Disruption of VGLUT1 in Cholinergic Medial Habenula Projections Increases Nicotine Self-Administration

Abstract

Cholinergic projections from the medial habenula (MHb) to the interpeduncular nucleus (IPN) have been studied for their complex contributions to nicotine addiction and have been implicated in nicotine reinforcement, aversion, and withdrawal. While it has been established that MHb cholinergic projections corelease glutamate, no direct evidence has demonstrated a role for this glutamate projection in nicotine consumption. In the present study, a novel floxed Slc17a7 [vesicular glutamate transporter 1 (VGLUT1)] mouse was generated and used to create conditional knock-out (cKO) mice that lack VGLUT1 in MHb cholinergic neurons. Loss of Slc17a7 expression in ventral MHb cholinergic neurons was validated using fluorescent in situ hybridization, and immunohistochemistry was used to demonstrate a corresponding reduction of VGLUT1 protein in cholinergic terminals in the IPN. We also used optogenetics-assisted electrophysiology to evoke EPSCs in IPN and observed a reduction of glutamatergic currents in the cKO, supporting the functional disruption of VGLUT1 in MHb to IPN synapses. cKO mice exhibited no gross phenotypic abnormalities and displayed normal thigmotaxis and locomotor behavior in the open-field assay. When trained to lever press for food, there was no difference between control and cKO. However, when tested in a nicotine self-administration procedure, we found that the loss of VGLUT1-mediated glutamate corelease led to increased responding for nicotine. These findings indicate that glutamate corelease from ventral MHb cholinergic neurons opposes nicotine self-administration, and provide additional support for targeting this synapse to develop potential treatments for nicotine addiction.

Keywords: acetylcholine; corelease; glutamate; interpeduncular nucleus; medial habenula; nicotine.

Figure 1.

cKO of Slc17a7 (VGLUT1) from cholinergic neurons in MHb. A, Schematic of Kiaa^Cre; Rosa26^ZSGreen reporter mouse line with Cre expression driven by Kiaa1107 regulatory elements and ZsGreen expression dependent on Cre recombination. B, Native ZsGreen fluorescence counterstained with DAPI in MHb (outlined); scale bar: 200 μm. C, Schematic of Cre recombination of the floxed Slc17a7 (VGLUT1) locus in the cKO (Kiaa^Cre; Slc17a7^flox/flox) mouse line. D, Fluorescent in situ hybridization of Cre, Chat, and Slc17a7 expression in MHb of control and cKO mice at two bregma points; scale bar: 100 μm. E, Higher-magnification images from white squares in D; scale bar: 50 μm. Densitometric quantification (without background subtraction) in ventral and dorsal MHb of (F) Cre, (G) Slc17a7 (VGLUT1), and (H) Chat signals. Only Slc17a7 was significantly reduced in ventral MHb of cKO (**p = 0.006); n = 3 mice per group.

Figure 2.

Loss of VGLUT1 in central IPN of cKO mice. A, Immunohistochemistry for ChAT, VGLUT1, and VGLUT2 in the IPN of control (Slc17a7^flox) and cKO mice (Kiaa^Cre; Slc17a7^flox/flox); bottom row shows ChAT and VGLUT1 merge; scale bar: 100 μm. Densitometric quantification (without background subtraction) in central and lateral IPN of (B) ChAT, (C) VGLUT1, and (D) VGLUT2 signals. Only VGLUT1 was significantly reduced in cKO and only in the central IPN (**p = 0.0040); n = 4 mice per group.

Figure 3.

VGLUT2-expressing projections from MHb to IPN. A, Fluorescent in situ hybridization from Kiaa^Cre mouse showing Cre and Slc17a6 expression in the MHb (outlined); scale bar: 100 μm. B, High-resolution image showing expression of Cre, Slc17a6 (VGLUT2), and DAPI in MHb of Kiaa^Cre mouse; scale bar: 10 μm. Yellow arrows indicate some of the cells containing both Cre and Slc17a6 mRNA. C, Image of MHb from Slc17a6^Cre (VGLUT2) mouse injected with AAV1-Ef1α-DIO-ChR2:mCherry bilaterally into the MHb (outlined); scale bar: 100 μm. D, Immunohistochemistry of IPN from Slc17a6^Cre (VGLUT2-Cre) mouse injected bilaterally with AAV1-Ef1α-DIO-ChR2:mCherry in MHb. VGLUT2^Cre MHb terminals in IPN represented by mCherry expression, stained with VGLUT1 and ChAT; scale bar: 100 μm.

Figure 4.

Reduced glutamate transmission from MHb to IPN in VGLUT1 cKO mice. A, Schematic of electrophysiological preparation, with bilateral injections of AAV1-Ef1α-DIO-ChR2:mCherry in MHb of control (Kiaa^Cre) or cKO (Kiaa^Cre; Slc17a7^flox/flox) mice. Slice recordings using optogenetic stimulation performed in the IPN 3+weeks after injection. B, Images from control mouse of native Cre-dependent mCherry fluorescence in MHb (left) and fibers in IPN (center, right); scale bar: 100 μm. C, Whole-cell recordings in IPN with single-pulse optogenetic stimulation of MHb terminals led to oEPSC amplitudes that were reduced in the cKO (left, **p = 0.001). Representative traces before and after DNQX in control (black) and cKO (blue; right). D, oEPSC amplitude following train stimulation (1 s) did not differ significantly different between control and cKO groups (left). Representative traces before and after mecamylamine in control (black) and cKO (blue; right). Note that bars in panels C, D represent mean ± SEM, individual cells are represented by gray circles (control) or blue squares (cKO).

Figure 5.

Increased nicotine self-administration in cKO mice. A, Total distance traveled (left) and distance traveled across test segments (right) in open field assay showed no significant differences between genotype. B, Total time in center (left) and time in center across test segments (right) did not differ between genotype. C, Active and inactive lever presses during food training across test session did not differ by genotype. D, Active and inactive lever presses for nicotine show increased self-administration for cKO mice (Sidak’s *p < 0.05, ***p < 0.001). E, cKO mice earned more total nicotine infusions in first three nicotine test sessions (t test, *p < 0.05). F, Nicotine infusions earned by control and cKO mice in dose–response paradigm (Sidak’s, **p < 0.005).