D Nowicka

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.

Parvalbumin‐containing neurons, perineuronal nets and experience‐dependent plasticity in murine barrel cortex

The ability to undergo experience‐dependent plasticity in the neocortex is often limited to early development, but also to particular cortical loci and specific experience. In layers II–IV of the barrel cortex, plasticity evoked by removing all but one vibrissae (univibrissa rearing) does not have a time limit except for layer IV barrels, where it can only be induced during the first postnatal week. In contrast, deprivation of every second vibrissa (chessboard deprivation) removes time limits for plasticity. The mechanism permitting plasticity in response to chessboard deprivation and halting it in reply to univibrissa rearing is unknown. Condensation of chondroitin sulfate proteoglycans into perineuronal nets and an increase in intracortical inhibition mediated by parvalbumin‐containing interneurons are implicated in closing the critical period for ocular dominance plasticity. These factors could also be involved in setting up the critical period in barrels in a way that depends on a particular sensory experience. We therefore examined changes in density of parvalbumin‐containing cells and perineuronal nets during development of mouse barrel cortex and after brief univibrissa and chessboard experience in adolescence. We observed a progressive increase in the density of the two markers across cortical layers between postnatal day 10 and 20, which was especially pronounced in the barrels. Univibrissa rearing, but not chessboard deprivation, increased the density of perineuronal nets and parvalbumin‐containing cells in the deprived barrels, but only those that immediately neighbour the undeprived barrel. These data suggest the involvement of both tested factors in closing the critical period in barrels in an experience‐dependent manner.

Calcyclin (S100A6) Binding Protein (CacyBP) Is Highly Expressed in Brain Neurons

The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting. Polyclonal antibodies against recombinant CacyBP purified from E. coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach. CacyBP immunoreactivity was also detected in lung and kidney. Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0.34 ng/μg protein, respectively. Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined. Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex. The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood. In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21. The results presented here show that CacyBP is predominantly a neuronal protein.

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.

Disturbance of perineuronal nets in the perilesional area after photothrombosis is not associated with neuronal death.

Perineuronal nets (PNNs) are a condensed form of extracellular matrix that covers the surface of a subset of neurons. Their presence limits neuronal plasticity and may protect neurons against harmful agents. Here we analyzed the relationship between spatiotemporal changes in PNN expression and cell death markers after focal cortical photothrombotic stroke in rats. We registered a substantial decrease in PNN density using Wisteria floribunda agglutinin staining and CAT-315 and brevican immunoreactivity; the decrease occurred not only in the lesion core but also in the perilesional and remote cortex as well as in homotopic contralateral cortical regions. Fluoro Jade C and TUNEL staining in perilesional and remote areas, however, showed a low density of dying cells. Our results suggest that the PNN reduction was not a result of cellular death and could be considered an attempt to create conditions favorable for synaptic remodeling.

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.

Aging somatosensory cortex displays increased density of WFA-binding perineuronal nets associated with GAD-negative neurons.

The mechanisms of aging in the brain and the subsequent decrease in cognitive abilities remain elusive. While most studies refer to research conducted in old and senile animals, little is known about the early symptoms of normal, healthy aging. In this study, we examined whether perineuronal nets (PNNs), a special form of extracellular matrix (ECM) tightly associated with neurons that is thought to be involved in limiting neuronal plasticity, undergo changes in density during early aging. Using histochemistry and immunohistochemistry, we found that in middle-aged mice (1-year-old), the density of WFA-binding PNNs in the somatosensory cortex as well as in the visual cortex was increased in comparison to that in young adults (3-month-old). Moreover, in the somatosensory cortex, this increase was not associated with any of the GABAergic neuron types that were examined. We propose that early age-related changes in neuronal plasticity may be associated with this increase and can be conceptualized as the spreading of structural brakes for synaptic rearrangements.

Dissociation of synaptic zinc level and zinc transporter 3 expression during postnatal development and after sensory deprivation in the barrel cortex of mice.

In the neocortex, synaptic zinc level is regulated by sensory experience. Previously, we found that trimming of mystacial vibrissae resulted in an increase of synaptic zinc level in corresponding deprived barrels in the cortex of mice. The present study focused on the relationship between synaptic zinc and zinc transporter 3 (ZnT3) protein expression in the barrel cortex of mice during postnatal development and after sensory deprivation of selected vibrissae. Using immunocytochemistry and western blot analysis, we found that ZnT3 expression is delayed as compared with the onset of synaptic zinc and presynaptic markers, such as synapsin I and synaptophysin. Further, neither long-term deprivation in young mice nor short deprivation in adult mice, that resulted in an increase of synaptic zinc level, produced alterations in ZnT3, synapsin I or synaptophysin expression in deprived barrels. These results suggest that in the barrel cortex ZnT3, synapsin I or synaptophysin are not determinant for the activity-dependent regulation of the synaptic zinc level.

Matrix metalloproteinase inhibition counteracts impairment of cortical experience‐dependent plasticity after photothrombotic stroke

Matrix metalloproteinases (MMPs) are fine modulators of brain plasticity and pathophysiology. The inhibition of MMPs shortly after ischaemic stroke reduces the infarct size and has beneficial effects on post‐stroke behavioural recovery. Our previous studies have shown that photothrombotic cortical stroke disrupts use‐dependent plasticity in the neighbouring cortex. The aim of the present study was to check whether the inhibition of MMPs after photothrombosis rescued the plastic capacity of the barrel cortex. To induce plasticity in adult mice, a unilateral deprivation of all vibrissae except row C was applied. The deprivation started immediately after stroke and lasted 7 days. This procedure, in control (non‐stroke) animals, results in an enlargement of functional representation of the spared row, as shown with [14C]2‐deoxyglucose uptake mapping. In mice with stroke induced by photothrombosis in the vicinity of the barrel cortex, vibrissae deprivation did not result in an enlargement of the cortical representation of the spared row C of vibrissae, which confirmed our previous results. However, when mice were injected with the broad‐spectrum inhibitor of MMPs FN‐439 (10 mg/kg, i.v.) immediately before a stroke, an enlargement of the representation of the spared row similar to the enlargement found in sham mice was observed. These results indicate the involvement of MMPs in the impairment of use‐dependent plasticity in the vicinity of an ischaemic lesion.

Spatio-temporal pattern of N-methyl-D-aspartate receptor NR1 mRNA expression during postnatal development of visual structures of the rat brain.

N‐methyl‐D‐aspartate (NMDA) excitatory amino acid receptors (NMDAR) play a pivotal role in various physiological and pathological responses of the central nervous system (CNS), including such phenomena of neuronal plasticity as cortical development. Hence, it could be suggested that expression of genes coding for NMDAR components can be differentially regulated throughout the development. To test this notion we analyzed with in situ hybridization mRNA levels of NMDAR subunit designated as NR1, during time of development (1–6 weeks postnatally) spanning the critical period of functional formation of major visual structures of the rat brain, namely visual cortex (VC), superior colliculus (SC), and lateral geniculate nucleus (LGN). The highest levels of NR1 mRNA were found in the VC and SC at the onset of the critical period, i.e., 21 days postnatally, whereas in the LGN a tendency toward a similar pattern of expression was observed. Analysis of spatial distribution of NR1 mRNA in the SC and VC revealed that an adult‐like laminar pattern of expression was also achieved between 14 and 21 days postnatally. This pattern of expression corroborates with developmentally regulated changes in an overall density of cell bodies in these areas.