Soft-diet feeding impairs neural transmission between mitral cells and interneurons in the mouse olfactory bulb

Accepted Manuscript Title: Soft-diet feeding impairs neural transmission between mitral cells and interneurons in the mouse olfactory bulb Authors: Tomohiro Noguchi, Chizuru Utsugi, Makoto Kashiwayanagi PII: DOI: Reference:

S0003-9969(17)30236-4 http://dx.doi.org/doi:10.1016/j.archoralbio.2017.07.015 AOB 3953

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Archives of Oral Biology

Received date: Revised date: Accepted date:

30-4-2017 21-7-2017 23-7-2017

Please cite this article as: Noguchi Tomohiro, Utsugi Chizuru, Kashiwayanagi Makoto.Soft-diet feeding impairs neural transmission between mitral cells and interneurons in the mouse olfactory bulb.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2017.07.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Soft-diet feeding impairs neural transmission between mitral cells and interneurons in the mouse olfactory bulb

Tomohiro Noguchia, Chizuru Utsugib, and Makoto Kashiwayanagia

a

Department of Sensory Physiology, Asahikawa Medical University, 078-8510

Asahikawa, Japan; bDental Asahikawa Association Douhoku Oral Health Center, 0700029 Asahikawa, Japan

Correspondence: Makoto Kashiwayanagi, Department of Sensory Physiology, Asahikawa Medical University, Asahikawa 078-8510, Japan. Fax: +81-166-68-2339; e-mail: [email protected] Funding: This work was supported by a grant from the Japan Society for the Promotion of Science (No. 24770064 to MK). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors declare that no competing interests exist.

Graphical abstract

Highlights    

A soft diet impaired neurogenesis in the subventricular zone and olfactory functions sIPSCs at the mitral cells of mice fed a soft diet were measured The soft-diet feeding changed the intervals between sIPSCs and their peak amplitudes The soft-diet feeding in mice attenuates the neural functions at the olfactory bulb

Abstract (Objective) The subventricular zone in mice generates a lot of neuroblasts even during adulthood. These neuroblasts migrate to the olfactory bulb and differentiate into inhibitory interneurons such as granule cells and periglomerular cells.

Olfactory

sensory neurons receive information from various odorants and transmit it to the olfactory bulb.

Our previous study showed that soft-diet feeding impairs

neurogenesis in the subventricular zone, in turn leading to the reduction of odorinduced behaviors and Fos-immunoreactivities, the latter of which are markers of 2

neural activity, at the olfactory bulb after exposure to odors.

Release of GABA from

inhibitory interneurons at the olfactory bulb induces inhibitory currents at the mitral cells, which are output neurons from the olfactory bulb.

(Design) In the present

study, we measured spontaneous inhibitory postsynaptic currents (sIPSCs) at the mitral cells of mice fed a soft diet in order to explore the effects of changes in texture of diets on neural function at the olfactory bulb.

(Results) The soft-diet feeding

extended the intervals between sIPSCs and reduced their peak amplitudes. (Conclusions) The present results suggest that soft-diet feeding in mice attenuates the neural functions of inhibitory interneurons at the olfactory bulb.

Keywords: soft diet, hard diet, mitral cell, olfactory bulb, neural function

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1. Introduction Mastication has been shown to be related to brain function in humans and animals (Weijenberg et al., 2011). For example, mastication influences cognitive processing time, as evaluated by reaction time and the latency of event-related potential waveforms during an auditory oddball paradigm (Sakamoto et al., 2009). A five-year prospective cohort study of elderly subjects showed that tooth loss predicts the development of mild memory impairment (Okamoto et al., 2015). Mastication also relates to dementia in Alzheimer’s patients.

An investigation of a

potential association between a history of oral disease and the development of dementia caused by Alzheimer’s disease indicates that participants with the fewest teeth had the highest prevalence of dementia (Stein et al., 2007).

In addition, a case-

control study targeting identical twins showed that a history of tooth loss before age 35 is a significant risk factor for Alzheimer’s disease (Gatz et al., 2006).

Mastication

induces long-term increases in blood perfusion of the trigeminal principal nucleus in humans (Viggiano et al., 2015). These results suggest that mastication has a significant effect on brain function in humans (Miura et al., 2003). Compared to mice fed a hard diet, those fed a soft diet performed more poorly on tests of working memory and spatial memory (Kato et al., 1997; Yamamoto and Hirayama, 2001), suggesting that texture of diets correlates with brain function also in experimental animals.

In a previous study, we showed that ingestion of a hard diet

induced remarkable excitation of neurons at the principal sensory trigeminal nucleus (Pr5) in mice, which receive oral somatosensory information via the trigeminal nerves, whereas ingestion of a soft diet did not (Utsugi et al., 2014a).

These results

suggest that sensory inputs induced by mastication are also impaired by feeding animals only a soft diet (powdered food).

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Neurogenesis occurs in the subventricular zone and hippocampus throughout life (Ming and Song, 2011).

Neurons generated in the subventricular zone migrate

via the rostral migratory stream to the main olfactory bulb (Lois and Alvarez-Buylla, 1994) and accessory olfactory bulb (Honda et al., 2009). These cells differentiate into granule cells and periglomerular cells in the olfactory bulb (Lois and AlvarezBuylla, 1994; Carlen et al., 2002; 2003). Newly generated neurons migrated from the subventricular zone play important roles in odor discrimination (Gheusi et al., 2000) and odor memory (Rochefort et al., 2002) in the olfactory bulb. In mice, soft-diet feeding reduced neurogenesis in the subventricular zone, decreased the number of newly generated neurons in the olfactory bulb and accessory olfactory bulb, and impaired olfactory functions (Utsugi et al., 2014a; 2014b). Granule cells and periglomerular cells, which originate from the subventricular zone, make inhibitory synapses with mitral cells and modify odor information.

Through

an electrophysiological technique, GABAA receptor-mediated reciprocal inhibitory postsynaptic currents (IPSCs), which reflect prolonged GABA release from granule cells (Schoppa et al., 1998), are recorded.

In the present study, spontaneous IPSCs

(sIPSCs) recorded from mitral cells of the olfactory bulb of male mice fed a soft or hard diet were studied to explore the effects of changes in diet texture on neural functions.

2. Materials & Methods All experiments were carried out in accordance with the Guidelines for the Use of Laboratory Animals of the Asahikawa Medical University and approved by the Committee of Asahikawa Medical University for Laboratory Animal Care and Use (approval ID: 11014).

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2.1. Animals A total of 25 bulb/c male mice (from 24 to 44 weeks old) were used.

Five

mice were fed 25 g of hard or soft diet per week for 1 month, with the animals separated into different cages according to diet.

The mice were obtained from the

Animal Institute of Asahikawa Medical University.

The hard and soft diets were

purchased from Sankyo Laboratory Co. (Sapporo, Japan). The soft diet was actually the same hard diet reduced to powder.

2.2. Electrophysiological analysis of mitral cells The mice, all of whom were deeply anesthetized with pentobarbital sodium (150 mg/kg), were perfused through the heart with ice-cold sucrose-based Ringer’s solution (234 mM sucrose, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 10 mM MgCl2, 11 mM glucose, pH 7.4) oxygenated with 95% O2 and 5% CO2 mixed gas.

The olfactory bulbs and brain were removed and cut into

parasagittal slices at a thickness of 200 m with a vibrating slicer (DOSAKA EM, Kyoto, Japan).

The slices were incubated in normal Ringer’s solution (125 mM

NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, 11 mM glucose, pH 7.4) saturated with O2/CO2 gas at 37°C for 30 min and equilibrated in the same solution at room temperature until use. All recordings were performed at room temperature.

Each brain slice was

fixed to the glass at the bottom of a recording chamber filled with normal Ringer’s solution saturated with O2/CO2 gas.

Individual mitral cells in the slice were

observed by infrared differential interference contrast video microscopy through an E600FN microscope (Nikon, Tokyo, Japan).

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Glass electrodes were filled with CsCl

intracellular solution (115 mM CsCl, 15 mM NaCl, 2 mM MgCl2, 2 mM Na2ATP, 0.5 mM EGTA/CsOH, 10 mM HEPES, 10 mM tetraethylammonium chloride, pH 7.2/CsOH). Glass electrodes with resistances of 5.7 ± 0.11 M (n = 60; mean ± SE) were made from borosilicate glass capillaries with an inner filament (GD-1.5, Narishige Co., Tokyo, Japan) using a two-stage electrode puller (PP853, Narishige Co.) and then heat polished.

The seal resistance achieved with the tips of the glass

electrodes on the cell surface was 9.6 ± 0.6 G (n = 57). Current signals were acquired through an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) with a 10 kHz low-pass filter, and were digitized by a Digidata 1320A digitizer (Molecular Devices) controlled via Clampex software (Molecular Devices) at a sampling frequency of 20 kHz.

Compensation of the

capacitive current and series resistance was performed manually using a built-in circuit of the amplifier.

Under the voltage clamp condition with a holding potential

of −80 mV, sIPSCs were recorded from mitral cells, which were identified by their cellular shape and location in the olfactory bulb.

Excitatory activity of neurons was

blocked by 20 M 6-cyano-7-nitroquinoxaline-2,3-dione , 20 M 2-amino-5phosphonovaleric acid and 10 M tetrodotoxin. sIPSCs were detected with templates comprising rising (time constants of 1, 3, and 5 msec) and decaying (10 msec time constant) exponentials.

They were then collected for analysis when their

peak amplitude was above 20 pA. For analyzing the effect of soft-diet feeding on synaptic activity in the olfactory bulb, the peak amplitude and the instantaneous frequency of the recorded sIPSCs were measured (Table 1).

Spontaneous events of mice with bicuculline (n = 2) and

without it (n = 3) were pooled from 7 cells each.

Similarly, spontaneous events of

soft-diet (n = 4) and hard-diet (n = 4) mice were pooled from 7 and 13 cells, 7

respectively.

2.3. Statistical analysis Differences in membrane resistance and capacitance, and in decay constants were compared using Student's t-tests. In the present study, only sIPSCs with amplitudes above 20 pA were analysed, making it likely that the data were not distributed normally. Therefore, changes in sIPSC peak amplitudes and sIPSC interevent intervals were compared with Mann Whitney U-tests.

3. Results 3.1. Inhibition of sIPSCs by bicuculline To demonstrate that the recorded signals were originated from GABAergic synapses, we first electrophysiologically recorded the sIPSCs of mitral cells under the pharmacological suppression of excitatory postsynaptic currents. bicuculline partially inhibited sIPSCs at the mitral cells (Fig. 1A).

Application of As can be seen in

the enlarged typical sIPSCs without and with bicuculline, bicuculline reduced the amplitude and width of the sIPSCs (Fig. 1B).

Bicuculline extended the average

interval between sIPSCs, and attenuated both the average peak amplitude and decay constant of sIPSCs.

3.2 Soft-diet feeding impaired functions of neurons in the olfactory bulb If a soft diet affects the functions of interneurons in the olfactory bulb, an alteration of inhibitory input into mitral cells should be observed.

We therefore

measured the sIPSCs of mitral cells of mice fed a soft or hard diet.

The sIPSCs

recorded from mitral cells of the soft-diet feeding mice showed reduced frequency

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and amplitude than those of the hard-diet feeding mice (Fig. 2A).

In typical sIPSCs,

the soft-diet feeding reduced the amplitude of typical sIPSCs but did not change the time course.

Similar to the case with the application of bicuculline, soft-diet feeding

extended the average interval and attenuated the average amplitude of sIPSCs. However, the decay constants of sIPSCs in the soft-diet feeding mice were similar to those of the hard-diet feeding mice.

4. Discussion Decreases in neurogenesis at the subventricular zone impair the various functions related to olfaction. Ablation of adult neurogenesis induced by irradiation of the subventricular zone impairs long-term odor memory rewarded with water (Lazarini et al., 2009).

The blocking of neurogenesis by cytarabine reduces

oscillations in the olfactory bulb (Breton-Provencher et al., 2009).

Olfactory

perceptual learning is also impaired with aging, and this impairment is associated with a reduction of neurogenesis (Morenoet al., 2003).

In our previous study, we showed

that mice fed a soft diet showed low neurogenesis and did not avoid the odor of 50% butyric acid, while mice fed only a hard diet or a hard diet after a soft one showed normal or recovered neurogenesis, respectively, and avoided the odor in either case. This suggests that the decrease in adult neurogenesis induced by a soft diet impaired odor cognition for avoidance.

Thus, the decrease in neuroblasts migrated from the

subventricular zone changes the functions of neurons at the olfactory bulb. In electrophysiological experiments, chewing has been shown to change neural function at the hippocampus.

Chewing on a wooden stick restored attenuation of the

long-term potentiation induced by restrained stress in CA1 hippocampal neurons (Ono et al., 2009). The rescue of stress-suppressed long-term potentiation by chewing

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failed to occur in rats treated with histamine H1 receptor antagonist before exposure to stress and chewing (Ono et al., 2009). This suggests that chewing restores the attenuation of long-term potentiation by stress via the histaminergic nervous system. In the present study, we showed that soft-diet feeding impaired inhibitory inputs from interneurons to the mitral cells at the olfactory bulb. Granule cells are GABAergic interneurons in the olfactory bulb and inhibit mitral cells via GABAA receptor subunit 1/3 (Panzanelli et al., 2009), which is an ionotropic receptor that gates chloride channels.

There are two currents having

different kinetics in sIPSCs, which are generated by GABA released from granule cells via GABAA receptors.

In a CA1 pyramidal cell, fast sIPSCs and slow sIPSCs

are recorded (Capogna and Pearce, 2011). One type of GABAA receptor is antagonized by bicuculline, while the other type is insensitive to it (Johnston, 2013). As shown in the present study, bicuculline shortened sIPSCs recorded from mitral cells at the olfactory bulb, suggesting that bicuculline inhibits slow sIPSCs in mitral cells. The time constants of sIPSCs recorded in soft-diet mice were similar to those in hard-diet mice. This suggested that a soft-diet feeding inhibited both fast and slow sIPSCs.

It is therefore possible that a decrease in newly generated granule cells

migrated from the subventricular zone by soft-diet feeding changes the characteristics of GABA released from granule cells, although it is also possible that soft-diet feeding affects GABAA receptors in mitral cells. A prolonged trigeminal stimulation enhances GABAergic transmission in the spinal trigeminal nuclei (Viggiano at el., 2004).

Changes in odorant sensory inputs

alter GABA biosynthesis levels in interneurons with modulation of DNA secondary structure formation in the olfactory bulb (Parrish-Aungst et al., 2011; Wang et al., 2017). These results indicate activity-dependent regulation of inhibition in

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GABAergic neurons (Lau and Murthy, 2012).

In mice and rats, retronasal

stimulation by odorants induces odor responses (Scott et al., 2007; Rebello et al., 2015). Therefore, it is possible that difference in retronasal sensory inputs of diets affects GABA synthesis in the olfactory bulb.

However, ingestion of a hard and soft

diet induces almost the same neural excitation at the olfactory bulb (Utsugi et al., 2014a). The result suggests that reduction of sIPSCs at the olfactory bulb by softdiet feeding is not induced by reduction of odor inputs by soft-diet feeding. In mice, impairment of spatial memory and decreases in neurogenesis at the hippocampus by molar tooth loss are reduced by an enriched environment containing running wheels and colored tunnels (Kondo et al., 2016).

In our previous study, we

showed that hard-diet feeding leads to the recovery of neurogenesis at the subventricular zone and olfactory functions in mice (Ustugi et al., 2014a). Therefore, in future studies we will need to explore the effects of an enriched environment or hard-diet feeding on soft-diet-impaired inhibitory functions of interneurons at the olfactory bulb.

Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 15K07962 to MK).

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Figure Legends Figure 1

Bicuculline reduced sIPSCs recorded from mitral cells.

A, Typical recordings of sIPSCs from mitral cells without or with bicuculline. Membrane resistance and capacitance showed no significant difference between control and + bicuculline (t-test, p > 0.05).

Rm: 0.33 + 0.22 in control, 0.19 + 0.08

G in + bicuculline. Cm: 44 +16 pF in control, 40 + 9 pF in +bicuculline. mean + 95% confidence interval.

B, Ensemble average of 738 sIPSCs in control (black) and

of 55 sIPSCs in + bicuculline (gray).

Inset, comparison of sIPSC time course.

C,

Increases in the average sIPSC inter-event interval of + bicuculline (p < 0.01, MannWhitney U-test). D, Decreases in the average sIPSC peak amplitude of + bicuculline (p < 0.01, Mann-Whitney U-test).

E, Decreases in the average sIPSC decay constant

of + bicuculline (p < 0.01, Student’s t-test). Number of animals and cells were shown in Table 1.

Figure 2 Soft-diet feeding reduced sIPSCs recorded from mitral cells. A, Typical recordings of sIPSCs from mitral cells of mice fed a hard or soft diet.

By

using the whole-cell patch-clamp technique, sIPSCs were pooled from 13 cells (hard diet) and 7 cells (soft diet).

Membrane resistance and capacitance showed no

significant difference between hard- and soft-diet mice (t-test, p > 0.05).

Rm: 0.18

+ 0.08 (hard, 13 cells), 0.36 + 0.28 G (soft, 7 cells). Cm: 44 + 12 pF (hard, 13 cells), 37 + 22 pF (soft, 7 cells).

Mean + 95% confidence interval.

B, Ensemble

averages of 2945 sIPSCs in hard (black) and of 586 sIPSCs in soft (gray). comparison of sIPSC time course.

Inset,

C, Increases in the average sIPSC inter-event

interval of soft-diet mice (p < 0.01, Mann-Whitney U-test). D, Decreases in the average sIPSC peak amplitude of soft-diet mice (p < 0.01, Mann-Whitney U-test). 16

E, The average sIPSC decay constants of hard- and soft-diet mice. Number of animals and cells were shown in Table 1.

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Table 1. Numbers of animals and cells used for analyses. Mice n of n of mice cells With 2 7 bicuculline Without 3 7 Bicuculline Soft diet 4 7 Hard diet 4 13

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n of sIPSCs 55 738 586 2946