Hypothalamic neurotensin projections promote reward by enhancing glutamate transmission in the VTA

Abstract

The lateral hypothalamus (LH) sends a dense glutamatergic and peptidergic projection to dopamine neurons in the ventral tegmental area (VTA), a cell group known to promote reinforcement and aspects of reward. The role of the LH to VTA projection in reward-seeking behavior can be informed by using optogenetic techniques to dissociate the actions of LH neurons from those of other descending forebrain inputs to the VTA. In the present study, we identify the effect of neurotensin (NT), one of the most abundant peptides in the LH to VTA projection, on excitatory synaptic transmission in the VTA and reward-seeking behavior. Mice displayed robust intracranial self-stimulation of LH to VTA fibers, an operant behavior mediated by NT 1 receptors (Nts1) and NMDA receptors. Whole-cell patch-clamp recordings of VTA dopamine neurons demonstrated that NT (10 nm) potentiated NMDA-mediated EPSCs via Nts1. Results suggest that NT release from the LH into the VTA activates Nts1, thereby potentiating NMDA-mediated EPSCs and promoting reward. The striking behavioral and electrophysiological effects of NT and glutamate highlight the LH to VTA pathway as an important component of reward.

Figure 1.

A, Schematic of the optical ICSS paradigm. Each active nosepoke produced a train of 20 Hz blue light pulses (5 ms each) to stimulate lateral hypothalamic axons terminating in the VTA. B, Images showing cannula placement aimed at the VTA. TH (red) indicates midbrain dopamine neurons; green represents ChR2-expressing neurons (AAV). In the merged image: *The cannula location. The arrow indicates cannula path toward the VTA. Scale bar, 500 μm. C, Average nosepoke rates for optical stimulation of LH to VTA fibers. Mice perform significantly more active nosepokes (black) than inactive nosepokes (white) (p < 0.001, n = 10, Mann–Whitney test). D, Extinction experiment depicting operant responding at the active nosepoke. Base, Baseline session to which active nosepokes were normalized; Extinct, extinction experiments in which the laser was turned off 10 min into the hour-long trial, nosepoke cues remained present; Reinst, reinstatement sessions in which each active nosepoke produced optical stimulation in the absence of nosepoke cues; Rev, reversal experiments during which the active and inactive nosepokes were switched. Active nosepokes during extinction, but not reinstatement or reversal, were significantly lower than baseline (p = 0.006, n = 10, Dunnett's multiple-comparisons test).

Figure 2.

A, SR48692 reduced optical self-stimulation of LH to VTA fibers. Day 1, baseline; Day 2, vehicle (0.1% DMSO in saline); Day 3, SR48692, the Nts1 antagonist. Left, Raw nosepoke data. SR48692, n = 5: 116 ± 37 active, 9 ± 3 inactive; vehicle, n = 3: 393 ± 155 active, 12 ± 2 inactive (p < 0.05 active nosepoke comparison; p > 0.05 inactive nosepoke comparison; two-way ANOVA, Bonferroni post hoc test). Right, Normalized active nosepokes. SR48692, 33.0 ± 9.3% baseline; vehicle, 99.0 ± 19.6% baseline (p = 0.036, Mann–Whitney test). B, Cumulative nosepokes of a representative mouse after infusion of vehicle (blue) and SR48692 (red), the Nts1 receptor antagonist. Each square represents one nosepoke. C, Frequency distribution of internosepoke intervals. Blue represents vehicle; and red, Nts1 antagonist. The 30 s internosepoke interval (hyphenated line) separates the bimodal distribution into high- and low-frequency nosepoking. Bout, three or more nosepokes with and interpoke-interval <30 s. D, E, Raster plots of bout activity from the representative animal. Each red tick represents one nosepoke; each line is one bout within a given session.

Figure 3.

AP5 reduces optical self-stimulation of LH to VTA fibers. Using a between-subject design, animals received either AP5, the NMDA receptor antagonist, or vehicle before the optical ICSS session. Groups did not differ in baseline operant responding. AP5, n = 4: 48 ± 19 active, 7 ± 4 inactive; vehicle, n = 4: 290 ± 104 active, 14 ± 6 inactive; p = 0.035; two-way ANOVA, Bonferroni post hoc test.

Figure 4.

A, B, Single-cell examples of evoked NMDA-mediated current in a VTA neuron recorded via whole-cell, voltage-clamp experiments at a holding potential of 40 mV. NMDA currents were measured 25 ms after the stimulus artifact. The NT active peptide fragment (8–13) was bath applied for 10 min before being washed out. A, NT 10 nm. B, 100 nm. C, D, Population responses to varied doses of NT. C, 10 nm (red): 119.2 ± 1.3%, n = 6, p < 0.001. D, 100 nm (green): 85.1 ± 2.2%, n = 5, p < 0.001; 300 nm (blue): 66.6 ± 4.5%, n = 5, p < 0.001; 500 nm (purple): 77.7 ± 1.2%, n = 5, p < 0.001. The shaded regions indicate the data points averaged for analysis. E, Average dose-responses of NMDA-mediated current to NT. One-way ANOVA with Tukey's post hoc analysis indicated that all concentrations were significantly different from one another (p < 0.001 for all comparisons, except 100 vs 500 nm in which p < 0.01). F, Example traces of NT-induced potentiation at 10 nm (red) and inhibition by 300 nm NT (blue). Approximately 12 sweeps were averaged per trace. The stimulus artifacts were removed from the trace examples.

Figure 5.

A, B, The Nts1 antagonist, SR48692, was bath applied 10 min before and washed out 10 min after NT superfusion. Antagonist concentrations were higher than those of the agonist in each experiment to ensure receptor occupancy by the antagonist. A, SR48692 100 nm blocked NT-induced potentiation at 10 nm (99.2 ± 1.3%, n = 5, p = 0.028 measured 12 min after NT application). B, SR48692 500 nm attenuated NT-induced reduction at 100 nm (89.7 ± 0.9%, n = 8, p < 0.001). Comparisons were conducted between minutes 16 and 22. C, D, Effect of NT on NMDA-mediated EPSCs in Nts1 knock-out mice. C, 10 nm (91.1 ± 2.2%, n = 7, p = 0.006 compared with the effect of NT 10 nm in wild-type). D, 100 nm (85.6 ± 2.4%, n = 4, p = 0.008 compared with the effect of NT 100 nm in wild-type). Analysis was conducted between minutes 16 and 22.

Figure 6.

A, B, Dose-responses of evoked AMPA-mediated EPSCs to various NT concentrations: 10 nm (red), 74.4 ± 3.5%, n = 9, p < 0.001; 100 nm (green), 60.9 ± 8.7%, n = 4, p < 0.001; 500 nm (blue), 59.3 ± 3.3%, n = 6, p < 0.001; holding potential = −70 mV. One-way ANOVA indicated that the treatment was a significant factor (p = 0.009). Tukey's post hoc analysis revealed that the 10 nm effect was significantly different from both 100 and 500 nm (p < 0.05); however, 100 and 500 nm were not statistically different from one another (p > 0.05). Average values were determined between minutes 16–22 for A, D, E, G, and H, as indicated by the shaded region. C, Response of a single neuron to electrical stimulation in the absence (black) and presence (red) of 10 nm NT. The stimulus artifact was removed from the trace example. D, SR48692, the Nts1 antagonist, was bath applied before, during, and after NT application. AMPA-mediated current reduction of 72.5 ± 5.4%; n = 5 was not statistically distinct from the 74.4 ± 3.5% current reduction observed by 10 nm NT in the absence of the antagonist (p = 0.570). E, AMPA-mediated currents were recorded at a holding potential of 40 mV in the presence of NMDA-receptor antagonist, AP5 (71.2 ± 4.3%, n = 6, p < 0.001 from baseline, p = 0.310 vs AMPA-mediated currents recorded at −70 mV). F, Paired-pulse ratios of two AMPA EPSC peaks evoked 50 ms apart at a holding potential of −70 mV. Red represents cells perfused with 10 nm NT; blue, 100 nm (no significant difference, p = 0.631 and p = 0.475, respectively). G, H, Effect of NT on AMPA-mediated EPSCs in NT 1 receptor knock-out mice. G, 10 nm (97.1 ± 6.1%, n = 5, p = 0.114 from baseline, p < 0.001 compared with the effect of NT 10 nm in wild-type). H, 100 nm (80.9 ± 1.3%, n = 4, p < 0.001 from baseline, p = 0.068 compared with effect of NT 100 nm in wild-type).

Figure 7.

A, Illustration demonstrates that the current carried by both the AMPA and NMDA receptors, the early- and late-phase components of the EPSC, respectively, was measured 2.5 ms after the stimulus artifact. The NMDA component alone was measured 25 ms after the stimulus artifact (black). The AMPA-mediated current revealed by bath application of NMDA antagonist AP5 decays quickly (gray). Analysis was conducted between minutes 32 and 40 for all panels. B, Bar graph plotting average values of potentiation measured at the two points shown in A. NMDA = 123.4 ± 5.2%, AMPA + NMDA = 116.4 ± 3.5%; n = 8, p = 0.037. C, D, Raw data, single-cell responses to AP5 (50 μm), NMDAR antagonist, and DNQX (10 μm), AMPAR antagonist when measured at different time points. E, F, NT response in a population of VTA dopamine neurons. E, NMDA only (123.4 ± 5.2%). F, Combined AMPA + NMDA peak (116.4 ± 3.5%); n = 8.

Figure 8.

A, A graphic representation of the AAV construct: ChR2 and EYFP cassettes are under control of the CaMKII promoter. B, Horizontal hemisection of mouse brain (50 μm): rostral is at the top of image; midline is along the right side. Neurotrace (blue) was used to visualize background tissue. AAV-induced ChR2-EYFP signal is shown in green (no antibody). TH antibody was visualized on the red channel to highlight dopamine neurons. Scale bar, 500 μm. Inset, VTA dopamine neurons surrounded by ChR2-expressing terminals. Scale bar, 20 μm. Example trace shows the NMDA current optically evoked by aiming a fiber optic directly at the recorded neuron in the VTA and the AMPA current measured in the presence of NMDA antagonist, AP5. Blue bar represents 5 ms flash of blue light. C, D, Comparison of electrically evoked EPSCs (nonspecific, NS) versus EPSCs evoked by optically stimulating LH to VTA fibers. Recordings were performed in separate experiments; however, electrical and optical stimulation was delivered at 0.1 Hz each. Nonspecific terminal stimulation: 119.2 ± 1.3%, n = 6; LH terminal stimulation: 70.1 ± 6.1%, n = 6 (p < 0.001). Analysis was conducted between minutes 32 and 40 in C–F as indicated by shaded regions. E, F, The experiment shown in C and D was repeated by recording alternating electrical and optical responses in the same cell. Each neuron received 0.1 Hz stimulation; however, every other pulse was delivered by a bipolar stimulating electrode or a fiber optic coupled to a laser, such that each method of stimulation was delivered at 0.05 Hz. Nonspecific terminal stimulation: 117.1 ± 5.6%; LH terminal stimulation: 81.8 ± 4.8%, n = 8 (p < 0.001).

Figure 9.

A, A hemisection of a horizontal mouse brain section used for recording the effect of NT-containing LH terminal activation in the VTA. Scale bar, 500 μm. NT-containing neurons in the LH were selectively infected with ChR2 via the Cre-lox system in mice. A double-floxed ChR2 virus was injected into the LH of Nts-Cre mice. Optical stimulation of LH neurotensin-containing axon terminals in the VTA produced NMDA currents (black) and AMPA currents (red). B, Nts1 antagonist, SR48692, reduced optically evoked NMDA currents to 67.2 ± 4.5% (n = 6, p < 0.001). C, SR48692 did not significantly reduce electrically evoked NMDA currents (98.4 ± 1.4%, n = 9, p = 0.963).