Synaptotagmin isoforms differentially regulate glutamate and GABA release in the lateral habenula
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eLife Assessment
This paper addresses a key question regarding the molecular mechanisms underlying GABA and glutamate release from co-releasing neurons projecting from the entopeduncular nucleus (EPN) to the lateral habenula (LHb) in mice. The authors conclude that the two neurotransmitters are released from separate vesicle pools and rely on distinct molecular machinery; these conclusions contrast with previous functional studies at the same synapse, suggesting that GABA and glutamate are co-packaged within the same vesicles. The study employs useful electrophysiological and imaging approaches, however, a key limitation is the use of Cre lines that also label a purely glutamatergic EPN population projecting to the LHb. This inadequate methodology complicates the interpretation of the data and weakens the central conclusions regarding neurotransmitter co-release mechanisms.
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Abstract
Neurotransmitter co-transmission contributes to diverse physiological processes throughout the mammalian brain, including sensory integration, motivational control, and social behaviors. Projections from the globus pallidus internus (GPi; the entopeduncular nucleus, EPN, in rodents) to the lateral habenula (LHb) are well-characterized by the co-transmission of both GABA and glutamate. These dual-release inputs modulate behavioral states in chronically learned helpless (cLH) rats, influencing both the onset and recovery of pathological phenotypes. Here, we employed confocal 3D reconstructions that confirmed the presence of both vesicular transporters VGAT and VGLUT2 in EPN axon terminals within the LHb. Further investigation revealed that GABA and glutamate are packaged in distinct vesicle populations within individual presynaptic terminals. Notably, the calcium (Ca²⁺) sensors Synaptotagmin-2 (Syt2) and Synaptotagmin-3 (Syt3) are highly expressed in the EPN whereas expression of the canonical Ca²⁺ sensor, Synaptotagmin 1 (Syt1), is downregulated. Additionally, using confocal microscopy, we observed selective spatial correlations of Syt2 and VGLUT2 and between Syt3 and VGAT in LHb axon terminals. These observations strongly suggested that Syt2 serves as the predominant Ca²⁺ sensor for glutamatergic vesicle fusion, and Syt3 serves as the predominant Ca²⁺ sensor for GABAergic vesicle fusion in the LHb. To test this hypothesis, we employed targeted antisense oligonucleotide (ASO) knockdown of Syt2 and Syt3 in EPN neurons and measured LHb glutamatergic and GABAergic currents. Syt2 knockdown resulted in an increase in mEPSC frequency, amplitude, half-width and decay, suggesting increased glutamate vesicle release probability and increased glutamate vesicle packing. However, Syt2 knockdown had no influence on mIPSCs amplitude or frequency. On the other hand, Syt3 knockdown had no apparent effect on glutamate release but caused an increase in mIPSC frequency suggesting increased quantal release probability of GABA. Together, these findings identify a molecular mechanism by which synaptotagmin isoforms govern differential glutamate and GABA release at EPN dual-transmitter terminals in the LHb. These results provide evidence for presynaptic mechanisms regulating excitatory–inhibitory balance within this brain structure and importantly provide molecular targets for pharmacological intervention.
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eLife Assessment
This paper addresses a key question regarding the molecular mechanisms underlying GABA and glutamate release from co-releasing neurons projecting from the entopeduncular nucleus (EPN) to the lateral habenula (LHb) in mice. The authors conclude that the two neurotransmitters are released from separate vesicle pools and rely on distinct molecular machinery; these conclusions contrast with previous functional studies at the same synapse, suggesting that GABA and glutamate are co-packaged within the same vesicles. The study employs useful electrophysiological and imaging approaches, however, a key limitation is the use of Cre lines that also label a purely glutamatergic EPN population projecting to the LHb. This inadequate methodology complicates the interpretation of the data and weakens the central conclusions regarding …
eLife Assessment
This paper addresses a key question regarding the molecular mechanisms underlying GABA and glutamate release from co-releasing neurons projecting from the entopeduncular nucleus (EPN) to the lateral habenula (LHb) in mice. The authors conclude that the two neurotransmitters are released from separate vesicle pools and rely on distinct molecular machinery; these conclusions contrast with previous functional studies at the same synapse, suggesting that GABA and glutamate are co-packaged within the same vesicles. The study employs useful electrophysiological and imaging approaches, however, a key limitation is the use of Cre lines that also label a purely glutamatergic EPN population projecting to the LHb. This inadequate methodology complicates the interpretation of the data and weakens the central conclusions regarding neurotransmitter co-release mechanisms.
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Reviewer #1 (Public review):
Summary:
White et al. explore the role of synaptotagmin isoforms in mediating neurotransmitter release from EPN terminals in the LHb. The authors show a relatively high expression of Syt2 and Syt3 in the EPN relative to other Syt isoforms. The authors then perform a series of experiments to show that Syt2 preferentially regulates glutamatergic transmission while Syt3 regulates GABAergic transmission.
Strengths:
Interesting, timely topic.
Weaknesses:
While interesting, the study is rather preliminary. There are a number of issues the authors need to address.
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Reviewer #2 (Public review):
Summary:
This is an important study of the molecular mechanisms of GABA vs. glutamate release by coreleasing neurons that project from the EPN to LHB. The conclusion is that separate pools of vesicles release each transmitter and use different molecular machinery to do so. This is in contrast to and in disagreement with functional studies of the same synapse that conclude that the transmitters are copackaged.
As detailed below, the study has a major flaw. It uses an incorrect Cre line, which is also expressed in a purely glutamatergic population in the EPN that also projects to the LHB. In addition, there is little quantification and validation of important tools and no histological confirmation of the sites of expression of viral-encoded proteins.
Strengths:
The strength of the study is in the importance of …
Reviewer #2 (Public review):
Summary:
This is an important study of the molecular mechanisms of GABA vs. glutamate release by coreleasing neurons that project from the EPN to LHB. The conclusion is that separate pools of vesicles release each transmitter and use different molecular machinery to do so. This is in contrast to and in disagreement with functional studies of the same synapse that conclude that the transmitters are copackaged.
As detailed below, the study has a major flaw. It uses an incorrect Cre line, which is also expressed in a purely glutamatergic population in the EPN that also projects to the LHB. In addition, there is little quantification and validation of important tools and no histological confirmation of the sites of expression of viral-encoded proteins.
Strengths:
The strength of the study is in the importance of the question addressed and in the ambition of the tools used.
Weaknesses:
(1) The study uses Vglut2-IRES-Cre mouse to gain control over EPN to LHB projections. However, as has been shown by several groups, this line is not exclusive to the EPN co-releasing population. It is also expressed in glutamatergic EPN PV neurons that project solely to the EPN. Therefore, all of the studies here are contaminated with analysis of a purely glutamatergic Vglut2+ projection. This calls into question all the conclusions about the differential localization and function of synaptic proteins.
(2) It is unclear from the paper, but it seems that some experiments may have been done with no Cre control, which likely led to contamination in neighboring brain regions, some of which project to LHB as well.
(3) Histology: There is no histology shown for the mice used in the study. This is a crucial point. We need to see that the injection was clean and specific for each mouse used in the study (although, given the use of Vglut2-Cre, it cannot be specific to the coreleasing population). Whole-brain histology is necessary.
(4) ASO KO: Unfortunately, there is little validation of the ASO KO. The effects shown in Figure S2 show a very small effect, if any. There appear to be no statistics. The functional effects in the main figure are also relatively subtle.
(5) Other concerns: There are many typos and errors, including in important claims.
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