Dysbindin-1 modulates prefrontal cortical activity and schizophrenia-like behaviors via dopamine/D2 pathways

Abstract

Dysbindin-1 regulates D2-receptor trafficking and is implicated in schizophrenia and related cognitive abnormalities, but whether this molecular effect mediates the clinical manifestations of the disorder is unknown. We explored in dysbindin-1-deficient mice (dys-/-) (1) schizophrenia-related behaviors, (2) molecular and electrophysiological changes in medial prefrontal cortex (mPFC) and (3) the dependence of these on D2-receptor stimulation. Dysbindin-1 disruption altered dopamine-related behaviors and impaired working memory under challenging/stressful conditions. Dys-/- pyramidal neurons in mPFC layers II/III were hyperexcitable at baseline but hypoexcitable following D2 stimulation. Dys-/- were also respectively more and less sensitive to D2 agonist- and antagonist-induced behavioral effects. Dys-/- had reduced expression of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and CaMKKβ in mPFC. Chronic D2 agonist treatment reproduced these changes in protein expression, and some of the dys-/- behavioral effects. These results elucidate dysbindin's modulation of D2-related behavior, cortical activity and mPFC CaMK components, implicating cellular and molecular mechanisms of the association of dysbindin with psychosis.

Figures

Figure 1

Dysbindin mutant mice show faster acquisition to criteria but impaired working memory performance in a prefrontal cortex-dependent T-maze task. (a) Latency to retrieve the hidden food pellet and (b) days needed to reach criterion, by dys+/+, +/– and –/– littermates during the discrete paired-trial T-maze task; *P < 0.05 and #P = 0.07 vs dys+/+, §§§P < 0.0005 vs day 1. (c, d) Percentage of correct choices displayed by the same dys+/+, +/– and –/– mice during the discrete paired-trial variable-delay T-maze test with different intra-trial delays randomly presented (4, 30, 60 and 240 s) and an inter-trial delay of 20 s under normal conditions (c) and during the mild stress of placement in a new home cage 15 min before testing (d). The dotted line corresponds to chance levels (50%) of correct choices. *P < 0.05 vs +/+ mice. Values represent mean±s.e.m. in all the figures. A similar number of the dys–/– (N=11), +/– (N=10) and +/+ (N=11) mice tested reached the criteria (100, 90 and 100%, respectively; P = 0.6). Acoustic startle and prepulse inhibition (PPI) are increased in dysbindin mutant mice. (e) Animal movements displayed by dys+/+, +/– and –/– littermates during no stimulus trials or following the presentation of a 120-dB stimulus (Startle). (f) Percent PPI of the acoustic startle response displayed by the same mice after the presentation of 74, 78, 82, 86 and 90 dB prepulse sound stimuli. Ns: +/+ = 15, +/–= 12, –/–= 16. *P < 0.05 and **P < 0.005 vs +/+. For all genotypes, PPI progressively increased with higher prepulse intensities (F4,160 = 81.00, P < 0.0001).

Figure 2

Dopamine (DA) D2-receptor modulation of acoustic startle and prepulse inhibition (PPI) in wild-type and dysbindin mutant mice. Animal movement displayed by dys+/+, +/– and –/– littermates following the presentation of (a, b) a 120-dB stimulus (Startle) or (c, d) of no stimulus. (a, c) After acute injection with vehicle (Vehicle) or one of two doses of the D2 antagonist eticlopride, that is 0.1mgkg–1 (Etic. 0.1) or 0.5mgkg–1 (Etic. 0.5); (b, d) after acute injection with vehicle (Vehicle) or one of two doses of the D2 agonist quinpirole, that is 0.5mgkg–1 (Quin. 0.5) or 1 mg/kg (Quin. 1). (e–j) Percent PPI of the acoustic startle response displayed by (e, f) dys+/+, (g, h) dys+/– and (i, j) dys–/– littermates after the presentation of 74, 78, 82, 86 and 90 dB prepulse sound stimuli and after acute injection with (e, g, i) vehicle (Vehicle) or one of the two doses of eticlopride or (f, h, j) with vehicle (Vehicle) or one of the two doses of quinpirole. Etic. Ns: +/+ = 14, +/– = 21, –/– = 15; Quin. Ns: +/+ = 10, +/– = 16, –/–= 10. *P < 0.05, **P < 0.005 and ***P < 0.0005 vs vehicle-treated mice.

Figure 3

Enhanced excitability of dysbindin null mutant mice pyramidal neurons in medial prefrontal cortex (mPFC) layer II/III. (a, b) Repetitive firings were evoked by various depolarizing steps, and action potential numbers were plotted against the depolarizing currents injected into the pyramidal cells and fast-spiking (FS) interneurons in mPFC. Representative traces are shown on the left and quantifications are shown on the right. (a) Firing frequency from dys–/– layer II/III pyramidal neurons was much higher than that from +/+ at the same injection currents. (b) The FS interneurons were identified by their shape, location and FS characters. No difference in firing frequency was observed between dys–/– and +/+ layer II/III FS interneurons. (c) Sample traces of spontaneous excitatory post-synaptic currents (sEPSCs) recorded from the layer II/III pyramidal neurons (upper panel) and FS interneurons (lower panel) from dys+/+ and –/– mice. (d) Quantification of sEPSCs recorded from the layer II/III pyramidal neurons and FS interneurons from dys+/+ and –/– mice. The frequency of sEPSCs from dys–/– pyramidal neurons was remarkably higher than that from +/+ pyramidal neurons. In contrast, the frequency of sEPSCs from dys–/– FS interneurons was decreased compared with +/+. No differences were observed in the amplitude and rise time as well as decay time of sEPSCs between dys–/– and +/+ mice. *P < 0.05, **P < 0.01. Effect of quinpirole on excitability of pyramidal neurons and FS interneurons in layer II/III. (e) Sample traces showing the changes in firing rates recorded in layer II/III pyramidal neurons in the mPFC upon application of quinpirole. Current clamp recordings were performed by holding the membrane potentials at their resting potentials. A depolarizing pulse (1.5 s) was applied to evoke typically 7–16 spikes in the baseline every 5 min. Note the decrease in firing frequency after treatment with quinpirole in both dys+/+ and –/– neurons. (f) Time course of the quinpirole effect. *P < 0.05, **P < 0.01. (g) The effect of quinpirole on the excitability of FS interneurons. A depolarizing pulse (1.5 s) was applied to evoke about 13–19 spikes in the baseline every 5 min. Note the increase in firing frequency after treatment with quinpirole in both dys+/+ and –/– neurons. No differences were observed in firing frequency after exposure to quinpirole between dys+/+ and –/– FS interneurons.

Figure 4

Dysbindin disruption and chronic dopamine (DA)/D2 stimulation modulate medial prefrontal cortex (mPFC) Ca2+/calmodulin-dependent protein kinase (CaMK) components similarly. (a) CaMKII and (b) CaMKKβ protein levels in the mPFC of dys+/+, +/– and –/– littermates by immunoblot analysis. CaMKII Ns: +/+ = 10, +/– = 24, –/– = 19; CaMKKβ Ns: +/+ = 5, +/– = 10, –/– = 7. *P < 0.05, **P < 0.005, ***P < 0.0005 and #P = 0.06 vs +/+. Sample western blots are shown in Supplementary Figure 6. There was no significant difference between dys–/–, +/– and +/+ mice on mPFC CaMKIV (F2,19 = 0.55, P = 0.59; Supplementary Figure 7) and CaMKKα (F2,19 = 0.54, P = 0.59; Supplementary Figure 7) protein immunoreactivity. (c) CaMKII and (d) CaMKKβ protein levels in the mPFC of B6 mice after 15 daily repeated treatment with vehicle (Vehicle) or one of two doses of the D2 agonist quinpirole, that is 0.5mgkg–1 (Quin. 0.5) or 1mg kg–1 (Quin. 1). CaMKII Ns: vehicle = 7, Quin. 0.5 = 5, Quin. 1 = 5; CaMKKβ Ns: vehicle = 9, Quin. 0.5 = 4, Quin. 1 = 5. Results are expressed as percentage of the +/+ or vehicle group for each experiment. *P < 0.05, **P < 0.005 and #P = 0.05 vs vehicle-treated mice. Sample western blots are shown in Supplementary Figure 6. Chronic D2 stimulation did not affect mPFC CaMKIV (F2,15 = 1.74, P = 0.21; Supplementary Figure 8), CaMKI (F2,15 = 0.06, P = 0.94; Supplementary Figure 8) and CaMKKα (F2,15 = 0.53, P = 0.60; Supplementary Figure 8) protein immunoreactivity.

Figure 5

Chronic dopamine (DA) D2-receptor stimulation impairs working memory performance. (a) Days needed to reach criterion and (b) latency to retrieve the hidden food pellet displayed by B6 littermates after at least 15 daily repeated treatment with vehicle (Vehicle) or 0.5mgkg–1 (Quin. 0.5) of quinpirole during the discrete paired-trial T-maze task. Ns: vehicle = 8, Quin. 0.5 = 7; **P < 0.001 vs vehicle-treated mice, §§§P < 0.0005 vs day 1. All mice used reached the criterion. The habituation performance of the quinpirole-treated mice was different from their vehicle-treated littermates on day 1 (P < 0.001), but not on day 2 (P = 0.16). It is worth noting that a similar pattern was present for the dys–/– mice (Figure 1b). (c) Percentage of correct choices displayed by the same vehicle- and quinpirole-treated B6 littermates during the discrete pairedtrial variable-delay T-maze test with different intra-trial delays randomly presented (4, 30, 60 and 240 s) and an inter-trial delay of 20 s. Both groups displayed the same delay-dependent performance, progressively increasing the number of mistakes with longer delays (F3,52 = 18.01, P < 0.0001). The dotted line corresponds to chance levels (50%) of correct choices. *P < 0.05 vs vehicle-treated mice. Prepulse inhibition (PPI) is increased in chronically quinpirole-treated mice. (d) Animal movement displayed by chronically vehicle- and quinpirole-treated littermates during no stimulus trials or following the presentation of a 120-dB stimulus (Startle). (e) Percent PPI of the acoustic startle response displayed by the same mice after the presentation of 74, 78, 82, 86 and 90 dB prepulse sound stimuli. Ns: vehicle = 8, Quin. 0.5 = 7. *P < 0.05 vs vehicle-treated mice. In both groups, PPI progressively increased with higher prepulse intensities (F4,48 = 25.41, P < 0.0001).