Developmental expression of insulin-like growth factor-II receptor immunoreactivity in the rat central nervous system.

A polyclonal antiserum raised against the insulin-like growth factor-II (IGF-II) receptor has been used to map the distribution of this receptor in the developing rat central nervous system (CNS). Transiently high levels of receptor immunoreactivity were found in the developing brain, particularly in the cortex and hypothalamus. The amount of receptor immunostaining in these areas decreases toward the time of birth, and levels are approximately equivalent to those in the adult by postnatal day 7. The choroid plexus, cerebral vasculature, ependymal cells, retina, and pituitary contain high levels of receptor immunoreactivity throughout embryogenesis and adulthood. Some mesodermally derived tissues, such as bone, also demonstrate transient expression of IGF-II receptor during fetal development. These data are consistent with potential roles for IGF-II in CNS development, in the development of specific mesodermal tissues, and in specific regions of the postnatal CNS.

Distribution of insulin-like growth factor II receptor immunoreactivity in rat tissues.

Antibodies specific for the insulin-like growth factor II (IGF-II) receptor were used to study its distribution in a number of rat tissues and cell lines in order to determine which cells might be responsive to local or circulating IGF-II. In cultured 18-54,SF and B104 neuroblastoma cells, plasma membrane and cytoplasmic staining corresponding to Golgi apparatus could be seen, consistent with the glycoprotein nature of this receptor. Antibody binding was also seen in the central nervous system, confined primarily to the choroid plexus, and the vascular and ependymal elements. Some staining was seen in the parenchyma of the brain, in addition to binding around nerve sheaths and axon bundles. There were high levels of immunoreactivity in all three lobes of the pituitary, including vascular and cellular elements. In liver, highest levels of immunoreactivity occurred in the sinusoidal cells. In lung, IGF-II receptor immunostaining was seen in the alveoli and around the bronchioles. Staining in kidney was observed in glomeruli, tubules, and Bowman's capsules. Lower levels of immunostaining were seen in skeletal muscle, located primarily around the muscle sheaths. Localization of IGF-II receptor to cells of known function in different tissues will help elucidate the role of this ligand-receptor system in regulating growth and metabolism.

Structural and immunohistochemical characterization of insulin-like growth factor I and II receptors in the murine central nervous system.

The description of the cellular localization of insulin-like growth factor (IGF) receptors in the central nervous system (CNS) remains incomplete, as do the descriptions of changes in their characteristics with respect to different developmental stages. We, therefore, performed affinity labeling studies in microsomal membrane preparations of adult and fetal rat brain and liver tissues with [125I]IGF-I and [125I]IGF-II. These studies demonstrated tissue- and developmental stage-specific structural variants of type I receptor alpha-subunits as well as type II receptors. The adult rat brain type I alpha-subunit had an apparent mol wt (Mr) of 127,000, whereas those of adult and fetal rat liver measured 140,000. Fetal rat brain microsomes, however, had two types of type I receptor alpha-subunits measuring 130,000 and 120,000 Mr. The larger subunit from fetal brain consistently migrated at an apparent Mr of 3,000, greater than subunits from adult brain. Both type I and II receptors were more abundant in fetal liver and brain than in adult tissues. Affinity labeling was also performed directly to monolayers of cultured fetal brain neurons and newborn astrocytes. These studies detected both type I and II receptors on the surfaces of both types of cells. However, only the high Mr (140,000) form of the type I alpha-subunit was detected in cultured CNS cells, suggesting that expression of low Mr variant receptors is altered in vitro. Type II receptors were demonstrated by immunohistochemistry in adult rat hypothalamic neurons. However, the majority of neurons did not react with type II receptor antibody. This finding implies that only a minority of hypothalamic neurons are capable of responding to IGF-II via type II receptors. On the other hand, all astrocytes had striking type II receptor immunoreactivity. This signifies a more general biological role for this receptor in astrocytes compared with neurons. These results suggest that different tissue-, developmental stage-, and cell-specific processes are mediated by IGF receptors and suggests new directions in which to explore potential biological actions for these receptor-ligand systems in the CNS.

Localization and structural characterization of insulin-like growth factor receptors in mammalian retina.

Insulin-like growth factors (IGFs) are peptide mitogens, structurally related to insulin, whose biological actions in the CNS are incompletely known. The retina is largely uncharacterized with respect to IGF receptors. We, therefore, studied IGF receptors in bovine and murine retinal tissues by immunohistochemistry, autoradiographic localization, and affinity labeling. Notable IGF-II receptor immunoreactivity was found in retinal pigment epithelium (RPE), with intermediate levels in choroid, low levels in the inner and outer plexiform layers and outer nuclear layer, and very low levels in other regions. Autoradiographic localization using [125I]IGF-II confirmed the IGF-II receptor immunohistochemistry. Autoradiographic localization using [125I]IGF-I labeled the nuclear layers and the photoreceptor region. Affinity labeling disclosed differences in the apparent mol wt of IGF-I and IGF-II receptors from bovine eye tissues and those from liver and brain. IGF-I receptor alpha-subunits (the IGF-binding subunit) migrated at: liver, 139,000; brain, 125,000; RPE, 125,000 and 135,000 (two sizes); and retina, 125,000 and 135,000. IGF-II receptors migrated at: liver, 245,000; brain, 235,000; RPE, 240,000; and retina, 230,000. We conclude that mammalian retina contains both IGF-I and -II receptors, which differ from those found in other tissues and have a characteristic spatial distribution within the retina.

On neuronal homology: a comparison of similar axons in Musca, Sarcophaga, and Drosophila (Diptera: Schizophora).

Thoracic axons occurring in Musca and Sarcophaga are similar to those previously reported in the giant fiber pathway of Drosophila. Serial section reconstruction of both species has shown that the cervical giant fiber descending from the brain into the thoracic ganglion and the thoracic motor axon innervating the tergotrochanteral muscle follow courses matching those of similar axons in Drosophila. Likewise in both Musca and Sarcophaga a thoracic axon establishes axoaxonal synapses onto dorsal longitudinal muscle motor neurons. This axon is similar in both course and synaptic configuration to the peripherally synapsing interneuron in Drosophila. Although these similarities suggest that the three axon pairs are homologous in all three fly species, Several differences are also observed. Thus this system of identified axons may be a useful model for investigating phylogenetic variation in specific neuronal form and connectivity.

Apoptosis of sinusoidal endothelial cells occurs during liver preservation injury by a caspase-dependent mechanism.

BACKGROUND: Cold ischemia/warm reperfusion (CI/WR) liver injury remains a problem in liver transplants. Sinusoidal endothelial cells (SEC) are a target of CI/WR injury, during which they undergo apoptosis. Because caspase proteases have been implicated in apoptosis, our aim was to determine whether liver CI/WR injury induces a caspase-dependent apoptosis of SEC. METHODS: Rat livers were stored in the University of Wisconsin (UW) solution for 24 hr at 4 degrees C and reperfused for 1 hr at 37 degrees C in vitro. Apoptosis was quantitated using the TUNEL assay, and caspase 3 activation determined by immunohistochemical analysis. Rat liver orthotopic liver transplants (OLT) were also performed using livers stored for 30 hr. RESULTS: Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive hepatocytes were rare and did not increase during CI/WR injury. In contrast, TUNEL positive SEC increased 6-fold after reperfusion of livers stored under cold ischemic conditions, compared with controls or livers stored but not reperfused. Immunohistochemical analysis demonstrated active caspase 3 only in endothelial cells after CI/WR injury. When IDN-1965, a caspase inhibitor, was given i.v. to the donor animal and added to UW solution and the reperfusion media, TUNEL positive endothelial cells were reduced 63+/-11% (P<0.05). Similarly, the duration of survival after OLT was significantly increased in the presence of the inhibitor. CONCLUSION: During liver CI/WR injury: 1) selective apoptosis of endothelial cells occurs; 2) caspase 3 is activated only in endothelial cells; and 3) a caspase inhibitor reduces endothelial cell apoptosis and prolongs animal survival after OLT. The pharmacologic use of caspase inhibitors could prove useful in clinical transplantation.

Effects of intranigral substance P and neurokinin A on striatal dopamine release--I. Interactions with substance P antagonists.

The functional role of striatonigral neurokinins were studied by analysing the effects of intranigral injections of substance P and neurokinin A on the extracellular level of dopamine and dihydroxyphenylacetic acid in the striatum, as measured by in vivo microdialysis in rats. Two substance P antagonists, substance P D-Pro2 D-Trp7,9 and substance P D-Arg1 D-Trp7,9 Leu11 were tested and analysed for their ability to block the neurokinin effects. Unilateral injections of substance P (0.00007-7.0 nmol injected in 0.2 microliter) as well as neurokinin A (0.009-9.0 nmol) into the substantia nigra, pars reticulata of halothane anaesthetized rats produced long-lasting increases in ipsilateral striatal dopamine and dihydroxyphenylacetic acid levels. The dose-response relationship for substance P on dopamine was biphasic, with maximal effects occurring after the middle dose (0.007-0.07 nmol). The dose-response relationship for neurokinin A was monophasic. Intranigral injections of substance P D-Pro2 D-Trp7,9 (0.07-0.7 nmol) or substance P D-Arg1 D-Trp7,9 Leu11 (0.07-0.7 nmol) produced a decrease in striatal dopamine, but an increase in striatal dihydroxyphenylacetic acid. At a low dose (0.07 nmol) substance P D-Pro2 D-Trp7,9 enhanced the dopamine increase produced by intranigral substance P (0.07 nmol) or neurokinin A (0.09), while at a high dose (0.7 nmol) it blocked both substance P and neurokinin A effects. Both doses of substance P D-Arg1 D-Trp7,9 Leu11 (0.07 and 0.7 nmol) blocked the substance P- but not the neurokinin A-induced increase in striatal dopamine. Immunohistochemical analysis revealed that high doses of substance P (7.0 nmol) and neurokinin A (0.9 and 9.0 nmol), as well as substance P D-Pro2 D-Trp7,9 and substance P D-Arg1 D-Trp7,9 Leu11 (0.07 and 0.7 nmol), induced a restricted loss of tyrosine hydroxylase in dendrites and cells, and neuropeptide K in terminals, at the site of injection. Further analysis shows that co-administration of substance P (0.07 nmol) or neurokinin A (0.09 nmol) did not modify the extent of the depletion of both immunoreactivities induced by substance P D-Arg1 D-Trp7,9 Leu11 (0.7 nmol). The extent of the effect produced by substance P D-Arg1 D-Trp7,9 Leu11 (0.7 nmol) was, however, smaller than the spread of intranigral injection of [125I]Bolton-Hunter-labelled substance P D-Arg1 D-Trp7,9 Leu11, and it is suggested that the "neurotoxic" effects of the substance P antagonists are not primarily involved in their abilities to inhibit striatal dopamine release and block the stimulation of dopamine after intranigral substance P and neurokinin A.(ABSTRACT TRUNCATED AT 400 WORDS)

Lowicryl K4M embedding of brain tissue for immunogold electron microscopy.

We present methods for embedding brain tissue in Lowicryl K4M embedding medium and localizing antigens using postembedding immunogold techniques. After perfusion fixation with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, blocks of rat brain were placed in 2% aqueous uranyl acetate for 1 hour, dehydrated in 50%, 70%, and 95% ethanol, infiltrated with Lowicryl/ethanol mixtures (1:2 for 10 min, 1:1 for 15 min) and 100% Lowicryl (20 min and 25 min). Polymerization was carried out under UV light for 24-48 hours at room temperature. Several neural antigens, including three different synaptic vesicle proteins and an enzyme associated with the postsynaptic density, were localized by this technique, indicating that this procedure may have wide applicability.

Distribution of neuropeptide K-immunoreactivity in the rat central nervous system.

The distribution of neuropeptide K (NPK), a 36-residue amidated peptide originally isolated from porcine brain, is described in the rat CNS by immunohistochemical methods. Antibodies were generated in rabbits to N-terminus and C-terminus regions of the peptide and the distribution of immunoreactive cell bodies and fibers was mapped in colchicine-treated and normal rat brains. Major areas of cell body staining included the medial habenular nucleus, the ventromedial nucleus of the hypothalamus, the interpeduncular nucleus, the lateral dorsal tegmental nucleus, the nucleus raphe pallidus, and the nucleus of the solitary tract. Some of the areas of dense NPK-fiber immunoreactivity included the ventral pallidum, the caudate-putamen, certain areas of the hypothalamus, the central and medial amygdaloid nuclei, the entopeduncular nucleus, the habenular nuclei, the substantia nigra pars reticulata, the caudal part of the spinal nucleus of the trigeminal nerve, the nucleus of the solitary tract and the dorsal horn of the spinal cord. A striking similarity exists between this pattern of immunoreactive staining and that described for substance P, suggesting that the tachykinin systems do not exist independently in the brain. The possible roles for multiple tachykinins in the brain are discussed.

Pancreastatin distribution and plasma levels in the pig.

Pancreastatin is a peptide isolated from porcine pancreas which has insulin-suppressive actions in vitro and sequence homology with chromogranin A. Using radioimmunoassay and immunocytochemistry we investigated whether pancreastatin has a more widespread distribution and a possible endocrine role in the pig. Pancreastatin immunoreactivity was found in plasma, adrenal gland, pancreas, anterior pituitary and throughout the gastrointestinal tract. The immunoreactivity was colocalized with chromogranin immunoreactivity in endocrine cells and ultrastructurally (in the pancreas) to storage granules. Characterization of pancreastatin-like immunoreactivity, using gel permeation and high performance liquid chromatography, separated 3 different pancreastatin-like immunoreactive forms: one molecular form, indistinguishable from synthetic pancreastatin 1-49, was predominant in pancreas and thyroid and released into the circulation postprandially. However, a high dose (greater than 1 nmol/l) infusion of pancreastatin 33-49 (the biologically active moiety in vitro) into conscious pigs had no effect on either basal or glucose-stimulated insulin secretion.

Adjustment of connectivity in rat neocortex after prenatal destruction of precursor cells of layers II-IV.

Prenatal administration of cytotoxic drugs during proliferation of precursor cells of neurons in granular and supragranular layers of the rat cerebral cortex prevents these layers from forming and causes malformations of some cells in the surviving layers. But it does not prevent the surviving layers from establishing normal efferent connections, nor does it prevent afferent fibers from colonizing the cortex and establishing a bilaminar pattern of synaptic connections, partly in an abnormal position.

Expression of GFAP immunoreactivity during development of long fiber tracts in the rat CNS.

Astrocyte maturation in the developing corpus callosum and dorsal columns of the spinal cord was studied immunocytochemically in the rat, using antiserum to glial fibrillary acidic protein (GFAP) with a view to determining the relationships of astrocytes to the advancing axons of the corpus callosum and corticospinal tract. Between the eighteenth and nineteenth days of gestation, when the corpus callosum commences forming, most of the GFAP staining in the cerebral hemispheres is contained in radial processes, but some staining of glial cell bodies is also seen in the ventricular zone. At the region of interhemispheric fusion, where the corpus callosum will form, an accumulation of astrocytic processes demonstrable electron microscopically shows light immunocytochemical staining for GFAP. These processes do not adopt a stereotyped orientation. Rather, the overall impression as one moves towards the midline, is of radially disposed processes being disrupted and disoriented by the growing callosal axons at the fusion of the hemispheres. At no time can any orderly arrangement of GFAP-containing processes be seen which might indicate that the processes are serving to guide the growing axons across the midline. There is no immunoreactive staining of cell bodies or processes ventral to the corpus callosum, except in postnatal animals. Prior to the arrival of corticospinal axons in the spinal cord on the first postnatal day (PO)21, GFAP immunoreactivity is greatest in radial processes of the lateral funiculi and in the dorsal median septum. Oblique or vertical processes increase in the cuneate fasciculus from P0 tot P4 but do not appear in the gracile fasciculus until P4. Virtually no stained processes appear in the region to be traversed by the principal corticospinal tract, nor later in the tract itself until late in postnatal development. Only by 3 weeks postnatal is the adult pattern of GFAP staining observed in the corticospinal tract. These results also indicate that the expression of GFAP immunoreactivity is a relatively late phenomenon in astrocytes associated with advancing axons and implies that this aspect of astrocytic maturation is unrelated to any guidance that the immature astrocytes might provide for the growing axons.

Occurrence of neuropeptide K-like immunoreactivity in ventral horn cells of the chicken spinal cord during development.

The possible occurrence of NPK-LI in the ventral horns of the embryonic chicken spinal cord was investigated by means of the indirect immunofluorescence method. The results showed a transient appearance of NPK-LI in cells of the lateral motor column between day 5 of incubation and hatching. After this they disappeared and in the ventral horns NPK-LI remained only in fibers. The results are discussed in terms of a possible trophic action of NPK during development.

Developmental expression of proenkephalin mRNA and peptides in rat striatum.

The development of proenkephalin (PE) gene expression in the rat striatum was examined at the mRNA and peptide levels. Immunocytochemistry was performed with antisera generated to the PE-specific peptide product Met-enkephalin-Arg-Gly-Leu (MERGL). The distribution of immunostaining was compared with the distribution of PE mRNA, determined by in situ hybridization with an oligonucleotide probe. PE mRNA first appeared at E16 in the caudal ventrolateral striatum, followed at E17-18 by the appearance of MERGL immunoreactivity in a similar distribution. The anatomical gradients of PE gene expression were similar to the pattern of histogenesis of striatal neurons, suggesting that the timing of PE gene expression is related to the time of neuronal withdrawal from the mitotic cycle. The relation of the development of PE gene expression to the known patterns of striatal histogenesis, neurochemical compartmentalization and dopaminergic innervation is discussed.

Neuropeptide K-like immunoreactivity in human dental pulp.

Nerve fibres displaying such immunoreactivity were revealed by indirect immunofluorescence. Neuropeptide K-like immunoreactive fibres, entering the pulp within large nerve trunks, were distributed around blood vessels as well as in the stroma. Some immunoreactive fibres were also observed in the para-odontoblastic region. In view of the biological activity of neuropeptide K, it is tentatively proposed that it may act in the dental pulp as a regulatory peptide involved in neurogenic inflammation, blood flow regulation and sensory transmission.

First clinical trial of a novel caspase inhibitor: anti-apoptotic caspase inhibitor, IDN-6556, improves liver enzymes.

OBJECTIVE: To evaluate the safety of IDN-6556, a novel anti-apoptotic pan-caspase inhibitor, administered in single and multiple ascending doses in normal volunteers and patients with hepatic dysfunction. MATERIALS AND METHODS: IDN-6556 was administered as a 30-minute intravenous infusion in rising doses to 3 groups: Group A, normal volunteers, given as a single infusion, Group B, normal volunteers, given q.i.d. for 7 days, Group C, patients with hepatic impairment (elevated transaminases, alanine transaminase, ALT and aspartate transaminase, AST), given q.i.d. for 7 days. RESULTS: The drug was well tolerated up to 10 mg/kg/infusion for a single dose, and 1.5 mg/kg/infusion q.i.d. for 7 days, with the dose-limiting adverse event of phlebitis or inflammation at the site of the infusion. This toxicity was predicted from animal studies. Clinically and statistically meaningful dose-related falls in transaminases were seen in all but 1 of the hepatic impaired patients. Two-way ANOVA analyses of the changes for all the IDN-6556 groups combined versus placebo were: ALT absolute change: p < 0.0001 and % change: p = 0.012, AST absolute and % changes: p < 0.0001. After discontinuation of the drug (after 7 days of dosing), the transaminases rapidly returned to the pre-treatment levels. CONCLUSIONS: Following intravenous administration of a novel anti-apoptotic caspase inhibitor, adverse events were mild-to-moderate in severity, resolved in a few days and did not result in any subject terminating treatment prematurely. The effects in hepatic impaired patients appear to be consistent with both the administration and subsequent abrupt withdrawal of an effective hepatoprotective drug that delays cell death in hepatocytes.

The early formation of the corpus callosum: a light and electron microscopic study in foetal and neonatal rats.

A light and electron microscopic study of the developing corpus callosum was carried out in foetal and neonatal rats in order to determine the mode of growth of the earliest callosal axons across the midline and to investigate the potential role played by non-neuronal cells during the formation of the tract. The axons of the corpus callosum first cross the midline between the 18th and 19th days of gestation by traversing the anterodorsal aspect of the pre-existing hippocampal commissure. Prior to the appearance of the callosal axons at the midline, there is an aggregation of astrocyte processes anterior and dorsal to the hippocampal commissure. Careful examination of these processes in different planes of section shows that they are not organized in any obvious way that would provide a clearly defined path for the growing axons; nor are there any preferentially oriented extracellular spaces at the midline. No specialized membrane contacts could be seen between non-neuronal cell processes and the early callosal axons. Thus, there is no overt morphological evidence for an active role of non-neuronal cells in axon guidance in the initial formation of the corpus callosum. The development of the corpus callosum is accompanied by the formation of a temporary cavum septi pellucidi, which is always closed to the subarachnoid space. The cavum persists during the first postnatal week, after which time it becomes populated by cells of the lateral septal nuclei. Macrophages are present within the cavum and may play a role in its formation. Macrophages are also found within parts of the corpus callosum. No obvious degeneration of axons is seen in the corpus callosum during its early development.

Applications of monoclonal antibodies to neuroscience research.

The preceding discussion documents the diverse ways in which monoclonal antibodies have contributed to neuroscience research. They provide highly specific reagents to membrane-associated proteins, such as pumps, channels, receptors, and cell-adhesion molecules, that are useful for purifying these proteins, studying their structures at high resolution, and mapping their distributions. In many cases, the specific reagents were obtained using only partially purified antigens. Monoclonal antibodies to cytoskeletal proteins, organelles, and protein kinases have revealed that specific molecules are concentrated in anatomically distinct regions of the cell. A protein kinase has been shown to be a major postsynaptic constituent in many synapses. Individual proteins, such as actin, tubulin, and calmodulin appear to have different antigenic epitopes shielded in different parts of the cell. Monoclonal antibodies have provided a diversity of cell-type-specific reagents in both vertebrate and invertebrate nervous systems. They seem likely to be useful in identifying functionally related subpopulations of neurons and describing neural cell lineages. They will also serve to identify molecules that are important in regulating cell migration in the cerebellum, in marking cell position in the retina, and directing axon growth. This review also documents many purposes for which monoclonal antibodies are poorly suited or must be used with caution: A monoclonal antibody to a protein does not always reveal every place where that molecule is located. Pre- or post-translational microheterogeneity can expose different epitopes on the protein, such as may occur on the Na+-channel. Other proteins within the cell may shield antigenic sites on proteins such as calmodulin. Monoclonal antibodies can bind to epitopes on unrelated molecules (Nigg et al 1982, Lane & Koprowski 1982). This is revealed in some cases as multiple bands on immunoblots. Some cross-reactivity, however, may have a functional basis. For example, structural homology is clearly the basis for the antigenic epitopes that are shared among the five classes of intermediate filaments (Pruss et al 1981). The epitope that appears to be shared between the muscarinic and alpha 1-adrenergic receptors may be conserved because the two receptors modulate common effectors. The cross-reactivity between these receptors was only recognized because very specific and sensitive assays exist for each. It is quite possible that these same antibodies also bind sites on many other types of receptors. Mapping the distribution of this epitope may therefore have little relationship to the actual distribution of the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of cytotoxic deletions of somatic sensory cortex in fetal rats.

Pregnant rats were injected on the 14th day of gestation with the cytotoxic drug methylazoxymethanol acetate. This compound causes the death of neural precursor cells that were synthesizing DNA at the time of injection. After birth, the progeny of treated mothers grew to maturity with a neocortex that was greatly reduced in area by the death of all cells, particularly at the frontal and occipital poles but at medial and lateral margins of neocortex as well. In the remaining cortex layers II through IV failed to develop. The experiment deprived growing thalamocortical axons, which innervate the somatic sensory cortex late in development, of part of their normal target area and of a substantial number of their definitive target cells. It also deprived them of any cues they might have received from these target cells migrating through them as the axons accumulate beneath the cortical plate. Anatomical experiments indicated that, despite these defects, thalamocortical axons could still colonize the sensorimotor areas and form synapses in their typically bilaminar pattern, though the outer, denser lamina of terminations occurred abnormally at the level of the apices of layer V pyramidal cell bodies. Receptive field mapping of single and multiunit responses in the somatic sensory region showed brisk responses and receptive fields of normal size. It also indicated the formation of a body map that was topographically intact except for deletions at its periphery; that is, a total map was not compressed into a smaller area. This suggests that somatic sensory thalamocortical fibers recognize only remaining cortical target cells in appropriate fields. Moreover, successful ones among them seem to recognize neighborhood relations and conserve synaptic space at the expense of those that would have innervated the deleted peripheral parts of the area. Pyramidal neurons in the remaining cortical layers and in ectopic islands of cells that had incompletely migrated from the neuroepithelium, probably on account of destruction of radial glial cell precursors, were shown by retrograde labeling to send their axons only to appropriate subcortical targets. Pyramidal neurons, though recognized as such, also adopted a variety of abnormal orientations, such as inversion, apparently in an attempt to grow apical dendrites toward major zones of synaptic terminations.

Ceramide induces caspase-independent apoptosis in rat hepatocytes sensitized by inhibition of RNA synthesis.

Ceramide has been implicated as a second messenger in intracellular signaling pathways leading to apoptosis in nonhepatic cells. To determine whether ceramide can mediate hepatocyte apoptosis, the cytotoxicity of ceramide was determined in rat hepatocytes. The rat hepatocyte cell line, RALA255-10G, and primary rat hepatocytes were completely resistant to toxicity from 10 to 100 micromol/L C2 ceramide. Resistance was not the result of a failure to take up ceramide, because ceramide treatment did cause nuclear factor-kappaB (NF-kappaB) activation. Because ceramide may mediate cell death from tumor necrosis factor alpha (TNF-alpha), the ability of RNA synthesis inhibition and NF-kappaB inactivation to sensitize hepatocytes to ceramide toxicity was examined. RALA hepatocytes were sensitized to ceramide toxicity by coadministration of actinomycin D (ActD). Cell death occurred by apoptosis as determined by the presence of morphological evidence of apoptosis, caspase activation, poly(ADP-ribose) polymerase (PARP) degradation, and DNA hypoploidy. Despite the induction of apoptosis associated with caspase activation, cell death from ActD/ceramide was not blocked by caspase inhibition. Inhibition of NF-kappaB activation also sensitized RALA hepatocytes to ceramide toxicity, but to a lesser extent than for TNF-alpha. Thus, unlike many nonhepatic cell types, rat hepatocytes are resistant to cell death from ceramide because of the transcriptionally dependent up-regulation of a protective gene(s). The ability of ActD and NF-kappaB inactivation to sensitize RALA hepatocytes to ceramide toxicity suggests that ceramide may act as a downstream mediator of TNF-alpha toxicity.