Biomedical subjects

R K Filipkowski

Publications and source records attributed to R K Filipkowski.

16 recordsLinked to original sources

PubMed2001

Kainate-induced genes in the hippocampus: lessons from expression patterns.

Kainate, the analog of the excitatory amino acid L-glutamate, upon binding to non-NMDA glutamate receptors, causes depolarization of neurons followed by severe status epilepticus, neurodegeneration, plasticity and gliosis. These events are best observed in hippocampus, the limbic structure implicated in learning and long-term memory formation. Neurons in all hippocampal structures undergo hyper-activation, however, whereas the cells in the CA subfields degenerate within 2--3 days following the application of kainate, the granule cells of the dentate gyrus are resistant to any form of neurodegeneration and even initiate new synaptic contacts. These physiological and histological changes are modulated by short-term and long-term alterations in gene expression. Perhaps close examination of the changing spatio-temporal patterns of mRNAs of various genes may help in generating a clearer picture of the molecular events leading to complex cognitive functions.

Animals↗

PubMed2001

Expression of c-Fos, Fos B, Jun B, and Zif268 transcription factor proteins in rat barrel cortex following apomorphine-evoked whisking behavior.

Apomorphine-evoked expression of transcription factor proteins: c-Fos, Fos B, Jun B, and Zif268 (also named Krox-24, NGFI-A, Egr-1), was investigated in rat somatosensory (barrel) cortex. The effect of the N-methyl-D-aspartate receptor antagonist MK-801 on their expression was also analyzed. Apomorphine is a dopamine receptor agonist, eliciting motor activity, including enhanced whisking leading to the activation of vibrissae representation in the barrel cortex. Rats had their whiskers clipped on one side of the snout. The Zif268 levels were markedly reduced by this procedure alone. In contrast, apomorphine (5.0 mg/kg) evoked marked c-Fos elevation, less pronounced changes in Jun B and Zif268 and no change in Fos B. The greatest apomorphine-evoked c-Fos accumulation was observed in layers IV and V/VI of non-deprived barrel cortex and was not significantly influenced by MK-801 injection at 0.1 mg/kg. A higher dose of MK-801 (1.0 mg/kg) produced abnormalities in locomotor behavior and diminished c-Fos levels on the non-deprived side to the ones observed in the sensory stimulus-deprived cortex. We conclude that the response of the somatosensory cortex is selective with respect to both the gene activated and its cortical layer localization. Furthermore, sensory stimulation provides a major but not the only component to apomorphine-evoked barrel cortex gene activation.

Afferent Pathways↗

PubMed2000

Functional analysis of the leader peptide of the yeast gene CPA1 and heterologous regulation by other fungal peptides.

The 25-amino-acid leader peptide present at the 5' end of yeast CPA1 mRNA is responsible for the translational repression of that gene by arginine. We show here that the active domain of the yeast peptide is highly specific and extends over amino acids 6-23. The region between amino acids 6-21 is well conserved between similar peptides present upstream of CPA1-homologous genes in other fungi. The Neurospora crassa arg-2 peptide represses the expression of CPA1, whereas the peptide from Aspergillus nidulans has only a weak regulatory effect. Such results suggest that the N- and C-terminal amino acids of the peptide may influence its regulatory activity. We also show that the transcription start sites of CPA1 are not modified when the cells are grown in the presence of arginine, nor in a strain carrying an inactive peptide.

Amino Acid Sequence↗

PubMed2000

Inducing gene expression in barrel cortex--focus on immediate early genes.

Using only their vibrissae, rats and mice are able to recognize and differentiate surfaces not distinguishable by primates using their fingertips. It has been shown that sensory stimulation elicits the expression of immediate-early genes (IEG), e.g., c-fos and zif268, in the sensory cortex of rats and mice. Though most of these findings come from visual system, mice and rats rely more on their vibrissal system which also offers many advantages for designing precise and precisely controlled experiments. In this review, new models for the selective and simple stimulation of vibrissae are presented and discussed. The data demonstrating IEG expression in the vibrissal system is also reviewed.

Animals↗

PubMed1999

Neuronal excitation-driven and AP-1-dependent activation of tissue inhibitor of metalloproteinases-1 gene expression in rodent hippocampus.

Understanding of biological function of AP-1 transcription factor in central nervous system may greatly benefit from identifying its target genes. In this study, we present several lines of evidence implying AP-1 in regulating expression of tissue inhibitor of metalloproteinases-1 (timp-1) gene in rodent hippocampus in response to increased neuronal excitation. Such a notion is supported by the findings that timp-1 mRNA accumulation occurs in the rat hippocampus after either kainate- or pentylenetetrazole-evoked seizures with a delayed, in comparison with AP-1 components, time course, as well as with spatial overlap with c-Fos protein (major inducible AP-1 component) expression. Furthermore, AP-1 sequence derived from timp-1 promoter is specifically bound by hippocampal AP-1 proteins after treating the rats with either pro-convulsive agent. Finally, timp-1 promoter responds to excitatory activation both in vivo, in transgenic mice harboring the timp-LacZ gene construct, and in vitro in neurons of the hippocampal dentate gyrus cultures. These findings suggest that the AP-1 transcription factor may exert its role in the brain through affecting extracellular matrix remodeling.

Animals↗

PubMed1999

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↗

PubMed1998

Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning.

The calcium-calmodulin-dependent kinase II (CaMKII) is required for hippocampal long-term potentiation (LTP) and spatial learning. In addition to its calcium-calmodulin (CaM)-dependent activity, CaMKII can undergo autophosphorylation, resulting in CaM-independent activity. A point mutation was introduced into the alphaCaMKII gene that blocked the autophosphorylation of threonine at position 286 (Thr286) of this kinase without affecting its CaM-dependent activity. The mutant mice had no N-methyl-D-aspartate receptor-dependent LTP in the hippocampal CA1 area and showed no spatial learning in the Morris water maze. Thus, the autophosphorylation of alphaCaMKII at Thr286 appears to be required for LTP and learning.

2-Amino-5-phosphonovalerate↗

PubMed1998

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↗

PubMed1998

The dorsal hippocampus is essential for context discrimination but not for contextual conditioning.

The authors describe how (a) the timing of hippocampal lesions and (b) the behavioral-representational demands of the task affect the requirement for the hippocampus in contextual fear conditioning. Post- but not pretraining lesions of the hippocampus greatly reduced contextual fear conditioning. In contrast, pretraining lesions of the hippocampus abolished context discrimination, a procedure in which mice are trained to discriminate between 2 similar chambers (shock context vs. no-shock context). Whereas either contextual- or cue-based strategies can be used to recognize an aversive context, discrimination between similar contexts is optimally acquired by contextual (hippocampal)-based strategies. In keeping with the lesion results, Nf1(+/-)/Nmdar1(+/-) mutant mice, which have spatial learning deficits, are impaired in context discrimination but not in contextual conditioning. Together, these data dissociate hippocampal and nonhippocampal contributions to contextual conditioning, and they provide direct evidence that the hippocampus plays an essential role in the processing of contextual stimuli.

Analysis of Variance↗

PubMed1997

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↗

PubMed1995

Kainate-evoked secondary gene expression in the rat hippocampus.

Kainate treatment provides a model to study elevated expression of genes whose function may be related to neuronal plasticity. In particular, expression of components of AP-1 transcription factor, i.e. Fos and Jun proteins, has been widely investigated in this system. While AP-1 has been repeatedly implicated in various plasticity-related phenomena, very little is known about its downstream gene targets. In the experiments reported here we have analyzed whether genes recently identified as kainate-induced in the rat dentate gyrus and coding for secretogranin II, clathrin heavy chain and heat shock cognate protein 70 can be characterized by a secondary, i.e. possibly inducible transcription factor-dependent mode of activation. Using in situ hybridization and northern studies we have found that expression of all three genes occurs in all hippocampal regions activated following kainate treatment, the time-course of this activation is delayed when compared to mRNA accumulation of AP-1 components, and finally the expression of all three genes is significantly blocked by a cycloheximide-protein synthesis inhibitor. These results suggest that indeed the genes examined are characterized by their secondary mode of activation.

Animals↗

PubMed1995

Elevated cathepsin D expression in kainate-evoked rat brain neurodegeneration.

Expression patterns of cathepsin D (lysosomal aspartic protease) and glial fibrillary acidic protein (GFAP, a marker of reactive astroglia) were determined by Northern blot analysis and immunohistochemistry in the rat brain during neurodegeneration accompanying kainate-evoked seizures. The level of cathepsin D mRNA in the hippocampus, limbic cortex, and temporo-parieto-occipital neocortex was shown to increase, starting at 6 h after kainate treatment, and reaching peak values at 3-7 days after the neurotoxin administration. A similar time course of elevated accumulation was noted for GFAP mRNA in these structures. Immunohistochemical analysis performed 3 days after kainate treatment showed that the increased cathepsin D levels were confined mainly to the degenerating neurons in the susceptible brain areas, while the elevated GFAP immunoreactivity was observed in reactive astrocytes. Although cathepsin D and GFAP expression levels were elevated by kainate administration, their expression patterns revealed significant differences with regard to both intensity and site of induction.

Animals↗

PubMed1994

Dynamic changes in the composition of the AP-1 transcription factor DNA-binding activity in rat brain following kainate-induced seizures and cell death.

Kainate, a potent excitatory and neurotoxic agent, has also proved useful in studies on other glutamate-driven phenomena, such as neuronal plasticity. Long-term effects of kainate are apparently dependent on its influence on the expression of various genes, including those encoding the AP-1 transcription factor, consisting of proteins belonging to the Fos and Jun families. In our studies we analysed c-fos, fos B, c-jun, jun B and jun D mRNA levels as well as a functional feature of AP-1, its DNA-binding activity, in the rat brain following systemic injection of kainate. Two phases of elevated AP-1 DNA-binding activity were observed in the hippocampus and entorhinal cortex, and were correlated with period of seizures (2 and 6 h after kainate injection) and neuron damage (48-72 h). At 72 h after kainate treatment DNA fragmentation, believed to be diagnostic of apoptotic processes typical of programmed cell death phenomena, was noted. Two and six hours after the treatment, AP-1 consisted predominantly of Fos B, c-Fos, Fra-2 and Jun B, while at 72 h Jun D constituted the major AP-1 component in place of Jun B, and no c-Fos was detected. Only a slight AP-1 increase was seen 24 h after kainate treatment. In the sensory cortex, only the late phase of AP-1 elevation was detected. Contrary to AP-1, no effect of kainate on levels of two other transcription factors, CREB/ATF (cAMP-responsive element binding proteins) and OCT (octamer element DNA-binding activity) was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

DNA fragmentation in rat brain after intraperitoneal administration of kainate.

Cell death occurs in many neuropathological conditions. However, the mechanisms governing this process(es) remain generally unknown. In this report we studied whether excitotoxic neuronal death evoked by kainic acid (KA) in rat brain is associated with ladder-like DNA fragmentation. DNA was isolated from hippocampi, entorhinal and sensory cortices at various times following intraperitoneal KA (10 mg kg-1) injections. Typical oligonucleosome-sized DNA fragmentation was observed in all three structures at 18 h and 72 h following KA administration. These findings were further confirmed by in situ nick-translation. DNA fragmentation is believed to be diagnostic for apoptosis. The clear ladders of DNA fragmentation appeared after 18 h, although slight degradation was observed as early as 12 h after KA administration.

Animals↗

PubMed1994

A segment of mRNA encoding the leader peptide of the CPA1 gene confers repression by arginine on a heterologous yeast gene transcript.

The expression of the yeast gene CPA1, which encodes the small subunit of the arginine pathway carbamoylphosphate synthetase, is repressed by arginine at a translational level. CPA1 mRNA contains a 250-nucleotide-long leader which includes a 25-codon upstream open reading frame (uORF). Oligonucleotide site-directed mutagenesis of this uORF as well as sequencing of constitutive cis-dominant mutations has suggested that the leader peptide product of the CPA1 uORF is an essential negative element for repression of the CPA1 gene by arginine. In this work, a series of deletions affecting the regions 5' and 3' to the uORF in the leader sequence was constructed. The arginine-dependent repression of CPA1 was little affected in these constructions, indicating that these regions are not essential for the regulatory response. This conclusion was further supported by the finding that inserting the mRNA segment encoding the leader peptide sequence of CPA1 in the leader sequence of another gene, namely, GCN4, places this gene under arginine repression. Similarly, the behavior of fusions of the leader sequence of CPA1 with those of ARG4 or GAL10 confirmed that the regions of this leader located upstream and downstream from the uORF are dispensable for the regulation by arginine. Finally, a set of substitution mutations which modify the uORF nucleotide sequence while leaving unchanged the corresponding amino acid sequence was constructed. The mutations did not affect the repression of CPA1 by arginine. The data presented in this paper consequently agree with the conclusion that the leader peptide itself is the main element required for the translational repression of CPA1.

Amino Acid Sequence↗

PubMedDate not supplied

Tactile experience induces c-fos expression in rat barrel cortex.

Understanding gene expression that is responsive to sensory stimulation is central to elucidate molecular mechanisms underlying neuronal plasticity. In this study we demonstrate two new methods of stimulating whiskers that provide major sensory input to rat neocortex. In the first paradigm, animals were placed on the top of a cylinder and their vibrissae were brushed by hand. In the second paradigm, animals were placed for a brief period of time into a new, wired cage resulting in vibrissae stimulation when they explored the new environment. Both approaches induced c-Fos expression in barrel cortex corresponding to the stimulated vibrissae, especially in layer IV. Layers II/III and V/VI also showed c-Fos induction, but there were no detectable changes in layer VIb. The majority of c-Fos-expressing cells are probably not inhibitory neurons, because they do not show parvalbumin staining. Both paradigms, in contrast to the previous methods, are simple to use and do not require anesthesia, restraint of animals, or elaborate experimental setups.

Animals↗