Dynamics of olfactory bulb input and output activity during odor stimulation in zebrafish

The processing of odor-evoked activity in the olfactory bulb (OB) of zebrafish was studied by extracellular single unit recordings from the input and output neurons, i.e., olfactory receptor neurons (ORNs) and mitral cells (MCs), respectively. A panel of 16 natural amino acid odors was used as stimuli. Responses of MCs, but not ORNs, changed profoundly during the first few hundred milliseconds after response onset. In MCs, but not ORNs, the total evoked excitatory activity in the population was initially odor-dependent but subsequently converged to a common level. Hence, the overall population activity is regulated by network interactions in the OB. The tuning widths of both ORN and MC response profiles were similar and, on average, stable over time. However, when analyzed for individual neurons, MC response profiles could sharpen (excitatory response to fewer odors) or broaden (excitatory response to more odors), whereas ORN response profiles remained nearly unchanged. Several observations indicate that dynamic inhibition plays an important role in this remodeling. Finally, the reliability of odor identification based on MC population activity patterns improved over time, whereas odor identification based on ORN activity patterns was most reliable early in the odor response. These results demonstrate that several properties of MC, but not ORN, activity change during the initial phase of the odor response with important consequences for odor-encoding activity patterns. Furthermore, our data indicate that inhibitory interactions in the OB are important in dynamically shaping the activity of OB output neurons.     

Odorants suppress a voltage-activated K+ conductance in rat olfactory neurons.

Stimulation of olfactory receptor neurons (ORNs) with odors elicits an increase in the concentration of cAMP leading to opening of cyclic nucleotide-gated (CNG) channels and subsequent depolarization. Although opening of CNG channels is thought to be the main mechanism mediating signal transduction, modulation of other ion conductances by odorants has been postulated. To determine whether K+ conductances are modulated by odorants in mammalian ORNs, we examined the response of rat ORNs to odors by recording membrane current under perforated-patch conditions. We find that rat ORNs display two predominant types of responses. Thirty percent of the cells responded to odorants with activation of a CNG conductance. In contrast, in 55% of the ORNs, stimulation with odorants inhibited a voltage-activated K+ conductance (IKo). In terms of pharmacology, ion permeation, outward rectification, and time course for inactivation, IKo resembled a delayed rectifier K+ conductance. The effect of odorants on IKo was specific (only certain odorants inhibited IKo in each ORN) and concentration dependent, and there was a significant latency between arrival of odorants to the cell and the onset of suppression. These results indicate that indirect suppression of a K+ conductance (IKo) by odorants plays a role in signal transduction in mammalian ORNs.     

Odorant Responsiveness of Squid Olfactory Receptor Neurons

In the olfactory organ of the squid, Lolliguncula brevis there are five morphological types of olfactory receptor neurons (ORNs). Previous work to characterize odor sensitivity of squid ORNs was performed on only two of the five types in dissociated primary cell cultures. Here, we sought to establish the odorant responsiveness of all five types. We exposed live squid or intact olfactory organs to excitatory odors plus the activity marker, agmatine (AGB), an arginine derivative that enters cells through nonselective cation channels. An antibody against AGB was used to identify odorant‐activated neurons. We were able to determine the ORN types of AGB‐labeled cells based on their location in the epithelium, morphology and immunolabeling by a set of metabolites: arginine, aspartate, glutamate, glycine, and glutathione. Of 389 neurons identified from metabolite‐labeled tissue, 3% were type 1, 32% type 2, 33% type 3, 15% type 4, and 17% type 5. Each ORN type had different odorant specificity with type 3 cells showing the highest percentages of odorant‐stimulated AGB labeling. Type 1 cells were rare and none of the identified type 1 cells responded to the tested odorants, which included glutamate, alanine and AGB. Glutamate is a behaviorally attractive odorant and elicited AGB labeling in types 2 and 3. Glutamate‐activated AGB labeling was significantly reduced in the presence of the adenylate cyclase inhibitor, SQ22536 (80 μM). These data suggest that the five ORN types differ in their relative abundance and odor responsiveness and that the adenylate cyclase pathway is involved in squid olfactory transduction. Anat Rec, 291:763‐774, 2008. © 2008 Wiley‐Liss, Inc.     

Administration of transforming growth factor-alpha enhances anatomical and behavioral recovery following olfactory nerve transection

Although replacement of olfactory receptor neurons (ORNs) and subsequent reinnervation of the olfactory bulb occur following ORN injury, the intrinsic and extrinsic factors that contribute to the regulation of this dynamic process have not yet been fully identified. Recent research indicates that several growth factors have neurogenic effects on ORNs in vitro, and that chronic in vivo administration of either basic fibroblast growth factor, epidermal growth factor, or transforming growth factor-alpha (TGF-alpha) following chemical lesion can enhance the normal rate of ORN reinnervation of the olfactory bulb. The primary goal of the present experiments was to further assess the extent to which growth factor-related enhancements in the rate of anatomical recovery during ORN reconstitution and subsequent reinnervation of olfactory bulb are accompanied by enhancements in the rate of recovery of odor-guided behavior.A series of experiments in rats was conducted to initially characterize the time course of the anatomical and behavioral recovery normally observed following ORN reconstitution as a consequence of olfactory nerve transection, and to subsequently characterize the anatomical and behavioral effects of TGF-alpha administration on this normal rate of recovery. Consistent with a host of prior studies, olfactory nerve transection produced consistent and substantial deafferentation of olfactory bulb followed by a time-dependent anatomical recovery which was significantly enhanced by administration of TGF-alpha. The effect of TGF-alpha on functional recovery following olfactory nerve transection was also assessed using an odor-guided fear conditioning task. ORN lesioned animals receiving injections of TGF-alpha during recovery were found to display enhanced conditioned responding to an olfactory stimulus compared to untreated subjects. Further behavioral analyses suggested that this enhanced functional recovery was likely not due to non-specific effects of TGF-alpha on cognition or motor activity, but rather to enhanced olfactory input to the CNS.Future studies will likely reveal the exact mechanism of action mediating the anatomical and concomitant behavioral effects of this growth factor. Since ORNs are one of only a few populations of neurons capable of regeneration or replacement, the continued study of the cellular and molecular factors that coordinate this regenerative process may ultimately lead to the development of therapeutic strategies to promote an enhanced functional recovery following injury to other neuronal populations.     

Sparsening and temporal sharpening of olfactory representations in the honeybee mushroom bodies

We explored the transformations accompanying the transmission of odor information from the first-order processing area, the antennal lobe, to the mushroom body, a higher-order integration center in the insect brain. Using Ca2+ imaging, we recorded activity in the dendrites of the projection neurons that connect the antennal lobe with the mushroom body. Next, we recorded the presynaptic terminals of these projection neurons. Finally, we characterized their postsynaptic partners, the intrinsic neurons of the mushroom body, the clawed Kenyon cells. We found fundamental differences in odor coding between the antennal lobe and the mushroom body. Odors evoked combinatorial activity patterns at all three processing stages, but the spatial patterns became progressively sparser along this path. Projection neuron dendrites and boutons showed similar response profiles, but the boutons were more narrowly tuned to odors. The transmission from projection neuron boutons to Kenyon cells was accompanied by a further sparsening of the population code. Activated Kenyon cells were highly odor specific. Furthermore, the onset of Kenyon cell responses to projection neurons occurred within the first 200 ms and complex temporal patterns were transformed into brief phasic responses. Thus two types of transformations occurred within the MB: sparsening of a combinatorial code, mediated by pre- and postsynaptic processing within the mushroom body microcircuits, and temporal sharpening of postsynaptic Kenyon cell responses, probably involving a broader loop of inhibitory recurrent neurons.     

Sensory processing in the Drosophila antennal lobe increases reliability and separability of ensemble odor representations

Here we describe several fundamental principles of olfactory processing in the Drosophila melanogaster antennal lobe (the analog of the vertebrate olfactory bulb), through the systematic analysis of input and output spike trains of seven identified glomeruli. Repeated presentations of the same odor elicit more reproducible responses in second-order projection neurons (PNs) than in their presynaptic olfactory receptor neurons (ORNs). PN responses rise and accommodate rapidly, emphasizing odor onset. Furthermore, weak ORN inputs are amplified in the PN layer but strong inputs are not. This nonlinear transformation broadens PN tuning and produces more uniform distances between odor representations in PN coding space. In addition, portions of the odor response profile of a PN are not systematically related to their direct ORN inputs, which probably indicates the presence of lateral connections between glomeruli. Finally, we show that a linear discriminator classifies odors more accurately using PN spike trains than using an equivalent number of ORN spike trains.     

Functional asymmetries in cockroach ON and OFF olfactory receptor neurons

The ON and OFF olfactory receptor neurons (ORNs) on the antenna of the American cockroach respond to the same changes in the concentration of the odor of lemon oil, but in the opposite direction. The same jump in concentration raises impulse frequency in the ON and lowers it in the OFF ORN and, conversely, the same concentration drop raises impulse frequency in the OFF and lowers it in the ON ORN. When the new concentration level is maintained, it becomes a background concentration and affects the responses of the ON and OFF ORNs to superimposed changes. Raising the background concentration decreases both the ON-ORN's response to concentration jumps and the OFF-ORN's response to concentration drops. In addition, the slopes of the functions approximating the relationship of impulse frequency to concentration changes become flatter for both types of ORNs as the background concentration rises. The progressively compressed scaling optimizes the detection of concentration changes in the low concentration range. The loss of information caused by the lower differential sensitivity in the high concentration range is partially compensated by the higher discharge rates of the OFF ORNs. The functional asymmetry of the ON and OFF ORNs, which reflects nonlinearity in the detection of changes in the concentration of the lemon oil odor, improves information transfer for decrements in the high concentration range.     

Dendritic patterning by Dscam and synaptic partner matching in the Drosophila antennal lobe

In the olfactory system of Drosophila melanogaster, axons of olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons typically target 1 of approximately 50 glomeruli. Dscam, an immunoglobulin superfamily protein, acts in ORNs to regulate axon targeting. Here we show that Dscam acts in projection neurons and local interneurons to control the elaboration of dendritic fields. The removal of Dscam selectively from projection neurons or local interneurons led to clumped dendrites and marked reduction in their dendritic field size. Overexpression of Dscam in projection neurons caused dendrites to be more diffuse during development and shifted their relative position in adulthood. Notably, the positional shift of projection neuron dendrites caused a corresponding shift of its partner ORN axons, thus maintaining the connection specificity. This observation provides evidence for a pre- and postsynaptic matching mechanism independent of precise glomerular positioning.     

Presence of Ca2+-dependent K+ channels in chemosensory cilia support a role in odor transduction

Olfactory receptor neurons (ORNs) respond to odorants with changes in the action potential firing rate. Excitatory responses, consisting of firing increases, are mediated by a cyclic AMP cascade that leads to the activation of cationic nonselective cyclic nucleotide-gated (CNG) channels and Ca2+-dependent Cl- (ClCa) channels. This process takes place in the olfactory cilia, where all protein components of this cascade are confined. ORNs from various vertebrate species have also been shown to generate inhibitory odor responses, expressed as decreases in action potential discharges. Odor inhibition appears to rely on Ca2+-dependent K+ (KCa) channels, but the underlying transduction mechanism remains unknown. If these channels are involved in odor transduction, they are expected to be present in the olfactory cilia. We found that a specific antibody against a large conductance KCa recognized a protein of approximately 116 kDa in Western blots of purified rat olfactory ciliary membranes. Moreover, the antibody labeled ORN cilia in isolated ORNs from rat and toad (Caudiverbera caudiverbera). In addition, single-channel recordings from inside-out membrane patches excised from toad chemosensory cilia showed the presence of 4 different types of KCa channels, with unitary conductances of 210, 60, 12, and 29 and 60 pS, high K+-selectivity, and Ca2+ sensitivities in the low micromolar range. Our work demonstrates the presence of K+ channels in the ORN cilia and supports their participation in odor transduction.     

Biophysical mechanisms underlying olfactory receptor neuron dynamics

The responses of olfactory receptor neurons (ORNs) to odors have complex dynamics. Using genetics and pharmacology, we found that these dynamics in Drosophila ORNs could be separated into sequential steps, corresponding to transduction and spike generation. Each of these steps contributed distinct dynamics. Transduction dynamics could be largely explained by a simple kinetic model of ligand-receptor interactions, together with an adaptive feedback mechanism that slows transduction onset. Spiking dynamics were well described by a differentiating linear filter that was stereotyped across odors and cells. Genetic knock-down of sodium channels reshaped this filter, implying that it arises from the regulated balance of intrinsic conductances in ORNs. Complex responses can be understood as a consequence of how the stereotyped spike filter interacts with odor- and receptor-specific transduction dynamics. However, in the presence of rapidly fluctuating natural stimuli, spiking simply increases the speed and sensitivity of encoding.     

Immunohistochemical analysis of the development of olfactory organs in two species of turtles Pelodiscus sinensis and Mauremys reevesii

The nasal cavity of turtles is composed of the upper and lower chambers, lined by the upper and lower chamber epithelia, respectively. In many turtles including the Reeve’s turtle Mauremys reevesii, the upper chamber epithelium contains ciliated olfactory receptor neurons (ORNs) and the lower chamber epithelium contains microvillous ORNs. However, in the olfactory organ of the Chinese soft-shelled turtle Pelodiscus sinensis, both the upper and lower chamber epithelia contain ciliated ORNs. In the present study, we immunohistochemically examined the developmental process of olfactory organs in soft-shelled turtle and the Reeve’s turtle to clarify the developmental origins of the lower chamber epithelium in these turtles. Obtained data indicate that olfactory organs of these turtles have identical origin and follow similar process of development, suggesting that, in the lower chamber epithelium of the nasal cavity, ciliated ORNs differentiate in soft-shelled turtle whereas microvillous ORNs differentiate in the Reeve’s turtle.     

Highly specific olfactory receptor neurons for types of amino acids in the channel catfish

Odorant specificity to l-alpha-amino acids was determined electrophysiologically for 93 single catfish olfactory receptor neurons (ORNs) selected for their narrow excitatory molecular response range (EMRR) to only one type of amino acid (i.e., Group I units). These units were excited by either a basic amino acid, a neutral amino acid with a long side chain, or a neutral amino acid with a short side chain when tested at 10(-7) to 10(-5) M. Stimulus-induced inhibition, likely for contrast enhancement, was primarily observed in response to the types of amino acid stimuli different from that which activated a specific ORN. The high specificity of single Group I ORNs to type of amino acid was also previously observed for single Group I neurons in both the olfactory bulb and forebrain of the same species. These results indicate that for Group I neurons olfactory information concerning specific types of amino acids is processed from receptor neurons through mitral cells of the olfactory bulb to higher forebrain neurons without significant alteration in unit odorant specificity.     

Specificities of olfactory receptor neuron responses to amino acids in the black bullhead catfish (Ameiurus melas)

In vivo investigations of catfish olfactory receptor neurons (ORNs) were previously limited to studying responses of spontaneously active cells. The olfactory organ, however, also contains ORNs that lack spontaneous activity and respond to amino acids. To record electrical activity of ORNs that were inactive prior to stimulation, we bathed the olfactory organ with low conductive, highly purified water that reduces shunting and enables detection of action potentials from ORNs distant to the electrode. After stimulation with amino acids, these ORNs elicited either phasic–tonic or tonic only activities. The spike frequency of the phasic activity consisted of transient frequencies up to 108 Hz that lasted <450 ms. All tonic activities saturated at action potential frequencies of 17–21 Hz. Their durations were dose dependent over several log units of concentration as they closely followed that of the suprathreshold amino acid stimulation. Specificities of 44 ORNs were investigated with ten different amino acids tested at 10^?4?M. Thirteen ORNs were excited by only one amino acid, l-norvaline, and 22 additional ORNs were excited by l-norvaline and l-methionine. Nine ORNs were excited by >2 amino acids that included l-norvaline. In 29 of 31 neurons responding to >1 amino acid, the duration of the responses to the most stimulatory amino acid was at least double compared to that to the other amino acids. The amplitude of electro-olfactogram (EOG) correlated significantly with the number of ORNs activated by the same amino acids confirming that the EOG represents the sum of ORN receptor potentials.     

Activity-dependent modification of inhibitory synapses in models of rhythmic neural networks

The faithful production of rhythms by many neural circuits depends critically on the strengths of inhibitory synaptic connections. We propose a model in which the strengths of inhibitory synapses in a central pattern-generating circuit are subject to activity-dependent plasticity. The strength of each synapse is modified as a function of the global activity of the postsynaptic neuron and by correlated activity of the pre- and postsynaptic neurons. This allows the self-assembly, from random initial synaptic strengths, of two cells into reciprocal oscillation and three cells into a rhythmic triphasic motor pattern. This self-assembly illustrates that complex oscillatory circuits that depend on multiple inhibitory synaptic connections can be tuned via simple activity-dependent rules.     

Dopamine reduces odor- and elevated-K(+)-induced calcium responses in mouse olfactory receptor neurons in situ

Although D2 dopamine receptors have been localized to olfactory receptor neurons (ORNs) and dopamine has been shown to modulate voltage-gated ion channels in ORNs, dopaminergic modulation of either odor responses or excitability in mammalian ORNs has not previously been demonstrated. We found that <50 microM dopamine reversibly suppresses odor-induced Ca2+ transients in ORNs. Confocal laser imaging of 300-microm-thick slices of neonatal mouse olfactory epithelium loaded with the Ca(2+)-indicator dye fluo-4 AM revealed that dopaminergic suppression of odor responses could be blocked by the D2 dopamine receptor antagonist sulpiride (<500 microM). The dopamine-induced suppression of odor responses was completely reversed by 100 microM nifedipine, suggesting that D2 receptor activation leads to an inhibition of L-type Ca2+ channels in ORNs. In addition, dopamine reversibly reduced ORN excitability as evidenced by reduced amplitude and frequency of Ca2+ transients in response to elevated K(+), which activates voltage-gated Ca2+ channels in ORNs. As with the suppression of odor responses, the effects of dopamine on ORN excitability were blocked by the D2 dopamine receptor antagonist sulpiride (<500 microM). The observation of dopaminergic modulation of odor-induced Ca2+ transients in ORNs adds to the growing body of work showing that olfactory receptor neurons can be modulated at the periphery. Dopamine concentrations in nasal mucus increase in response to noxious stimuli, and thus D2 receptor-mediated suppression of voltage-gated Ca2+ channels may be a novel neuroprotective mechanism for ORNs.     

AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training

Two intermingled hypothalamic neuron populations specified by expression of agouti-related peptide (AGRP) or pro-opiomelanocortin (POMC) positively and negatively influence feeding behavior, respectively, possibly by reciprocally regulating downstream melanocortin receptors. However, the sufficiency of these neurons to control behavior and the relationship of their activity to the magnitude and dynamics of feeding are unknown. To measure this, we used channelrhodopsin-2 for cell type-specific photostimulation. Activation of only 800 AGRP neurons in mice evoked voracious feeding within minutes. The behavioral response increased with photoexcitable neuron number, photostimulation frequency and stimulus duration. Conversely, POMC neuron stimulation reduced food intake and body weight, which required melanocortin receptor signaling. However, AGRP neuron-mediated feeding was not dependent on suppressing this melanocortin pathway, indicating that AGRP neurons directly engage feeding circuits. Furthermore, feeding was evoked selectively over drinking without training or prior photostimulus exposure, which suggests that AGRP neurons serve a dedicated role coordinating this complex behavior.