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O Steward

Publications and source records attributed to O Steward.

At least 37 records · Page 2Linked to original sources
PubMed1998

No evidence for disruption of normal patterns of mRNA localization in dendrites or dendritic transport of recently synthesized mRNA in FMR1 knockout mice, a model for human fragile-X mental retardation syndrome.

Recent studies have revealed that FMRP, the gene product of the fragile-X gene FMR1, is an RNA-binding protein. These and other data have led to the idea that FMRP may play a role in targeting mRNAs for transport to synaptic sites. The present study evaluated whether a null mutation of FMR1 disrupts the patterns of localization of three mRNAs that are present constitutively in dendrites (the mRNAs for MAP2, CAMII kinase and dendrin), or disrupt the rapid dendritic transport of the mRNA for activity-regulated cytoskeletal protein (ARC), coded for by an immediate-early gene. In situ hybridization analyses revealed that the patterns of mRNA localization in dendrites and the dendritic transport of ARC mRNA are indistinguishable from normal in FMR1 knockout mice. These results indicate that FMRP does not play an obligatory role in targeting this set of mRNAs to dendrites, although it might be involved in targeting other dendritic mRNAs yet to be identified.

Animals↗
PubMed1998

Differential intracellular sorting of immediate early gene mRNAs depends on signals in the mRNA sequence.

This study characterizes the differential targeting of recently synthesized immediate early gene (IEG) mRNAs to neuronal cell bodies versus dendrites and tests the hypothesis that this targeting is based on signals in the encoded proteins. A single electroconvulsive seizure induces the expression of a number of IEG mRNAs in granule cells of the dentate gyrus. Most of these IEG mRNAs remain in the cell body, including two that are characterized in the present study (the mRNAs for NGFI-A and COX-2). In contrast, the mRNA for Arc moved rapidly into dendrites at an apparent rate of approximately 300 micron/hr. Inhibiting protein synthesis with cycloheximide did not disrupt the differential mRNA sorting, demonstrating that the differential targeting of mRNAs is not dependent on translation.

Animals↗
PubMed1998

Mechanisms of motor recovery after subtotal spinal cord injury: insights from the study of mice carrying a mutation (WldS) that delays cellular responses to injury.

UNLABELLED: Partial lesions of the mammalian spinal cord result in an immediate motor impairment that recovers gradually over time; however, the cellular mechanisms responsible for the transient nature of this paralysis have not been defined. A unique opportunity to identify those injury-induced cellular responses that mediate the recovery of function has arisen from the discovery of a unique mutant strain of mice in which the onset of Wallerian degeneration is dramatically delayed. In this strain of mice (designated WldS for Wallerian degeneration, slow), many of the cellular responses to spinal cord injury are also delayed. We have used this experimental animal model to evaluate possible causal relationships between these delayed cellular responses and the onset of functional recovery. For this purpose, we have compared the time course of locomotor recovery in C57BL/6 (control) mice and in WldS (mutant) mice by hemisecting the spinal cord at T8 and evaluating locomotor function at daily postoperative intervals. The time course of locomotor recovery (as determined by the Tarlov open-field walking procedure) was substantially delayed in mice carrying the WldS mutation: C57BL/6 control mice began to stand and walk within 6 days (mean Tarlov score of 4), whereas mutant mice did not exhibit comparable locomotor function until 16 days postoperatively. INTERPRETATION AND CONCLUSION: (a) The rapid return of locomotor function in the C57BL/6 mice suggests that the recovery resulted from processes of functional plasticity rather than from regeneration or collateral sprouting of nerve fibers. (b) The marked delay in the return of locomotor function in WldS mice indicates that the processes of neuroplasticity are induced by degenerative changes in the damaged neurons. (c) These strains of mice can be effectively used in future studies to elucidate the specific biochemical and physiological alterations responsible for inducing functional plasticity and restoring locomotor function after spinal cord injury.

Animals↗
PubMed1998

Synaptic activation causes the mRNA for the IEG Arc to localize selectively near activated postsynaptic sites on dendrites.

Polyribosomal complexes beneath postsynaptic sites on dendrites provide a substrate for local translation of particular mRNAs, but the signals that target mRNAs to synapses remain to be defined. Here, we report that high frequency activation of the perforant path projections to the dentate gyrus causes newly synthesized mRNA for the immediate-early gene (IEG) Arc to localize selectively in activated dendritic segments. Newly synthesized Arc protein also accumulates in the portion of the dendrite that had been synaptically activated. The targeting of Arc mRNA was not disrupted by locally inhibiting protein synthesis, indicating that the signals for mRNA localization reside in the mRNA itself. This novel mechanism through which newly synthesized mRNA is precisely targeted to activated synapses is well suited to play a role in the enduring forms of activity-dependent synaptic modification that require protein synthesis.

Afferent Pathways↗
PubMed1997

Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches.

Recent studies have sought to identify the genes involved in excitotoxic neurodegeneration. Here we report that certain strains of mice, including strains that are used for gene targeting studies, do not exhibit excitotoxic cell death after kainic acid seizures. Kainic acid produced excitotoxic cell death in the CA3 and CA1 subfields of the hippocampus in 129/SvEMS and FVB/N mice, in the same pattern as described in rats. C57BL/6 and BALB/c mice exhibited excitotoxic cell death only at very high doses of kainate, and then only in a very restricted area, although they exhibited comparable seizures. Hybrids of 129/SvEMS x C57BL/6 mice created using embryonic stem cells from 129/SvEMS mice also did not exhibit excitotoxic cell death. These results demonstrate that C57BL/6 and BALB/c strains carry gene(s) that convey protection from glutamate-induced excitotoxicity. This differential susceptibility to excitotoxicity represents a potential complication for gene targeting studies.

Animals↗
PubMed1997

Genetic influences on cellular reactions to CNS injury: the reactive response of astrocytes in denervated neuropil regions in mice carrying a mutation (Wld(S)) that causes delayed Wallerian degeneration.

This study compares the reactive changes in astrocytes in denervated neuropil regions in normal mice and in mice carrying the Wld(S) mutation which leads to delayed Wallerian degeneration. In situ hybridization and immunocytochemical techniques were used to define the time course of changes in the levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA in the denervated neuropil of the hippocampus after unilateral aspiration lesions of the entorhinal cortex. In control mice, GFAP mRNA levels increased rapidly in the denervated neuropil to a peak that was about tenfold higher than control at 2-4 days, decreased between 6 and 8 days postlesion, and then increased again to a second peak at 10 days postlesion. Increases in immunostaining for GFAP were evident by 2 days, remained elevated until 12 days postlesion and then decreased slowly. In mice carrying the Wld(S) mutation, the upregulation of GFAP mRNA levels in the denervated laminae was substantially delayed. Strikingly absent was the dramatic increase in labeling at 2-4 days postlesion which was such a prominent feature of the response in control animals. Peak labeling in the denervated laminae was not seen until 10-12 days postlesion. The development of a well-defined band of intensely immunostained and hypertrophied astrocytes in the denervated zone was also delayed in the Wld(S) animals, although there were modest increases in immunostaining as early as 2 days postlesion that were seen throughout the hippocampus ipsilateral to the lesion. These results suggest that degenerative changes in axons and synaptic terminals are the principal trigger for upregulating GFAP expression in the denervated neuropil, although other signals also play a role in the early postlesion response.

Animals↗
PubMed1997

Genetic influences on cellular reactions to brain injury: activation of microglia in denervated neuropil in mice carrying a mutation (Wld(S)) that causes delayed Wallerian degeneration.

This study examines the relationship between the appearance of degenerative changes in synaptic terminals and axons and the activation of microglia in denervated neuropil regions of normal mice of the C57BL/6 strain and mutant mice (Wld(S)), in which Wallerian degeneration is substantially delayed. The time course of degenerative changes in synaptic terminals and axons was assessed using selective silver staining. Microglial cells were identified by immunostaining for Mac-1, a monoclonal antibody to the CR3 complement receptor, and by histochemical staining for nucleoside diphosphatase (NDPase). Increased argyrophilia, indicative of degenerative changes, was evident as early as 1 day postlesion in normal mice, but was not seen until 6-8 days in mice with the Wld(S) mutation. Microglial activation in normal C57BL/6 mice was evident by 24 hours postlesion, as evidenced by increased immunostaining for Mac-1, increased histochemical staining for NDPase, and morphological changes indicative of an activated phenotype (short, thick processes). Quantitative evaluation of immunostaining for Mac-1 revealed that peak activation occurred between 2 and 6 days postlesion with a return to a quiescent phenotype by 12 days. In contrast, the microglial response was significantly delayed and prolonged in mice bearing the Wld(S) mutation. Activated microglia were not seen within the deafferented area until 6 to 8 days postlesion and peak activation occurred between 12 and 20 days postlesion. These data suggest that the response of microglia in denervated neuropil zones is triggered by the same types of degenerative changes that cause increased argyrophilia as detected by selective silver staining methods.

Acid Anhydride Hydrolases↗
PubMed1997

Multiple subcellular mRNA distribution patterns in neurons: a nonisotopic in situ hybridization analysis.

Previous studies have established that most of the mRNAs that neurons express are localized in the cell body and very proximal dendrites, whereas a small subset of mRNAs is present at relatively high levels in dendrites. It is not clear, however, whether particular mRNAs have the same subcellular distribution in different types of neurons or whether different types of neurons sort mRNAs in different ways. The present study was undertaken to address these questions. Nonisotopic in situ hybridization techniques were used to define the subcellular localization of representative mRNAs including beta-tubulin, low-molecular-weight neurofilament protein (NF-68), high-molecular-weight microtubule-associated protein (MAP2), growth-associated protein 43 (F1/GAP43), the alpha subunit of calcium/calmodulin-dependent protein kinase II (alpha CaMII kinase), and poly (A+) mRNA. The mRNAs for beta-tubulin, neurofilament 68, and F1/GAP43 were restricted to the region of the cell body and very proximal dendrites in most neurons. In some neuron types, however, labeling for NF-68 extended for considerable distances into dendrites. In some neurons that express MAP2, the mRNA was present at the highest levels in the proximal third to half of the dendritic arbor, whereas in other neurons the highest levels of labeling were in the cell body. In most neurons that express alpha CaMII kinase, the highest levels of the mRNA were in the cell body, but labeling was also present throughout dendrites. However, in a few types of neurons, alpha CaMII kinase mRNA was largely restricted to the cell body. The fact that there are no general rules for mRNA localization that apply to all neuron types implies the existence of neuron type-specific mechanisms that regulate mRNA distribution.

Animals↗
PubMed1997

Signals that regulate astroglial gene expression: induction of GFAP mRNA following seizures or injury is blocked by protein synthesis inhibitors.

Previous studies have revealed that a single electroconvulsive seizure (ECS) strongly induces glial fibrillary acidic protein (GFAP) expression in astrocytes in the hippocampal dentate gyrus. The signals that trigger this induction are not known, but circumstantial evidence suggests the hypothesis that GFAP expression may be induced as a result of the induction of growth factor expression by dentate granule cells that also occurs as a result of the ECS and other types of seizures. The present study tests one prediction of this hypothesis by evaluating whether increases in GFAP mRNA levels after ECS are blocked by inhibiting protein synthesis at various times after the ECS. We report that the upregulation of GFAP expression following ECS is blocked by protein synthesis inhibitors given 5 min before or up to 12 h after a single ECS. This temporal gradient suggests an intermediate step involving the increased expression of a protein growth factor.

Animals↗
PubMed1997

High frequency transcranial magnetic stimulation mimics the effects of ECS in upregulating astroglial gene expression in the murine CNS.

The present study evaluates the consequences of high frequency (25 hz) trans-cranial magnetic stimulation on the expression of glial fibrillary acidic protein (GFAP) in the murine CNS. Trains of transcranial magnetic stimulation (1-30 trains at 25 Hz, 10 s duration) were delivered to mice via 5-cm diameter round coils. The stimulation produced stimulus-locked motor responses but did not elicit behavioral seizures. GFAP mRNA levels were evaluated 12, 24, 36, 48 h, 4 days, and 8 days following stimulation by in situ hybridization. Following multiple 25 Hz trains, there were dramatic increases in the levels of GFAP mRNA in the hippocampal dentate gyrus; more modest increases were observed in the cerebral cortex. The selective increases in GFAP mRNA in the dentate gyrus were similar to those observed following single electroconvulsive seizures (ECS). These results indicate that trans-cranial magnetic stimulation can be used to modulate astroglial gene expression, inducing the first stage of a reactive response that is similar to what occurs following nervous system injury.

Analysis of Variance↗
PubMed1996

Protein synthesis within dendrites: glycosylation of newly synthesized proteins in dendrites of hippocampal neurons in culture.

There is increasing evidence that certain mRNAs are present in dendrites and can be translated there. The present study uses two strategies to evaluate whether dendrites also possess the machinery for protein glycosylation. First, precursor labeling techniques were used to conjunction with autoradiography to visualize glycosyltransferase activities that are characteristic of the rough endoplasmic reticulum (RER) (mannose) or the Golgi apparatus (GA) (galactose and fucose) in dendrites that had been separated from their cell bodies and in intact neurons treated with brefeldin A or low temperature. Second, immunocytochemical techniques were used to define the subcellular distribution of proteins that are considered markers of the RER (ribophorin I) and GA (p58, alpha-mannosidase II, galactosyltransferase, and TGN38/41). Autoradiographic analysis revealed that isolated dendrites incorporated sugar precursors in a tunicamycin-sensitive and protein synthesis-dependent manner. Moreover, when intact neurons were pulse-labeled with 3H-labeled sugars at low temperature or after treatment with brefeldin A, labeling was distributed over proximal and sometimes distal dendrites. Immunolabeling for RER markers was predominantly localized in cell bodies but extended for a considerable distance into dendrites of all neurons. Immunolabeling for GA markers was confined to the cell body in approximately 70% of the neurons, but in 30% of the neurons, the staining extended into proximal and middle dendrites. These results indicate that the machinery for glycosylation extends well into dendrites in many neurons.

Animals↗
PubMed1996

Genetic influences on cellular reactions to spinal cord injury: activation of macrophages/microglia and astrocytes is delayed in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration.

Reactive changes in macrophages/microglia and astrocytes were evaluated following spinal cord injury in normal mice of the C57BL/6J strain and in mice carrying a mutation (WldS) which delays the onset of Wallerian degeneration in damaged axons. Crush injuries were produced at the T8 level by using an extradural approach; animals were allowed to survive for 2 days to 12 weeks, and spinal cords were prepared for immunocytochemistry using antibodies against Mac1 and glial fibrillary acidid protein (GFAP). In normal mice, Mac1-positive macrophages accumulated at the injury site by 4 days and immunostaining of these cells peaked at 6-8 days. Cells in the gray matter near the crush site and in the ascending dorsal column also exhibited increased Mac1 staining that was prominent at 1 week and remained high at 2-4 weeks. In mice carrying the WldS mutation, the accumulation of macrophages at the injury site and the increase in immunostaining of these cells were delayed, as were the increases in immunostaining in the gray matter and dorsal columns. Both normal and mutant mice exhibited pronounced increases in glial fibrillary acidic protein immunostaining at the edge of the crush site and for some distance both rostral and caudal to the injury; increased immunostaining was also prominent along the ascending dorsal columns. The center of the crush site, which contained connective tissue, remained completely unstained for GFAP. In normal mice, immunostaining for GFAP reached a peak at 1 week postinjury and then declined. In mice carrying the WldS mutation, increases in GFAP immunostaining did not reach a peak until 2-3 weeks postinjury. These results indicate that activation of macrophages, microglia, and astrocytes is delayed and prolonged in mice carrying the WldS mutation.

Animals↗
PubMed1996

Genetic influences on cellular reactions to spinal cord injury: a wound-healing response present in normal mice is impaired in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration.

Progressive tissue necrosis is a process unique to the injured mammalian spinal cord which often leads to gradually increasing cavitation and enlargement of the lesion. To evaluate the role of neuronal degeneration in initiating this response, histopathological changes were compared in C57BL and WldS (delayed Wallerian degeneration mutation) mice. The spinal cord was crushed at T8, producing a primary lesion at the site of the trauma and a secondary lesion extending rostrocaudally in the dorsal columns (where long ascending and descending fiber tracts undergo Wallerian degeneration). Cavitation was relatively mild at both sites and developed mainly at the margins of the lesions. In striking contrast to spinal cord injury in rats, progressive necrosis did not occur in mice; instead, the primary and secondary lesion sites became filled in by macrophages and fibroblasts embedded in a well-vascularized collagenous stroma. Quantitative image analysis revealed that the primary lesion decreased dramatically in size and cavitation between 2 and 3 weeks in C57BL, whereas in WldS the reduction in size and cavitation began later (at 4 weeks) and was less complete. The initial development of the secondary lesion began later and its healing was less complete in WldS than C57BL. These results are consistent with the hypothesis that neuronal damage, including Wallerian degeneration, triggers inflammatory responses leading to tissue repair. For this reason, any delay in neuronal degeneration, as in the WldS mutation, results in deficient tissue repair as reflected in the larger size of both primary and dorsal column lesions.

Animals↗
PubMed1996

Homosynaptic and heterosynaptic changes in driving of dentate gyrus interneurons after brief tetanic stimulation in vivo.

This study addressed changes in interneuron driving in the dentate gyrus (DG) of urethane-anaesthetized rats in response to tetanic stimulation of the perforant path (PP) or the converging dentate commissural pathway (CP). Using an extracellular tungsten electrode, we recorded from putative interneurons in the DG that fired to stimulation of both the PP and the CP. Conditioning trains (400 Hz, 17.5 ms) were delivered to each pathway individually and to the two pathways together. The primary measure of synaptic drive was the latency of interneuron discharge. High-intensity PP tetany, CP tetany, and paired tetany consistently reduced firing latency to CP driving (P < .05 for all three), indicating an LTP-like increase in synaptic activation through the CP. High-intensity PP tetany decreased latency to PP driving in only two of seven cases. Heterosynaptic changes occurred frequently in individual experiments. Activity-mediated adjustments in synaptic driving of inhibitory interneurons could play a role in normal physiological function.

Animals↗
PubMed1996

The process of reinnervation in the dentate gyrus of adult rats: physiological events at the time of the lesion and during the early postlesion period.

Destruction of the entorhinal cortex (EC) triggers a number of cellular and molecular responses in the denervated hippocampus and dentate gyrus. The signals that trigger these changes are not known but could include physiological events that occur during the production of the injury or during the early postlesion period. Of particular interest is whether experimental lesions induce seizures and/or spreading depression (SD), both of which have been shown to dramatically alter neuronal and glial gene expression. In the present study, acute neurophysiological techniques were used to evaluate whether seizures or SD occur during the production of EC lesions. Chronic recording techniques were used to monitor electroencephalographic (EEG) activity during the first 24 h after the injury in order to evaluate the extent of postlesion seizures. One or more episodes of SD occurred in 9 of 13 animals during the production of electrolytic EC lesions. However, hippocampal seizures were not observed except for very brief episodes of seizure activity at the onset of an episode of SD. Chronic recordings of postlesion EEG activity revealed that spontaneous electrographic seizures occurred during the first 24 h postlesion in all animals. The spontaneous electrographic seizures were approximately 30 s in duration and were not accompanied by motor convulsions. The first seizures occurred within several hours after the lesion, and seizures continued to occur periodically (at an average frequency of 0.42 per hour) over the 24-h recording period. Seizures occurred on the side of the brain ipsilateral to the lesion in all animals and occurred on the side contralateral to the lesion in 3 of 5 animals. These results indicate that EC lesions produce physiological events that occur variably in different animals; these processes may account for some of the variability in the cellular responses to this "standardized" injury.

Age Factors↗
PubMed1996

The role of postlesion seizures and spreading depression in the upregulation of glial fibrillary acidic protein mRNA after entorhinal cortex lesions.

Unilateral lesions of the entorhinal cortex have been shown to lead to dramatic increases in GFAP mRNA levels in denervated zones in the hippocampus and dentate gyrus and sometimes (but not always) in nondenervated zones in the contralateral hippocampus and dentate gyrus. The variable distribution of the increases in GFAP mRNA expression suggests that the events which trigger changes in GFAP mRNA levels occur to a variable extent in individual animals. The companion paper characterizes two candidate triggering events: spreading depression (SD) that occurs to a variable extent at the time of the lesion and recurrent seizures that occur during the early postlesion interval. The goal of the present study was to evaluate whether individual differences in the extent or spatial distribution of lesion-induced increases in GFAP mRNA are related to the occurrence of either SD or seizures. We quantified the increases in GFAP mRNA levels in individual animals that had been monitored physiologically to define the incidence of SD and postlesion seizures. The results revealed that the quantitative extent of the increases in GFAP mRNA in denervated zones and was not related to either SD or postlesion seizures. The increases in GFAP mRNA in nondenervated zones also were not related to episodes of spreading depression that occurred at the time of lesion production but were related to the spontaneous seizures that developed during the first 24 h postlesion after the animals had recovered from the surgical anesthesia. Taken together, these data indicate that physiological events that occur during the early postlesion interval can play an important role in determining the pattern and extent of altered cellular gene expression in response to an injury.

Animals↗