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

The piriform cortex (PCx) is essential for learning of odor information. The current view postulates 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 few local NMDA-spikes delivered at theta frequency, while global spike timing dependent plasticity protocols (STDP) failed to induce LTP in these distal synapses. An inverse result was observed for more proximal apical IC synapses; they undergo plasticity with STDP but are refractive to local NMDA-spike protocols. These results are consistent with a self-potentiating mechanism of odor information via NMDA-spikes which can form branch-specific memory traces of odors.


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The piriform cortex (PCx) is a main cortical station in olfactory processing, receiving direct odor 21 information from the olfactory bulb via the lateral olfactory tract (LOT) as well as higher brain regions 22 and is thought to be important for odor discrimination and recognition. Pyramidal neurons in the PCx 23 serve as the main integration units within which the discrete molecular information channels are 24 hypothesized to be recombined to form an "odor object". PCx pyramidal neurons receive two spatially 25 distinct inputs which terminate on different compartments of their apical dendrites: first, the direct 26 afferent excitatory inputs from olfactory bulb (OB) via the LOT, which terminate mainly on distal 27 apical dendrites (layer Ia). Second, intracortical excitatory inputs (IC) from local PCx neurons and 28 other cortical areas, which target the more proximal portions of the apical dendritic tree (layers Ib and 29 II) and basal dendrites (Bekkers and Suzuki, 2013;Haberly, 1985;Haberly, 2001;Haberly and Price, 30 1977; Isaacson, 2010; Neville and Haberly, 2004;Suzuki and Bekkers, 2006). 31 Synaptic plasticity rules have crucial role in determining the way cortical networks acquire, organize 32 and store information. It is postulated that PCx mediates learning and recall of olfactory information 33 (Franks and Isaacson, 2005;Haberly, 1985;Haberly, 2001;Saar et al., 1998Saar et al., , 2001. Previous studies 34 have shown that similar to neocortical and hippocampal pyramidal neurons (Bliss and Collingridge,35 1993; Feldman, 2012; Sjostrom et al., 2007), IC synapses of PCx pyramidal neurons undergo plasticity 36 changes. NMDA-R dependent long-term potentiation (LTP) was robustly observed in IC synapses 37 both when stimulated by theta rhythm alone (Haberly et al., 1994), or when paired with either a burst 38 of LOT activation Haberly, 1990, 1993;Kanter et al., 1996), or back-propagating action 39 potentials using spike timing dependent protocols (STDP) (Johenning et al., 2009). In line with the 40 importance of IC plasticity changes in learning, it was shown that following olfactory rule learning in-41 vivo, PCx pyramidal neurons exhibited increase in IC synaptic strength and excitability (Lebel et al., Lisman and Spruston, 2010;Sandler et al., 2016;Sjostrom and Hausser, 2006;Sjostrom et al., 2008). 82 In this work we tested both global spike timing dependent plasticity protocols as well as local 83 induction protocols mediated by dendritic NMDA-spikes (Kumar et al., 2018). 84 Location dependent STDP in apical and basal dendrites of PCx pyramidal neurons. 85 To directly test the location dependent capability of PCx dendrites to undergo LTP with an STDP 86 protocol, we performed whole-cell patch-clamp voltage recordings from the soma of layer II pyramidal 87 neurons as determined from the Dodt contrast image and somatic firing pattern (Suzuki and Bekkers,88 2006). Neurons were loaded with calcium sensitive dye OGB-1 (200 µM) and CF633 (200 µM) to 89 enable visualization of dendrites using a confocal microscope. We used focal synaptic stimulation to 90 activate synaptic inputs in distal LOT (299 ± 6.29 µm from soma) synapses located in layer Ia ( Figure   91 1A) or more proximally (139.33 ± 5.15 µm from soma) in layer Ib ( Figure 1E Figure 1B), this STDP protocol also failed to potentiate distally located layer Ia inputs (104.99 ± 103 3.86% of control; p = 0.7577, n=3). In sharp contrast, using the same protocol but activating IC instead 104 of LOT synapses at more proximal apical dendritic locations induced robust potentiation of layer Ib 105 inputs, (Figure 1F and 1G; 168.93 ± 6.54% of control EPSP; p = 0.01387, n = 6).

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Thus, the STDP burst protocol is efficient in inducing robust LTP in IC inputs both at proximal apical 115 locations and basal synapses but fails altogether to induce LTP at LOT synapses located on distal 116 apical dendrites.

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Local NMDA-spikes induce LTP of LOT inputs in distal dendrites of PCx pyramidal neurons, 118 but fail to potentiate proximal IC inputs. 119 We recently reported the initiation of local NMDA-spikes in distal apical dendrites of PCx pyramidal    To test this possibility, we focally stimulated distal basal dendrites using visually positioned theta 169 electrode placed at single basal dendrites with an average distance of 144.83 ± 9.21 µm from the soma 170 ( Figure 7A). Similar to apical dendrites, dendritic NMDA-spikes were evoked in basal dendrites of 171 PCx neurons ( Figure 7B). The average spike threshold recorded at the soma was 10.2 ± 1.6 mV. The 172 dendritic spike amplitude and area as measured at the soma was 27.2 ± 2.5 mV and 2999.5 ± 434.7 173 mV*ms respectively (n = 6 cells). 174 Next, we tested if these NMDA-spikes can mediate potentiation of basal dendrite inputs. Using the 175 same NMDA-spike induction protocol used for apical dendrites significantly potentiated the IC 176 synapses on basal dendrites ( Figure 7C; 140.64 ± 4.5 % of the control; p = 0.006526, n = 6 cells).

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However, the degree of potentiation was smaller compared to that of LOT inputs in distal apical 178 dendrites ( Figure 7D). EPSPs which were sub-threshold for NMDA-spikes failed to induce significant 179 potentiation of the EPSPs (108.73 ± 3.13 % of the control EPSP, p = 0.8253 n = 5; Figure 7F and   Learning mechanisms in piriform cortex. 254 The anatomical arrangement of the olfactory bulb LOT inputs is such that these inputs terminate ). The specificity to odor is enabled by strengthening the connectivity only between neurons that 263 respond to the same set of LOT inputs. Upon exposure to the odor these neurons will fire, and as a 264 result the connectivity between these neurons will be strengthened by associative LTP mechanisms. 265 Upon repeated exposures to the odor, a stronger and more robust activation of this odor-specific 266 neuronal ensemble will take place due to the potentiated IC recurrent connections (Haberly, 2001).

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Experimental evidence supports the strengthening of IC inputs by an associative LTP mechanism, 268 where IC and LOT inputs are co-activated (Johenning et al., 2009;Kanter and Haberly, 1993). In                           No significant change in EPSP amplitudes was observed, 108.73 ± 3.13 % of control (p = 0.825, n= 5).

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In the box plots, the grey dots represent average EPSPs of each experiment and the diamond represents   Committee.

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Acute coronal brain slices 300 µm thick, were prepared from the anterior piriform cortex of Wistar rats 460 (male and female) 4-6 weeks old or mice 7-12 weeks old. The entire brain was removed and placed in injections were made through the thinned skull using a hydraulic micromanipulator (M0-10 Narishige).

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Following the surgery, Ketoprofen and Buprenorphine were administered to the animal for 2 509 consecutive days. The animals were then returned to their home cage for a period of minimum 3 weeks 510 to ensure full recovery from the surgery and expression of the injected virus.

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Long-term plasticity induction protocols 512 Control EPSPs were acquired at 0.033 Hz before (10-15 minutes) and after the induction protocol.

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Stability of the recording was ensured by monitoring the resting membrane potential and only 514 experiments in which the membrane potential was not changed more than 3 mV were included. Spike  All relevant data are included in the paper and/or its supplementary information files. Additional data 541 will be made available from the corresponding author upon reasonable request.