A receptor-independent outward currents, reflecting the hyperpolarization component of spikelets . FF recruitment of the spikelet-mediated inhibition curtails the integration time window of concomitant excitatory postsynaptic potentials (EPSPs) and dampens their temporal integration. In contrast with GABAergic-mediated feed-forward inhibition, the depolarizing component of spikelets transiently increases the peak amplitude of EPSPs, and thus postsynaptic spiking probability. Therefore, spikelet transmission can propagate within the BC network to generate synchronous inhibition of Purkinje cells, which can entrain cerebellar output for driving temporally precise behaviors.In the cerebellar cortex, molecular layer interneurons use chemical and electrical synapses to form subnetworks that fine-tune the spiking output of the cerebellum. Although electrical synapses can entrain activity within neuronal assemblies, their role in feed-forward circuits is less well explored. By combining whole-cell patch-clamp and 2-photon laser scanning microscopy of basket cells (BCs), we found that classical excitatory postsynaptic currents (EPSCs) are followed by GABA GABAergic interneurons play important but diverse roles in gating, routing and modulating excitatory information flow within neural circuits through inhibitory chemical synapses. These different computational functions are classified according to canonical wiring motifs : feed-baInterneurons communicate with each other through both chemical and electrical synapses. While chemical synapses can regulate interneuron firing in the same way as for principal neurons , the preOwing to the low-pass filtering properties of the cell membrane, presynaptic APs are heavily filtered and consequently detected as ‘spikelets’ in electrically coupled postsynaptic cells. The relative contribution of the depolarizing component  or the aIn the cerebellar cortex, MLIs are known to mediate feed-forward inhibition (FFI) onto each other and Purkinje cells (PCs) by the means of GABAergic synapses. Coherent firing of MLIs is required for encoding precise movement kinematics . SpecifiWe found that electrical stimulation of parallel fiber (PF) excitatory inputs reliably elicited excitatory postsynaptic currents (EPSCs), followed rapidly by an outward current that was sensitive to gap junction antagonists and Cx36 knockdown, consistent with suprathreshold, PF-mediated recruitment of an electrically coupled neuron delivering a spikelet to its neighbor. Paired whole-cell recordings from electrically coupled BCs showed that the spikelets were predominantly inhibitory due to the depolarized presynaptic resting potential. Consequently, the synaptic recruitment of an electrically coupled MLI neighbor displays some of the characteristic features of FFI: temporal shortening of single EPSPs and dampening of temporal summation. Unlike chemical FFI, however, we demonstrate that EPSP-evoked spikelets can amplify synchronous compound synaptic responses, and consequently increase the probability of AP firing over a brief time window. Thus, synapse-evoked spikelet recruitment in a FF motif can act as a mechanism for temporal contrast enhancement of information flow within neural circuits.MLIs found in the inner third of the molecular layer of the cerebellar cortex are more likely to be BCs, defined by their basket-like axonal projections targeting PC somata and axon initial segment . BecauseA receptor blocker, gabazine . Despite (10 µM ) reducedWe also examined the frequency of observing PF-mediated outward currents in the absence of Cx36 expression  are 4-fold less likely to exhibit such outward currents  and exhibit 5-fold smaller outward currents (2.2 ± 0.4 vs. 12.7 ± 1.8 pA), consistent with the lower electrical coupling probabilities and amplitudes observed previously in P18-23 rats . Togethei.e., filtered presynaptic APs) arising from electrically coupled neighbors, which are generated by suprathreshold PF-evoked EPSPs. If true, then extracellular stimulation of a PF beam outside the dendritic tree of the recorded BC, in the presence of gabazine, would depolarize a coupled neighbor to threshold . Spikelet transmission was isolated by blocking GABAergic transmission with gabazine, which did not affect CCs . EvokingSpikelets recorded from different cell types throughout the brain have been shown to differ in their waveforms, and notably in the balance between excitation and inhibition . In immai.e., without whole-cell dialysis), we compared the peak inward and outward current amplitudes of spikelets from paired recordings when altering presynaptic membrane potential to those evoked by off-beam stimulation and direct stimulation (from i.e. more depolarized). In order to estimate the resting membrane potential of unperturbed electrical partners , in agreement with previous estimates . This deIn the inner-third MLI population of young rats, connections by electrical synapses were found to be more common than chemical ones . We re-eAR antagonist did not alter the outward current amplitude, consistent with a larger fraction of electrical than chemical connections. In the remaining connections the fractional block by 10 μM gabazine was ~50% on average  and the axon initial segment of PCs. Both the capacitive and K-currents generate a local field that sufficiently hyperpolarizes the AIS membrane potential and reduces the probability of PC spiking . In thatSpikelet waveforms have been shown to depend on a variety of physiological parameters across different cell types, including resting membrane potential  and actiConsistent with a net inhibitory action, we showed that FF recruitment of spikelets narrows the half-width of concomitant EPSPs and dampens their temporal summation. However, because the spikelets comprised a significant depolarizing component, perhaps due to the strong average CC , we also examined their ability to enhance EPSP depolarization. When stimulating PFs within the dendritic tree of BCs, inward current components of spikelets were not reliably detectable. Nevertheless, we found that the transient excitatory drive of spikelets significantly increased the peak amplitude of the compound synaptic potential when both signals were coincident (within a 4–6 ms window). Thus, unlike cFFI, electrical feed-forward modulation (eFFM) shortens the temporal window for EPSP-spike coupling, thus improving spike precision, while simultaneously enhancing spike probability . The degHere we did not examine the combined contribution of cFFI and eFFI, as its true contribution requires an accurate estimate of the GABAR reversal potential, which has not been performed in mature MLIs. Moreover, differential recruitment of cFFI and eFFI in MLIs under physiological spatio-temporal PF activation will require future experiments in awake animals. Nevertheless, we found that BCs are more likely to transmit spikelets than chemical inhibition to their neighbors  followed by a rapid disinhibition . In support of the sagittal propagation of spikelets, we find no correlation in CC over intersomatic distances up to 60 µm . Experiments were performed on acute brain slices prepared from postnatal day 30–60 (~44 days on average) WT CB6F1 mice, which were bred in house from a cross between female BALB/cByJ and male C57BL/6J obtained from Charles River Laboratories. Experiments in 2, 4 MgCl2, 1.25 NaH2PO4, 24 NaHCO3, 25 glucose, 230 sucrose, and 0.5 ascorbic acid. The solution was bubbled with 95% O2 and 5% CO2. Slices were cut from the dissected cerebellar vermis using a vibratome (Leica VT1200S), and incubated at 32°C for 30 min in a solution containing (in mM): 85 NaCl, 2.5 KCl, 0.5 CaCl2, 4 MgCl2, 1.25 NaH2PO4, 24 NaHCO3, 25 glucose, 75 sucrose and 0.5 ascorbic acid. Slices were then transferred to an external recording solution containing (in mM): 125 NaCl, 2.5 KCl, 1.5 CaCl2, 1.5 MgCl2, 1.25 NaH2PO4, 24 NaHCO3, 25 glucose and 0.5 ascorbic acid, and maintained at room temperature for up to 6 hr.Acute parasagittal slices (200 μm) of cerebellar vermis were prepared as follows: Mice were rapidly killed by decapitation, after which the brains were removed and placed in an ice-cold solution containing (in mM): 2.5 KCl, 0.5 CaClA receptors. Whole-cell patch recordings were performed using a Multiclamp 700B amplifier (Molecular Devices) at elevated temperatures (~32°C). We used fire-polished thick-walled glass patch electrodes (tip resistances of 4–6 MΩ). Patch pipettes were backfilled with the following internal solution (in mM): 115 KMeSO3, 40 HEPES, 1 EGTA, 6 NaOH, 4.5MgCl2, 0.49 CaCl2, 0.3 NaGTP, 4 NaATP, 1 K2-phospocreatine and 0.02 Alexa-594 or 0.04 Alexa-488 ; 115 CsMeSO3, 40 HEPES, 1 EGTA, 6 NaOH, 4.5MgCl2, 0.49 CaCl2, 0.3 NaGTP, 4 NaATP, 1 Tris-phospocreatine and 0.02 Alexa-594 . Series resistance was 14.0 ± 5.8  and always under 30 MΩ. All values of membrane potential were corrected for liquid junction potential, estimated to be −8 mV for K+-based internal solution and −11 mV for Cs+-based internal solutions  of cerebellar vermis. Unless otherwise stated  and 4, 1olutions . HoldingD-AP5 (D-(-)−2-Amino-5-phosphopentanoic acid), SR 95531 (2–3-Carboxyprobyl)−3-amino-6(4-methoxyphenyl pyridazinium bromide), and QX-314 chloride were purchased from Abcam, Cambridge, UK. NBQX  was purchased from Tocris Bioscience, Bristol, UK. Alexa Fluor 488 and 594 were purchased from Life Technologies, Carlsbad, California, USA. Mefloquine hydrochloride and DMSO (Dimethyl sulfoxide) were bought from Sigma-Aldrich, St Louis, Missouri, USA.www.neuromatic.thinkrandom.com) written within the Igor Pro environment . Peak amplitudes of synaptic responses recorded in voltage clamp or current clamp were measured as the difference between a baseline amplitude, estimated from 10 ms immediately preceding the stimulation artifact, and the mean amplitude over a 200 µs time window centered around the peak of the mean response. Due to the long-lasting nature of the after-hyperpolarization component of spikelets, the baseline subtracted peak amplitude of the inhibitory component was averaged over a time window of 3 ms, centered around the peak amplitude of the mean response. For the experiments in which we performed direct stimulation of neighboring MLIs, the lack of a synaptic delay required subtraction of the stimulus artifact, which we performed using a function in Neuromatic. In brief, the decaying inward component of the artifact was subtracted by first fitting an extrapolated double exponential function between the time of the peak of the averaged inward component of the artifact to just before the spikelet. The fitted function was then subtracted from all the traces. The remaining mean artifact, comprising the outward component and rising inward component of the mean artifact, were subtracted directly from all traces.Electrophysiological signals were low-pass filtered at 10 kHz, digitized at 100 kHz using an analog-to-digital converter , then acquired and analyzed using the Neuromatic analysis package  spontaneous EPSCs or spikelet activity were not analyzed.Measurement of distances between extracellular stimulation pipette locations and somata  or betweData are expressed as averages ± SEM unless otherwise indicated. Statistical tests were performed using a non-parametric Mann-Whitney two-sample rank test routine for unpaired comparisons, or Wilcoxon matched-pairs signed rank test for paired comparisons unless otherwise stated. Kruskal-Wallis tests, followed by Dunn's multiple comparison tests, were employed to compare multiple groups within a single experiment. Linear correlations were determined by Pearson correlation coefficient. Statistical significance was defined as p<0.05. All statistical tests were two-tailed, and performed in GraphPad Prism 6.2, 1 mM MgCl2, 10 µM gabazine (SR-95531) and 50 µM D-AP5 to block GABAAR and NMDAR, respectively. In our attempt to block the PF-mediated outward current . In the experiments shown in  current , we alsol values .Stable EPSC responses from parallel fibers (on-beam stimulation) were obtained by positioning a monopolar glass electrode  on top of the slices, above the dendrites of the patched MLIs . Stimulus intensities were set to 5–10 V above threshold for detecting an EPSC , referree.g., BC somata in the inner third of the molecular layer were identified and whole-cell patch clamped using infrared Dodt contrast  and a QIClick digital CCD camera  mounted on an Ultima multiphoton microscopy system  based on an Olympus BX61W1 microscope, equipped with a water-immersion objective . Two-photon excitation of Alexa-488 and Alexa-594 was performed at 780 nm for pairs of BCs. When Alexa-594 alone was used (single-cell patch-clamp), it was excited at 840 nm. A transmitted light PMT was mounted after the Dodt tube in order to acquire a pulsed IR laser-illuminated contrast image simultaneously with the 2P-LSM image. When the whole morphology of one cell could not be captured within a single stack of images in which the focal plane was varied  by the hyperpolarization value (100 ms average) recorded in the injected cell. Bidirectional CCs for a given BC-BC pair were calculated from the average of the two unidirectional CCs. Spikelet transmission was assessed by detecting postsynaptic inward currents in response to presynaptic AP firing since outward currents could arise from either spikelets and/or GABAergic currents. Inward current amplitudes were estimated from each sweep, following the baseline procedure described above, by averaging data points over a 200 µs window and occurring within a 2 ms window starting just after the time of the peak of the presynaptic AP. Inward current amplitudes were considered detectable if the amplitude distribution of peak inward currents was larger than a prestimulus amplitude distribution , measured from a reflected time window identical to that used for peak amplitudes. If inward currents were not detected, then outward currents in the postsynaptic responses were used to infer the presence of a GABAergic synapse. However, when an electrical synapse was detected, the outward current in postsynaptic responses could be caused by a mix of electrical and chemical inputs. In that case, peak amplitudes of postsynaptic outward currents were compared at different holding membrane potentials of the postsynaptic cell (to change the electromotive force for GABAergic inputs), and/or before and after application of gabazine. The presence of a GABAergic synapse was inferred when the peak amplitude distributions of outward currents in these different conditions were significantly different from each other. When an electrical connection was present, bias current in the presynaptic cell was manually adjusted, to compensate for passive flow of different holding currents from the postsynaptic cell.e = ne/npairs and for chemical: fc = nc/nunidirectional; where ne and nc are the number of bidirectional electrical and unidirectional chemical synapses, respectively). We assumed the frequency distributions could be described by binomial statistics, and therefore calculated a standard deviation (SD = f*(1-f)) and a standard error (SEM = SD/√n). We performed similar error calculations for all observation frequencies . Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:eLife policy at this time is not to ask for additional experiments if the manuscript can be revised to state only those conclusions that are well supported by the existing data. We focused our discussion on the 5 issues below that we intend as a guide to your revision. Please also address other points of discussion or clarification raised in the original reviews that are included below.The reviewers were interested in the topic and the proposed mechanism. However, we noted several issues in our discussion that need to be more carefully addressed in the text because the data in our opinion was not definitive. As noted above, Essential revisions:1) Weak effects of KO/mefloquine. These effects were rather modest given that they are central to the proposed mechanism. The authors should discuss the possibility that ephaptic coupling could account for some component of the phenomenon, and the KO data need to be better integrated into the main text.2) Comparison of GJs vs GABA in inhibiting the network. The reviewers were not convinced regarding the relative contribution of these components, which require a more balanced discussion.3) Conclusions regarding resting membrane potential. Here again the reviewers were not convinced by the data presented. The authors should only make conclusions that are well supported by the data.4) To better clarify the impact of your work, please point out similarities and differences to previous studies in the Discussion section.5) The computation of connection probabilities needs to be better clarified.We think these revisions can be made without increasing the length of the manuscript by eliminating redundancies in the text.Reviewer #1:In the manuscript "Feed-forward recruitment of electrical synapses enhances synchronous spiking in the mouse cerebellar cortex" Hohne and colleagues describe a mechanism in the cerebellar cortex by which electrical coupling of inhibitory interneurons produces a specialized feedforward motif that enhances temporal summation at very short time intervals while shortening the window of integration by inhibiting temporal summation outside this brief window. Together, these combined mechanisms are proposed to support a globalize and temporally precise inhibition of Purkinje neurons that could enhance synchrony thought to be important for information transmission downstream. The manuscript presents a progression of experiments describing the phenomenology and mechanism of the electrical coupling of basket cells, including evidence that outward currents observed in BCs are mediated in part by gap junctions; the spatial relationship of stimulation and the observation of electrical spikelet mediated outward currents; analog effects on electrical transmission; relative prevalence of electrical and chemical inhibition; and finally, the effect of electrical coupling on temporal summation of EPSPs. The data are thought provoking and of high quality and contribute to an active literature on MLI organization and physiology.That said, the study has some shortcomings, which should be addressable with mainly text clarifications or additional analyses. I recognize the impossibility of collecting new data in the short term, thus where new data suggested below are not obtainable, interpretations should be tempered and narrowed.1) The primary takeaway is that for independent PF beams, BC networks can detect coincident excitation that will generate a global precise inhibition of downstream Purkinje neurons. For this to be true, the temporal windowing contrast enhancement effect shown in Figure 6 needs to be robust to the presence of chemical inhibition. To my read, gabazine was present, leaving this uncertainty unresolved. Superimposed chemical and electrical inhibition could have predictable or unpredictable effects on the network level phenomenon reported here, raising the importance of empirical data in this light. For instance, Figure 3 shows that membrane potential of the presynaptic partner in an electrical pair affects electrical transmission. If effective GABAergic inhibition caused hyperpolarization of the presynaptic partner the magnitude of electrical inhibition would be expected to be reduced. Of course, this prediction may not pan out, but the main point is that it is difficult to predict how robust the phenomena reported here are to a full suite of intact circuit mechanisms.2) The identification of gap junctions as mediating the outward currents measured is surprisingly weak. In Figure 1 they show that the outward current is unlikely mediated by unclamped K currents from direct stimulation, since it is robust to Cs internal and moreover the gap junctional antagonist mefloquine reduces the magnitude and frequency of observing these currents. However, a very important dataset is relegated to a supplementary figure that is in turn poorly integrated into the findings, specifically, experiments performed in Cx-36 knockouts shown in Figure 2. Although the title of the figure (S3) is 'Outward currents are smaller in SCs than BCs' the main text refers to the figure in the context of testing for the presence of outward currents in Cx-36 knockouts. The data are a bit of a mess with different age ranges used, precluding a clean interpretation. The data show that the amplitude of the outward currents recorded in Cx-36KO mice at P90-150 are about the amplitude as those recorded in wildtypes at P30-60 (the range used in the rest of the paper). There is a clear decrement in the amplitude of the putative electrical inhibition between wildtype and knockouts at a given age range, however, supporting the conclusion that this is gap-junction mediated. Given the extensive literature on ephaptic signaling in this part of the molecular layer, some caution is warranted in interpreting these as a purely jap-junctional mediated mechanism. Together, the mixing of experimental conditions and the persistence of the outward current in the knockouts seems to undermine one of the main takeaways of the study, sowing confusion and doubt. The authors should make some effort at better explaining and integrating this dataset into the main part of the study.3) I have no reason to doubt the authors on the description of the inward and outward currents shown in Figure 1, or their sensitivity and insensitivity to blockade by receptor blockers, but the results as presented are very qualitative. Please report analyses in the text. For example, "Bath application of 10 μm .… NBQX eliminated both the EPSC and subsequent outward current (n = 6/6 experiments) should provide amplitude info +/- drug, as should the gabazine experiment before that.4) Figure 3 data presents an interesting experiment testing how RMP of one or the other MLI in a coupled pair influences the spikelet polarity. They identify CC with paired recordings and see that the membrane potential of the presynaptic cell has a strong effect on the spikelet waveform but not vice versa. Some confusion in the exposition: The example in B shows bidirectional coupling but they then focus in unidirectional coupling. It is unclear if the cells used in D-F are unidirectionally or bidirectionally coupled or whether the holding potential manipulations of the 'pre and post' synaptic cells are filtered into the other. This overall raises the question of whether the correlations they show in H are based solely on the membrane potential of the presynaptic cell or on the differential membrane potentials between the two coupled cells. This could be clarified with edits.5) For data shown in Figure 4, please report the number of cases where chemical inhibition was distinguished with pharmacology vs driving force. Also, I am missing how the probability distributions of chemical and electrical connections are compared and quantified. The results report that the probability of bidirectional transmission of spikelets was 0.52 +/- 0.06 (31/60 pairs). What does the error here represent? Similar with the GABAergic synapse: 0.40 +/- 0.05% (34/86 pairs). Finally, these probability distributions are compared with an unpaired t-test and shown to be not significantly different, but I am having difficulty following the mechanics of this analysis.Reviewer #2:In this study from the DiGrigorio lab, electrical synapses within the basket cell (BC) network were examined for their capacity to drive precisely timed activity, and thus inhibition of Purkinje cells. The most interesting conclusions are contained in Figure 5, which shows that narrow band synaptic activation of BCs can drive excitation of neighboring BCs with a temporal precision exceeding that of conventional EPSPs. The basis for this precision is that the excitation comes by way of spikelets and each spikelet is curtailed by the potent electrotonic spread of spike afterhyperpolarizations. An important conclusion then is that the well-known rapid feedforward inhibition of Purkinje cells driven by parallel fiber activation has both a chemical component, and, via the BC network, an electrical component. Thus, the authors coin the acronym eFFM, electrical feedforward modulation. This is good quality electrophysiology from a top lab, so I no major technical concerns. I do feel that apart from the above, the majority of the work is demonstrating rather incremental or expected things. For example, the generation of postsynaptic inhibition through the spikelet waveform is well described elsewhere. Also, I am not so convinced by the RMP estimate.The use of the ratio of inward to outward current to guesstimate prejunctional resting membrane potential seems not very strong. This ratio is affected by other things besides RMP, for example: during the whole cell recording there can be washout of currents that underlie AHPs, there may be alterations in junctional conductance (which will filter the fast inward current more than the slow outward current). Indeed for the direct stim experiments, the slope of the graph in 5H is nearly twice that of the -60 mV graph. Do you really think the RMP is nearly -50 mV? If the prejunctional cell population is firing chronically, how would that impact this estimate?Reviewer #3:In this report, Hoehne et al., examine a novel type of feedforward inhibition mediated by an electrically coupled interneuron network in the cerebellum. They find that basket cells, a type of molecular layer interneuron, show curtailed synaptic excitation due to the offsetting effect of an after-hyperpolarization of a shared spikelet, generated in a neighboring electrically coupled basket cell. Interestingly, the depolarizing phase of the spikelet can cause increased firing in the postsynaptic cell. The combination of these opposing effects producing a type of contrast enhancement. Overall, this is an elegant, well-executed study. It is important for two reasons. First, the uniqueness of the underlying mechanism of electrical feedforward inhibition (FFI) whose influence differs from that mediated by chemical connections. Second, molecular layer inhibition is an important feature for organizing the output of the cerebellum, thus, contrast enhancement as described in this report could have a substantial effect on information coding in the cerebellum's circuits. The authors should consider revising their manuscript in several places to improve clarity and increase the impact of their work. These criticisms do not take away from overall enthusiasm.1) The authors argue that electrical coupling is mediated by gap junctions but the effects of Cx36 knockout are very limited . The Marty group showed that MLI electrical coupling was absent in Cx36 KO mice so this is a very important caveat to the interpretation of their results. Did the authors consider the effects of Cx36 KO on temporal summation or on contrast enhancement? The authors should elaborate on the possibility of developmental compensation or alternative mechanisms that could explain this phenomenon in the Discussion section (ephaptic coupling?). Lastly, the presentation of the Cx36 data seems out of place in the Results section. Perhaps this was due to the lack of explanation in their methodology .2) The authors attempt to compare the prevalence of electrical vs chemical FFI is confusing. Why not simply state the rate of identifying electrically and chemically connected pairs? Then report the rate of spikelet coupling . Again, could spikelet coupling reflect a different mechanism? Last, is the prevalence of electrical vs chemical FFI best described by the rate of electrically and chemically connected pairs of basket cells?3) Can the authors always be sure the presynaptic coupled neuron is a basket cell? It looks like the stimulating pipette is place throughout the molecular layer where stellate cells (and their neurites) also reside.4) Could temperature-dependent effects on spike width and after-hyperpolarization duration contribute to differences with results from the Marty group ? This may also be true for the resting membrane potential in the presynaptic cell which the authors clearly show affects the polarity of influence.5) EPSP half-widths in 5B and 5D seem to be different (responses appear much more prolonged in 5B). Does this mean that many responses in 5D in the LS condition have coupled spikelets ? How does this affect the interpretation of their results? Essential revisions:1) Weak effects of KO/mefloquine. These effects were rather modest given that they are central to the proposed mechanism. The authors should discuss the possibility that ephaptic coupling could account for some component of the phenomenon, and the KO data need to be better integrated into the main text.We recognize that vague wording and presentation of critical control experiments supporting the mechanism of GABAergic-independent feed-forward inhibition may have obfuscated reader. To correct this and other major concerns, we have added new main figures, performed additional analysis, clarified the description of results, and considered alternative hypotheses for our findings in the Discussion section.1) We now emphasize that the 70% block by mefloquine is consistent with a gap-junction mediated mechanism, as we used a sub-saturating concentration in order to prevent significant off-target effects.i.e. peak amplitude of inward and outward spikelet currents), suggesting that spikelets are mediated by gap junctions.2) We now show that the coupling coefficient, a proxy of gap junction coupling, is positively correlated with the size of spikelets (3) Regarding the less than complete knock down of electrical coupling in the Cx36 KO mice, we now: a) highlight that there is still a 3-fold reduction in the outward current when compared to age-matched controls, b) discuss literature supporting the possibility that expression of Cx45 might compensate for Cx36 knock down, and c) discuss differences between our study and those of previous publications. In particular, we discuss the possibility that the use of older animals (>P90) vs. the 3 weeks old animals used in previous studies could allow for compensatory mechanisms to be expressed.4) We now included a thorough discussion of the potential for spikelet transmission by ephaptic coupling. In brief, the large amplitude of BC spikelet currents is not compatible with the direct ephaptic currents generated between BC terminals and PC axon initial segments . Nor are they compatible with the sodium channel current evoked by ephaptic coupling between PC axons, since our spikelets are insensitive to internal QX314 and alterations in postsynaptic membrane potentials .2) Comparison of GJs vs GABA in inhibiting the network. The reviewers were not convinced regarding the relative contribution of these components, which require a more balanced discussion.AR antagonists did not statistically alter AP-evoked outward currents in paired BC recordings in 13 out of 21 cases. When Gabazine did have an effect, it blocked by only 50%.Indeed, it is difficult to assess the relative contribution of chemical and electrical synapses to inhibitory transmission given the current data set. We have now added a paragraph in the Discussion section to highlight the limitations of our ability to assign a specific physiological role. Nevertheless, the observed higher frequency of electrical synapses implies a prominent and important mechanism. In the first submission there was an error in our spreadsheet, and upon correction, the higher frequency of electrical synapses is actually statistically significant. Moreover, we also now show that saturating concentrations of GABAWe feel it is also important to highlight that spikelet-mediated inhibition, unlike chemical inhibition, is also coupled to a rapid initial depolarizing component that we show increases spike probability in addition to narrowing the time window for spiking, and they can propagate across multiple cells, ensuring entrainment of the network not otherwise possible with chemical inhibition.Finally, we now mention in the Discussion section that future in vivo experiments are necessary to assess the physiological contribution of feed-forward spikelet transmission.3) Conclusions regarding resting membrane potential. Here again the reviewers were not convinced by the data presented. The authors should only make conclusions that are well supported by the data.We have carefully rewritten the Results sections so as not to overstate our conclusions. But the well-known voltage dependence of the relative hyperpolarization component, we feel, is a good indicator that MLIs have a depolarized resting potential. This is also consistent with the high propensity to evoke spikes in neighboring cells. But, independent of the resting membrane potential, the prominent biphasic spikelet is a recording of an unperturbed cell and likely to represent physiologically relevant characteristics.4) To better clarify the impact of your work, please point out similarities and differences to previous studies in the Discussion section.In the Discussion section, we now specifically comment on differences between our conclusions and those described previously. In brief, beyond the novel demonstration that spikelets are recruited in a feed-forward manner, we also show that the biphasic spikelet transmission is likely more physiologically relevant. We hypothesize that previous work in the same cell type did not observe the hyperpolarization component due to their hyperpolarized holding membrane potentials.5) The computation of connection probabilities needs to be better clarified.We have re-written the text and provided a new table to clarify the frequency of different criteria used to estimate connection probabilities of chemical and electrical connections between BCs.We think these revisions can be made without increasing the length of the manuscript by eliminating redundancies in the text.We have also made an effort to reduce redundancies to a minimum. However, to address all the comments of the reviewers, the Discussion section is indeed longer.eLife.In summary, we are thankful for the reviewers’ comments as they have led to a greatly improved manuscript by encouraging us to revise analyses, calculations and presentation of the results. We have increased the number of main figures from 6 to 8, and hope that together with our additional changes we have made a more convincing argument that feed-forward recruitment of spikelet transmission if mediated by gap junctions and provides a powerful mechanism for synchronizing neural activity in ways not possible by chemical inhibition. We hope that the current manuscript is now suitable for publication in Reviewer #1:In the manuscript "Feed-forward recruitment of electrical synapses enhances synchronous spiking in the mouse cerebellar cortex" Hohne and colleagues describe a mechanism in the cerebellar cortex by which electrical coupling of inhibitory interneurons produces a specialized feedforward motif that enhances temporal summation at very short time intervals while shortening the window of integration by inhibiting temporal summation outside this brief window. […] The data are thought provoking and of high quality and contribute to an active literature on MLI organization and physiology.That said, the study has some shortcomings, which should be addressable with mainly text clarifications or additional analyses. I recognize the impossibility of collecting new data in the short term, thus where new data suggested below are not obtainable, interpretations should be tempered and narrowed.We thank the reviewer for the encouraging words and describe below how we have addressed the concerns.1) The primary takeaway is that for independent PF beams, BC networks can detect coincident excitation that will generate a global precise inhibition of downstream Purkinje neurons. For this to be true, the temporal windowing contrast enhancement effect shown in Figure 6 needs to be robust to the presence of chemical inhibition. To my read, gabazine was present, leaving this uncertainty unresolved. Superimposed chemical and electrical inhibition could have predictable or unpredictable effects on the network level phenomenon reported here, raising the importance of empirical data in this light. For instance, Figure 3 shows that membrane potential of the presynaptic partner in an electrical pair affects electrical transmission. If effective GABAergic inhibition caused hyperpolarization of the presynaptic partner the magnitude of electrical inhibition would be expected to be reduced. Of course, this prediction may not pan out, but the main point is that it is difficult to predict how robust the phenomena reported here are to a full suite of intact circuit mechanisms.ARs.The reviewer is correct in that the data presented do not differentially address the effect of chemical and electrical inhibition on feed-forward modulation of BC spiking. Indeed, a proper exploration of this would require experiments to be performed in a fully intact circuit (in vivo), where physiological GC activity might differentially recruit electrical FFI or chemical FFI. We have now pointed out these shortcomings of our study and indicate that future experiments would be required to dissect the true relative contribution. Even in vitro, the precise dissection is challenging due to the uncertainty in reversal potential for GABAIn subsection “Electrical synapse-mediated spikelets dynamically modulate BC EPSP-spike coupling”, we added the following text:“Here we did not examine the combined contribution of cFFI and eFFI, as its true contribution requires an accurate estimate of the GABAR reversal potential, which has not been performed in mature MLIs. Moreover, differential recruitment of cFFI and eFFI in MLIs under physiological spatial temporal PF activation will require future experiments in awake animals. Nevertheless, we found that BCs are more likely to transmit spikelets than chemical inhibition to their neighbors , which could account for the narrowed excitatory responses of BCs versus SCs in vivo . Recent in vivo findings show that BC spikes have a stronger influence on PC spiking than SCs , implying that the specific timing of BCs will strongly influence PC output.”We also highlighted the important difference between chemical inhibition and electrical synapses: namely the depolarizing – synchronizing component, and the electrical propagation within the BC network, which are likely to entrain a greater fraction of BCs than chemical inhibition alone.In subsection “Electrical synapse-mediated spikelets dynamically modulate BC EPSP-spike coupling”, we added the following text:“Nevertheless, we found that the transient excitatory drive of spikelets significantly increased the peak amplitude of the compound synaptic potential when both signals were coincident (within a 4-6 ms window). Thus, unlike cFFI, electrical feed-forward modulation (eFFM) can shorten the temporal window for EPSP-spike coupling, thus improving spike precision, while simultaneously enhancing spike probability .”In subsection “Electrical connectivity in the cerebellar cortex is tuned for rapid output synchrony”, we added the following text:“This high degree of electrical connectivity can provide a global network mechanism spanning > 100 µm in the sagittal plane  for generating a FF modulation of PC spiking patterns through synchronous inhibition  followed by a rapid disinhibition . It should be noted that chemical inhibition alone does not propagate within the network, but spikelet-mediated inhibition does.”We also would like to point out to the reviewer that it seems likely that the concurrence of both chemical inhibition and action potentials within the presynaptic cell would simply amplify the presynaptic hyperpolarization, which in turn is transmitted as inhibition to the electrically coupled receiving cell. Alternatively, if chemical inhibition is transmitted to both the pre and postsynaptic cell, then indeed the hyperpolarizing component of the transmitted spikelet would be occluded, but the receiving cell would still experience inhibition. Nevertheless, the most important difference of electrical (spikelet) and chemical inhibition, is that the spikelet inhibition will be coupled to a depolarizing component, which will be increased under hyperpolarized conditions of the presynaptic cell , and will be very rapidly transmitted, indeed faster than the synaptic delay for inhibition. Thus, in all cases, the depolarizing component will be transmitted to the postsynaptic cell. In this light, the temporal contrast enhancement figure provides important and physiologically relevant intuition as to how brief BC synchrony can be generated, with or without additional chemical inhibition.2) The identification of gap junctions as mediating the outward currents measured is surprisingly weak. In Figure 1 they show that the outward current is unlikely mediated by unclamped K currents from direct stimulation, since it is robust to Cs internal and moreover the gap junctional antagonist mefloquine reduces the magnitude and frequency of observing these currents. However, a very important dataset is relegated to a supplementary figure that is in turn poorly integrated into the findings, specifically, experiments performed in Cx-36 knockouts shown in Figure 2. Although the title of the figure is 'Outward currents are smaller in SCs than BCs' the main text refers to the figure in the context of testing for the presence of outward currents in Cx-36 knockouts.We have now moved the results regarding the KO to the main Figure 2 and corrected the title.The data are a bit of a mess with different age ranges used, precluding a clean interpretation. The data show that the amplitude of the outward currents recorded in Cx-36KO mice at P90-150 are about the amplitude as those recorded in wildtypes at P30-60 (the range used in the rest of the paper).We thank the reviewer for pointing out the shortcomings in the presentation of our results, which perhaps contributed to an apparent underwhelming conclusion. Nevertheless, we are confident that gap junctions are the likely mechanism for the following reasons, which we now emphasized in the Results section and Discussion section.1) Regarding the incomplete block of the outward currents by mefloquine: in order to minimize off-target effects, we used 20 mM mefloquine, which is expected to block gap junctions by 70% . The outward current was reduced to 33%, consistent with an entirely gap junction-mediated mechanism.We added the following text to subsection “PF-triggered outward current mediated by electrical synapses in cerebellar basket cells”: “In brain slices prepared from Cx36 KO mice, we found a 45 % reduction in the frequency of observing outward currents, and the amplitude of the remaining events was 3-fold smaller than age-matched recordings (4.2 ± 0.8 vs. 12.7 ± 1.8 pA).”2) We apologize for the confusion resulting from showing Cx36 data in a Supplementary figure. To correct this, we moved the Cx36 KO data from a supplementary figure to a main figure , and now only show age-matched comparisons, which highlight the 3-fold reduction in outward current in Cx36 mice. We previously misstated the Cx36 reduction as only two-fold, rather than three-fold.3) We also now describe the strong correlation between coupling coefficient and spikelet amplitude in a main figure . This result shows that the larger the coupling coefficient, the larger the spikelets. They also further support a gap junction origin of the spikelet transmission between BCs.There is a clear decrement in the amplitude of the putative electrical inhibition between wildtype and knockouts at a given age range, however, supporting the conclusion that this is gap-junction mediated. Given the extensive literature on ephaptic signaling in this part of the molecular layer, some caution is warranted in interpreting these as a purely jap-junctional mediated mechanism.Indeed, it is possible that the remaining inward and outward current in mefloquine or in Cx36 KOs could result from transmission of an action potential between closely juxtaposed dendrites or axons. Ephaptic transmission has been shown to involve different mechanisms.In Blot et al., 2014, BC-PC ephaptic transmission is thought to occur between the axon terminal structures, pinceau, and the axon initial segment of PCs. Both the capacitive and K-currents generate a local field that sufficiently hyperpolarizes the AIS membrane potential and reduces the probability of PC spiking. Their simulations suggest that the ephaptic-mediated hyperpolarization is likely around ~ 1 µV at the somata, and thus undetectable. Thus, the large mV changes we observe between two BCs seem incompatible with such a coupling, as it would likely have to be between two axon terminals or crossings between BC dendrites.In contrast, Han et al., 2018 measure ephaptic currents in PC-PC pairs that are 10's of pA, but are due to regenerative activation of local AIS sodium conductances in the post-PC, as they are blocked by intracellular perfusion of QX314 (sodium channel antagonist), and eliminated by postPC hyperpolarization to -70 mV . In the new Figure 5 we show that the amplitude of the spikelet is insensitive to postsynaptic manipulation in paired recordings of BC-BC pairs. Moreover, all the outward current experiments in Figure 1 were performed in the presence of QX-314. Kim et al. also nicely showed that the extracellular field was only generated within 20 µm of the high conductance density region of the AIS , and not the soma or axon. Thus, the spikelets that we report, or even the 30% remaining unblocked current (in the presence of mefloquine or Cx36 KO), are unlikely mediated by this sodium current-dependent ephaptic mechanism.Together, the mixing of experimental conditions and the persistence of the outward current in the knockouts seems to undermine one of the main takeaways of the study, sowing confusion and doubt. The authors should make some effort at better explaining and integrating this dataset into the main part of the study.Again, we apologize if some of the explanation or logical flow of our arguments was not clear, but we believe that we have performed multiple experiments to support gap-junction mediated FFM. In addition to changes in the Results section and figures (see above), we also modified the discussion to clearly state the reasons for our conclusion, and the possibility of other interpretations (ephaptic). See Discussion section:“Extracellular stimulation of PFs in parasagittal slices in the presence of gabazine produced outward currents of 10 pA in BCs and less than 3 pA in SCs . We performed several experiments whose results were consistent with spikelet transmission through gap junctions as the origin of GABAergic-independent outward currents: (1) sub-saturating concentrations (20 µM) of the gap junction blocker mefloquine blocked the outward current by 70 %, as expected for a current entirely mediated by gap junctions . (2) We observed a 3-fold reduction in peak amplitude of the outward current in Cx36 KO mice . The incomplete knockdown could be due to genetic compensation by Cx45, which is known to also be expressed in MLIs . Nevertheless, we only observed one of five BC-BC pairs in Cx36 KO mice that showed bidirectional spikelets and a CC greater than 2 % , as opposed to the nearly 60 % BC-BC pairs . (3) To confirm that the outward currents were mediated by spikelets, we showed that stimulation of PFs outside the dendritic field of the recorded cell and direct stimulation of MLIs in NBQX both evoked currents with a rapid inward component and an outward component similar to that following EPSCs. (4) These extracellularly-evoked spikelets were very similar to those evoked by single APs propagating between electrically coupled BC pairs. And finally, (5) the strength of electrical coupling between two BCs, estimated from 400 ms step depolarizations, was positively correlated with spikelet amplitude , confirming that spikelets were mediated by gap junctions.Electrical coupling between neurons has also been shown to be mediated without a gap junction or chemical synapse, and is referred to as ephaptic coupling. We cannot directly rule out that the remaining spikelet currents in mefloquine or in Cx36 KOs could result from transmission of an AP between closely juxtaposed dendrites or axons. BC-PC ephaptic transmission is thought to occur between the axon terminal of BCs (pinceau) and the axon initial segment of PCs. Both the capacitive and K-currents generate a local field that sufficiently hyperpolarizes the AIS membrane potential and reduces the probability of PC spiking . In this study, simulations suggest that the ephaptic-mediated hyperpolarization is likely around ~ 1 µV at the somata, and thus unlikely to account for the large mV changes we observe between two BCs. In contrast, ephaptic currents in PC-PC pairs are 10's of pA, but are due to regenerative activation of local AIS sodium conductances in the post-PC, which is eliminated by post-PC hyperpolarization to -70 mV . We showed that the synapse-evoked outward current was insensitive to 1 mM of the sodium channel blocker QX-314 ; and, in BC-BC paired recordings, the amplitude of the spikelet is insensitive to postsynaptic manipulation of membrane potential . Both results are inconsistent with a sodium dependent ephaptic mechanism.”3) I have no reason to doubt the authors on the description of the inward and outward currents shown in Figure 1, or their sensitivity and insensitivity to blockade by receptor blockers, but the results as presented are very qualitative. Please report analyses in the text. For example, "Bath application of 10 μm .… NBQX eliminated both the EPSC and subsequent outward current (n = 6/6 experiments) should provide amplitude info +/- drug, as should the gabazine experiment before that.We have added a summary panel  describing the NBQX blockade of the outward current. See added text in subsection “PF-triggered outward current mediated by electrical synapses in cerebellar basket cells”: “Bath application of 10 µM of the AMPA receptor (AMPAR) antagonist, NBQX, eliminated both the EPSC and the subsequent outward current to below detectable levels .”AR antagonist did not alter detectably the outward current amplitude, and, in the remaining connections, the fractional block by 10 µM gabazine was ~50% on average . The following text was added to the Results section:Unfortunately, we did not characterize PF-evoked outward currents with and without gabazine, however, we did examine in some BC-BC pairs the effect of gabazine on the outward currents . We found that, in 13 out of 21 unidirectional connections from BC-BC pairs, the GABAAR antagonist did not alter the outward current amplitude. In the remaining connections the fractional block by 10 µM gabazine was ~50 % on average .”“If we analyzed the gabazine only experiments, we found that in 13 out of 21 unidirectional connections, the GABA4) Figure 3 data presents an interesting experiment testing how RMP of one or the other MLI in a coupled pair influences the spikelet polarity. They identify CC with paired recordings and see that the membrane potential of the presynaptic cell has a strong effect on the spikelet waveform but not vice versa. Some confusion in the exposition: The example in B shows bidirectional coupling but they then focus in unidirectional coupling. It is unclear if the cells used in D-F are unidirectionally or bidirectionally coupled or whether the holding potential manipulations of the 'pre and post' synaptic cells are filtered into the other. This overall raises the question of whether the correlations they show in H are based solely on the membrane potential of the presynaptic cell or on the differential membrane potentials between the two coupled cells. This could be clarified with edits.We have clarified in the text and in the methods that bidirectional coupling coefficients (CCs) are values that represent the average of the two unidirectional CCs. Unidirectional CCs are calculated from the ratio of the membrane potential in the receiving cell over the hyperpolarization amplitude of the cell in which the current injection was delivered.Because current injections were made to maintain resting potentials in current clamp, and the voltage clamp circuit ensured a constant membrane potential, the resting membrane potentials were essentially decoupled, allowing for an accurate determination of the membrane potential dependence on the spikelet shape, without influencing the membrane potential of the electrically connected cell. In Figure 5E and F the receiving cell is held at -70mV, which might certainly influence the resting membrane potential of electrically-coupled neighbors.5) For data shown in Figure 4, please report the number of cases where chemical inhibition was distinguished with pharmacology vs driving force.This is a good suggestion. We have now added a supplementary table indicating the number of unidirectional connections tested for each method .Also, I am missing how the probability distributions of chemical and electrical connections are compared and quantified. The results report that the probability of bidirectional transmission of spikelets was 0.52 +/- 0.06 (31/60 pairs). What does the error here represent? Similar with the GABAergic synapse: 0.40 +/- 0.05% (34/86 pairs). Finally, these probability distributions are compared with an unpaired t-test and shown to be not significantly different, but I am having difficulty following the mechanics of this analysis.e = ne/npairs and for chemical: fc = nc/nunidirectional connections). We assumed the frequency distributions could be described by binomial statistics, and therefore calculated a standard deviation (SD = f*(1-f)) and a standard error (SEM = SD/<inline-graphic mime-subtype="png" mimetype="image" xlink:href="media/image1.png" />n). We performed similar error calculations for all observation frequencies .”Perhaps this was not well explained. The standard error we show is calculated from the standard deviation of a binomial distribution centered on the observed frequency of each connection type. Once having the SEM, we could perform a simple unpaired two-tailed t-test. This was originally briefly described in the figure legend, but now also described in the Materials and methods section: “To calculate the error in our estimate of the frequency of observing electrical and chemical synapses in paired recordings, we first computed the observed frequencies from our data set .Indeed, it is possible that washout might alter the voltage-dependence of the AHP, and thus shift our calibration. The most likely candidate would be washout of calcium buffers that affect calciumdependent potassium conductances. However, we have included 1 mM EGTA in the pipette which approximates the concentration of the slow on-rate calcium binding protein parvalbumin. Nevertheless, the spikelets initiated from unperturbed cells have a large outward component consistent with a large presynaptic AHP, which is associated with APs initiated from resting membrane potentials much more depolarized than potassium current reversal potentials. Moreover, the fact that we reliably stimulate synapse-evoked spikes also suggests a depolarized resting membrane potential.We now refer to the caveats of our membrane potential estimation in the discussion, and emphasize the bipolarity of the spikelets rather than the absolute value membrane potential, subsection “Electrical synapse-mediated spikelets dynamically modulate BC EPSP-spike coupling”:“Comparison of spikelets evoked in an unperturbed, electrically coupled neighbor to those evoked in paired recordings with presynaptic membrane potential manipulations is consistent with a depolarized BC resting membrane potential .”Indeed for the direct stim experiments, the slope of the graph in 5H is nearly twice that of the -60 mV graph. Do you really think the RMP is nearly -50 mV?We also find this result to be striking. We now state that our data are consistent with a resting membrane potential more depolarized than -60 mV, provided that the voltage dependence of the spikelet shape is similar for whole-cell dialyzed and unperturbed cells, although previous studies also estimated resting membrane potentials to be more depolarized than -60 mV .We have added the following sentence to the Results section:“Provided that the voltage-dependence of the shape of the presynaptic AP is similar in patchdialyzed and unperturbed cells, these data indicate that unpatched MLIs have a depolarized resting membrane potential of at least -60 mV (i.e. BCs could be more depolarized).”If the prejunctional cell population is firing chronically, how would that impact this estimate?Because we could perform stimulus aligned averages, the background spikelet activity did not generally affect our experiments. Indeed, there were one or two cells out of the over 100 recorded cells that were not analyzed due to high background spikelet activity. We have added this criterion to the Materials and methods section.Reviewer #3:1) The authors argue that electrical coupling is mediated by gap junctions but the effects of Cx36 knockout are very limited . The Marty group showed that MLI electrical coupling was absent in Cx36 KO mice so this is a very important caveat to the interpretation of their results.Indeed, we were disappointed that the Cx36 KO mice did not completely eliminate spikelet transmission, although we did observe a 3-fold reduction of currents in age-matched controls. This was incorrectly described in the first manuscript as 2-fold. We also made several changes to figures and text to justify our conclusion despite the remaining currents. Please see response to comment number #2 of reviewer 1. In short, it is possible that because our animals are 3-4 months old, perhaps the remaining currents are mediated by another gap junction protein Cx45, also shown to be expressed in SCs.Did the authors consider the effects of Cx36 KO on temporal summation or on contrast enhancement?We agree that this would be an ideal experiment. But because Cx36 did not eliminate electrical coupling, instead we compared temporal summation at sites with and without eFFM. We felt that under the circumstances this would be a cleaner approach. Moreover, we could select dendritic locations in which PF stimulation evoked EPSPs that did not evoke a spikelet, thus allowing us to precisely probe the effect of spikelets evoked at different times within the EPSP, which itself doesThe authors should elaborate on the possibility of developmental compensation or alternative mechanisms that could explain this phenomenon in the Discussion (ephaptic coupling?).Indeed, Cx45 is expressed in MLIs and might underlie residual or compensatory spikelet transmission. Please see response to comment 2, reviewer 1 for full description of how we address this issue now in the manuscript, including the possibility of ephaptic coupling.Lastly, the presentation of the Cx36 data seems out of place in the Results. Perhaps this was due to the lack of explanation in their methodology .This is a good suggestion, we now show the Cx36 data in the main Figure 2 and more clearly introduce BC-BC paired recordings in the Results section:“In order to confirm that extracellular stimulation indeed evoked a presynaptic AP which could then propagate across electrical synapses formed by gap junctions to produce a spikelet in the postsynaptic cell, we performed paired BC-BC recordings. Hyperpolarizing current pulses were used to confirm the presence and the strength of coupling coefficients .”We also changed “coupled pairs” to “electrically coupled cells.”2) The authors attempt to compare the prevalence of electrical vs chemical FFI is confusing. Why not simply state the rate of identifying electrically and chemically connected pairs?We felt the use of “prevalence” was accurate, since it is often used to refer to the percentage of a population that is affected by a disease, and inherently also a frequency. But instead we replaced it with the words “more often” or “frequency” to avoid any confusion.Then report the rate of spikelet coupling .Perhaps our text was confusing, but the frequency of observing spikelets in BC-BC pairs is less than the detection of electrical coupling. We think this is for two reasons: (1) transient spikelet propagation through gap junctions are filtered more than the steady-state current components of long step depolarizations, and (2) some BC-BC pairs may not be directly connected, but connected through a third MLI. Nevertheless, we have added new panels to the new Figure 4 (D-F), which show a positive correlation between spikelet amplitude and coupling strength, as would be expected from being mediated by gap junctions.Again, could spikelet coupling reflect a different mechanism?Please see our responses to reviewer 1 for a thorough discussion of this issue.Last, is the prevalence of electrical vs chemical FFI best described by the rate of electrically and chemically connected pairs of basket cells?We no longer use the word “prevalence,” but either use “more often” or “frequency.”3) Can the authors always be sure the presynaptic coupled neuron is a basket cell? It looks like the stimulating pipette is place throughout the molecular layer where stellate cells (and their neurites) also reside.Indeed, the presynaptic cell could be a SC or a BC, but Rieubland show that electrical coupling of putative BC-SC paired recordings occurs at a much lower frequency (13% versus 61% putative BC-BC pairs) and have lower CC. We therefore refer to extracellularly evoked spikelets as coming from MLIs more generally rather than BCs. However, we do mention that more often than not, the spikelets are mediated by neighboring basket cells, based on the prominent electrical coupling and low probability of coupling between BCs and SCs .In the Discussion section we updated the text:“Our results indicate that low stimulation intensity of small PF beams leads to the recruitment of APs in electrically coupled MLIs that generate spikelets in the postsynaptic BCs , but more rarely and less prominently in SCs . Because of the low frequency (13 %) of BC-SC electrical coupling , we propose that eFFI is mostly provided by other BCs.”4) Could temperature-dependent effects on spike width and after-hyperpolarization duration contribute to differences with results from the Marty group ? This may also be true for the resting membrane potential in the presynaptic cell which the authors clearly show affects the polarity of influence.Alcami, 2018 show that there were no differences in spikelet shape if experiments are performed at 21 or 34 °C, so a temperature-dependence seems unlikely to account for the difference. However, as suggested by the reviewer, the most likely difference is due to the hyperpolarized holding potential (-70 mV in some cases), which we predict should have a small AHP. Interestingly, when they recorded a spikelet, the prominent outward current was reminiscent of our recordings . Finally, we cannot rule out the possibility that in P11-13 rats, the AHP is less prominent. We have added the following text to the Discussion section.“Comparison of spikelets evoked in an unperturbed, electrically coupled neighbor to those evoked in paired recordings with presynaptic membrane potential manipulations is consistent with a depolarized BC resting membrane potential . We postulate that previous recordings showing mostly depolarizing spikelets in cerebellar MLIs were due to either hyperpolarized holding potentials  or an intrinsic difference in the amplitude of the AHP recorded from brain5) EPSP half-widths in 5B and 5D seem to be different (responses appear much more prolonged in 5B). Does this mean that many responses in 5D in the LS condition have coupled spikelets ? How does this affect the interpretation of their results?The large distribution in EPSP decays is perhaps due to cable filtering when performing synaptic stimulation in different parts of the dendrite, similar to what we previously showed for stellate cells  However, it is also possible that BCs have different numbers of electrically coupled neighbors, which could decrease their input resistance  and thus accelerate the decay of the EPSPs.It seems unlikely that EPSCs without outward currents could have significantly altered half-widths, but we cannot strictly rule it out.We have added the following text to the Results section:“The variability in the EPSC decay in response to low stimulation could be due to the variability in cable filtering when eliciting synapse stimulation in different locations within the dendrite, or the variability in the number of electrically coupled cells, which have been shown to decrease input resistance . But we cannot rule out that a small spikelet may accelerate some EPSPs without showing an outward current. Nevertheless, recruiting an additional spikelet, on average, accelerated EPSP decays”


Literature also shows racial disparities in the severity of COVID-19 which may confound the effect of CHD on outcomes.3This review directly compares studies and case reports of patients with ages ranging from 3 months to 76 years. Discrepancy in ages is important to consider since COVID-19 is known to affect infants less than 1 year of age more severely.4 Lewis et al reported that out of 53 CHD patients, there appeared to be no correlation between the complexity of the CHD and the subsequent infection-related cardiac decompensation displayed by the patient.4 This may explain why patients with similar CHDs experience different disease trajectories. Therefore, using only the CHD status is not a sufficient indicator for poor outcomes and markers of symptom progression such as raised cardiac troponins upon admission may prove more useful.5 This is particularly relevant since CHD patients with infection-induced myocardial injury are at a higher risk of complications.5 Treatment protocols used to manage infections should also be considered amongst the studies included in the review. For example, Non-steroidal anti-inflammatory drugs use in early infection is thought to be controversial due to reports of causing more complications when administered for respiratory tract infections.6Contrary to this review, there is evidence that individual defects such as single ventricular defects are not sufficient cause alone for worse COVID-19 outcomes.1 Massin et al demonstrates that a significant proportion of children with CHD have associated non-cardiac comorbidities including genetic conditions, with the most common being trisomy 18.7 These comorbidities make it difficult to assess the direct effect of CHDs on outcomes. Similarly, Agarwal et al show that adults with CHD are twice as likely to have non-cardiac comorbidities compared to their non-CHD counterparts.8 Additionally, for patients below the age of 40, the more severe the CHD, the higher the risk of developing non-cardiac comorbidities.8From the studies included in the review, 24% of the patients had comorbidities with the most frequent being genetic conditions, type 2 diabetes mellitus, and chronic kidney disease.In conclusion, in order to determine causality or a significant correlation between COVID-19 prognosis and CHD, this review needs to stratify CHD according to type of defect and severity, whilst controlling for confounders such as age and comorbidities.


BD5 is a new binary data format based on HDF5 . It can be used for representing quantitative biological dynamics data obtained from bioimage informatics techniques and mechanobiological simulations. Biological Dynamics Markup Language (BDML) is an XML (Extensible Markup Language)-based open format that is also used to represent such data; however, it becomes difficult to access quantitative data in BDML files when the file size is large because parsing XML-based files requires large computational resources to first read the whole file sequentially into computer memory. BD5 enables fast random  access to quantitative data on disk without parsing the entire file. Therefore, it allows practical reuse of data for understanding biological mechanisms underlying the dynamics. Recent advances in bioimage informatics and mechanobiological simulation techniques have led to the production of a large amount of quantitative data of spatiotemporal dynamics of biological objects ranging from molecules to organisms . A wide http://www.hdfgroup.org/HDF5/). It is widely used for describing various kinds of large-scale biological data [The XML-based BDML format has the advantages of machine/human readability and extensibility. However, it is often problematic for accessing and retrieving data when the size of the BDML file becomes too large . This problem arises because parsing an XML-based file often requires large computational resources to first read the whole file sequentially into computer memory. In fact, many sets of quantitative data stored in the SSBD:database   were divcal data –9.Here, we describe the development of the BD5 data format, based on HDF5, for representing quantitative biological dynamics data in a manner that enables quick access and retrieval.Here, we extended BDML to support HDF5-based storage of quantitative biological dynamics data. In contrast to XML documents, HDF5 format can allow random  access to parts of the file without parsing the entire contents. Therefore, HDF5 is a more efficient file format for accessing and retrieving the contents of the file.datasets and groups. Datasets are array-like objects that store numerical data, whereas groups are hierarchical containers that store datasets and other groups. Detailed information on BD5 is available at http://ssbd.qbic.riken.jp/bdml/. Here, we summarize the major BD5 datasets and groups. BD5 format has one container named data  corresponding to an index number of a time-ordered sequence,trackInfo dataset for the information of tracking of one object to another.object and feature groups. For a fixed time interval, the index will correspond to each sequential time point. For example, if the time interval is 2 minutes, group 0 will have t = 0 and group 1 will have t = 1, while tScale is 2 and tUnit is minute (Each of the numbered groups corresponds to an index of a time-ordered sequence that has s minute . For irrobject group has numbered dataset(s) corresponding to the reference number of the biological object(s) predefined under the objectDef dataset. Each row of the numbered object includes an identifier of the object and its spatiotemporal information such as time point and xyz-coordinates  that allows us to connect the xyz-coordinates together to form a line or a face within a biological object. To represent multiple biological objects from different measurements, different numbered datasets are used (Each rdinates . To repr is used . The mulare used . The datfeature group has numbered dataset(s) corresponding to the reference number of the object(s) predefined in the objectDef dataset. Each row of the numbered object includes an identifier of the object, an identifier of the feature (fID) predefined in featureDef, and the value of the feature , in BDML format.To allow the use of BD5 to describe quantitative data, we needed to update the BDML format so that it could be used to describe the corresponding meta-information. The latest version of BDML (version 3.0) can handle an external file by using the  element . The bd5To evaluate the performance of the BD5 format, we first compared the file access time between XML- and HDF5-based files . We measured the time for accessing coordinate data at a randomly selected time point in the BDML and BD5 files (334 pairs of files) by using a Python-based program . The accFile size can be a critical benchmark for a data format because the transfer of large files often fails. Therefore, we next compared the disk space requirement between the XML- and HDF5-based files by comparing the size of BDML and BD5 files (347 pairs of files) . We founIn addition, we determined the relationships between access time and file size for BDML and BD5 files . In BD5,https://github.com/openssbd/BD5_samples. The codes include sample codes that use NumPy arrays to describe five different entities  for representing biological objects and to write this information to a BD5 file. We also provided sample codes that read segmented regions of interest (ROIs) stored in a TIFF image file, and the ROIs are then saved in a BD5 file. Moreover, we provide a sample code to read data stored in CSV-formatted file and write the data to a BD5 file, and sample codes to read a BD5 file for further analysis using quantitative dataset. These sample codes will help developers of bioimaging software tools to understand and support BD5 format.Sample programs and codes for writing and reading BD5 files using Python are provided as Jupyter notebooks at object and feature groups correspond to the reference numbers of the objects and features predefined in the objectDef and featureDef datasets. It also checks the consistency of the dimensions declared and the actual dimensions used within the datasets. It provides type checking of the data and error warnings if the data do not conform to the BD5 specification. The Python source code is available at https://github.com/openssbd/bd5lint/.We also provide a program, bd5lint, for detecting bugs and inconsistencies in BD5 files. The program checks the structure of BD5 files and checks that the ordered numbered datasets in the http://www.codesynthesis.com/products/xsd/) is required for the BDML schema to C++ data-binding compiler. The source codes of the tool BDML2BD5 and a Docker container (bdml2bd5-docker) to build the executable are available at GitHub .To support data storage in older BDML files, we provide a C++-based software tool named BDML2BD5. By using this tool, BDML files can be converted into BD5 files. To compile the tool, the HDF5 library is required for HDF5 data writing, and CodeSynthesis XSD (https://www.hdfgroup.org/downloads/hdfview/). This tool can compensate for the lack of human readability.In this study, we developed a new BD5 data format based on HDF5 for representing quantitative biological dynamics data. Compared with BDML, which is based on XML, the BD5 format has two advantages: (a) fast access to and retrieval of quantitative data owing to random access to the HDF5-based file, and (b) fast transfer of files containing large quantitative data because the file size is dramatically reduced. A drawback of the BD5 is that human readability is low when compared with the BDML format. BD5 files cannot be opened by text editors because the file is binary formatted. However, the HDF group provides a software tool named HDFView that enables the user to open and read all HDF5-based files  dramatically reduced the file size, but slowed down access to the data because of the decompression cost. Therefore, we did not apply it to the BD5 files, but it can be a practical approach to reduce file size when larger datasets are produced. Another approach is to divide and store a dataset into multiple BD5 files followed by bundling these BD5 files together. To bundle multiple BD5 files, we can use external links supported by HDF5, or we can extend BDML format to handle multiple BD5 files. We will determine the specification or provide guidelines for dividing and bundling datasets in BD5 data format in the future.Compared to the BDML format, BD5 file size is considerably smaller, but future advances in imaging techniques and devices will certainly produce larger datasets. One approach to tackle this is to use the HDF5 compression filter. We found that the filter , which is one of the major databases for sharing bioimage data and quantitative biological dynamics data [https://github.com/HDFGroup/h5serv). This enables SSBD:database to provide a web service for users to access quantitative data stored in BD5 files (http://ssbd.qbic.riken.jp/restfulapi/). Because HDF5 and XML are supported by many software platforms, BD5 is a promising data format for storing quantitative biological dynamics data.BD5 format has already been used in the latest version of SSBD:database (ics data . At the https://docs.openmicroscopy.org/ome-model/) and segmentation channels in Cell Feature Explorer . However, not all data can be represented as an ROI on a microscopy image. For example, in an automated cell lineage tracing study of Caenorhabditis elegans, each nucleus was represented as a sphere with center and radius, independently of the z-stack images [Like BDML, BD5 can represent quantitative biological dynamics data that is associated with, but independent of, microscopy images. Such data has often been represented as ROIs on the corresponding microscopy images; for example, the ROIs in the OME data model , and we are actively collaborating with the OME community (https://www.openmicroscopy.org/). We will continue to contribute to the bioimaging community by developing standard data formats for storing quantitative data of biological dynamics.To promote the support of BD5 format by software developers, we provide sample Python programs and Jupyter notebooks with explanations for writing and reading BD5 files. These sample programs will facilitate the understanding and use of BD5 format. For the standardization of this format, it is necessary to align our approach and our proposed format with those used by international projects and to support other open and widely used platforms. We have started to support the BD5 format on the Galaxy platform , e.g., h


On Days 0 and 28 (±2) all dogs in each household were treated with the allocated product according to label directions, and all household cats received spinetoram (Cheristin®). On Day 0 and at weekly intervals through Day 56 (±2), on-animal and premises flea burdens were enumerated, a veterinary dermatologist scored integumental changes using canine atopic dermatitis extent and severity index (CADESI)-4 and flea allergy dermatitis (FAD) scales, and owners scored pruritus using the validated canine pruritus severity scale (CPSS).Post-launch field investigations of recently-approved flea control products establish an efficacy baseline and in subsequent years can detect any efficacy decline suggestive of emerging resistance. As part of a continuing program of yearly assessment of flea control products in west central Florida, this study, using client-owned dogs, investigated the efficacy of lotilaner and spinosad in controlling fleas and in alleviating dermatologic signs likely associated with flea infestations. Forty-four qualifying households were randomized to either a lotilaner , and 100% from Week 6 through the final assessment (Week 8) when all study dogs were flea-free. For both groups, at each timepoint, flea counts on dogs and in traps were significantly reduced compared to the initial assessment (p < 0.001), as were improvements in median CADESI-4, FAD and CPSS scores (p ≤ 0.001). At Week 4, the geometric mean flea count on dogs in the lotilaner group (0.1) was significantly lower than that of dogs in the spinosad group (0.6) (p = 0.027), significantly fewer dogs in the lotilaner group were found to have fleas (p = 0.034), and mean owner-rated pruritus scores were significantly lower (p = 0.025).Under field conditions favoring heavy flea challenge, two consecutive monthly treatments of dogs with either lotilaner or spinosad produced a 100% reduction in canine flea infestations and dramatic improvements in dermatologic lesions and pruritus, based on scoring by a veterinary dermatologist and by dog owners. Household flea burdens were driven to extinction in all but one home in each treatment group. These conditions offer an excellent opportunity to monitor the performance of flea control treatments under severe flea infestation pressure. Over the last three decades, the Clinical Parasitology Group in the Department of Diagnostic Medicine/Pathobiology at Kansas State University (KSU) has undertaken regular investigations in this region of many of the leading veterinarian recommended on-animal and oral flea control products. While immediate post-launch studies on flea control products are important to establish a baseline, studies in subsequent years can determine if efficacy continues to match the baseline and detect any loss of efficacy that could be an indicator of emerging resistance.The isoxazolines are the most recent innovation to be approved for flea and tick control. The field studies required for USA registration of the first three isoxazolines—afoxolaner, fluralaner and sarolaner—compared these products with spinosad . SpinosaTo date, studies in west central Florida have investigated the efficacy of afoxolaner, fluralaner and sarolaner , 2018. H22.1e.g., heartworm preventives), no other veterinary drugs or treatments were to be given to any dog during the post-treatment period of the study.Referrals from Animal Dermatology South, New Port Richey, FL, and advertisements on Facebook allowed identification of 44 private residences that qualified for inclusion in the study. Household enrollment was initiated on May 15, 2018 and the final assessment of the last enrolled house was on July 20, 2018. Each qualifying home had at least one healthy, non-fractious dog, with no conditions that could affect study results. Homes with up to four additional healthy dogs or cats could qualify for the study . All enrolled pets were required to spend at least 12 h per day indoors. To meet label requirements for spinosad, dogs in enrolled homes had to be at least 14 weeks of age and weigh at least 2 kg. To ensure a valid challenge, at least five fleas had to be observed, using area flea counts, on at least one household dog, and at least five fleas collected in two intermittent-light flea traps left overnight at a pre-enrollment visit. Owners had to agree not to use any other topical or premise flea control products and to not bring any other dogs or cats into the household for the duration of the study. With the exceptions of maintaining a stabilized dose rate of any medication used to manage chronic conditions and administration of scheduled prophylactic treatments  at a minimum dose rate of 20 mg/kg. Group 2 dogs were treated orally with a spinosad tablet  at a minimum dose rate of 30 mg/kg. Dogs had food available ad libitum or had been fed before or soon after dosing. All dogs and cats were weighed prior to each treatment to ensure proper dosing. Treatments were administered according to label directions on two occasions, once on Day 0, and once on Days 28–30. Dosing was completed by the veterinary student staff or by pet owners under staff observation. Cats in all enrolled households were treated topically with an 11.2% spinetoram spot-on solution (Cheristin®) administered according to label directions.Each home that met all enrollment criteria was assigned a random number using Excel  and blocked into groups of two. The higher random number within each block was assigned to group 1 and the lower to group 2. Group 1 dogs were treated orally with a lotilaner chewable tablet . However, all qualifying and non-qualifying animals in each home were administered group-appropriate treatments.2.3The flea population on each dog was assessed using a validated visual area count of five anatomic locations on each dog - the dorsal midline, the tail head, left lateral, right lateral, and inguinal region . Countin2.4A board-certified veterinary dermatologist completed blinded clinical examinations of each study dog on Day 0 using the canine atopic dermatitis extent and severity index (CADESI)-4 and a flea allergy dermatitis (FAD) assessment tool . At the 2.5All study dogs that received at least one treatment were monitored for adverse events. Any event observed by study staff or reported by owners was recorded in the study file. All adverse events were reported to the sponsoring company which then informed the relevant regulatory authority.2.6Efficacy within each treatment group at each assessment was defined as the percent reduction from baseline in geometric mean live adult on-dog flea counts and household intermittent-light flea trap counts. For flea count data in each group, descriptive statistics  were presented for collections on Day 0 and Weeks 1 through 8. The percent change from Day 0 was calculated based on the geometric mean and the arithmetic mean for each group at Weeks 1 through 8. For geometric mean calculations, a natural log transformation was applied to flea/house count plus 1, and 1 was subtracted from the resulting mean value prior to calculation of efficacy. Percentage of control achieved by each product was calculated using the formula:The SAS Proc Mixed Procedure  was used for the statistical analysis. Possible differences between groups were assessed by repeated measures analysis of variance modeling of the natural log transformed counts with group, week and group by week interaction as fixed effects. Comparisons between groups at each week were generated from this model using linear contrast statements. The Wilcoxon signed rank test was used to evaluate within group comparisons from Day 0 to each week. The number and percentage of cases with zero flea counts were tabulated for each week and group. Possible differences between groups were assessed by the chi-square test.For dermatology assessments in each group, descriptive statistics  were presented for scores collected on Day 0 and for subsequent study weeks. Possible differences between groups were assessed by repeated measures analysis of covariance modeling of the ranked values. The Day 0 value was included as a covariate with group, week and group by week interaction as fixed effects. Comparisons of median scores between groups at each assessment were generated from this model using linear contrast statements. The Wilcoxon signed rank test was used to evaluate within group comparisons from Day 0 to each week.33.1There were 44 qualifying households enrolled into the study, 22 for each group. Within these households were 33 and 36 dogs qualifying for the lotilaner and spinosad groups, respectively. Overall group demographics were well balanced, except for the ratio of female to male which was 1.06 and 0.385 in the lotilaner and spinosad groups, respectively. The average age for dogs in each group was 6.5 years, with a similar range in each group (4 months to 15 years). Average weight in lotilaner-treated dogs was 17.7 kg (range 2.4–54.6) and in spinosad-treated dogs was 19.0 kg (2.3–58.2). The proportion of households with multiple dogs was 63.6 and 72.6% for the lotilaner and spinosad groups, respectively. There were five households in the lotilaner group that also had cats, and six in the spinosad group. Five dogs  in the study were reported to be receiving heartworm prophylaxis. There were 25 different dog breeds and cross breeds represented, with the most common breed descriptions being Pit Bull (including Pit Bull cross) (15) and Chihuahua (including Chihuahua cross) (14).Three dogs in the lotilaner group, all from separate households, were lost to follow up, one after Week 2, one after Week 3 and one after Week 4. In the spinosad group eight dogs were lost to follow up, one after the Week 2 visit, three dogs from one household after Week 3, two from separate households after Week 4, and two from a single household after Week 6. In all cases, the reasons for dogs being lost to follow up were due to protocol violations, including owners moving or being unavailable, and none were attributed to adverse events or to treatment failure. All data recorded from the non-completing dogs were included in the analyses up to the point dogs were lost from the study. Occasional data points were missing from dogs in each group because of owner failure to make or keep appointments and neither the owner nor the dog(s) were available. Within groups there was some between-week variation in the numbers of dogs with flea counts, CPSS scores and dermatology scores. These variations arose when the dog was available for evaluation, but the primary owner was not present.3.2At study entry geometric mean dog flea counts were similar between the two groups  . There wFor both groups at all assessments, compared to study entry, the reductions in geometric mean flea counts were significant (p < 0.001). One week after the first treatment there was a significantly greater percentage of dogs on which no fleas were found in the spinosad group (88.9%) than in the lotilaner group (66.7%) (p = 0.025) . At WeekFor intermittent light trap results, at study entry geometric mean flea counts were 22.2 and 19.2 in the spinosad and lotilaner groups, respectively . At all Ctenocephalides felis felis.At the enrollment visit for one household five fleas were initially counted in the retrieved traps. However, under microscopic examination at KSU one of the counted fleas was found to be a wingless fly  with a general shape and size very similar to a flea. Although not meeting the entry criterion of a trap count of five fleas, the household was retained in the study because the live flea counts of 78 and 19 on the two dogs that were present was considered adequate to ensure that there was a substantial flea challenge at study initiation. Other than this one instance of initial incorrect identification, all 6099 fleas collected in the traps throughout the study were identified as During the study three households met the description of a red-line home (an increase in premises trap counts of at least 20% over day 0 within 1–4 weeks post-treatment) . In one 3.3Median CADESI-4 scores at enrollment were 21.0 and 19.5 in the lotilaner and spinosad groups, respectively . BenchmaIn each group median FAD assessment scores at study entry were 9.0, declining to 1.0 at Week 8 . Scores Using the owner-assessed CPSS, the range of 0–1.9 is regarded as being indicative of a normal dog . At stud3.4Ancylostoma caninum as the cause of the diarrhea, and the dog recovered following treatment with a combination product containing praziquantel, pyrantel pamoate and febantel .There were three reported adverse events in the lotilaner group, none of which were attributed to treatment, although the role of treatment as a cause of transient anorexia reported for two dogs in a single home one to two days post-treatment could not be entirely discounted. In another household, diarrhea was reported by the owner to have occurred eight hours after the dog was treated. A fecal test identified infection with the hookworm In the spinosad group there were six reports of gastrointestinal events, one in each of six dogs. In one household each of two dogs vomited within two hours following the first treatment, and the owner also reported an observation of “inappropriate defecation” by the dogs. The dogs were not re-dosed until the next scheduled treatment. No fleas were found on either dog at any post-treatment assessment and there was no vomiting or other adverse event following the second scheduled treatments. In a second household two dogs vomited two hours following their second monthly treatment and were not redosed. Flea counts were zero on those dogs in the two weeks following this treatment, after which this home was non-responsive to requests for appointments and was withdrawn from the study. In a third household, nine days after treatment the owner reported transient episodes of vomiting after the dog had been fed table scraps, while in a fourth household an enrolled dog vomited 24 h after treatment. All reported events were mild to moderate in degree and dogs recovered uneventfully without treatment.4The on-animal flea count methodology used in this and previous in-home investigations conducted by these authors, detects approximately 23.5% of the total dog flea burden . TherefoLotilaner and spinosad both demonstrated rapid and sustained efficacy in controlling fleas under extreme natural flea challenge, including in red-line homes. The efficacy response to each product, with associated improvement in dermatology (including CPSS) scores, can be attributed to the rapid knockdown of fleas  that was sustained throughout each month following treatment. Laboratory studies with spinosad have demonstrated that 100% of fleas infesting dogs are killed within four hours post-treatment, with flea killing evident within one hour after treatment . The onsIt is of interest that of the 69 dogs in the current study, 66 (95.7%) were scored by their owners on the CPSS as being abnormally pruritic at enrollment  . In two From the many field reports describing comparisons of spinosad with other flea control products, our study is just the second to find a significant improvement over spinosad . MeadowsFor lotilaner, these results align with laboratory and earlier field studies. In a laboratory study, fleas were observed to become moribund on lotilaner-treated dogs as soon as one hour after treatment, and by two hours the geometric mean live flea count reduction was 64.0%, with many of the live fleas classified as moribund . The morThe ability of both products to rapidly and continuously kill on-dog fleas resulted in a progressive reduction to extinction or near-extinction of emergent fleas. Of interest were the on-dog and intermittent-light trap flea counts from one, specific spinosad-group household. At baseline, the dog had too many fleas to count (recorded as the maximum of 250) and trap counts identified 524 fleas. At Week 1, trap counts had increased to 1722, making this a true red-line home, but only two fleas were identified on the dog. At Week 2, trap counts had declined substantially, and by Week 4 (when the owner moved to an assisted-living care facility) the combined count from both traps was 26, at which time the dog was free of fleas. The findings of large-scale flea emergence early in the study, low post-treatment counts on the dog, and the decline in flea trap counts demonstrates the “vacuuming effect” of a monthly flea treatment with a product such as spinosad or lotilaner. Fleas that emerge and infest a treated dog are rapidly killed, before laying any eggs, so that there is no addition to household immature flea life-stage bio-mass. There is therefore a progressive depletion in environmental flea burden. Even though flea trap counts remained high due to a large preexisting immature flea life-stage biomass in the early post-treatment period, this dog showed a progressive improvement in all dermatology variables until being withdrawn because of the owner’s circumstances.Both isoxazolines and spinosad have repeatedly demonstrated high sustained efficacy and residual insecticidal activity that are mediated through binding to different target sites . The resTreatments were administered according to schedule to all dogs and cats in qualifying households in which observations were continued to Week 4 and beyond. Those treatments included non-study dogs and cats. Such treatment of all dogs and cats in a household is an important factor in minimizing household flea biomass and therefore in controlling flea challenge to household pets.Dirofilaria immitis, and the economic circumstances that place prophylaxis beyond the means of some dog owners. We feel it is incumbent on the pharmaceutical suppliers of heartworm preventives and practicing veterinarians to address this problem as a means of reducing the risk of heartworm infection of all dogs.An unexpected finding from our routine medical history screening questionnaire was the very limited use of heartworm preventives in the enrolled canine population. Just five dogs  were receiving this prophylaxis despite being in an area in which heartworm infection is regarded as presenting significant risk to canine health. These failures in use of heartworm preventives are likely due to a combination of factors, including inadequate education of dog owners of the risks of their dog’s infection with Both products were well tolerated, with a few observations of post-treatment vomiting by some spinosad-group dogs. This is a well-recorded effect of spinosad treatment that can occur in a small proportion of dogs and was not considered to present any safety risk. There was no reduction in efficacy observed in dogs that vomited at approximately two hours post treatment and were not re-dosed. For lotilaner, the study confirmed the safety that has been reported from early laboratory work demonstrating a wide safety margin, including in young puppies, and from field reports . The saf5Under field conditions favoring heavy flea challenge in the subtropical climate of west central Florida, two consecutive monthly treatments of dogs with either lotilaner or spinosad were safe and produced a 100% reduction in canine flea infestations leading to marked improvement in dermatologic lesion scores and owner-scored pruritus. In the 38 enrolled households that completed the 2-month study, premises flea burdens were driven to extinction in all but one home in each treatment group.® and Cheristin™ that were evaluated in this investigation. WGR has acted as a paid consultant for Elanco. MC has provided lectures sponsored by Elanco. BHH has had projects and lectures funded by Elanco. There were no conflicting interests that could have influenced the conduct and reporting of these studies.The studies reported here were funded by Elanco Animal Health, Greenfield, IN. AJR and TMK are employees of Elanco Animal Health. MWD has had research projects funded at Kansas State University and lectures sponsored by Elanco Animal Health, manufacturers of Credelio, ComfortisElanco Animal Health Incorporated, Greenfield, IN, USA.This study was funded by a grant from The datasets supporting the conclusions of this article are included within the article. Raw data from this study has not been shared at this time because we anticipate additional publications exploring some of the detailed and complex dermatologic results.MWD and BHH were primary authors of study design, served as primary study investigators and assisted in manuscript revision. WGR drafted the manuscript and assisted in study design and was the primary study monitor. VS coordinated and supervised data collection and entry. AH, BS, CB, LP, NS, SAW and TSB were responsible for animal handling, collection of data and data entry. MC was responsible for designing and completing blinded dermatology assessments and assisting in study design. AJR and TMK assisted in design of study, monitoring of study and manuscript revision. All authors reviewed and approved the final manuscript.The protocol was reviewed and approved as complying with the regulations set forth by the Kansas State University Institutional Animal Care and Use Committee. This research protocol IACUC # 4086 was approved 10 May 2018. Each pet owner was required to receive a written and verbal explanation of the study protocol and the study products to be used, and was required to sign an informed consent form prior to enrolling their dog(s) into this field trial.Not applicable.