Positron Emission Tomography Studies of Pig Brain Activity While Exposed to Pheromones and other Odors[1]

 

 

D. L. ANDERSON*, M. BELLON*, B. CHATTERTON*, R. L. MCPHERSON‡, AND J. J. MCGLONE‡ [2]

*Royal Adelaide Hospital, Adelaide, South Australia

‡Texas Tech University, Lubbock, TX 79409

 

ABSTRACT:  The purpose of this study was to determine if Positron Emission Tomography (PET) technology could be used as an effective method of investigating olfactory activation by biologically relevant and non-relevant odors for the domestic pig (Large White gilts).  Brain activation was estimated by PET imaging of 18F.d-glucose (18FDG) uptake in brain tissue.  The cerebellum was used as a reference (“fixed uptake”) tissue to compare other regions based on the relative count density of pixels in the computer-reconstructed images.  Compared with control, olfactory stimulation with a non-biologically-relevant odor (amyl acetate) caused a 22% increase in olfactory bulb activity.  A biologically-relevant maternal pheromone (simulated skin secretions) caused a 52% increase in 18FDG uptake, compared to control pigs.  Both olfactory signals increased activation of the hypothalamus over the control (nil) level of activation.  The parietal lobe was also activated in the pigs with the biologically relevant but not in the pig with the control odor exposure.  In conclusion, PET technology can be used to study olfactory activation of the pig brain. Regional differences in brain activation could be demonstrated for three states of olfactory exposure: control situation (no odor), control odor exposure (non-biologically-relevant odor) and biologically-relevant odor exposure.    

 

Key Words:  Pigs, Positron Emission Tomography, Brain, Pheromone.

 

 

Introduction

Positron Emission Tomography (PET) has been used in pig models in recent years to image activity in organs such as the liver and kidneys (Piert et al., 1999; Juillard et al., 2000).  However, there are few studies of pig brain activation, and to our knowledge, no previously reported studies of pig olfactory activity using PET.   Use of PET technology to study pig brain olfactory activation is a way to study important behavioral and physiological mechanisms.  By using PET scan technology repeated samples may be taken on the same animals and data can be generated on whole-organ physiological activation. The use of 18FDG as a measure of real-time brain activation was recently described in the pig.   Poulsen et al. (1997) concluded that the pig was a suitable model for PET studies of cerebral blood flow, cerebral oxygen consumption and glucose metabolism.  Since then, a number of papers have used the domestic pig as a model for brain activity, particularly related to Parkinson’s disease and dopamine metabolism using tracers other than 18 FDG (Danielsen et al., 2000; Ishizu et al., 2000).  The objectives of this study were 1) to determine the feasibility of PET technology to measure olfactory activation of the pig brain and; 2) to determine if relative activation of the olfactory bulb could be distinguished by exposure using biologically relevant and non-relevant odors. 

 

 

Materials and Methods

Four Large White gilts 14 and 35 d of age taken directly from the nursing sow were used for all studies.  Prior to being transferred to prepared pet cages for the study, all gilts were weighed and observations of general health were made. Gilts that had an unusually high or low BW, or that were in poor physical health were excluded from the study.  A total of four gilts were evaluated: one control (nil), one odor control (amyl acetate), and two pigs experienced pheromones.

On the day of the study, the gilts were transported in separate animal carriers to the Institute of Medical and Veterinary Science Research Theatres in Adelaide (Australia).  Each of the gilts was housed separately in a large pet carrier with straw bedding and all were fasted but had ad-libitum access to water.  Handling of the gilts was kept to a minimum and the period of time between weaning to the start of the study was no longer than 6 h.

At the time of delivery of the radioactive 18FDG, the first of the study gilts were sedated using a combination of Ketamine and Diazepam given intramuscularly in the neck. The prepared dose of 18FDG was drawn up and the 10 mL dose of olfactory test agent allocated to that animal was measured out away from the designated research area.

After sedation, the gilts were wrapped securely and intravenous access was gained via the ear vein of the pig. The femoral vein was used in one case because the ear vein was not accessible.  At 30 s before injection of the dose of 18FDG, the test agent was painted onto the snout of the pig. The 18FDG was then given and the IV line removed. The test agent was continually applied for a further 15 min (primary uptake phase of 18FDG). The gilt was then returned to the holding cage and left for 30-45 min to complete the uptake phase of the 18FDG. After completion of the uptake phase the gilt was euthanized via intra-cardiac injection of Pentobarbitone (5-10 mL) and transported to the PET facility within the department of nuclear medicine, Royal Adelaide Hospital (Australia).

Gilts were scanned in a dedicated full-ring PET scanner (C-PET, Adac Laboratories, Milpetas CA).  The useful field of view is 56 cm in diameter; with a scan length of 25 cm. Resolution at the centre of the field of view is approximately 5 mm FWHM.

Data collection was by analysis of regional counts in the digitally reconstructed images using proprietary data acquisition (ADAC) software (v 3.0) (Adac Laboratories, Milpetas CA). A fixed size region of interest was drawn over relevant structures on the appropriate “slices”, and the counts recorded. The cerebellum served as the control region for levels of activity.  The cerebellum is not considered to be involved in olfaction or perception of olfactory signals, and is frequently used as an (assumed) fixed reference.  Brain activity (uptake of 18FDG) was expressed relative to the cerebellum.  The relative activity of brain regions after different treatments were compared to two control situations, no odor (“nil”), a biologically non-relevant odor, or amyl acetate was used.  We were interested in showing which brain regions were activated by olfactory stimulation, if any, and if the control odor and the biologically-relevant odor caused brain activation at different levels. 

The maternal pheromone was a commercial product called “Suilence” (Ceva Santi Animale Libourne, France).  The pheromone is a mixture of molecules intended to mimic those found on the skin of sows during lactation. For odor application, amyl acetate was applied to the snout with a brush (10 mL, MW 130.19, Sigma Chemical, St. Louis, MO).  While the odor was brushed, pigs were breathing normally.  The act of brushing was used for all gilts except the nil gilt.  

 

 

Results and Discussion

The olfactory bulb of the resting, non-odor-exposed gilt showed less activity (82%) than the cerebellum (selected as the control area).   This finding may be interpreted that the un-stimulated olfactory bulb was less metabolically active than the resting cerebellum.  The olfactory: cerebellar ration was 22% greater than the nil gilt when the gilt was exposed to amyl acetate.  These data support the hypothesis that olfactory bulb activation can be demonstrated with PET technology. 

The relevant maternal pheromone showed a further increase in uptake (2% and 48%) compared with the control odor Compared with activity after the control odor (amyl acetate); activity in the parietal lobe, the hypothalamus and to a small extent, the temporal lobe was increased after the maternal pheromone.

The trends shown in pig brain activation (Table 1) were encouraging in that appropriate regions of the brain were activated.  Olfactory signals should cause increased glucose uptake in the olfactory bulb, the temporal lobe of the neocortex and the underlying amygdala, and the hypothalamus.   Brushing of the snout should cause activation of the somatosensory cortex through the sense of touch. 

These data support the hypotheses that 1) pigs can sense the maternal pheromone Suilence by olfaction, 2) that the maternal pheromone is a biologically-relevant odor to young pigs that activates the brain beyond the levels observed by a non-biologically-relevant odor.  Results also demonstrate that olfactory-induced activation of the pig brain was able to be demonstrated using PET technology. Olfactory activation and activation of other areas of the brain associated with olfactory stimulation (temporal cortex) and with the brushing against the snout (somatosensory cortex) were able to be clearly distinguished using PET technology (Figure 1).

 

 

 

Implications

The use of PET technology to study brain activation is an important tool for the animal sciences.  Brain activation is important in feeding, sexual, and social behaviors.  PET technology could be used to study basic brain mechanisms associated with these important behaviors.  In addition, a central research question in the science of animal welfare has to do with cognitive perceptions of farm animals.  PET technology may provide a window into how the brains of farm animals process information about how they perceive their environment.

 

 

 

Literature Cited

Danielsen, E. H., P. Cumming, F. Andersen, D. Bender, T. Brevig, L. Falborg, A. Gee, N.M. Gillings, S. B. Hansen, F. Hermansen, J. Johansen, T. E. Johansen, A. Dahl-Jrgensen, H. A. Jrgensen, M. Meyer, O. Munk, E.B. Pedersen, P. H. Poulsen, A. B. Rodell, M. Sakoh, C. Z. Simonsen, D. F. Smith, L. Ostergard, J. Zimmer, and A. Gjedde.  2000.  The DaNeX study of embryonic mesencephalic, dopaminergic tissue grafted to a minipig model of Parkinson’s disease: preliminary findings of effect of MPTP poisoning on striatal dopaminergic markers.  Cell Tranplant (Cell transplatation). 9:247.

Ishizu, K., D. F. Smith, D. Bender, E. Danielsen, S. B. Hansen, D. F. Wong, P. Cumming, and A. Gjedde.  2000.  Positron emission tomography of radioligand binding in porcine striatum in vivo:  halpoperidol inhibition linked to endogenous ligand release.  Synapse.  38:87.

Juillard, L. M., F. Janier, D. Fouque, M. Lionnet, D. Le Bars, L. Cinotti, P. Barthez, C. Gharib, and M. Laville.  2000.  Renal blood flow measurement by positron emission tomography using 15O-labeled water. Kidney Int. 57:2511. 

Piert, M., H. Machulla, G. Becker, A. Stahlschmidt, M. Patt, P. Aldinger, P. D. Dissmann, H. Fischer, R. Bares, H. D. Becker, and W. Lauchart.  1999.  Introducing fluorine-18 fluoromisonidazole positron emission tomography for the localization and quantification of pig liver hypoxia.  Eur. J. Nucl. Med. 26:95.

Poulsen, P. H., D. F. Smith, L. Ostergaard, E. H. Danielsen, A. Gee, SB Hansen, J. Astrup, and A. Gjedde.  1997.  In vivo estimation of cerebral blood flow, oxygen consumption and glucose metabolism in the pig by [15O] water injection, [15O] oxygen inhalation and dual injections of [18F] fluorodeoxyglucose.  J. Neurosci. Methods.  77:19.

 

 

 

Table 1.  Relative activation of regions of pig brains by PET scan when experiencing nothing (nil), a control odor (amyl acetate), or 10 mL of Suilence (a synthetic maternal pheromone).  (Most table figures are expressed as brain activity relative to the cerebellum, a neutral brain area not thought to be involved in olfaction). 

 

 

 

 

 

Neocortex region

 

Pig #

 

Treatment

 

Cere-bellum

 

Olfactory bulb

 

Hypothalamus

 

Parietal

 

Temporal

4

control (nil)

1.0

0.82

1.10

1.24

1.08

2

amyl acetate

1.0

1.001

1.22

1.12

1.16

% change over nila

--

+22%

+11%

-10%

+7%

1

pheromone

1.0

1.48

1.24

1.43

1.11

3

pheromone

1.0

1.02

1.34

1.35

1.23

 

Mean

1.0

1.25

1.29

1.39

1.17

% change over nilb

--

+52%

+17%

+12%

+8%

% change over controlc

--

+25%

+5.7%

+24%

+1%

a Amyl acetate vs. control (nil).

b Pheromone vs. control (nil).

c Pheromone vs. amyl acetate.

 

 

Figure 1.  PET scan image of pig #3.   Regions of greater neural activity are shown in light colors and regions of less activation are successively darker.  The white arrows indicate activation of the parietal lobe – a region involved with somatosensory and somatomotor stimulation and memory.

 

 

 

 

 

 

 

 

 

 

 

 


[1] The authors thank Ceva Sante Animale for donation of the experimental maternal pheromone and Dominique Thibaud for technical assistance.   

[2] Correspondence: Dr. John McGlone, Texas Tech University, Animal Science Building room 123, Lubbock TX 79409; Phone: (806) 742-2826; Fax: (806) 742-2335; E-mail: Dr. John McGlone