Mark Stopfer, PhD, Head, Unit on Sensory Coding and Neural Ensembles
Iori Ito, PhD, Postdoctoral Fellow
Joby Joseph, PhD, Postdoctoral Fellow
Baranidharan Raman, PhD, Postdoctoral Fellow
Nobuaki Tanaka, PhD, Postdoctoral Fellow
Stacey Brown, MS, Technician
Kui Sun, MD, Technician
Rose Chik Ying Ong, MS, Graduate Student
Jeffrey Tang, College Student
Ryan Tsou, College Student

All animals need to know what is going on in the world around them; thus, brain mechanisms have evolved to gather and organize sensory information and to build transient and sometimes enduring internal representations of an animal's surroundings. Using relatively simple animals and focusing primarily on olfaction, we combine electrophysiological, anatomical, behavioral, and other techniques to examine the ways intact neural circuits, driven by sensory stimuli, process information. In the past year, our research program has begun to investigate the mechanisms—including transient oscillatory synchronization and slow temporal firing patterns of ensembles of neurons—that underlie information coding and decoding; how multimodal stimuli are integrated into unified perceptions; and how innate sensory preferences are determined. Our work reveals basic mechanisms by which sensory information is transformed, stabilized, and compared as it makes its way through the nervous system.

Encoding a temporally structured olfactory stimulus with a temporally structured neural code

Brown, Joseph, Stopfer

A variety of sensory neural systems use spatiotemporal coding mechanisms to represent stimuli. These time-varying response patterns elicited by the stimuli sometimes endure longer than the stimuli themselves. We were interested in determining whether the temporal structure of a stimulus could interfere with or even disrupt the spatiotemporal structure of the neural representation. We investigated this potential confound in the locust olfactory system. When biologically relevant, non-pheromonal odors were presented in trains of nearly overlapping pulses, as occurs naturally in odor plumes, responses of first-order interneurons (the projection neurons) changed reliably, significantly, and often greatly with pulse position, as responses to one pulse interfered with subsequent responses. Thus, the temporal structure of the stimulus did indeed interfere with the temporal structure of the neural representation in the individual neurons.

However, in the locust olfactory system (as in the vertebrate system), large numbers of individual projection neurons converge upon follower cells such that coding appears to be achieved by means of an ensemble mechanism. Further, the oscillatory synchronization mechanism engaged by odorants results in a shutter-like parsing of projection neuron activity into a series of discrete time bins, each the duration of a single oscillatory cycle (about 50 milliseconds). We designed an analysis that was guided by our knowledge of how the olfactory system decodes the output of the projection neurons. Using a custom-built multi-unit amplifier, we made simultaneous recordings from large groups of projection neurons. Next, guided by follower neuron responses to the projection neuron ensemble, we devised a statistical means for combining the responses of an ensemble of projection neurons as a series of discrete 50-millisecond time bins. By examining projection neuron activity in this biologically relevant way and using a novel statistical analysis, we found that we could accurately classify the odorants while also characterizing the temporal properties of the stimulus. Further, using an extracellular recording technique, we found that second-order, follower neurons (Kenyon cells) showed firing patterns consistent with the information in the projection neuron ensemble. Thus, we established that ensemble-based spatiotemporal coding can disambiguate complex and potentially confounding temporally structured sensory stimuli, providing an invariant response to a stimulus presented in various ways.

Central and peripheral plasticity in early olfactory processing

Joseph, Brown, Stopfer

As information moves through the brain, it is dramatically transformed in myriad ways. Our previous work suggested that one of the general mechanisms responsible such restructuring is neural plasticity. We investigated the mechanisms underlying the temporally invariant neural codes described above and found evidence for both excitatory and inhibitory plasticity.

To investigate mechanisms underlying the temporally invariant responses, we delivered repeated, rapid pulses of odors with timing designed to mimic features of natural plumes and monitored, in intact animals, neural activity in several locations: olfactory receptor neurons, first-order interneurons, and second-order interneurons. At each location, we sought to understand responses in terms of the interactions of plasticity occurring at earlier sites. We also sought to understand the potential value to the animal of the restructurings.

We found that interneuronal responses to natural forms of odor stimuli are shaped by at least two interacting, plastic mechanisms: rapid adaptation in the receptors and relatively enduring facilitation of inhibition within the downstream targets of the receptors. Peripheral adaptation appears to make the olfactory system relatively insensitive to stimuli that repeat very rapidly. Central facilitation of inhibition increases the reliability and sparseness of stimuli that are encountered repeatedly but relatively slowly. Further, these mechanisms constrain the projection neuron ensemble to provide relatively stable output to its downstream followers, thereby allowing the encoding of information about odor identity and concentration with firing patterns that are not confounded by the timing patterns of the stimulus.

One of our goals is to determine how follower neurons decode this time-varying ensemble activity. Intracellular and extracellular recordings from Kenyon cells showed that their firing rates change dramatically throughout trains of odor pulses in a timing-dependent manner: for brief interpulse intervals, the great majority of action potentials fire at the beginning of the train and again following the train's conclusion. We found that the Kenyon cell firing threshold can be met when projection neurons fire at relatively low rates but are highly synchronized by the oscillatory mechanism of the antennal lobe (as occurs during the onset of the pulse train). On the other hand, the threshold can be met when the instantaneous firing rate of the projection neuron ensemble is high in the absence of pronounced synchronization (as occurs following the offset of the train).

Taken together, our investigations suggest that the non-associative plasticity elicited by odor plumes leads to responses in the projection neuron ensemble that combine an instantaneous report of sensory input with a record of recent input, allowing extraction of high-level features.

Spike train integration: contributions of synchronous neural activity to sensory representation

Ito, Ong, Raman, Tsou

Accumulating evidence suggests that in many animals, from insects to mammals, olfactory information is represented by temporally structured synchronized firing of a spatially distributed population of neurons in olfactory systems. We used new techniques to determine whether non-pheromone odors induce oscillatory activity in the moth olfactory system. Further, we delivered odorants in temporal patterns such as moths would encounter in nature: as brief pulses, as moths would experience in an odor plume in flight, and as very lengthy pulses, as moths would experience when sampling food from and forming learned associations about flowers.

All odors tested (a plant essential oil and 13 pure chemical odors, including alcohols, aliphatics, aromatics, a ketone, and terpenoids) at a wide range of concentrations induced strongly synchronized neural activity as revealed by local field potential (LFP) oscillations in the first and second olfactory relays, antennal lobe, and mushroom body. A wide range of stimulus pulse durations all evoked clear oscillatory activity. Interestingly, relatively brief, plume-like pulses (less than 750 milliseconds) produced only fast oscillations (30-40 Hz), whereas long pulses (greater than 1 second), such as those that effectively induce associative learning, produced an oscillatory burst that was initially fast and then slow (10-20 Hz). This is the first clear evidence that moths, like locusts, produce oscillatory responses to odors, the first indication that brief and lengthy presentations of the same odorant elicit somewhat different types of neural responses and that the oscillatory mechanism can operate stably in fast and slow modes.

We examined the mechanisms underlying neural synchronous oscillations. Odor-elicited action potentials in antennal lobe neurons were very tightly phase-locked to the LFP, and the same antennal lobe neurons were phase-locked during fast and slow oscillations, indicating that a single neural network is responsible for both fast and slow response modes. Further, intracellular recordings from the interneurons showed subthreshold membrane potential oscillations that were highly correlated with the LFP recorded from the mushroom body. Blocking GABAergic inhibition from local neurons onto projection neurons by means of local injections of picrotoxin into the antennal lobe reversibly abolished both fast and slow oscillations in the LFP recorded downstream in the mushroom body; local saline injections had no such effect. The results show that, in the moth, odor stimuli lead to coordinated, oscillatory synchronous firings of projection neurons in the antennal lobe and that such coordinated activity is transmitted to the secondary center, the mushroom body.

The moth's Kenyon cells respond to odor stimuli with very sparse spiking, with spikes highly phase-locked to LFP oscillations, indicating that Kenyon cells are influenced by the oscillatory timing machinery of the antennal lobe and favor synchronous inputs from projection neurons. Together, these results demonstrate that the odor-coding and -decoding scheme identified in the locust, which includes oscillatory synchrony among projection neurons and sparse responses in Kenyon cells, applies to moths as well. In the moth, odor representations in the antennal lobe are relatively dense, consisting of sustained, lengthy, and bursty trains of spikes in projection neurons; in the mushroom body, odor representations have become very sparse, consisting of a rare spikes in Kenyon cells, which fire mainly after the onset and offset of the odor. Some Kenyon cells showed both on- and off-responses to odor pulses, often in an odor-specific fashion, but some showed only on- or off-responses, and off-responses followed long rather than brief stimuli.

To begin to examine the long-noted connection between mushroom body activity and odor perception and memory, we used an associative conditioning paradigm to train moths to extend the proboscis following pairing of odor with sugar-water reward. Our behavioral findings suggest that the early phase of odor response gives rise to perceptions that differ from those of the late phase. These results provide behavioral support for the hypothesis that olfactory stimuli are indeed represented by precisely timed, sparse, and spatially distributed Kenyon cell firings. Our results also suggest the testable hypothesis that coincident Kenyon cell and reward pathway activity is required for effective learning.

Sensory processing in naive locusts: behavior and physiology

_Sun, Chiraboga, ¹ Feldman ² _

We wished to determine how the sensory capacities of animals develop. Through their innate sensory preferences, animals often demonstrate the existence of inborn information. But it is not known how this information is encoded and how it differs from information acquired through direct experience.

Building on the work of several summer interns, we found that newly hatched locusts, literally just crawling out of their hatching cups, immediately move toward fresh grass. In a series of behavioral studies using thousands of locusts, we established that the hatchlings choose real grass over visually similar but odorless plastic grass; that they choose paper rubbed with fresh grass over clean paper of the same color; and that they choose paper dabbed with colorless monomolecular odorants that are components of grass odor over paper with other colorless odorants (even when each odorant had been diluted so that all vapor pressures were identical). These results indicate that naive locusts, which had never eaten, touched, or otherwise encountered their natural food source, have a built-in preference for the odor of their natural food.

This preference may be attributable to a peripheral mechanism; for example, perhaps hatchling locusts have a surplus of odor receptors for grass odors. We tested this hypothesis by making electro-antennograms from hatchlings and found that the antennae respond equally well to grass and an assortment of non-plant odors (as do, as we found, the antennae of adult locusts). The same result obtained when we diluted the odors to provide equal vapor pressures. In addition, we found that, in hatchling antennae, sensory adaptation occurs for grass odors with the same timing and extent as for non-plant odors. Thus, we established that the innate sensory preference must be encoded centrally, in the brain.

We therefore investigated sensory responses in the hatchling brain. By recording LFPs in the mushroom bodies of hatchlings, we found that the oscillatory synchronization mechanism is already intact (although the oscillation frequency is significantly slower than in the adult). Recording intracellularly from antennal lobe neurons, we found odor-specific temporal patterns in the distributed responses of projection neurons. The responses appeared similar to those of adults, although less complex: response patterns were shorter in duration and contained fewer alternating excitatory and inhibitory components. We also found no evidence in the antennal lobe for specialized neurons responding only to grass odors. Thus, representations of grass and other odors in the hatchling brain are broadly, spatiotemporally distributed even though they are somewhat different from and apparently more rudimentary than those of the adult brain.

Information reformatting through multiple interneuronal relays in the Drosophila olfactory system

Tanaka

The responses of primary sensory neurons to sensory stimuli are usually relatively simple compared with those of interneurons in the brain. Neural representations of the sensory stimuli are formed through the complex activity of interneurons. Such activity is determined not only by simple sensory input but also by parallel inputs from interacting networks of neurons in the brain. To explore the reformatting mechanisms and functions of these networks, we are employing a combination of genetic and electrophysiological tools in Drosophila.

Drosophila is useful for this task for several reasons: (1) several existing transgenic lines allow us to visualize specific types of neurons and to modulate their activities; (2) a great deal of information about olfactory sensory neurons has already accumulated; and (3) the anatomy of the olfactory neural network has been studied in detail. However, given Drosophila's small size, electrophysiological study of function has only recently been possible.

We have established techniques to record intracellularly from olfactory interneurons in the Drosophila brain. By means of genetically encoded fluorescence, we visualized specific types of olfactory interneurons in transgenic flies and succeded in recording from them intracellularly. We found that different odorants elicited a variety of response patterns in different cell types. Using genetic techniques, we are now examining the mechanisms underlying these olfactory responses by conditionally inhibiting synaptic transmission of specific types of neurons.

Cellular and behavioral analysis of multimodal integration

Joseph

Strong behavioral evidence suggests that insects, like other animals, integrate information from several modalities, such as olfaction and vision. Using anatomical and electrophysiological techniques, we are exploring neural mechanisms of multimodal integration by identifying neurons in the locust brain that respond to several stimulation modes. In addition, we are working to identify behaviors that correlate with and are perhaps caused by the activities of these neurons.

The contributions of olfactory receptor neuron response dynamics to spatiotemporal sensory coding

Raman, Joseph, Tang

Like mitral cells of the olfactory bulb, groups of projection neurons of the antennal lobe respond to odorants with spatially distributed, temporally complex firing patterns that change with and thus contain information about the odorants that elicit them. Several lines of evidence suggest that interactions between the excitatory projection neurons and inhibitory local neurons are at least partly responsible for these patterns, that is, these early-stage odor representations, thus shedding light on the underlying neural mechanisms. Yet, computational models incorporating these interactions could not reproduce the full complexity of projection neuron firing patterns.

Recent work from genetically modified Drosophila found that olfactory receptor neurons may respond to odors with differently timed excitation or even with inhibition. We constructed a simplified computational model of the locust antennal lobe that included complex heterogeneous input from receptors modeled after those observed in Drosophila. The model generated more realistic, temporally complex spatiotemporal response patterns, particularly when several, rapid stimuli simulating odor plumes were presented. Thus, heterogeneous, temporally structured input from olfactory receptor neurons may contribute significantly to the spatiotemporal complexity of the projection neuron ensemble odor code. We are now evaluating the extent of heterogeneity in the responses of locust olfactory receptor neurons to odor presentations as well as the potential contribution of these patterned responses to the complexity of the odor code.

¹ April Chiriboga, BS, former Technician
² Jacklyn Feldman, former high school student

Publication Related to Other Work

For further information, contact stopferm@mail.nih.gov.