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1

Cycling D2 knock-out mice as a tool to investigate the role of adult neurogenesis

100%
Filipkowski R. K.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
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nr S
The function of adult brain neurogenesis remains elusive, although it has been suggested to play a key role in learning and memory. In the previous studies, we employed cyclin D2 knock-out (cD2 KO) mice and demonstrated apparent complete deficiency in generating new neurons accompanied by minor morphological abnormalities of the brain, including smaller hippocampal formation (Kowalczyk et al. 2004). We have shown that these mice perform and learn in several behavioral tasks: context and trace fear conditioning, novel object recognition, Morris water maze, and spatial tests in IntelliCage (Jaholkowski et al. 2009). Recently, we determined that cD2 KO mice also perform surprisingly well in two-way avoidance and Barnes maze. Additionally, cD2 KO mice were subjected to hippocampus-dependent behavioral tests not requiring learning. Mutant mice showed significant impairment in several species-typical behaviors: nest construction, digging, and marble burying. Moreover, cD2 KO mice were more active in the open field and motility chamber, and showed increased explorative behavior in IntelliCage. Notably, similar deficits in species-typical behaviors and an increase in locomotor activity were previously shown in rodents with hippocampal lesions. Recently, we have also investigated alcohol consumption in cD2 KO mice and shown that they consumed significantly more of 8-16% ethanol and demonstrated a significantly higher preference for 4 – 16% ethanol (Jaholkowski et al. 2011). We conclude that either morphological abnormalities of the hippocampal formation or the impairment in the adult brain neurogenesis (or both) alter hippocampus-dependent behaviors and ethanol consumption in cD2 KO mice without influencing their learning abilities. These results show that new neurons are not necessary for learning and suggest their possible role in several species-typical behaviors as well as in ethanol self-administration.
2

Behavioral, ultrasonic, and cardiovascular responses of male wistar rats in different social and emotional contexts after ultrasonic playback

100%
Filipkowski R. K.
Acta Neurobiologiae Experimentalis
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2019
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tom 79
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nr Suppl.1
Rats are social animals that use ultrasonic vocalizations (USV) to communicate. USV are usually divided into 50 kHz calls which accompany appetitive states, and 22 kHz vocalizations which are usually associated with aversive states. Both kinds of states are known to affect animals’ heart rate (HR). Also, the polyvagal theory claims that both cardiovascular parameters and USV emission is affected by the autonomous system, as they share a common signaling pathway. The aim of the study was to evaluate the changes in behavior, HR, and USV emission after playback of ultrasounds. Male Wistar rats were housed in pairs or separately for 4 weeks, and some of the animals underwent fear conditioning. Animals were implanted with DSI telemetry transmitters for acquisition of cardiovascular parameters. After recovery, rats were subjected to ultrasounds playback consisting of initial 10 min of static silence and five sets of 10 s sounds (50 or 22 kHz), either natural, collected from animals, or artificial tones, separated by 5 min silence intervals. Video, audio, and cardiovascular parameters were collected. Surprisingly, presentation of both 50 and 22 kHz sounds induced approach behavior. Both single- and pair-housed animals responded with a larger number of USV to both natural and artificial 50 kHz sounds playback rather than to 22 kHz sounds. The emitted USV were, almost exclusively, within the 50 kHz range. Animal HR levels decreased gradually during the experimental session. Single‑housed animals had, in general, higher HR than paired rats. There was an impact of every kind of ultrasonic presentation on HR levels; in general, 50 kHz ultrasonic playback caused a sudden increase in HR, whereas 22 kHz presentations evoked a HR drop. Surprisingly, USV and artificial tones had similar effects on HR and USV responses. Social context did not appear to alter rats’ USV emission. The results following fear conditioning are being analyzed. Also, in a separate set of experiments, rats ultrasonic responses were analyzed following presentation of a defined number of pre‑recorded USV.
3

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

100%
Filipkowski R. K.
Acta Neurobiologiae Experimentalis
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2000
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tom 60
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nr 3
4

Neurodegeneration-associated expression of cathepsin D.

51%
Hetman M., Filipkowski R. K., Danysz W., Kaczmarek L.
Acta Neurobiologiae Experimentalis
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1997
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tom 57
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nr 5
5

Immunolocalization of AP-1 and CRE-DNA binding proteins in the rat brain

51%
Filipkowski R. K., Nishizawa M., Kaczmarek L.
Acta Neurobiologiae Experimentalis
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1995
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tom 55
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nr 5
6

Genetic models to study adult neurogenesis

51%
Filipkowski R. K., Kiryk A., Kowalczyk A., Kaczmarek L.
Acta Biochimica Polonica
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2005
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tom 52
|
nr 2
In the central nervous system (CNS) generation of new neurons continues throughout adulthood, when it is limited to the olfactory bulb and hippocampus. The knowledge regarding the function of newly-generated neurons remains limited and is vigorously investigated using diverse approaches. Among these are genetically modified mice, most of them of knock-out type (KO). Results from 23 diverse KO mouse models demonstrate the importance of particular proteins (growth factors, nitric oxide synthases, receptors, cyclins/cyclin-associated proteins, transcription factors, etc.) in adult neurogenesis (ANGE) as well as separate it from developmental neurogenesis. These results bring us closer to revealing the function of newly generated neurons in adult brains.
7

Fear conditioning affects reaction to ultrasonic signals in SHR and wistar rats

45%
Wardak A., Polowy R., Grymanowska A., Filipkowski R. K., Olszynski K. H.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
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nr Suppl.1
INTRODUCTION: Ultrasonic vocalizations (USV) are means of communication between rats. We are studying them by presenting USV or artificial tones from a speaker (playback experiments) and observing vocal (rat’s own USV), behavioral, and cardiovascular (heart rate, HR) reactions. We used Wistar rats, which are common in USV experiments, and SHR (spontaneously hypertensive rats), whose USV habits have not been investigated. AIM(S): We are especially interested in the role of the autonomic nervous system. Therefore, we are investigating the impact of fear conditioning which affects autonomic balance in Wistar rats and SHR with higher activity of the sympathetic system. METHOD(S): Three different protocols were used (1x, 6x, or 10x; 1 mA, 1 s shocks) and later, the animals were presented with 50 kHz (appetitive) or 22 kHz (aversive) USV. On the day of conditioning, Wistar rats emitted 22 kHz USV immediately after the first electric impulse, while SHR remained silent typically to the sixth-eight shock. Levels of freezing were similar in both strains. On the following day, during ultrasonic signals presentation, after 50 kHz USV playback, SHR did not show a rise in HR nor an increase in their own USV emission, which were both observed in Wistar rats. Both strains responded to 22 kHz USV by a decrease in HR, independently of fear protocol. During the conditioning test, the day after playback experiments, Wistar rats showed lower HR following 1x conditioning. Also, a dramatic rise in numbers of USV was observed in some of 6x animals. Only the HR of 1x conditioned Wistar rats was lower than in the control (not conditioned) group (HR of 6x and 10x Wistar rats did not differ from control group), while in SHR, all conditioned groups tended to have higher HR than controls. CONCLUSIONS: We confirm that fear conditioning affects the reaction to ultrasonic signals in SHR and Wistar rats. Presumably the autonomic nervous system participates in reactions to USV playback; however, further research with pharmacological agents is essential. To our best knowledge, these are the first studies about USV in SHR. FINANCIAL SUPPORT: This work was supported by National Science Centre, Poland, grant no. 2015/19/B/ NZ4/03393.
8

Defensive conditioning-related functional geterogeneity among nuclei of rat amygdala

45%
Savonenko A., Filipkowski R. K., Werka T., Zielinski K., Kaczmarek L.
Acta Neurobiologiae Experimentalis
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1999
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tom 59
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nr 3
9

Mechanism of adult neurogenesis in mice lacking cyclin D2

45%
Kowalczyk A., Wielkopolska E., Filipkowski R. K., Rylski M., Kaczmarek L.
Acta Neurobiologiae Experimentalis
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2005
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tom 65
|
nr 3
10

Ultrasonic and cardiovascular responses to ultrasonic sounds and vocalizations

45%
Filipkowski R. K., Polowy R., Malz M., Gawrys O., Olszynski K. H.
Acta Neurobiologiae Experimentalis
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2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Ultrasonic vocalizations (USV) of adult rats are thought to be means of social communication and are divided into two categories, 55 kHz and 22 kHz, signaling, respectively, appetitive and aversive states. These states are also known for changes in the heart rate (HR). The autonomous system has a role in both USV shaping and HR response. A common signaling pathway via the vagus nerve connects the laryngeal muscles and the heart. This causes an overlap in HR parameters and many behavioral reactions (the polivagal theory). AIM(S): The aim of this study was to investigate USV emissions and HR changes in rats evoked by USV presentation. METHOD(S): Ten weeks old Wistar male rats were housed in pairs or separately for 4 weeks. Telemetry transmitters for HR acquisition were implanted in the peritoneum with the detector placed in the aorta. Rats were exposed to five 10‑s sets of sounds (counterbalanced): 55-kHz, 22-kHz USV (both natural, collected from other animals), 55-kHz, 22-kHz tones, 22-kHz uninterrupted tone (all three artificial, software‑generated) separated with 5 min silence intervals. HR and USV emitted were registered. RESULTS: Rats of both groups responded with USV mostly and more often to 55-kHz tones and vocalizations than during presentation of 22-kHz sounds. The responses were, almost exclusively, within 55 kHz range. In general, single-housed rats vocalized more often than pair-housed ones but the effect was not strong. Also, HR changes were more pronounced following presentation of natural USV. During 55-kHz USV presentation, there was an elevation of HR in single-housed animals, while in pair-housed animals, this elevation was preceded by a transient HR drop. During 22-kHz USV presentation, a decrease in HR in both groups was observed, although it was more clear in paired-housed rats. CONCLUSIONS: Social context may have an impact on HR levels and USV emissions in response to ultrasounds presentations. However, it does not seem to influence the distinction between artificial and natural USV. FINANCIAL SUPPORT: The work was supported by National Science Centre, Poland, grant no. 2015/19/B/ NZ4/03393.
11

Nuclear betta-catenin is constitutively present in thalamic neurons

45%
Misztal K., Wisniewska M., Klejman M., Filipkowski R. K., Kuznicki L.
Acta Neurobiologiae Experimentalis
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2009
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tom 69
|
nr 3
Wnt signaling is one of the most important mechanisms engaged in embryonic development. In the adult organism it is present in regenerating tissues. The key mediator in the signaling is β-catenin, which translocates to the nucleus and triggers transcription of the Lef1/Tcf target genes. Recently, expression of some of the Wnt pathway components has been observed in the thalamus of the adult brain. We show that β-catenin and Lef1 are constitutively present in nuclei of thalamic neurons in vivo. We also demonstrate that β-catenin accumulates in about 40% of thalamic cells in vitro, without any prior stimulation. This phenomenon does not depend on soluble factors produced by glia cells or cortical neurons, because nor glia cells co-culture neither cortical conditioned medium affects the number of β-catenin positive cells. These observations support a new idea that Wnt/β-catenin signaling is an intrinsic feature of thalamic neurons, independent on thalamo-cortical interaction what was proposed before. Whether the nuclear localization of β-catenin in thalamic neurons is a consequence of autocrine Wnt stimulation or a specifi c regulation inside the cells remains to be determined.
12

C-Fos expression in mouse brain after fear conditioning; effect of fyn mutation

45%
Filipkowski R. K., Kogan J., Frankland P., Silva A. J., Kaczmarek L.
Acta Neurobiologiae Experimentalis
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1997
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tom 57
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nr 5
13

Gene expression in neuronal apoptosis - looking for a therapeutic window

45%
Figiel I., Filipkowski R. K., Hetman M., Kaminska B., Kaczmarek L.
Biotechnologia
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1996
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nr 4
14

Glutamate, glutamine and GABA levels in the rat hippocampus in the pharmacological models of autism: a microdialysis study

39%
Diamandakis D., Zieminska E., Hilgier W., Filipkowski R. K., Polowy R., Toczylowska B., Lazarewicz J. W.
Acta Neurobiologiae Experimentalis
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2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: The disorders of the glutamatergic neurotransmission have been implicated in the pathogenesis of autism, but data on brain content of glutamate (Glu) in patients and animal models are inconsistent. AIM(S): Aim of this study is to evaluate changes in the brain content of Glu, glutamine (Gln) and GABA in the rat models of pharmacologically-induced autism. METHOD(S): The rat females at the 11th day of gestation were given orally 800 mg/kg b.w. of valproic acid (VPA) or 500 mg/kg b.w. of thalidomide (THAL). The pups at PND 9 were submitted to ultrasonic vocalization (USV) test, and at PND 30, under anesthesia, to in vivo unilateral microdi alysis of the hippocampus with a calcium-containing medium. The samples of dialysate representing the basal level followed by a 40 min pulse of 100 mM KCl were collected. The contralateral hippocampi were prepared and homogenized. After derivatization of the amino acids with o-phtalaldehyde, the samples were submitted to HPLC analysis with a fluorescence detection. RESULTS: The results of USV tests showed that the pups prenatally treated with VPA, and to a greater extent with THAL, less frequently produced USV calls, which is regarded as impairment in social communication, a symptom characteristic of autism. In the male rats of the VPA and THAL groups, a total content of Glu increased to 143% and 158%, respectively, and also Gln and GABA contents were significantly elevated. All these values remained unchanged in the female rats. Basal levels of Glu, Gln and GABA in the dialysates of the hippocampi in the experimental groups did not differ from controls, however in VPA‑treated male rats during application of 100 mM KCl a reduction by 59% of Gln concentration and tendency to increase GABA level were found. CONCLUSIONS: The results demonstrate increased content of glutamate in the hippocampus of rats in two chemical models of autism, support a hypothesis on the role of the glutamatergic disturbances in the pathogenesis of autism. FINANCIAL SUPPORT: This study was supported by the Polish National Science Centre, grant no. 2014/15/B/ NZ4/04490.
15

Looking for beta-catenin target genes in mature neurons

39%
Wisniewska M. B., Klejman M., Michowski W., Dabrowski M., Filipkowski R. K., Kuznicki J.
Acta Neurobiologiae Experimentalis
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2007
|
tom 67
|
nr 3
16

Are early immediate genes activated in learninginduced plasticity?

39%
Radwanska A., Filipkowski R. K., Konopacki F. A., Wilczynski G. M., Zakrzewska R., Kossut M.
Acta Neurobiologiae Experimentalis
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2007
|
tom 67
|
nr 3
17

Alteration of mTOR signalling pathway in rats prenatally exposed to valproic acid

39%
Adamczyk A., Cieslik M., Gassowska-Dobrowolska M., Jesko H., Filipkowski R. K., Boguszewski P. M.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Autism is a neurodevelopmental disorder characterised by impaired social interaction, deficits in communication and stereotyped behaviours with synaptic dysfunction suggested as the major causative factor. However, the molecular mechanisms of synapses impairment remain unclear. The most recent studies point to mTOR, a regulator of protein synthesis at spines, as a potential molecular basis of autism. AIM(S): Here, we investigated whether the Akt/mTOR pathway is damaged in rats prenatally exposed to valproic acid (VPA), an animal model exhibiting autistic-like behaviour. METHOD(S): Pregnant Wistar rats were injected i.p. with a single dose of 400 mg/kg VPA on embryonic day 12.5. Autism‑like behaviours were verified by measuring ultrasonic vocalizations and elevated plus maze test. Gene expression and protein levels were analysed using real-time PCR and western blot methods, respectively. RESULTS: Our behavioural investigations have shown impaired communication and increased anxiety in VPA group. Along with the behavioural changes we observed alteration of mTOR signalling in the cerebral cortex, hippocampus and cerebellum of autistic model rats. Enhancement of phospho-mTOR protein level was the most pronounced in the hippocampus, where the phosphorylation of mTOR targets was observed: increased p-4E-BP1, and reduced phospho-p70S6K. These changes were accompanied by an increase in p-Akt protein level. Activation of mTOR in the cerebellum caused an increase of p-4E-BP1, but not of p70S6K. The mild but significant rise in phosphorylated mTOR in the cerebral cortex did not lead to any changes in p70 or 4E-BP1 phosphorylation. Synaptosomes isolated from VPA subjects revealed significant abnormalities in their ultrastructure including unidentified electron-dense matrix material as well as fragile and malformed the post-synaptic densities. CONCLUSIONS: These results suggested that altered signalling via Akt/mTOR/p70S6K/p-4E-BP1 may result in disturbed spine protein synthesis and thereby lead to synaptic dysfunction. FINANCIAL SUPPORT: Supported from MMRC statutory theme 8.
18

Glutamate, glutamine and GABA levels in the rat hippocampus in the pharmacological models of autism: MRS and NMR study

39%
Zieminska E., Filipkowski R. K., Polowy R., Orzel J., Toczylowska B., Gorka M. J., Lazarewicz J. W.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: An imbalance in excitatory/inhibitory neurotransmission has been implicated in the pathogenesis of autism. AIM(S): We tested this hypothesis by measuring with Magnetic Resonance Spectroscopy (MRS) and Nuclear Magnetic Resonance (NMR) the content of glutamate (Glu), glutamine (Gln) and GABA in the rat hippocampus in two pharmacological models of autism. METHOD(S): The rat females at the 11th day of gestation were given orally 800 mg/kg of valproic acid (VPA) or 500 mg/kg of thalidomide (THAL). The pups at PND 9 were submitted to ultrasonic vocalization (USV) test, and at PND 30, to MRS studies using the 7 T Bruker BioSpec 70/30 Avance III system. Spectrum was acquired with the short echo time PRESS sequence (TR/TE=2500/20 ms, 512 averages, 2048 points, scan time=17 min) with VAPOR water suppression, the outer volume suppression, frequency drift correction (flip angle 5°) and eddy current correction. Metabolite concentrations were estimated using the LCModel software. The amino acids from homogenates of rat hippocampi were extracted for NMR studies using the HCl-Bligh and Dyer procedure. Three-trimethylsilyl propionic acid (1 mM) was used as an internal reference signal. All NMR spectra were acquired at 25°C on a Avance III HD 500 MHz (Bruker) spectrometer. RESULTS: The results of USV tests showed that the “autistic pups” produced less calls/animal (VPA-122, THAL-33) as compared to control animals (295). MRS studies demonstrated increase by 21% and 20% in Glu content in the hippocampus of male rats from both, VPA- and THAL-treated groups, whereas Gln and GABA were on the control levels. NMR studies showed gender-dependent differences in Glu content in VPA-group (by 36%) and THAL‑group vs. control (by 16%); increased level of Gln in males from both groups (by 47% and 74%) and increased (by 86%) level of GABA in male VPA‑treated rats. CONCLUSIONS: These results are consistent with a hypothesis on the role of the imbalance in glutamatergic vs. GABAergic neurotransmission in the pathogenesis of autism. FINANCIAL SUPPORT: This study was supported by the Polish National Science Centre, grant no. 2014/15/B/ NZ4/04490.
19

Irregular nuclear shapes of neural stem cells in adult murine dentate gyrus

39%
Grymanowska A. W., Martin A., Olszynski K. H., Wilczynski G. M., Magalska A., Filipkowski R. K.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: In adult hippocampal neurogenesis, stem cells and their derivative progenitors are generated. They differentiate into neurons as they migrate from the subgranular zone to the granule cell layer of the dentate gyrus, maintaining homeostatic tissue regeneration and supporting brain plasticity. Depending on the stage of the neurogenesis, different subpopulations of cells of the neurogenic lineage can be distinguished, i.e. neural stem cells (NSC, type 1), intermediate progenitor cells (type 2a and type 2b), neuroblasts (type 3) and granule neurons (type 4). Little is known about the architecture of nuclei in these cells, while the cell nucleus is known to be highly organized with numerous functional and structural domains as well as dynamic organization of chromatin governed by epigenetic mechanisms which were shown to respond to external signals. AIM(S): We aimed to distinguish type 1 through 4 cells and investigate their nuclear shape. METHOD(S): Transgenic Nestin-GFP mice were used. Cell types were identified with immunohistochemistry and morphological features: type 1 (GFP+, one long neural process), type 2a (GFP+), type 2b (GFP+/DCX+), type 3 (DCX+), type 4 (NeuN+). Confocal microscopy was used to collect z‑stack files of the nuclei of different cell types. RESULTS: We observed irregularity in shape of the nuclei in type 1 cells (NSC) with the presence of nuclear envelope invaginations. When selected layers were analyzed, NSC nuclei turned out to have reduced circularity, roundness and solidity when compared with other cell types. CONCLUSIONS: The irregularity observed and nuclear envelope invaginations seem to be characteristics of the “stemness” as the shape of the nucleus becomes more regular with successive stages of neurogenesis. The biological significance of the observed phenomenon is not yet clear and further studies are necessary to better understand the process of adult neurogenesis at the nuclear level. FINANCIAL SUPPORT: The work was supported by National Science Centre, Poland, grant no. 2014/14/M/NZ4/00561.
20

Maternal immune activation alters synaptic protein expression and causes social interaction impairment in rat offspring

39%
Cieslik M., Gassowska-Dobrowolska M., Jesko H., Boguszewski P. M., Filipkowski R. K., Adamczyk A.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Emerging epidemiology data indicate that maternal immune activation (MIA) resulting from inflammatory stimuli such as bacterial infections during pregnancy may constitute a risk factor for multiple neurodevelopmental diseases including autism spectrum disorders (ASD). Genetic and environmental variation, inflammation during early development, and their interaction can influence synaptic dysfunction in ASD. However, the molecular links between infection-induced fetal development alterations and the risk of ASD are still unclear. AIM(S): The aim of this study was to investigate the effect of MIA on the expression and protein level of key synaptic proteins along with the autism-associated behavior in male rat offspring. METHOD(S): Pregnant Wistar rat dams were injected intraperitoneally (i.p.) at gestational day 9.5 with 0.1 mg/kg lipopolysaccharide (LPS), which induces immune response similar to that against gram-negative bacteria. RESULTS: Our data shown impaired social interaction, tested by the play behaviors (Tickling test on post-natal day PND 45–50). However, we did not observe any changes in ultrasonic vocalization (9–11 PND) and bedding preference (PND 15) in MIA offspring. Along with the social interaction changes, MIA has induced presynaptic protein alterations in adolescent rat offspring. These alterations included decreased level of synaptobrevin and syntaxin-1, the key components of SNARE complex, as well as higher level of synapsin. Together with changes in presynaptic proteins, MIA induced reduction in PSD-95 and down-regulation of SHANK family proteins. Moreover, alteration in the protein level of phospho-Akt, and 4E-BP1 was found in MIA subjects. CONCLUSIONS: It is possible that variations of Akt/ mTOR pathway are responsible for aberrant synthesis of key synaptic proteins. The altered synthesis of these proteins would generate changes in synaptic structure and function, contributing to ASD-like behaviors. FINANCIAL SUPPORT: Supported by the statutory theme 8.
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