The spino-bulbar-cerebellar pathway: Activation of neurons projecting to the lateral reticular nucleus in the rat in response to noxious mechanical stimuli.

It is now well established that the cerebellum receives input from nociceptors which may serve to adjust motor programmes in response to pain and injury. In this study, we investigated the possibility that spinoreticular neurons (SRT) which project to a pre-cerebellar nucleus, the lateral reticular nucleus (LRt), respond to noxious mechanical stimulation. Seven adult male rats received stereotaxic injections of the b subunit of cholera toxin in the LRt. Following a 5 day interval, animals were anesthetised with urethane and a noxious mechanical stimulus was applied to the right hind paw. Animals were fixed by perfusion 5min following application of the stimulus. Retrogradely labelled SRT neurons of the lumbar spinal cord were examined for immunoreactivity for phosphorylated ERK (pERK) and the neurokinin-1 (NK-1) receptor. Approximately 15% of SRT cells in deep laminae (IV-VII and X) expressed pERK ipsilateral to the site of the stimulus. Around 60% of SRT cells with the NK-1 receptor expressed pERK but 5% of pERK expressing cells were negatively labelled for NK-1. It is concluded that a significant proportion of SRT cells projecting to the LRt respond to noxious mechanical stimuli and that one of the functions of this pathway may be to provide the cerebellum with nociceptive information.

Alpha-synuclein in motor neuron disease: an immunohistologic study.

Alpha-synuclein (ASN) has been implicated in neurodegenerative disorders characterized by Lewy body inclusions such as Parkinson's disease and dementia with Lewy bodies. Lewy body-like inclusions have also been observed in spinal neurons of patients with amyotrophic lateral sclerosis (ALS) and reports suggest possible ASN abnormalities in ALS patients. We assessed ASN immunoreactivity in spinal and brain tissues of subjects who had died of progressive motor neuron disorders (MND). Clinical records of subjects with MND and a comparison group were reviewed to determine the diagnosis according to El-Escariol Criteria of ALS. Cervical, thoracic and lumbar cord sections were stained with an antibody to ASN. A blinded, semiquantitative review of sections from both groups included examination for evidence of spheroids, neuronal staining, cytoplasmic inclusions, anterior horn granules, white and gray matter glial staining, corticospinal tract axonal fiber and myelin changes. MND cases, including ALS and progressive muscular atrophy, displayed significantly increased ASN staining of spheroids ( P< or =0.001), and glial staining in gray and white matter ( P< or =0.05). Significant abnormal staining of corticospinal axon tract fibers and myelin was also observed ( P< or =0.05 and 0.01). Detection of possible ASN-positive neuronal inclusions did not differ between groups. Significant ASN abnormalities were observed in MND. These findings suggest a possible role for ASN in MND; however, the precise nature of this association is unclear.

Site-specific mutagenesis in a homogeneous polyglutamine tract: application to spinocerebellar ataxin-3.

In recent years, nine neurodegenerative diseases have been found to be caused by the expansion of a CAG-triplet repeat in the coding region of the respective genes, resulting in lengthening of an otherwise harmless polyglutamine tract in the gene products. To facilitate structural studies of these disease mechanisms, a general protocol is described that allows site-specific mutations to be introduced into the polyglutamine tract. Based on 'cassette mutagenesis', this protocol involves engineering unique restriction sites into the flanking regions of the CAG repeat and subsequently replacing the wild-type CAG repeat with a double-stranded synthetic DNA fragment containing the desired mutations. This method was applied to the spinocerebellar ataxin-3 protein, such that the wild-type amino acid sequence -Q(3)KQ(22)- was replaced by a -Q(9)CQ(9)- sequence. In this case, the incorporated cysteine residue can be exploited for various chemical modifications, lending the host glutamine repeat to many structural and biophysical techniques for the resolution of a specific residue. The method reported here bypasses many problems that can arise from PCR-based mutagenesis methods.

Modulation of responses of feline ventral spinocerebellar tract neurons by monoamines.

Ventral spinocerebellar tract neurons located in laminae V-VII of cat lumbar spinal cord were tested for the effects of ionophoretically applied monoamines and receptor selective agonists. Extracellularly recorded responses, monosynaptically evoked by group I afferents in a muscle nerve, were compared before, during, and after ionophoresis. They were analyzed with respect to changes in the number of evoked spikes and in the latency. Both serotonin (5-HT) and noradrenaline (NA) were found to facilitate responses of all neurons tested. Ionophoresis of three serotonin subtype receptor agonists (5-carboxamidotryptamine maleate, 5 methoxytryptamine HCl, and alpha-methyl 5-hydroxytryptamine) and of two NA receptor agonists (phenylephrine and isoproterenol) likewise had a facilitatory effect. However, three other 5-HT receptor agonists (8-hydroxy-dipropylaminotetraline hydrobromide), 2-methyl 5-hydroxytryptamine, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl and two NA receptor agonists (tizanidine and clonidine) had the opposite effect because they depressed responses of the tested neurons. These results show that information forwarded by means of the ventral spinocerebellar tract may be modulated by monoamines and that several receptor subtypes, located pre- or postsynaptically, may be involved. The results also demonstrate that transmission by means of group I muscle afferents may not only be facilitated by monoamines but also depressed by selective receptor subtype activation.

Serotoninergic and noradrenergic axons make contacts with neurons of the ventral spinocerebellar tract in the cat.

Contacts between monoaminergic fibers and electrophysiologically identified neurons of the ventral spinocerebellar tract were investigated in the cat. Five neurons were labeled intracellularly with rhodamine dextran, and monoaminergic fibers were revealed with antibodies against serotonin and dopamine beta-hydroxylase. The distribution of appositions between monoaminergic varicosities and the soma and the whole length of dendrites of these neurons was examined by using a three-channel confocal microscope. The analysis showed that close appositions between monoaminergic fibers and labeled processes occurred over the whole surface of the neurons. The highest percentage of such appositions was found on proximal dendrites, for both serotonin (37%) and noradrenaline (57%). The total number of serotoninergic contacts (66-134 per neuron) by far exceeded that of noradrenergic contacts (3-36 per neuron). Contacts between serotoninergic fibers and two neurons were analyzed by using electron microscopy. These neurons were labeled intracellularly with horseradish peroxidase, and serotoninergic varicosities were identified by immunocytochemistry. Six of 10 serially analyzed boutons in apposition to proximal dendrites were found to form morphologic synapses. The identification of the remaining four was inconclusive. These results indicate that many of the appositions seen in confocal microscopy may represent direct synaptic contacts. They also indicate that monoaminergic neurons may modulate activity of neurons of the ventral spinocerebellar tract by direct postsynaptic actions in addition to any effects evoked by means of volume transmission.

Substance P and enkephalin containing fibers in the developing nucleus dorsalis of the human spinal cord.

A peroxidase-antiperoxidase immunostaining method was employed to determine the initial stage of appearance and localization of substance P (SP) and enkephalin (ENK) in the nucleus dorsalis of the developing human spinal cord. Both SP- and ENK-positive fibers started to appear from the 10th week of gestation in regions surrounding the nucleus dorsalis. SP-positive fibers then reached the nucleus at 13 weeks and from 26 weeks to term, three strands of SP-positive fibers, which were predominantly originated from the superficial layers of the dorsal horn, penetrated into the nucleus dorsalis from its medial, median and lateral aspects. From 26 weeks onwards, ENK-positive fibers, originated from the superficial and the adjacent layers of the spinal cord, formed a thicker medial and a thinner lateral bundle projecting into the nucleus dorsalis. Our results show that both SP- and ENK-positive fibers started to appear at around 10 weeks and a consistent pattern of immunoreactivity was established by around 26-30 weeks of gestation.