Supralinear dendritic integration in murine dendrite-targeting interneurons

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    In this manuscript, Griesius et al analyze the dendritic integration properties of NDNF and OLM interneurons, and the current dataset suggests that even though both cell types display supralinear NMDA receptor-dependent synaptic integration, this may be associated with dendritic calcium transients only in NDNF interneurons. These findings are important because they could shed light on the functional diversity of different classes of interneurons in the mouse neocortex and hippocampus, which in turn can have major implications for understanding information flow in complex neural circuits. They are considered as being currently incomplete, however, due to: (i) the large variability and small sample size of multiple datasets, which prevents a finer evaluation of cellular and molecular mechanisms accounting for the difference in the integrative properties of different interneuron types; (ii) lack of control experiments to rule out that the effect of the NMDA antagonist AP5 on synaptic integration is not confounded by potential phototoxicity damage; (iii) lack of a precise control of the uncaging location.

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Abstract

Non-linear summation of synaptic inputs to the dendrites of pyramidal neurons has been proposed to increase the computation capacity of neurons through coincidence detection, signal amplification, and additional logic operations such as XOR. Supralinear dendritic integration has been documented extensively in principal neurons, mediated by several voltage-dependent conductances. It has also been reported in parvalbumin-positive hippocampal basket cells, in dendrites innervated by feedback excitatory synapses. Whether other interneurons, which support feed-forward or feedback inhibition of principal neuron dendrites, also exhibit local non-linear integration of synaptic excitation is not known. Here we use patch-clamp electrophysiology, and 2-photon calcium imaging and glutamate uncaging, to show that supralinear dendritic integration of near-synchronous spatially clustered glutamate-receptor mediated depolarization occurs in NDNF-positive neurogliaform cells and oriens-lacunosum moleculare interneurons in the mouse hippocampus. Supralinear summation was detected via recordings of somatic depolarizations elicited by uncaging of glutamate on dendritic fragments, and, in neurogliaform cells, by concurrent imaging of dendritic calcium transients. Supralinearity was abolished by blocking NMDA receptors (NMDARs) but resisted blockade of voltage-gated sodium channels. Blocking L-type calcium channels abolished supralinear calcium signalling but only had a minor effect on voltage supralinearity. Dendritic boosting of spatially clustered synaptic signals argues for previously unappreciated computational complexity in dendrite-projecting inhibitory cells of the hippocampus.

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  1. eLife assessment

    In this manuscript, Griesius et al analyze the dendritic integration properties of NDNF and OLM interneurons, and the current dataset suggests that even though both cell types display supralinear NMDA receptor-dependent synaptic integration, this may be associated with dendritic calcium transients only in NDNF interneurons. These findings are important because they could shed light on the functional diversity of different classes of interneurons in the mouse neocortex and hippocampus, which in turn can have major implications for understanding information flow in complex neural circuits. They are considered as being currently incomplete, however, due to: (i) the large variability and small sample size of multiple datasets, which prevents a finer evaluation of cellular and molecular mechanisms accounting for the difference in the integrative properties of different interneuron types; (ii) lack of control experiments to rule out that the effect of the NMDA antagonist AP5 on synaptic integration is not confounded by potential phototoxicity damage; (iii) lack of a precise control of the uncaging location.

  2. Reviewer #1 (Public review):

    The manuscript by Griesius et al. addresses the dendritic integration of synaptic input in cortical GABAergic interneurons (INs). Dendritic properties, passive and active, of principal cells have been extensively characterized, but much less is known about the dendrites of INs. The limited information is particularly relevant in view of the high morphological and physiological diversity of IN types. The few studies that investigated IN dendrites focused on parvalbumin-expressing INs. In fact, in a previous study, the authors examined dendritic properties of PV INs, and found supralinear dendritic integration in basal, but not in apical dendrites (Cornford et al., 2019 eLife).

    In the present study, complementary to the prior work, the authors investigate whether dendrite-targeting IN types, NDNF-expressing neurogliaform cells, and somatostatin(SOM)-expressing O-LM neurons, display similar active integrative properties by combining clustered glutamate-uncaging and pharmacological manipulations with electrophysiological recording and calcium imaging from genetically identified IN types in mouse acute hippocampal slices.

    The main findings are that NDNF IN dendrites show strong supralinear summation of spatially- and temporally-clustered EPSPs, which is changed into sublinear behavior by bath application of NMDA receptor antagonists, but not by Na+-channel blockers. L-type calcium channel blockers abolished the supralinear behavior associated calcium transients but had no or only weak effect on EPSP summation. SOM IN dendrites showed similar, albeit weaker NMDA-dependent supralinear summation, but no supralinear calcium transients were detected in these INs. In summary, the study demonstrates that different IN types are endowed with active dendritic integrative mechanisms, but show qualitative and quantitative divergence in these mechanisms.

    While the research is conceptionally not novel, it constitutes an important incremental gain in our understanding of the functional diversity of GABAergic INs. In view of the central roles of IN types in network dynamics and information processing in the cortex, results and conclusions are of interest to the broader neuroscience community.

    The experiments are well designed, and closely follow the approach from the previous publication in parts, enabling direct comparison of the results obtained from the different IN types. The data is convincing and the conclusions are well-supported, and the manuscript is very well-written.

    I see only a few open questions and some inconsistencies in the presentation of the data in the figures (see details below).

  3. Reviewer #2 (Public review):

    Summary:

    Griesius et al. investigate the dendritic integration properties of two types of inhibitory interneurons in the hippocampus: those that express NDNF+ and those that express somatostatin. They found that both neurons showed supralinear synaptic integration in the dendrites, blocked by NMDA receptor blockers but not by blockers of Na+ channels. These experiments are critically overdue and very important because knowing how inhibitory neurons are engaged by excitatory synaptic input has important implications for all theories involving these inhibitory neurons.

    Strengths:

    (1) Determined the dendritic integration properties of two fundamental types of inhibitory interneurons.

    (2) Convincing demonstration that supra-threshold integration in both cell types depends on NMDA receptors but not on Na+ channels.

    Weaknesses:

    It is unknown whether highly clustered synaptic input, as used in this study (and several previous studies), occurs physiologically.

  4. Reviewer #3 (Public review):

    Summary:

    The authors study the temporal summation of caged EPSPs in dendrite-targeting hippocampal CA1 interneurons. There are some descriptive data presented, indicating non-linear summation, which seems to be larger in dendrites of NDNF expressing neurogliaform cells versus OLM cells. However, the underlying mechanisms are largely unclear.

    Strengths:

    Focal 2-photon uncaging of glutamate is a nice and detailed method to study temporal summation of small potentials in dendritic segments.

    Weaknesses:

    (1) NMDA-receptor signaling in NDNF-IN. The authors nicely show that temporal summation in dendrites of NDNF-INs is to a certain extent non-linear. However, this non-linearity varies massively from cell to cell (or dendrite to dendrite) from 0% up to 400% (Figure S2). The reason for this variability is totally unclear. Pharmacology with AP5 hints towards a contribution of NMDA receptors. However, the authors claim that the non-linearity is not dependent on EPSP amplitude (Figure S2), which should be the case if NMDA-receptors are involved. Unfortunately, there are no voltage-clamp data of NMDA currents similar to the previous study. This would help to see whether NMDA-receptor contribution varies from synapse to synapse to generate the observed variability? Furthermore, the NMDA- and AMPA-currents would help to compare NDNF with the previously characterized PV cells and would help to contribute to our understanding of interneuron function.

    (2) Sublinear summation in NDNF-INs. In the presence of AP5, the temporal summation of caged EPSPs is sublinear. That is potentially interesting. The authors claim that this might be dependent on the diameter of dendrites. Many voltage-gated channels can mediate such things as well. To conclude the contribution of dendritic diameter, it would be helpful to at least plot the extent of sublinearity in single NDNF dendrites versus the dendritic diameter. Otherwise, this statement should be deleted.

    (3) Nonlinear EPSP summation in OLM-IN. The authors do similar experiments in dendrite-targeting OLM-INs and show that the non-linear summation is smaller than in NDNF cells. The reason for this remains unclear. The authors claim that this is due to the larger dendritic diameter in OLM cells. However, there is no analysis. The minimum would be to correlate non-linearity with dendritic diameter in OLM-cells. Very likely there is an important role of synapse density and glutamate receptor density, which was shown to be very low in proximal dendrites of OLM cells and strongly increase with distance (Guirado et al. 2014, Cerebral Cortex 24:3014-24, Gramuntell et al. 2021, Front Aging Neurosci 13:782737). Therefore, the authors should perform a set of experiments in more distal dendrites of OLM cells with diameters similar to the diameters of the NDNF cells. Even better would be if the authors would quantify synapse density by counting spines and show how this density compares with non-linearity in the analyzed NDNF and OLM dendrites.

    (4) NMDA in OLM. Similar to the NDNF cells, the authors claim the involvement of NMDA receptors in OLM cells. Again there seems to be no dependence on EPSP amplitude, which is not understandable at this point (Figure S3). Even more remarkable is the fact that the authors claim that there is no dendritic calcium increase after activation of NMDA receptors. Similar to NDNF-cell analysis there are no NMDA currents in OLMs. Unfortunately, even no calcium imaging experiments were shown. Why? Are there calcium-impermeable NNDA receptors in OLM cells? To understand this phenomenon the minimum is to show some physiological signature of NMDA-receptors, for example, voltage-clamp currents. Furthermore, it would be helpful to systematically vary stimulus intensity to see some calcium signals with larger stimulation. In case there is still no calcium signal, it would be helpful to measure reversal potentials with different ion compositions to characterize the potentially 'Ca2+ impermeable' voltage-dependent NMDA receptors in OLM cells.