Role of laterodorsal tegmentum projections to nucleus accumbens in reward-related behaviors

Abstract

The laterodorsal tegmentum (LDT) is associated with reward considering that it modulates VTA neuronal activity, but recent anatomical evidence shows that the LDT also directly projects to nucleus accumbens (NAc). We show that the majority of LDT-NAc inputs are cholinergic, but there is also GABAergic and glutamatergic innervation; activation of LDT induces a predominantly excitatory response in the NAc. Non-selective optogenetic activation of LDT-NAc projections in rats enhances motivational drive and shifts preference to an otherwise equal reward; whereas inhibition of these projections induces the opposite. Activation of these projections also induces robust place preference. In mice, specific activation of LDT-NAc cholinergic inputs (but not glutamatergic or GABAergic) is sufficient to shift preference, increase motivation, and drive positive reinforcement in different behavioral paradigms. These results provide evidence that LDT-NAc projections play an important role in motivated behaviors and positive reinforcement, and that distinct neuronal populations differentially contribute for these behaviors.

Fig. 1

LDT stimulation drives a predominantly excitatory response in the NAc. a Strategy used for LDT-NAc optogenetic manipulation. b Representative immunofluorescence showing YFP staining in the LDT and c in terminals in the NAc; scale bar = 100 μm. d Representative immunofluorescence for eYFP (green) and ChAT, EAAC1 or GAD65/67 (red); scale bar = 50 μm. e Respective quantification of double and triple positive cells, indicative of transfected neurons (n = 6 animals, 1way ANOVA). f Electrophysiological strategy for cell recording in the LDT and in the NAc. NAc neurons were separated into pMSNs, pCINs, and pFS. g LDT neurons increase firing rate in response to optical activation of LDT cell bodies (80 pulses of 10ms at 20 Hz) (n = 5 animals; 25 LDT cells; 1way ANOVA). h 48% of LDT recorded cells increase their firing rate during stimulation. i NAc cells increase firing rate in response to LDT optical stimulation (n = 9 animals; 65 cells; 1way ANOVA). j Around half of recorded cells in the NAc show an increase in the firing rate upon stimulation, 34% present no change and 12% decrease activity. k Heatmap representation of percentage of cell responses in the NAc when LDT terminals are stimulated. Each row represents a neuron. l Average distribution of the firing rate of NAc neurons showing an increase in activity during stimulation period (KS test). m 52% of recorded pMSNs increase their activity (29/56 cells), 34% did not change firing rate (19/56 cells) and 14% decrease their activity (8/56 cells); 67% pCINs (2/3 cells) and 50% pFS (3/6 cells) interneurons increase and 33% pCINs (1/3 cells) and 50% pFS (3/6 cells) interneurons do not change their activity. Values are shown as mean ± s.e.m. **p < 0.01, ***p < 0.001. ac- anterior commissure; 4V: 4th ventricle

Fig. 2

LDT-NAc optical activation induces preference and increases motivation. a Schematic representation of the two-choice task. Pressing stim- lever yields one food pellet and pressing stim+ lever delivers one pellet + optical stimulation of LDT-NAc inputs (80 10 ms pulses at 20 Hz). b Time-course representation of the responses in ChR2 (n = 24) and YFP (n = 12) rats. Optogenetic activation of LDT-NAc terminals focuses responses for the lever associated with the laser-paired reward (stim+) over an otherwise equivalent food reward (stim-) in ChR2 animals, but not in control YFP group. c In pellet extinction conditions, both groups decrease responses for both levers. d In laser extinction conditions, ChR2 animals still manifest preference for the stim+ lever, despite no stimulation being given; YFP animals do not manifest preference. e In the progressive ratio task animals have to increasingly press a lever more times to obtain the same reward (stimulation associated with stim+ lever). f Cumulative presses performed during the progressive ratio task show that ChR2 animals press more on stim+ lever. g Increase in breakpoint for stim+ lever in ChR2 animals, indicative of enhanced motivation. h Food pellets earned during the PR task. i CPP and m RTPP paradigms, in which one chamber is associated with laser stimulation (ON side). j Total time spent in the OFF and ON sides in YFP and ChR2 groups. k Ratio of preference after CPP conditioning and l time difference, showing preference for the ON side on ChR2 but not on YFP animals. n Representative tracks for a ChR2 and a YFP animal during the RTPP. o Percentage and p difference of time spent on the ON and OFF sides, showing preference for the side associated with stimulation. Values are shown as mean ± s.e.m.*refers to difference between ChR2 stim+ and stim- levers, RM 2way ANOVA; ^refers to difference between ChR2 stim+ and YFP stim+ levers, RM 2way ANOVA. *p< 0.05; **p < 0.01; ***p < 0.001

Fig. 3

LDT-NAc optical inhibition decreases preference and motivation. a Schematic representation of the two-choice task. Pressing stim- lever yields one food pellet and pressing stim+ lever delivers a pellet + optical inhibition of LDT-NAc inputs (4s at constant light). b Time-course representation of the responses in NpHR (n = 10) and YFP (n = 4) rats. Optogenetic inhibition of LDT-NAc terminals shifts preference for the non-stimulated lever (stim-) in NpHR animals, but no preference is observed in YFP group. c In pellet extinction conditions, both groups decrease responses for both levers. d In laser extinction conditions, pressing either lever originates the delivery of a pellet, and stim+ no longer yields laser stimulation. NpHR animals still prefer stim- lever; YFP animals do not manifest preference. e Progressive ratio task. f Cumulative presses performed during the progressive ratio task show that NpHR animals press less on stim+ lever. g Decrease in breakpoint for stim+ lever in NpHR animals. h Number of food pellets earned during progressive ratio sessions. i CPP and m RTPP paradigms, in which one chamber is associated with NpHR-mediated inhibition of LDT-NAc projections (ON side). j Total time spent in the OFF and ON sides. k Ratio of preference after CPP conditioning and l time difference on the ON and OFF sides, showing no preference/avoidance in any of the groups. n Representative tracks for an NpHR and a YFP animal during RTPP. o Percentage and p difference of time spent on the ON and OFF sides, showing no difference between groups. Values are shown as mean ± s.e.m. *refers to difference between NpHR stim+ and stim- levers, RM 2way ANOVA; ^refers to difference between NpHR stim+ and YFP stim+ levers, RM 2way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

Activation of LDT-NAc cholinergic terminals increases motivation and induces preference. a Strategy used for optogenetic manipulation of LDT cholinergic terminals in the NAc. DIO-ChR2 was injected in the LDT of ChAT-cre mice, and optical stimulation was performed in the terminals in the NAc. b LDT cholinergic terminals stimulation increase NAc net firing rate (n = 5 animals; 47 cells; RM 1way ANOVA). c The majority of NAc cells show an increase in the firing rate during stimulation, 21% present no change and 9% decrease activity. d Heatmap representation of percentages of cell responses in the NAc when LDT cholinergic terminals are stimulated. e Average distribution firing rate of NAc neurons showing an increase in activity during stimulation period (KS test). f Percentage of responses of each NAc cell type to cholinergic LDT terminal activation. g Time-course representation of the responses in the two-choice task of ChAT-ChR2 (n = 8) and ChAT-eYFP (n = 6) mice. Activation of LDT-NAc cholinergic terminals enhances responses for stim+ lever in ChAT-ChR2 animals, but not in control ChAT-YFP group. h Cumulative presses performed during the progressive ratio task show that ChAT-ChR2 animals press more on stim+ lever. i Increased breakpoint for stim+ lever in ChAT-ChR2 animals, indicative of enhanced motivation. j Number of pellets consumed during progressive ratio sessions. k–l In the CPP, ChAT-ChR2 (n = 6, two animals lost cannula) animals prefer the ON chamber, i.e., the one associated with NAc-LDT cholinergic stimulation, whereas no preference is seen in control group (n=4, two animals lost cannula). m In the RTPP test, ChAT-ChR2 animals also prefer the ON chamber. Values are shown as mean ± s.e.m. *refers to difference between ChAT-ChR2 stim+ and stim- lever, RM 2way ANOVA; ^refers to difference between ChAT-ChR2 stim+ and ChAT-YFP stim+ lever, RM 2way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

Optogenetic activation of LDT-NAc glutamatergic inputs during reward-related behaviors. a Strategy used for optogenetic manipulation of LDT glutamatergic terminals in the NAc. DIO-ChR2 was injected in the LDT of VGluT-cre mice, and optical stimulation was performed in the terminals in the NAc. b Stimulation of glutamatergic terminals increase NAc net firing rate (n = 4 animals; 42 cells; RM 1way ANOVA). c 45% of NAc cells show an increase in the firing rate during stimulation, 32% present no change and 23% decrease activity. d Heatmap representation of percentages of cell responses in the NAc upon stimulation of glutamatergic terminals. e Average firing rate of NAc neurons showing an increase in activity from the baseline during stimulation (KS test). f Percentage of responses of each NAc cell type to glutamatergic LDT terminal activation. g Time-course representation of the responses in the two-choice task of VGluT-ChR2 (n = 11) and VGluT-YFP (n = 6) mice on stim+ and stim- lever. VGluT-ChR2 animals prefer the stim+ over stim- lever; no preference is observed in VGluT-YFP animals. h Cumulative presses performed during the progressive ratio task, showing increased presses in stim+ lever in VGluT-ChR2 animals. i Breakpoint is increased on VGluT-ChR2 animals for the stim+ in comparison to stim- lever, though not statistically different from control group. j Number of pellets consumed during progressive ratio sessions. k Total time spent in the ON and OFF chambers in the CPP test, showing no preference. l Ratio of preference between ON and OFF chambers. m Percentage of time spent in each side of the RTPP test, showing decreased time spent in the ON chamber in VGlut-ChR2 animals (n = 7, four animals lost canula) but not in VGlut-YFP group. Values are shown as mean ± s.e.m. *refers to difference between VGluT-ChR2 stim+ and stim- lever, RM 2way ANOVA; ^refers to difference between VGluT-ChR2 stim+ and VGluT-YFP stim+ lever, RM 2way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 6

Selective activation of LDT-NAc GABAergic inputs during reward-related behaviors. a Strategy used for optogenetic manipulation of LDT glutamatergic terminals in the NAc. DIO-ChR2 was injected in the LDT of VGAT-cre mice, and optical stimulation was performed in the terminals in the NAc. b Stimulation of GABAergic terminals decrease NAc net firing rate (n = 6 animals; 53 cells; RM 1way ANOVA). c The majority of NAc cells decreased the firing rate during stimulation, 27% presented no change and 11% increased activity. d Heatmap representation of percentages cell responses in the NAc upon stimulation of GABAergic terminals. e Average firing rate of NAc neurons showing a decrease in activity from the baseline during optical inhibition (KS test). f Percentage of responses of each NAc cell type to GABAergic LDT terminal activation. g Time-course representation of the responses during the two-choice task of VGAT-ChR2 (n = 7) and VGAT-YFP (n = 10) mice on stim+ and stim- lever. VGAT-ChR2 animals prefer the stim- over stim+ lever; no preference is observed in VGAT-YFP animals. h Cumulative presses performed during the progressive ratio task, showing increased presses in stim- lever in VGAT-ChR2 animals. i Breakpoint for stim+ lever is decreased in VGAT-ChR2 animals, indicative of decreased motivation for the laser-paired reward. j Number of pellets earned during progressive ratio sessions. k Total time spent in ON and OFF chambers in the CPP (n_VGAT-ChR2 = 6, one animal lost cannula; n_VGAT-YFP = 7, four animals lost cannula). l Ratio of preference between ON and OFF chambers in the CPP test, showing no differences between groups. m Percentage of time spent in each side of the RTPP, showing no differences between groups. Values are shown as mean ± s.e.m. *refers to difference between VGAT-ChR2 stim+ and stim- lever, RM 2way ANOVA; ^refers to difference between VGAT-ChR2 stim+ and VGAT-YFP stim+ lever; RM 2way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001