Interplay of beta2* nicotinic receptors and dopamine pathways in the control of spontaneous locomotion

Abstract

Acetylcholine (ACh) is a known modulator of the activity of dopaminergic (DAergic) neurons through the stimulation of nicotinic ACh receptors (nAChRs). Yet, the subunit composition and specific location of nAChRs involved in DA-mediated locomotion remain to be established in vivo. Mice lacking the beta2 subunit of nAChRs (beta2KO) display striking hyperactivity in the open field, which suggests an imbalance in DA neurotransmission. Here, we performed the selective gene rescue of functional beta2*-nAChRs in either the substantia nigra pars compacta (SNpc) or the ventral tegmental area (VTA) of beta2KO mice. SNpc rescued mice displayed normalization of locomotor activity, both in familiar and unfamiliar environments, whereas restoration in the VTA only rescued exploratory behavior. These data demonstrate the dissociation between nigrostriatal and mesolimbic beta2*-nAChRs in regulating unique locomotor functions. In addition, the site-directed knock-down of the beta2 subunit in the SNpc by RNA interference caused hyperactivity in wild-type mice. These findings highlight the crucial interplay of nAChRs over the DA control of spontaneous locomotion.

Fig. 1.

Lentiviral restoration of functional β2*-nAChRs in the DAergic pathways of β2KO mice. (A) Midbrain DA pathways from SNpc (A9; green) and VTA (A10; blue) innervating caudate putamen (CPu) or NuAcc and PFC. Shown in red are cholinergic afferents from LDTg and PPTg to DA nuclei. The magnifications show DA striatal terminal (left), with cholinergic interneurons in red, and cholinergic inputs over DA nuclei and GABA interneuron (right). β*, β2*-nAChRs. (B) Map of the bicistronic lentiviral reexpression vector [PGK-β2-Ires-eGFP]. LTR, long terminal repeat; FLAP, sequence comprising central polypurine tract and central termination sequence (see SI Text); PGK, mouse phosphoglycerate kinase promoter; beta2, mouse WT β2 nicotinic ACh receptor subunit cDNA; IRES2, internal ribosome entry sequence; eGFP, enhanced green fluorescent protein; WPRE, woodchuck hepatitis B virus posttranscriptional regulatory element; 3′PPT, 3′polypurine tract; ΔU3, deletion of U3 portion of 3′LTR. (C) Coronal sections (−3 mm from bregma) showing the site of lentivirus injection in SNpc-RESC (Left) and VTA-RESC mice (Right). eGFP (green) indicates the virally transduced area and TH (red) stains DA neurons of the SNpc and VTA. Shown in the magnification are lentivirus-transduced DA neurons, showing colocalization of eGFP and TH (arrows), or eGFP-positive non-TH neurons and glial cells (arrowheads). (D–F) [¹²⁵I]-epibatidine autoradiography demonstrating restoration of high-affinity β2*-nAChRs binding sites in SNpc-RESC and VTA-RESC mice. (D) Saggital sections (1 mm lateral from the bregma suture). (E) Coronal sections at −3 mm from bregma containing the SNpc and VTA [fasciculus retroflexus (fr; arrow) shows non-β2* binding of [¹²⁵I]-epibatidine]. (F) Coronal sections at + 0.62 mm from bregma, showing region-specific reexpression in the CPu projections of SNpc-RESC and in the NuAcc of VTA-RESC mice. Arrows indicate reexpression areas at CPu and NuAcc (D and F) or at SNpc and VTA (E).

Fig. 2.

Nigral β2*-nAChRs control striatal DA release. (A) DA extracellular concentrations (pg/20 μl) in the striatum of WT, β2KO, and SNpc-RESC mice under basal conditions (100 min) and after an i.p. injection of saline (0.1 ml/10g, arrow; WT, n = 11; KO, n = 10; SNpc-RESC, n = 9). (B) Area under the curve (AUC) values from 20–100 min for basal concentrations of DA in WT, KO, and SNpc-RESC mice. *, P < 0.05 (Dunnett vs. WT mice).

Fig. 3.

Quantitative analysis of open-field behavior and its predictions. Total distance traveled (A) and time spent in fast (B) or slow (C) movements during a 30-min session in the open field. SNpc-RESC were restored to WT levels of distance traveled and fast, but not slow, movements. VTA-RESC showed only restoration of slow movements. One-way ANOVA, followed by Tukey's Multiple Comparison test: WT, n = 19; KO, n = 20; SNpc-RESC, n = 12; VTA-RESC, n = 10; *, P < 0.05; **, P < 0.01. (D) Mean percentage of variation in five open-field parameters in β2KO compared with WT (0). Bars indicate decrease or increase in each parameter. The total percentage of variation is indicated in the top line. In each bar the respective contribution of β2*-nAChRs from SNpc, VTA, or other regions is indicated. These values were calculated based on the percentage of restoration of each parameter achieved by the reexpression experiments. (E) Predicted variation of open-field parameters in a WT mouse with elimination of β2*-nAChRs in the SNpc, considering the percentage of variation for each parameter according to the values obtained in D.

Fig. 4.

Silencing of the β2 subunit in the SNpc by a lentivirus-delivered short inhibitory RNA. (A) Map of the lentiviral RNAi vectors [U6-shRNA-Ubiq-EGFP], silencing vector (shβ2), or control vector (shScr). U6, polymerase III promoter to drive the transcription of sh shRNAs; sense, shRNA target sequences (see Methods); loop, sequence from an endogenous miRNA (5′ GTGAAGCCACAGATG 3′); antisense, complementary to the sense sequence, used to form the sh double strand RNA; Ubiq, human ubiquitin promoter; eGFP, green fluorescent protein. All other regions indicated in the diagram are identical to the reexpression vector in Fig. 1B. (B) [¹²⁵I]-epibatidine autoradiography showing decrease in β2*-nAChRs in the SNpc after the injection of shβ2 compared with control side injected with shScr (Left, rostral coronal section at −2.9 mm; Right, caudal section at −3.3 mm from Bregma). (C) Mean values of [¹²⁵I]-epibatidine binding in SNpc of mice injected with shβ2 or shScr or not injected (NI). Binding was quantified at four consecutive coronal sections per mouse (between −2.9 and −3.3 mm from bregma). One-way ANOVA followed by Tukey's Multiple Comparison test (shβ2, n = 8; shScr, n = 6; NI, n = 4); *, P < 0.01. (D–F) Behavioral analysis during 30 min in the open field. Shown are the total distance traveled (D) and time spent in fast (E) or slow (F) movements of WT mice bilaterally injected with shScr or shβ2 in the SNpc. In shβ2-injected mice, distance traveled and time in navigation were increased from the shScr group (mean ± SEM; two-tailed Student's t test; shβ2, n = 8; shScr, n = 8; *, P < 0.05). Dashed lines indicate the values predicted for the shβ2 group (see Fig. 3E).

Fig. 5.

Nigrostriatal β2*-nAChRs modulate nocturnal activity in a familiar environment. Total distance traveled in activity boxes during 24 h (dark phase: 8:00 p.m. to 8:00 a.m.), starting at 3:00 p.m. (A and B) β2KO mice display enhanced motor activity compared with WT mice during the dark phase (P < 0.01; repeated measures ANOVA followed by Fisher LSD test). (A) SNpc-RESC mice recovered normal (WT) levels of activity during the dark phase (WT, n = 8; KO, n = 8; SNpc-RESC, n = 11). (B) VTA-RESC mice activity was similar to the KO group during the whole test (WT, n = 9; KO, n = 11; VTA-RESC, n = 9; scale in y axis as in A).

Fig. 6.

Endogenous cholinergic control through β2*-nAChRs over DA pathways and spontaneous locomotion: a model. Cholinergic afferents (red) from PPTg or LDTg arrive to DA nuclei at VTA and SNpc and control DA release through β2*-nAChRs either at the nigrostriatal (green) or mesocorticolimbic (blue) pathways. Glutamatergic corticostriatal inputs (orange) stimulate medium spiny neurons (MSNs) both at dorsal and ventral striatum. DA action at MSNs controls the basal ganglia outputs and the manifestation of fast or slow movements, depending on DA balance between nigrostriatal vs. mesolimbic circuits.