Transfected rat high-molecular-weight neurofilament (NF-H) coassembles with vimentin in a predominantly nonphosphorylated form

A fully encoding cDNA for the high-molecular-weight rat neurofilament protein (NF-H) has been isolated from a lambda gt11 library, sequenced and subcloned into eukaryotic expression vectors. Sequence analysis shows that rat NF-H has an overall homology of 72 and 88% with human and mouse NF-H, respectively. The head and rod domains are almost entirely identical, and the divergences are due to differences in the long C-terminal extensions of the molecule. The consensus phosphorylation sequence for neurofilaments Lys-Ser-Pro (KSP) is present 52 times. The predicted molecular mass of the protein is 115 kDa, 42% lower than that observed by SDS-PAGE. Upon transfection into vimentin-containing fibroblasts, such as L tk-, L929, and 3T6 cells, NF-H is seen distributed with vimentin by light and electron microscopic examinations indicating that copolymers of NF-H and vimentin are formed in these cells. Only a negligible proportion of the cells is positive when stained with a number of antibodies directed against phosphorylated NF-H epitopes. This is in contrast with the middle molecular weight NF protein (NF-M) transfected into L tk- and L929 cells, which can readily be detected by antibodies against phosphorylated neurofilament epitopes. The mobilities of the transfected protein on 1- and 2-dimensional gels confirm that NF-H is predominantly in a nonphosphorylated form. These results indicate that phosphorylation of NF-H, but not NF-M, on the KSP sequence is due to protein kinases, which are not present in fibroblasts and are presumably NF-H specific. The stable NF-H-expressing cell lines can therefore be used to study these putative neurofilament kinases in vitro and in vivo.     

Elevated intrathecal antibodies against the medium neurofilament subunit in multiple sclerosis

Neurofilaments are cytoskeletal proteins localized within axons, which may interact with the immune system during and following tissue destruction in multiple sclerosis (MS). Antibodies against the medium neurofilament subunit synthesized intrathecally may reflect axonal damage in MS patients. Both immunoglobulin G (IgG) and M (IgM) responses against the purified native medium subunit of neurofilaments (NFM) using enzyme-linked immunosorbent assay (ELISA) were determined in paired serum and cerebrospinal fluid samples obtained from 49 MS patients, 16 normal controls (CN), 21 control patients with miscellaneous diseases (CD) and 14 patients with neurodegenerative disorders (CDEG). Intrathecal production of IgM and IgG antibodies to NFM were elevated in MS patients compared with the CN or CD groups (p < 0.04 for IgM, p < 0.01 for IgG). The increase was present in all the MS courses (relapsing-remitting, primary and secondary progressive). Similar local anti-NFM IgG and IgM synthesis occurred in the MS and CDEG groups. MS patients with short and long disease duration did not differ in terms of their anti-NFM IgM and IgG responses. Repeated examinations showed stable intrathecal anti-NFM production. Intrathecal IgG and IgM antibodies against NFM were increased in MS patients and may serve as a potential marker for axonal pathology. The extent of anti-NFM levels did not correspond to any individualized clinical profiles of MS patients.     

In situ localization of NF-H neurofilament subunit mRNAs in rat brain

The expression of NF-H neurofilament subunit mRNAs was investigated in the rat brain at different ontogenic stages. The levels of NF-H mRNAs vary 15-fold among brain regions with the highest level in the brainstem. In situ localization studies revealed that the NF-H mRNAs are mainly concentrated in the brainstem motoneuron nuclei. By increasing the sensitivity of the hybridization method, NF-H mRNAs could also be localized in neurons present in the cortex, thalamus and hippocampus areas. Minor amounts of NF-H mRNAs were already detected at 17-day embryonic stages.     

Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats

A new panel of greater than 300 monoclonal antibodies (mAbs) was prepared to the high, middle, and low Mr rat neurofilament (NF) subunits (NF-H, NF-M and NF-L, respectively). NF proteins were purified both from native, i.e., phosphorylated rat NFs and from enzymatically dephosphorylated rat NFs. The resulting mAbs were used to biochemically and immunochemically distinguish and characterize distinct and differentially phosphorylated isoforms of NF subunits. By immunoblot, all mAbs specific for NF-L and some mAbs specific for NF-M detected their specific NF subunit regardless of whether or not the NFs had been treated with alkaline phosphatase, and such antibodies were termed "phosphate-independent" or P[ind] mAbs. The other mAbs were specific for NF-M, NF-H, or for both NF-M and NF-H, and they recognized epitopes in the COOH termini of these subunits. Significantly, the latter mAbs could discriminate different isoforms of NF-M and NF-H, depending on the phosphorylation state of each variant. Such mAbs were assigned to one of 4 distinct categories on the basis of their performance in immunoblots of progressively dephosphorylated rat NF samples and by immunohistochemistry of various adult rat nervous tissues: (1) P[-] mAbs preferentially stained neuronal perikarya and dendrites, and they recognized only extensively dephosphorylated (and nonphosphorylated) NF-H; (2) P[+] mAbs stained axons more strongly than perikarya, and primarily blotted phosphorylated, but not nonphosphorylated, forms of NF-H and NF-M; (3) P[++] mAbs stained axons almost to the exclusion of perikarya, and in blots recognized only the extensively phosphorylated forms of NF-H and NF-M (i.e., subunits subjected to limited enzymatic dephosphorylation); (4) P[ ] mAbs also predominantly stained axons, but the briefest alkaline phosphatase treatment abolished the NF-M and NF-H immunobands produced by these mAbs. Two-dimensional gel analysis and immunoblotting of total proteins from adult rat dorsal root ganglion verified mAb specificity in situ, and showed that differentially phosphorylated isoforms of NF-M and NF-H occur in vivo. This provided additional evidence that mAbs can detect all 4 phosphorylation-dependent endogenous isoelectric variants of NF-H and NF-M.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monoclonal antibodies to the heavy neurofilament subunit (NF-H) of Torpedo cholinergic neurons.

Previous studies from our laboratory suggest that Alzheimer's disease sera contain a repertoire of antibodies to the heavy neurofilament subunit (NF-H) and that a subpopulation of these antibodies bind specifically to epitopes highly enriched in NF-H isolated from the purely cholinergic electromotor neurons of Torpedo. In the present study, we prepared and characterized monoclonal antibodies (MAbs) that bind to epitopes specifically enriched in Torpedo cholinergic neurons. This was performed by a differential enzyme-linked immunosorbent assay (ELISA) in which MAbs were selected that bind to epitopes much more abundant in the NF-H protein of Torpedo cholinergic neurons than in NF-H from the chemically heterogeneous Torpedo spinal cord. This yielded four MAbs, three of which (TC4, TC8, and TC21) were found to be specific to NF-H and one (TC15) that reacts with both NF-H and the medium-size neurofilament subunit NF-M. Dephosphorylation abolishes the binding of MAbs TC4 and TC15 to Torpedo cholinergic NF-H, partially reduces that of MAb TC21 and has no effect on the binding of MAb TC8. This suggests that the antigenic sites specific to Torpedo cholinergic NF-H contain phosphorylated as well as non phosphorylated epitopes. All the MAbs cross-react with rat brain NF-H.     

Delayed phosphorylation of the largest neurofilament protein in rat optic nerve development

Monoclonal antibodies selectively reacting with the high molecular weight neurofilament proteins (NF 150K and NF 200K) on immunoblots of bovine spinal cord extracts were obtained upon immunization of mice with chicken brain antigen and with highly purified NF 150K or NF 200K isolated from bovine spinal cord by anion exchange chromatography. Antibodies reacting with NF 200K or with both NF 150K and NF 200K were selected for this study. The antibodies were screened on immunoblots for reactivity with phosphorylated epitopes by dilution of the supernatants in sodium potassium phosphate as well as by treatment of nitrocellulose transfers with alkaline phosphatase. Abolishment of staining under these conditions was taken as evidence of reactivity with phosphorylated epitopes. With phosphate/phosphatase-sensitive antibodies, NF 200K immunoreactivity was a late event in rat optic nerve development. It was first observed at day 18 on immunoblots of sodium dodecyl sulfate extracts analyzed by gel electrophoresis. Conversely, with phosphate/phosphatase-insensitive antibodies, NF 200K immunoreactivity was already present on day 10, the earliest age in this study. With one monoclonal reacting with phosphorylated NF 150K and NF 200K, NF 150K immunoreactivity was already present on day 10. It is proposed that NF 200K expression precedes NF 200K phosphorylation in development.     

Regulation of nonphosphorylated and phosphorylated forms of neurofilament proteins in the prefrontal cortex of human opioid addicts

The neurofilament (NF) proteins (NF-H, NF-M, and NF-L for high, medium, and low molecular weights) play a crucial role in the organization of neuronal shape and function. In a preliminary study, the abundance of total NF-L was shown to be decreased in brains of opioid addicts. Because of the potential relevance of NF abnormalities in opioid addiction, we quantitated nonphosphorylated and phosphorylated NF in postmortem brains from 12 well-defined opioid abusers who had died of an opiate overdose (heroin or methadone). Levels of NF were assessed by immunoblotting techniques using phospho-independent and phospho-dependent antibodies, and the relative (% changes in immunoreactivity) and absolute (changes in ng NF/microg total protein) amounts of NF were calculated. Decreased levels of nonphosphorylated NF-H (42-32%), NF-M (14-9%) and NF-L (30-29%) were found in the prefrontal cortex of opioid addicts compared with sex, age, and postmortem delay-matched controls. In contrast, increased levels of phosphorylated NF-H (58-41%) and NF-M (56-28%) were found in the same brains of opioid addicts. The ratio of phosphorylated to nonphosphorylated NF-H in opioid addicts (3.4) was greater than that in control subjects (1.6). In the same brains of opioid addicts, the levels of protein phosphatase of the type 2A were found unchanged, which indicated that the hyperphosphorylation of NF-H is not the result of a reduced dephosphorylation process. The immunodensities of GFAP (the specific glial cytoskeletol protein), alpha-internexin (a neuronal filament related to NF-L) and synaptophysin (a synapse-specific protein) were found unchanged, suggesting a lack of gross changes in glial reaction, other intermediate filaments of the neuronal cytoskeletol, and synaptic density in the prefrontal cortex of opioid addicts. These marked reductions in total NF proteins and the aberrant hyperphosphorylation of NF-H in brains of opioid addicts may play a significant role in the cellular mechanisms of opioid addiction.     

Transmembrane orientation of an early biosynthetic form of acetylcholine receptor delta subunit determined by proteolytic dissection in conjunction with monoclonal antibodies

The transmembrane topology of acetylcholine receptor (AChR) delta subunit, synthesized in vitro and co-translationally integrated into dog pancreas rough microsomal membranes, was studied using limited proteolysis and domain-specific immunoprecipitation. Forty-four kilodaltons (kd) of the 65-kd delta subunit comprise a single fragment that is inaccessible to exhaustive proteolytic digestion from the cytoplasmic surface of the membrane by trypsin, chymotrypsin, thermolysin, and pronase. Previously, we have shown that this 44-kd "protected" fragment contains the amino terminus of the intact molecule and all of the core oligosaccharides (Anderson, D.J., P. Walter, and G. Blobel (1982) J. Cell Biol. 93: 501-506). Here we demonstrate that this domain can be further dissected into a 26-kd fragment, together with low molecular weight material, when the membranes are rendered permeable to trypsin by low concentrations of deoxycholate (Kreibich, G., P. Debey, and D. D. Sabatini (1973) J. Cell Biol. 58: 436-462). This 26-kd fragment contains all of the core oligosaccharides present on the intact subunit and therefore constitutes at least part, if not all, of the extracellular domain. The remaining low molecular weight material may derive from the membrane-embedded domain; our data imply that as much as 18 kd may be internal to the lipid bilayer. On the other hand, part of the cytoplasmic pole of AChR-delta can be recovered as a discrete, 12-kd fragment upon mild trypsinization of intact vesicles. We have used this 12-kd fragment to identify anti-AChR-delta monoclonal antibodies (mAbs) that react with the cytoplasmic domain of this subunit. Partial proteolytic fragmentation of the AChR in vitro translation products, in topologically well defined rough microsomes, may be used as a general assay to characterize the domain specificity of anti-AChR mAbs. For example, in the case of AChR-beta, we were able to identify two mAbs that recognize extracellular and cytoplasmic fragments, respectively.     

Traumatically induced reactive change as visualized through the use of monoclonal antibodies targeted to neurofilament subunits.

Reactive axonal change has long been recognized as a feature of traumatic brain injury. To date, the histological methods used to identify reactive axons have been of limited utility, and they have not provided insight into the initial intraaxonal event that triggers reactive change. In this investigation, monoclonal antibodies to the 68, 150, and 200 kilodalton (kD) neurofilament subunits have been used to follow the progression of reactive axonal change. Anesthetized rats and cats were subjected to moderate traumatic brain injury. One to 72 hours (h) postinjury, their brains were processed for the light (LM) and electron (EM) microscopic immunocytochemical visualization of the various neurofilament subunits. Although all of the chosen antibodies revealed some degree of immunoreactivity within the reactive axon, the 68 kD antibody revealed a dramatic increase in immunoreactivity following injury. Within one h of injury, intensely 68 kD-immunoreactive axonal segments were observed with LM, and parallel EM microscopic analyses demonstrated that this increased immunoreactivity was associated with an increased number of 68 kD-immunoreactive neurofilaments, the majority of which coursed in an axis parallel to the axon's course. Over 2-6 h postinjury, these 68 kD-immunoreactive filaments demonstrated increasingly disordered alignment in relation to the axon's long axis, withdrawing from the focus of injury while becoming encompassed by an expanding organelle cap. It is posited that this increased 68 kD immunoreactivity is associated with a traumatically-induced increase in subunit exchange which contributes to cytoskeletal dysfunction leading to organelle accumulation, focal swelling and ultimate axonal detachment.     

Axonal maturaion in development—I. Characterization of monoclonal antibodies reacting with axon-specific neurofilament epitopes

Monoclonal antibodies reacting with the high molecular weight neurofilament polypeptides (NF 150K and NF 200K) were obtained upon immunization with NF 150K and NF 200K isolated from bovine spinal cord by anion exchange chromatography. The five monoclonal antibodies obtained with NF 200K stained only axons. With three monoclonals the reactivity was abolished by digestion with phosphatase and by dilution of the supernatants in sodium potassium phosphate. The nine monoclonal antibodies obtained upon immunization with NF 150K stained both high molecular weight neurofilament polypeptides on immunoblots of bovine and rat spinal cord extracts with the exception of one monoclonal only reacting with the homologous antigen. The antibodies could be divided into two groups, axon-specific and conventional. Conventional antibodies decorated neurofilaments regardless of their location, i.e. axons, perikarya and dendrites. With all these antibodies the immunostaining was not affected by phosphatase digestion of neurofilament protein nor by dilution of the supernatants in sodium potassium phosphate. Axon-specific antibodies reacting with both NF 150K and NF 200K in rat spinal cord only stained the heterologous antigen (NF 200K) in rat optic nerve and sciatic nerve extracts. We suggest that some axon-specific neurofilament antibodies recognize neurofilament modifications other than phosphorylation; or, alternatively that they react with phosphorylated epitopes not accessible to phosphate or to exogenous phosphatases. Furthermore, we suggest that some neurofilament modifications do not occur uniformly throughout the nervous system.     

Temporal and topographic relationships between the phosphorylated and nonphosphorylated epitopes of the 200 kDa neurofilament protein during development in vitro

The ontogeny of the triplet of neurofilament proteins (NF), and the phosphorylated and nonphosphorylated derivatives of the 200 kDa neurofilament subunit (NF200P, NF200D) have been investigated in dissociated cultures prepared from gestational day 13 mouse spinal cord and dorsal root ganglia (DRG), using immunocytochemical methods. Neurofilament-like immunoreactivity (NF-LI), as detected with antiserum, occurred in the somata and processes of all neurons from day 1 in culture, and reached a maximum density and intensity at days 16-20. The first labeling of neurons by NF200D antibodies occurred at day 3, and was confined to DRG cells. Only a small, proximal portion of the axons from these cells exhibited NF200D-LI. At later stages, however, this immunoreactive region extended to include progressively more distal parts. Spinal cord neurons first became NF200D-positive at day 9; however, many NF200D-negative neurons still remained in mature cultures. Also at these later stages, some axons were stained for less than their full length with the NF200D antibody. NF200P-LI was first apparent at day 17, in smooth and varicose axons and only where NF-LI was also present. In contrast, NF200P- and NF200D-LI were usually localized in mutually exclusive populations of axons and other fibers. In some, predominantly thick axons, however, the proximal segment was NF200D-positive, whereas the distal part exhibited solely NF200P-LI. In contrast to NF70 and NF150, the 200 kDa neurofilament is dilatory in its appearance in most neurons in culture. The development of the nonphosphoderivative precedes that of the phosphoderivative, and the respective ontogenies are specific for different neuronal types. Posttranslational phosphorylation of NF200 seems therefore to occur at a later stage of development than the induction of NF200 itself, while there is a wide variation in its rates of phosphorylation during passage down different axons.     

Ultrastructural evidence for the existence of a direct hypothalamic-vagal descending pathway

The ultrastructural changes in the presynaptic nerve terminals in the dorsal vagal nucleus were studied in rats with bilateral electrolytic lesions placed in the dorso-medio-caudal hypothalamic area in the sagittal plane of the dorsomedial nuclei, just behind these nuclei in the P_3,5–P_3,7 interval. On days 3,4 and 5 following placement of the hypothalamic lesions, degeneration of some of the presynaptic profiles in the dorsal vagal nucleus was found in the experimental animals. The data obtained provide evidence for the existence of descending hypothalamic axons terminating in the medulla oblongata on the neurons of the dorsal vagal nucleus. The possible involvement of the descending hypothalamic-vagal nerve pathway in the control of endocrine pancreas is discussed.     

Expression of neurofilament subunits in neurons of the central and peripheral nervous system: an immunohistochemical study with monoclonal antibodies

The extent to which all neurofilament (NF) subunits (NF68, NF150, NF200) are expressed by different populations of mature CNS and PNS neurons is controversial. We addressed this issue in immunohistochemical studies of mature bovine tissues using monoclonal antibodies specific for each bovine NF subunit. All three NF subunits were detected in the perikarya and neurites of both CNS and PNS neurons; they were seen in nearly all PNS neuronal perikarya, and in all identifiable CNS and PNS axons. Most, but not all, CNS neuronal perikarya contained each of these NF antigens. CNS neurons devoid of immunodetectable NF antigens were generally small. The presence of low levels of NF antigens in neurons with scant perikaryal cytoplasm may account for the apparent absence of NF immunoreactivity in some classes of neurons, although other explanations, such as microheterogeneity among NF proteins, could account for this finding. NF antigens were also seen in some cells of the diffuse neuroendocrine system (adrenal chromaffin cells and cells of the pars distalis and pars intermedia), but not in other cell types. We suggest that the expression of all three NF subunits is a common feature of CNS and PNS neurons and their processes, and of some cells of the diffuse neuroendocrine system. These findings have implications for hypotheses concerning the structure and function of the intermediate filaments of neurons, and for hypotheses concerning neurodegenerative diseases involving NF proteins.     

2,5-Hexanedione-induced changes in the neurofilament subunit pools of rat peripheral nerve

Axon atrophy is the principle morphological feature of the peripheral neuropathy induced by 2,5-hexanedione (HD). Axon caliber is determined by a stationary neurofilamentous cytoskeleton that is maintained through dynamic interactions with mobile neurofilament (NF) subunits. To determine the effects of HD on the stationary and mobile NF pools, groups of rats were exposed to HD at dosing schedules (175 mg/kg x 101 days or 400 mg/kg x 26 days) that produced moderate levels of neurological deficits and, as assessed by previous studies, prevalent axon atrophy in peripheral nerve. Sciatic and tibial nerves from HD-intoxicated rats and their age-matched controls were triton-extracted and separated by differential centrifugation into a high-speed pellet (P1) of NF polymer and a corresponding supernatant fraction (S1), which presumably contained mobile monomer. Cytoskeletal proteins (NF-L, NF-M, NF-H and beta-tubulin) in each fraction were determined by immunoblot analysis. Results show that regardless of HD dose-rate, triton-soluble NF subunits in the supernatant fractions were significantly reduced, whereas triton-insoluble proteins in the corresponding pellets were inconsistently affected. Beta-tubulin also exhibited inconsistent fractional changes, while abnormal higher molecular weight NF proteins were detected primarily in the triton-insoluble fraction. Studies with antibodies directed against phosphorylated (RT97) and non-phosphorylated (SMI32) epitopes on NF-H did not reveal major changes in subunit phosphorylation. These results suggest that HD intoxication is primarily associated with depletion of soluble NF proteins, which could produce axon atrophy through disruption of cytoskeletal turnover and maintenance.     

The developmental expression of neurofilament and glial filament proteins in the human pituitary gland: an immunohistochemical study with monoclonal antibodies

Motoneurons were recorded intracellularly in the isolated perfused spinal cord of 10 - 16-day chick embryos. Inhibitory postsynaptic potentials (IPSPs) were present in motoneurones of all ages studied and could be evoked by both ventral white column and dorsal root stimulation. IPSPs produced by orthodromic stimulation displayed many features of mature vertebrate motoneuronal IPSPs including the chloride dependence and sensitivity to currents passed through the cell membrane. Strychnine and chloride-free solution produced marked disinhibitory effects in the spinal cord indicating the presence of inhibitory synapses in interneuronal circuits of at least 11-day and older embryos. Possible sources of descending inhibitory influences on motoneurones and some functional aspects are discussed. The results support the hypothesis that the inhibition starts in the embryonic chick spinal cord rather early, before the 10th day of development.     

Light subunit of neurofilament triplet protein in the cerebrospinal fluid after subthalamic nucleus stimulation for Parkinson's disease

Constantinescu R, Holmberg B, Rosengren L, Corneliusson O, Johnels B, Zetterberg H. Light subunit of neurofilament triplet protein in the cerebrospinal fluid after subthalamic nucleus stimulation for Parkinson’s disease.
Acta Neurol Scand: 2011: 124: 206–210.
© 2010 John Wiley & Sons A/S. Objectives – Cerebrospinal fluid (CSF) levels of neurofilament triplet protein (NFL), a non‐specific marker of neuronal damage, are normal in Parkinson’s disease (PD) but increased after brain trauma and in several neurological disorders. Using longitudinal CSF‐NFL measurements as an indicator of neuronal damage, this study investigated the impact of deep brain stimulation (DBS) of the subthalamic nucleus (STN) on the brain, directly following the surgical intervention and in chronically treated patients with PD. Materials and methods – CSF‐NFL levels were measured consecutively in eight patients with PD before and after STN‐DBS treatment. Results – CSF‐NFL levels were normal prior to STN‐DBS and increased sharply during the first 2 weeks post‐operatively, but normalized after 12 months or more. Conclusion – The STN‐DBS procedure leads to an acute but limited neuronal damage, as expected. However, normal CSF‐NFL levels at 12 months post‐operatively and beyond suggest the absence of any long‐term neuronal damage caused by long‐term STN‐DBS stimulation.     

Levels of the light subunit of neurofilament triplet protein in cerebrospinal fluid in Huntington's disease

BackgroundNeurofilaments are major structural elements of neuronal cells. The light subunit of neurofilament triplet protein (NFL) has been shown to be increased in several neurological diseases (e.g. vascular, infectious, neurodegenerative), indicating axonal damage.MethodsIn this study we analyzed the NFL levels in all (N = 35) available cerebrospinal fluid (CSF) samples from a clinical trial in Huntington's disease (HD) and compared them to age and gender matched controls.ResultsThe CSF–NFL levels were significantly higher in HD subjects compared with age and genders matched controls, and were correlated with scores on the Unified Huntington's Disease Rating Scale Total Functional Capacity assessment. The potential of CSF–NFL levels as a disease activity marker in HD needs to be further investigated.     

Electrophysiological evidence for the existence of GABAA receptors in cultured frog melanotrophs

The neurotransmitter GABA exerts a biphasic effect on alpha-melanocyte-stimulating hormone (alpha-MSH) secretion from pars intermedia cells: GABA induces a rapid and transient stimulation followed by a sustained inhibition of alpha-MSH release. In the present study, we have investigated the effect of GABA on the electrophysiological properties of frog melanotrophs in primary culture using the patch-clamp technique in the whole cell configuration. In all cells tested, GABA stimulated an inward current and induced depolarization. A transient period of intense firing was consistently observed at the onset of GABA administration. During the depolarization phase, the membrane potential reached a plateau corresponding to the Cl- equilibrium potential. When repeated hyperpolarizing pulses were applied, an increase of membrane conductance was observed throughout the response evoked by GABA. The effect of GABA was abolished by the chloride channel blocker picrotoxin, and by antagonists of GABAA receptors (bicuculline and SR 95531). The depolarizing action of GABA was mimicked by muscimol, an agonist of GABAA receptors. Taken together, our results indicate that the rapid and transient stimulation of alpha-MSH release induced by GABA can be accounted for by activation of a chloride conductance which causes membrane depolarization. These data support the notion that the transient stimulation of alpha-MSH secretion induced by GABA can be accounted for by membrane depolarization which provokes activation of voltage-operated calcium channels. Since no evidence was found for GABA-induced hyperpolarization, the intracellular mechanisms leading to the strong inhibitory effect of GABA on alpha-MSH secretion remain to be elucidated.     

Early and late appearance of neurofilament phosphorylated epitopes in rat nervous system development: in vivo and in vitro study with monoclonal antibodies

Neurofilament phosphorylation in rat nervous system development was studied by indirect immunofluorescence with monoclonal antibodies reacting with phosphorylated epitopes in tissue sections and in primary dissociated cultures. The antibodies either decorated neurofilaments shortly after their appearance or after a considerable delay (from 4 to 9 days in vivo and from 12 to 27 days in vitro), thus suggesting the existence of at least two classes of phosphorylated epitopes. With most antibodies there was a good correlation between in vivo and in vitro findings as to the early or late appearance of phosphorylated epitopes. Monoclonal NE14 was the main exception in that immunoreactivity with this antibody was present in 1-day cultures, while it only occurred 4 days after the first appearance of neurofilaments in vivo. The effect of phosphorylation on neurofilament structure and function remains to be determined. Neurofilament expression is an early phenomenon in ontogeny coinciding with neuronal differentiation. It is possible that late phosphorylation events may stabilize the axonal cytoskeleton following the massive loss of axons that occurs in several fiber tracts during late fetal and neonatal life.