0306-4522/91S3.W+ 0.00 Pcr8amonPressptc IBRO

Neuroscience Vol. 42, No. I, pp. l-18, 1991 Printedin GreatBritain

THE BASOLATERAL AMYGDALA-VENTRAL STRIATAL SYSTEM AND CONDITIONED PLACE PREFERENCE: FURTHER EVIDENCE OF LIMBIC-STRIATAL INTERACTIONS UNDERLYING REWARD-RELATED PROCESSES B. J. Evanrn,*t

K. A. Moaara,* A. O’BRIEN* and T. W. ROBBE@

*Departments of Anatomy and ~Ex~~rn~~ Psychology, University of Cambridge, Downing Street, Cambridge CB2 3DY, U.K. Abstract-The effects on the expression of a conditioned plaoe preference of bilateral, excitotoxic amino acid-induced lesions of the basolateral region of the amygdala, or the ventral striatum, or asymmetric,

unilateral lesions of both structures were studied. The place preference was conditioned by exposing hungry rats to sucrose in a distinctive environment. Following acquisition, bilateral quisqualate-induced lesions of the basolateral amygdala, as well as bilateral quinolinate-induced lesions of the ventral striatum, abolished the conditioned place preference. Bilateral ventromedial, but not dorsolateral, quinolinateinduced caudate-putamen lesions attenuated the place preference. Combining a unilateral lesion of the basolateral arnygdala with a contralateral lesion of the ventral striatura also disrupted the conditioned place preference. These data provide further support for the hypothesis that the basolateral amygdala and ventral striatum are important parts of a neural system subserving stimulus-reward associations.

The conditioned place preference (CPP) paradigm is widely used to measure the rewarding properties of drugs, such as psychostimulants and opiates, electrical self-stimulation of the brain and also natural rewards, such as sweet solutions or a sexual partner.5,8~31,39,52.55*56 In this paradigm, the constellation of stimuli comprising a distinctive environment or “place”, if reliably associated with the primary reward, will subsequently elicit approach hehaviour towards and rn~n~n~~ of contact with this en~ro~ent in the absence of the primary reward.“as6 These cues presumably gain conditioned incentive properties by a process of Pavlovian conditioning. The dopaminergic innervation of the ventral striaturn, which arises in the midbrain ventral tegmental area,” has become a focal point of studies on the neural basis of such reward-related, or incentive motivational processes. Psychostimulant drugs such as n-amphetamine or cocaine, which enhance dopaminergic transmission, are not only self-administered directly into the ventral striatum,29 but will support the acquisition of a CPP. 5*t39,47*48,s2 However, while it is clear that enhancing dop~nergic tr~s~ssion in the ventral striatum facilitates approach responses to such drug-associated conditioned incentives, it is unclear whether this neurochemical mechanism mediates similar responses to more natural rewards, such tTo whom correspondence should be addressed.

CPP, conditioned place preference; CR, conditioned reinforcement; MLR, mesencephalic locomotor region.

Abbreviations:

as food or a sexual partner. Furthermore, it seems unlikely that stimulus-reward associations are formed in the ventral striatum, and more likely that they gain access to response output systems via this route, where modulation by the dopaminergic system can occur 6,lS,33,34.35,41 By contrast, there is considerable evidence that the amygdala, particularly its basolateral part, is fundamentally involved in stimulus-reward associations. As early as 1956, Weiskran~ suggested that the behavioural changes follo~ng amygdala lesions may indicate a role for the amygdala in the association of environmental stimuli with a variety of biologically important aspects of events, thus mediating the impact of their reinforcing value. More specifically, Spiegler and MishkinM demonstrated a marked impairment in remembering stimulus--reward associations over short delays by monkeys with amygdala lesions, which was not due to difficulties in object recognition. GaiTan and Harrison subsequently demonstrated that amygdalectomy profoundly disrupted the association of arbitrary stimuli (tones) with the intrinsic, incentive value of a primary reward (in this case food), arguing that the amygdala mediates the control over behaviour by secondary (or conditioned) reinforcers.1g-21 Recent neuroanatomical data have indicated that experiments on the role of the amygdala in stimulus-reward associations and on the ventral striatum dopamine system in the neurobiology of reward may be closely related. Thus, in contrast to earlier views that the amygdala and other telencephalic “limbic”

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structures influence emotional behaviour primarily through projections to the hypothalamus and brainstem,‘,‘0*24,49 efferent projections from the basolateral amygdala to the ventral striatum”*25~28~37,40,43,59 may be of major importance in mediating the impact of limbic processes on behavioural output.6,‘5,4’ On the basis of these neuroanatomical data and the demonstration of parallel projections to the ventral striatum from the subiculum and limbic frontal cortex,“~25~2*~2*a~37~40~57~58 the ventral striatum has been called a “limbic-motor interface”, that is, a site where affective processes occurring in limbic structures gain access to response systems.25,3M5,37Indeed, Mogenson and his colleagues have demonstrated that hippocampal and amygdaloid electrical stimulation affects the activity of ventral striatal neurons in a way modulated by coincident manipulation of dopamine transmission there.“s58d2 Furthermore, locomotor responses induced by pharmacological stimulation of the amygdala or hippocampus, or by exposure to a novel environment, are also affected by manipulation of the ventral striatum or the targets of its outflow, namely the ventral pallidum or mesencephalic locomotor region (MLR).33,36*62 In our own work, we have extended these observations to more complex situations in which the control over instrumental responses by conditioned reinforcers has been studied. Thus, excitotoxic amino acid-induced lesions of the basolateral amygdala were seen to attenuate the acquisition of a new response with a conditioned reinforcer in thirsty animals-a stringent test of conditioned reinforcement (CR),6 Subsequently, infusion of D-amphetamine dose-dependently increased responding for the CR, but to a lesser maximum extent in amygdalalesioned than in the control animals, which reflected the attenuated control over behaviour by CR in the former.6 In a more integrated behavioural context, similar amygdala lesions markedly impaired responding by male rats for a sexual reward, an oestrous female, presented under a second-order schedule in which responding was maintained by the contingent presentation of a visual CR which had gained salience by prior association with sexual interaction.‘4*‘5,4’ Again, n-amphetamine infused into the ventral striatum ameliorated the lesioninduced decrease in instrumental behaviour, provided CR was contingently presented during the session.15 Neither manipulation affected behaviour elicited by the primary reward, i.e. the consummatory sexual responses of mounting, intromitting’4,‘5 and ingestion.6v4’ This mediation by amygdaloid and ventral striatal mechanisms of the effects of CRs on instrumental behaviour seems likely to be related to the effects of manipulations of the same structures on the presumably Pavlovian process of CPP, although both the sensory control and nature of the mediating responses in CPP are not well established.” For the purpose of this article we will assume that these

effects can be related to a single process of incentivemotivation.39,4’ While the neuroanatomical, neurophysiological and our recent behavioural data are all consistent with the hypothesis that parts of the amygdala and the ventral striatum comprise a neural system concerned with incentive motivational processes, there is no direct evidence that the behavioural consequences of amygdala lesions or ventral striatal dopaminergic manipulations reflect the serial and integrated actions of related parts of such a system. In the present experiments, we have addressed this issue by investigating not only the behavioural consequences of bilateral, cell body lesions of the basolateral amygdala or ventral striatum, but also of an asymmetric lesion, or “disconnection” procedure in which a unilateral lesion of the amygdala has been combined with a contralateral lesion of the ventral striatum. This latter procedure disconnects the route of communication between the basolateral amygdala and ventral striatum by destroying either its origin or termination within each hemisphere. Powerful effects of such disconnection procedures have been reported in analysing auditory and visual interaction with the amygdala in primates. ‘9-2’We have used these procedures to study the effects of amygdala and ventral striatal lesions on a place preference conditioned by exposure to the high incentive ingestive reward, sucrose.‘6 EXPERIMENTAL

PROCEDURES

Subjects and housing

A total of 151 experimentally naive, adult male rats nomegicus) of the Lister Hooded strain (OLAC, Bicester, England) was used. They weighed approx. 250 g at the start of the experiments and were housed in pairs in a temperature-controlled room, maintained on a reversed light-dark cycle (12: 12 h light/dark, lights off at 08:OO).All testing took place in the dark phase. The subjects received their food (15 pellets SDS Rat Maintenance Diet per cage, approx. 10 g per rat) in the early evening, at the conclusion of the day’s testing, unless otherwise stated in the behavioural method. Animals were weighed each day and their body weights were shown to remain fairly constant throughout the duration of the experiment. Water was freely available unless otherwise stated in the behavioural method. (Rams

Apparatus The apparatus was made entirely from 3-mm Perspex (Plexiglas). The overall dimensions were 60 x 100 x 30 cm; this was subdivided into three compartments, two measuring 30 x 80 x 3Ocm and one 60 x 20 x 3Ocm, the whole apparatus being closed by a lid made of clear Perspex. The larger compartments had floor, walls, door and screen all either black or white. The black compartment had wood shavings on the floor while the white compartment contained fine sawdust. The end wall of these compartments each had a hole drilled, through which could be introduced a drinking spout attached to a burette. At the opposite end in the middle of the wall was a 12 x 12 cm vertically opening (guillotine) door which gave access to the third, smaller grey compartment. In each of the larger compartments an appropriately shaded screen measuring 18 x 14cm was placed IOcm before the doorway to minimize a rat’s ability to monitor visually the larger compartments whilst remaining in the grey compartment. The smaller compartment was

Limbic-striatal grey with a bare floor, and provided a starting chamber as well as a neutral environment. General behavioural method

The behavioural method consisted of three discrete phases: (i) pre-exposure phase, (ii) conditioning phase, (iii) test phase. Each phase was ended with a preference test. Prior to pre-exposure, all animals were handled daily. Any alterations to the general behavioural method are described under the appropriate individual experiments. Pre-exposure phase (three days). The purpose of this phase was three-fold: (i) to accustom rats to the taste of a 20% sucrose solution, (ii) to familiarize the subjects with the place preference apparatus and (iii) to accustom the animals to entering larger chambers from the grey compartment. At lights-off and prior to testing, all water bottles were removed from the home cages. At the end of the day, the animals were given free access to a 20% sucrose solution for 30 min in the home cage prior to feeding, to accustom them to the taste of 20% sucrose. The water bottles were then returned. Every day, each animal was placed into the grey compartment for 3 min. Subsequently, both guillotine doors were lifted and the animal allowed free access to the entire apparatus for a further 15 min. On the third such pre-exposure, a preference test was carried out in which the animals were given free access to the entire apparatus for 15 min, following 3 min in the grey compartment. During the 15 min, various indices of exploratory behaviour were recorded. Each compartment was subdivided into regions of equal floor area by line markings on the clear Perspex lid. Transitions, separably for “between compartments” and “between floor areas within compartments” were recorded, as was rearing behaviour. A transition was recorded as such when the entire head and body of a rat (but not necessarily the tail) had crossed the appropriate line. A “rear” was recorded when the animal moved from a stance with all four paws on the ground to a vertical one where both front paws were higher than a horizontal line level with the tops of the doorways. The data were recorded manually on a sheet representing the 15min time course, subdivided into 5-s epochs. The total time spent in each compartment was subsequently calculated to establish each animal’s initial preference. The animals were divided into two counterbalanced groups for conditioning on the basis of this initial preference test, matched for time spent on each side: “black” and “white”. Conditioning phase (15 days). In this phase a place preference was conditioned by compartment-selective exposure to sucrose solution in a counterbalanced procedure. On the odd numbered days (1, 3, 5, etc. to 13) each rat was restricted to its “paired” compartment according to the group to which it was assigned after pre-exposure, i.e. either black or white. This was accomplished by dividing the grey compartment in half with a grey Perspex divider in order to allow access to only one large compartment from each half of the grey compartment. Black and white animals were run concurrently, being placed in the appropriate side of the grey compartment for 3 min until, by lifting the guillotine doors, being allowed free access to that side of the apparatus for a further 15 min. On each of these days the animals were allowed to drink freely from a drinking spout connecting to a burette containing 20% sucrose (w/v), introduced through the holes at the end of the large compartments. The volume drunk by each animal was recorded on each day. On the alternate, even numbered days (2, 4, 6, etc. to 14) an identical protocol was followed except that the animals were restricted to the alternative, unpaired side of the apparatus and no burettes were presented. The order in which the subjects were exposed to the apparatus was reversed after two days. A preference test was carried out on day 15 as described in the pre-exposure phase (three days) above. The time spent in each compartment over the duration of the test and also

interactions

3

in 5-rain time bins was calculated. This indicated the postconditioning place preference. Animals did not continue to the final stage if they showed no absolute preference for their paired side compared to the unpaired side over both the whole test and the first 5-min epoch. Following this, the remaining animals were assigned to two counterbalanced groups for surgery: lesion and sham. These groups were matched for time spent on their paired and unpaired sides, and for their conditioning group (either black or white). Test phase (13 dnys). The effects of various neural manipulations and appropriate control procedures on the acquired place preference and associated ingestive and locomotor activity were investigated. Prior to surgery, the animals were given one more conditioning trial, i.e. exposure to sucrose in their paired side. The volume of sucrose drunk by each animal was recorded as the pre-operative ingestion level. The two groups were then taken for surgery. The subjects were allowed at least one week to recover, with food available ad Iibitum for all but the last day, when the controlled feeding schedule was reinstated. A preference test was subsequently carried out (day 11). On the following day, the subjects’ post-operative ingestion level was measured by giving free access to sucrose from the burette in the appropriate side of the apparatus. Surgical methods The subjects were anaesthetized with Avertin (tribromoethanol, Fluka A.G., F.R.G., in tertiary amyl alcohol: 1 ml/100 g body weight, intraperitoneal) and placed in a stereotaxic frame (David Kopf, Tujunga, California, U.S.A.), with the incisor bar set as detailed under the appropriate manipulation. Infusions of the appropriate excitotoxic amino acids were made via 30-gauge stainless steel cannulae attached to 5 ~1 syringes (Precision Sampling Ltd., Dynatech, Baton Rouge, Louisiana, U.S.A.) over a period of 1 min, with the cannula being left in place for a further 2-3 min. Experiment 1: lesions of the basolateral amygdnla. Bilateral lesions of the basolateral amygdala were made by infusing 1 ~1 of quinolinic acid (2,3-pyridinediacarboxylic acid, Sigma) as a 0.12 M solution in phosphate buffer, at each injection site. Two injections were made sequentially at the following coordinates in order to lesion the basolateral amygdala throughout its rostrocaudal extent: bregma +0.2 and -0.8; lateral f4.5; ventral -8.Omm from cortical surface (incisor bar + 5 mm). Experiment 2: lesions of the striatum. Bilateral lesions were made in the appropriate part of the striatum by infusing 2 ~1 quisqualic acid (/?-[3,5-dioxo-1,2,4-oxadiazolinin-2-yl]-L-alanine, Sigma) as a 0.12 M solution in phosphate buffer, at each injection site. The coordinates used were: ventral striatum: bregma +3.4; lateral k 1.7; ventral -7.2 mm from cortical surface (after Pellegrino et al., 1979, incisor bar + 5 mm); ventromedial caudate-putamen: bregma +2.5; lateral +3.5; ventral -5.5 mm from cortical surface (after Pellegrino et al., 1979; incisor bar + 5 mm); dorsolateral caudate-putamen: bregma +0.2; lateral +3.5; ventral -4.6 mm from cortical surface (after Paxinos and Watson,3* incisor bar -3.3 mm). Experiment 3: asymmetric lesion procedure. Asymmetric lesions were made by combining a unilateral basolateral amygdala lesion with a contralateral ventral striatal lesion, each being made by infusing the appropriate excitotoxin as described in Experiments 1 and 2a above. Control procedures. Unilateral basolateral amygdala lesions were made as for bilateral lesions, except that only the left or right site was used, chosen at random. Sham operations for each neural manipulation were made and consisted of identical infusions of nhosuhate buffer vehicle (PH 7.&7.4). Lesion assessment

At the end of behavioural testing, the subjects were perfused transcardially, under barbiturate anaesthesia, with

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B. J. E~ERITTet al.

5Oml 0.9% (w/v) NaCl at room temperature, followed by 15Oml 10% (w/v) formal saline. Brains were removed and post-fixed for 24 h and then transferred to a 10% (w/v) solution of sucrose in 0.2 M phosphate buffer and left for 48 h or until the brain sank. After blocking, sections were cut at 60pm on a freezing microtome, and every third section was mounted on egg-albumin-coated glass slides. Following oven drying, sections were stained with Cresyl Violet and coverslipped under Permamount. Lesions were detlned as regions of neuronal loss, which were usually associated with areas of intense gliosis. These

regions were mapped onto standardized sections of the rat brain.B Striatal lesioned brains were aiso processed for the demonstration of NADPH diaphorase after the method of Ellison et ~1.” since this procedure facilitates analysis of the extent of the lesion in both dorsal and ventral striatum. Stati9ticul analysis obvious results were subjected to a two or three factor analysis of variance (ANOVA), with neural manip~ation (lesion) as the between-subject variable and compartment

(side) as the within-subject variable, with time period (5min bins-time), or test period (pre- or post-operative) introduced as additional within-subject factors.3o Where appropriate, post hoc comparisons to chance levels were made using the Wilcoxon matched pairs signed ranks test statistic T.” Results are presented as means f standard errors. RESULTS

Experiment 1: the effects of bilateral quinolinic acidinduced iesions ofthe basalateral amygdala on conditioned place preference Specific method. Fifty-three animals were used. Eight animals were eliminated after conditioning, having not reached the criterion for conditioning stated in the method. Thus, 45 subjects proceeded to the test phase, 13 bilateral lesion, 14 bilateral sham, nine unilateral lesion and nine unilateral sham animals.

Fig. 1. Photomicro~aphs of control (a, b) and quinolinate-lesions (c, d) amygdalae stained with Cresyl Violet. In a, the distinctive basolateral region of the amygdala can be seen (BL, basal magnocellular and overlying lateral nucleus), medial to the external capsule (ec). This area is shown at larger magnification in b. In c, the effects of infusing quinolinate can be seen. There is marked gliosis and, as is shown in d, no neurons can be seen in the basal magnocellular nucleus. The lesions frequently include, as here, lateral parts of the central nucleus and the accessory basal nucleus. The piriform cortex and endopiriform nucleus are aiso occasionally damaged.

Limbic-striatal

5

interactions

Fig. 2. Schematic representation of a bilateral, quinolinate-induced excitotoxic lesion of the basolateral amygdala. The black area in the temporal lobes represents the region of neuronal loss (for description, see text). This representation of the lesion is typical for the group. BL, basolateral amygdala; C, central nucleus; Co, cortical nucleus; M, medial nucleus; GP, globus pallidus; H, hippocampus; IC, internal capsule; CPU, caudateputamen.

Subsequently, three animals died, one from each lesion group, and one unilateral sham animal. Therefore, 42 subjects proceeded to behavioural tests and lesion assessment. L&on assessment. In all cases, lesioned animals received either a bilateral or a unilateral lesion as intended. Typically, lesioned animals sustained more or less complete damage to the basolateral parts of the amygdala, throughout its rostrocaudal extent. There was also occasional damage to the central nucleus, particularly its lateral part, but not the medial or cortical nuclei. The pyriform cortex and perirhinal, temporal cortex was also variably

damaged in some animals. The overlying globus pallidus and striatum were unaffected by the infusions of quinolinic acid. The histological appearance of this kind of amygdala lesion is shown in Fig. 1 and the extent of a typical lesion is shown in Fig. 2. On the basis of post hoc histology, no animals were excluded from the behavioural_analysis. Thus, analysis was performed on 25 bilaterally operated animals, 12 lesions and 13 shams, and 17 unilaterally operated animals, nine lesions and eight shams. Behavioural results. Bilateral lateral amygdala

lesions of the baso-

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B. J.

=1

EVERITTet

A

Fig. 3. The effects on conditioned place preference of (A) bilateral q~nolinate-indu~d lesions of the baso~ater~ amygdala, (8) bilateral q~~ualate-indu~ lesions of the ventral striatum and (C) asymmetric lesions of the basolatera1 amygdala and contralateral ventral striatum. The data are shown as percentage of time spent in the paired and unpaired compartments after subtraction of time spent in the neutral compartment. The latter remained relatively constant (at about 250 s of the 900 s test). S, sham; L, lesion.

Place preference. The data are presented here, and throughout, as the time spent in each of the larger chambers as a percentage of the time spent in both. These percentage data did not alter the significance levels of analysis of absolute times in any case. The effect of the lesion on the significant preoperative place preference is shown in Fig. 3 (panel A). The previously established place preference was completely abolished in the lesioned animals, whereas the sham-operated animals showed a quantitatively smaller but qualitatively identical preference to their pre-operative data. There was a significant lesion x side interaction [F(1,23) = 7.68, P = 0.011. Further analysis revealed that this was due to the difference between the sham group preference, which is sig~fieantly different from chance (Wilcoxon’s T = 10.0, P -C0.01) and the lesion group preference, which is not (T = 28.5, P > 0.05). This effect was maintained over each 5-min epoch, i.e. there was no interaction with time (lo < 1).

al.

Ingestive and locomotor responses. Analysis of unconditioned measures of ingestive behaviour (ingestion by volume) and body weight, did not reveal any effects of the lesion, or the surgical procedure, on either factor (F < 1 in each case). Horizontal (locomotor activity) and vertical (rearing) exploratory behaviour in the sham animals was distributed differentially amongst compartments over the total test and each time bin, with activity highest on the unpaired side. This effect was attenuated in the lesioned animals. Analysis of these data showed that the differential locomotor activity in the shams was due to an increase of activity on the unpaired side, and that this was not observed in lesioned animals. This effect was also maintained throughout the test period, i.e. over each epoch. Thus, there were significant lesion x side interactions in the analysis of “total” and “within-compartment” activity [F( 1,23) = 5.04, P = 0.03 and F (1,23) = 6.19, P = 0.02], but not of “between-compartment” activity (F < 1). There was a significant effect of time [for total locomotor activity, F(2,46) = 23.7, P < O.OOOOl] which indicated an overall decrease in activity as the test progressed, but this was regardless of side or lesion. There was a greater level of rearing on the unpaired side in the sham animals which was atten~t~ by the lesion, but the lesion x side interaction just failed to reach significance [F(1,23) = 3.98, P = 0.061. Behavioural results-unilateral lesions of the base lateral amygdala. There was no effect of the lesion on the significant pre-operative place preference (F c: 1, see Table 1) and no interaction with any other factor. The unilateral lesions also had no effect on ingestion or body weight, nor on any aspect of exploratory behaviour (F < 1). Experiment 2a: the effects of bilateral, q~~q~a~~c acid-induced lesions of the ventral striatum on conditioned place preference Spectjic method. Twenty-eight animals were used. Following the conditioning phase, two rats were

Table 1. Effects of unilateral excitotoxin-induced basolateral amygdala, unilateral ventral striatal, bilateral ventromedial dorsal striatal or bilateral dorsolateral dorsal striatal lesions on conditioned mace nreference Lesion

Percentage time paired side

Percentage time unpaired side

Sham Unilateral BLA Sham Unilateral VS Sham Bilateral ventromcdial DS Sham Bilateral dorsolateral DS

63 + 9 60+7 66.5&4 64*7 60&9 52& 11* 57&4 59&7

37+ 12 40+8 33.5 + 9 3656 4oi7 5oi9 43+6 41* 5

BLA, basolateral amygdala; VS, ventral striatum; DS, dorsal striatum. Data are percentage times spent on paired and unpaired sides after correction for time spent on neutral side, +S.E.M. * = P < 0.01. Note that time spent on the “neutral” side of the apparatus (range = 236309 s) did not change significantly in any group as a result of lesion or sham surgery. In groups having a significant preference, time spent on the “paired” side ranged from 376 to 445 s and time on the “unpaired” side ranged from 181 to 259s. Following bilateral ventromedial caudateputamen lesion, time on the “paired” side was reduced to 339 s and time on the “unpaired” side increased to 313 s.

Limbic-striatal as they had not achieved the criterion for conditioning; thus 26 animals proceeded to the test phase. During the period following stereotaxic surgery two animals (both from the lesion group) died. Therefore, 24 animals (12 lesions and 12 shams) proceeded to behavioural tests and lesion assessment. Lesion assessment. Quisqualate-induced lesions were targeted at the nucleus accumbens and lesioned animals generally sustained radical damage to much of the ventral striatum. This typically included more or less complete damage to the entire nucleus accumbens, but also variable and small amounts of the overlying (i.e. ventral part) of the caudateputamen and/or the underlying olfactory tubercle. The channels of ventral pallidal neurons lying between the nucleus accumbens and olfactory tubercle were also sometimes destroyed by the excitotoxin. The lesions did not encroach upon the preoptic area, nor on the medial and lateral septal nuclei. The loss of neurons in lesioned brains was accompanied by intense gliosis and general shrinkage of this area, such that the ventral pial surface of the brain appeared closer to the caudateputamen than in control brains (Fig. 4). No lesioned animal was excluded post hoc from the behavioural analysis on the basis of histology. Thus, the following analyses were performed on 24 animals: 12 shams and 12 lesions. The extent of a typical lesion is shown in Fig. 5.

eliminated

interactions

1

The lesion group showed a significant decrease in rearing behaviour compared with the sham group (rears/min in paired, unpaired and neutral sides, respectively: shams: 3.6, 4.4 and 3.4; lesions: 2.3, 2.4, 1.9) [F(l,22) = 7.29, P = 0.0131. Similar trends in inter-compartment activity as seen with locomotor behaviour were observed, but these did not reach statistical significance (F < 2). Behavioural results: unilateral lesions of the ventral striatum. There was no effect of unilateral ventral

striatal lesions on the significant pre-operative preference (n = 6, F < 1, see Table 1).

place

Experiment 2b: the eflects of bilateral quisqualic acidinduced lesions of the ventromedial caudate-putamen on conditioned place preference

Specific method. Eighteen animals were used and all exhibited a significant place preference following conditioning. Thus, 18 subjects proceeded to surgery and test phase: eight lesions and 10 shams. Two animals died following surgery. Therefore, 16 animals (seven lesions, nine shams) proceeded to behavioural tests and lesion assessment. Lesion assessment. The typical extent of a ventromedial caudateputamen lesion is shown in Fig. 7. It can be seen that the area of neuronal loss involved largely ventromedial regions of the caudateputamen, extending dorsally to central areas, but not encroaching on dorsolateral aspects. The globus Behavioural results. Place preference. The effects of the lesion on the pallidus was never affected by the quisqualate infusions. No lesioned animal was excluded post pre-operative significant place preference are shown in Fig. 3. Following surgery, there was a significant hoc from the behavioural analysis on the basis of lesion x side interaction [F(2,44) = 11.9, P < O.OOl] neurohistology and so the following analysis was indicating the significant, though slightly reduced performed on nine shams and seven ventromedial post-operative preference in the sham group (T = 7.0, caudate-putamen lesioned rats. Behavioural results. P < 0.01) compared to the lesion group preference which did not differ significantly from chance. Thus, Place preference. Table 1 shows the effect of bithe conditioned place preference was completely lateral lesions of the ventromedial caudate-putamen abolished following bilateral lesions of the ventral on the conditioned place preference. There was a striatum. significant difference between lesion and sham group Ingestive and locomotor responses. Pre-surgery preferences [F(2,28) = 3.6, P = 0.041, the lesion group showing no significant preference, as compared to ingestion levels were not significantly different chance levels, whilst the sham group had maintained between groups but, after surgery, the lesion group showed a small but significant decrease in the volume its pre-operative preference, although this was someof sucrose ingested as compared to the shams during what reduced (T = 5, P c 0.01). a 15min ingestion test [shams: 12.8 + 0.8 ml; lesions: Ingestive and locomotor responses. Analysis 9.6 + 1.2 ml; F(1,22) = 8.84, P = 0.0071. revealed a significant lesion x side interaction due to an alteration in the distribution of activity in Post-operatively there was no significant difference between groups in overall locomotor activity. the lesioned animals similar to that seen in ExperHowever, the sham animals showed less activity in iment 2a. This was reflected in total [F(2,28) = 3.4, P = 0.0461 and within- [F(2,28) = 3.5, P = 0.0451, but the paired compartment compared to the unpaired compartment and this differential distribution of not between-compartment activity. activity was attenuated in the lesioned group (Fig. 6). Experiment 2~: the effects of bilateral quisqualic acidThis resulted in a significant lesion x side interaction, induced lesions of the dorsolateral caudate-putamen on with the lesioned animals showing greater activity conditioned place preference in the paired compartment compared with shams Specific method. The previous experiments had [F(2,44) = 8.26, P = O.OOl]. This pattern of activity shown no evidence of any inherent factors in the reflected a significant change in within-compartment apparatus adverse to conditioning in either the white [F(2,44) = 5.66, P = 0.007] but not between-compartment activity in the lesioned animals. or black compartments, and so in this experiment the

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B. J. EVERITT et al.

‘ , ,

Limbic-striatal

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Fig. 5. Schematic representation of a bilateral, q~~u~t~indu~ exeitotoxic lesion of the ventral striatum. The black area represents the region of neuronal loss (for description, see text). This representation of the lesion is typical for the group. pre-exposure phase and initial preference test were omitted. Twenty-six animals underwent the conditioning phase (eight pairings on each side). Of these, 24 acquired a significant place preference and were assigned to matched groups for the test phase, consisting of 12 lesions and 12 shams. One sham animal died during surgery. Therefore, 23 animals (12

lesions and 11 shams) proceeded to hehavioural tests and lesion analysis. Lesion assess~enr. As can he seen in Fig. 8, the area of neuronal loss in a typical quisqualate-induced lesion was restricted to dorsal and dorsolateral parts of the caudate-putamen. Neuronal loss did not extend into ventromedial areas of the striatum, nor into

Fig. 4. Photomicrographs of control (a, b) and quisqualate-lesioned (c, d) ventral striatum stained with Cresyl Violet. In a, the area of the nucleus accumbens (NAS) around the anterior commissure (ac) can be seen. This is shown at higher magnification in b where the typical ventral striatal neurons are visible. In c the marked gliosis around the anterior commissure is shown and in d the clear neuronal loss. Notice that the ventral surface of the brain is now closer to the anterior commissure as a result of shrinkage of the neural tissue following loss of accumbens neurons. IC, island of Calleja; CT, olfactory tubercle.

B. J. Evsmun et al.

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Fig. 6. The effects of quisqualate-induced

ventral striatal

lesions on locomotor activity within and between each compartment.

the globus pallidus. On the basis of post hoc neurohistology, one lesioned animal was shown to have sustained only a unilateral lesion and so was excluded from the behavioural analysis. The following analysis was therefore performed on 11 shams and 11 dorsolateral caudat~putamen lesion animals. The extent of a typical lesion is shown in Fig. 9. Behauio~ru6 results.

Place preference. Prior to surgery, both groups demonstrated significant place preferences compared to chance levels (lesions: T = 0, n = 11, P < 0.001; shams: T = 0, n = 11, P < 0.001; Table 1). Postoperatively, there was no significant difference between the groups [F(2,40) = 0.16, n.s.1. However, there was a significant reduction in the magnitude of the place preference displayed pre- and postsurgery in the sham group and a similar trend was seen in the lesion group, although this was not signi~cant [shams: F(2,40) = 3.88, P = 0.028; lesions: F(2,40) = 2.83, P = 0.071. Ingestive and locomotor response. There was no significant difference in any aspect of locomotor activity between groups; both lesions and shams exhibited the same distribution of activity between compartments as seen in sham animals in the previous experiments, i.e. reduced locomotion in the paired compartment. There were no significant differences between the groups on any other behavioural variables measured, including ingestion. Experiment 3: the effects of asymmetric lesions of the baso~utera~ ~ygda~a and ventral striatMm on conditio~ed place preference

In Experiments 1 and 2, both bilateral amygdala and also ventral striatal lesions were shown to abolish a previously established place preference, while unilateral lesions of either structure were without significant effect. To investigate the hypothesis that these structures form part of a neural system mediating the processes underlying CPP, an asymmetric lesion procedure was adopted. Thus, a unilateral basolateral

amygdala lesion was combined with a lesion of the contralateral ventral striatum. SpeciJic method, Twenty-six animals were used, with the pre-exposure phase omitted entirely, animals being assigned randomly to conditioning groups. The earlier experiments indicated that the apparatus does not generate a consistent initial side preference and it was decided, therefore, that omitting the pre-exposure period might result in better conditioning by reducing latent inhibition.30b The conditioning phase consisted of eight conditioning trials. One animal was eliminated, having not achieved the criterion for conditioning. Thus, 25 animals proceeded to surgery: 13 shams and 12 lesions. One animal, from the sham group, died during surgery leaving 24 animals for behavioural testing. Lesion assessment. Typically, lesioned animals sustained unilateral lesions of the basolateral amygdala throughout its rostrocaudal extent, and lesions of the ventral striatum, comprising the whole of the nucleus accumbens, variable parts of the olfactory tubercle and the ventromedial caudate-putamen. Thus, the unilateral lesions seen in this experiment resembled closely those described in Experiments 1 and 2a above. Five animals had no lesion at either one or both sites and were therefore excluded from the behavioural analysis. Thus, analysis was performed on 19 animals, 12 shams and seven lesions. Behavioural results.

Place preference. The results of post-operative preference tests are shown in Fig. 3C. In both groups, the post-operative preference was smaller than that seen pre-operatively, but the post-operative preference for the sham group differed significantly from chance (T = 11, n = 12, P < 0.05), whereas the lesion preference did not (T = 13, n = 7, P > 0.05). There was a significant lesion x side interaction [F( 1,17)= 6.77, P = 0.021, indicating the complete attenuation of place preference in the lesioned animals. Ingestive and locomotor responses. There were no significant effects of the asymmetric lesions on sucrose ingestion or on body weight. However, the lesion group tended to ingest less sucrose following deprivation than did shams [F(1,17) = 3.10, P = 0.091. Nor were there any significant effects on measures of exploratory behaviour (locomotor activity or rearing) over the whole test or 5-min epochs. However, there were significant effects of time on locomotor activity and rearing [F(2,34) = 12.58, P < 0.01, and F(2,34) = 8.99, P < 0.011, indicating a decrease in the overall level of exploratory behaviour during the test, regardless of lesion or side. Efsects qf satiation on conditioned place preference

The effects of satiation on the conditioned place preference were studied in the sham-operated animals from Experiment 2b. Rats were re-conditioned by allowing access to sucrose or nothing in each of two conditioning tests. Prior to the place preference test, rats were allowed free access to food in their home

Limbic-striatal

interactions

Fig. 7. Schematic representation of a bilateral, q~squ~at~indu~

excitotoxic lesion of the v~~orn~i~ dorsal striatum. The black area represents the region of neuronal loss (for description, see text). This ~pr~ntatio~ of the lesion is typical for the group.

cages for 24 h and then tested in the preference apparatus in the usual way. The results showed that the preference in satiated rats was not different from that seen in the animals when tested in a food-deprived state (mean time in paired and unpaired sides, respectively, satiated: 381 s and 190 s; deprived: 392 s and 185 s; F < 1). DlsCUSSION

The effects of various neural manip~ations on a place preference conditioned by exposure to sucrose

were studied. Significant pre-operative

place prefer-

ences were abolished in subjects receiving bilateral lesions of the basolateral amygdala. A similar abolition of place preference followed lesions of the ventral striatum, both those involving the nucleus accumbens and those involving the ventromedial caudate-putamen. Bilateral amygdala lesions had no effect on unconditioned measures of ingestive behaviour, but reduced exploratory behaviour in a manner consistent with the loss of the place preference. However, the ventral st~a~l-l~ion~ animals showed a slight reduction in sucrose ingestion following 23-h food deprivation, changes in locomotor activity in the paired and unpaired compartments of

12

B. J. EVERITTet al.

Limbic-striatal interactions

13

Fig. 9. Schematic representation of a bilateral, quisqualate-induced excitotoxic lesion of the dorsolateral dorsal striatum. The black area represents the region of neuronal loss (for description, see text). This representation of the lesion is typical for the group. the apparatus compared to sham-operated controls, and reductions in rearing, although in each ease the accnmbens-lesioned animals were more impaired than those receiving lesions to the ventromedial caudate-putamen. Unilateral amygdala and ventral striatal lesions, bilateral lesions of the dorsolateral striatum and all sham operations had no effect on the previously established CPP.

The asymmetric lesion procedure, which combined a unilateral lesion of the basolateral amygdala with a contralateral lesion of the ventral striatum, also abolished the conditioned place preference. In this experiment, the post-operative place preference in sham-operated animals was quantitatively smaller than in the above, bilateral lesion experiments, and comparison of the magnitude of the effects of

Fig. 8. Photomicrographs of a control (a, b) and quisqualate-lesioned (c, d) dorsolateral dorsal striatum stained with Cresyl Violet. The area of neuronal loss caused by infusion of quisqualate is shown in c (cf. a). The border of the area of neuronal loss is quite sharp and indicated by arrowheads. The loss of neurons in the lesioned brain is also shown at higher ma~i~cation in d (cf. b).

14

B. J. Evarun ef al.

bilateral versus ascetic lesions is made dillicult by the possibility of a floor effect. The results following the disconnection procedure strongly support the view that the amygdala and ventral striatum interact serially in this behavioural context, although careful consideration of the behavioural and neuroanatomical results is necessary before discussing this further. Behavioural considerations

Both amygdala and ventral striatal lesions abolished the CPP. Behavioural measures taken during the preference tests, as well as control experiments, indicated that this was a rather selective effect of the lesions. Furthe~ore, although the consequences of the lesions in terms of abolition of the CPP were similar, it was clear from other measures that the two preparations were different. Measures of the ingestion of sucrose following 23-h deprivation demonstrated that there was no deficit in primary motivation following basolateral amygdala lesions and that this could not underlie the loss of the CPP. The hypodipsia which is well known to follow electrolytic lesions of the amygdaia,4*q,‘8~42 is not seen after excitotoxic amino acid-induced, axon sparing lesions whether water, saccharine or sucrose is provided.i2 This emphasizes the irn~~~~ of using the methods employed here to lesion this structure when attempting to define the functional consequences of damage to the amygdala itself, rather than to fibres of passage. Although there was a significant reduction in sucrose ingestion following ventral striatal lesions, this was minor and it is difficult to envisage how it could underlie such a large impairment in CPP. Indeed, in a control experiment, sham-lesioned rats that were given a CPP test after free-feeding showed a place preference which was not significantly different from that observed when they were tested under mildly deprived conditions. No changes in sucrose ingestion followed other striatal or the asymmetric lesions. In controls, locomotor activity and rearing were differentially distributed between compartments during the preference test in that they reared more and were more active in the unpaired side when sucrose was no longer presented in the paired side; this presumably reflects an adaptive response to reinforcer omission. Overall, amygdala-lesioned rats showed no absolute change in activity and thus the change in CPP could not be attributed to a gross change in locomotor activity. However, there were differences in the dist~bution of this activity between compartm~ts in the apparatus. Thus, the amygdalalesioned rats were fess active in the unpaired side and also reared less in this environment than did controls during the preference tests conducted one week after surgery. Rats with lesions of the ventral striatum or ventromedial caudate-putamen similarly showed no gross change in levels of locomotor activity, but did show a quite different distribution of activity between sides in being significantly more active on the paired

side during the extinction test, such that there was no difference between activity on paired and unpaired sides, as usually seen in controls. Whereas amygdalalesioned animals showed a simple loss of the differential distribution in activity between sides shown by controls, ventral striatal- and ventromedial caudate-putamen-lesioned animals showed an inappropriate increase in activity on the side where least activity was seen in controls. This perhaps suggests an increase in activity independent of control by environmental contingencies. No changes in activity were seen following lesions of the dorsolateral striatum or the as~met~c lesions. The latter observation is important since it suggests that the loss of CPP following asymmetric lesions reflects disruption of a discrete process subserved by interactions between amygdala and striatum, and that the hyperactivity seen following bilateral ventral striatal lesions may have been rather a non-specific, release phenomenon. One possible explanation for the attenuation or abolition of place preference following the lesions is that it was consequent upon impaired discrimination by the animals of the sensory characteristics of the paired and unpaired ~ompar~ents. The data on this point are not entirely str~ghtfo~ard, partly because measures of sensory di~~mination or threshold usually depend upon reinforced discrimination which may be disrupted indirectly by the effects of amygdala lesions on stimulus-reward associations. The bulk of the literature1~21~27~32~44 indeed suggests the latter interpretation. In our own work we have shown that amygdala lesions can reduce the efficacy of conditioned reinforcement in the acquisition of a new response, while not affecting the ability of the same stimulus to control discriminated approach behaviour to a water dipper.‘j However, amygdala lesions do impair such di~rimination when approaching a sucrose reward.16 In both of these situations the light and dipper noise act potentially as conditioned incentives for approach, but also as possible discriminative stimuli which occasion an instrumental panel push. It is possible that the amygdala lesion impairs only the former process. This would predict that discrimination which depends on predominantly stimulus-response rather than stimulus-reward associations, e.g. conditional discriminations, would be more resistant to amygdala lesions and demonstrate that rats with amygdala lesions discriminate the stimuli in sensory terms. This prediction is bohtered by the relative lack of effect of ventral striatal dopamine depletion on a conditional visuo-spatial discrimination,41a although further research on this issue is clearly required. Using either sucrose or water as the primary rewards, amygdala lesions markedly attenuated the control over instrumental behaviour by conditioned reinforcement in thirsty animals (Ref. 6: Burns, Robbins and Eve&t, unpublished observations). Such results are consistent with data from a variety of experiments

Limbic-striatal interactions which indicate the special role of the amygdala in stimulus-reward associations’5~‘e2’~*7~4’~” and indicate that alterations in discrimination following amygdala lesions are not necessary for the observed impairment in such associations. Therefore, we suggest that the association of arbitrary environmental cues with reward (in this case sucrose) and the subsequent ability of those cues to direct conditioned approach depends on processes occurring in the amygdala. Further, control by the conditioned incentives over approach responses which bring the animal into contact with an environment where reinforcement is likely to occur, depends not only upon the ventral striatum, but on interactions between this structure and the basolateral amygdala. The consequences of amygdala-ventral striatal disconnection strongly support this view. Neuroanatomical considerations

There is now abundant evidence demonstrating rich connections between the ventral striatum and “limbic” structures such as the allocortical hippocampal formation, juxtallocortical limbic cortical structures including the anterior cingulate and other regions of the medial frontal cortex, as well as the basolateral amygdala (which may now be viewed as a quasi-cortical structure7).2~1’~28*281,37,40~43 In the past, the major output from telencephalic components of the limbic system was seen primarily to target hypothalamic and brainstem autonomic structures.‘.24 This rich system of projections appeared to be perfectly organized to underlie integrated endocrine, autonomic and behavioural responses to salient environmental events which together characterize emotional responses.44 However, it has never been clear from such an account as to how complex behavioural patterns, especially instrumental actions, might be recruited through connections between, for example, the basolateral amygdala, ventromedial hypothalamus and the medullary vagal complex. 49 The demonstration of direct interactions between the central amygdala and brainstem in mediating fear-potentiated startle” is indicative of the nature of the behavioural and autonomic responses likely to be mediated by such a system. However, the description by Heimer and WilsonZ5 or the relatively discrete entity of the ventral striatum, incorporating as it does the nucleus accumbens septi, ventromedial caudateputamen and olfactory tubercle, together with the definition of its limbic allocortical afferents,LL,2*,28a,37,U),43 has radically altered the perception of the limbic system and the routes by which affective processes occurring in limbic structures might gain access to higher order components of the motor system,3S35 in a way parallel to the hypothalamic and brainstem targets*0s49which dominated such concepts hitherto. Until recently, the majority of tests of the hypothesis that this targeting of outflow from the hippocampus and amygdala to the ventral striatum indeed represents the route

15

by which affective processes result in behavioural responses, involved relatively simple locomotor responses to novelty, or to electrical or chemical stimulation of the hippocampus, amygdala or ventral striatum itself.33-M~s741 While these data clearly indicate that such a system of connections could indeed act serially to alter response output, they do not address directly the issue of the kinds of situations in which such a system might be important. In particular, they do not specify the sorts of processes occurring in the amygdala and, indeed, other limbic afferents, nor the way that they might affect different kinds of behavioural response. Indeed, in the present experiments the disconnection procedure had no effect on locomotor activity, which might then represent a distinct outcome of ventral striatal activity. The results of our earlier experiments clearly indicated that the basolateral amygdala is involved in the association of stimuli with reward as measured by the acquisition of a new response with conditioned reinforcement or the maintenance of responding by conditioned reinforcers under a second-order schedule.6*‘5,4’The experiments reported here have reached a similar conclusion using a third method of studying stimulus-reward associations. Previous experiments demonstrated that responding impaired by lesions of the amygdala could be “gain-amplified”, albeit from reduced post-lesion baselines, by coincident enhancement of dopaminergic transmission in the ventral striatum.6.‘5~41While being strongly indicative of an interaction between amygdala and ventral striatum it is entirely plausible that the influences on behavioural output involve quite separate, though perhaps related, processes. However, the results of the present experiments using the place preference paradigm strengthen the view that these structures do indeed interact to determine approach responses to conditioned incentives. Thus, it is difficult to argue against such an interaction when considering the behavioural consequences of the disconnection surgery in which contralateral lesions were made of one of each pair of the implicated structures. If the ventral striatum does represent a nodal point for the convergence of limbic afferents onto a putative motor integrating system, then the way in which ventral striatal efferents gain access to elements of the motor system must be considered. There are many alternatives. In the past, much emphasis has been placed on projections of the nucleus accumbens to the pars compacta of the substantia nigra, thus providing a route by which activity in the dorsal striatum, concerned as it is with the initiation of movement, can be influenced!lb More recently, however, the rich outflow from the ventral striatum to the ventral pallidum has been emphasized. 2,3M5 This projection, which parallels dorsal striatal projections to the dorsal pallidum, has highlighted a more direct means by which the ventral striatum can influence motor responses, since the ventral pallidum projects directly to the subthalamic

B. J. EVERITT et al.

16

nucleus, substantia nigra and the ~d~c~o-pontine region22*23*3M6,H) which appears homologous with the MLR. This has heen the major focus of attention of the studies of Mogenson and colleagues who have shown that Locomotor activity generated by excitatory amino acid-induced stimulation of the hippocampus or amygdala, or by blocking GABA transmission in the ventral pallidum, is readily blocked by MLR lesions or procaine infusion into the region. 33-36, s7-61 There have to date been few experiments on more integrated behavioural responses following manipulations of this extensive neural system. Lesions of the ventral palhdum resulted in a lowering of the “break point” in responding for cocaine and heroin self-administration.26 However, this interesting result is tempered by the location of the lesions, which appear to be more in the bed nucleus of the stria terminalis than in the ventral pallidum (Fig. 7 of Ref. 26). Indeed, it must be borne in mind that the ventral pallidum is an exceptionally difficult structure to lesion selectively. It is but one component of an extremely complex group of structures comprising the basal forebrain, the others being the extended amygdala and the magnocellular corticopetal neurons which include the cholinergic nucleus basalis of Meynert.* Many studies claiming to have lesioned the latter structure following ibotenic acid infusions into the basal forebrains have, in fact, damaged the cholinergic neurons there to a relatively minor extent, but have destroyed the sub- and post-commissurai parts of the ventral pallidurn in its entirety.17 It will prove interesting in due course to re-appraise the often profound effects of these lesions in the context of reward-related and/or response-selection processes, rather than in terms of mnemonic processes as has been the case hitherto. Lesions of the brainstem MLR target of ventral pallidal projections block the acquisition of both an amphetamine- and a morphine-induced CPP, although the same lesions had no effect on CPP if

made after a~uisition.3 The authors argue that the results indicate the importance of the MLR in the unconditioned, incentive motivational responses to opiates and stimulants, the effects of which are mediated by forebrain sites such as the ventrai striaturn, but depend on outputs channelled through the MLR in order to elicit the appetitive responses of approach and exploration on which CPP acquisition depends. It is not clear whether similar approaches to natural rewards depend upon this motor outflow from the ventral striato-pallidal system, nor whether outputs relayed to the mediodorsal thalamus22~23 should also be considered in this context, since by this route limbic-stbatal processes may not only m-enter limbic circuitry, 35 but also influence response selection at a higher level which involves the frontalprefrontal cortex, including the supplementary motor area. CONCLUSION

The results of the experiments reported here strongly suggest that the basolateral parts of the amygdala and the ventral striatum to which they project form an essential substrate for incentive motivational responses to stimuli which have gained salience through their predictive association with a natural, ingestive reward, sucrose. The nature of the conditioned place preference paradigm is such that the discrete processes subserved by each structure or, indeed, both in combination are difficult to define. However, when taken in conjunction with data from this and other laboratories reviewed above, the results are consistent with the view that the amygdala is involved in the association of environmental stimuli with reward, while the ventral striatum is involved in motor response output directed towards such stimuli. ~ck~owle~ge~ents-The research reported here was supported by a Medical Research Council Project Grant to B.J.E. and T.W.R. We thank Keith Page for his help with histology.

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