present method is exemplified on two coronal sections of the midbrain, in which 5-HT immunoreactive nerve cell bodies have been visualized. The reliability of the method is shown by the fact that it makes it possible not only to recognize groups B7, B8 and B9 of Dahlström and Fuxe [5] but also to assess the existence of two subpopulations within group B7 as previously suggested by Fuxe and Jonsson [11] based on subjective evaluations. Furthermore, the suggestion could be made that a small assembly of 5-HT nerve cell bodies in the dorsal part of the mesencephalic tegmentum may form a new 5-HT cell group. Hence, the present method offers the advantage of assessing the existence of transmitter-identified nerve cell groups on the basis of objective criteria and to describe them in a quantitative fashion. Therefore, it allows quantitation of morphological changes occurring in transmitter-identified neurons under different experimental and pathological conditions.

Serotonin nerve terminals in adult rat neocortex

Axonal processes which take up and retain exogenous tritiated serotonin ([ 3H]5-HT) have been demonstrated in the fronto-parietal cortex of adult rats, by means of high resolution radioautography. Prolonged topical superfusion with relatively high concentrations of [ 3H]5-HT allowed detection of a maximal number of axonal reactions in the upper 3 layers of cortex. Comparison of results obtained from normal rats and animals pretreated with 6-hydroxydopamine or bearing midbrain raphe lesions established the specificity of this labeling. All reactive axons seemed to arise from the serotonin nerve cell bodies in groups B7 and B8 of Dahlström and Fuxe 15. In electron microscope radioautographs, the serotonin fibers appeared as tenuous, naked axonal processes (0.1–0.5 μm in diameter) exhibiting small enlargements (0.7 μm in mean diameter) spaced at frequent intervals (1–3 μm). These boutons contained occasional mitochondria, small, round, agranular ‘synaptic’ vesicles and large granular vesicles. Within axons, [ 3H]5-HT was concentrated in the boutons, and to a much lesser extent in connecting segments. This reactive pattern resembled that revealed by the fluorescence technique for endogenous serotonin. Preferential accumulations of the tracer by mitochondria and vesicular organelles indicated that these elements could sequester exogenous serotonin. Large granular vesicles were not necessarily visible in random thin sections of the labeled varicosities, and thus could not serve as the unique criterion for electron microscopic identification of 5-HT terminals. Moreover, these organelles are known to be present in other types of nerve endings. Topometric analysis of serial thin sections nevertheless demonstrated that large granular vesicles were potentially detectable in every 5-HT containing bouton, and also enabled extrapolation of their average number at 7 per varicosity. This low number makes it unlikely that large granular vesicles primarily represent storage sites. They could rather serve as a carrier for particle-bound enzymes essential to the local metabolism of serotonin or its precursors. A very small fraction of the serotonin varicosities exhibited the membrane differentiations of typical synaptic terminals. Extensive sampling in serial thin sections revealed junctional complexes in only 5% of labeled boutons, as opposed to 50% of unlabeled nerve endings in the sorrounding neuropil. The data do not preclude the possibility that other monoaminergic neurons also share similar characteristics. It is probable that endogenous serotonin can be liberated from all axonal varicosities including those lacking strictu senso synaptic relationships. The overall configuration and ultrastructural features of cortical serotonin fibers suggest intrinsic dynamic properties which could assume particular significance in terms of function, plasticity and regrowth.