Behavioral Neuroscience1995, Vol. 109, No. 3,404-412

Copyright 1995 by the American Psychological Association, Inc.0735-7044/95/S3.00

8-OH-DPAT Microinjected in the Region of the Dorsal Raphe NucleusBlocks and Reverses the Enhancement of Fear Conditioning and

Interference With Escape Produced by Exposure to Inescapable Shock

Steven F. Maier, Ruth E. Grahn, and Linda R. WatkinsUniversity of Colorado at Boulder

Prior work suggests that inhibition of the dorsal raphe nucleus (DRN) either during exposure toinescapable electric shock (IS) or during later behavioral testing might block the usual behavioralconsequences of IS. The 5-HTiA agonist 8-OH-DPAT was microinjected into the region of theDRN either before exposure to IS or before testing for fear conditioning and escape learningconducted 24 hr later. IS potentiated fear conditioning and interfered with escape performance.These effects were completely prevented by intra-DRN administration of 8-OH-DPAT at eitherpoint. Low but not high systemic doses of 8-OH-DPAT had a similar effect, supporting the ideathat the effective site of action is presynaptic. The relation between these data and other effects of8-OH-DPAT is discussed.

A number of recent experiments suggest that the dorsalraphe nucleus (DRN) may play a critical role in mediating thebehavioral consequences of exposure to inescapable shock(IS). Maier et al. (1993) reported that lesions of the DRNmade before exposure to IS prevented both the subsequentfacilitation of fear conditioning and the interference withescape learning that usually follows IS. DRN lesions had noeffect on these measures by themselves, but blocked the impactof IS on these tasks. On the basis of these and other data,Maier et al. (1993) argued that sequelae of IS such asincreased fear conditioning and poor escape learning might beat least partially produced by excessive DRN activity occurringat the time of testing and that this exaggerated DRN responseto the test situation might occur because the DRN is tempo-rarily sensitized by the prior IS-induced increases in its activity(see Maier, 1993, for a more extended discussion and ratio-nale).

Lesion studies have numerous limitations. In addition,Maier et al. (1993) could not determine whether DRN activityat the time of IS or at the time of behavioral testing 24 hr laterwas critical. A preferable procedure would be to inhibit DRNactivity only before IS or only before later behavioral testing.DRN cells contain GABAA receptors (Harandi et al., 1987;Steinbusch & Nieuwenhuys, 1983) and the DRN is normallyunder GABAergic restraint (Scatton et al., 1986). BecauseGABAergic inhibition is potentiated by benzodiazepine li-

Steven F. Maier, Ruth E. Grahn, and Linda R. Watkins, Depart-ment of Psychology, University of Colorado at Boulder.

This research was supported by National Institute of Mental HealthGrant MH50479 and RSDA MH 00314 to Steven F. Maier. We wouldlike to thank Sarah H. Horwood and Kelly L. Nichols for excellentassistance in conducting the experiments.

Some of the methods used are identical to those employed by Maieret al. (1993), and much of this description is similar or identical to thatin Maier et al. (1993).

Correspondence concerning this article should be addressed toSteven F. Maier, Department of Psychology, Campus Box 345,University of Colorado, Boulder, Colorado 80309-0345.

gands (Costa et al., 1975), Maier, Kalman, and Grahn (1994)microinjected chlordiazepoxide into the region of the DRNeither before IS or before fear conditioning and shuttleboxescape testing 24 hr later. Chlordiazepoxide blocked theeffects of IS when administered at either time point, therebysupporting a role for enhanced DRN activity during both ISexposure and later testing.

In addition to GABAA receptors, the soma and dendrites ofDRN neurons contain receptors of the 5-HT1A class (Sotelo etal., 1991). These receptors are inhibitory "autoreceptors"(Radja et al., 1991), and thus systemic and intra-DRN adminis-tration of agonists for the receptor inhibit DRN electricalactivity (Sprouse & Aghajanian, 1987) as well as 5-HT synthesis/metabolism (Hamon et al., 1988) and release (Sharp, Bram-well, & Grahame-Smith, 1989) in projection regions of theDRN. A presynaptic site for these actions is further confirmedby the fact that 5-HTiA agonists do not inhibit 5-HT releasewhen applied directly to neurons in the projection regions(Sharp & Hjorth, 1990).

The influence of partial and selective 5-HT1A agonists onIS-induced behavioral changes has been explored (Drugan,Crawley, Paul, & Skolnick, 1987; Giral, Martin, Soubrie, &Simon, 1988; Martin, Benninger, Hamon, & Puech, 1990;Martin, Tissier, Adrian, & Puech, 1991). The general effect hasbeen that partial agonists such as buspirone and the selectiveagonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)block or reverse the escape deficit that follows IS. However,the experiments conducted thus far were not designed todetermine whether 8-OH-DPAT would be effective whenadministered before IS only or before testing only. In addition,the site of action is unclear. Most of the experiments employedsystemic drug injection, and there are postsynaptic 5-HT1A

receptors in regions such as hippocampus and septum (Pazos& Palacios, 1985). Indeed, Martin et al. (1990, 1991) havesuggested a postsynaptic site of action for 5-HTIA agonistblockade of IS effects, rather than the DRN somatodendriticautoreceptor. In their experiments intraseptal injection waseffective, but DRN injection was not. In addition, DRN lesions

404

8-OH-DPAT BLOCKS INTERFERENCE WITH ESCAPE 405

did not eliminate the impact of systemic 8-OH-DPAT (Martinet al., 1990,1991).

However, the behavioral procedures used by Martin et al.(1990,1991) are quite different than those used by Maier et al.(1993, 1994) and may produce a different phenomenon medi-ated by different systems. In addition, the drugs were repeat-edly administered between IS and testing, thereby potentiallyallowing for receptor adaptations. Indeed, even a singleadministration of 8-OH-DPAT can desensitize 5-HTiA recep-tors in the DRN if the dose is large (Beer, Kennett, & Curzon,1990; Kennett, Morrow, Dourish, & Curzon, 1987).

The present series of experiments was therefore undertakento explore the impact of 8-OH-DPAT on the enhanced fearconditioning and escape deficits that follow IS using theprocedures of Maier et al. (1993, 1994) and employing singleinjections before either IS or later behavioral testing.

Experiment 1

The purpose of Experiment 1 was to determine the effects of8-OH-DPAT microinjected into the region of the DRN onboth fear conditioning and escape learning after IS. Theprocedures were identical to those employed in our laboratory(Maier, 1990; Maier et al., 1993, 1994). That is, both fearconditioning and shuttlebox escape performance were mea-sured in the same subjects. The standard escape trainingprocedure used in this laboratory involves five escape trials onwhich a single crossing (FR-1) of the shuttlebox is required toterminate shock, followed by trials on which two crossings arerequired (FR-2). The typical result is that prior IS interfereswith the acquisition or performance of FR-2 shuttlebox escaperesponding but has no effect on FR-1 escape (see Maier,Albin, & Testa, 1973, for a rationale). Maier (1990) placedsubjects that had received IS or control treatment 24 hr earlierin a shuttlebox and first assessed freezing for 10 min. Freezinghas been argued to be a measure of fear conditioned to cuesthat are present (Blanchard & Blanchard, 1971; Fanselow &Lester, 1988). The subjects were then exposed to one or two (indifferent experiments) FR-1 shock escape trials and freezingwas then measured for 20 min. After observation of postshockfreezing the rats received the four (or three) remaining FR-1escape trials, followed by FR-2 escape trials.

In the study described above (Maier, 1990), there was nofreezing by the end of the 10-min observation period precedingshock in the shuttlebox. The one or two shocks led to largeamounts of freezing, but this effect was greatly enhanced in theIS subjects. Fanselow (Fanselow & Lester, 1988) has arguedthat freezing always reflects fear conditioned to cues presentduring shock rather than an unconditioned reaction to theshock itself. Thus this measure indicates that there is exagger-ated fear conditioning 24 hr after IS.

Method

Subjects. The subjects were 34 male Holtzman rats (Harlan Labs,Indianapolis, IN). They were 90-100 days old at the time of theexperiment. They were housed individually, maintained on a 12-hrlight-dark cycle, with experimentation occurring during the light partof the cycle. All procedures were in accord with protocols approved by

the University of Colorado Institutional Animal Care and Use Commit-tee.

Apparatus. Inescapable shocks and restraint were administered inPlexiglas tubes, 17.5 cm long and 7.0 cm in diameter. The rat's tailextended from the rear of the tube and was taped to a Plexiglas rod.Shock was administered to the tail through fixed electrodes. Escapetesting and fear measurement were conducted in shuttleboxes measur-ing 46.0 x 20.7 x 20.0 cm (L x W x H). The end walls were aluminumand the side walls and tops were clear Plexiglas. The floor wasconstructed of stainless steel rods, 0.3 cm in diameter and spaced 1.4cm apart. An aluminum wall with a 5.5 x 7.5-cm (W x H) archway cutout of it divided the shuttleboxes into two equal compartments. Theshuttleboxes were housed within sound- and light-attenuating enclo-sures equipped with a ventilation fan and 28-V houselight. The front ofeach enclosure was left open during behavioral observation. Scrambledshocks were delivered to the grid floors by shockers modeled after theGrason-Stadler Model 700. There was no light in the room in whichthe shuttleboxes were located and the observer was not visible fromthe shuttleboxes.

Surgery. Rats were anesthetized with a mixture of 60 mg/kgketamine and 13 mg/kg xylazine. Each rat was implanted with a13-mm, 26-g stainless steel cannula that terminated 1 mm above theactual drug injection site (see below). The patency of the cannulalumen was maintained by a stainless steel stylet (Medwire Corp,316SS8T) inserted into each cannula at the time of surgery. Thecannulas were implanted above the DRN according to the atlas ofPaxinos and Watson (1986). Each cannula was aimed at anterior-posterior +0.7 mm, medial-lateral 0.0 mm, and dorsal-ventral +4.5mm relative to interaural zero. Cannulas were secured to the skull byflowing dental acrylic around the cannula and stainless steel screwsaffixed to the surrounding skull.

Procedure. The experiment was conducted 12-17 days after sur-gery. The rats were divided into 4 groups. Two groups received IS onDay 1 and the other two groups were restrained (R) in the Plexiglastubes for an equivalent period of time. All groups received shuttleboxfear and escape testing 24 hr later (Day 2). One of the IS and one ofthe R groups received microinjections of 1 u,g 8-OH-DPAT (ResearchBiochemicals, Boston, MA) 10 min before initiation of IS or R. Theother IS and R group received vehicle before IS or R testing.8-OH-DPAT or vehicle (deoxygenated 0.9% sodium chloride) wasadministered by a microinjection apparatus consisting of a 10-|xlHamilton glass syringe attached to a 33-gauge microinjection needlevia a length of calibrated PE-20 tubing (fluid movement of 10.25 mmthrough the tubing equalled 1 u.1 delivered volume). The microinjec-tion needle extended 1 mm beyond the internal end of the pre-implanted guide cannula when fully inserted. Microinjections wereperformed over a 2-min period, after gently restraining the subject in asoft towel. A sterile stylet was replaced in the cannula followingcompletion of the injection procedure.

The inescapable shock session consisted of 100 5-s 1.0-mA shocksdelivered on a random-interval 1-min schedule. Shuttlebox testingoccurred 24 hr later. If the animals were the first of the day to betested, an animal not involved in the experiment was first given escapetraining in the shuttlebox so that feces and urine from shocked animalswould be present in the tray below the grid floor. The session beganwith 10 min of behavioral observation by an experimenter unaware ofgroup membership. The procedure was modeled after that used byFanselow and colleagues (e.g., Fanselow & Helmstetter, 1988). Theexperimenter observed each animal 1 s every 8 s in time to a signal andscored it as freezing or not freezing on a computer terminal. In orderto be scored as freezing, all four paws had to be on the grids and therehad to be an absence of all movement of the body and vibrissae beyondthat required for respiration. This is a very distinctive behavior that iseasy to recognize and rarely occurs in nonfear situations. The 10-minobservation period was followed by 2 FR-1 escape trials. Shock

406 S. MAIER, R. GRAHN, AND L. WATKINS

intensity was 0.8 mA and shocks occurred with an average intertrialinterval of 60 s. Shocks terminated automatically after 30 s if escapehad not occurred, and a 30-s latency was assigned. The 2 FR-1 escapetrials were followed by a 20-min observation period, conducted asbefore. The 20-min post-FR-1 observation period was followed by 3more FR-1 escape trials and then 25 FR-2 escape trials. The observerwas unaware of group membership in all cases.

Histological verification. Two to three days following behavioraltesting, rats were anesthetized with 55 mg/kg sodium pentobarbital.To verify the site of experimental drug injection, the same type ofmicroinjector was used to deliver 1 u.1 saturated Evans Blue vital dye 1mm beyond the internal extent of the pre-implanted guide cannula.The rats were then transcardially perfused with heparinized saline.Following fixation of the brain in the skull with 10% formalin, 30%sucrose solution for at least 3 days, the brains were removed andcryostat sectioned (40 u,m) at -20 °C. Sections mounted on gelatin-treated slides were stained with cresyl violet and cover-slipped to allowaccuracy of cannula placement to be assessed using light microscopy.Data from animals not found to have microinjection sites within thecaudal DRN were excluded from analyses.

Statistical analyses. Data were analyzed by repeated measuresanalysis of variance (ANOVA) followed by Newman-Keuls analyses(alpha set at .05), which make all possible pairwise comparisons.

Results

Data from one subject was lost. There was too little freezingbefore the presentation of shock in the shuttlebox in this andthe following experiments to allow meaningful analysis. Freez-ing occurring after the first two shocks in the shuttlebox ispresented in Figure 1. As in prior studies the two shocks led tosubstantial freezing, which extinguished over the 20-min obser-vation period. Also consistent with previous work, animals thathad received IS 24 hr earlier showed enhanced levels offreezing compared with controls. Microinjection of 8-OH-DPAT 24 hr earlier had no effect by itself, but it completelyblocked the enhancement of freezing produced by IS whenadministered before IS. The effects of 8-OH-DPAT, F(l, 29) =

iNIB

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2 4 6 8

2 MEM BLOCKS

Figure 1. Mean number of observation periods for which freezing wasobserved after two shocks in the shuttlebox, across blocks of 2 min.Subjects had received either restraint (R) or inescapable shock (IS) 24hr earlier, and either 8-OH-DPAT (DPAT) into the region of thedorsal raphe nucleus or saline (SAL) before IS or R.

30-T

~ 25-

£& 15-

IS-DPAT

R-DPAT

IS-SAL

R-SAL

BS AS 1 2 3 4 5

BLOCKS OF 5 TRIALS

Figure 2. Mean shuttlebox escape latencies for two FR-1 trialspreceding the observation period (BS), the three FR-1 trials after theobservation period (AS), and five trial blocks of FR-2 trials. Subjectshad received either restraint (R) or inescapable shock (IS) 24 hrearlier, and either 8-OH-DPAT (DPAT) into the region of the dorsalraphe nucleus or saline (SAL) before IS or R.

11.77,;? < .002, IS,F(1, 29) = 5.08,;? < .04, the interactionbetween 8-OH-DPAT and IS, F(l, 29) = 4.47,^ < .05, trialblocks, F(9, 261) = 157.60,/> < .0001, the interaction between8-OH-DPAT and trials, F(9, 261) = 2.89, p < .003, and theinteraction between IS, 8-OH-DPAT, and trials, F(9, 261) =2.20, p < .03, were all reliable. Subsequent Newman-Keulstests (p < .05) revealed that the IS-saline group differed fromall of the others, which did not differ among themselves.

Shuttlebox performance is shown in Figure 2. Latencies areplotted separately for the 2 FR-1 trials before the freezingobservation period, the 3 FR-1 trials after the observationperiod, and the 25 FR-2 trials in blocks of 5 trials. There wereno differences in FR-1 latencies before the observation period.Thus the differences between groups in freezing cannot beattributed to differential shock durations on FR-1 trials.Latencies on the remaining 3 FR-1 trials were elevated in theIS-saline group, although this effect was not significant, F(l,29) = 2.34, p = .14, and is not typically observed. FR-2latencies were elevated in the IS-saline group, confirming theusual interfering effects of prior IS. Because trials terminateautomatically after 30 s, the data indicate that almost all thesubjects in this group failed to escape on almost all trials.8-OH-DPAT administered 24 hr earlier appeared to interferewith performance in the R group, but it nevertheless blockedthe interference produced by IS when given before IS. Theeffects of IS, F(l, 29) = 8.4,p < .01, 8-OH-DPAT, F(l, 29) =5.14, p < .05, the interaction between IS and 8-OH-DPAT,F(l, 29) = 26.08,p < .0001, and the interaction between trialblocks and 8-OH-DPAT, F(4, 116) = 2.50, p < .06, were allreliable. Newman-Keuls post hoc tests indicated that theIS-saline group differed from all of the others, which did notdiffer among themselves.

Experiment 2

Experiment 1 indicates that 8-OH-DPAT microinjected inthe region of the DRN before IS completely eliminates both

8-OH-DPAT BLOCKS INTERFERENCE WITH ESCAPE 407

o

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4 6 8

2 MIN BLOCKS

10

Figure 3. Mean number of observation periods for which freezing wasobserved after two shocks in the shuttlebox, across blocks of 2 min.Subjects had received either restraint (R) or inescapable shock (IS) 24hr earlier, and either 8-OH-DPAT (DPAT) into the region of thedorsal raphe nucleus or saline (SAL) before shuttlebox testing.

the enhancement of fear conditioning and the interferencewith FR-2 escape behavior that occurs 24 hr later. The purposeof Experiment 2 was to determine the impact of 8-OH-DPATmicroinjected in the region of the DRN before behavioraltesting rather than before IS.

Method

All aspects of the design and procedure were identical to those inExperiment 1. The only difference was that 8-OH-DPAT or salinevehicle was microinjected 5-10 min before shuttlebox testing ratherthan before IS or restraint. There were 35 subjects distributed betweenthe four groups.

Results

The amount of freezing observed after the two FR-1 shocksis displayed in Figure 3. IS again potentiated freezing relativeto controls and 8-OH-DPAT had no effect on freezing by itself.However, 8-OH-DPAT administered before testing substan-tially reduced the enhancement of freezing in IS subjects. Theeffects of IS, F(l, 30) = 23.01, p < .0001, 8-OH-DPAT, F(l,30) = 7.41,p < .01, trial blocks, F(9, 270) = 129.48,;? < .0001,and the interaction between IS and trial blocks, F(9, 270) =3.80, p < .0002, were reliable. Newman-Keuls post hoccomparisons indicated that the IS-saline group differed fromall of the others, which did not differ among themselves.

Shuttlebox latencies are shown in Figure 4. There were nogroup differences in FR-1 escape latencies either before orafter the observation period. IS interfered with FR-2 escapebehavior, and this effect was blocked by 8-OH-DPAT. Theeffects of IS, F(\, 30) = 10.35, p < .003, the interactionbetween IS and 8-OH-DPAT, F(\, 30) = 5.69, p < .03, trialblocks, F(4,120) = 8.13, p < .0001, the interaction between ISand trials, F(4, 120) = 3.26, p < .02, and the interactionbetween IS, 8-OH-DPAT, and trials, F(4,120) = 2.82,p < .03,were significant. Newman-Keuls comparisons indicate that the

IS-saline group differs from all of the others, which do notdiffer among themselves.

Experiment 3

The 8-OH-DPAT microinjected in the region of the DRNblocked the effects of IS measured here, whether it wasadministered before IS or before later behavioral testing. Thisblockade presumably occurred because 8-OH-DPAT activatedinhibitory 5-HT1A somatodendritic receptors in the DRN,thereby reducing serotonergic neurotransmission. It is there-fore interesting to consider what the effects of systemicallyadministered 8-OH-DPAT in the present paradigm might be.As noted previously, 5-HTiA receptors occur postsynapticallyin a number of regions, as well as in the DRN. Interestingly,somatodendritic DRN 5-HT1A receptors are far more sensitiveto 8-OH-DPAT than are postsynaptic 5-HT1A receptors suchas those in hippocampus (Sprouse & Aghajanian, 1988). Thuslow systemic doses of 8-OH-DPAT selectively activate DRN5-HTJA receptors, with postsynaptic action increasing as doseincreases (Sharp, 1992). The net effect is that low systemicdoses inhibit serotonergic neurotransmission, and as doseincreases the net effect switches to an increase in serotonergicactivity (Sharp, 1992). These considerations suggest that sys-temic 8-OH-DPAT might be expected to have an inverteddose-response relationship with regard to blockade of ISeffects. Low doses that activate only somatodendritic autore-ceptors should block IS-induced behavioral changes, whereashigher doses that also activate postsynaptic sites might not.Experiment 3 was designed to assess this possibility.

Method

Subjects. The subjects were 64 rats as in the previous experiments.Procedure. On Day 1 four groups received IS as above, and four

were restrained. Shuttlebox testing proceeded 24 hr later as inExperiments 1 and 2. One of the IS and one of the restrained groups

30-i

25 -

aJ2.g

i 5-

BS AS 1 2 3 4 5

BLOCKS OF 5 TRIALS

Figure 4. Mean shuttlebox escape latencies for two FR-1 trialspreceding the observation period (BS), the three FR-1 trials after theobservation period (AS), and five trial blocks of FR-2 trials. Subjectshad received either restraint (R) or inescapable shock (IS) 24 hrearlier, and either 8-OH-DPAT (DPAT) into the region of the dorsalraphe nucleus or saline (SAL) before shuttlebox testing.

408 S. MAIER, R. GRAHN, AND L. WATKINS

received an intraperitoneal injection of saline vehicle 5-10 min beforeshuttlebox testing, one of each received 0.001 mg/kg 8-OH-DPAT, oneof each received 0.01 mg/kg, and one of each was given 0.10 mg/kg.Thus the design was a 2 (IS versus restraint) x 4 (0, 0.001, 0.01, 0.10mg/kg) factorial with 8 rats per group.

Results

IS once again potentiated postshock freezing (Figure 5).None of the doses of 8-OH-DPAT had a direct effect onfreezing and all blocked the enhancement of freezing pro-duced by IS. A repeated measures ANOVA revealed that theeffects of IS, F(l, 56) = 8.28, p < .006, 8-OH-DPAT,F(l, 56) = 6.62,p < .008, trial blocks, F(9, 504) = 302.43,p <.0001, and the interaction between 8-OH-DPAT and trialblocks, F(27, 504) = 2.16, p < .0008, were significant.Newman-Keuls post hoc comparisons (p < .05) indicate thatthe IS-saline group differs from all of the others, which do notdiffer among themselves.

Shuttlebox performance is depicted in Figure 6. The data forone subject was lost. There were no group differences in FR-1escape latencies, either before or after the observation period.IS interfered with FR-2 escape behavior, and this was com-pletely blocked by the 0.001-mg/kg dose, partially blocked bythe 0.01-mg/kg dose, and unaffected by the 0.10-mg/kg dose.The 0.10-mg/kg dose itself interfered with FR-2 shuttlebehavior. The effects of IS, F(l, 55) = 8.07, p < .007,8-OH-DPAT, F(3, 56) = 7.88, p < .0002, and the interactionbetween 8-OH-DPAT and trial blocks, F(12, 220) = 2.77, p <.002, were significant. Newman-Keuls post hoc comparisonsindicated that of all possible pairwise comparisons the IS-saline and IS-O.lO-mg/kg groups differed from the restrained-0.001, restrained-0.01, and IS-0.001 groups. In addition, therestrained-. 10 and IS-.01 groups differed from the restrained-0.001 and IS-0.10 groups. No other differences were signifi-cant.

(3

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2 4 6 8

2 MIN BLOCKS

10

Figure 5. Mean number of observation periods for which freezing wasobserved after two shocks in the shuttlebox, across blocks of 2 min.Subjects had received either restraint (R) or inescapable shock (IS) 24hr earlier, and either saline (SAL) or 0.001, 0.01, or 0.10 mg/kgsystemically administered 8-OH-DPAT before shuttlebox testing.

BS AS

BLOCKS OF 5 TRIALS

Figure 6. Mean shuttlebox escape latencies for two FR-1 trialspreceding the observation period (BS), the three FR-1 trials after theobservation period (AS), and five trial blocks of FR-2 trials. Subjectshad received either restraint (R) or inescapable shock (IS) 24 hrearlier, and either saline (SAL) or 0.001, 0.01, or 0.10 mg/kgsystemically administered 8-OH-DPAT before shuttlebox testing.

In sum, all of the systemic 8-OH-DPAT doses blocked thepotentiation of freezing produced by IS, but the propensity ofsystemic 8-OH-DPAT to block the interference with FR-2escape behavior was an inverse function of dosage. The lowdose of 8-OH-DPAT had no effect by itself in R subjects, butblocked the interference with FR-2 escape produced by IS.The medium dose had a small but nonsignificant effect in Rsubjects, but also blocked the IS effect. The high dose of8-OH-DPAT itself interfered with FR-2 shuttle escape, and soits lack of effect in IS animals is difficult to interpret.

General Discussion

Microinjection of the selective 5-HTtA agonist 8-OH-DPATin the region of the DRN blocked both the potentiation of fearconditioning and the FR-2 escape deficit that follows exposureto IS. This effect occurred whether 8-OH-DPAT was adminis-tered before IS or before later testing. 8-OH-DPAT is verylikely to have produced blockade of these behavioral effects ofIS by activating inhibitory somatodendritic 5-HTiA autorecep-tors in the DRN. 8-OH-DPAT is highly selective for the5-HTiA receptor (Hjorth et al., 1982; Middlemiss & Fozard,1983), and there are few if any 5-HT1A receptors in the generalregion of the DRN other than those on the soma and dendritesof DRN neurons (Palacios & Dietl, 1988).

The dose-response relationship that was found to occurfollowing systemic administration of 8-OH-DPAT constitutesfurther support for the blockade of IS-induced effects beingmediated at a presynaptic site of action at the DRN. Somato-dendritic DRN 5-HT1A receptors are more sensitive to ago-nists than are postsynaptic 5-HT1A receptors (Sprouse &Aghajanian, 1987). A systemic administration of the low doseused in the present study (0.001 mg/kg) is sufficient to activateDRN receptors as indexed by inhibition of 5-HT releasemeasured in a projection region of the DRN (Sharp, Bram-well, Hjorth, & Grahame-Smith, 1989), but is not sufficient to

8-OH-DPAT BLOCKS INTERFERENCE WITH ESCAPE 409

activate postsynpatic 5-HT1A receptors (Dourish, Hutson, &Curzon, 1985). The middle dose (0.01 mg/kg) is ambiguous,and the high dose (0.10 mg/kg) is clearly sufficient to activatepostsynaptic receptors. The low dose blocked both the IS-induced potentiation of freezing and shuttle deficit. Further-more, the high dose did not block the shuttle deficit and itselfinterfered with FR-2 escape. This pattern can most easily beexplained by positing a presynaptic site for the 8-OH-DPATblockade of the IS-induced behavioral changes. It also suggeststhat postsynaptic 5-HT]A receptor stimulation interferes withFR-2 shuttle escape behavior. Indeed, there is data to supportthe proposition that stimulation of postsynpatic 5-HT1A recep-tors located in the dorsal periaqueductal gray interferess withescape behavior (Beckett, Lawrence, Marsden, & Marshall,1992; Graeff, Silveira, Nogueria, Audi, & Oliveira, 1993). Thiswould explain why the 0.10-mg/kg dose interfered with FR-2escape in R subjects—it was sufficient to activate postsynaptic5-HTiA receptors in regions such as the dorsal periaqueductalgray that themselves interfere with escape.

These results and conclusions are at odds with those ofMartin and his colleagues, who found that the effects of8-OH-DPAT on IS-induced shuttle deficits were not reducedby DRN lesions and that intraseptal, but not intra-DRNmicroinjection, was effective in eliminating interference withshuttle escape (Martin et al., 1990, 1991). However, theprocedures used in these studies were quite different fromthose used here. Rats were first exposed to IS using scrambledgridshocks of 15-s duration, rather than fixed electrode shockas employed in our experiments. Shuttlebox testing began 2days later and occurred on each of 3 successive days. Theshuttlebox shocks terminated after 3 s if an escape response(FR-1) had not occurred, and the measure recorded wasescape failures. The 8-OH-DPAT was injected 6 hr after ISand then twice a day thereafter, with each shuttlebox sessionbeing preceded by an injection by 30 min.

One obvious difference between the present study and thoseof Martin and colleagues (1990, 1991) is the dosing pattern of8-OH-DPAT employed to prevent the effects of IS. Physiologi-cal responses to 8-OH-DPAT vary dramatically depending ondose and schedule. Single systemic doses have been shown toproduce hyperphagia (Bendotti & Samanin, 1986; Dourish etal., 1985), induce stereotypy and forepaw treading (Dourish etal., 1985), reduce synthesis of 5-HT in forebrain structures(Hjorth & Magnusson, 1988), and decrease 5-HT levels inhippocampus (Sharp, Bramwell, Hjorth, & Grahame-Smith,1989).

Repeated administration of 8-OH-DPAT is not as wellcharacterized. The effects of one administration of 8-OH-DPAT have been shown to alter the effects of a secondadministration 24 hr later (Beer et al., 1990; Kennett et al.,1987). The second administration appears to be less effectivein inducing hyperphagia (Kennett et al., 1987) and binding of8-OH-DPAT to the 5-HT1A receptor in the raphe is reduced(Beer et al., 1990). Larsson and colleagues (Larsson, Renyi,Ross, Svensson, & Angeby-Moller, 1990) examined the effectsof repeated 8-OH-DPAT administration on several behaviorsand biochemical indices. Repeated 8-OH-DPAT did not alterpresynaptic 5-HT1A receptor-mediated indices such as 5-HTaccumulation and reduced synthesis in forebrain structures,

whereas postsynaptic receptor-mediated events such as hypo-thermia and the cage-leaving response were reduced bychronic administration of 8-OH-DPAT.

Martin and colleagues have proposed that the activityexerted by their repeated administration of 8-OH-DPAT waspostsynaptically mediated, because DRN manipulations hadno effect (Martin et al., 1990). Because this observation isconcordant with Larsson and colleagues' findings, it wouldappear that the behavioral phenomenon studied by Martin isindeed different from the phenomenon reported in the presentstudies that has been shown to be sensitive to DRN manipula-tions (Maier et al., 1993, 1994). The systemic doses of8-OH-DPAT effective in the present experiments were muchsmaller than those found to be effective in the Martinexperiments. Low doses of 8-OH-DPAT (0.015-0.060 mg/kg)have been suggested to act preferentially at somatodendriticautoreceptors, whereas larger doses (0.125—4.00 mg/kg) in-duce postsynaptic effects such as the components of theserotonin syndrome (Dourish et al., 1985). The dose used inthe Martin experiments (1 mg/kg) corresponds to postsynapticactivation, and the dose found effective in blocking the effectsof inescapable shock in the present experiments (0.001 and0.01 mg/kg) probably acted preferentially at somatodendriticautoreceptors in the DRN.

Changes associated with repeated administration of 8-OH-DPAT and the different effects of large versus small doses of8-OH-DPAT suggest that the phenomena studied by Martinand colleagues is postsynaptic receptor mediated, whereas thatof Maier and colleagues is a presynaptic effect. It is onlypossible to speculate concerning the differences in procedurebetween the laboratories that might be critical to the produc-tion of fundamentally different phenomena. A likely possibilityis the different nature of the stressors employed—inescapablegridshock delivered to freely moving animals and fixed elec-trode shock administered to restrained subjects. This factordeserves further study and direct comparison.

The effectiveness of intra-DRN injection of 8-OH-DPATreported in the present studies is consistent with a number ofother findings. Maier (1993) and Maier et al. (1993,1994) haveargued that uncontrollable aversive events induce intense"anxiety" and that the continued presence of this anxietyduring later behavioral testing is a critical mediator of thebehavioral sequelae of IS. Both behavioral and pharmacologi-cal treatments designed to reduce anxiety do block and reverselearned helplessness effects (Drugan, Ryan, Minor, & Maier,1984; Maier et al., 1994), and IS but not equal amounts ofescapable shock produce anxiety that persists for 48-72 hr asmeasured by the social interaction test (Short & Maier, 1993).Although the literature is not without contradictions, thegeneral pattern is that a single intra-DRN administration of8-OH-DPAT reduces anxiety (De Vry, Glaser, Schuurman,Schreiber, & Traber, 1991), just as it reduced the behavioralproducts of IS measured here. For example, 8-OH-DPATinjected in the region of the DRN has been reported todecrease anxiety as measured by social interaction and toincrease punished responding in a conflict test (Higgins, Jones,& Oakley, 1992). As in Experiment 3 here, the effects ofsystemic 8-OH-DPAT on anxiety measures are conflicting. Ithas been suggested that this may be because the behavioral

410 S. MAIER, R. GRAHN, AND L. WATKINS

effects of 8-OH-DPAT acting at the DRN and median raphenucleus are often opposed to each other and because postsyn-paptic effects can counter the impact of 8-OH-DPAT at theDRN (Soubrie, 1989). As found here with regard to learnedhelplessness effects, the dose-response relationship betweensystemic 8-OH-DPAT and anxiolysis is often U shaped (DeVry et al., 1991). In addition, DRN lesions reduce anxiety asmeasured by social interaction (File, Hyde, & Macleod, 1979),and they block learned helplessness effects produced by fixedelectrode IS (Maier et al., 1993). To the extent that anxiety andlearned helplessness are intimately related, the literatureconcerning the DRN and 8-OH-DPAT would thus appear tobe consistent with the present findings. Finally, learned help-lessness has also been related to depression, and a singleinfusion of 8-OH-DPAT into the region of the DRN reducesdepression as measured in the forced swimming test (Cervo,Grignaschi, & Samanin, 1988).

It is possible that intra-DRN administration of 8-OH-DPATblocked and reversed the effects of IS on fear conditioning andFR-2 escape because it modulated some other process thatalters freezing and shuttle responding. General activity andpain sensitivity/reactivity are obvious possibilities. Perhaps8-OH-DPAT in the DRN increases activity or reduces thenoxiousness of electric shock, thereby reducing freezing aftershock and increasing shuttle escape performance. However,DRN manipulations have very little effect on general activity.Neither DRN lesions (Jacobs & Cohen, 1976) nor intra-DRNinjection of 8-OH-DPAT (Cervo et al., 1988) have an effect onopen field activity. In addition, neither manipulation altersshuttle escape latencies in control subjects. DRN manipula-tions do interact with pain processes. However, stimulationrather than inhibition of the DRN reduces pain reactivity ornoxiousness (Oliveras, Guilbaud, & Besson, 1979), and reduc-tions in DRN activity would be expected to interfere with anyIS-induced analgesia rather than potentiating it.

As already discussed, the data reported here are consistentwith the idea that activation of the DRN by IS is important inthe cascade of events that leads to potentiation of fearconditioning and escape failures observed 24 hr later and alsosuggest an important role for increased DRN activity at thetime of testing, perhaps in response to the shocks used intesting. We have elsewhere (Maier, 1993; Maier et al., 1993,1994) suggested ways in which increased 5-HT release in DRNprojection regions at the time of testing might mediate thebehavioral effects of IS and mechanisms by which DRNactivation at the time of IS might sensitize the DRN to behyperreactive during later testing. This discussion need not berepeated here. However, it should be noted that the dataconcerning the role of 5-HT in mediating learned helplessnesseffects is as inconsistent as the data concerning 8-OH-DPATand the DRN. There are experiments that suggest thatincreased 5-HT is critical (Brown, Rosellini, Samuels, & Riley,1982; Edwards, Johnson, Anderson, Turano, & Henn, 1986),that decreased 5-HT activity is critical (Petty & Sherman, 1983;Sherman & Petty, 1980), and that 5-HT is not involved(Anisman, Irwin, & Sklar, 1979; Hamilton, Zacharko, &Anisman, 1986). Unfortunately, behavioral procedures andparameters differ among these studies as radically as they dobetween the present experiments and those of Martin and his

colleagues. Different stressor conditions may well producedifferent phenomena with different causes. It will requiresystematic behavioral studies to isolate the conditions thatdetermine when and how 5-HT plays a key role.

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Received August 31, 1994Revision received October 17,1994

Accepted December 7,1994

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