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Development of techniques to reveal mechanisms underlying stereotypies in Mongolian gerbils

Christel Moons (UGent)
(2008)
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(UGent) , (UGent) and (UGent)
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Abstract
Although for SPECT scanning an animal has to be anaesthetized, it provides a functional assessment of the molecules of interest, which can be directly linked to the behaviour as it was observed prior to the start of the physiological data collection. Finally, the species of choice is described. When kept in standard laboratory housing, the Mongolian gerbil (Meriones unguiculatus) consistently develops stereotypic digging and bar-gnawing. For both of these behaviours the motivational mechanism has been extensively studied. The expression of bar-gnawing in young animals seems to show a steep increase after weaning, especially if no new litter was present yet in the parental home cage, while stereotypic digging seems to be associated with the lack of the possibility to retreat via a tunnel into a dark chamber. To date, only one detailed definition has been described for stereotypic digging in the Mongolian gerbil and it relies on a minimum cut-off duration. It has been developed and until now mainly used for studies in juvenile gerbils, rarely for adults. This PhD centres on a few questions regarding the use of different working definitions of the digging stereotypy in the Mongolian gerbil and the feasibility of telemetry and SPECT. These questions have been introduced in chapter II and are revisited below. 1) In laboratory Mongolian gerbils, what is the impact of using the current definition for stereotypic behaviour - developed in juvenile animals - on observations in adults? Chapter III describes two observational studies (A and B) each employing a different working definition for the digging stereotypy (WDa and WDb), the former based on the description of the behaviour in a laboratory cage while for the latter, we first determined the morphology of digging behaviour in a more natural environment. The aim was to test to what extent the datasets differ when compared with a third and often used definition (WD12; Wiedenmayer 1992). The latter definition describes the digging stereotypy as digging behaviour lasting at least 12 seconds. For both studies, adopting WD12 caused a decrease in total duration (A: - 12.53 %, B: - 22.00 %) and number (A: - 56.05 %, B: - 63.08 %) of stereotypic digging bouts. Consequently, the average bout duration increased (A: + 85.77 %, B: + 100.78 %). Although absolute values were affected, strong correlations existed between WDa and WDb datasets on the one hand and WD12 on the other hand, indicating definitions yielded qualitatively similar results. It is argued that in adult gerbils there appears to be no morphological evidence to differentiate between stereotypic digging bouts of ≤ 12 s and of > 12 s. Furthermore, data from one of the observational studies also show that some adult gerbils consistently develop stereotypic behaviour combining sequential bouts of digging and bar-gnawing. Using working definitions that apply duration cutoffs to define a behaviour could distort the results when quantifying such combined stereotypy. 2) Is it feasible to use implantable telemetry in juvenile Mongolian gerbils? Because it was our intention to use telemetry in weanling gerbils during subsequent studies, chapter IV investigates whether subcutaneous implantation of telemetric devices is preferable to intraperitoneal surgery with respect to animal welfare. A detailed description is provided of anaesthetic and surgical techniques during a pre-trial involving subcutaneous (n = 10, aged 21-29 days) and intraperitoneal (n = 10, aged 19-34 days) implantation of dummy telemetry transmitters (1.9 cm³, 3.6 g after shortening of leads). Body weight was measured and analysed over four-day intervals. Three animals died during the subcutaneous procedure but none post-surgery. Two animals from the intraperitoneal procedure died in the first three days post-surgery, probably because the intestine was strangled by the excess lead that was coiled in the abdomen. In the other implanted animals, growth decreased during the first four days and the difference in body weight remained significant compared to control animals at least until the last observation day. Chapter IV showed that the implantation of a telemetric device in weanling gerbils is feasible and is best executed through the intraperitoneal technique. However, when continuing the experiments with fully functional transmitters, two technical problems were encountered. First, we observed a progressive reduction in signal quality that is inherent to using young, developing animals: as the animal’s body elongates, the connective tissue that has developed around the electrode leads prevents the lead tips from moving simultaneously. Second, animal losses were suffered because of the physical presence of the transmitter in the abdominal cavity, similar to what was seen during the pilot trial. Because too much time was needed to evaluate the success of each remedial measure applied, we decided to focus on the SPECT technique instead. 3) a) Is it feasible to use 99mTc-ECD in Mongolian gerbils for perfusion imaging using SPECT? b) Is it possible to use 123I-FP-CIT to image and quantify the dopamine transporter in Mongolian gerbils? To date, there is no evidence in the literature documenting the use of SPECT in Mongolian gerbils. Chapter V reports on a pilot trial in which 99mTechnetium-labelled Ethylcysteinate Dimer tracer (24.42 ± 5.92 MBq) was injected in the femoral vein of four gerbils to provide brain perfusion images that allow anatomical identification of DAT-rich regions that were imaged in another four gerbils using 123I-labelled FP-CIT tracer (44.33 ± 11.66 MBq). Furthermore, the optimal scan time for FP-CIT was established in one gerbil. Scans were performed using a conventional triple head gamma camera adapted with multi-pinhole collimators which rendered a resolution of 2.0 mm. The optimal scan time for DAT-imaging was 4.5 hours. The study was successful in obtaining brain perfusion images as well as in demonstrating regional binding of FP-CIT to the basal ganglia, DAT-rich areas in the brain. 4) Are there special precautions that can be taken to refine the SPECT technique in a small laboratory rodent species such as the Mongolian gerbil? Two elements of SPECT in particular are risk factors for causing animal discomfort: the manipulations to inject radiotracers in the femoral vein and body heat loss resulting from the anaesthesia at the time of scanning. Chapter VI describes refinement measures as they were developed for use in the experiment described in chapter V. First, the highest chance of successfully performing the intravenous tracer injection is obtained by briefly anesthetizing the animal, placing it in a restraining device and - simultaneously with moment of regaining consciousness - identify the femoral vein and inject the tracer. Both the chemical and physical restraint phase was short, resulting in a maximum handling time of 5 minutes. Second, prevention of body heat loss was obtained using a heating device, which was constructed based on a circulation system of water continuously heated to gerbil body temperature (38 °C). Both refinements were implemented to minimize discomfort in the experimental animals. 5) What is the influence of age on 123I-FP-CIT binding to dopamine transporters in a population of Mongolian gerbils housed under standard laboratory conditions? Using SPECT, the difference in specific binding index between four age categories (50d, 80d, 107d and 206d) was examined in chapter VII. An increase was observed between category 50d and 80d, while onwards a decrease was seen. However, statistical analysis revealed no change in DAT-binding index between 50d, 80d, and 107d. On the other hand, compared to the 107d age category animals of the 206d age category showed significantly less binding of radioligand to DAT (P < 0.01). Furthermore, a statistical trend was observed in that the binding index in the 80d age category was smaller than in that of 206d (P < 0.1). This study supports the idea that DAT-density, as measured by 123I-FP-CIT binding to DAT, is age-dependent in Mongolian gerbils housed under laboratory circumstances. Given the fact that (1) gerbils consistently develop stereotypic digging when housed in laboratory cages without specific enrichment controlling the motivation for the behaviour and (2) the involvement of the dopaminergic system in the occurrence of stereotypic behaviour has been demonstrated in the existing literature, the question arises whether there is an association between the occurrence of stereotypies and the level of radioligand binding to DAT. Finally, chapter VIII presents - aside from a general discussion on the previous chapters - a number of questions, which can be answered using telemetry and SPECT. Two of these questions represent the research directions as was discussed in the introduction: (1) Are there rewarding properties that can be attributed to stereotypies, particularly in the sense that they serve a coping function?; (2) Which are the predisposing factors associated with the expression of stereotypic behaviour?; (3) Is symptom severity in stereotypies related to different levels of dopaminergic activity?. This thesis has contributed to future stereotypy research in a multidisciplinary way. First, we discussed the risks associated with using different working definitions to quantify the occurrence of the digging stereotypy in Mongolian gerbils. Second, in the same species we investigated the feasibility of implantable telemetry in weanling animals and identified a number of pitfalls. Finally, we demonstrated that it is possible to use SPECT in Mongolian gerbils, mores specifically to document cerebral perfusion and DAT-density.
Keywords
Stereotypies, Mongolian gerbils, SPECT, ethology, bio-telemetry

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Please use this url to cite or link to this publication:

MLA
Moons, Christel. “Development of Techniques to Reveal Mechanisms Underlying Stereotypies in Mongolian Gerbils.” 2008 : n. pag. Print.
APA
Moons, C. (2008). Development of techniques to reveal mechanisms underlying stereotypies in Mongolian gerbils. Ghent University. Faculty of Veterinary Medicine, Merelbeke, Belgium.
Chicago author-date
Moons, Christel. 2008. “Development of Techniques to Reveal Mechanisms Underlying Stereotypies in Mongolian Gerbils”. Merelbeke, Belgium: Ghent University. Faculty of Veterinary Medicine.
Chicago author-date (all authors)
Moons, Christel. 2008. “Development of Techniques to Reveal Mechanisms Underlying Stereotypies in Mongolian Gerbils”. Merelbeke, Belgium: Ghent University. Faculty of Veterinary Medicine.
Vancouver
1.
Moons C. Development of techniques to reveal mechanisms underlying stereotypies in Mongolian gerbils. [Merelbeke, Belgium]: Ghent University. Faculty of Veterinary Medicine; 2008.
IEEE
[1]
C. Moons, “Development of techniques to reveal mechanisms underlying stereotypies in Mongolian gerbils,” Ghent University. Faculty of Veterinary Medicine, Merelbeke, Belgium, 2008.
@phdthesis{948392,
  abstract     = {Although for SPECT scanning an animal has to be anaesthetized, it provides a functional assessment of the molecules of interest, which can be directly linked to the behaviour as it was observed prior to the start of the physiological data collection. Finally, the species of choice is described. When kept in standard laboratory housing, the Mongolian gerbil (Meriones unguiculatus) consistently develops stereotypic digging and bar-gnawing. For both of these behaviours the motivational mechanism has been extensively studied. The expression of bar-gnawing in young animals seems to show a steep increase after weaning, especially if no new litter was present yet in the parental home cage, while stereotypic digging seems to be associated with the lack of the possibility to retreat via a tunnel into a dark chamber. To date, only one detailed definition has been described for stereotypic digging in the Mongolian gerbil and it relies on a minimum cut-off duration. It has been developed and until now mainly used for studies in juvenile gerbils, rarely for adults.
This PhD centres on a few questions regarding the use of different working definitions of the digging stereotypy in the Mongolian gerbil and the feasibility of telemetry and SPECT. These questions have been introduced in chapter II and are revisited below. 
1) In laboratory Mongolian gerbils, what is the impact of using the current definition for stereotypic behaviour - developed in juvenile animals - on observations in adults?
Chapter III describes two observational studies (A and B) each employing a different working definition for the digging stereotypy (WDa and WDb), the former based on the description of the behaviour in a laboratory cage while for the latter, we first determined the morphology of digging behaviour in a more natural environment. The aim was to test to what extent the datasets differ when compared with a third and often used definition (WD12; Wiedenmayer 1992). The latter definition describes the digging stereotypy as digging behaviour lasting at least 12 seconds. For both studies, adopting WD12 caused a decrease in total duration (A: - 12.53 %, B: - 22.00 %) and number (A: - 56.05 %, B: - 63.08 %) of stereotypic digging bouts. Consequently, the average bout duration increased (A: + 85.77 %, B: + 100.78 %). Although absolute values were affected, strong correlations existed between WDa and WDb datasets on the one hand and WD12 on the other hand, indicating definitions yielded qualitatively similar results. It is argued that in adult gerbils there appears to be no morphological evidence to differentiate between stereotypic digging bouts of ≤ 12 s and of > 12 s. Furthermore, data from one of the observational studies also show that some adult gerbils consistently develop stereotypic behaviour combining sequential bouts of digging and bar-gnawing. Using working definitions that apply duration cutoffs to define a behaviour could distort the results when quantifying such combined stereotypy.
2) Is it feasible to use implantable telemetry in juvenile Mongolian gerbils?
Because it was our intention to use telemetry in weanling gerbils during subsequent studies, chapter IV investigates whether subcutaneous implantation of telemetric devices is preferable to intraperitoneal surgery with respect to animal welfare. A detailed description is provided of anaesthetic and surgical techniques during a pre-trial involving subcutaneous (n = 10, aged 21-29 days) and intraperitoneal (n = 10, aged 19-34 days) implantation of dummy telemetry transmitters (1.9 cm³, 3.6 g after shortening of leads). Body weight was measured and analysed over four-day intervals. Three animals died during the subcutaneous procedure but none post-surgery. Two animals from the intraperitoneal procedure died in the first three days post-surgery, probably because the intestine was strangled by the excess lead that was coiled in the abdomen. In the other implanted animals, growth decreased during the first four days and the difference in body weight remained significant compared to control animals at least until the last observation day.
Chapter IV showed that the implantation of a telemetric device in weanling gerbils is feasible and is best executed through the intraperitoneal technique. However, when continuing the experiments with fully functional transmitters, two technical problems were encountered. First, we observed a progressive reduction in signal quality that is inherent to using young, developing animals: as the animal’s body elongates, the connective tissue that has developed around the electrode leads prevents the lead tips from moving simultaneously. Second, animal losses were suffered because of the physical presence of the transmitter in the abdominal cavity, similar to what was seen during the pilot trial. Because too much time was needed to evaluate the success of each remedial measure applied, we decided to focus on the SPECT technique instead.
3) a) Is it feasible to use 99mTc-ECD in Mongolian gerbils for perfusion imaging using SPECT?
b) Is it possible to use 123I-FP-CIT to image and quantify the dopamine transporter in Mongolian gerbils?
To date, there is no evidence in the literature documenting the use of SPECT in Mongolian gerbils. Chapter V reports on a pilot trial in which 99mTechnetium-labelled Ethylcysteinate Dimer tracer (24.42 ± 5.92 MBq) was injected in the femoral vein of four gerbils to provide brain perfusion images that allow anatomical identification of DAT-rich regions that were imaged in another four gerbils using 123I-labelled FP-CIT tracer (44.33 ± 11.66 MBq). Furthermore, the optimal scan time for FP-CIT was established in one gerbil. Scans were performed using a conventional triple head gamma camera adapted with multi-pinhole collimators which rendered a resolution of 2.0 mm. The optimal scan time for DAT-imaging was 4.5 hours. The study was successful in obtaining brain perfusion images as well as in demonstrating regional binding of FP-CIT to the basal ganglia, DAT-rich areas in the brain.
4) Are there special precautions that can be taken to refine the SPECT technique in a small laboratory rodent species such as the Mongolian gerbil?
Two elements of SPECT in particular are risk factors for causing animal discomfort: the manipulations to inject radiotracers in the femoral vein and body heat loss resulting from the anaesthesia at the time of scanning. Chapter VI describes refinement measures as they were developed for use in the experiment described in chapter V. First, the highest chance of successfully performing the intravenous tracer injection is obtained by briefly anesthetizing the animal, placing it in a restraining device and - simultaneously with moment of regaining consciousness - identify the femoral vein and inject the tracer. Both the chemical and physical restraint phase was short, resulting in a maximum handling time of 5 minutes. Second, prevention of body heat loss was obtained using a heating device, which was constructed based on a circulation system of water continuously heated to gerbil body temperature (38 °C). Both refinements were implemented to minimize discomfort in the experimental animals.
5) What is the influence of age on 123I-FP-CIT binding to dopamine transporters in a population of Mongolian gerbils housed under standard laboratory conditions?
Using SPECT, the difference in specific binding index between four age categories (50d, 80d, 107d and 206d) was examined in chapter VII. An increase was observed between category 50d and 80d, while onwards a decrease was seen. However, statistical analysis revealed no change in DAT-binding index between 50d, 80d, and 107d. On the other hand, compared to the 107d age category animals of the 206d age category showed significantly less binding of radioligand to DAT (P < 0.01). Furthermore, a statistical trend was observed in that the binding index in the 80d age category was smaller than in that of 206d (P < 0.1). This study supports the idea that DAT-density, as measured by 123I-FP-CIT binding to DAT, is age-dependent in Mongolian gerbils housed under laboratory circumstances. Given the fact that (1) gerbils consistently develop stereotypic digging when housed in laboratory cages without specific enrichment controlling the motivation for the behaviour and (2) the involvement of the dopaminergic system in the occurrence of stereotypic behaviour has been demonstrated in the existing literature, the question arises whether there is an association between the occurrence of stereotypies and the level of radioligand binding to DAT.
Finally, chapter VIII presents - aside from a general discussion on the previous chapters - a number of questions, which can be answered using telemetry and SPECT. Two of these questions represent the research directions as was discussed in the introduction: (1) Are there rewarding properties that can be attributed to stereotypies, particularly in the sense that they serve a coping function?; (2) Which are the predisposing factors associated with the expression of stereotypic behaviour?; (3) Is symptom severity in stereotypies related to different levels of dopaminergic activity?.
This thesis has contributed to future stereotypy research in a multidisciplinary way. First, we discussed the risks associated with using different working definitions to quantify the occurrence of the digging stereotypy in Mongolian gerbils. Second, in the same species we investigated the feasibility of implantable telemetry in weanling animals and identified a number of pitfalls. Finally, we demonstrated that it is possible to use SPECT in Mongolian gerbils, mores specifically to document cerebral perfusion and DAT-density.},
  author       = {Moons, Christel},
  isbn         = {9789058641625},
  keywords     = {Stereotypies,Mongolian gerbils,SPECT,ethology,bio-telemetry},
  language     = {eng},
  pages        = {152},
  publisher    = {Ghent University. Faculty of Veterinary Medicine},
  school       = {Ghent University},
  title        = {Development of techniques to reveal mechanisms underlying stereotypies in Mongolian gerbils},
  year         = {2008},
}