Ultrastructure of aberrant serotonin-immunoreactive fibers in the caudate putamen complex of the aged rat

Degeneration of neurons in the central nervous system is associated with morphological changes. Previous observations made at the light microscopical level indicated degeneration of serotonin-immunoreactive (IR) fibers in the aged rat brain. In this study, a comparison at the ultrastructural level was made between serotonin-IR normal thin and aberrant swollen varicose fibers in the caudate-putamen complex of the aged rat. Ultrastructural features such as the size and content of the thin varicose fibers resembled those in the caudate-putamen complex of the young rat as reported by others. The aberrant profiles were swollen, reaching a size of 6 microns. Their vesicles varied in size and were no longer uniformly round. Moreover, distorted mitochondria and membrane-filled vacuolelike structures were a common feature of the aberrant profiles. These changes are indicative of a degenerative process and give further evidence that, whereas many serotonergic fibers are preserved at high age, other serotonergic fibers are degenerating in the caudate-putamen complex of the aged rat.     

Morphological, neurochemical, and behavioral studies on serotonergic denervation and graft-induced reinnervation of the rat hippocampus.

A procedure was developed to conduct simultaneously immunocytochemical and neurochemical studies on the serotonergic system in adjacent 300-micron-thick slices of rat hippocampus. This procedure was applied to correlate morphological (innervation pattern and density), neurochemical (5-hydroxytryptamine and 5-hydroxyindolacetic acid levels and [3H]5-hydroxytryptamine uptake and release) and behavioral (spatial learning) effects of neurotoxin-induced denervation and reinnervation by grafting fetal mesencephalic raphe cells. Intracerebroventricular injections of a low dose of 5,7-dihydroxytryptamine caused a discrete serotonergic denervation of the hippocampus. Eleven months after lesioning, 5-hydroxytryptamine and 5-hydroxyindolacetic acid levels and [3H]5-hydroxytryptamine uptake capacity were decreased by 50-60%. By this time, the residual fibers displayed an enhanced vulnerability towards K(+)-induced depolarization. Grafting of a fetal raphe cell suspension resulted in a reinnervation of the host hippocampus. The pattern of reinnervation was comparable to control innervation and the density was supranormal at the level of the graft. As observed semiquantitatively, the innervation density decreased with distance from the core of the graft. Neurochemical studies showed that the fibers were capable of synthesizing, metabolizing and releasing 5-hydroxytryptamine. The turnover of 5-hydroxytryptamine in both the denervated and the reinnervated hippocampus was comparable to that in control tissue. Previous behavioral testing of the denervated and of the denervated and implanted animals did not reveal any effect on spatial learning, either in an individual or in a social test paradigm. The latter data substantiate the notion that interference with the hippocampal serotonergic innervation does not hamper adequate spatial learning.     

Serotonergic fibres degenerating in the aging rat brain or sprouting from grafted fetal neurons are not affected by the neurotrophic ACTH analogue Org 2766.

The effects of chronic treatment with the purported neurotrophic factor ACTH(4-9) analogue Org 2766 were studied on age-related degeneration of serotonergic fibres and on gliosis in the rat hippocampus and caudate putamen complex. In addition, the potential growth-promoting effects of Org 2766 were investigated on fetal serotonergic cells implanted in a previously denervated hippocampus of young adult rats. Chronic treatment of rats from the age of 11 months to 17-18 months did not affect the incidence of aberrant serotonergic fibres in the caudate-putamen complex or the fibres densities in the hippocampus or the caudate-putamen complex. Gliosis was unaffected by Org 2766 treatment as indicated by increased number and staining intensity of glial fibrillary acidic protein-immunoreactive cell bodies in both brain areas. Grafting of fetal raphe cells in young adult rats caused a time-dependent reinnervation of the previously denervated hippocampus. The reinnervation was not affected by treatment of the rats with Org 2766 for 4 weeks following implantation.     

Calcium ionophore-induced acrosome reaction correlates with fertilization rates in vitro in patients with teratozoospermic semen

The aim of this study was to determine the relationship between calcium ionophore A23187-induced acrosome reaction (AR) and sperm fertilizing ability. Semen samples remaining after preparation for standard IVF were studied in 109 patients who had sperm concentrations > or =20 x 10(6)/ml. Ionophore-induced AR was performed on motile spermatozoa selected by centrifugation on a Percoll gradient. Semen analysis was performed using standard methods. Patients with higher (>50%, n = 76) fertilization rates had significantly higher ionophore-induced AR than patients with lower (<50%, n = 33) fertilization rates (49 +/- 14 versus 38 +/- 21%, P < 0.05). When the data from all patients were analysed by logistic regression, only the percentage sperm motility in insemination medium and ionophore-induced AR were significantly related to fertilization rates. Similar results were also obtained when the data from a subgroup of patients with poor (<15% normal) sperm morphology were analysed. However, when patients with normal sperm morphology > or =15% were analysed separately, only sperm count and the percentage of spermatozoa with progressive motility in semen were significantly related to fertilization rates. In conclusion, ionophore- induced AR was significantly related to fertilization rates in vitro mainly in patients with teratozoospermic semen. Tests for ionophore- induced AR may provide additional information about sperm fertilizing ability but may not indicate specific defects of the physiological AR.      

Effects of hypothermia and gender on survival and behavior after perinatal asphyxia in rats

Previous studies in rats have demonstrated that perinatal asphyxia (PA) produces long-term morphological alterations, particularly affecting hippocampus. neostriatum, and cerebral cortex. These changes were prevented by applying hypothermia during the asphyctic insult. Because these cerebral areas are involved in cognitive and motor functions, the aim of the present study was to determine whether periods of PA during normothermia or hypothermia produces long-term behavioral impairments in rats of both sexes. The cognitive and motor functions were studied using the spatial Morris water maze (MWM) task at 1.5 months, and the open field at 5 months, respectively. The present study revealed that female rats had a higher survival rate than males after PA in normothermic conditions (p < 0.014). and that hypothermia drastically prolonged the time of survival in both sexes (p < 0.001). There were no differences in learning and memory functions between groups or male and female rats when tested with MWM. Rats subjected to hypothermia treatment did not show differences in the MWM compared to controls. A lower locomotor activity in the open field test was only observed in male rats that suffered 15 and 20 min of PA in normothermia (p < 0.05). Hypothermia treatment prevented this hypoactivity. PA in females, even if severe, did not affect the motor activity. The data of both behavioral tests showed differences between sexes, i.e., the female rats learned the MWM task slower, and were more active in the open field. This work lends further support for the hypothesis that hypothermia can prevent mortality as well as long-term sequelae induced by PA.     

Interchromosomal duplications of the adrenoleukodystrophy locus: a phenomenon of pericentromeric plasticity

A 9.7 kb segment encompassing exons 7-10 of the adrenoleukodystrophy (ALD) locus of the X chromosome has duplicated to specific locations near the pericentromeric regions of human chromosomes 2p11,10p11, 16p11 and 22q11. Comparative sequence analysis reveals 92-96% nucleotide identity, indicating that the autosomal ALD paralogs arose relatively recently during the course of higher primate evolution (5-10 million years ago). Analysis of sequences flanking the duplication region identifies the presence of an unusual GCTTTTTGC repeat which may be a sequence-specific integration site for the process of pericentromeric- directed transposition. The breakpoint sequence and phylogenetic analysis predict a two-step transposition model, in which a duplication from Xq28 to pericentromeric 2p11 occurred once, followed by a rapid distribution of a larger duplicon cassette among the pericentromeric regions. In addition to facilitating more effective mutation detection among ALD patients, these findings provide further insight into the molecular basis underlying a pericentromeric-directed mechanism for non- homologous interchromosomal exchange.      

The connections between the septum-diagonal band complex and histaminergic neurons in the posterior hypothalamus of the rat. Anterograde tracing with Phaseolus vulgaris-leucoagglutinin combined with immunocytochemistry of histidine decarboxylase

The connections between nuclei of the septum-diagonal band complex and the clusters of histaminergic neurons in the posterior hypothalamic region were studied with a dual-labeling procedure in which anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin was combined with immunohistochemistry of histidine decarboxylase. Phaseolus vulgaris-leucoagglutinin was injected in the medial and lateral septal nuclei, and in various parts of the nuclei of the diagonal band of Broca. The fibers arising from the medial and lateral septal nuclei traverse the vertical limb of the diagonal band and, in part, join the medial forebrain bundle in the preoptic area. Other fibers descend diffusely through the lateral hypothalamus to the posterior hypothalamus, or course in a bundle of fibers ensheathing the fornix. The nuclei of the diagonal band project via the medial forebrain bundle and the diffuse pathway to the posterior hypothalamic region. All the nuclei of the septum-diagonal band complex, with the exception of the medial and lateral parts of the nucleus of the horizontal limb of the diagonal band, project to clusters of histaminergic neurons. These projections exhibit the following arrangement: along the axis lateral septal nucleus-medial septal nucleus-vertical limb of the diagonal band-medial part of the horizontal limb of the diagonal band, the septohypothalamic fibers decrease in density and distribute to fewer clusters of histaminergic neurons. Varicosities on the labeled fibers are formed in close proximity to the cell bodies and dendrites of the histaminergic neurons.     

No colocalization of immunoreactivities for VIP and neuronal NOS, and a differential relation to cGMP-immunoreactivity in bovine penile smooth muscle

The distribution of immunoreactivity (IR) for the neuropeptide vasoactive intestinal polypeptide (VIP) and neuronal nitric oxide synthase (nNOS) in the bovine retractor penis muscle (RP) and penile artery (PA) was studied by using two different methods. The distribution of these immunoreactivities was also compared with that of the immunoreactivity for cyclic guanosine monophosphate (cGMP). In both tissues the nerve fibers and terminals immunoreactive for VIP had a distribution that was completely different from that of the nerve fibers and terminals immunoreactive for nNOS. This contrasts with the previous observations in penile smooth muscle of other species. In the RP, as well as in the PA, many of the VIP-IR fibers were also immunoreactive for neurofilaments (NF), whereas the nNOS-IR fibers were consistently devoid of NF-IR. Stimulation with sodium nitroprusside, a nitric oxide donor, considerably increased cGMP-IR in the smooth muscle cells in both RP and PA, and in several nerve fibers in PA. Many of these cGMP-IR nerve fibers exhibited nNOS-IR, whereas none of them was immunoreactive for VIP. Our results suggest that the degree of coexistence of VIP-IR and nNOS-IR in the nerve fibers and terminals innervating penile smooth muscle show wide species differences. They also suggest that the mechanisms by which VIP could be involved in neurogenic penile erection may vary between species.     

Artificial insemination and in-vitro fertilization using donor spermatozoa: a report on 15 years of experience

Donor insemination (DI) using cryopreserved semen commenced at The Royal Women's Hospital in 1976. Over the next 15 years we performed 5953 treatment cycles to achieve 816 pregnancies (13.7% per cycle) and 706 live births. In-vitro fertilization (IVF) using donor spermatozoa commenced in 1986. Over the next 5 years we performed 303 treatment cycles for 185 couples. Including subsequent transfer of cryopreserved embryos, a total of 33% of couples achieved a successful pregnancy by IVF. Statistical analysis indicated that, for DI pregnancies, the most important semen variable was the percentage post-thaw motility, whilst for normal fertilization in IVF it was the pre-freeze motility. These results may be explained by the compensatory effects of post-thaw processing of spermatozoa for IVF, but not for DI in our clinic.