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. 2014 Jun 5;8:193.
doi: 10.3389/fnbeh.2014.00193. eCollection 2014.

Olfactory Bulb Encoding During Learning Under Anesthesia

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Free PMC article

Olfactory Bulb Encoding During Learning Under Anesthesia

Alister U Nicol et al. Front Behav Neurosci. .
Free PMC article

Abstract

Neural plasticity changes within the olfactory bulb are important for olfactory learning, although how neural encoding changes support new associations with specific odors and whether they can be investigated under anesthesia, remain unclear. Using the social transmission of food preference olfactory learning paradigm in mice in conjunction with in vivo microdialysis sampling we have shown firstly that a learned preference for a scented food odor smelled on the breath of a demonstrator animal occurs under isofluorane anesthesia. Furthermore, subsequent exposure to this cued odor under anesthesia promotes the same pattern of increased release of glutamate and gamma-aminobutyric acid (GABA) in the olfactory bulb as previously found in conscious animals following olfactory learning, and evoked GABA release was positively correlated with the amount of scented food eaten. In a second experiment, multiarray (24 electrodes) electrophysiological recordings were made from olfactory bulb mitral cells under isofluorane anesthesia before, during and after a novel scented food odor was paired with carbon disulfide. Results showed significant increases in overall firing frequency to the cued-odor during and after learning and decreases in response to an uncued odor. Analysis of patterns of changes in individual neurons revealed that a substantial proportion (>50%) of them significantly changed their response profiles during and after learning with most of those previously inhibited becoming excited. A large number of cells exhibiting no response to the odors prior to learning were either excited or inhibited afterwards. With the uncued odor many previously responsive cells became unresponsive or inhibited. Learning associated changes only occurred in the posterior part of the olfactory bulb. Thus olfactory learning under anesthesia promotes extensive, but spatially distinct, changes in mitral cell networks to both cued and uncued odors as well as in evoked glutamate and GABA release.

Keywords: anesthesia; microdialysis; mitral cells; multiarray electrophysiology; neurotransmitters; olfactory bulb; olfactory learning; social transmission of food preference.

Figures

Figure 1
Figure 1
Training and testing of mice in social transmission of food preference paradigms. Behavioral procedures were conducted over a 5 day period. In the 3 days preceding training and testing, mice were submitted to a restricted feeding regime in which they were allowed free access to food in a fixed 2 h period each day. The food provided in this period was the normal daily diet in powdered form rather than the pellets to which they were accustomed. Access to drinking water was unrestricted during the restricted feeding regime. In days 2 and 3 of this regime the animals spent 30 min of the feeding period individually in the test arena (a 33 × 33 × 25 cm high white plastic container). In this period of habituation to the test environment, ∼2 g of the powdered food was provided in a feed cup—a plastic cylinder (4 cm diameter × 5 cm high) fixed in the center of a plastic dish (6 cm diameter × 2 cm high) to catch any spillage (Figure 1). On day 4, Demonstrator mice were “loaded” by being given unrestricted access to either a flavored food or the normal food, both in powdered form. Observer mice were anesthetized, and exposed to the training odor by one of two procedures; either (1) carried on the breath of an anesthetized demonstrator, or (2) introduced to the anesthetic gas with or without CS2. On day 5, mice were given a 30 min simultaneous choice test to determine their food preference. Mice which consumed none of either food in the preference test were excluded from further analysis.
Figure 2
Figure 2
Changes in in vivo glutamate and GABA following olfactory learning. (A) Food preference test of observer mice where they were given a choice between cocoa- or cumin-scented foods, 24 h after exposure to the demonstrator’s breath when both observer and demonstrator were anesthetized (Training procedure 1). Histograms show relative consumption of cocoa-scented food (% from total intake). Relative consumption of the two foods was significantly different across the groups (p = 0.0007). While plain food-trained mice (controls, whose demonstrator had been given normal plain food) showed no significant preference for either food, cocoa-trained mice ate almost exclusively cocoa-scented food and cumin-trained animals ate nearly none (p-values given in the Figure are relative to cumin-trained mice). (B) Immediately after being tested for food preference, mice were used for microdialysis experiment. A microdialysis probe was placed in the posteromedial region of the olfactory bulb and 15 min microdialysis samples (25 µl) were taken. Odors were presented, via an odor containing bag, on samples 5 and 10. (C) In test mice (both cocoa- and cumin-trained animals) the concentrations of both glutamate and GABA sampled during exposure to the cued food odor were significantly increased relative to baseline (dotted line), while there was no such increase when exposed to the uncued odor. In the control (plain-trained) mice on the other hand there were no differences in concentrations of either transmitter during exposure to the two odors. (D) For test mice, the amount of cued food consumed during the preference test was positively correlated with concentrations of GABA collected during exposure to the cued but not uncued odor during microdialysis sampling (right panel). In the control mice, there was no such relationship between release of either transmitter during exposure to a food odor, and the quantity of that food consumed in the preference test (left panel).
Figure 3
Figure 3
Changes in neuronal activity during olfactory learning under anesthesia. (A) Neuronal activity was sampled from neurons across a 6 × 4 array of tungsten microelectrodes (tip separation 350 µ) advanced into the olfactory bulb from the dorsal surface. Spikes from individual neurons were sorted from multineuronal activity at multiple channels across the array. The averaged waveform is shown for spikes generated by a single neuron at the position highlighted on the array. Odors were presented for 10 s, by switching the normal anesthetic gas supply with one carrying an odor. (B) The times of occurrence of spikes generated by the neuron depicted in (A) are shown during presentations of the coriander food odor. The histogram shows the average response across 10 presentations of this odor. (C) Each odor was presented 10 times before training, and 10 times after training. Training comprised 10 presentations of the ginger food odor combined with CS2. The response during training was elevated relative to the response to ginger alone before training. This enhanced response achieved significance after training when ginger was presented in the absence of CS2. The response to coriander fell from pre- to post-training. (D) In the pilot behavioral simulation of social transfer of odor preference, mice were anesthetized and presented with odors carried in the anesthetic gas; ginger, CS2, or ginger and CS2 combined. When tested for preference between coriander and ginger flavored foods, mice which had been exposed to the ginger odor or CS2 separately preferred coriander over ginger. In those exposed to ginger and CS2 combined, this preference was overcome, and the mice consumed similar amounts of both foods.
Figure 4
Figure 4
Spatial distribution of olfactory bulb neural responses during learning. (A) In order to establish the spatial distribution of olfactory bulb mitral cell responses and learning-related changes, the microelectrode array was partitioned mediolaterally and anteroposteriorly (photograph and schematic illustrate the localization of the 24-electrode arrays). There was no significant variation in multiunit responses, or in learning-related changes, across the mediolateral dimension of the array. Histograms in (B) show that the increase (mean ± sem) in the size of the multiunit responses to the combined presentation of ginger and CS2, and the learning-related increase in the size of the multineuron response to ginger was restricted to the posterior half of the array.

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