Transforming growth factor-β1 and -β2 promote neurite sprouting and elongation of cultured rat hippocampal neurons

Transforming growth factor-β (TGF-β) is known as a potent regulator of cell proliferation and differentiation. In the present study, we investigated the effects of TGF-β1 and -β2 on the survival, neurite sprouting and process elongation of primary cultured hippocampal neurons obtained from rat embryos. Addition of TGF-β1 little affected the total number of surviving neurons, but clearly increased the number of neurons bearing processes, indicating that TGF-β1 promotes neurite sprouting rather than neuronal survival. Furthermore, TGF-β1 significantly promoted the elongation of axon-like processes, but did not affect the process branching and the number of dendrite-like processes. TGF-β2 also promoted the neurite sprouting and stimulated the elongation of axons without affecting the branching. The effects of TGF-β2 were very similar to those of TGF-β1 in terms of both effective concentrations (0.1–1 ng/ml) and maximal effects. It is possible that TGF-β1 and -β2 play roles in the formation of neuritic networks in the central nervous system.     

Muscarinic modulation of TREK currents in mouse sympathetic superior cervical ganglion neurons

Muscarinic receptors play a key role in the control of neurotransmission in the autonomic ganglia, which has mainly been ascribed to the regulation of potassium M‐currents and voltage‐dependent calcium currents. Muscarinic agonists provoke depolarization of the membrane potential and a reduction in spike frequency adaptation in postganglionic neurons, effects that may be explained by M‐current inhibition. Here, we report the presence of a riluzole‐activated current (I_RIL) that flows through the TREK‐2 channels, and that is also inhibited by muscarinic agonists in neurons of the mouse superior cervical ganglion (mSCG). The muscarinic agonist oxotremorine‐M (Oxo‐M) inhibited the I_RIL by 50%, an effect that was abolished by pretreatment with atropine or pirenzepine, but was unaffected in the presence of himbacine. Moreover, these antagonists had similar effects on single‐channel TREK‐2 currents. I_RIL inhibition was unaffected by pretreatment with pertussis toxin. The protein kinase C blocker bisindolylmaleimide did not have an effect, and neither did the inositol triphosphate antagonist 2‐aminoethoxydiphenylborane. Nevertheless, the I_RIL was markedly attenuated by the phospholipase C (PLC) inhibitor ET‐18‐OCH3. Finally, the phosphatidylinositol‐3‐kinase/phosphatidylinositol‐4‐kinase inhibitor wortmannin strongly attenuated the I_RIL, whereas blocking phosphatidylinositol 4,5‐bisphosphate (PIP₂) depletion consistently prevented I_RIL inhibition by Oxo‐M. These results demonstrate that TREK‐2 currents in mSCG neurons are inhibited by muscarinic agonists that activate M1 muscarinic receptors, reducing PIP₂ levels via a PLC‐dependent pathway. The similarities between the signaling pathways regulating the I_RIL and the M‐current in the same neurons reflect an important role of this new pathway in the control of autonomic ganglia excitability.     

A light and electron microscopic study of the rat superior cervical ganglion cells by intracellular HRP-labeling

Horseradish peroxidase (HRP) was injected intracellularly into single neurons of the isolated rat superior cervical ganglia. Intracellular iontophoresis of HRP did not seem to damage the sympathetic neurons or to affect synaptic transmission. Under the light microscope, 9 of the 27 HRP-labeled sympathetic neurons exhibited varicosites in their dendrites, but not in their axons; the varicose dendrites came into close contacts with adjacent non-labeled neurons. With the electron microscope, the varicose dendrites of 3 separate neurons lightly stained with HRP, morphological features of synapses could be identified at the contact site: clusters of vesicles in the varicose dendrites, intercellular space of about 20 nm separating the apposed membranes, and an intermediate density on the postjunctional membrane. These findings suggest at the ultrastructural level the occurrence of dendro-dendritic and dendro-somatic synapses in mammalian sympathetic ganglia.     

Transforming growth factor-β1 exhibits delayed gene expression following focal cerebral ischemia

Transforming growth factor-β1 (TGF-β1) is a pleiotropic peptide growth factor. The expression of TGF-β1 mRNA in the focal ischemic cortex of rats was studied by means of Northern hybridization. A moderately low level of constitutively expressed TGF-β1 mRNA was detected following ohm surgery or in the contralateral (nonlschemic) cortex. A significant increase of TGF-β1 mRNA level in the ischemic cortex was observed at 2 days (3.2-fold increase compared to sham-operated animals, p < 0.01, N = 4) following permanent occlusion of the middle cerebral artery (PMCAO). The elevated TGF-μ1 mRNA expression was plateaued for up to 16 days (3–6-fold increase, p < 0.01) following PMCAO- This temporal profile for TGF-β1 mRNA expression in focal stroke was significantly delayed compared to that of TNF-α, IL-1β and IL-6 MRNA expressions as demonstrated previously which peaked at 12 h and decreased to almost basal levels by S days following PMCAO. Interestingly, the TGF-β1 mRNA expression profile was remarkably parallel with that of monocyte/macrophage accumulation in the ischemic cortex, as well as with the increased formation of extracellular matrix in the focal ischemic brain. These data suggest that TGF-β1 may play a role in anti-inflammatory process and in tissue remodeling following ischernic brain injury.     

Transforming growth factor-βs inhibit mitogen-stimulated proliferation of astrocytes

We have studied the influence of three members of the transforming growth factor-β (TGF-β) family of multifunctional growth factors on the proliferation of cultured astrocytes isolated from newborn mouse cerebral cortex. Although TGF-βs 1, 2, and 3 cause only a small reduction in the low level of astrocyte proliferation occurring in chemically defined medium, they each inhibit the effects of five astrocyte mitogens (bFGF, EGF, PDGF, IL-1α, and IL-2). Inhibition is observed when astrocytes are exposed to mitogen and TGF-r3 at the same time and when they are exposed to TGF-β prior to, and separately from, mitogen. This latter effect appears to be due to the binding of TGF-βs to astrocyte-secreted extracellular matrix. These findings raise the possibility that TGF-β may co-operate with other growth factors to control astrocyte proliferation in vivo.     

Transforming growth factor-β1 inhibits the proliferation of rat astrocytes induced by serum and growth factors

A number of cytokines and growth factors may affect astrocyte proliferation and functions. Transforming growth factor-β1 (TGF-β1) is a pleiotropic cytokine which exerts multiple effects on growth and differentiation of different cell types. TGF-β1 is present in low amounts in the normal brain. TGF-β1 gene expression, however, is increased in the central nervous system (CNS) in several pathological conditions. In this study we examined the in vitro effects of TGF-β1 on the proliferative response of rat astrocytes to serum and growth factors. Astrocyte cultures were established from the cerebellum and cortex of newborn Lewis rats. The proliferative response of these cultures to serum and growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), IGF-2, interleukin 1 (IL-1)] was studied by [³H]-thymidine incorporation test in the presence or absence of TGF-β1. TGF-β1 significantly inhibited the proliferative response of astrocyte cultures to both autologous and heterologous serum. In addition, a strong inhibition of bFGF-, EGF-, and PDGF-induced proliferation was observed. The effect of TGF-β1 on the proliferative response to IL-1 was less evident but still significant. No effect was observed when TGF-β1 was added to IGF-1 and IGF-2 stimulated cultures. These data confirm previous reports showing a down-regulating activity of TGF-β on astrocyte proliferation and suggest that this cytokine may play physiological and pharmacological roles in the regulation of reactive astrocytosis in the CNS. © 1995 Wiley-Liss, Inc.     

Quantitative analysis of the number and distribution of neurons richly innervated by GABA-immunoreactive axons in the rat superior cervical ganglion

The superior cervical ganglion of rats contains a considerable number of nerve fibers with GABA-like immunoreactivity which show a nonuniform distribution within the ganglion. The topography of these fibers has been analyzed by using antibodies raised against GABA-BSA-glutaraldehyde complexes. GABA-positive axons and axon varicosities accumulated around a subpopulation of principal ganglion cells forming basketlike patterns. These neurons richly innervated by GABA-positive axons (RIG-neurons) in turn were aggregated in patches with strong immunoreactivity. The size and packing density of the patches containing RIG-neurons and GABA-positive axons approaching them had rostral-to-caudal and medial-to-lateral gradients. Similar patterns were found in right and left ganglia. In five ganglia, a quantitative analysis revealed on average 1,344 RIG-neurons per ganglion representing about 5% of the total neuron population, with small variations (standard deviation 122) despite the highly variable shape of the ganglia. The distribution of RIG-neurons resembles that of neurons sending their axons into the internal carotid nerve. To check this possible correlation, HRP was injected into the eye and applied to the transected external carotid nerve. Double staining for the retrogradely transported peroxidase and GABA immunohistochemistry revealed that RIG-neurons formed a small subpopulation of retrogradely labelled neurons in both experiments. This suggests that RIG-neurons innervate various target organs. This conclusion is in agreement with the observation that RIG-neurons also exist in other sympathetic ganglia. Data presented suggest that sympathetic ganglion cells can be classified on the basis of non-uniform innervation patterns formed by axons that use different neurotransmitters.     

Tumor necrosis factor enhancement of transient outward potassium currents in cultured rat cortical neurons

The effect of recombinant human tumor necrosis factor-α (TNF) on voltage-gated membrane currents of cultured neurons derived from embryonic rat cerebral cortex was studied using the whole-cell patch-clamp technique. Treatment of neurons with TNF resulted in an increase in outward potassium current density, dependent upon the concentration of TNF and the incubation time, without affecting other membrane currents such as barium and N-methyl-D-aspartate (NMDA). Long exposures (12–48 hr) to TNF (10–100 ng/ml) increased transient outward potassium current (A-current) density without affecting the parameters of activation and inactivation of the current. Prolonged exposures to TNF diminished its increasing effect on the A-current. Since the increase of A-current density induced by TNF is inhibited by both the anti-TNF receptor antibody and cycloheximide treatment, the effect of TNF might be mediated through receptors and by de novo synthesis of the channel protein itself and/or modulating proteins associated with the channel activities. Results indicate that phosphatidylcholine-specific phospholipase C and protein kinase C, but not ceramide, are involved in the signal transduction. In toxicological experiments, TNF had no neurotoxicity. Moreover, a 12 hr pretreatment of TNF protected neurons against NMDA-induced neurotoxicity. This protective effect of TNF was canceled by 4-aminopyridine, an A-current blocker, suggesting that the increase of A-current densities induced by TNF contributes to the neuroprotection. J. Neurosci. Res. 50:990–999, 1997. © 1997 Wiley-Liss, Inc.     

Localization and developmental changes of the expression of two inward rectifying K-channel proteins in the rat brain

We have raised affinity-purified polyclonal antibodies specific for the inward rectifying K⁺ channel (IRK1/Kir2.1) and the G protein-activated inward rectifying K⁺ channel (GIRK1/Kir3.1) examined their distributions in the rat brain immunohistochemically. The regional expression pattern of the IRK1 and GIRK1 proteins were similar to those of mRNA of the previous in situ hybridization study. The subcellular distribution was studied in the cerebellum, cerebral cortex and hippocampus. In the cerebellum, the IRK1 protein was clearly detected in the somata and proximal dendrites of Purkinje cells, while the GIRK1 protein was present in the somata and clustered dendrites of granule cells. In the cerebral cortex and hippocampus, both IRK1- and GIRK1-immunoreactivities were detected in the somata and apical dendrites of the pyramidal cells. The presence of IRK1 or GIRK1 proteins in the axons could not proved by the present study. The developmental changes of the expression pattern of the GIRK1 protein were also investigated in the hippocampus and in the cerebellum of postnatal day (P) 7 to P17 rats. The GIRK1 protein was detected neither in the subgranular zone of the dentate gyrus nor in the proliferative zone of the external granule cell layer of the cerebellum, in which granule cell precursors are reported to proliferate, while it was clearly detected in the adjacent layer in which postmitotic but immature cells exist. These results imply that the expression of the GIRK1 protein starts just after the neuronal precursors finished the last mitotic cell division. ©1997 Elsevier Science B.V. All rights reserved.     

Age-related increase in c-fos expression in the sympathetic neurons of the rat superior cervical ganglion

C-fos expression was studied immunocytochemically in sympathetic neurons of the rat superior cervical ganglion (SCG). Fos-like immunoreactivity was confined to the principal neurons of the ganglion and was exclusively localized within their nuclei. In 2-month-old rats, the immunoreactivity was detected in 1.2% of the principal neurons with a density of 4.95 Fos-positive cells/mm2 of ganglion area. This proportion increased with age and reached a value which was 6.5-fold higher in the 26-month-old rats than that in the young adult. A density of 24.5 Fos-positive cells/mm2 of ganglion area was seen in the 26-month-old animals. The age-enhanced c-fos expression suggests that Fos may be involved in regulation of the genetic events associated with the adaptive changes in neuronal activity of the sympathetic ganglion during aging.     

Age-related increase inc-fos expression in the sympathetic neurons of the rat superior cervical ganglion

C-fos expression was studied immunocytochemically in sympathetic neurons of the rat superior cervical ganglion (SCG). Fos-like immunoreactivity was confined to the principal neurons of the ganglion and was exclusively localized within their nuclei. In 2-month-old rats, the immunoreactivity was detected in 1.2% of the principal neurons with a density of 4.95 Fos-positive cells/mm² of ganglion area. This proportion increased with age and reached a value which was 6.5-fold higher in the 26-month-old rats than that in the young adult. A density of 24.5 Fos-positive cells/mm² of ganglion area was seen in the 26-month-old animals. The age-enhancedc-fos expression suggests that Fos may be involved in regulation of the genetic events associated with the adaptive changes in neuronal activity of the sympathetic ganglion during aging.     

Principal neurons as local circuit neurons in the rat superior cervical ganglion: The synaptology of the neuronal processes revealed by intracellular injection of biocytin

To analyze the local circuitry of the sympathetic ganglion, the synaptic relations of the neuronal processes of the principal neurons in the rat superior cervical ganglion were investigated by correlated light and electron microscopy combined with intracellular injection of biocytin. Intracellular iontophoresis of biocytin followed by avidin-biotinylated horseradish peroxidase cytochemistry allowed complete visualization of the neuronal processes of the principal neurons. The stained principal neurons have a single process (axon), which leaves the ganglion, and several intraganglionic processes (dendrites), some of which show specific terminal arborizations. Some terminals of the dendritic collaterals formed pericellular plexuses or intercellular glomerular plexuses. Electron microscopically, the dendrites and their collaterals contain numerous small vesicles. Synaptic membrane specializations were observed between the stained dendritic collaterals and unlabeled neurites. These may be both preganglionic axon terminals and processes of principal neurons. The likely direction of neurotransmission often could not be determined because of the bidirectional synaptic structures. Our findings show that the dendritic collaterals of principal neurons appear to make both post- and presynaptic contacts with both the principal neurons and the preganglionic axons. It is suggested that the principal neurons might participate in local circuits involving not only preganglionic axons but also neighboring principal neurons. © 1993 Wiley-Liss, Inc.     

Substance P in the superior cervical ganglion and the submaxillary gland of the rat

Substance P has been detected in the superior cervical ganglion and submaxillary gland of the rat by radioimmunoassay. Decentralization, section of the carotid sinus nerve, or deefferentation does not affect the level of substance P in the rat superior cervical ganglion. Superior cervical ganglionectomy does not affect the amount of substance P in the submaxillary gland; however, section of the chorda tympani or duct ligation significantly reduces substance P in the submaxillary gland. It is concluded that substance P in the superior cervical ganglion is not located in the preganglionic sympathetic neurons, afferents from the glossopharyngeal nerve or post-ganglionic neurons projecting to the submaxillary gland or carotid body. Substance P does not appear to be associated with sympathetic innervation of the submaxillary gland, but it is anatomically and functionally related to the chorda tympani and possibly involved in the parasympathetic innervation of the gland. Substance P may also be present in gland cells of the submaxillary gland.     

Elimination of the fast transient in superior cervical ganglion neurons with expression of KV4.2W362F: molecular dissection of IA.

Electrophysiological and molecular studies have revealed considerable heterogeneity in voltage-gated K(+) currents and in the subunits that underlie these channels in mammalian neurons. At present, however, the relationship between native K(+) currents and cloned subunits is poorly understood. In the experiments here, a molecular genetic approach was exploited to define the molecular correlate of the fast transient outward K(+) current, I(Af), in sympathetic neurons and to explore the functional role of I(Af) in shaping action potential waveforms and controlling repetitive firing patterns. Using the biolistic gene gun, cDNAs encoding a dominant negative mutant Kv4.2 alpha-subunit (Kv4.2W362F) and enhanced green fluorescent protein (EGFP) were introduced into rat sympathetic neurons in vitro. Whole-cell voltage-clamp recordings obtained from EGFP-positive cells revealed that I(Af) is selectively eliminated in cells expressing Kv4.2W362F, demonstrating that Kv4 alpha-subunits underlie I(Af) in sympathetic neurons. In addition, I(Af) density is increased significantly in cells overexpressing wild-type Kv4.2. In cells expressing Kv4.2W362F, input resistances are increased and (current) thresholds for action potential generation are decreased, demonstrating that I(Af) plays a pivotal role in regulating excitability. Expression of Kv4.2W362F and elimination of I(Af) also alters the distribution of repetitive firing patterns observed in response to a prolonged injection of depolarizing current. The wild-type superior cervical ganglion is composed of phasic, adapting, and tonic firing neurons. Elimination of I(Af) increases the percentage of adapting cells by shifting phasic cells to the adapting firing pattern, and increased I(Af) density reduces the number of adapting cells.     

嗅鞘细胞对培养脊髓神经元生长状态的影响

目的研究嗅鞘细胞(OECs)对体外培养脊髓背根神经节神经元生长状态的影响。方法取新生大鼠脊髓背根神经节细胞与嗅鞘细胞共培养,在显微镜下观察神经元生长发育情况,染色后进行细胞计数,并测定细胞活性。结果共培养组细胞密度明显高于对照组,神经元胞体大而饱满,突起较长,细胞活性较高。结论嗅鞘细胞可明显促进体外培养脊髓背根神经节神经元的生长,提高细胞活性。     

Differentiation of α-adrenergic responsiveness in the neonatal rat iris after decentralization or extirpation of the superior cervical ganglion

The effects of stimulating α-adrenergic receptors in the iris of neonatal rats (up to 30 days of age) whose superior cervical ganglion (SCG) has been decentralized or extirpated 3 days after birth were investigated by measuring changes in pupil diameter. The responses of these experimental animals were compared with those of normal animals of similar age. α-adrenergic stimulation was affected by topical application of noradrenaline (NA) after blockade of β-adrenoceptors with dichloroisoproterenol and cholinoceptors with atropine. The results reveal that although decentralization of the SCG depletes the NA content of the ipsilateral iris as demonstrated by radio-chemical assay and formaldehyde-induced fluorescence, it does not affect the development of α-adrenoceptor responsiveness. Even the more severe disruption of the innervation caused by extirpation of the SCG did not affect the responsiveness of the ipsilateral iris.     

Immunocytochemical demonstration of β-endorphin and β-lipotropin in cultured human spinal ganglion neurons

Human fetal spinal ganglion neurons isolated and cultured in vitro were found to contain immunoreactive β-endorphin and β-lipotropin as demonstrated by immunoperoxidase and immunofluorescence techniques. The specificity of the immunoreactions was confirmed by the negative staining by prior absorption of the specific antisera with added peptides. The culture system described may provide a valuable model system in which cellular mechanisms underlying the functions of opioid peptides can be investigated.