Multitasking in the olfactory system: context-dependent responses to odors reveal dual GABA-regulated coding mechanisms in single olfactory projection neurons

Abstract

Studies of olfaction have focused mainly on neural processing of information about the chemistry of odors, but olfactory stimuli have other properties that also affect central responses and thus influence behavior. In moths, continuous and intermittent stimulation with the same odor evokes two distinct flight behaviors, but the neural basis of this differential response is unknown. Here we show that certain projection neurons (PNs) in the primary olfactory center in the brain give context-dependent responses to a specific odor blend, and these responses are shaped in several ways by a bicuculline-sensitive GABA receptor. Pharmacological dissection of PN responses reveals that bicuculline blocks GABAA-type receptors/chloride channels in PNs, and that these receptors play a critical role in shaping the responses of these glomerular output neurons. The firing patterns of PNs are not odor-specific but are strongly modulated by the temporal pattern of the odor stimulus. Brief repetitive odor pulses evoke fast inhibitory potentials, followed by discrete bursts of action potentials that are phase-locked to the pulses. In contrast, the response to a single prolonged stimulus with the same odor is a series of slow oscillations underlying irregular firing. Bicuculline disrupts the timing of both types of responses, suggesting that GABAA-like receptors underlie both coding mechanisms. These results suggest that glomerular output neurons could use more than one coding scheme to represent a single olfactory stimulus. Moreover, these context-dependent odor responses encode information about both the chemical composition and the temporal pattern of the odor signal. Together with behavioral evidence, these findings suggest that context-dependent odor responses evoke different perceptions in the brain that provide the animal with important information about the spatiotemporal variations that occur in natural odor plumes.

Fig. 1.

Local interneurons and projection neurons are the two main types of neurons involved in processing olfactory information in the glomeruli of the AL. Both cell types are readily identifiable in neuropil recordings, based on their differential responses to electrical stimulation of the antennal nerve and their distinctly different responses to treatment with GABA receptor antagonists.A, Serial reconstructions of one local interneuron (LN) and one projection neuron (PN) from two preparations, showing the overlap of their arborizations in the male-specific macroglomerular complex (MGC). PNs with arborizations in the MGC respond selectively to the female sex pheromone (Hansson et al., 1991;Christensen et al., 1996). LNs are wide-field amacrine neurons that are confined to the AL and are mostly, if not exclusively, GABAergic. PNs have an axon that exits the lobe (dashed line) and terminates in higher centers in the protocerebrum (PR). AN, Antennal nerve;OE, oesophageal canal; OL, optic lobe.B, Intracellular records illustrate the different postsynaptic responses evoked in an LN (top records) and a PN (bottom records) by brief electrical stimulation of the ipsilateral AN (asterisks). Shock artifacts are clipped. Records on theleft show responses in normal saline, and records on theright show the effects of the GABA_A receptor antagonist bicuculline (100 μm). Each record shows multiple consecutive sweeps using a stimulus pulse frequency of 10 Hz. Under normal conditions, the LN exhibited a short-latency excitatory response that followed high-frequency stimulation, whereas the PN exhibited a multiphasic response with an early IPSP (I₁) that became increasingly attenuated with successive stimulus pulses. Repetitive stimulation also led to a small reduction in the latency of the first spike in the PN, but no such time shift was observed in the initial LN spike. A 10 min bath application of bicuculline had no effect on the latency of the early spike response of the LN, whereas the same treatment eliminated the I₁ phase of the PN response, leading to a marked desynchronization and reduced latency of the PN spike.C, The responses of 12 PNs in as many males to short intermittent pulses of female sex pheromone illustrate how a time-varying odor stimulus modulates the temporal pattern of PN spike activity (stacked dot raster plots). The intermittent stimulus pattern shown here consisted of four 100 msec pulses separated by 100 msec intervals, and the timing of the voltage commands to the stimulus device are shown beneath the plots as a series of black bars. Each stimulus pulse evoked a train of spikes that was phase-locked to the intermittent stimulus pattern. Note too that the duration of every spike train copied the duration of the odor pulse, and that the temporal pattern of activity in each spike train is variable, both within and between cells, unlike the responses of PNs reported for other insect species (see Discussion).

Fig. 2.

Effects of injected current and Cl⁻ ion substitution on the postsynaptic potentials recorded in a moth PN. A, Electrical stimulation of the antennal nerve (asterisk) evoked the characteristic triphasic response in the PN, and each phase responded differently to a shift in the resting membrane potential. B, The two inhibitory phases (I₁ andI₂) displayed distinctly different reversal potentials in response to injected current. Plots of IPSP amplitude versus membrane potential (relative to rest) reveal that the later relatively prolonged I₂ phase of the PN response (○—○) had a reversal potential that was nearly 10 mV more negative than that of the early fast I₁ phase (•—•).C, I₁ and I₂ evoked by antennal nerve shock (asterisks) were affected differently by changes in the value of E_Cl. Reducing extracellular chloride by substituting 18.7 mmNa₂SO₄ for 37.5 mm NaCl (top records) or increasing intracellular chloride by passing hyperpolarizing current (bottom records) led to a decrease and reversal of I₁, but not of I₂ (broken traces). Either treatment caused I₁ to become depolarizing, accompanied by increased excitability in the PN (E). Effect inbottom records is shown immediately after current was discontinued. The effects of both treatments were highly reproducible and readily reversible after only a few minutes of recovery time. The I₁ phase recovered fully after a return to normal saline solution for 5 min (top) or cessation of hyperpolarizing current for 5 min (bottom). Resting potential is indicated by dashed horizontal lines.

Fig. 3.

Intracellular responses of a PN to pressure ejection of GABA and several GABA receptor agonists (at 100 mm) into the antennal lobe neuropil. A brief (50 msec) GABA pulse evoked a rapid onset and prolonged hyperpolarization in the PN that could prevent spiking for >10 sec. The GABA_A agonist muscimol mimicked the response to GABA. In contrast, baclofen, a potent GABA_B agonist, evoked only a very small and delayed membrane hyperpolarization, whereas cis-4-aminocrotonic acid (CACA), a potent GABA_C agonist, had no detectable effect. Onset of the pressure pulse is indicated by thedashed vertical line through the records. Spikes in all but the top record are clipped.

Fig. 4.

Comparison of intracellular PN responses to applied GABA and ACh (both at 100 mm) and the effect of pretreatment with bicuculline (100 μm) pressure ejected into the neuropil. Onset of transmitter pulse is indicated by thedashed vertical line through the records. Pressure ejection of GABA evoked rapid, prolonged, and dose-dependent membrane hyperpolarization. A 100 msec pulse of bicuculline immediately preceding a 100 msec pulse of GABA completely and reversibly abolished the GABA-evoked hyperpolarization. In contrast to GABA, a 100 msec pulse of ACh evoked a membrane depolarization and increased spiking activity. Pretreatment with bicuculline did not block, but instead led to a marked facilitation of, the excitatory response to ACh. The responses to GABA and ACh were reproducible and reversible. Spikes in all records are clipped.

Fig. 5.

Summary of responses to GABA and ACh and the effects of pretreatment with 100 μm BMI. For both transmitters, amplitude of evoked membrane hyperpolarization was used as a measure of inhibitory responses, and number of evoked spikes above background was used as a measure of excitatory responses. In the majority of cases, responses to GABA were inhibitory and blocked by BMI, whereas responses to ACh were excitatory and facilitated by BMI.Numbers in parentheses are replicates, and all data are mean ± SEM.

Fig. 6.

Temporal pattern of action potentials in response to a single odor pulse was strongly dependent on the duration of the odor pulse, and this pattern increased in complexity as the stimulus duration increased. Stimulus is the species-specific sex pheromone blend (stimulus pulses indicated by bar beneath each record). A relatively brief 300 msec odor pulse evoked a train of spikes that lasted for 309 msec before being abruptly halted by a strong membrane hyperpolarization. As the stimulus duration was lengthened from 1 to 2 to 5 sec, each successive pulse evoked a greater number of alternating inhibitory and excitatory potentials, and the response of PNs became increasingly oscillatory. These oscillations could be a consequence of extensive synaptic interconnection with GABAergic LNs in the antennal lobe and suggest the emergence of a different computational mechanism with prolonged stimulation.

Fig. 7.

Temporal response to odor also depended on the temporal pattern of input to the PN. A, Responses to stimulation with a series of five 100 msec odor pulses at 500 msec intervals. Intermittent stimulation evoked a discrete burst of spikes for each pulse (top trace). This temporal pattern was completely disrupted after bath application of 100 μmbicuculline for 5 min (middle trace). Blocking the fast GABA-mediated chloride conductance in the PN revealed the underlying excitatory input to the neuron, as indicated by increased tonic spiking activity. Note, however, that the slower later hyperpolarizing phase that signals the end of the response was delayed but not blocked by bicuculline. The stimulus-modulated spike pattern returned after a switch back to normal saline solution for 10 min (bottom trace). B, Responses in the same PN to a single 500 msec pulse show that the same stimulus could evoke a different temporal pattern of activity in the PN (top trace). In this case, the initial burst of spikes was followed by a series of periodic fluctuations in membrane potential accompanied by irregular spiking activity. The membrane fluctuations and temporal spiking pattern were eliminated by bicuculline (middle trace). Again, this effect was readily reversible; a temporally patterned response returned after washout (bottom trace), but the temporal pattern of action potentials was not the same as that before bicuculline treatment.

Fig. 8.

Model of two opposing parallel pathways converging on a glomerular PN based on the current pharmacological, anatomical, and electrophysiological data. Two feedforward pathways and one feedback pathway to the PN are shown, but others yet to be characterized may exist. According to this model, bicuculline blocks the inhibitory input to the PN, mediated through the population of GABAergic local interneurons (LNs) (only one shown). Bicuculline blocks both the short-latency IPSP generated by feedforward inhibition (Fig. 1B) and the slow oscillations (Fig. 7B) possibly generated by a feedback pathway to the same pool of inhibitory LNs. Output synapses that could mediate this effect have been identified in PNs. Bicuculline does not block the excitatory input pathway (+), however, regardless of whether the PN receives direct input from primary afferent axons (as shown) or indirect input through other intercalated neurons. The fact that I₁ always precedes the excitation in these PNs is additional evidence for an indirect excitatory input.