Profiles of serotonin receptors in the developing human thalamus.

The critical importance of the thalamus and its serotonergic innervation with respect to neuropsychiatric syndromes is increasingly recognized. This study investigates the localization of serotonin (5-hydroxytryptamine; 5-HT) receptors by immunohistochemistry in the thalamic nuclei of human fetuses aged 21 to 32 weeks of gestation. Results indicate that, already at 21 weeks of gestation, two 5-HT receptors are present in the dorsomedial nucleus of the developing thalamus: 5-HT2A receptors are localized in neurons and 5-HT2C receptors in fibers. By 31 and 32 weeks of gestation, 5-HT1A and 5-HT4 receptors are also detected in neuronal fibers of the same nucleus. At this later developmental stage, the percentage of 5-HT2A labeled neurons has significantly increased in the dorsomedial nucleus, and 5-HT2C positive neurons are observed in the centromedian and lateroventral thalamic nuclei as well. In contrast, neither neuronal cells nor fibers display any immunoreactivity for 5-HT3 or 5-HT6 receptors at any of the ages examined. Our observation that 5-HT1A, 5-HT2A, 5-HT2C and 5-HT4 receptors are present in the human thalamus prenatally indicates that 5-HT may play a role during fetal development. Disrupted development of the thalamic serotonergic system during this gestational period may contribute to the pathophysiology of neuropsychiatric disorders.

Development of the human insular cortex: differentiation, proliferation, cell death, and appearance of 5HT-2A receptors.

The development of the human insula was studied in the foetuses from 21 to 32 gestation weeks, using silver staining, immunohistochemistry of proliferative cell nuclear antigen (PCNA), activated caspase-3, and TdT-mediated dUTP-biotin nick end labeling (TUNEL) techniques. To test whether the insula also has a significant role in psychiatry behavior, we also mapped the major receptor of serotonin, 5HT-2A, in the developing insula as well. Opercular formation was evident by 21 weeks gestation. At this time, the cortical layers in the insula had started to organize, with silver impregnated pyramidal and stellate cells demonstrated various processes. By 25 gestation weeks, gyri in the insula were observed. PCNA positive cells decreased in density from 21 gestation weeks onwards while an increase of TUNEL positive cells was evident from 25 to 32 weeks of gestation. Activated capase-3 positive cells were detected in the insula, along with TUNEL positive cells, confirming possibly apoptosis. Serotonin 2A receptor appeared robustly in the 31/32 gestation week specimens. Our study showed early differentiation in the insula, when compared with other parts of the human cortex in the literature. In addition, proliferation as well as apoptosis were demonstration. Expression of 5HT (serotonin) 2A receptor positive cells in development was indicative of the insula as a significant psychiatric center.

bcl2, bax, and nestin in the brains of patients with neurodegeneration and those of normal aging.

This study was conducted by employing specimens from the frontal cortices of Alzheimer, multiple-infarct dementia patients, and those of normal aging (age matched to patients). The objective was to evaluate and compare the bcl2, bax, and nestin patterns in these three groups. Using immunocytochemistry, it was observed that bcl2 and bax active sites were colocalized in 45% of cells in Alzheimer, 52% of cells in multiple infarct, and 30% of cells in normal aging. bcl2 and bax could also be separately located in cells of all three groups. bax cells were most prominent in number in Alzheimer patients and least prominent in normal aging. nestin was found in all three groups but was most prominent in the multiple-infarct patients. Both astrocytes and neurons demonstrated positive nestin sites. The difference in pattern between groups will lead to further understanding of cellular changes in neurodegenerative patients and those of normal aging.

Myelination of the pig's brain: a correlated MRI and histological study.

Minipigs, 2, 4, 6 months old, were used to evaluate the relationship between myelination in the fiber tracts of the central nervous system (CNS) of this animal during development. Histological results showed an increased density of the myelinated fibers as well as branching of these fibers in the areas studied, including the cortical white matter, olfactory tract, the corticospinal tract, the fasciculus cuneatus and the spinal V nucleus from 2 to 6 months old. By 6 months, the pig was sexually matured. Concomitantly, there was an increase in high signal-intensity regions (sites) in the magnetic resonance T(1)-weighted images as myelination progressed. There is a good correlation between the histologically observed progress of myelination and the T(1)-weighted images in the development of the CNS of the pig.

Retinal twin cones or retinal double cones in fish: misnomer or different morphological forms?

Scanning retinal electronmicroscopy studies demonstrated the presence of both twin cones (two identical cones adhered together) and double cones (two non-identical cones adhered together) in the retinae of the major forms of fish, which included actinopterygi, elasmobranch, and teleosts. The complexity of these visual cells in the retinae of different groups of fishes was revealed for the first time and reflected on the diversities of functions of the fish retina in general.

The complexity of the visual cells and visual pathways of the sturgeon.

The visual cells in the retinae of the sturgeon were studied by scanning electron microscopy and transmission electron microscopy. Our investigations revealed the presence of rods, two types of single cones, one type of double cone (two nonidentical cone components adhered together), and one type of twin cone (two identical cone components adhered together). In some of the cones, large glycogen bodies were present in the inner segments and all cones contained oil droplets. Such cone morphology was very similar to that described in the retinae of higher vertebrates, for example the chicken. DiI tracing of retinofugal pathways following uniocular injection demonstrated their bilateral localization and extensive termination in the diencephalon and mesencephalon of both sides. Fibers also crossed over from one side to another through commissures, including the posterior commissure. The complexity of the pathway surpassed that of the teleosts and further indicated the evolutionary importance of this fish.

Three-dimensional reconstruction of brain surface anatomy based on magnetic resonance imaging diffusion-weighted imaging: a new approach.

Fifty normal noninfarct patients and 12 cases with infarcts of the cerebrum were examined with routine magnetic resonance imaging and echo-planar diffusion-weighted imaging. The diffusion-weighted three-dimensional images were reconstructed with volume-rendering processing on workstation. Precentral gyrus, postcentral gyrus, superior parietal lobule, superior frontal gyrus, precentral sulcus, central sulcus, postcentral sulcus, intraparietal sulcus and superior frontal sulcus were best shown of all structures with an arbitrary score of 2.61-2.77. Supramarginal gyrus, middle frontal gyrus, inferior frontal gyrus and lateral sulcus were clearly shown in the majority of the cerebra with average scores of 2.0-2.49; angular gyrus, inferior frontal sulcus and superior temporal gyrus were not demonstrated satisfactorily and their average scores were 1.67-1.89. Middle temporal gyrus, inferior temporal gyrus, superior temporal sulcus and inferior temporal sulcus were difficult to identify, and thus had average scores of 0.87-1.26. Brain surface structures were better displayed in the older group of individuals than in the younger group. The structures in the 12 cases with acute or chronic cerebrum infarcts were also satisfactorily demonstrated with this new technique.

Substitution for natural musk in Pien Tze Huang does not affect its hepatoprotective activities.

Previous studies showed that Pien Tze Huang, a Chinese folk medicine well known for its therapeutic activity in treating liver diseases, protected the liver against carbon tetrachloride (CCl4)-induced damage in mice. In the present study, natural musk, one of the important ingredients of Pien Tze Huang, was replaced by a formulated substitute, and the new formulation of Pien Tze Huang was shown to have similar chromatographic patterns to the original Pien Tze Huang in gas chromatography-mass spectrometry and high performance liquid chromatography. When used in treating mice with CCl4- or galactosamine-induced liver damage, both the original and new formulations of Pien Tze Huang were found to be able to suppress to a similar extent both the histopathological changes in the liver and the elevation of serum alanine aminotransferase and aspartate aminotransferase. Necrosis, cellular ballooning, microvesicular steatosis and lymphocytes infiltration were all significantly reduced in the damaged liver. In hepatoma cells, both formulations activated the activator protein 1 (AP1) enhancer sequence, indicating that both of them were able to act through the JNK signal transduction pathway. The results of the present study showed that the substitution for natural musk does not affect the hepatoprotective activities of Pien Tze Huang. It is also postulated that both formulations protect the liver through regulating signal transduction in the cell.

Growth arrest-specific 2 gene expression during patellar tendon healing.

We examined the cellular and molecular processes involved in patellar tendon healing following induced injury. A wound was surgically created at the center of the patellar tendon of adult rats. The wound site was examined at selected time intervals by immunohistochemical and in situ hybridization techniques. It was found that, between the 2nd and 7th day postoperation, fibroblast-like cells invaded the wound site. DiI-labelling experiments suggested that the majority of cells that occupied the wound originated from the edges of the wound. Furthermore, immunohistochemical studies revealed that at the wound site a meshwork of fibronectin developed that can support the migration of the DiI-labelled cells. We also examined the spatial and temporal expression patterns of the growth arrest specific 2 (GAS2) gene during patellar tendon healing. GAS2 was found strongly expressed in the tenocytes of unoperated patellar tendons. The gene was also expressed in the intact regions of operated tendons but not in the fibroblast-like cells that occupied the wound site, when examined 2 days postoperation. In addition the strip of intact tendon directly opposite the wound site also did not express GAS2. Examination of the experimental tendon at the 3rd month, when cells had completely occupied the wound site, revealed that Gas2 was expressed by all cells found in the wound. Bromodeoxyuridine (BrdU) incorporation analysis revealed that the presence of Brdu-positive cells in the wound indirectly correlated with the absence of Gas2 expression. We speculate that the GAS2 gene might play a role in regulating tenocyte proliferation during tendon healing.

Substance P and enkephalin containing fibers in the developing nucleus dorsalis of the human spinal cord.

A peroxidase-antiperoxidase immunostaining method was employed to determine the initial stage of appearance and localization of substance P (SP) and enkephalin (ENK) in the nucleus dorsalis of the developing human spinal cord. Both SP- and ENK-positive fibers started to appear from the 10th week of gestation in regions surrounding the nucleus dorsalis. SP-positive fibers then reached the nucleus at 13 weeks and from 26 weeks to term, three strands of SP-positive fibers, which were predominantly originated from the superficial layers of the dorsal horn, penetrated into the nucleus dorsalis from its medial, median and lateral aspects. From 26 weeks onwards, ENK-positive fibers, originated from the superficial and the adjacent layers of the spinal cord, formed a thicker medial and a thinner lateral bundle projecting into the nucleus dorsalis. Our results show that both SP- and ENK-positive fibers started to appear at around 10 weeks and a consistent pattern of immunoreactivity was established by around 26-30 weeks of gestation.

Fibroblast growth factor-8b-stimulated myogenic cell proliferation is suppressed by the promyelocytic leukemia gene.

Muscle cell growth is regulated by growth-promoting and -inhibiting factors. In this study, the physiological effects of fibroblast growth factor (FGF)-8b and the promyelocytic leukemia (PML) gene on G8 myogenic cells were examined. FGF-8b was found to strongly stimulate myogenic cell proliferation. Signal transduction assays using AP-1/SEAP and E-box/SEAP reporters revealed that the transcriptional factors junB/c-fos and c-myc were involved in FGF-8b-stimulated G8 cell growth. Besides examining factors that positively stimulate myogenic cell growth, we also examined genes that negatively affect cell growth. PML is a growth suppressor gene and we studied its expression in G8 cells under different growth conditions. Immunohistochemical staining revealed that in the presence of low serum, PML was expressed in approximately 23.2% of all cultured G8 cells. However, under normal culture conditions (10% serum), PML expression dropped to about 2.6%. We found that the PML gene acted antagonistically to FGF-8b, as the overexpression of PML in G8 cells significantly inhibited FGF-8b-stimulated cell proliferation. It also inhibited AP-1 and E-box transactivation. However, we believe that PML functions as a stress-response gene in G8 cells rather than as a gene normally involved in regulating muscle development.

Neurotransmitters, neuropeptides and calcium binding proteins in developing human cerebellum: a review.

Many endogenous neurochemicals that are known to have important functions in the mature central nervous system have also been found in the developing human cerebellum. Cholinergic neurons, as revealed by immunoreactivities towards choline acetyltransferase or acetylcholinesterase, appear early at 23 weeks of gestation in the cerebellar cortex and deep nuclei. Immunoreactivities gradually increase until the first postnatal month. Enkephalin is localized in the developing cerebellum, initially in the fibers of the cortex and deep nuclei at 16-20 weeks and then also in the Purkinje cells, granule cells, basket cells and Golgi cells at 23 weeks onward. Another neuropeptide, substance P, is localized mainly in the fibers of the dentate nucleus from 9 to 24 weeks but substance P immunoreactivity declines thereafter. GABA, an inhibitory neurotransmitter of the central nervous system, starts to appear at 16 weeks in the Purkinje cells, stellate cells, basket cells, mossy fibers and neurons of deep nuclei. GABA expression is gradually upregulated toward term forming networks of GABA-positive fibers and neurons. Catecholaminergic fibers and neurons are also detected in the cortex and deep nuclei at as early as 16 weeks. Calcium binding proteins, calbindin D28K and parvalbumin, make their first appearance in the cortex and deep nuclei at 14 weeks and then their expression decreases toward term, while calretinin appears later at 21 weeks but its expression increases with fetal age. The above findings suggest that many neurotransmitters, neuropeptides and calcium binding proteins (1) appear early during development of the cerebellum; (2) have specific temporal and spatial expression patterns; (3) may have functions other than those found in the mature neural systems; and (4) may be able to interact with each other during early development.

Bmp-4 requires the presence of the digits to initiate programmed cell death in limb interdigital tissues.

The effects of Bmp-4 on interdigital cell death were investigated in the mouse. Affi-Gel beads, loaded with recombinant Bmp-4 protein, were transplanted into the interdigital tissues of day 12.5 hindlimb, ex utero. It was established that Bmp-4 could induce precocious interdigital cell death. Using in situ hybridization, the expression patterns of bmp-4 and alk-6 receptor were established. Both genes were found coexpressed in the interdigital region of 12.5- and 13. 5-day hindlimbs. This suggests that Bmp-4 may act in an autocrine fashion. We have also studied the effects of Bmp-4 on 12.5-day interdigital tissue cultures. In all specimens examined, the interdigital tissues produced cartilage instead of participating in cell death. The addition of exogenous Bmp-4 to the interdigital cultures did not induce apoptosis but instead enhanced chondrogenesis. The discrepancy between the effects of Bmp-4 in vitro and ex utero was attributed to the presence of digits. When a flanking digit was left attached to the interdigital tissues, in vitro, Bmp-4 promoted apoptosis instead of chondrogenesis. In sum, the results suggest that Bmp-4 is a multifunctional protein and its effect on the interdigital tissues is dependent on the modulating influence of the digits.

Intravenous injections of the ribosome inactivating protein trichosanthin did not affect methionine enkephalin and beta-endorphin levels in the mouse brain and pituitary.

The effect of trichosanthin on levels of the neuropeptides beta-endorphin (beta-EP) and methionine enkephalin (met-EK) in the mouse brain and pituitary was investigated. Mature male ICR mice were divided into two groups. One group received intravenous injections of physiological saline and served as the control. Another group received daily intravenous injections of trichosanthin (0.2 mg/25g/injection) for three consecutive days. The animals were sacrificed four hours after the last injection. Their brains were dissected into three regions: A (thalamus and hypothalamus), B (cerebral cortex) and C (cerebellum and brainstem) and their pituitaries were collected. The samples were then extracted and assayed for beta-EP and met-EK by specific radioimmunoassays. It was found that there were no statistically significant changes in the levels of the two neuropeptides in the pituitary and the brain regions except for the level of beta-EP in brain region A.

Delay of the denervation process in skeletal muscle by sensory ganglion graft and its clinical application.

We examined the effects of dorsal root ganglion isografts on the denervation process of skeletal muscle. A segment of sciatic nerve was removed from each of 25 inbred Wistar-Kyoto rats. Fifteen were set aside as controls. In the remaining 10 rats, isogeneic cervical dorsal root ganglia were grafted to the severed distal stump of the common peroneal nerve. Between day 72 and day 286 postoperatively, both controls and recipients were killed after twitch and tetanic tension recording of the extensor digitorum longus was performed. The wet muscle weight and the twitch and tetanic tensions of the denervated extensor digitorum longus in the graft group were significantly greater than those in the control group. The mean area of the denervated tibialis anterior muscle fibers in the graft group also was significantly larger than that in the control group. In electron and light microscopic images, nerve cells along the periphery of each dorsal root ganglion were found surviving with regenerating axons throughout the experimental period. Numerous myelinated axons were observed in the common peroneal nerve of the graft group, and there were significantly more axonal branches in the extensor digitorum longus of the graft group than in the extensor digitorum longus of the control group. Thus sensory nerve fibers from the grafted dorsal root ganglia had certain beneficial effects to slow the denervation process, presumably secreting trophic factors into the denervated muscle. Clinically, we have transferred avulsed dorsal root ganglia in cases of total brachial plexus avulsion directly into denervated skeletal muscle. This procedure, accompanied by nerve crossing procedures, will probably keep denervated skeletal muscle in a better condition until regenerating motor axons from the repair site reach their target muscle.

Immunologic localization and kinetic characterization of a Na+/Ca2+ exchanger in neuronal and non-neuronal cells.

The plasma membrane Na+/Ca2+ exchanger is believed to play a role in the regulation of Ca2+ fluxes in neurons, though the lack of specific inhibitors has limited the delineation of its precise contribution. We recently reported the development of antibodies against a 36-kDa brain synaptic membrane protein which immunoprecipitated exchanger activity from solubilized membranes. In the present study we examined the kinetics of the Na+/Ca2+ exchanger in primary neurons in culture, in a neuronal hybrid cell line (NCB-20), and in a fibroblast-like cell line (CV-1) to see whether the level of exchanger activity correlated with the degree of immunostaining produced by our antibodies. The Vmax was determined for each cell type and found to be highest in primary neurons. Exchanger activity increased in primary neurons between days 1 and 6 in culture, but no such time-dependent change occurred in either of the cell lines. Immunoblot analysis of the three cell types probed with the anti-36-kDa protein antibodies revealed significantly greater immunostaining in the primary neurons compared with the other two cell types. Intensity of staining of neurons also increased significantly between days 1 and 6 in culture. Immunocytochemistry showed significant labelling of the primary neurons on the neuritic processes and points of contact between cells. The NCB-20 and CV-1 cells showed considerably lower levels of immunoreactivity. The antibodies immunoextracted approximately 90% of the exchanger activity in the primary neurons and approximately 70 and 50% of the activity in NCB-20 and CV-1 cells respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve regeneration in predegenerated basal lamina graft: the effect of duration of predegeneration on axonal extension.

We used predegenerated acellular grafts to bridge proximal and distal stumps of transected nerves and studied how the duration of predegeneration might affect axonal regeneration. Predegenerated acellular grafts were prepared by transecting the tibial nerve of donor rats and, after a period of degeneration, freeze-thawing a 40-mm long segment of the distal stump. Five degeneration periods were used: 0 days (for fresh grafts), 3 days, 1 week, 4 weeks, and 8 weeks. Fresh cellular grafts not treated with freeze-thawing were also used for comparison. Each graft was then transplanted to an isogeneic recipient rat, in which it was used to bridge the proximal stump of the transected left tibial nerve and the distal stump of the transected right tibial nerve. Six weeks were allowed for the regeneration of axons in all grafts. The regeneration was then assessed by studying transverse sections of the grafts, to determine the maximum length that the axons had regenerated, and the packing density of axons (percentage of sampled areas occupied by axons). The results show that axons had grown to the maximum length in the 4-week predegenerated grafts, and had the highest packing density in the 1-week predegenerated grafts. Regeneration in the fresh acellular (0-day predegenerated) and 8-week predegenerated grafts, especially the latter, was poor. We examine the results with reference to time-dependent events of Wallerian degeneration and propose that there are beneficial effects of multiple factors on the grafts during the first 4 weeks of predegeneration, causing a slow but significant improvement in their capability to support axonal growth. The subsequent rapid deterioration of such capability may be related to structural changes in the extracellular scaffold.

Trichosanthin, a ribosome inactivating and anti-HIV protein, does not alter the concentration of beta-endorphin in the mouse brain.

Trichosanthin was administered intramuscularly into mature male ICR mice at a dosage of 0.2mg/25g, daily for three days. The mice were sacrificed four hours after the last injection. Their brains were dissected out and divided into three regions: A(thalamus and hypothalamus), B(cerebral cortex) and C(cerebellum and brainstem). Their pituitaries were also removed. The pituitaries and the various brain regions were extracted with hot 1M acetic acid. The extracts were lyophilized and reconstituted in radioimmunoassay (RIA) buffer before assay for beta-endorphin by RIA. It was found that there was a significant elevation of beta-endorphin concentration after trichosanthin treatment only in brain region A. The data suggest that trichosanthin does not adversely affect the brain.

Reinnervation of denervated skeletal muscles by grafted dorsal root ganglion.

We examined whether or not the cervical dorsal root ganglion (DRG) of the rat, when isografted and connected to the distal stump of the severed common peroneal nerve, could survive, project axons to the denervated leg muscles, and exert beneficial influences to delay the degeneration of the denervated muscles. Rats in which the muscles were similarly denervated but no DRG was grafted served as the control. After a postoperative period of 72 to 286 days, histological study showed that nerve cells at the superficial part of the grafted DRG survived. Indirect electrical stimulation via the distal stump of the common peroneal nerve produced no contraction of the muscles, indicating that no functional neuromuscular contacts had been reestablished. Direct stimulation of the denervated muscles did elicit contraction, and the isometric twitch and tetanic tensions were significantly much higher in the experimental rats with a grafted DRG than in the control rats. Cholinesterase-silver staining indicated the presence of axons in the denervated muscles, but the axons did not terminate on endplates. Compared with the control muscles, the experimental muscles had significantly more axons, and had atrophied less as indicated by muscle wet weight and histological appearance. These results indicate that the sensory axons can delay the weakening and atrophy of muscles after denervation. We suggest that the sensory axons may exert certain trophic influence on the denervated muscle fibers, though the actual mechanism is unknown.