Differential seizure-induced and developmental changes of neurexin expression.
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Aging↗
Biomedical subjects
L Kaczmarek
Publications and source records attributed to L Kaczmarek.
Rapid phosphorylation of Elk-1 transcription factor and activation of MAP kinase signal transduction pathways in response to visual stimulation.
The AP-1 transcription factor, which is composed of various combinations of Fos and Jun proteins, is believed to be a key participant in molecular processes that guide activity-dependent changes in gene expression. In this study, we investigated the activity of different MAP kinases that have been implicated in AP-1 activation. We examined the activities of ERK, JNK/SAPK, and p38 MAPK along with their nuclear targets (Elk-1 and c-Jun) in rat visual cortex after light stimulation. The transcription factor Elk-1 (a possible regulator of c-fos expression) was found to be transiently modified by phosphorylation when visual stimulation was applied after a period of dark rearing. In vitro kinase assay with Elk-1 as substrate showed that light stimulation activated MAPK/ERK in visual cortex but not frontal cortex. Furthermore, ERK activation was temporally matched to onset of Elk-1 phosphorylation. The activity of JNK1 (c-Jun N-terminal kinase 1) was elevated at 2-6 h after visual exposure and was also temporally correlated to increase of endogenous P-c-Jun levels and its appearance within the AP-1 DNA-binding complex. The activities of p38 MAP kinases did not change significantly. These results demonstrate the differential engagement of MAPK signaling pathways following sensory stimulation and their relative effects upon AP-1 expression in the intact brain.
Animals↗
Defensive conditioning-related functional heterogeneity among nuclei of the rat amygdala revealed by c-Fos mapping.
The amygdala is a complex forebrain structure proposed to play a pivotal role in fear conditioning circuitry. In this study, c-Fos immunomapping was applied to investigate the functional activation of particular amygdalar nuclei following a 50-trial training session of two-way active avoidance reaction. To dissect distinctive responses displayed by the animals and to cluster them into groups of correlated behaviors, factor analysis was employed. The training procedure resulted in an increase of c-Fos expression within the cortical, medial, lateral and basolateral, but not central, nuclei. The expression in the cortical nucleus correlated negatively with grooming behavior, whereas c-Fos immunolabeling of the other three subdivisions of the amygdala could be associated with the number of intertrial responses. No correlation was observed between c-Fos expression and avoidance reactions performed or the amount of shock received by the animal. The results obtained with c-Fos mapping of various regions of rat amygdala, combined with a fine dissection of behavioral repertoire, imply that there are specific functional links between particular parts of the structure and distinctive behaviors that reflect various emotional states of the animal.
Aggression↗
Synthesis, and cytotoxic activity of some novel indolo[2,3-b]quinoline derivatives: DNA topoisomerase II inhibitors.
A series of new 5H-indolo[2,3-b]quinoline derivatives bearing methoxy and methyl groups at C-2 and C-9 was synthesized (according to the modified Graebe-Ullmann reaction). These compounds were evaluated for their antimicrobial and cytotoxic activity and tested as inhibitors of DNA topoisomerase II. Lipophilic and calf thymus DNA binding properties of these compounds were also established. In the SAR studies we used quantum-mechanical methodology to analyze the molecular properties of the drugs. All of the 5H-indolo[2,3-b]quinolines tested were found to inhibit the growth of gram-positive bacteria and pathogenic fungi at MIC ranging between 2.0 and 6.0 microM. They showed also cytotoxic activity in vitro against several human cancer cell lines of different origin (ID50 varied from 0.6 to 1.4 microM), and stimulated the formation of topoisomerase-II-mediated pSP65 DNA cleavage at concentration between 0.2 and 0.5 microM. The most active indolo[2,3-b]quinolines which had the greatest contribution to the increase in the Tm of DNA displayed also the highest DNA binding constants and the highest cytotoxic activity. The differences in DNA binding properties and cytotoxic activity seem to be more related to steric than electrostatic effects.
Anti-Bacterial Agents↗
Brain as a unique antisense environment.
During the last few years, antisense oligodeoxyribonucleotides (asODN) have become a commonly used tool for blocking of gene expression in the mammalian central nervous system. Successful gene inhibition has been reported for such diverse targets as those encoding neurotransmitter receptors, neuropeptides, trophic factors, transcription factors, cytokines, transporters, ion channels, and others. This review presents a discussion of recent studies on ODN in the brain, with a focus on specific approaches taken by the researchers in this field and especially on peculiar features of this organ as a milieu for asODN action. It is concluded that from the presented literature survey no coherent view on how to rationally design ODN for brain studies has emerged.
Animals↗
Sensory regulation of immediate-early genes c-fos and zif268 in monkey visual cortex at birth and throughout the critical period.
The postnatal development of ocular dominance columns (ODCs) in monkey visual cortex provides an exquisite model for studying mechanisms of experience-guided neuronal plasticity. While the presence of columns at birth in Old World monkeys is now well established, it remains unclear whether cortical neurons at this early stage are capable of modulating gene expression in response to changing sensory conditions. Using a set of monocular deprivation and stimulation protocols, we examined activity-driven expression of the immediate-early genes (IEGs) c-fos and zif268 during the critical period of development. We observed well-delineated patterns of ODCs produced by sensory regulation of both IEGs throughout the critical period, starting as early as the first postnatal day. The expression levels are similar in layers II/II, IVC and VI throughout development, with no selective decline in the thalamorecepient layer (layer IVC) of adult monkeys. A narrow strip of non-columnar c-Fos expression was observed at the border of layers IVC and V. Our results show that neurons in monkey visual cortex are equipped at birth with the molecular machinery for coupling sensory inputs to active genomic responses and that this responsivity extends throughout the critical period. The findings are discussed within the context of a possible role for IEGs in sensory-driven cortical plasticity during development.
Animals↗
Kainate-evoked changes in dystrophin messenger RNA levels in the rat hippocampus.
Dystrophin and dystroglycan messenger RNAs are expressed in specific brain areas, including regions of the cortex and the hippocampus, and in such neurons dystrophin has been localized to postsynaptic densities. In the present study we examined by in situ hybridization the effect of neuronal activation and neurotoxicity induced by kainate and pentylenetetrazole administered in vivo on dystrophin and dystroglycan expression in the rat brain. Kainate injection resulted in a transient but dramatic decrease in dystrophin transcript levels in the dentate gyrus granule cells, neurons not affected by kainate neurotoxicity, 6 h after injection. There was also a strong, concomitant increase in dystrophin messenger RNA levels in the CA3 subfield. At 24-72 h after kainate injection, the dystrophin transcript in the dentate granule cells returned to control levels, while it decreased gradually in the CA subfields, coinciding with the neurodegeneration observed in these areas. Comparable results were obtained with pan-dystrophin probes and probes specific to the short, G-dystrophin (Dp71) isoform that predominates in the dentate gyrus. This indicates that any dystrophin transcript that might be expressed in these areas responds to kainate in the same manner. In contrast, kainate insult had no significant effect on the dystroglycan messenger RNA levels in these hippocampal areas at 6 h post-injection. At later times. however, there was a gradual decrease in the dystroglycan messenger RNA in those areas which respond to the kainate insult with extensive neuronal death. For comparison, seizures which are not associated with progressive neurodegeneration were induced by pentylenetetrazole: in this situation the dystrophin and dystroglycan messenger RNA levels remained unchanged in all areas of the hippocampal formation. Since activation of glutamate receptors is thought to be involved in some forms of synaptic plasticity in the adult hippocampus, our data indicate that the dystrophin gene behaves as a candidate plasticity-related gene responding to glutamate.
Animals↗
Plasticity- and neurodegeneration-linked cyclic-AMP responsive element modulator/inducible cyclic-AMP early repressor messenger RNA expression in the rat brain.
In order to explore the role of CREM (cyclic-AMP responsive element modulator) gene expression in the function of the central nervous system, the gene transcripts were investigated in the rat brain in several conditions linked to increased neuronal activity. Up-regulation of CREM messenger RNA levels in the hippocampus was found to follow intraperitoneal administration of kainate (10 mg/kg). This increase was observed in both the dentate gyrus and hippocampus proper (CA subfields) and reached its maximum at 6 h after the treatment. Intrahippocampal injection of N-methyl-D-aspartate (200 nmol) resulted in elevated CREM messenger RNA expression as well. A similar increase of the messenger RNA abundance was also observed in the retrosplenial cortex after treating the female rats with a high dose (5 mg/kg) of dizocilpine maleate, an N-methyl-D-aspartate receptor antagonist. All these conditions are linked to neuronal excitation and neurodegeneration. However, an increase in CREM messenger RNA accumulation was also observed in the visual cortex after exposure of dark-adapted animals to the light, a procedure linked to neuronal plasticity. In the latter condition, it was found that CREM messenger RNA reached its highest levels at 6 h, i.e. later than the maximal increase of expression of immediate early genes such as c-fos, jun B and zif268, observed 45 min following the onset of visual stimulation. The ICER (inducible cyclic-AMP early repressor) form of CREM messenger RNA was identified to be induced by the light exposure. Finally, it was also found that cycloheximide, an inhibitor of protein synthesis, overinduces CREM/ICER gene expression. Together, these data suggest that CREM/ICER may be responsive to neuronal activation. Furthermore, given that CREM products have been shown previously to down-regulate expression of immediate early genes in vitro, they suggest that ICER may function as a molecular switch involved in down-regulation of immediate early gene expression in the rat brain.
Animals↗
Orthovanadate induces cell death in rat dentate gyrus primary culture.
The aim of this study was to define the effects of a potent inhibitor of tyrosine phosphatases, sodium orthovanadate (0.1-100 microM for up to 48 h), on dentate gyrus cells (DGC) in culture. Treatment with 100 microM orthovanadate evoked a delayed form of cell death. To examine the possible involvement of apoptosis in orthovanadate-induced cell death, biochemical and morphological alterations were compared with those of necrotic death induced by sodium azide. Phase-contrast microscopy and nuclear condensation analysis showed that orthovanadate and azide each evoked cell death by distinct pathways. TUNEL assay was positive in both cases. Application of a protein synthesis inhibitor, cycloheximide, did not prevent cytotoxicity caused by either orthovanadate or azide and potentiated the effects of vanadate. We conclude that orthovanadate-induced death of DGC bears features of apoptosis.
Animals↗
Activity-dependent regulation of cytochrome b gene expression in monkey visual cortex.
The recent demonstration that certain mitochondrial subunits of cytochrome oxidase (CO) are regulated by neuronal activity has stimulated interest in the molecular processes that coordinate nuclear and mitochondrial gene expression following synaptic stimulation. We have studied the constitutive expression and activity-guided regulation of cytochrome b (cyt b), a gene that is encoded by mitochondrial DNA and that was cloned by subtractive hybridization from the lateral geniculate nucleus in the monkey. We have found cyt b mRNA expression in monkey striate cortex to be similar to that of CO activity with regard to the laminar profile and the presence of blobs in the supragranular layers. Layers 2/3, 4C, and 6 contained large numbers of stained cells, many of which were judged to be excitatory neurons, because they showed a Zif268-immunopositive nucleus. We have also found that removal of functional activity reduced cyt b mRNA content in area V1. Columns of reduced cyt b staining were visible after 3 days and were especially striking after 6 days of monocular deprivation. After 3 months of deprivation, the columns lost their contrast and became blurred. Our principal finding, that neuronal activity regulates cyt b levels, suggests that expression of a mitochondrial gene can be affected in a manner similar to that of several known nuclear genes. The differences in cyt b mRNA levels and CO activity after long-term deprivation suggests that some form of differential control is exerted on cyt b. Cyt b expression, therefore, may be used as a marker of altered mitochondrial transcription that is guided by the metabolic demands of active neurons.
Animals↗
Increased expression of cathepsin D in retrosplenial cortex of MK-801-treated rats.
Single administration of a high dose of an uncompetitive NMDA receptor antagonist-dizocilpine maleate (MK-801)-results in transient neuronal vacuolization and cell death in retrosplenial cortex in rodents. In this study expression of cathepsin D (CatD), a major lysosomal aspartic protease, was investigated in brains of female rats treated with 1, 5, or 10 mg/kg of MK-801. Northern blot analysis demonstrated that the CatD mRNA level was moderately increased in retrosplenial cortex 24 h-7 days after the treatment. Concomitantly, increased CatD immunoreactivity was observed, predominantly in the degenerating neurons in layer III of retrosplenial cortex. Neuronal response was spatially distinguished from glial reactivation marked by increased mRNA and protein levels of glial fibrillary acidic protein, as demonstrated by Northern blot and immunohistochemistry in retrosplenial cortex 24 h-7 days after MK-801 treatment. These data suggest that activation of the lysosomal proteolytic system of neurons may play a role in MK-801-evoked neurodegeneration.
Animals↗
Sensory regulation of immediate-early gene expression in mammalian visual cortex: implications for functional mapping and neural plasticity.
The expression of immediate-early genes that code for transcription factors has been extensively studied in the brain with regard to imaging functional activity. The components of the AP-1 transcription factor--in particular, c-Fos--and Zif268 have been widely used for this purpose. However, the precise details by which they are induced after synaptic stimulation remain unknown. Furthermore, the roles of these two proteins in neurons remains speculative and include such varied functions as short-term maintenance of cellular homeostasis to long-term changes that guide cortical plasticity. Current efforts at elucidating the physiological roles of AP-1 and Zif268 rely on assessing their expression in response to different conditions of sensory and pharmacological stimulation. In this review, we have examined the expression patterns of these transcription factors in the mammalian visual cortex under different conditions, with particular emphasis on the constitutive levels and how they change after visual deprivation and stimulation. A synthesis of this information offers further insight into their likely functions and the extent to which transcription factors may represent patterns of neural activity as a possible prelude to plastic events.
Animals↗
Pharmacokinetics of antisense analogues in the central nervous system.
A thorough evaluation of the pharmacokinetical properties of oligodeoxyribonucleotides (ODN) is a first step towards their rational application as gene expression blockers in the central nervous system (CNS). In this paper we present our own data, as well as those of other authors, on tissue distribution, stability, retention and cellular uptake of phosphodiester, phosphorothioate, and end-capped analogues of ODN introduced into the CNS. ODN are easily distributed within nervous tissue, and their tissue penetration depends on anatomical conditions. Retention of radioactivity delivered with ODN within nervous tissue is higher for phosphodiesters than for phosphorothioates. On the other hand, the tissue stability of phosphorothioates is substantially greater than the tissue stability of phosphodiesters as well as that of end-capped ODN. If the elimination process of ODN is also due to their degradation, it is apparently accomplished by endonucleases, because the recovery of end-capped ODN (resistant to exonucleases) was similar to unprotected phosphodiesters. The uptake of ODN by nerve cells is rather poor, although we have shown that phosphorothioates at least can be internalized by nerve cells in vivo. ODN are metabolized by nerve cells, which results in the formation of unidentified molecules of higher molecular weight than ODN themselves.
Animals↗
Cellular and molecular correlates of glutamate-evoked neuronal programmed cell death in the in vitro cultures of rat hippocampal dentate gyrus.
An excessive neuronal stimulation through glutamate receptors is known to result in excitotoxic cell death of apoptotic (programmed) character. Granule cells of hippocampal dentate gyrus are believed to be particularly resistant to excitotoxic insults, despite the fact that pyramidal neurons of the hippocampus proper are apparently the most vulnerable brain cells. In this study, we report that neurons derived from the rat 5-day-old dentate gyrus, and maintained in vitro for 6 days, may undergo apoptosis after treatment with L-glutamate, in a dose-dependent manner-with up to 80% of neurons displaying features of programmed cell death after 24 h exposure to 0.5 mM glutamate. This conclusion is based on morphological evaluation of the cultures, nuclear staining with Hoechst 33258 and acridine orange revealing chromatin abnormalities, as well as terminal transferase labeling of DNA fragmentation. Since apoptosis is believed to be an active process involving gene expression, immunocytochemical of c-Fos and c-Jun transcription factor proteins was performed. Elevated expression of both proteins was found to follow quickly (within 1 h) after addition of glutamate. However, this effect was not dose-dependent, thus it does not provide clear correlations to the programmed cell death. In conclusion, this study reports on the establishment of a novel apoptotic model of excitotoxicity, and invites further efforts to investigate a basis for in vitro susceptibility and in vivo resistance of dentate gyrus granule cells to excitotoxic insult evoking apoptosis.
Animals↗
Interaction among preschoolers with and without disabilities: effects of across-the-day peer intervention.
We examined the effects of a peer-mediated intervention package that taught typically developing children to be more aware of communicative attempts to classmates with disabilities, to use a small set of facilitative strategies ("Stay, play, talk"), and to distribute strategy use across the school day. A multiple baseline design across subjects was instituted with two cohorts of preschool children. Following baseline observations, a total of 8 target children with moderate development disabilities were eventually paired with trained peers who received "buddy training." One trained peer were taught facilitative strategies and encouraged to use them during classroom activities, consistent improvements in social interaction on the part of the trained peers and target children were demonstrated. Similar or more frequent interactions were demonstrated when trained peers were reassigned to different target children during generalization probes. In addition, treatment effects were revealed when comparing sequential analyses applied to the specific communicative behaviors across experimental conditions, in changes in target children's sociometric ratings, and in social validity judgments of videotapes from before and after treatment. This peer intervention approach has promise for improving the communicative interaction and social integration of children with disabilities attending inclusive preschools.
Child↗
Glutamate receptors in cortical plasticity: molecular and cellular biology.
Glutamate receptors (GluRs) provide the major excitatory input to cortical neurons. Four main subtypes of GluRs are distinguished, namely, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate, and metabotropic receptors. All of them have been implicated in neuronal plasticity, and this paper reviews data that may be pertinent to the role played by GluRs in neocortical plasticity both in adult animals as well as during postnatal development. Emphasis is given to receptor distribution analyzed by various means, such as physiological responses, ligand binding as revealed by receptor autoradiography, and expression of receptor subunits at both mRNA and protein (immunoreactivity) levels. Possible mechanisms of involvement of GluRs in plastic changes on cortical neuron response are reviewed, and data on up- and downregulation of GluRs in neocortical plasticity are summarized. Functional studies involving either activation or blocking, and effects of such manipulation on cortical plasticity are discussed.
Animals↗
Kainate-evoked modulation of gene expression in rat brain.
Kainate is a glutamate analog that produces neuronal excitation resulting in seizures within hours following its intraperitoneal injection into adult rats. Then, at 2-3 days after the treatment, neurodegeneration of apoptotic character can be observed in limbic system. As a consequence, plastic reorganization and glial reactivation phenomena occur. These physiological and pathological responses are reflected by specific changes in gene expression, that can be dissected according to their spatio-temporal patterns. The early phase of gene expression observed in all hippocampal subfields appears to reflect a sudden burst of spiking activity. Changes in mRNA levels restricted to dentate gyrus are suggestive of a link to neuronal plasticity. The late gene expression response implies its correlation either to neuronal cell death or glial reactivation, depending on cellular localization of gene products. Thus analysis of the temporal and spatial gene expression pattern in the hippocampus after kainate treatment may provide clues revealing specific phenomena to which gene expression could be attributed.
Animals↗
Visual stimulation regulates the expression of transcription factors and modulates the composition of AP-1 in visual cortex.
It is believed that long-term changes in neuronal function are orchestrated by transcription factors, such as AP-1 and ZIF 268, which are in turn regulated by synaptic stimulation. To further our understanding of the functional effects of such expression, we have examined the DNA-binding activities of both AP-1 and ZIF 268 by way of electrophoretic mobility shift assays (EMSA) on nuclear extracts from visual cortices of rats treated with selective light exposure. Visual stimulation after dark rearing increased the DNA-binding activities of both AP-1 and ZIF 268 to their highest levels within 2 hr. ZIF 268 thereafter dropped to levels similar to that observed in naive animals, whereas AP-1 DNA-binding activity continued to remain elevated even after 24 hr of stimulation. The components of the AP-1 complex, when assessed by EMSA-supershift analysis, showed considerable variability under different conditions of exposure. FosB and JunD were the major constituents of AP-1 in both naive and dark-reared animals. Brief visual stimulation (2 hr) added c-Fos, c-Jun, and JunB to this complex, whereas prolonged stimulation (6-24 hr) reduced c-Fos and c-Jun levels significantly, leaving only FosB, JunB, and JunD as the major components of AP-1. These results suggest that transcriptional control by AP-1 may be generated by selective combinatorial interactions of different members of the Fos and Jun families and that are guided by activity-dependent processes.
Activating Transcription Factor 2↗