Amygdalar neurotransmission alterations in the BTBR mice model of idiopathic autism

Abstract

Autism Spectrum Disorders (ASD) are principally diagnosed by three core behavioural symptoms, such as stereotyped repertoire, communication impairments and social dysfunctions. This complex pathology has been linked to abnormalities of corticostriatal and limbic circuits. Despite experimental efforts in elucidating the molecular mechanisms behind these abnormalities, a clear etiopathogenic hypothesis is still lacking. To this aim, preclinical studies can be really helpful to longitudinally study behavioural alterations resembling human symptoms and to investigate the underlying neurobiological correlates. In this regard, the BTBR T⁺ Itpr3^tf/J (BTBR) mice are an inbred mouse strain that exhibits a pattern of behaviours well resembling human ASD-like behavioural features. In this study, the BTBR mice model was used to investigate neurochemical and biomolecular alterations, regarding Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF), together with GABAergic, glutamatergic, cholinergic, dopaminergic and noradrenergic neurotransmissions and their metabolites in four different brain areas, i.e. prefrontal cortex, hippocampus, amygdala and hypothalamus. In our results, BTBR strain reported decreased noradrenaline, acetylcholine and GABA levels in prefrontal cortex, while hippocampal measurements showed reduced NGF and BDNF expression levels, together with GABA levels. Concerning hypothalamus, no differences were retrieved. As regarding amygdala, we found reduced dopamine levels, accompanied by increased dopamine metabolites in BTBR mice, together with decreased acetylcholine, NGF and GABA levels and enhanced glutamate content. Taken together, our data showed that the BTBR ASD model, beyond its face validity, is a useful tool to untangle neurotransmission alterations that could be underpinned to the heterogeneous ASD-like behaviours, highlighting the crucial role played by amygdala.

Fig. 1. NA, DA, DOPAC, HV, Ach, Glutamate, GABA, NGF and BDNF levels in PFC of BTBR (black bar) and BL6 (white bar) mice.

A NA levels (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, ***P = 0.0008 BTBR vs. BL6; B DA amount (fmol/mg) in the PFC of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; C DOPAC levels (fmol/mg) in the PFC of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; D HVA levels (fmol/mg) in the PFC of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; E Ach levels (uM) in the PFC of BL6 (n = 5) and BTBR (n = 4) mice. Unpaired Student’s t-test, *P = 0.0176 BTBR vs. BL6; F Glutamate levels (uM/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; G GABA levels (uM/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, *P = 0.0290 BTBR vs. BL6; H NGF expression levels of BTBR (n = 5) and BL6 (n = 4). Unpaired Student’s t-test, n.s. Quantification of the optical band density of NGF normalized for optical band density of β-actin housekeeping gene; I BDNF expression levels of BTBR (n = 5) and BL6 (n = 4). Unpaired Student’s t-test, n.s. Quantification of the optical band density of BDNF normalized for optical band density of β-actin housekeeping gene.

Fig. 2. NA, DA, DOPAC, HV, Ach, Glutamate, GABA, NGF and BDNF levels in HIPP of BTBR (black bar) and BL6 (white bar) mice.

A NA amount (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; B DA levels (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; C DOPAC amount (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, *P = 0.0323 BTBR vs. BL6; D HVA amount (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; E Ach levels (uM) of BL6 (n = 5) and BTBR (n = 4) mice. Unpaired Student’s t-test, n.s.; F Glutamate levels (uM/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; G GABA levels (uM/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, *P = 0.0221 BTBR vs. BL6; H NGF expression levels of BTBR (n = 5) and BL6 (n = 4). Unpaired Student’s t-test, *P = 0.0361 BTBR vs. BL6. Quantification of the optical band density of NGF normalized for optical band density of β-actin housekeeping gene; I BDNF expression levels of BTBR (n = 5) and BL6 (n = 4). Unpaired Student’s t-test, *P = 0.0352 BTBR vs. BL6. Quantification of the optical band density of BDNF normalized for optical band density of β-actin housekeeping gene.

Fig. 3. NA, DA, DOPAC, HV, Ach, Glutamate, GABA, NGF and BDNF levels in HYP of BTBR (black bar) and BL6 (white bar) mice.

A NA amount (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; B DA levels (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; C DOPAC amount (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; D HVA amount (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; E Ach levels (uM) of BL6 (n = 5) and BTBR (n = 4) mice. Unpaired Student’s t-test, n.s.; F Glutamate levels (uM/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; G GABA levels (uM/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, n.s.; H NGF expression levels of BTBR (n = 5) and BL6 (n = 4). Unpaired Student’s t-test, n.s. Quantification of the optical band density of NGF normalized for optical band density of β-actin housekeeping gene; I BDNF expression levels of BTBR (n = 5) and BL6 (n = 4). Unpaired Student’s t-test, n.s. Quantification of the optical band density of BDNF normalized for optical band density of β-actin housekeeping gene.

Fig. 4. NA, DA, DOPAC, HV, Ach, Glutamate, GABA, NGF and BDNF levels in AMY of BTBR (black bar) and BL6 (white bar) mice.

A NA levels (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, *P = 0.0221 BTBR vs. BL6; B DA levels (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, *P = 0.0266 BTBR vs. BL6; C DOPAC levels (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, **P = 0.0100 BTBR vs. BL6; D HVA levels (fmol/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, *P = 0.0469 BTBR vs. BL6; E Ach levels (uM) of BL6 (n = 5) and BTBR (n = 4) mice. Unpaired Student’s t-test, *P = 0.0286 BTBR vs. BL6; F Glutamate levels (uM/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, *P = 0.0366 BTBR vs. BL6; G GABA levels (uM/mg) of BL6 (n = 5) and BTBR (n = 5) mice. Unpaired Student’s t-test, *P = 0.0244 BTBR vs. BL6; H NGF expression levels of BTBR (n = 5) and BL6 (n = 4). Unpaired Student’s t-test, *P = 0.0167 BTBR vs. BL6. Quantification of the optical band density of NGF normalized for optical band density of β-actin housekeeping gene; I) BDNF expression levels of BTBR (n = 5) and BL6 (n = 4). Unpaired Student’s t-test, n.s. Quantification of the optical band density of BDNF normalized for optical band density of β-actin housekeeping gene.

Fig. 5. Correlation between repetitive behaviours and hyperlocomotion with amygdalar NA levels and between non social behaviours and amygdalar GABA amount in BTBR and BL6 mice.

A Correlation between poking holes (nr) and amygdalar NA (fmol/mg of tissue). Pearson correlation, r = −0.8857; **P = 0.0015; B Correlation between distance travelled (mt) and amygdalar NA (fmol/mg of tissue). Pearson correlation, r = −0.8608; **P = 0.0029; C Correlation between non social behaviours frequency (nr) and amygdalar GABA (uM/mg of tissue). Pearson correlation, r = −0.7203; *P = 0.0188.