Associative cortex features in the first olfactory brain relay station

Abstract

Synchronized firing of mitral cells (MCs) in the olfactory bulb (OB) has been hypothesized to help bind information together in olfactory cortex (OC). In this survey of synchronized firing by suspected MCs in awake, behaving vertebrates, we find the surprising result that synchronized firing conveys information on odor value ("Is it rewarded?") rather than odor identity ("What is the odor?"). We observed that as mice learned to discriminate between odors, synchronous firing responses to the rewarded and unrewarded odors became divergent. Furthermore, adrenergic blockage decreases the magnitude of odor divergence of synchronous trains, suggesting that MCs contribute to decision-making through adrenergic-modulated synchronized firing. Thus, in the olfactory system information on stimulus reward is found in MCs one synapse away from the sensory neuron.

Figure 1

(A) Saggital MRI of the mouse's head showing the location of the electrodes. The inset is a Nissil stained saggital section of an adult mouse's OB. (Bi) Time course for trials in the odor discrimination task (Slotnick and Restrepo, 2005). When the mouse inserts its head into the odor chamber, an odor valve (OV) opens directing the odor into the air stream and simultaneously a final valve (FV) opens directing the air stream to exhaust (“OV+FV on”). At time zero FV turns off (“FV off”) eliciting an abrupt odor onset at approximately 0.3 sec measured with a PID (see Methods). The animal must lick for the rewarded odor on the water delivery tube at least once for four 0.5 sec periods (blue blocks) in the response area (RA). The red bar shows the range of decision times (Doucette and Restrepo, 2008). If the animal licks correctly it receives water during a Water Reward (WR) period. (Bii) Sniffing behavior during the odor discrimination task. Mice increase sniff frequency in anticipation of odor presentation, and decrease sniff frequency steadily during odor presentation. As the decision is made, sniff frequency is reduced to basal levels for correct rejections and below basal levels following the water reward for hits (rewarded trials) (11 sessions across 5 animals, significant difference starting at 1.68 seconds post FV off, ranksum test p<0.05). Data is displayed as mean ± SEM. (C) Percent correct responses as a function of the block number (20 trials per block, 10 rewarded and 10 unrewarded). The mice learn to refrain from licking to the unrewarded odor.

Figure 2

Precise synchronized firing between SMCs. (A) Scatterplots for three single-units recorded from electrodes 5, 1 and 2 (spike shapes shown on left and microelectrode layout shown on lower right). Spikes synchronized within <250 μsec between units 5 and 1: red (asterisk), between 5 and 2: black (asterisk). (B) 1 to 3. Lag histograms. The y-axis shows the number of spikes per trial in the reference unit that lag by the time delay denoted by each bin when compared to spikes in the partner unit. (B1) and (B2) for the units in channels 5 and 1 (1.8% of spikes are synchronized). (B3) histogram for one single-unit and a multi-unit (6.4% synchronized spikes). Red lines are lag histograms calculated after spikes were shuffled randomly ± one mean interspike interval. (B4) Average autocorrelogram for the synchronized spike trains (from 2857 multi-unit pairs in the RA). An autocorrelogram calculated after shifting the reference unit spikes by a random time within ± one interspike interval was subtracted from the data and the result was normalized by dividing by the shifted autocorrelogram. The number of animals in this study is 8, the number of units recorded from was 345 (SU) and 820 (MU), and the number of pairs recorded from was 578 SU×SU, 1620 MU×SU and SU×MU and 4391 MU×MU. Recording was performed in 67 sessions (39 first day and 28 reversals).

Figure 3

(Ai) Scatterplot for synchronized spike firing for all blocks in a session. Each block has 20 trials (10 rewarded and 10 unrewarded). As shown, the synchronized trains develop an excitatory response to the rewarded odor and an inhibitory response to the unrewarded odor. (Aii) Side-by-side comparison of the synchronized spike firing in hit trials in blocks 1-2 and 7-8. (B) Odor-induced change in rate of synchronized firing for the data in A. Firing rate was calculated as the firing rate in the RA minus rate in the previous 2 seconds. Rewarded red and unrewarded blue, mean±SEM, n=10 trials (*significant difference, P<0.05, corrected for multiple comparisons by FDR). (C) Behavioral percent correct responses for the same session.

Figure 4

Odor responses of spikes synchronized between two multi-units. Spike trains are shown for one block of the session where the mice learned to differentiate between odors A (rewarded) and AB (unrewarded) (Figure 4A) and in a second session where the odors were reversed for reward (AB rewarded, A unrewarded) (Figure 4B). Synchronized spikes were those firing in both units within < 250 μsec and the block chosen was the best block where the odor responses to the two odors were most divergent. (Ai) synchronized spike trains for divergent odor responses in best block (trials: 10 rewarded, 10 unrewarded, red bar is the RA -0.5 to 2.5 sec- for the rewarded odor). (Aii) z-score cumulative histogram in best block for 68 spike trains for multi-unit odor divergent responses (broken lines) and for 48 odor divergent synchronized spike trains. Z-score was calculated as the RA firing rate minus the rate for the 2 seconds preceding the RA divided by the standard deviation of the rate in the preceding interval. The magnitude of the difference in z-score between rewarded and unrewarded odor did not correlate with when the best (most divergent) block occurred (correlation coefficient of 0.05, p-value of 0.76). Synchronized spike trains (Bi) and z-score cumulative histogram (Bii) for odor reversal session including 31 multi-units (broken lines) and 6 multi-unit synchronized pairs (solid lines, n=6) (odor A –blue-unrewarded and odor AB –red-rewarded). (C) p-value for a ranksum test reporting on the difference in the Euclidean distance between rewarded and unrewarded odor responses in principal component (PC) space. PC analysis was calculated for the time course of synchronized spike firing in multi-units in all trials within the best block (see Figure S3 for the results of the PC analysis). The response to the rewarded and unrewarded odors diverges at ∼1 sec. The animal makes a decision to stop licking for the unrewarded odor at ∼1.25 sec (blue line, determined by a ranksum test of the difference in licks in best blocks with > 85% correct responses).

Figure 5

Cumulative histograms of the responses of synchronized firing of pairs of multi-units to odors in the odor discrimination task shown separately for trials where the animal makes the correct behavioral decision (hits, blue and correct rejections, CR, black) or an incorrect decision (false alarm in green and miss in red). Responsiveness was calculated on a trial-per-trial basis in divergent blocks that included at least one mistake as a z-score defined in the Methods. A positive z-score indicates that the synchronized firing rate increased upon exposure to the odor. An ANOVA with a post-hoc test indicated that the z-scores for miss were not different from the z-scores for hits and that the responses from false alarms did not differ from correct rejections. There was a significant difference between hits/misses and correct rejections/false alarms. In order to ensure the incorrect trials mirrored the correct trials we also did an ANOVA where we only included false alarms where the animal licks for 80% or more of the time in the 2 sec response area and misses where the animal licked less than 20% of the time in the response area. The ANOVA test yielded the same differences/lack of differences between hits, misses, correct rejections and false alarms. The number of trials included are: 1,431 hits, 193 misses, 1,219 correct rejections and 378 false alarms.

Figure 6

(A) Odor responsiveness of synchronized spike trains for unit pairs was recorded during the odor discrimination behavioral task (as in Figures 3 and 4). This panel shows as a function of distance between recording electrodes the percent of unit pairs whose synchronized spike trains were responsive to odors (red) and the percent of unit pairs whose synchronized spike trains were differentially responsive to the rewarded and unrewarded odors (blue). The number of pairs used to calculate percent values at each distance are (in order of ascending distance): 742, 380, 143 and 35. (B) Percent of synchronized spikes in a reference unit shown as a function of distance between the electrode recording the reference unit spike train and the electrode recording the partner unit spike train (only MU×MU pairs were included). The values shown are the mean of percent of spikes that are synchronized ± standard deviation. The number of pairs used to calculate each point are (in the order of ascending distance) 1498, 790, 280 and 64.

Figure 7

Adrenergic blockade. (A) z-score for 20 unit odor responses that were significantly divergent (broken lines) and for 14 odor divergent synchronized pairs (solid lines) (red: rewarded, blue: unrewarded, calculated in the best block). (B) Percent odor divergent synchronized pairs with a significant difference between rewarded and unrewarded trials in the percent of synchronized spikes. p-values: * p=0.016, **p=0.0016 (Chi-Squared test, n= 48 control, 14 adrenergic). The rest of the divergent synchronized pairs changed synchronized firing due solely to an increase in firing rate of the reference unit. (C) d' calculated as the difference in z-score between rewarded and unrewarded unit odor responses. The inset illustrates how d' was calculated (red: control, green: adrenergic block). (D) d' for synchronized spike trains. The two histograms do not differ in C (p>0.05 in K-S test), but differ in D (p= 0.01). All synchronized pairs were from multi-units.