Neurogenetic approaches to axon sprouting in the hippocampus.
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Animals↗
Biomedical subjects
J L Noebels
Publications and source records attributed to J L Noebels.
Glutamate transporter mRNA expression in proliferative zones of the developing and adult murine CNS.
Neuronal migration, differentiation, and synapse formation are developmental processes within the CNS significantly influenced by ionotropic and metabotropic glutamate receptor activity. Extracellular glutamate concentrations mediating this activity are regulated by transport proteins localized in neuronal and glial cell membranes. We have used in situ hybridization analysis with subtype-specific antisense-oligonucleotides to study the distribution of glia-specific excitatory amino acid transporter (mEAAT1 and mEAAT2) mRNAs during the later stages of embryogenesis and postnatal CNS development. Distinct but overlapping embryonic and postnatal patterns of localization were observed for the two transporter transcripts. Both mEAAT1 and mEAAT2 mRNAs were found during the peak period of gliogenesis (E15-E19) in the telencephalic and mesencephalic CNS proliferative zones. The overall expression of mEAAT1 mRNA diminished after the completion of cell migration, whereas mEAAT2 mRNA expression increased significantly during postnatal development. Interestingly, mEAAT2 transcript expression continued in the subventricular zone postnatally and persisted in this proliferative zone in the adult brain. From PO onward, mEAAT1 mRNA was present predominantly in the cerebellar Purkinje cell layer and at a much lower abundance in the cortex, hippocampus, basal nuclei, and septum, whereas from P7 onward, mEAAT2 mRNA expression increased throughout most of the neuraxis. Postnatally, transcripts for mEAAT1 and mEAAT2 were found in cell bodies, processes, and commissural white matter tracts of the CNS. The divergent temporal and spatial expression of EAAT subtypes and their persistence in mature fiber tracts and radial glia layers reveal that specific EAATs are likely to play multiple distinct roles in the developing and adult CNS, including the regulation of cell proliferation, axon-glia interactions, and neuronal survival.
ATP-Binding Cassette Transporters↗
Selective failure of brain-derived neurotrophic factor mRNA expression in the cerebellum of stargazer, a mutant mouse with ataxia.
In search of the possible involvement of neurotrophic factors in inherited neurological disease, we examined brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) mRNA expression patterns in the ataxic mutant mouse stargazer (stg). Using in situ hybridization, we found a selective and near total reduction in BDNF mRNA in the cerebellar granule cell layer. NT-3 or NGF mRNA expression in the cerebellum was normal. Northern blot analysis demonstrated a 70% reduction in BDNF mRNA in the whole cerebellum. BDNF mRNA levels in other mutant brain regions were unchanged. Absence of BDNF mRNA in granule cells was observed at postnatal age (P15), coincident with the onset of ataxia, and expression levels failed to follow the developmental increase found in the wild type at later ages (P20 and P30). Despite the severe BDNF reduction, in situ hybridization patterns for both the full-length and the truncated BDNF TrkB receptor mRNA were unaltered. No major cytoarchitectural abnormalities were apparent in the stg/stg cerebellum. BDNF expression in a related ataxic mutant, tottering, was unaltered. These data show that BDNF can be regulated selectively in distinct brain regions, possibly by differential activation of its multiple promoters. Absence of cerebellar granule cell BDNF mRNA in stg/stg mice demonstrates that sustained expression of this neurotrophin is not required for cell survival in the developing cerebellar cortex. Our data, in contrast, suggest a role of BDNF in maturation of specific cerebellar neurons and pathways. Early failure of cerebellar BDNF expression may be related to the ataxic phenotype in stg mice.
Animals↗
Molecular and cellular plasticity in developing epileptic brain.
Defined transgenic models of epilepsy in the mouse represent unique opportunities to examine interactions between synchronous synaptic activity and cellular programs of brain development. We are beginning to acquire a list of the kinds of genes favoring sudden, intermittent aberrant discharges in central neurons, and we have found that, rather than arising from a few gene superfamilies regulating membrane excitability, they are involved in many diverse functions of the cell. Whereas some primary gene defects impinge directly on membrane electrogenesis and neurotransmitter signaling at synapses, others are too far removed from these processes to clearly visualize the steps by which they promote epileptogenesis. We have tantalizing evidence that several, and probably all, epilepsy genes entrain some degree of secondary molecular and cellular plasticity, and we can guess that these downstream rearrangements may account for the delayed onset of epileptic phenotypes in some syndromes. It is too early to tell whether these, or other induced changes, provide the basis for the reversibility of some epilepsies. The diversity of epilepsy genes and their intervening cellular phenotypes promise to provide a rich source of novel molecular targets for therapeutic discovery and will have a lot to teach us in the future about the developmental potential of neural circuits in the mammalian brain.
Animals↗
Molecular characterization of a high-affinity mouse glutamate transporter.
The complete coding sequence of a mouse glutamate transporter (mEAAT2) has been cloned by polymerase chain reaction (PCR) from adult whole-brain total RNA. Southern hybridization analysis of PCR products amplified from templates derived from various murine adult tissues demonstrated that the transcript for mEAAT2 was specific to the central nervous system. High-affinity transport of D-aspartate, Km value (17 +/- 5 microM), was determined in a vaccinia/T7 RNA polymerase expression system. The deduced amino-acid sequence of mEAAT2 shares 96 and 93% identity with the rat and human EAAT2 homologues, respectively.
ATP-Binding Cassette Transporters↗
Aberrant expression of neuropeptide Y in hippocampal mossy fibers in the absence of local cell injury following the onset of spike-wave synchronization.
Stargazer mutant mice inherit a recessive neuronal excitability phenotype featuring frequent non-convulsive spike-wave seizures that arise from synchronous bursting in neocortical, thalamic and hippocampal networks. Immunocytochemistry reveals that granule cells in the mutant dentate gyrus aberrantly express neuropeptide Y (NPY) at multiple ages following the developmental onset of seizures. The ectopic NPY is selectively concentrated in the mossy fibers, co-localizing with the releasable dense core vesicle pool. The NPY content of native NPY+local circuit neurons is also elevated in the mutant CNS. There is no concurrent elevation of hippocampal 72 kDa heat shock protein (HSP72), glial fibrillary acidic protein (GFAP) or NADPH-diaphorase, three markers that are induced during cellular injury, and no evidence of granule cell loss. Since mossy fiber NPY expression appears after the developmental onset of spike-wave discharges and can be induced in wild type granule cells by electrical stimulation, the altered peptide phenotype is likely to reflect transynaptic gene induction triggered by synchronous bursting. These results link a specific pattern of repetitive synaptic input with selective molecular plasticity in dentate granule cells that may contribute to dynamic modifications in hippocampal network excitability.
Action Potentials↗
Hypomyelination alters K+ channel expression in mouse mutants shiverer and Trembler.
Voltage-gated K+ channels are localized to juxtaparanodal regions of myelinated axons. To begin to understand the role of normal compact myelin in this localization, we examined mKv1.1 and mKv1.2 expression in the dysmyelinating mouse mutants shiverer and Trembler. In neonatal wild-type and shiverer mice, the focal localization of both proteins in axon fiber tracts is similar, suggesting that cues other than mature myelin can direct initial K+ channel localization in shiverer mutants. In contrast, K+ channel localization is altered in hypomyelinated axonal fiber tracts of adult mutants, suggesting that abnormal myelination leads to channel redistribution. In shiverer adult, K+ channel expression is up-regulated in both axons and glia, as revealed by immunocytochemistry, RNase protection, and in situ hybridization studies. This up-regulation of K+ channels in hypomyelinated axon tracts may reflect a compensatory reorganization of ionic currents, allowing impulse conduction to occur in these dysmyelinating mouse mutants.
Aging↗
Genetic epilepsy model derived from common inbred mouse strains.
The recombinant inbred mouse strain, SWXL-4, exhibits tonic-clonic and generalized seizures similar to the commonest epilepsies in humans. In SWXL-4 animals, seizures are observed following routine handling at about 80 days of age and may be induced as early as 55 days by rhythmic gentle tossing. Seizures are accompanied by rapid, bilateral high frequency spike cortical discharges and followed by a quiescent post-ictal phase. Immunohistochemistry of the immediate early gene products c-Fos and c-Jun revealed abnormal activation within cortical and limbic structures. The seizure phenotype of SWXL-4 can be explained and replicated fully by the inheritance of susceptibility alleles from its progenitor strains, SWR/J and C57L/J. Outcrosses of SWXL-4 with most other common inbred strains result in F1 hybrids that have seizure at least as frequently as SWXL-4 itself. Quantitative trait locus mapping reveals a seizure frequency determinant, Szf1, near the pink-eyed dilution locus on chromosome 7, accounting for up to 32% of the genetic variance in an F2 intercross between SWXL-4 and the linkage testing strain ABP/Le. These studies demonstrate that common strains of mice such as SWR and C57L contain latent epilepsy susceptibility alleles. Although the inheritance of susceptibility may be complex, these results imply that a number of potentially important and practical, noninvasive models for this disorder can be constructed and studied in crosses between common mouse strains.
Animals↗
Analysis of voltage-gated and synaptic conductances contributing to network excitability defects in the mutant mouse tottering.
1. Intracellular current- and voltage-clamp recordings were carried out in CA3 pyramidal neurons from hippocampal slices of adult tg/tg mice and their coisogenic C57BL/6J (+/+) controls with the use of the single-electrode switch-clamp technique. The principal aim of this study was to investigate the mechanisms responsible for the tg gene-linked prolongation (mean 60%) of a giant synaptic response, the potassium-induced paroxysmal depolarizing shift (PDS) at depolarized membrane potentials (Vm -47 to -54 mV) during synchronous network bursting induced by 10 mM potassium ([K+]o). 2. To examine the role of intrinsic voltage-dependent conductances underlying the mutant PDS prolongation, neurons were voltage clamped by the use of microelectrodes filled with 100 mM QX-314 or QX-222 chloride (voltage-gated sodium channel blockers) and 2 M cesium sulphate (potassium channel blocker). The whole-cell currents active during the PDS showed a significantly prolonged duration (mean 34%) at depolarized Vms in tg/tg compared with +/+ cells, indicating that a defect in voltage-dependent conductances is unlikely to completely account for the mutant phenotype. 3. Bath application of 40 microM (DL)-2-aminophosphonovalerate (DL-APV) produced a 30% reduction in PDS duration in both genotypes but failed to significantly alter the tg gene-linked prolongation compared with the wild type. These data indicate that the mutant PDS abnormality does not result from a selective increase of the N-methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic component. 4. Blockade of gamma-aminobutyric acid-A (GABAA) transmission with picrotoxin (50 microM) or bicuculline (1-5 microM) completely eliminated the difference in PDS duration between the genotypes. Furthermore, although both GABAA receptor antagonists increased the mean PDS duration in +/+ neurons, they did not significantly alter it in tg/tg neurons. These findings are consistent with a reduction in GABAA receptor-mediated synaptic inhibition during bursting in the tg CA3 hippocampal network. 5. To test this hypothesis, bursting CA3 pyramidal neurons were loaded intracellularly with chloride by the use of KCl-filled microelectrodes to examine the effect of reversing the hyperpolarizing chloride-dependent GABAA receptor-mediated inhibitory postsynaptic component of the PDS. Chloride loading prolonged PDS duration in both genotypes, but the increase was greater in +/+ than in tg/tg neurons, indicating that a smaller GABAA inhibitory postsynaptic potential (IPSP) component was reversed in the mutant.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Genetic mapping and evaluation of candidate genes for spasmodic, a neurological mouse mutation with abnormal startle response.
Spasmodic (spd) is a recessive mouse mutation characterized by a prolonged righting reflex, fine motor tremor, leg clasping, and stiffness. Using an intersubspecific backcross that segregates spd, we placed spd on Chr 11 with the following gene order: Adra-1-3.8 +/- 2.1 cM-Pad-1-6.3 +/- 2.7-(spd, Anx-6, Csfgm, Glr-1, Il-3, Il-4, Il-5, Sparc)-9.1 +/- 2.4-D11 Mit5-2.2 +/- 1.5-Asgr-1. This localization eliminated the alpha 1-adrenergic receptor (Adra-1) and the alpha 1 and gamma 2 subunits of the GABAA receptor as candidate genes. Two other promising candidate genes, annexin VI (Anx-6) and a glutamate receptor (Glr-1), were mapped to within 2.1 cM of the spd locus. Although no recombination was observed between spd and Anx-6 or Glr-1, no evidence was obtained for a lesion in either gene. The presence of normal Anx-6 and Glr-1 mRNA transcripts was confirmed by Northern blot analysis, in situ hybridization, and DNA sequence analysis. The localization of Anx-6 and Glr-1 extends the known synteny homology between human chromosome 5q21-q31 and mouse Chr 11 and reveals the probable chromosomal location of the human counterpart to spd. Synteny homology and phenotypic similarities suggest that spasmodic mice may be a genetic model for the inherited human startle disease, hyperekplexia (STHE).
Alternative Splicing↗
Localization and modulatory actions of zinc in vertebrate retina.
Zinc ions are colocalized with glutamatergic synaptic vesicles in vertebrate photoreceptors and may act as a diffusible molecular switch regulating neurotransmitter signaling at two distinct sites in the outer retina. In the dark, extracellular zinc acts presynaptically at rods and cones to minimize the depletion of tonically released glutamate, and selectively reduces GABA-mediated depolarization in horizontal cells, accelerating the response kinetics of the second-order cells. The discovery of zinc ions in photoreceptors provides a mechanism for gain control, kinetics modulation, and the balance of rod vs cone output at the first synapse in the visual system.
Animals↗
Developmental analysis of hippocampal mossy fiber outgrowth in a mutant mouse with inherited spike-wave seizures.
Neural firing patterns are an essential determinant of normal axon terminal growth and synaptic connectivity in developing afferent pathways, but the trophic role of synchronous activity in associative neural networks is less well defined. We examined the ontogeny of inherited synchronous hippocampal network discharges and mossy fiber innervation patterns at sequential stages of development in the stargazer (stg) mutant, a single-locus mouse mutation expressing generalized spike-wave epilepsy. Brief bursts of 6/sec repetitive discharges arise spontaneously on postnatal days 17-18 and persistently activate neocortical and hippocampal networks throughout adulthood. We found a striking pattern of mossy fiber recurrent axon collateral sprouting in the inner molecular layer of dentate gyrus in the adult stg hippocampus. Sprouting is not apparent until 4-6 weeks following seizure onset, but then steadily intensifies with continued synchronous activation. In the adult mutant, axon outgrowth is accompanied by a mild selective loss of hilar interneurons without gliosis. These data indicate that hypersynchronous stimulation during late postnatal brain development is linked, following a prolonged latent period, to significant fiber outgrowth and synaptic reorganization within the hippocampal formation. Since the pattern of synchronous activation in the stg mutant strongly resembles that seen in human spike-wave absence epilepsy, the synaptic plasticity described in this model has important implications for normal brain development in this common disorder.
Aging↗
A burst-dependent hippocampal excitability defect elicited by potassium at the developmental onset of spike-wave seizures in the Tottering mutant.
Hippocampal CA3 pyramidal neurons in the adult epileptic mutant mouse tottering (tg) show normal intrinsic membrane properties, yet fire abnormally prolonged paroxysmal depolarizing shifts (PDS) during in vitro exposure to elevated extracellular potassium solutions. Intracellular recordings in immature mutants reveal that this network burst abnormality is present during the developmental period that coincides with the onset of seizures in the mutant (19-20 postnatal days), and is significantly more pronounced at this age than at adulthood. These data are inconsistent with the hypothesis that the mutant PDS prolongation represents a secondary consequence of a prolonged history of repeated seizures and suggest that it may reflect a cellular epileptogenic phenotype more directly related to the primary neuropathological expression of the tg gene.
Aging↗
Elevated expression of type II Na+ channels in hypomyelinated axons of shiverer mouse brain.
Type I and type III Na+ channels are localized mainly in neuronal cell bodies in mouse brain. Type II channels are preferentially localized in unmyelinated fiber tracts but are not detectable in normally myelinated fibers. In shiverer mice, which lack compact myelin due to a defect in the myelin basic protein gene, elevated expression of type II Na+ channels was observed in the hypomyelinated axons of large-caliber fiber tracts such as the corpus callosum, internal capsule, fimbria, fornix, corpus medullare of the cerebellum, and nigrostriatal pathway by immunocytochemical analysis with subtype-specific antibodies. No difference was observed in the localization of type I and type III Na+ channels between wild-type and shiverer mice. These findings support the hypothesis that type II Na+ channels are preferentially localized in axons of brain neurons and suggest that their density and localization are regulated by myelination. The selective increase in the number of type II channels in hypomyelinated fiber tracts may contribute to the hyperexcitable phenotype of the shiverer mouse.
Animals↗
Sodium channel density in hypomyelinated brain increased by myelin basic protein gene deletion.
Trophic control over the expression and membrane distribution of voltage-dependent ion channels is one of the principal organizing events underlying the maturation of excitable cells. The myelin sheath is a major structural determinant of regional ion channel topography in central axons, but the exact molecular signals that mediate local interactions between the oligodendrocyte and axolemma are not known. We have found that large caliber fibre pathways in the brain of the mutant mouse shiverer (shi, gene on chromosome 18), whose developmental fate of myelination is averted by deletion of five exons in the myelin basic protein gene, have a striking excess of sodium channels. As cytoplasmic membranes of shiverer oligodendroglia still adhere to axons, the evidence indicates that myelin basic protein or a myelin basic protein-dependent glial transmembrane signal associated with compact myelin formation, rather than a simple glial-axon contact inhibition or an intrinsic genetic program of neuronal differentiation, could be critical in downregulating sodium channel density in axons. Here we use the shiverer mutant to show that mature central nervous system projection neurons with large caliber unmyelinated fibres sustain functional excitability by increasing sodium channel density. This axon plasticity, triggered by the absence of a single glial protein, contributes to the unexpectedly mild degree of neurological impairment in the mutant brain without myelin, and may be a potentially inducible mechanism determining the recovery of function from dysmyelinating disease.
Animals↗
Genetic and phenotypic heterogeneity of inherited spike-wave epilepsy: two mutant gene loci with independent cerebral excitability defects.
Two recessive gene loci controlling cerebral excitability in the mouse (tg, chr 8 and stg, chr 15) share generalized neocortical spike-wave seizures as a common mutant phenotype. Although the primary molecular defects are unknown, homozygous tg mutants display a gene-linked hyperplasia of central noradrenergic axons originating in the locus ceruleus, and early selective lesions of these fibers correct the epileptic phenotype in the adult. In contrast, we find that stg homozygotes, despite a more severe seizure disorder, show no alterations in regional noradrenergic fiber innervation, and seizure frequency is unaffected by neonatal noradrenergic depletion. These mutations demonstrate that excessive synchronous neuronal discharges alone are insufficient to trigger abnormal growth of locus ceruleus fibers, and reveal the existence of two distinct intervening brain neuromodulatory mechanisms, norepinephrine (NE)-dependent and NE-independent, underlying the inheritance of this common pattern of epilepsy.
Animals↗
Synchronous hippocampal bursting reveals network excitability defects in an epilepsy gene mutation.
A mutation at the tottering locus (tg, recessive, on chromosome 8) stimulates noradrenergic locus coeruleus axon terminal outgrowth and predisposes the brain to generalized spike-wave epilepsy in the young mouse. In an isolated synaptic circuit studied in vitro, the hyperinnervated mutant hippocampal pyramidal neurons respond normally when individually activated; however, latent neuronal signaling defects emerge during synchronous network bursting, revealing two conditional excitability phenotypes: a voltage-dependent prolongation of a complex synaptic response, the paroxysmal depolarizing shift, and a beta-adrenoreceptor-linked attenuation of the afterhyperpolarization. In this target brain region, the tg locus transforms neuronal excitability without altering measured intrinsic membrane properties, indicating that gene control of inherited epileptic traits may be mediated in part by activity-dependent modulation of network behavior favoring synchronous neuronal firing.
Animals↗