Synergistic effects of the peptide fragment D-NAPVSIPQ on ethanol inhibition of synaptic plasticity and NMDA receptors in rat hippocampus.

The L1 cell adhesion molecule has been implicated in ethanol teratogenesis as well as NMDAR-dependent long-term potentiation (LTP) of synaptic transmission, a process thought to be critical for neural development. Ethanol inhibits LTP at least in part by interacting with NMDA receptors. Ethanol also inhibits L1-mediated cell adhesion in a manner that is prevented by an octapeptide, D-NAPVSIPQ (D-NAP), as well as long chain alcohols such as 1-octanol. Here we analyzed the effects of D-NAP and 1-octanol on ethanol modulation of LTP induced by theta burst stimulation in two subfields of the rat hippocampus, the dentate gyrus and area CA1. When theta burst stimulation was delivered in ethanol (50 mM), LTP was inhibited by about 50%. Surprisingly, when D-NAP (10(-7) M) and ethanol were co-applied or applied sequentially, LTP was completely absent. The effects of D-NAP were persistent, since delivery of a second theta burst stimulation following washout of D-NAP and ethanol elicited minimal plasticity. Application of D-NAP alone had no effect on LTP induction or expression. The synergistic effect of D-NAP on ethanol inhibition of LTP was concentration-dependent since D-NAP (10(-10) M) had an intermediate effect, while D-NAP (10(-13) M) had no effect on ethanol suppression of LTP. These observations were also replicated with a different ethanol antagonist, 1-octanol, in area CA1. To address the mechanisms underlying this long-lasting suppression of LTP, the sensitivity of pharmacologically isolated NMDAR extracellular field potentials to combinations of D-NAP and ethanol was determined. D-NAP (10(-7)M) alone had no effect on NMDA extracellular field potentials; however, the peptide significantly increased the inhibitory action of ethanol on NMDA extracellular field potential. The findings suggest that D-NAP and 1-octanol selectively interact with NMDA receptors in an ethanol-dependent manner, further implicating the L1 cell adhesion molecule in alcohol-related brain disorders.

Sleep and wakefulness in c-fos and fos B gene knockout mice.

G-protein coupled receptor (GPCR) stimulation has been implicated in the regulation of sleep. Upon stimulation of a GPCR an intracellular cascade involving second and third messengers is initiated. The latter include the fos-family of immediate early genes (IEGs). Although there is considerable evidence indicating that IEGs are expressed in response to sleep, the effects of their deletion on sleep is not known. The present study examined sleep-wakefulness in mice lacking the c-fos or fos B genes. Null c-fos mice compared to their wildtype (WT) and heterozygote (het) siblings had more wakefulness and less slow wave sleep (SWS); REM sleep was not affected. The null c-fos mice also had increased delta activity (0.3-4 Hz). In contrast, the null and heterozygote fos B mice had less REM sleep, but the time spent in SWS or wakefulness was not different from their wild-type (WT) siblings. In the null c-fos mice, the increased wakefulness and the reduction in SWS could not be due to a systemic alteration in temperature since the core temperature was similar in all mice. By demonstrating that these IEGs are involved in sleep, we suggest that the deletion of specific genes, even within a family of genes, can have a specific effect on sleep.

Antagonists of alcohol inhibition of cell adhesion.

Increasing evidence suggests that alcohols act within specific binding pockets of selective neural proteins; however, antagonists at these sites have not been identified. 1-Alcohols from methanol through 1-butanol inhibit with increasing potency the cell-cell adhesion mediated by the immunoglobulin cell adhesion molecule L1. An abrupt cutoff exists after 1-butanol, with 1-pentanol and higher 1-alcohols showing no effect. Here, we demonstrate surprisingly strict structural requirements for alcohol inhibition of cell-cell adhesion in L1-transfected NIH 3T3 fibroblasts and in NG108-15 neuroblastoma x glioma hybrid cells treated with BMP-7, an inducer of L1 and neural cell adhesion molecule. The target site discriminates the tertiary structure of straight-chain and branched-chain alcohols and appears to comprise both a hydrophobic binding site and an adjacent hydrophilic allosteric site. Modifications to the 2- and 3-carbon positions of 1-butanol increased potency, whereas modifications that restrict movement about the 4-carbon abolished activity. The effects of ethanol and 1-butanol on cell-cell adhesion were antagonized by 1-pentanol (IC(50) = 715 microM) and 1-octanol (IC(50) = 3.6 microM). Antagonism by 1-octanol was complete, reversible, and noncompetitive. 1-Octanol also antagonized ethanol inhibition of BMP-7 morphogenesis in NG108-15 cells. 1-Octanol and related compounds may prove useful in dissecting the role of altered cell adhesion in ethanol-induced injury of the nervous system.

Alcohol inhibition of cell adhesion in BMP-treated NG108-15 cells.

BACKGROUND: The L1 cell adhesion molecule is expressed as alternatively spliced neuronal and nonneuronal isoforms. We have reported that in transfected fibroblasts, ethanol variably inhibits cell-cell adhesion mediated by the nonneuronal isoform of human L1. In contrast, ethanol consistently inhibits morphogenetic changes and cell-cell adhesion in NG108-15 cells treated with OP-1 (BMP-7), a powerful inducer of L1 and N-CAM gene expression. METHODS: All studies were performed by using NG108-15 cells cultured in serum-free medium. Cell morphology was assessed by a quantitative assay of cell clustering. Cell adhesion was measured by a short-term re-aggregation assay, and isoforms of L1 were characterized by RT-PCR and sequencing. RESULTS: We show that ethanol inhibits the morphogenetic effects of BMP-2, BMP-4, BMP-5, and BMP-6, each of which increases the expression of L1 and N-CAM. Pretreatment of NG108-15 cells with 25-100 mM ethanol did not induce tolerance to ethanol's inhibition of OP-1 morphogenesis or cell-cell adhesion. Ethanol or anti-L1 Fab fragments partially inhibited cell-cell adhesion in OP-1-treated NG108-15 cells. The combination of ethanol and Fab fragments did not inhibit cell-cell adhesion more than Fab fragments alone. As in L1-transfected fibroblasts, a series of n-alcohols displayed a cutoff between butanol and pentanol for inhibition of cell-cell adhesion in OP-1-treated NG108-15 cells. RT-PCR and direct sequencing revealed that the neuronal isoform was the sole or predominant L1 isoform in OP-1-treated NG108-15 cells. CONCLUSIONS: These data suggest that ethanol inhibits cell-cell adhesion in OP-1-treated NG108-15 cells by interacting directly or indirectly with the neuronal isoform of L1.

Characterization of ethanol-sensitive and insensitive fibroblast cell lines expressing human L1.

Ethanol inhibits L1-mediated cell-cell adhesion in fibroblast cell lines stably transfected with human L1. Here we show that this action of ethanol is present in only a subset of transfected NIH/3T3 and L cell clonal cell lines. All L1-expressing cell lines had higher levels of cell adhesion than cell lines transfected with empty vector. In all ethanol-sensitive cell lines, L1-mediated adhesion was inhibited by ethanol (IC50 5-10 mM), 2 mM butanol, but not 5 mM pentanol. In contrast, ethanol-insensitive cell lines were not inhibited by up to 200 mM ethanol, 2 mM butanol, or 5 mM pentanol. Ethanol sensitivity or insensitivity was a stable property of each cell line and was not associated with differences in electrophoretic mobility, abundance, or cell surface localization of L1. Fab fragments prepared from anti-L1 polyclonal antisera inhibited cell adhesion only in the ethanol-sensitive cell lines. These data suggest that L1 may exist in an alcohol-sensitive or an alcohol-insensitive state that may be governed by host cell factors.

Matrix metalloproteinases in the cerebrospinal fluid of patients with Lyme neuroborreliosis.

Neurologic manifestations of Lyme disease include meningitis, encephalopathy, and cranial and peripheral neuropathy. There are no sensitive markers for neuroborreliosis, and diagnosis is often based on clinical presentation and cerebrospinal fluid (CSF) abnormalities, including intrathecal antibody production. Matrix metalloproteinase (MMP) activity in CSF was compared in patients with neuroborreliosis, patients with diverse neurologic disorders, and healthy controls. The CSF of 17 of 18 healthy subjects and 33 of 37 patients with neurologic symptoms and normal CSF and imaging studies contained only MMP2. The CSF of several patients with neurologic disorders contained MMP2, MMP9, and gelatinolytic activity at 130 and 250 kDa. The 130-kDa MMP was found without the 92-kDa MMP9 in the CSF of 11 (79%) of 14 patients with neuroborreliosis and only 7 (6%) of 118 control patients (P < .001). This pattern of CSF gelatinase activity may be a useful marker for neuroborreliosis.

Treatment of occupational cramp with botulinum toxin: diffusion of toxin to adjacent noninjected muscles.

Over a 5-year period, 40 patients, 11 with musician's and 29 with writer's cramp, were treated with botulinum toxin A using a precise injection technique in which the hollow-bore electromyography (EMG) needle was positioned by both standard EMG and by muscle twitch evoked by stimulating current passed through it. Moderate to complete improvement in dystonia occurred in 28 patients (70%) after the first injection and in 34 patients (85%) after the second injection with better outcome in nonmusicians than in musicians. Of note, weakness of uninjected muscles, immediately adjacent to those injected, was found in 25/40 patients (63%). The most common patterns of toxin spread were from flexor digitorum sublimis to profundus, extensor carpi radialis to extensor digitorum communis, and extensor indicis proprius to extensor pollicis brevis. Spread to, and weakness of, adjacent uninjected muscles was a major factor contributing to suboptimal outcome in 6/39 (15%) such patients.

Alcohol inhibits cell-cell adhesion mediated by human L1.

Mental retardation, hydrocephalus, and agenesis of the corpus callosum are observed both in fetal alcohol syndrome (FAS) and in children with mutations in the gene for the cell adhesion molecule L1. We studied the effects of ethanol on cell-cell adhesion in mouse fibroblasts transfected with human L1. L1-transfected fibroblasts exhibited increased cell-cell adhesion compared with wild-type or vector-transfected controls. Ethanol potently and completely inhibited L1-mediated adhesion both in transfected L cells and NIH/3T3 cells. Half-maximal inhibition was observed at 7 mM ethanol, a concentration achieved in blood and brain after ingesting one alcoholic beverage. In contrast, ethanol did not inhibit the adhesion of fibroblasts transfected with vector alone or with N-CAM-140. L1-mediated cell-cell adhesion was inhibited with increasing potency by n-propanol and n-butanol, but was not inhibited at all by n-alcohols of 5 to 8 carbons, acetaldehyde, or acetate, suggesting that ethanol interacts directly with a small hydrophobic pocket within L1. Phenylalanine, teratogenic anticonvulsants, and high concentrations of glucose did not inhibit L1-mediated cell-cell adhesion. Ethanol also inhibited potently the heterotypic adhesion of rat cerebellar granule cells to a monolayer of L1-transfected NIH/3T3 cells, but had no effect on their adhesion to N-CAM-140 or vector-transfected NIH/3T3 cells. Because L1 plays a role in both neural development and learning, ethanol inhibition of L1-mediated cell-cell interactions could contribute to FAS and ethanol-associated memory disorders.

Ulnar neuropathy and dystonic flexion of the fourth and fifth digits: clinical correlation in musicians.

Peripheral nerve lesions are sometimes associated with focal dystonia. We diagnosed ulnar neuropathy in 28 of 73 (40%) cases of occupational cramp in musicians. Focal slowing of ulnar conduction across the elbow was identified in 15 of 19 (79%) patients using the near nerve technique and in 5 of 17 (29%) patients using surface recording. Ulnar neuropathy was present in 24 of 31 (77%) cases with flexion dystonia of the fourth and fifth digits and only 4 of the remaining 42 (10%) cases with other patterns of focal dystonia. Focal dystonia improved in 13 of 14 patients whose ulnar neuropathy improved and appeared or worsened in 2 patients following ulnar nerve injury. These data, together with our recent observation of a dystonic pattern of antagonist bursting in patients with isolated ulnar neuropathy (Muscle Nerve 1995, 18:606-611), suggest that ulnar neuropathy may initiate or sustain a specific dystonia, flexion of the fourth and fifth digits, by inducing a central disorder of motor control.

Haplotype analysis at the DYT1 locus in Ashkenazi Jewish patients with occupational hand dystonia.

Genetic haplotypes at five marker loci that are closely linked to the DYT1 gene on chromosome 9q were determined in 10 Ashkenazi Jewish patients with focal hand dystonia (eight with musician's cramp, two with writer's cramp). The founder haplotype associated with > 90% of cases generalized dystonia in the Ashkenazi Jewish population could not be constructed from any of the twenty chromosomes. Potential haplotypes were determined, and no common haplotype was discerned in these patients. These findings argue against a role for the founder mutation in the DYT1 gene in the etiology of occupational hand dystonia in this ethnic group. Further, if the DYT1 gene is involved in these later onset dystonias, there is no evidence for a common mutation in the Ashkenazic Jewish population. It appears that excessive, repetitive use, possibly in combination with ulnar neuropathy, may serve as the inciting cause of some focal dystonias.

Does ulnar neuropathy predispose to focal dystonia?

We have observed a high incidence of ulnar neuropathy in musicians with dystonic flexion of the ipsilateral little and ring fingers. To investigate the relationship between ulnar neuropathy and focal dystonia, we compared the patterns of surface EMG activity in extensor digitorum communis (EDC4) and flexor digitorum superficialis (FDS4) during tapping of the ring finger in normal controls and patients with ulnar neuropathy or focal dystonia. Ten of 10 normal subjects exhibited well-formed alternating EMG bursts in EDC4 and FDS4 separated by clear silent periods. Seven of 7 patients with dystonic flexion of the little and ring fingers showed loss of silent periods between poorly formed bursts in FDS or EDC. Surprisingly, 9 of 10 patients with ulnar neuropathy showed burst pattern abnormalities qualitatively similar to those observed in the dystonic patients. These data suggest that ulnar neuropathy alters the execution of a motor task involving multiple peripheral nerves.

Time course of phosphorylated CREB and Fos-like immunoreactivity in the hypothalamic supraoptic nucleus after salt loading.

Phosphorylation of the cAMP response element binding protein (CREB) precedes the induction of immediate early gene expression. Using antibodies that distinguish CREB from phosphorylated CREB (PCREB), we studied the appearance of PCREB-like immunoreactivity (PCREB-LI) and Fos-LI in the hypothalamic supraoptic nucleus (SON) of rats treated with hypertonic or normal saline and uninjected controls. Fifteen minutes after injection, increased numbers of PCREB-LI cells were seen in both normal and hypertonic saline-treated rats as compared with uninjected controls. Forty-five minutes after injection, levels of c-fos mRNA in the SON were elevated in hypertonic saline-treated rats as compared with normal saline-treated rats, and were minimally detectable in uninjected rats. At this time period, the hypertonic saline-treated rats showed increased number of Fos-LI cells in the SON, whereas normal saline-treated rats showed little or no Fos-LI cells. The discrepancy between levels of PCREB-LI and c-fos mRNA suggests that injection of hypertonic saline may activate additional transcriptional factors besides CREB. The lack of Fos-LI in the presence of modest increases in c-fos mRNA in normal saline-treated rats implies that levels of c-fos mRNA must exceed a threshold before increases in Fos-LI cells are detectable by immunostaining of the SON. Such a threshold might permit neuronal cells to activate diverse genes, through phosphorylation of CREB, without inducing the constellation of Fos-responsive genes.

Neuroprotective effect of human osteogenic protein-1 in a rat model of cerebral hypoxia/ischemia.

Possible neuroprotective actions of osteogenic protein-1 (OP-1) were evaluated in a rat model of cerebral hypoxia/ischemia. Intraperitoneal injection of 50 micrograms of OP-1 prior to bilateral carotid ligation and transient hypoxia in 12-day-old rats reduced cerebral infarct area from 44.8 +/- 3.3% in vehicle-injected controls to 29 +/- 4.9%. Treatment of 14-day-old rats with 20 micrograms of OP-1 1 h after hypoxia reduced mortality from 45% to 13%. OP-1 may represent a novel class of neuroprotective agents.

Idiopathic striopallidodentate calcification with prominent supranuclear abnormality of eye movement.

We present a 63-year-old man with idiopathic striopallidodentate calcifications who exhibited a marked supranuclear defect of eye movement in addition to an extrapyramidal movement disturbance and dementia. The resulting clinical disorder resembled progressive supranuclear palsy.

Ethanol inhibits neural cell-cell adhesion.

Gestational exposure to ethanol causes defects in neuronal migration, fasciculation, and synaptogenesis, developmental events that depend on the patterned expression and function of cell adhesion molecules (CAMs). Recombinant human osteogenic protein-1 (hOP-1) increases cell-cell adhesion and promotes cell clustering in proliferating neuroblastoma x glioma hybrid NG108-15 cells by strongly inducing N-CAM and L1. Here we show that concentrations of ethanol achieved during social drinking inhibit hOP-1-induced cell clustering without affecting cell proliferation, the induction and cell surface expression of N-CAM and L1, or the alternative splicing and sialylation of N-CAM. This inhibition was reproduced by other alcohols in proportion to their chain length, but not by teratogenic anticonvulsants or phenylalanine. Ethanol inhibition of hOP-1 morphogenesis was inversely proportional to the concentration of hOP-1 and, hence, to the levels of N-CAM and L1. Low concentrations of ethanol (IC50 5-10 mM) inhibited cell-cell adhesion in hOP-1-treated cells, and this action too was reproduced more potently by propanol and butanol. Ethanol may perturb brain and skeletal development by inhibiting CAM-mediated cell-cell interactions.