Olfactory stimuli trigger lateral excitatory interactions among glomeruli
antennal lobes receive olfactory input from two peripheral organs, the antennae and the maxillary palps. The palps contain ~120 ORNs which fall into six distinct types not found in the antennae (de Bruyne et al., 1999
; Goldman et al., 2005
). The antennae contain an additional 43 ORN types not found in the palps (de Bruyne et al., 2001
; Hallem et al., 2004
). Thus, each glomerulus receives direct ORN input exclusively from either the palps or the antennae. Within the antennal lobe, the six palp glomeruli are intermingled with the 43 antennal glomeruli (Couto et al., 2005
; Fishilevich and Vosshall, 2005
). This anatomy provides a convenient way to independently manipulate inputs to two groups of glomeruli.
In order to determine whether interactions between glomeruli shape PN odor responses, we began by acutely severing the antennal nerves. This manipulation leaves the palp ORNs intact, and allows us to test whether antennal PNs receive lateral input from palp glomeruli (). Consistent with previous reports (Berdnik et al., 2006
), we found that ablating ORN input to some glomeruli does not induce morphological rearrangement of the remaining ORN axons, even five days post-surgery (). However, we cannot exclude the possibility that removing ORN input to some glomeruli induces more subtle synaptic plasticity of inter-glomerular connections. Therefore we performed PN recordings immediately after severing the antennal nerves in order to rule out a role for any such plasticity.
Olfactory stimuli trigger excitatory interactions among glomeruli
Whole-cell patch-clamp recordings from PNs typically show abundant spontaneous synaptic input (). In contrast, PNs postsynaptic to antennal glomeruli in antennae-less flies show no spontaneous activity (). Nevertheless, odor stimulation of the maxillary palps evokes a depolarization in these PNs (n=6 PNs in 6 flies; ). The magnitude of this depolarization varied across cells, but was sufficient to produce a train of spikes in a PN postsynaptic to glomerulus VC3, for example (). We confirmed that each of the PNs we recorded from innervated an antennal glomerulus by filling the cell with biocytin (see Methods).
We averaged the membrane potential across six presentations of the same odor in the same cell () before averaging across experiments (). All these odors strongly activate one or more palp ORN types (de Bruyne et al., 1999
), and all elicited a substantial depolarization in each of the antennal PNs we recorded from. The solvent we use to dilute our odors (paraffin oil) evoked no response (). Removing both antennae and maxillary palps abolished odor-evoked depolarizations (n = 3), demonstrating that the maxillary palps mediate this response ().
These results demonstrate that antennal PNs receive lateral synaptic input from palp ORNs, and that the net effect of this indirect input is excitatory. It is possible that some inter-glomerular synapses hyperpolarize PNs, but if so, this inhibition is evidently obscured by a larger excitatory component. It is important to note that ORN input was removed acutely (~10–20 minutes before recording). Therefore, the lateral excitation we observed cannot reflect remodeling of the antennal lobe circuitry.
Total lateral synaptic excitation to a PN is substantial
What is the total impact of all lateral synaptic input to a PN? In order to answer this question we selectively silenced a single ORN type. By recording from PNs postsynaptic to the “silent” glomerulus while stimulating the antennae and palps with odors, we should be able to observe the total lateral input to that PN.
In order to silence a single ORN type, we used flies with mutations in one of two odorant receptor genes, Or43b
and Or10a. Or43b
is expressed in ORNs that project to glomerulus VM2, and Or10a
is expressed in ORNs that project to glomerulus DL1 (Couto et al., 2005
; Fishilevich and Vosshall, 2005
). The Or43b1
null allele was produced by gene targeting, and has been described previously (Elmore et al., 2003
). The Or10af03694
allele results from a pBac insertion in the gene (Thibault et al., 2004
), but has not been characterized previously. We first verified that this mutation does not disrupt ORN axon targeting (Supplemental Fig. S1A
). We then confirmed that both the Or43b1
mutations virtually abolish ORN odor responses (Supplemental Fig. S1B–D
). We occasionally observed very weak responses to a few specific odors in both mutants (Supplemental Fig. S2
). These weak responses may be mediated by Or83b, a protein with homology to odorant receptors that is expressed in most ORNs, and which is required for trafficking receptors to the cell membrane (Larsson et al., 2004
). We did not use these odors in our stimulus set when we analyzed PN responses in these mutants (–). Note that these mutations also reduce the rate of spontaneous ORN spikes (Supplemental Fig. S1C
PNs postsynaptic to “silent” ORNs show reduced activity but normal morphology
Odor tuning of PN responses postsynaptic to normal versus “silent” ORNs
We then recorded from PNs postsynaptic to these “silent” ORNs (). In flies with the mutation that silences VM2 ORNs, we targeted VM2 PNs using an enhancer trap line (Tanaka et al., 2004
) to specifically label these cells with GFP (NP5103-Gal4,UAS-CD8:GFP;Or43b1
). We recorded from one PN per fly and confirmed the glomerular identity of each recorded PN by imaging the biocytin fill post hoc
. As expected from the decrease in spontaneous activity in mutant ORNs, spontaneous spiking in VM2 PNs was reduced compared to wild type (). Similarly, in flies with mutant DL1 ORNs, we recorded from DL1 PNs using an enhancer trap line (Tanaka et al., 2004
) to specifically label these cells with GFP (Or10af03694;
). Again, spontaneous spiking was reduced in these PNs (). We confirmed that these PNs show normal dendrite morphology in the absence of functional ORNs (), consistent with previous reports (Wong et al., 2002
; Berdnik et al., 2006
In flies with silent VM2 ORNs, all VM2 PNs were depolarized by every odor we tested (n=10 cells in 10 flies), with the exception of 4-methyl phenol, which failed to elicit any activity in two cells. These depolarizations were typically large enough to trigger a train of action potentials (). Similarly, in flies with silent DL1 ORNs, all DL1 PNs were depolarized by every odor we tested (n=10 cells in 10 flies). Again, most responses were large enough to elicit a train of spikes. We also saw similar spiking responses to odors in cell-attached mode (prior to going whole-cell), demonstrating that these depolarizations are not an artifact of intracellular dialysis.
Comparing odor responses in PNs postsynaptic to normal versus “silent” ORNs
As expected, most odors evoked a larger response in wild-type flies than in mutant flies. For example, ethyl butyrate elicits vigorous activity in normal VM2 ORNs (Supplemental Fig. S1
) and in wild-type VM2 PNs. When the VM2 ORNs are silenced, this odor elicits a much smaller response in VM2 PNs (). Similarly, methyl salicylate evokes a very strong response in DL1 ORNs (Supplemental Fig. S1
), and in wild-type DL1 PNs, but only a small response in a DL1 PNs postsynaptic to silent ORNs (). We also noticed that in PNs postsynaptic to silent ORNs, odor-evoked responses were often more transient than in wild-type PNs ().
Some odor responses, however, were relatively unaffected by odorant receptor mutations. For example, 2,3-butanedione evokes very little response in wild-type VM2 ORN (Supplementary Fig. S1
). When these ORNs are silenced, the response of VM2 PNs to this odor is virtually unaltered (). Similarly, ethyl acetate does not excite wild-type DL1 ORNs (Supplementary Fig. S1
). Accordingly, the response of DL1 PNs to ethyl acetate is undiminished by silencing their presynaptic ORNs ().
How does the size of total lateral input to a PN depend on the odor stimulus? We quantified odor responses by computing mean firing rates over the 500-ms duration of the odor stimulus, and we plotted these response magnitudes for each odor stimulus to produce tuning curves (). These plots show that different odors evoke different amounts of lateral excitatory input to each PN. However, the odor tuning of PNs postsynaptic to silent ORNs is completely different from the normal odor tuning of these PNs. For both glomeruli, the odor tuning of wild type and mutant PNs showed no significant correlation (both comparisions Pearson’s r2<0.05, p>0.4).
We also noted that, in the absence of direct ORN inputs, both VM2 PNs and DL1 PNs are broadly tuned to odors. This suggests that each of these glomeruli receives indirect excitatory input from multiple ORN types, not just one or two. We wondered whether these two glomeruli receive indirect input from similar or different populations of ORNs. To assess this, we compared the responses of PNs in these two “silent” glomeruli to the same odors. Overall, the responses of these two PN types are significantly correlated (; Pearson’s r2 = 0.31, p<0.05). However, some odors elicit different amounts of lateral input to these glomeruli. For example, butyric acid elicits a larger response in VM2 than in DL1 (p=0.05, t-test, n=6 for each glomerulus). These results are consistent with the idea that these two glomeruli receive indirect input from overlapping populations of ORNs, but that the indirect inputs to these PNs are not identical.
Characterizing lateral input to many glomeruli originating from a single glomerulus
Because total lateral excitatory input to VM2 and DL1 PNs is broadly tuned and significantly correlated, it is likely that two glomeruli receive indirect input from many of the same ORNs. This, in turn, implies that each ORN type provides indirect input to many glomeruli. To test this prediction directly, we designed experiments to measure the spread of excitation across the antennal lobe evoked by activation of a single ORN type. We used two approaches to selectively stimulate one ORN type (). These two approaches have complementary strengths and weaknesses, but yielded similar results. In the first method (experiment 1), we took advantage of a mutation in Or83b
. This gene is expressed in most ORNs and encodes a chaperone protein required for trafficking odorant receptors to ORN dendrites (Larsson et al., 2004
; Benton et al., 2006
). Mutating Or83b
abolishes odor responses in all maxillary palp ORNs (and many antennal ORNs; Supplemental Fig. S3
). Thus, all ORN input to the antennal lobes is abolished by removing the antennae of mutant flies. In these flies, we then rescued normal function in the maxillary palp ORNs that project to glomerulus VA7l. This was done by expressing Or83b
under the control of the odorant receptor gene promoter corresponding to the VA7l ORNs (Or46-Gal4/UAS-Or83b;Or83b2
) (Fishilevich and Vosshall, 2005
). We verified the specificity of this rescue by making extracellular ORN recordings from the maxillary palps. Each sensillum in the palp contains exactly two ORNs, and a VA7l ORN is always paired with an ORN that projects to another glomerulus (de Bruyne et al., 1999
; Goldman et al., 2005
). In the palps of “rescued” flies, we encountered only silent sensilla, or sensilla containing exactly one spontaneously active ORN (n
=51 sensilla). This ORN always displayed odor tuning that matched the odor tuning of wild-type VA7l ORNs (, Supplemental Fig. S4
). We verified that the rescued ORNs correctly target glomerulus VA7l by co-expressing CD8:GFP with Or83b (). Furthermore, biocytin fills show that PNs postsynaptic to neighboring glomeruli do not inappropriately invade glomerulus VA7l (). This argues that the antennal lobe circuitry is grossly normal in the Or83b2
Two strategies for stimulating a single ORN type
This approach permits us to selectively stimulate exactly one ORN type. However, it has the drawback that most ORN types are inactive during the development of the fly, which could conceivably produce subtle changes in the antennal lobe circuitry. To address this issue, we used a second method to stimulate one ORN type under conditions in which almost all ORNs are normal and active throughout the life of the fly; this ensures that normal antennal lobe circuitry is preserved. In this approach (experiment 2, ), we sought to identify a panel of odors that only stimulates one ORN type. Because most ORNs respond to many different odors it is difficult to find such an odor set. To simplify the problem, we cut the antennal nerves just prior to recording, leaving only the six maxillary palp ORN types. Additionally, we used flies bearing the Δ85
mutation, which abolishes most odor responses in two of the six maxillary palp ORN types (Supplemental Figs. S5
). Thus, in antennae-less flies in a Δ85
background there are only four functional maxillary palp ORN types. We screened a large panel of odors and identified a set of 14 that exclusively stimulates the VM7 ORNs, while evoking no response from the other three functional ORN types in this genotype. This is demonstrated by local field potential recordings from the maxillary palp. When VM7 ORNs are functional, all odors in this set evoke a local field potential response. These responses are abolished by the Or42af04305
mutation (Thibault et al., 2004
), which renders VM7 ORNs nonfunctional (; see also Supplemental Figs. S5
). In summary, both experiments 1 and 2 permit selective stimulation of one ORN type.
In experiment 1 (genetically rescuing VA71 ORNs), we recorded from a total of 72 PNs postsynaptic to non-rescued glomeruli. PNs were selected at random from the dorsal cluster of PN cell bodies in the antennal lobe. Only one PN was recorded in each fly, which allowed us to unambiguously determine the glomerular identity of each PN after filling it with biocytin. Together, these 72 PNs targeted 24 out of the 49 glomeruli in the antennal lobe. In every one of these PNs, we observed a depolarization while stimulating the VA71 ORNs with odors. This implies that the VA71 ORNs broadcast indirect excitatory input to most glomeruli. As expected, the tuning of the lateral excitatory input to PNs always reflected the tuning of the VA71 ORNs (). Odors that excited VA71 ORNs produced depolarizations in PNs, whereas inhibition of VA71 ORNs led to a small hyperpolarization. This hyperpolarization probably represents an interruption in tonic lateral excitation driven by spontaneous action potentials in VA71 ORNs.
Odor stimulation of one ORN type evokes lateral input to many PNs
In experiment 2 (selective odor stimulation of VM7 ORNs), we recorded from a total of 15 PNs. We observed odor-evoked depolarizations in every one of these PNs. This suggests that the VM7 ORNs send lateral excitation indirectly to most glomeruli. And as expected, the tuning of the lateral excitatory input always reflected the tuning of the VM7 ORNs (). The average magnitude of this depolarization was about half of that observed in experiment 1.
In both experiment 1 (VA71-only) and experiment 2 (VM7-only), the magnitude of lateral depolarization did not scale linearly with ORN firing rate. Odors that evoked only a small ORN response produced a near-maximal lateral depolarization in PNs. Odors that evoked a larger ORN response saturated the lateral circuitry. This is shown by plotting the area under the membrane potential deflection (computed after low-pass filtering the membrane potential) versus ORN firing rate (). The nonlinearity of these curves illustrates both the sensitivity and the saturation of inter-glomerular excitatory circuits. To confirm that the lateral depolarizations in experiment 2 are driven by the VM7 ORNs, we combined the Δ85 mutation with an odorant receptor gene mutation that silences the VM7 ORNs (Or42af04305;Δ85). In this genotype, we saw essentially no odor responses in any PNs (, open circles, n=3).
These results demonstrate that ORN inputs from one glomerular processing channel can easily saturate the lateral excitatory circuitry of the antennal lobe. How are inputs from multiple ORN types integrated by this circuitry? We used an odor blend to investigate how odor-evoked signals from two ORN inputs are combined. In antennae-less Δ85 flies, the odor 2-butanone (10−5 dilution) activates only the VM7 ORNs (), while the odorant methyl salicylate (10−2 dilution) activates only the VA71 ORNs (data not shown). In isolation, these stimuli appear to saturate the lateral excitatory circuitry of the antennal lobe: stimulating VM7 PNs with 2-butanone evokes a maximal lateral depolarization in experiment 2, and stimulating VA71 PNs with methyl salicylate evokes a maximal lateral depolarization in experiment 1 (, magenta traces). What happens when these two inputs are integrated? When we blended the two odors to stimulate both VA71 and VM7 ORNs simultaneously in antennae-less Δ85 flies, we observed lateral depolarizations that were significantly larger than the response to either odor alone (; p≤0.01 for both comparisons, n=8 PNs, paired t-tests). This shows that multiple ORN inputs are effectively integrated by the lateral excitatory circuitry of the antennal lobe. However, the response to the blend was significantly smaller than that predicted by a linear sum of the responses evoked by stimulating each ORN type individually (; p<10−4, paired t-test). This demonstrates that saturation occurs across ORN input channels, not just within an ORN input channel.
Lateral excitatory circuits sub-linearly summate inputs from multiple ORN types
For a given ORN input (either VA71 or VM7), different PNs showed substantially different amounts of lateral depolarization in response to the same odor. In experiment 1, for example, the response evoked by the strongest odor (4-methyl phenol) ranged from 1.7 mV to 11.8 mV. Responses in some PNs were large enough to elicit a few spikes, but responses in other PNs responses were much weaker. This suggests that different PNs receive different amounts of lateral excitatory input from any given glomerulus. Are these connections random, or are they a stereotyped function of glomerular identity? To address this question, we compared the responses of PNs corresponding to the same glomeruli recorded in different flies. In our data set from experiment 1, there were 11 glomeruli that we hit at least 3 times. In experiment 2, we recorded selectively from PNs in only three glomeruli by using an enhancer trap line to label these cells with GFP (NP3481-Gal4,UAS-CD8:GFP;+/+;Δ85). This allowed us to obtain multiple recordings from the same PN type.
Overall, we found that the strength of lateral excitatory connections was relatively stereotyped across flies. In quantitative terms, differences we observed between glomeruli were larger than the variability we observed for a given glomerulus across flies. For example, in experiment 1, selective stimulation of VA71 ORNs produced a significantly greater depolarization in VC4 PNs compared to VA1v PNs (, p<0.05; VC4 n=7, VA1v n=8). In experiment 2, selective stimulation of VM7 ORNs produced a significantly greater depolarization in glomerulus DL5 than in glomerulus VM2 or DM6 (; p<0.05; DL5 n=5, VM2 n=3, DM6 n=7). We also noted that the nonlinear relationship between the lateral depolarization and ORN firing rate had a similar sensitivity and exponential shape across different glomeruli; it is the saturation level that differs (). Together, these results demonstrate that the amount of lateral excitatory input to a PN depends on the identity of the glomerulus where its dendrite is located. The magnitude of lateral depolarization is not correlated with the input resistance of the cell (Pearson’s r2=0.027), ruling out one trivial explanation for this systematic difference.
The strength of lateral connections is heterogeneous and stereotyped across flies
We next asked whether the magnitude of glomerular cross-talk varied systematically with distance. shows the relative connection strength for all 24 glomeruli we recorded from in experiment 1, mapped in relation to the location of the only glomerulus receiving direct ORN input (VA71). Connection strength is defined as the relative response to 4-methyl phenol, which is the strongest stimulus in our odor set for the VA71 ORNs. This map illustrates that there is no obvious relationship between inter-glomerular distance and the strength of lateral excitatory connections. We also asked if there is a correlation between connection strength and the morphological class of the ORN type corresponding to each glomerulus. Previous studies have shown that ORNs housed in the three major morphological classes of sensilla (basiconic, coeloconic, and trichoid) tend to project to nearby glomeruli and therefore define several zones in the antennal lobe (Couto et al., 2005
; Fishilevich and Vosshall, 2005
). shows that the zones defined by these sensillum classes receive similar overall levels of lateral excitation. Finally, we tested whether there is a relationship between the odor tuning of the normal ORN input to a glomerulus and the strength of excitatory lateral input it receives from VA71. Comprehensive odor tuning data for 16 of the colored glomeruli in has been compiled by other investigators (Hallem and Carlson, 2006
). We used this data set to compute the correlation coefficient (Pearson’s r
) between ORN odor responses for every pair-wise combination of these 16 glomeruli, and then plotted the difference in lateral depolarization for each pair as a function of this correlation. We found there is no relationship between glomerular tuning and depolarization strength ().
Lateral excitation is broadly distributed throughout the antennal lobe