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1

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

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Wardak A., Polowy R., Grymanowska A., Filipkowski R. K., Olszynski K. H.
Acta Neurobiologiae Experimentalis
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2019
|
tom 79
|
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.
2

Ultrasonic and cardiovascular responses to ultrasonic sounds and vocalizations

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Filipkowski R. K., Polowy R., Malz M., Gawrys O., Olszynski K. H.
Acta Neurobiologiae Experimentalis
|
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.
3

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

86%
Diamandakis D., Zieminska E., Hilgier W., Filipkowski R. K., Polowy R., Toczylowska B., Lazarewicz J. W.
Acta Neurobiologiae Experimentalis
|
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.
4

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

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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.
5

Stimulus‑seeking in rats is accompanied by increased c‑Fos expression in hippocampal CA1 as well as short 22 kHz and flat 50 kHz calls

72%
Robakiewicz I., Polak M., Rawska M., Alberski D., Polowy R., Wytrychiewicz K., Syperek M., Matysiak J., Filipkowski R. K.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
|
nr 3
We determined CA1 hippocampal field to be involved in self‑exposure, a type of novelty‑seeking behaviour that has also been associated with short 22 kHz and flat 50 kHz ultrasonic vocalizations (USV) in adult male Long‑Evans rats. Rats were habituated for three days to a self‑exposure cage with two nose‑poke holes. On day four, the animals from the experimental group were allowed to turn the cage light off for 5 s with a nose‑poke (test/self‑exposure session), while rats from control‑yoked group had changing light conditions coupled and identical to the experimental animals. The experimental rats performed more nose‑pokes during self‑exposure session than animals from the control group. This effect was accompanied by a higher density of c‑Fos‑positive nuclei in the hippocampal CA1. There were no significant group differences in c‑Fos expression in other brain regions analysed. However, possible involvement of several other structures in self‑exposure (i.e., CA3, the dentate gyrus, amygdala, prefrontal cortex, and nucleus accumbens) is also discussed, as their correlational activity, reflected by c‑Fos immunoactivity, was observed in the experimental rats. During test sessions, there were more nose‑pokes accompanied by short 22 kHz calls and 50 kHz calls performed by the rats of the experimental group than of the control group. The CA1 region has previously been associated with novelty; short 22 kHz USV and flat 50 kHz USV could be associated with self‑exposure, also they appear to be emitted correlatively.
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