Neurotransmitters and neuropeptides in the developing human central nervous system. A review.

This is a review on the ontogenesis of major neurotransmitters and neuropeptides in the developing human central nervous system. In general, the molecules under study appeared early in development, usually in the first trimester. Cholinergic neurons were found to be present around the time of neuropeptide formation. The newly formed neuropeptidergic fibers extended towards the cholinergic centers where both might interact. In the major centers of the central nervous system, neuropeptides were also noted to colocalize with various neurotransmitters. For example, in the facial nucleus, enkepahlin and substance P fibers coexisted with cholinergic and catecholaminergic neurons, suggesting complex interactions. In the interpeduncular nucleus, peptidergic neurons acting as interneurons clearly modulated the afferent input to this nucleus. In the hippocampus and in sensory organs such as the retina, there were indications that neuropeptides and gamma-amino butyric acid coexisted. We hypothesize that interactions of neurotransmitters and peptides in neurons and fibers early in development play an indispensable role in the morphogenesis of the human central nervous system.

Apoptosis in astrocytomas with different grades of malignancy.

Apoptosis, a form of programmed cell death was studied in astrocytomas with varying stages of malignancy, including low grade astrocytoma, anaplastic astrocytoma and glioblastoma multiforme. Apoptosis was visualized by employing the TUNEL technique and the evaluation of nuclear morphology and this was correlated with a study of bcl 2 expression. A decrease in the percentage of apoptotic cells and bcl 2 expression were evident with increasing malignancy. Furthermore, there was an increase in the proportion of apoptotic cells of the early stage in the more advanced form of astrocytoma. This is a study which demonstrates a possible direct inverse relationship between the severity of apoptosis and the stages of malignancy.

Tyrosine hydroxylase- and dopamine-beta-hydroxylase-positive neurons and fibres in the developing human cerebellum--an immunohistochemical study.

Six human fetuses of gestational ages 16-28 weeks were employed. The immunocytochemical avidin-biotin-peroxidase complex method combined with the silver Bodian technique was used to evaluate the presence of tyrosine hydroxylase and dopamine-beta-hydroxylase neurons and afferent and efferent fibres in the cerebellum during development. Our results illustrated that by 16-18 weeks, immunoreactivity of the Purkinje cells and the granule cells was evident. By 23 weeks, the positive Purkinje cells were tightly packed together and the perinuclear granules began to extend into the processes. The positive cells next to Purkinje cells were the basket cells and stellate cells. By 26-28 weeks, all positive cells increased in number and size. Mossy and climbing fibres appeared early in development (16-18 weeks of gestation) and were seen synapsing with the positive granule cells. At the same time, some parallel fibres were observed. At later stages, the tyrosine hydroxylase- and dopamine-beta-hydroxylase-positive Purkinje cells were surrounded by abundant climbing fibres, while parallel fibres were also evident in the molecular layer. In the deep cerebellar nuclei, positive tyrosine hydroxylase and dopamine-beta-hydroxylase neurons were present by 16-18 weeks of development. Those in the dentate nucleus were more polymorphic but smaller in size. Some afferent fibres were also spotted around 16-18 weeks of gestation and their numbers increased later. Positive efferent fibres were present by 26 weeks. All these observations point to an early presence of tyrosine hydroxylase and dopamine-beta-hydroxylase components in cerebellar development.

The human primary olfactory pathway: fine structural and cytochemical aspects during development and in adults.

Despite increasing knowledge about the biophysiology of the human olfactory system, understanding of the development of this pathway in humans lags considerably behind that of other vertebrates. Developmental studies have largely concentrated on the generation of cell types in the olfactory epithelium during the first trimester, while detailed ultrastructural observations usually describe the adult morphology. In this review, we have shown that contrary to what has been generally assumed, the surface of the human olfactory epithelium is heterogeneous and that its olfactory nerves differ ultrastructurally from those of other vertebrates studied. The development of the human primary olfactory pathway is discussed in terms of the appearance of olfactory bulb laminae, synaptogenesis and the expression of specific cell markers, such as the S-100 protein and olfactory marker protein (OMP). Positive immunohistochemical staining for N-cadherin in human fetuses suggests that growth of olfactory axons to their target may be mediated by cell adhesion molecules. The overall data presented here indicate that this pathway develops more precociously in humans than in rodents. Whether this translates also to earlier functional maturity remains to be elucidated.

Acetylcholinesterase-containing neurons, substance P and enkephalin fibers in the ventral horns of developing human embryos and fetuses.

The presence of the acetylcholinesterase neurons and substance P-like and enkephalin-like fibers in the various nuclear columns of the ventral horns of the spinal cords was studied in the developing human by acetylcholinesterase histochemistry and substance P and enkephalin immunohistochemistry. Acetylcholinesterase-positive neurons initially appeared in the lateral neuronal columns and eventually were also observed in the medial columns as well as the median columns at various levels of the spinal cord by 10 weeks' gestation. Acetylcholinesterase-positive neurons in the lower sacral levels were not detected until 11-12 weeks' gestation. Diffused substance P- and enkephalin-like fibers were demonstrated as early as 10 weeks' gestation but did not align with any particular nuclear column until after 15 weeks' gestation. These fibers further increased in length and adopted reticular branching patterns and many of these tended to surround the cell bodies of the nuclear columns. Possible interaction of acetylcholinesterase neurons and substance P and enkephalin fibers would commence by 15 weeks' gestation.

Immunocytochemical localization of cyclooxygenase in the rat lens.

The localization of rat lens fatty acid cyclooxygenase (prostaglandin synthase) was studied using indirect immunofluorescent and indirect streptavidin-biotin immunoperoxidase staining techniques. Both methods employed monoclonal and polyclonal antibodies directed against fatty acid cyclooxygenase, the key enzyme in the conversion of arachidonic acid to prostaglandins. The immunocytochemical studies demonstrate that (1) fatty acid cyclooxygenase is present in the rat lens epithelial cell layer; (2) the enzyme appears predominantly in the cytoplasm; (3) there is an apparent higher concentration of the enzyme in the region designated as germinative and transitional zones, and meridional rows; and (4) the enzyme appears to be absent in the lens capsule and in the nucleus of the lens. The presence of the cyclooxygenase enzyme in the lens epithelium, especially the relative intense staining in the epithelial mitogenic region, suggests that oxygenation of polyunsaturated fatty acids by the fatty acid cyclooxygenase may have an important role in cellular differentiation.

Immunohistochemical localization of substance P, enkephalin and serotonin in the developing human retina.

The localization of substance P (SP), enkephalin (ENK) and serotonin (5-HT) in the retinae of 12 human embryos/fetuses ranging in age from 6-30 weeks was determined immunohistochemically using the PAP method. At the 6 week stage [crown rump length (CRL) unknown due to incomplete specimen], the developing retina consisted of a single undifferentiated cell mass from which immunoreactive cells were absent. By 90 mm CRL (10th week of gestation), the retina was composed of an outer neuroblastic layer, an inner plexiform layer and an innermost layer of ganglion cells. At this stage, SP, ENK and 5-HT positive cells were detected solely in the outer neuroblastic layer. By 140 mm CRL (17 weeks), the retina consisted of cell layers similar in number and type to those of the adult retina. In specimens 140-216 mm CRL (gestation ages 17-24 weeks), SP, ENK and 5-HT neurons were present in the outer nuclear, inner nuclear and inner plexiform layers. In addition, 5-HT positive neurons and fibers were evident in the outer plexiform layer. By 285-295 mm CRL (26-30 weeks), neurons in the ganglion cell layer and the fovea were also SP and ENK positive. In earlier specimens, the cell bodies alone were immunopositive and not until 142 mm CRL (17 weeks) were positive processes observed. Finally, the presence of SP, ENK and 5-HT immunopositive structures occurred in a sequence from outer to inner layers of the developing human retina.

Responses of astrocytes in culture after low dose laser irradiation.

The effect of Helium-Neon low dose laser on astrocytes was investigated in cultures of isolated astrocytes from albino neonatal rats. The laser appeared to inhibit the growth of astrocytes as exemplified by the smaller sizes of the cells and the decreased leucine uptake in each cell after treatment. Temporary decrease in the number of mitoses was also observed, but this trend was reversed soon after. Electron microscopic studies revealed an increase in buddings from cell bodies and processes (branches) after irradiation.

Development and localization of enkephalin and substance P in the nucleus of tractus solitarius in the medulla oblongata of human fetuses.

The presence of enkephalin and substance P-positive neurons and fibers were studied by immunohistochemistry (peroxidase-antiperoxidase or avidin-biotin-peroxidase complex methods) in 26 human fetuses ranging from 11 weeks of gestation to 40 weeks of gestation. Enkephalin-positive neurons were localized in the commissural, medial and intermediate subnuclei as early as 11-12 weeks' gestation. Positive enkephalin fibers were localized around 12 weeks' gestation and in many subnuclei, notably the medial, commissural, intermediate, ventrolateral, ventral and dorsolateral subnuclei. Substance P-positive neurons were localized in the commissural and medial subnuclei around gestation age 13 weeks. Positive substance P fibers appeared even earlier, around 11 weeks of gestation in many subnuclei, notably the medial, intermediate, ventral, ventrolateral and dorsolateral subnuclei. Increase in both enkephalin- and substance P-positive fibers was evident in the later stages of development (e.g. around 26 weeks of gestation). The importance of the early appearance of enkephalin and substance P neurons and fibers of the pain pathways in the major subnuclei connecting with the cardiovascular, gastrointestinal and respiratory functions in the human has to be stressed.

Localization of acetylcholinesterase positive neurons and substance P and enkephalin positive fibers by histochemistry and immunohistochemistry in the sympathetic intermediate zone of the developing human spinal cord.

Localization of acetylcholinesterase positive neurons and substance P and enkephalin fibers were studied by histochemistry and immunohistochemistry in the intermediate sympathetic zone of the spinal cords of 39 human embryos/fetuses from gestation ages five to 40 weeks. Acetylcholinesterase positive neurons were observed in the nucleus intermediolateralis pars principalis as early as the fifth week of gestation. By the ninth to 13th weeks of gestation, positive neurons were also seen in the nuclei intermedialis pars funicularis, intercalatus spinalis and intercalatus pars paraependymalis. Increase in amount of these positive acetylcholinesterase neurons was demonstrated till term. Substance P and enkephalin fibers were initially observed by the eighth gestation week in the intermediolaterlis pars principalis nucleus and positive fibers were then detected in the nucleus intermedialis pars funicularis as well as the nucleus intercalatus spinalis by the 14th week of gestation. By the 26th week of gestation, all the major nuclei intermediolateralis par principalis, intermedialis pars funicularis, intercalatus spinalis and intercalatus pars paraependymalis has substance P and enkephalin fibers. Initial demonstration of acetylcholinesterase positive neurons appeared to be at an earlier stage than that of our substance P and enkephalin positive fibers.

Immunohistochemical studies on the development of 5-HT (serotonin) neurons in the nuclei of the reticular formations of human fetuses.

5-HT-positive neurons were detected by the PAP immunohistochemical methods in different nuclei of the reticular formations of human fetuses as early as 10 weeks of gestation. The majority of positive 5-HT cells were located in dorsal raphe and central superior nuclei, and there was a reduction of these neurons per 40-microns section as the fetus aged.

Scanning electron microscopy of the development of layer I of the human visual cortex (area 17).

The development of layer I of the human visual cortex was studied. Tissues were obtained from 14 aborted and stillborn human fetuses, ranging in age from 13 to 32 fetal weeks. The middle third of the rostrocaudal area 17 above the left calcarine sulcus was selected for observation. The sections were cut perpendicular to the pial surface of the cortex. The specimens were processed for scanning electron microscopy. At 13-16 weeks the Cajal-Retzius neurons were the predominant neuronal elements of layer I, with a small amount of fibers present. In addition, it was found that at 13 weeks the folding plasma membranes of the glial cells formed primary myelin sheaths which wrapped around the nerve cell bodies and processes. Axosomatic and axodendritic contacts were present in the cortices of the fetuses. The nerve fibers of layer I increased progressively with age. However, at 17 weeks, some degenerating apical dendrites were occasionally found. These degenerating dendrites appeared fragmented and beaded. At 21-23 weeks, layer I was well developed with all its essential components present. However, the upper half of the cortical plate was still undifferentiated at this age. At 26-32 weeks, layer I had a prominent plexiform lamina. This lamina was poor in neurons and rich in fibers. In summary, during layer I ontogenesis, there were the following major changes: (1) a change from higher to lower neuronal density: (2) changes in morphology of neurons; (3) initial appearance of synapses early in development; (4) early onset of glial wrapping; (5) presence of degenerating fibers during ontogenesis, and (6) increase in fiber proportion as fetus aged.

Localization of substance P and enkephalin by immunohistochemistry in the spinal cord of human fetus.

The peroxidase-antiperoxidase method was used to study the distribution of substance P and enkephalin during development of the spinal cords of human fetuses. Thirty-seven cases were collected, ranging from 5- to 40-weeks-old (fetal ages). Both types of transmitters were present initially around the fifth week in the mantle layer of the base of the dorsal horn, around the tenth week at the anterior gray and the intermediate gray and around the sixth week at the marginal layer at the base of the ventrolateral funiculus. Substance P- and enkephalin-positive sites at the marginal layers at the base of the dorsolateral funiculus were evident in the same area at 5-6 weeks. The positive fibers in the dorsal horn were initially located in the superficial layers. By the eleventh week, the positive sites spread to other surface layers at the lateral sides of the dorsal horns bilaterally at all spinal levels above the sacral. In the sacral levels adjacent to the conus medullaris, the spreading to surface layers was not apparent bilaterally until the seventeenth week. By weeks 18-26 the positive sites penetrated deeper in the dorsal horn and by week 27 assumed the adult path. The enkephalin cell bodies were present in the Rexed layers I and II of the dorsal horn and the substance P-positive sites were apparent in the dorsal ganglia of the 28-40-week-old fetuses.

Olfactory marker protein is present in olfactory receptor cells of human fetuses.

Light microscopic observations indicated that cellular differentiation of the primary olfactory pathway in human fetuses was relatively advanced by the end of the first trimester. However, immunohistochemical staining showed that olfactory marker protein was present in the receptor cells only at about 28 weeks post-conception. At about 32 weeks, modest amounts of the protein could be found in the peripheral olfactory nerve layer and a few glomeruli, with increased staining observed in subsequent weeks. The results show conclusively that human fetal olfactory neurons synthesize olfactory marker protein during the later half of gestation.