Plasticity of olfactory bulb inputs mediated by dendritic NMDA-spikes in rodent piriform cortex

  1. Amit Kumar
  2. Edi Barkai
  3. Jackie Schiller

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    Evaluation Summary:

    This manuscript investigates the plastic properties of synapses impinging on pyramidal neurons in the piriform cortex from the lateral olfactory tract (LOT) and intracortical inputs. Their findings uncover some of the location and pathway-dependent plasticity rules and challenge the notion that LOT inputs (carrying direct odor information from the bulb) become "hardwired" after the critical period. The results provide novel information about how activity and experience alter synaptic communication in the olfactory circuit in a synapse-type specific manner.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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Abstract

The piriform cortex (PCx) is essential for learning of odor information. The current view postulates that odor learning in the PCx is mainly due to plasticity in intracortical (IC) synapses, while odor information from the olfactory bulb carried via the lateral olfactory tract (LOT) is ‘hardwired.’ Here, we revisit this notion by studying location- and pathway-dependent plasticity rules. We find that in contrast to the prevailing view, synaptic and optogenetically activated LOT synapses undergo strong and robust long-term potentiation (LTP) mediated by only a few local NMDA-spikes delivered at theta frequency, while global spike timing-dependent plasticity (STDP) protocols failed to induce LTP in these distal synapses. In contrast, IC synapses in apical and basal dendrites undergo plasticity with both NMDA-spikes and STDP protocols but to a smaller extent compared with LOT synapses. These results are consistent with a self-potentiating mechanism of odor information via NMDA-spikes that can form branch-specific memory traces of odors that can further associate with contextual IC information via STDP mechanisms to provide cognitive and emotional value to odors.

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  1. Version published to 10.7554/elife.70383 on eLife
  2. eLife

    Author Response:

    **Reviewer #3 (Public Review): **

    [...] This work challenges the notion that LOT inputs - inputs responsible for carrying information from the olfactory bulb as well as higher brain regions and thought to be important for odor recognition - onto pyramidal neurons become "hardwired" (LOT synapse stability in adulthood) after the olfactory critical period. Importantly, their data clearly demonstrate that LOT inputs onto distal apical dendrites can undergo LTP when these inputs are co-activated with other LOT inputs capable to generate local NMDA-spikes. These data help to reconcile seemingly conflicting previous findings.

    Weaknesses:

    We thank the reviewer for his important comments. Below are our answers point by point.

    Major issues:

    – Novelty: 1) It is well established that postsynaptic depolarizations given by dendritic spikes (i.e. NMDA-spikes) can trigger LTP in several cell types (i.e. Major et al., 2013; Golding et al, 2012; Remy and Spruston, 2007; Gambino et al., 2014). 2) The finding that LOT inputs onto distal apical layer 2 pyramidal neuron dendrites from PCx do not trigger LTP when global STDP protocols are used corroborates previously published findings [Johenning et al., 2009]. 2) The demonstration that IC inputs trigger LTP via global STDP protocols in proximal distal dendrites also corroborates previous findings [Johenning et al., 2009].

    We agree with the reviewer that NMDA-spike evoked potentiation was described before including by our group (Gordon et al. 2006). The main claim of the manuscript concerns an erroneous dogma with regard to the plasticity capabilities of LOT synapses in PCx. For piriform cortex the dogma in the literature is that LOT inputs do not undergo plasticity changes in adulthood. Thus a main novelty of this manuscript is to describe for the first time that LOT synapses do undergo large and robust LTP with local NMDA-spikes. Such a focal plasticity rule can have important impacts as to how odor information is learnt and represented in pyramidal neurons of piriform cortex which we discuss in the manuscript page 11-12 lines 269-299 (in non-tracked version of the manuscript).

    – The most interesting/puzzling finding is how even applying up to 23 NMDA-spikes at 4Hz at more proximal apical locations via activation of IC synapses completely failed to induce LTP of these IC inputs. However, global STDP can effectively trigger plasticity in these synapses. Unfortunately, the authors didn't explore the mechanisms of why distally located LOT synapses can trigger strong LTP via NMDA-spikes, but those more proximally located IC synapses cannot. These mechanisms should be explored, especially since they challenged the local depolarization and calcium influx properties of LTP induction. An experiment to study the role of active conductances that can explain these puzzling results should be designed, including imaging of local calcium concentration using the same tools as those described in the Methods section (although not presented in the results section) and measurements of local voltage changes using dendritic patch recordings (a technique for which the lab is well known).

    We thank the reviewer for this comment following which we performed additional experiments to clarify the point. As suggested by reviewer # 2, NMDA-spikes in the proximal apical dendrites that served for the induction protocol of the original version of the manuscript were indeed smaller than the full-blown NMDA spikes that can be generated in proximal location (average peak amplitudes of 32.7±4.6 mV and 47.4±5.8 mV and average area under curve of 3725±461 mVms and 7741±974 mVms for small and full blown NMDA-spikes respectively; see also Kumar et al. 2018). The reason we used smaller NMDA-spikes was to avoid initiation of BAPs during the induction period. However, following the reviewer’s comment to clarify this point we did further experiments: 1. We examined the ability of full blown proximal NMDA-spikes to induce plasticity in IC synapses. A similar NMDA-spike induction protocol (4-10 NMDA-spikes at 4Hz) also induced potentiation of proximal IC synapses (127 ± 4.39 microns from soma) (Figure 6A-D), but to a smaller extent compared to distal LOT synapses and even smaller than potentiation induced by STDP protocol in these synapses. Post induction, the proximal IC EPSP amplitude was 148.48 ± 4.1% of the control (Figure 6F; p = 0.00204; n = 9; p=0.0013 for comparison of proximal versus distal NMDA-spike potentiation; p=0.0127 for comparison with STDP in IC synapses). The number of NMDA spikes needed for this potentiation was between 4-10 NMDA spike repetitions. 2. To control for the contribution of these BAPs, we repeated the induction protocol but instead of using NMDA-spikes we used pairing BAPs and local EPSPs for 5 repetitions (1 EPSP paired with 3BAPs at 150 Hz repeated 5 times at 4 Hz). In this case we did not observe potentiation of these proximal IC synapses (Figure 6G), thus we concluded NMDA-spikes were crucial for the potentiation of IC synapses with the NMDA-spike protocol. 3. We measured the local calcium transients in active spines and neighboring shafts following STDP protocol activation (pairing BAPS and EPSPs) compared to local NMDA spikes in proximal IC. Interestingly we find, calcium transients evoked by NMDA-spikes both in shafts and spines, were significantly larger than those evoked by STDP stimulation (Figure 6Eand 6H; p<0.0001) despite the degree of potentiation with STDP protocol was higher (p=0.0127) compared to the NMDA-spike protocol. This results indicate the amount of calcium entry per se is not the only variable determining the degree of potentiation. See for example Gordon et al. 2006 where we showed that in distal basal dendrites of layer 2-3 neocortical neurons BDNF was a necessary requirement to gate plasticity in addition to calcium entry.

    These new experiments were added to the revised version and are replacing the previous experiments (page 7 lines 153-168 in non-tracked version of the manuscript and new Figure 6).

    – A demonstration that NMDA-spikes can occur in vivo in the apical and basal dendrites of PNs from PCx (i.e. during odor discrimination and plasticity task) would greatly strengthen their in vitro findings indicating that LTP can be triggered in LOT-synapses and IC synapses directed to basal dendrites when driven by NMDA-spikes. This is important since LOT synaptic contacts onto distal tuft dendrites of pyramidal neurons are few (~ 200 total contacts, Miyamichi et al, 2011) and sparse (Davison and Ehlers 2011). Hence, for the reported NMDA-spike-dependent plasticity observed in vitro to be the modus operandi for plasticity and memory formation in vivo the need for a significant amount of synchronously activated LOT inputs directed to >20 clustered spines in the apical dendrites of PNs from PCx would be required according to the presented data. Or at least, provide a more extended discussion on this issue.

    The reviewer raises an important point, following which we have extended the discussion with regard to the probability of NMDA-spikes to occur in-vivo. Our calculations are based on the following estimations:

    1. Typically, pyramidal neurons from layer 2B in adult mice have 10 terminal apical branches (Moreno-Velasquesz et al. 2021).

    2. The size of the LOT band is ~ 100 microns (Bekkers and Suzuki. 2013; Moreno-Velasquesz et al. 2021).

    3. Typical number of spine density is at least 1 spine/ micron, would result in at least ~ 100 spines per single terminal branch at LOT band. This band is almost exclusively innervated by LOT axons (see our results with baclofen blockade in Kumar et al. 2018 and Bekkers and Suzuki. 2013; Giessel and Datta. 2015). Thus, per terminal branch there are ample of LOT synapses given that ~10 synapses are needed to initiate a local NMDA spike. However, a critical question relates to the statistics of LOT activation during a natural odor stimulation.

    4. Srinivasan and Stevens. 2018, estimated each piriform neuron receives ~ 0.64 synapses from one glomerulus. We assumed 110 glomeruli are activated by a typical odor, which translates to 70 synapses per neuron following (Moreno-Velasquesz et al. 2021; Srinivasan and Stevens. 2018).

    5. For the probability calculations we assumed the connectivity of LOT inputs to layer 2B pyramidal neurons is random and independent (Giessel and Datta. 2015; Moreno-Velasquesz et al. 2021). we calculated the probability that at least one terminal dendritic branch will be simultaneous activated by 10 random LOT axons (the estimated number for NMDA-spike initiation) in any given neuron to be 15% (see Figure supplemental 3).

    These estimations show that there is a fair chance NMDA spikes will be initiated in dendrites of layer 2B pyramidal neurons in the pyriform cortex following odor stimulation. It should be stressed that these calculations are based on multiple assumption that were only partially tested experimentally, and thus serve only as proof of principle that dendritic NMDA spikes can serve for odor representation in pyriform pyramidal neurons. We agree that ultimately one should validate the occurrence of NMDA-spikes in piriform cortex pyramidal neurons by recording from dendrites in-vivo, however we feel this is beyond the scope of the present manuscript and will require an extensive experimental effort which we intend to pursue in the future.

    We have added a discussion on page 12 lines 300-317 (in non-tracked version of the manuscript) along with a new Figure supplement 3 showing a graph of our calculations.

  3. eLife

    Evaluation Summary:

    This manuscript investigates the plastic properties of synapses impinging on pyramidal neurons in the piriform cortex from the lateral olfactory tract (LOT) and intracortical inputs. Their findings uncover some of the location and pathway-dependent plasticity rules and challenge the notion that LOT inputs (carrying direct odor information from the bulb) become "hardwired" after the critical period. The results provide novel information about how activity and experience alter synaptic communication in the olfactory circuit in a synapse-type specific manner.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    With this manuscript, Kumar at al. study how plasticity can be achieved at the synapses between the lateral olfactory tract (LOT) and pyramidal neurons in the piriform cortex. These pyramidal neurons receive LOT inputs distally and intracortical input more proximally. The authors use patch clamp electrophysiology and electrical stimulation or optogenetics to show that local dendritic NMDA spikes are required to induce plasticity at these synapses, whereas a protocol (spike-timing dependent plasticity, STDP) that pairs pre- and post-synaptic activity is ineffective. On the other hand, they confirm previous findings that the STDP pairing protocol induces plasticity at intracortical synapses, whereas NMDA spikes are ineffective in this case. Finally, an STDP pairing protocol and NMDA spikes, when applied separately, were both able to induce plasticity at synapses on the basal dendrites although at a lesser degree than in the apical dendrites.

    The results are clearly described in the text and figures, and support the conclusions drawn by the authors. This work should therefore clarify some discrepancies in the previous literature about plasticity of excitatory inputs to pyramidal neurons of the piriform cortex, as discussed in the paper. It also fits well with reports from other neurons that show different plasticity requirements for spatially segregated inputs arriving to a specific neuron.

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript uses whole-cell recordings and local synaptic stimulation to characterize the plasticity rules in pyramidal neurons in piriform cortex. From previous work it was typically thought that the ascending sensory inputs to piriform from olfactory bulb were plastic early during development but then became largely fixed during adulthood, with learning depending instead on changes in intracortical synapses. The authors previously showed that distal dendrites show robust NMDA-dependent dendritic spikes, a finding that also overturned previous thinking for these neurons. Here they show that even a small number of distal NMDA spikes causes high levels of plasticity that can more than double the strength of synaptic inputs from olfactory bulb. Plasticity is demonstrated in several ways via different paradigms including selective ChR2 activation of LOT inputs and glutamate uncaging. Intracortical synapses, in contrast, do not change strength with NMDA spikes but are instead enhanced by spike-timing protocols. Ascending and intracortical synapses thus follow distinct rules for engaging plasticity. The data are clearly described and the effects are robust and compelling. Overall the results add to our knowledge of how activity and experience alter synaptic communication in the olfactory circuit. The authors propose that this form of plasticity could shape cortical odor processing in distinct ways than changes in intracortical synapses.

  6. eLife

    Reviewer #3 (Public Review):

    The paper by Kumar, Barkai, and Schiller, entitled "Plasticity of olfactory bulb inputs mediated by dendritic NMDA-spikes in piriform cortex" aims to understand LTP induction in intracortical (IC) and lateral olfactory tract (LOT) synapses onto layer 2 pyramidal neurons from the piriform cortex (PCx). Their findings uncover some of the location and pathway-dependent plasticity rules and challenge the notion that LOT inputs (carrying direct odor information from the bulb) become "hardwired" after the critical period. More specifically, Kumar et al., show:

    1. LOT inputs onto distal apical (PCx) layer 2 pyramidal neuron dendrites do not trigger LTP when global STDP protocols are being used. However, strong LTP is induced when these inputs are co-activated with other LOT inputs capable of generating local NMDA-spikes delivered at theta frequency.

    2. In contrast to LOT inputs, IC inputs directed to more proximal apical dendrites trigger the opposite: locally activated NMDA-spikes fail to trigger LTP, whereas a global STDP protocol – where synaptic inputs are followed by backpropagating action potentials – can effectively trigger LTP.

    3. IC inputs in basal dendrites can potentiate both global STDP induction protocols and local NMDA-spikes

    Strengths:

    Understanding the dendritic mechanisms by which the PCx performs odor discrimination, recognition and memory are fundamental for understanding single-cell and network computations in odor processing. More specifically, this works focuses on understanding the synapses and pathways responsible for these plasticity changes, and the conclusions are clear and well-substantiated by the provided data.

    This work challenges the notion that LOT inputs - inputs responsible for carrying information from the olfactory bulb as well as higher brain regions and thought to be important for odor recognition - onto pyramidal neurons become "hardwired" (LOT synapse stability in adulthood) after the olfactory critical period. Importantly, their data clearly demonstrate that LOT inputs onto distal apical dendrites can undergo LTP when these inputs are co-activated with other LOT inputs capable to generate local NMDA-spikes. These data help to reconcile seemingly conflicting previous findings.

    Weaknesses:

    Major issues:

    – Novelty: 1) It is well established that postsynaptic depolarizations given by dendritic spikes (i.e. NMDA-spikes) can trigger LTP in several cell types (i.e. Major et al., 2013; Golding et al, 2012; Remy and Spruston, 2007; Gambino et al., 2014). 2) The finding that LOT inputs onto distal apical layer 2 pyramidal neuron dendrites from PCx do not trigger LTP when global STDP protocols are used corroborates previously published findings [Johenning et al., 2009]. 2) The demonstration that IC inputs trigger LTP via global STDP protocols in proximal distal dendrites also corroborates previous findings [Johenning et al., 2009].

    – The most interesting/puzzling finding is how even applying up to 23 NMDA-spikes at 4Hz at more proximal apical locations via activation of IC synapses completely failed to induce LTP of these IC inputs. However, global STDP can effectively trigger plasticity in these synapses. Unfortunately, the authors didn't explore the mechanisms of why distally located LOT synapses can trigger strong LTP via NMDA-spikes, but those more proximally located IC synapses cannot. These mechanisms should be explored, especially since they challenged the local depolarization and calcium influx properties of LTP induction. An experiment to study the role of active conductances that can explain these puzzling results should be designed, including imaging of local calcium concentration using the same tools as those described in the Methods section (although not presented in the results section) and measurements of local voltage changes using dendritic patch recordings (a technique for which the lab is well known).

    – A demonstration that NMDA-spikes can occur in vivo in the apical and basal dendrites of PNs from PCx (i.e. during odor discrimination and plasticity task) would greatly strengthen their in vitro findings indicating that LTP can be triggered in LOT-synapses and IC synapses directed to basal dendrites when driven by NMDA-spikes. This is important since LOT synaptic contacts onto distal tuft dendrites of pyramidal neurons are few (~ 200 total contacts, Miyamichi et al, 2011) and sparse (Davison and Ehlers 2011). Hence, for the reported NMDA-spike-dependent plasticity observed in vitro to be the modus operandi for plasticity and memory formation in vivo the need for a significant amount of synchronously activated LOT inputs directed to >20 clustered spines in the apical dendrites of PNs from PCx would be required according to the presented data. Or at least, provide a more extended discussion on this issue.

  7. Version published to 10.1101/2021.05.25.445578v1 on bioRxiv