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Dysfunctional dopaminergic neurotransmission in asocial BTBR mice.

Squillace M, Dodero L, Federici M, Migliarini S, Errico F, Napolitano F, Krashia P, Di Maio A, Galbusera A, Bifone A, Scattoni ML, Pasqualetti M, Mercuri NB, Usiello A, Gozzi A - Transl Psychiatry (2014)

Bottom Line: Recent psychosocial and neuroimaging studies have highlighted reward-processing deficits and reduced dopamine (DA) mesolimbic circuit reactivity in ASD patients.However, the neurobiological and molecular determinants of these deficits remain undetermined.DA D1 receptor-dependent behavioural and signalling responses were found to be unaltered in BTBR mice, whereas dramatic reductions in pre- and postsynaptic DA D2 and adenosine A2A receptor function was observed in these animals.

View Article: PubMed Central - PubMed

Affiliation: Ceinge Biotecnologie Avanzate, Naples, Italy.

ABSTRACT
Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by pronounced social and communication deficits and stereotyped behaviours. Recent psychosocial and neuroimaging studies have highlighted reward-processing deficits and reduced dopamine (DA) mesolimbic circuit reactivity in ASD patients. However, the neurobiological and molecular determinants of these deficits remain undetermined. Mouse models recapitulating ASD-like phenotypes could help generate hypotheses about the origin and neurophysiological underpinnings of clinically relevant traits. Here we used functional magnetic resonance imaging (fMRI), behavioural and molecular readouts to probe dopamine neurotransmission responsivity in BTBR T(+) Itpr3(tf)/J mice (BTBR), an inbred mouse line widely used to model ASD-like symptoms owing to its robust social and communication deficits, and high level of repetitive stereotyped behaviours. C57BL/6J (B6) mice were used as normosocial reference comparators. DA reuptake inhibition with GBR 12909 produced significant striatal DA release in both strains, but failed to elicit fMRI activation in widespread forebrain areas of BTBR mice, including mesolimbic reward and striatal terminals. In addition, BTBR mice exhibited no appreciable motor responses to GBR 12909. DA D1 receptor-dependent behavioural and signalling responses were found to be unaltered in BTBR mice, whereas dramatic reductions in pre- and postsynaptic DA D2 and adenosine A2A receptor function was observed in these animals. Overall these results document profoundly compromised DA D2-mediated neurotransmission in BTBR mice, a finding that is likely to have a role in the distinctive social and behavioural deficits exhibited by these mice. Our results call for a deeper investigation of the role of dopaminergic dysfunction in mouse lines exhibiting ASD-like phenotypes, and possibly in ASD patient populations.

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Impaired Drd2-mediated postsynaptic reactivity and signalling in BTBR mice. Representative coronal sections showing Drd2 mRNA expression in the striatum of B6 (a) and BTBR mice (b). The histogram (c) illustrates Drd2 mRNA levels in BTBR (n=7) after densitometric quantification of autoradiograms. Values are expressed as percentage variation with respect to B6 control mice (n=4). (d) Cataleptic response induced by 0.5 mg kg−1 of haloperidol in B6 (n=4; vehicle, n=4) and BTBR animals (0.5 mg kg−1, n=6; vehicle, n=4). Striatal PKA-dependent P-Ser845-GluR1 (e) and P-Ser235/236-S6 (f) protein levels in B6 and BTBR mice treated with 0.5 mg kg−1 haloperidol (P-Ser845-GluR1: B6, n=5; BTBR, n=6; P-Ser235/236-S6: n=6 each strain) or vehicle (P-Ser845-GluR1: B6, n=5; BTBR, n=4; P-Ser235/236-S6: n=6 each strain). The top panels in e and f show representative blots comparing the different strains and treatment groups. *P<0.05, **P<0.01, ***P<0.0001, compared with vehicle-treated group within strain (one-way analysis of variance). All values are expressed as mean±s.e.m. PKA, protein kinase A.
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fig5: Impaired Drd2-mediated postsynaptic reactivity and signalling in BTBR mice. Representative coronal sections showing Drd2 mRNA expression in the striatum of B6 (a) and BTBR mice (b). The histogram (c) illustrates Drd2 mRNA levels in BTBR (n=7) after densitometric quantification of autoradiograms. Values are expressed as percentage variation with respect to B6 control mice (n=4). (d) Cataleptic response induced by 0.5 mg kg−1 of haloperidol in B6 (n=4; vehicle, n=4) and BTBR animals (0.5 mg kg−1, n=6; vehicle, n=4). Striatal PKA-dependent P-Ser845-GluR1 (e) and P-Ser235/236-S6 (f) protein levels in B6 and BTBR mice treated with 0.5 mg kg−1 haloperidol (P-Ser845-GluR1: B6, n=5; BTBR, n=6; P-Ser235/236-S6: n=6 each strain) or vehicle (P-Ser845-GluR1: B6, n=5; BTBR, n=4; P-Ser235/236-S6: n=6 each strain). The top panels in e and f show representative blots comparing the different strains and treatment groups. *P<0.05, **P<0.01, ***P<0.0001, compared with vehicle-treated group within strain (one-way analysis of variance). All values are expressed as mean±s.e.m. PKA, protein kinase A.

Mentions: We next investigated postsynaptic Drd2 mRNA expression and function in BTBR and B6 mice. Consistent with previous measurements in the substantia nigra, Drd2 mRNA levels in the striatum and nucleus accumbens appeared comparable in the two strains (P=0.89, Student's t-test; Figure 5a, b and c). To probe the functional responsivity of postsynaptic Drd2 receptors, we measured in the two cohorts the cataleptic effect produced by the Drd2-like antagonist haloperidol.49 As expected, robust haloperidol-induced catalepsy was observed in B6 mice (one-way ANOVA: F(1,6)=14.967, P=0.0083; Figure 5d). Interestingly only marginal effects were found in BTBR mice (F(1,8)=5.228, P=0.0516; Figure 5d) thus highlighting a significant hypo-functionality of postsynaptic Drd2 receptors in these animals. We next investigated the efficiency of postsynaptic Drd2 signalling at MSN sites. By blocking the negative control exerted by Drd2 activity upon adenilate cyclase, the use of haloperidol can unmask adenosine A2aR-dependent modulation of cyclic adenosine monophosphate/PKA-dependent phosphorylation of GluR1 on Serine 845 (P-Ser845-GluR1).34,57 As expected, a significant increase in haloperidol-dependent striatal P-Ser845-GluR1 levels was observed in B6 mice (one-way ANOVA: F(1,9)=7.732, P=0.0214), whereas in BTBR animals phosphoprotein levels were unaltered compared with vehicle-treated controls (F(1,8)=0.484, P=0.5064) (Figure 5e). Because basal P-Ser845-GluR1 levels are concomitantly affected by Drd1 and A2aR receptor activation58 and as Drd1-mediated signalling appeared to be functional in BTBR (Figure 3f), the discrepant PKA-related posphoprotein levels observed in the two strains (Figure 5f) led us to investigate the presence of an impaired A2aR intracellular signalling in BTBR mice. This hypothesis was tested by measuring the changes of phosphorylation levels of ribosomal protein S6 on Ser-235/236 in response of haloperidol (0.5 mg kg−1) administration, a selective A2aR/PKA-related target associated to Drd2 blockade.59 Statistical analysis of the P-Ser-235/236-S6 levels (Figure 5f) revealed a blunted adenosine A2aR/PKA mediated signalling in the striatum of BTBR mice (one-way ANOVA: F(1,10)=2.784, P=0.1262) whereas a significant phosphorylation increase was found in B6 animals (F(1,10)=45.079, P<0.0001). Collectively these data corroborate the presence of profoundly dysfunctional D2dr- and A2aR-mediated neurotransmission in BTBR, and provide a plausible mechanistic explanation for the blunted striatal fMRI responses observed with GBR 12909 in these animals.


Dysfunctional dopaminergic neurotransmission in asocial BTBR mice.

Squillace M, Dodero L, Federici M, Migliarini S, Errico F, Napolitano F, Krashia P, Di Maio A, Galbusera A, Bifone A, Scattoni ML, Pasqualetti M, Mercuri NB, Usiello A, Gozzi A - Transl Psychiatry (2014)

Impaired Drd2-mediated postsynaptic reactivity and signalling in BTBR mice. Representative coronal sections showing Drd2 mRNA expression in the striatum of B6 (a) and BTBR mice (b). The histogram (c) illustrates Drd2 mRNA levels in BTBR (n=7) after densitometric quantification of autoradiograms. Values are expressed as percentage variation with respect to B6 control mice (n=4). (d) Cataleptic response induced by 0.5 mg kg−1 of haloperidol in B6 (n=4; vehicle, n=4) and BTBR animals (0.5 mg kg−1, n=6; vehicle, n=4). Striatal PKA-dependent P-Ser845-GluR1 (e) and P-Ser235/236-S6 (f) protein levels in B6 and BTBR mice treated with 0.5 mg kg−1 haloperidol (P-Ser845-GluR1: B6, n=5; BTBR, n=6; P-Ser235/236-S6: n=6 each strain) or vehicle (P-Ser845-GluR1: B6, n=5; BTBR, n=4; P-Ser235/236-S6: n=6 each strain). The top panels in e and f show representative blots comparing the different strains and treatment groups. *P<0.05, **P<0.01, ***P<0.0001, compared with vehicle-treated group within strain (one-way analysis of variance). All values are expressed as mean±s.e.m. PKA, protein kinase A.
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fig5: Impaired Drd2-mediated postsynaptic reactivity and signalling in BTBR mice. Representative coronal sections showing Drd2 mRNA expression in the striatum of B6 (a) and BTBR mice (b). The histogram (c) illustrates Drd2 mRNA levels in BTBR (n=7) after densitometric quantification of autoradiograms. Values are expressed as percentage variation with respect to B6 control mice (n=4). (d) Cataleptic response induced by 0.5 mg kg−1 of haloperidol in B6 (n=4; vehicle, n=4) and BTBR animals (0.5 mg kg−1, n=6; vehicle, n=4). Striatal PKA-dependent P-Ser845-GluR1 (e) and P-Ser235/236-S6 (f) protein levels in B6 and BTBR mice treated with 0.5 mg kg−1 haloperidol (P-Ser845-GluR1: B6, n=5; BTBR, n=6; P-Ser235/236-S6: n=6 each strain) or vehicle (P-Ser845-GluR1: B6, n=5; BTBR, n=4; P-Ser235/236-S6: n=6 each strain). The top panels in e and f show representative blots comparing the different strains and treatment groups. *P<0.05, **P<0.01, ***P<0.0001, compared with vehicle-treated group within strain (one-way analysis of variance). All values are expressed as mean±s.e.m. PKA, protein kinase A.
Mentions: We next investigated postsynaptic Drd2 mRNA expression and function in BTBR and B6 mice. Consistent with previous measurements in the substantia nigra, Drd2 mRNA levels in the striatum and nucleus accumbens appeared comparable in the two strains (P=0.89, Student's t-test; Figure 5a, b and c). To probe the functional responsivity of postsynaptic Drd2 receptors, we measured in the two cohorts the cataleptic effect produced by the Drd2-like antagonist haloperidol.49 As expected, robust haloperidol-induced catalepsy was observed in B6 mice (one-way ANOVA: F(1,6)=14.967, P=0.0083; Figure 5d). Interestingly only marginal effects were found in BTBR mice (F(1,8)=5.228, P=0.0516; Figure 5d) thus highlighting a significant hypo-functionality of postsynaptic Drd2 receptors in these animals. We next investigated the efficiency of postsynaptic Drd2 signalling at MSN sites. By blocking the negative control exerted by Drd2 activity upon adenilate cyclase, the use of haloperidol can unmask adenosine A2aR-dependent modulation of cyclic adenosine monophosphate/PKA-dependent phosphorylation of GluR1 on Serine 845 (P-Ser845-GluR1).34,57 As expected, a significant increase in haloperidol-dependent striatal P-Ser845-GluR1 levels was observed in B6 mice (one-way ANOVA: F(1,9)=7.732, P=0.0214), whereas in BTBR animals phosphoprotein levels were unaltered compared with vehicle-treated controls (F(1,8)=0.484, P=0.5064) (Figure 5e). Because basal P-Ser845-GluR1 levels are concomitantly affected by Drd1 and A2aR receptor activation58 and as Drd1-mediated signalling appeared to be functional in BTBR (Figure 3f), the discrepant PKA-related posphoprotein levels observed in the two strains (Figure 5f) led us to investigate the presence of an impaired A2aR intracellular signalling in BTBR mice. This hypothesis was tested by measuring the changes of phosphorylation levels of ribosomal protein S6 on Ser-235/236 in response of haloperidol (0.5 mg kg−1) administration, a selective A2aR/PKA-related target associated to Drd2 blockade.59 Statistical analysis of the P-Ser-235/236-S6 levels (Figure 5f) revealed a blunted adenosine A2aR/PKA mediated signalling in the striatum of BTBR mice (one-way ANOVA: F(1,10)=2.784, P=0.1262) whereas a significant phosphorylation increase was found in B6 animals (F(1,10)=45.079, P<0.0001). Collectively these data corroborate the presence of profoundly dysfunctional D2dr- and A2aR-mediated neurotransmission in BTBR, and provide a plausible mechanistic explanation for the blunted striatal fMRI responses observed with GBR 12909 in these animals.

Bottom Line: Recent psychosocial and neuroimaging studies have highlighted reward-processing deficits and reduced dopamine (DA) mesolimbic circuit reactivity in ASD patients.However, the neurobiological and molecular determinants of these deficits remain undetermined.DA D1 receptor-dependent behavioural and signalling responses were found to be unaltered in BTBR mice, whereas dramatic reductions in pre- and postsynaptic DA D2 and adenosine A2A receptor function was observed in these animals.

View Article: PubMed Central - PubMed

Affiliation: Ceinge Biotecnologie Avanzate, Naples, Italy.

ABSTRACT
Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by pronounced social and communication deficits and stereotyped behaviours. Recent psychosocial and neuroimaging studies have highlighted reward-processing deficits and reduced dopamine (DA) mesolimbic circuit reactivity in ASD patients. However, the neurobiological and molecular determinants of these deficits remain undetermined. Mouse models recapitulating ASD-like phenotypes could help generate hypotheses about the origin and neurophysiological underpinnings of clinically relevant traits. Here we used functional magnetic resonance imaging (fMRI), behavioural and molecular readouts to probe dopamine neurotransmission responsivity in BTBR T(+) Itpr3(tf)/J mice (BTBR), an inbred mouse line widely used to model ASD-like symptoms owing to its robust social and communication deficits, and high level of repetitive stereotyped behaviours. C57BL/6J (B6) mice were used as normosocial reference comparators. DA reuptake inhibition with GBR 12909 produced significant striatal DA release in both strains, but failed to elicit fMRI activation in widespread forebrain areas of BTBR mice, including mesolimbic reward and striatal terminals. In addition, BTBR mice exhibited no appreciable motor responses to GBR 12909. DA D1 receptor-dependent behavioural and signalling responses were found to be unaltered in BTBR mice, whereas dramatic reductions in pre- and postsynaptic DA D2 and adenosine A2A receptor function was observed in these animals. Overall these results document profoundly compromised DA D2-mediated neurotransmission in BTBR mice, a finding that is likely to have a role in the distinctive social and behavioural deficits exhibited by these mice. Our results call for a deeper investigation of the role of dopaminergic dysfunction in mouse lines exhibiting ASD-like phenotypes, and possibly in ASD patient populations.

Show MeSH
Related in: MedlinePlus