5,7-Dihydroxytryptamine lesions in the fornix—fimbria attenuate latent inhibition

BEHAVIORALAND NEURALBIOLOGY59, 194--207 (1993)

5,7-Dihydroxytryptamine Lesions in the Fornix-Fimbria Attenuate Latent Inhibition HELEN J. CASSADAY,1 STEPHEN N. MITCHELL, JONATHAN H. WILLIAMS,1 AND JEFFREY A. GRAY2 Department of Psychology, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, England

Lubow, 1989, for review). Disruption of LI has been proposed as a model of the attentional disorder found in acute schizophrenia (Gray, Feldon, Rawlins, Hemsley, & Smith, 1991a; Schmajuk, 1987; Solomon, Crider, Winkelman, Turi, Kamer, & Kaplan, 1981; Weiner, 1990). LI is abolished in both animals (Solomon et al., 1981; Weiner, Lubow, & Feldon, 1981, 1984, 1988) and h u m a n subjects (Gray, Hemsley, Feldon, Gray, & Rawlins, 1991b; Gray, Pickering, Hemsley, Dawling, & Gray, 1992) treated with the psychotogenic (Connell, 1958) indirect dopamine agonist, amphetamine, and in acute schizophrenics (Baruch, Hemsley, & Gray, 1988; Gray et al., 1991b). The present experiment investigated the role of hippocampal 5-hydroxytryptamine (5HT) systems in LI in the rat. Solomon and co-workers (Solomon, Kiney, & Scott, 1978; Solomon, Nichols, Kieruan, Kamer, & Kaplan, 1980) have shown that LI is attenuated by central 5HT depletion. The role of 5HT systems in LI may be related to schizophrenic attention disorder via the LSD (lysergic acid diethylamide) model of schizophrenia (Claridge, 1978) since one action of LSD is to decrease 5HT turnover (Rosecrans, Lovell, & Freedman, 1967) by an action at the autoreceptor (Aghajanian, Foote, & Sheard, 1968). There is much evidence that dopaminergic and serotonergic systems tend to be functionally opposed to one other, so that an increase in the activity of one has equivalent effects to a decrease in the activity of the other (Barnes, Costall, & Naylor, 1987; Costall, Hui, & Naylor, 1979; Trulson & Jacobs, 1979). J u s t such an opposition has already been suggested for LI by experiments in Solomon's laboratory. These have shown that LI is abolished both by amphetamine (Solomon et al., 1981) and by electrolytic lesions of the medial raphe nucleus, origin of a major part of the serotonergic innervation of the forebrain (Solomon et al., 1980). In addition,

When animals are preexposed to a stimulus without consequence they are subsequently slower to associate this stimulus with an important event, such as footshock. This retarding effect of stimulus preexposure is called latent inhibition and can be demonstrated in a variety of classical and instrumental paradigms and in a wide range of species, including man. Latent inhibition is disrupted in acute schizophrenics and by amphetamine treatment in both rat and man. The present study investigated the role of hippocampal 5HT terminals in latent inhibition using a conditioned suppression procedure with male Sprague-Dawley rats. Microinjections of 5,7-dihydroxytryptamine in the fornix-fimbria significantly reduced hippocampal indoleamine levels and attenuated latent inhibition of conditioned suppression. This finding supports the hypothesis that the destruction of mesolimbic 5-hydroxytryptamine terminals reduces latent inhibition. This result is discussed in terms of the possible involvement of reduced serotonergic function in schizophrenic attentional disorder. In addition to the predicted lesion effect, biochemical analyses indicated that experimental treatments in the latent inhibition procedure altered neurotransmitter turnover: utilization ratios for 5-hydroxytryptamine and/or dopamine were increased in preexposed relative to nonpreexposed animals in four of the six brain regions sampled. ©1993AcademicPress, Inc. INTRODUCTION Latent inhibition (LI) consists in a retardation of learning if the stimulus to be conditioned (CS) has previously been presented without consequence (see 1 Present address: Department of Experimental Psychology, University of Oxford, Oxford OXl 3UD, England. Fax: 0865 310447. 2 H.J.C. held a Science and Engineering Research Council Studentship. We gratefully acknowledge support from Squibb Pharmaceutical Co. We thank Dr. H. Hodges, Dr. J. Feldon, Dr. M. Snape, and Tim King for advice and help in various stages of the research. Address correspondence and reprint requests to Helen J. Cassaday at present address. 194 0163-1047/93 $5.00 Copyright © 1993 by AcademicPress, Inc. All rights of reproduction in any form reserved.

5,7-DHT LESIONS ATTENUATELATENTINHIBITION it has been demonstrated that the abolition of LI produced by 5,7-dihydroxytryptamine (5,7-DHT) lesions of the medial raphe is reversible by haloperidol, providing further evidence for the reciprocal interaction of the dopaminergic and serotonergic systems (Loskutova, Luk'yanenko, & II'yuchenok, 1990). Recent pharmacological experiments show that LI may be abolished by compounds that affect 5HT transmission (Cassaday, Hodges, & Gray, 1991). Agents active at 5HT binding sites provide tools to reduce 5HT neuronal firing rate (5HTla) and release at the terminal (5HTlb) and to block postsynaptic 5HT2 receptors (Brazell, Marsden, Nisbet, & Routledge, 1985; Leysen, Gommeren, Van Gompel, Wynants, Janssen, & Laduron, 1985). The present study reports an attempt to identify specific 5HT pathways mediating the effects of medial raphe lesions and serotonergic compounds on LI. Solomon et al. (1980) found that electrolytic lesions of the dorsal and medial raphe differentially affected LI. Medial but not dorsal raphe lesions disrupted LI of two-way active avoidance using a tone CS. Biochemical analysis showed that medial raphe lesions significantly reduced 5HT levels in the septohippocampal system while dorsal raphe lesions did not. This suggests that the 5HT projection to limbic regions is involved in LI. If so, direct disruption of the 5HT projection to the hippocampus should also attenuate LI; the present experiment tested this hypothesis using a 5,7-DHT lesion to the fornixfimbria. The hippocampus has been hypothesized to be involved in attentional mechanisms (Schmajuk, 1984, 1987) and hippocampal lesions have been consistently reported to abolish LI (Weiner, 1990). However, most of the existing literature concerns the effects ofnonspecific lesions to the septohippocampal system. In such cases, damage to fibers of passage might equally account for those deficits observed. ]:n contrast to this view, it has been found that simply placing an electrode in the dorsal hippocampus is sufficient to disrupt LI (DeVietti, Emmerson, & Wittman, 1982). The attenuation produced by the mere placement of an electrode (no stimulating current was passed) suggests that widespread damage to fibers of passage is not necessary to show effects on LI subsequent to hippocampal lesion. This suggests t h a t LI is very sensitive to interventions affecting dorsal hippocampus. The present study used microinjections of 5,7DHT in the fornix-fimbria to deplete 5HT in dorsal hippocampus (Williams & Azmitia, 1981), without damaging 5HT terminals in the septum or norad-

195

renergic (NA) terminals in the hippocampus (McNaughton, Azmitia, Williams, Buchan, & Gray, 1980; Williams & Azmitia, 1981). Thus, we sought to assess the effects on LI of 5HT depletion produced by selective neurotoxic lesion, at a site where effects on LI after mechanical damage are well documented. The effects of these 5,7-DHT lesions on LI were studied using a conditioned suppression of drinking procedure adopted from Weiner et al. (1984). We compared the disruption of drinking after the presentation of a tone (CS) associated with shock (UCS) in groups preexposed (PE) or not preexposed (NPE) to the tone. Normal animals with previous experience of the tone without consequence (preexposed) show reduced learning relative to controls, which do not have such preexposure. The 5,7DHT lesion was predicted to abolish the effects of stimulus preexposure and thus result in relatively better associative learning (reflected in increased suppression) in the PE group. Learning was measured both as a suppression ratio, which reflects the animal's immediate response to the CS and is highly sensitive to performance differences in the first 60-s stimulus onset, and as the total licks during presentation of the CS, which reflect the overall response to the tone. The NPE groups provided a control for other possible lesion effects, not specifically related to the hypothesis under investigation, on the conditioned suppression measure of learning. MATERIALS AND METHODS

Animals Forty-nine male Sprague-Dawley rats were used (Bantin and Kingman, UK). The rats were caged in pairs, both of the same experimental condition. The cage dimensions were 40 x 28 x 20 cm. They were maintained under a 10/14 h light/dark cycle (lights on 9:00 AM), during which food and water were available ad libitum prior to water deprivation. Water deprivation was introduced over the second week after arrival. Prior to surgery, the rats were given 6 days of pretraining to establish stable baseline drink rates and then placed on ad lib water once more. The animals weighed 225-360 g at surgery and 300-450 g at behavioral testing.

Drugs The 5,7-DHT (Sigma) was injected as 5 t~g free base in 0.4 ill saline containing 0.2 m g / m l ascorbic acid. Desipramine (Sigma) was dissolved in saline and administered to all animals 20 min prior to anesthesia (10 m g / k g ip) to protect noradrenergic

196

CASSADAYET AL.

neurons (Azmitia, Buchan, & Williams, 1978; McNaughton et al., 1980).

Surgery Surgery was performed using a Kopf stereotaxic instrument with 3 ml/kg ip. "Equithesin" anesthesia (sodium pentobarbital 9.7 mg/ml, chloral hydrate 42 mg/ml, MgSO4, ethanol, and propylene glycol). Pilot studies had been carried out using the in vivo dye trypan blue in 16 animals to determine the optimal injection coordinates for different weight ranges (see Williams & Azmitia, 1981). Injections were made 1.1 mm lateral, 4.9-5.0 mm below the surface of the skull, and 1.1-1.3 mm posterior to bregma. All coordinates were measured with the stereotaxic arm positioned at 15° to the median plane. Depth was adjusted to 4.9 mm for animals with a lambda-bregma distance less than 6.2 ram. Distance from bregma was adjusted to 1.1 mm for animals with a lambda-bregma distance less than 6.5 mm, and to 1.3 mm for animals with a lambda-bregma distance greater than 7.3 mm. The range of lambda-bregma distances in the present experiment was 6.0-8.7 mm. A 3-mm2 flap of skull was removed to allow a cannula (30 gauge) to be lowered into the brain unilaterally at a lateral angle of 15 ° to the median plane to give a single midline injection at the coordinates described above. Five micrograms 5,7DHT (free base) was injected in 0.4/~1 vehicle over 8 min. The cannula was left in place for 2 min after the infusion. Control animals were treated identically except that they received microinjections of ascorbate-saline vehicle only. While under anesthetic, animals were given 2.5 ml physiological glucose-saline (sc). Four animals died after surgery. The remaining 45 rats were randomly allocated to the two behavioral treatments (PE or NPE), giving rise to four groups: Vehicle-PE (n = 11), Vehicle-NPE (n = 11), DHT-PE (n = 12), and DHT-NPE (n = 11).

Apparatus The apparatus consisted of three experimental chambers set in sound-attenuating boxes with ventilation fans. Two of the chambers were made of metal with internal dimensions 24.5 x 21.5 x 20.0 cm. One chamber was wooden with internal dimensions 22.5 x 24.0 x 15.0 cm. Each animal was tested in only one chamber and experimental conditions were counterbalanced as far as possible across boxes. Each of the chambers was illuminated

by a houselight in the roof and positioned over a tray of sawdust. Drinking bottles (Oasis Pet Waterers) were inserted into the chambers through holes of diameter 3.0 cm, positioned 1.5 cm above the grid floor. Surrounding metal plates of diameter 1.5 cm, again 1.5 cm above the grid floor, provided a second pole to complete a circuit when the animals contacted the water spout. The pulses generated by the intermittent contact of licking were registered by a drinkometer (Campden Instruments, Model 453). When water was not available, a plastic door covered the holes. The preexposed to-be-conditioned stimulus was a 3.0-kHz tone (sound level 90 dB, including background) of 15.0 s duration. The tone was produced by an audiogenerator (Campden Instruments, Model 258), via loudspeakers attached to the roof of each chamber. Three constant current generators with inbuilt scramblers (AIM Biosciences, Cambridge) were calibrated to produce a 0.37-mA shock (alternating current) via the grid floors. The grids had bar spacing of 1.0 and 1.5 cm for the metal and wooden chambers, respectively. Shock levels were measured as mean current delivered at the bars with a 10-k~ resistance in parallel to represent the rat. The equipment was operated by a Commodore CBM 3040 computer, which also recorded the data generated at test.

Behavioral Procedure Pretraining was given in order to establish stable baseline drink rates. The experimental phase followed the last day of pretraining and consisted of four procedural stages: preexposure, acquisition, reshaping, and test, carried out on consecutive days. Thus while preexposure and acquisition were 24 h apart, test was conducted 48 h after acquisition because of the intervening reshaping day.

Pretraining Water deprivation was introduced gradually, 1 week after delivery. The rats were initially deprived overnight and access to water was further limited each day to allow adaptation to a 23-h deprivation schedule. They were handled in pairs for approximately 20 min/day over this 7-day period. The animals were then accustomed to drinking in the experimental apparatus. Each rat was allocated to a chamber into which it was placed on consecutive days until it had made 600 licks or 10 min had elapsed without commencing drinking. Three animals were trained at any one time. The subjects were then returned to the home cage and allowed

5,7-DHT LESIONSATTENUATELATENTINHIBITION access to water for 1 h. Water was given 15 min after the last squad had finished. This phase of the experiment was split so that the animals were pretrained both pre- and postoperatively, for 6 and 4 days, respectively. After 2 weeks to recover from sttrgery with water available ad libitum, the animals were reintroduced to water deprivation and given the 4 days additional pretraining before testilag in the LI procedure.

Preexposure Animals were placed in their respective experimental chambers without access to water ("off-thebaseline": the spouts were covered by plastic doors). PE rats were given 30 15.0-s tone presentations with a fixed inter-tone interval (ITI) of 50.0 s. NPE animals were placed in the experimental chambers for the equivalent time period (32.5 min) without presentation of the tone. Animals were run in groups of three in a semirandom sequence of PE and NPE squads.

Acquisition In an off-the-baseline procedure, all the subjects were given two tone-shock pairings, spaced over 15 rain. After 5 min, the tone was presented for 15 s tbllowed immediately by a 1-s footshock. A second pairing of tone and shock followed 5 rain later and the animals were left in the apparatus for an additional 5 min. Again, animals were run in squads of three in the same order as in preexposure.

Reshaping This session was inserted to reinstate drinking in the experimental apparatus and ensure stable baseline drink rates. The procedure was identical to that used in the pretraining, except that rats were removed after 200 s from the time they began drinking.

Test Each rat was placed in its chamber with access 1~othe water spout. After rats had made 90 licks the l~one was presented for 600 s. Since each animal determined tone onset, rats were tested individually 1~o avoid interference between boxes. The latencies to complete licks 80-90 (A) and 90-100 (B) were recorded. If animals failed to make an additional 10 licks after tone presentation, they were assigned a score of 600 s for the B period. In addition to the latency measures, the number

197

of licks in each minute during the 10-min tone presentation was also recorded.

Dissections Eighteen to twenty-one days after the completion of behavioral testing the animals were killed by cervical dislocation in a counterbalanced sequence of experimental conditions over 3 days. The brains were rapidly removed and dissected on an ice-cooled dissection block (Heffner, Hartman, & Selden, 1980). Six brain regions were sampled bilaterally: frontal cortex, septum, amygdala, nucleus accumbens, hypothalamus, and hippocampus. The samples were weighed, frozen in liquid nitrogen, and stored at -70°C until biochemical analysis by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Two control dissections (all regions) were excluded from the biochemistry (only) because of a technical failure at this stage. These were both from the Vehicle-PE condition.

Tissue Preparation and HPLC Brain samples were sonicated on ice in 0.1 M perchloric acid (containing 0.1 mM EDTA) and centrifuged at 13,000g at 4°C for 10 min. The supernatants were then removed, refrozen in liquid nitrogen, and stored at -70°C. The HPLC system consisted of an ACS 351 series pump (HPLC Technology), on-line degasser (ERC 3510, Erma Inc.), Chromspher C18 cartridge column (8 ~ m particle size), guard column, and saturation precolumn (all from Chrompack UK Ltd.). Electrochemical detection was accomplished with an LC2A detector (BAS Inc.) maintained at + 0.75 V with respect to an Ag/AgC1 reference electrode. Chromatographic separation and electrochemical detection were performed at 10°C. The mobile phase consisted of a citrate-phosphate buffer containing 1.5 mM octane sulphonic acid, 12% methanol, and 1 mM EDTA at pH 2.65. The flow rate was 0.5 ml/min. Peaks were displayed, integrated, and stored using a Shimadzu C-R3A coupled to an FDD1A disk drive (Dyson Instruments Ltd.). All constituents (BDH) of the HPLC mobile phase were Analar or HPLC grade.

Data Analysis Behavior. Baseline drink rates (A) and suppression ratios were analyzed using 2 × 2 Analyses of Variance (ANOVAs) with main factors of Preexposure and Lesion condition. The suppression ratio is calculated as A/(A + B) for each rat. A suppres-

198

CASSADAY ET AL.

TABLE 1 Latency Measures: Times to Complete Licks 80-90 (A) and 90-100 (B) and Suppression Ratios A/(A + B) Group

A ± SE

PE-VEH NPE-VEH PE-DHT NPE-DHT

3.07 2.27 7.23 2.40

± ± ± -+

0.67 0.13 5.00 0.23

B _+ SE 418.06 600.00 518.06 542.96

± 73.34 _+ 00.00 _+ 50.54 + 31.22

SR _+ SE 0.07 0.00 0.07 0.00

+ 0.04 ± 0.00 _+ 0.06 _+ 0.00

Note. Mean times (-+ SEM; SE) to complete licks 80-90 (A) and 90-100 (B) are expressed in seconds (n = 11-12 per group). Mean suppression levels ± SEM (SR ± SE) are derived from A/(A + B). Groups: PE, preexposed; NPE, nonpreexposed; VEH, vehicle-treated; DHT, 5,7-DHT treated.

sion ratio approaching 0.00 indicates complete suppression (i.e., good conditioning to the tone), while a suppression ratio of 0.50 indicates no change in response rate from the pretone to the tone-on period (i.e., a failure to learn about the tone-shock relationship). The number of licks made over the 600 s of test were analyzed using a 2 x 2 x 10 ANOVA with main factors of Preexposure and Lesion condition and a repeated measurement factor of Blocks (the 10 1-min intervals of tone presentation). Post hoc comparisons were made by t test based on the pooled error term derived from the appropriate stratum of the ANOVA. Because the parametric approach tends to be too liberal in analyses of data which are not normally distributed, the lick totals were also subjected to rank sum factorial analYsis (Meddis,

1984) with lesion condition (Vehicle, DHT) and preexposure condition (PE, NPE) as factors. The specific prediction that the 5,7-DHT lesions would reduce LI (see Introduction) was tested via a single statistic representing the expected form of the interaction. Biochemistry. The 34 neurochemical measures were subjected to a two-factor (Lesion, Preexposure) Multivariate Analysis of Variance (MANOVA). A design including the within-subjects factor Region (hippocampus, septum, nucleus accumbens, hypothalamus, amygdala, frontal cortex) could not be used since hippocampal homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) levels were not detectable. Subsequently, 2 x 2 univariate ANOVAs were performed for each neurotransmitter and metabolite, together with utilization ratios for DA and 5HT, for each brain region individually. These analyses were followed by t tests based on the relevant error term. The dependent variables were levels of dopamine (DA) and its metabolites, DOPAC and HVA, noradrenaline (NA), 5HT, and the 5HT metabolite, 5-hydroxyindoleacetic acid (5HIAA), in each of the six brain regions. The utilization ratio measure is provided by the ratio of metabolite to transmitter levels and gives an index of neurotransmitter turnover. Ratios greater than 1 indicate that metabolism exceeds synthesis and ratios less than 1 indicate that the rate of metabolism falls short of that of synthesis. RESULTS Behavior

400

300 0

2 SEM

2OO E Z

1O0

0 VEH-PE

VEFI-NPE

DHT-PE

DHT-NPE

FIG. 1. LI after 5,7-DHT lesion to the fornix-fimbria. Ordinate shows the total number of licks made during the 10-min tone at test. Fewer licks reflect better learning of the tone-shock association. Groups: PE, preexposed; NPE, nonpreexposed; VEH, vehicle-treated; DHT, 5,7-DHT-treated. Bar shows 2 standard errors of the mean, derived from the interaction term of the ANOVA.

The A and B periods, together with the mean suppression ratio scores, are presented in Table 1. Figure 1 presents the mean total number of licks. Figure 2 shows the pattern of drinking over time during tone presentation in the test session, revealed by the licks measure. It can be seen that there were substantial differences between the groups which emerged principally after the first 4 min of tone presentation at test. Baseline drink rates. Analysis of variance showed that experimental treatments did not affect pre-CS licking (A period; see Table 1): there was no effect of Preexposure [F(1, 41) = 1.08], Lesion [F(1, 41) = 0.63] or their interaction [F(1, 41) = 0.56]. Licks. The 5,7-DHT lesion abolished LIo Analysis of variance showed that the Lesion x Preexposure interaction was significant both overall [F(1,

199

5,7-DHT LESIONS ATTENUATE LATENT INHIBITION

90

ca

25

70

•=

60

~5

50

]~

40

~ Z

30 20

-~2 SEM

~ 1 ~, / , //,e' / /

lo

I 1

2

7

//

,___

DHT

PE - - e - - DHT NPE "'

"

/

Q

//"

pEVEH VEH

i-,IPE

,,' /

3 4 5 6 7 8 9 Blocks (1 min intervals)

PE controls were also significantly less suppressed than NPE controls (showing LI) over this period; but this difference was not significant in the lesion condition and was in the reverse direction (greater suppression in the PE group); thus LI was clearly absent. Lesioned animals in the NPE condition were significantly less suppressed than NPE controls only at the penultimate interval (block 9; Fig. 2).

10

FIG. 2. LI after 5,7-DHT lesion to the fornix-fimbria. Ordinate shows the number of licks made over the 10-min tone presentation at test. Fewer licks reflect better learning of the tene-shock association. Groups: PE, preexposed; NPE, nonpreexposed; VEH, vehicle-treated; DHT, 5,7-DHT-treated. Bar shows 2 standard errors for comparisons between groups within blocks.

41.) = 4.79, p < .04] and in interaction with Blocks [F(9, 369) = 2.31, p < .02]. As predicted, the number of licks during tone presentation was less in the DHT-PE than in the Vehicle-PE condition [t(41) =: 1.99, p < .05, one-tailed], while the number of licks in the DHT-NPE was nonsignificantly greater than in the Vehicle-NPE condition [t(41) = 1.12]. Post hoc tests also confirmed that LI was present in the vehicle-treated group [t(41) = 2.57, p < .02, one-tailed] and absent in the DHT-treated group [t(41) = 0.51]. Analysis of rank data provided furtiler confirmation that the lesion significantly at~ n u a t e d LI. There was a significant Lesion × Preexposure interaction in the predicted direction [z = 2.35, p < .01, one-tailed], i.e., the interaction reflected the fact that LI was present in the vehicletreated group but absent in the DHT-treated group. The mean ranks (low ranks reflect heavy suppression) and the median number of licks for each group are reported in Table 2. A change in the degree of LI should be reflected in changes in the amount of learning in the PE group with a minimal change in the NPE group. Table 2 shows that the attenuation of LI indeed resulted from a decrease in the median number of licks in the DHT-PE group. Overall, drinking increased over time during tone presentation (Fig. 2). Statistically, there was a main effect of Blocks [F(9, 369) = 6.09, p < .001] and a significant linear trend effect of Blocks [F(1, 41) -9o19, p < .005]. The PE controls were significantly less suppressed than PE-lesioned animals over the last 180 s of tone presentation (blocks 8-10; Fig. 2).

Suppression ratios. The Preexposure effect was not significant [F(1, 41) = 2.94, p < .1]. No other effects approached significance. Biochemistry

Tables 3 and 4 show the levels of indole- and catecholamines, respectively, for each brain region assayed, and the results of the relevant statistical analyses. The 5,7-DHT lesion significantly depleted hippocampal 5HT [F(1, 39) = 12.85, p < .001] and 5HIAA [F(1, 39) = 16.45, p < .001] by 75 and 78% of vehicle-injected control levels, without affecting levels of other transmitters in the hippocampus, and, except in the amygdala, there were no Lesion effects in the other tissues sampled. In the amygdala, 5HIAA levels were significantly reduced (Table 3) after 5,7-DHT lesion [F(1, 39) = 7.29, p < .01]. The MANOVA confirmed that there was no effect of lesion in the other brain regions sampled [Wilks lambda = 0.271; F(34, 6) = 0.47, p > .9]. Unexpectedly, the MANOVA showed that the LI procedure itself had significant effects on the neurochemical measures [Wilks lambda = 0.017; F(34, 6) = 10.22, p < .01]. Post hoc univariate ANOVAs indicated that PE, irrespective of the lesion condition, increased 5HIAA levels significantly in the amygdala, frontal cortex, and hypothalamus (Table 3) and increased the 5HT utilization ratio in the TABLE 2 Rank Mean and Median Number of Licks during Presentation of the Tone CS Group

Rank mean

Median

Semi-interquartile range

PE-VEH NPE-VEH PE-DHT NPE-DHT

29.5 15.0 22.6 24.8

305 1 19 2

382.5 1 67 312.5

Note. Ranks and median number of licks during tone presentation (n = 11-12 per group). Scores ranked independently of experimental condition to compute mean ranks. Groups: PE, preexposed; NPE, nonpreexposed; VEH, vehicle-treated; DHT, 5,7-DHT-treated.

200

CASSADAY ET AL.

TABLE 3 Regional Indoleamine Levels after LI Procedure with 5,7-DHT Treatment Group PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA

E D D x E PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E D D x E PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E

5HT (A) Hippocampus 316 -+ 11 343 -+ 17 274 _+ 25 223 -+ 30 NS F(1, 39) = 12.85 p < .001 NS (B) Nucleus accumbens 835 -+ 34 796 -+ 46 737 -+ 58 886 -+ 41

5HIAA 340 355 301 242

-+ 13 -+ 09 -+ 21 -+ 26

NS F(1, 39) = 16.45 p < .001 NS

D × E

NS

NS

NS NS

NS NS

D D x E

NS

D

NS

D × E

NS

546 511 505 426

-+ 18 _+ 22 _+ 33 -+ 27

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E D

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E

(C) Amygdala 741 _+ 39 801 -+ 40 782 -+ 31 713 + 34

713 671 648 645

Group

_+ 44 _+ 13 -+ 19 _+ 18

F(1, 39) = p < F(1, 39) = p < NS

5.82 .02 7.29 .01

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E

5HT

5HIAA

(D) Septum 438 _+ 41 532 -+ 31 547 -+ 41 547 + 33

556 563 603 525

-+ 29 -+ 23 -+ 26 -+ 29

NS NS

NS NS

NS

NS

(E) Frontal cortex 569 -+ 22 522 -+ 23 589 -+ 23 586 +- 33 NS NS NS

320 305 339 294

F(1, 39) = 6.05 p < .02 NS NS

(F) Hypothalamus 803 -+ 33 866 -+ 38 870 _+ 23 889 +- 40 NS

-+ 15 -+ 09 -+ 10 -+ 16

926 805 875 806

+- 48 +- 15 -+ 22 +- 43

D

NS

F(1, 39) = 8.39 p < .01 NS

D x E

NS

NS

Note. Mean (-+ SEM) indoleamine levels are shown as ng/g tissue wet wt (n = 9-12 per group). Groups: PE, preexpesed; NPE, nonpreexposed; VEH, vehicle-treated; DHT, 5,7-DHT-treated. Data were analyzed by ANOVA (main effects: E, Exposure; D, 5,7-DHT; interaction: D x E, DHT x Exposure). NS, not significant. s e p t u m a n d h y p o t h a l a m u s ( T a b l e 5). I n a d d i t i o n , PE increased DOPAC levels in the nucleus accumb e n s a n d h y p o t h a l a m u s ( T a b l e 4) a n d i n c r e a s e d t h e D A u t i l i z a t i o n r a t i o in t h e n u c l e u s a c c u m b e n s , a m y g d a l a , s e p t u m , a n d h y p o t h a l a m u s ( T a b l e 5). The M A N O V A also showed an almost significant i n t e r a c t i o n b e t w e e n P r e e x p o s u r e a n d L e s i o n [Wilks lambda = 0.049; F ( 3 4 , 6) = 3.45, p = .06]. U n i variate ANOVAs showed significant Lesion x P r e e x p o s u r e i n t e r a c t i o n s for t h r e e n e u r o c h e m i c a l v a r i a b l e s , a l l of w h i c h i n v o l v e d t h e D A s y s t e m . Bec a u s e of t h e l a r g e n u m b e r of u n i v a r i a t e A N O V A s c o n d u c t e d (45 in all; see T a b l e s 3 - 5 ) a n d t h e a b s e n c e of a p r i o r i p r e d i c t i o n s , t h e t h r e e s i g n i f i c a n t L e s i o n x Preexposure interactions in the univariate analy s e s m i g h t h a v e a t t a i n e d t h e 5% s i g n i f i c a n c e l e v e l b y c h a n c e a l o n e (the s t r o n g e s t w a s s i g n i f i c a n t a t p < .03). T h e r e l e v a n t d a t a a n d s t a t i s t i c a l r e s u l t s a r e p r e s e n t e d i n T a b l e 6.

DISCUSSION T h e s e r e s u l t s a r e i m p o r t a n t i n t w o r e s p e c t s : first, t h e y p r o v i d e e v i d e n c e t h a t s e l e c t i v e d e s t r u c t i o n of t h e 5 H T a f f e r e n t s to t h e h i p p o c a m p u s c a n a b o l i s h LI; second, t h e y d e m o n s t r a t e t h a t p r e e x p o s u r e d u r i n g t h i s LI p r o c e d u r e (or a s e c o n d a r y c o n s e q u e n c e of s u c h p r e e x p o s u r e , see below) h a s e n d u r i n g effects upon at least two different n e u r o c h e m i c al systems i n w i d e s p r e a d r e g i o n s of t h e b r a i n . W e d i s c u s s e a c h of t h e s e f i n d i n g s in t u r n .

Specificity and Extent of 5,7-DHT Lesion M i c r o i n j e c t i o n s of 5 , 7 - D H T i n t h e f o r n i x - f i m b r i a s i g n i f i c a n t l y r e d u c e d h i p p o c a m p a l i n d o l e a m i n e levels, b o t h 5 H T a n d 5 H I A A , to 75 a n d 78% of c o n t r o l levels, respectively. This depletion was less t h a n t h o s e p r e v i o u s l y r e p o r t e d a f t e r t h i s lesion. F o r ex-

201

5,7-DHT LESIONS ATTENUATE LATENT INHIBITION

Regional

Catecholamine

Group

DA

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E D DxE

6.14 7.75 7.18 8.10

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E

7169 6397 6347 7247

D D x E

DOPAC

1.24 0.91 1.29 1.12

with 5,7-DHT Treatment HVA

(A) Hippecampus ND ND ND ND

ND ND ND ND

NA

320 358 339 313

NS NS NS ± 318 +- 436 ± 538 _+ 184

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E D D x E

503 704 552 431

± ± ± ±

248 083 037 039

NS NS NS ± ± + ±

65 74 44 58

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E

276 297 263 263

± ± _+ ±

50 96 76 34

23 33 06 16

NS NS NS ± ± _+ ± NS NS NS

726 596 613 667

= 4.74 p <.04 NS NS

(C) Amygdala 174 + 109 75 _+ 011 58 +_ 008 57 +- 005

64 42 39 42

NS NS NS

NS NS F(1, 39) = 5.26 p < .03

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E D D x E

(B) Nucleus accumbens 2015 ± 160 1490 ± 109 1685 ± 142 1611 ± 136 ~1,39)

NS F(1, 39) = 4.14 p < .05 492 394 262 323

± -+ ± ±

18 15 10 23

NS NS NS

NS

]PE-VEH NPE-VEH PE-DHT NPE-DHT .&NOVA E D D x E

D D x E

± ± +-+

TABLE 4 Levels after LI Procedure

(D) Septum 231 ± 25 241 ± 27 244 _+ 28 153 ± 18

108 116 109 98

(F) Hypothalamus 93 ± 23 46 ± 04 79 ± 14 46 ± 08 F(1, 39) = 9.49 p < .01 NS NS

261 273 206 202

60 59 53 61

NS

NS NS

NS NS

± ± ± ±

25 05 05 09

453 510 466 466

± ± _+ ±

± ± -+ -+

± ± ± ±

_+ ± -+ -+

44 57 16 19

21 26 17 16

NS NS NS 11 06 08 09

770 834 791 685

± ± -+ ±

70 46 62 40

NS NS NS

4.0 4.2 3.9 3.2

258 277 270 253

NS NS NS 27 24 23 26

± ± ± ±

NS

NS NS NS

NS NS NS 10 17 10 16

46 49 44 49

NS NS NS

NS NS F(1, 39) = 4.10 p < .05 (E) Frontal cortex 25 _+ 5.0 21 ± 6.4 22 _+ 3.0 21 _+ 2.9

± ± ± ±

± ± -+ ±

11 10 08 14

NS NS NS 3.2 3.5 2.5 2.9

2126 2059 2082 1877

± ± -+ ±

NS

NS

NS NS

NS NS

100 081 094 05-2

Note. Mean (± SEM) catecholamine levels are shown as n g / g tissue wet wt (n = 9-12 per group). ND not detectable. Groups: PE, preexposed; NPE, nonpreexposed; VEH, vehicle-treated; DHT, 5,7-DHT-treated. Data were analyzed by ANOVA (main effects: E, Exposure; D, 5,7-DHT; interaction: D x E, DHT x Exposure). NS, not significant.

202

CASSADAY ET AL.

TABLE 5 Regional Utilization Ratios after LI Procedure with 5,7-DHT Treatment Group

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E D D x E PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E D D × E PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E D D x E

5HT (A) Hippocampus 1.082 -+ 0.05 1.067 _+ 0.08 1.161 _+ 0.09 1.155 _+ 0.07

DA

Group

NS

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E

NS NS

D D x E

(B) Nucleus acctunbens 0.866 _+ 0.05 0.869 _+ 0.06 0.925 _+ 0.08 0.734 -+ 0.03 NS NS NS (C) Amygdala 0.741 _+ 0.06 0.653 + 0.04 0.653 -+ 0.03 0.604 _+ 0.03 NS NS NS

ND ND ND ND

0.383 0.335 0.381 0.313

-+ -+ -+ -+

0.03 0.03 0.04 0.03

F(1, 39) = 4.57 p < .04 NS NS 0.485 0.335 0.394 0.321

-+ -+ -+ -+

0.07 0.03 0.03 0.02

F(1, 39) = 8.74 p < .01 NS NS

PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E D D x E PE-VEH NPE-VEH PE-DHT NPE-DHT ANOVA E D D x E

5HT (D) 1.356 1.074 1.156 0.967

Septum -+ 0.14 -+ 0.05 _+ 0.08 _+ 0.03

F(1, 39) = 9.53 p < .01 NS NS (E) Frontal cortex 0.566 _+ 0.19 0.599 _+ 0.18 0.589 -+ 0.17 0.503 -+ 0.15

DA

0.682 0.538 0.706 0.580

-+ -+ + -+

F(1, 39) = 8.80 p < .01 NS NS 2.114 1.684 1.783 1.739

+ + -+ -+

NS

NS

NS NS

NS NS

(F) Hypothalamus 1.180 -+ 0.11 0.944 -+ 0.04 1.014 + 0.04 0.912 -+ 0.04 F(1, 39) = 8.61 p < .01 NS NS

0.04 0.03 0.07 0.04

0.432 0.234 0.380 0.282

-+ +-+ -+

0.32 0.21 0.09 0.14

0.08 0.02 0.06 0.04

F(1, 39) = 9.50 p < .01 NS NS

Note. Mean ( - SEM) utilization ratios (see text) for 5HT and DA (n = 9-12 per group). ND, not detectable. Groups: PE, preexposed; NPE, nonpreexposed; VEH, vehicle-treated; DHT, 5,7-DHT-treated. Data were analyzed by ANOVA (main effects: E, Exposure; D, 5,7-DHT; interaction: D x E, DHT × Exposure). NS, not significant.

ample, Williams, Meara, and Azmitia (1990) found that it reduced [3H]5HT uptake to 40-50% of control levels. However, the use of a different measure of 5HT function renders direct comparison between these studies difficult. Furthermore, the present analyses used tissue from whole hippocampus. Williams and Azmitia (1981) report that the procedure used here depletes dorsal hippocampal 5HT more than ventral, consistent with the known anatomy of 5HT afferents to the hippocampal formation (Azmitia & Segal, 1978); thus, the true extent of damage to dorsal hippocampal 5HT afferents is likely to be underestimated by our measures. The biochemical results confirmed that the lesion had been selective to 5HT fibers projecting to the hippocampus. There were no main effects of the lesion on the levels of NA, nor of DA or its metabolites in any brain region assayed, an important observation given the known involvement of DA systems in LI

(Solomon et al., 1981; Weiner et al., 1981, 1984, 1988). Nor were there any significant effects of the lesion on 5HT levels in regions other than the hippocampus, including the septal area, which is traversed by the 5HT afferents destined for the hippocampus (Azmitia & Segal, 1978). The lesion did, however, give rise to a reduction of 5HIAA levels in the amygdala. Since fornix-fimbria 5HT fibers do not appear to project to the amygdala (Azmitia & Segal, 1978), this is unlikely to have been a direct effect of the lesion; it is more consistent with the possibility that hippocampal 5HT terminals modulate extrahippocampal 5HT function via a reciprocal feedback loop (Doty, 1989). The interval to taking the brains for neurochemistry (35-45 days after surgery) is also important since 5,7-DHT lesions of the limbic 5HT pathways may be followed by structural and functional recovery of 5HT terminals. Clewans and Azmitia

5,7-DHT LESIONS ATTENUATE LATENT INHIBITION

TABLE

Effects of 5,7-DHT Lesion on LI

6

Summary of Interactions between L e s i o n and Preexposure Nucleus accumbens PE-VEH NPE-VEH PE - D H T NPE - D H T ANOVA E D DxE

DAlevel 7169 6397 6347 7247

± ± ± ±

318 436 538 184

NS NS F(1, 39) = 4.14 p < .05

Septum

DA level

PE-VEH NPE-DHT PE-DHT NPE-DHT ~uNOVA E D D×E

503 704 552 431

± ± ± ±

50 96 76 34

NS NS F(1, 39) = 5.26 p < .03

203

DOPAC level 231 241 244 153

± ± ± ±

25 27 28 18

NS NS F(1, 39) = 4.10 p < .05

Note. Mean ( ± SEM) levels of DA and DOPAC in septum and nucleus accumbens are shown as n g / g tissue wet wt. Groups: PE, preexposed; NPE, nonpreexposed; VEH, vehicle-treated; DHT, 5,7-DHT-treated. Data were analyzed by ANOVA (main effects: E, Exposure; D, 5,7-DHT; interaction: D x E, DHT x Exposure). NS, not significant.

(1984) investigated the time course of such recovery after 5,7-DHT lesions to the cingulum bundle, measured by changes in tryptophan hydroxylase activity. Functional recovery appeared to be mediated by the collateral sprouting of intact fibers in the fornix-fimbria 28-90 days after injection of the neurotoxin. In the present experiment, the converse process may have occurred, collateral sprouting of intact 5HT fibers in the cingulum bundle providing partial compensation for an initially greater loss of hippocampal 5HT innervation caused by the fornixfimbria 5,7-DHT lesion. Behavioral testing itself was not run at the time of probable minimum hippocampal 5HT function: tryptophan hydroxylase levels have been reported to be minimal 7 days after such lesions (Clewans & Azmitia, 1984). Although our animals were used as soon as possible after recovery from the stress of surgery, the delay was at least 2 weeks. If, however, these effects--recovery of neurochemical function and/or less than optimal timing of behavioral testing--influenced our results, they were insufficient to prevent the observed changes in behavior, which closely approximated those predicted.

Behavioral changes due to the 5,7-DHT lesion and preexposure were evident principally in measurements of total licks over the whole 10-min period of tone presentation at test. As shown in Figures 1 and 2, LI (i.e., less suppression of licking in the PE relative to the NPE condition) was clearly present in the vehicle-treated controls, and equally clearly absent in the lesioned animals. The lesion increased suppression only in the PE condition, so that PE animals with the lesion licked less during the tone than their vehicle controls. The lesion did not significantly affect licking in the NPE condition (except in the ninth minute of tone presentation), but there was a nonsignificant tendency toward decreased suppression. Thus, the increased suppression caused by the lesion in the PE condition could not have been due to a measurement floor effect, reflecting, e.g., a general increase in learning efficiency. There were no effects of the lesion on the measurement of lick rate (time to complete licks 80-90) before the introduction of the tone in the test session. Thus, the differential effect of the lesion upon PE animals probably reflects a change in the reaction to the tone CS rather than a general change in licking behavior independent of this reaction. We conclude, therefore, that destruction of the 5HT afferents to the hippocampus abolished LI (as measured by the number of licks during 10 min CS presentation), and that this effect, as predicted, was due to changes in the effect of preexposure rather than to changes in the behavior of the NPE animals. Our results confirm and extend previous reports that systemic pharmacological interruption of normal 5HT function (Cassaday et al., 1991; Solomon et al., 1978), electrolytic destruction of the medial raphe (Solomon et al., 1980), and mechanical lesions of the hippocampus are all capable of disrupting LI. Similarly, both electrolytic and 5,7-DHT lesions to the raphe have been found to disrupt LI (see Weiner, 1990, for review). Our findings extend these previous reports by showing that limited destruction of the 5HT innervation of the hippocampus is sufficient to disrupt LI. These results raise the possibility that reduction of hippocampal 5HT function is the main mechanism by which generalized disruption of central 5HT systems reduces LI. This conclusion contrasts sharply with the findings of Tsaltas, Preston, Rawlins, Winocur, and Gray (1984) that 6-hydroxydopamine lesions of the dorsal ascending noradrenergic bundle which depleted hippocampal NA to less than 10% of control levels did not reduce LI. Despite some contrary evidence, it

204

CASSADAY ET AL.

has been consistently reported that there is no effect of NA depletion on LI when conventional procedures are used (see Weiner, 1990, for review). This contrast between the effects of noradrenergic and serotonergic depletion on LI indicates that the effects of the 5,7-DHT lesions observed in the present study do not merely result from a nonspecific disruption of hippocampal function (cf. DeVietti et al., 1982), but may reflect a specific involvement of 5HT in LI. The present findings cannot exclude the possibility that 5HT terminal fields outside the hippocampus contribute to LI: for example, the apparent reduction of amygdalar 5HT levels may have contributed to the disruption of LI. However, the finding that electrolytic lesions of the dorsal raphe do not affect LI (Solomon et al., 1980) probably excludes a necessary role for serotonergic synapses in the nucleus accumbens or the amygdala (Azmitia & Segal, 1978; Weiner, 1990). The present study found clear abolition of LI measured as a function of total licks over the 10-min period of tone presentation at test. The suppression ratio showed no significant effects of either preexposure or lesion. This is probably due to the overall marked degree of suppression in the present study, which may reflect a delayed consequence of the stress of surgery. Other cases in which the licks measure revealed effects to which the suppression ratio was insensitive involved strong suppression, associated either with surgery (C.-T. Tai, personal communication) or due to increased anxiety consequent upon LSD treatment (see Cassaday, 1990). However, in other experiments, both in our laboratory (Cassaday et al., 1991) and elsewhere (Weiner et al., 1981, 1984), the suppression ratio has proved a sensitive measure of both LI and of the effects of drugs on LI. It is possible, therefore, that the suppression ratio and the total licks during test measure different processes each separately sensitive to LI. The suppression ratio, for example, may measure the initial strength of tone-shock association and total licks, the rapidity with which this association is extinguished in the absence of further shocks, or suffers from generalization decrement with increasing tone duration. This interpretation of our results is strengthened by the fact that neither the effect of PE in the controls, nor the abolition of this effect by the lesion, was manifest until the fifth minute of tone presentation, and did not become significant until the eighth (Fig. 2). Hence, the 5HT innervation of the hippocampus may be particularly involved in the effect of preexposure upon the resistance to extinction or to generalization decrement of a CS-UCS association. Clearly,

further studies are needed to test whether LI measured during an extinction or generalization test differs from LI measured in other ways.

Effects of Preexposure on Neurochemical Variables Both multivariate and univariate ANOVAs indicated that the 30 preexposures to the to-be-conditioned stimulus as part of the LI procedures had long-lasting effects on a variety of neurochemical variables. These effects were unexpected. Nonetheless, they were highly significant; pervasive, appearing in five different brain structures (amygdala, hypothalamus, septum, nucleus accumbens, and frontal cortex), and affecting two neurochemical systems, the serotonergic and dopaminergic; and consistent, taking the direction of increased metabolism and/or utilization of the transmitter in PE groups in every case. The fact that Preexposure effects were associated with changes in DA function in brain regions (amygdala, hypothalamus, septum, and nucleus accumbens) which show stress-induced increases in monoaminergic turnover (Lane, Sands, Co, Cherek, & Smith, 1982) suggested the possibility that they were stress related. First, the effective predictability of the shock was reduced in PE groups given prior unreinforced experience of the tone. Predictable shock is known to be generally less stressful than unpredictable and classical contingencies have been found to reduce the effects of shock on monoamine levels (Schutz, Schutz, Orsingher, & Izquierdo, 1979). It is difficult to draw conclusions about the neurochemical consequences of preexposure per se independent of the animals motivational state since the effects of preexposure are measured via modulation of that state, here conditioned fear. In both appetitive and aversive procedures, the predictability of the UCS by the CS is likely to influence the degree to which learning is stressful. Second, exposure to the salient tone used to produce LI may have sensitized the preexposed animals via nonassociative mechanisms to the two shocks delivered at acquisition (cf. Antelman, Knopf, Kocan, Edwards, Ritchie, & Nemeroff, 1988), thus accounting for the difference in transmitter function between PE and NPE groups. However, given the independent evidence (Gray et al., 1991a; Weiner, 1990) that manipulations of both serotonergic and dopaminergic systems can modulate LI, as demonstrated in a variety of procedures, it is tempting to speculate that these neurochemical effects of preexposure reflect changes in attention to the CS. The observed effects of preexposure on neurochemical measures occurred equally in the control

5,7-DHT LESIONS ATTENUATE LATENT INHIBITION

and 5,7-DHT lesion groups, so it is unlikely that t:he observed neurochemical changes were related ~ the rats' behavior at test (since the rats' total licks depended on both their preexposure and lesion condition). Rather, it appears that the fornix-fimbria 5,7-DHT lesions did not prevent the changes in neurotransmitter function associated with preexposure. This reinforces the possibility that hippocampal 5HT depletion diminished the expression of LI (see previous section), perhaps by increasing the rate of extinction of the CS-UCS association or the rate of generalization decrement during the test. The present study gave slight evidence of interactions between the effects of the fornix-fimbria 5,7DHT lesion and preexposure on measures of DA function in the nucleus accumbens and septum (see Table 6). The weight placed on these results must [~e small in view of the (marginal) nonsignificance of the Preexposure x Lesion interaction in the MANOVA, and the consequent possibility that they reflect type I statistical error (since only 3 of 34 univariate ANOVAs showed a significant interact:ion at the 5% level). With these cautions in mind, the Preexposure x Lesion interaction in nucleus accumbens DA levels nevertheless deserves consideration. Evidence from Solomon's laboratory (Solomon & Staton, 1982) suggests that the disruption of LI by elevated dopaminergic transmission is mediated in the nucleus accumbens, and several workers have subsequently proposed that increased nucleus accumbens DA function is the "final common path" by which a variety of treatments attenuate LI (Gray et al., 1991a; Weiner, 1990). In line with this proposal, Young, Gray, and Joseph (1992) have recently reported that CS preexposure reduces the DA release elicited in nucleus accumbens by a tone CS paired with a shock UCS, using an LI procedure ,fimilar to the one used here. It is tempting to speculate that the abolition of LI caused by fornix-fimbria 5,7-DHT lesions in the present study was mediated indirectly by the observed alterations in nucleus accumbens DA. However, in view of the 3week interval between behavioral testing and biochemical analyses, the nature and meaning of the association between total licks and nucleus accumbens DA levels at the time of testing are highly uncertain. Clearly, further studies are needed to elucidate possible interactions between hippocam]pal 5HT, nucleus accumbens DA, and preexposure in the development and expression of LI.

Significance for the Neural Basis of Schizophrenia The psychotogenic, indirect DA agonist, amphetamine, blocks LI in the conditioned-suppression pro-

205

cedure used here in an inverse dose-dependent manner consistent with mediation by DA release in the nucleus accumbens (Weiner, Izraeli-Telerant, & Feldon, 1987; Weiner et al., 1988). We have shown a similar, inversely dose-dependent blockade of LI by oral amphetamine in normal human volunteers (Gray et al., 1992). This' indicates a functional equivalence between the procedures used to demonstrate LI with animal and human subjects, respectively. The same human paradigm has also revealed loss of LI in acute schizophrenics, and its subsequent normalization with continued neuroleptic medication (Baruch et al., 1988; Gray et al., 1991b). These converging lines of evidence suggest that blockade of LI in rats tested in the conditioned suppression procedure is functionally related to acute schizophrenia (Gray et al., 1991a, 1991b). In this light, the results of the present experiment indirectly support the proposal that the attentional deficits observed in acute schizophrenia (Baruch et al., 1988) may result from diminished central 5HT function, as suggested by the LSD model of schizophrenia (Claridge, 1978). Within the LI model of attention disorder, the present result suggests that a reduction in the efficiency of the hippocampal 5HT input underlies, at least in part, the cognitive abnormalities characteristic of the acute phase of schizophrenia. This hypothesis is not incompatible with the dopamine hypothesis of schizophrenia, according to which these abnormalities are due to increased dopaminergic transmission. However, the site at which such an increase in dopaminergic transmission affects LI is unlikely to lie in the hippocampal formation, which receives only a sparse dopaminergic innervation (Verney, Baulac, Berger, Alvarez, Vigny, & Helle, 1985). Thus, taken together, these findings indicate the need for further exploration of the anatomical and physiological interrelations between the hippocampal formation and nucleus accumbens in the development and expression of LI. REFERENCES Aghajanian, G. K., Foote, W. E., & Sheard, M. H. (1968). Lysergic acid diethylamide sensitive neuronal units in the midbrain raphe. Science, 161, 706-708. Antelman, S. M., Knopf, S., Kocan, D., Edwards, D. J., Ritchie, J. C., & Nemeroff, C. B. (1988). One stressful event blocks multiple actions of diazepam for up to at least a month. Brain Research, 445, 380-385. Azmitia, E. C., Buchan, A. M., & Williams, J. H. (1978). Structural and functional restoration by collateral sprouting of hippocampal 5HT axons. Nature, 274, 374-376. Azmitia, E. C., & Segal, M. (1978). Autoradiographic analysis of

206

CASSADAY ET AL.

the differential ascending projections of the dorsal and median raphe in the rat. Journal of Comparative Neurology, 179, 641-668. Barnes, J. C., Costall, B., & Naylor, R. J. (1987). Influence of medial raphe nucleus lesions on behavioural responding to dopamine infusion into the rat amygdala. British Journal of Pharmacology, (Proceedings Supplement), 91, 428P. Baruch, I:, Hemsley, D. R., & Gray, J. A. (1988). Differential performance of acute and chronic schizophrenics on a latent inhibition task. Journal of Nervous and Mental Disease, 176, 598-606. Brazell, M. P., Marsden, C. A., Nisbet, A. P., & Routledge, C. (1985). The 5HT1 receptor agonist RU 24969 decreases 5hydroxytryptamine (5HT) release and metabolism in the rat frontal cortex in vitro and in vivo. British Journal of Pharmacology, 86, 209-216. Cassaday, H. J. (1990). The effects of pharmacological and neu-

rotoxic manipulation of serotonergic function on latent inhibition in the rat. Unpublished Ph.D. thesis, University of London. Cassaday, H. J., Hodges, H., & Gray, J. A. (1991). The effects of pharmacological and neurotoxic manipulation of serotenergic activity on latent inhibition in the rat: Implications for the neural basis of acute schizophrenia. In G. B. Cassano and H. S. Akiskal (Eds.), Serotonin-related psychiatric syndromes: Clinical and therapeutic links (pp. 99-105). London: Royal Society of Medicine Services Limited: International Congress and Symposium Series. Claridge, G. (1978). Animal models of schizophrenia: The case for LSD-25. Schizophrenia Bulletin, 4, 186-209. Clewans, C. S., & Azmitia, E. (1984). Tryptophan hydroxylase in hippocampus and midbrain following unilateral injection of 5,7-dihydroxytryptamine. Brain Research, 307, 125-133. Connell, P. H. (1958). Amphetamine psychosis. Maudsley Monograph 5. London: Oxford Univ. Press. Costall, B., Hui S.-C. G., & Naylor, R. J. (1979). The importance ofserotonergic mechanisms for the induction of hyperactivity by amphetamine and its antagonism by intra-accmnbens (3,4-Dihydroxy-phenylamino)-2-imidazoline (DPI). Neuropharmacology, 18, 605-609. DeVietti, T. L., Emmerson, R. Y., & Wittman, T. K. (1982). Disruption of latent inhibition by placement of an electrode in dorsal hippocampus. Physiological Psychology, 10, 46-50. Doty, R. W. (1989). Schizophrenia: A disease of interhemispheric processes at forebrain and brainstem levels? Behavioural Brain Research, 34, 1-33. Gray, J. A., Feldon, J., Rawlins, J. N. P., Hemsley, D. R., & Smith, A. D. (1991a). The neuropsychology of schizophrenia. Behavioral and Brain Sciences, 14, 1-20. Gray, J. A., Hemsley, D. R., Feldon, J., Gray, N. S., & Rawlins, J. N. P. (1991b). Schiz bits: Misses, mysteries and hits. Behavioral and Brain Sciences, 14, 56-71. Gray, N. S., Pickering, A. D., Hemsley, D. R., Dawling, S., & Gray, J. A. (1992). Abolition of latent inhibition by a single 5 mg dose of D-amphetamine in man. Psychopharmacology, 167, 425-430. Heffner, T. G., Hartman, J. A., & Selden, L. S. (1980). A rapid method for the regional dissection of the rat brain. Pharmacology Biochemistry and Behavior, 13, 453-456. Lane, J. D., Sands, M. P., Co, C., Cherek, D. R., & Smith, J. E. (1982). Biogenic monoamine turnover in discrete rat brain

regions is correlated with conditioned emotional response and its conditioning history. Brain Research, 240, 95-108. Leysen, J. E., Gommeren, W., Van Gompel, P., Wynants, J., Janssen, P. F. M., & Laduron, P. M. (1985). Receptor-binding properties in vitro and in vivo of ritanserin. Molecular Pharmacology, 27, 600-611. Loskutova, L. V., Luk'yanenko, F. Ya., & II'yuchenok, R. Yu. (1990). Interaction of serotonin- and dopaminergic systems of the brain in mechanisms of latent inhibition in rats. Neuroscience and Behavioral Physiology, 20, 500-505. Lubow, R. E. (1989). Latent inhibition and conditioned attention theory. Cambridge/New York: Cambridge Univ. Press. McNaughton, N., Azmitia, E. C., Williams, J. H., Buchan, A., & Gray, J. A. (1980). Septal elicitation of hippocampal theta rhythm after localized de-afferentation of serotonergic fibres. Brain Research, 200, 259-269. Meddis, R. (1984). Statistics using ranks: A unified approach. Oxford: Blackwell. Rosecrans, J. A., Lovell, R. A., & Freedman, D. X. (1967). Effects of lysergic acid diethylamide on the metabolism of brain 5hydroxytryptamine. Biochemical Pharmacology, 16, 20112021. Schmajuk, N. A. (1984). Psychological theories of hippocampal function. Physiological Psychology, 12, 166-183. Schmajuk, N. A. (1987). Animal models of schizophrenia: The hippocampally lesioned animal. Schizophrenia Bulletin, 12, 317-327. Schutz, R. A., Schutz, M. T. B., Orsingher, O. A., & Izqnierdo, I. (1979). Brain dopamine and noradrenalin levels in rats submitted to four different aversive behavioural tests. Psychopharmacology, 63, 289-292. Solomon, P. R., Kiney, C. A., & Scott, D. R. (1978). Disruption of latent inhibition following systemic administration of parachlorphenylalanine (PCPA). Physiology and Behavior, 20, 265-271. Solomon, P. R., Nichols, G. L., Kiernan, J. M., III, Kamer, R. S., & Kaplan, L. J. (1980). Differential effects of lesions in medial and dorsal raphe of the rat: latent inhibition and septohippecampal serotonin levels. Journal of Comparative and Physiological Psychology, 94, 145-154. Solomon, P. R., Crider, A., Winkelman, J. W., Turi, A., Kamer, R. M., & Kaplan, L. J. (1981). Disrupted latent inhibition in the rat with chronic amphetamine or haloperidol-induced supersensitivity: Relationship to schizophrenic attention disorder. Biological Psychiatry, 16, 519-537. Solomon, P. R., & Staten, D. M. (1982). Differential effects of microinjections of D-amphetamine into the nucleus accumbens or the caudate putamen on the rat's ability to ignore an irrelevant stimulus. Biological Psychiatry, 17, 743-756. Trulson, M. E., & Jacobs, B. L. (1979). Long-term amphetamine treatment decreases brain serotonin metabolism: Implications for theories of schizophrenia. Science, 205, 1295-1297. Tsaltas, E., Preston, G. C., Rawlins, J. N. P., Winocur, G., & Gray, J. A. (1984). Dorsal bundle lesions do not affect latent inhibition of conditioned suppression. Psychopharmacology, 84, 549-555. Verney, C., Baulac, M., Berger, B., Alvarez, C., Vigny, A., & Helle, K. B. (1985). Morphological evidence for a dopaminergic terminal field in the hippocampal formation of young and adult rat. Neuroscience, 14, 1039-1052.

5,7-DHT LESIONS ATTENUATE LATENT INHIBITION Weiner, I. (1990). Neural substrates of latent inhibition: The switching model. Psychological Bulletin, 108, 442-461. Weiner, I., Lubow, R. E., & Feldon, J. (1981). Chronic amphetamine and latent inhibition. Behavioural Brain Research, 2, 285-286. Weiner, I., Lubow, R. E., & Feldon, J. (1984). Abolition of the expression but not the acquisition of latent inhibition by chronic amphetamine in rats. Psychopharmacology, 83, 194199. Weiner, I., Izraeli-Telerant, A., & Feldon, J. (1987). Latent inhibition is not affected by acute or chronic administration of 6 mg/kg DL-amphetamine. Psychopharmacology, 91, 345351. Weiner, I., Lubow, R. E., & Feldon, J. (1988). Disruption of latent

207

inhibition by acute administration of low doses of amphetamine. Pharmacology Biochemistry and Behavior, 30, 871878. Williams, J. H., & Azmitia, E. C. (1981). Hippocampal serotonin reuptake and nocturnal locomotor activity after microinjections of 5,7-DHT in the fornix-fimbria. Brain Research, 207, 95-107. Williams, J. H., Meara, J. R., & Azmitia, E. C. (1990). Effects of 5,7-dihydroxytryptamine injections in the fornix-fimbria on locomotor activity in photocell cages and the open field. Behavioural Brain Research, 40, 37-44. Young, A. M. J., Gray, J. A., & Joseph, M. H. (1992). Dopamine function in selective attention and conditioning: a microdialysis study using latent inhibition. Journal of Psychopharmacology, 6, ~112.