Inhibitory basal ganglia nuclei differentially innervate pedunculopontine nucleus subpopulations and evoke opposite motor and valence behaviors

Reviewer #1 (Public review):

Summary:

Fallah and colleagues characterize the connectivity between two basal ganglia output nuclei, the SNr and GPe, and the pedunculopontine nucleus, a brainstem nucleus that is part of the mesencephalic locomotor region. Through a series of systematic electrophysiological studies, they find that these regions target and inhibit different populations of neurons, with anatomical organization. Overall, SNr projects to PPN and inhibits all major cell types, while the GPe inhibits glutamatergic and GABAergic PPN neurons, and preferentially in the caudal part of the nucleus. Optogenetic manipulation of these inputs had opposing effects on behavior - SNr terminals in the PPN drove place aversion, while GPe terminals drove place preference.

Strengths:

This work is a thorough and systematic characterization of a set of relatively understudied circuits. They build on the classic notions of basal ganglia connectivity and suggest a number of interesting future directions to dissect motor control and valence processing in brainstem systems.

Weaknesses:

Characterization of the behavioral effects of manipulations of these PPN input circuits could be further parsed, for a better understanding of the functional consequences of the connections demonstrated in the ephys analyses.

All the cell type recording studies showing subtle differences in the degree of inhibition and anatomical organization of that inhibition suggest a complex effect of general optogenetic manipulation of SNr or GPe terminals in the PPN. It will be important to determine if SNr or GPe inputs onto a particular cell type in PPN are more or less critical for how the locomotion and valence effects are demonstrated here.

Reviewer #2 (Public review):

Summary:

Fallah et al carefully dissect projections from SNr and GPe - two key basal ganglia nuclei - to the PPN, an important brainstem nucleus for motor control. They consider inputs from these two areas onto 3 types of downstream PPN neurons: GABAergic, glutamatergic, and cholinergic neurons. They also carefully map connectivity along the rostrocaudal axis of the PPN.

Strengths:

The slice electrophysiology work is technically well done and provides useful information for further studies of PPN. The optogenetics and behavioral studies are thought-provoking, showing that SNr and GPe projections to PPN play distinct roles in behavior.

Weaknesses:

Although the optogenetics and behavioral studies are intriguing, they are somewhat difficult to fit together into a specific model of circuit function. Perhaps the authors can work to solidify the connection between these two arms of the work. Otherwise, there are a few questions whose answers could add context to the interpretation of these results:

(1) Male and female mice are used, but the authors do not discuss any analysis of sex differences. If there are no sex differences, it is still useful to report data disaggregated by sex in addition to pooled data.

(2) There is some lack of clarity in the current manuscript on the ages used - 2-5 months vs "at least 7 weeks." Is 7 weeks the time of virus injection surgery, then recordings 3 weeks later (at least 10 weeks)? Please clarify if these ages apply equally to electrophysiological and behavioral studies. If the age range used for the test is large, it may be useful to analyze and report if there are age-related effects.

(3) Were any exclusion criteria applied, e.g. to account for missed injections?

(4) 28-34degC is a fairly wide range of temperatures for electrophysiological recording, which could affect kinetics.

(5) It would be good to report the number of mice used for each condition in addition to n=cells. Statistically, it would be preferable not to assume that each cell from the same mouse is an independent measurement and to use a nested ANOVA.

Reviewer #3 (Public review):

Summary:

The study by Fallah et al provides a thorough characterization of the effects of two basal ganglia output pathways on cholinergic, glutamatergic, and GABAergic neurons of the PPN. The authors first found that SNr projections spread over the entire PPN, whereas GPe projections are mostly concentrated in the caudal portion of the nucleus. Then the authors characterized the postsynaptic effects of optogenetically activating these basal ganglia inputs and identified the PPN's cell subtypes using genetically encoded fluorescent reporters. Activation of inputs from the SNr inhibited virtually all PPN neurons. Activation of inputs from the GPe predominantly inhibited glutamatergic neurons in the caudal PPN, and to a lesser extent GABAergic neurons. Finally, the authors tested the effects of activating these inputs on locomotor activity and place preference. SNr activation was found to increase locomotor activity and elicit avoidance of the optogenetic stimulation zone in a real-time place preference task. In contrast, GPe activation reduced locomotion and increased the time in the RTPP stimulation zone.

Strengths:

The evidence of functional connectivity of SNr and GPe neurons with cholinergic, glutamatergic, and GABAergic PPN neurons is solid and reveals a prominent influence of the SNr over the entire PPN output. In addition, the evidence of a GPe projection that preferentially innervates the caudal glutamatergic PPN is unexpected and highly relevant for basal ganglia function.

Opposing effects of two basal ganglia outputs on locomotion and valence through their connectivity with the PPN.

Overall, these results provide an unprecedented cell-type-specific characterization of the effects of basal ganglia inputs in the PPN and support the well-established notion of a close relationship between the PPN and the basal ganglia.

Weaknesses:

The behavioral experiments require further analysis as some motor effects could have been averaged out by analyzing long segments. Additional controls are needed to rule out a motor effect in the real-time place preference task. Importantly, the location of the stimulation is not reported even though this is critical to interpret the behavioral effects.

There are some concerns about the possible recruitment of dopamine neurons in the SNr experiments.

Author Response:

Reviewer #1 (Public review):

Summary:

Fallah and colleagues characterize the connectivity between two basal ganglia output nuclei, the SNr and GPe, and the pedunculopontine nucleus, a brainstem nucleus that is part of the mesencephalic locomotor region. Through a series of systematic electrophysiological studies, they find that these regions target and inhibit different populations of neurons, with anatomical organization. Overall, SNr projects to PPN and inhibits all major cell types, while the GPe inhibits glutamatergic and GABAergic PPN neurons, and preferentially in the caudal part of the nucleus. Optogenetic manipulation of these inputs had opposing effects on behavior - SNr terminals in the PPN drove place aversion, while GPe terminals drove place preference.

Strengths:

This work is a thorough and systematic characterization of a set of relatively understudied circuits. They build on the classic notions of basal ganglia connectivity and suggest a number of interesting future directions to dissect motor control and valence processing in brainstem systems.

We thank the reviewers for these positive comments.

Weaknesses:

Characterization of the behavioral effects of manipulations of these PPN input circuits could be further parsed, for a better understanding of the functional consequences of the connections demonstrated in the ephys analyses.

We will further analyze our behavioral data to reveal more nuanced functional effects.

All the cell type recording studies showing subtle differences in the degree of inhibition and anatomical organization of that inhibition suggest a complex effect of general optogenetic manipulation of SNr or GPe terminals in the PPN. It will be important to determine if SNr or GPe inputs onto a particular cell type in PPN are more or less critical for how the locomotion and valence effects are demonstrated here.

This is a really interesting future direction and we will expand on these points in the discussion.

Reviewer #2 (Public review):

Summary:

Fallah et al carefully dissect projections from SNr and GPe - two key basal ganglia nuclei - to the PPN, an important brainstem nucleus for motor control. They consider inputs from these two areas onto 3 types of downstream PPN neurons: GABAergic, glutamatergic, and cholinergic neurons. They also carefully map connectivity along the rostrocaudal axis of the PPN.

Strengths:

The slice electrophysiology work is technically well done and provides useful information for further studies of PPN. The optogenetics and behavioral studies are thought-provoking, showing that SNr and GPe projections to PPN play distinct roles in behavior.

We appreciate the reviewer’s positive evaluation.

Weaknesses:

Although the optogenetics and behavioral studies are intriguing, they are somewhat difficult to fit together into a specific model of circuit function. Perhaps the authors can work to solidify the connection between these two arms of the work.

We will expand on these topics in the discussion.

(1) Male and female mice are used, but the authors do not discuss any analysis of sex differences. If there are no sex differences, it is still useful to report data disaggregated by sex in addition to pooled data.

While we do not have sufficient n for a well-powered analysis of sex differences in behavior, we find that both male and female mice increase movement in response to SNr axon stimulation and decrease movement in response to GPe axon stimulation. We will expand on this further in the revised manuscript.

(2) There is some lack of clarity in the current manuscript on the ages used - 2-5 months vs "at least 7 weeks." Is 7 weeks the time of virus injection surgery, then recordings 3 weeks later (at least 10 weeks)? Please clarify if these ages apply equally to electrophysiological and behavioral studies. If the age range used for the test is large, it may be useful to analyze and report if there are age-related effects.

7 weeks is the youngest age at which mice used for electrophysiology were injected, and all were used for electrophysiology between 2-5 months. For behavior, the youngest mice used were 11 weeks old at time of behavior (8 weeks old at injection). Mice in the GPe-stimulated condition were 110 ± 7.4 SEM days old and mice in the SNr-stimulated condition 132 ± 23.4 SEM days old. We will add these details to the revised manuscript.

In addition, we have correlated distance traveled at baseline and during stimulation with age for both SNr and GPe stimulated conditions. Baseline distance traveled did not correlate with age, but there was a trend toward more movement during stimulation with older mice in the SNr axon stimulation group. We will discuss this in the revised manuscript.

(3) Were any exclusion criteria applied, e.g. to account for missed injections?

All injection sites and implant sites were within our range of acceptability, so we did not exclude any mice for missed injections.

(4) 28-34degC is a fairly wide range of temperatures for electrophysiological recording, which could affect kinetics.

This is an important consideration. We have checked our main measurement of current amplitude in the condition where we found significant differences between rostral and caudal PPN (SNr to Vglut2 PPN neurons) against temperature and found no correlation (Pearson’s r value = -0.0076). Similarly, we found no correlation between baseline (pre-opto) firing frequency and temperature (r = -0.068).

(5) It would be good to report the number of mice used for each condition in addition to n=cells. Statistically, it would be preferable not to assume that each cell from the same mouse is an independent measurement and to use a nested ANOVA.

For electrophysiology, the number of mice used in each experiment was 6 (3 male, 3 female). In the manuscript ‘N’ represents number of mice and ‘n’ represents number of cells. Because of the unpredictability of how many healthy cells can be recorded from one mouse, our data were planned to be collected with n=cells, and are underpowered for a nested ANOVA. However, rostral and caudal data were collected from the same mice. While we do not have sufficient paired data for each parameter, analyzing one of our main and most important findings with a paired comparison (with biological replicates being mice) shows a statistically significant difference in the inhibitory effect of SNr axon stimulation on firing rate between rostral and caudal glutamatergic neurons (p=0.031, Wilcoxon signed rank test).

Reviewer #3 (Public review):

Summary:

The study by Fallah et al provides a thorough characterization of the effects of two basal ganglia output pathways on cholinergic, glutamatergic, and GABAergic neurons of the PPN. The authors first found that SNr projections spread over the entire PPN, whereas GPe projections are mostly concentrated in the caudal portion of the nucleus. Then the authors characterized the postsynaptic effects of optogenetically activating these basal ganglia inputs and identified the PPN's cell subtypes using genetically encoded fluorescent reporters. Activation of inputs from the SNr inhibited virtually all PPN neurons. Activation of inputs from the GPe predominantly inhibited glutamatergic neurons in the caudal PPN, and to a lesser extent GABAergic neurons. Finally, the authors tested the effects of activating these inputs on locomotor activity and place preference. SNr activation was found to increase locomotor activity and elicit avoidance of the optogenetic stimulation zone in a real-time place preference task. In contrast, GPe activation reduced locomotion and increased the time in the RTPP stimulation zone.

Strengths:

The evidence of functional connectivity of SNr and GPe neurons with cholinergic, glutamatergic, and GABAergic PPN neurons is solid and reveals a prominent influence of the SNr over the entire PPN output. In addition, the evidence of a GPe projection that preferentially innervates the caudal glutamatergic PPN is unexpected and highly relevant for basal ganglia function.

Opposing effects of two basal ganglia outputs on locomotion and valence through their connectivity with the PPN.

Overall, these results provide an unprecedented cell-type-specific characterization of the effects of basal ganglia inputs in the PPN and support the well-established notion of a close relationship between the PPN and the basal ganglia.

We thank the reviewer for their positive comments.

Weaknesses:

The behavioral experiments require further analysis as some motor effects could have been averaged out by analyzing long segments.

We will further analyze our motor effects in the revised manuscript.

Additional controls are needed to rule out a motor effect in the real-time place preference task.

This is an important point. Our use of unilateral stimulation in the RTPP task reduces potential motor effects, and our supplemental videos show that the mice can easily escape and enter the stimulated zone. However, we can't completely rule out a motor component. To delve into this further, we analyzed mouse speed in the RTPP task. We find that in both SNr and GPe stimulation conditions, the maximum speed of the mouse is not different in the stimulated vs unstimulated zone. We will further analyze mouse speed at the transition into and out of the stimulated zone to identify any acute motor effects in this experiment.

Importantly, the location of the stimulation is not reported even though this is critical to interpret the behavioral effects.

The implant locations were generally over the middle-to-rostral PPN and we will clarify this in the revised manuscript. These locations are shown in figure 7B.

There are some concerns about the possible recruitment of dopamine neurons in the SNr experiments.

We are very interested in this possibility and plan to discuss this with more clarity in a revised manuscript.