In Vivo Expression of Inducible Nitric Oxide Synthase in Cerebellar Neurons

Abstract: In the CNS, nitric oxide (NO) functions as both neuromodulator and neurotoxic agent. In vivo neuronal expression of NO synthase (NOS) has been attributed to constitutive NOS—both the neuronal and the endothelial types. The other class of NOS—the inducible NOS (iNOS)—is known to mediate toxic effects of NO in various tissues. In this study, we show for the first time that direct intracerebellar injection of endotoxin and cytokine (lipopolysaccharide and interferon-γ) induced in vivo neuronal expression of the iNOS gene, as demonstrated by fluorescent in situ hybridization and immunohistochemical staining analyzed by confocal laser-scanning microscopy. This raises the possibility that neuronal iNOS might contribute significantly to the vulnerability of the brain to various insults.     

Ras-Regulated Hypophosphorylation of the Retinoblastoma Protein Mediates Neuronal Differentiation in PC12 Cells

Abstract: To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12- O -tetradecanoylphorbol 13-acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin-6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa-Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras-dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of PC12 cells.     

Consequences of food-attraction conditioning in Helix: a behavioral and electrophysiological study

Food-attraction conditioning is a learning phenomenon by which adult Helix pomatia acquire the ability to locate food through exposure to that particular food. Food-conditioned snails can be distinguished from naive snails during their approach to food. Naive snails keep their tentacles upright — whereas food-conditioned animals bend the tentacles down-ward, in a horizontal orientation, pointed in the direction of the food.Tentacle musculature is innervated by two peritentacular nerves (PTn), each projecting to approximately one hemi-section of the tentacle wall. Stimulating the peritentacular nerves caused the tentacles to bend downward in a manner reflecting the full complement of tentacle movements performed by conditioned snails.The neural correlate of tentacle movements was investigated in isolated ganglion preparations with the posterior tentacles attached. PT nerve activity was recorded while the olfactory epithelia were stimulated with natural food odors. Preparations obtained from conditioned animals responded with a substantial increase in unit activity (mean increase 280%) to stimulation with odor of the conditioned food but not to other odors. Preparations from naive animals did not respond to food odor stimulation. The electrophysiological results demonstrated that plasticity due to conditioning the snails in vivo survived dissection and could be monitored in vitro.Abbreviations ext PTn external peritentacular nerve - int PTn internal peritentacular nerve     

Growth of Ectopic Dendrites on Cortical Pyramidal Neurons in Neuronal Storage Diseases Correlates with Abnormal Accumulation of GM2 Ganglioside

Abstract: Ganglioside analysis and quantitative Golgi studies of the cerebral cortex of cats with ganglioside and nonganglioside lysosomal storage diseases reveal a correlation between the amount of accumulated GM2 ganglioside and the extent of ectopic dendrite growth on cortical pyramidal neurons. This correlation was not observed with any of the other gangliosides assayed for, including GM1 ganglioside. These results suggest a specific role for GM2 ganglioside in the initiation of ectopic neurites on pyramidal cells in vivo and are consistent with the developing hypothesis that different gangliosides have specific roles in different cell types dependent upon the receptor or other effector molecules with which they may interact.     

Cell Cycle Arrest of Proliferating Neuronal Cells by Serum Deprivation Can Result in Either Apoptosis or Differentiation

Abstract: Apoptotic cell death plays a critical role in the development of the nervous system. The death of mature nondividing neurons that fail to receive appropriate input from the target field has been extensively studied. However, the mechanisms mediating the extensive cell death occurring in areas of the developing brain where proliferating neuroblasts differentiate into mature nondividing neurons have not been analyzed. We show here that the cell cycle arrest of a proliferating cell of neuronal origin by removal of serum results in either apoptotic cell death or differentiation to a mature nondividing neuronal cell. The proportion of cells undergoing death or differentiation is influenced in opposite directions by treatment of the cells with cyclic AMP and retinoic acid. This suggests that following the withdrawal of signals stimulating neuroblast cell division, neuronal cells either can cease to suppress a constitutive suicide pathway and hence die by apoptosis or, alternatively, can differentiate into a mature neuronal cell. Regulation of the balance between apoptosis and neuronal differentiation could therefore play a critical role in controlling the numbers of mature neurons that form.     

1H Nuclear Magnetic Resonance Spectroscopy Study of Cerebral Glutamate in an Ex Vivo Brain Preparation of Guinea Pig

Abstract: Cerebral glutamate was monitored in a superfused cerebral cortical preparation by ¹H NMR spectroscopy using a semiselective spin-echo sequence N -acetyl aspartate (NAA) as an internal concentration reference. During controlled metabolic conditions, the cerebral ¹H NMR-detected glutamate-to-NAA ratio was ∼ 20–30% lower than expected from the ratio of neutralized perchloric acid extracts of the preparations. Inhibition of respiration in the presence of glucose did not change the ¹H NMR glutamate-to-NAA ratio in brain slice preparation. In contrast, either complete depletion of ATP during cyanide poisoning together with 0 m M glucose, anoxia in the absence of glucose, or treatment with nigericin or with a protonophore, carbonyl cyanide- m -fluorophenylhydrazone, increased ¹H NMR-detected glutamate/NAA in the cerebral preparations without a change in the relative and absolute concentration ratios determined from the tissue acid extracts. Spin-spin relaxation times of glutamate and NAA peaks in anoxic slices were 749 ± 89 and 729 ± 94 ms, respectively, and thus, the portion of glutamate that could not be detected by ¹H NMR was quantified in absolute terms. It was calculated that an increase in the glutamate-to-NAA ratio from 0.55 ± 0.02 to 0.67 ± 0.02 during aglycemic anoxia corresponded to some 6 mmol/kg of tissue dry weight of glutamate from the total concentration of 28 mmol/kg dry weight. It is suggested that this 22% of total glutamate pool is present in a noncytoplasmic compartment during controlled metabolic state.     

Neuronal types in the spinal dorsal gray of the turtle Chrysemys d'orbigny: a Golgi study

At thoracic and lumbar levels the spinal dorsal gray of young specimens of the turtle Chrysemys d'orbigny consists of a cell-free neuropil and an aggregation of perikarya termed here the lateral column of the dorsal horn (LCDH). Nerve cell clusters also occur in the dorsal commissure. The main neuropil area can be divided into a thin superficial layer containing some myelinated fibers (neuropil area Ib) and a compact core composed of unmyelinated axon terminals, dendritic branches, and thin glial processes (neuropil area II). A looser neuropil area is located at the horn base (neuropil area III). The so-called marginal zone of de Lange represents a fourth synaptic field termed here neuropil area Ia. The LCDH consists of neurons of different size and shape. Two peculiar nerve cell types have been recognized in the dorsal horn: giant and bitufted neurons. The former exhibits a large dendritic arbor, which after passing through neuropil areas II and Ib projects into neuropil area Ia and the adjacent white matter. Most frequently Golgi-stained giant neurons have perikarya and dendritic domains on the same side (ipsilateral giant neurons). There are also heterolateral giant neurons whose dendritic branches invade the opposite horn. Bitufted neurons are characterized by the presence of two main dendritic shafts connecting neuropil area II of both dorsal horns. At neuropil levels the major dendritic branches ramify profusely giving rise to short tortuous terminal processes. Perikarya of bitufted neurons occur in the dorsal commissure. The LCDH also contains many small and medium-sized neurons. These are oriented in two main directions: parallel or radial with respect to the dorsal horn surface. The population of horizontally oriented neurons comprises two subtypes termed here alpha and beta. Radially oriented neurons are pleomorphic, defying precise, unequivocal classification.     

Laminin and Neuropeptide Y Are Increased by Synapsin Transfection in Cultured NG108-15 Neuroblastoma/Glioma Hybrid Cells

Abstract: We have investigated the presence and expression of laminin and neuropeptide Y (NPY) in several NG108-15 cell lines transfected with synapsin Ib, IIa, or IIb. The content of laminin, a basal membrane glycoprotein that promotes adhesion and induces neurite outgrowth and neuronal differentiation, was increased in all transfected cell lines examined. In cells that were chemically differentiated with prostaglandin E₁ plus 3-isobutyl-1-methylxanthine, laminin levels were increased even further. The content of NPY, suggested to be a neurotransmitter/neuromodulator in peripheral sympathetic neurons as well as in central neurons, was also increased in all transfected cell lines examined. Immunohistochemical analysis combined with confocal laser microscopy showed that NPY staining was granular and very often enriched in neuritic varicosities. The distribution and the staining pattern of NPY were consistent with storage of NPY in large dense-cored vesicles. The results indicate that, in differentiated neurons, the synapsins increase the levels of a neuropeptide transmitter stored in large dense-cored vesicles and of an extracellular matrix protein associated with neuronal maturation.