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The effects of ethanol on chick embryo sensory and spinal cord neurons growing on one of several biological substrates (poly-D-lysine, laminin, or neuron-produced neurite-promoting materials) were examined. Ethanol inhibited process formation by the neurons in a dose-dependent manner and inhibited the production of neurotrophic factors. Neuronal attachment to the substrates, survival of attached neurons, and receptor interactions of sensory neurons with nerve growth factor were not influenced by ethanol. It appears that ethanol alters certain metabolic characteristics of developing neurons.

Counts of callosal neurons retrogradely labeled by horseradish peroxidase (visualized using multiple substrates) were obtained in areas 17 and 18 of five kittens reared with their eyelids bilaterally sutured and of three kittens which had undergone bilateral enucleation on postnatal days 1--4. These counts were compared with those obtained in normal adult cats. The normal adult distribution of the callosal neurons results from the gradual postnatal reduction of a more widespread juvenile population. Binocular visual deprivation by lid suturing dramatically decreases the final number of callosal neurons and narrows their region of distribution (callosal zone) in areas 17 and 18. A less severe reduction in the final number of callosal neurons is caused by bilateral enucleation, which also increases the width of the callosal zone compared to that of normal cats. Thus, visual experience is necessary for the normal stabilization of juvenile callosal connections. However, since some callosal neurons form connections in the absence of vision, other influences capable of stabilizing juvenile callosal neurons also exist. These influences are probably antagonized by destabilizing influences or inhibited, when the eyes are intact.

We have examined the consequences of surface interactions with glial and nonglial cells on the in vitro growth of CNS neurons. When cerebellar or spinal cord cells were plated onto monolayers highly enriched in cortical astrocytes or sciatic nerve Schwann cells, neurons generally grew as single cells and showed relatively little tendency to aggregate. Similarly, neurites showed little tendency to fasciculate. In contrast, when plated onto fibroblast, heart muscle-fibroblast, or astrocyte-free meningeal monolayers, neurons rapidly aggregated, and neurite outgrowth was primarily in large fascicles. There were no glia detectable in the majority of aggregates or fascicles, suggesting that aggregation and fasciculation were due to interactions between neurons. Neurite outgrowth over 24 hr was also greater on astrocytes than on nonglia. Whether or not aggregation and fasciculation occurred was due to surface properties of the glial and nonglial cells. When neurons were added to astrocyte and nonglial monolayers growing in medium conditioned by a large excess of co-cultured nonglia or astrocytes, respectively, the pattern of neuronal growth was determined by the type of monolayer with which the neurons were in contact. Moreover, the initial growth of neurons on heat-killed astrocytes was indistinguishable from growth on living astrocytes. The pattern of neuronal growth on these different monolayers suggests that neurons are more adherent to glia than to other neurons but are more adherent to other neurons than to nonglia. Such an adherence hierarchy could explain the consistent finding of an apposition of neurons to glial surfaces during neuronal migration and axon outgrowth. Our findings also suggest that the interaction of axons with the non-neuronal milieu through which they grow may play an important role in regulating fasciculation, a process which has generally been treated as due primarily to axon-axon interactions.

Neurons are born in the ventricular walls of the vertebrate central nervous system. From there, the young neurons migrate to their final destinations, where differentiation occurs. Neuronal migration has been described during the ontogeny of the avian and mammalian brain. Whereas in mammals most neurogenesis occurs during early development, in the adult avian forebrain wide-spread neurogenesis continues to occur. How do neurons born in adulthood reach their final destination? We report here that small elongated cells, born in the ventricular zone adjacent to the lateral ventricle, differentiate into mature neurons 20-40 days later, after migrating over distances of up to 5 mm. Migration rates are highest (28 micron h-1) when young neurons migrate through regions which are rich in radial glia. The adult vertebrate brain offers unique opportunities for studying factors that regulate neuronal migration, pathfinding and differentiation.

Explanation of the embryonic rat superior cervical ganglion initiated a retrograde response in noradrenergic neurons. By the 4th day of explanation, tyrosine hydroxylase activity per cell decreased to 62% of the day 0 control value. The rate of neurite outgrowth during the same period increased by 66%. By the 6th day both tyrosine hydroxylase activity and rate of neurite outgrowth returned to initial values. This inverse relationship is consistent with a proposed reordering of synthetic priorities that occurs in the parental cell body during axonal regeneration.

The development of immunoreactivity (IR) in the nervous system of asexually reproducing Microstomum lineare has been studied by a combination of simultaneous and double immunostaining with antisera to 5-HT and RF-amide, as well as with monoclonal antibodies to SCPB (molluscan small cardioactive peptide). Immunoreactivity appears in a distinct sequential order. 5-HT antigenicity in the postpharyngeal commissure indicates the initiation of the development of a new zooid. The development of a new brain and pharyngeal plexus always starts in connection to the parental nerve cords. Significantly different developmental patterns are observed for the IR to 5-HT and RF-amide, whereas IR to SCPB has the same localization as that to RF-amide, but appears both weaker and later during the development. Influences of the immunoreactive substances on the asexual reproduction and the feeding behaviour are discussed.

Afferent and efferent innervation of the chicken basilar papilla was studied during development. Observations from the 10-day embryonic stage (E 10) and the 8-day post-hatch (P 8), were focussed on some characteristic features of the synaptogenesis. The presynaptic specializations (synaptic bodies: SB) appear early (E 10) in hair cells, opposite afferent dendrites. They are found at all developmental stages in tall hair cells (THC), whereas they become rare in short hair cells (SHC). The maturation of afferent endings, especially those connected to THC, is characterized around the E 15 stage, by an over abundant sprouting and branching. The development of the efferent system indicates two stages. Starting at E 14, some small efferent synapses are seen at the base of the THC and SHC. From E 18, the large synapses extend to the base of SHC. These results are discussed in the light of homologies with the mammalian inner and outer cochlear hair cells.

The capacity of neurons to grow new synapses following partial denervation has been studied in the brain of aged rats and compared to that of younger animals. Lesion induced synapse formation is reduced in aged rats in the hippocampus and septum, two brain areas which show particularly robust growth responses in younger animals. The rate of growth as well as the final magnitude of the response is diminished in aged animals. A possible mechanism for the decreased growth response in aged animals is discussed in light of current models of reactive synaptogenesis. The loss of a compensatory growth response in the aged animal may be one of the factors which contribute to decreased brain plasticity and the slower and poorer recovery from brain damage following injury.

Colchicine, injected into the amniotic cavity of 3 to 10 day-old chick embryos, has the following effects on the developing retina: 1. Cells in arrested metaphase accumulate in the ventricular portion of the matrix region. If colchicine is applied at the end of the first week of incubation, the kinetic migration of the cell nuclei is inhibited. 2. The cells lose their typical slender, bipolar shape to become ovoid to spherical. These morphological changes are more obvious when colchicine acts during the first week of incubation. 3. The formation of temporary gap junctions between the matrix cells of the retina occurs during the first week of development. When colchicine is administered around the 3rd day of incubation, the assembly of intramembranous particles (IMPs) during gap junctions formation is incomplete. These results suggest that an intact microtubular system within the submembranous cytoskeleton is essential for the assembly of these intercellular contacts.

A mouse monoclonal antibody (MAb 6B9, isotype IgM) was raised against autopsy tissue samples from the central nervous system (CNS) of multiple sclerosis (MS) patients. By immunofluorescence microscopy, MAb 6B9 intensely stains most or all cells in fetal rats. However, MAb 6B9 differentially stains various cell types in adult rats. Neurons, ependymal cells, and adrenal chromaffin cells are stained intensely, whereas astrocytes and oligodendrocytes are not stained. The 6B9-reactive antigen (6B9 antigen) is sensitive to periodic acid, but insensitive to treatment with protease, RNase, or hyaluronidase. Results from immunofluorescence microscopy on semithin sections and cultured neuroblastoma cells indicate that 6B9 antigen is intracellular. This is supported by immunoelectron microscopy, where labeling for 6B9 antigen appears in the cytoplasm distinct from any identifiable organelle. Further studies on 6B9 antigen should reveal its chemical nature as well as the significance of developmental changes in its distribution.

When embryonic central nervous system neurons are seeded at low densities with Eagle's basal medium supplemented with the serum substitute N1, glucose, and glutamine, neuronal survival for even 24 h requires the additional supply of exogenous pyruvate--and so does the survival of many peripheral nervous system neurons. Pyruvate can be replaced by alpha-ketoglutarate or oxaloacetate, but not by Krebs cycle substrates that are not keto acids. Most other alpha-keto acids tested (though not beta- or gamma-keto acids) also mimic pyruvate. The apparent equivalence to pyruvate of all these compounds includes identical ED50 values (300 microM for embryonic avian fore-brain neurons, 30-40 microM for rat hippocampal neurons), and also identical susceptibilities to the pyruvate-sparing effects of other low-molecular-weight agents present in Dulbecco's modified Eagle's medium or in astroglia conditioned medium. The substitute alpha-keto acids, however--unlike pyruvate, alpha-ketoglutarate, or oxaloacetate--support cell survival only in the presence of alpha-amino acids that transaminate to alpha-ketoglutarate, oxaloacetate, or pyruvate. The alpha-keto acids, therefore, operate as acceptors of amino groups from appropriate donors to generate Krebs cycle-relevant substrates. Consistent with this view, [14C]glutamate did not generate appreciable 14CO2 unless accompanied by a suitable alpha-keto acid.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for studies of organic solvent effects on the development of neurones and astrocytes is described. Chick embryos in ovo were exposed to a low concentration of carbon tetrachloride vapours. After the exposure, primary cultures of neurones and astrocytes were prepared from the brain hemispheres of the embryos. Toxic effects of the solvent were analyzed in the cultures during the course of cellular differentiation as changes in cell growth and morphology, respiratory activity, and biochemical properties characteristic of each cell type. The development of neurones from embryos exposed from day 5 to day 8 to 25 p.p.m. of carbon tetrachloride was not affected by the exposure. On the other hand, growth rate and respiratory activity of astrocytes from embryos exposed from day 11 to day 14 to 25 p.p.m. of CCl4 were affected by the exposure, whereas no morphological changes or changes in the cyclic AMP levels could be observed. Consequently, low concentrations of carbon tetrachloride seem to affect the development of astrocytes rather than that of neurones in our experimental system.

The influence of the organophosphate, soman (pinacolyl methylphosphonofluoridate), on neuritic growth and substrate utilization by mammalian autonomic neural tissue was studied using explants of the rat superior cervical ganglion as a model. Soman produced a dose-dependent decrease in neuritic outgrowth from explants of this tissue maintained in a serum-free medium. Acetylcholinesterase in the explant as well as in culture media also decreased. In contrast, the effects of soman on explant metabolism were modest. Total protein and DNA content of the tissue was not affected. Only marginal changes in substrate utilization were detected; glucose use was unaltered, lactate production increased 20% with the highest soman tested. Soman increased the content of phosphocreatine in ganglion explants. This increase occurred in the absence of changes in the oxidation-reduction state of NAD calculated from pyruvate/lactate ratios. The results indicated that soman inhibited neuritic outgrowth from explants of the rat superior cervical ganglion in the absence of major effects on substrate utilization by this tissue.

The early development of descending pathways from the brain stem to the spinal cord has been studied in Xenopus laevis tadpoles. The relatively protracted development of this permanently aquatic amphibian as well as its transparency during development make this animal particularly attractive for experimental studies. Between the 5th and 10th myotome the spinal cord was crushed with a thin needle and dry horseradish peroxidase (HRP) crystals were applied. After a survival time of one day the tadpoles were fixed and the brain and spinal cord were stained as a whole according to a modification of the heavy metal intensification of the DAB-reaction, cleared in cedarwood oil and examined as wholemounts. At stage 28 (the neural tube has just closed) the first brain stem neurons projecting to the spinal cord were found in what appear to be the nucleus reticularis inferior and -medius. At this stage of development the first, uncoordinated swimming movements can be observed. At stage 30/31 (the tailbud is visible) both Mauthner cells project to the spinal cord as well as the interstitial nucleus of the fasciculus longitudinalis medialis situated in the mesencephalon. Towards stage 35/36 (the tail is now clearly visible), a more extensive reticulospinal innervation of the spinal cord appears, now including cells of the nucleus reticularis superior. At this stage also the first vestibulospinal and raphespinal projections were found. At stage 43/44 (the tadpoles have now a well-developed tail) the pattern of reticulospinal projections appears to be completed with the presence of labeled neurons in the nucleus reticularis isthmi. From stage 43/44 on, the number of HRP-positive cells is steadily increasing. At stage 47/48, when the hindlimb buds appear, the descending projections to the spinal cord are comparable with the adult situation except for the absence of a rubrospinal and a hypothalamospinal projection. The observations demonstrate that already very early in development reticulospinal fibers and, somewhat later, Mauthner cell axons and vestibulospinal fibers innervate the spinal cord. Furthermore, a caudorostral gradient appears to exist with regard to the development of descending projections to the spinal cord. However, the interstitial nucleus of the fasciculus longitudinalis medialis forms an exception to this rule.

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4-5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1-2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour.(ABSTRACT TRUNCATED AT 250 WORDS)

The postnatal fate of the first-generated neurons of the cat cerebral cortex was examined. These neurons can be identified uniquely by 3H-thymidine exposure during the week preceding the neurogenesis of cortical layer 6. Previous studies in which 3H-thymidine birthdating at embryonic day 27 (E27) was combined with immunohistochemistry have shown that these neurons are present in large numbers during fetal and early postnatal life within the subplate (future white matter), that they are immunoreactive for the neuron-specific protein MAP2 and for the putative neurotransmitters GABA, NPY, SRIF, and CCK. Here, the same techniques were used to follow the postnatal location and disappearance of the early generated subplate neuron population. At birth (P0), subplate neurons showing immunoreactivity for GABA, NPY, SRIF, or CCK are present in large numbers and at high density within the white matter throughout the neocortex, and the entire population can be observed as a dense MAP2-immunoreactive band situated beneath cortical layer 6. Between P0 and P401 (adulthood), the MAP2-immunostained band disappears so that comparatively few MAP2-immunoreactive neurons remain within the white matter. There is a corresponding decrease in the number and density of neurons stained with antibodies against neurotransmitters. In each instance, these neurons could be double-labeled by the administration of 3H-thymidine at E27, indicating that they are the remnants of the early generated subplate neuron population. The major period of decrease occurs during the first 4 postnatal weeks, and adult values are attained by 5 months. Within the white matter of the lateral gyrus (visual cortex), the density of immunostained neurons decreases dramatically: MAP2, 82%, SRIF, 81%, and NPY, 96%. While SRIF-immunoreactive neurons compose a nearly constant percentage of MAP2-immunoreactive neurons in the white matter between P0 (22%) and P401 (23%), those immunoreactive for NPY decline from 18 to 4%. These changes occur during the same period in which there is less than a twofold increase in white matter area. These observations indicate that the interstitial neurons of the adult neocortical white matter are the oldest neurons of the cerebral cortex since most if not all are derived from the subplate neuron population. In addition, a quantitative analysis suggests that the postnatal decline in subplate neuron density cannot be accounted for solely through dilution by differential growth of the white matter and most likely reflects an absolute decrease in subplate neuron number.(ABSTRACT TRUNCATED AT 400 WORDS)

The anlages of the medial-basal hypothalamus (MBH), septopreoptic area (POA), Rathke's pouch, and the parietal cortex (CC) of rats (at 12.5, 14.5 and 16.5 days of gestation) were transplanted singly or in combination into the third ventricle of adult female rats, and the development of neurons in the grafts was investigated immunohistochemically with the use of antisera to tyrosine hydroxylase (TH), somatostatin (SRIH), ACTH, methionine enkephalin-Arg6-Gly7-Leu8 (Enk-8), rat corticotropin-releasing factor (rCRF), rat hypothalamic growth hormone-releasing factor (rhGRF), and luteinizing hormone-releasing hormone (LHRH). TH and all the peptides examined except LHRH were detected in distinct neurons in MBH grafts and in cografts of MBH plus Rathke's pouch from 12.5-day-old embryos. SRIH, rCRF, Enk-8, and TH were found in POA grafts from embryos of the same age. Although immunoreactive LHRH was first detected in neurons in POA grafts from 16.5-day-old embryos, it appeared in cografts of POA and MBH from 12.5-day-old embryos. The immunoreactive fibers developed in the grafts expressed the same characteristic behaviors as in intact brain; the fibers containing hormonal substances formed complexes with the vasculature like in the organum vasculosum laminae terminalis (OVLT) or in the median eminence, while the fibers containing neurotropic signals formed fiber networks surrounding other nerve cell bodies as if they synaptically associate. In CC grafts, the neurons contained TH, SRIH, rCRF, or Enk-8, and their axonal processes formed fiber networks. These findings suggest that all the hypothalamic neurons examined are committed by 12.5 days of gestation to develop maintaining transmitter phenotype and target recognition capacity.

Neurites of superior cervical ganglion neurons from embryonic, perinatal, and adult rats extended at different rates when placed in tissue culture on similar collagen substrata. Using high resolution cinematography and a time-lapse video recording system, we concluded that these differences arise from variations in individual growth cone behavior. Growth cones of embryonic and perinatal neuronal origin exhibited high peak rates of advance and filopodial and lamellipodial excresences. Perinatal cones differed from embryonic ones in that they were somewhat larger, advanced in straighter paths, and retracted less, consequently translocating at 14 to 29 microns/hr compared with 8 to 22 microns/hr for embryonic cones (ranges of 4-hr means). The growth cones of neurons obtained from adult rats had scant cytoplasm and short branched filopodia, lacked definitive lamellipodia, and traversed the terrain at 4 to 13 microns/hr due to lack of high peak rates of advance and more time spent in stationary or minimal advance phases. Periodic pauses lasting 10 to 20 min, occurring every 20 to 90 min, interrupted the forward advance of growth cones of all ages. During pauses or slow forward movement, the growth cone displayed numerous filopodia whereas, during brief episodes when embryonic and perinatal growth cones moved at peak rates of 200 microns/hr or more, the cone periphery was predominantly lamellipodial. We conclude that the predominance of a lamellipodial or filopodial conformation correlates with the rate of growth cone advance and that age-dependent variations in neurite extension rates are related to differences in growth cone form and pattern of translocation. This is the first documentation of differing behavior of single growth cones of neurons of varying developmental ages in culture.

To date, there are no reports of growth of significant numbers of axons into or across a lesion of the mammalian spinal cord. However, recent studies showing that CNS axons will grow into PNS environments indicate that comparable growth into spinal cord lesions could be achieved if ischemic necrosis could be prevented and the lesion site repopulated by astrocytes and ependymal cells rather than by the macrophages, lymphocytes, and fibroblasts that generally accumulate at sites of CNS injury. To examine this possibility, we made a laminectomy at T5 in rats and crushed the spinal cord for 2 s with a smooth forceps (leaving the dura mater intact to prevent ingrowth of connective tissue). At 1 week, the lesion was filled with mononuclear cells, degenerating nerve fibers, and capillaries that were oriented parallel to the long axis of the spinal cord. By 2 weeks, longitudinally oriented cords of ependymal cells and astrocytes had migrated into the lesion from the adjacent spinal cord, and similarly oriented nerve fibers had begun to grow into the lesion along these capillaries and cellular cordons. The mononuclear cells had now assumed phagocytic activity and were engorged with myelin and other cellular debris. After 3 weeks, the astrocytes had elaborated thick cell processes. The nerve fibers in the lesion were still oriented longitudinally but had increased in number and were often arranged in small fascicles. These observations provide the first histological evidence of growth of nerve fibers into a lesion of the rat spinal cord. We conclude that the intrinsic regenerative capacity of the spinal cord can be expressed if ischemic necrosis and collagenous scarring are prevented and the spinal cord parenchyma is first reconstructed by its nonneuronal constituents.

Nucleus laminaris (NL) is a third-order auditory nucleus in the avian brain stem which receives spatially segregated binaural inputs from the second-order nuclei magnocellularis. The examination of the development of dendritic structure in NL revealed a number of events: In the initial period of dendritic growth (E 8--9) there is no gradient of dendritic morphology or apparent size. Starting about E 9--10, there is a spatiotemporal gradient of proliferation of numerous fine dendritic processes, from rostromedial to caudolateral, corresponding with the morphological lamination of NL, and possibly with the onset of cell death. This is followed by a spatiotemporal gradient of the elimination of the overproliferated processes, from rostromedial to caudolateral possibly coinciding with the cessation of cell death. A result of the spatiotemporal gradients of dendritic process proliferation and elimination is a spatial gradient in the morphology (extension, branching) of the remaining "mature" dendrites. At E 15 there is only a slight spatial gradient of total dendritic size across NL; this gradient is larger at E 19, and by P 25 there is a 13-fold change in dendritic size from rostromedial to caudolateral. Regression analyses suggest the size gradient begins to form when NL activity becomes driven by cochlear activity, at about E 14. The progressive formation of the size gradient is largely the result of two factors: the growth of dendritic trees, and the loss or primary dendrites. The growth rate of the dendritic trees of NL cells was found to be very highly correlated with the intensities of the sound frequencies to which the cells respond. From E 15 to P 25 there is a 50% loss of the "mature" primary dendrites of NL neurons. The separate dorsal and ventral dendritic size gradients seen at E 15 realign to coincide at E 19, and the moderate correlation of dorsal and ventral dendritic sizes seen at E 15 and E 19 is significantly increased at P 25, indicating a developmental process of sharpening in the relationship of the dorsal and ventral dendritic organizations in the nucleus. The data suggest that a key element in the regulation of dendritic size and structure in n. laminaris may be the activity of the afferents to the cells.

The growth of axons in the developing nervous system depends on the elongation of the microtubules that form their principal longitudinal structural element. It is not known whether individual microtubules in the axon elongate at their proximal ends, close to the cell body, and then move forward into the lengthening axon, or whether tubulin subunits are transported to the tip of the axon and assembled there onto the free ends of microtubules. The former possibility is supported by studies of slow axonal transport in mature nerves from which it has been deduced that microtubule assembly occurs principally at the neuronal cell body. By contrast, the polarity of microtubules in axons, which have their 'plus' or 'fast-growing' ends distal to the cell body, suggests that assembly occurs at the growing tip, or growth cone, of the axon. We have addressed this question by topically applying Colcemid (N-desacetyl-N-methylcolchicine), and other drugs which alter microtubule stability, to different regions of isolated nerve cells growing in tissue culture. We find that the sensitivity to these drugs is greatest at the growth cone by at least two orders of magnitude, suggesting that this is a major site of microtubule assembly during axonal growth.

Neural and non-neural cellular processes have been studied in organotypic cultures of spinal cord and olfactory bulb by means of the freeze-fracturing technique. Identification of specific cellular elements in replicas has been achieved by comparison with thin-sectioned material in which differences in shape and contents are evident. Freeze-fracturing reveals that neural growth cones may be distinguished from glial pseudopodia by the low number of intramembranous particles within their plasma membrane; the counts of particles within the growth cone membrane average 85/microm(2) (for the inner leaflet) as opposed to hundreds per square micrometer in glial pseudopodia. Whereas the intramembranous particle number in glial pseudopodia is only slightly lower than in their perikaryal plasmalemma, the number of particles in outgrowing axons increases about eightfold from the periphery towards the perikaryon. Furthermore, with prolonged time of growth in culture, the particle density in the young nerve fibers increases by about the same factor. The same phenomenon, i.e. a low intramembranous particle level at earlier stages and an increase in numbers as the nerve fiber matures, is observed in fetal nerve tissue in vivo. These findings suggest that the plasmalemma of the outgrowing nerve, and especially of the growth cone, is immature and that maturation is accompanied by the insertion of intramembranous particles. Furthermore, these data indicate that the chemistry of the growth cone membrane is distinct from that of the neuron soma which may be significant for the mechanisms of guidance and recognition in the growing nerve tip.

Nerve growth factor (NGF)-untreated (naive) and neurite-bearing NGF-treated ("primed") PC12 rat pheochromocytoma cells were used as model system to study the role of phospholipid methylation in the NGF mechanism of action. The neurite-bearing cultures were deprived of NGF for 3 h before experimentation. Under both experimental conditions, the cells were labelled with [methyl-3H]methionine and then challenged with NGF for time periods ranging from 5 s to 30 min. Methylated phospholipids were extracted and then resolved and identified by TLC as phosphatidyl mono-, di-, and trimethyl ethanolamine. Quantification of the amount of radioactivity incorporated into each of the phospholipids indicated that NGF does not significantly alter phospholipid methylation either in naive or in neurite-bearing cells. Furthermore, using a methyltransferase inhibitor, it was found that neurite outgrowth still occurs when phospholipid methylation is almost completely blocked. These results indicate that phospholipid methylation does not play a primary role in the mechanism of action of NGF.

In many cases, in a fixed neuronal circuit, the complete morphological and biochemical development of a neuron depends on the impulses it receives from the neuron before. The possible involvement of this phenomenon to learning is studied in this work.

One of the dogmas of neurobiology has it that when nerve cells in the vertebrate brain die, they are not replaced by new ones. The author finds to the contrary. He has shown that when the adult canary needs to learn new songs, it does grow some new neurons. The finding could eventually lead to the discovery of ways to repair lesions in the human brain.

Action potentials of embryonic nerve and muscle cells often have a different ionic dependence and longer duration than those of mature cells. The action potential of spinal cord neurons from Xenopus laevis exhibits a prominent calcium component at early stages of development that diminishes with age as the impulse becomes principally sodium dependent. Whole-cell voltage-clamp analysis has been undertaken to characterize the changes in membrane currents during development of these neurons in culture. Four voltage-dependent currents of cells were identified and examined during the first day in vitro, when most of the change in the action potential occurs. There are no changes in the peak density of the calcium current (ICa), its voltage dependence, or time to half-maximal activation; a small increase in inactivation is apparent. The major change in sodium current (INa) is a 2-fold increase in its density. In addition, more subtle changes in the kinetics of the macroscopic sodium current were noted. The peak density of voltage-dependent potassium current (IKv) increases 3-fold, and this current becomes activated almost twice as fast. No changes were noted in the extent of its inactivation. The calcium-dependent potassium current (IKc) consists of an inactivating and a sustained component. The former increases 2-fold in peak current density, and the latter increases similarly at less depolarized voltages. The changes in these currents contribute to the decrease in duration and the change in ionic dependence of the impulse.

The development of a population of cerebrospinal-fluid-contacting neurons in the spinal cord of the Xenopus embryo ('Kolmer-Agduhr' cells) has been followed by using an immunocytochemical procedure that identifies GABA in fixed nervous tissue. Stained Kolmer-Agduhr cells containing GABA first appeared at stage 25 and their numbers increased steadily with the developmental age of the embryo. The Kolmer-Agduhr neurons had ascending ipsilateral axons that often terminated in growth cones. These axons and growth cones could be stained by the GABA antiserum from the earliest stages of outgrowth from the Kolmer-Agduhr cell body. We measured the angle of the earliest axons' outgrowth relative to the rostrocaudal axis of the spinal cord. The initial outgrowth of axons was always rostral over a narrow range of angles. This observation is inconsistent with the hypothesis of random initial outgrowth followed by later selection of the correct orientation, which would predict that axons would initially grow out over a wide range of angles. Instead, it suggests that, even from the earliest moments, axon outgrowth from the Kolmer-Agduhr cells is directed rostrally in a specific stereotyped manner.

The motile activity of growth cones of specific identified neurons is inhibited by the neurotransmitter serotonin, although other identified neurons are unaffected. As a consequence, affected neurons are unable to form electrical synapses, whereas other neurons whose growth is unaffected can still interconnect. This result demonstrates that neurotransmitters can play a prominent role in regulating neuronal architecture and connectivity in addition to their classical role in neurotransmission.

Granule cells of the rat dentate gyrus which are denervated by unilateral destruction of the entorhinal cortex are reinnervated in part by proliferation of surviving pathways from the contralateral entorhinal cortex. The cells of origin of these lesion-induced projections were identified by retrograde labeling with horseradish peroxidase and were the same cell type which normally project to the ipsilateral dentate gyrus

The ontogenesis of the synaptic function was investigated in the central noradrenaline (NA), dopamine (DA), serotonin (5-HT), acetylcholine (ACh) and GABA nervous systems of developing rats. Monoamines histochemically first appeared on days 12 to 14 in the fetal brain and may exert a trophic action on target neurons as a "differentiation signal". The presynaptic functions such as the high affinity uptake and depolarization-induced, Ca2+-dependent release of L-[3H]DA, and [2H]DA, and [3H]DA, and [3H]5-HT were observed in synaptosomes and slices from the fetus (18 days of gestation) and brain of newborn rats. Monoamine-stimulated activity of adenylate cyclase and specific binding of ligands in NA(alpha 1, alpha 2, beta 1, beta 2), DA, 5-HT, ACh (muscarinic and nicotinic) and GABA receptors indicated dynamic changes through postnatal development. Behavioral findings suggest when neurotransmitter receptors become sensitive and reach functional maturity (NA and DA, already at birth; 5-HT and muscarinic ACh, 15 to 20 days; GABA, 12 to 13 days (mouse)). In addition, differences in behavioral responsiveness to drugs were observed in rats at various developing stages, probably due to the interaction between plural neuronal systems. Finally, the brain undergoing rapid differentiation seems to be most sensitive to hormones and centrally acting drugs. Thus, permanent alteration in central functions may occur when some classes of drugs, dose-dependently, are administrated to animals at the perinatal stage.

We studied (a) the distribution and properties of fast and slow 125I-nerve growth factor (125I-NGF) binding sites in cultured human neuroblastoma (NB) cell lines that were categorized as responsive (N+) or unresponsive (N-) to NGF by neurite outgrowth, (b) whether fast or slow sites mediate actions of NGF, and (c) whether NGF-mediated conversion of fast to slow sites occurs in human NB and pheochromocytoma PC 12 cells. In human NB SH-SY5Y cells, the slow sites were trypsin resistant and binding was of high affinity. Loss of binding to the slow sites had a half-time of 25 to 30 min at 37 degrees C and was very slow at 4 degrees C. In contrast, the fast sites were trypsin sensitive and binding was of lower affinity; its dissociation half-time was less than 1 min at 4 degrees C and 37 degrees C. The association rate constants of both sites were about 0.8 to 1.2 X 10(7) M-1 sec-1. Some human NB cells had both fast and slow sites. The N+ human NB lines SH-SY5Y and LA-N-5 had only slow sites. Despite the virtual elimination of fast sites by trypsin in NB MC-IXC cells, remaining slow sites could still efficiently bind 125I-NGF. These observations showed that fast sites are not required for slow site binding, neurite outgrowth, or other demonstrated actions of NGF in some NB cells. In PC 12 cells, 125I-NGF initially bound to fast sites was not directly transferred to slow sites as required for NGF-mediated conversion. The association rate constants of fast and slow sites in PC12 cells were both about 2 X 10(7) M-1 sec-1. The association kinetics were consistent with simple bimolecular reactions in both NB and PC12 cells. The combined evidence in NB and PC12 cells did not support the hypothesis of NGF-mediated conversion of fast to slow sites.

Rat cortical explants were cultured in the presence or absence of the mitotic inhibitor cytosine arabinoside to determine whether or not it affects the composition of the nonneuronal cell population within the outgrowth zone. Ultrastructural morphometric analysis of the incidence of fibroblasts, fibrous astrocytes, and protoplasmic astrocytes-epithelial cells at 18 days in vitro, revealed statistically significant decreases in the incidence of fibroblasts and fibrous astrocytes in the explants treated with the inhibitor compared with control explants. Coupled with earlier findings of enhanced neurite outgrowth and decreased nonneuronal cell proliferation that follows such treatment, it appears that cytosine arabinoside may potentiate neurite outgrowth by altering the composition, as well as the number, of nonneuronal cells in the outgrowth zone. These data indicate that fibroblasts and fibrous astrocytes may limit the regenerative response of severed axons in the mammalian central nervous system.

Growth-associated protein, GAP-43, is a polypeptide that is induced in neurons when they grow axons. We show by means of subcellular fractionation and immunohistochemical localization that GAP-43 is a component of neuronal growth cones as well as growing neurites; it is similar to a major phosphoprotein, pp46, of a growth cone-enriched subcellular fraction. These conclusions are consistent with the possibility that the induction of GAP-43/pp46 is an important event in the establishment of a productive growth state in which a neuron is competent to extend an axon.

To determine whether production of new neurons of a particular type is regulated by the presence of previously differentiated neurons of the same type, we ablated all tyrosine hydroxylase immunoreactive (THIR) cells from larval frog retina with the neurotoxin 6-hydroxydopamine, and examined the retinas in subsequent weeks for newly generated THIR neurons. Three weeks after neurotoxin administration, new THIR cells appeared near the zone of neural proliferation at the ciliary margin at a higher density than that of normal retina, while the densities of other amacrine cell types, serotonin (t-HT) immunoreactive and substance P immunoreactive (SPIR), remained the same as controls. Thus the production of new retinal TRIR cells is selectively up-regulated following ablation of previously differentiated cells of this type.