It requires more than a quick look at the abstract and the figures to fully understand a research paper and its limitations. One way to get there is to write a summary or critical review of a paper. In a contribution to (informal) post-publication review, I will select neuroscience papers that are, in my opinion, worth the time needed to write up a review. other reviews: On precise balance in the hippocampus ; on simulations of calcium imaging datasets .
Simplified scheme of a L5 pyramidal neuron. From Wikipedia, used under CC BY 4.0 license (modified).
What are the papers about? The mammalian cortex is a layered structure that harbors different neuronal types in different layers. One of the most prevalent and most fascinating neuronal types is the layer 5 (L5) pyramidal neuron (schematic drawing above). While the cell body resides deep in layer 5, several hundred micrometers below the surface, it sends a so-called ‘apical trunk’ dendrite up to superficial layers, where it branches into a so-called ‘apical tuft’. The apical tuft receives input which is fundamentally different from input received at the soma. This, together with the long distance between apical tuft and soma, poses a simple, but difficult-to-answer question: how tightly are electrical activity at the soma, the apical trunk and the apical tuft coupled, and how do the electrical compartments interact? The two papers address exactly this question. – Their main finding is that calcium activity at the soma and the apical dendrite are very tightly coupled, no matter what happens to the mouse.
Why this question is important: Six months ago, I wrote a blog post which explained why it would be so interesting to see uncoupled activity of soma and the apical dendrite. Several studies reported – using indirect methods – that there was strong uncoupling happening during sleep or motor learning, but the few studies working on this topic that used direct methods have rather shown tight coupling between apical and somatic activity (Helmchen et al., 1999; Kerlin et al., 2018), with only few outlier events.
These two papers try to address this question again, in a different brain region, in various behavioral conditions, and probably more systematically and in a more targeted way than previous studies.
The method: Both teams performed simultaneous calcium imaging in two optical layers from L5 neurons in the primary visual cortex (V1) of transgenic GCaMP6 mice. The two layers covered either soma and apical trunk, or apical trunk and apical tuft.
Calcium does only partially reflect the electric events that happen in the respective location. Calcium events are an imperfect, non-linear and supra-threshold mirror of electrical activity, and, in addition, there are calcium events independent of electrical activity. Still, calcium imaging is the best available method to investigate somato-dendritic coupling, since theoretically better-suited methods (dendritic patching, voltage imaging) do not work reliably in awake animals for now.
I also would like to highlight the section “Limitations of the use of calcium imaging to assess dendritic activity in awake behaving mice” from the Francioni and Rochefort paper, which discusses in a bit more detail the challenges for dendritic calcium imaging. I think it is a very good idea to include this part in the main paper and not in the methods section, because this is an experiment that requires careful analysis due to motion artifacts, unwanted signal from spines or axonal boutons, imperfect calcium indicators and other problems.
Paper 1 (Harnett lab): The main result of the Beaulieu-Laroche et al. paper is that somatic activity and activity measured in the distal apical trunk (distance between dendritic and somatic regions: ca. 350 μm) are strongly correlated in V1. One would expect that this tight coupling becomes loose when the dendritic tree is bombarded by more input. Surprisingly, however, they find that this tight coupling is independent of visual stimulation or whether the mouse is running or sitting still.
I haven’t talked to the authors, but from this exploratory study design, which tested different conditions (visual input vs. no black screen, running vs. pausing), I would guess that they were hoping to see a behavioral regime where the tight somato-dendritic coupling breaks down, due to strong excitatory apical inputs, due to a modulation of the leakiness of the apical trunk, due to shunting inhibition or another weird phenomenon on the molecular level. The fact that nothing of this happens seems a bit disappointing from the perspective of the study director.
To better understand these calcium imaging measurements, the authors performed calibration experiments in vitro (slices), where they measured both calcium signals in somata and dendrites and patched the respective locations with micropipettes to record the electrical signals. Somehow, the whole-cell recordings did not wash out the GCaMP from the neurons (Fig. S3), which was a bit surprising to me. Using these slice experiments, they conclude that the high somato-dendritic correlations are either due to burst-firing somata that trigger (active) dendritic electrogenesis, or due to dendritic spikes that elicit somatic firing. The authors are unfortunately unable to discern these two possibilities.
To make things a bit more complex, a side-figure shows that single L5 spikes are not seen using the GCaMP6f reporter (but are seen using an OGB-1 calcium dye, see Fig. S3G,H). The authors find that single somatic L5 spikes also do not trigger dendritic electrogenesis, which seems to explain that the calcium signal is correlated also in this regime of single spikes. Indirectly, this suggests that there is no tight coupling of calcium activity between soma and apical dendrite if there is only one somatic spike.
Paper 2 (Rochefort lab): Francioni and Rochefort perform similar calcium imaging experiments. Due to technical reasons (using a piezo instead of a tunable lens), their two imaging planes are closer to each other (<200 μm). As a main result, the authors come to the conclusion that calcium activity is highly correlated not only between soma and (proximal) apical trunk, but also between the proximal and distal trunk and between distal trunk and the apical tuft; and also between processes within a single apical tuft. Including not only the apical trunk but also the apical tuft is an important extension compared to the paper from the Harnett lab. As another difference, they use for some of their experiments GCaMP6s instead of GCaMP6f to have a more sensitive readout of somatic activity.
Very similar to Beaulieu-Laroche et al., Francioni and Rochefort report that this tight somato-dendritic coupling is not affected by visual stimulation or by locomotion, also for the apical tuft. Actually, they report that “less than 3% of the total number of transients” were branch-specific, and that those few events were mostly low-amplitude signals.
The authors then analyze the data more carefully and find that quite some calcium events that can be seen in the soma do not propagate into the apical trunk, and many events in the apical trunk do not propagate into the apical tuft. They quantify the overall loss from somatic activity to the dendritic tuft to ca. 40%. In my opinion, this suggests that most of the calcium activity is generated first in the soma and then propagates into the apical compartment, where it is (according to Beaulieu-Laroche et al.) amplified by active conductances, but not always (as seen by Francioni and Rochefort). One remaining question is why the attenuation from soma to apical trunk has not also been observed by Beaulieu-Laroche et al., but this is probably due to technical reasons, for example the calcium indicator.
Consequences for understanding the role of apical dendrites: During the last 20 years, many people, in particular neuroscientists coming from slice physiology or from theory, have speculated about active roles of dendrites and the computational independence of dendritic segments. Although these two studies (hopefully) are only the beginning of more detailed research covering other brain regions and other behavioral conditions with similar methodological approaches, the findings seem to dampen the enthusiasm about dendritic processing a bit.
Francioni and Rochefort make it very clear, that “this result is consistent with two possible mechanisms: either (1) global apical tuft activation systematically triggers somatic action potentials, or (2) global tuft calcium transients are triggered by back-propagating action potentials alone or in conjunction with tuft synaptic inputs.”
Both options would be possible. In my opinion, however, their paper points rather towards the second option. First, they see that 40% of somatic events do not make it to the apical tuft; these are very likely back-propagating action potentials that get stuck in the middle of their journey in the apical dendrite. Second, given that they do not see the other phenomenon prominently (dendritic events that do not make it to the soma, <3% of events), it seems very likely that a very large fraction of all dendritic events are evoked by back-propagating action potentials.
This suggests a (to-be-confirmed) picture of L5 pyramidal neurons that are driven by basal instead of apical inputs. This is consistent with recent experiments that micro-dissected the dendritic tree, although of L2/3 neurons (Park, Papoutsi et al., bioRxiv, 2019). Somatic spikes would then propagate into the apical tree and, if elicited at high-enough frequency, recruit active dendritic sodium and calcium channels. The activation of the dendritic conductances could in addition depend on local apical input and play several roles, for example: 1) enhancement of the somatic spikes (bursting), 2) triggering of synaptic weight changes in the apical dendrite, 3) triggering of transmitter release to signal from apical locations to their respective pre-synapse. However, it would be unlikely that apical input by itself generates a calcium spike that activates the soma.
Conclusion: The studies by Francioni and Rochefort and Beaulieu-Laroche et al. contribute a lot to a better understanding of the function of apical dendrites of L5 pyramidal neurons. Even though the main result might seem like a partial disappointment at first glance, this kind of systematic study (instead of a study strongly driven by an hypothesis) seems to be the right thing to me. And more of the same, with different behavioral conditions, other cortical regions and possibly a more detailed analysis of the rare dendritic events seems to be the way to go. These two very interesting studies provide important information about somato-dendritic coupling, but the apical dendrite of L5 neurons still largely remains mysterious.