Local inhibition modulates odor- evoked

articles

© 2002 Nature Publishing Group http://neurosci.nature.com

Local inhibition modulates odorevoked synchronization of glomerulus-specific output neurons Hong Lei, Thomas A. Christensen and John G. Hildebrand Arizona Research Laboratories, Division of Neurobiology, University of Arizona, PO Box 210077, Tucson, Arizona 85721-0077, USA Correspondence should be addressed to T.A.C. ([email protected])

Published online: 13 May 2002, DOI:10.1038/nn859 At the first stage of olfactory processing in the brain, synchronous firing across glomeruli may help to temporally bind multiple and spatially distributed input streams activated by a given odor. This hypothesis, however, has never been tested in an organism in which the odor-tuning properties of several spatially identifiable glomeruli are known. Using the sphinx moth, an insect that meets these specific criteria, we recorded odor-evoked responses simultaneously from pairs of projection neurons (PNs) innervating the same or different glomeruli in the macroglomerular complex (MGC), which is involved in processing pheromonal information. PNs that branched in the same glomerulus and were activated by the same pheromone component also showed the strongest coincident responses to each odor pulse. Glomerulus-specific PN pairs were also inhibited by the pheromone component that selectively activated PNs in the neighboring glomerulus, and about 70% of all intraglomerular pairs showed increased synchronization when stimulated with a mixture of the two odorants. Thus, when two adjacent glomeruli receive their inputs simultaneously, the temporal tuning of output from each glomerulus is enhanced by reciprocal and inhibitory interglomerular interactions.

Temporal summation of synaptic inputs can greatly increase the probability of activating target neurons in networks that process sensory information1,2. Studies in the mammalian visual and somatosensory systems have shown that synchronized spiking across a distributed ensemble of neurons may also serve to temporally bind the different features of a complex stimulus, thus reinforcing the population code that is read postsynaptically3,4. Likewise, in the olfactory systems of both vertebrates and invertebrates, temporal patterning is centrally involved in encoding chemosensory information5–9. In some cases, network oscillations modulate the patterns of synchronized spiking activity evoked by odors6–9, whereas in others, the timing of odor-evoked synchrony is neither oscillatory nor odor specific10–12. Thus the functional importance of precise timing relationships between the different neural elements that encode olfactory information is not known for any organism. In the insect antennal lobe, the structural and functional analog of the vertebrate olfactory bulb, the first synapses occur in anatomically discrete and functionally distinguishable modules called glomeruli (reviewed in ref. 13). A glomerulus is the site of convergence of numerous olfactory receptor cells (ORCs) expressing one or a few types of odorant receptors14,15, and each receptor probably recognizes only a limited number of physical determinants associated with a given odor molecule16–18. Different odorants thus activate different subsets of glomeruli, producing a specific spatial representation of each odorant at the earliest stages of processing in the brain13,18,19. Information about the specific chemical and spatiotemporal features of the odor stimulus is then transmitted in multiple, parallel channels to higher centers through glomerular PNs. GABA (γ-aminobutyric nature neuroscience • volume 5 no 6 • june 2002

acid) contributes to the precision of spike timing in olfactory PNs in both vertebrates and invertebrates20–22, and both fast and slow inhibitory postsynaptic potentials (IPSPs) from GABAergic local interneurons are prominent components of PN responses in diverse animal species (reviewed in ref. 13). In the vertebrate olfactory bulb, for example, dendrodendritic reciprocal synapses between mitral/tufted (M/T) cells and granule cells provide a basis for recurrent inhibition of M/T cell activity18,20. Numerous studies have also suggested that the tuning specificity of a glomerulus (its ‘odor contrast’ in analogy to retinal processing for vision) may be enhanced further by lateral inhibition mediated by reciprocal synaptic connections between glomeruli18,23–26. This idea, however, remains controversial on the grounds that it is not yet known which features of an olfactory stimulus provide the local contrast27. In a recent study, simultaneous extracellular recordings from pairs of M/T cells presumed to innervate different glomeruli showed synchronized firing in response to some olfactory stimuli9. In that study, pairs of M/T cells innervating the same glomerulus were not examined, and the full range of odorants represented in these glomeruli was not known9. A rigorous test for odor-evoked synchrony across olfactory glomeruli requires that the tuning characteristics of several readily identifiable glomeruli be well defined. Here we used intracellular methods to record simultaneously from pairs of PNs in the MGC of the male sphinx moth—an insect olfactory system that meets these criteria. PNs that innervated the same glomerulus and responded selectively to the same odorant (a component of the sex pheromone) showed greater syn557

articles


Table 1. Physiological features of paired MGC-PN recordings

Responses to olfactory stimuli were obtained in a total of 75 experiments involving simultaneous intracellular recordings from pairs of MGC-PNs. In 34 pairs, both neurons showed primarily excitatory responses to only one odorant; cumulus neurons responded to C15; toroid neurons responded to BAL. Pheromone-responsive pairs were then classified according to their response specificity (C15/C15, BAL/BAL or C15/BAL). Each pair of neurons was then scored for four traits associated with synchronous firing, as measured by the correlation coefficient between PNs. Expression of odor-evoked synchrony (% synchronous events relative to total spikes) is indicated by the filled triangles (large, 75–100%; medium, 50–74%; small, 15–49%). Expression of the remaining traits (% increase in correlation) is indicated by the filled circles (large, >100%; medium, 50–100%; small, 5–49%). None of the pairs showed oscillatory synchrony; six pairs showed all other traits (indicated by asterisks). None of the C15/BAL pairs showed blend-dependent effects on synchrony. n, not tested.

chronization of spike discharges as compared to PNs that innervated neighboring glomeruli and fired in response to different pheromone components. Furthermore, we found that odor-evoked synchrony between PNs from one MGC glomerulus was augmented by local inhibitory input from the neighboring MGC glomerulus encoding the other key component of the pheromone. These findings support a crucial role for interglomerular interactions in shaping the glomerular representations for odors in the brain. Although these lateral synaptic interactions serve to enhance the contrast between odor signals, the underlying mechanism differs from ‘classical’ lateral inhibition as seen in other sensory systems.

Fig. 1. PNs innervating the same glomerulus responded selectively to the same odorant and showed stimulussynchronized responses to repetitive stimulation. (a) Innervation patterns in the antennal lobe corresponding to each of the different pairs of PNs analyzed. The most easily identifiable glomeruli of the MGC are the cloud-shaped cumulus and the ring-shaped toroid, and MGC-PNs in this study innervated only one of these two structures. MGCPNs in M. sexta do not innervate any of the other, sexually isomorphic glomeruli in the antennal lobe. (b) Intracellular responses to a train of five brief (200-ms) pulses of the single pheromone components C15 (10 ng), BAL (10 ng) or the mixture of the two odorants (blend; 10 ng each). Three pairs of PNs are shown: two cumulus PNs selective for C15 (left), two toroid PNs selective for BAL (center) and one PN of each type (right). Note that PNs were depolarized by one odorant and hyperpolarized by the other. (c) Detail of the opposing responses to the two odorants. The two traces in each column show responses recorded from a single neuron in each pair. In each case, one odorant depolarized the PN, whereas the other evoked membrane hyperpolarization (asterisks) and/or stopped spike activity completely. (d) Cross correlations between three pairs of PNs that innervated the same or different glomeruli (average of five responses to the pheromone blend). The correlograms showed greater odor-evoked synchrony between PNs when both innervated the cumulus (left; r = 0.60; n = 11) or the toroid (center; r = 0.51; n = 15), but significantly less co-activity (r = 0.27) between interglomerular pairs (right; P = 0.02, Kruskal-Wallis ANOVA).

558

RESULTS Male Manduca sexta moths are attracted to a blend of two related odorants (BAL and C15; see Methods) that represent key components of the female sex pheromone 11,22. Each odorant is detected by a different population of ORCs, and the filtered information is then relayed to two principal glomeruli in the MGC, where the first steps of synaptic processing take place28–30. Our results are based on olfactory responses recorded simultaneously from 34 pairs of MGC-PNs in as many moths (Table 1). In 26 pairs, spiking activity in both PNs was triggered selectively by the same odorant (Fig. 1): the primary excitatory stimulus for these PNs was either C15 (n = 11 pairs, Fig. 1a and b, left) or BAL (n =

a

b

c d

nature neuroscience • volume 5 no 6 • june 2002

articles

Counts Counts

Counts

Synchronous spikes (% total)


Synchronized spike/ bin

Fig. 2. Time-series analysis of responses to a d repeated odor pulses recorded simultaneously 5 PN1 from pairs of PNs. (a) Raster plots show the 10 ng BAL PN2 4 responses of two toroid PNs to five consecutive PN1 x PN2 pulses of BAL. Lower raster series shows only the 3 synchronous events between the two neurons 2 1s (PN 1 × PN 2). A 5-ms bin was used, reflecting Single-pulse trials (10 ng C15) 1 ng BAL instantaneous spike frequencies in the 200 Hz b #1 1 #2 range. For some pulses, spontaneous spiking #3 0 resumed in the two PNs before the onset of the 0 0.1 0 .2 0.3 0 .4 2 Time (s) subsequent odor pulse (time course of stimulus 1 ng BAL 1 train is indicated beneath rasters). The transient PN 1 0 Trains of pulses window of synchrony between PNs was in every 3 case tightly modulated by stimulus dynamics, and 2 PN 2 the duration of each window matched the dura1 10 ng BAL 0 tion of the odor pulse. (b) Time course of PN 1 0 120 160 200 240 280 320 odorant-evoked synchrony between two cumulus 4 PNs selective for C15. Raster plots depict the PN 2 2 occurrence of PN synchrony (top) in the responses to three consecutive odor pulses, and 0 40 80 120 160 post-stimulus time histograms (PSTHs; 5-ms bins) c Response time (ms) f summarize the results across trials. The PSTHs 100 * Cumulus * * 80 illustrate the absence of periodicity in synchro60 nous events whether odor pulses were separated 40 Toroid by 1 min (top) or 1 s (middle and bottom). No c/c t/t c/t 20 periodicity was seen whether data were aligned 0 1st 2nd 3rd 4th 5th pulse with respect to odor onset (middle) or to the first e PN-PN synchrony (%) Latency, onset to peak (ms) synchronous event in each pair of responses (botd c/c d 30 d e 100 tom). (c) The occurrence of synchrony remained t/t cd bc c/t ILP 80 consistent across repeated odor pulses separated c ab 20 ab b 60 by 1 s, but in accordance with Fig. 1, the percenta a 40 age of synchronous spikes between PNs (relative 10 CMB 20 to total) was greater for intraglomerular PN pairs 0 0 (c/c or t/t) than for interglomerular pairs (c/t) (c, 1 10 1 10 10 10 1 10 1 1 10 1 Dosage (ng) Dosage (ng) cumulus; t, toroid). All values are mean ± s.d.; within each pulse, a significant difference between two means is indicated by an asterisk (KruskallWallis test; P < 0.05; n = 11, 15 and 8, for cumulus, toroid and mixed pairs, respectively). (d) Odorant-evoked synchrony is modulated by stimulus concentration. PSTHs show the averaged responses to five consecutive 150-ms odor pulses (5-ms bins) and illustrate the time course of synchronous firing between a pair of toroid PNs in response to stimulation with BAL at a dosage of 1 ng (shaded) or 10 ng (black). Stimulation with the higher concentration resulted in both an increased frequency and reduced latency of synchronous events. Intracellular records show the responses from both PNs to a single pulse consisting of 1 ng (top traces) or 10 ng (bottom traces) of BAL. Stimulus intensity and the calculated correlation coefficients between PNs were positively correlated (r = 0.35 and 0.48 for the 1- and 10-ng stimuli, respectively). Small tick marks in each trace reflect cross-talk between the two recording electrodes. Stimulus time course is indicated by the solid bar beneath the traces. (e) Summary of dose-dependent effects on synchrony between PNs in each group. Half of all PN/PN pairs in this study were tested with two concentrations of the pheromone blend (1- and 10-ng dosages). Left, synchrony was expressed as the percentage of synchronous events relative to the total spike counts in each pair of PNs. A statistically significant effect of concentration was seen in both intraglomerular PN/PN groups (cumulus pairs, n = 7; toroid pairs, n = 8) as well as in the interglomerular pairs (n = 2) (Kruskal-Wallis test followed by Mann-Whitney U-test; P < 0.05). Right, dose-dependent effect on the latency from stimulus onset to peak of synchronous firing between PNs. Again, the higher concentration shifted this latency to significantly lower values for both intraglomerular groups of PNs, as well as for the interglomerular group (P < 0.05). All values are mean ± s.d. Means shown with the same letter are not significantly different (P > 0.05). (f) Confocal-microscopic montage showing the anatomy of a pair of PNs from the interglomerular group, recorded simultaneously in the moth antennal lobe. Both PNs were stained with LY (see Methods). The uniglomerular arborizations of each neuron are located in different glomeruli: one innervates the cumulus (the C15 neuron, larger soma) and the other has branches confined to the toroid (the BAL neuron, smaller soma). As found in previous studies, each PN gave rise to a single axon that projected to the ipsilateral inferior lateral protocerebrum (ILP). Each axon also extended sparse collateral branches into the ipsilateral calyces of the mushroom bodies (CMB).

15 pairs, Fig. 1a and b, center). In eight additional PN pairs, the primary stimulus that triggered spiking was different for each neuron (Fig. 1a and b, right). We identified MGC-PNs through electrical stimulation of the antennal nerve or iontophoretic injection of Lucifer Yellow (LY) stain (Fig. 2f). As found previously28,29, PNs with arborizations confined to the glomerulus known as the ‘cumulus’ were selectively depolarized by C15, whereas those with branches in the ‘toroid’ glomerulus were depolarized by BAL (Figs. 1 and 2f). In this study, all neurons that innervated the toroid also showed nature neuroscience • volume 5 no 6 • june 2002

inhibitory responses to C15; that is, they were hyperpolarized by the odorant representing the primary excitatory input to the adjacent glomerulus. Similarly, all cumulus neurons were hyperpolarized by BAL (Fig. 1c)29. We investigated how interglomerular inhibition might be involved in modulating the output from these two identified glomeruli. Cross-correlations within and between glomeruli Comparison of two simultaneously impaled PNs showed striking similarities in their responses to repeated odorant pulses, and 559

articles

this occurred whether the two neurons were tuned to the same or to different odorants (Fig. 1b). Without exception, both PNs in each pair fired discrete bursts of action potentials in response to consecutive pulses of the excitatory odorant, and each burst of spikes was truncated by a distinct membrane hyperpolarization (Fig. 1b and c)22. Time-series analysis showed further that the temporal relationships between PN spike trains differed markedly depending on the odor-tuning properties of the two neurons recorded. Olfactory stimulation with the pheromone blend (C15 + BAL) evoked synchronous firing in pairs of PNs innervating the same glomerulus (correlation coefficients, mean ± s.d.: 0.60 ± 0.15 for cumulus pairs, n = 11; 0.51 ± 0.26 for toroid pairs, n = 15), but significantly less synchrony between PNs innervating different glomeruli (0.27 ± 0.10, n = 8, P = 0.02; Fig. 1d). These differences were evident in spite of the substantial overlap between spike trains in each pair of PNs (Fig. 1b). In addition, synchronized spiking did not appear spontaneously but only in response to olfactory stimulation. In all cases, we found more synchrony between two PNs innervating the same glomerulus and less synchrony between PNs innervating two neighboring glomeruli, even though the two glomeruli encoded information about chemically similar odorants. Stimulus-locked modulation of PN synchrony We also measured the influence of stimulus dynamics on the time course of synchrony between PNs (Fig. 2a and b). In accordance with our earlier studies, a stimulation protocol that produced multiple odor pulses separated by 1-s periods of clean air (much like a natural odor plume12) yielded no evidence of an odor-specific or periodic pattern of synchrony in the responses of paired cumulus PNs or toroid PNs (Fig. 2b, middle and bottom). Instead, spiking patterns were closely matched to the dynamics of the stimulus itself12. Synchronous firing occurred during a time window that started at the onset of the response and continued for a period that approximated the duration of the stim560

PN 2

Time (s) Time (s)

Counts/bin


Counts/bin

Fig. 3. Time course of synchronous firing a 5 C15 0.35 b between PNs. (a) PSTHs from two toroid 20 mV PN 1 4 PNs (black, red) show excitatory responses PN 2 0.25 to stimulation with BAL, but suppression of 3 + 2 50 background activity with C15 stimulation 6 2 alone (stimulus onset was at time = 0; dura0.15 0 tion = 200 ms). (b) Dynamic correlation + + 1 2 100 analysis (color contour plots) show that PN 1 6 0.05 0 spikes in the two PNs showed the greatest 0 0.05 0.15 0.25 0.35 0.05 0.15 0.25 0.35 2 + + synchrony at the onset of their response win200 6 0.35 5 dows. Color scale represents the normalized BAL 0 correlation calculated over five consecutive 4 2 300 + + responses (range = –1 to +1). (c) Effect of 0.25 6 3 stimulus duration on patterns of odor-evoked 0 synchrony between PNs. Raw data traces 2 0.15 + + + 2 400 (top) show responses of a pair of toroid PNs 6 1 to stimulation with a 50-ms pulse of BAL. 0 Spikes in the PNs became synchronized near 0.05 0 response onset. The moment at which the 0 0.1 0.2 0.3 0.4 0.05 0.15 0.25 0.35 0.05 0.15 0.25 0.35 time lag between spikes in the two PNs Time (s) Time (s) Time (s) Blend exceeded 5 ms is indicated by the asterisk. Each panel shows the near-coincidence histograms (top graphs) and the superimposed PSTHs (bottom graphs) for the two PNs stimulated with BAL pulses of 50, 100, 200, 300 and 400 ms, respectively (stimulus time course indicated by solid bar beneath each panel). All traces were averaged over five trials. The times at which positive correlations above 0.3 occurred are indicated in each panel (+). Several conditions are readily apparent. The first and largest coincidence peak reliably reflects stimulus onset, regardless of stimulus duration. The distribution of spike activity broadens as a function of stimulus duration, and longerduration stimuli may also evoke a greater number of smaller coincidence peaks (bottom panel). This latter effect could be due to the greater probability that the stimulus time course will be non-uniform as the period of stimulation is prolonged.

ulus pulse (Fig. 2a). Although the two neurons often continued to fire after the odor pulse had ended, and their spike trains overlapped in time, we saw no synchronous firing except during this window defined by the stimulus duration. To test whether the absence of periodic patterning was due to the effects of adaptation, we also tested an inter-stimulus interval of 1 min. Again, we found no evidence for an odor-specific pattern of synchrony that was repeated from trial to trial (Fig. 2b, top). Neither did we find any significant change in the strength of PN/PN coherence over repeated trials using this pulsatile stimulation protocol (Fig. 2c; Table 1). Once again, intraglomerular PN pairs showed a higher percentage of synchronous events per stimulus than did the interglomerular pairs (Fig. 2c; Table 1). Concentration-dependent effects on PN synchrony Of the 15 toroid pairs studied, eight were tested with two stimulus concentrations (1- and 10-ng pulses of BAL). Of these eight pairs of PNs, all 16 neurons were sensitive to changes in stimulus intensity (Fig. 2d). In each case, the 1-ng stimulus evoked fewer synchronous events than the 10-ng stimulus (mean correlation coefficients for the 1- and 10-ng stimuli were 0.24 and 0.64, respectively; P < 0.0005; Mann-Whitney U-test; Fig. 2d and e; Table 1). For cumulus pairs tested with C15 (n = 7), the corresponding values were 0.22 and 0.69 (P < 0.0005). Again, we saw no significant change in synchrony over repeated trials with the same stimulus, and the temporal pattern of synchrony evoked at both odor intensities always reflected stimulus dynamics with great temporal precision (Fig. 2d). In addition to increased synchrony (Fig. 2e, left), stimulation with the elevated odor concentration resulted in significant reductions in the latency of the response in individual PNs and in the latency to synchronous events between PNs (Fig. 2e, right). The 10-ng stimulus advanced the response onset by as much as 50 ms as compared with the 1-ng stimulus, and this pattern was generally consistent over repeated stimulus trials (Fig. 2e, right). nature neuroscience • volume 5 no 6 • june 2002

articles

Correlation

Counts/bin

PN 2


Counts/bin

Counts/bin

Fig. 4. Blend-enhanced synchronization of PNs inner- a vating the same glomerulus. In about 70% of all cases examined, synchrony between glomerulus-specific PNs was enhanced by stimulation with the pheromone blend. (a) Cross-correlograms (10-ms bins) calculated from the simultaneous responses of two toroid PNs to a BAL stimulus. The number of synchronous events between the two neurons was markedly greater in response to a mixture of BAL + C15 (Blend, right panel) than to BAL – – – – – – alone (middle panel), and there was little or no coTime (s) Time (s) Time (s) activity between PNs in the absence of an odor stimulus b C15 (control, left panel). (b) Dynamic correlation analysis showed the time course of synchrony. Again, synchrony occurred reproducibly at response onset. The blendenhanced correlation is evident from the broader distriBAL bution of warm colors in the surface plots, and this correlation was seen across the five consecutive odor BAL Blend 30 pulses (magnitude of correlation represented by color Blend 20 scale, top right). Mean-rate histograms (inset) show that 10 enhanced co-activity in response to the blend is not sim0 ply a function of increased spike activity evoked by PN # PN 1 combining the two odorants. (c) Summary of blendenhanced effects on synchrony between PNs in each Time (s) group. Left, synchrony was expressed as the percentage of synchronous events relative to the total spike counts Latency, response onset PN-PN synchrony (%) c in both PNs. The blend effect occurred in both the to peak synchrony (ms) cumulus (7 out of 11) and toroid (11 out of 15) groups c/c ab abd 30 c 100 b b t/t but was statistically significant only in the former group d 25 c/t 80 (Kruskal-Wallis test followed by Mann-Whitney U-test; P b ab 20 < 0.05). Interglomerular synchrony evoked by the blend ab 60 a (n = 8) was also significantly weaker than intraglomerular 15 40 synchrony. Right, differential effects of the individual and 10 blended odorants on the latency from stimulus onset to 20 5 synchronous firing between PNs. The odor blend shifted 0 0 this latency to lower values for both the cumulus and C15 Blend BAL Blend Blend C15 Blend BAL Blend Blend toroid (intraglomerular) groups of PNs, but the effect was significant only in the latter group (P < 0.05). For the interglomerular group, the latency was not significantly different from that measured in the other two groups. All values are mean ± s.d. Means sharing the same letter were not significantly different (P > 0.05).

Dynamics of PN/PN synchronization Peristimulus-time histograms (PSTHs) showed that the time course of responses in two PNs selective for the same odorant were often nearly indistinguishable (Fig. 3a). Nevertheless, dynamic correlation analysis of the underlying pattern of PN firing showed that the peak period of synchrony between the two neurons occurred only during a narrow window near the onset of the response (Fig. 3b). This brief period of co-activity (hereafter referred to as ‘onset synchrony’) was followed first by a period of desynchronized spiking, and then by complete suppression of all spiking until the next stimulus pulse. Onset synchrony between PNs also occurred before the peak response as measured by mean spike rate (Fig. 3c), indicating that the peaks of synchronous firing did not reflect random coincident firing between the two neurons. Onset synchrony always accompanied the rising phase of the overlapping PSTHs from the two PNs, and it occurred at all stimulus durations tested, from 50 to 400 ms (Fig. 3c). Peaks in synchrony occasionally re-appeared during the falling phase of the odor response (possibly signaling stimulus offset), but the timing of these peaks was less predictable than it was during the rising phase (Fig. 3c). Blend-enhanced effects on synchrony In nature, olfactory stimuli are typically mixtures of odorants. We therefore examined whether the blend of BAL and C15 (a mixture nature neuroscience • volume 5 no 6 • june 2002

that defines the sex pheromone in this insect) would evoke patterns of PN synchrony that were different from the patterns evoked by the individual odorants. This question is particularly intriguing in light of the finding that in the PNs studied here, input from the neighboring glomerulus was inhibitory (Fig. 1c) and could thus perform a modulatory function when the two adjacent glomeruli were activated simultaneously. In 7 of 11 cumulus pairs and 11 of 15 toroid pairs, the timing of PN/PN synchrony was enhanced by the mixture of odorants (Fig. 4a). The blend evoked significantly greater synchrony between PNs innervating the same glomerulus (P = 0.05), and it also led to a broadening of the temporal window during which synchrony occurred. This effect was reproducible over repeated trials (Fig. 4b). Comparing the ‘blend effect’ across the three groups of PN pairs, it is evident once again that the degree of synchronous firing between interglomerular PNs was significantly smaller than that seen in either of the intraglomerular groups (Fig. 4c, left; P < 0.05). Furthermore, in about 50% of PN pairs in the latter two groups, we saw no apparent difference in spike rate when the primary excitatory stimulus or the blend containing that odorant was used (Fig. 4b, inset), indicating that blend-enhanced synchrony was not a simple consequence of increased PN spiking. We next examined the responses evoked by the C15+BAL blend to determine whether the blended stimulus had an effect on latency to PN synchrony, as seen with elevated stimulus 561

articles


a

b

c

Fig. 5. Correlation between PNs in one glomerulus is modulated by inhibitory input from the neighboring glomerulus. (a) Simultaneous PN/PN recordings showed a multiphasic postsynaptic response to a blend of BAL + C15 (bar shows time course of a 200-ms pulse). In both neurons the response consisted of a pronounced IPSP (arrow) that immediately preceded a burst of action potentials22. This example is from two toroid PNs that were both depolarized by BAL but hyperpolarized by C15 as in Fig. 1b. The IPSPs evoked by C15 directly preceded the onset of synchronous firing evoked by BAL. The PNs remained synchronized for a period that matched the stimulus duration, and shortly thereafter became desynchronized (asterisk). Spiking in both PNs then ceased and the neurons returned to their resting states. (b) Relationship between spike synchrony in a pair of PNs innervating one glomerulus, and the strength of inhibitory synaptic input from the neighboring glomerulus. The correlation between PN pairs is plotted against the mean amplitude of the IPSP evoked by the odorant that activates the adjacent glomerulus (y = 0.4 + 0.3x – 0.04x2; P = 0.01). (c) Schematic circuit diagram relating the two glomerular networks examined in this olfactory system (round synapses are excitatory; triangular synapses are inhibitory). The BAL- and C15-selective populations of olfactory receptor cells (ORCs) transmit odor information to the toroid and the cumulus, respectively. ORCs can excite PNs monosynaptically and/or indirectly through disinhibitory pathways involving a diverse population of about 300 GABAergic local interneurons (LN1–n). In the presence of the conspecific pheromone blend, the two MGC glomeruli first act as filters that provide specific spatial addresses for inputs from the two classes of ORCs that are simultaneously activated. The GABAergic LNs (providing the major inhibitory input to PNs) then serve at least two important processing functions at this early stage: (i) they organize the spatial pattern of activity in the activated glomeruli through specific interglomerular linkages, and (ii) they organize the timing of output signals simultaneously from each glomerulus through the modulation of synchrony between PNs (PN1 and 2 in toroid, PN3 and 4 in cumulus).

coefficient for each pair of PNs, we found a strongly positive correlation between these two variables (Fig. 5b).

DISCUSSION concentrations (Fig. 2e). Stimulation with the blend of odorants also resulted in reductions in the latency of the responses of individual PNs and in the latency to onset synchrony between PNs (Fig. 4c, right). This shift in latency occurred in both intraglomerular groups, but was statistically significant only in the toroid pairs (P < 0.05). The odor-blend stimulus did not reduce the latency of response when synchrony was measured across glomeruli. Modulation of synchrony by interglomerular inhibition GABA is centrally involved in the precision of spike timing in glomerular PNs of both vertebrates and invertebrates, and fast IPSPs from GABAergic local neurons (LNs) are commonly seen in PNs of moths22,30. If PN/PN synchrony is not modulated by oscillations (which could arise from inhibitory feedback loops in the glomerular neuropil), we reasoned that feed-forward inhibitory connections could serve to synchronize PNs. We therefore tested whether the activity of one glomerulus was modulated by inhibition from its neighbor. Simultaneous intracellular recordings from neurons stimulated with the blend of odorants detected multiphasic responses comprising virtually identical, fast-onset IPSPs, followed by depolarizing EPSPs that gave rise to trains of action potentials in both neurons (Fig. 5a). In 12 pairs of odorant-matched PNs, we measured the amplitudes of the IPSPs evoked by the primary excitatory odorant to the neighboring glomerulus31. When the average values were plotted against the correlation 562

It has been proposed for the mammalian olfactory bulb that neural synchronization across glomerular outputs may enhance the representation of a complex olfactory stimulus by integrating the different signal streams activated by the odor into a unified olfactory ‘image’ at the level of the sensory cortex9,18. In the sphinx moth, we have shown that synchrony does indeed occur across glomeruli, but we found that even when a blend of odorants was used as a stimulus, the firing patterns of PNs from the same glomerulus always showed the highest correlations (Fig. 1d). A number of studies have also suggested that lateral inhibition (mediated by reciprocal connections between M/T and granule cells in the olfactory bulb) may sharpen odor tuning in a glomerulus (in direct analogy to contrast enhancement in a retinal ganglion cell)18,23–25, but this is not universally accepted27. In an attempt to resolve these issues, we used intracellular recordings from glomerular output neurons to examine the dynamic interactions that occur within and between two adjacent glomeruli with identified odor tuning. In the absence of odor, MGC-PNs in moths typically fire action potentials sporadically, and firing between PNs is asynchronous (Fig. 1b). When presented with pulses of the correct olfactory stimulus, however, PNs synchronize their discharges in response to each stimulus pulse (Fig. 1d), with PNs from the same glomerulus showing the highest correlations (Fig. 2a). The enhanced precision of PN/PN spiking is therefore a potential means of strengthening the spatial representation of the stimulus, which according to many recent imaging studies can be defined nature neuroscience • volume 5 no 6 • june 2002


articles

by the specific glomerulus (or combination of glomeruli) encoding it19,32–34. In view of this capacity to modulate spike timing on a millisecond time scale, we propose that the MGC glomeruli act as multifunctional coding modules in the brain, participating in the simultaneous and parallel encoding of the different attributes of the stimulus (quality, quantity and spatiotemporal features)11,12,22 inherent in a dynamic odor plume35. Our results showing tight correlations in spike timing between PNs with the same tuning characteristics provide new support for the longheld hypothesis that olfactory glomeruli represent the fundamental coding modules in early olfactory processing13,16,19,20,25. What coding functions might synchrony perform? In other sensory systems, there is evidence that the incorporation of intercellular timing relations into a neural-population model improves the accuracy of ensemble coding and thus facilitates stimulus identification36,37. In motor cortex, the synchronization of spike activity between interneurons also facilitates the encoding of arm movements by ensembles of neurons38. How might neural synchronization function in encoding olfactory information? Recent results from olfactory-bulb slice preparations show that synchronous firing is consistently greater for intra- than for interglomerular pairs of M/T cells, but these studies did not involve odor stimulation39,40. Our results here provide the first evidence from intracellular recordings that PNs innervating the same glomerulus and tuned to the same odorant are more tightly synchronized than pairs of PNs that process different olfactory inputs. This then raises the question: what specific function might intraglomerular synchrony serve? One suggestion is that it temporally integrates information streams from select subsets of the functionally diverse population of output neurons that arise from a single glomerulus41. Neural-ensemble recordings in the moth MGC11 recently showed that each brief stimulus pulse triggers a transient burst of activity across the coding ensemble, but specific features of the stimulus are encoded in the precise temporal relationships superimposed on the ensemble. For example, different subsets of PNs synchronize at different stimulus concentrations, suggesting a functional partitioning within the glomerulus11. According to this organizational scheme, the complete ensemble could encode the odor signal as well as monitor its concentration dynamics because different subsets of PNs would synchronize as the ambient concentration of the stimulus changed. Synchronous firing across PNs may therefore help to reinforce the spatially organized segregation of odorant-specific signals encoded in each glomerulus13–20. Recent studies in the moth brain indicate that such segregation of odorant-specific pathways is maintained even at higher levels of sensory integration in the protocerebrum42. Thus, in the moth olfactory system, evidence from both intracellular and neural-ensemble recording studies indicates that the chemical identity of an odor is encoded spatially, according to which glomeruli are activated (or inhibited) by the stimulus. Other key features of the stimulus (including odor intensity, dynamics and the quality of specific odorant blends) are encoded in specific temporal patterns of activity superimposed on the spatial ensemble11,12,29,31. It has been suggested that if the target neurons in higher centers function as coincidence detectors, then synchronous input to these centers may facilitate the decoding of these different stimulus features18. Interglomerular inhibition shapes odor representations In all experiments involving BAL and C15, PNs that were depolarized by one of these odorants were hyperpolarized by the other nature neuroscience • volume 5 no 6 • june 2002

odorant, and more than half of these PN pairs showed significantly greater synchronization in response to the blend of the two odorants (Fig. 4; Table 1). This finding provides new evidence that the temporal patterning of output from a given glomerulus may be further modulated by inhibitory input arising from neighboring glomeruli18,23–25. Earlier single-unit studies in moths showed that the timing of spike discharges in MGC-PNs is modulated by bicuculline-sensitive, GABAA-like synapses from LNs22,30,43. When these synapses are blocked pharmacologically, the ability of MGC-PNs to resolve intermittent odor pulses falls dramatically. A testable circuit model that can help explain the possible organization of these inhibitory interglomerular interactions is shown in Fig. 5c. This model incorporates much of our current knowledge of the connectivity that constitutes the processing networks in MGC glomeruli (reviewed in ref. 22). The model shows that when a blend of odorants is being processed, inhibition from one glomerulus could reset the timing of spike discharges in PNs activated in the neighboring glomerulus, thus aiding in their synchronization. There are both similarities and differences between this organization and that seen in the vertebrate retina. Like retinal ganglion cells, PNs in one glomerulus show enhanced temporal tuning in the presence of inhibition from the neighboring glomerulus; unlike ganglion cells, PNs do not show a broadening of their odor tuning in the absence of this inhibition. Although we cannot rule out the possibility that other cellular or synaptic mechanisms promote synchronous firing between PNs26,39,40, we believe that the BAL-evoked IPSP in cumulus PNs and the C15-evoked IPSP in toroid PNs (Fig. 1c) could serve this resetting function22,31. Pharmacological experiments to test this hypothesis are now in progress. It must be noted that the principles outlined here for the glomerular processing of pheromonal information are probably not unique to these specialized odorants or to the sexually dimorphic glomeruli dedicated to them. Data from both the MGC44,45 and non-pheromonal glomeruli46 are consistent with the fundamental hypothesis that each glomerulus is an identifiable, functional unit dedicated to the processing of odorant-specific information. Likewise, both pheromonal and non-pheromonal odorants activate an odor-specific ‘mosaic’ of antennal-lobe glomeruli in a reproducible pattern (reviewed in ref. 19). Using multichannel electrode arrays11, we are now probing the hypothesis of glomerular chemotopy by recording ensemble responses to a wide array of odorants that are known to trigger activity from both broadly and narrowly tuned ORNs on the moth antennae47. We propose that in the context of a blended olfactory stimulus (as typically found in nature), reciprocal inhibitory interactions between glomeruli provide a temporal mechanism for strengthening the spatial representation of a complex stimulus by synchronizing PNs, both within and between the population of activated glomeruli. Thus, although a single odorant evokes synchronization among outputs from the same glomerulus, the blending of several odorants increases the probability of lateral inhibitory interactions between neighboring glomeruli, thereby augmenting the temporal tuning of synchronous output from each participating glomerulus. Finally, do our results support the suggestion that lateral inhibition operates between glomeruli in the olfactory system18,27? Lateral-inhibitory interactions between MGC glomeruli do indeed help to shape the temporal representations of pheromonal stimuli, but not in the same sense that lateral inhibition in the retina, for example, enhances the local spatial variations in an antagonistic center-surround receptive field. In analogy to ‘on563


articles

center’ retinal ganglion cells that are hyperpolarized by illumination of the surround48, MGC-PNs innervating one glomerulus (for example, the toroid) are hyperpolarized by the olfactory input to the neighboring glomerulus (the cumulus). Unlike the receptive field organization of retinal ganglion cells, however, the responses of toroid PNs are not suppressed by uniform stimulation of the entire receptive field (that is, stimulation with a blend of BAL and C15, as in Fig. 4). Rather, the blend of odorants specifically leads to an enhancement of synchronized firing between glomerulus-specific PNs in both the cumulus and the toroid. Thus, reciprocal inhibition between glomeruli at this level of processing in the olfactory pathway serves to emphasize the presence of the individual constituents of the blend, rather than to exaggerate the difference (or ‘contrast’) between the two odorant molecules. Although this latter function may be reserved for higher levels of processing in the protocerebrum, inhibition in the antennal lobe is important for synchronizing glomerular output, thus enhancing the transmission of weak olfactory signals and increasing stimulus contrast relative to background odors in the animal’s environment.

METHODS Preparation. Manduca sexta (L.) (Lepidoptera: Sphingidae) were reared in the laboratory on artificial diet under a long-day photoperiod, and adult male moths, 1–3 days after emergence, were prepared for experiments as described previously28,29. For electrophysiological recording, the moth was restrained in a plastic tube with its head fully exposed. The labial palps, proboscis and cibarial musculature were then removed to allow access to the brain. To eliminate movement, the head was isolated and pinned to a wax-coated glass Petri dish with the antennal lobes facing upward. Tracheae and a small part of the sheath overlying one antennal lobe were then removed with fine forceps. The preparation was continuously superfused with physiological saline solution containing 150 mM NaCl, 3 mM CaCl2, 3 mM KCl, 10 mM TES buffer (pH 6.9) and 25 mM sucrose. Intracellular recording and staining. Intracellular recording and dyemarking were performed with borosilicate glass microelectrodes filled with a 4% solution of Lucifer Yellow CH (LY) (Sigma) in 0.2 M LiCl and having resistances of 100–400 MΩ (ref. 29). We recorded simultaneous activity from two antennal-lobe neurons by independently controlling two microelectrodes at penetration sites separated by 50–200 µm, depending on whether we wished to record from PNs innervating the same or different glomeruli. The signals from both electrodes were visualized on an oscilloscope and recorded on FM tape. After carrying out this physiological characterization, we injected cells with LY by passing hyperpolarizing current (up to 1.5 nA) for 5–15 min. At the completion of an experiment, the brain was excised and immersed in formaldehyde fixative solution (2.5% formaldehyde in 0.1 M sodium phosphate buffer with 3% sucrose added). Brains were fixed overnight, dehydrated through a graded series of ethanol solutions and cleared with methyl salicylate. Cleared brains were viewed as whole mounts with a laser-scanning confocal microscope (Bio-Rad MRC-600, Cambridge, Massachusetts) equipped with a Nikon Optiphot-2 microscope and both 15-mW krypton/argon and 100-mW argon laser-light sources. Optical sections were 2 or 5 µm thick. Preparations were then returned to 100% ethanol and embedded in Spurr’s resin. When higher-resolution images were needed, histological sections of the brain were cut at 48 µm with a sliding microtome. Sensory stimulation and neuron identification. Orthodromic electrical stimulation of the antennal nerve and olfactory stimulation of the antenna were used to generate postsynaptic responses in antennal lobe neurons. Hook electrodes were placed beneath the antennal nerve, and repetitive 0.1-ms shocks were delivered as a test for monosynaptic input. Previous studies reported distinct differences in the physiological profiles of local and projection neurons in the moth antennal lobe: LNs more often receive direct antennal input, and the mean half-width of action 564

potentials in LNs is about twice that of PNs43. These criteria were used as reliable indicators of PNs in all experiments. Olfactory stimuli were delivered to the preparation as reported previously43. Pulses of air from a constant air stream were diverted through a glass syringe containing a piece of filter paper to which was applied a single odorant (1 or 10 ng) or a blend of two odorants (1 or 10 ng of each). The odor stimulus was pulsed by means of a solenoid-activated valve controlled by an electronic stimulator (W-P Instruments, Sarasota, Florida). In every experiment, the outlet of the stimulus syringe was positioned about 2 cm from and orthogonal to the center of the antennal flagellum ipsilateral to the impaled antennal lobe. Stimulus durations varied from 50 ms to 5 s, and multiple odor pulses were separated by intervals of 1 s or 1 min. The odor stimuli used were: (i) E,Z-10,12-hexadecadienal (bombykal, BAL), the primary 16-carbon aldehyde component of the female’s sex pheromone; (ii) E,Z-11,13-pentadecadienal (C15), a 15-carbon aldehyde mimic of the second essential component of the sex pheromone; and (iii) a mixture of BAL and C15 (blend). Although we substituted C15 for the natural pheromone component, we refer to both BAL and C15 as pheromone components. Data analysis. Analog signals stored on FM tape were digitized at 20 KHz per channel using Autospike (Syntech, Silversum, the Netherlands) or Axoscope software (Axon Instruments, Foster City, California). Time stamps representing the occurrence of each action potential in an intracellular trace were then analyzed with Neuroexplorer (Nex Technologies, Winston-Salem, North Carolina). Cross-correlograms and correlation coefficients were calculated (5-ms bins) to help quantify synchrony between two PNs. These data were corrected by subtracting shiftpredictor values to control for synchrony related solely to the timing of the stimulus49. Dynamic correlations (or joint post-stimulus-time histograms) were used to analyze the time course and temporal patterning of PN synchronization50. The resulting matrix illustrates the magnitude and timing of synchrony, with perfect synchrony between two PNs occurring along the main diagonal. Near-coincidence histograms were extracted from the main diagonal of each matrix using a bin width of 5–10 ms. Latency measurements (time between stimulus onset and first synchronous events) were also used as a means to quantify the effects of stimulus concentration and blending odorants. Acknowledgments We thank K. Daly, V. Pawlowski and B. Smith for discussions and comments and H. Stein and A.A. Osman for technical assistance. Supported by grants and contracts from National Institutes of Health (NIDCD).

Competing interests statement The authors declare that they have no competing financial interests.

RECEIVED 4 JANUARY; ACCEPTED 1 APRIL 2002 1. Vaadia, E., Ahissar, E., Bergman, H. & Lavner, Y. Correlated activity of neurons: a neural code for higher brain functions? in Neuronal Cooperativity (ed. Kruger, J.) 205–279 (Springer-Verlag, Berlin, 1991). 2. Rieke, F., Warland, D., de Ruyter van Steveninck, R. & Bialek, W. Spikes: exploring the neural code (MIT Press, Cambridge, Massachusetts, 1997). 3. Murthy, V. N. & Fetz, E. E. Synchronization of neurons during local field potential oscillations in sensorymotor cortex of awake monkeys. J. Neurophysiol. 76, 3968–3982 (1996). 4. Singer, W. & Gray, C. M. Visual feature integration and the temporal correlation hypotheses. Annu. Rev. Neurosci. 18, 555–586 (1995). 5. Adrian, E. D. Olfactory reactions in the brain of the hedgehog. J. Physiol. (Lond.) 100, 459–473 (1942). 6. Gelperin, A. Oscillatory dynamics and information processing in olfactory systems. J. Exp. Biol. 202, 1855–1864 (1999). 7. Laurent, G., Wehr, M. & Davidowitz, H. Temporal representations of odors in an olfactory network. J. Neurosci. 16, 3837–3847 (1996). 8. Mellon, D. & Wheeler, C. J. Coherent oscillations in membrane potential synchronize impulse bursts in central olfactory neurons of the crayfish. J. Neurophysiol. 81, 1231–1241 (1999). 9. Kashiwadani, H., Sasaki, Y. F., Uchida, N. & Mori, K. Synchronized oscillatory discharges of mitral/tufted cells with different molecular receptive ranges in the rabbit olfactory bulb. J. Neurophysiol. 82, 1786–1792 (1999).



articles

10. Kay, L. M. & Laurent, G. Odor- and context-dependent modulation of mitral cell activity in behaving rats. Nat. Neurosci. 2, 1003–1009 (1999). 11. Christensen, T. A., Pawlowski, V. M., Lei, H. & Hildebrand, J. G. Multi-unit recordings reveal context-dependent modulation of synchrony in odorspecific neural ensembles. Nat. Neurosci. 3, 927–931 (2000). 12. Vickers, N. J., Christensen, T. A., Baker, T. C. & Hildebrand, J. G. Odourplume dynamics influence the brain’s olfactory code. Nature 410, 466–470 (2001). 13. Christensen, T. A. & White, J. Representation of olfactory information in the brain. in The Neurobiology of Taste and Smell, Vol. 2 (eds. Finger, T. E., Silver, W. L. & Restrepo, D.) 201–232 (Wiley, New York, 2000). 14. Ressler, K. J., Sullivan, S. L. & Buck, L. B. Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79, 1245–1255 (1994). 15. Vassar, R. et al. Topographic organization of sensory projections to the olfactory bulb. Cell 74, 309–318 (1994). 16. Lancet, D. Vertebrate olfactory reception. Ann. Rev. Neurosci. 9, 329–355 (1986). 17. Singer, M. S. & Shepherd, G. M. Molecular modeling of ligand-receptor interactions in the OR5 olfactory receptor. Neuroreport 5, 1297–1300 (1994). 18. Mori, K., Nagao, H. & Yoshihara, Y. The olfactory bulb: coding and processing of odor molecule information. Science 286, 711–715 (1999). 19. Galizia, C. G. & Menzel, R. The role of glomeruli in the neural representation of odours: results from optical recording studies. J. Insect Physiol. 47, 115–130 (2001). 20. Shipley, M. T. & Ennis, M. Functional organization of olfactory system. J. Neurobiol. 30, 123–176 (1996). 21. Stopfer, M., Bhagavan, S., Smith, B. & Laurent, G. Impaired odor discrimination on desynchronization of odour-encoding neural assemblies. Nature 390, 70–74 (1997). 22. Christensen, T. A., Waldrop, B. R. & Hildebrand, J. G. Multitasking in the olfactory system: context-dependent responses to odors reveal dual GABAregulated coding mechanisms in single olfactory projection neurons. J. Neurosci. 18, 5999–6008 (1998). 23. Rall, W., Shepherd, G. M., Reese, T. S. & Brightman, M. W. Dendrodentritic synaptic pathway for inhibition in the olfactory bulb. J. Exp. Neurol. 14, 44–56 (1966). 24. Buonviso, N. & Chaput, M. A. Response similarity to odors in olfactory bulb output cells presumed to be connected to the same glomerulus: electrophysiological study using simultaneous single-unit recordings. J. Neurophysiol. 63, 447–454 (1990). 25. Shepherd, G. M. & Greer, C. A. Olfactory bulb. in The Synaptic Organization of the Brain (ed. Shepherd, G.M.) 139–169 (Oxford, New York, 1990). 26. Desmaisons, D., Vincent, J. D. & Lledo, P. M. Control of action potential timing by intrinsic subthreshold oscillations in olfactory bulb output neurons. J. Neurosci. 19, 10727–10737 (1999). 27. Laurent, G. A systems perspective on early olfactory coding. Science 286, 723–728 (1999). 28. Hansson, B. S., Christensen, T. A. & Hildebrand, J. G. Functionally distinct subdivisions of the macroglomerular complex in the antennal lobes of the sphinx moth Manduca sexta. J. Comp. Neurol. 312, 264–278 (1991). 29. Heinbockel, T., Christensen, T. A. & Hildebrand, J. G. Temporal tuning of odor responses in pheromone-responsive projection neurons in the brain of the sphinx moth Manduca sexta. J. Comp. Neurol. 409, 1–12 (1999). 30. Waldrop, B., Christensen, T. A. & Hildebrand, J. G. GABA-mediated synaptic


31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

42. 43. 44. 45. 46. 47. 48. 49. 50.

inhibition of projection neurons in the antennal lobes of the sphinx moth Manduca sexta. J. Comp. Physiol. A 161, 23–32 (1987). Christensen, T. A. & Hildebrand, J. G. Coincident stimulation with pheromone components improves temporal pattern resolution in central olfactory neurons. J. Neurophysiol. 77, 775–781 (1997). Johnson, B. A., Woo, C. C. & Leon, M. Spatial coding of odorant features in the glomerular layer of the rat olfactory bulb. J. Comp. Neurol. 393, 457–471 (1998). Belluscio, L. & Katz, L. C. Symmetry, stereotypy, and topography of odorant representations in mouse olfactory bulbs. J. Neurosci. 21, 2113–2122 (2001). Fuss, S. H. & Korsching, S. I. Odorant feature detection: activity mapping of structure response relationships in the zebrafish olfactory bulb. J. Neurosci. 21, 8396–8407 (2001). Murlis, J., Elkinton, J. S. & Cardé, R. T. Odor plumes and how insects use them. Annu. Rev. Entomol. 37, 505–532 (1992). Deadwyler, S. A., Bunn, T. & Hampson, R. E. Hippocampal ensemble activity during spatial delayed-nonmatch-to-sample performance in rats. J. Neurosci. 16, 354–372 (1996). Nicolelis, M. A. et al. Simultaneous encoding of tactile information by three primate cortical areas. Nat. Neurosci. 1, 621–630 (1998). Maynard, E. M. et al. Neuronal interactions improve cortical population coding of movement direction. J. Neurosci. 19, 8083–8093 (1999). Schoppa, N. E. & Westbrook, G. L. Glomerulus-specific synchronization of mitral cells in the olfactory bulb. Neuron 31, 639–651 (2001). Carlson, G. C., Shipley, M. T. & Keller, A. Long-lasting depolarizations in mitral cells of the rat olfactory bulb. J. Neurosci. 20, 2011–2021 (2000). Christensen, T. A. Anatomical and physiological diversity in the central processing of sex-pheromonal information in different moth species. in Insect Pheromone Research: New Directions (eds. Cardé, R.T. & Minks, A.K.) 184–193 (Chapman & Hall, New York, 1997). Lei, H., Anton, S. & Hansson, B. S. Olfactory protocerebral pathways processing sex pheromone and plant odor information in the male moth Agrotis segetum. J. Comp. Neurol. 432, 356–370 (2001). Christensen, T. A., Waldrop, B. R., Harrow, I. D. & Hildebrand, J. G. Local interneurons and information processing in the olfactory glomeruli of the moth Manduca sexta. J. Comp. Physiol. A 173, 385–399 (1993). Rospars, J. P. & Hildebrand, J. G. Anatomical identification of glomeruli in the antennal lobes of the male sphinx moth Manduca sexta. Cell Tissue Res. 270, 205–227 (1992). Rospars, J. P. & Hildebrand, J. G. Sexually dimorphic and isomorphic glomeruli in the antennal lobes of the sphinx moth Manduca sexta. Chem. Senses 25, 119–129 (2000). King, J. R., Christensen, T. A. & Hildebrand, J. G. Response characteristics of an identified, sexually dimorphic olfactory glomerulus. J. Neurosci. 20, 2391–2399 (2000). Shields, V. D. C. & Hildebrand, J. G. Responses of a population of antennal olfactory receptor cells in the female moth Manduca sexta to plant-associated volatile organic compounds. J. Comp. Physiol. 186, 1135–1151 (2001). Barlow, H. B., Hill, R. M. & Levick, W. R. Retinal ganglion cells responding selectively to direction and speed of image motion in the rabbit. J. Physiol. (Lond.) 173, 377–407 (1964). Nowak, L. G., Munk, M. H. J., James, A. C., Girard, P. & Bullier, J. Crosscorrelation study of the temporal interactions between areas V1 and V2 of the macaque monkey. J. Neurophysiol. 81, 1057–1074 (1999). Aertsen, A. M. H. J., Gerstein, G. L., Habib, M. K. & Palm, G. Dynamics of neuronal firing correlation: modulation of “effective connectivity”. J. Neurophysiol. 61, 900–917 (1989).

565