In vitro formation of corticospinal synapses in an organotypic slice co-culture.

In order to study biological properties of the corticospinal tract, we have reconstructed this system in an in vitro slice culture preparation. Motor cortex and spinal cord slices, prepared from newborn rats, were co-cultured on pored membranes for 16-24 days. Anterograde labeling with biocytin showed that substantial neural connections had formed between the cortex and spinal cord slices. Retrograde labeling with horseradish peroxidase or 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate demonstrated that the parent cells were located primarily in the deeper layer of the cortex, as is found in vivo. Stimulation of the deep layer of the cortex elicited extracellular postsynaptic responses and intracellular excitatory postsynaptic potentials (EPSPs) in the co-cultured spinal cord that were mediated by the 1-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/ kainate-type glutamate receptor. The intracellular injection of biocytin after EPSPs were recorded showed that one-third of these cells were large stellate cells, which are thought to be motoneurons, while a large portion of the remaining labeled cells were bipolar cells of smaller sizes. Using this reconstructed in vitro preparation, we recorded field EPSPs (fEPSPs) along a 100-microm-interval lattice in the spinal gray matter, which allowed the quantitative evaluation of synapse formation. The fEPSP amplitudes were more than two-fold larger when the forelimb cortex was co-cultured with cervical cord rather than lumbar cord. However, hindlimb cortex did not show this preference. The fEPSP amplitudes were more than twice as large when the dorsal side of the spinal cord was adjacent to the cortex than the ventral side. In summary, we have reconstructed the corticospinal projection and synapses in vitro using cortical and spinal explants. This system allows for an efficient quantitative evaluation of synapse formation and for studies of postsynaptic cells. Our results suggest that synapse formation shows preferences along and perpendicular to the neuraxis of the spinal cord.

A new splicing variant for human tyrosine hydroxylase in the adrenal medulla.

It has been reported that several mRNA isoforms of tyrosine 3-monooxygenase (tyrosine hydroxylase; TH) occur only in primates. New TH isoforms produced by skipping of exon 3 in the adrenal medulla of patients with progressive supranuclear palsy (PSP) have recently been reported, J. Neurochem. 67 (1996) 19. Here, we looked for the presence of new TH isoforms in control brains and adrenal medulla and in brains from patients with PSP. We found a novel type of TH mRNA in the adrenal medulla from one of the control subjects. The mRNA lacked exon 4, resulting in a premature stop codon at amino acid 147. This result suggests the importance of alternative splicing in the regulation of TH activity.

alpha-Synuclein affects the MAPK pathway and accelerates cell death.

Insoluble alpha-synuclein accumulates in Parkinson's disease, diffuse Lewy body disease, and multiple system atrophy. However, the relationship between its accumulation and pathogenesis is still unclear. Recently, we reported that overexpression of alpha-synuclein affects Elk-1 phosphorylation in cultured cells, which is mainly performed by mitogen-activated protein kinases (MAPKs). We further examined the relationship between MAPK signaling and the effects of alpha-synuclein expression on ecdysone-inducible neuro2a cell lines and found that cells expressing alpha-synuclein had less phosphorylated MAPKs. Moreover, they showed significant cell death when the concentration of serum in the culture medium was reduced. Under normal serum conditions, the addition of the MAPK inhibitor U0126 also caused cell death in alpha-synuclein-expressing cells. Transfection of constitutively active MEK-1 resulted in MAPK phosphorylation in alpha-synuclein-expressing cells and improved cell viability even under reduced serum conditions. Thus, we conclude that alpha-synuclein regulates the MAPK pathway by reducing the amount of available active MAPK. Our findings suggest a mechanism for pathogenesis and thus offer therapeutic insight into synucleinopathies.

The human hand motor area is transiently suppressed by an unexpected auditory stimulus.

OBJECTIVE: To study the effect of a loud auditory stimulus on the excitability of the human motor cortex. METHODS: Ten normal volunteers participated in this study. The size of responses to transcranial magnetic or electrical cortical stimulation (TMS or TES) given at different times (ISIs) after a loud sound were compared with those to TMS or TES alone (control response). Different intensities and durations of sound were used at several intertrial intervals (ITIs). In addition, we examined how the presence of a preceding click modulated the effect of a loud sound (prepulse inhibition). The incidence of startle response evoked by various stimuli was also studied. RESULTS: A loud auditory stimulus suppressed EMG responses to TMS when it preceded the magnetic stimulus by 30-60 ms, whereas it did not affect responses to TES. This suggests that the suppression occurred at a cortical level. Significant suppression was evoked only when the sound was louder than 80 dB and longer than 50 ms in duration. Such stimuli frequently elicited a startle response when given alone. The effect was not evoked if the ITI was 5 s, but was evoked when it was longer than 20 s. A preceding click reduced the suppression elicited by loud sounds. CONCLUSIONS: Auditory stimuli that produced the greatest effect on responses to TMS had the same characteristics as those which yielded the most consistent auditory startle. We suggest that modulation of cortical excitability occurs in parallel with the auditory startle and both may arise from the same region of the brain-stem.

Air-puff-induced facilitation of motor cortical excitability studied in patients with discrete brain lesions.

Air-puff stimulation applied to a fingertip is known to exert a location-specific facilitatory effect on the size of the motor evoked potentials elicited in hand muscles by transcranial magnetic stimulation. In order to clarify its nature and the pathway responsible for its generation, we studied 27 patients with discrete lesions in the brain (16, 9 and 2 patients with lesions in the cerebral cortex, thalamus and brainstem, respectively). Facilitation was absent in patients with lesions affecting the primary sensorimotor area, whereas it was preserved in patients with cortical lesions that spared this area. Facilitation was abolished with thalamic lesions that totally destroyed the nucleus ventralis posterolateralis (VPL), but was preserved with lesions that at least partly spared it. Lesions of the spinothalamic tract did not impair facilitation. The size of the N20-P25 component of the somatosensory evoked potential showed a mild correlation with the amount of facilitation. The facilitation is mainly mediated by sensory inputs that ascend the dorsal column and reach the cortex through VPL. These are fed into the primary motor area via the primary sensory area, especially its anterior portion, corresponding to Brodmann areas 3 and 1 (possibly also area 2), without involving other cortical regions. The spinothalamic tract and direct thalamic inputs into the motor cortex do not contribute much to this effect. Some patients could generate voluntary movements despite the absence of the facilitatory effect. The present method will enable us to investigate in humans the function of one of the somatotopically organized sensory feedback input pathways into the motor cortex, and will be useful in monitoring ongoing finger movements during object manipulation.

PQBP-1, a novel polyglutamine tract-binding protein, inhibits transcription activation by Brn-2 and affects cell survival.

A novel gene, designated PQBP-1, which encodes a 265 residue protein that binds to the polyglutamine tract of the brain-specific transcription factor Brn-2, was identified. PQBP-1, which also interacts with the polyglutamine tract of triplet repeat disease gene products, binds with a higher affinity to an expanded polyglutamine tract. PQBP-1 has several functional domains, including hepta- and di-amino acid repeat sequences rich in polar residues essential for its interaction with the polyglutamine tract, a WWP/WW domain which binds to proline-rich motifs in other proteins, a putative nuclear localization signal sequence and a C2domain implicated in Ca2+-dependent phospholipid signaling. PQBP-1 is located in the nucleus and inhibits transcriptional activation by Brn-2. Overexpression of PQBP-1 in P19 embryonic carcinoma cells suppresses their growth rate and enhances their susceptibility to various stresses including serum deprivation, retinoic acid treatment and UV irradiation. Northern blot and in situ hybridization analyses revealed that PQBP-1 is a ubiquitous protein and is expressed primarily in neurons throughout the brain, with abundant levels in hippocampus, cerebellar cortex and olfactory bulb. These results suggest that PQBP-1 mediates important cellular functions under physiological and pathological conditions via its interaction with polyglutamine tracts.

Cloning and mapping of ZNF231, a novel brain-specific gene encoding neuronal double zinc finger protein whose expression is enhanced in a neurodegenerative disorder, multiple system atrophy (MSA).

A novel brain-specific gene, neuronal double zinc finger protein (ZNF231), was cloned and mapped. We used the high-density cDNA filter method to analyze the gene-expression profile in brains with multiple system atrophy (MSA). MSA is a sporadic progressive neurodegenerative disease characterized clinically by cerebellar symptoms, parkinsonism, autonomic dysfunction, or their various combinations, but its pathogenesis has yet to be clarified. In total, 8300 cDNA clones were screened, and a novel gene, ZNF231, was identified, whose expression was elevated in cerebella of patients with MSA. Its transcript is approximately 16 kb long and encodes an open reading frame of 3926 amino acid residues that has several interesting motifs; two glycine-proline dipeptide repeats (aa 22-32 and aa 61-74), a pair of homologous C8 double zinc finger motifs (aa 169-226 and aa 465-521), a leucine zipper motif (aa 561-582), a SH3 domain-binding motif (aa 825-831), two nuclear targeting signals (aa 1011-1028 and aa 1071-1091), two glutamine-rich domains (aa 2428-2473 and aa 3775-3804), and a histidine-rich domain (aa 3597-3682). These features suggest that the new gene encodes a nuclear protein or transcription regulator. Northern blot and RT-PCR analyses showed that its expression is specific to the brain and apparently restricted to the neurons. Elevation of ZNF231 expression may be involved in the pathogenesis of multiple system atrophy. The gene for ZNF231 is located on chromosome 3p21.

Polar amino acid-rich sequences bind to polyglutamine tracts.

Polyglutamine tracts are found in different proteins including transcription factors and cofactors as well as in triplet repeat disease gene products. To characterize the protein motif that binds to the polyglutamine tract, we screened a human embryonic brain cDNA library with the polyglutamine tract of Brn-2 as bait using the yeast two-hybrid method. All six isolated clones encoding polyglutamine tract binding proteins were rich in polar amino acids. Three of these clones could form polar helical structures. These observations suggest that polar amino acid-rich sequences are essential for binding to the polyglutamine tract.

Substance P is a possible neurotransmitter in the rat spinothalamic tract.

In order to shed some light on the neurotransmitters in the spinothalamic tract (STT), we examined, biochemically and immunohistochemically, the contents of various neurotransmitter candidates in the terminal field of the STT after cervical hemi-chordotomy (HC) and dorsal quadrant-chordotomy (dQC) in the rat. Substance P (SP), calcitonin gene-related peptide (CGRP), enkephalin, neuropeptide Y, neurotensin, oxytocin and dynorphin A were analyzed immunohistochemically. The contents of neuropeptides (SP, CGRP and cholecystokinin octapeptide) were measured by radioimmunoassay and those of amino acids (aspartic acid, glutamic acid, gamma-aminobutyric acid (GABA) and glycine) and noradrenaline were determined using high-performance liquid chromatography. Cervical hemi-chordotomy, but not dQC, caused significant decreases of the SP-like immunoreactivity in and SP content of the ventral thalamus on the ipsilateral side, compared with that on the contralateral side and of rats subjected to sham-operation. However, neither HC nor dQC resulted in any changes in the ventral thalamic contents of other putative neurotransmitters examined. These results suggest that, in rats, the STT contains SP and that SP-positive fibers run in the ventral half of the ascending spinal tract at the cervical level.

Suppression of motor cortical excitability by electrical stimulation over the cerebellum in ataxia.

We studied the effect of electrical stimulation over the cerebellum on electromyographic responses evoked by magnetic stimulation over the cerebral motor cortex in 41 normal volunteers and 32 patients with ataxia due to various disorders. In all the normal subjects, stimulation over the cerebellum significantly reduced the size of electromyographic response in the first dorsal interosseous muscle evoked by magnetic cortical stimulation, when the cerebellar stimulus preceded the cortical stimulus by 5, 6, and 7 msec. This suppression was absent or reduced in ataxic patients who had atrophy of the cerebellar hemispheres as demonstrated by magnetic resonance imaging and in patients with dysfunction of the cerebellothalamocortical pathway who had lesions in the superior cerebellar peduncle or in the motor thalamus. In contrast, suppression was normal in ataxic patients who had pontine lesions that affected the pontocerebellar afferent pathway to the cerebellum. Results were also normal in patients without cerebellar ataxia, such as those with Parkinson's disease, sensory ataxia, and cerebrovascular disease without ataxia. We conclude that electrical stimulation activates cerebellar structures that suppress motor cortical excitability through a cerebellothalamocortical pathway and that the afferent systems to the cerebellum make no or little contribution to the effect. The technique described here would be useful for distinguishing ataxia due to lesions of cerebellar afferent pathway from other types of cerebellar ataxia.

Inherited amyloid polyneuropathy type IV (gelsolin variant) in a Japanese family.

We describe a Japanese family with familial amyloid polyneuropathy type IV. The family originates from central Japan, Nagano prefecture, and is unrelated to Finnish or other Caucasian populations. Of 42 members in three generations, 14 individuals (5 men, 9 women) are affected by corneal lattice dystrophy, cranial neuropathy, mild peripheral neuropathy, and skin changes. Polarizing microscopy and immunohistochemistry studies of skin biopsy samples demonstrated abundant amyloid deposits, which bound an antigelsolin monoclonal antibody. Direct sequence analysis of a DNA fragment spanning codon 187 of plasma gelsolin complementary DNA and restriction analysis using a modified polymerase chain reaction demonstrated a single base substitution, guanine to adenine, at nucleotide position 654, which is identical to the mutation in Finnish familial amyloid polyneuropathy type IV. This strongly suggests that the mutation causes the familial amyloid polyneuropathy type IV phenotype regardless of ethnic background.

Topographical distribution of gamma-aminobutyric acid within the cat thalamus in relation to the basal ganglia, as determined by mass fragmentometry.

Chemical ionization mass spectrometry was introduced for the assay of GABA in the cat brain. The method is quite simple, sensitive, and specific for quantitative analysis. Study of the regional distribution of the GABA content within the thalamus disclosed that the ventromedial nucleus (VM) of the thalamus had a high concentration of GABA. The VM receives the afferent projection from the zona reticulata of the substantia nigra. The result, together with the results obtained by physiological as well as pharmacological studies, supports the hypothesis that the transmitter substance of the nigrothalamic pathway is GABA.

Inhibitory effects of acetylcholine on neurones in the feline nucleus reticularis thalami.

1. Short iontophoretic pulses of acetylcholine (ACh) inhibited almost every spontaneously active cell encountered in the nucleus reticularis thalami of cats anaesthetized with a mixture of halothane, nitrous oxide and oxygen. On 200 cells the mean current needed to eject an effective inhibitory dose of ACh was 67 +/- 2 nA. When the ACh-evoked inhibition was mimicked by gamma-aminobutyric acid (GABA) or glycine on the same cell, the current required to release ACh was found to be approximately twice as great as that required to release an equally effective dose of GABA or glycine. 2. ACh inhibitions developed with a latency which was very much shorter than that for ACh excitation in cells of the ventrobasal complex. The latency of the ACh-evoked inhibition was as rapid as the onset and offset of the excitation of the same cells glutamate and their inhibition by GABA or glycine. 3. The firing pattern of ACh-inhibited neurones in the nucleus reticularis was characterized by periods of prolonged, high frequency bursts, and their mean firing frequency was 22 Hz. Raster dot displays and interspike interval histograms showed that whereas ACh suppressed the spikes that occurred between bursts much more readily than those that occurred during bursts, all spikes were equally sensitive to the depressant action of GABA and glycine. Large doses of ACh provoked or exaggerated burst activity. 4. ACh-evoked inhibition was extremely sensitive to blockade by short iontophoretic applications of atropine, which had no effect on the inhibitions evoked on the same cell equipotent doses of GABA or glycine. The ACh-evoked inhibitions were also antagonized by dihydro-beta-erythroidine released with slightly larger currents. When tested on the same cell, small iontophoretic applications of picrotoxin and bicuculline methoiodide blocked the inhibition evoked by GABA but had no effect on that evoked by ACh. Iontophoretic strychnine only rarely affected the inhibition evoked by ACh, while readily blocking the inhibition evoked on the same cell by an equipotent dose of glycine. In two cats, intravenous strychnine (1-2 mg/kg) had no effect on the ACh-evoked inhibition, while greatly reducing the sensitivity of the cell under study to glycine. 5. Only four out of forty-eight ACh-inhibted cells tested were inhibited by iontophoretic applications of either guanosine or adenosine 3':5'-phosphate. 6. Cells of the nucleus reticularis have been shown to have an inhibitory action on the thalamic relay cells, which are excited by ACh. It is suggested that the presence of both ACh excited and inhibited cells in different nuclei of the thalamus could be of considerable functional significance in gating sensory transmission through the thalamus.