L. Virgo

Induction of the immediate early gene c-jun in human spinal cord in amyotrophic lateral sclerosis with concomitant loss of NMDA receptor NR-1 and glycine transporter mRNA.

The aetiology of the sporadic form of amyotrophic lateral sclerosis (ALS) is poorly understood although abnormalities in glutamate and glycine transport have been implicated which both could contribute to a neurodegenerative process mediated through the N-methyl-D-aspartate (NMDA) receptor. In this study we have used in situ hybridization to investigate whether any changes in the expression of NMDA receptors, the glycine transporter or glutamate-mediated injury responses are detectable in ALS. Two immediate early genes were investigated as markers of neuronal injury responses, c-jun and zif-268, both constitutively expressed in the spinal cord. Levels of c-jun mRNA were most abundant in intermediate grey and layer IX of the ventral horn containing motor neurones. This pattern was markedly changed in ALS with large increases (2-3 fold) in c-jun mRNA occurring in dorsal and ventral horn. The marked increase in c-jun mRNA was also substantiated by slot blot analysis of tissue homogenates of spinal cord and a parallel induction of zif-268 mRNA was also seen. NMDA receptor NR-1 mRNA was widely distributed in control spinal cord with the highest concentrations occurring in layers IX, X, intermediate grey and dorsal horn. The ALS cases showed a selective decrease in the level of NR-1 mRNA in the ventral region (50%) whilst no significant decrease was detected in the dorsal region. Quantitation of tissue homogenates with dorsal and ventral regions combined also yielded a significant decrease of 40% which supports the analysis from in situ hybridization densitometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Ampa receptor subunit mrna expression in control and Als spinal cord

The distribution of glutamate receptor subunits in human spinal cord has yet to be fully elucidated. The aim of this study was to examine the distribution of mRNAs for the subunits of the AMPA type of glutamate receptor (GluR A, B, C and D) in control human spinal cord using in situ hybridization and to examine in parallel the expression of these mRNAs in patients with sporadic amyotrophic lateral sclerosis (ALS). We also quantitated mRNA levels for these subunits in spinal cord homogenates. The relative abundances of the receptor subunits were as follows: GluR A > GluR B > GluR D > GluR C with A, B and D expressed in foci corresponding to the medial, lateral and ventral clusters of motor neurones. Quantitation of homogenates revealed a significant decrease (38%) in GluR A mRNA in ALS spinal cord compared with controls, and a 67% decrease of GluR B mRNA compared with controls. Levels of GluR C and D were too low to analyse densitometrically.

Cholecystokinin messenger RNA deficit in frontal and temporal cerebral cortex in schizophrenia

No consistent markers of pathology have been established yet in schizophrenia, although abnormalities in frontal and temporal structures are indicated from positron emission tomography (PET) studies. We have used in situ hybridization to investigate functional changes focusing on the quantitation of cholecystokinin (CCK) mRNA, whose product has been shown to be depleted in schizophrenia. CCK mRNA and G(o) alpha-subunit mRNA were measured in eight schizophrenic and eight control subjects matched for age and postmortem delay. The study revealed a marked decrease in CCK mRNA of 83% in frontal cortex (BA10) and 63% in superior temporal cortex (BA22) in schizophrenia with no change in G(o) alpha-subunit mRNA in either region. This study was extended to a further series of eight patients to determine the reproducibility of this effect and to quantitate laminar changes in CCK mRNA. Quantitation of CCK mRNA in inner cortical layers (layer V/VI) was carried out in frontal and temporal cortex in comparison with G(o) alpha-subunit mRNA, which is also concentrated in this region; this study showed a similar selective decrease in CCK mRNA in frontal and temporal cortex of 47% and 51%, respectively. A confirmatory decrease in CCK mRNA was also obtained by slot blot analysis of CCK mRNA in tissue extracts of frontal cortex by reference to levels of beta-tubulin mRNA, CCK mRNA:beta-tubulin mRNA was significantly decreased (67%) in schizophrenic tissue compared to control tissue. There was no significant correlation of CCK mRNA loss with neuroleptic treatment or duration of illness.

Changes in expression of NMDA receptor subunits in the rat lumbar spinal cord following neonatal nerve injury

The vulnerability of motoneurones to glutamate has been implicated in neurological disorders such as amyotrophic lateral sclerosis but it is not known whether specific receptor subtypes mediate this effect. In order to investigate this further, the expression of N‐methyl‐ d‐aspartate (NMDA) receptor subunits was studied during the first three post‐natal weeks when motoneurones are differentially vulnerable to injury following neonatal nerve crush compared to the adult. Unilateral nerve crush was carried out at day 2 after birth (P2) which causes a decrease of 66% in motoneurone number by 14 days (P14). To study receptor expression in identified motoneurones, serial section analysis was carried out on retrogradely labelled common peroneal (CP) motoneurones by combined immunocytochemistry and in situ hybridization (ISH). mRNA levels were also quantified in homogenates from lumbar spinal cords in which the side ipsilateral to the crush was separated from the contralateral side. The NR1 subunit of the NMDA receptor was widely distributed in the spinal cord being expressed most strongly in motoneurone somata particularly during the neonatal period (P3–P7). The NR2 subunits were also expressed at higher levels in the somata and dendrites of neonatal motoneurones compared to older animals. NR2B mRNA was expressed at low to moderate levels throughout the studied period whereas NR2A mRNA levels were low until P21. Following unilateral nerve crush, an initial decrease in NR1 mRNA occurred at one day after nerve crush (P3) in labelled CP motoneurones ipsilateral to the crush which was followed by a significant increase in NR1 subunit expression at 5 days post‐injury. This increase was bilateral although reaching greater significance ipsilateral to the crush compared with sham‐operated animals. A significant increase in NR1 and NR2B mRNA post injury was also detected in spinal cord homogenates. In addition, the changes in levels of NR1 and NR2B mRNA were reflected by comparable bilateral changes at P7 in receptor protein determined by quantitative immunocytochemical analysis of NR1 and NR2 subunit expression in identified CP motoneurones indicating a co‐ordinated regulation of receptor subunits in response to injury.

Distribution of the N-methyl-d-aspartate glutamate receptor subunit NR2A in control and amyotrophic lateral sclerosis spinal cord

The distribution of the different glutamate receptor subunits in human spinal cord has yet to be fully elucidated. The aim of this study was to examine the distribution of the N-methyl-D-aspartate (NMDA) glutamate receptor modulatory subunit NR2A, in control human spinal cord and to examine in parallel the expression of the mRNA in amyotrophic lateral sclerosis (ALS). The aetiology of ALS is poorly understood, although abnormalities in glutamate and glycine transport have been reported as well as alterations in NMDA receptors including the NR1 subunit; suggesting a role for glutamate in the disease process. We have used the technique of in situ hybridisation to localise this receptor subunit to the laminae of human spinal cord and have found that it shows a widespread distribution similar to that previously reported for the universal NMDA receptor subunit NR1. Quantitation of mRNA expression in control and ALS cases showed a significant widespread loss of NR2A from both dorsal and ventral horns with losses of 55% and 78%, respectively, in ALS as compared to control. These results were substantiated by analysis of spinal cord homogenates, which showed a significant total decrease of 50% in ALS spinal cord as compared to control.

Characterisation of the distribution of choline acetyltransferase messenger RNA in human spinal cord and its depletion in motor neurone disease.

We have characterised the distribution of choline acetyltransferase (ChAT) mRNA in spinal cord from normal and motor neurone disease/amyotrophic lateral sclerosis (MND) subjects by in situ hybridisation. High concentrations of ChAT-mRNA were detected in 4 main regions of spinal cord, layer IX of the ventral horn, layer III of the dorsal horn, the intermediate grey matter and layer X around the central canal. ChAT mRNA was most highly concentrated in layers IX and III. Substantial decreases in ChAT mRNA were detected in ventral grey matter (layer IX) of cervical and lumbar cord in all cases of MND. Smaller and more variable changes in ChAT mRNA were seen in MND in other regions of spinal cord which indicates that these changes may arise as secondary processes. The value of this technique in understanding the pathophysiology of MND is discussed.

Changes in expression of NR-1 and c-jun mRNA in rat lumbar spinal cord after neonatal common peroneal nerve crush

We have examined the expression of the NR-1 subunit of the glutamate NMDA receptor and the immediate early gene c-jun in lumbar spinal cord following neonatal common peroneal nerve crush. The expression of these two genes was studied up to 12 days post-injury (crush occurring at neonatal day P2). The levels of both NR-1 and c-jun mRNA were increased in spinal cord ipsilateral to the site of crush, the induction of mRNA was shown to occur in a time-dependent manner, peaking at 5 days post-injury. The level of NR-1 mRNA showed the most substantial change following nerve crush, increasing 5 times from 4 h to 5 days post-crush. An increase in expression of NR-1 was also observed in spinal cord contralateral to the injury, although quantitatively this was a smaller effect. These results indicate that early postnatal injury causes a significant increase in the expression of NR-1 mRNA which is most marked at 5 days after injury. This period coincides with that of maximum cell death and indicates that the selective induction of NR-1 could underlie the mechanism of this cell death.

Application of in situ hybridization to studies of messenger RNA in the nervous system: functional correlations and their quantitation

With a tissue as heterogeneous and complex in its organization as the nervous system, in situ hybridization offers an approach of considerable potential. The complexity of this tissue means that a technique with high resolving power is needed to identify changes in gene expression that occur in a small group of cells. The CNS is extremely heterogeneous, it contains a dozen classical transmitters and more than 40 neuropeptides all in discrete cell clusters. This gives rise to a diversity of functional cell types each with its own strict localization and interaction with other cells. Activation of specific pathways may then result in only highly localized changes in metabolism which would be difficult to detect with tissue homogenates where the transmitter being studied may only be present in 1% of cells or less. The technique of in situ hybridization however allows the detection of highly localized changes in mRNA levels in specific cell types. The applications are numerous, from tracing pathways, localization of function to particular cell groups and localization of mRNAs of unknown function. Further, abnormal gene expression in neurological disorders may give some insight into the pathogenesis of disease and examples of these different applications will be given in the text. To satisfy the requirements for in situ hybridization it is essential to preserve the tissue during preparative treatments in order to be able to accurately define the cell types containing the mRNA being studied. Many of the mRNA species of particular interest are often far from abundant e.g. receptors and neuropeptides. Thus in studies of the nervous system, resolution, structural integrity and quantitation are key issues and these aspects will be discussed in detail together with the particular applications for which these approaches have been used.