In situ hybridization study of the distribution of choline acetyltransferase mRNA and its splice variants in the mouse brain and spinal cord.

Choline acetyltransferase is the enzyme that catalyzes the biosynthesis of the neurotransmitter acetylcholine. Seven types of mRNA for choline acetyltransferase that differ in the 5'-noncoding region are transcribed from the cholinergic gene locus from different promoter regions and produced by alternative splicing in the mouse. Digoxigenin-labeled riboprobes and in situ hybridization histochemistry were used to investigate the expression of N1, R1, R2, R3, R4 and total choline acetyltransferase mRNA in the mouse CNS. The relative levels of choline acetyltransferase transcripts differed dramatically in distinct subdivisions of the mature cholinergic nervous system. Neurons hybridizing with antisense riboprobes for all of the five investigated splice variants (R1, R2, R3, R4 and N1) as well as those hybridizing with riboprobe for the common protein-coding region were found in a number of expected regions in the CNS. They include the basal forebrain, striatum, pontomesencephalic tegmentum, motor and autonomic nuclei of the brainstem, and spinal cord. Neurons with a moderate to very high level of expression of R1 and R2 splice variants were distributed in both the forebrain and brainstem nuclei. On the other hand, R3, R4 and N1 splice variants revealed a moderate to high level of expression in the brainstem motor and autonomic nuclei and ventral and lateral horns of the spinal cord compared to a low expression level in forebrain cholinergic structures. No expression of the N1, R1, R2, R3 and R4 splice variants was detectable in the neurons of the cerebral cortex, hippocampus and medial habenular nucleus. With the riboprobe for the common protein-coding region, the neurons of the medial habenular nucleus could be labeled at high level, while intrinsic cortical neurons were labeled at low level. Hippocampus revealed no significant hybridization for total choline acetyltransferase mRNA. These findings strongly suggested that: (1) R1 and R2 were the major splice variants expressed in the neurons of forebrain nuclei; (2) R1, R2, R3, R4 and N1 splice variants were almost equally expressed in the brainstem motor and autonomic nuclei and ventral and lateral horns of the spinal cord; (3) inferring from a paucity of other isoforms, M type choline acetyltransferase mRNA is a splice variant predominantly expressed in the cerebral cortex and medial habenular nucleus.

Target velocity signals of visual tracking in vermal Purkinje cells of the monkey.

Discharges of Purkinje cells were recorded from the vermis, lobules VI and VII, of a monkey trained to track a visual target. When the monkey tracked a sinusoidally oscillating target, cellular activity changes in phase with the velocity signal of the eye movement. When the monkey fixated a stationary point, almost identical modulation in activity occurred, reflecting the velocity signal of the motion of the retinal image of the target. The data suggest that the vermis participates in the control of smooth pursuit eye movements by providing the oculomotor system with the actual target velocity information which is the sum of eye velocity and retinal image velocity signals.

Production of U beam from RIKEN 18 GHz electron cyclotron resonance ion source.

For the RIKEN radio isotope factory (RIBF) project, we produced the multicharged uranium beam with two methods. To produce lower charge state U ion beams (14+-20+) we used the UF(6) gas as an ionized gas. The typical beam intensity of U(14+-20+) was 2-1 particle microA at the extraction voltage of 14 kV. To produce higher charge state U ion beam (U(35+)), we chose the sputtering method. The beam intensity was 70 particle nA at the extraction voltage of 5.4 kV. Using this method, we successfully produced multicharged U beam continuously for one month without break for RIBF commissioning.

Vesicular acetylcholine transporter-immunoreactive axon terminals enriched in the pontine nuclei of the mouse.

Information to the cerebellum enters via many afferent sources collectively known as precerebellar nuclei. We investigated the distribution of cholinergic terminal-like structures in the mouse precerebellar nuclei by immunohistochemistry for vesicular acetylcholine transporter (VAChT). VAChT is involved in acetylcholine transport into synaptic vesicles and is regarded as a reliable marker for cholinergic terminals and preterminal axons. In adult male mice, brains were perfusion-fixed. Polyclonal antibodies for VAChT, immunoglobulin G-peroxidase and diaminobenzidine were used for immunostaining. In the mouse brain, immunoreactivity was seen in almost all major cholinergic cell groups including brainstem motoneurons. In precerebellar nuclei, the signal could be detected as diffusely beaded terminal-like structures. It was seen heaviest in the pontine nuclei and moderate in the pontine reticulotegmental nucleus; however, it was seen less in the medial solitary nucleus, red nucleus, lateral reticular nucleus, inferior olivary nucleus, external cuneate nucleus and vestibular nuclear complex. In particular, VAChT-immunoreactive varicose fibers were so dense in the pontine nuclei that detailed distribution was studied using three-dimensional reconstruction of the pontine nuclei. VAChT-like immunoreactivity clustered predominantly in the medial and ventral regions suggesting a unique regional difference of the cholinergic input. Electron microscopic observation in the pontine nuclei disclosed ultrastructural features of VAChT-immunoreactive varicosities. The labeled bouton makes a symmetrical synapse with unlabeled dendrites and contains pleomorphic synaptic vesicles. To clarify the neurons of origin of VAChT-immunoreactive terminals, VAChT immunostaining combined with wheat germ agglutinin-conjugated horseradish peroxidase retrograde labeling was conducted by injecting a retrograde tracer into the right pontine nuclei. Double-labeled neurons were seen bilaterally in the laterodorsal tegmental nucleus and pedunculopontine tegmental nucleus. It is assumed that mesopontine cholinergic neurons negatively regulate neocortico-ponto-cerebellar projections at the level of pontine nuclei.

Expression of glutamine synthetase and cell proliferation in human idiopathic epiretinal membrane.

BACKGROUND/AIM: The mechanisms of the cellular origin and cell proliferation in the idiopathic epiretinal membrane (ERM) are unsolved. The aim of this study was to examine the expression of cell cycle related molecules and glutamine synthetase (GS), which is expressed in Müller cells and their processes, in ERM tissues. METHODS: The ERMs were surgically removed using pars plana vitrectomy. Formalin fixed, paraffin embedded ERM tissues were analysed by immunohistochemistry with anti-cyclin D1, p27 (KIP1), proliferating cell nuclear antigen (PCNA), and GS antibodies. RESULTS: The histopathological findings showed that all the ERMs consisted of oval or spindle mononuclear cells with thin collagen-like tissues. Immunoreactivity for GS was detected in collagen-like tissues of ERM, presenting a continuous, isodense pattern. GS immunopositive cells in all cases expressed PCNA in their nuclei. Nuclear immunoreactivity for cyclin D1 was noted in the ERM constituent cells, whereas p27 (KIP1) positive nuclei were not detected. CONCLUSION: Cyclin D1 and PCNA were expressed in the idiopathic ERM, which was mainly derived from Müller cells and extensions of their processes.

Development of a pepper-pot emittance meter for diagnostics of low-energy multiply charged heavy ion beams extracted from an ECR ion source.

Several fluorescent materials were tested for use in the imaging screen of a pepper-pot emittance meter that is suitable for investigating the beam dynamics of multiply charged heavy ions extracted from an ECR ion source. SiO2 (quartz), KBr, Eu-doped CaF2, and Tl-doped CsI crystals were first irradiated with 6.52-keV protons to determine the effects of radiation damage on their fluorescence emission properties. For such a low-energy proton beam, only the quartz was found to be a suitable fluorescent material, since the other materials suffered a decay in fluorescence intensity with irradiation time. Subsequently, quartz was irradiated with heavy (12)C(4+), (16)O(4+), and (40)Ar(11+) ions, but it was found that the fluorescence intensity decreased too rapidly to measure the emittance of these heavy-ion beams. These results suggest that a different energy loss mechanism occurs for heavier ions and for protons.

Multisystem degeneration: drugs and square wave jerks.

We studied a 55-year-old woman with macrosquare wave jerks and macrosaccadic oscillations with a vertical component. The oscillations almost completely disappeared after administration of diazepam, clonazepam, thiamylal, or phenobarbital. A disorder of GABAergic tonic inhibitory system from the substantia nigra to superior colliculus probably plays an important the pathogenesis of these abnormal ocular movements. role in the pathogenesis of these abnormal ocular movements.

Oculopharyngeal muscular dystrophy in a Japanese family with a short GCG expansion (GCG)(11) in PABP2 gene.

Clinicopathological and molecular genetic findings on a new Japanese family with oculopharyngeal muscular dystrophy are reported. The family has 54 members, ten of whom are affected (seven male and three female), in 3 generations. Three affected males, one affected female and one unaffected female of seven living siblings in the third generation were examined. Bilateral ptosis developed in the 4th and 5th decades in the three male cases, and in the 7th decade in the female, and this was followed by diplopia, nasal voice, dysphagia and muscle weakness. In addition, severe external ophthalmoplegia, dysphonia, and proximal amyotrophy were prominent in this family. Electromyographs revealed myogenic/neurogenic changes, and computed tomography disclosed selective muscle wasting with fatty replacement, predominantly in the lower extremities. Muscle biopsy in the four affected patients showed variation in fiber size, and the presence of small angulated fibers and occasional rimmed vacuoles. Electron microscopic examination revealed an accumulation of filamentous inclusions in muscle fiber nuclei. DNA analysis identified that (GCG)(6) in the PABP2 gene was expanded to (GCG)(11) in the four affected cases examined. All studies were negative in the one unaffected. These results confirm that OPMD is caused by GCG short expansion and provides insights into the genetic mechanisms which may contribute to adult onset myopathy, confined to oculopharyngeal muscles.

Quantitative analysis of the oculocardiac reflex by traction on human extraocular muscle.

The oculocardiac reflex was quantitatively studied in 15 patients with strabismus. The reflex was observed in all patients when the medial rectus and inferior oblique muscles were stretched; the medial rectus muscle had a lower threshold than the inferior oblique. Bradycardia was evoked in 7 of the 15 patients when the lateral rectus was tractioned with tensions of 50 g and 600 g. The oculocardiac reflex was a graded phenomenon as a function of tension applied to the extraocular muscles. As tension was increased, bradycardia occurred rapidly and became deep. Systemic administration of atropine prevented completely the bradycardia from occurring. The results suggest that the response of the extraocular muscles to stretch are critically mediated through a polysynaptic path to the heart, resulting in suppression of the heart rate.

Saccade-related activity of periaqueductal gray matter of the monkey.

Single unit activity was recorded extracellularly from the periaqueductal gray matter (PAG) and the superior colliculus of three monkeys during spontaneous saccades and fixation. Most saccade-related cells were found in the dorsal and lateral regions of the PAG and they paused with saccades. The pause preceded the onset of saccades by 34.5 +/- 6.8 msec (n = 31). In 90% of the 31 PAG cells, the saccade-related modulation in activity occurred in all directions. There was a linear relationship between pause duration and saccade duration with correlation coefficients larger than 0.70 in most cells. Superior colliculus cells showed bursts preceding onset of saccades. The lead times averaged 25.3 msec (n = 35). There was no linear relationship between burst duration and saccade duration. These results suggest that the dorsal and lateral regions of PAG play an important role in the saccadic system, probably through long lead burst units in the deep layer of the superior colliculus and/or pontine reticular formation.

Stability of ocular counterrolling and Listing's plane during static roll-tilts.

PURPOSE: To investigate whether habituation occurs in ocular counterrolling (OCR), how stable shifted Listing's plane is, and what effects visual stimulation and alertness exert on the OCR and on Listing's plane. METHODS: Two monkeys (Macaca fuscata) were engaged in this experiment. A dual scleral search coil method was used for recording three-dimensional eye movements. Spontaneous eye movements were recorded for 2 hours in dark and light, while each monkey was held in different static roll positions (up to +/-34 degrees) with its head fixed. Eye movements were also recorded during the monkeys' drowsy periods in the dark. RESULTS: In alert conditions, OCR gains showed some fluctuations but did not change consistently for 2 hours in seven of seven sessions in the dark and in seven of nine sessions in the light. The OCR gains in the light did not differ from those in the dark. The thickness of shifted Listing's plane during the static roll-tilt was also stable for 2 hours and was within the range of the thickness of Listing's plane in the upright position in the light. During drowsy periods, the thickness of Listing's plane increased, and the gain in OCR decreased. CONCLUSION: Ocular counterrolling shows no habituation for 2 hours of static roll-tilts in the alert monkey. In the same condition, Listing's plane consistently maintains its precision. Visual input does not affect OCR, but alertness is necessary to keep the stability of OCR and Listing's plane.

Pupillary light reflex in amblyopia.

The pupillary light reflex of 15 strabismic and anisometropic amblyopes, and eight subjects who had recovered from functional amblyopia was studied by using an infrared electropupillogram. Ten of the fifteen amblyopes had significantly longer latencies of contraction when the amblyopic eyes were stimulated than when the normal eyes were stimulated. However, there was no relationship between the delay in pupillary light reflexes and reduced visual acuity of amblyopic eyes. The amplitudes and maximum velocities of the contraction were not altered significantly in amblyopic and normal eyes. All of the subjects who had recovered showed no significant difference of the latencies of the pupillary responses to stimulation between normal and amblyopic eyes. These findings indicate that a retinal mechanism in amblyopic eyes may be responsible for the abnormally long pupillary light reflex latency.

Extrinsic and intrinsic connections of the cat's lateral suprasylvian visual area.

The lateral suprasylvian visual area (LS) is known to have numerous interconnections with visual cortical areas as well as with subcortical structures implicated in visually-guided behaviors. In contrast, little data is available regarding connections within the LS itself. In order to obtain information about intra-areal connections and to re-investigate LS connectivity with various cortical and subcortical areas, the traces (biocytin or WGA-HRP) was injected into various loci along the medial and lateral banks of the LS. The anterograde tracer, biocytin injections into both medial and lateral bank produced label contained within the respective bank that extended rostrally and caudally from the infection site. In addition, following medical bank injections, considerable label was distributed throughout the fundus and, to a lesser extent, in the lateral bank. In contrast, no label could be detected in the medial bank after lateral bank injections, and, although label was observed in the fundus, it was restricted to the most lateral aspects. Moderate labeling could be observed in the medial bank following the tracer injection into the most rostral aspect of the lateral bank. It is likely that input derived from various visual cortical areas which project to the medial bank of the LS has access to this intra-areal circuitry. This may provide a route by which visual cortical information can be relayed to other cortical and subcortical structures involved in visually-guided behaviors such as the anterior ectosylvian visual cortex, striatum, and the deep layers of the superior colliculus, despite the fact that these structures themselves do not receive substantial direct projections from the visual cortical areas that are associated with the medial bank. Examination of the laminar location of the cells-of-origin of striate and extrastriate projections to LS using retrograde trace, WGA-HRP, revealed that the supragranular laminae of areas 17, 18 and 19 were the source of LS afferents whereas afferents from the other cortical areas (e.g., 20a, 20b, 21a, 21b, 7 and anterior ectosylvian visual area) were from both supra- and infragranular laminae. In addition, all LS subregions received intra-areal afferent projections from all LS cortical laminae. Thus, although rather clear hierarchical relationship between LS and visual cortical areas appears to exist, the interconnections among LS subregions provide no clear evidence of simple hierarchical relationships between regions LS or may have feed-forward and feed-back pathways.

Prognosis of non-small cell lung cancer patients with positive pleural lavage cytology after a thoracotomy: results of the survey conducted by the Japan Clinical Oncology Group.

OBJECTIVE: The purpose of this study was to clarify the prognosis of non-small cell lung cancer patients without pleural effusion whose intrapleural cancer cells were detected by a cytologic examination of pleural lavage fluid obtained immediately after a thoracotomy. METHOD: A questionnaire survey on the survival of the patients with positive pleural lavage cytology from January 1985 to December 1994 was performed by the Japan Clinical Oncology Group. RESULTS: According to the data collected from 15 institutions, 1890 non-small cell lung cancer patients without pleural effusion underwent pleural lavage cytology immediately after thoracotomy and 142 (7.8%) of them were found to have intrapleural cancer cells detected by the cytological analysis. The information of survival on 113 patients was available. This comprised of 64 males and 49 females with a mean age of 64.6 years. The predominant histologic type was adenocarcinoma (74%). Out of these 113 patients, 109 (97%) underwent a surgical resection. The 5-year survival rate was 30% in all patients, 49% in pathological stage I (n=35), 23% in stage II (n=20) and 26% in stage IIIA (n=34). CONCLUSION: Patients with a positive pleural lavage cytology in pathological stage I or II appear to have a poor 5-year survival rate.

Prognosis of resected non-small cell lung cancer patients with carcinomatous pleuritis of minimal disease.

OBJECTIVE: The purpose of this study was to clarify the prognosis of resected non-small cell lung cancer (NSCLC) patients with carcinomatous pleuritis of minimal disease which might be considered as the next advanced stage of positive pleural lavage cytology. METHOD: The data were collected from a questionnaire survey on the survival of the patients with carcinomatous pleuritis found at thoracotomy from 1985 to December 1994 which was conducted by the Japan Clinical Oncology Group (JCOG). RESULTS: Out of 227 patients with carcinomatous pleuritis found at thoracotomy who had available information on a survival, 100 patients who underwent a resection of the primary tumor had carcinomatous pleuritis of minimal disease defined based on the criteria of the Japan Lung Cancer Society. The mean malignant fluid volume (+/-S.E.) was 37.1 (6.3) ml and the mean number of pleural disseminated nodules was 5.6 (0.9). A lobectomy was performed in 79 patients, a pneumonectomy in 11 and a limited resection in ten. The 3- and 5-year survival rates were 31.8 and 22.8%, respectively. CONCLUSIONS: The prognosis of resected NSCLC patients with carcinomatous pleuritis of minimal disease was unexpectedly good. This indicates that no fine line may exist between positive pleural lavage cytology findings and the aforementioned lesion.

Pupillary abnormalities in schizophrenic patients during long-term administration of psychotropic drugs: dissociation between light and near vision reactions.

Pupillographic studies were made of the reactions to light and near vision in 12 schizophrenic patients under long-term administration of psychotropic drugs. The results showed a significant reduction in the light reaction, while the near vision reaction was preserved. The pupillographic study revealed not only reduction in amplitude of the light reaction but also changes in dynamic aspects of the reaction, i.e., prolonged latency time, shortened constriction time, and half redilatation time after the light stimulus. The mechanisms underlying the dissociation between light reaction and near vision reaction induced by long-term administration of psychotropic drugs are obscure, but both the peripheral and central actions of these drugs may be involved.

The origin of high and regular discharge rates of eye-movement-related neurons in the region of the interstitial nucleus of Cajal.

Burst-tonic neurons in the region of the interstitial nucleus of Cajal (INC) that show a close correlation to vertical eye movement have been known to exhibit high and regular discharge rates, not only during fixation in alert animals, but also during sleep. Since they receive major input from vertical semicircular canals, we examined in this study whether or not the source of the high and regular discharge rates was the primary vestibular afferents. Infusion of lidocaine into the middle ear bilaterally resulted in a significant decrease of mean discharge rates and an increase in the coefficient of variation of the mean rates. However, burst-tonic neurons in cats that had received bilateral labyrinthectomy 6 weeks previously still exhibited high and regular discharge rates similar to those of normal cats. These results indicate that high and regular discharges of eye-position-related INC cells are maintained largely by input from primary vestibular afferents in normal cats. However, such characteristic discharges could also be maintained centrally in the brainstem without peripheral vestibular input in labyrinthectomized cats.

Possible downward burster-driving neurons related to the anterior semicircular canal in the region of the interstitial nucleus of Cajal in alert cats.

It is well known that vestibular nystagmus evoked by head rotation occurs in the plane specific to that in which head rotation was applied in three-dimensional space. Although burster-driving neurons (BDN) have been demonstrated for a quick phase of horizontal nystagmus, it is not yet known where the counterpart for vertical nystagmus is located. We analyzed the activity of a class of neurons in the region within, and in the close vicinity of, the interstitial nucleus of Cajal (INC) in alert cats. Their activity gradually increased during an upward slow phase evoked by nose-down pitch. This increased activity was further followed by burst discharge shortly before and during the downward quick phase. Gradually increased activity was also evoked by contralateral roll. These results suggest that the gradually increased activity was evoked by activation of the contralateral anterior canal. Many of these cells were fired by electrical stimulation of the contralateral vestibular nerve with short latencies. These cells also showed burst discharge shortly before and during downward saccades induced by visual stimuli, and the number of spikes during bursts was correlated with saccade amplitudes. Although all had irregular resting discharges, eye-position-related activity was rarely obtained. The characteristic behavior of these cells is very similar, except for their on-directions, to the behavior of horizontal BDNs, suggesting that these INC cells are a candidate for downward BDNs related to the anterior canal.

Ocular torsion produced by unilateral chemical inactivation of the interstitial nucleus of Cajal in chronically labyrinthectomized cats.

Unilateral lesions of the rostral midbrain involving the interstitial nucleus of Cajal (INC) have been known to produce ocular torsion in alert animals including humans, which has been assumed to be the result of an impaired otolith-ocular reflex. We examined the effects of chemical deactivation of the INC using a gamma-aminobutyric acid (GABA) agonist (muscimol) in cats that had received bilateral labyrinthectomy, and compared the results with those in normal cats. Ocular torsion with a magnitude similar to that observed in normal cats appeared in chronically labyrinthectomized cats after unilateral muscimol infusion into the INC, indicating that ocular torsion following unilateral INC deactivation can be produced by a mechanism independent of the otolith-ocular reflex.

Morphologic alterations and cytokinetic studies of tracheal autograft epithelium in rabbits.

BACKGROUND: Although tracheobronchoplasty has been used widely in the field of thoracic surgery, few details of the morphologic changes in and cytokinetics of the graft epithelium have been reported. The aim of this study was to focus on these aspects in autografted rabbit tracheas. METHODS: Resected cervical tracheas were anastomosed immediately after removal, retrieved on postoperative days 1 through 28, and examined morphologically. Mitotic and bromodeoxyuridine-labeling indices of the graft epithelium were analyzed. RESULTS: On postoperative days 1 to 4, the graft epithelium showed focal desquamation at the anastomoses. Ciliated cells disappeared during postoperative days 4 to 7 and then increased gradually. Nonciliated cells retained a somewhat columnar shape on postoperative days 4 to 7, except at denuded foci. Thereafter, the grafts were covered completely with pseudostratified mucociliary epithelium. On postoperative day 4, both indices were maximal and appeared higher at the anastomotic than midgraft sites. CONCLUSIONS: Most of the graft epithelium was preserved during acute ischemia and then started to regenerate. The increased regenerative activity near the anastomoses may be attributable to mechanical damage or different nutritional conditions.