The role of anterior insular cortex inputs to dorsolateral striatum in binge alcohol drinking
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Evaluation Summary:
Haggerty et al. reported findings examining how changes in brain function are involved in alcohol binge drinking, with a selective focus on the synaptic and circuit alterations that occur in the anterior insular cortex inputs within the dorsolateral striatum. They show that chronic alcohol drinking produces glutamatergic synaptic adaptations and by stimulating this circuit, binge drinking could be reduced without altering either water consumption or general performance for select reinforcing, anxiogenic or locomotor behaviors. The results of this study may specifically improve our understanding of the neurocircuitry mediating a common alcohol use disorder associated behavior referred to as "front-loading" or excessive drinking during the very beginning of the session.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
How does binge drinking alcohol change synaptic function, and do these changes maintain binge consumption? The anterior insular cortex (AIC) and dorsolateral striatum (DLS) are brain regions implicated in alcohol use disorder. In male, but not female mice, we found that binge drinking alcohol produced glutamatergic synaptic adaptations selective to AIC inputs within the DLS. Photoexciting AIC→DLS circuitry in male mice during binge drinking decreased alcohol, but not water consumption and altered alcohol drinking mechanics. Further, drinking mechanics alone from drinking session data predicted alcohol-related circuit changes. AIC→DLS manipulation did not alter operant, valence, or anxiety-related behaviors. These findings suggest that alcohol-mediated changes at AIC inputs govern behavioral sequences that maintain binge drinking and may serve as a circuit-based biomarker for the development of alcohol use disorder.
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Author Response
Reviewer #2 (Public Review):
This is an interesting and well-performed study that adds to the literature base. The authors investigated the role of a discrete brain pathway in binge drinking of alcohol. They adopted a multidisciplinary approach that overall suggested that alcohol-induced changes at synapses of anterior insula (AI) cortex inputs to the dorsolateral striatum (DLS) maintain binge drinking. Further, they suggest this may be a biomarker for the development of alcohol use disorder (AUD).
Strengths:
- Extends previous studies and builds further evidence for AI→DLS involvement in aberrant alcohol intake.
- Adopts elegant approaches to isolate the defined connections. This included in vivo optogenetic stimulations (both open and closed loop), recording of defined synapses in slice preparations, applying in …
Author Response
Reviewer #2 (Public Review):
This is an interesting and well-performed study that adds to the literature base. The authors investigated the role of a discrete brain pathway in binge drinking of alcohol. They adopted a multidisciplinary approach that overall suggested that alcohol-induced changes at synapses of anterior insula (AI) cortex inputs to the dorsolateral striatum (DLS) maintain binge drinking. Further, they suggest this may be a biomarker for the development of alcohol use disorder (AUD).
Strengths:
- Extends previous studies and builds further evidence for AI→DLS involvement in aberrant alcohol intake.
- Adopts elegant approaches to isolate the defined connections. This included in vivo optogenetic stimulations (both open and closed loop), recording of defined synapses in slice preparations, applying in vivo optogenetic stimulation parameters to isolated brain slices
- Well-controlled for the most part, although at times the authors assert "specific" effects without unequivocal proof. For example, the insula also projects to the ventral striatum and this pathway has been implicated in regulation of alcohol intake in rodent models (Jaramillo et al., 2018), and is activated in heavy drinking humans during high threat related alcohol cue presentation (Grodin et al., 2018).
- Measures the microstructure of drinking behavior in subjects.
- Employed an artificial neural network and machine learning to interrogate data. After training the network it could predict both the fluid consumed (water vs alcohol) and the virus type based on drinking microstructure data.
- Applied a series of behavioral tests to confirm that stimulating the defined pathway was not in and of itself reinforcing, anxiogenic or altered locomotion.
Weaknesses:
- Only used male mice, in humans binge drinking in females is a major problem and rates of AUD between males and females have been converging in recent times (Grant et al., 2015).
We took age-matched female mice that were injected with AAV-ChR2 into AIC and had them undergo the same 3 weeks of Drinking in the Dark to replicate the male data displayed in Figure 1 with an experimental focus on AIC inputs. We then performed whole cell patch clamp electrophysiology in DLS brain slices from these female mice. We measured optically evoked input-output responses (oEPSCs), AMPA/NMDA current ratios (oNMDA/oAMPA), and paired pulse ratios (oPPR). These data are presented in supplemental figure 4. In contrast to males, we did not observe any effect of alcohol consumption on AIC inputs into the DLS of female mice compared to males. We also combined both male and female datasets to statistically determine if we had sex differences for these specific measures by the existence of a main effect and/or a sex x fluid interaction. We report these statistics in text from lines 180 to 195, where we note that we did not have a sex x fluid effect for oEPSCs but did note that we had a sex x fluid effect for our oNMDA/oAMPA synaptic plasticity measure. This finding further justifies the behavioral data and circuit manipulations being conducted in solely male mice.
While this is a fascinating sex difference and important data for the field, this manuscript is not specifically about exploring sex differences per se. We believe we have done our due diligence and correctly reported the existence of sex differences, or the possibility of sex differences, but the electrophysiological findings that we later modulate in vivo are only present in males. We point out that future work is needed to determine the contribution of circuit-specific changes in females at these synapses. Ultimately it will take much more work to fully elucidate sex difference circuit-specific mechanisms that we feel are far beyond the scope of this manuscript.
- At times over-interpreted, especially with regards to specificity.
We are not exactly sure what the reviewer is referring to with “regards to specificity,” but we have done our best to address what we think they are asking and hope that we have adequately addressed this critique. We added sentences (lines 173-178) regarding alcohol-induced plasticity at other inputs to DLS that were not tested and (lines 442 - 446) how we are not sure whether these synapses control consumption of other non-alcohol substances (but point out our prior sucrose drinking data from Muñoz et al., Nat. Comm. 2018).
- Lacks a mechanism, although the authors do acknowledge this.
This is just a first step towards discovering a mechanism. We previously identified an unusually alcohol-sensitive synapse and are now elucidating its behavioral role and some associated plasticity at that synapse that may be part of a mechanism. With our new single session alcohol data to compare our 3 week drinking data to, we are closer to beginning the process of discovering a mechanism. Additional work that is beyond the scope of this manuscript is needed.
- I would like some more discussion about the potential for this to be a biomarker in humans.
We have removed language in the body of the manuscript and expanded on the implications of our findings at the end of our results and discussion from lines 514 to 548.
Reviewer #3 (Public Review):
Haggerty et al. assess how the projection from the agranular insular cortex to the dorsolateral striatum contributes to binge drinking in mice. The authors use whole-cell patch-clamp electrophysiology to examine synaptic adaptations following binge drinking (Drinking-in-the-Dark) in male mice, finding a constellation of changes that include increased AMPA and NMDA receptor function at insula synapses onto striatal projection neurons. They go on to assess a causal role for this projection in regulating binge drinking using optogenetics, finding that stimulating insula->striatal transmission in vivo reduces total ethanol consumed during DID, along with several specific behavioral measurements of drinking microstructure. One of the most interesting of these findings is a decrease in "front-loading", or drinking during the very beginning of the session, a phenotype that has been associated with problematic drinking and alcohol use disorder in humans. Finally, the authors use machine learning to build a predictive model that can reliably discern stimulated mice from controls. These studies improve our understanding of the neurocircuitry that mediates binge drinking and synaptic and circuit adaptations that occur following binge drinking. Experiments are blinded and performed in a rigorous manner, including physiological validation experiments in support of the in vivo optogenetic manipulation. Despite many strengths, there are significant limitations and gaps in the electrophysiology studies included in this version of the manuscript. As acknowledged by the authors, there are curious findings that are seemingly at odds with each other, and further studies addressing cell type specificity and/or feedforward inhibition would significantly improve the interpretation of this work. Furthermore, the manuscript would be significantly improved by an expanded Introduction containing more specific background information along with a standalone Discussion to place these findings within the broader literature. Lastly, a major limitation of these studies is the low number of mice used for the in vivo optogenetic control experiments and the exclusion of female mice throughout.
Major concerns:
- Expanded Introduction and Discussion. The Introduction does not discuss and/or downplays historical literature investigating neuroadaptations following binge drinking. Studies examining changes in glutamate receptor function within striatal circuits should be discussed in greater detail, rather than the broad pass and review citation included. Behavioral studies examining how the function of the insula and DLS regulate ethanol exposure should also be discussed, especially including work examining the insula to accumbens pathway. It would also be worthwhile to reference human studies implicating the insula and DLS in AUDs.
We have expanded the introduction and discussion to include these topics.
- It is difficult to form a comprehensive picture of the electrophysiological changes reported in Figure 1. The data seems to indicate increased AMPAR function, even more increased NMDAR function, decreased glutamate release probability, and decreased population spikes. These conflicting findings are acknowledged and there are two possible factors mentioned in the manuscript - differential engagement of MSN populations and changes in feedforward inhibition through local interneurons. I disagree with the authors' dismissal of potential MSN subtype-specific effects contributing to these discrepancies. Although AIC inputs innervate D1 and D2 MSNs comparably under control conditions, it is quite possible that the pathways are differentially altered following DID, as has been observed in many reports of alcohol or drug exposure (e.g. Cheng et al. Biological Psychiatry 2017). On the other hand, I wholeheartedly agree with the authors that AIC-driven feedforward inhibition through local interneurons (or even MSNs) could explain the curious divergence between the synaptic and population-level changes depicted in Figure 1. I think additional experiments addressing to help connect the dots are critical in interpreting the changes described in this manuscript. The authors could consider targeted recordings from specific cell types (e.g. D1, D2, and/or interneurons), measurements of AMPA/NMDA receptor subunit stoichiometry, and/or additional experiments in conditions where feedforward transmission is blocked (e.g. PTX or TTX/4AP).
The reviewer has excellent points that will help elucidate a mechanism. Many of these suggestions are planned experiments in our laboratory, but are, in our opinion, beyond the scope of the present manuscript. Please see our response to Reviewer #2’s 3rd stated weakness. We have revised the text to incorporate some of the points raised here.
- N=2 mice in the ICSS experiment in Figure 4J is not sufficient to interpret, and including error bars on this data set is misleading. There also appears to be a difference in distance traveled between GFP and ChR2 mice in Figure 4C, but statistics are not reported. It is also hard to understand what that might mean given the way these data are normalized.
For this revised manuscript we reran this experiment with 6 animals per group and updated Figure 4 I and J and the accompanying methods section titled “Intracranial self-stimulation” to reflect the change. We also note that the new, correctly powered experiment confirmed the previous claim that AIC inputs to the DLS do not modulate operant responding behaviors.
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Evaluation Summary:
Haggerty et al. reported findings examining how changes in brain function are involved in alcohol binge drinking, with a selective focus on the synaptic and circuit alterations that occur in the anterior insular cortex inputs within the dorsolateral striatum. They show that chronic alcohol drinking produces glutamatergic synaptic adaptations and by stimulating this circuit, binge drinking could be reduced without altering either water consumption or general performance for select reinforcing, anxiogenic or locomotor behaviors. The results of this study may specifically improve our understanding of the neurocircuitry mediating a common alcohol use disorder associated behavior referred to as "front-loading" or excessive drinking during the very beginning of the session.
(This preprint has been reviewed by eLife. We …
Evaluation Summary:
Haggerty et al. reported findings examining how changes in brain function are involved in alcohol binge drinking, with a selective focus on the synaptic and circuit alterations that occur in the anterior insular cortex inputs within the dorsolateral striatum. They show that chronic alcohol drinking produces glutamatergic synaptic adaptations and by stimulating this circuit, binge drinking could be reduced without altering either water consumption or general performance for select reinforcing, anxiogenic or locomotor behaviors. The results of this study may specifically improve our understanding of the neurocircuitry mediating a common alcohol use disorder associated behavior referred to as "front-loading" or excessive drinking during the very beginning of the session.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Haggerty et al. reported findings that complement excellent previous work by the same group further exploring the mechanisms mediating binge alcohol drinking. The authors used a combination of ex-vivo electrophysiology and optogenetic techniques with several behavioral procedures to demonstrate that (1) binge alcohol drinking produces glutamatergic synaptic adaptations selective to anterior insular cortex (AIC) inputs within the dorsolateral striatum (DLS); and (2) AIC→DLS projection mediates alcohol binge drinking but not water consumption. Moreover, optogenetic manipulation of AIC→DLS pathway does not alter operant or anxiety-like behaviors. In general, this is an important study and the selective investigation of the AIC→DLS projection is critical to the field.
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Reviewer #2 (Public Review):
This is an interesting and well-performed study that adds to the literature base. The authors investigated the role of a discrete brain pathway in binge drinking of alcohol. They adopted a multidisciplinary approach that overall suggested that alcohol-induced changes at synapses of anterior insula (AI) cortex inputs to the dorsolateral striatum (DLS) maintain binge drinking. Further, they suggest this may be a biomarker for the development of alcohol use disorder (AUD).
Strengths:
1. Extends previous studies and builds further evidence for AI→DLS involvement in aberrant alcohol intake.
2. Adopts elegant approaches to isolate the defined connections. This included in vivo optogenetic stimulations (both open and closed loop), recording of defined synapses in slice preparations, applying in vivo optogenetic …Reviewer #2 (Public Review):
This is an interesting and well-performed study that adds to the literature base. The authors investigated the role of a discrete brain pathway in binge drinking of alcohol. They adopted a multidisciplinary approach that overall suggested that alcohol-induced changes at synapses of anterior insula (AI) cortex inputs to the dorsolateral striatum (DLS) maintain binge drinking. Further, they suggest this may be a biomarker for the development of alcohol use disorder (AUD).
Strengths:
1. Extends previous studies and builds further evidence for AI→DLS involvement in aberrant alcohol intake.
2. Adopts elegant approaches to isolate the defined connections. This included in vivo optogenetic stimulations (both open and closed loop), recording of defined synapses in slice preparations, applying in vivo optogenetic stimulation parameters to isolated brain slices
3. Well-controlled for the most part, although at times the authors assert "specific" effects without unequivocal proof. For example, the insula also projects to the ventral striatum and this pathway has been implicated in regulation of alcohol intake in rodent models (Jaramillo et al., 2018), and is activated in heavy drinking humans during high threat related alcohol cue presentation (Grodin et al., 2018).
4. Measures the microstructure of drinking behavior in subjects.
5. Employed an artificial neural network and machine learning to interrogate data. After training the network it could predict both the fluid consumed (water vs alcohol) and the virus type based on drinking microstructure data.
6. Applied a series of behavioral tests to confirm that stimulating the defined pathway was not in and of itself reinforcing, anxiogenic or altered locomotion.Weaknesses:
1. Only used male mice, in humans binge drinking in females is a major problem and rates of AUD between males and females have been converging in recent times (Grant et al., 2015).
2. At times over-interpreted, especially with regards to specificity.
3. Lacks a mechanism, although the authors do acknowledge this.
4. I would like some more discussion about the potential for this to be a biomarker in humans. -
Reviewer #3 (Public Review):
Haggerty et al. assess how the projection from the agranular insular cortex to the dorsolateral striatum contributes to binge drinking in mice. The authors use whole-cell patch-clamp electrophysiology to examine synaptic adaptations following binge drinking (Drinking-in-the-Dark) in male mice, finding a constellation of changes that include increased AMPA and NMDA receptor function at insula synapses onto striatal projection neurons. They go on to assess a causal role for this projection in regulating binge drinking using optogenetics, finding that stimulating insula->striatal transmission in vivo reduces total ethanol consumed during DID, along with several specific behavioral measurements of drinking microstructure. One of the most interesting of these findings is a decrease in "front-loading", or drinking …
Reviewer #3 (Public Review):
Haggerty et al. assess how the projection from the agranular insular cortex to the dorsolateral striatum contributes to binge drinking in mice. The authors use whole-cell patch-clamp electrophysiology to examine synaptic adaptations following binge drinking (Drinking-in-the-Dark) in male mice, finding a constellation of changes that include increased AMPA and NMDA receptor function at insula synapses onto striatal projection neurons. They go on to assess a causal role for this projection in regulating binge drinking using optogenetics, finding that stimulating insula->striatal transmission in vivo reduces total ethanol consumed during DID, along with several specific behavioral measurements of drinking microstructure. One of the most interesting of these findings is a decrease in "front-loading", or drinking during the very beginning of the session, a phenotype that has been associated with problematic drinking and alcohol use disorder in humans. Finally, the authors use machine learning to build a predictive model that can reliably discern stimulated mice from controls. These studies improve our understanding of the neurocircuitry that mediates binge drinking and synaptic and circuit adaptations that occur following binge drinking. Experiments are blinded and performed in a rigorous manner, including physiological validation experiments in support of the in vivo optogenetic manipulation. Despite many strengths, there are significant limitations and gaps in the electrophysiology studies included in this version of the manuscript. As acknowledged by the authors, there are curious findings that are seemingly at odds with each other, and further studies addressing cell type specificity and/or feedforward inhibition would significantly improve the interpretation of this work. Furthermore, the manuscript would be significantly improved by an expanded Introduction containing more specific background information along with a standalone Discussion to place these findings within the broader literature. Lastly, a major limitation of these studies is the low number of mice used for the in vivo optogenetic control experiments and the exclusion of female mice throughout.
Major concerns:
Expanded Introduction and Discussion. The Introduction does not discuss and/or downplays historical literature investigating neuroadaptations following binge drinking. Studies examining changes in glutamate receptor function within striatal circuits should be discussed in greater detail, rather than the broad pass and review citation included. Behavioral studies examining how the function of the insula and DLS regulate ethanol exposure should also be discussed, especially including work examining the insula to accumbens pathway. It would also be worthwhile to reference human studies implicating the insula and DLS in AUDs.
It is difficult to form a comprehensive picture of the electrophysiological changes reported in Figure 1. The data seems to indicate increased AMPAR function, even more increased NMDAR function, decreased glutamate release probability, and decreased population spikes. These conflicting findings are acknowledged and there are two possible factors mentioned in the manuscript - differential engagement of MSN populations and changes in feedforward inhibition through local interneurons. I disagree with the authors' dismissal of potential MSN subtype-specific effects contributing to these discrepancies. Although AIC inputs innervate D1 and D2 MSNs comparably under control conditions, it is quite possible that the pathways are differentially altered following DID, as has been observed in many reports of alcohol or drug exposure (e.g. Cheng et al. Biological Psychiatry 2017). On the other hand, I wholeheartedly agree with the authors that AIC-driven feedforward inhibition through local interneurons (or even MSNs) could explain the curious divergence between the synaptic and population-level changes depicted in Figure 1. I think additional experiments addressing to help connect the dots are critical in interpreting the changes described in this manuscript. The authors could consider targeted recordings from specific cell types (e.g. D1, D2, and/or interneurons), measurements of AMPA/NMDA receptor subunit stoichiometry, and/or additional experiments in conditions where feedforward transmission is blocked (e.g. PTX or TTX/4AP).
N=2 mice in the ICSS experiment in Figure 4J is not sufficient to interpret, and including error bars on this data set is misleading. There also appears to be a difference in distance traveled between GFP and ChR2 mice in Figure 4C, but statistics are not reported. It is also hard to understand what that might mean given the way these data are normalized.
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