ErbB4 signaling in dopaminergic axonal projections increases extracellular dopamine levels and regulates spatial/working memory behaviors

Abstract

Genetic variants of Neuregulin 1 (NRG1) and its neuronal tyrosine kinase receptor ErbB4 are associated with risk for schizophrenia, a neurodevelopmental disorder characterized by excitatory/inhibitory imbalance and dopamine (DA) dysfunction. To date, most ErbB4 studies have focused on GABAergic interneurons in the hippocampus and neocortex, particularly fast-spiking parvalbumin-positive (PV+) basket cells. However, NRG has also been shown to modulate DA levels, suggesting a role for ErbB4 signaling in dopaminergic neuron function. Here we report that ErbB4 in midbrain DAergic axonal projections regulates extracellular DA levels and relevant behaviors. Mice lacking ErbB4 in tyrosine hydroxylase-positive (TH+) neurons, but not in PV+ GABAergic interneurons, exhibit different regional imbalances of basal DA levels and fail to increase DA in response to local NRG1 infusion into the dorsal hippocampus, medial prefrontal cortex and dorsal striatum measured by reverse microdialysis. Using Lund Human Mesencephalic (LUHMES) cells, we show that NRG/ErbB signaling increases extracellular DA levels, at least in part, by reducing DA transporter (DAT)-dependent uptake. Interestingly, TH-Cre;ErbB4^f/f mice manifest deficits in learning, spatial and working memory-related behaviors, but not in numerous other behaviors altered in PV-Cre;ErbB4^f/f mice. Importantly, microinjection of a Cre-inducible ErbB4 virus (AAV-ErbB4.DIO) into the mesencephalon of TH-Cre;ErbB4^f/f mice, which selectively restores ErbB4 expression in DAergic neurons, rescues DA dysfunction and ameliorates behavioral deficits. Our results indicate that direct NRG/ErbB4 signaling in DAergic axonal projections modulates DA homeostasis, and that NRG/ErbB4 signaling in both GABAergic interneurons and DA neurons contribute to the modulation of behaviors relevant to psychiatric disorders.

Figure 1

ErbB4 mRNA and protein is expressed in soma and axons of midbrain DAergic neurons. (a) Double-fluorescence in situ hybridization (RNAscope) for ErbB4 (white) and TH (green) transcripts in midbrain coronal sections from wild-type C57BL/6J mice; anatomical region corresponds to area highlighted in green in the adjacent scheme. The boxed area in (a) is enlarged in the two panels on the right (a ^I–II) to visualize the numerous DA neurons abundantly expressing ErbB4 transcripts (arrowheads); nuclei were labeled with DAPI (blue). (band c) Representative immunofluorescence images of dissociated primary midbrain neurons isolated from (b) wild-type C57BL/6J or (c) ErbB4-KO mice. (b ^I–III) Higher magnification of the area demarked in b shows that ErbB4 receptor puncta (arrowheads) distribute on the cell soma and along DAT-positive axonal processes that are positive for the axon hillock marker Ankyrin G (ankG). (b ^IV–VI) A second magnified area from the same neuron shows ErbB4 immunoreactive puncta along a more distal DAergic Tau-positive axonal process. ErbB4 immunoreactivity is specific, because somatic (c) and axonal (c ^I–IIIandc ^IV–VI) puncta are absent from DAT-positive mesencephalic neurons isolated from ErbB4-KO mice. For panels (b) and (c), 9 DAT-positive neurons from C57BL/6J WT and 10 DAT-positive neurons derived from ErbB4-KO mice were analyzed in two independent cell culture preparations, respectively. Scale bars=200 μm (A and a ^I–II), 100 μm (Band C), 10 μm (b ^I–VI and c ^I–VI).

Figure 2

NRG1-mediated increases in extracellular DA requires direct ErbB4 signaling in DAergic neurons in vivo. Local delivery of NRG1 (left shaded area) and measurements of extracellular DA were performed using reverse microdialysis in the (a) dorsal hippocampus, (b) mPFC and (c) dorsal striatum of freely moving adult TH-Cre;ErbB4^f/f (left, red lines) or PV-Cre;ErbB4^f/f KO mice (right, green lines), and their corresponding ErbB4^f/f littermate controls (black lines). Samples were collected for 15 min to account for low DA levels in the hippocampus and mPFC. Local ErbB4 activation with NRG1 (1 nm, 15 min) elicits a robust increase of extracellular DA in control ErbB4^f/f mice (controls for TH-Cre;ErbB4^f/f, hippocampus: 245.6±33.8%, n=6, mPFC: 208.9±19.2%, n=6, striatum: 179.7±9.1, n=6) (controls for PV-Cre;ErbB4^f/f, hippocampus: 288.0±59.4%, n=6; mPFC: 193.6±8.5%, n=6, striatum: 166.1±6.0, n=6) and in PV-Cre;ErbB4^f/f mutant mice (hippocampus: 328.2±71.0%, n=6, mPFC: 217.1±10.6%, n=6, striatum: 167.8±6.0%. n=6), but not in TH-Cre;ErbB4^f/f mice (hippocampus: 94.8±8.7%, n=7, F(1,11)=11.73, P=0.0057; mPFC: 102.2±1.6%, n=6, F(1,10)=20.02, P=0.0012; striatum: 97.3±4.8%, n=6, F(1,10)=30.83, P=0.0002). The functionality of DA processes in each anatomical area was assayed 60 min after the NRG1 application when DA levels had returned to baseline by delivering a depolarizing KCl pulse (50 mm, 15 min). Extracellular DA levels increased in all genotypes, indicating that dopaminergic processes retained normal capacity for depolarization-dependent release. Data represent the mean±s.e.m. of the percentage of baseline variation. In c, DA increases in striatum during the KCl pulse were plotted on a second y axis shown on the right side of the graph. *P<0.05, **P<0.01 and ***P<0.005.

Figure 3

NRG/ErbB4 signaling increases extracellular DA levels by reducing DAT uptake efficiency. Experiments were performed in differentiated LUHMES cells, which exhibit numerous properties of DAergic neurons, to determine if NRG/ErbB signaling cell-autonomously regulates extracellular DA levels. (a) Cells were treated for 30 min with either vehicle (V), 1 nm NRG1, or 10 μm PD158780+1 nm NRG1 (PD+NRG1); DA levels in conditioned media are plotted as ratios of post- over pre-treatment values. (n=8/treatment). NRG1 increased DA levels relative to baseline (vehicle vs NRG1: 1.010±0.019 vs 1.280±0.019, n=8/treatment; F(2,21)=88.77, P<0.0001), and this increase was blocked by PD158780 (PD+NRG1: 0.889±0.025). (b) The DAT blocker GBR12935 increased DA in the media (F(2,15)=31.09, P<0.0001, n=6/treatment) and occluded any further NRG1-mediated increases of DA (P>0.05). Extracellular DA levels in LUHMES cell media were measured 30 min following the vehicle (V) control, and again 30 min later after addition of either more vehicle (V+V, open squares) or NRG1 (V+NRG1, solid squares). In parallel samples, cultures were initially treated for 30 min with 100 nm GBR12935 (G) and followed by 30 min treatment with GBR12935 and NRG1 (G+NRG1; solid circles). The solid bar represents either vehicle or GBR12935 treatment during 1 h of treatment, and the open bar represents addition of NRG1 (1 nm) or vehicle to the media (n=6/treatment). (c) NRG1 treatment (20 min) dose-dependently reduced [³H]-MPP+ uptake in LUHMES cells (0 nm: 100±1.7%, n=16; 0.2 nm: 91.3±2.0%, n=13; 2 nm: 77.8±2.8%, n=20; and 20 nm: 78.6±2.7%, n=20). One-way analysis of variance (ANOVA) F(3,65)=18.10, P<0.0001. (d) [³H]-MPP⁺ and [³H]-DA uptake in LUHMES treated for 20 min with vehicle (V), 2 nm NRG1 (N) or 10 μm PD158780+2 nm NRG1 (PD+N) during 20 min. NRG1 treatment reduced [³H]-MPP⁺ (72.2±2.0%, n=25) and [³H]-DA uptake (74.4±4.2%, n=18) similarly and uptake was blocked by co-application of PD158780 ([³H]-DA: 89.8±2.0%, n=17; [³H]-MPP⁺: 90.0±1.8%, n=23), consistent with an effect of NRG1 on transporter function. Two-way ANOVA revealed a primary effect of NRG1 treatment on DAT-mediated uptake (F(2,119)=63.95, P<0.0001). Data in (cand d) are plotted as percentages, with 100% defined as uptake under vehicle-treatment conditions, and represent mean±s.e.m. *P<0.05, **P<0.01 and ***P<0.005.

Figure 4

TH-Cre;ErbB4f/f mice exhibit altered DA homeostasis and display spatial learning memory deficits. (a) TH-Cre;ErbB4^f/f mice (red) show elevated basal extracellular dopamine (DA) levels in the dorsal hippocampus (KO vs control: 1.22±0.11 nm vs 0.65±0.12 nm; U=3, P=0.0076; Mann-Whitman U-test) and mPFC (3.07±0.36 nm vs 1.46±0.19 nm; U=1, P=0.0043), but reduced levels in dorsal striatum (10.0±1.13 nm vs 15.11±1.42 nm; U=5, P=0.0411), compared to their corresponding control littermates (black). By contrast, PV-Cre;ErbB4^f/f mice (green) did not show altered DA levels (KO vs control: 0.70±0.10 nm vs 0.73±0.09 nm (hippocampus); 1.19±0.07 nm vs 1.12±0.16 nm (mPFC); 14.23±1.32 nm vs 14.72±1.48 nm (striatum); all P>0.05). (b–e) Assessment of behaviors that differ between TH-Cre;ErbB4^f/f (red) and their littermate controls (ErbB4^f/f, black). (b) Spontaneous alternation (in %) during the T-maze test (n=14/genotype). (c) Memory for familiar/unfamiliar environments in the Y-maze test, plotted as preference (in %) for the novel arm (n=12/genotype). (d) Spatial learning and memory in the Barnes maze during training days 1–4, including latency time to reach the escape target (left panel) and number of errors (right panel). (e) Barnes maze test at day 5 (test day), showing number of incorrect nose pokes (errors, left), time spent in the correct zone (middle) and number of nose pokes plotted as a function of target location (right) (n=12/genotype). (f–j) Performances were indistinguishable between TH-Cre;ErbB4^f/f (red) and their littermate controls (ErbB4^f/f) in a variety of tasks, including: (f) Novelty-induced horizontal locomotor activity in the open field (cumulative distance traveled during 5-min intervals); (g) Amphetamine-induced horizontal locomotor activity (total distance traveled in 1 h following vehicle, 0.5, 1.5, 2.5 and 3.5 mg/kg amphetamine i.p. injection); (h) Pre-pulse inhibition of the acoustic startle response (40 ms sound, 120 dB intensity); (i) Basal anxiety assessed as time spent in open vs closed arms in the elevated plus maze; and (j) Contextual and cued fear memory (unconditioned stimulus (US): 0.5 mA foot shock, 2 ms). Data are represented as means±s.e.m. *P<0.05, **P<0.01 and ***P<0.005.

Figure 5

Rescuing ErbB4 expression in midbrain DAergic neurons of TH-Cre;ErbB4^f/f mice restores behavioral deficits and dopamine (DA) balance. (a) Schematic depiction of AAV-ErbB4.DIO (ErbB4.DIO) or AAV-GFP.DIO (GFP.DIO) stereotaxic bilateral microinjections (0.5 μl/hemisphere) into the midbrain of 2-month-old TH-Cre;ErbB4^f/f mice to cre-dependently express either ErbB4 or GFP in DAergic neurons. (b) Spontaneous alternation in the T-maze (left) was restored after injections of AAV-ErbB4.DIO (ErbB4.DIO vs GFP.DIO: 85.4±4.3% vs 60.4±7.6%, n=16/group; U=67, P=0.0158) as was time spent in unfamiliar arms during the Y-maze test (right; ErbB4.DIO vs GFP.DIO: 42.7±2.7% vs 34.5±2.1%; n=12/ErbB4.DIO; n=11/GFP.DIO; t(21)=2.380, P=0.0269). (c) Basal extracellular DA levels in the mPFC (ErbB4.DIO vs GFP.DIO: 1.4±0.2 nm vs 2.7±0.1 nm, n=6/group; U=0, P=0.0022) and striatum (ErbB4.DIO vs GFP.DIO: 13.7±0.7 nm vs 10.2±0.8 nm; n=6/group; U=3, P=0.0152) were normalized after AAV-ErbB4.DIO injections. (d) Relative extracellular DA levels in TH-Cre;ErbB4^f/f mice [% of baseline] increased after local delivery of 1 nm NRG1 (shaded area) in the mPFC (ErbB4.DIO vs GFP.DIO: 204.1± 8.0% vs 80.5±7.7%, n=6/group; two-way ANOVA treatment F(1,10)=38.14, P=0.0001) and striatum (ErbB4.DIO vs GFP.DIO: 214.5±22.9% vs 93.4±6.2%, n=6/group; F(1,10)=8.8, P=0.0141) 10 weeks following AAV microinjection. Data are represented as means±S.E.M. *P<0.05, **P<0.01, ***P<0.0001.