Task-specific roles of local interneurons for inter- and intraglomerular signaling in the insect antennal lobe

  1. Debora Fusca
  2. Peter Kloppenburg

  • Curated by eLife

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    Evaluation Summary:

    The question investigated - to understand the computational significance of different types of local interneurons in neural circuits - is an important and significant problem. Here authors elucidate the role of the two types of LNs, by combining whole-cell patch clamp recordings with calcium imaging via single cell dye injection. This method enables them to monitor calcium dynamics of the different axons and branches of single LNs in identified glomeruli of the antennal lobe, while the membrane potential can be recorded at the same time. They conclude that non-spiking LNs with graded responses show glomerular restricted responses to odorants and spiking LNs show similar responses across glomeruli.

    (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 #1 agreed to share their name with the authors.)

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Abstract

Local interneurons (LNs) mediate complex interactions within the antennal lobe, the primary olfactory system of insects, and the functional analog of the vertebrate olfactory bulb. In the cockroach Periplaneta americana , as in other insects, several types of LNs with distinctive physiological and morphological properties can be defined. Here, we combined whole-cell patch-clamp recordings and Ca 2+ imaging of individual LNs to analyze the role of spiking and nonspiking LNs in inter- and intraglomerular signaling during olfactory information processing. Spiking GABAergic LNs reacted to odorant stimulation with a uniform rise in [Ca 2+ ] i in the ramifications of all innervated glomeruli. In contrast, in nonspiking LNs, glomerular Ca 2+ signals were odorant specific and varied between glomeruli, resulting in distinct, glomerulus-specific tuning curves. The cell type-specific differences in Ca 2+ dynamics support the idea that spiking LNs play a primary role in interglomerular signaling, while they assign nonspiking LNs an essential role in intraglomerular signaling.

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  1. Version published to 10.7554/elife.65217 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    The manuscript provides very high quality single-cell physiology combined with population physiology to reveal distinctives roles for two anatomically dfferent LN populations in the cockroach antennal lobe. The conclusion that non-spiking LNs with graded responses show glomerular-restricted responses to odorants and spiking LNs show similar responses across glomeruli generally supported with strong and clean data, although the possibility of selective interglomerular inhibition has not been ruled out. On balance, the single-cell biophysics and physiology provides foundational information useful for well-grounded mechanistic understanding of how information is processed in insect antennal lobes, and how each LN class contributes to odor perception and behavior.

    Thank you for this positive feedback.

    Reviewer #2 (Public Review):

    The manuscript "Task-specific roles of local interneurons for inter- and intraglomerular signaling in the insect antennal lobe" evaluates the spatial distribution of calcium signals evoked by odors in two major classes of olfactory local neurons (LNs) in the cockroach P. Americana, which are defined by their physiological and morphological properties. Spiking type I LNs have a patchy innervation pattern of a subset of glomeruli, whereas non-spiking type II LNs innervate almost all glomeruli (Type II). The authors' overall conclusion is that odors evoke calcium signals globally and relatively uniformly across glomeruli in type I spiking LNs, and LN neurites in each glomerulus are broadly tuned to odor. In contrast, the authors conclude that they observe odor-specific patterns of calcium signals in type II nonspiking LNs, and LN neurites in different glomeruli display distinct local odor tuning. Blockade of action potentials in type I LNs eliminates global calcium signaling and decorrelates glomerular tuning curves, converting their response profile to be more similar to that of type II LNs. From these conclusions, the authors infer a primary role of type I LNs in interglomerular signaling and type III LNs in intraglomerular signaling.

    The question investigated by this study - to understand the computational significance of different types of LNs in olfactory circuits - is an important and significant problem. The design of the study is straightforward, but methodological and conceptual gaps raise some concerns about the authors' interpretation of their results. These can be broadly grouped into three main areas.

    1. The comparison of the spatial (glomerular) pattern of odor-evoked calcium signals in type I versus type II LNs may not necessarily be a true apples-to-apples comparison. Odor-evoked calcium signals are an order of magnitude larger in type I versus type II cells, which will lead to a higher apparent correlation in type I cells. In type IIb cells, and type I cells with sodium channel blockade, odor-evoked calcium signals are much smaller, and the method of quantification of odor tuning (normalized area under the curve) is noisy. Compare, for instance, ROI 4 & 15 (Figure 4) or ROI 16 & 23 (Figure 5) which are pairs of ROIs that their quantification concludes have dramatically different odor tuning, but which visual inspection shows to be less convincing. The fact that glomerular tuning looks more correlated in type IIa cells, which have larger, more reliable responses compared to type IIb cells, also supports this concern.

    We agree with the reviewer that "the comparison of the spatial (glomerular) pattern of odor-evoked calcium signals is not necessarily a true apples-to-apples comparison". Type I and type II LNs are different neuron types. Given their different physiology and morphology, this is not even close to a "true apples-to-apples comparison" - and a key point of the manuscript is to show just that.

    As we have emphasized in response to Essential Revision 1, the differences in Ca2+ signals are not an experimental shortcoming but a physiologically relevant finding per se. These data, especially when combined with the electrophysiological data, contribute to a better understanding of these neurons’ physiological and computational properties.

    It is physiologically determined that the Ca2+ signals during odorant stimulation in the type II LNs are smaller than in type I LNs. And yes, the signals are small because small postsynpathetic Ca2+ currents predominantly cause the signals. Regardless of the imaging method, this naturally reduces the signal-to-noise ratio, making it more challenging to detect signals. To address this issue, we used a well-defined and reproducible method for analyzing these signals. In this context, we do not agree with the very general criticism of the method. The reviewer questions whether the signals are odorant-induced or just noise (see also minor point 12). If we had recorded only noise, we would expect all tuning curves (for each odorant and glomerulus) to be the same. In this context, we disagree with the reviewer's statement that the tuning curves do not represent the Ca2+ signals in Figure 4 (ROI 4 and 15) and Figure 5 (ROI 16 and 23). This debate reflects precisely the kind of 'visual inspection bias' that our clearly defined analysis aims to avoid. On close inspection, the differences in Ca2+ signals can indeed be seen. Figure II (of this letter) shows the signals from the glomeruli in question at higher magnification. The sections of the recordings that were used for the tuning curves are marked in red.

    Figure II: Ca2+ signals of selected glomeruli that were questioned by the reviewer.

    1. An additional methodological issue that compounds the first concern is that calcium signals are imaged with wide-field imaging, and signals from each ROI likely reflect out of plane signals. Out of plane artifacts will be larger for larger calcium signals, which may also make it impossible to resolve any glomerular-specific signals in the type I LNs.

    Thank you for allowing us to clarify this point. The reviewer comment implies that the different amplitudes of the Ca2+ signals indicate some technical-methodological deficiency (poorly chosen odor concentration). But in fact, this is a key finding of this study that is physiologically relevant and crucial for understanding the function of the neurons studied. These very differences in the Ca2+ signals are evidence of the different roles these neurons play in AL. The different signal amplitudes directly show the distinct physiology and Ca2+ sources that dominate the Ca2+ signals in type I and type II LNs. Accordingly, it is impractical to equalize the magnitude of Ca2+ signals under physiological conditions by adjusting the concentration of odor stimuli.

    In the following, we address these issues in more detail:

    1. Imaging Method

    2. Odorant stimulation

    3. Cell type-specific Ca2+ signals

    4. Imaging Method:

    Of course, we agree with the reviewer comment that out-of-focus and out-of-glomerulus fluorescence can potentially affect measurements, especially in widefield optical imaging in thick tissue. This issue was carefully addressed in initial experiments. In type I LNs, which innervate a subset of glomeruli, we detected fluorescence signals, which matched the spike pattern of the electrophysiological recordings 1:1, only in the innervated glomeruli. In the not innervated ROIs (glomeruli), we detected no or comparatively very little fluorescence, even in glomeruli directly adjacent to innervated glomeruli.

    To illustrate this, FIGURE I (of this response letter) shows measurements from an AL in which an uniglomerular projection neuron was investigated in an a set of experiments that were not directly related to the current study. In this experiment, a train of action potential was induced by depolarizing current. The traces show the action potential induced fluorescent signals from the innervated glomerulus (glomerulus #1) and the directly adjacent glomeruli.

    These results do not entirely exclude that the large Ca2+ signals from the innervated LN glomeruli may include out-of-focus and out-of-glomerulus fluorescence, but they do show that the bulk of the signal is generated from the recorded neuron in the respective glomeruli.

    Figure I: Simultaneous electrophysiological and optophysiological recordings of a uniglomerular projection using the ratiometric Ca2+ indicator fura-2. The projection neuron has its arborization in glomerulus 1. The train of action potentials was induced with a depolarizing current pulse (grey bar).

    1. Odorant Stimulation: It is important to note that the odorant concentration cannot be varied freely. For these experiments, the odorant concentrations have to be within a 'physiologically meaningful' range, which means: On the one hand, they have to be high enough to induce a clear response in the projection neurons (the antennal lobe output). On the other hand, however, the concentration was not allowed to be so high that the ORNs were stimulated nonspecifically. These criteria were met with the used concentrations since they induced clear and odorant-specific activity in projection neurons.

    2. Cell type-specific Ca2+ signals:

    The differences in Ca2+ signals are described and discussed in some detail throughout the text (e.g., page 6, lines 119-136; page 9, lines 193-198; page 10-11, lines 226-235; page 14-15, line 309-333). Briefly: In spiking type I LNs, the observed large Ca2+ signals are mediated mainly by voltage-depended Ca2+ channels activated by the Na+-driven action potential's strong depolarization. These large Ca2+ signals mask smaller signals that originate, for example, from excitatory synaptic input (i.e., evoked by ligand-activated Ca2+ conductances). Preventing the firing of action potentials can unmask the ligand-activated signals, as shown in Figure 4 (see also minor comments 8. and 10.). In nonspiking type II LNs, the action potential-generated Ca2+ signals are absent; accordingly, the Ca2+ signals are much smaller. In our model, the comparatively small Ca2+ signals in type II LNs are mediated mainly by (synaptic) ligand-gated Ca2+ conductances, possibly with contributions from voltage-gated Ca2+ channels activated by the comparatively small depolarization (compared with type I LNs).

    Accordingly, our main conclusion, that spiking LNs play a primary role in interglomerular signaling, while nonspiking LNs play an essential role in intraglomeular signaling, can be DIRECTLY inferred from the differences in odorant induced Ca2+ signals alone.

    a) Type I LN: The large, simultaneous, and uniform Ca2+ signals in the innervated glomeruli of an individual type I LN clearly show that they are triggered in each glomerulus by the propagated action potentials, which conclusively shows lateral interglomerular signal propagation.

    b) Type II LNs: In the type II LNs, we observed relatively small Ca2+ signals in single glomeruli or a small fraction of glomeruli of a given neuron. Importantly, the time course and amplitude of the Ca2+ signals varied between different glomeruli and different odors. Considering that type II LNs in principle, can generate large voltage-activated Ca2+ currents (larger that type I LNS; page 4, lines 82-86, Husch et al. 2009a,b; Fusca and Kloppenburg 2021), these data suggest that in type II LNs electrical or Ca2+ signals spread only within the same glomerulus; and laterally only to glomeruli that are electrotonically close to the odorant stimulated glomerulus.

    Taken together, this means that our conclusions regarding inter- and intraglomerular signaling can be derived from the simultaneously recorded amplitudes and the dynamics of the membrane potential and Ca2+ signals alone. This also means that although the correlation analyses support this conclusion nicely, the actual conclusion does not ultimately depend on the correlation analysis. We had (tried to) expressed this with the wording, “Quantitatively, this is reflected in the glomerulus-specific odorant responses and the diverse correlation coefficiiants across…” (page 10, lines 216-217) and “ …This is also reflected in the highly correlated tuning curves in type I LNs and low correlations between tuning curves in type II LNs”(page 13, lines 293-295).

    1. Apart from the above methodological concerns, the authors' interpretation of these data as supporting inter- versus intra-glomerular signaling are not well supported. The odors used in the study are general odors that presumably excite feedforward input to many glomeruli. Since the glomerular source of excitation is not determined, it's not possible to assign the signals in type II LNs as arising locally - selective interglomerular signal propagation is entirely possible. Likewise, the study design does not allow the authors to rule out the possibility that significant intraglomerular inhibition may be mediated by type I LNs.

    The reviewer addresses an important point. However, from the comment, we get the impression that he/she has not taken into account the entire data set and the DISCUSSION. In fact, this topic has already been discussed in some detail in the original version (page 12, lines 268-271; page 15-16; lines 358-374). This section even has a respective heading: "Inter- and intraglomerular signaling via nonspiking type II LNs" (page 15, line 338). We apologize if our explanations regarding this point were unclear, but we also feel that the reviewer is arguing against statements that we did not make in this way.

    a) In 11 out of 18 type II LNs we found 'relatively uncorrelated' (r=0.43±0.16, N=11) glomerular tuning curves. These experiments argue strongly for a 'local excitation' with restricted signal propagation and do not provide support for interglomerular signal propagation. Thus, these results support our interpretation of intraglomerular signaling in this set of neurons.

    b) In 7 out of 18 experiments, we observed 'higher correlated' glomerular tuning curves (r=0.78±0.07, N=7). We agree with the reviewer that this could be caused by various mechanisms, including simultaneous input to several glomeruli or by interglomerular signaling. Both possibilities were mentioned and discussed in the original version of the manuscript (page 12, lines 268-271; page 15-16; lines 358-374). In the Discussion, we considered the latter possibility in particular (but not exclusively) for the type IIa1 neurons that generate spikelets. Their comparatively stronger active membrane properties may be particularly suitable for selective signal transduction between glomeruli.

    c) We have not ruled out that local signaling exists in type I LNs – in addition to interglomerular signaling. The highly localized Ca2+ signals in type I LNs, which we observed when Na+ -driven action potential generation was prevented, may support this interpretation. However, we would like to reiterate that the simultaneous electrophysiological and optophysiological recordings, which show highly correlated glomerular Ca2+ dynamics that match 1:1 with the simultaneously recorded action potential pattern, clearly suggest interglomerular signaling. We also want to emphasize that this interpretation is in agreement with previous models derived from electrophysiological studies(Assisi et al., 2011; Fujiwara et al., 2014; Hong and Wilson, 2015; Nagel and Wilson, 2016; Olsen and Wilson, 2008; Sachse and Galizia, 2002; Wilson, 2013).

    In light of the reviewer's comment(s), we have modified the text to clarify these points (page 14, lines 317-319).

    Reviewer #3 (Public Review):

    To elucidate the role of the two types of LNs, the authors combined whole-cell patch clamp recordings with calcium imaging via single cell dye injection. This method enables to monitor calcium dynamics of the different axons and branches of single LNs in identified glomeruli of the antennal lobe, while the membrane potential can be recorded at the same time. The authors recorded in total from 23 spiking (type I LN) and 18 non-spiking (type II LN) neurons to a set of 9 odors and analyzed the firing pattern as well as calcium signals during odor stimulation for individual glomeruli. The recordings reveal on one side that odor-evoked calcium responses of type I LNs are odor-specific, but homogeneous across glomeruli and therefore highly correlated regarding the tuning curves. In contrast, odor-evoked responses of type II LNs show less correlated tuning patterns and rather specific odor-evoked calcium signals for each glomerulus. Moreover the authors demonstrate that both LN types exhibit distinct glomerular branching patterns, with type I innervating many, but not all glomeruli, while type II LNs branch in all glomeruli.

    From these results and further experiments using pharmacological manipulation, the authors conclude that type I LNs rather play a role regarding interglomerular inhibition in form of lateral inhibition between different glomeruli, while type II LNs are involved in intraglomerular signaling by developing microcircuits in individual glomeruli.

    In my opinion the methodological approach is quite challenging and all subsequent analyses have been carried out thoroughly. The obtained data are highly relevant, but provide rather an indirect proof regarding the distinct roles of the two LN types investigated. Nevertheless, the conclusions are convincing and the study generally represents a valuable and important contribution to our understanding of the neuronal mechanisms underlying odor processing in the insect antennal lobe. I think the authors should emphasize their take-home messages and resulting conclusions even stronger. They do a good job in explaining their results in their discussion, but need to improve and highlight the outcome and meaning of their individual experiments in their results section.

    Thank you for this positive feedback.

    References:

    Assisi, C., Stopfer, M., Bazhenov, M., 2011. Using the structure of inhibitory networks to unravel mechanisms of spatiotemporal patterning. Neuron 69, 373–386. https://doi.org/10.1016/j.neuron.2010.12.019

    Das, S., Trona, F., Khallaf, M.A., Schuh, E., Knaden, M., Hansson, B.S., Sachse, S., 2017. Electrical synapses mediate synergism between pheromone and food odors in Drosophila melanogaster . Proc Natl Acad Sci U S A 114, E9962–E9971. https://doi.org/10.1073/pnas.1712706114

    Fujiwara, T., Kazawa, T., Haupt, S.S., Kanzaki, R., 2014. Postsynaptic odorant concentration dependent inhibition controls temporal properties of spike responses of projection neurons in the moth antennal lobe. PLOS ONE 9, e89132. https://doi.org/10.1371/journal.pone.0089132

    Fusca, D., Husch, A., Baumann, A., Kloppenburg, P., 2013. Choline acetyltransferase-like immunoreactivity in a physiologically distinct subtype of olfactory nonspiking local interneurons in the cockroach (Periplaneta americana). J Comp Neurol 521, 3556–3569. https://doi.org/10.1002/cne.23371

    Fuscà, D., and Kloppenburg, P. (2021). Odor processing in the cockroach antennal lobe-the network components. Cell Tissue Res.

    Hong, E.J., Wilson, R.I., 2015. Simultaneous encoding of odors by channels with diverse sensitivity to inhibition. Neuron 85, 573–589. https://doi.org/10.1016/j.neuron.2014.12.040

    Husch, A., Paehler, M., Fusca, D., Paeger, L., Kloppenburg, P., 2009a. Calcium current diversity in physiologically different local interneuron types of the antennal lobe. J Neurosci 29, 716–726. https://doi.org/10.1523/JNEUROSCI.3677-08.2009

    Husch, A., Paehler, M., Fusca, D., Paeger, L., Kloppenburg, P., 2009b. Distinct electrophysiological properties in subtypes of nonspiking olfactory local interneurons correlate with their cell type-specific Ca2+ current profiles. J Neurophysiol 102, 2834–2845. https://doi.org/10.1152/jn.00627.2009

    Nagel, K.I., Wilson, R.I., 2016. Mechanisms Underlying Population Response Dynamics in Inhibitory Interneurons of the Drosophila Antennal Lobe. J Neurosci 36, 4325–4338. https://doi.org/10.1523/JNEUROSCI.3887-15.2016

    Neupert, S., Fusca, D., Kloppenburg, P., Predel, R., 2018. Analysis of single neurons by perforated patch clamp recordings and MALDI-TOF mass spectrometry. ACS Chem Neurosci 9, 2089–2096.

    Olsen, S.R., Bhandawat, V., Wilson, R.I., 2007. Excitatory interactions between olfactory processing channels in the Drosophila antennal lobe. Neuron 54, 89–103. https://doi.org/10.1016/j.neuron.2007.03.010

    Olsen, S.R., Wilson, R.I., 2008. Lateral presynaptic inhibition mediates gain control in an olfactory circuit. Nature 452, 956–960. https://doi.org/10.1038/nature06864

    Sachse, S., Galizia, C., 2002. Role of inhibition for temporal and spatial odor representation in olfactory output neurons: a calcium imaging study. J Neurophysiol. 87, 1106–17.

    Shang, Y., Claridge-Chang, A., Sjulson, L., Pypaert, M., Miesenbock, G., 2007. Excitatory Local Circuits and Their Implications for Olfactory Processing in the Fly Antennal Lobe. Cell 128, 601–612.

    Wilson, R.I., 2013. Early olfactory processing in Drosophila: mechanisms and principles. Annu Rev Neurosci 36, 217–241. https://doi.org/10.1146/annurev-neuro-062111-150533

    Yaksi, E., Wilson, R.I., 2010. Electrical coupling between olfactory glomeruli. Neuron 67, 1034–1047. https://doi.org/10.1016/j.neuron.2010.08.041

  3. eLife

    Evaluation Summary:

    The question investigated - to understand the computational significance of different types of local interneurons in neural circuits - is an important and significant problem. Here authors elucidate the role of the two types of LNs, by combining whole-cell patch clamp recordings with calcium imaging via single cell dye injection. This method enables them to monitor calcium dynamics of the different axons and branches of single LNs in identified glomeruli of the antennal lobe, while the membrane potential can be recorded at the same time. They conclude that non-spiking LNs with graded responses show glomerular restricted responses to odorants and spiking LNs show similar responses across glomeruli.

    (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 #1 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    The manuscript provides very high quality single-cell physiology combined with population physiology to reveal distinctives roles for two anatomically dfferent LN populations in the cockroach antennal lobe. The conclusion that non-spiking LNs with graded responses show glomerular-restricted responses to odorants and spiking LNs show similar responses across glomeruli generally supported with strong and clean data, although the possibility of selective interglomerular inhibition has not been ruled out. On balance, the single-cell biophysics and physiology provides foundational information useful for well-grounded mechanistic understanding of how information is processed in insect antennal lobes, and how each LN class contributes to odor perception and behavior.

  5. eLife

    Reviewer #2 (Public Review):

    The manuscript "Task-specific roles of local interneurons for inter- and intraglomerular signaling in the insect antennal lobe" evaluates the spatial distribution of calcium signals evoked by odors in two major classes of olfactory local neurons (LNs) in the cockroach P. Americana, which are defined by their physiological and morphological properties. Spiking type I LNs have a patchy innervation pattern of a subset of glomeruli, whereas non-spiking type II LNs innervate almost all glomeruli (Type II). The authors' overall conclusion is that odors evoke calcium signals globally and relatively uniformly across glomeruli in type I spiking LNs, and LN neurites in each glomerulus are broadly tuned to odor. In contrast, the authors conclude that they observe odor-specific patterns of calcium signals in type II nonspiking LNs, and LN neurites in different glomeruli display distinct local odor tuning. Blockade of action potentials in type I LNs eliminates global calcium signaling and decorrelates glomerular tuning curves, converting their response profile to be more similar to that of type II LNs. From these conclusions, the authors infer a primary role of type I LNs in interglomerular signaling and type III LNs in intraglomerular signaling.

    The question investigated by this study - to understand the computational significance of different types of LNs in olfactory circuits - is an important and significant problem. The design of the study is straightforward, but methodological and conceptual gaps raise some concerns about the authors' interpretation of their results. These can be broadly grouped into three main areas.

    1. The comparison of the spatial (glomerular) pattern of odor-evoked calcium signals in type I versus type II LNs may not necessarily be a true apples-to-apples comparison. Odor-evoked calcium signals are an order of magnitude larger in type I versus type II cells, which will lead to a higher apparent correlation in type I cells. In type IIb cells, and type I cells with sodium channel blockade, odor-evoked calcium signals are much smaller, and the method of quantification of odor tuning (normalized area under the curve) is noisy. Compare, for instance, ROI 4 & 15 (Figure 4) or ROI 16 & 23 (Figure 5) which are pairs of ROIs that their quantification concludes have dramatically different odor tuning, but which visual inspection shows to be less convincing. The fact that glomerular tuning looks more correlated in type IIa cells, which have larger, more reliable responses compared to type IIb cells, also supports this concern.

    2. An additional methodological issue that compounds the first concern is that calcium signals are imaged with wide-field imaging, and signals from each ROI likely reflect out of plane signals. Out of plane artifacts will be larger for larger calcium signals, which may also make it impossible to resolve any glomerular-specific signals in the type I LNs.

    3. Apart from the above methodological concerns, the authors' interpretation of these data as supporting inter- versus intra-glomerular signaling are not well supported. The odors used in the study are general odors that presumably excite feedforward input to many glomeruli. Since the glomerular source of excitation is not determined, it's not possible to assign the signals in type II LNs as arising locally - selective interglomerular signal propagation is entirely possible. Likewise, the study design does not allow the authors to rule out the possibility that significant intraglomerular inhibition may be mediated by type I LNs.

  6. eLife

    Reviewer #3 (Public Review):

    To elucidate the role of the two types of LNs, the authors combined whole-cell patch clamp recordings with calcium imaging via single cell dye injection. This method enables to monitor calcium dynamics of the different axons and branches of single LNs in identified glomeruli of the antennal lobe, while the membrane potential can be recorded at the same time. The authors recorded in total from 23 spiking (type I LN) and 18 non-spiking (type II LN) neurons to a set of 9 odors and analyzed the firing pattern as well as calcium signals during odor stimulation for individual glomeruli. The recordings reveal on one side that odor-evoked calcium responses of type I LNs are odor-specific, but homogeneous across glomeruli and therefore highly correlated regarding the tuning curves. In contrast, odor-evoked responses of type II LNs show less correlated tuning patterns and rather specific odor-evoked calcium signals for each glomerulus. Moreover the authors demonstrate that both LN types exhibit distinct glomerular branching patterns, with type I innervating many, but not all glomeruli, while type II LNs branch in all glomeruli.

    From these results and further experiments using pharmacological manipulation, the authors conclude that type I LNs rather play a role regarding interglomerular inhibition in form of lateral inhibition between different glomeruli, while type II LNs are involved in intraglomerular signaling by developing microcircuits in individual glomeruli.

    In my opinion the methodological approach is quite challenging and all subsequent analyses have been carried out thoroughly. The obtained data are highly relevant, but provide rather an indirect proof regarding the distinct roles of the two LN types investigated. Nevertheless, the conclusions are convincing and the study generally represents a valuable and important contribution to our understanding of the neuronal mechanisms underlying odor processing in the insect antennal lobe. I think the authors should emphasize their take-home messages and resulting conclusions even stronger. They do a good job in explaining their results in their discussion, but need to improve and highlight the outcome and meaning of their individual experiments in their results section.

  7. Version published to 10.1101/2020.11.26.369686v1 on bioRxiv