Zona incerta neurons projecting to the ventral tegmental area promote action initiation towards feeding

Abstract

Key points: The zona incerta (ZI) and ventral tegmental area (VTA) are brain areas that are both implicated in feeding behaviour. The ZI projects to the VTA, although it has not yet been investigated whether this projection regulates feeding. We experimentally (in)activated the ZI to VTA projection by using dual viral vector technology, and studied the effects on feeding microstructure, the willingness to work for food, general activity and body temperature. Activity of the ZI to VTA projection promotes feeding by facilitating action initiation towards food, as reflected in meal frequency and the willingness to work for food reward, without affecting general activity or directly modulating body temperature. We show for the first time that activity of the ZI to VTA projection promotes feeding, which improves the understanding of the neurobiology of feeding behaviour and body weight regulation.

Abstract: Both the zona incerta (ZI) and the ventral tegmental area (VTA) have been implicated in feeding behaviour. The ZI provides prominent input to the VTA, although it has not yet been investigated whether this projection regulates feeding. Therefore, we investigated the role of ZI to VTA projection neurons in the regulation of several aspects of feeding behaviour. We determined the effects of (in)activation of ZI to VTA projection neurons on feeding microstructure, food-motivated behaviour under a progressive ratio schedule of reinforcement, locomotor activity and core body temperature. To activate or inactivate ZI neurons projecting to the VTA, we used a combination of canine adenovirus-2 in the VTA, as well as Cre-dependent designer receptors exclusively activated by designer drugs (DREADD) or tetanus toxin (TetTox) light chain in the ZI. TetTox-mediated inactivation of ZI to VTA projection neurons reduced food-motivated behaviour and feeding by reducing meal frequency. Conversely, DREADD-mediated chemogenetic activation of ZI to VTA projection neurons promoted food-motivated behaviour and feeding. (In)activation of ZI to VTA projection neurons did not affect locomotor activity or directly regulate core body temperature. Taken together, ZI neurons projecting to the VTA exert bidirectional control overfeeding behaviour. More specifically, activity of ZI to VTA projection neurons facilitate action initiation towards feeding, as reflected in both food-motivated behaviour and meal initiation, without affecting general activity.

Keywords: DREADD; feeding; motivation; tetanus toxin light chain; ventral tegmental area; zona incerta.

Figure 1. TetTox‐GFP is expressed in ZI‐region neurons projecting to the VTA

A, to selectively inactive ZI neurons projecting to the VTA, CAV2Cre was injected into the VTA and Cre‐dependent TetTox‐GFP was injected into the ZI. An AAV‐hSyn‐mCherry virus was injected together with CAV2Cre to visualize the injection site in the VTA. B, immunofluorescence of mCherry (red) in the VTA following virus injection of CAV2Cre/AAV‐hSyn‐mCherry (bregma −5.30 mm). C, TetTox‐GFP mRNA expression in the ZI and the zone medioventral to the ZI, together representing the ZI‐region (bregma −2.12 mm). Because we often observed spread of virus infection to this region which includes the DMH, we use the term ZI‐region to refer to data of rats with proper targeting of the ZI, but in which there was variable viral spread through surrounding brain regions. D, ZI neurons projecting to the VTA stained for GFP. E, ZI projection terminals in the VTA stained for GFP. F, absence of ZI projection terminal staining in the paraventricular thalamus (PVT), following staining for GFP. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2. Effect of inactivation of ZI‐region neurons projecting to the VTA on responding for sucrose under a PR schedule of reinforcement and locomotor activity

Rewards (A), active lever presses (ALPs) (B) and inactive lever presses (ILPs) (C) in PR testing, averaged per week. Baseline (BL): PR performance 1 week before virus injections (average of five sessions is shown). PR testing recommenced 4 weeks after virus injections, and was assessed under ad libitum feeding and food restriction (FR) in the home cage (average of three sessions per week is shown). Statistical analyses were performed by two‐way RM ANOVAs, followed by post hoc analyses. Ad libitum: rewards and ALPs, F _group ≥ 51.274, P< 0.001, post hoc P < 0.01 for all weeks; ILPs, F _group = 10.175, P = 0.008, post hoc P < 0.05 at weeks 4–6. FR: rewards and ALPs, F _group ≥ 21.836, P ≤ 0.001, post hoc P < 0.02 for all weeks; ILPs, F _group = 0.866, P = 0.370. D, locomotor activity in arbitrary units (a.u.) averaged per week. Ad libitum and FR: F _group ≥ 2.683, P ≥ 0.127. E, locomotor activity over 24 h averaged over the weekend data of weeks 6–9. F _group = 2.337, P = 0.152. Data are shown as the mean ± SD. n = 8 for controls and n = 6 for TetTox rats. ^# P < 0.07, ^* P < 0.05, ^** P < 0.01, ^*** P < 0.06 compared to controls. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3. Effect of inactivation of ZI‐region neurons projecting to the VTA on homeostatic feeding

A, chow intake per day, averaged per week, under ad libitum feeding and during food restriction (FR). Baseline (BL): paired t test t _group = 0.775, P = 0.454; ad libitum: RM ANOVA F _group = 22.444, P < 0.001, post hoc P < 0.05 at weeks 3–9. FR: RM ANOVA F _group = 3.697, P = 0.079. B, average chow intake per day during the light and dark phase over the course of ad libitum feeding. RM ANOVA F _{circadian‐phase × group} = 29.066, P < 0.001. Post hoc: P < 0.001 for total and dark, P = 0.004 for light. C, percentage (%) of chow intake during the dark and light phase over the course of ad libitum feeding. RM ANOVA F _{circadian‐phase × group} = 0.210, P = 0.655. F _{circadian‐phase} = 139.55, P < 0.001. Number of meals (D) and (E) meal size averaged over the 24 h weekend data of weeks 6–9. Number of meals: RM ANOVA F _{circadian‐phase × group} = 10.886, P = 0.003. Post hoc P < 0.01 for total and dark, P = 0.090 for light. Meal size: RM ANOVA F _{circadian‐phase × group} = 2.707, P = 0.111. F _group = 4.161, P = 0.064. F, body weight averaged per week. BL: paired t test t _group = 0.004, P = 0.997; ad libitum: RM ANOVA F _{time × group} = 6.813, P = 0.015, post hoc P < 0.05 at weeks 7–11. FR: RM ANOVA F _{time × group} = 1.002, P = 0.353; F _group = 3.708, P = 0.078. G, chow intake per 100 g of body weight per day, averaged per week. BL: paired t test t _group = 0.917, P = 0.379; ad libitum: RM ANOVA F _group = 26.280, P < 0.001, post hoc P < 0.05 at weeks 3–7. FR: RM ANOVA F _group = 0.317, P = 0.585. Data are shown as the mean ± SD. n = 8 for controls and n = 5‐6 for TetTox rats. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 compared to controls. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4. Effect of inactivation of ZI‐region neurons projecting to the VTA on body temperature

A, core body temperature during ad libitum feeding and food restriction (FR), averaged per week. RM ANOVA F _{time × group} = 3.754, P = 0.026. Post hoc P < 0.01 in weeks 3–8, and P < 0.05 in weeks 2 and 9. B, core body temperature over 24 h averaged over the weekend data of weeks 6–9. RM ANOVA total: F _{time × group} = 3.154, P < 0.001; dark: F _group = 17.962, P = 0.001; light: F _group = 3.581, P = 0.083. C and D, correlation between food intake and chow intake during the dark and light phase, respectively. E, average core body temperature during ad libitum feeding and FR. RM ANOVA F _{feeding‐condition × group} = 2.440, P = 0.144. RM ANOVA F _{feeding‐condition} = 51.808, P < 0.001. Weekend data of weeks 6–9 were analysed. n = 8 for controls and n = 5‐6 for TetTox rats. Data are shown as the mean ± SD. A and B, ^** P < 0.01 compared to controls. E, ^*** P < 0.001 for ad libitum vs. FR. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5. DREADD hM3D(Gq)‐mCherry is selectively expressed in ZI neurons projecting to the VTA

A, to selectively inactive ZI neurons projecting to the VTA, CAV2Cre was injected into the VTA and Cre‐dependent DREADD hM3D(Gq)‐mCherry was injected into the ZI. An AAV‐hSyn‐YFP virus was injected together with CAV2Cre to visualize the injection site in the VTA. B, immunofluorescence of DREADD hM3D(Gq)‐mCherry (red) positive neurons in the ZI. C, immunofluorescence of GFP (green) in the VTA following virus injection of CAV2Cre/AAV‐hSyn‐YFP (bregma −5.30 mm). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6. Effect of chemogenetic activation of ZI to VTA projection neurons on responding for sucrose under a PR schedule of reinforcement

Rewards (A), active lever presses (ALPs) (B) and inactive lever presses (ILPs) (C) in PR testing following saline and CNO injection. Statistical analyses were performed using paired t tests. t _treatment ≥ 2.449, P ≤ 0.031 for ALPs and rewards. n = 13. Data are shown as the mean ± SD. ^* P < 0.05 for saline vs. CNO. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7. Effect of chemogenetic activation of ZI to VTA projection neurons on feeding

Effects of CNO vs. saline treatment on (A) cumulative number of meals per hour. RM ANOVA F _{treatment × hour} = 0.863, P = 0.486. F _treatment = 5.256, P = 0.041; (B) first meal interval. Paired t test t _treatment = 3.100, P = 0.009; and (C) size of the first, second and rest of meals. RM ANOVA F _{treatment × hour} = 6.329, P = 0.006. Post hoc P < 0.05 for first meal size. D, satiety ratio (first meal interval/first meal size; a measure of post‐meal satiety). Paired t test t _treatment = 0.268, P = 0.268. E, 0–1 h and 2–6 h cumulative food intake (FI). RM ANOVA F _{treatment × hour} = 7.603, P = 0.017. Post hoc P < 0.05 at 0–1 h. n = 13. Data are shown as the mean ± SD. ^* P < 0.05 and ^** P < 0.01 for saline vs. CNO. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 8. Effect of chemogenetic activation of ZI to VTA projection neurons on locomotor activity and core body temperature

The effect of CNO vs. saline treatment on (A) the delta change in locomotor activity and (B) core body temperature, in the presence and absence of food. RM ANOVA F _treatment ≥ 0.072, P ≥ 0.265. n = 13. Data are shown as the mean ± SD. [Color figure can be viewed at wileyonlinelibrary.com]