Effects of 5-HT1A Receptor Overexpression in the Frontal Cortex on Autism-Like Behavior and the Expression of 5-HT1A, 5-HT7 Receptors and BDNF in BTBR Mice

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Abstract

Autism spectrum disorders (ASD) are the most common neurodevelopmental disorders, however, their mechanisms are still poorly understood. Serotonin (5-HT) and brain-derived neurotrophic factor (BDNF) are known as key players in the regulation of brain plasticity and behavior. Among the variety of 5-HT receptors, the most interesting is the 5-HT1A receptor, which is the main regulator of the brain 5-HT system functioning. In this work, we investigated the effect of 5-HT1A receptor overexpression in the frontal cortex induced by the administration of the adeno-associated virus pAAV-Syn-HTR1A-eGFP to BTBR T+ Itpr3tf/J (BTBR) mice, a model of autism, on autism-like behavior and on the expression of the Htr1a gene transcription factor – Freud-1 (encoded by the Cc2d1a gene), its intracellular signal transducer ERK1/2 (encoded by the Mapk3 gene), 5-HT₇ receptors, mature BDNF, proBDNF and TrkB and p75NTR receptors. Overexpression of the 5-HT1A receptor had no effect on time in the center and locomotor activity in the open field test, social behavior in the three-chamber test, immobility time in the tail suspension test, and associative learning in the “operant wall” paradigm, but it enhanced the severity of stereotyped behavior in the marble burying test. 5-HT1A receptor overexpression in the frontal cortex did not affect the mRNA and protein levels of 5-HT₇ receptors, mature BDNF, proBDNF and TrkB and p75NTR receptors in the cortex and hippocampus of BTBR mice. However, overexpression caused an increase in the protein level of the transcription factor Freud-1 in the hippocampus without changing the mRNA level of Cc2d1a in the frontal cortex and hippocampus. No changes in the pERK/ERK ratio were found in both investigated brain structures. Thus, the results of this study indicate a possible disruption in interactions of: 5-HT1A receptors with downstream intracellular signal transducers; 5-HT system, BDNF and TrkB receptors; and 5-HT1A and 5-HT₇ receptors in the frontal cortex of BTBR mice.

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About the authors

E. M. Kondaurova

Institute of Cytology and Genetics, Siberian Branch of RAS

Author for correspondence.
Email: chudabest@gmail.com
Russian Federation, Novosibirsk

Yu. D. Grigorieva

Institute of Cytology and Genetics, Siberian Branch of RAS

Email: chudabest@gmail.com
Russian Federation, Novosibirsk

I. I. Belokopytova

Institute of Cytology and Genetics, Siberian Branch of RAS

Email: chudabest@gmail.com
Russian Federation, Novosibirsk

E. A. Kulikova

Institute of Cytology and Genetics, Siberian Branch of RAS

Email: chudabest@gmail.com
Russian Federation, Novosibirsk

A. S. Tsybko

Institute of Cytology and Genetics, Siberian Branch of RAS

Email: chudabest@gmail.com
Russian Federation, Novosibirsk

N. V. Khotskin

Institute of Cytology and Genetics, Siberian Branch of RAS

Email: chudabest@gmail.com
Russian Federation, Novosibirsk

T. V. Ilchibaeva

Institute of Cytology and Genetics, Siberian Branch of RAS

Email: chudabest@gmail.com
Russian Federation, Novosibirsk

N. K. Popova

Institute of Cytology and Genetics, Siberian Branch of RAS

Email: chudabest@gmail.com
Russian Federation, Novosibirsk

V. S. Naumenko

Institute of Cytology and Genetics, Siberian Branch of RAS

Email: chudabest@gmail.com
Russian Federation, Novosibirsk

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2. Fig. 1. (a) Maps of AAV-Syn-HTR1A-eGFP and AAV-Syn-eGFP plasmids. ITR, inverted terminal repeat; EGFP, green fluorescent protein gene; Syn, synapsin promoter; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; hGH polyA, human growth hormone polyadenylation signal. (b) Photomicrograph of brain sections after injection of AAV-Syn-HTR1A-eGFP. Cell nuclei were stained with bis-benzimide solution (Hoechst 33258, 5 μg/ml in PBS, Sigma-Aldrich, Burlington, MA, USA). Scale bar, 100 μm.

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3. Fig. 2. Experimental scheme.

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4. Fig. 3. Expression of 5-HT1A receptors. Levels of Htr1a gene mRNA (a) and 5-HT1A protein (b) in the frontal cortex and hippocampus of BTBR mice. Gene expression is presented as the number of cDNA copies of the corresponding gene per 100 copies of Polr2a cDNA. The protein level was estimated as relative chemiluminescence units and normalized to relative chemiluminescence units of GAPDH. *p < 0.05, ***p < 0.001.

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5. Fig. 4. Results of behavioral testing: a – number of buried balls in the “ball burying” test; b – sniffing time in the three-chamber test; c – time of immobility in the “tail suspension” test; d – distance traveled and d – time spent in the center in the “open field” test. *p < 0.05.

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6. Fig. 5. Dynamics of behavioral changes in BTBR mice in a home cage over four days: motor activity (a), water (b) and food (c) consumption levels. ***p < 0.001, **p < 0.005 compared to the first day.

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7. Fig. 6. The number of pellets received (a) and the number of nose pokes in the “operant wall” test (b).

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8. Fig. 7. Expression of 5-HT7 receptors. Levels of Htr7 gene mRNA (a) and 5-HT7 protein (b) in the frontal cortex and hippocampus of BTBR mice. Gene expression is presented as the number of cDNA copies of the corresponding gene per 100 copies of Polr2a cDNA. The protein level was estimated in relative chemiluminescence units and normalized to the relative chemiluminescence units of GAPDH.

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9. Fig. 8. Levels of Bdnf gene mRNA (a) and BDNF proteins (b) and its precursor proBDNF (c) in the frontal cortex and hippocampus of BTBR mice. Gene expression is presented as the number of cDNA copies of the corresponding gene per 100 copies of Polr2a cDNA. Protein levels were estimated in relative chemiluminescence units and normalized to relative chemiluminescence units of GAPDH.

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10. Fig. 9. Expression of BDNF receptors. Levels of Ntrk2 gene mRNA (a) and TrkB protein (b), levels of Ngfr gene mRNA (c) and p75NTR protein (d) in the frontal cortex and hippocampus of BTBR mice. Gene expression is presented as the number of cDNA copies of the corresponding gene per 100 copies of Polr2a cDNA. Protein levels were estimated in relative chemiluminescence units and normalized to relative chemiluminescence units of GAPDH.

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11. Fig. 10. Expression of the transcription factor Freud-1. Levels of Cc2d1a gene mRNA (a) and Freud-1 protein (b) in the frontal cortex and hippocampus of BTBR mice. Gene expression is presented as the number of cDNA copies of the corresponding gene per 100 copies of Polr2a cDNA. The protein level was estimated in relative chemiluminescence units and normalized to the relative chemiluminescence units of GAPDH. *** p < 0.001.

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12. Fig. 11. Expression of the intracellular mediator ERK1/2. The level of Mapk3 gene mRNA (a) and the ratio of ERK and pERK proteins (b) in the frontal cortex and hippocampus of BTBR mice. Gene expression is presented as the ratio of Mapk3 expression levels to Polr2a expression levels. The protein level was estimated in relative chemiluminescence units and normalized to the relative chemiluminescence units of GAPDH.

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