Target cell-specific synaptic dynamics of excitatory to inhibitory neuron connections in supragranular layers of human neocortex
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Evaluation Summary:
The authors made paired recordings from synaptically-connected excitatory and inhibitory neurons in slices of human neocortex and used posthoc molecular methods to identify major classes of the recorded interneurons. The principal finding is that, as found previously in rodent cortex, short-term plasticity of the synaptic connections from excitatory to inhibitory neurons depends on the molecular identity of the inhibitory neurons. This is important, as it suggests that many rodent studies carried out over the past decades are physiologically relevant to humans.
(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
Rodent studies have demonstrated that synaptic dynamics from excitatory to inhibitory neuron types are often dependent on the target cell type. However, these target cell-specific properties have not been well investigated in human cortex, where there are major technical challenges in reliably obtaining healthy tissue, conducting multiple patch-clamp recordings on inhibitory cell types, and identifying those cell types. Here, we take advantage of newly developed methods for human neurosurgical tissue analysis with multiple patch-clamp recordings, post-hoc fluorescent in situ hybridization (FISH), machine learning-based cell type classification and prospective GABAergic AAV-based labeling to investigate synaptic properties between pyramidal neurons and PVALB- vs. SST-positive interneurons. We find that there are robust molecular differences in synapse-associated genes between these neuron types, and that individual presynaptic pyramidal neurons evoke postsynaptic responses with heterogeneous synaptic dynamics in different postsynaptic cell types. Using molecular identification with FISH and classifiers based on transcriptomically identified PVALB neurons analyzed by Patch-seq, we find that PVALB neurons typically show depressing synaptic characteristics, whereas other interneuron types including SST-positive neurons show facilitating characteristics. Together, these data support the existence of target cell-specific synaptic properties in human cortex that are similar to rodent, thereby indicating evolutionary conservation of local circuit connectivity motifs from excitatory to inhibitory neurons and their synaptic dynamics.
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Author Response
Reviewer #2 (Public Review):
Kim et al. examined the properties of neuronal connections responsible for inhibitory cell activation to show that the characteristics examined were similar in humans and rodents. This is important, as it suggests that the many rodent studies carried out over the past decades are physiologically relevant to humans.
Strengths
- Human brain tissues are difficult to obtain, hence the study provides valuable insights
- An impressive multipronged approach was used for cell classifications
- Despite the lack of novel findings, the revelation of the similarities between human and rodent synapses is important and has far-reaching implications. This important finding suggests the knowledge generated from rodent research is, at least partly, physiologically relevant to and transferrable to humans.
Author Response
Reviewer #2 (Public Review):
Kim et al. examined the properties of neuronal connections responsible for inhibitory cell activation to show that the characteristics examined were similar in humans and rodents. This is important, as it suggests that the many rodent studies carried out over the past decades are physiologically relevant to humans.
Strengths
- Human brain tissues are difficult to obtain, hence the study provides valuable insights
- An impressive multipronged approach was used for cell classifications
- Despite the lack of novel findings, the revelation of the similarities between human and rodent synapses is important and has far-reaching implications. This important finding suggests the knowledge generated from rodent research is, at least partly, physiologically relevant to and transferrable to humans.
Weaknesses
- The study is descriptive by design, and hence provides limited conceptual advances, especially with the retrospect that synaptic properties are similar between humans and rodents (although see strength #3). For example, very similar findings and techniques have already recently been reported by a number of the same authors in the Campagnola et al., Science 2022 paper.
We agreed that stimulus protocols of connectivity assays with multiple patch-clamp recordings in this study had been adapted from the recent publication (Campagnola et al., Science 2022). In this previous study, especially for human synaptic connectivity data, the main cell type categorization was at the level of excitatory and inhibitory neurons which identified based on morphological features and observed PSP characteristics (e.g., direction of membrane potential changes) when it connected each other. However, we went further to identify interneuron subclasses in the connectivity assays using virally labeled slice cultures and post-hoc HCR staining in addition to intrinsic classifier, which is not investigated from the recent publication (Campagnola et al., 2022). Therefore, following scientific findings and their implications are not the same shown in the previous study and we think this study provides a significant advance of our understanding in human cortical circuits organization.
- Despite the fact that normal physiology was reported, the use of pathological human brain tissue could affect the results.
We agreed that the use of pathological human brain tissue to investigate normal physiology is not ideal, however, as mentioned in the METHODS below (section of “Acute slice preparation”), our surgically resected neocortical tissues show minimal pathology, and we believe these tissue preparations can be used to address normal physiological properties of human neurons. Importantly, we saw no effect of disease state (epilepsy vs. tumor) on the intrinsic or synaptic properties that we measured. Our METHODS state that “Surgically resected neocortical tissue was distal to the pathological core (i.e., tumor tissue or mesial temporal structures). Detailed histological assessment and using a curated panel of cellular marker antibodies indicated a lack of overt pathology in surgically resected cortical slices (Berg et al., 2021).”. We also state in the RESULTS that “These tissues were distal to the epileptic focus or tumor, and have shown minimal pathology when examined (Berg et al., 2021). Brain pathology was evaluated using six histological markers that were independently scored by three pathologists. Surgically resected tissues have been used extensively to characterize human cortical physiology and anatomy (Berg et al., 2021).”. Lastly, this is the best possible human tissue available for us to conduct physiological experiments. It is an unavoidable caveat of this work that our healthy brain tissue was derived from a donor brain exhibiting a serious disease.
- The manuscript may not be easy to understand for the uninvited, because many concepts and abbreviations were not properly introduced.
Thank you for pointing this oversight out. We updated our manuscript and made sure that we fully describe all abbreviations. We now changed the abbreviation of MPC back to multiple patch-clamp recording, and some other abbreviations such as LAMP5, SLC17A7, DLX are now better explained. We have also changed the order of multiple figures (i.e., Figure 5 – Figure supplements to Figure 3 – Figure supplements) and removed some complicated figures (e.g., Figure 1 – Figure supplement 1) to present the data in a fashion that can be understood by a more general reader.
- The statistical treatment is not ideal, so some conclusions may not be valid.
We performed additional statistical analyses as suggested and implemented in the text of the RESULTS.
Furthermore, we also made additional Figure supplements (Figure 4 – Figure supplement 3, Figure 4 – Figure supplement 4, Figure 6 – Figure supplement 2, and Figure 6 – Figure supplement 3) to support our conclusions.
- The mixed usage of acute and cultured slices is not ideal and likely affects the outcome.
We agree that the mixed usage of acute and cultured slices is not ideal, and it could affect the interpretation of outcome. Therefore, we performed additional analyses to see if there is any correlated change of synaptic property (i.e., paired pulse ratio) along the days after slice culture (now implemented in Figure 4 – Figure supplement 4 and Figure 6 – Figure supplement 3) and we didn’t find any significant correlation. However, we noticed the short-term synaptic dynamics are rather differentiated between acute and slice culture condition shown in Figure 4 – Figure supplement 1d. We think this is due to sampling bias rather than tissue preparation difference and these points are now more carefully described in the DISCUSSION as “This difference we observed in this study, i.e., more facilitating synapses were detected in slice cultures than in acute slices, could either reflect an acute vs. slice culture difference. However, we believe it is more likely to reflect a selection bias for PVALB neurons when patching in unlabeled acute slices, and that the AAV-based strategy with a pan-GABAergic enhancer allows a more unbiased sampling of interneuron subclasses whose properties are preserved in culture. In support of this, PPR analysis as a function of days after slice culture shows no relationship to acute versus slice culture preparation (Figure 4 – Figure supplement 4, Figure 6 – Figure supplement 3). Furthermore, we have observed that viral targeting of GABAergic interneurons greatly facilitates sampling of the SST subclass in the human cortex compared to unbiased patch-seq experiments (Lee et al., 2022), and this selection bias likely explains synapse type sampling differences in cultured slices compared to acute preparations.”.
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Evaluation Summary:
The authors made paired recordings from synaptically-connected excitatory and inhibitory neurons in slices of human neocortex and used posthoc molecular methods to identify major classes of the recorded interneurons. The principal finding is that, as found previously in rodent cortex, short-term plasticity of the synaptic connections from excitatory to inhibitory neurons depends on the molecular identity of the inhibitory neurons. This is important, as it suggests that many rodent studies carried out over the past decades are physiologically relevant to humans.
(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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Reviewer #1 (Public Review):
Kim and coauthors have performed multiple simultaneous whole cell recordings in living slices of human neocortex obtained from neurosurgical resection in order to study the properties of synaptic connections from excitatory pyramidal neurons onto various types of inhibitory interneurons. Strengths of the study include the unique ability to study biophysical properties of human synapses, and the sophisticated in situ hybridization and other approaches used to identify the class of the postsynaptic interneurons. The main finding of the study is that a key principle identified in rodent neocortex: that fast-spiking parvalbumin-positive neurons receive initially depressing synapses, whereas other categories of interneurons receive more initially facilitating synapses, is conserved in the human. The authors also …
Reviewer #1 (Public Review):
Kim and coauthors have performed multiple simultaneous whole cell recordings in living slices of human neocortex obtained from neurosurgical resection in order to study the properties of synaptic connections from excitatory pyramidal neurons onto various types of inhibitory interneurons. Strengths of the study include the unique ability to study biophysical properties of human synapses, and the sophisticated in situ hybridization and other approaches used to identify the class of the postsynaptic interneurons. The main finding of the study is that a key principle identified in rodent neocortex: that fast-spiking parvalbumin-positive neurons receive initially depressing synapses, whereas other categories of interneurons receive more initially facilitating synapses, is conserved in the human. The authors also make important technical contributions to our ability to study synapses in human tissue including a slice culture technique that prolongs the use of these valuable samples, and a multi-pronged approach to characterizing interneuron identity. The main weaknesses of the current version of the manuscript relate to incomplete analyses and a somewhat confusing presentation that leave in question the relative importance of interneuron identity vs. other factors in determining the degree of synaptic facilitation and depression.
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Reviewer #2 (Public Review):
Kim et al. examined the properties of neuronal connections responsible for inhibitory cell activation to show that the characteristics examined were similar in humans and rodents. This is important, as it suggests that the many rodent studies carried out over the past decades are physiologically relevant to humans.
Strengths
1. Human brain tissues are difficult to obtain, hence the study provides valuable insights
2. An impressive multipronged approach was used for cell classifications
3. Despite the lack of novel findings, the revelation of the similarities between human and rodent synapses is important and has far-reaching implications. This important finding suggests the knowledge generated from rodent research is, at least partly, physiologically relevant to and transferrable to humans.Weaknesses
1. The …
Reviewer #2 (Public Review):
Kim et al. examined the properties of neuronal connections responsible for inhibitory cell activation to show that the characteristics examined were similar in humans and rodents. This is important, as it suggests that the many rodent studies carried out over the past decades are physiologically relevant to humans.
Strengths
1. Human brain tissues are difficult to obtain, hence the study provides valuable insights
2. An impressive multipronged approach was used for cell classifications
3. Despite the lack of novel findings, the revelation of the similarities between human and rodent synapses is important and has far-reaching implications. This important finding suggests the knowledge generated from rodent research is, at least partly, physiologically relevant to and transferrable to humans.Weaknesses
1. The study is descriptive by design, and hence provides limited conceptual advances, especially with the retrospect that synaptic properties are similar between humans and rodents (although see strength #3). For example, very similar findings and techniques have already recently been reported by a number of the same authors in the Campagola et al., Science 2022 paper.
2. Despite the fact that normal physiology was reported, the use of pathological human brain tissue could affect the results.
3. The manuscript may not be easy to understand for the uninvited, because many concepts and abbreviations were not properly introduced.
4. The statistical treatment is not ideal, so some conclusions may not be valid.
5. The mixed usage of acute and cultured slices is not ideal and likely affects the outcome.
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