Imaging odor-evoked activities in the mouse olfactory bulb using optical reflectance and autofluorescence signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain. Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex(1). IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group(2). The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues(3), it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group(4). The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of ?100μm(3) constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB(5, 6, 7) which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet. Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.     

Pharmacological analysis of ionotropic glutamate receptor function in neuronal circuits of the zebrafish olfactory bulb

Although synaptic functions of ionotropic glutamate receptors in the olfactory bulb have been studied in vitro, their roles in pattern processing in the intact system remain controversial. We therefore examined the functions of ionotropic glutamate receptors during odor processing in the intact olfactory bulb of zebrafish using pharmacological manipulations. Odor responses of mitral cells and interneurons were recorded by electrophysiology and 2-photon Ca(2+) imaging. The combined blockade of AMPA/kainate and NMDA receptors abolished odor-evoked excitation of mitral cells. The blockade of AMPA/kainate receptors alone, in contrast, increased the mean response of mitral cells and decreased the mean response of interneurons. The blockade of NMDA receptors caused little or no change in the mean responses of mitral cells and interneurons. However, antagonists of both receptor types had diverse effects on the magnitude and time course of individual mitral cell and interneuron responses and, thus, changed spatio-temporal activity patterns across neuronal populations. Oscillatory synchronization was abolished or reduced by AMPA/kainate and NMDA receptor antagonists, respectively. These results indicate that (1) interneuron responses depend mainly on AMPA/kainate receptor input during an odor response, (2) interactions among mitral cells and interneurons regulate the total olfactory bulb output activity, (3) AMPA/kainate receptors participate in the synchronization of odor-dependent neuronal ensembles, and (4) ionotropic glutamate receptor-containing synaptic circuits shape odor-specific patterns of olfactory bulb output activity. These mechanisms are likely to be important for the processing of odor-encoding activity patterns in the olfactory bulb.     

Lactosamine differentially affects olfactory sensory neuron projections to the olfactory bulb

During embryonic development, olfactory sensory neurons extend axons that form synapses with the dendrites of projection neurons in glomeruli of the olfactory bulb (OB). The glycosyltransferase beta3GnT1 regulates the expression of 1B2-reactive lactosamine glycans that are mosaically distributed among glomeruli. In newborn beta3GnT1-/- mice, lactosamine expression is lost, and many glomeruli fail to form. To determine the role of lactosamine in OB targeting, we analyzed the trajectories of specific OR axon populations and their reactivity with 1B2 in beta3GnT1-/- mice. mI7 axons and P2 axons, both of which are weakly 1B2+ in wild-type mice, fail to grow to their normal positions in the glomerular layer during early postnatal development and never recover in adult mutant mice. In contrast, many M72 axons, which are always lactosamine negative in wild-type mice, survive but are misguided to the extreme anterior OB in neonatal mutant mice and persist as heterotypic glomeruli, even in adult null mice. These results show that the loss of lactosamine differentially affects each OR population. Those that lose their normal expression of lactosamine fail to form stable connections with mitral and tufted cells in the OB, disappear during early postnatal development, and do not recover in adults. Neurons that are normally lactosamine negative, survive early postnatal degeneration in beta3GnT1-/- mice but extend axons that converge on inappropriate targets in the mutant OB.     

Connectivity and dynamics in the olfactory bulb

Dendrodendritic interactions between excitatory mitral cells and inhibitory granule cells in the olfactory bulb create a dense interaction network, reorganizing sensory representations of odors and, consequently, perception. Large-scale computational models are needed for revealing how the collective behavior of this network emerges from its global architecture. We propose an approach where we summarize anatomical information through dendritic geometry and density distributions which we use to calculate the connection probability between mitral and granule cells, while capturing activity patterns of each cell type in the neural dynamical systems theory of Izhikevich. In this way, we generate an efficient, anatomically and physiologically realistic large-scale model of the olfactory bulb network. Our model reproduces known connectivity between sister vs. non-sister mitral cells; measured patterns of lateral inhibition; and theta, beta, and gamma oscillations. The model in turn predicts testable relationships between network structure and several functional properties, including lateral inhibition, odor pattern decorrelation, and LFP oscillation frequency. We use the model to explore the influence of cortex on the olfactory bulb, demonstrating possible mechanisms by which cortical feedback to mitral cells or granule cells can influence bulbar activity, as well as how neurogenesis can improve bulbar decorrelation without requiring cell death. Our methodology provides a tractable tool for other researchers.     

Quinolinic acid stimulates synaptosomal glutamate release and inhibits glutamate uptake into astrocytes

Quinolinic acid (QA) is an endogenous neurotoxin involved in various neurological diseases, whose action seems to be exerted via glutamatergic receptors. However, the exact mechanism responsible for the neurotoxicity of QA is far from being understood. We have previously reported that QA inhibits vesicular glutamate uptake. In this work, investigating the effects of QA on the glutamatergic system from rat brain, we have demonstrated that QA (from 0.1 to 10mM) had no effect on synaptosomal L-[3H]glutamate uptake. The effect of QA on glutamate release in basal (physiological K+ concentration) or depolarized (40 mM KCl) conditions was evaluated. QA did not alter K+-stimulated glutamate release, but 5 and 10mM QA significantly increased basal glutamate release. The effect of dizolcipine (MK-801), a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor on glutamate release was investigated. MK-801 (5 microM) did not alter glutamate release per se, but completely abolished the QA-induced glutamate release. NMDA (50 microM) also stimulated glutamate release, without altering QA-induced glutamate release, suggesting that QA effects were exerted via NMDA receptors. QA (5 and 10mM) decreased glutamate uptake into astrocyte cell cultures. Enhanced synaptosomal glutamate release, associated with inhibition of glutamate uptake into astrocytes induced by QA could contribute to increase extracellular glutamate concentrations which ultimately lead to overstimulation of the glutamatergic system. These data provide additional evidence that neurotoxicity of QA may be also related to disturbances on the glutamatergic transport system, which could result in the neurological manifestations observed when this organic acid accumulates in the brain.     

Inhibition in the fish olfactory bulb

Inhibition in the olfactory bulb of the carp was studied by recording potentials from secondary neurons intracellularly. Three types of inhibition — trace, early, and late — can arise in neurons of the olfactory bulb. Trace inhibition corresponds to hyperpolarization about 20 msec in duration, which is closely connected with the spike, but it is not after-hyperpolarization but an IPSP. Early and late inhibition correspond to IPSPs of different parameters. The first has a latency of 0–50 msec (relative to the spike) and a duration of 60–400 msec; the corresponding values for the second are 100–400 msec and 0.5–3 sec. The possible mechanisms of these types of inhibition are discussed.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 3, No. 6, pp. 650–656, November–December, 1971.     

Electrical signaling in the olfactory bulb

The olfactory bulb employs lateral and feedback inhibitory pathways to distribute odor information across parallel assemblies of mitral and granule cells. The pathways involve dendritic action potentials that can interact with a variety of voltage-dependent conductances and synaptic transmission to produce complex and dynamic patterns of activity. Electrical coupling also helps to ensure proper coordination and synchronization of these patterns. These mechanisms provide numerous options for dynamic modulation and control of signaling in the olfactory bulb.     

Guidepost neurons for the lateral olfactory tract: Expression of metabotropic glutamate receptor 1 and innervation by glutamatergic olfactory bulb axons

The guidepost neurons for the lateral olfactory tract, which are called lot cells, are the earliest‐generated neurons in the neocortex. They migrate tangentially and ventrally further down this tract, and provide scaffolding for the olfactory bulb axons projecting into this pathway. The molecular profiles of the lot cells are largely uncharacterized. We found that lot cells specifically express metabotropic glutamate receptor subtype‐1 at a very early stage of development. This receptor is functionally competent and responds to a metabotropic glutamate receptor agonist with a transient increase in the intracellular calcium ion concentration. When the glutamatergic olfactory bulb axons were electrically stimulated, lot cells responded to the stimulation with a calcium increase mainly via ionotropic glutamate receptors, suggesting potential neurotransmission between the axons and lot cells during early development. Together with the finding that lot cells themselves are glutamatergic excitatory neurons, our results provide another notable example of precocious interactions between the projecting axons and their intermediate targets. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Autoradiographic localization of high affinity uptake sites for 3H-D-aspartate in the rat olfactory bulb

In order to reveal excitatory amino acid-ergic neuronal connections in the rat olfactory bulb, uptake sites for the tritiated D-aspartic acid were analyzed by high resolution autoradiography. Light microscopy revealed both cellular and terminal-like uptake. Based on electron microscopy, overwhelming majority of the cellular uptake was assigned to glial cells. A fairly high number of labelled terminals appeared in the surroundings of the mitral cell somata, within the deepest portion of the external plexiform layer, in the internal plexiform layer and in the outer half of the granule cell layer. Labelled terminals synapsed onto likely granule cell dendrites or spines, at asymmetric membrane thickenings. These results suggest that, although the output neurons may not utilize glutamic or aspartic acid as their transmitters, these amino acids may, however, contribute to the bulbar neurotransmission, as mediator substances of a subgroup of centrifugal fibers to the olfactory bulb.     

Functional regeneration of the olfactory bulb requires reconnection to the olfactory nerve in Xenopus larvae

Larvae of the South African clawed frog (Xenopus laevis) can regenerate the telencephalon, which consists of the olfactory bulb and the cerebrum, after it has been partially removed. Some authors have argued that the telencephalon, once removed, must be reconnected to the olfactory nerve in order to regenerate. However, considerable regeneration has been observed before reconnection. Therefore, we have conducted several experiments to learn whether or not reconnection is a prerequisite for regeneration. We found that the olfactory bulb did not regenerate without reconnection, while the cerebrum regenerated by itself. On the other hand, when the brain was reconnected by the olfactory nerve, both the cerebrum and the olfactory bulb regenerated. Morphological and histological investigation showed that the regenerated telencephalon was identical to the intact one in morphology, types and distributions of cells, and connections between neurons. Froglets with a regenerated telencephalon also recovered olfaction, the primary function of the frog telencephalon. These results suggest that the Xenopus larva requires reconnection of the regenerating brain to the olfactory nerve in order to regenerate the olfactory bulb, and thus the regenerated brain functions, in order to process olfactory information.     

A model of olfactory adaptation and sensitivity enhancement in the olfactory bulb

It has been suggested that the olfactory bulb, the first processing center after the sensory cells in the olfactory pathway, plays a role in olfactory adaptation, odor sensitivity enhancement by motivation and other olfactory psychophysical phenomena. In a mathematical model based on the bulbar anatomy and physiology, the inputs from the higher olfactory centers to the inhibitory cells in the bulb are shown to be able to modulate the response, and thus the sensitivity of the bulb to specific odor inputs. It follows that the bulb can decrease its sensitivity to a pre-existing and detected odor (adaptation) while remaining sensitive to new odors, or increase its sensitivity to interested searching odors. Other olfactory psychophysical phenomena such as cross-adaptation etc. are discussed as well.     

Flexible categorization in the mouse olfactory bulb

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Effects of a glutamate uptake inhibitor on glutamate release induced by veratridine and ischemia

It has been postulated that a reversal of glutamate reuptake (“uptake reverse”) may contribute to glutamate release during cerebral ischemia. We tested this hypothesis by studying the effect of threo-3hydroxy- -aspartic acid (THA), a glutamate uptake inhibitor, on extracellular glutamate accumulation measured by microdialysis during 4-vessel ischemia (20 min). The inhibitory effect of THA on sodium-dependent glutamate uptake was measured in vitro on rat hippocampal slices (K_i = 45 ± 11 μM). We examined in vivo the effect of THA (400 μM in the dialysis solution) on the extracellular glutamate release from the rat hippocampus, during veratridine depolarization and ischemia. THA decreased the amount of glutamate appearing in the extracellular space during veratridine depolarization (61%). In contrast, the glutamate release induced by ischemia was not affected by THA. We conclude that a reversal of the sodium-dependent uptake contributes to an increase in extracellular glutamate during veratridine depolarization. In contrast, glutamate release occurring during ischemia is not mediated by uptake reverse.     

Axon mis-targeting in the olfactory bulb during regeneration of olfactory neuroepithelium

During development, primary olfactory axons typically grow to their topographically correct target zone without extensive remodelling. Similarly, in adults, new axons arising from the normal turnover of sensory neurons essentially project to their target without error. In the present study we have examined axon targeting in the olfactory pathway following extensive chemical ablation of the olfactory neuroepithelium in the P2-tau:LacZ line of mice. These mice express LacZ in the P2 subpopulation of primary olfactory neurons whose axons target topographically fixed glomeruli on the medial and lateral surfaces of the olfactory bulb. Intraperitoneal injections of dichlobenil selectively destroyed the sensory neuroepithelium of the nasal cavity without direct physical insult to the olfactory neuron pathway. Primary olfactory neurons regenerated and LacZ staining revealed the trajectory of the P2 axons. Rather than project solely to their topographically appropriate glomeruli, the regenerating P2 axons now terminated in numerous inappropriate glomeruli which were widely dispersed over the olfactory bulb. While these errors in targeting were refined over time, there was still considerable mis-targeting after four months of regeneration.     

Heat shock protein responses to aging and proteotoxicity in the olfactory bulb

The olfactory bulb is one of the most vulnerable brain regions in age‐related proteinopathies. Proteinopathic stress is mitigated by the heat shock protein (Hsp) family of chaperones. Here, we describe age‐related decreases in Hsc70 in the olfactory bulb of the female rat and higher levels of Hsp70 and Hsp25 in middle and old age than at 2–4 months. To model proteotoxic and oxidative stress in the olfactory bulb, primary olfactory bulb cultures were treated with the proteasome inhibitors lactacystin and MG132 or the pro‐oxidant paraquat. Toxin‐induced increases were observed in Hsp70, Hsp25, and Hsp32. To determine the functional consequences of the increase in Hsp70, we attenuated Hsp70 activity with two mechanistically distinct inhibitors. The Hsp70 inhibitors greatly potentiated the toxicity of sublethal lactacystin or MG132 but not of paraquat. Although ubiquitinated protein levels were unchanged with aging in vivo or with sublethal MG132 in vitro, there was a large, synergistic increase in ubiquitinated proteins when proteasome and Hsp70 functions were simultaneously inhibited. Our study suggests that olfactory bulb cells rely heavily on Hsp70 chaperones to maintain homeostasis during mild proteotoxic, but not oxidative insults, and that Hsp70 prevents the accrual of ubiquitinated proteins in these cells.

     

Inhibition in the olfactory bulb of the lizard

Inhibition in neurons of the lizard olfactory bulb was investigated by intracellular recording. The hyperpolarization arising in the neurons after the spike in the response to orthodromic and antidromic activation is similar in composition and reflects the development of early and late IPSPs, differing from one another in latency, duration, and mechanism of generation. The early IPSP is evidently generated by the functioning of dendrodendritic synapses, formed by dendrites of the interglomerular cell on the membrane of the apical dendrites of the secondary neurons, whereas synapses generating the late IPSP are located on the basal dendrites and are formed by endings of the granular cells. The mechanisms of generation of the early and late IPSPs in the secondary neurons are discussed. A classification of neurons of the lizard olfactory bulb is given on the basis of analysis of their intracellular activity.