An Anatomical and Physiological Basis for Flexible Coincidence Detection in the Auditory System

Reviewer #1 (Public Review):

Kreeger and colleagues have explored the balance of excitation and inhibition in the cochlear nucleus octopus cells of mice using morphological, electrophysiological, and computational methods. On the surface, the conclusion, that synaptic inhibition is present, does not seem like a leap. However, the octopus cells have been in the past portrayed as devoid of inhibition. This view was supported by the seeming lack of glycinergic fibers in the octopus cell area and the lack of apparent IPSPs. Here, Kreeger et al. used beautiful immunohistochemical and mouse genetic methods to quantify the inhibitory and excitatory boutons over the complete surface of individual octopus cells and further analysed the proportions of the different subtypes of spiral ganglion cell inputs. I think the analysis stands as one of the most complete descriptions of any neuron, leaving little doubt about the presence of glycinergic boutons.

Kreeger et al then examined inhibition physiologically, but here I felt that the study was incomplete. Specifically, no attempt was made to assess the actual, biological values of synaptic conductance for AMPAR and GlyR. Thus, we don't really know how potent the GlyR could be in mediating inhibition. Here are some numbered comments:

(1) "EPSPs" were evoked either optogenetically or with electrical stimulation. The resulting depolarizations are interpreted to be EPSPs. However previous studies from Oertel show that octopus cells have tiny spikes, and distinguishing them from EPSPs is tricky. No mention is made here about how or whether that was done. Thus, the analysis of EPSP amplitude is ambiguous.

(2) For this and later analysis, a voltage clamp of synaptic inputs would have been a simple alternative to avoid contaminating spikes or shunts by background or voltage-gated conductances. Yet only the current clamp was employed. I can understand that the authors might feel that the voltage clamp is 'flawed' because of the failure to clamp dendrites. But that may have been a good price to pay in this case. The authors should have at least justified their choice of method and detailed its caveats.

(3) The modeling raised several concerns. First, there is little presentation of assumptions, and of course, a model is entirely about its assumptions. For example, what excitatory conductance amplitudes were used? The same for inhibitory conductance? How were these values arrived at? The authors note that EPSGs and IPSGs had peaks at 0.3 and 3 ms. On what basis were these numbers obtained? The model's conclusions entirely depend on these values, and no measurements were made here that could have provided them. Parenthetical reference is made to Figure S5 where a range of values are tested, but with little explanation or justification.

(4) In experiments that combined E and I stimulation, what exactly were time timecourses of the conductance changes, and how 'synchronous' were they, given the different methods to evoke them? (had the authors done voltage clamp they would know the answers).

(5) Figure 4G is confusing to me. Its point, according to the text, is to show that changes in membrane properties induced by a block of Kv and HCN channels would not be expected to alter the amplitudes of EPSCs and IPSCs across the dendritic expanse. Now we are talking about currents (not shunting effects), and the presumption is that the blockers would alter the resting potential and thus the driving force for the currents. But what was the measured membrane potential change in the blockers? Surely that was documented. To me, the bigger concern (stated in the text) is whether the blockers altered exocytosis, and thus the increase in IPSP amplitude in blockers is due BOTH to loss of shunting and increase in presynaptic spike width. Added to this is that 4AP will reduce the spike threshold, thus allowing more ChR2-expressing axons to reach the threshold. Figure 4G does not address this point.

(6) Figure 5F is striking as the key piece of biological data that shows that inhibition does reduce the amplitude of "EPSPs" in octopus cells. Given the other uncertainties mentioned, I wondered if it makes sense as an example of shunting inhibition. Specifically, what are the relative synaptic conductances, and would you predict a 25% reduction given the actual (not modeled) values?

(7) Some of the supplemental figures, like 4 and 5, are hardly mentioned. Few will glean anything from them unless the authors direct attention to them and explain them better. In general, the readers would benefit from more complete explanations of what was done.

Reviewer #2 (Public Review):

Summary:

Kreeger et.al provided mechanistic evidence for flexible coincidence detection of auditory nerve synaptic inputs by octopus cells in the mouse cochlear nucleus. The octopus cells are specialized neurons that can fire repetitively at very high rates (> 800 Hz in vivo), yield responses dominated by the onset of sound for simple stimuli, and integrate auditory nerve inputs over a wide frequency span. Previously, it was thought that octopus cells received little inhibitory input, and their integration of auditory input depended principally on temporally precise coincidence detection of excitatory auditory nerve inputs, coupled with a low input resistance established by high levels of expression of certain potassium channels and hyperpolarization-activated channels.

In this study, the authors used a combination of numerous genetic mouse models to characterize synaptic inputs and enable optogenetic stimulation of subsets of afferents, fluorescent microscopy, detailed reconstructions of the location of inhibitory synapses on the soma and dendrites of octopus cells, and computational modeling, to explore the importance of inhibitory inputs to the cells. They determined through assessment of excitatory and inhibitory synaptic densities that spiral ganglion neuron synapses are densest on the soma and proximal dendrite, while glycenergic inhibitory synaptic density is greater on the dendrites compared to the soma of octopus cells. Using different genetic lines, the authors further elucidated that the majority of excitatory synapses on the octopus cells are from type 1a spiral ganglion neurons, which have low response thresholds and high rates of spontaneous activity. In the second half of the paper, the authors employed electrophysiology to uncover the physiological response of octopus cells to excitatory and inhibitory inputs. Using a combination of pharmacological blockers in vitro cellular and computational modeling, the authors conclude that glycine in fact evokes IPSPs in octopus cells; these IPSPs are largely shunted by the high membrane conductance of the cells under normal conditions and thus were not clearly evident in prior studies. Pharmacological experiments point towards a specific glycine receptor subunit composition. Lastly, Kreeger et. al demonstrated with in vitro recordings and computational modeling that octopus cell inhibition modulates the amplitude and timing of dendritic spiral ganglion inputs to octopus cells, allowing for flexible coincidence detection.

Strengths:

The work combines a number of approaches and complementary observations to characterize the spatial patterns of excitatory and inhibitory synaptic input, and the type of auditory nerve input to the octopus cells. The combination of multiple mouse lines enables a better understanding of and helps to define, the pattern of synaptic convergence onto these cells. The electrophysiology provides excellent functional evidence for the presence of the inhibitory inputs, and the modeling helps to interpret the likely functional role of inhibition. The work is technically well done and adds an interesting dimension related to the processing of sound by these neurons. The paper is overall well written, the experimental tests are well-motivated and easy to follow. The discussion is reasonable and touches on both the potential implications of the work as well as some caveats.

Weaknesses:

While the conclusions presented by the authors are solid, a prominent question remains regarding the source of the glycinergic input onto octopus cells. In the discussion, the authors claim that there is no evidence for D-stellate, L-stellate, and tuberculoventral cell (all local inhibitory neurons of the ventral and dorsal cochlear nucleus) connections to octopus cells, and cite the relevant literature. An experimental approach will be necessary to properly rule out (or rule in) these cell types and others that may arise from other auditory brainstem nuclei. Understanding which cells provide the inhibitory input will be an essential step in clarifying its roles in the processing of sound by octopus cells.

The authors showed that type 1a SGNs are the most abundant inputs to octopus cells via microscopy. However, in Figure 3 they compare optical stimulation of all classes of ANFs, then compare this against stimulation of type 1b/c ANFs. While a difference in the paired-pulse ratio (and therefore, likely release probability) can be inferred by the difference between Foxg1-ChR2 and Ntng1-ChR2, it would have been preferable to have specific data with selective stimulation of type 1a neurons.