The twitcher mouse: an enzymatically authentic model of human globoid cell leukodystrophy (Krabbe disease)

The Twitcher is a new neurological mutant of mouse which shows clinical and histopathological features similar to those of human and canine globoid cell leukodystrophy (Krabbe disease). Brain and liver tissues of affected mice showed a profound deficiency of galactosylceramidase and lactosylceramidase I activities. A distinct group with intermediate enzyme activities could be identified among the phenotypically normal mice, consistent with their being heterozygotes. The Twitcher mouse is, therefore, not only a clinically and morphologically, but also an enzymatically, authentic model of human globoid cell leukodystrophy. This is the first enzymatically authentic murine model of human sphingolipidosis.     

Suppressed UDP-galactose: Ceramide galactosyltransferase and myelin protein mRNA in twitcher mouse brain

The developmental changes in expression of steady-state mRNA that encode proteins that are important for myelination (myelin basic protein, myelin-associated glycoprotein, proteolipid protein, UDP-galactose:ceramide galactosyltransferase) and glial fibrillary acidic protein were investigated in the brain of the twitcher mouse, a model of human globoid cell leukodystrophy. This disease is caused by a mutation in the gene encoding the lysosomal enzyme, galactosylceramidase, which catalyzes the degradation of the myelin lipid galactosylceramide. Before postnatal day (PND) 20, the levels of myelin protein mRNA were similar in twitcher and normal mice. With progression of demyelination after PND 25-30, myelin protein mRNA levels gradually decreased. The period of maximum expression of the myelin protein genes in twitcher mice was, however, similar to that of normal control mice. mRNA levels for the gene that encodes the enzyme UDP-galactose:ceramide galactosyltransferase which is responsible for catalyzing the final step in galactosylceramide synthesis, was exceptionally downregulated from the early stages of the disease. The increase of glial fibrillary acidic protein (GFAP) mRNA levels preceded morphological evidence of demyelination. J. Neurosci. Res. 51:536–540, 1998. © 1998 Wiley-Liss, Inc.     

The twitcher mouse: normal pattern of early myelination in the spinal cord

The pattern of early myelination was investigated in the dorsal columns of the cervical spinal cord in the twitcher, an authentic murine model of human globoid cell leukodystrophy, and their littermates. There were no differences in the number of myelinated fibers until the day 20 postnatal. However, myelin sheath in the homozygous affected twitchers at the day 20 were thinner than those of heterozygous and normal littermates, while at the day 10 no significant differences were detected. These observations indicated that in the twitcher mouse, despite the generic deficiency of galactosylceramidase, myelination progresses normally in early stages and then hypomyelination becomes apparent before myelin breakdown.     

Hyperexcitability in organotypic mouse hippocampal explants

1. We have developed an organotypic hippocampal tissue culture model for analyses of sustained hyperexcitability in which repetitive electric stimulation of the dentate area enhances the amplitude and complexity of evoked normal and epileptiform field potentials recorded extracellularly from CA3/2 areas of neonatal mouse hippocampal explants.

2. In explants where spontaneous field potentials are not detectable at the onset of the experiment, brief repetitive electric stimulation elicits self-sustained epileptiform discharges that continue for the duration of the recorded period (2–10 hours).

3. Lowering the extracellular Ca⁺⁺ level to 0.1–0.2 mM markedly attenuates these discharges and repetitive stimulation during a 2–4 hr period fails to elicit hyperexcitability. When tested after return to normal media repetitive stimulation can elicit hyperexcitability.

4. Raising the extracellular K⁺ levels to 8–9 mM enhances the complexity of evoked as well as spontaneous field potentials and, in some cases, elicits self-sustained epileptiform discharges in the absence of repetitive electric stimulation.     

Udpgalactose:Ceramide galactosyltransferase in cultured oligodendrocytes: An enzymological and immunological study

The developmental expression of UDPgalactosexeramide galactosyltransferase (CGalT), an enyme marker of one myelinogenic activity in nervous tissue, was studied in cultured oligodendrocytes. The activity of CGalT in cultures followed a characteristic pattern of developmental changes. In the primary cultures these changes could be represented by a biphasic curve with a maximum of enzymatic activity at about the 25th day in culture. After purifying the oligodendrocytes from the primary cultures and replating them in culture dishes, similar developmental changes of CGalT were observed. In the subultures prepared from 20-day-old primary cultures the activity of CGalT per oligodendrocyte increased from 1.3 × 10⁻⁶ nmol/hr on day 4 to 3.7 × ⁻⁶ nmol/hr on day 21. Immunocytochemical studies with the antiserum against rat brain CGalT showed the presence of CGalT⁺ oligodendrocytes after 7 days in the primary culture (earliest time studied), later on the number of CGalT⁺ oligodendrocytes increased until 28 days (latest time examined). In the subcultures of purified oligodendrocytes the bulk of oligodendrocytes was stained by the anti-CGalT antibodies after 15 days. These results suggest that the initial expression of CGalT in oligodendroglial cultures involves an increase of the number of CGalT⁺ oligodendrocytes and of the amount of enzyme protein per cell.     

Development of serotonin, substance P and thyrotrophin-releasing hormone in mouse medullary raphe grown in organotypic tissue culture: developmental regulation by serotonin

Substance P (SP) and thyrotrophin-releasing hormone (TRH) are co-localized with serotonin (5-HT) in cells of the medullary raphe nuclei. In order to examine the factors that control development of multiple neurotransmitters within individual brain nuclei, we have grown presumptive raphe nuclei in organotypic tissue culture, an environment in which mammalian embryonic brain is easily accessible and manipulable. Tissue was obtained from E13 mice. A discrete midline segment of the rhombencephalon was dissected intact or was separated into 'rostral' (RR) and 'medullary' (MR) fragments. Tissue was explanted onto collagen coverslips and grown for up to two weeks in Maximow depression chambers. Tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, was barely detectable at explantation. During the first week in culture, however, TPH activity increased 7-fold. After two weeks, TPH activity increased almost 2.5-fold above the one-week level. Immunocytochemical analysis of the cultures confirmed a widespread distribution of 5-HT-positive cells and fibers throughout the explant. SP, monitored by radioimmunoassay, was detected after two days in culture, and attained a level of 111.7 +/- 9.8 pg/culture after two weeks. TRH activity was similarly elevated after two weeks in vitro. Therefore, developmental increases in TPH, SP, and TRH occurred in culture, mimicking the condition in vivo. RR and MR fragments, when grown apart on separate coverslips, developed 1.57-2.26 times the TPH activity that developed in the undivided piece. Inclusion of 1 microM pargyline in the fragments restored TPH to control levels. The effect of pargyline was blocked by methiothepin, suggesting autoreceptor-mediated regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The location of neuromuscular junctions on regenerating adult mouse muscle in culture

The location of neuromuscular junctions which form in vitro between regenerating adult mouse muscle fibres and sections of embryonic mouse spinal cord was examined. The position of the original motor end-plates on the explanted muscle fibres was determined by using either rhodamine-labelled α-bungarotoxin (RαBT) binding to the acetylcholine receptors, or by stains to demonstrate acetylcholinesterase (AChE) also located at the end-plate. In this culture system, the explanted muscle fibres degenerate and regenerate to form new myotubes which develop cross-striations and contractions. The location of the newly-formed neuromuscular junctions in these mature cultures was then demonstrated using RαBT-binding to acetylcholine receptors, silver impregnation and cholinesterase techniques. Less than half the new neuromuscular junctions were at the original end-plate areas indicating that, at least in this system, junctions can form at sites other than those of the original end-plate.     

Neurotrophin-4 selectively promotes survival of striatal neurons in organotypic slice culture

The neurotrophins (NGF, BDNF, NT-3, and NT-4) provide trophic support to subpopulations of neurons in the central and peripheral nervous systems. We examined organotypic slices of neonatal mouse striatum maintained in medium supplemented with neurotrophins or with CNTF to determine which of these factors influence the survivability of striatal neurons. Neuron counts at the end of the culture period revealed that NT-4 was the only factor that had a significant effect on neuronal survival, suggesting that NT-4 is a trophic factor for striatal neurons in organotypic slices.     

Growth of the WW strain of Theiler virus in mouse central nervous system organotypic culture

Summary Myelinated organotypic cultures of mouse central nervous system (CNS) provide a favorable milieu for growth of the WW strain of Theiler virus (TV), WW-TV induced a cytopathic effect characterized by neuronal destruction and swelling of myelin sheaths. Tissue culture medium and homogenates of infected cultures produced neurologic disease in mice, and TV. was demonstrated in cultures by indirect immunoflourescence and electron microscopy. Ultrastructurally, severe alterations in neurons and the presence of cytoplasmic inclusions containing paracrystalline arrays of 30-nm virus particles were evident in astrocytes. Demyelination appeared to be secondary to lysis of oligodendrocytes by virus. CNS organotypic cultures provide an environment for the study of TV-induced demyelination.     

Histogenesis of the vestibular sensory epithelium in organotypic culture of mouse embryo otocysts: A tritiated thymidine autoradiographic study

The cytogenesis in the vestibular sensory epithelium of mouse embryo otocysts grown in organ culture was examined using tritiated thymidine autoradiography. Pulse-labeling with [³H]-thymidine was applied either in vivo, at various intervals before explantation, or in vitro at specific stages of development. Observations of the development in vitro showed that the cytogenesis and cytodifferentiation of vestibular sensory cells were disturbed by explantation. By varying the intervals between the [³H]thymidine exposure and the date of explantation, we were able to demonstrate that explantation itself caused a significant decrease in the mitotic and the postmitotic phases of the hair cell precursors. Labeling of the expiants excised on day 13 of gestation revealed that precursors of the hair cells were progressively losing their mitotic capacity in vitro. In contrast with this finding, precursors of the supporting cells were less affected by explantation and culture conditions and they retained the capacity for survival, proliferation and differentiation.     

Development of mouse spinal cord in tissue culture: IV. Effects of embryonic extracts on neuron formation and migration

Possible influences upon patterns of neurogenesis expressed in vitro were examined quantitatively by the use of microfragment cultures of embryonic day 10 mouse neural tube. Crude extracts were prepared either from whole embryos (day 13 or 15 of gestation) or from embryonic brains (day 18 of gestation) and added to the culture medium for the first 10 days of culturing. Neuronal outgrowth zones surrounding individual microfragments were reduced in area (indicating restricted neuronal migration) and in number of neurons present (indicating restricted production of neurons) following treatment with either of the extracts. The severity of reductions observed were related to the developmental age of embryonic tissue used for preparing the extract, as greatest reduction resulted from addition of embryonic day 18 brain extracts and to concentration employed, higher doses further restricting neuronal outgrowth. By increasing the concentrations of extract the proportional number of large-sized neurons forming the outgrowth zones became greater relative to the small neuron contribution, indicating an enhanced survival for this neuronal population. The formation and migration of astroglial precursor cells was not affected by the addition of any of the extracts. The number of neurons remaining within the original portions of neural tube microfragments was not significantly altered following culturing in the presence of embryonic extract. This suggested that the reduction in neuron number in the outgrowth zone actually reflected a decreased neuron production and was not simply the result of a retention of neurons within the remaining portion of the microfragment. The results suggest the presence of substances within mouse embryos that have regulatory effects on aspects of development of the central nervous system. Indications are that survival and maturation of postmitotic neuroblasts are promoted in vitro while the formation of additional neuronal progenitor cells may be partially inhibited by the addition of embryonic mouse extracts to the medium. We propose that an endogenous negative feedback mechanism may be involved in the coordination of patterns of neurogenesis.     

Rat fetal ventral mesencephalon grown as solid tissue cultures: influence of culture time and BDNF treatment on dopamine neuron survival and function

Free-floating roller tube (FFRT) cultures of fetal rat and human nigral tissue are a means for tissue storage prior to grafting in experimental Parkinson's disease. In the present study, FFRT cultures prepared from embryonic-day-14 rat ventral mesencephalon were maintained for 4, 8, 12, or 16 days in vitro (DIV) in the presence or absence (controls) of BDNF [100 ng/ml]. The dopamine content in the culture medium, analyzed by HPLC, was significantly higher (4–5 fold) in the BDNF group at DIV 8 and DIV 12 compared to the corresponding control levels (40 pg/ml). The number of tyrosine hydroxylase immunoreactive neurons was significantly higher for BDNF treated cultures (2729±300) at DIV 8, as compared to controls (1679±217). At DIV 12, the culture volume was significantly increased by BDNF (1.05±0.12 vs. 0.71±0.04 mm³). Similar results were obtained for total protein. Western blot analysis demonstrated increasing signals for GFAP with increasing time in culture, but levels for control and BDNF treated cultures did not differ at any time-point investigated. In conclusion, it is suggested that the time window for effective storage of dopaminergic tissue prior to grafting can be extended by using the FFRT culture technique and that the in vitro storage may be further prolonged by treatment with BDNF.     

Depolarizing stimuli increase tyrosine hydroxylase in the mouse locus coeruleus in culture

The influence of membrane depolarization on the development and regulation of brain noradrenergic neurons was studied in explant cultures of the mouse locus coeruleus (l.c.). Exposure to the depolarizing agents veratridine or elevated K+ significantly increased the catalytic activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. The effects of veratridine were prevented by tetrodotoxin, suggesting that transmembrane Na+ influx was necessary for the rise in TH. Morphometric analysis indicated that the rise in TH activity was not accompanied by altered TH-positive cell number or cell diameter. Rather, TH fluorescence intensity increased in each neuron, suggesting that depolarization increased TH per neuron. Immunoblot and densitometric analysis indicated that depolarization did, indeed, increase TH immunoreactive protein. Moreover, depolarization elevated enzyme activity in cultured neurons expressing the normal developmental increase in TH, as well as those in which plateau levels had already been attained. We conclude that depolarization and/or Na+ influx regulates a critical transmitter macromolecule in brain neurons, as in the periphery, by altering enzyme molecule number.     

Proteostasis network alteration in lysosomal storage disorders: Insights from the mouse model of Krabbe disease

In Krabbe disease, a mutation in GALC gene causes widespread demyelination determining cell death by apoptosis, mainly in oligodendrocytes and Schwann cells. Less is known on the molecular mechanisms induced by this deficiency. Here, we report an impairment in protein synthesis and degradation and in proteasomal clearance with a potential accumulation of the misfolded proteins and induction of the endoplasmic reticulum stress in the brain of 6-day-old twitcher mice (TM) (model of Krabbe disease). In particular, an imbalance of the immunoproteasome function was highlighted, useful for shaping adaptive immune response by neurological cells. Moreover, our data show an involvement of cytoskeleton remodeling in Krabbe pathogenesis, with a lamin meshwork disaggregation in twitcher oligodendrocytes in 6-day-old TM. This study provides interesting protein targets and mechanistic insight on the early onset of Krabbe disease that may be promising options to be tested in combination with currently available therapies to rescue Krabbe phenotype.     

Distribution of proteolipid protein and myelin basic protein in cultured mouse oligodendrocytes: primary vs. secondary cultures.

The distribution of proteolipid protein (PLP) and myelin basic protein (MBP) was examined in differentiating oligodendrocytes of primary and secondary mouse brain cell cultures by single- and double-label indirect immunofluorescence. In primary cultures, MBP and PLP were differentially located in oligodendrocytes. MBP became concentrated as fine punctate dots lining the edges of processes and as coarse grains in flattened sheet-like structures. PLP was distributed diffusely throughout cell bodies and processes but was limited to the perimeter of sheets and some processes within sheets. To compare the detailed distribution of PLP and MBP in the absence of underlying cells, a simple method for the growth of isolated oligodendrocytes in secondary cultures was developed. Cells were maintained in primary culture for 39-41 days, harvested by scraping, enriched for oligodendrocytes, and plated at low cell density. After 1 week, isolated oligodendrocytes had developed long processes and large flattened membranous sheets. MBP and PLP were differentially localized in these cell structures. The sheets contained fine-grained patches of MBP, which were surrounded by networks of MBP- processes. In contrast, PLP was initially seen throughout the cell bodies and processes. In older cultures, PLP became strikingly concentrated in curvilinear membranous profiles. The observations show that PLP and MBP are differentially located in cultured mouse oligodendrocytes. Furthermore, the precise distribution of these myelin-specific antigens is dependent on culture conditions.     

The use of networks of dissociated rat dorsal root ganglion neurons to induce myelination by oligodendrocytes in culture

A neuronal culture system has been developed that has demonstrated to induce myelin formation by added oligodendrocytes. Networks of dissociated dorsal root ganglion neurons were prepared by suppressing non-neuronal cells (i.e. fibroblasts and Schwann cells) with a continuous 2 week exposure to 10⁻⁵ M fluorodeoxyuridine in the culture medium. After drug withdrawal, neuroglial cells were introduced in optic nerve implants from 1–2 week-old rats. These added glial cells migrated extensively over the unensheathed neurities and central myelin was formed by 2 weeks after the implant addition.     

The actions of substance P and serotonin on myenteric neurons in tissue culture

Intracellular recordings were made from guinea-pig myenteric neurons in tissue culture. The responses to substance P (SP) and serotonin were compared. Putative transmitters were ejected by pressure from micropipettes. The response to SP was, in the majority of cases, a prolonged depolarization, with a latency of 1-5 s and a duration of 1-2 min. During the depolarization there was an increase in the input resistance of the cells, and the excitability was augmented. In two neurons the response to SP was hyperpolarization followed by depolarization. In about 30% of the cells the response to SP was relatively fast, with a duration of less than 10 s and a latency of less than 100 ms. These responses did not show desensitization. Serotonin induced relatively fast responses only, with durations of 0.3-3 s and latencies of 20-50 ms. These responses were usually depolarizing, but in 3 cells were hyperpolarizing. Two types of behaviour were observed during repetitive ejection of serotonin. Some cells showed strong desensitization while in others there was no desensitization and there was even summation. The actions of SP and serotonin were compared by their ejection in the same experiments. The same general properties described above were observed. It is concluded that the two putative transmitters act via distinct response mechanisms. The SP-induced depolarizations in tissue culture resemble in several respects the slow synaptic potentials recorded previously in myenteric neurons in freshly isolated preparations.     

Survival and death of mature avian motoneurons in organotypic slice culture: trophic requirements for survival and different types of degeneration

We have developed an organotypic culture technique that uses slices of chick embryo spinal cord, in which trophic requirements for long-term survival of mature motoneurons (MNs) were studied. Slices were obtained from E16 chick embryos and maintained for up to 28 days in vitro (DIV) in a basal medium. Under these conditions, most MNs died. To promote MN survival, 14 different trophic factors were assayed. Among these 14, glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor were the most effective. GDNF was able to promote MN survival for at least 28 DIV. K(+) depolarization or caspase inhibition prevented MN death but also induced degenerative-like changes in rescued MNs. Agents that elevate cAMP levels promoted the survival of a proportion of MNs for at least 7 DIV. Examination of dying MNs revealed that, in addition to cells exhibiting a caspase-3-dependent apoptotic pattern, some MNs died by a caspase-3-independent mechanism and displayed autophagic vacuoles, an extremely convoluted nucleus, and a close association with microglia. This organotypic spinal cord slice culture may provide a convenient model for testing conditions that promote survival of mature-like MNs that are affected in late-onset MN disease such as amyotrophic lateral sclerosis.     

Development of a mouse model of ethanol addiction: naltrexone efficacy in reducing consumption but not craving

Summary. The aim of the present study was validating pharmacologically a mouse model of alcohol addiction. Mice (n = 60) were offered ethanol (5% and 10%) and water in a free choice paradigm consisting of four phases: free choice (10 weeks), withdrawal (2 weeks), re-exposure (2 weeks) and quinine- adulteration (2 weeks). Control mice (n = 10) had access to water. They were housed individually with food ad libitum. The animals’ behaviour was evaluated at the beginning of the treatment and during the withdrawal period. After the exposure to the model, mice received i.p. naltrexone (0.0; 0.125; 2.0 and 16.0 mg/kg) or saline. Mice were characterized as: addicted (n = 15, preference for ethanol without reducing intake when ethanol were adulterated with quinine); heavy drinker (n = 14, preference for ethanol but reduced intake when ethanol were adulterated); and light drinker (n = 16, no preference for ethanol). Naltrexone reduced ethanol intake in the heavy and light groups (p ≤ 0.01 and p ≤ 0.05, respectively, compared to saline-treated group) with no effect on water intake. It is discussed that naltrexone may be acting in the positive reinforcing properties of ethanol but does not seem to have anti-craving properties. It was concluded that the addicted mice had a compulsive behavior manifested by the continued ethanol intake even under aversive conditions and under naltrexone treatment suggesting that this model might be useful to study addiction.