Demonstrating the ability of GABAergic cells in the zona incerta to modulate motivation

Reviewer #1 (Public review):

Summary:

This manuscript by Laura Korobkova and Brian Dias describes an interesting study of the role of GABAergic neurons in the zona incerta (ZI) in incentive motivation for reward.

The authors report that DREADD inhibition of ZI neurons reduced the effort breakpoint in a progressive ratio task, which measures the intensity of incentive motivation to obtain food rewards. In other tests, chemogenetic inhibition did not alter food consumption or memory.

Conversely, DREADD excitation of ZI neurons increased incentive motivation in the progressive ratio task, expressed as a higher breakpoint for food rewards.

Korobkova and Dias report that prior stress exposure to a series of stressors (e.g., forced swim & water submersion, restraint, mild footshock) by itself reduced the breakpoint for food reward under vehicle, though it did not impair the ability to learn an instrumental response. However, DREADD excitation of ZI neurons in previously stressed mice increased the breakpoint to normal levels equivalent to the never-stressed group. This important finding indicates the ability of ZI stimulation to rescue the incentive motivational deficit induced by prior stress.

In fiber photometry studies using vGAT-CRE mice to specifically identify GABA neurons, Korobkova and Dias report that ZI GABA neurons are excited by sensory signals, including neutral cues. However, after reward conditioning, ZI GABA neurons increase their activation to the CS+ cue that predicts reward, but not to the CS- cue that doesn't. ZI neurons also respond in an instrumental reward task during both lever press and reward delivery. The authors conclude that ZI neurons respond to sensory stimuli, but specifically code the motivational significance of reward-related stimuli.

In optogenetic studies, the authors find that ZI GABA neuron stimulation during a reward CS+ enhances motivated responding to obtain reward, particularly in females, but not stimulation outside the CS+. This suggests the ZI stimulation in females may specifically enhance the incentive salience of the CS+, namely the cue's ability to trigger an increase in 'wanting' for the reward. However, that effect was not found here in males.

Altogether, this is a fine contribution to the literature, and the authors deserve congratulations on their study and manuscript.

Strengths:

This is a powerful and creative set of studies that clarifies the roles of ZI neurons in sensory processing and especially in incentive motivation for rewards. The use of multiple methods and test situations to triangulate on reward motivation functions gives a well-rounded perspective on ZI function. The discovery of incentive motivation roles for ZI neurons is intriguing and improves understanding of ZI, which traditionally has been a relatively understudied brain structure. The finding that ZI stimulation may rescue stress-induced deficits in motivation is especially notable and may have therapeutic implications.

Weaknesses:

Minor: This version of the manuscript focuses the introduction and discussion specifically on ZI GABA neurons. The ZI may be primarily GABAergic, but also contains other neurons, and DREADD studies may have used the hSyn promoter, which would impact all types of ZI neurons. Other studies here did more specifically target GABA neurons using vGAT Cre mice and specific targeting. The manuscript might be slightly improved by distinguishing in the discussion a bit more clearly which effects implicate GABA neurons specifically, and which effects might include other neurons too, to more clearly parse out the relative roles of GABA vs broader neuronal populations in ZI.

Reviewer #2 (Public review):

Summary:

This paper describes a study that uses a combination of observational and experimental techniques to investigate the hypothesis that the zona incerta is a neural loci where sensory information is integrated to interpret the motivational value of reward-associated cues. They show that manipulation of GABAergic neurons in this region bidirectionally modulates responding during a progressive ratio test, that activating these neurons recovers motivational deficits incurred by chronic stress, and that they fire in response to reward-associated visual or auditory cues. They also showed that activity in these neurons is not necessary for incentive salience of reward-associated cues, because inactivating them did not prevent Pavlovian-instrumental transfer. However, activating them did enhance responding during the presentation of reward-associated cues in females but not in males.

Strengths:

The study has a very systematic and elegant approach to assess how this region responds first to intrinsic motivation and then to motivation-enhancing effects of reward-associated cues.

Weaknesses:

Males and females are used throughout, but sample sizes are generally too small to make a meaningful interpretation of sex differences (which is not the focus of the study, but is worth bearing in mind). In the last experiment, the lack of discrimination between CS+ and CS- conditions across training for males confounds any interpretation of sex-differences in the outcomes.

The ZI is known to be a region where there is notable convergence of neural inputs from a diverse and heterogenous range of sensory and other cortical inputs. To my knowledge, this is the first study that has directly tested whether it may serve to encode motivational/incentive properties of reward-associated cues. The outcomes are not definitive - it appears that they are sufficient but not necessary. However, this study represents an important first step - the ZI also has notable heterogeneity in the genetic identity of neurons, and properly dissecting the function of ZI microcircuits will likely require characterising function based on more than one molecular marker. This is addressed by the authors in the discussion.

In summary, this study will have a significant impact on our understanding of how motivation is calculated based on complex environmental signals.

Reviewer #3 (Public review):

Summary:

The authors investigated the role of the zona incerta in motivation and cue-reward associations. Using chemogenetic and optogenetic manipulations of the ZI, they altered motivation in cued and uncued variants of the progressive ratio task and rescued deficits in motivation induced by chronic stress. They further use fiber photometry to demonstrate that the ZI tracks the formation of cue-reward associations.

Strengths:

(1) The authors fill an important gap in the literature linking sensory input to motivation via the zona incerta.

(2) The authors demonstrate that ZI tracks cue value rather than just tracking sensory input.

(3) The authors demonstrate that the ZI excitation rescues stress-induced suppression of motivation.

(4) The authors perform several important control tasks, demonstrating that their findings are not a result of alterations in locomotor activity, food consumption, or memory.

Weaknesses:

In Figure 1D and E (inhibitory vs excitatory DREADDS), the control groups in the Gi group appear to have more elevated breakpoints than the control groups in the Gq group, although a statistical comparison between the two is not reported. It is not clear if this is because the two groups were given a different reinforcement schedule, this should be made clearer.

In Figure 1E, it is important to note that although the authors found a significant planned comparison between Gq VEH and Gq CNO, the interaction was not significant, nor were comparisons to mice injected with control virus. Thus, activation of ZI GABA neurons appears to be a relatively weak effect.

In Figure 5, the authors see what is likely a significant difference in lever presses during acclimation between the Gi and GFP groups, which they state is an expected difference. However, it is difficult to see why this would be expected. While Gi:CNO manipulation yielded lower breakpoints in Figure 1D, it did not yield lower FR1 responding for food in Fig S3 (although this was FR1 for food dispenser visits rather than lever press). One reason I ask is that the authors highlight the differences in CS+/CS- between groups, but the biggest difference between groups appears to be in acclimation, which may be driving the group x block interaction.

In Figure 6, the authors demonstrate that optogenetic stimulation during cue light increases the breakpoint in females, but not in males. They suggest that this may be because the males did not sufficiently discriminate the cue light before optogenetic manipulation began. If this were the case, then the authors would need to use "cue discrimination" as a factor to determine if it is a better predictor than sex.

The authors' work demonstrates that chemogenetic inhibition of GABAergic ZI cells reduces uncued motivation for reward but enhances cued responses under extinction. The authors state that this is a paradoxical finding that suggests that the ZI operates within a redundant motivation network. However, a critical difference between the two tasks is that one measures motivation for food while the other measures persistent responding under food extinction, which are not the same process. Thus, a simpler explanation is that ZI inhibition reduces motivation and impairs extinction.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript by Laura Korobkova and Brian Dias describes an interesting study of the role of GABAergic neurons in the zona incerta (ZI) in incentive motivation for reward.

The authors report that DREADD inhibition of ZI neurons reduced the effort breakpoint in a progressive ratio task, which measures the intensity of incentive motivation to obtain food rewards. In other tests, chemogenetic inhibition did not alter food consumption or memory.

Conversely, DREADD excitation of ZI neurons increased incentive motivation in the progressive ratio task, expressed as a higher breakpoint for food rewards.

Korobkova and Dias report that prior stress exposure to a series of stressors (e.g., forced swim & water submersion, restraint, mild footshock) by itself reduced the breakpoint for food reward under vehicle, though it did not impair the ability to learn an instrumental response. However, DREADD excitation of ZI neurons in previously stressed mice increased the breakpoint to normal levels equivalent to the never-stressed group. This important finding indicates the ability of ZI stimulation to rescue the incentive motivational deficit induced by prior stress.

In fiber photometry studies using vGAT-CRE mice to specifically identify GABA neurons, Korobkova and Dias report that ZI GABA neurons are excited by sensory signals, including neutral cues. However, after reward conditioning, ZI GABA neurons increase their activation to the CS+ cue that predicts reward, but not to the CS- cue that doesn't. ZI neurons also respond in an instrumental reward task during both lever press and reward delivery. The authors conclude that ZI neurons respond to sensory stimuli, but specifically code the motivational significance of reward-related stimuli.

In optogenetic studies, the authors find that ZI GABA neuron stimulation during a reward CS+ enhances motivated responding to obtain reward, particularly in females, but not stimulation outside the CS+. This suggests the ZI stimulation in females may specifically enhance the incentive salience of the CS+, namely the cue's ability to trigger an increase in 'wanting' for the reward. However, that effect was not found here in males.

Altogether, this is a fine contribution to the literature, and the authors deserve congratulations on their study and manuscript.

Strengths:

This is a powerful and creative set of studies that clarifies the roles of ZI neurons in sensory processing and especially in incentive motivation for rewards. The use of multiple methods and test situations to triangulate on reward motivation functions gives a well-rounded perspective on ZI function. The discovery of incentive motivation roles for ZI neurons is intriguing and improves understanding of ZI, which traditionally has been a relatively understudied brain structure. The finding that ZI stimulation may rescue stress-induced deficits in motivation is especially notable and may have therapeutic implications.

Weaknesses:

Minor: This version of the manuscript focuses the introduction and discussion specifically on ZI GABA neurons. The ZI may be primarily GABAergic, but also contains other neurons, and DREADD studies may have used the hSyn promoter, which would impact all types of ZI neurons. Other studies here did more specifically target GABA neurons using vGAT Cre mice and specific targeting. The manuscript might be slightly improved by distinguishing in the discussion a bit more clearly which effects implicate GABA neurons specifically, and which effects might include other neurons too, to more clearly parse out the relative roles of GABA vs broader neuronal populations in ZI.

The current version of our manuscript notes “Our chemogenetic manipulations targeted all GABAergic ZI neurons, and emerging evidence suggests molecular heterogeneity within this population that may map onto distinct functional roles (Arena et al., 2024; Wilt et al., 2025). Future work to first profile the molecular heterogeneity of GABAergic cells in the ZI and then using intersectional strategies to target defined ZI sub-populations will be essential to providing a more nuanced view of ZI GABAergic influences on motivation.” Our revision will make sure to discuss newer literature demonstrating cellular heterogeneity of ZI and emphasize that this heterogeneity will provide more nuanced contributions of ZI cells beyond the studied GABAergic population on motivation.

Reviewer #2 (Public review):

Summary:

This paper describes a study that uses a combination of observational and experimental techniques to investigate the hypothesis that the zona incerta is a neural loci where sensory information is integrated to interpret the motivational value of reward-associated cues. They show that manipulation of GABAergic neurons in this region bidirectionally modulates responding during a progressive ratio test, that activating these neurons recovers motivational deficits incurred by chronic stress, and that they fire in response to reward-associated visual or auditory cues. They also showed that activity in these neurons is not necessary for incentive salience of reward-associated cues, because inactivating them did not prevent Pavlovian-instrumental transfer. However, activating them did enhance responding during the presentation of reward-associated cues in females but not in males.

Strengths:

The study has a very systematic and elegant approach to assess how this region responds first to intrinsic motivation and then to motivation-enhancing effects of reward-associated cues.

Weaknesses:

Males and females are used throughout, but sample sizes are generally too small to make a meaningful interpretation of sex differences (which is not the focus of the study, but is worth bearing in mind). In the last experiment, the lack of discrimination between CS+ and CS- conditions across training for males confounds any interpretation of sex-differences in the outcomes.

The goal of our study was not to determine sex differences in motivation mediated by the ZI but rather demonstrate a general role of GABAergic cells in the ZI on motivation. As such, while all our experiments used male and female mice and our statistical analyses did not uncover any sex differences in most experiments, our sample sizes of each sex are too small to definitively make any statements about sex differences. As noted already in our Discussion, the sex-specific cue-utilization behavioral strategy in our optogenetic experiment warrants further investigation as a contributing factor to motivation that may or may not be influenced by GABAergic cells in the ZI. It bears mentioning that, to our knowledge, none of the recently published literature on the role of the zona incerta in learning, memory and appetitive behavior that is cited in this manuscript (including our own prior work) has uncovered sex differences in the contributions of the zona incerta to these behaviors.

The ZI is known to be a region where there is notable convergence of neural inputs from a diverse and heterogenous range of sensory and other cortical inputs. To my knowledge, this is the first study that has directly tested whether it may serve to encode motivational/incentive properties of reward-associated cues. The outcomes are not definitive - it appears that they are sufficient but not necessary. However, this study represents an important first step - the ZI also has notable heterogeneity in the genetic identity of neurons, and properly dissecting the function of ZI microcircuits will likely require characterising function based on more than one molecular marker. This is addressed by the authors in the discussion.

In summary, this study will have a significant impact on our understanding of how motivation is calculated based on complex environmental signals.

Reviewer #3 (Public review):

Summary:

The authors investigated the role of the zona incerta in motivation and cue-reward associations. Using chemogenetic and optogenetic manipulations of the ZI, they altered motivation in cued and uncued variants of the progressive ratio task and rescued deficits in motivation induced by chronic stress. They further use fiber photometry to demonstrate that the ZI tracks the formation of cue-reward associations.

Strengths:

(1) The authors fill an important gap in the literature linking sensory input to motivation via the zona incerta.

(2) The authors demonstrate that ZI tracks cue value rather than just tracking sensory input.

(3) The authors demonstrate that the ZI excitation rescues stress-induced suppression of motivation.

(4) The authors perform several important control tasks, demonstrating that their findings are not a result of alterations in locomotor activity, food consumption, or memory.

Weaknesses:

In Figure 1D and E (inhibitory vs excitatory DREADDS), the control groups in the Gi group appear to have more elevated breakpoints than the control groups in the Gq group, although a statistical comparison between the two is not reported. It is not clear if this is because the two groups were given a different reinforcement schedule, this should be made clearer.

In our revision, we will be sure to insert language re-emphasizing that the Gi and Gq experiments were performed using different reinforcement schedules, FR3 and FR1, respectively.

In Figure 1E, it is important to note that although the authors found a significant planned comparison between Gq VEH and Gq CNO, the interaction was not significant, nor were comparisons to mice injected with control virus. Thus, activation of ZI GABA neurons appears to be a relatively weak effect.

In our revision, we will insert language to acknowledge that reducing motivation after inhibiting GABAergic ZI cell activity is stronger than increasing motivation seen after stimulating the activity of these cells.

In Figure 5, the authors see what is likely a significant difference in lever presses during acclimation between the Gi and GFP groups, which they state is an expected difference. However, it is difficult to see why this would be expected. While Gi:CNO manipulation yielded lower breakpoints in Figure 1D, it did not yield lower FR1 responding for food in Fig S3 (although this was FR1 for food dispenser visits rather than lever press). One reason I ask is that the authors highlight the differences in CS+/CS- between groups, but the biggest difference between groups appears to be in acclimation, which may be driving the group x block interaction.

In a revision, we will revise the language to state that the acclimation difference that is more pronounced in the GFP control group vs the Gi group is to be expected because we had already shown that inhibition of GABAergic cells in the ZI would reduce lever pressing. We will also report a planned comparison of CS+ versus CS− responding that omits the acclimation block, which shows discrimination between sound (CS+) and light (CS−) in the Gi group but not in the GFP group, confirming that the cue effect is not driven by the acclimation difference. We will also note that acclimation responding is non-reinforced and effortful, whereas FR1 dispenser visits (Fig. S3) are reinforced and low-effort, which is why the two measures dissociate.

In Figure 6, the authors demonstrate that optogenetic stimulation during cue light increases the breakpoint in females, but not in males. They suggest that this may be because the males did not sufficiently discriminate the cue light before optogenetic manipulation began. If this were the case, then the authors would need to use "cue discrimination" as a factor to determine if it is a better predictor than sex.

In a revision, we will add an analysis that includes cue discrimination as a factor, to test whether it is a better predictor of the optogenetic effect on breakpoint than sex.

The authors' work demonstrates that chemogenetic inhibition of GABAergic ZI cells reduces uncued motivation for reward but enhances cued responses under extinction. The authors state that this is a paradoxical finding that suggests that the ZI operates within a redundant motivation network. However, a critical difference between the two tasks is that one measures motivation for food while the other measures persistent responding under food extinction, which are not the same process. Thus, a simpler explanation is that ZI inhibition reduces motivation and impairs extinction.

Our revision will include discussion of this important point and tie it into our previous work (Venkataraman et al. 2019 and 2021 – cited in this version) that included extinction-like protocols, albeit in classical (not operant) conditioning protocols.