Evgeni Nikolaev

Important role of matrix metalloproteinase 9 in epileptogenesis

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling–induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.

The antitumorigenic response of neural precursors depends on subventricular proliferation and age.

Glioblastomas, the most aggressive primary brain tumors, occur almost exclusively in adult patients. Neural precursor cells (NPCs) are antitumorigenic in mice, as they can migrate to glioblastomas and induce tumor cell death. Here, we show that the antitumor effect of NPCs is age‐dependently controlled by cell proliferation in the subventricular zone (SVZ) and that NPCs accumulating at a glioblastoma are diverted from their normal migratory path to the olfactory bulb. Experimentally induced cortical glioblastomas resulted in decreased subventricular proliferation in adult (postnatal day 90) but not in young (postnatal day 30) mice. Adult mice supplied fewer NPCs to glioblastomas and had larger tumors than young mice. Apart from the difference in proliferation, there was neither a change in cell number and death rate in the SVZ nor a change in angiogenesis and immune cell density in the tumors. The ability to kill glioblastomas was similar in NPCs isolated from young and adult mice. The proliferative response of NPCs to glioblastomas depended on the expression of D‐type cyclins. In young mice, NPCs express the cyclins D1 and D2, but the expression of cyclin D1 is lost during aging, and in adult NPCs only cyclin D2 remains. In young and adult cyclin D2‐deficient mice we observed a reduced supply of NPCs to glioblastomas and the generation of larger tumors compared with wild‐type mice. We conclude that cyclin D1 and D2 are nonredundant for the antitumor response of subventricular NPCs. Loss of a single D‐type cyclin results in a smaller pool of proliferating NPCs, lower number of NPCs migrating to the tumor, and reduced antitumor activity.

Matrix Metalloproteinase (MMP) 9 Transcription in Mouse Brain Induced by Fear Learning

Background: Matrix metalloproteinase 9 is involved in fear-associated memory formation wherein transcriptional regulation is poorly known. Results: Overexpression and promoter binding activity of AP-1 factors regulate MMP-9 transcription, preceding elevated enzymatic activity in mouse brain. Conclusion: c-Fos and c-Jun AP-1 components positively regulate MMP-9 transcription in fear learning. Significance: The novel tools and approaches in vivo allowed us to explore MMP-9 transcription in mouse brain. Memory formation requires learning-based molecular and structural changes in neurons, whereas matrix metalloproteinase (MMP) 9 is involved in the synaptic plasticity by cleaving extracellular matrix proteins and, thus, is associated with learning processes in the mammalian brain. Because the mechanisms of MMP-9 transcription in the brain are poorly understood, this study aimed to elucidate regulation of MMP-9 gene expression in the mouse brain after fear learning. We show here that contextual fear conditioning markedly increases MMP-9 transcription, followed by enhanced enzymatic levels in the three major brain structures implicated in fear learning, i.e. the amygdala, hippocampus, and prefrontal cortex. To reveal the role of AP-1 transcription factor in MMP-9 gene expression, we have used reporter gene constructs with specifically mutated AP-1 gene promoter sites. The constructs were introduced into the medial prefrontal cortex of neonatal mouse pups by electroporation, and the regulation of MMP-9 transcription was studied after contextual fear conditioning in the adult animals. Specifically, −42/-50- and −478/-486-bp AP-1 binding motifs of the mouse MMP-9 promoter sequence have been found to play a major role in MMP-9 gene activation. Furthermore, increases in MMP-9 gene promoter binding by the AP-1 transcription factor proteins c-Fos and c-Jun have been demonstrated in all three brain structures under investigation. Hence, our results suggest that AP-1 acts as a positive regulator of MMP-9 transcription in the brain following fear learning.

Defensive conditioning-related increase in AP-1 transcription factor in the rat cortex

In the studies reported herein, electrophoretic mobility shift assay (EMSA) and immunocytochemistry have been applied to document increased levels of AP-1 transcription factor, and its major component, c-Fos in the rat brain following behavioral training of two-way active avoidance. A single training session (50 trials) provoked elevation of AP-1 in the visual, sensory and limbic cortex but not in the hippocampus. A session following long term training (10 sessions, up to asymptotic level of performance) had much smaller effect on AP-1 levels in the visual cortex than single training session. The long term training was used to ensure that observed effects were related to acquisition of the reaction rather than simply to behavioral performance. Supershift EMSA analysis with antibodies directed at individual AP-1 components revealed that AP-1 extracted from the brains of trained as well as naive animals is composed of the same proteins, i.e., in order of relative level within the protein family: c-Fos, Fos B, Fra-2, and Jun D, Jun B, c-Jun. These studies reinforce the notion that transcription factors as regulators of gene expression-and AP-1 in particular-may respond to behavioral stimulation and furthermore may play a role in acquisition of behavioral reactions.

The effect of age on the dynamics and the level of c-Fos activation in response to acute restraint in Lewis rats

Recent studies have reported an age-related increase of anxiety in rodents with a concomitant decrease in neuronal activity in some of the key structures of the fear/anxiety circuit. In the present study we present evidence that distinct parts of this circuit are differentially affected by age in Lewis rats. The effect of ageing is observed both at the actual level of neuronal activation and its time-course. While the structures belonging to the HPA axis react with a bigger neuronal activation and almost no change in the shape of dynamics curve in response to restraint, the structures involved in higher processing of emotional cues (amygdala and hippocampus) become deficiently activated with age despite their generally higher basal level of activation.

Behavioural evaluation of long-term neurotoxic effects of NMDA receptor antagonists.

High doses of NMDA antagonists e.g. (+)MK-801 evoke neurodegeneration in retrosplenial cortex in rodents. To assess functional consequences of such treatment, three paradigms of two-way active avoidance learning (with visual or auditory conditioned stimuli) and additionally a spatial learning paradigm — radial maze — were used. Female rats were treated i.p. with 5 mg/kg of (+)MK-801. Recumbence, severe hypothermia and loss of body weight were observed for 3–7 days. Despite that, there were no statistically significant differences in performance of avoidance reaction between saline and (+)MK-801 treated animals trained 10–40 days after the drug administration. However, in the radial maze test (+)MK-801 impaired reference (but not working) memory in the experiment that started 8 days after the treatment. Similar effect was observed on reversal learning. The clinically used NMDA receptor antagonist memantine at the doses of 20 and 40 mg/kg had also no such long term negative effect on working memory during training (even positive effect was seen at 20 mg/kg) but at 40 mg/kg impaired learning on the first day of reversal. This indicates that (+)MK-801 neurotoxicity in the retrosplenial cortex is connected with subtle alterations in the learning performance that may be seen in some tests only. Moreover, memantine doses greatly exceeding therapeutically relevant range produce minimal functional alteration. An additional experiment revealed that the same dose of memantine results in two fold higher serum levels of the antagonist in female than male rats. Hence, considering that profiling studies are done in male rats, a safety factor of over 16 fold can be calculated for memantine.

D1 receptors involved in the acquisition of sexual experience in male rats

As we found previously, changes in ultrasonic vocalizations in the 50-kHz band emitted before a female is introduced into a copulatory chamber (precontact vocalizations-PVs) and shortening of ejaculation latency (EL) in subsequent copulatory sessions are correlated and reflected acquisition of sexual experience in male rats. In the present experiments, we investigate the role of the D1 receptor in this phenomenon. First, the effects of a D1 antagonist and D1 agonists injected subcutaneously during the first sexual contacts were analyzed. The antagonist (SCH-23390) inhibited changes in PVs and EL as well as rearing (R) and mount latency (ML). Both agonists (SKF-38393 and selective SKF-82957) inhibited PVs also at doses where no effects on EL and ML were observed. Repeated injections of five agonists, with or without socio-sexual contacts, had an effect on later PVs and R (long-term effect) but not on copulatory performance during the subsequent four sessions. In the last experiment, a D1 agonist (SKF-82957, 0.5 microg) was injected bilaterally into nucleus accumbens (N.Acc) before each of the first five sessions. The D1 agonist in N.Acc facilitated initiation of copulation in treatment-naïve rats. Repeated administration of the D1 agonist into the brain had long-lasting effects on PVs, R and ML, probably as a result of long-term changes in neuronal structures involved in acquisition of sexual experience, in this part of the nucleus accumbens. Repeated D1 agonist administration probably inhibits/reverses sensitization processes in dopamine structures. The results indicate a role of D1 receptors in acquisition of socio-sexual experience and suggest independent neuronal pathways for changes in PVs and EL.

Blockade of androgen receptor in the medial amygdala inhibits noncontact erections in male rats

Our previous work demonstrated that androgens in the medial amygdala (MeA) of castrated male rats maintained noncontact erections (NCEs), which occur during exposure to an inaccessible receptive female, for one week after implantation. The present experiments investigated the effects of implantation into the MeA of either flutamide (F), a blocker of androgen receptors, or of 1,4,6-androstatrien-3,17-dione (ATD), which blocks aromatization of testosterone. One day after implantation of F, fewer males displayed NCEs, and had longer latencies to the first NCE and fewer NCEs, and spent less total time in genital grooming, compared to the control group. ATD had only weak facilitative effects on some measures of NCEs. These results suggest that androgen receptors in the MeA play a major role in the regulation of NCEs and that the MeA is one of the neuronal structures that regulate male sexual arousal. Furthermore, it is sensitive to relatively fast changes in the level of androgen receptors stimulation.

Age increases anxiety and reactivity of the fear/anxiety circuit in Lewis rats.

A growing body of data indicates that changes in emotional behavior occur with age. Young Lewis rats are known to display hypofunction of the HPA axis. With age the reactivity of this axis is thought to increase with a concomitant rise in anxiety. In the current study, we investigate how and if the pattern of neuronal activation (measured as c-Fos protein expression) in Lewis rat brains changes with age and in response to novel environments differing in aversiveness. We found that distinct parts of the fear/anxiety circuit (i.e., the amygdalar complex, hippocampus and hypothalamus) undergo diverse age-related changes in response to behavioral challenges. While in the hypothalamus an increase in responsivity to mild stressors was observed with age, no such effect was present in the hippocampus. The amygdalar complex (especially the medial and cortical nuclei) on the other hand exhibited an age-dependent decrease in neuronal activation to mild stressors. This was accompanied by a marked increase in anxiety not correlated with a decline in locomotor activity.

Cardiac Nerve Growth Factor Overexpression Induces Bone Marrow–derived Progenitor Cells Mobilization and Homing to the Infarcted Heart

Reparative response by bone marrow (BM)-derived progenitor cells (PCs) to ischemia is a multistep process that comprises the detachment from the BM endosteal niche through activation of osteoclasts and proteolytic enzymes (such as matrix metalloproteinases (MMPs)), mobilization to the circulation, and homing to the injured tissue. We previously showed that intramyocardial nerve growth factor gene transfer (NGF-GT) promotes cardiac repair following myocardial infarction (MI) in mice. Here, we investigate the impact of cardiac NGF-GT on postinfarction BM-derived PCs mobilization and homing at different time points after adenovirus-mediated NGF-GT in mice. Immunohistochemistry and flow cytometry newly illustrate the temporal profile of osteoclast and activation of MMP9, PCs expansion in the BM, and liberation/homing to the injured myocardium. NGF-GT amplified these responses and increased the BM levels of active osteoclasts and MMP9, which were not observed in MMP9-deficient mice. Taken together, our results suggest a novel role for NGF in BM-derived PCs mobilization/homing following MI.