novel restraint system for neuroendocrine. j. schultz-darken 1, r. m. pape 1, p. l. tannenbaum 2, w....

Effective and well-tolerated drug deliveryand restraint systems are essential forstudies of the neural mechanisms regulatingphysiology and behaviour While suchsystems are available for large primatessuch as macaques (Friday amp Lipkin 1990)and baboons (Coelho amp Carey 1990)suitable delivery and restraint systems forsmall primates are not as well developed

(Blum et al 1983 OrsquoByrne 1988 OrsquoByrne ampMorris 1988)

Common marmosets Callithrix jacchusare anthropoid New World primates and areincreasingly being used in biomedicalresearch (Scott 1994 Owen et al 1997Abbott et al 2003 Mansfield 2003) Themarmosetrsquos small size (350ndash400 g) tractabi-lity high fecundity potential (typically withtwin births every 5ndash6 months) and rapidmaturation (adult by 18 months of age)provide clear advantages over the larger lessfecund and slower-maturing macaques

Novel restraint system for neuroendocrinestudies of socially living common marmoset monkeys

N J Schultz-Darken1 R M Pape1 P L Tannenbaum2 W Saltzman3

amp D H Abbott14

1National Primate Research Center and 4Department of Obstetrics and Gynecology University ofWisconsin Madison Wisconsin 53715 2Johnson amp Johnson Pharmaceutical Research andDevelopment 1000 Rt 202 Raritan NJ 08869 and 3Department of Biology University of CaliforniaRiverside CA 92521 USA

Summary

We describe a novel soft jacket and sling-harness restraint that permits species-typicalpostures for small-bodied primates such as the common marmoset (Callithrix jacchus)during long-term (6 h) continuous restraint The restraint system is straightforward to useand manipulate it is easily repaired and the materials used are readily available The softjacket allows for increased versatility and longevity and the sling-harness provides forgreater movement and much longer duration of continuous restraint (up to 3 days)compared to a previously described more conventional chair restraint for small-bodiedprimates The new restraint system prevents the normal diurnal decrease in plasma cortisollevels across the daylight hours however it does not disrupt ovulatory cycles Unlike thepreviously available techniques therefore this new restraint system is applicable to manyneurobiological and neuroendocrine studies involving small-bodied non-human primatesand is especially suited to investigations requiring the maintenance of relationships withinsocial groups

Keywords Marmoset restraint long-term sampling reproductive neuroendocrinologybehavioural adaptation methods cortisol

Accepted 24 February 2004 copy Laboratory Animals Ltd Laboratory Animals (2004) 38 393ndash405

Correspondence to Nancy J Schultz-Darken PhD NationalPrimate Research Center University of Wisconsin 1220Capitol Court Madison WI 53715ndash1299 USAE-mail darkenprimatewiscedu

conventionally used in biomedical research(Abbott 1992) Marmosets make excellentsubjects for studies in neurobiology neuro-endocrinology and behaviour because theyadapt easily to a variety of experimentalprocedures including venepuncture (Hearn1977 1983) venous cannulation (OrsquoByrne1988) hypothalamic pushndashpull perfusion(Abbott et al 1998) operant testing (Pryceet al 1993) learning paradigms (Collins etal 1998) functional magnetic resonanceimaging (Ferris et al 2001) and social testingprocedures (Saltzman et al 1996 Bakeret al 1999) These monkeys also appear tobe comfortable in small enclosed spaces(Ferris et al 2001)

Common marmosets share numerousbiochemical physiological and behaviouralsimilarities with humans For examplemarmosets exhibit a 28-day ovarian cycleestablish complex long-lasting socialrelationships and live in small family-basedsocial units (Abbott 1992) Offspring remainwithin their natal group into adulthood andall group members contribute to infant careincluding extensive carrying and post-weaning provisioning of infants (French 1997Tardif 1997) In contrast to humans howeverthe behaviourally dominant female in mar-moset social groups (the mother in familygroups) is usually the only female to conceiveand give birth (Abbott 1984 1987 1993) Atthe same time the ovulatory cycles of subor-dinate females in social groups are suppressedin response to a combination of behaviouralvisual and olfactory cues from the dominantfemale so that subordinate females are usual-ly anovulatory and hypo-oestrogenemic(Abbott et al 1988 1993 1997 Barrett et al1990 1993) The common marmoset there-fore provides a specialized primate model fordetermining mechanisms mediating thesocial regulation of reproduction

To investigate the neuroendocrinemechanisms underlying the social controlof ovulation in marmosets we needed tosignificantly refine previously describedrestraint techniques to permit long-term (upto 3 days) assessment and manipulation ofhypothalamic and pituitary function Animproved methodology would also providenew opportunities for neurobiological inves-

tigation in a wide range of studies utilizingcommon marmosets and other small-bodiedprimates Our immediate goal was todevelop a well-tolerated restraint systemthat would permit a moderate degree ofmovement while enabling repeated intra-venous and hypothalamic sampling of themarmoset in close proximity to its socialgroup members A backpack previouslydescribed for use with marmosets did nothave the versatility necessary for ourproposed studies (Ruiz de Elvira amp Abbott1986) as it was designed to accommodateonly osmotic mini-pumps housed in smallfluid-filled (5 ml) chambers Furthermore apreviously described restraint system formarmosets was limited to a maximum of6 h of continuous use (OrsquoByrne amp Morris1988) Since the previous restraint systemdid not allow for species-typical restingpostures it could not accommodate theminimum of 3 days of continual restraintrequired for hypothalamic perfusion studies(Gearing amp Terasawa 1988)

To permit such long-term restraint wedeveloped a soft jacket that serves as both(1) a pocket for an indwelling intravenouscannula and (2) an integral part of a novelsling-harness restraint This system permit-ted such complex techniques as hypothala-mic pushndashpull perfusion with simultaneoussampling from an intravenous cannula Ourfindings demonstrated that marmosetsquickly adapted to 3 days of continuousrestraint in this novel system and validatedthe systemrsquos usefulness in neurobiologicalinvestigations using the marmoset

Materials and methods

AnimalsSubjects were 17 captive-born adult (2ndash8years of age) female common marmosets(Callithrix jacchus) housed at the NationalPrimate Research Center University ofWisconsin (NPRC) as previously described(Saltzman et al 1998) NPRC is fully accred-ited by AAALAC as part of the University ofWisconsinndashMadison Graduate SchoolAnimal protocols and experiments wereapproved by the Graduate School Animal

394 Schultz-Darken et al

Laboratory Animals (2004) 38

Care and Use Committee of the Universityof Wisconsin Madison The animals weremaintained according to recommendations ofthe Guide for the Care and Use of LaboratoryAnimals (US National Research Council) andthe United States Animal Welfare Act withits subsequent amendments

The 11 ovary-intact and six bilaterallyovariectomized female subjects were eitherpair-housed with males or housed in socialgroups (Abbott 1984 Saltzman et al 1994)Ovary-intact females exhibited regularovulatory cycles Bilateral ovariectomywas performed using a ventral midlineapproach 129 43 days (mean SEM) priorto first restraint We formed social groupsby introducing four unrelated adults ofeach sex into a large observation room(363 212 218 cm) containing nest boxesand tree branches Individuals were removedin cases of behavioural incompatibility(usually within 3ndash14 days) and the resultingstable group comprised one dominant

female and 1ndash3 subordinate females alongwith 1ndash3 males as previously described(Saltzman et al 1998) Social groupsremained together with a stable hierarchyfor 2 months to over 3 years as described inan earlier study (Abbott amp George 1991)

Sling-harness restraintJacket for sling-harness restraintThe jacket was substantially improved froman initial backpack system designed for mar-mosets by Ruiz de Elvira and Abbott (1986)The outer layer of heavy sailcloth (GallagherTent and Awning Madison WI USA) waslined with brushed flannel and a 5 cm wideVelcro strap fastened the back flap Thenew materials now provided the pliabilityversatility and longevity absent fromprevious designs

This new version of the jacket incorporatedthe addition of a sailcloth sleeve onto theback flap of the jacket (Fig 1) containing

Long-term restraint for marmosets 395


Fig 1 Design of the jacket (A) Front view including the standard frontal length (Lf7 cm) width (W 8 cm)measured at a point about 5 mm below the bottom of the spaces for the arms with a slightly tapered lowerportion to accommodate the width of the monkeyrsquos abdomen (B) Back view illustrating the jacket configura-tion that houses an intravenousatrial cannula without the cylindrical sleeve for attachment to the restraintapparatus The dotted area represents the Velcro attachment on the underside of the outer flap affixed toVelcro sewn on to the back of the jacket thus permitting an adjustable width across the back The standardlength (Lb) of each back section is 12 cm The part of the jacket underneath the Velcro fastening (dotted area)also includes the pouch (approximately 25 cm 40 cm) for housing an intravenousatrial cannula (C) Backview illustrating the jacket configuration with the additional cylindrical sleeve for attachment to the restraintapparatus The design is identical to that housing the intravenousatrial cannula except that an additionalcylindrical sleeve (S length 125 cm diameter 15 cm) was sewn onto the outer flap of the back of thejacket to accommodate attachment to the angled support arm on the sling-harness restraint (Fig 2)

a thin aluminium bar (115 18 02 cm)The bar provided increased stability andrigidity to the sleeve when it was fitted onto the angled part of the support bar of thesling-harness restraint (Fig 2) The newjacket still contained a pocket on the backfor housing the exteriorized portion of anintravenous cannula similar to that

described by Ruiz de Elvira and Abbott(1986) To fit the jacket to an adult mar-moset one person manually restrained themarmoset using leather gloves whileanother slipped the open jacket up theanimalrsquos forelimbs and over the shouldersfastening the Velcro attachment around theanimalrsquos back (Fig 1)



Fig 2 Side view of a marmoset in the sling-harness restraint The entire restraint is supported by a stain-less steel base (ssb length of the right side of the base 35 cm) The neck collar (nc) is attached for headrestraint during hypothalamic pushndashpull perfusion and the monkey has an intracranial cannula maintainedin precision placement by a small microdrive unit (md Narishige Scientific Instrument Laboratory TokyoJapan) The jacket ( ja) shown is the configuration for sling-harness restraint (Fig 1C) The sleeve (sl) on theback of the jacket has been slipped down the angled part of the support arm (sa) and stainless steel gripshave been fastened above (ssg1) and below (ssg2) the sleeve to hold it in position Immediately prior tohypothalamic pushndashpull perfusion the neck collar is attached to a stainless steel holding bar (hb) by twonuts and bolts placed through pre-drilled 05 cm holes (not shown) in the side of the neck collar Theholding bar has an adjustable attachment (aah) on the angled part of the support arm below the jacketattachment The monkeyrsquos feet are supported by a stainless steel mesh platform (mp) that has an adjustableattachment (aap) near the base of the vertical part of the support arm The monkeyrsquos left hand is resting onthe food tray (ft) that holds a food dish (fd 5 cm in diameter and 6 cm deep)

Sling-harness restraint apparatusThe sling-harness restraint consisted ofa stainless steel base (approximately35 35 cm) upon which an angled supportbar (12 cm diameter stainless steel flattened on one side) was mounted (Fig 2)A marmoset wearing the jacket was fittedto the restraint by slipping the sleeve of thejacket over the angled part of the supportbar until the top of the support bar protrudedapproximately one centimetre above thetop of the sleeve (Figs 2 and 1C) Anadjustable stainless steel grip (which hadbeen slipped over the support bar prior toattachment of the sling-harness jacket) was then moved up the support bar andtightened into position at the base of thejacketrsquos sleeve to prevent the jacket slidingfarther down the support bar (Fig 2) Anadditional stainless steel grip was thenplaced over the top one centimetre of thesupport bar and tightened into position toprevent the jacket sliding off the top of thesupport bar The marmoset was then freeto move into a variety of postures includ-ing standing squatting or huddling as itsfeet were supported by an adjustable stain-less steel mesh platform (255 160 cm)attached to the lower (vertical) part of thesupport bar

A food tray adjustable for both heightand distance from the restrained animalwas affixed to the front of this platformThe food tray and platform were moved as necessary to accommodate the manypositions assumed by marmosets includingthose during sleep Soft towels were positioned on the platform to facilitate ahuddling posture and during periods ofdim lighting when the main room lightswere off (1800ndash0600 h) a soft folded towelwas positioned on top of the cleaned foodtray to provide ventral support to the headand neck A huddled body posture is one ofmany that are typical and well-described forcommon marmosets (Stevenson amp Rylands1988)

To minimize the marmosetrsquos ability tomanipulate an acutely implanted hypothala-mic cannula a neck collar was fitted to themarmoset when the animal was first placedin the sling-harness restraint The two

halves of the circular plastic neck collar(outer diameter 10 cm inner diameter 35or 31 cm depending on the neck size of themarmoset depth 05 cm) were held togetherby an lsquoO-ringrsquo A tongue-in-groove arrange-ment enabled the two halves of the collarto be slotted together To minimize headmovement during hypothalamic pushndashpullperfusion the neck collar was attached to astainless steel holding bar (the positionshown in Fig 2) by placement of two nutsand bolts through pre-drilled 05 cm holes inone side of the neck collar The holding barhad an adjustable attachment to the angledsupport bar at a point below the marmosetrsquosjacket and above the platformrsquos attachmentto the base of the restraint An adjustablehinge on the attachment of the holding barto the support arm provided the neededflexibility for setting a locked position ofthe neck collar that was well-tolerated by the marmoset

Training of animals in the sling-harnessrestraintMonkeys commenced their adaptation tothe sling-harness restraint well before(220 69 days) they were first used ina hypothalamic pushndashpull perfusionexperiment Training comprised a seriesof trials of increasing duration and wasperformed in a procedure room separatefrom the animalsrsquo regular housing roomsDuring these trials restrained animalsremained in full view of their cage mateswhich had been placed inside a small cage(61 cm 46 cm 61 cm equipped withperches sleeping box and bedding) posi-tioned to within 03 m of the front of thesling-harness restraint Training began withone hour of restraint on day 1 4 h on day 28 h on day 3 or 4 and overnight restraint(24 h) starting on day 7 or 8 This schedulefacilitated optimal adaptation to therestraint During training marmosetslearned to feed themselves and to drinkfluids offered manually from a syringeOnce adapted to the restraint (see Results)each marmoset underwent an additional 8 hrestraint session once every 6 months tomaintain adaptation The head was notrestrained during training





Maintenance of animals in the sling-harness restraint

Food and fluids were offered hourly torestrained animals during lights-on(0600ndash1800 h) Food items included a spe-cialized marmoset diet (ZuPreem ShawneeMission KS USA) marmoset jelly (MazuriPurina Mills International (PMI) St LouisMO USA) a variety of fruit (apples bananasor grapes) miniature marshmallows andliquid Ensure (a human dietary supplementrich in calories vitamins and mineralsAbbott Laboratories Columbus OH USA)We assessed the fluid intake supplied by thefluids or fruits offered (as derived fromBogert Briggs and Calloway 1973) salinePlasmalyte 10 dextrose solution (all fromBaxter Health Care Corp Deerfield ILUSA) tap water and Ensure were recordedas specific millilitres of fluid one cm3 pieceof apple or one grape was equated to 5 ml offluid and one cm3 piece of banana or onepiece of marmoset jelly was equated to 2 mlof fluid

Monitoring of animals in the sling-harness restraint

Observations were recorded at least hourlyduring lights-on (0600ndash1800 h) and once(at 2200 h) during dim lighting when themain room lights were off At each timedobservation we recorded (1) the amount offood and fluid intake (2) behaviour andposture (3) deposition of urine and faecesand (4) ambient temperature Commentson the animalrsquos posture occurrence of

struggling and vocalizations were alsorecorded on a behavioural assessment sheetand were later ranked on a scale of 1through 8 (with 1 rated the least and 8 ratedthe most agitated see Table 1) These behavioural assessments were used to gauge the adaptation of the marmosets tothe restraint

Blood samplingCortisol levels have served as an index ofgeneralized stress in marmosets (Smith ampFrench 1997) In order to evaluate stress inour animals during long-term restraint forhypothalamic pushndashpull perfusion foursubjects (three ovary-intact females in themid-follicular phase of the ovarian cycleand one ovariectomized female) underwentcatheterization of the right internal jugularvein (modified from OrsquoByrne (1988) poly-ethylene PE10 tubing Becton DickinsonSparks MD USA) under Saffan anaesthesia(20 mgkg im alphaxolonealphadolone15 mg5 mg Veterinary Drug CoDunnington York UK) at least one weekprior to hypothalamic pushndashpull perfusionAfter catheterization the subjects returnedto their home cages wearing a jacket specif-ically designed to hold a dorsally exterior-ized intravenous cannula tubing inside aVelcro-sealed pocket To maintain cannulapatency the implanted polyethylene tubingwas completely filled with heparinizedsaline (30 Uml) and flushed daily Bloodsamples (01 ml) were collected from each ofthe four marmosets at approximatelyhourly intervals (between 1200 h and

Table 1 Assessment scale for behavioural responses of marmosets restrained in the sling-harness assembly

Score Behavioural category

1 Quiet marmoset was calm and moved body in a relaxed manner2 Mostly quiet agitated only initially involving mild struggling3 Mostly quiet with brief intermittent mild agitation4 Became quiet after initial struggle then became increasingly agitated over time5 Mild agitation for about half of the restraint period6 Moderate agitation during half of the restraint period7 Restless and agitated during most of the restraint period8 Extremely agitated during most of the restraint period

2200 h) during hypothalamic pushndashpullperfusion on the third consecutive day inthe sling-harness restraint and were assayedfor cortisol All blood samples wereimmediately placed on ice and centrifugedat 2000 rpm for 10 min and the plasmafraction was removed and stored at 20Cuntil assayed

To monitor ovarian cycles blood samples(01ndash03 ml) were collected twice weeklyfrom the femoral vein of three ovary-intactfemales during brief (2ndash5 min) restraintwithout anaesthesia (Hearn 1977 Saltzmanet al 1994) for analysis of plasma proges-terone concentrations All blood sampleswere immediately placed on ice and cen-trifuged at 2000 rpm for 10 min and theplasma fraction was removed and stored at20C until assayed

To control the onset of ovarian cyclesin intact females cloprostenol sodium(a prostaglandin F2-alpha analogue 10 gIM Estrumate Shering-Plough CorpKenilworth NJ USA) was injected for 1ndash3consecutive days during the luteal phase(14ndash39 days following ovulation) in order toinduce luteolysis and terminate the lutealphase or early pregnancy (Summers et al1985) In our marmoset colony ovulationusually occurs 8ndash12 days after cloprostenoltreatment (Saltzman et al 1998) In thepresent study subjects received cloprostenol4ndash5 days prior to a pushndashpull perfusion ses-sion Plasma progesterone determinationsfrom the twice-weekly blood samples takenafter cloprostenol injection and beforepushndashpull perfusion confirmed that eachovary-intact subject responded to clo-prostenol and was in the follicular phase ofthe ovarian cycle (plasma progesterone30 nmoll Harlow et al 1983) Ovulationoccurs on the day before a sustained rise ofplasma progesterone above 30 nmoll(Harlow et al 1983) Follicular phase dura-tion was determined as the time from theday plasma progesterone levels fell below30 nmoll until the day of ovulation

Hormone assaysPlasma cortisol levels were measured induplicate aliquots using a GammaCoat

radioimmunosssay kit (DiaSorin IncStillwater MN) as described previously(Saltzman et al 1998) Assay sensitivityand inter- and intra-assay coefficients ofvariation (CVs) were 0041 nmoltube 75and 33 respectively Plasma from twice-weekly blood samples was assayed induplicate for progesterone using an enzymeimmunoassay (Saltzman et al 1994) Assaysensitivity and inter- and intra-assay CVswere 795 pmolwell 129 and 46respectively

Statistical analysisData are expressed as mean SEM unlessotherwise noted The behavioural scores ofrestrained marmosets volume of fluidintake and rates of urine and faecal excre-tion were analysed utilizing the Friedmannon-parametric ANOVA followed by theWilcoxon signed rank test for pairwisecomparisons One-way ANOVAs withrepeated-measures design (Sokal amp Rohlf1995) were used to analyse (1) the log-transformed plasma cortisol values and (2)the differing durations between cloprostenolinjection and estimated day of ovulationbefore during and after hypothalamicpushndashpull perfusion A probability value of005 (two-tailed) was used to determinesignificance

Results

Responses of marmosets to sling-harnessrestraintAll 17 female marmosets adapted wellto the sling-harness restraint The mostreactive behavioural response score of lsquo8rsquo(Table 1) was achieved during the first hourof training This score was attained by onlyone female and was well above the meanscore of 41 (06) for this session Thebehavioural response scores in this firsttraining session exceeded (P 0015) thosein all subsequent restraint periods (Fig 3)The highest scoring female in the first train-ing session also achieved the highest scoresin all succeeding sessions lsquo6rsquo during the 4 hand 8 h training sessions (mean values of 24(04) and 32 (03) respectively) and lsquo5rsquo



during overnight training and pushndashpullexperiments (mean values of 24 (03) and20 (03) respectively Fig 3) As indicatedby the mean scores the majority of animalsadapted more easily to the restraint systemthan the continually highest scoring femaleAll 17 animals however did successfullycomplete the training schedule and wereused subsequently in hypothalamicpushndashpull perfusion sessions (22 03sessions per animal range 1ndash4 sessionsper animal) During the actual pushndashpullperfusion procedure subjects appeared tobe mostly calm and alert The subjectsvisually and vocally interacted with theirgroup mates ate napped and slept duringthe night

Fluid consumption during restrainttraining and long-term restraint stabilized at

approximately 4ndash5 mlh These values werewithin the daily fluid maintenancerequirements for captive adult marmosets(1 ml per kcal of dietary energy intakeKerr 1972 Panel on Nonhuman PrimateNutrition 1978) During the initial one hourof training however restrained marmosetsconsumed a larger volume of fluid(96 06 ml) because of frequent (every5ndash10 min) offerings of Ensure and fruitemployed as positive reinforcement Urinewas voided approximately every 1ndash2 h andfaeces were deposited every 3ndash10 h through-out all restraint periods reflecting a relative-ly stable excretion and defaecation rate

Plasma cortisol levels during restraint forhypothalamic pushndashpull perfusionAs an assessment of stress in femalemarmosets undergoing continuousrestraint hypothalamic pushndashpullperfusion and repeated blood samplingfrom an intravenous cannula we deter-mined plasma cortisol levels on the thirdday of restraint during pushndashpull perfusionData from ovary-intact females in the earlyfollicular phase and the single ovariec-tomized female are shown separately (Fig4) as circulating cortisol levels in ovariec-tomized females can be lower than those inovary-intact females (Saltzman et al 1998)Plasma cortisol levels in ovary-intactfemales (514 [505ndash523] mollback-transformed mean of all time pointscombined [95 CI]) did not changesignificantly (F[714] 088 NS) during thehours sampled (1300ndash2200 h) and approxi-mated those found during morning(0845ndash0915 h and 1145 h) sampling ofovary-intact females under baselineconditions (Saltzman et al 1994 Fig 4)Nevertheless plasma cortisol levels inrestrained females did not show theexpected decrease in values across the day(Fig 4 Smith amp French unpublished data)Cortisol levels during restraint andpushndashpull perfusion however failed toreach the high values exhibited by femaleswounded during the establishment of thesocial dominance hierarchy in the 2 daysfollowing group formation (Fig 4)



Fig 3 Behavioural responses (mean SEM) of17 female marmosets undergoing sling-harnessrestraint training or hypothalamic pushndashpull perfu-sion Behavioural definitions are given in Table 1P 0015 versus all other training sessions andpushndashpull perfusion (Friedman one-way ANOVApost-hoc Wilcoxon paired t-test)



Fig 4 Plasma cortisol levels (A) on the third day of continuous restraint during hypothalamic pushndashpullperfusion (three ovary-intact females [solid circles back-transformed means (95 confidence interval)]and one ovariectomized female [open circles individual values] from 1300ndash2200 h and (B) in morning(0900 h [stippled bar mean (95 confidence interval)] and 1145 h [diagonal-lined bar]) baselinesamples during the early follicular phase in ovary-intact female marmosets and following aggression andwounding during the morning of the second day following social group formation (0900 h hatched bar)in ovary-intact females (data for (B) are modified from Saltzman et al 1994) Note that marmoset plasmacortisol values are typically much higher than those found in Old World primates (Coe et al 1992)

Fig 5 Plasma progesterone concentrations in twice-weekly blood samples from a representative ovary-intact female before during and after restraint for hypothalamic pushndashpull perfusion (PPP) Note themaintenance of regular ovulatory cycles from 4 months before through 2 months after restraint and PPPFemale marmosets in our colony usually ovulate within 8ndash12 days of a prostaglandin analogue(cloprostenol) injection (Saltzman et al 1998) We considered animals to have ovulated on the dayimmediately preceding a sustained elevation of plasma progesterone above 30 nmoll (dotted horizontalline) The arrows indicate the days on which cloprostenol was given to terminate the luteal phase or earlypregnancy and initiate a new follicular phase

Ovarian function during restraint forhypothalamic pushndashpull perfusionRestraint and pushndashpull perfusion did notresult in any obvious disruption of ovulatorycycles in the three ovary-intact females thatwere monitored during their ovulatorycycles immediately before during andimmediately after restraint and pushndashpullperfusion Figure 5 illustrates a representa-tive example of the reliable and regularovulatory cycles and plasma progesteronelevels shown by one of these three femalesOver all the cycles monitored follicularphase duration ranged from 7 to 14 daysfairly typical for marmosets in this colony(Saltzman et al 1998) There was also nosignificant (F[24] 054 NS) delay in thetime taken to ovulate during the cycleinvolving restraint for hypothalamicpushndashpull perfusion (Table 2)

Discussion

This novel long-term restraint systemprovides a practical solution to the chal-lenges posed by increasingly complex neuro-biological and neuroendocrinologicalresearch performed on common marmosetsThe restraint system is particularly usefulfor studies in which little disturbance of thesocial environment can be permittedincluding investigation of the neural mecha-nisms regulating social control of femalereproductive neuroendocrinology (Abbottet al 1998) and the neuroendocrine basis ofparental behaviour (Pryce 1993)

The previously described backpack formarmosets (Ruiz de Elvira amp Abbott 1986)could not be modified to accommodatelong-term restraint as it was not sufficiently

pliable and produced axillary chafing(D H Abbott unpublished results) Incontrast the jacket described here is highlyversatile and pliable enabling the develop-ment of a long-term restraint system forassessment of neuroendocrine mechanismseither directly (eg hypothalamic pushndashpullperfusion Abbott et al 1998) or indirectlyfrom dynamic changes in circulatinghormone concentrations (eg plasmaluteinizing hormone [LH] levels) The jacketand sling-harness restraint are applicable toother neurobiological procedures includingmicrodialysis or electrophysiological record-ing from specific brain regions The jacketmaterials (sailcloth brushed flannel andVelcro) are easily obtained by most laborato-ries and together with the relatively simplejacket design will enable neurobiologicaland neuroendocrine studies in marmosetsand other small nonhuman primates in avariety of laboratory settings

The previously described marmosetrestraint system (OrsquoByrne amp Morris 1988)did not readily facilitate neuroendocrinestudies of marmosets It restrained thesesmall arboreal primates in an upright sitting posture that is not species-typical for marmosets The restraint was based ona design for macaques which are moreterrestrial have ischial callosities and canreadily adopt an upright sitting postureThe previous restraint also permitted amaximum of only 6 h of continuousrestraint (OrsquoByrne 1988) This time periodis far shorter than the length of restraint(2 days) required for long-term assessmentof neuroendocrine function (Gearing ampTerasawa 1988) Our new jacket and sling-harness restraint system provides up to 3 days of continuous restraint for assessmentof neuropeptide and neurotransmitter release(Abbott et al 1997 1998)

Restraint and hypothalamic pushndashpull per-fusion did however cause some activation ofthe hypothalamicndashpituitaryndashadrenal (HPA)axis Although plasma cortisol levels onthe third day of continuous restraint forpushndashpull perfusion and jugular cannulationsampling were similar to those foundpreviously in morning basal samples fromfemale marmosets (Saltzman et al 1994



Table 2 Duration of follicular phase before during andafter restraint for hypothalamic pushndashpull perfusion(PPP) in three female marmosets

Follicular phase Timing of ovulatory cycle duration (days)

Immediately before PPP 83 07During PPP 93 18Immediately after PPP 107 17

Mean SEM

Abbott et al 1997 Saltzman et al 1998)they did not exhibit the expected decreaseacross the daytime hours (Saltzman et al1994 unpublished data Smith amp French1997) This absence of a diurnal decrease incirculating cortisol levels suggests at leastpartial activation of the HPA axis in femalemarmosets restrained for hypothalamicpushndashpull perfusion Adaptation of the mon-keys to the restraint (Wade amp Ortiz 1997)the amount of movement permitted while inthe restraint (Wade amp Ortiz 1997) and theclose visual proximity of cage mates(Tilbrook et al 1999) may all minimize theelevation of circulating cortisol levels inrestrained animals These restraint-associatedelevations in circulating cortisol levels wereof a substantially smaller magnitude thanthose observed in female marmosets follow-ing wounding incurred during establishmentof intra-sexual social dominance hierarchiesover 3 days of new group formation(Saltzman et al 1994) The latter durationwas similar to that experienced duringrestraint for pushndashpull perfusion Cortisolelevations during long-term restraint in otherlaboratory-housed animals are usually notas muted as those demonstrated by femalemarmosets (eg rhesus monkeys Normanet al 1994 sheep Tilbrook et al 1999 ratsTan amp Nagata 2002)

Activation of the HPA axis however wasnot associated with any significant inhibi-tion of ovulatory cycles in restrained femalemarmosets Regular cycles were maintainedin ovary-intact females before during andafter restraint for hypothalamic pushndashpullperfusion in contrast to the inhibitoryeffects of long-term restraint on ovulatoryfunction observed in female rhesus monkeys(Chen et al 1992) Furthermore we havealready demonstrated that our restraint andhypothalamic pushndashpull perfusion systemdoes not inhibit hypothalamic GnRHrelease in female marmosets (Abbott et al1997 1998) in contrast to the inhibitorycontributions made by other restraintsystems towards the release of pituitary LHin female marmosets (OrsquoByrne et al 1989)rhesus monkeys (Norman et al 1994) sheep(Turner et al 2002) and rats (Kam et al2000) It is still not clear however whether

activation of the HPA axis by restraint iscausally involved in suppression of repro-ductive function (Norman 1994 Cameron1997 Tilbrook et al 2002) Thus our novelrestraint system appears to be appropriatefor investigating the neuroendocrine regula-tion of female reproduction but not adrenalfunction in common marmosets

Acknowledgments We thank S Brice J KempE Duhr J A Vanderloop and the animal care andveterinary staff of NPRC for their care and mainte-nance of the marmosets D J Mohr for surgicalassistance F H Wegner D J Wittwer G Schefflerand S Jacoris for assistance with the hormoneassays and E Terasawa for assistance with thepushndashpull perfusion procedure We also thankL Endlich for the drawing in Fig 2 This research wassupported by grants from the National Institutes ofHealth (RR000167 HD07678 MH11417 andMH53709) and National Science Foundation (IBN92-21771) This is publication number 43-016 of theNPRC

References

Abbott DH (1984) Behavioural and physiological sup-pression of fertility in subordinate marmoset mon-keys American Journal of Primatology 6 169ndash86

Abbott DH (1987) Behaviourally mediated suppres-sion of reproduction in female primates Journal ofZoology (London) 213 455ndash70

Abbott DH (1992) Reproduction in female marmosetmonkeys Callithrix jacchus In ReproductiveBiology of South American Vertebrates (HamlettWC ed) New York Springer-Verlag pp 245ndash61

Abbott DH (1993) Social conflict and reproductivesuppression in marmoset and tamarin monkeysIn Primate Social Conflict (Mason WAMendoza SP eds) Albany SUNY Press pp 331ndash72

Abbott DH George LM (1991) Reproductiveconsequences of changing social status in femalecommon marmosets In Primate Responses toEnvironmental Change (Box HO ed) New YorkChapman and Hall pp 295ndash310

Abbott DH Hodges JK George LM (1988) Socialstatus controls LH secretion and ovulation infemale marmoset monkeys (Callithrix jacchus)Journal of Endocrinology 117 329ndash39

Abbott DH Barrett J George LM (1993) Comparativeaspects of the suppression of female reproductionmarmoset and tamarin monkeys In Marmosetsand Tamarins Systematics Behaviour andEcology (Rylands AB ed) Oxford OxfordUniversity Press pp 152ndash63

Abbott DH Saltzman W Schultz-Darken NJ Smith TE (1997) Specific neuroendocrine mechanisms not involving generalised stress medi-



ate social regulation of female reproduction incooperatively breeding marmoset monkeys Annalsof the New York Academy of Sciences 807 219ndash38

Abbott DH Saltzman W Schultz-Darken NJTannenbaum PL (1998) Adaptations to subordinatestatus in female marmoset monkeys ComparativeBiochemistry and Physiology C 119 261ndash74

Abbott DH Barnett DK Colman RJ Yamamoto MESchultz-Darken NJ (2003) Aspects of commonmarmoset basic biology and life history importantfor biomedical research Comparative Medicine53 339ndash50

Baker JV Abbott DH Saltzman W (1999) Socialdeterminants of reproductive failure in malecommon marmosets housed with their natalfamily Animal Behaviour 58 501ndash13

Barrett J Abbott DH George LM (1990) Extension ofreproductive suppression by pheromonal cues insubordinate female marmoset monkeys Callithrixjacchus Journal of Reproduction and Fertility90 411ndash18

Barrett J Abbott DH George LM (1993) Sensory cuesand the suppression of reproduction in subordinatefemale marmoset monkeys Callithrix jacchusJournal of Reproduction and Fertility 97 301ndash10

Blum B Israeli J Kulikoowski JJ Gur M Carden D(1983) Stereotaxic head restraint for chronic andsemi-chronic marmoset and macaque monkeysBrain Research Bulletin 11 399ndash403

Bogert LJ Briggs GM Calloway DH (1973) Nutritionand Physical Fitness 9th edn Philadelphia PAWB Saunders Table 2A Nutritive values of foodsin average servings or common measures

Cameron JL (1997) Stress and behaviorally inducedreproductive dysfunction in primates Seminars inReproduction and Endocrinology 15 37ndash45

Chen MD OrsquoByrne KT Chiappini SE Hotchkiss JKnobil E (1992) Hypoglycemic rsquostressrsquo andgonadotropin-releasing hormone pulse generatoractivity in the rhesus monkey role of the ovaryNeuroendocrinology 56 666ndash73

Coe CL Savage A Bromley LJ (1992) Phylogeneticinfluences on hormone levels across the primateorder American Journal of Primatology 28 81ndash100

Coelho AM Jr Carey KD (1990) A social tetheringsystem for nonhuman primates used inlaboratory primates Laboratory AnimalScience 40 388ndash94

Collins P Roberts AC Dias R Everitt BJ Robbins TW(1998) Perseveration and strategy in a novel spatialself-ordered sequencing task for nonhumanprimates effects of excitotoxic lesions anddopamine depletions of the prefrontal cortexJournal of Cognitive Neuroscience 10 332ndash54

Ferris CF Snowdon CT King JA Duong TQZiegler TE Urgurbil K Ludwig RSchultz-Darken NJ Wu Z Olson DP Sullivan JTannenbaum PL Vaughan JT (2001) Functionalimaging of brain activity in conscious monkeys

responding to sexually arousing cues NeuroReport12 2231ndash6

French JA (1997) Proximate regulation of singularbreeding in callitrichid primates In CooperativeBreeding in Mammals (Solomon NG French JAeds) New York Cambridge University Presspp 34ndash75

Friday KE Lipkin EW (1990) Long-term parenteralnutrition in unrestrained nonhuman primates anexperimental model American Journal of ClinicalNutrition 51 470ndash6

Gearing M Terasawa E (1988) Luteinizing hormonereleasing hormone (LHRH) neuroterminalsmapped using the pushndashpull perfusion methodin the rhesus monkey Brain Research Bulletin21 117ndash21

Harlow CR Gems S Hodges JK Hearn JP (1983) Therelationship between plasma progesterone and thetiming of ovulation and early embryonic develop-ment in the marmoset monkey (Callithrixjacchus) Journal of Zoology (London) 201 273ndash82

Hearn JP (1977) Restraining device for small monkeys Laboratory Animals 11 261ndash2

Hearn JP (1983) The common marmoset (Callithrixjacchus) In Reproduction in New WorldPrimates New Models in Medical Science (Hearn Jed) Lancaster MTP Press pp 181ndash215

Kam K Park Y Cheon M Son GH Kim K Ryu K(2000) Effects of immobilization stress onestrogen-induced surges of luteinizing hormoneand prolactin in ovariectomized rats Endocrine12 279ndash87

Kerr GR (1972) Nutritional requirements of subhu-man primates Physiological Reviews 52 415ndash67

Mansfield K (2003) Marmoset models commonlyused in biomedical research ComparativeMedicine 53 383ndash92

Norman RL McGlone J Smith CJ (1994) Restraintinhibits luteinizing hormone secretion in the follicular phase of the menstrual cycle in rhesusmacaques Biology and Reproduction 50 16ndash26

OrsquoByrne KT (1988) Long-term blood samplingtechnique in the marmoset Laboratory Animals22 151ndash3

OrsquoByrne KT Morris KD (1988) A restraint system forthe common marmoset (Callithrix jacchus)Laboratory Animals 22 148ndash50

OrsquoByrne KT Lunn SF DIxson AF (1989) Naloxonereversal of stress-induced suppression of LHrelease in the common marmoset Physiology andBehavior 45 1077ndash80

Owen S Thomas C West P Wolfensohn S Wood M(1997) Report on primate supply for biomedicalscientific work in the UK Eupren UK WorkingParty Laboratory Aniimals 31 289ndash97

Panel on Nonhuman Primate Nutrition (1978)Nutrient Requirements of Nonhuman PrimatesWashington DC National Academy of Sciences



Pryce CR (1993) The regulation of maternalbehaviour in marmosets and tamarinsBehavioural Processes 30 201ndash24

Pryce CR Doumlbeli M Martin RD (1993) Effects of sexsteroids on maternal motivation in the commonmarmoset (Callithrix jacchus) development andapplication of an operant system with maternalreinforcement Journal of Comparative Psychology107 99ndash115

Ruiz de Elvira MC Abbott DH (1986) A backpacksystem for long-term osmotic minipump infusionsinto unrestrained marmoset monkeys LaboratoryAnimals 20 329ndash34

Saltzman W Schultz-Darken NJ Scheffler GWegner FH Abbott DH (1994) Social and repro-ductive influences on plasma cortisol in femalemarmoset monkeys Physiology and Behavior56 801ndash10

Saltzman W Schultz-Darken NJ Abbott DH (1996)Behavioural and endocrine predictors ofdominance and tolerance in female commonmarmosets Callithrix jacchus Animal Behavior51 657ndash74

Saltzman W Schultz-Darken NJ Wegner FHWittwer DJ Abbott DH (1998) Suppression ofcortisol levels in subordinate female marmosetsreproductive and social contributions Hormonesand Behavior 33 58ndash74

Scott L (1994) Marmosets as models for man In TheMarmoset Role in Pharmaceutical Development(Fowler JSL ed) Ipswich UK Pharmaco LSRpp 1ndash5

Smith TE French JA (1997) Psychosocial stress andurinary cortisol excretion in marmoset monkeys(Callithrix kuhli) Physiology and Behavior62 225ndash32

Sokal RR Rohlf FJ (1995) Biometry 3rd edn NewYork WH Freeman

Stevenson MF Rylands AB (1988) The marmosetsgenus Callithrix In Ecology and Behavior ofNeotropical Primates Vol 2 (Mittermeier RARylands AB Coimbra-Filho AF da FonescaGAB eds) Washington DC World Wildlife Fundpp 131ndash222

Summers PM Wennink CJ Hodges JK (1985)Cloprostenol-induced luteolysis in the marmosetmonkey (Callithrix jacchus) Journal ofReproduction and Fertility 73 133ndash8

Tan Z Nagata S (2002) Superimposed cold stress-induced hypothalamicndashpituitaryndashadrenal responseduring long-duration restraint stress Journal ofUOEH 24 361ndash73

Tardif SD (1997) The bioenergetics of parental behav-iour and the evolution of alloparental behaviour inmarmosets and tamarins In Cooperative Breedingin Mammals (Solomon NG French JA eds)Cambridge Cambridge University Press pp 11ndash33

Tilbrook AJ Canny BJ Serapiglia MD Ambrose TJClarke IJ (1999) Suppression of the secretion ofluteinizing hormone due to isolationrestraintstress in gonadectomized rams and ewes is influ-enced by sex steroids Journal of Endocrinology160 469ndash81

Tilbrook AJ Turner AI Clarke IJ (2002) Stress andreproduction central mechanisms and sex differ-ences in non-rodent species Stress 5 83ndash100

Turner AI Canny BJ Hobbs RJ Bond JD Clarke IJTilbrook AJ (2002) Influence of sex and gonadalstatus of sheep on cortisol secretion in response to ACTH and on cortisol and LH secretion inresponse to stress importance of different stres-sors Journal of Endocrinology 173 113ndash22

Wade CE Ortiz RM (1997) Urinary excretion of cor-tisol from rhesus monkeys (Macaca mulatta)habituated to restraint Contemporary Topics inLaboratory Animal Science 36 55ndash7



conventionally used in biomedical research(Abbott 1992) Marmosets make excellentsubjects for studies in neurobiology neuro-endocrinology and behaviour because theyadapt easily to a variety of experimentalprocedures including venepuncture (Hearn1977 1983) venous cannulation (OrsquoByrne1988) hypothalamic pushndashpull perfusion(Abbott et al 1998) operant testing (Pryceet al 1993) learning paradigms (Collins etal 1998) functional magnetic resonanceimaging (Ferris et al 2001) and social testingprocedures (Saltzman et al 1996 Bakeret al 1999) These monkeys also appear tobe comfortable in small enclosed spaces(Ferris et al 2001)

Common marmosets share numerousbiochemical physiological and behaviouralsimilarities with humans For examplemarmosets exhibit a 28-day ovarian cycleestablish complex long-lasting socialrelationships and live in small family-basedsocial units (Abbott 1992) Offspring remainwithin their natal group into adulthood andall group members contribute to infant careincluding extensive carrying and post-weaning provisioning of infants (French 1997Tardif 1997) In contrast to humans howeverthe behaviourally dominant female in mar-moset social groups (the mother in familygroups) is usually the only female to conceiveand give birth (Abbott 1984 1987 1993) Atthe same time the ovulatory cycles of subor-dinate females in social groups are suppressedin response to a combination of behaviouralvisual and olfactory cues from the dominantfemale so that subordinate females are usual-ly anovulatory and hypo-oestrogenemic(Abbott et al 1988 1993 1997 Barrett et al1990 1993) The common marmoset there-fore provides a specialized primate model fordetermining mechanisms mediating thesocial regulation of reproduction

To investigate the neuroendocrinemechanisms underlying the social controlof ovulation in marmosets we needed tosignificantly refine previously describedrestraint techniques to permit long-term (upto 3 days) assessment and manipulation ofhypothalamic and pituitary function Animproved methodology would also providenew opportunities for neurobiological inves-

tigation in a wide range of studies utilizingcommon marmosets and other small-bodiedprimates Our immediate goal was todevelop a well-tolerated restraint systemthat would permit a moderate degree ofmovement while enabling repeated intra-venous and hypothalamic sampling of themarmoset in close proximity to its socialgroup members A backpack previouslydescribed for use with marmosets did nothave the versatility necessary for ourproposed studies (Ruiz de Elvira amp Abbott1986) as it was designed to accommodateonly osmotic mini-pumps housed in smallfluid-filled (5 ml) chambers Furthermore apreviously described restraint system formarmosets was limited to a maximum of6 h of continuous use (OrsquoByrne amp Morris1988) Since the previous restraint systemdid not allow for species-typical restingpostures it could not accommodate theminimum of 3 days of continual restraintrequired for hypothalamic perfusion studies(Gearing amp Terasawa 1988)

To permit such long-term restraint wedeveloped a soft jacket that serves as both(1) a pocket for an indwelling intravenouscannula and (2) an integral part of a novelsling-harness restraint This system permit-ted such complex techniques as hypothala-mic pushndashpull perfusion with simultaneoussampling from an intravenous cannula Ourfindings demonstrated that marmosetsquickly adapted to 3 days of continuousrestraint in this novel system and validatedthe systemrsquos usefulness in neurobiologicalinvestigations using the marmoset

Materials and methods

AnimalsSubjects were 17 captive-born adult (2ndash8years of age) female common marmosets(Callithrix jacchus) housed at the NationalPrimate Research Center University ofWisconsin (NPRC) as previously described(Saltzman et al 1998) NPRC is fully accred-ited by AAALAC as part of the University ofWisconsinndashMadison Graduate SchoolAnimal protocols and experiments wereapproved by the Graduate School Animal








































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novel restraint system for neuroendocrine. j. schultz-darken 1, r. m. pape 1, p. l. tannenbaum 2, w....

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