Id2 GABAergic interneurons comprise a neglected fourth major group of cortical inhibitory cells
Curation statements for this article:-
Curated by eLife
eLife assessment
This is a valuable contribution to the effort to provide genetic access to and characterization of the major classes of interneurons in the mammalian neocortex. The authors develop an improved strategy for intersectionally targeting a fourth (and final) major category of diverse interneurons in the mouse, including the previously studied neurogliaform cells. They provide a detailed characterization of these cells and show convincingly that their genetic strategy can be used to identify and manipulate these cells, both in vitro and in vivo.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Neuroscience (eLife)
Abstract
Cortical GABAergic interneurons (INs) represent a diverse population of mainly locally projecting cells that provide specialized forms of inhibition to pyramidal neurons and other INs. Most recent work on INs has focused on subtypes distinguished by expression of Parvalbumin (PV), Somatostatin (SST), or Vasoactive Intestinal Peptide (VIP). However, a fourth group that includes neurogliaform cells (NGFCs) has been less well characterized due to a lack of genetic tools. Here, we show that these INs can be accessed experimentally using intersectional genetics with the gene Id2 . We find that outside of layer 1 (L1), the majority of Id2 INs are NGFCs that express high levels of neuropeptide Y (NPY) and exhibit a late-spiking firing pattern, with extensive local connectivity. While much sparser, non-NGFC Id2 INs had more variable properties, with most cells corresponding to a diverse group of INs that strongly expresses the neuropeptide CCK. In vivo, using silicon probe recordings, we observed several distinguishing aspects of NGFC activity, including a strong rebound in activity immediately following the cortical down state during NREM sleep. Our study provides insights into IN diversity and NGFC distribution and properties, and outlines an intersectional genetics approach for further study of this underappreciated group of INs.
Article activity feed
-
-
Author Response
Reviewer #2 (Public Review):
Machold and colleagues develop and describe an intersectional genetic mouse (Id2Cre:Dlx5/6FlpE) that allows for the targeting of a cortical interneuron subpopulation predominantly consisting of the neurogliaform cell subtype (NGFCs). The strategy is a modification of that previously published by the authors (Id2cre:Nkx2-1Flpo; Valero et al., 2021) in which a subset of deep layer 6 NGFCs with distinct embryonic origins were targeted. Conversely, using the NDNF transgenic mouse lines previous studies, including thosefrom the Rudy laboratory, have clearly shown the prevalence of NGFCs in the outermost cortical Layer 1 region. Thus, the Id2Cre:Dlx5/6FlpE mouse poses an advantage over these previous approaches permitting the targeting of NGFCs in Layers 2-5. NGFCs in these regions have been …
Author Response
Reviewer #2 (Public Review):
Machold and colleagues develop and describe an intersectional genetic mouse (Id2Cre:Dlx5/6FlpE) that allows for the targeting of a cortical interneuron subpopulation predominantly consisting of the neurogliaform cell subtype (NGFCs). The strategy is a modification of that previously published by the authors (Id2cre:Nkx2-1Flpo; Valero et al., 2021) in which a subset of deep layer 6 NGFCs with distinct embryonic origins were targeted. Conversely, using the NDNF transgenic mouse lines previous studies, including thosefrom the Rudy laboratory, have clearly shown the prevalence of NGFCs in the outermost cortical Layer 1 region. Thus, the Id2Cre:Dlx5/6FlpE mouse poses an advantage over these previous approaches permitting the targeting of NGFCs in Layers 2-5. NGFCs in these regions have been hitherto difficult to study in an expedited manner.
The manuscript is of the resource/toolbox type and the authors are thorough in their description of the distribution and molecular characteristics of the ID2 neurons labelled by this intersectional approach. Furthermore, the authors perform a series of in vivo experiments. These entail the identification of NGFCs, the assessment of their influence on other neuronal populations, and the ability to delineate their activity during various network and behavioral states. Indeed, the authors reveal an activity pattern that is unique to NGFCs across epochs of specific network states. Therefore, this clearly demonstrates the applicability of the ID2Cre:Dlx5/6Flpe mouse to study the role of L2-5 NGFCs in a whole brain setting and these in vivo experiments constitute a major strength of the current study.
However, as with many transgenic mice, they are not always perfect, and the authors are very transparent regarding the additional, albeit a relatively smaller number of reported non-NGFCs particularly those of the CCK IN subtype. Indeed, clear morpho- functional divergence is revealed by the authors between these ID2 IN subpopulations. Furthermore, it is possible that this variability may differ across varying cortical regions. Thus, careful consideration of this caveat is necessary when using this mouse for future in vitro and in vivo studies. Related to this matter is a concern regarding the framing of the manuscript. The authors term the ID2 mixed population as the "4th group" since they do not express PV, SST, and VIP. One could argue this is a matter of semantics but to combine IN types that display distinct morphological and physiological properties into a single "group" based on one molecular feature is not consistent with that proposed by the widely accepted Petilla terminology (Ascoli et al., 2008).
We agree that the definition of “group” here for INs delineated by the molecular markers PV, SST, VIP and Id2 is oversimplified, but in practice, the use of the corresponding genetic tools (e.g., Pvalb-Cre, Sst-Cre etc.) has resulted in widespread adoption of this marker-based organization of IN diversity. For example, PV+ INs targeted with PV-Cre encompass both basket cells and chandelier cells that while sharing some electrophysiological properties (e.g., fast-spiking behavior) are completely distinct morphologically, and innervate different subcellular compartments (soma vs. axon initial segment). The same is true for SST INs, in that there appear to be at least three main subtypes – Martinotti, non-Martinotti, and long range projecting – each with distinct axonal projections and electrophysiology. Thus, while the molecular targeting approaches developed to date have greatly facilitated functional studies of IN subtypes, they have prioritized marker expression over the other aspects of IN diversity outlined in the Petilla framework.
Of interest to many who investigate cortical INs is the ability to genetically target specific subtypes during development. To this end, a potential and welcome addition to the manuscript would be an analysis (perhaps restricted to distribution/molecular characterization) highlighting whether the Id2cre:Dlx5/6Flpe strategy allows genetic access to layer 2-5 NGFCs during postnatal development following maternal tamoxifen administration.
We agree that a method to target NGFC at early postnatal ages would be useful; however, the expression of Id2 is dynamic during development, and is robust in ventricular zone progenitors at embryonic stages (Neuman et al., 1993 Dev. Biol. PMID 8224536) so maternal tamoxifen administration is likely to result in nonspecific labeling. Furthermore, we found that multiple doses of tamoxifen were necessary to achieve decent labeling of the Id2 IN population in adult animals, a protocol that would be difficult to perform in pregnant dams or early postnatal animals due to pup lethality.
Regardless, the experiments in the current study are, in general, well performed and clearly presented with the authors' conclusions supported by the results. Thus, it is clear that further refinements to genetic strategies are obviously required to exclusively target NGFCs throughout the cortical depth. Nevertheless, in the interim, the approach described in this current manuscript will be of use to the neuroscience community and help to further unravel the physiological role of this relatively understudied neuronal subtype.
-
eLife assessment
This is a valuable contribution to the effort to provide genetic access to and characterization of the major classes of interneurons in the mammalian neocortex. The authors develop an improved strategy for intersectionally targeting a fourth (and final) major category of diverse interneurons in the mouse, including the previously studied neurogliaform cells. They provide a detailed characterization of these cells and show convincingly that their genetic strategy can be used to identify and manipulate these cells, both in vitro and in vivo.
-
Reviewer #1 (Public Review):
The authors have previously suggested that virtually all cortical interneurons are positive for one of three markers: Pvalb, Sst, or Htr3. Here they present convincing evidence that the Htr3a group, includes a significant fraction that does not in fact express this marker in the adult and has distinct properties. By mining existing single-cell RNAseq results, the authors conclude that these cells express the marker Id2. Because some excitatory neurons and non-neuronal cells also express this marker, they selectively target these Id2+ interneurons by combining a pan-interneuron driver and an Id2-creER driver. They show that these neurons are present across cortical layers and account for about 18% of all interneurons. Based on morphological and electrophysiological analyses, this group includes non-VIP …
Reviewer #1 (Public Review):
The authors have previously suggested that virtually all cortical interneurons are positive for one of three markers: Pvalb, Sst, or Htr3. Here they present convincing evidence that the Htr3a group, includes a significant fraction that does not in fact express this marker in the adult and has distinct properties. By mining existing single-cell RNAseq results, the authors conclude that these cells express the marker Id2. Because some excitatory neurons and non-neuronal cells also express this marker, they selectively target these Id2+ interneurons by combining a pan-interneuron driver and an Id2-creER driver. They show that these neurons are present across cortical layers and account for about 18% of all interneurons. Based on morphological and electrophysiological analyses, this group includes non-VIP neurons in layer 1, neurogliaform cells in layers 2-6, and a small population of CCK basket cells. These in vitro characterizations are well done and will make it straightforward for others to adopt the same or related genetic strategies to target these cells for other functional or mechanistic studies. Since it was not previously possible to genetically target most of these cells, they have been less studied than the other populations of interneurons, so the present study fills an important gap in the field.
The authors also use optogenetics and silicon probe multielectrode recordings to characterize the state-dependent firing and impact of these neurons in vivo. By monitoring sleep, and wake state, the authors show convincingly that these cells reduce their firing during NREM sleep and show a rebound increase in firing after this reduction. The authors then stimulate these neurons optogenetically and show that the firing of other neurons is more likely to be reduced than increased, as expected if these neurons provide broad inhibitory output. The magnitude of this effect is a bit difficult to assess from the current presentation of these data and so it is not clear whether the authors' suggestion that recruitment of these neurons "might drive a widespread switch in the activity of all other cortical neurons" is supported, or whether the effects on circuit activity are more subtle. Regardless of this concern, this is an important study for our understanding of the properties and functions of cortical interneurons.
-
Reviewer #2 (Public Review):
Machold and colleagues develop and describe an intersectional genetic mouse (Id2Cre:Dlx5/6FlpE) that allows for the targeting of a cortical interneuron subpopulation predominantly consisting of the neurogliaform cell subtype (NGFCs). The strategy is a modification of that previously published by the authors (Id2cre:Nkx2-1Flpo; Valero et al., 2021) in which a subset of deep layer 6 NGFCs with distinct embryonic origins were targeted. Conversely, using the NDNF transgenic mouse lines previous studies, including those from the Rudy laboratory, have clearly shown the prevalence of NGFCs in the outermost cortical Layer 1 region. Thus, the Id2Cre:Dlx5/6FlpE mouse poses an advantage over these previous approaches permitting the targeting of NGFCs in Layers 2-5. NGFCs in these regions have been hitherto difficult to …
Reviewer #2 (Public Review):
Machold and colleagues develop and describe an intersectional genetic mouse (Id2Cre:Dlx5/6FlpE) that allows for the targeting of a cortical interneuron subpopulation predominantly consisting of the neurogliaform cell subtype (NGFCs). The strategy is a modification of that previously published by the authors (Id2cre:Nkx2-1Flpo; Valero et al., 2021) in which a subset of deep layer 6 NGFCs with distinct embryonic origins were targeted. Conversely, using the NDNF transgenic mouse lines previous studies, including those from the Rudy laboratory, have clearly shown the prevalence of NGFCs in the outermost cortical Layer 1 region. Thus, the Id2Cre:Dlx5/6FlpE mouse poses an advantage over these previous approaches permitting the targeting of NGFCs in Layers 2-5. NGFCs in these regions have been hitherto difficult to study in an expedited manner.
The manuscript is of the resource/toolbox type and the authors are thorough in their description of the distribution and molecular characteristics of the ID2 neurons labelled by this intersectional approach. Furthermore, the authors perform a series of in vivo experiments. These entail the identification of NGFCs, the assessment of their influence on other neuronal populations, and the ability to delineate their activity during various network and behavioral states. Indeed, the authors reveal an activity pattern that is unique to NGFCs across epochs of specific network states. Therefore, this clearly demonstrates the applicability of the ID2Cre:Dlx5/6Flpe mouse to study the role of L2-5 NGFCs in a whole brain setting and these in vivo experiments constitute a major strength of the current study.
However, as with many transgenic mice, they are not always perfect, and the authors are very transparent regarding the additional, albeit a relatively smaller number of reported non-NGFCs particularly those of the CCK IN subtype. Indeed, clear morpho-functional divergence is revealed by the authors between these ID2 IN subpopulations. Furthermore, it is possible that this variability may differ across varying cortical regions. Thus, careful consideration of this caveat is necessary when using this mouse for future in vitro and in vivo studies. Related to this matter is a concern regarding the framing of the manuscript. The authors term the ID2 mixed population as the "4th group" since they do not express PV, SST, and VIP. One could argue this is a matter of semantics but to combine IN types that display distinct morphological and physiological properties into a single "group" based on one molecular feature is not consistent with that proposed by the widely accepted Petilla terminology (Ascoli et al., 2008).
Of interest to many who investigate cortical INs is the ability to genetically target specific subtypes during development. To this end, a potential and welcome addition to the manuscript would be an analysis (perhaps restricted to distribution/molecular characterization) highlighting whether the Id2cre:Dlx5/6Flpe strategy allows genetic access to layer 2-5 NGFCs during postnatal development following maternal tamoxifen administration.
Regardless, the experiments in the current study are, in general, well performed and clearly presented with the authors' conclusions supported by the results. Thus, it is clear that further refinements to genetic strategies are obviously required to exclusively target NGFCs throughout the cortical depth. Nevertheless, in the interim, the approach described in this current manuscript will be of use to the neuroscience community and help to further unravel the physiological role of this relatively understudied neuronal subtype.
-
Reviewer #3 (Public Review):
This is an interesting and carefully done study that will be of considerable value to the field of cortical interneurons. The main result is the development of a novel intersectional genetic strategy to identify and manipulate neurogliaform cells (NGFCs), an interneuron subtype that has been somewhat under-explored to date (but perhaps not quite as enigmatic as implied by the authors). The new strategy, using Id2-CreER transgenic mice crossed with a pan-interneuronal Flp line, appears to label all interneurons which do not express PV, Sst, or VIP, and thus defines a fourth subclass of interneurons. The main members of this subclass are NPY-expressing NGFCs. The strategy allows the targeting of NGFCs in all cortical layers, in contrast to previous strategies using the NDNF-Cre mice which target mostly Layer 1 …
Reviewer #3 (Public Review):
This is an interesting and carefully done study that will be of considerable value to the field of cortical interneurons. The main result is the development of a novel intersectional genetic strategy to identify and manipulate neurogliaform cells (NGFCs), an interneuron subtype that has been somewhat under-explored to date (but perhaps not quite as enigmatic as implied by the authors). The new strategy, using Id2-CreER transgenic mice crossed with a pan-interneuronal Flp line, appears to label all interneurons which do not express PV, Sst, or VIP, and thus defines a fourth subclass of interneurons. The main members of this subclass are NPY-expressing NGFCs. The strategy allows the targeting of NGFCs in all cortical layers, in contrast to previous strategies using the NDNF-Cre mice which target mostly Layer 1 NGFCs (and possibly also other Layer 1 subtypes). The same strategy also labels a relatively small population of non-NGF Id2 cells belonging to the CCK-expressing subtype(s).
In the first stage of the study, the authors characterize the labeled neurons by their expression of protein markers (most notably NPY and CCK), by their dendritic and axonal morphology, and by their electrophysiological properties. This characterization is detailed and rigorous and the observed characteristics are consistent with what is already known about the properties of NGFCs. The weaknesses here are that the morphological features are not analyzed quantitatively, the definition of electrophysiological subtypes remains somewhat subjective, and the authors do not attempt a multivariate analysis that could provide a data-driven parcellation into subtypes.
The authors then go two steps further. First, they use ex-vivo recordings to demonstrate that presumed CCK+ neurons (identified by their firing pattern as "non-late-spiking), but not NGFCs (identified by their "late-spiking" phenotype), are sensitive to endocannabinoids released from postsynaptic pyramidal cells upon depolarization of the latter. This DSI ("depolarization suppression of inhibition") is a well-studied property of hippocampal CCK+ basket cells, so its demonstration adds to the validation of the intersectional strategy in targeting this subtype in the neocortex. Somewhat surprisingly, the authors do not attempt to demonstrate in their ex-vivo experiments what may be the best-known property of NGFCs - their propensity to preferentially activate GABAB receptors.
The authors then perform in-vivo silicon probe recordings in which Id2 cells are "optotagged" with ChR2 and can thus be identified in extracellular recordings. These in-vivo recordings are probably the first ever from identified NGFCs below layer 1, although some uncertainty remains about the identification of optotagged cells as NGFCs vs CCK-expressing interneurons. They find several differences between firing patterns of NGFCs and other interneurons or pyramidal cells (identified by their extracellular spike waveforms), the most dramatic being a pronounced "rebound" of NGFC firing during slow-wave sleep immediately after a DOWN-to-UP state transition. While the functional significance of these findings is not clear, these experiments provide proof of concept that this fourth (and last?) interneuron subclass can be identified, recorded, and manipulated in freely behaving animals.
In summary, while adding only modestly to our knowledge of NGFCs and CCK-expressing interneurons per se, this work provides an important new tool that will no doubt be used in future studies to target cortical NGFCs and CCK interneurons for in-vivo and ex-vivo recordings, for optogenetic manipulations and for calcium or voltage imaging using genetically-encoded probes. In this sense, the current study is a breakthrough into what may truly be "the last frontier" of cortical interneurons.
-
-