Neuronal responses to intragastric Neuronal responses to intragastric administration of amino acids in the administration of amino acids in the

rat amygdala and lateral rat amygdala and lateral hypothalamic areahypothalamic area

アミノ酸溶液の胃内投与に対する扁桃体およびアミノ酸溶液の胃内投与に対する扁桃体および視床下部外側野ニューロンの応答様式視床下部外側野ニューロンの応答様式

Davaasuren Munkhzul

System Emotional Science, Graduate School of Medical and

Pharmaceutical Science, University of Toyama, Toyama, Japan,

A.B.

Effects of foods on emotion

Behavioral responses to a bitter taste solution

Another example of emotion induced by foods (2)Another example of emotion induced by foods (2)

Negative emotion

Speculation from these photos suggests that exteroceptive sensory signals, especially taste sensation, induce emotion; taste sensation of palatable foods induces pleasant emotion, and consequently promotes feeding behavior. So, feeding behavior is a kind of appetitive behavior driven by positive emotion.

Pleasant feeling; Taste good.

Pleasant feeling; A sense of well-being or satisfaction?

Exteroceptive sensory signals

Schematic illustration of sites of actions of foods

Visceral sensory signals

Recent studies reported that visceral sensory signals induce not only satiation but also rewarding effects (Sclafani, 2008).

Thus,　 foods stimulate both the taste system and gastrointestinal (GI) tract to induce emotion. In the same way, umami compounds in the foods are also supposed to stimulate both the taste system and GI tract. I will show you this possibility in the next slide.

IntroductionIntroduction

1. 1. Intragastric infusion of umami substances, monosodium glutamate (MSG) and inosine monophosphate (IMP), altered activities of the vagus nerve and limbic system (Tsurugizawa et al., 2010; Kitamura et al., 2011), and is reported to be rewarding (Uematsu et al., 2009).

2. 2. On the other hand, recent studies suggest that abnormality of vagal functions is related to socio-emotional disturbance including depression, social disturbance, aggression, etc. in humans.

3. 3. We hypothesized that visceral sensory signals might induce emotion such as a sense of well-being, and that deficits in this process would induce socio-emotional disturbance.

4. 4. These findings also suggest that umami compounds might affect socio-emotional behaviors through the vagus nerve.

IntroductionIntroduction

• On the other hand, the amygdale (CeA) and lateral hypothalamic

area (LHA), which are major structures in the limbic system, play

an important role in emotion, and receive strong inputs from the

nucleus of solitary tract (NTS) that receives afferent inputs from

the vagus nerve (Berthoud et al. 2000).

• In the present study, we recorded neuronal activity from the

central amygdala (CeA) and lateral hypothalamic area (LHA), and

investigated changes in neuronal activity during intragastric

administration of amino acids including umami substance,

glutamate.

Purpose of present studyPurpose of present study

• To investigate the gut-brain communication, neuronal activity was recorded from central nucleus amygdala (CeA) and lateral hypothalamic area (LHA) during intragastric infusion of amino acids and umami substance.

Materials and MethodMaterials and Method

Subjects:• 15 adult male Wistar rats,　 270-320g

Surgery:• Chronic electrode implatantion • Intragastric cannula implantation

Umami solutions: • Glutamate solution group: GA (glutamate arginine), MSG

(monosodium glutamate), MSG+IMP, and Dashi (dried bonito solution) (60mM, 12, 5, 50%, 83.5%)

• Other solution group: AH (arginine HCl), NaCl, Saline (60mM, 0.9%)

An experimental set up for recording amygdalar and hypothalamic neurons during

intragastric infusion

Syringe pump

Solutions (10 ml/kg)

30 cm

30 cm

Video image

Red lamp

CCD camera

Neuronal activity

Mirror

60 mM NaCl, 60mM GA, 60mM AH0.9% Saline60 mM MSG60 mM MSG + 　 IMP22.5 % Dashi (dried bonito solution)etc.

Intragastric tube

Tetrodes

Rat

(Japanese soup stock made from dried bonito)

• Recording schedule

…

… Amino acid injection

Saline injection(Checking stretch receptor-related responses)

. PAUSE-5min Saline-injection-30sec .

-10 0 10 min 50 -5 0 5 min

Materials and MethodMaterials and Method

10 min – 20minInject 1ml/min/kg (60 mM) solution for 10min (GA, AH, Saline, MSG or

NaCl. One solution per day)

Data Analysing method• Single unit activities were sorted by the Offline Sorter program. • In each cluster of neuronal activities, interspike interval histogram was

constructed to confirm that absolute refractory period was longer than 1 ms. • Finally, superimposed waveforms of the isolated units were drawn to check

wave form consistency throughout the recording sessions, and then were transferred to the Neuroexplorer program.

Materials and MethodMaterials and MethodPC 2

PC 1

Rang

e +/-

2 mV

Unit a Unit b

Data Analysis• Pre and post administration period were defined as 10 min before and after the onset of

the administration, respectively.• Perievent histograms aligned with each administration of solution were constructed in each

neuron (bin size=60s). • The baseline firing rate of a neuron was calculated from the pre administration period. • Significant responses were defined as those with activities exceeding 2 standard

deviations above or below the mean firing rates. • In each solution, latency and duration of the neuronal response were computed.

00.51

1.52

2.53

3.54

4.55

- 20 - 10 0 10 20 30 40 50

Latency

Period 1 Period 2 Period 3 Period 4 Period 5

0 50minTime course

Inhibitory duration

Firi

ng r

ate

(spi

kes/

sec)

Administration period

Exc duration

Materials and MethodMaterials and Method

0

0.2

0.4

0.6

0.8

1

1 .2

1 .4

1 .6

1 .8

2

- 20 - 1 0 0 1 0 20 30 40 50

t im e (m in )

Excitatory neuronExcitatory neuron

Inhibitory neuronInhibitory neuron

Firi

ng r

ate

(spi

kes/

sec)

Administration period

Administration period

Table.1 Summary of the neuronal responsesTable.1 Summary of the neuronal responsesIn Central Amygdala and Lateral Hypothalamus.In Central Amygdala and Lateral Hypothalamus.

GA AH MSG Saline NaCl MSG-IMP Dashi

CeA

Excitatory 15 1 10 1 5 1 2

Inhibitory 15 2 5 2 1 1 1

Ex+In 4 1 3 2 1 1 1

Non-resp 9 4 9 4 2

Total-103 43 8 27 9 9 3 4

LH

Excitatory 1

Inhibitory 3 1

Ex+In 2 1

Non-resp 3

Total-11 9 2

ResultsResults

0

500

1000

1500

2000

2500

CeA LH

0

200

400

600

800

1000

GA AH

Response Latency (sec)

Resp

on

se Du

ration

(sec)

Duration Latency

[Fig. 1 A-B] Comparison of mean duration & latency response between CeA and LH (Type mixed)

The result of 2-way ANOVA: Significant main effect of Region (p < 0.05)

The result of 2-way ANOVA: n.s.

GA AH

CeA LH

GA AH GA AH

A B

ResultsResults

0

1

2

3

4

-20 -10 0 10 20 30 40 50time (min)

A. CeA responses

0

10

20

30

40

-20 -10 0 10 20 30 40 50

time (min)

Fig.2 Examples of CeA and LH neuronal responsesResultsResults

B. B. LHA responses

Intragastric infusion (60 mM GA, 10 ml/kg)

Intragastric infusion (60 mM GA, 10 ml/kg)Firi

ng r

ate

(spi

kes/

sec)

[Fig 3] Mean duration of Excitatory responses in each time window in each Solution

****** ************

****

(**p<0.01, ***p<0.001, Bonferroni's correction)

The result of 2-way repeated measured ANOVA: Significant main effect of Time (p < 0.001), Significant interaction of Time x Solution (p < 0.05)

0

100

200

300

400

500

600

GAMSGNaClMSG+IMPDashi

0 - 10 min 10 – 20 min 20 – 30 min 30 – 40 min 40 – 50 min

Response D

uration (sec)

ResultsResults

Fig.4 Examples of excitatory responses in the rat amygdalar (CeA)Fig.4 Examples of excitatory responses in the rat amygdalar (CeA)

B.

A. Intragastric infusion (60 mM MSG, 10 ml/kg)

00.20.40.60.81.0

Intragastric infusion (22.5% dried bonito solution, 10 ml/kg)

0

0.2

0.4

0.6

0.8

-10 0 10 20 30 40 Min

-10 0 10 20 30 40 Min

Firi

ng r

ate

(spi

kes/

sec)

ResultsResults

0

50

100

150

200

250

300

350

400

450 GAAHMSGSaline

[Fig.5] Mean duration of Inhibitory responses in each time window in each Solution

0 - 10 min 10 – 20 min 20 – 30 min 30 – 40 min 40 – 50 min

Response D

uration (sec)

The result of 2-way repeated measured ANOVA: n.s.

ResultsResults

01020304050

-20 -10 0 10 20 30 40 50time (min)

01234

-20 -10 0 10 20 30 40 50time (min)

Fig.6 An example of Inhibitory response in the CeA

ResultsResults

Intragastric infusion (60 mM MSG, 10 ml/kg)

Intragastric infusion (0,9% saline, 10 ml/kg)

Firi

ng r

ate

(spi

kes/

sec)

0

500

1000

1500

2000

GA AH MSG Saline NaCl MSG+IMP Dashi

Resp

on

se D

uratio

n (sec)

Solution

[Fig.7] Mean duration response of each Solution (CeA, Type mixed)

⇒The result of one-way anova: n.s.

0

200400600800

100012001400

Glu Solutions Others

[Fig 8] After groups combined…

Resp

on

se D

uratio

n (sec)

****

(** p<0.01, student t test)

ResultsResults

[Fig. 9] Mean Latency response to each Solution (CeA, Type mixed)

0200400600800

100012001400

GA AH MSG Saline NaCl MSG+IMPDashi

Resp

on

se La

tenc

y (sec)

Solution

⇒The result of one-way anova: n.s.

0100200300400500600700800900

Glu Solutions Others

[Fig.10] After groups combined…

Resp

on

se Laten

cy (sec)

(p>0.05, student t test)

ResultsResults

=> Saline injection elicited significant responses in only 2 neurons.

Fig.11 Responses to saline injection (1bin=30s)

0

0.5

1

1 .5

2

2.5

- 5 0 5

t ime (min )

firin

g ra

te (H

z)

0

0.2

0.4

0.6

0.8

1

1 .2

1 .4

1 .6

1 .8

- 5 0 5

t ime (m in )

firin

g ra

te (H

z)

injectioninjection

00

ResultsResults

Result SummaryResult Summary

In CeA• 103 neurons responded excitatorily and/or Inhibitorily to at least one of the

amino acid solutions.• Mean duration was longer in the solutions including glutamate than the other

solutions.• Mean response latencies to the solutions were not significantly different

among the solutions.• Response durations of the excitatory responses were significantly longer in

the 2nd period window than other response window.• However, inhibitory response duration was not significantly different.• Saline injection elicited significant responses in only 2 neurons among the10

neuron tested.

In LHA• 11 neurons responded to at least one of the amino acid solutions.• Mean response duration was significantly longer in the LHA than CeA.

• The results suggest that the LHA and CeA receive interoreciptive information from the gut.

• More CeA neurons responded to the solutions, suggesting that CeA is more involved in gut-derived (interoceptive) information processing than LHA.

• Saline injection result suggest that CeA’s neuronal responses were not ascribed to stretch receptor of the vagus, but to chemical information from the gut.

DiscussionDiscussion

Thank you …Thank you …