Acute ICV insulin injection does not alter physiological parameters such as food intake, body weight and peripheral blood glucose level.

<p>Bar graphs represent food intake <i>(A)</i> and body weight <i>(B)</i> (mean ± SEM) measured on NaCl ICV injection days (NaCl) and 14 mU insulin ICV injection days (Insulin). The means were not statistically different (paired t-test, p>0.05, ns, n = 18). <i>(C)</i> Bar graphs represent the peripheral blood glucose levels (mean ± SEM) measured from tail blood samples 1 h after ICV NaCl injection (NaCl) and 1 h after ICV insulin injection (Insulin). The means were not statistically different (paired t-test, p>0.05, ns; n = 11).</p

Comparison of the mean (± SEM) respiratory frequency in Hz between satiated NaCl (n = 10) and fasted insulin (n = 10) rats during food odor presentation.

<p>Four 10 sec periods were considered: Pre-odor (−10 sec to 0 sec); Odor presentation (start: 0 sec to 10 sec, middle: 10 sec to 20 sec, end: 20 sec to 30 sec). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051227#s3" target="_blank">Results</a> are presented for each experimental condition: in the fasted state after 14 mU insulin ICV injection; in the satiated state after NaCl ICV injection. For the four periods, the means of respiratory frequency observed in two experimental conditions were not statistically different. (Two-way repeated measures ANOVA, p>0.05, ns).</p

Overall course of the behavioral experiments.

<p><i>(A)</i> Olfactory detection experiment. Animals were tested daily in the two-tube experimental device over 16 days (D1–D16). <i>Habituation:</i> On the first 3 days (D1–D3), rats were trained to drink pure water from both tubes (W/W) of the experimental cage. <i>Aversion acquisition:</i> On the following 3 days (D4–D6), rats had access to water odorized with isoamyl-acetate (ISO) diluted at 10⁻⁵ in both tubes (10⁻⁵/10⁻⁵). ISO 10⁻⁵ consumption >0.5 mL was paired with an intraperitoneal injection of LiCl (LiCl IP) to induce a conditioned olfactory aversion (COA) to ISO. <i>Aversion test:</i> On D7, the COA efficiency was tested by giving the animals a choice between water odorized with ISO 10⁻⁵ and pure water (10⁻⁵/W). On Habituation, Aversion acquisition and Aversion test days, animals were trained to receive a daily NaCl ICV injection (NaCl ICV). <i>Olfactory detection test:</i> During the Olfactory detection test period (D8–D11), rats were offered a choice between ISO 10⁻⁹ or ISO 10⁻⁸ and pure water (10⁻⁹/W, 10⁻⁸/W). For a given odorant dilution, the animals were tested on two consecutive days: once 1 h after NaCl ICV injection (D8 and D10) and once 1 h after a 14 mU insulin ICV injection (D9 and D11). <i>Aversion re-test:</i> On D12, the COA stability was assessed by giving the rats the choice between ISO 10⁻⁵ and pure water (10⁻⁵/W). <i>Aversion retention</i>: During the Aversion retention period (D15–D16), three days after the Aversion re-test, rats were offered again the choice between ISO 10⁻⁵ and pure water (10⁻⁵/W) and the animals were tested on two consecutive days: once 1 h after a 14 mU insulin ICV injection (D15) and once after a NaCl ICV injection (D16). (<i>B</i>) Sniffing experiment. Animals were tested daily in a whole-body plethysmograph over 7 days. The rats were first allowed to familiarized with the recording chamber for 4 days (Habituation), D1–D4 without (D1, D2) or with (D3, D4) food odor stimulation. During the sniffing test period (Food odor detection test, D5–D7), the animals were tested either in the fasted state (at 10:00 a.m.), 1 h after 14 mU insulin ICV injection (D5); in the fasted state, 1 h after NaCl ICV injection (D6); in the satiated state, 1 h after NaCl ICV injection (at 4:00 p.m.) (D7).</p

IR quantification in the main OB. Bar graphs represent an arbitrary unit of the IR densitometric value obtained by quantification of the IR-Cy3 immunofluorescent signal on frozen OB sections (mean ± SEM).

<p><i>(A)</i> Specificity of the anti-IR antibody. ANOVA of densitometric values revealed significant effect of the antibody used (p<0.001). The mean densitometric value obtained with a monoclonal mouse antibody directed against the β-subunit of the IR (anti-IR), was significantly higher than the mean densitometric value obtained when using either the mouse IgG1 (isotype control) or with the omission of the primary antibody (no anti-IR) (SNK, compared to isotype control # p<0.05, compared to No anti-IR § p¹<0.05; n = 6). The mean densitometric value obtained with the omission of the primary antibody was significantly higher than the mean densitometric value obtained with the isotype control (SNK, # p<0.05). (B–E) Mean IR densitometric values of the OB sections as a function of feeding states (B), zones (C), regions (D) and layers (E). Repeated measures ANOVA revealed significant effects of zones (ANOVA, p<0.0001), regions (ANOVA, p<0.001) and layers (ANOVA, p<0.0001; n = 10 including 5 fasted rats and 5 satiated rats) but no significant effect of the feeding state (ANOVA, p>0.05). <i>(B)</i> Feeding states: The mean IR densitometric values of the OB sections of fasted (F) and satiated (S) animals are similar. <i>(C)</i> Zones: The mean IR densitometric value was significantly higher in the caudal posterior zone (PZ) compared to the rostral anterior zone (AZ) and the intermediate zone (IZ) (SNK, compared to AZ # p<0.05, compared to IZ § p<0.05). <i>(D)</i> Regions: The mean IR densitometric value was significantly lower in the dorsomedial (DM) and the ventromedial (VM) regions of the main OB compared to the dorsolateral (DL) and ventrolateral (VL) regions (SNK, compared to DL # p<0.05, compared to VL § p<0.05). <i>(E)</i> Layers: The mean IR densitometric values measured in the nerve (NL), glomerular (GL), external plexiform (EPL), mitral cell (MCL) and granular cell (GCL) layers of the main OB were all statistically different from each other (SNK, # p<0.05).</p

Insulin ICV injection (14 mU) decreases olfactory detection in fasted rats.

<p>(<i>A</i>) Validation of the COA paradigm in the two-tube experimental device. Before the conditioning, during the last day of habituation (Hab.), both tubes were filled with pure water, and during the first day of aversion acquisition (Av.), both tubes were filled with ISO 10⁻⁵. A repeated measure ANOVA revealed a significant effect of the aversive conditioning (p<0.0001). For habituation and aversion acquisition, when two identical drinks were proposed, bar graphs represent the olfactory detection index corresponding to the number of licks of the first sampled tube normalized to the total number of licks (mean ± SEM). The mean olfactory detection indexes for habituation and aversion were not statistically different (SNK, p>0.05, ns). After conditioning, rats were given the choice between pure water and ISO 10⁻⁵ for the aversion test (Aversion test) and re-test (Aversion re-test). Bar graphs represent the olfactory detection index corresponding to the number of licks of the pure-water tube normalized to the total number of licks (mean ± SEM). The mean olfactory detection indexes for the aversion test and re-test were not statistically different (SNK, p>0.05, ns). The mean olfactory detection indexes were significantly different after conditioning (SNK, compared to Hab. # p¹<0.05, compared to Av. § p¹<0.05; n = 18). The dashed line represents the chance level (50%). (<i>B</i>) Insulin injection before acquisition does not alter aversion retention. Bar graphs represent the olfactory detection index corresponding to the percentage of licks of the pure water tube normalized to the total number of licks (mean ± SEM) when rats were given the choice between pure water and ISO 10⁻⁵ 1 h after ICV NaCl injection (NaCl) and 1 h after ICV insulin injection (Insulin). The mean olfactory detection indexes were not statistically different (paired t-test, p¹>0.05, ns; n = 11). (<i>C</i>) Insulin ICV injection (14 mU) decreases olfactory detection. Bar graphs represent the olfactory detection index when rats had the choice between pure water and odorized water at ISO 10⁻⁸ and ISO 10⁻⁹ 1 h after ICV NaCl injection (NaCl) and 1 h after ICV insulin injection (Insulin). The mean olfactory detection indexes were significantly different for odor dilutions (ANOVA, p¹<0.005) and ICV treatments (p¹<0.005; n = 18). Post-hoc comparisons showed that insulin ICV injections significantly decreased the olfactory detection index for both ISO 10⁻⁹ and ISO 10⁻⁸ odor dilutions (SNK, compared to NaCl for each odor # p¹<0.05; n = 18) and that the olfactory detection index is higher for ISO 10⁻⁸ compared to ISO 10⁻⁹ for each ICV treatment (SNK, § p¹<0.05; n = 18) (<i>D</i>) Insulin ICV injection (14 mU) does not change locomotor activity. Bar graphs represent the number of side changes in the two-tube experimental device (mean ± SEM) when rats had the choice between pure water, ISO 10⁻⁸ and ISO 10⁻⁹ 1 h after ICV NaCl injection (NaCl) and 1 h after ICV insulin injection (Insulin). The mean side changes were not statistically different for odors or ICV treatments (ANOVA, p>0.05, ns; n = 18). ¹Established using transformed data.</p

Insulin ICV injection (14 mU) alters olfactory sniffing behavior for a food-odor.

<p><i>(A)</i> Bar graphs represent evolution of the mean (± SEM) respiratory frequency during food odor presentation (30 sec), in fasted animals treated with NaCl (n = 10) or insulin (n = 10). Four periods of 10 seconds have been defined: Pre-odor (−10 sec to 0 sec); Odor presentation (start: 0 sec to 10 sec, middle: 10 sec to 20 sec, end: 20 sec to 30 sec). The mean of respiratory frequency of fasted-NaCl animals increased during the presentation of a food odor (Odor presentation, start) compared to Pre-odor period. In Fasted-Insulin rats, no change in respiratory frequency was observed upon odor presentation (Two-way repeated measures ANOVA followed by post-hoc comparisons; * p<0.05 significant differences). <i>(B)</i> Individual example of typical time-course of respiration frequency during food odor presentation in two conditions (Fasted rat treated with NaCl or insulin).</p

The effects of feeding state and 14 mU insulin ICV injection on blood and OB insulin levels.

<p>Bar graphs represent insulin concentrations (mean ± SEM) in the olfactory bulb (OB, full bars, left scale) and plasma (hatched bars, right scale) of fasted (n = 5) and satiated (n = 5) animals 1 h after ICV NaCl injection (Fasted NaCl, Satiated NaCl) or 1 h after ICV insulin injection (Fasted Insulin). The mean OB and plasma insulin levels were statistically different in satiated compared with fasted animals (Mann-Whitney test, * p<0.01), and the mean OB insulin levels were statistically different in fasted animals receiving 14 mU ICV insulin compared with fasted animals receiving NaCl ICV (Mann-Whitney test, # p<0.01).</p

IR mRNA and protein levels in the OB are not modulated by feeding states.

<p><i>(A)</i> Bar graphs represent the relative abundance of IR mRNA in the OB of satiated (gray bar; n = 6) and fasted (white bar; n = 6) rats as determined by semi-quantitative RT-PCR and using β-actin mRNA for normalization (mean ± SEM). Representative Western blots <i>(B)</i> and quantitative densitometric histograms <i>(C)</i> of the IR protein levels in the OB of satiated (gray bar, S; n = 4) and fasted rats (white bar, F; n = 4) (mean ± SEM). Bar graphs represent ratios of the IR subunits (IRα, and IRβ) protein levels after normalization against actin. The means were not statistically different (Mann-Whitney test, p>0.05).</p

IR localization in the OB layers.

<p>Representative images of the various layers of the main OB of adult rats obtained from frontal frozen sections (14 µm) stained with the nuclear marker DAPI <i>(A, E, I, M, Q, U)</i> and with double immunostaining of the blood vessel marker laminin <i>(B, F, J, N, R, V)</i> and IR <i>(C, G, K, O, S, W</i>). The signals were visualized on an epifluorescence microscope using sequential channel scanning with a merged overlay <i>(D, H, L, P, T, X)</i>. In the nerve layer <i>(A–D)</i>, IR immunostaining is restricted to the endothelium of blood vessels (white arrowheads). In the glomerular layer <i>(E–H)</i>, IR immunostaining is abundant is the neuropil of some glomeruli (full line). Some glomeruli are not labeled (dotted line). In the external plexiform layer <i>(I–L)</i>, a punctiform IR immunostaining is scattered on labeled fibers (arrows). In the mitral cell layer <i>(M–P)</i>, between the dotted lines, IR immunostaining is present in most cell bodies, although some mitral cells are not labeled (dotted circle). The labeled cell bodies are consistently surrounded by blood microvessels (arrowhead). <i>(Q–T)</i> Enlargement of a mitral cell body (dotted line) surrounded by three blood microvessels (arrowheads). In the granular cell layer <i>(U–X)</i>, IR immunostaining is located in clusters of labeled granular cell bodies (arrows). The labeled clusters are often located close to blood microvessels (arrowhead).</p