Adolescent stress leads to glutamatergic disturbance through dopaminergic abnormalities in the prefrontal cortex of genetically vulnerable mice

Abstract

Background: Stress during the adolescent period influences postnatal maturation and behavioral patterns in adulthood. Adolescent stress-induced molecular and functional changes in neurons are the key clinical features of psychiatric disorders including schizophrenia.

Objective: In the present study, we exposed genetically vulnerable mice to isolation stress to examine the molecular changes in the glutamatergic system involving N-methyl-d-aspartate (NMDA) receptors via dopaminergic disturbance in the prefrontal cortex (PFc).

Results: We report that late adolescent stress in combination with Disrupted-in-Schizophrenia 1 (DISC1) genetic risk elicited alterations in glutamatergic neurons in the PFc, such as increased expression of glutamate transporters, decreased extracellular levels of glutamate, decreased concentration of d-serine, and impaired activation of NMDA-Ca²⁺/calmodulin kinase II signaling. These changes resulted in behavioral deficits in locomotor activity, forced swim, social interaction, and novelty preference tests. The glutamatergic alterations in the PFc were prevented if the animals were treated with an atypical antipsychotic drug clozapine and a dopamine D1 agonist SKF81297, which suggests that the activation of dopaminergic neurons is involved in the regulation of the glutamatergic system.

Conclusion: Our results suggest that adolescent stress combined with dopaminergic abnormalities in the PFc of genetically vulnerable mice induces glutamatergic disturbances, which leads to behavioral deficits in the young adult stage.

Keywords: Adolescent stress; Dopaminergic neuron; Genetic vulnerability; Glutamatergic neuron; Prefrontal cortex.

Fig. 1

Behavioral abnormalities in the DM. a Aberrant locomotor activity. b Increased immobility in the forced swim test. c Deficits in sociability in the social interaction test. d Impaired visual memory in the novelty preference test. CTL wild-type mice without adolescent isolation, DM DISC1 mutant mice exposed to a 3-week period of isolation. N = 13–17 for a, N = 20 for b, N = 13–17 for c and d. Values are means ± SE. Statistical differences were determined using a two-way ANOVA with repeated measures (group, F_{(1, 28)} = 68.15, P < 0.05 for a), and the t test for b to d (*P < 0.05)

Fig. 2

Glutamatergic disturbances in the DM. a Levels of phospho-NR1/total-NR1 in the PFc. b Levels of phospho-CaMK II/total-CaMK II in the PFc. c Extracellular levels of glutamate at baseline in the PFc. d Extracellular levels of glutamate upon high K⁺ stimulation in the PFc. Extracellular levels of glutamate were measured by in vivo microdialysis. e Levels of GLAST in the PFc. f Levels of GLT-1 in the PFc. p- phospho-, t- total-. N = 7 for a, b, e, and f; N = 6–7 for c and d. Values are means ± SE. Statistical differences were determined using the t test for a, b, c, e, and f, and the two-way ANOVA with repeated measures (group, F_{(1, 12)} = 12.39, P < 0.05 for d), (*P < 0.05)

Fig. 3

Effects of a competitive glutamate transport inhibitor TBOA on behavioral and neurochemical abnormalities in the DM. a Effects of TBOA on the impaired performances in the forced swim test. b Normalization effect of TBOA on the decreased extracellular levels of glutamate at baseline in the PFc of the DM. c Normalization effect of TBOA on the decreased rate of CaMK II phosphorylation in the PFc of the DM after the forced swim test. p- phospho-, t- total-, Veh treated with vehicle, TBOA treated with DL-threo-β-benzyloxyaspartate. N = 12–14 for a, N = 7–9 for b, N = 5 for c. Values are means ± SE. Statistical differences were determined using a one-way ANOVA (group, F_{(2, 37)} = 6.45, P < 0.05 for a; F_{(2, 23)} = 10.87, P < 0.05 for b; F_{(2, 12)} = 17.35, P < 0.05 for c), followed by Bonferroni post hoc tests (*P < 0.05)

Fig. 4

Serine system disturbance in the DM. a Concentration of d-serine in the PFc of the DM. b The mRNA expression levels of serine racemase in the PFc of the DM. c The mRNA expression levels of d-amino acid oxidase in the PFc of the DM. N = 8 for a, N = 14 for b, N = 13–14 for c. Values are means ± SE. Statistical differences between two groups were analyzed with the t test (*P < 0.05)

Fig. 5

Effects of a partial NMDA receptor glycine-site agonist DCS and an inhibitor of d-amino acid oxidase MPC on behavioral and neurochemical abnormalities in the DM. a Effects of DCS on the impaired performances in the forced swim test. b Normalization effect of DCS on the decreased rate of CaMK II phosphorylation in the PFc of the DM after the forced swim test. c Effects of MPC on the impaired performances in the forced swim test. d Normalization effect of MPC on the decreased rate of CaMK II phosphorylation in the PFc of the DM after the forced swim test. e Effects of MPC on the decreased concentration of d-serine in the PFc of the DM. p- phospho-, t- total-, Veh treated with vehicle, DCS treated with D-cycloserine, MPC treated with 5-methylpyrazole-3-carboxylic acid. N = 12 for a, N = 10 for b, N = 17–19 for c, N = 6 for d, N = 10–13 for e. Values are means ± SE. Statistical differences were determined using one-way ANOVA (group, F_{(2, 33)} = 6.54, P < 0.05 for a; F_{(2, 27)} = 42.24, P < 0.05 for b; F_{(2, 51)} = 6.33, P < 0.05 for c; F_{(2, 15)} = 13.32, P < 0.05 for d; F_{(2, 33)} = 3.50, P < 0.05 for e), followed by Bonferroni post hoc tests (*P < 0.05)

Fig. 6

Effects of an atypical antipsychotic drug CLZ on behavioral and neurochemical abnormalities in the DM. a Effects of CLZ on aberrant locomotor activity. b Effects of CLZ on the impaired performance in the forced swim test. c Effects of CLZ on the impaired performance in the social interaction test. d Effects of CLZ on the impaired performance in the novelty preference test. e Effects of CLZ on the impaired performance in the prepulse inhibition test. f Normalization effect of CLZ on the decreased rate of CaMK II phosphorylation in the PFc of the DM after the forced swim test. p- phospho-, t- total-, Veh treated with vehicle, CLZ treated with clozapine. N = 11–12 for a, N = 9–11 for b, N = 11–12 for c to e, N = 7 for f. Values are means ± SE. Statistical differences were determined using a two-way ANOVA with repeated measures (group, F_{(2, 32)} = 7.31, P < 0.05 for a; F_{(2, 32)} = 15.68, P < 0.05 for e), and one-way ANOVA (group, F_{(2, 27)} = 7.92, P < 0.05 for b; F_{(2, 32)} = 16.70, P < 0.05 for c; F_{(2, 32)} = 25.41, P < 0.05 for retention of d; F_{(2, 32)} = 7.93, P < 0.05 for prepulse 74 in e; F_{(2, 32)} = 16.91, P < 0.05 for prepulse 78 in e; F_{(2, 32)} = 14.49, P < 0.05 for prepulse 86 in e; F_{(2, 18)} = 21.79, P < 0.05 for f), followed by Bonferroni post hoc tests (*P < 0.05)

Fig. 7

Effects of an atypical antipsychotic drug CLZ on dopaminergic disturbance in the DM. a Effects of CLZ on the extracellular levels of dopamine at baseline in the NAc. b Effects of CLZ on the levels of dopamine upon METH challenge in the NAc. c Effects of CLZ on the extracellular levels of dopamine at baseline in the PFc. d Effects of CLZ on the levels of dopamine upon METH challenge in the PFc. Effects of CLZ on the decreased levels of TH (e) and increased levels of D2R (f) in the PFc of the DM. Veh treated with vehicle, CLZ treated with clozapine. N = 7 for a and b, N = 7–8 for c and d, N = 6 for e and f. Values are means ± SE. Statistical differences were determined using a two-way ANOVA with repeated measures (group, F_{(2, 18)} = 3.25, P > 0.05 for b; F_{(2, 19)} = 0.39, P > 0.05 for d), and a one-way ANOVA (group, F_{(2, 18)} = 0.50, P > 0.05 for a; group, F_{(2, 19)} = 6.25, P < 0.05 for c; F_{(2, 15)} = 8.49, P < 0.05 for e; F_{(2, 15)} = 26.83, P < 0.05 for f), followed by Bonferroni post hoc tests (*P < 0.05)

Fig. 8

Effects of a dopamine-D1 receptor agonist SKF on behavioral and neurochemical abnormalities in the DM. a Effects of SKF on impaired performance in the forced swim test. b Effects of SKF on the decreased NR1 phosphorylation in the PFc of the DM after the forced swim test. c Normalization effect of SKF on the decreased CaMK II phosphorylation in the PFc of the DM after the forced swim test. Veh treated with vehicle, SKF treated with SKF81297. N = 8–10 for a, N = 7 for b, N = 8 for c. Values are means ± SE. Statistical differences were determined using a one-way ANOVA (group, F_{(2, 27)} = 12.75, P < 0.05 for a; F_{(2, 18)} = 13.94, P < 0.05 for b; F_{(2, 21)} = 15.85, P < 0.05 for c), followed by Bonferroni post hoc tests (*P < 0.05)

Fig. 9

Adolescent stress leads to glutamatergic disturbance through alterations in dopaminergic signaling in the prefrontal cortex of the DM. Our results suggest that presynaptic and postsynaptic glutamate and dopamine transmissions in the PFc are impaired by adolescent stress in genetically susceptible animals. In the presynaptic glutamate transmission in the PFc, (1) the upregulation of glial glutamate transporters (GLAST, GLT-1), and (2) the decreased extracellular levels of dopamine and glutamate are observed. In the postsynaptic glutamate transmission in the PFc, (3) the functions of the NMDA receptor and serine system are impaired. These abnormalities of glutamate and dopamine transmissions, and a disturbance of the serine system induced (4) the abnormality of NMDA-CaMK II signaling via dopamine D1 receptor and (5) behavioral deficits. TBOA DL-threo-β-benzyloxyaspartate (a competitive glutamate transport inhibitor), DCS D-cycloserine (a partial NMDA receptor glycine-site agonist), CLZ clozapine (an atypical antipsychotic drug), SKF SKF81297 (a dopamine-D1 receptor agonist), MPC 5-methylpyrazole-3-carboxylic acid (an inhibitor of d-amino acid oxidase), DAO d-amino acid oxidase