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From M.A. Bozarth (1987). Neuroanatomical boundaries of the reward-relevant opiate-receptor field in the ventral tegmental area as mapped by the conditioned place preference method in rats. Brain Research, 414: 77-84.

Neuroanatomical Boundaries of the Reward-Relevant
Opiate-Receptor Field in the Ventral Tegmental Area
As Mapped by the Conditioned Place Preference Method in Rats

Michael A. Bozarth

Center for Studies in Behavioral Neurobiology

Department of Psychology
Concordia University
Montreal, P.Q. H3G 1M8 CANADA

 

Abstract

The conditioned place preference produced by morphine microinjected into the ventral tegmental area was studied in rats. Cannula placements were varied along the rostro-caudal plane to determine the approximate anatomical focus of morphine’s rewarding effect. Microinjections within a 1.4 mm range produce a significant change in place preference suggesting that morphine injected into this zone is rewarding. Injection sites rostral and caudal to this zone were ineffective as were injections ventral to this region. The approximate anatomical boundaries of the reward-relevant opiate receptor-field within the ventral tegmental area correspond well with the distribution of the A10 dopamine-containing cell bodies.
 

A number of studies suggest that the rewarding effects of opioids may involve an action in the ventral tegmental area (see 1, 7). Opioids are reinforcing when directly injected into this region, as demonstrated by studies of intracranial self-administration (4, 36). Dopamine-depleting lesions of this system block the acquisition of intravenous heroin self-administration (8), and narcotic antagonist microinjections into this region appear to attenuate the reinforcing effects of intravenous heroin (9). Opioids microinjected into the ventral tegmental area also produce a conditioned place preference (6, 25, 26). This method offers independent corroboration of a drug’s rewarding effect by measuring the association developed between specific environmental stimuli and the rewarding action of a drug (28); when tested in the drug-free condition, animals will increase the amount of time spent in the compartment of a shuttle box that has been associated with the rewarding effect of a drug. Conditioned place preferences have been demonstrated with a variety of drugs that have well documented rewarding actions (see 2). In addition, a conditioned place preference has been reported following the injection of an enkephalinase inhibitor into the ventral tegmental area (12).

Intracranial self-administration studies are perhaps the most direct demonstration of the rewarding effect of central morphine injections. This technique has been used to directly compare the rewarding effect of morphine injected into various brain regions (6). There is, however, a serious limitation to the use of this technique to determine the neuroanatomical boundaries of the population of opiate receptors that are responsible for opiate reward within a given brain region.

In intracranial self-administration studies, the animal controls the number of infusions and hence the total volume of drug injected into its brain. Animals with a high rate of self-administration have a larger field of effective drug spread than animals with low response rates; even microinjections through cannulae that are distal to the site of drug action may be rewarding when significant concentrations of drug have diffused to the reward-relevant receptors (see Figure 1). Therefore, it is difficult to estimate the distance of cannula placements to the reward-relevant receptors using intracranial self-administration studies (see 1).

 

Drug Diffusion

Figure 1: Because changes in the infusion volume produce a concomitant change in the field of effective drug action, attempts to extrapolate the anatomical boundaries of the receptor field mediating drug reward from cannula placements must use a fixed infusion volume. Reprinted from (1).

 

This problem can be overcome by injecting each animal with the same volume of drug and assessing reward using the conditioned place preference technique. Cannula placements can then be meaningfully compared to determine the location of the reward-relevant receptors. With this approach, the anatomical limits of the brain regions mediating reward from a given drug can be more precisely defined in relation to various microinjection sites. This anatomical localization can then direct electrophysiological and neurochemical studies designed to delineate the mechanisms of drug reward.

Methods

Subjects

Experimentally naive, male, Long-Evans rats (weighing 350 to 400 g) were unilaterally implanted with 22 gauge guide cannulae aimed at the ventral tegmental area. With the upper incisor bar 5 mm above the interaural line, the coordinates ranged from 2.0 to 4.4 mm posterior to bregma, 0.6 mm lateral to the midsagittal suture, and 7.8 to 8.2 mm ventral from dura. Sodium pentobarbital (60 mg/kg, i.p.) was used as the anesthetic with atropine sulfate (0.04 mg/kg, s.c.) and penicillin G (30,000 units, i.m.) given prophylactically. Obturators were fitted approximately 0.25 mm beyond the tips of the guide cannulae and remained in place except during drug infusions. All testing occurred during the light phase of a 12-hour light/12-hour dark cycle of illumination. Rats were individually housed and had free access to food and water in their home cages.

Apparatus and Procedure

Place preference was measured in a shuttle box (25 x 36 x 35 cm) with a plywood floor on one side and a plywood floor covered with wire mesh on the other. The amount of time spent on each side of the box was automatically recorded. Rats were allowed access to the entire shuttle box for 15 minutes per day on five consecutive days; the last day served as an indication of the animals’ initial place preferences. After these preconditioning trials, they received four daily injections of morphine while being forced to remain on their nonpreferred sides for 30 minutes. Following the four days of conditioning, the rats were injected with drug vehicle and tested again for their place preferences (15 minutes).

An electrolytic microinfusion transducer system (3) was used to unilaterally inject morphine sulfate into the ventral tegmental area immediately before each of the four conditioning trials. A 150 mA infusion current delivered 250 ng of morphine sulfate (750 pmoles morphine base) dissolved in 500 nl of Ringer’s solution. Infusions were delivered over 28 seconds through a 28 gauge injection cannula that extended 0.5 to 1.0 mm beyond the guide cannula. An additional 30 seconds was allowed for drug diffusion before the injection cannula was removed from the guide cannula. Ringer’s solution (500 nl) was injected prior to the test trial.

Histological Analysis

Following completion of the behavioral testing, the rats were deeply anesthetized with sodium pentobarbital (approximately 90 mg/kg, i.p.) and perfused intracardially with isotonic saline followed by a 10% formalin solution. The brains were removed and fixed in formalin for at least three days. The brains were then frozen and sectioned along a coronal place at 40 micron intervals. After staining with formol-thionin, the brain sections were viewed at approximately 10 times magnification, and the cannula placements were identified according to the brain atlas of Pellegrino, Pellegrino, and Cushman (23). The most dorsal penetration of the cannula was used as the reference point. Changes in place preference were then plotted as a function of the number of millimeters that the cannulae were posterior to bregma on de Groot’s (10) plane of sectioning.

Because the determination of cannula placements was of central importance to this study, special care was taken regarding the method used to classify placements. The initial groupings were done with the knowledge of the place preference scores for some of the subjects. Next, two additional judges blindly rated the placements for 72% of the animals. The reliability coefficient (15; see also 37) of these ratings was found to be 0.979 indicating a high degree of interjudge reliability.

Results

Figure 2 shows the changes in place preference following the conditioning trials. The scores were derived by subtracting the amount of time spent on the conditioning side during the last preconditioning trial from the time spent on the conditioning side after the conditioning trials. Positive scores indicate an increase in preference for the conditioning side, while negative scores show a decrease in the amount of time spent on the conditioning side. The scatterplot was used to determine the anatomical intervals for grouping the data for subsequent analysis.

 

Changes in Place Preference

Figure 2: Changes in place preference as a function of cannula placements plotted for individual subjects. Positive scores indicate an increase and negative scores a decrease in the amount of time spent in the compartment associated with morphine microinjections.

 

Typical cannula placements are illustrated in Figure 3. The amount of tissue damage resulting from these infusions was minimal and probably less than that usually observed after infusions using the microsyringe method of microinjection. Most cannula placements were on the lateral border of the interpeduncular nucleus just medial to the substantia nigra. Several subjects with cannulae more dorsal than those illustrated in the figure were eliminated from this study.

 

Cannulae Dispersion

Figure 3: The medio-lateral and dorso-ventral range of cannula placements used in this study. The open circles on the left side illustrate the ventral tegmental cannula placements; the filled circles on the right side show the approximate distribution of the A9 (lateral) and the A10 (medial) dopamine- containing cell bodies. Note that the nominal ventral tegmental cannula placements are along the lateral edge of the interpeduncular nucleus. Abbreviations: ip, interpeduncular nucleus; ml, medial lemniscus; pvg, periventricular gray substance; sn, substantia nigra; v, ventricle. (The coronal brain section is based on 23, and the location of dopamine-containing cells is adapted from 32.)

 

Ten animals with injection cannulae that were ventral to this region were also tested. These cannulae probably terminated in the ventral cerebral vasculature or cistern as evidenced by the frequent appearance of cerebral spinal fluid flowing up the guide cannulae (placements ranged from 2.6 to 4.0 mm posterior to bregma). The mean change in place preference for this group was 45.7 (SEM = 55.8) indicating that infusions into this region were not effective in producing a change in place preference. This finding is important, albeit fortuitous, because it eliminates the possibility that morphine infusions into the ventral tegmental area were rewarding because they had entered the cerebral vasculature or ventral cistern and were transported to a distal site of action.

DETERMINATION OF ANATOMICAL BOUNDARIES

To facilitate statistical comparisons of the effect of cannula placement on conditioned place preference, the scores illustrated in Figure 2 were grouped into anatomical zones at approximately 0.6 mm intervals. This was guided by visual inspection of the scatterplot which suggested that placements rostral to 2.4 mm posterior to bregma and caudal to 3.8 mm posterior to bregma were ineffective in producing a change in place preference. (No subjects were tested with cannulae 4.0 mm posterior to bregma.) To decrease the differences in the number of subjects in each zone, the effective range from 2.4 to 3.8 mm posterior to bregma was also divided into two groupings. The results of this analysis are shown in Figure 4. An analysis of variance (37) demonstrated a significant difference among rats implanted with cannulae in the various zones throughout the ventral tegmental area [F (3,58) = 10.267, p < .001]. A Newman-Keuls’ test was performed for specific comparisons among the various groups (37). Both the 2.4 to 3.0 mm and the 3.2 to 3.8 mm zones were reliably different from the rostral and caudal placements defined by the anatomical groupings (p’s < .01).

Analysis by Anatomical Zones

Figure 4: Mean (± SEM) changes in place preference for the various anatomical zones. See Table 1 for details of the analysis.

 

Because the number of subjects tested in each group ranged from 9 to 20 (and hence the cell frequencies for the analysis of variance were very different), a more conservative approach to data analysis might be to analyze each group separately for changes in place preference. This was done by a series of t-tests and by using Fisher’s method for specific comparisons (17) to provide protection against Type I statistical errors. t-tests for correlated measures revealed that rats with cannula placements in the 2.4 to 3.0 mm and the 3.2 to 3.8 mm groups showed a significant change in place preference, while rats with placements rostral or caudal to this region did not change their preferences as a result of the drug injections (see Table 1). Thus, the results of both approaches to statistical analysis yield the same conclusion.
Table I
Statistical Evaluation Of The Data
Illustrated In Figure 4
Zone n Mean ± SEM t, p n2
1.8-2.2  9 -15.0 ± 42.7 -0.351, p > 0.70 1.3%
2.4-3.0 17 271.4 ± 51.0 5.322, p < 0.001 62.5%
3.2-3.8 20 272.1 ± 51.3 5.304, p < 0.001 58.4%
4.0-4.6 16 -6.8 ± 41.0 -0.166, p > 0.70 0.2% 
 

Figure 5 is a histological reconstruction of the range of cannula placements tested in this experiment. The zone where morphine microinjections produced a change in place preference extended throughout the ventral tegmental area covering approximately a 1.4 mm range. This region has previously been shown to contain a moderate level of opiate receptor binding (11, 13, 24, 31) as well as enkephalin cell bodies and/or terminals (14, 30, 34). Published reports mapping the distribution of dopamine-containing cells (18, 22, 35) have been based on König and Klippel’s (16) brain atlas using a different angle of sectioning than that used in the present study. This makes precise comparisons with the present study tenuous. However, in an unpublished report using de Groot’s plane of sectioning, fluorescence histochemical mapping of the A10 and A10 dopamine-containing cell bodies revealed a distribution from approximately 2.4 to 4.0 mm posterior to bregma (32). This suggests a close correspondence between the location of the ventral tegmental (and substantia nigra) dopamine-containing cell bodies and the zone where morphine injections are effective in producing a change in place preference.

 

Opiate Reward Map

Figure 5: Histological reconstruction of the approximate anatomical boundaries of the reward-relevant opiate-receptor field. The striped area indicates the zone where morphine infusions produced a change in place preference. The dorsal limits of the mid portion of the zone have been extrapolated from Phillips and LePiane (25). Abbreviations: dmh, dorsomedial nucleus of the hypothalamus; hp, habenulo-interpeduncular tract; ip, interpeduncular nucleus; lm, medial lemniscus; mp, posterior mamillary nucleus; mt, mamillothalamic tract; p, pons; pvg, periventricular gray substance; vmh, ventromedial nucleus of the hypothalamus; vtn, ventral tegmental nucleus of Tsai (adapted from 23).

 

Discussion

The results of this experiment confirm the finding that injections of morphine into the ventral tegmental area are rewarding. In an earlier report, Phillips and LePiane (25) found that bilateral injections of morphine produced a conditioned place preference that was blocked by naloxone. They also found that if the injections were made 2.5 mm dorsal to the ventral tegmental area, no change in place preference was shown. The present study extended these findings by showing that unilateral injections are also effective in producing a conditioned place preference and by establishing the rostro-caudal boundaries of the ventral tegmental opiate-receptor field that is capable of producing this effect. This latter finding was the primary reason for selecting this technique of assessing opiate reward, because it overcomes the problem associated with variable infusion volumes that accompany the intracranial self-administration technique (see 1).

There was a great deal of variability in changes in place preferences produced by morphine injections into sites that appeared very similar. This is possibly due, in part, to differences in drug diffusion with even seemingly similar cannula placements. Another factor that may account for this variability in responsiveness to morphine infusions is the fact that only about 80% of the animals receiving systemic opiate injections show a change in place preference (M. Bozarth, unpublished observations; see also 2). Thus, even effective sites of chemical stimulation might be expected to produce a place preference in only some fraction of the total subjects tested. It is interesting to note that the magnitude of the change in place preference was about the same as that produced by bilateral infusions of morphine into the ventral tegmentum (25).

The anatomical boundaries derived in the present study are not, of course, finely demarcated zones indicating the actual boundaries of the reward-relevant opiate-receptor field. Several factors make these zones only approximations to the actual location of the receptor field mediating opiate reward. First, the extent of drug dispersion following these microinjections has not been determined, although it is probably on the order of 0.25 to 0.5 mm from the injection site (see 20, 21). The data analysis, which is based on histological zones grouped at 0.6 mm intervals, contains placements that are separated by only a few tenths of a millimeter and would obviously have partially overlapping fields of effective drug spread. Second, even if the extent of drug spread were known, the portion of the reward-relevant receptor population that must be chemically activated to produce a change in place preference remains unknown. If a large percentage of the target receptors must be activated, then the zone identified in the present study may represent the midpoint of a broad homogeneous field. If, on the other hand, only a small proportion of the total receptor field capable of mediating this response needs to be stimulated by the morphine infusions, then the zone demarcated by this technique comes closer to actually mapping the extent of the reward-relevant receptors in the ventral tegmental area. Third, the histological analysis did not provide a high degree of anatomical resolution. The injection cannulae used in this study had a diameter of approximately 0.36 mm, and determination of the brain section that represented the deepest penetration of these cannulae was difficult. The high degree of interjudge reliability, however, suggests consistency in the histological groupings, although such determinations of cannula placements are only a reference point for the data analysis.

The results of this study offer corroborative support to the notion that the ventral tegmental area contains an opiate-receptor field involved in morphine’s rewarding action. The fact that naloxone blocks the development of a conditioned place preference from central morphine injections (25) demonstrates that the activation of opiate receptors is necessary for this rewarding action (see 1). The failure of injections dorsal to the ventral tegmentum to produce a change in place preference (25) eliminates the possibility that drug was diffusing up the cannula shaft and was spreading to a distal site of action. The present study showed that infusions rostral or caudal to the ventral tegmental area were also ineffective thus eliminating axonal streaming (i.e., drug diffusion along axonal projection, see 29) as a possible explanation of this effect. Furthermore, because injections ventral to this region did not result in a conditioned place preference, drug dispersion mediated by the ventral cistern or cerebral vasculature cannot account for the rewarding effect of these infusions.

Also important in studies involving anatomical mapping of central drug effects is the use of a low drug dose. By using 250 ng morphine injections, the present study provides greater anatomical resolution than studies employing larger doses. For example, a 10 mg dose is 40 times greater than the morphine dose found to be effective in this study. Proportionally greater drug spread would accompany the higher injection dose because the extent of drug diffusion is controlled, in part, by the concentration of the injected drug. Investigators should be cautious in arbitrarily selecting what may be unnecessarily high drug doses for microinjection studies. This practice may prohibit meaningful anatomical localization of central drug effects.

The finding that the approximate anatomical boundaries of the reward-relevant opiate-receptor field correspond well with the distribution of dopamine-containing cell bodies is in accord with earlier studies suggesting a role for dopamine in opioid reward. Lesions of the ventral tegmental dopamine system disrupt the acquisition of intravenous heroin self-administration (8). The conditioned place preference produced by systemic heroin injections is blocked by drugs that block dopamine receptors (5, 27) and is attenuated by lesions of the ventral tegmental dopamine system (33).

In summary, morphine infusions into the ventral tegmental area produce a change in place preference that is both pharmacologically and anatomically specific (see 1). The zone of reward-relevant opiate receptors has tentatively been defined as extending about 1.4 mm from 2.4 to 3.8 mm posterior to bregma along de Groot’s plane. The fact that this zone corresponds to the approximate location of the mesolimbic dopamine cell bodies is in accord with the notion that opiate reward involves the activation of the ventral tegmental dopamine system (1, 7).

ACKNOWLEDGMENTS

Martha Asselin is thanked for conducting the behavioral experiments and rating the cannula placements. Roy A. Wise is thanked for rating the cannula placements and for comments on an earlier version of this paper. This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and from the National Institute on Drug Abuse (U.S.A.). The author is a University Research Fellow sponsored by NSERC.

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