Author response: Chronic exposure to odors at naturally occurring concentrations triggers limited plasticity in early stages of Drosophila olfactory processing
Year: 2023
Type: peer-review
Abstract: Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract In insects and mammals, olfactory experience in early life alters olfactory behavior and function in later life. In the vinegar fly Drosophila, flies chronically exposed to a high concentration of a monomolecular odor exhibit reduced behavioral aversion to the familiar odor when it is reencountered. This change in olfactory behavior has been attributed to selective decreases in the sensitivity of second-order olfactory projection neurons (PNs) in the antennal lobe that respond to the overrepresented odor. However, since odorant compounds do not occur at similarly high concentrations in natural sources, the role of odor experience-dependent plasticity in natural environments is unclear. Here, we investigated olfactory plasticity in the antennal lobe of flies chronically exposed to odors at concentrations that are typically encountered in natural odor sources. These stimuli were chosen to each strongly and selectively excite a single class of primary olfactory receptor neuron (ORN), thus facilitating a rigorous assessment of the selectivity of olfactory plasticity for PNs directly excited by overrepresented stimuli. Unexpectedly, we found that chronic exposure to three such odors did not result in decreased PN sensitivity but rather mildly increased responses to weak stimuli in most PN types. Odor-evoked PN activity in response to stronger stimuli was mostly unaffected by odor experience. When present, plasticity was observed broadly in multiple PN types and thus was not selective for PNs receiving direct input from the chronically active ORNs. We further investigated the DL5 olfactory coding channel and found that chronic odor-mediated excitation of its input ORNs did not affect PN intrinsic properties, local inhibitory innervation, ORN responses or ORN-PN synaptic strength; however, broad-acting lateral excitation evoked by some odors was increased. These results show that PN odor coding is only mildly affected by strong persistent activation of a single olfactory input, highlighting the stability of early stages of insect olfactory processing to significant perturbations in the sensory environment. Editor's evaluation Does exposure to odors induce some type of adaptive plasticity at the early layers of the olfactory circuit? In this valuable work, the authors use naturalistic concentrations of odor to provide convincing evidence that there is actually very little plasticity in the projection neurons at the glomerular layer of the system. Their findings show small increases in responses to low concentrations of the exposed odor. https://doi.org/10.7554/eLife.85443.sa0 Decision letter eLife's review process Introduction In many animals, early sensory experience modifies the structure and function of sensory systems. For example, visual experience is required for the normal development of the mammalian visual system (Espinosa and Stryker, 2012). One prominent hypothesis is that an important function of sensory plasticity is to adapt circuit function to the current statistical distribution of sensory inputs in the environment, allowing for more efficient sensory codes (Barlow, 1961; Fiser et al., 2010; Gilbert et al., 2009; Pienkowski and Eggermont, 2011). This hypothesis requires sensory driven plasticity to be stimulus- and cell-specific; in other words, neurons encoding specific stimuli that occur very frequently (or very rarely) in the environment should be selectively affected by plasticity (Das et al., 2011; Kreile et al., 2011; Sachse et al., 2007; Sengpiel et al., 1999; Wilson et al., 1985; Zhang et al., 2001). The circuit mechanisms that would implement such use-dependent, input-specific plasticity are not well understood. The orderly structure of the olfactory system provides a useful experimental model for investigating the synaptic and circuit mechanisms mediating stimulus-selective sensory plasticity. In insect and vertebrate olfactory circuits, sensory information is organized in anatomically discrete synaptic compartments, called glomeruli. Each glomerulus receives direct input from only a single class of primary olfactory receptor neurons (ORNs), all expressing the same olfactory receptor, and, thus, all sensitive to the same chemical feature(s) (Ressler et al., 1994; Vassar et al., 1994; Vosshall and Stocker, 2007; Vosshall et al., 2000). Furthermore, the dendrites of each second-order uniglomerular projection neuron (PN) arborize in only a single glomerulus, so each PN receives direct input from only a single class of ORNs (Stocker et al., 1990). In the vinegar fly Drosophila melanogaster, the majority of odorant receptors and ORN subtypes have been mapped to their corresponding glomeruli in the brain (Couto et al., 2005; Fishilevich and Vosshall, 2005; Silbering et al., 2011), and the odor tuning profiles for a large subset of the odorant receptors have been characterized (Silbering et al., 2011; de Bruyne et al., 1999; de Bruyne et al., 2001; Hallem and Carlson, 2006; Hallem et al., 2004). As a result, specific odors can be used to selectively target neural activation of defined olfactory channels (Olsen et al., 2010; Schlief and Wilson, 2007). Together with the highly compartmentalized organization of the circuit, these features make the fly olfactory system a powerful experimental model for studying the specificity of sensory plasticity. Passive odor experience in early life, in the absence of explicit coupling to reward or punishment, can alter olfactory circuit structure and function, including olfactory preference or discrimination ability (Mandairon and Linster, 2009; Mandairon et al., 2006a). For instance, chronic odor exposure in rodents can trigger changes in the structural connectivity and physiological response properties of neurons in the olfactory bulb, the first central processing area for odors in the brain (Wilson et al., 1985; Liu and Urban, 2017; Liu et al., 2016; Todrank et al., 2011; Woo et al., 2006). In insects, passive odor experience has also been found to impact olfactory processing in the antennal lobe, the insect analog of the olfactory bulb (Golovin and Broadie, 2016; Twick et al., 2014; Bai and Suzuki, 2020). Chronic exposure to high concentrations of monomolecular odor, stimuli which are typically strongly aversive, reduces behavioral avoidance toward the familiar odor selectively when it is reencountered (Das et al., 2011; Devaud et al., 2001; Devaud et al., 2003); in parallel, structural alterations in glomerular volume are also observed (Das et al., 2011; Sachse et al., 2007; Devaud et al., 2001; Devaud et al., 2003; Golovin et al., 2019; Kidd et al., 2015). Odor experience-dependent reductions in olfactory aversion have been interpreted as a form of long-term behavioral habituation to overrepresented stimuli in the environment and are attributed to reduced PN responses in flies chronically exposed to odor (Das et al., 2011; Sachse et al., 2007; see also Kidd et al., 2015). Importantly, since behavioral plasticity is odor-specific and does not generalize to other odors, structural and functional neural plasticity are also believed to be glomerulus-specific (and thus odor-specific), acting to selectively reduce the olfactory sensitivity of only those PNs activated by the overrepresented stimuli. However, since past studies exposed animals to intense monomolecular odors (Das et al., 2011; Sachse et al., 2007; Devaud et al., 2001; Devaud et al., 2003; Golovin et al., 2019; Chodankar et al., 2020; e.g. from odor sources of 10% isoamyl acetate [v/v] or 20% ethyl butyrate [v/v]), which typically excite many classes of ORN inputs, stringent testing of this idea has not been possible. Arriving at a systematic understanding of how odor experience modifies the structure and function of olfactory circuits has been challenging for several reasons. First, diverse protocols are used for odor exposure, which vary in the degree of control over odor delivery, odor concentration, timing, as well as context (availability of food, mates, etc). Second, different studies focus on different odors and glomeruli, and the high dimensionality of chemical stimulus space and olfactory circuits presents unique challenges to methodical exploration. Finally, nearly all studies, in insects or in mammals, use very high concentrations of monomolecular odorants during the exposure period, at intensities that are not encountered in the natural world (see Discussion). Odors at these concentrations broadly activate many classes of ORNs, complicating the evaluation of the contributions of direct and indirect activity for triggering plasticity in each olfactory processing channel. Some of the major outstanding questions include: (1) How does olfactory plasticity modify circuit function in the context of odor environments that could be realistically encountered in the natural world? (2) To what extent is plasticity selective for the olfactory channel(s) which directly detect overrepresented odors? (3) Are the rules governing olfactory plasticity the same or different across glomeruli? The goals of this study were to investigate the impact of olfactory experience on odor coding in the Drosophila antennal lobe using a physiologically plausible olfactory environment that strongly but selectively increases neural activity in a single class of ORNs. This experimental design allowed us to readily distinguish the effects of direct versus indirect chronic activity on specific classes of PNs, which convey neural output from the antennal lobe to higher olfactory centers in the fly brain. This distinction is important because it allows us to unambiguously evaluate whether olfactory plasticity selectively affects only the chronically active glomerulus. Investigating three different glomerular channels, we unexpectedly observed that, rather than reduce PN sensitivity, chronic odor exposure resulted in mild increases in PN sensitivity, particularly in response to weak odors. PN responses to moderate or strong stimuli were mostly unaffected. Changes in odor responses, when present, were observed broadly, both in glomeruli that receive either direct or indirect activity from the chronically active ORN class. These results diverge from current models suggesting that odor-specific behavioral plasticity arises from a glomerulus-specific long-term adaptation of PN responses in the antennal lobe (Das et al., 2011; Sachse et al., 2007; Chodankar et al., 2020; Sudhakaran et al., 2012) and motivate the search for other circuit mechanisms mediating how olfactory experience alters behavior toward familiar odors. Results Chronic activation of direct ORN input modestly increases PN responses to weak stimuli To investigate how odors that are overrepresented in the environment are encoded by the fly olfactory system, we chronically exposed flies to 1 s pulses of a specific monomolecular odorant, introduced into the bottle in which they normally grow (Figure 1A). We chose to use the odors at concentrations previously shown to selectively activate a single class of ORNs (Olsen et al., 2010; Hong and Wilson, 2015) in order to simplify the investigation of odor coding in postsynaptic PNs receiving direct versus indirect chronic excitation. An additional criterion was that the odor stimuli drive strong, consistent, and saturating levels of firing in PNs receiving direct input from the activated ORN class (in other words, using a higher concentration of odor would not reliably drive higher firing rates in the PN type that is directly excited by that stimulus). Photoionization measurements of the odor concentration in the rearing bottle demonstrated that the stimulus amplitude changed <20% over 12 hr (Figure 1B), after which the odor source was refreshed. The largest decrease in delivered odor concentration occurred within the first 2 hr and slowed significantly thereafter. Odors were pulsed to minimize neural adaptation to the odor, and the interval between odor pulses delivered to the bottle was 20 s. Pilot recordings showed that, at this interstimulus interval, the odors reliably activated cognate PNs to saturating or near saturating firing rates over many trials (Figure 1C–D, and data not shown; see also Figure 2Q). In addition, PN response duration to the 1 s stimulus increased modestly with successive odor presentations. Thus, although the odor stimuli to which we exposed flies were of significantly lower concentration than what has been previously used to investigate the effect of chronic odor exposure on PNs (Das et al., 2011; Sachse et al., 2007; Kidd et al., 2015), these stimuli drove high, reliable, near saturating levels of firing in their corresponding PN type (Figure 1C, data not shown). On average, this odor exposure protocol more than doubled the average firing rate of the targeted PN and elicited more than a million extra spikes in the activated PN over the course of the 2-day period of exposure (see Materials and methods). Figure 1 Download asset Open asset Chronic stimulation of olfactory neurons in a controlled odor environment. (A) Schematic of experimental setup for chronic odor exposure. The valve was opened for 1 s every 20 s to deliver odor. See Materials and methods (chronic odor exposure) for details. (B) Photoionization detector measurement of odor concentration at the center of the fly bottle ("X" in A) during chronic odor exposure to 2-butanone (10–4) over the course of 24 hr. Each trace is the odor profile averaged across 45 consecutive odor pulses (collected over 15 min), sampled every 2 hr over a 24 hr period. (C) Raster plots of the spiking responses in an example DL5 projection neuron (PN) to a 1 s pulse of either solvent (paraffin oil, gray) or E2-hexenal (10–7, black) in consecutive trials spaced 20 s apart. Recordings were established from a fly immediately after 2 days of chronic exposure to E2-hexenal (10–7) as in (A). Spontaneous (open circles) and stimulus-evoked (filled circles) firing rates are plotted for each trial. (D) Peristimulus time histograms of measurements in (C) show that the odor environment reliably evokes high levels of PN firing across presentation trials. The average odor-evoked response across all trials is in black. Responses to presentation of solvent are overlaid in gray. Figure 2 Download asset Open asset Chronic excitation of direct presynaptic olfactory receptor neuron (ORN) input can modestly enhance projection neuron (PN) sensitivity to weak stimuli. (A) Schematic of experimental setup for (B–F). Recordings were established from DL5 PNs that receive direct presynaptic input from the ORN class (ab4a) chronically activated by the rearing odor (E2-hexenal, 10–7), n=7–19 cells. (B) Odor-evoked depolarization in DL5 PNs in response to varying concentrations of E2-hexenal from flies chronically exposed to E2-hexenal or solvent (paraffin oil). (C) Left: mean odor-evoked depolarization to each stimulus in (B) in the 500 ms after nominal stimulus onset. Right: mean normalized odor-evoked depolarization across all stimuli. Within each stimulus, responses were normalized to the mean odor-evoked depolarization in the solvent-exposed group. (D) Peristimulus time histograms of odor-evoked spiking responses in DL5 PNs from (B). (E) Left: mean odor-evoked firing rates to each stimulus from (D) in the 500 ms after nominal stimulus onset. Right: mean normalized odor-evoked firing rates across all stimuli, computed analogously as in (C). (F) Mean input resistance of DL5 PNs recorded from E2-hexenal- or solvent-exposed flies. (G) Experimental setup for (H–J). Recordings were established from VM7 PNs that receive direct presynaptic input from the ORN class (pb1a) chronically activated by the rearing odor (2-butanone, 10–4), n=6–11 cells. (H–I) As in (B–C) but describing odor-evoked depolarization in VM7 PNs in response to a concentration series of 2-butanone from flies chronically exposed to 2-butanone or solvent. (J) Mean input resistance of VM7 PNs recorded from 2-butanone- or solvent-exposed flies. (K) Experimental setup for (L–P). Recordings were established from VA6 PNs that receive direct presynaptic input from the ORN class (ab5a) chronically activated by the rearing odor (geranyl acetate, 10–4), n=7–11 cells. (L–P) As in (B–F) but describing odor-evoked responses to varying concentrations of geranyl acetate in VA6 PNs from flies chronically exposed to geranyl acetate or solvent. (Q–S) Response curves in DL5 (Q), VM7 (R), and VA6 (S) PNs from odor-exposed and control flies to a concentration series of the cognate odor for each glomerulus. Results from panels (A–P) are replotted here and extended with measurements at additional stimulus concentrations. n=3–19 cells. All plots are mean ± SEM across flies in each experimental condition. One cell was recorded per fly. Two-tailed p-values report the fraction of resampled absolute differences of means (between simulated experimental groups) which are greater than the absolute observed difference between the means of experimental groups (odor-exposed versus solvent-exposed; see Materials and methods). Starred (*) p-values are significant at the level of α=0.05, corrected for multiple comparisons (Bonferroni adjustment). See Supplementary file 1 for the full genotype and number of cells in every condition. Figure 2—source data 1 Source data for Figure 2B–C, D–E, H–I, L–M and N–O. https://cdn.elifesciences.org/articles/85443/elife-85443-fig2-data1-v2.xlsx Download elife-85443-fig2-data1-v2.xlsx Using these conditions, newly eclosed flies were chronically exposed for 2 days to E2-hexenal (10–7), which selectively activates ORNs projecting to glomerulus DL5, or to solvent (as a control) (see Materials and methods). On day 3, we established fluorescently guided, whole-cell current clamp recordings from uniglomerular PNs receiving direct input from the DL5 glomerulus (hereafter referred to as DL5 PNs, Figure 2A), and measured their responses to a concentration series of E2-hexenal. DL5 PNs were identified and targeted for recording based on their expression of green fluorescent protein (GFP), mediated by a genetic driver that specifically labels this cell type (see Materials and methods). DL5 PN responses elicited by moderate to high concentrations of E2-hexenal (>10–8) were unchanged in E2-hexenal exposed flies, as compared to controls (Figure 2B and Q). However, low concentrations of E2-hexenal (10–10–10–9) elicited increased levels of odor-evoked membrane depolarization in DL5 PNs from E2-hexenal exposed flies compared to solvent-exposed flies (Figure 2B). The heightened depolarization of DL5 PNs by weak stimuli corresponded to higher average rates of odor-evoked spiking (Figure 2D). We quantified these effects by calculating the total depolarization and average evoked firing rate during the first 500 ms after nominal stimulus onset (Figure 2C and E). To determine if any differences were arising by chance, we used permutation testing in which we iteratively shuffled the experimental labels of each measurement (E2-hexenal versus solvent exposure) within each stimulus. p-Values were calculated directly from the fraction of 10,000 shuffled trials in which the absolute difference between the simulated group means was larger than or equal to the actual observed absolute mean difference (see Materials and methods). This statistical analysis confirmed that E2-hexenal exposure increased odor-evoked firing rates in DL5 PNs to weak but not strong levels of stimulation (Figure 2E). When firing rates in E2-hexenal exposed flies were normalized to the control rate within each stimulus, we observed an overall increase in odor-evoked DL5 PN firing rate due to E2-hexenal exposure (Figure 2E-F). Differences in the amount of membrane depolarization between odor- and solvent-exposed groups were not statistically significant at any stimulus concentration (Figure 2C), suggesting a small, but systematic increase in membrane depolarization was nonlinearly amplified by its interaction with the firing threshold in DL5 PNs. We next asked whether these results generalize to other glomeruli. Using the same approach, we exposed flies to either 2-butanone which selectively activates ORNs projecting to glomerulus VM7 (Figure or geranyl acetate which selectively activates ORNs projecting to glomerulus VA6 (Figure The concentrations of these odors were chosen because each stimulus selectively and reliably high firing rates in its corresponding to the level of E2-hexenal (10–7) activation of DL5 PNs. we chronically exposed flies groups) for 2 days to each of these stimuli and measured the responses in each PN type to the glomerulus receiving direct input from the activated to a concentration series of each odor (Figure and the DL5 glomerulus, VM7 PNs in flies increases in odor-evoked depolarization in response to direct stimulation as compared to control flies, and these effects were more at weak concentrations Figure and VM7 PN spikes are small and in to those of other PNs, and odor-evoked spikes on large could not be reliably across all firing rates in data for VM7 PNs we report odor responses only in of membrane However, chronic activation of direct ORN input to VA6 PNs by exposure to geranyl acetate did not alter PN odor responses across the of concentrations of the familiar odor geranyl acetate, at the level of membrane depolarization firing rate (Figure and These concentrations elicited levels of membrane depolarization from to which were to that at which other PN types plasticity after chronic exposure. these results that, in some glomeruli DL5 and chronic activation of direct ORN input modestly PN odor responses to weak stimuli, in an overall of the of stimulus concentrations encoded by PN However, this effect does not to be across all glomeruli. Chronic excitation of ORNs in other glomeruli alters PN response properties olfactory input is compartmentalized into channels organized each glomerular odor processing on an of local neurons that lateral and inhibitory glomeruli Thus, PN odor responses both direct input from its presynaptic ORN and indirect input, arising from activity in other glomeruli and local lateral observed that chronic activation of an ORN class that provides direct input to a PN can some plasticity in that we next asked whether this plasticity is selective for those PNs directly postsynaptic to the chronically active ORNs, or whether PNs that receive only indirect activity from the chronically active ORN are similarly To this we odor responses in VM7 and VA6 PNs from flies chronically exposed to E2-hexenal Figure and which provides direct excitation to the DL5 glomerulus. Figure Download asset Open asset Chronic excitation of olfactory receptor neurons in other glomeruli alters projection neuron (PN) response (A) Experimental setup for and Recordings were established from VM7 PNs that receive indirect activity from the ORN class (ab4a) chronically activated by the rearing odor (E2-hexenal, 10–7), cells. (B) Odor-evoked depolarization in VM7 PNs in response to varying concentrations of 2-butanone from flies chronically exposed to E2-hexenal or solvent (paraffin oil). (C) Left: mean odor-evoked depolarization to each stimulus from (B) in the 500 ms after nominal stimulus onset. Right: mean normalized odor-evoked depolarization across all stimuli. Within each stimulus, responses were normalized to the mean odor-evoked depolarization in the solvent-exposed group. (D) Mean input resistance of VM7 PNs recorded from E2-hexenal- or solvent-exposed flies. (E) Left: mean in VM7 PN responses to each stimulus from calculated over a s after stimulus Right: mean normalized across all stimuli. Within each stimulus, responses were normalized to the mean in the solvent-exposed group. (F) as but for VM7 PNs from Figure with chronic activation of direct ORN are from VM7 PN recordings in flies chronically exposed to 2-butanone or solvent n=6–11 cells. (G) Mean of of membrane in VM7 PNs computed over the s stimulus in E2-hexenal- or solvent-exposed flies. Mean firing rate of VM7 PNs during the s stimulus onset in E2-hexenal- or solvent-exposed flies. Experimental setup for Recordings were established from VA6 PNs that receive indirect activity from the ORN class (ab4a) chronically activated by the rearing odor (E2-hexenal, 10–7), As in (B–C) but for odor-evoked depolarization in VA6 PNs to varying concentrations of geranyl As in (D) but for VA6 PNs. As in (B–C) but for odor-evoked spiking in VA6 PNs in response to varying concentrations of geranyl acetate to from flies chronically exposed to E2-hexenal (10–7) or solvent (paraffin oil). All plots are mean ± SEM across flies, in each experimental condition. p-Values are as in Figure See Supplementary file 1 for the full genotype and number of flies in every condition. Figure data 1 Source data for Figure and Download Chronic indirect excitation evoked plasticity with varying properties in different PNs. In E2-hexenal exposed flies, PNs mildly responses to weak stimuli (Figure and This effect was small but observed in both VM7 and VA6 PNs at the level of odor-evoked depolarization (Figure and In VM7 PNs, the firing rate also higher in E2-hexenal exposed flies as compared to controls (Figure Finally, chronic indirect excitation the response properties of some PNs. For example, odor-evoked depolarization in VM7 PNs from flies a more and after as compared to controls (Figure and or as compared to flies that chronic direct excitation Figures and This effect does not to generalize to all glomeruli. VA6 PNs, for instance, exhibit different response characterized by an of excitation that odor (Figure In recordings from VA6 PNs in E2-hexenal exposed flies, this excitation was across multiple odor as compared to solvent-exposed controls (Figure of these however, were within the of and so the overall impact of chronic indirect excitation on VA6 firing rates was mild (Figure these demonstrated that excitation of a single ORN class can to changes in PN odor response properties in multiple glomeruli, including in glomeruli not receiving direct synaptic input from the chronically activated ORN class. This result local lateral in the antennal lobe in PN plasticity after chronic exposure to odor. Chronic exposure to intense monomolecular odor can affect PN odor responses similarly to chronic exposure at naturalistic concentrations The of results so from those of some past studies, which that chronic odor exposure reductions in PN sensitivity (Das et al., 2011; Sachse et al., was in how PN odor responses were measured versus in past the major difference in study is the odor environment in which flies are past studies the odor a of monomolecular odor into the environment of the we introduced monomolecular odors at lower concentrations as pulses into the was that odors at these lower concentrations activate their corresponding PN type very strongly but are at a concentration that could occur in a natural thus, the more models how an odor overrepresented in a natural environment impact olfactory However, we next to how exposure to intense monomolecular odor affects PN We introduced into the bottle of the flies a small either 20% geranyl acetate more than in Figure or solvent The of the was with to
Source: eLife
Institution: California Institute of Technology
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4381572035