Neuronal glutamate transporters control reciprocal inhibition and gain modulation in D1 medium spiny neurons
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This manuscript reports important findings that help to understand the function of glutamate transporters and their effects on synaptic function at D1- and D2-MSNs within the dorsolateral striatum. These findings were evaluated to be of interest and well-executed. Overall, the majority of claims are supported by high quality data, but the evidence for some underlying mechanisms and region specificity were incomplete in the manuscript's current form.
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Abstract
Understanding the function of glutamate transporters has broad implications for explaining how neurons integrate information and relay it through complex neuronal circuits. Most of what is currently known about glutamate transporters, specifically their ability to maintain glutamate homeostasis and limit glutamate diffusion away from the synaptic cleft, is based on studies of glial glutamate transporters. By contrast, little is known about the functional implications of neuronal glutamate transporters. The neuronal glutamate transporter EAAC1 is widely expressed throughout the brain, particularly in the striatum, the primary input nucleus of the basal ganglia, a region implicated with movement execution and reward. Here, we show that EAAC1 limits synaptic excitation onto a population of striatal medium spiny neurons identified for their expression of D1 dopamine receptors (D1-MSNs). In these cells, EAAC1 also contributes to strengthen lateral inhibition from other D1-MSNs. Together, these effects contribute to reduce the gain of the input-output relationship and increase the offset at increasing levels of synaptic inhibition in D1-MSNs. By reducing the sensitivity and dynamic range of action potential firing in D1-MSNs, EAAC1 limits the propensity of mice to rapidly switch between behaviors associated with different reward probabilities. Together, these findings shed light on some important molecular and cellular mechanisms implicated with behavior flexibility in mice.
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Author Response
Reviewer #1 (Public Review):
This manuscript reports new findings about the role of the glutamate transporter EAAC1 in controlling neural activity in the striatum. The significance is two-fold - it addresses gaps in knowledge about the functional significance of EAAC1, as well as provides a potential explanation for how EAAC1 mutations contribute to striatal hyperexcitability and OCD-associated behaviors. The manuscript is clearly presented, and the well-designed experiments are rigorously performed and analyzed. The main results showing that EAAC1 deletion increases the dendritic arbor of MSN D1 neurons and increases excitatory synaptic connectivity, as well as reduces D1-to-D1 mediated IPSCs are convincing. These results clearly demonstrate that EAAC1 deletion can alter excitatory and inhibitory synaptic function. …
Author Response
Reviewer #1 (Public Review):
This manuscript reports new findings about the role of the glutamate transporter EAAC1 in controlling neural activity in the striatum. The significance is two-fold - it addresses gaps in knowledge about the functional significance of EAAC1, as well as provides a potential explanation for how EAAC1 mutations contribute to striatal hyperexcitability and OCD-associated behaviors. The manuscript is clearly presented, and the well-designed experiments are rigorously performed and analyzed. The main results showing that EAAC1 deletion increases the dendritic arbor of MSN D1 neurons and increases excitatory synaptic connectivity, as well as reduces D1-to-D1 mediated IPSCs are convincing. These results clearly demonstrate that EAAC1 deletion can alter excitatory and inhibitory synaptic function. Modelling the potential consequences for these changes on D1 MSN neural activity, and the behavior changes are interesting. Minor weaknesses include incomplete support for the conclusions about how EAAC1 regulates GABAergic transmission.
We would like to take this opportunity to thank the reviewer. New sets of pharmacology experiments now address the minor concern about supporting the conclusions about the regulation of GABAergic transmission by EAAC1. The revised manuscript also includes new behavioral assays that allow us to examine in more depth the cell- and region-specificity of the effects of EAAC1.
Reviewer #2 (Public Review):
The manuscript by Petroccione et al., examines the modulatory role of the neuronal glutamate transporter EAAC1 on glutamatergic and GABAergic synaptic strength at D1- and D2-containing medium spiny neurons within the dorsolateral striatum. They find that pharmacological and genetic disruption of EAAC1 function increases glutamatergic synaptic strength specifically at D1-MSNs. They show that this is due to a structural change in release sites, not release probability. They also show that EAAC1 is critical in maintaining lateral inhibition specifically between D1-MSNs. Taken together, the authors conclude that EAAC1 functions to constrain D1-MSN excitation. Using a computational modeling technique, they posit that EAAC1's modulatory role at glutamatergic and GABAergic inputs onto D1-MSNs ultimately manifests as a reduction of gain of the input-output firing relationship and increases the offset. They go on to show that EAAC1 deletion leads to enhanced switching behavior in a probabilistic operant task. They speculate that this is due to a dysregulated E/I balance at D1-MSNs in the DLS. Overall, this is a very interesting study focused on an understudied glutamate transporter. Generally, the study is done in a very thorough and methodical manner and the manuscript is well written.
We thank the reviewer for the thorough analysis and insightful comments on the manuscript. Our point-to-point responses to the concerns raised on the initial submission of this work are reported below:
Major Comments/Concerns:
Regional/Local manipulations in behavior study: The manuscript would be greatly improved if they provided data linking the ex vivo electrophysiological findings within the DLS with the behavior. Although they are using a DLS-dependent task, they are nonetheless, using a constitutive EAAC1 KO mouse. Thus, they cannot make a strong conclusion that the behavioral deficits are due to the EAAC1 dysfunction in the DLS (despite the strong expression levels in the DLS).
Corrected - We concur with the reviewer. To address this concern, we performed new experiments to assess the cell- and regional-specificity of the effects of EAAC1 on task-switching behaviors.
First, we repeated the behavioral assays described in Fig. 8 in two mouse lines (D1Cre/+:EAAC1f/f and A2ACre/+:EAAC1f/f) lacking EAAC1 expression in D1- or D2-MSNs, respectively (Supp. Fig. 8-1). As in the case of EAAC1+/+ and EAAC1-/- mice, when the switch time was short (<15 s), D1Cre/+:EAAC1f/f and A2ACre/+:EAAC1f/f mice collected a similar number of rewards (Supp. Fig. 8-1K, L) and performed a similar number of lever presses (Supp. Fig. 8-1M, N). As the switch time increased (30-75 s), D1Cre/+:EAAC1f/f mice collected more rewards than A2ACre/+:EAAC1f/f mice, at low and high reward probabilities (Supp. Fig. 8-1L, N). Overall, the task switching behavior of D1Cre/+:EAAC1f/f mice was similar to that of EAAC1-/- mice, whereas that of A2ACre/+:EAAC1f/f mice was similar to that of EAAC1+/+ mice (cf. Supp. Fig. 8 and Supp. Fig. 8-1). This suggests that loss of expression of EAAC1 from D1-MSNs is sufficient to reproduce the task switching behavior of EAAC1-/- mice. Because EAAC1 limits excitation onto D1-MSNs (Fig. 2, 3) and lateral inhibition between D1-MSNs (Fig. 4-6), these findings suggest that increased excitation onto D1-MSNs and reciprocal inhibition among D1-MSNs limit execution of reward-based behaviors with task-switching intervals >30s.
Second, as noted by the reviewer, another potential limitation of the experiments performed on constitutive EAAC1-/- mice is that , on their own, they do not allow us to say whether they are due to changes in E/I onto D1MSNs within a specific domain of the striatum like the DLS. Although the DLS is recruited during task-switching, reward-based flexibility in executive control relies on neuronal activity in the VMS (Wallis 2007; Gu et al. 2008). Therefore, we asked whether limiting excitation in D1-MSNs and strengthening D1-D1 lateral inhibition via EAAC1 in the VMS could also alter reward-based task-switching behaviors. To address this question, we repeated the task switching test in EAAC1f/f mice that received stereotaxic injections of a Cre-dependent viral construct (AAV-D1Cre) that we used to remove EAAC1 expression from D1-MSNs in the DLS or VMS, respectively (Supp. Fig. 8-2). The results showed that the task switching behaviors of EAAC1f/f mice receiving AAV-D1Cre injections in the DLS or VMS were similar to each other and to those of EAAC1-/- mice, while being statistically different from those of EAAC1+/+ mice. This finding is important, as it suggests that: (i) the DLS and VMS are both recruited for the execution of task switching behaviors; (ii) the modulation of E/I onto D1-MSNs by EAAC1 may not be limited to the DLS but could extend to the VMS.
Third, we performed further tests to examine the regional-specificity of the effects of EAAC1 in D1-MSNs. D1 receptor expressing cells are present not only throughout the striatum, but also in the substantia nigra (pars compacta and reticulata; SN) and ventral tegmental area (VTA) (Cadet et al. 2010; Savasta, Dubois, and Scatton 1986; Boyson, McGonigle, and Molinoff 1986; Wamsley et al. 1989). To determine whether lack of EAAC1 in D1expressing cells in the SN/VTA could also contribute to increased compulsivity, we repeated the task switching behavioral assays in EAAC1f/f mice that received injections of AAV-D1Cre in the SN/VTA (Supp Fig. 8-3). The task switching behavior of these mice was similar to that of EAAC1+/+ , not EAAC1-/- mice, suggesting that altering EAAC1 expression in D1-MSNS of the DLS/VMS, but not the SN/VTA, is implicated with the control of task switching of reward-based behaviors in mice.
The results of these new sets of experiments are included in the revised version of the manuscript and their implications are reported in the Discussion section of the paper.
Statistics used in the study: There are some missing details regarding the precise stats using for the different comparisons. I am particularly concerned that the electrophysiology studies that were a priori designed as a 2-factor analysis did not have 2-way ANOVAs performed, but rather a series of t-tests. For example, in Figure 3b, the two factors are 1) cell type and 2) genotype. Was a 2-way ANOVA performed? It is hard for me to tell from the text.
Corrected - We apologize for any potential confusion. The statistical analysis for the experiments included in this work includes paired and unpaired t-tests, one-way ANOVA, two-way ANOVA, and ANOVA for repeated measures tests followed by post hoc t-test comparisons (reported in the text). To ensure both accuracy and readability of the manuscript, we report the results of the statistical comparisons in the main text of the manuscript, but also provide a fully detailed statistical analysis across all datasets performed in the data repository for this manuscript deposited on Open Science Framework. We revised the methods section to clarify the use of different statistical tests and values reported in the manuscript.
Moderate Concerns:
Control mice: I am moderately concerned that littermates were not used for controls for the EAAC1 KO, but rather C57Bl/6NJ presumably ordered from a vendor. It has been shown that issues like transit and rearing conditions can have long term effects on behavior. Were the control mice reared in house? How long was the acclimation time before use?
Corrected - Sorry for the potential confusion. The EAAC1-/- mice are bred in house and have been backcrossed with C57BL/6J for more than 10 generations. We perform backcrossing regularly and routinely in our animal colony. The C57BL/6J are also bread in house. They are replaced every 10 generations to avoid genetic drift. Therefore, there is no concern about transit from vendors and rearing affecting the results of our experiments. This information has been added to the Methods section of the paper.
OCD framework: I generally find the OCD framework unnecessary, particularly in the Introduction. Compulsive behaviors are not restricted to OCD. Indeed, the link between the behavioral observations and OCD phenotype seems a bit tenuous. In addition, studying the mechanisms of behavioral flexibility in and of itself is interesting. I do not think such a strong link needs to be made to OCD throughout the entirety of the paper. The authors should consider tempering this language or restricting it to the discussion and end of the abstract.
Corrected - We concur with the reviewer and have revised the manuscript accordingly. At the end of the Abstract, we refer only to behavior flexibility. We have toned down our emphasis on OCD in the Introduction, broadening the genetic link between the gene encoding EAAC1 (SLC1A1) and neuropsychiatric diseases like OCD, ADHD and ASD. This is now limited to a single sentence. We also revised the Discussion section because we agree with the reviewer on the fact that compulsive behaviors are not limited to OCD.
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eLife assessment
This manuscript reports important findings that help to understand the function of glutamate transporters and their effects on synaptic function at D1- and D2-MSNs within the dorsolateral striatum. These findings were evaluated to be of interest and well-executed. Overall, the majority of claims are supported by high quality data, but the evidence for some underlying mechanisms and region specificity were incomplete in the manuscript's current form.
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Reviewer #1 (Public Review):
This manuscript reports new findings about the role of the glutamate transporter EAAC1 in controlling neural activity in the striatum. The significance is two-fold - it addresses gaps in knowledge about the functional significance of EAAC1, as well as provides a potential explanation for how EAAC1 mutations contribute to striatal hyperexcitability and OCD-associated behaviors. The manuscript is clearly presented, and the well-designed experiments are rigorously performed and analyzed. The main results showing that EAAC1 deletion increases the dendritic arbor of MSN D1 neurons and increases excitatory synaptic connectivity, as well as reduces D1-to-D1 mediated IPSCs are convincing. These results clearly demonstrate that EAAC1 deletion can alter excitatory and inhibitory synaptic function. Modelling the …
Reviewer #1 (Public Review):
This manuscript reports new findings about the role of the glutamate transporter EAAC1 in controlling neural activity in the striatum. The significance is two-fold - it addresses gaps in knowledge about the functional significance of EAAC1, as well as provides a potential explanation for how EAAC1 mutations contribute to striatal hyperexcitability and OCD-associated behaviors. The manuscript is clearly presented, and the well-designed experiments are rigorously performed and analyzed. The main results showing that EAAC1 deletion increases the dendritic arbor of MSN D1 neurons and increases excitatory synaptic connectivity, as well as reduces D1-to-D1 mediated IPSCs are convincing. These results clearly demonstrate that EAAC1 deletion can alter excitatory and inhibitory synaptic function. Modelling the potential consequences for these changes on D1 MSN neural activity, and the behavior changes are interesting. Minor weaknesses include incomplete support for the conclusions about how EAAC1 regulates GABAergic transmission.
-
Reviewer #2 (Public Review):
The manuscript by Petroccione et al., examines the modulatory role of the neuronal glutamate transporter EAAC1 on glutamatergic and GABAergic synaptic strength at D1- and D2-containing medium spiny neurons within the dorsolateral striatum. They find that pharmacological and genetic disruption of EAAC1 function increases glutamatergic synaptic strength specifically at D1-MSNs. They show that this is due to a structural change in release sites, not release probability. They also show that EAAC1 is critical in maintaining lateral inhibition specifically between D1-MSNs. Taken together, the authors conclude that EAAC1 functions to constrain D1-MSN excitation. Using a computational modeling technique, they posit that EAAC1's modulatory role at glutamatergic and GABAergic inputs onto D1-MSNs ultimately manifests …
Reviewer #2 (Public Review):
The manuscript by Petroccione et al., examines the modulatory role of the neuronal glutamate transporter EAAC1 on glutamatergic and GABAergic synaptic strength at D1- and D2-containing medium spiny neurons within the dorsolateral striatum. They find that pharmacological and genetic disruption of EAAC1 function increases glutamatergic synaptic strength specifically at D1-MSNs. They show that this is due to a structural change in release sites, not release probability. They also show that EAAC1 is critical in maintaining lateral inhibition specifically between D1-MSNs. Taken together, the authors conclude that EAAC1 functions to constrain D1-MSN excitation. Using a computational modeling technique, they posit that EAAC1's modulatory role at glutamatergic and GABAergic inputs onto D1-MSNs ultimately manifests as a reduction of gain of the input-output firing relationship and increases the offset. They go on to show that EAAC1 deletion leads to enhanced switching behavior in a probabilistic operant task. They speculate that this is due to a dysregulated E/I balance at D1-MSNs in the DLS.
Overall, this is a very interesting study focused on an understudied glutamate transporter. Generally, the study is done in a very thorough and methodical manner and the manuscript is well written.
Major Comments/Concerns:
1. Regional/Local manipulations in behavior study: The manuscript would be greatly improved if they provided data linking the ex vivo electrophysiological findings within the DLS with the behavior. Although they are using a DLS-dependent task, they are nonetheless, using a constitutive EAAC1 KO mouse. Thus, they cannot make a strong conclusion that the behavioral deficits are due to the EAAC1 dysfunction in the DLS (despite the strong expression levels in the DLS).2. Statistics used in the study: There are some missing details regarding the precise stats using for the different comparisons. I am particularly concerned that the electrophysiology studies that were a priori designed as a 2-factor analysis did not have 2-way ANOVAs performed, but rather a series of t-tests. For example, in Figure 3b, the two factors are 1) cell type and 2) genotype. Was a 2-way ANOVA performed? It is hard for me to tell from the text.
Moderate Concerns:
3. Control mice: I am moderately concerned that littermates were not used for controls for the EAAC1 KO, but rather C57Bl/6NJ presumably ordered from a vendor. It has been shown that issues like transit and rearing conditions can have long term affects on behavior. Were the control mice reared in house? How long was the acclimation time before use?4. OCD framework: I generally find the OCD framework unnecessary, particularly in the introduction. Compulsive behaviors are not restricted to OCD. Indeed, the link between the behavioral observations and OCD phenotype seems a bit tenuous. In addition, studying the mechanisms of behavioral flexibility in and of itself is interesting. I don't think such a strong link needs to be made to OCD throughout the entirety of the paper. The authors should consider tempering this language or restricting it to the discussion and end of the abstract.
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