A major role for intracortical circuits in the strength and tuning of odor-evoked excitation in olfactory cortex

Abstract

In primary sensory cortices, there are two main sources of excitation: afferent sensory input relayed from the periphery and recurrent intracortical input. Untangling the functional roles of these two excitatory pathways is fundamental for understanding how cortical neurons process sensory stimuli. Odor representations in the primary olfactory (piriform) cortex depend on excitatory sensory afferents from the olfactory bulb. However, piriform cortex pyramidal cells also receive dense intracortical excitatory connections, and the relative contribution of these two pathways to odor responses is unclear. Using a combination of in vivo whole-cell voltage-clamp recording and selective synaptic silencing, we show that the recruitment of intracortical input, rather than olfactory bulb input, largely determines the strength of odor-evoked excitatory synaptic transmission in rat piriform cortical neurons. Furthermore, we find that intracortical synapses dominate odor-evoked excitatory transmission in broadly tuned neurons, whereas bulbar synapses dominate excitatory synaptic responses in more narrowly tuned neurons.

Figure 1

Selective silencing of intracortical excitation in rat piriform cortex. A, Baclofen abolishes ASSN-mediated synaptic transmission but has no effect on LOT-mediated EPSPs. (A₁) Recording schematic. (A₂) LOT-mediated fEPSPs exhibit paired-pulse facilitation, whereas ASSN-mediated fEPSPs weakly depress. Cortical baclofen (500 µM) application abolishes the ASSN-mediated fEPSP, while the LOT-mediated fEPSP is unaffected. (A₃) Summary (n=4) of LOT (●) and ASSN (○) fEPSPs in response to baclofen and subsequent application of NBQX (50 µM). B, Baclofen abolishes polysynaptic EPSCs and IPSCs but has no effect on monosynaptic LOT EPSCs. (B₁) Recording schematic. (B₂) EPSCs (−80 mV) and IPSCs (+10 mV) evoked by LOT stimulation in the same cell before and during baclofen application. Dotted traces from control are superimposed. Grey boxes, amplitude measurement regions plotted in (B₃) for IPSC (○) and monosynaptic EPSC (●). C, Baclofen consistently blocks odor-evoked IPSCs but has variable effects on EPSCs and the suppression of odor-evoked responses is reversed by subsequent application of a GABA_B receptor antagonist. (C₁) Recording schematic. (C₂) Traces from one cell in response to odor presentation (2 s, amyl acetate). Baclofen abolished odor-evoked IPSCs, while EPSCs were only partially blocked. (C₃) Fraction of the odor-evoked EPSC and IPSC blocked by baclofen for 27 odor-cell pairs (n=7 cells). (C₄) Traces of odor (cineole)-evoked currents in one cell under control conditions (black), in the presence of baclofen (red), and following subsequent application of the antagonist CGP55845 (CGP, 10 µM, green). (C₅) Summary of the effects of baclofen (BAC) and CGP rescue on odor-evoked EPSC and IPSC charge normalized to control conditions (CON, n=11 odor-cell pairs, 3 cells).

Figure 2

Contribution of intracortical (ASSN) and sensory afferent (LOT) inputs to odor-evoked excitation in piriform cortex pyramidal cells. A, The strength of odor-evoked excitation is positively correlated with the recruitment of intracortical input. (A₁) Baclofen-sensitive charge (ASSN component) vs. total EPSC charge for all odor-cell pairs with correlation (r) and p value. (A₂) Fraction of baclofen-sensitive charge (ASSN component) vs. total EPSC charge for all odor-cell pairs. B, Baclofen-insensitive charge (LOT component) is not correlated with odor-evoked excitation (total EPSC charge). C, Within each cell, baclofen-sensitive (ASSN) excitatory responses are not correlated with the baclofen-insensitive (LOT) component. Each color represents odor-cell pairs from an individual cell (n=4 cells that responded to ≥4/8 odors; Spearman’s correlation p>0.05 for all cells).

Figure 3

Silencing intracortical inputs has different effects on odor-evoked excitation depending on the EPSC tuning properties of individual cells. A, Baclofen has weak effects on odor-evoked EPSCs in cells that receive selective excitation. Anatomical reconstruction (A₁) and synaptic responses (A₂) of a representative pyramidal cell that received broadly-tuned odor-evoked inhibition (IPSCs, +10 mV) and selective excitation (EPSCs, −80 mV). 1/8 odorants elicited excitation under control conditions (black) that was largely unaffected by baclofen (red). For display purposes, only 4 odor responses are shown. ○, significant odor response, ⊘, lack of response (see Experimental Procedures). Scale bars: 50 pA for IPSCs, 25 pA for EPSCs. B, Baclofen strongly blocks odor-evoked EPSCs in cells receiving broadly-tuned excitation. Anatomy (B₁) and synaptic responses (B₂) of a representative cell that received widespread inhibition and broadly-tuned excitation. 7/8 odorants elicited excitation under control conditions (4 odors displayed). Same scale as A₂. Odors 1–4: cineole, amyl acetate, R-limonene, phenylethyl alcohol.

Figure 4

Intracortical inputs underlie broadly-tuned odor-evoked excitation in piriform cortex. A, Broadly-tuned cells receive stronger intracortical input than selectively responding cells. (A₁) A strong correlation between EPSC tuning and the baclofen-sensitive charge (ASSN) component of EPSCs across all odor-cell pairs. (A₂) The baclofen-sensitive (ASSN) fraction of odor-evoked excitation displays a similar strong correlation with EPSC tuning. B, The baclofen-insensitive (LOT) charge component of odor-evoked excitation in the same cells is not correlated with EPSC tuning. C, The strength of odor-evoked excitation (total charge) is positively correlated with EPSC tuning. A–C, n=27 odor-cell pairs, 7 cells. D, Blocking intracortical input increases the selectivity of odor-evoked excitation for broadly-tuned neurons, while narrowly-tuned cells are unaffected.