Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr 16:12:29.
doi: 10.3389/fncir.2018.00029. eCollection 2018.

The Effect of Single Pyramidal Neuron Firing Within Layer 2/3 and Layer 4 in Mouse V1

Jochen F Meyer  1 , Peyman Golshani  2 , Stelios M Smirnakis  3
Affiliations

The Effect of Single Pyramidal Neuron Firing Within Layer 2/3 and Layer 4 in Mouse V1

Jochen F Meyer et al. Front Neural Circuits. .

Abstract

The influence of cortical cell spiking activity on nearby cells has been studied extensively in vitro. Less is known, however, about the impact of single cell firing on local cortical networks in vivo. In a pioneering study, Kwan and Dan (Kwan and Dan, 2012) reported that in mouse layer 2/3 (L2/3), under anesthesia, stimulating a single pyramidal cell recruits ~2.1% of neighboring units. Here we employ two-photon calcium imaging in layer 2/3 of mouse V1, in conjunction with single-cell patch clamp stimulation in layer 2/3 or layer 4, to probe, in both the awake and lightly anesthetized states, how (i) activating single L2/3 pyramidal neurons recruits neighboring units within L2/3 and from layer 4 (L4) to L2/3, and whether (ii) activating single pyramidal neurons changes population activity in local circuit. To do this, it was essential to develop an algorithm capable of quantifying how sensitive the calcium signal is at detecting effectively recruited units ("followers"). This algorithm allowed us to estimate the chance of detecting a follower as a function of the probability that an epoch of stimulation elicits one extra action potential (AP) in the follower cell. Using this approach, we found only a small fraction (<0.75%) of L2/3 cells to be significantly activated within a radius of ~200 μm from a stimulated neighboring L2/3 pyramidal cell. This fraction did not change significantly in the awake vs. the lightly anesthetized state, nor when stimulating L2/3 vs. underlying L4 pyramidal neurons. These numbers are in general agreement with, though lower than, the percentage of neighboring cells (2.1% pyramidal cells and interneurons combined) reported by Kwan and Dan to be activated upon stimulating single L2/3 pyramidal neurons under anesthesia (Kwan and Dan, 2012). Interestingly, despite the small number of individual units found to be reliably driven, we did observe a modest but significant elevation in aggregate population responses compared to sham stimulation. This underscores the distributed impact that single cell stimulation has on neighboring microcircuit responses, revealing only a small minority of relatively strongly connected partners.

One sentence summary: Patch-clamp stimulation in conjunction with 2-photon imaging shows that activating single layer-2/3 or layer-4 pyramidal neurons produces few (<1% of local units) reliable single-cell followers in L2/3 of mouse area V1, either under light anesthesia or in quiet wakefulness: instead, single cell stimulation was found to elevate aggregate population activity in a weak but highly distributed fashion.

Keywords: calcium imaging; cortex; electrical stimulation; functional connectivity; in vivo patch-clamp recording; mouse V1; two-photon microscopy.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(a) Left: Group of layer-2 OGB-labeled neurons in a Viaat-Cre x Ai9 mouse whose interneurons are labeled with tdTomato (yellow); pyramidal neurons appear green. The whole-cell patched cell (arrow) appears orange because it is filled with both OGB and Alexa 594 from the pipette solution. This allowed us to confirm the identity of the neurons we stimulated in whole-cell patch. The scale bar is 50 μm. Right: Coronal view of L2/3 and L4 of OGB-labeled area V1 showing the tip of a patch pipette after recording from a L4 neuron ~370 μm below the pia in cell-attached mode. Interneurons are labeled red. The dashed area represents the range of depths we recorded the responses of L2/3 neurons from. (b) Average projection of the top 130 μm of a z-stack containing a whole-cell patched L2/3 neuron passively filled with Fluoro-Ruby dextran. It displays the typical apical dendrite pattern of an L2/3 pyramidal cell projected in the x-y plane, including spines seen on its superficial dendritic segments (see top right). We compared the firing patterns and spike wave shapes of all patched neurons with those of confirmed pyramidal cells to ensure that we were only stimulating pyramidal units. The bottom right panel shows a typical distribution of ISIs from one of the stimulated pyramidal cells. (c) Voltage trace of the neuron in whole-cell patch shown in panel (a) during electrical stimulation. Note the high reliability with which APs are elicited during electrical stimulation over the course of ~45 min of stimulation (see panel d). (d) Voltage traces during stimulation of the same neuron at the beginning of a recording (top), and 45 min later (bottom), demonstrating that patched neurons can be stimulated reliably over long periods of time. The resistance drifted somewhat over time, but the number of elicited spikes remained similar. Stimulated neurons always fired multiple spikes (12–15 on average) per stimulation epoch (see section Methods/Results). Note that the percentage of followers per stimulated cell did not depend on the average number of elicited spikes per recording (see Supplementary Results). (e) Top Trace: Typical Calcium activity spontaneously generated by the L2/3 neuron from (d). Bottom Trace: Timestamps of recorded APs. Note the close correspondence between the calcium signal and underlying APs. (f) On average, the ΔF/F amplitude of the calcium signal (y-axis) corresponds well with the number of APs (x-axis). The upper and lower box bounds depict the 25th and 75th percentile, respectively, while whiskers extend from the 5th to the 95th percentile.
Figure 2
Figure 2
(A) Top Row: (i): Cyan: histogram of the distribution of z-values derived from comparisons between the real responses to electrical stimulation and 5,000 iterations of randomly shuffled responses (see section Methods). Red Line: mean of the distribution. Dashed Lines: Thresholds for the z-value mean, above which excited followers are identified, and below which inhibited followers are identified. This is derived from the sham stimulation experiments, to yield high specificity, i.e., no sham followers (B). (ii) Analogous to (i) for an inhibited follower cell. The red line (mean of the distribution) now lies below the lower threshold, below which inhibited followers are identified. (iii) Analogous to (i,ii) for a typical non-follower (neutral) cell. Note that mean z-value of the cyan histogram did not cross either threshold. Bottom Row: Corresponding average responses (±sem) of the excited (i), inhibited (ii), and neutral (iii) neurons shown in the top row. For (i,ii) trials which contributed to significance (see section Methods) were averaged. Green line: stimulation onset. Red line: stimulation end. Green shaded areas (i,ii) indicate which frames were averaged to compute the response (400 ms after stimulus offset). Orange shaded area in (iii) analogous for a non-follower. (B) Distribution of all z-value means from all 1,069 cells that received sham stimulation. z-value means never exceeded 2.1 (“excitatory-follower threshold”) or fell below −2.3 (“inhibitory-follower threshold”). (C) Simulation shows that our follower algorithm was more sensitive than a previous method by Kwan and Dan (2012). It identified more neurons correctly as followers at almost all levels of simulation, identifying essentially all followers that have an additional spike elicited >60% of the time upon stimulation. The sensitivity drops sharply below that, so that the algorithm identifies only ~50% of followers that have one additional spike elicited ~40% of the time upon stimulation. (D) Simulation of activity modulation when a calcium signal equivalent to 1 AP was added to 10, 40, 50, or 100% of all trials. Red trace: average ΔF/F signal (±sem) of an example neuron after addition of 1-AP calcium transients. Black trace: control with no added activity. Our algorithm identified this neuron as a follower when at least 40% of all trials received a simulated extra AP. The red and green dashed lines represent the periods where the single cell would have been stimulated electrically (analogous to A). Note that here electrical stimulation does not occur but instead an extra action potential is inserted at stimulus offset. Note that because the frame rate was lower than the calcium signal rise time, it can appear as though the calcium signal starts to rise within a frame before the time stamp of the AP, due to alignment jitter. The light green and light orange shaded areas indicate the frames that were averaged to calculate the simulated stimulus response (400 ms). (E) The blue histograms at the bottom represent the mean z-values across all 75 cells (one FOV) receiving simulated 1-AP calcium signals in 10, 40, 50, or 100% of all trials. Here, we combined data from 2 spontaneous activity recordings that had 190 simulated trials, which was the typical number in our experiments. This shows that our algorithm identifies ~50% of neurons as followers when at least 40% of the trials receive one extra AP. Red arrows indicate the z-scores corresponding to the simulated panels shown in (D). Dashed Line: Threshold for excited follower identification.
Figure 3
Figure 3
(A) Four out of nineteen (21%) stimulated L2/3 pyramidal cells were able to influence at least one neighboring unit significantly (“effective stimulators”) under anesthesia, compared to 5/14 (35.7%) in the awake state. When stimulating L4 neurons, we recorded 3/14 (21.4%) effective stimulators. (B) Left: Blue dots represent data from all L2/3 anesthetized recordings, red from L2/3 awake recordings, and green from L4 awake recordings. The average percentage of follower cells per recording is low regardless of layer and brain state (0.56–0.75%). Right: Same conventions but considering only data sets which had at least one significant follower. Error bars represent standard error of the mean. (C) Black dots: Mean z-scores and error bars (sem) of simulated datasets as a function of the probability of eliciting one AP per stimulation epoch. Blue Dot: Mean and sem of the mean z-score across the excited followers from all real stimulation experiments (including L2/3 and L4 stimulation), showing that on average, the statistical significance of identified follower cells is similar to cells with simulated excitation in 50% of trials. (D) Black dots: Mean (± sem) ΔF/F-values of “relevant” (trials that contribute to significance: see section Methods) trials of all simulated cells as a function of percent simulated trials. Blue Dot: Mean (± sem) ΔF/F-value of “relevant” trials across all excited followers including L2/3 and L4 stimulation. The ΔF/F response of excited followers was similar to simulated followers when an AP was added in 50% of their trials.
Figure 4
Figure 4
(A) For each FOV, we averaged the z-scores of all cells, for both sham experiments (n = 19) and real stimulation experiments (n = 47). Data from L2/3 anesthetized, L2/3 awake and L4 awake stimulation experiments were pooled together, because there were no significant differences between them. Box plots show the median (central horizontal line), 25th and 75th percentiles (upper and lower box boundary), and 5 and 95th percentiles (bottom, top whiskers, respectively). Medians were significantly different (p = 0.025, Wilcoxon ranksum test). (B) Percentage of followers per FOV as a function of the median z-score of all cells in each FOV. Red circles: percent of excited followers. Blue circles: percent inhibited followers. Gray Circles: datasets without followers. Star: A single dataset with both inhibited and excited followers. The population influence of a stimulated cell generally matched the type of followers present in the respective FOV.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Beltramo R., D'urso G., Dal Maschio M., Farisello P., Bovetti S., Clovis Y., et al. . (2013). Layer-specific excitatory circuits differentially control recurrent network dynamics in the neocortex. Nat. Neurosci. 16, 227–234. 10.1038/nn.3306 - DOI - PubMed
    1. Brecht M., Schneider M., Sakmann B., Margrie T. W. (2004). Whisker movements evoked by stimulation of single pyramidal cells in rat motor cortex. Nature 427, 704–710. 10.1038/nature02266 - DOI - PubMed
    1. Carillo-Reid L., Yang W., Bando Y., Peterka D. S., Yuste R. (2016). Imprinting and recalling cortical ensembles. Science 353, 691–694. 10.1126/science.aaf7560 - DOI - PMC - PubMed
    1. Chao H. T., Chen H., Samaco R. C., Xue M., Chahrour M., Yoo J., et al. . (2010). Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes. Nature 468, 263–269. 10.1038/nature09582 - DOI - PMC - PubMed
    1. Clopath C., Bonhoeffer T., Hüebener M., Rose T. (2017). Variance and invariance of neuronal long-term representations. Philos. Trans. R. Soc. Lond. B Biol. Sci. 372:20160161 10.1098/rstb.2016.0161 - DOI - PMC - PubMed

Publication types

LinkOut - more resources