Models of Anxiety: Ultrasonic UNIT 5.18

Vocalizations of Isolated Rat PupsLucianne Groenink,’ P» Monika Verdouw,’ Ruud van Oorschot, andBerend Olivier”2’3

‘Psychopharmacology, Department of Pharmaceutical Sciences, Utrecht Institute forPharmaceutical Sciences and Rudolf Magnus Institute of Neuroscience, Utrecht»The Netherlands2PsychoGenics lnc, Tarrytown, New York3Deptartment of Psychiatry, Yale University School of Medicine, New Haven, Connecticut

ABSTRACT

Described in this unit is the ukrasonic distress vocalization test in rat pups. This testis a reliable method for detecting anxiolytic properties of test compounds. In this test,ultrasonic vocalizations (30 to 50 kHz) are elicited by separating rat pups of 9 to 11 days ofage from their mother and littermates for a brief period of time. The test can be performedunder two different stress conditions. Pups are placed in isolation in either a warm (37°C)or cold (18°C> environment forS min, The total number and duration of ultrasonic callsemitted by the pups during this period is used as an index of anxiety. Establishedanxiolytic compounds, ineluding benzodiazepines, serotoninlA receptor agonists, andselective serotonin reuptake inhibitors (SSRIs), consistently reduce the number andcumulative duration of these ultrasonic distress vocalizations. Curr Protoc. Pharmacol.43:5A8.U5.18,14. © 2008 by John Wiley & Sons, mc.

Keywords: anxiety • ultrasonic vocalization • distress • rat pup - negative geotaxis

When rat pups of 9 to Ii days are separated from their mother and littermates, they BASICemit ultrasonic calls in the range of 30 to 50 kHz. This ultrasonic vocalization (USV) PROTOCOLcan be used as a measure of distress or anxiety. Measurement of the number of vocalizations during a short testing period (5 min) can be used to quantify the amountof distress (anxiety). While the ultrasonic cries are reduced by drugs with anxiolyticproperties, including selective serotonin reuptake inhibitors (SSRI5), anxiogenic drugsincrease these vocalizations. The paradigm also distinguishes drug classes with posidve reinforcing properties, such as mu and delta opioid receptor agonists and (indirect)dopamine receptor agonists. Noradrenergic drugs, however, are not reliably detected inthis test.

Described in this unit are the basic methods for measurement and quantification ofultrasonic calls in rat pups under two different stress conditions by placing the pups oneither a cold plate (18°C) or a warm plate (37°C). The protocol describes how to applythis distressvocalization paradigrn to measure the psychoactive (anx.iolytic) effcts oftest substances, and to analyze side effticts of these test agents usi.ng a negative geotaxistest and rectal temperature measurement.

NOTE: All protocois using live animai.s must first be reviewed. and approved by aninstitutional A.n i.mal Care and Use Comm.ittee (.1ACUC) or must conform to governmentalregulations refarding the care ard use of laboratory animais,

Anm.aI Niodelsof Disease

Current Prntocois ie Pharn cuioçv 5,18. C5. 1814. December2008Published unline Dec.mnbr fls: inve (www,inrscience,iley,cum),[4(21: 1.0,1002/0171 14i.755.eh0518s43

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U!troniccahzations

Rat Pups

S1&2

Materials

Young adult male and female Wistar rats, age 10 to 12 weeks, for breedingprogram, or pregnant dams, gestation day 9 (e.g., Harlan/CPB Zeist)

Vehicle for dissolving test eompoundTest compoundSilicon oil

100 x 50 x 60—em and 425 x 266 x 180—mm cagesCustom made ultrasonie voealization testing cylinder: Plexiglas cvlinder (19-em

id.. 15-em height) with a lid through which a mierophone ean be inserted(Fig. 518.1)

Audio apparatus—eomplete system consists of:Microphones, or mini-3-bat detector (Ultra Sound Advice:

http://www ulrrasoutukidi’ice.co.uk)Control unit. ineluding amplifler, audio filters, and software for data aequisition

and processing, such as Avisoft-SASLabPro (Avisoft Bioacoustics;http://ww.avisofi.com), Sonotrack (Metris B.V.: hrtp://www,netris.nt),ultrasonic vocalization detector (Mcd Assoeiates). or UltraVox (Noldusinformation Technology)

Alternatively. a svstem can be built with separate components, ineludingultrasonic mierophone with amplifler, audiofilters. analog-to-digitalconverter, oscilloscope. and customized signal-detection software(see, e.g., Fish et al., 2004; Satow et al., 2008)

Computer for data recording and statistical analysis software: eg.. SPSS or SAS(http://wwwsas.com)

Cold and hot plates: 20-em stainless steel plates held at either 1 8 or 3T C byeirculating thermostatted water

Refrigeratedlheating eirculators (e.g., Julabo F12-ED) to either eool (18C) orwarm water (3TC) and to circulate the thermostatted water through the steelplates

Separate warm plate for keeping nest at 37°CAnimal balance, aceuracy 0.1 g (e.g., Mettler, type PG5000)l-ml syringes25-G x 5/8-in. neediesWaterproof markers in various colorsInclined (20°) sereen (Fig. 5.18.2)Digital thermometer with thermistor probe (eg., Keithley, 871A) for rectal

temperature measurementStopwatch

NOTE: Most USV studies have been performed with pups from an inhouse breedingprogram. This is not absolutely necessary, as it is also possible to order pregnant dams(eg, Hodgson et al., 2008) However, prenatal stress could result in behavioral and neurochemical alterations in the offspring (Weinstock, 2007) and might affect test outeome,

Establish breeding conditians

1 •Pkce two male Wistar ets in separate 100 x 50 x 60—em cages each contamningfive females Maintai.n roups under a. reversed 12 hr IigheIi2 1w dark schedule for

- ti T t - t

aure 5 18 2 Nq: eyo ena apparaTus Negaie gntar easured‘:d suce ihcn C&CcI re flrrniV D jr ct€ . k ) Th i is p ta o c r 1Q d ir t’e e

j rr4 :_)

iimal I k

Figure 518.1 Ultrasonic pup vocalization measurement apparatus. The pup is placed on theplate. which is perfused with either warm (37 0) or cold (18 C) water. A microphone is insertedthrough the lid of the cylinder. A custom-made detector is shown in the upper right corner of thefigure: an oscilloscope is positioned just under t, and the therrriostat is shown on the left side ofthe figure.

This schedule mainrains a regular supplv ofpups for continued experimentation. A complete standard experiment requires 64 pups: eight pups/dose for one vehicle and threedoses of test compound. each performed on both cold (18G and warm (3TC) plares.Thus, if rats are optimallv producing litters of nine or more pups see sreps 5 and 6),1 weeks breeding will provide sujficient pups for one experiment.

4. Verify pregnancy visually (vaginal plug after mating and palpation on day 12) andhouse pregnant females singly in 425 x 266 x 180—mm cages with extra nestingmaterial a few days before delivery.

5. Note the day of birth as Day 0. On Day 1, reduce the number of pups in a litter to 9.Replace females that produce smaller litters.

Pups, regardless ofgender are used fir experiments on Days 9, 10, and 11.

Outline experimental plan

6. Randomly allocate eight pups of one litter to vehicle and three doses of a testcompound, according to a 2 x 4 balanced-block design. In alternating fashion, orderthe two blocks of four pups from one litter to be tested on either the warm or coldplate. Repeat foratotal of eight litters, each containing eightpups (see Table 5.18,1).

Since not all females deliver pups on the same day, the schedule allows for resting over3 successive days.

.4ssemble experimental apparatus

7. Place an ultrasonic microphone in the top of a test cylinder. Connect the microphoneto the control unit with an audio cable and activate the control unit.

8. Connect the control unit to a computer equipped with software for data acquisitionand processing (e.g., Avisoft-SASLAbPro, Sonotrack, UltraVox).

(‘alibrate the system as indicated bi, the supplier balancingpeak threshold andpeaks overthreshold. Basically set the peuk threshold ofyour system such that the background noiseis just detectabie, Then set the peaks over threshold two to three times higher than thenumber ofpeaks detected ut peuk threshold. Once you have set these rhresholds properlyin your experimenral setring, there is no iieed to calibrate your svsre,nfrequentlv.

9. On the day of the experiment, verify that the cold and hot plates are at their correct temperatures (l8 and 3TC, respectively). To verify if the system is properlydetecting ultrasound, shake a set of keys to produce high-pitched frequencies.

Measure body weight and treatpups with test compound10. Immediately before the experiment, remove the mother from one litter of pups and

transport the pups to the experimental room in their own nest cage. Place the nestcage on a warm plate to keep the nest temperature at -3TC.

11 -t 30 min betore testmg randomk remote a pop trom the nest eigh it md re..ordhe eight k. g ina 4oLkshed uch as J’able 18 1> Imect tht pup i p with ‘t.hIL1L

or LOSt compound at a volume of 10 rnl/kg body weight. Inject at a s.ite —5 mm tothe left or right of the navel, at a depth of 5 mm, using a 1-mi syringe and a 25-Ei,5/8-in. needle,

iizke the pap oct of the nest carefidly; do not sugvend it hv the tail, and hold it hy the neckwhiie holding the tail looselR

Renear steos 5fl( 1 2 everu 75 outS uit flfl’ SV S withi.!is4)mc. .

. -— -

vaO7atiOHS fl1

Rat Paus

Table 5.18.1 Drug Aflocation Scheme for the Rat Pup Ultrasonic Vocalization Test

. Anirnal .. Condirion”Nst numhLr DruLode’ \\u2ht) (olorodL

number (C or Vv)

01 01 A C LF

01 (12 S C RF

01 03 C C EB

01 04 D C RB

01 05 3 LFH

01 06 C W REN01 07 D W LBH01 08 A RBH

02 09 C C LF02 10 D C RF(>2 11 A C LB02 12 S C RB02 13 D W [FR

02 14 A W RFK

02 15 S W LBH02 16 C W RBH

03 17 C C LF03 18 A C RF03 19 D C LB03 20 S C RB03 21 A W LFH03 22 D W RFH

03 23 S W LI3H03 24 W RBH

04 25 D C LF04 26 S C RF04 27 C C LB04 28 A C R1304 29 S W LFH04 30 C W RFH04 31 A W LBH04 32 D W RBH

05 33 S C LF05 34 [) C RE05 45 A 13 L13

C RB1)5 37 D w LFH

3(1 REH(15 13 (1V [3H(15 0 8 (1V RF3I-1

41 13 LE

13 13 Li-3

- 13 13 1313

13 1 .1313

16 13 (1V

(1) (113 [3313131313 Animal Models

oflhsease

Oflh

5[18.5

Table 5.18.1 Drug Allocation Scheme for the Rat Pup Ultrasonic Vocalization Test,continued

Ammal ConditionNest number Drug code° Veight (g)

(C orColor code

07 49 D C LF07 50 C C RF01 51 B C LB07 52 A C RB07 53 C W LFH07 54 8 W RFH07 55 A W IBH07 56 D W RBH

08 57 8 C LF08 58 A C RF08 59 D C LB08 60 C C RB08 61 A W LFH08 62 D W RFH08 63 C W LBH08 64 S W RBH

Adnunistration of three doses of drug iB, C, and 0) and sehicle (A) oser 64 pups, dened from eight nesis

each containing etght pupsbTegting condinon ts designated as cold IC: 18 C) or warm (W, 37 C)

Color coding ts as follows LF, left front leg; RF right front leg: LEk leO back leg, REl, nght back leg,

t FH, left front head: RFH, right front head, LBH. leO back head; RBH, right back head,

Perform negalive geotaxis test

Depending on specific laboratory conditions, the negative geotaxis test may affect ultrasonic vocalization. When selling up the USV test, some pilot studies should be performedto determine the optima! order in which to perform steps 14 and 15.

14, At a time point 28 minutes after injection, carefully remove the pup from the nestand place it head-down on an inclined (20 ) screen (Fig. 5.18.2), Use a stopwatch tomeasure the time the pup needs to return its body 180 to a head-up position (bothforelegs must be placed above the hind legs). Repeat five times,

Pupa typica11v need to 10 sec to turn but a utqJf time should he set af ‘0 sec Thenegative geotaxis test weld information about muscie relasvtion and sensory or motordisturhances.

Ieasure ultrawni vocalizalion

S Immediately placc the pop in the ultrasonic voalization test vlinder 0fl elther theold or the ssaim plate (‘ee \nncipated Resuits lor discussion ot cold scrsus ssaîmondmonsi, Using the c mputer program. irnmediatels begin ncasunng the number

and durati n 1 ultia o i cali and ntinue neasurcment f r nin RLu rd thtart mc e in im an rc uncv (in kil ii act all

Dose of test compound or vehicleInjection/testing intervalDate, time of dayTest duration (300 sea).

Such data administration/recording may not avaiiable in all software programs. Runningmedium- to large-scale experinents withour such software to link the anïmal numbeîbody weight, treatment condition, etc, to the data file that holds the USV recording ofthat animal, howeve, is laborious and ermr-prone. As an example, Sonotrack (Metris)has this feature.

17. Store all data in a data file that allows the data to be fed into the statistical program(SAS or SPSS).

Measure rectal temperature

18. Dip the thermistor probe in silicon oil before inserting it into the rectum and holdstili until a stable rectal temperature is obtained for 10 sec.

19, Return the pup to the nest. Clean the plate with water and dry.

Repeat measurements for remaining animals

20. Repeat steps 14 to 19 for the remaining pups in the litter. Use each pup only once.

21. Repeat steps 10 to 19 for the other seven litters.

Analyze data

22. Express the number and duration of ultrasonic calls as mean ± standard error of themean (SEM). 1f a test compound suppresses ultrasonic calling, calculate an ED50;otherwise determine the lowest effective dose (LED; lowest dose that significantly reduces ultrasonic calling). Analyze data by analysis of variance, using test compound

.

Figure 51&3 Representative data from a rat pu.p USV test showing the effects of the benzodiazepine receptor agonist diazep.m on the number of USVs measured onder two temperatureconditions (18° or 37°C) Diazepam or vehicle was adminhtered intraperhoneaily 30 min before thetest, which lasted 5 min. Data are presented as the mean number of calls ± SEM from 8 determinations P <. 005 compared to vehicie, The experiment presented here used 84 pups accordingto the described protocol

ArOmal Modelsn/

5187

500

400

ci, 300-0(3

200(38:3z 100

_—_ 1 8C cold plate

— o- - 370C warm plate

-

- -0— —--—--- 0

treatment, litter, and test condition (warm or cold) as factors, followed by post-hoc t

tests.

A signifieance lei ei tp <0.05 is accepred. Analyze the ultrasanic vocalization, iegafivegeataxis. and remperarure rest a eparate experiments. Figure 5. 18,3 iliustrates there s uit.s of a tpical (JSV rest u ing diazeparn as an anxiolytic.

23. Calculate latency time for the negative geotaxis test as mean ± SEM. Calculatethe ED0 or LED. Analyze data by an analysis of variance. using test compoundtreatment and litter as eplanatorv factors, followed by post-hoc t tests.

.4 sii,’nilicanee level ofp <0.05 d accepted.

COMMENTARY

Background InformationRat pups erna uhrasonic vecalizations

<USVs in the 35- to 45-kHz range when separated from their mother and littermates. Asthese calls are associated with secial isolatien. the have heen deseribed as “distress‘oca1iza1ions” er “isolatien calls,” referringto cries emnted hy marnmahan infants acrossspecies. Rat pup USVs are of lar ngeal engin Roherts. 1975 and stimulate maternalretrieval (Allin and Banks, 1972: Smotherman et al.. 1974: Elwood. 1979) and maternal prolactin release (Terkel et al.. 1979:Voloschin and Tramezzani. 1984). The highfrequencv eau corresponds to a high auditorysensitivity of adult rats in a similar frequencvrange (Noirot. 1966). Calling develops gradually within a few days after birth, is maintained at relatively high levels until eye opening around Day 14 (Insel et al., 1988), wanesthereafter, and disappears completely aroundDay 18 (Allin and Banks, 1971).

As pups in the first weeks of life are blind,deaf, have no for, are poikilothermic, and depcnd on their morher lor feeding, temperaturecontrol, and excretion processes (BrouettcLahlou et al., 199’), they are not able to surviseii left unattended outside the nest. The USVtheretore is a ‘<tal means of communicationbetween the mother and her newborns. C sllinn is also enhanced hs <resstul stimuli uchas low temperatures A(lin and Banks, 1971Okon 1 9”7, iactile ,tiiuuiation iNoir t 19 ‘

Oswalt and Meter, 1975 t. nar briet maternalne ‘unter Shair ‘00 r unfarniliar Ifacrv stimuli (O,waie and Meter, 10 7S) fl

C t It ttk 11 lcaturt upoutrt ‘ce ue ot 1 Sla t cc L ii, ect p’tj Kaui

tiv’< t 9, t

Ho es er. not all ssrems are capable of measuring the frequency and amplitude of thecalls. Furthermore. systems ma differ in sensitivity. depending on the microphones. ampli[iers. and converters emploed. For instance.a bar detector must he tuned to the requiredfrequency and therefore can only pick up callsin a small frequenc band. which could resultin missed calls.

It is interesting to note that recent pharmacological studies indicate that the cumulativetime a pup spends vocalizing during the test isnot only a valuable, but sometimes also a moresensitive measure of anxiety than the numberof calls made in the vocalization test (Hodgsonet al., 2008).

PsychopharmacologyEstablished anxiolytic drugs belonging to

different pharmacological classes—in particular the benzodiazepines (Gardner. 1 985a,b;Inseletal., 1986; Gardnerand Budhram, 1987;Olivier et al., 1994, 1998a; Kehne et al.. 20(X);Hodgson et al., 2008), 5-HTIA receptor agonists (Hard and Engel. 1988; Mes and Olivier,1989; Olivier et al, 1993, 1994, l998b), andselective serotenin reuptake inhibitors (Mosand Olivier, 1988; Winslow and Insel. 1990:Olivier et al., 1998b; Kehne et al.. 2000;Hedgson er al , 2008)-— suppress rat pup t SVThe effect of tricyclic antidepressanis. such asdtsipramine and amitrvptilinc. is less COOsis

Lilt in this test Reported resuits wtth trivycllLanndepressants range from active t Wins(owandlnsel, 1990 Olisieretal , V)98h: I1odson

1 ‘005 tat,zic (iardncr, 1T d t al . 7)05j md base pr’duecdt r a t its in this est t 9 rik w ,nd

1 )9( 99in ‘ 1 ‘P90 r <v’‘3’r’. at ‘

.1’,atjtt1 t7 .

and putathe anxiolytics like the metabotropicNMDA receptor antagonists MPEP and MTEP(Winslow et al., 1990; Insel and Winslow,1991: Kehne et al., 1991; Hodgson et al.. 2008)and the CRF1 receptor antagonist CP154.526(Kehne et al, 2000: lijima and (‘haki. 20005)all reduce the USVs ernitted, The potentiallyanxiolytic vasopressin1greceptor antagonist.SSRI4.9415. however, did not alter the numher of l’S\ ([ijirna and Chaki. 2005). Someputatn.e anxiogenic agents. ‘uch as pentetra/ol. lindane. or CCK4. enhance calling (Inselet al.. 1986: Gardner and Budhram. 1987:RieraetaL. 1992: Rexetal., 1994 t. hereasanumher of other ps\ choacti e a!ents de oid ofanxieltic properties. such as neurolepties.pschostimulants. or musele relaxants. do notaffect calling tGardner. l985a: Kehoe andBoylan. 1992: Kehne et al.. 2000.

This pharmacolooical protile led to the suggestion that USV of rat pups is a sensitietest for mechanisticall dierse anxiokticstGardner. 1985a: Winslow and Insel. l991a.b:Ulivier et al.. 1994). There are. howeer. anumber of limitations to this assay. Noradrenergic drugs are not reliably detected in thistc..t. For example, clonidine. which displavsanxiolytïc and sedative effecis in hurnans. enhances calling in rat pups (Hard et al.. 1988:Insel et al.. 1988: Kehoe and Harris. 1989:Van der Poel et al.. 1991). It appears that clonidine is anxlogenic in rat pups hefore 16 daysof age. but is anxiolytic after Day 16 (Kehoeand Harris, 1989). Presumably. maturationalehanges in the noradrenergic system areresponsible for this change.

The ultrasonic distress vocalization test canalso he performed in mouse pups. Although theoom her of reported pharmacologïcal studies inmice is limited, dru effects reported thus farusmg the mouse are very similar to those notedin the tat. rhat is, benzodiazepines, SSRIs, anderotonin1 receptor agonlsts are acti’e in thenou’o. model(Fishetal ,2000, 2004e Furthert ri. th. ltrasL IIIL distre’»s ctcali,ation cao

helpful i detumimne the L’nequences c’fne irutatitns such is krock out. knockin.

a td t ar s cm 1 w h’s measure er ‘cia1 om“ :nimal ‘W”e.l c’ d

(X ‘i al. Y)0S

t t ilPatametersand1 “nubleshooting

-

‘—‘- --‘.

.. :-n, -1 r

Precautions must be taken when handlingthe pups before the actual experiment. Roughhandling evokes a high lesel of vocahianonthat will interfere with the test and provokeexeitement in the nest. Intraperitoneal injection ol’ pups should be practiced in advance.as .ome skill is required to inject pups correctl and consistently. 1f a pop falls out ofthe experimenter’s hand during the procedure. rcplace it immediately with the reservepop.

Maintain the environmental temperature ofthe experiment room at -22 C. Keep the nesttemperature constant at —37 C. as cooling ofthe nest affeets the test results.

Anticipated ResuitsA test agent bas anxiolt dc potential ifeither

the numher andlor the duration of calling onone or hoth of the platesil8 Cor 37 C is decreased. Ben/odiaLepines. such as diazeparn.chiordiatepoxide. and alprazolam. decreasethese parameters under both stress conditions.sx hereas partial benzodiazepine receptor agomsts. such as bretazenil and alpidem. onlyreduce calling on the warm plate (Olivieret al.. 1998a). Serotonergic anxiolytics. such as5-HTiA receptor agonists. also reduce cailing under both stress conditions. The SSRIsfluvoxamine and clomipramine are preferentially active on the cold plate. A 5-HTi receptor agonist. trifluoro-methylphenyl-piperazine(TFMPP), enhances calhng at low doses onthe cold plate, whereas 5-HT9 receptor antagonists, such as ketanserine, enhance calling atlower doses on both plates (Van der Poel et al..1992: Olivier et al.. l998b) Figure 5.18.4shos the effects of sarious compounds onhoth the IJSV and negative geotaxis tests.An increase in latency time in the negatiseceotaxis test suggests that the test compoundhas sedatise or muscle relaxant etfeets Insuch a Lase, a dcerease in ultrasonic socaltia

ins cannot olely he attohuted to inxiolyticproperties Similarly, ii a test ompound inluces hspothermia this may enhance 1 SV

1(1 ‘nce rnask potenti 1 a’ixiols lie properip u d 1 c drta dcire’et pop tra t mc vO ‘cli ciii e

t di inn.rit

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1000diepam

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600 1

18°C4001

rectal temperature

0 03 1 3

200 1 37°C

35

50

40

30j

20

10-10

negative geotaxs

A

B

800 i0)

0

600cl)

6

(cl

200

0

c 1000

800

0 03 1 3

8OH-DPATultrasonic distress calls

03

ltemperature37

HJ

cclcl)

0cl)(cl

>‘0ccl)ccl

(clcl)

ccl

ciIE

18°C

50•

40

30

20-

(clci

0

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6 0O5 01 02

37°C

0 005 01 02

negative geotaxis

ob ci 02

600

ketanserinultrasonic distress calls

400J 18C/\\\

577C///LNN.

rectal temperature

200

361

34 r

0 03 1 350 ————

negative geotaxis40 *

>30

20

cl)0

0

Dose mq kq p 0 e maq

ILJP) 8 qt

1000 -—D fluvoxamine rectal temperatureultrasonc distress calls

8001

1600-

18C0 03 1 3

T400

40negahve geotaxis

2Oo- 37C 20-1 10

3i3 6iooo—------— -

0

mipramne rectal temperatureultrasonc distress calls

8001 3618C

: 600i 1 *T3400-1 negative geotaxis

20:_37C

Dose (mg!kg Lp.) Oose (mgJkg i.p.)

Figure 5.18.4 (continued) Representative results of the USV and negative geotaxis tests inrat pup, (A) Diazepam, a GABAA benzodiazepine receptor agonist, significantly reduced USVsat the warm plate (LED 1 mg/kg) and the cold plate (LEO 1 mglkg). The negative geotaxislatency was significantly increased at the highest dose tested (LEO 3 mg/kg), In independenttests, the rectal temperature was measured (at room temperature). Diazepam decreased thisparameter (LEO < 1 mg/kg). (8) 8-Hydroxy-2-(2-N-propylamino)tetralin (8-OHDPAT), a 5-HTIAreceptor agonist, signficantly decreased USVs at the warm (LEO 0,1 mg/kg) and the coldplate (LED 0 05 mg kg). The latency of the negatve geotaxis was increased (LEO j 0.1 mglkg)The rectal temperature was decreased (LED 0.3 mg kg) (C) Ketansenn, a 5-HT receptor antagonst, showed enhanced USVs al lower doses and no effect at the hgher dosestested The atency of the negative geotaxs was ncreased (LEO 03 mg kg), rectal tempertrre was decreased LEO 0 3 mq kg) (D) Fluvoxamine, a specihc serotonm reuptake nbbtor decreased USVs al he arm (1 ED 1 mg ka) and the cold plate ([EO 1 mg kgF voxam ne hd not affect the negal ve geot’Ix or he e’ial emperature E) lmpramnet yhc anhdepressant lowered USVs tt ower aoses. bul had no effect al the rnghest doset.sted iltha 1gh he ffects ere cclv obsred c the old plate cnd hcd no effect cl he

cm o cle Rec en pe c re c d cgat tE’ rJ cxi ware n atfec ei ED Nest ef’e e

Time Considerations

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ature on ultra ound production b infant alhinorats. De: Ppo’hobiol. 4:49-1 6.

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Brouette-Lahlou, 1,. Vernet-Mauiy, E.. andVignouroux, M. 1992. Role of pups’ uitrasoniccalls in a particular maternal behasior in Wistarrats: Pups anogenital licking. Behav, Bram Res.50:137-154.

Dastur, F.N.. McGregor. IS., and Brown, R.E. 1999.Dopaminergic modulation of rat nup ultrasonicvocalizations. Eur 1. Pharmacol. 82:53-67.

Elsood, R. 1979. Uitrasounds and maternal behavior in the Mongohan gerbil. Des Psvchobiol.12:281-284.

Fish, E.W., Sekinda. M.. Ferrari. P.F.. Dirks, A..and Miczek. K.A. 2000. Distress ocaIizationsn maternally \eparated mouse piips:Modula110fl ‘via -HT

. 5-HTB andGABA., receptors.Psve’hnpiîorniorologv 149: 27” 2$Ç

1 ish, E.W., Faccidomo. S. Gupta. S.. and Miczek.K A. 2004. Anxiolytic like effects ot Fscitalopram, itaIopram, and R citalopram In maternally separated mouse pups. 1. Pha macol E.qJhcr 08 74-4%0

Hard, E. and Engel. J. 1988. Effects of 8-OH-DPAT 0fl ultrasonic vocalization and audio-genie immobility reaction in preweamng ratsiveurophannacoiogv 27:981-986.

Hard, E., Engel. J.. and Lindh, A.S. 1988, Effect ofclonidine on ultrasonic vocalizatiori in preweaning rats. J. j’veural Franam. 7:217-237.

llodgson, R.A., Guthrie, D.H.. and Varty, G.B.2008. Duration of ultrasonic socalizations inthe isolated rat pup as a hehasioral measure:Sensitiviry to anxiolytic and antidepressantdrugs. Pharmarol. Bio,hem. Behai: $8: 41—34$.

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Kehoe. P and Bolan, CH. 1992. Cocaine-inducedeffects on isolation stress in neonatal rats Behas\!eumsc i. 106’374-i’9

K’hee,P and Boylan.( 13 1994 Behasioralcllccts(kappa- pioid recept r ‘,tirnulation n nwna

al Is Behav 5kuroscz 08418

K hc . P od Harris, J Ç 1 89. Untc gen t esIr r i 11e rs r it,ris ruc “ .,tI

pop thha u fl11099 l0”

Of

I Piip—

A complete study of a single test compound

requires 64 pups. using three doses plus a con

trol group for each condition (cold and warm

plates). Assuming a constant supply of rat pupsfrom the breeding colony. injection requires a

few econds per pup and the negative geotaxis

test may require to 2.5 min (5 x 30 scc/test).

and the USV test 5 min for each pup. rherefore. a complete stud uslng 63 pups typically

takes 10 hr divided over 2 to 3 da’ss. On aer

age. onl\ one expenment can he performed per

week.

0 rdner ( . i o5a Enhibiu n )t Itr sonic disrcss ocalizate os n at pups hy Lhl rdiaicpo

idc and diazcpam D,u De Res 185 9

In Depression. Anxiety and Aggressiorr Preclinical and Clinical Interfaces, IR. Olivier and.1. Mos. eds.) pp 85 94 Medidact, Houten, rhesetherlands.

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\ irot, E. 1966. f ltrasounds in young rodents. 1.Changes ith age in albino mice. nim. Behas14,459 462.

\oirot, E. 1972. f Itrasounds and maternal behasiorm small rodents, Dei. Pschobzol. 5:371.’38,

Okon, E E. 1972. Factors affecung ultrasoundproduction in infant rodents. J Zool. 168:139-148.

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Osa1t, G.L. and Meier. G.W. 1975 Olfauorv,thermal, and tactual inlluenes on intantile oltrasonic vocalization in rats Dev Psshohiol8129 135

Rex, Barth, T. Voigt 1 P Domenev \ M.and Fink 11. 1994 t ttcus of cholecsstokininetrapeptide and sulfated cholecy stokmin c

topeptide in at model of anxiety Nz t 1”2 30 f)

‘s Smtclu ( aca ‘vi r,1a Fnd Ik r g cs Farr t ) to rc s. t

op 1 t ‘nu. i i t n dl s u da’

2/3 agonist in rodents: Detailed investigationswith a selective allosteric mGluRl Antagonist, FTIDC, (4 [1 (2 lluoropyrîdine 3-yl)5-methyl- 1H- 1 ,2,3-triaiol-4-yl-N-isopropy 1-N-methyl 3,6 dihvdropyridine- 112H 1-carboxa-mide). J. Pharmacol. Exp. [her Epub 22 Mayahead of pnnt

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Van der Poel, A.M.. Molewijk, H.E., Mos, 3., andflhivier. 8. 1992, Enhancement of ultrasonic distress vocalizations in infant rats: Effects of directand indirect noradrenergic receptor antagonistsand 5-HT2 receptor antagonists. In DestructiveDrives and Impulse Control, Preclinical Considerations. (B. Olivier and A. Manceaux, eds.)pp. 49-56, Medidact, Houten, The Netherlands.

Voloschin, L.M. and I’ramezzani, TH. 1984, Relationship of prolactin release in lactating ratsto mild ejection. sleep state, and ultrasonic socalization hy the pups. Endocrinolog} 114:618-

Weinstock, M 20(17 Gendcr differenc”s in the effccts of prenatal stress on bram development andhehaviour Veur( chem. Res. 32.1730 F40.

Winslow IT and Insel fR 1990 Serotonergicand catecholaminergic reuptake inhihitors havepposïte ctlects en the ultrasonic ist’lation calls

4 rat pups ‘VCu( ps hopharm u / rn “ 1

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Winslow. Heam, EE. Ferfuson, 1, Young,LL Matzuk, MM., and Insel. TR. 2000. Infantoca1iz ition adult aggression. and fear behasiorof an oxytocin nulI mutant meusc. Horm. Behav,37:145155,

Key ReferencesGardner, l95h. See ahoe.Delonpuon of a simple screeninQ ,flerhcld 10 t?lcO

sure the effecoi of anxiol rio drugs en t SV in ratpUp 5.

Insel and Winsloss. 1991. See ahose.Ra( -ktround data abt rat puri t SV and o,ne ps —

ohopharmaooloy of (.Sl .

()Iiieret al.. 1094. See abo’e.De,scripuon of the p svclwpharinacoloev of varlousdruk’ clasies in the t SV teit n rap pups.