Cell death in the nervous system.

Programmed cell death is an important process in many types of cell. In the central nervous system of vertebrates, up to 50% of neurons die during development. The fact that in many cases neuronal cell death depends on macromolecular synthesis suggests that there is a genetic program that must be activated for cell death to occur. In this review, general features of cell death are discussed; in addition, the role of putative death-associated genes is outlined. Finally, the influence of neurotrophic factors on cell death is described.

More real-time visual and haptic interaction with anatomical data.

The University of Colorado's Center for Human Simulation has developed fundamental algorithms for real-time visual and haptic interaction with polygonal and voxel base data sets, including those derived from the Visible Human Dataset. These algorithms are currently being used to create prototype simulators for surgery, needle insertion (anesthesiology, radiology, and rheumatology), dentistry, and ophthalmology. This paper briefly discusses our segmentation and classification effort as well as our ability to create texture-mapped polygonal models directly from the data: both are fundamental to the creation of anatomically based simulators.

6-hydroxydopamine induces the loss of the dopaminergic phenotype in substantia nigra neurons of the rat. A possible mechanism for restoration of the nigrostriatal circuit mediated by glial cell line-derived neurotrophic factor.

Intraparenchymal injections of the neurotoxin 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle in rats destroys the dopaminergic neurons in the pars compacta of the substantia nigra. In other transmitter systems it has been found that axotomy or neurotoxin exposure produces an initial loss of neurotransmitter phenotype, with cell death occurring over a much slower time course. To determine whether this also occurs in dopamine neurons after 6-OHDA, two approaches were utilized. First, the effect of injections of 6-OHDA into the medial forebrain bundle on nigral dopaminergic neurons was studied using combined fluorogold and immunocytochemical labeling. Four weeks after the 6-OHDA injection, there was an 85% reduction in the number of tyrosine hydroxylase (TH)-immunoreactive cells on the lesioned side. In contrast, there was only a 50% reduction in the number of fluorogold-labeled cells on the lesioned side. Second, the time course of the rescue of dopaminergic neurons after 6-OHDA by glial cell line-derived neurotrophic factor (GDNF) was determined using TH immunocytochemistry. Greater numbers of dopamine neurons were rescued 9 weeks after GDNF, compared with counts made 5 weeks after GDNF. Taken together, these results suggest loss of dopaminergic phenotype is greater than cell loss following 6-OHDA injections, and that GDNF restores the phenotype of affected cells.

Apparent apoptotic cell death in the olfactory epithelium of adult rodents: death occurs at different developmental stages.

In the olfactory epithelium of adult rodent, receptor neurons are generated continually. Despite the ongoing generation of new neurons, no corresponding increase occurs in the thickness of the mature olfactory epithelium. Thus, epithelial cell death must occur to offset the continual generation of new cells. In the present study, a sensitive method to label nicked DNA in dying cells was combined with immunocytochemistry to determine the identity of dying olfactory cells. In addition, the positions of putative apoptotic cells were mapped to provide additional information about the identity of dying cells. Double labeling experiments revealed that each of the olfactory cell types, i.e., basal cells (keratin-positive), immature neurons (GAP43-positive) and mature receptor neurons (olfactory marker protein (OMP)-positive), were positive for fragmented DNA, suggesting that they undergo apoptotic cell death. The results of the mapping study suggest that apoptotic cell death occurs primarily among GAP43-positive neurons.

Expression of the death-associated gene RP-8 in granule cell neurons undergoing postnatal cell death in the cerebellum of weaver mice.

RP-8 is one of several mRNAs elevated during apoptosis in immature thymocytes. We used in situ hybridization to look for RP-8 mRNA in the cerebella of weaver mice. In the weaver mouse cerebellar granule cells fail to differentiate and instead die during the first two weeks of postnatal development [30]. We demonstrate that the dying cells exhibit DNA degradation, a characteristic feature of apoptosis. RP-8 is expressed in the weaver cerebellum during the period of granule cell death, and is limited to cells in the same region where apoptotic granule cells are located. Thus, RP-8 expression, first associated with PCD in thymocytes, correlates with granule cell death in the weaver cerebellum.

Programmed cell death in developing grafts of fetal substantia nigra.

Intracerebral transplants of ventral mesencephalic (VM) tissue have been well characterized. VM grafts contain numerous tyrosine hydroxylase immunoreactive neurons which send axons into the host brain. Transplanted neurons in VM grafts develop normally in that they contain tyrosine hydroxylase and GAP43. An overlooked aspect of graft development is cell death. It has been suggested that cell death in VM grafts was mostly necrotic. However, recent work in this laboratory suggested that developing grafts contain numerous apoptotic cells. In the present paper morphological, histochemical, and molecular correlates of apoptosis were used to assay cell death during VM graft development. At early times (5-15 days) after grafting VM grafts contained numerous apoptotic cells. In older grafts (21 and 28 days) few apoptotic cells were observed. In situ end labeling of fragmented DNA with biotinylated dUTP showed that early grafts contained numerous positive cells. The expression of RP8, a molecular correlate of apoptotic cell death, occurred in early grafts, but was not detectable in older grafts. These results indicate that apoptosis is a normal part of VM graft development. As in naturally developing neural systems, cell death in grafts may function to eliminate cells that fail to connect to appropriate targets.

Expression of GAP43 mRNA in normally developing and transplanted neurons from the rat ventral mesencephalon.

These experiments were designed to determine whether the neuronal growth-related protein GAP43 is expressed at high levels by neurons that collateralize extensively or have long periods of synaptogenesis. We also evaluated the effects of target availability on GAP43 expression. Dopaminergic neurons of the rat ventral mesencephalon (VM) were chosen for investigation because they undergo extensive collateralization and synaptogenesis during postnatal development. Double label in situ hybridization histochemistry (ISHH) and immunocytochemistry (ICC) were used to measure changes in GAP43 mRNA levels within tyrosine hydroxylase (TH)-immunoreactive and -nonimmunoreactive neurons of the VM during postnatal development (p5-adult). TH neurons show higher levels of GAP43 mRNA than do non-TH neurons throughout normal postnatal development and in the adult. This result may be due to more extensive axonal arborization and synaptic remodeling on the part of TH neurons as they innervate the striatum. To test the effects of target availability on GAP43 utilization, grafts of embryonic (e15) VM were placed within previously 6-hydroxydopamine (6-OHDA)-lesioned striata and allowed to develop for 10-28 days. Levels of GAP43 mRNA in grafted TH neurons were reduced at all time points. The short distance to target in the graft paradigm may shorten the overall axonal process length, resulting in lower requirements for growth-related proteins such as GAP43. However, grafted non-TH neurons had elevated levels of GAP43 mRNA, perhaps attributable to prolonged target seeking by neurons that have been isolated from their normal targets.

The expression of GAP43 mRNA during the late embryonic and early postnatal development of the CNS of the rat: an in situ hybridization study.

GAP43 is a developmentally regulated phosphoprotein which is almost exclusively found in neurons. Numerous correlative studies have shown that GAP43 is expressed at high levels during neurite extension, axonal elongation and synaptogenesis. In this study we used in situ hybridization to examine GAP43 expression during late embryonic and early postnatal development. The highest relative levels of GAP43 at all stages were present in the neocortex. Levels in this and other regions peaked between postnatal days 5 and 10. These results indicate that high levels of GAP43 mRNA correlate most highly with the latter stages of axon outgrowth and with the early stages of synapse formation.

GAP-43 and 5B4-CAM immunoreactivity during the development of transplanted fetal mesencephalic neurons.

Developing neurons contain high levels of several proteins which are absent or relatively scarce in mature neurons. GAP-43 is a cytoplasmic protein primarily found within neurons; high levels of this protein are correlated with axonal elongation or regeneration. 5B4-CAM, a glycosylated transmembrane protein, is a member of the NCAM family present in growth cones and in plastic CNS structures. Antibodies directed against these two developmentally regulated proteins were used to characterize the time-course of maturation of transplanted fetal mesencephalic neurons. For our experiments unilateral injections of 6-hydroxydopamine were made into the nigrostriatal bundle in Sprague-Dawley rats. The effectiveness of the lesion was verified by apomorphine-induced rotation and by postmortem examination of the substantia nigra. Following behavioral testing, pieces of ventral mesencephalon obtained from E15 fetuses were transplanted into the caudoputamen ipsilateral to the lesion. Immunocytochemistry revealed high levels of GAP-43 and 5B4-CAM at 5, 11, and 15 days post-transplant but relatively lower levels by 3 weeks. At 13 weeks the immunoreactivity present within the transplant tissue was approximately equal to that found within the host striatal neuropil. This time-course of higher GAP-43 and 5B4-CAM immunoreactivities coincides with the time-course of neuritic outgrowth of dopamine containing cell populations within the ventral mesencephalon in situ as well as within ventral mesencephalic transplants. This implies that axon elongation occurs over a period similar to that which occurs during normal development. These data suggest that the effects of transplantation surgery and the altered environment of the host striatum do not significantly affect the time-course of development of ventral mesencephalic neurons.

Specific outgrowth from neurons of ventral mesencephalic grafts to the catecholamine-depleted striatum of adult hosts.

Grafts of fetal ventral mesencephalon including substantia nigra have been used to correct some motor deficits produced by unilateral destruction of the dopaminergic nigrostriatal pathway in rats. Histochemical studies have shown that dopaminergic neurons within the graft send processes from the graft to the host neuropil, wherein they form synapses. The results of numerous immunocytochemical studies indicate, however, that a large proportion of neurons in grafts are not catecholaminergic. Whether or not the nondopaminergic neurons in grafts project to the host brain is unknown. The purpose of the present study was to combine immunocytochemistry and retrograde tracing with fluorogold to identify the cell types which project from grafts to the host striatum. Tissue from the ventral mesencephalon of E15 fetuses was placed into the 6-hydroxydopamine denervated striatum of graft recipients. Six weeks to 6 months following transplantation, fluorogold was pressure injected under stereotaxic control immediately adjacent to the ventral mesencephalic grafts; after 4 days CNS tissue was prepared for light microscopic immunocytochemistry. Ventral mesencephalic grafts contained cell bodies immunoreactive for enkephalin, GAD, substance P, and serotonin in addition to those immunoreactive for tyrosine hydroxylase. Some cells of each immunochemically defined type were retrogradely labeled by the fluorogold injection into the host brain. Nevertheless, more catecholaminergic and serotonergic cells projected from grafts to the fluorogold injection site than did other cell types. Since many of the nonmonoaminergic neurons in grafts are probably projection neurons, our results suggest that the extent of neurite outgrowth from grafted cells is influenced by the surrounding target tissue.

Human fetal mesencephalic tissue grafted to dopamine-denervated striatum of athymic rats: light- and electron-microscopical histochemistry and in vivo chronoamperometric studies.

Human fetal mesencephalic tissue obtained from elective first-trimester abortions was grafted to 6-hydroxydopamine-denervated striatum of athymic (nude) rats. After 3-6 months, the transplants were evaluated by light and electron microscopy using antibodies against tryosine hydroxylase (TH), human specific Thy-1 (Thy-1), 5-hydroxytryptamine (5-HT), and laminin. In vivo chronoamperometric studies of K+-induced release of electroactive species were done prior to the histochemical evaluations. At the light microscopical level, Thy-1-immunoreactivity was evenly distributed throughout the entire transplants. Thy-1-immunoreactive nerve fibers were observed radiating from the graft into the host striatum. In sections that were double-stained with antibodies against Thy-1 and TH, such nerve fibers contained both markers. Also 5-HT-immunoreactive cells were found in the grafts with processes both in the grafts and radiating into host neuropil. Laminin immunohistochemistry showed an even distribution of capillaries in the graft with less density than in host brain, suggesting immaturity of graft tissue. At the ultrastructural level, TH-immunoreactive axons made symmetric contacts with unlabeled dendritic shafts and dendritic spines within the host brain. A few asymmetric contacts with TH-immunoreactive axons were seen. 5-HT-immunoreactive terminals made both symmetric and asymmetric contacts with unlabeled dendritic shafts and spines. In vivo chronoamperometry using local application of K+ revealed average signals that were lower on the transplanted side than in control striatum. However, close to the grafts significant amounts of the K+-evoked signal amplitudes were as large as 1.3 microM, and the ratio of the reduction to oxidation currents suggested release of a mixture of dopamine and 5-HT. Taken together, this study shows that human fetal mesencephalic tissue pieces survive grafting into nude rats, develop normal vascularization, and express coexistence of TH- and Thy-1-immunoreactivity. Human TH- and 5-HT-immunoreactive nerve fibers form synapses in host striatum and release monoamine neurotransmitters.

Human ventral mesencephalic xenografts to the catecholamine-depleted striata of athymic rats: ultrastructure and immunocytochemistry.

On the basis of animal studies, grafts of fetal human dopaminergic cells have been suggested as a therapy for Parkinson's disease. The purpose of this study was to characterize the ultrastructure and immunocytochemistry of human ventral mesencephalic xenografts placed into the catecholamine-depleted striata of athymic "nude" rats. Human fetal tissue was obtained from tissue fragments derived from elective abortions during the first trimester of pregnancy. Small pieces of the basal mesencephalon were grafted into the catecholamine-depleted striata of four athymic nude rats. The rats were allowed to survive from 3 to 6 months after grafting; following fixation, the striatal tissue containing the grafts was labeled with antibodies against tyrosine hydroxylase and serotonin. Immunocytochemistry revealed tyrosine-hydroxylase-like-immunoreactive (THLI) and serotoninlike-immunoreactive (5HTLI) cell bodies within the human grafts. Both 5HTLI and THLI fibers crossed the graft-host interface and innervated the previously lesioned striatum. Both types of fibers also entered the host cortex from the adjacent human graft. At the ultrastructural level, THLI and 5HTLI fibers and synaptic terminals were observed in the host neuropil. THLI and 5HTLI dendrites and axon terminals were also observed in the neuropil of the grafts themselves. THLI axon terminals are not normally present in the substantia nigra. The results of our study indicate that human xenografts can survive in the neuropil of the host striatum and form morphologically appropriate synapses within the host brain.

Abnormal expression of tyrosine hydroxylase-like immunoreactivity in intraocular transplants of rat caudate nucleus.

The purpose of the present study was to examine tyrosine hydroxylase (TH)-like immunoreactivity in single intraocular grafts of caudate nucleus, and in caudate grafts which were co-grafted with substantia nigra. Grafts of caudate obtained from fetal rats (crown-rump length: 15-17 mm) were placed into the anterior chamber of eye of adult female Sprague-Dawley rats, and were allowed to survive from 6 to 20 months. The host rats were then perfused with fixative and the tissue was prepared for immunocytochemistry for TH. In 7 out of 8 surviving caudate grafts, including those containing no substantia nigra, tyrosine TH-like immunoreactive (THLI) cell bodies were present. The results of our study suggest that TH is inappropriately expressed in caudate grafts placed in the anterior chamber of the eye.

Intracerebral xenografts of human mesencephalic tissue into athymic rats: immunochemical and in vivo electrochemical studies.

Intracerebral allografts of fetal neurons have been studied in both rodents and nonhuman primates. Such research has been directed towards problems in developmental neurobiology and in animal models of neurological diseases. Whether intracerebrally transplanted human fetal neurons are capable of forming synapses and releasing neurotransmitters are key questions in any application of this approach to human brain development and dysfunction. We studied these questions by examining the immunocytochemical and in vivo electrochemical properties of xenografts of human mesencephalic dopaminergic neurons placed into athymic "nude" rats. The transplanted neurons survive, continue to express human-specific Thy-1 immunoreactivity, and extend neuronal processes into the host brain where morphologically identifiable synapses form. Potassium-evoked release of monoamines occurs in the vicinity of the graft but is absent in more remote areas of the host neuropil. These results indicate that human fetal tissue fragments can provide a source of viable neuroblasts for transplantation. Further, synapses form between pre- and postsynaptic elements expressing different species-specific cell surface markers; thus, these markers do not play a determining role in synaptogenesis.

Direct demonstration of interactions between substance P immunoreactive terminals and tyrosine hydroxylase immunoreactive neurons in the substantia nigra of the rat: an ultrastructural study.

The results of many anatomical, physiological, and pharmacological studies suggest that substance P-containing neurons of the striatum project to the substantia nigra, and that substance P influences the activity of dopaminergic nigrostriatal neurons. The purpose of the present ultrastructural study was to employ dual immunocytochemical labeling to determine the morphological basis for the observed actions of substance P on nigral dopaminergic neurons. Substance P-like and tyrosine hydroxylase-like immunoreactivities were localized simultaneously at the ultrastructural level in the substantia nigra of the rat. A double label method was utilized which relied on a combination of the peroxidase-antiperoxidase method (Sternberger, 1979) for substance P, and immunogold or silver enhanced immunogold labeling for tyrosine hydroxylase. The present results indicate that tyrosine hydroxylase immunoreactive (THLI) dendrites in the substantia nigra receive synaptic input from terminals exhibiting substance P-like immunoreactivity. These findings support the idea that substance P is a major neurotransmitter in the striatonigral loop, and suggest that striatal substance P neurons act directly upon nigral dopaminergic cells.

The ultrastructure of enkephalin-immunoreactive neurons in the interpeduncular nucleus of the rat.

The interpeduncular nucleus of the rat is a complex structure, displaying diverse immunocytochemical and ultrastructural features. This nucleus contains opiate receptors, enkephalin-positive cell bodies and enkephalin-positive fibers. The ultrastructure of rat interpeduncular enkephalinergic neurons has not been described, nor has the role that these neurons play in the internal organization of the interpeduncular nucleus been established. The purpose of the present study was to describe the ultrastructure of enkephalinlike-immunoreactive (ELI) cells with particular emphasis on the subnuclear organization of their dendritic and terminal fields. Enkephalinlike-immunoreactive (ELI) cell bodies are present in the rostral and apical subnuclei of the interpeduncular nucleus (IPN), but are absent from the other subnuclei of the IPN. The rostral subnucleus also contains immunoreactive dendrites that are postsynaptic to nonreactive terminal boutons. Numerous ELI axon terminals were observed in the central and intermediate subnuclei. The results of our study suggest that enkephalinergic interneurons link the rostral IPN with more caudal regions of this nucleus.

Substantia nigra transplants into denervated striatum of the rat: ultrastructure of graft and host interconnections.

A number of recent experiments suggest that grafted dopaminergic neurons provide functional input to a host caudoputamen which previously had been deprived of its dopaminergic input. The purpose of the present study was to determine whether tyrosine hydroxylase immunoreactive processes which originate in the graft participate in morphologically identifiable synapses in the host neuropil. Prior to transplantation, adult Sprague-Dawley rats received unilateral injections of 6-hydroxydopamine into the medial forebrain bundle. Animals were screened for the success of striatal denervation by a test of apomorphine-induced rotation. Transplants of fetal substantia nigra then were placed into cavities in the caudoputamen. After a 6-8 month survival period, animals were perfused and prepared for tyrosine hydroxylase immunocytochemistry. No evidence of sprouting of the host catecholoaminergic system was observed, even after long survival times. Both pre- and postsynaptic immunoreactive elements were clearly present in the host caudoputamen. Immunoreactive axons made synaptic contact with unlabeled dendrites; immunoreactive dendrites were postsynaptic to unlabeled axon terminals. The present results suggest that both host-to-graft and graft-to-host synapses are present in the host caudoputamen.