Myelin-deficient Rat Research Articles

The attenuation of GABA sensitivity in the maturing myelin-deficient rat optic nerve

GABA (γ-aminobutyric acid) can modulate axonal excitability by activating GABA A receptors on some central nervous system axons. The effects of GABA on optic nerve axons decrease significantly during the course of myelination, suggesting that myelination may influence changes in GABA sensitivity. To test this hypothesis, we compared the depolarizing effect of GABA and the GABA A-receptor agonist, muscimol, on optic nerves of myelin-deficient (MD) rats and their unaffected siblings using a modified sucrose-gap technique. Optic nerves from both control and MD rats displayed relatively large GABA-induced depolarizations when studied at an early postnatal period. In both the control and MD rats, the GABA uptake inhibitor nipecotic acid led to a distinct depolarization suggesting endogenous release of GABA. Although the GABA-induced depolarization in the MD rat was significantly greater than that in the control rat at the third postnatal week, the response in the MD rat attenuated with maturation. These results demonstrate that the attenuation of the depolarization induced by GABA and nipecotic acid seen in the normal optic nerve also occurs in the MD rat optic nerve. This suggests that the attenuation of optic nerve sensitivity to GABA is not the result of myelination or interaction with myelin-associated proteins.

Ontogeny of glycerol phosphate dehydrogenasepositive oligodendrocytes in rat brain. Impaired differentiation of oligodendrocytes in the myelin deficient mutant rat

The ontogeny of oligodendrocytes in the myelin deficient (md) rat mutant and in control rats was explored immunohistochemically using an antiserum against the oligodendrocyte specific enzyme, glycerol phosphate dehydrogenase (GPDH), and the avidin-biotin complex technique. In control rats, GPDH was demonstrated to be expressed relatively early in oligodendrocyte differentiation, prior to either myelin basic protein or proteolipid protein expression. With development, oligodendrocytes containing GPDH increased in number, apparent staining intensity, cell soma area and process elaboration. Fewer GPDH+oligodendrocytes were observed in the brain of mutant rats than in unaffected littermates at all developmental ages, and major developmental increases in oligodendrocyte density were delayed. The density of GPDH+oligodendrocytes was reduced by about 40% in both the corpus callosum and in the cingulate cortex of P22–25 md mutants compared with control rats. The oligodendrocyte cell soma area was not influenced by the md condition, and increased 2-fold with development in rats of both genotypes. The area of coronal sections occupied by the corpus callosum increased about 2.5-fold with development, and was 30% smaller in mutant rats late in their lifespan than in unaffected littermates. The reductions in oligodendrocyte density reported here are of insufficient magnitude to fully account for biochemically measured reductions in oligodendrocyte gene expression accompanying the md trait, indicating that gene expression per oligodendrocyte is also impaired. Cell counts in control rats also revealed that oligodendrocytes are overproduced during development. Cell density and the total number of corpus callosum GPDH+oligodendrocytes per section were maximal at P22–25 and then decreased to adult values. These results suggest that glial cells, like neurons, may be generated in excessive numbers, and some subsequently die, as a normal concomitant of development.

Conduction properties of spinal cord axons in the myelin-deficient rat mutant

Spinal cords of myelin-deficient and normal age-matched (control) rats were removed and their conduction and pharmacological properties studied in an in vitro brain slice chamber. The conduction velocity of the myelin-deficient dorsal column axons was reduced to about 25% of control axons; however, the amyelinated myelin-deficient axons displayed refractory periods and the ability to sustain high-frequency action potential discharge similar to that of dorsal column axons in control rats. Pharmacological results suggest that the myelin-deficient dorsal column axons qualitatively express a normal complement of ion channels and receptors. The demonstration of a normal representation of channels and receptors on these axons supports the proposal that the oligodendrocyte, and not the axon, is the site of the primary defect in the myelin-deficient rat mutant. It is concluded that, unlike acutely demyelinated axons which display marked frequency-dependent conduction block, amyelinated axons of the myelin-deficient rat spinal cord develop compensatory mechanisms to stabilize action potential conduction.

Transferrin receptor expression in myelin deficient (md) rats.

The question of iron regulation in the brain is the subject of increasing interest as the evidence continues to accumulate that a loss of brain iron homeostasis plays a significant role in some neurodegenerative diseases. Most cells acquire iron through a specific receptor mediated process involving transferrin, the iron mobilization protein. It appears that in the brain, endothelial cells, neurons, and oligodendrocytes express the transferrin receptor. This study uses a strain of rats (myelin deficient, md) in which oligodendrocytes fail to mature, and examines the consequences of this genetic defect on the expression of the transferrin receptor in the brain. The affinity of transferrin for its receptor is similar between the cerebral cortex and cerebellum in both the normal and myelin deficient rats (Kd = 7.8-10.6 nM). The transferrin receptor density is normally 2-3 times higher in the cerebellum than in the cerebral cortex. In the myelin deficient rat strain, the density of the transferrin receptor is decreased in both the cerebrum (56%) and cerebellum (70%) compared to the littermate control animals. Because oligodendrocytes are the only cell type affected in this mutant, the results suggest that these cells are responsible for a considerable amount of the transferrin receptors that are expressed in the brain (excluding the endothelial cell contributions). These observations are consistent with the existing literature stating that oligodendrocytes are responsible for the majority of transferrin and transferrin mRNA which is expressed in the brain, and support the working hypothesis that imbalances in brain iron homeostasis, particularly during development, are associated with myelin disorders.

Reactive astrocytes in myelin-deficient rat optic nerve reveal an altered distribution of orthogonal arrays of particles (OAP).

Reactive astrocytes are a common feature of various pathological conditions within the CNS. Morphological changes of reactive astrocytes include an altered nucleus-cytoplasm relationship, nuclear indentations, an increased amount of intermediate filaments, and an immunologically immature phenotype. Additionally, the number of orthogonal arrays of particles (OAP) was found to be increased within parenchymal membranes of reactive astrocytes. This observation prompted us to investigate the distribution of astroglial OAP in the amyelinated CNS of the myelin-deficient (md) rat in which reactive astrocytes prevail. In the present freeze-fracture study, astroglial OAP were determined within endfoot and nonendfoot (parenchymal) membranes in the developing optic nerve of md rats and normal littermates at the age of 12, 23, 40, and 64 days postnatally (dpn). The endfoot OAP density in md astrocytes remained constant during the entire period of investigation. In myelinated littermates, OAP densities continuously increased up to adult values. In contrast, the parenchymal OAP density in md astrocytes increased during the entire period of investigation. In normal littermates, OAP densities remained constant during the first 40 dpn and thereafter increased rapidly. These observations suggest that the absence of myelinogenesis in the md mutant may be a stimulus for parenchymal membranes of reactive astrocytes to insert OAP or to assemble OAP subunits into complete arrays.

Functional capacities of transplanted cell-sorted adult oligodendrocytes.

Previous work has shown that transplanted dissociated glial cells derived from neonatal rats can myelinate areas of the myelin deficient (md) rat spinal cord. In the present studies we have examined the ability of adult glial cells, either as suspension of mixed glial cells or as 01 positive oligodendrocytes purified by cell sorting, to myelinate md rat axons following transplantation. Mixed glial cell preparations from adult rats myelinated large areas of either, dorsal, lateral or ventral columns. These areas contained clumped, transplanted oligodendrocytes. In addition, preparations containing over 90% 01 positive oligodendrocytes were also capable of myelinating large numbers of axons upon transplantation. These results suggest that adult oligodendrocytes may play an important role in remyelination of the adult CNS.

Transplantation of cultured premyelinating oligodendrocytes into normal and myelin-deficient rat brain.

Cultures of oligodendroglial cells at various stages of maturation, from progenitors to maturing oligodendrocytes, were prepared from neonatal rat brain primary cultures and then were prelabeled in the culture dish with the fluorescent dye, fast blue (FB). Single cell suspensions were grafted into normal or myelin-deficient rat brains. The normal as well as the myelin-deficient in vivo environment allowed cell survival, migration, and differentiation. The FB+ cells expressed the oligodendroglial markers, glycerol phosphate dehydrogenase, galactocerebroside, and myelin basic protein. In the normal rat transplanted cells were identifiable at all times studied up to 24 weeks. Extensive migration of FB+ cells was observed in whole-brain sagittal sections. Our results show that the plasticity of oligodendroglia differentiation, extensively studied in vitro, can now be investigated in the normal and myelin-deficient in vivo environment.

Rumpshaker: an X‐linked mutation affecting CNS myelination. A study of the female heterozygote

This study examines the myelin deficits found in the spinal cord and optic nerves of female mice heterozygotes for rumpshaker (rsh), an X-linked mutation causing hypomyelination. No clinical abnormalities were detected but morphological changes were evident, particularly in the spinal cord, which showed no evidence of resolving with age. In the spinal cord, scattered hypomyelinated axons, occasionally grouped in twos or threes, were the major feature; oligodendrocyte numbers were slightly elevated at all ages compared to normal male littermates and the total amount of myelin was reduced. Myelin protein composition of the sheaths was examined by immunostaining for myelin basic protein (MBP) and two peptide regions of PLP/DM-20 molecule; one being proteolipid protein (PLP)-specific and the other recognizing the c-terminal common to PLP-DM-20. The majority of myelin sheaths immunostained for MBP and PLP. Occasional MBP-positive sheaths failed to stain with PLP/DM-20 or PLP-specific antiserum. Therefore, at least two types of immunocytochemically-defined myelin sheaths are present in the heterozygotes. Changes in the optic nerves were much less obvious; glial cell numbers were increased but thinly myelinated axons were not detected although the total amount of myelin was reduced compared to normal littermates. In no instance were mosaic, amyelinated/hypomyelinated patches detected. Heterozygotes for rsh, therefore, are considerably different from those for other X-linked myelin mutations like the jimpy mouse and the myelin-deficient rat, both in regard to the severity of the lesions and their failure to recover with age.

Transplantation of labeled fetal spinal cord fragments into juvenile myelin‐deficient rat spinal cord

Minced and triturated fragments from the spinal cord of normal rat fetuses (15-18 days gestation) labeled with the fluorescent dye fast blue (FB) were successfully transplanted into juvenile myelin-deficient rat spinal cord under direct observation. Clusters of myelinated fibers were found subsequently in the recipient spinal cord, and, by fluorescence microscopy, clusters of FB-labeled cells were found at corresponding sites. The results indicate that the surgical approach used is suitable for transplantation of tissue fragments into a defined region of juvenile rat spinal cord, that FB can be used to locate the transplanted cells subsequently, and that FB does not interfere with maturation of the donor glia or with myelin formation.

Single Base Substitution in Codon 74 of the MD Rat Myelin Proteolipid Protein Genea

The myelin-deficient (md) rat is one of several X-linked animal mutants that have severe dysmyelination in the central nervous system. It appears in all to be the result of mutations in the myelin proteolipid protein gene which is located on the long arm of the X-chromosome. To identify the md rat mutation, we isolated and sequenced cDNAs corresponding to PLP and DM-20 mRNAs from the brain of hemizygous affected males. The only consistent sequence difference between these and normal rat sequences was the substitution of a C for an A at the first position of codon 74, resulting in a threonine to proline amino acid change. The presence of this helix-breaking amino acid in the second hydrophobic alpha-helical segment of the protein might be expected to influence its ability to interact with the membrane. PCR amplification and sequencing of the corresponding genomic regions were used to confirm the presence of the single base change in the hemizygote and both normal and mutant versions in the heterozygotes. It is interesting that this change, like those detected in other X-linked myelin disorders, involves an amino acid replacement within a hydrophobic alpha-helical segment of the PLP protein. Disruption of these structures apparently has severe consequences for the ability of PLP to contribute normally to myelination.

Alterations in Transferrin and Its Receptor in the Brain of Myelin‐Deficient Rats

Alterations in Transferrin and Its Receptor in the Brain of Myelin‐Deficient Rats

Axolemmal Abnormalities in Myelin Mutantsa

Evidence is reviewed that the paranodal axoglial junction plays important roles in the differentiation and function of myelinated axons. In myelin-deficient axons, ion flux across the axolemma is greater than that in myelinated fibers because a larger proportion of the axolemma is active during continuous, as opposed to saltatory, conduction. In addition, older myelin-deficient rats that have developed spontaneous seizures display small foci of node-like E-face particle accumulations in CNS axons as well as more diffuse regions of increased particle density and number. Assuming that the E-face particles represent sodium channels, such regions could underlie high sodium current density during activity, low threshold for excitation, and increased extracellular potassium accumulation. Depending on the degree of spontaneous channel opening, they could also represent sites of spontaneous generation of activity. The appearance of seizures and their gradual increase in frequency and severity could represent an increase in the number of such regions. In addition, diminution in the dimensions of the extracellular space during maturation would result in increased extracellular resistance, which, together with increasing axonal diameter, would tend to increase the likelihood of ephaptic interaction among neighboring axons as well as the likelihood of extracellular potassium rises to levels that could cause spontaneous activity.

Myelin formation following transplantation of normal fetal glia into myelin-deficient rat spinal cord

Structurally normal myelin sheaths develop in the spinal cord of juvenile myelin-deficient rats (mdr) 11 days after transplantation of normal fetal spinal cord fragments or cultured cells that do not yet express galactocerebroside. Cultures result in more extensive myelin formation, and in both cases the myelin that forms is located primarily at or near the site of transplantation. Myelin formation also occurs after transplantation of postnatal donor tissue, but the extent diminishes with donor age, and none was seen after transplantation of adult donor tissue over the two-week period studied. Injection of killed tissue, tissue derived from mouse donors or an extract of myelin also did not lead to myelin formation. The results imply that myelin formed in the host following transplantation was generated by oligodendrocytes newly differentiated from donor precursor cells rather than by donor oligodendrocytes that were already mature at the time of transplantation or by host oligodendrocytes that took up components of the injected material. We conclude that exogenous fetal glial cell precursors are able to survive, differentiate and form myelin in the environment of the juvenile mdr spinal cord.

The Myelin‐Deficient Rat Has a Single Base Substitution in the Third Exon of the Myelin Proteolipid Protein Gene

Several genetic disorders that occur in animals and in humans result in an inability to synthesize normal myelin. Some of these disorders are inherited in an X-linked manner. The localization of the myelin proteolipid protein (PLP) gene to the X chromosome has directed the study of X-linked myelination disorders toward PLP. The myelin-deficient rat is one such X-linked dysmyelinating mutant. From a cDNA library constructed from myelin-deficient rat brain mRNA, we have isolated and sequenced cDNAs corresponding to PLP and its alternatively spliced isoform, DM-20. An A to C transition was detected in these cDNAs, which results in a threonine to proline change at amino acid 74 in both PLP and DM-20. No other substitutions were seen in the cDNA sequences. Polymerase chain reaction amplification and sequencing of the corresponding genomic regions were used to confirm the single base change. This substitution occurs in a highly hydrophobic portion of the protein that is thought to be an alpha-helical transmembrane segment. The presence of a helix-breaking amino acid such as proline in this segment is likely to influence the ability of the protein to interact with the membrane.

X‐chromosome monosomy in the myelin‐deficient rat mutant

We have identified three examples of female Wistar rats exhibiting the tremor and seizures characteristic of the X-linked myelin deficiency (md) mutation, which is ordinarily seen only in males. Cytogenetic study of two of these animals has shown them to have 41 chromosomes instead of the normal 42. The missing chromosome was identified as an X chromosome by G-banding analysis. These animals thus have an XO genotype comparable to that in Turner's syndrome. Anatomically, one of the animals, which was studied in detail, showed no abnormality of the uterus, and the ovaries, although somewhat smaller than normal, were histologically indistinguishable from those in a normal female rat. No evidence of endocardial fibroelastosis was detected, nor was there any anomaly of the aorta. The myelin deficiency in the central nervous system was comparable to that in hemizygous mutant male rats. XO monosomy in the Wistar rat thus has little effect on phenotype and is more comparable to that in mice than to Turner's syndrome in man. The myelin-deficient rat is useful for studies of X-chromosome monosomy since XO females can readily be identified by the neurological syndrome characteristic of the md mutation.

Macromolecular structure of axon membrane and action potential conduction in myelin deficient and myelin deficient heterozygote rat optic nerves

The macromolecular structure of the axon membrane in optic nerves from 25-day-old male littermate control and myelin deficient (md) rats and 16-month-old md heterozygotic rats was examined with quantitative freeze-fracture electron microscopy. The axon membrane of control optic nerves displayed an asymmetrical partitioning of intramembranous particles (IMPs); P-fracture faces of myelinated internodal axon membrane were more particulate than those of pre-myelinated axons (approximately 1600 v 1100 microns-2, respectively), while relatively few IMPs (approximately 150 microns-2) were present on external faces (E-faces) of internodal or pre-myelinated axon membrane. Amyelinated axons of md optic nerves also exhibited an asymmetrical partitioning of IMPs; protoplasmic membrane face (P-face) IMP densities, taken as a group, exhibited a wide range (approximately 600-2300 microns-2) and, in most regions, E-faces displayed a relatively low IMP density (approximately 175 microns-2). Axons of greater than 0.4 microns diameter exhibited significantly greater mean P-face IMP density than axons less than 0.4 microns diameter. Aggregations of E-face IMPs (approximately 350 microns-2) were occasionally observed along amyelinated axon membrane from md optic nerves. Optic nerves from md heterozygote rats exhibit myelin mosaicism, permitting examination of myelinated and amyelinated axon membrane along the same tract. The axon membrane exhibits different ultrastructure in these two domains. Myelinated internodal axon membrane from md heterozygote optic nerves exhibits similar P- and E-face IMP densities to those of control internodal axolemma (approximately 1800 and 140 microns-2, respectively). Amyelinated axons in the heterozygote exhibit a membrane structure similar to amyelinated axons in md optic nerve. P-face IMP density of large diameter (greater than 0.4 microns) amyelinated axons from md heterozygote optic nerves is significantly greater than that of small calibre (less than 0.4 microns) axons. In most regions, amyelinated axon membrane exhibits a relatively low E-face IMP density (approximately 200 microns-2); however, focal aggregations (approximately 400 microns-2) of E-face particles are present. Electrophysiological recordings demonstrate that amyelinated axons in md optic nerves support the conduction of action potentials. Compound action potentials in md optic nerves exhibit a monophasic configuration, even at 20-days postnatal, similar to that of pre-myelinated optic nerve of 7-day-old normal rats. Moreover, conduction velocities in the amyelinated 20-day-old md optic nerve are similar to those displayed by pre-myelinated axons from 7-day-old optic nerves.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered cellular distribution of iron in the central nervous system of myelin deficient rats

Under normal conditions, iron is found predominantly in oligodendrocytes, the myelin producing cell, in the rat brain. A genetic mutant strain of rats known as myelin deficient rats is examined in the present study because their number of oligodendrocytes is decreased and those oligodendrocytes present are structurally abnormal. The levels of iron in the liver (major site of iron storage) and in the pons-cerebellum did not differ statistically between the myelin deficient rats and the littermate control rats, whereas only half of the iron normally found in the cerebrum-midbrain was present in the myelin deficient rat. Histologically, iron was found predominantly in oligodendrocytes in the littermate control rats, as expected. In the myelin deficient rat, iron staining was confined to astrocytes and microglia. The results of this study strongly suggest that iron uptake into the brain continues in the absence of normal oligodendrocytes and myelin. Furthermore, these data suggest that iron metabolism can be substantially altered, as indicated by the accumulation of iron in astrocytes and microglia, when normal or near normal levels of iron are quantitatively demonstrated. The response of astrocytes and microglia to sequester the iron (presumably through phagocytosis) in the absence of invasive damage represents, to our knowledge, a new functional observation for these cells. Based on these observations it is clear that iron histochemistry in combination with quantitative analysis is necessary to interpret data regarding iron physiology, at least in neurobiology, and iron accumulation by astrocytes and microglia may provide clues of altered iron metabolism despite normal iron levels.

Transcriptional Regulation Studies of Myelin-Associated Genes in <i>Myelin-Deficient</i>Mutant Rats

To identify and assess the consequences of the mutation in myelin-deficient (md) rats, the myelin proteolipid protein (PLP) gene and its expression were studied in md rats. Southern blots of the PLP gene demonstrated that no major deletions or insertions have occurred in this gene. In addition, the mutation in this gene does not result in a splicing defect in the RNAs, since all exons are represented in md PLP RNAs. These data are consistent with results in another laboratory indicating that a point mutation in the PLP gene in md rats results in a single amino acid alteration in the protein. To elucidate the molecular mechanisms producing reduced levels of PLP, myelin basic protein (MBP) and glycerol phosphate dehydrogenase (GPDH) mRNAs, and their corresponding proteins in md rats, in vitro transcription assays were performed. Transcription of the PLP gene in nuclei isolated from 23-day-old md rat brains was dramatically reduced relative to normal tissue. Thus, the single amino acid alteration in this protein alters the regulation of transcription of this gene. In contrast, the transcriptional activities of the MBP and GPDH genes in md rats were indistinguishable from normal animals. Thus, the lower level of MBP and GPDH mRNA and protein in md rats relative to normal results from a posttranscriptional event.

Myelin-deficient rat: a point mutation in exon III (A----C, Thr75----Pro) of the myelin proteolipid protein causes dysmyelination and oligodendrocyte death.

The expression of the proteolipid protein (PLP) gene of the myelin deficient (md) and normal rat was studied during the myelination period. The sizes of the PLP transcripts (1.6 and 3.2 kb) in the md and normal rat were identical although the md PLP messenger RNA level was extremely reduced as shown by in situ hybridization and Northern blot hybridization analysis. The structure of the md proteolipid protein gene was analyzed on the cDNA and genomic level. The molecular basis of the myelin deficiency phenotype has been elucidated: a point mutation in exon III (A----C transversion) verified by cDNA and genomic DNA sequencing causes a mutation of Thr75 to Pro and creates an additional AvaII restriction site in exon III of the md rat. The threonine to proline mutation located within the second transmembranal alpha-helix might induce a conformational change and thereby prohibit the integration of PLP into the membrane with the clinical manifestation of dysmyelination leading to premature death within 3-6 weeks.

Extracellular potassium activity and axonal conduction in spinal cord of the myelin-deficient mutant rat

We recorded somatosensory evoked potentials (SEPs), extracellular K + ionic activity ([K +] e), and K + clearance rates in the spinal cords of 14 myelin-deficient mutant rats and 16 normal male littermates at 16–41 days after birth. Tested under pentobarbital anesthesia (25 mg/kg ip) and hypothermic conditions (32–34°C), myelin-deficient rats had longer cortical SEP latencies (67 ± 20 ms) compared to those in normal siblings (48 ± 15 ms; P < 0.05). Mean baseline [K +] e levels were 2.6 ± 0.5 m M in myelin-deficient rats and 2.6 ± 0.8 m M in normal siblings. Clearance times of KCl solutions injected into the spinal cord were biphasic and exponential. The mean initial and secondary exponential half-times were 1.0 ± 0.5 and 2.7 ± 1.7 min for myelin-deficient rats and 0.8 ± 0.4 and 3.8 ± 3.2 min for normal siblings. Repetitive sciatic nerve stimulation (2–20 Hz, 2- to 6-s trains) produced 1–3 m M transient [K +] e rises in thoracic and lumbar cords of myelin-deficient rats. The [K +] e rises were largest in the dorsal spinal cord at 200–500 μm depth. The normal siblings had smaller or no stimulus-induced [K +] e rises. In myelindeficient rats, injection of 1 m M 4-aminopyridine (4-AP) solution into the thoracic spinal cord completely suppressed the stimulus-induced [K +] e and markedly increased spinal and cortical SEP amplitudes for several hours. In the normal siblings, the 4-AP injections transiently blocked spinal conduction for 20–30 min but thereafter enhanced cortical SEP amplitudes for 2–3 h. We conclude that sciatic nerve stimulation produces spinal cord [K +] e rises in myelin-deficient rat larger than those in the normal siblings, that the [K +] e transients represent increased K + release rather than impaired K + clearance, and that the K + ions come from 4-AP blockable sources.