Developmental expression of voltage-gated potassium channel beta subunits.

Expression of potassium channel beta subunits (Kvbeta) was determined in the developing mouse CNS using an antiserum against an amino acid sequence present in the C-terminus of Kvbeta1, Kvbeta2, and Kvbeta3. Using the anti-Kvbeta antiserum, we determined that Kvbeta expression is restricted to the spinal cord and dorsal root ganglia in the embryonic CNS. At birth, Kvbeta expression is detected in brainstem and midbrain nuclei, but was not detected in the hippocampus, cerebellum or cerebral cortex. During the first postnatal week, Kvbeta expression is present in hippocampal and cortical pyramidal cells and in cerebellar Purkinje cells. Expression of Kvbeta subunits reaches adult levels by the third postnatal week in all of the brain regions examined. A rabbit antiserum directed against a unique peptide sequence in the N-terminus of the Kvbeta1 protein demonstrates that this subunit displays a novel expression pattern in the developing mouse brain. Kvbeta1 expression is high at birth in all brain regions examined and decreases with age. In contrast, Kvbeta2 expression is low at birth and increases with age to reach adult levels by the third postnatal week. These findings support the notion that the differential regulation of distinct potassium channel beta subunits, in the developing mouse nervous system, may confer the functional diversity required to mediate both neuronal survival and maturation.

Neuronal death in cytokine-activated primary human brain cell culture: role of tumor necrosis factor-alpha.

We examined cytokine-mediated neuronal death in neuron-astrocyte cultures from second trimester human fetal cerebrum. In these cultures, high-output inducible nitric oxide synthase (NOS) and tumor necrosis factor-alpha (TNFalpha) are expressed in astrocytes after exposure to IL-1beta/IFNgamma. Neuronal cell death was evident at >/=48 h following cytokine stimulation. Neutralizing anti-TNFalpha antiserum inhibited ( approximately 48%) neurotoxicity in IL-1beta/IFNgamma-treated cultures, demonstrating a role for endogenously produced TNFalpha. Interestingly, the degree of neuroprotection conferred by superoxide dismutase or N-methyl D-aspartate (NMDA) receptor antagonists in these cultures was smaller and variable. Similarly, the effect of the NOS inhibitor, N(G)-monomethyl L-arginine (NMMA) on IL-1beta/IFNgamma-induced neuronal death was variable, showing no statistically significant effect when results from more than 30 independent cultures were averaged. Neurons die by apoptosis in cytokine-treated human fetal CNS cultures as shown by the characteristic nuclear morphology as well as positive labeling for TUNEL. Our results demonstrate a potent neurotoxicity mediated by the cytokine combination IL-1beta/IFNgamma in primary human neuron-astrocyte cultures and a crucial role for endogenous TNFalpha in mediating neurotoxicity in this system. These results firmly establish the neurotoxic potential of the inflammatory cytokines IL-1beta and TNFalpha in the human CNS.

Neuronal nitric oxide synthase expression in developing and adult human CNS.

Neuronal nitric oxide synthase (nNOS) is constitutively expressed by subpopulations of neurons in the CNS and is involved in neurotransmission, learning and memory, and neuronal injury. While the distribution of nNOS neurons has been characterized in the rodent CNS, the expression in human brain has not been well documented. We determined the expression of nNOS in second trimester human fetal and adult brain. In second trimester fetal brain, the nNOS neurons are concentrated in the developing cerebral cortex at the subplate zone and in layer VI, the striatum, and in certain brainstem nuclei. The nNOS neurons are sparsely distributed in the hippocampus, and virtually absent in the cerebellar cortex. The nNOS neurons in the subplate zone extend their processes radially, suggesting a developmental role, perhaps in guidance. The number and distribution of NADPH diaphorase-positive neurons corresponds to that of the nNOS neurons. While the distribution of nNOS neurons in the adult brain is similar to that found in fetal brain, the overall density is lower in the adult. The highest density of nNOS neurons is found in the striatum followed by the neocortex. A region-specific role for nNOS neurons in human brain and a potential developmental role for nNOS in the cerebral cortex are suggested by these data.

Beta-adrenergic and fibroblast growth factor receptors induce neuronal process outgrowth through different mechanisms.

The mechanisms that initiate and direct neuronal process formation remain poorly understood. We have recently described a neuronal progenitor cell line, AS583-8.E4.22 (AS583-8) which undergoes neurite formation in response to beta2-adrenergic and basic fibroblast growth factor (bFGF) receptor activation [Kwon, J.H. et al., (1996) Eur. J. Neurosci., 8, 2042-2055]. In the present study, a comparison of these responses revealed that isoproterenol (ISO), a beta-adrenergic receptor agonist, induces multiple, highly branched processes within 30 min while bFGF induces fewer, unbranched processes within 24 h. In contrast to the ISO response, bFGF induces mitogen-activated protein kinase activation and c-fos expression in the cell line and results in neurite outgrowth that is dependent on new mRNA and protein synthesis. Two-dimensional isoelectric focusing-sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cytoskeletal preparations revealed different patterns following ISO vs. bFGF exposure suggesting selective changes in protein expression and/or post-translational modifications. Immunoblot analysis of these preparations for beta-tubulin, tyrosinated alpha-tubulin and acetylated alpha-tubulin also revealed different patterns following each type of treatment. Follow-up confocal microscopy revealed that following ISO, the distribution of tyrosinated tubulin extends to the distal ends of processes whereas acetylated alpha-tubulin is diminished within distal ends. This pattern has been reported to be associated with enhanced microtubule dynamics, a state in which process outgrowth is facilitated. In contrast, following bFGF treatment the distributions of tyrosinated and acetylated alpha-tubulin were identical, a state associated with a diminution of microtubule dynamics. These results, a different time course of neurite formation, dependency on new gene expression and differential expression and cellular distribution of major cytoskeleton proteins suggest that neurite outgrowth induced by ISO vs. bFGF is mediated by two distinct intracellular effector mechanisms in AS583-8 cells. In addition, studies, using the differential distribution of post-translational modified alpha-tubulins in neurites of primary neuronal cultures as marker for the two distinct processes of neurite formation suggest, that similar mechanisms are present in vivo. Therefore, the AS583-8 cell line provides a useful model to study these signalling mechanisms that couple neurotransmitter and growth factor receptor activation to the cytoskeletal changes that mediate neurite formation.

Beta-adrenergic receptor activation promotes process outgrowth in an embryonic rat basal forebrain cell line and in primary neurons.

A clonal cell line, AS583-8.E4.22, from the embryonic day 15 rat basal forebrain was established using retrovirus-mediated transduction of a temperature-sensitive mutant of the simian virus 40 (SV40) large tumour antigen. The cell line expresses cytoskeletal and neurotransmitter features indicative of neuronal commitment. In response to agents that increase intracellular cAMP, including forskolin and catecholamines, the cell line exhibits rapid process outgrowth and growth cone formation that does not require new gene expression or protein synthesis. The neurite outgrowth induced by catecholamines is mediated by beta 2-adrenergic receptors and is characterized by a rapid, reversible redistribution of filamentous actin. Neurons from primary cultures of embryonic day 15 basal forebrain were also found to respond to beta-adrenergic receptor agonists by enhancing growth cone formation. These results suggest that catecholamines provide cues that induce cytoskeletal rearrangements leading to neuronal process outgrowth and growth cone formation in the developing basal forebrain and possibly other neuronal progenitor cell populations. The neuronal basal forebrain cell line provides an ideal model to study the signalling mechanisms underlying the catecholamine-induced process outgrowth.

BDNF-activated signal transduction in rat cortical glial cells.

Cortical glial cells in culture were found to be responsive to the neurotrophin brain-derived neurotrophic factor (BDNF), as evidenced by activation of multiple signal transduction processes. BDNF produced an increase in mitogen-activated protein (MAP) kinase tyrosine phosphorylation, MAP kinase activity, intracellular calcium concentration and c-fos expression in the glial cells. Only a subset of the glial cells responded to BDNF, as reflected in single-cell analysis of calcium transients and c-fos expression. BDNF had no detectable effect on glial mitotic activity, as measured by DNA synthesis. In parallel studies, nerve growth factor and neurotrophin-3 had no effect on signalling in these cultures. BDNF has previously been demonstrated to act via trkB receptors with a cytoplasmic tyrosine kinase domain (gp145trkB). Pretreatment of glial cultures with K252a, which at low concentrations specifically inhibits the trk tyrosine kinases, abolished BDNF effects on MAP kinase stimulation, suggesting that BDNF was acting through gp145trkB. However, subsequent studies showed that gp145trkB was expressed at extremely low levels in the cultures: gp145trkB mRNA transcripts could only be detected using the reverse transcription-polymerase chain reaction, and gp145trkB protein was not detected by either immunoblotting or immunocytochemistry. On the other hand, the glia expressed significantly higher levels of gp95trkB mRNA and protein, which represent truncated forms of trkB receptors lacking the tyrosine kinase domain. The results of these studies demonstrate that a subset of cultured CNS glia respond to BDNF with the activation of conventional signal transduction processes. The mechanism of BDNF-initiated signal transduction in glial cells most likely involves a relatively small number of gp145trkB receptors, but involvement of the more abundant truncated gp95trkB receptors cannot be excluded.

Conditional immortalization of neuronal cells from postmitotic cultures and adult CNS.

To determine whether postmitotic neurons can be immortalized by oncogenic transduction, we used two approaches involving conditional expression of a temperature-sensitive SV40 large T antigen (Tts). Initially, Tts was introduced into E17 rat embryonal hippocampal cells that were then cultured at the non-permissive temperature to enrich for postmitotic pyramidal neurons, and subsequently cloned at the permissive temperature. One clonal line (HMR10-3) expressed neuron-specific proteins upon differentiation, was capable of generating action potentials, and formed synapses with primary rat neurons in co-culture. Replating of these postmitotic cells at the permissive temperature resulted in reversible loss of neurofilament expression. Conditionally immortalized cell lines were also generated from the brain of an adult mouse carrying an inducible Tts transgene. These lines proliferated in a T antigen-dependent manner and expressed neuron-specific proteins upon differentiation at the non-permissive temperature. These results suggest that postmitotic neurons can be induced to enter the cell cycle without losing their commitment to a neuronal lineage.

Early nerve growth factor-induced events in developing rat septal neurons.

A culture system enriched for nerve growth factor (NGF) receptor bearing cells was developed to investigate signal transduction events activated by NGF in postmitotic central nervous system neurons. Cells from the septal region of embryonic rats at 16 days of gestation were grown on glass coverslips above a glial cell layer established from postnatal rat cortex. The separation of glial and neuronal planes in this "bilaminar" system permits the diffusion of glial-derived factors required by septal neurons for survival yet allows the investigation of NGF responses in a pure neuronal population. Approximately 15% of the neurons in this culture system were immunoreactive for the low affinity NGF receptor. NGF rapidly increased MAP kinase activity (2-5 min) and transiently induced expression of c-fos in septal neurons. NGF treatment also increased choline acetyltransferase activity, while the number of cholinergic neurons remained constant. Septal neuron survival depended on the presence of glial cells, but neuronal viability in the bilaminar system was unaffected by anti-NGF antiserum, indicating that glial-derived neurotrophic support is not mediated by NGF alone. These data suggest that the bilaminar culture system is a useful system for the study of early events in NGF-activated signal transduction and the nature of glial-derived trophic support of developing basal forebrain neurons.

Expression of neurotrophins and the low-affinity NGF receptor in septal and hippocampal reaggregate cultures: local physiologic effects of NGF synthesized in the septal region.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of a family of trophic factors designated the neurotrophins, each of which can bind to the low-affinity NGF receptor (LNGFR). To investigate the mechanisms that regulate the expression of the neurotrophins and the LNGFR in the developing brain, we grew cells from the embryonic mouse septum and hippocampus in reaggregating cell culture and compared neurotrophin and LNGFR expression in developing reaggregates with that seen in the developing septum and hippocampus in situ. NGF, BDNF, NT-3 and LNGFR were each expressed in septal and hippocampal reaggregates as well as the native septum and hippocampus. Additionally, the temporal expression profiles observed in reaggregates were generally similar to those seen in the respective brain regions in situ. In order to determine whether NGF can modulate neurotrophin or LNGFR expression, reaggregates were cultured in the continual presence of either exogenous NGF or anti-NGF antibodies. NGF-treated septal cultures expressed twice the level of LNGFR mRNA as was seen in untreated septal cultures; on the other hand, septal cultures grown in the presence of anti-NGF antibodies, to neutralize endogenously synthesized NGF, displayed a 3-fold decrease in LNGFR mRNA expression compared to untreated cultures. No effects of NGF or anti-NGF were observed on LNGFR expression in hippocampal reaggregates, or on neurotrophin mRNA expression in either reaggregate type. These results suggest that regulatory mechanisms intrinsic to the septal and hippocampal regions control neurotrophin and LNGFR expression. NGF is likely to be one of these regulatory cues since it acts locally in septal reaggregates to control the developmental expression of LNGFR mRNA. The possible roles of locally synthesized NGF and other neurotrophins in the development of septal neurons are discussed.

Immortal rat hippocampal cell lines exhibit neuronal and glial lineages and neurotrophin gene expression.

Clonal cell lines of rat embryonic hippocampal origin have been developed by using retroviral transduction of temperature-sensitive simian virus 40 large tumor antigens. The cell lines undergo morphological differentiation at the nonpermissive temperature and in response to differentiating agents. Immunocytochemical analysis indicates that various lines are derived from progenitors of neuronal, glial, and bipotential lineages. Selected neuronal lines differentiate in response to diffusible factors released by primary glia, and one line of glial lineage supports the maturation of primary neurons in culture. Selected cell lines exhibit different patterns of neurotrophin gene expression that change after differentiation. In some lines, the relative levels of neurotrophin 3 and brain-derived neurotrophic factor message expression may reflect the developmental or regional differential expression seen for these genes in the hippocampus in situ. These hippocampal cell lines, which express markers indicative of commitment to neuronal or glial lineages, are valuable for studies of development and plasticity in these lineages, as well as for studies of the regulation of neural trophic interactions.

Nucleus basalis lesions decrease alpha- and kappa-bungarotoxins binding in rat cortex.

A unilateral ibotenic acid lesion of the nucleus basalis magnocellularis in the rat, which is known to produce a reduction in cortical choline acetyltransferase activity and acetylcholine release, produces a decrease of 125I-alpha-bungarotoxin and 125I-kappa-bungarotoxin binding sites in the frontoparietal cortex of the lesioned hemisphere. This decrease can be observed at two weeks following the lesion and persists for up to twelve weeks. The results suggest that a population of bungarotoxin binding sites may have a presynaptic localization.

Pharmacokinetics and pharmacodynamics of physostigmine after intravenous administration in beagle dogs.

The time course of physostigmine (Phy), its metabolites and activity of cholinesterase (ChE) in plasma were studied after intravenous bolus administration of [3H]Phy (100 micrograms/kg) to beagle dogs. The maximal inhibition of ChE (78%) in plasma at 2 min correlated with the largest concentration of physostigmine (124 ng/ml). The concentration of physostigmine decreased by 88% to 16 ng/ml at 45 min when the activity of ChE was still 59% inhibited. Acetylcholinesterase activity in four regions of the brain (medulla, striatum, cerebellum and cortex) was not significantly different from controls at 70 +/- 5 min after administration of physostigmine. Concentrations of physostigmine and its metabolites determined by HPLC were not significantly different in different regions. In plasma, physostigmine was found, together with eseroline and two other metabolites M1 and M2. At 45 min, only 18% of total radioactivity was due to physostigmine and 52% was due to the major metabolite M1. On the contrary, in regions of the brain, metabolite M1 represented only 1.9-3.37% of total radioactivity at 70 +/- 5 min. Pharmacokinetic parameters, obtained in the dog, were compared to previously published data in rat and man. The elimination half-life (beta) was 30.7 min in the dog as compared to 15 min in rat and and 21.7 min in man. The Vd (ml/kg) was higher than total body water volume in all three species: dog (1832), rat (1352) and man (664), indicating sequestration of the drug in body compartments. Clearance (ml/min/kg) was found to be 41.2 in dog, which compares to 62 in rat and 22 in man.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of acetylcholinesterase from the caudate nucleus of the rat.

Acetylcholinesterase (AChE) can be released in the perfusate of rat caudate nucleus (CN) slices by two different modes of stimulation, with electrical stimulation at 5 Hz and with high concentrations of K+ (105 mM) using K+-propionate. We were unable to demonstrate AChE release with lower K+ concentrations (50 mM); however, at this concentration the drop in AChE activity seen in resting conditions was prevented. Practically all (95%) cholinesterase (ChE) found in the rat CN is AChE, which is represented by two major molecular forms (4S and 10S). In the perfusate, only AChE activity could be detected. A comparison of acetylcholine (ACh) and AChE release showed that maximal 3H-outflow and AChE release occurred at the same frequency (5 Hz), but the onset of AChE release was delayed. With high K+ (105 mM) depolarization, AChE release started after termination of the stimulation and continued for at least 50 min. These findings are consistent with the view that soluble form(s) of AChE can be slowly released from neurons under specific conditions of depolarization. In the caudate of the rat, the most likely sites for this release are processes of cholinergic interneurons. A hypothesis of AChE release is presented, and possible physiological and pathological implications of such a mechanism are discussed.