Profile of the regions on the alpha-chain of human acetylcholine receptor recognized by autoantibodies in myasthenia gravis.

Eighteen synthetic overlapping peptides encompassing the entire extracellular part (residues alpha 1-210) of the alpha-chain of human acetylcholine receptor (AChR) and a 19th peptide (residues alpha 262-276) corresponding to an extracellular connection between two transmembrane regions were prepared and used for the measurement, by solid-phase radioimmunoassay, of the binding of autoantibodies in plasma from myasthenia gravis (MG) patients. Autoantibodies were found to recognize only a limited number of the synthetic peptides. The regions recognized resided predominantly within the areas alpha 10-30, alpha 111-145 and alpha 175-198 and, less frequently, region alpha 45-77. Differences in the recognition profile of the peptides from patient to patient indicated that the autoantibody responses were under genetic control. However, by using a mixture of the appropriate peptides, it was possible to determine autoantibodies in all 15 myasthenia sera and to distinguish between these, normal human sera and other neurological or autoimmune diseases. The mapping of the continuous antigenic regions recognized by autoantibodies on the alpha-chain of human AChR has permitted a comparison of the regions recognized by autoantibodies and autoimmune T-cells from the same donor. It also provided a peptide-based direct antibody binding method for diagnosis of MG.

Possible role of the gamma-aminobutyric acid-A receptor system in the timing of the proestrous luteinizing hormone surge in rats.

To examine the role of the gamma-aminobutyric acid-A receptor-mediated system in the timing of the proestrous LH surge, we observed the free running activity rhythm and the timing of the LH surge simultaneously in blinded cycling female rats. Blood samples were obtained from unanesthetized freely moving rats through an intraatrial cannula. Five hours after the activity offset on the day of proestrus, bicuculline methiodide (BIC; 50 mg/kg x h) or saline was infused i.v. for 3 h into the freely moving rats. In the BIC group, the peak time of the surge occurred at 7.9 +/- 0.2 h after the activity offset, with a significant advance compared to the peak time in the saline group (i.e. 9.9 +/- 0.4 h), but neither BIC nor saline induced a significant phase shift in the circadian activity rhythm. We found that the infusion of BIC on the subjective morning of the proestrous day dissociates the timing of the LH surge from the circadian activity rhythm in rats.

Novel ipriflavone receptors coupled to calcium influx regulate osteoclast differentiation and function.

Ipriflavone (7-isopropoxyisoflavone) is an effective antiresorptive agent used to treat osteoporosis. However, the mechanism of its action on osteoclasts and their precursor cells is not well understood. To determine whether the mechanism involves direct effects on osteoclasts or their precursors, we examined the effects of ipriflavone on cytosolic free calcium ([Ca2+]i) in osteoclasts and their precursors and measured specific binding of 3H-labeled ipriflavone. Highly purified chicken osteoclast precursors, which spontaneously differentiate into multinucleated osteoclasts in 3-6 days, were loaded with fura-2, and the subcellular [Ca2+]i distribution was monitored by videoimaging. Ipriflavone induced a rapid increase in [Ca2+]i followed by a sustained elevation [EC50 = 5 x 10(-7) M, 263 +/- 74 nM (SE) (n = 8) above basal levels, by 10(-6) M ipriflavone, sustained phase]. The responses were the same in differentiated chicken osteoclasts and isolated rabbit osteoclasts. An influx of extracellular Ca2+ is likely to be responsible for the ipriflavone-induced change in [Ca2+]i because the response was abolished by 0.5 mM LaCl3, or by Ca-free medium containing EGTA. Moreover, high [Ca2+]i levels were detected adjacent to the cell membrane after ipriflavone addition. Ipriflavone induced Ca influx mainly through dihydropyridine-insensitive Ca2+ channels, because nicardipine (10(-7)M) and verapamil (10(-7)M) had no effects on ipriflavone-induced [Ca2+]i responses. [3H]Ipriflavone binding studies indicated the presence of specific ipriflavone binding sites (two classes), both in precursor cells [dissociation constant (Kd), 7.60 x 10(-8)M, 2.67 x 10(-6)M] and in mature osteoclasts (Kd, 4.98 x 10(-8)M, 3.70 x 10(-6)M). Specific ipriflavone binding was not displaced by various modulators of avian osteoclast function, such as estradiol (10(-8)M) or retinoic acid (10(-6)M), indicating that ipriflavone receptors differ from the receptors for these Ca-regulating hormones. The fusion of osteoclast precursor cells was significantly inhibited by ipriflavone, which led to dose-dependent inhibition of bone resorption and tartrate-resistant acid phosphatase activity. Novel specific ipriflavone receptors that are coupled to Ca2+ influx were demonstrated in osteoclasts and their precursor cells. These ipriflavone receptors may provide a mechanism to regulate osteoclast differentiation and function.

Decreased type IV collagen of skin and serum in patients with amyotrophic lateral sclerosis.

OBJECTIVE: To study type IV collagen of skin and serum in patients with ALS. BACKGROUND: Collagen abnormalities of skin have been reported in ALS patients. However, little is known concerning type IV collagen in ALS. METHODS: We studied type IV collagen immunoreactivity of skin and measured serum levels of the 7S fragment of the N-terminal domain of type IV collagen (7S collagen) in patients with ALS and control subjects. RESULTS: The basement membrane as well as blood vessels of skin in ALS patients was weakly positive for type IV collagen as compared with those of diseased control subjects. This weak immunostaining became more pronounced as ALS progressed. The optical density for type IV collagen immunoreactivity in ALS patients was significantly lower (p < 0.001) than in diseased control subjects and was significantly decreased with duration of illness (r = -0.85, p < 0.01). Serum 7S collagen levels in patients with ALS were significantly decreased (p < 0.01) as compared with those in diseased and healthy control subjects and were negatively and significantly associated with duration of illness (r = -0.81, p < 0.001). There was an appreciable positive correlation between concentrations of serum 7S collagen and the density for type IV collagen immunoreactivity in ALS patients (r = 0.81, p < 0.02). CONCLUSIONS: These data suggest that a metabolic alteration of type IV collagen may take place in the skin of ALS patients and that the decreased levels of serum 7S collagen may reflect a decreased type IV collagen immunoreactivity of skin in patients with ALS.

Loss of serotonin-containing neurons in the raphe of patients with myotonic dystrophy: a quantitative immunohistochemical study and relation to hypersomnia.

Hypersomnia occurs frequently in patients with myotonic dystrophy (MyD). We performed a quantitative immunohistochemical study of serotonin (5-HT)-containing neurons linked to hypersomnia in the dorsal raphe nucleus (DRN) and the superior central nucleus (SCN) in 8 patients with MyD, 5 of whom showed hypersomnia, and in 12 age-matched controls. The densities of 5-HT neurons in the DRN and the SCN were significantly lower in MyD patients with hypersomnia than in MyD patients without hypersomnia and controls. These data suggest that the loss of 5-HT neurons of the DRN and the SCN is associated with the presence of hypersomnia in MyD.

Loss of catecholaminergic neurons in the medullary reticular formation in myotonic dystrophy.

OBJECTIVE: To clarify the possible relation between the extent of involvement of catecholaminergic neurons and the presence of alveolar hypoventilation in patients with myotonic dystrophy (MyD). BACKGROUND: Respiratory insufficiency has been reported frequently in MyD patients. Recent data support the hypothesis that this respiratory failure results from a primary dysfunction of the CNS. METHODS: The authors performed a quantitative immunoreactive study of tyrosine hydroxylase immunoreactive (TH+) neurons linked to hypoventilation in the dorsal central medullary nucleus (DCMN), the ventral central medullary nucleus (VCMN), and the subtrigeminal medullary nucleus (SMN)--where the autonomic respiratory center is thought to be located--in eight MyD patients and in 10 age-matched control subjects. Alveolar hypoventilation of the central type was present in three of the MyD patients but not in the remaining MyD patients or the control subjects. RESULTS: The densities of TH+ neurons of the DCMN, the VCMN, and the SMN in MyD patients with hypoventilation were significantly lower than in those without hypoventilation (p < 0.02, p < 0.01, and p < 0.01, respectively) and control subjects (p < 0.01, p < 0.01, and p < 0.01, respectively). CONCLUSIONS: These data suggest that the loss of TH+ neurons of the DCMN, the VCMN, and the SMN is associated with the presence of hypoventilation in MyD and may be an important feature of MyD.

Neuronal loss in the medullary reticular formation in myotonic dystrophy: a clinicopathological study.

Respiratory insufficiency occurs frequently in patients with myotonic dystrophy (MyD). We have performed a quantitative study of neurons linked to respiratory function in the dorsal central medullary nucleus (DCMN), the ventral central medullary nucleus (VCMN), and the subtrigeminal medullary nucleus (SMN) in seven patients with MyD and eight age-matched controls. Alveolar hypoventilation of the central type occurred in three of the MyD patients but not in the remaining MyD patients or controls. The densities of neurons of the DCMN, the VCMN, and the SMN in MyD patients with hypoventilation were significantly lower than in MyD without hypoventilation and controls. These data suggest the neuronal loss of the DCMN, VCMN, and SMN is associated with the presence of hypoventilation in MyD and may be an important feature of MyD.

Decreased expression of full-length mRNA for cBCD541 does not correlate with spinal muscular atrophy phenotype severity.

Spinal muscular atrophy (SMA) is characterized by degeneration of spinal cord anterior horn cells and muscular atrophy and has three phenotypes based on clinical severity and age of onset. One of the responsible genes for SMA is the survival motor neuron (SMN) gene, which is homozygously absent or interrupted in more than 90% of SMA patients. The cBCD541 (BCD) gene is a highly homologous copy of the SMN gene, which has a single synonymous transition in the coding region and may compensate for the loss of the SMN gene. To evaluate the effects of the BCD gene expression on the phenotypes of SMA, we examined lymphocyte mRNA from 9 SMA patients lacking the SMN gene, 10 asymptomatic parents, and 15 control subjects. We amplified mRNA fragments containing exon 7 of the SMN or BCD genes using reverse transcription-polymerase chain reaction since the transcript lacking exon 7 encodes a putative protein with a different C-terminal end. We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript as an internal control, and the relative expression level of the SMN or BCD gene was shown as the ratio of SMN or BCD transcript to GAPDH transcript (S/G ratio). The mean S/G ratios of the patients were significantly lower than that of the parents and controls. However, among the patients examined in this study, there was no relationship between the S/G ratios and phenotypes of SMA. The results showed that the BCD gene expression was not related to the phenotypes of SMA. Furthermore, there was an overlap between the S/G ratios in patients and controls. As our discrimination study showed that the S/G ratio reflected the expression of the BCD transcripts in patients and the SMN transcripts in controls, this finding suggested that the BCD gene expression per se does not compensate for the loss of the SMN gene.

Pentobarbital stimulates the activity of the GnRH pulse generator interacting with opioid neurons in rats in proestrus.

In order to investigate the possibility that i.p. injection of pentobarbital sodium (PB, 32 mg/kg bw) potentiates the GnRH pulse generator activity, effects of i.v. infusions of an opiate receptor antagonist naloxone (NAL, 2 mg/h) on the pulsatile LH secretion were compared in saline (SAL)- and PB-injected rats in proestrus and diestrus 1. In SAL-injected rats in proestrus, NAL infusions significantly increased both the frequency and amplitude of LH pulses, and also the overall mean LH concentration. In PB-injected rats in proestrus, all the parameters of the pulsatile LH secretion were similar to those in SAL-injected rats in proestrus. The NAL infusion in PB-injected rats caused an increase in the frequency, but it was similar to that in SAL-injected rats. But, increases in the amplitude and the overall mean LH observed during NAL infusions in PB-injected rats were greater than in SAL-injected rats. In SAL-injected rats in diestrus 1, NAL infusions increased all the parameters, as in rats in proestrus. In PB-injected rats in diestrus 1, LH secretion was severely suppressed. NAL infusions recovered the pulsatile LH secretion, but the frequency and the overall mean LH of the secretion were smaller than those obtained during NAL infusions in SAL-injected rats. In addition, characteristic increases in the MUA (volleys), which occur in association with the initiation of an LH pulse and thus are considered to represent an increased activity of the GnRH pulse generator, appeared more frequently during NAL infusions in PB-injected rats in proestrus than in SAL-injected rats. These results suggest that the GnRH pulse generator in rats in proestrus, but not in rats in diestrus 1, is refractory to PB and further is potentiated by PB in the response to NAL. Together with the fact that this dosage of PB blocks the surge of LH secretion in rats in proestrus, the concept of the existence of separate neuronal mechanisms responsible for the surge and pulsatile secretion of LH are supported.

Fos expression by naloxone in LHRH neurons of the mediobasal hypothalamus and effects of pentobarbital sodium in the proestrous rat.

Because Fos is thought to be induced in neurons that are activated, we examined whether luteinizing hormone-releasing hormone (LHRH) neurons expressed Fos protein when they were stimulated by an opioid receptor antagonist naloxone (NAL), expecting to identify LHRH neurons which are regulated by opioid neurons directly or indirectly. Further, we examined whether an ovulation-blocking dosage of pentobarbital sodium (PB) would affect the NAL-induced Fos expression. Female rats were infused with naloxone (5 mg/kg/h) for 90 min (10.00-11.30) in the morning of proestrus, during which infusion blood sampling was done, and were killed by i.v. injection with an overdose of PB at 11.30-12.00. Dual immunoperoxidase/immunofluorescence staining for both Fos and LHRH revealed that some LHRH immunoreactive (ir) neurons in the forebrain expressed Fos-ir, associated with an increase in serum LH concentrations, but little co-localization was found in rats in proestrus which were infused with saline as the control. The proportion of LHRH-ir neurons which expressed Fos-ir was about 35-62% in the caudal part of the forebrain including the mediobasal hypothalamus, and this was larger than that (10%) in the rostral part of the forebrain including the preoptic area. PB injection (32 mg/kg bw, i.p.) 15 min prior to the beginning of NAL infusion significantly enhanced the increase in LH secretion due to NAL, and also enhanced Fos-ir expression in LHRH-ir neurons. Together with the well-established fact that PB blocks the LHRH surge generator and our previous findings that NAL stimulates the LHRH pulse generator even in the PB-blocked proestrous rat, these results strongly suggest that the LHRH pulse generator exists in the mediobasal hypothalamus which contains LHRH neurons that are responsive to NAL and express Fos protein.

LHRH pulse generator is stimulated by naloxone in the pentobarbital-blocked proestrous rat.

Previous studies by us and others led us to hypothesize that there are separate LHRH pulse and surge generators in the rat brain. The present study was designed to detect the activity of LHRH pulse generator by checking changes in LH secretion and the multiunit activity (MUA) of the arcuate-median eminence region of the hypothalamus during infusions of naloxone (NAL, 2 mg/h) in the proestrous rat in which the LHRH surge generator activity was blocked by pentobarbital sodium (PB, 32 mg/kg bw, ip). The animals were subjected to blood sampling in the morning (1000-1300 h) or afternoon (1400-1700), and injected with PB at 09.45 or 13.45, respectively. During saline infusions in the rat given PB injection at either 09.45 or 13.45, serum LH levels were low but fluctuated significantly, suggesting a pulsatile secretion in either the morning or the afternoon period. The pulse intervals were an average 28.2 min in the morning and 42.2 min in the afternoon. NAL infusions decreased the pulse interval significantly, to 22.0 min in the morning and to 27.0 min in the afternoon. In the electrophysiological experiment, characteristic increases in the MUA (volleys), which occur in association with the initiation of an LH pulse and therefore are considered to represent an increased activity of the LHRH pulse generator, appeared during NAL (5 mg/h) infusions in either the morning or the afternoon. These results strongly suggest that separate LHRH pulse and surge generators exist in the brain, and that, even during the critical period of proestrus, the activity of LHRH pulse generator is disclosed by PB, which, on the other hand, arrests the surge generator.

Thymoma: tumour type related to expression of epidermal growth factor (EGF), EGF-receptor, p53, v-erb B and ras p21.

As clinicopathological features may not be sufficient to predict the progression of thymoma, we have carried out what we believe to be the first immunohistochemical study describing the relationship between the different types of thymoma and the tumour stage, on the one hand, and the expression of epidermal growth factor (EGF), EGF-receptor (EGFR), p53, v-erb B and ras p21, on the other. The positive rates versus histological types and Masaoka's clinical stages in the 47 cases were as follows: p53 (non-invasive thymoma: 41.7%; malignant thymoma category I: 82.4%; malignant thymoma category II: 83.3%), EGF (non-invasive thymoma: 4.2%; malignant thymoma category I: 11.8%; malignant thymoma category II: 33.3%) and EGFR (non-invasive thymoma: 8.3%; malignant thymoma category I: 35.3%; malignant thymoma category II: 66.7%); p53 (stages I and II: 51.7%; stages III and IV: 77.8%), EGF (stages I and II: 3.4%; stages III and IV: 22.2%) and EGFR (stages I and II: 13.8%; stages III and IV: 44.4%). These data suggest that p53 may be implicated in the initial stages of tumorigenesis and that increased expression of EGF and EGFR may play a role in thymoma progression.

Bicuculline infusion advances the timing of Fos expression in LHRH neurons in the preoptic area of proestrous rats.

The effect of bicuculline (BIC) on Fos expression in lutenizing hormone-releasing hormone (LHRH) neurons was examined by immunocytochemistry. Proestrous rats were infused with saline (SAL 1400, n = 4) or BIC (BIC 1400, n = 5) for 3 h (10.00-13.00 h) and were killed at 1400 h. Three control rats (SAL 1700), which received saline infusion, were killed at 1700 h. In both the BIC 1400 group and the SAL 1700 group, many LHRH neurons in the preoptic area expressed Fos, but those in the SAL 1400 group did not. The distribution and proportion of LHRH neurons expressing Fos in the BIC 1400 group were identical to those in the SAL 1700 group. We conclude that GABAergic neurons play a critical role in inducing LH surge by controlling LHRH neuronal activity.

Electrophysiological study on the corticoreticular projection neurons of the cat.

Thirty-two motor cortical neurons of the cat which project on medullary reticular formation (RF) were electrophysiologically classified into 3 groups, i.e., 22 slow PT-RF neurons (collateral projections to RF from slow pyramidal tract (PT) neurons), 6 fast PT-RF neurons (those from fast PT neurons) and 4 RF (non-PT) neurons without any relation to PT neurons. Firings of slow PT-RF and RF (non-PT) neurons elicited by thalamic stimulation showed close correlation to the surface negative component of cortical evoked potentials. It was suggested that activities of these neurons are controlled mainly by excitatory thalamic inputs onto the distal and superficial part of their apical dendrites.

The inhibitory input from the substantia nigra to the mediodorsal nucleus neurons projecting to the prefrontal cortex in the cat.

The effects of stimulation of the substantia nigra pars reticulata (SNr) on the neurons of the mediodorsal nucleus (MD) projecting to the prefrontal cortex (PF) were studied in cats. The MD neurons projecting to the ventral part of the PF tended to be located in the ventral part of the MD, while those projecting to the dorsal part of the PF in the dorsal part. Spontaneous discharges of 30/57 tested MD neurons were suppressed by SNr stimulation at a latency ranging from 2 to 15 ms. The latency of the suppression corresponded well to that of antidromic responses of SNr neurons elicited by MD stimulation (from 1.4 to 14.0 ms). Intracellular recordings in a few MD neurons showed IPSP by SNr stimulation. The SNr is considered to exert an inhibitory effect on the MD neurons projecting to the PF.

The distribution of the globus pallidus neurons with input from various cortical areas in the monkeys.

Single neuron activities of the globus pallidus were recorded in awake monkeys. Electrical stimulation of various cortical areas (the prefrontal, premotor, supplementary motor and arcuate premotor areas, and the motor cortex) inhibited spontaneous discharge of pallidal neurons. This inhibitory response was considered to be mediated through the striatum. Considerable number of pallidal neurons responded to the stimulation of more than one cortical areas. However, the neurons receiving inputs from the motor cortex and/or arcuate premotor area and those receiving inputs from the prefrontal, premotor and/or supplementary motor areas were segregated. The former neurons were located ventrolaterally in the caudal part of the globus pallidus, while the latter ones were located dorsomedially in the rostral part of the globus pallidus.

The distribution of neurons in the substantia nigra pars reticulata with input from the motor, premotor and prefrontal areas of the cerebral cortex in monkeys.

We investigated the distribution of neurons in the substantia nigra pars reticulata (SNr) which received cortical input. The activities of single SNr neurons were studied extracellulary in awake monkeys. SNr neurons showed excitatory and/or inhibitory responses to cortical stimulation. These responses were considered to be mediated by the subthalamic nucleus and striatum, respectively. The neurons receiving inhibitory input from the motor, premotor and supplementary motor areas (Motor-related cortical areas) were located in the lateral part of the SNr, whereas those with input from the medial, dorsal and orbital areas of the prefrontal cortex (PFmdo) were frequently found in the rostro-medial part of this nucleus. SNr neurons with inhibitory input from the ventral periprincipal area (PSv) were mainly distributed in the intermedio-lateral portion, with some degree of overlap with input from other cortical areas. The distribution of the excitatory input was almost similar to that of inhibitory one, but the excitatory input from the PSv was much stronger than that from the PFmdo. Some SNr neurons receiving cortical input were proved to project to the thalamus. Our results support the existence of several parallel organization of the cortico-basal ganglia loop circuits [G.E. Alexander, M.R. DeLong, P.L. Strick, Parallel organization of functionally segregated circuits linking basal ganglia and cortex, Ann. Rev. Neurosci., 9, 1986, pp. 357-381.], but interaction between the loops can not be ignored.

Discharge patterns of pallidal neurons with input from various cortical areas during movement in the monkey.

Activities of pallidal neurons were studied in awake monkeys which were implanted with stimulating electrodes in the various cortical areas in the frontal lobe. Cortical inputs to each pallidal neuron were examined by inhibitory responses to stimulation through these electrodes. Discharge patterns of pallidal neurons were observed during performance of the reaction-time, delayed go/no-go discrimination and self-paced movement tasks. Most of the pallidal neurons with input from the arm of the motor cortex changed their firing rate in close relation to the arm movement (movement-related activity). Many of the neurons with input from the supplementary motor and cingulate areas showed sustained changes in discharge rate during the delay period in addition to movement-related activity. Most of the neurons with input from prefrontal cortex responded to light stimulus.

Cervical input to vestibulocollic neurons.

Extracellular responses of vestibulospinal neurons projecting to neck segments (vestibulocollic neurons) to stimulation of cervical afferents were studied in decerebellate cats anesthetized with chloralose or decerebrated intercollicularly. These neurons were antidromically activated by microstimulation in the C2 ventral horn ipsilateral or contralateral to the vestibular nuclei. Contralateral neck afferents produce in vestibulocollic neurons the same responses they produce in other vestibulo-spinal neurons, although the response (early excitation, inhibition) which predominates varies with type of preparation. In intercollicular decerebrate cats, the main effect of stimulating the contralateral C2 ganglion was inhibition (latency greater than or equal to 6 msec), often followed by late excitation. Early excitation, at a latency less than 6 msec, was rarely seen, although it occurred in half the vestibulospinal neurons projecting to the cervical enlargement. In contrast to the results in decerebrate cats, stimulation of the contralateral C2 ganglion produced early excitation in over one-third of the vestibulocollic neurons studied in chloralose-anesthetized cats. Results on vestibulocollic neurons were similar whether the cells projected ipsilaterally or contralaterally, or whether or not the cells also projected to the cervical enlargement (branching neurons). The influence of cervical afferents on vestibulocollic neurons demonstrates supraspinal paths by which cervical afferents may act upon the neck and might modify vestibular reflexes of the neck.

Acute autonomic and sensory neuropathy: a case report.

A female patient with acute autonomic and sensory neuropathy is described. Urinary disturbance developed rapidly and was followed by orthostatic syncope, absence of lacrimation, salivation and sweating, and sensory impairment. Muscle strength had been consistently normal despite diffuse muscular atrophy. Marked decrease in the number of small myelinated and unmyelinated fibres was revealed in biopsied sural nerve. Eighteen months after the onset, her autonomic symptoms have partially improved.