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The reinforcing effect of certain drugs of abuse could be caused by an action on one or a few circumscribed areas in the brain. Work based on such a proposition has yielded encouraging results for morphine, amphetamine, and cocaine (Bozarth & Wise, 1981; Goeders & Smith, 1983; Hoebel, Monaco, Hernandez, Aulisi, Stanley, & Lenard, 1983; Phillips & LePiane, 1980; Phillips, Mora, & Rolls, 1981). The evidence for this notion has been obtained with the technique of intracerebral microinjection. This technique enables us to link modified physiological activity in a particular brain area with specific changes in behavior, thus providing insight into the function of that area or into the behavioral significance of a drug action in that area. This technique has several pitfalls. Some of the pitfalls and methods to avoid them were previously reviewed by Routtenberg (1972). The major problem arises from diffusion to areas other than the one meant to be influenced by the injected substance. Also, interpretative problems arise from the high local concentration of the applied substance. This may cause nonspecific effects such as local anesthesia or irritation. With proper control procedures these uncertainties about the significance of data obtained with intracerebral injections can be largely removed. This will be illustrated in the following discussion on local injections of opiates and amphetamines and their effects on intracranial self-stimulation and other behaviors. Intracranial self-stimulation was used in such studies since it provides a stable baseline for determining the intensity and the duration of an effect from local drug injections. The method should provide a set of data that can later be used to guide experiments more directly concerned with the measurement of a reinforcing effect from a local injection.

Morphine, when given systemically, has a complex action on self-stimulation. It depresses self-stimulation for 1 to 2 hours and then enhances it (Adams, Lorens, & Mitchell, 1972; Lorens & Mitchell, 1973). Low doses can enhance self-stimulation without initial depression (Gerber, Bozarth & Wise, 1981; Glick & Rapaport, 1974), and the enhancement is particularly strong after repeated morphine treatments (Adams et al., 1972; Lorens & Mitchell, 1973). Long ago, Tatum, Seevers, and Collins (1927) suggested that the biphasic effects of opiates on behavior reflect actions on different systems in the brain. By exploring the brain with local, bilateral morphine injections in a variety of sites, it was found that injections into a certain site resulted in an enhancement of self-stimulation and that injections into another site produced a depression of self-stimulation with mixed effects from intermediate areas (see Figure 1). Apparently the locally injected morphine spreads over a large area and this complicates the picture, but the results are consistent with the idea that there exists an area mediating the excitation near the ventral tegmental area or caudal hypothalamus and an area mediating the depression within the pontine mesencephalon or a more caudal area. With a lower dose of 1 mg on each side, injections into the ventral tegmental area were more effective in inducing an immediate enhancement than injections into the caudal hypothalamus, and this indicates that the dopamine-containing cell bodies at A₉-A₁₀ represent the site where morphine acts to enhance self-stimulation (see Figure 2; Broekkamp, Phillips, & Cools, 1979a). An important question is to what extent this effect results from a local action and not from diffusion to other brain regions.
 

Figure 1: The effects of 5 mg (or 2 x 2.5 mg) morphine into different areas of the brain on hypothalamic self-stimulation. Median effects are given as percentage of the preinjection baseline rates. Reprinted with permission from Broekkamp, van den Bogaard, Heynen, Rops, Cools, and van Rossum, 1976. Copyright 1976 by Elsevier Biomedical Press.

Figure 2: Effects of self-stimulation of 2 x 1 mg morphine into the caudal hypothalamic area and into the ventral tegmental-substantia nigra area (VTA-SN). Bars on the left indicate the overall effect. Graphs on the right show the effect over time. Standard errors of the mean are presented for the overall data as well as statistical significance (** : p < 0.05). Groups: filled circles, controls; filled squares, caudal hypothalamus; open circles, VTA-SN. Reprinted with permission from Broekkamp, Phillips, and Cools, 1979a. Copyright 1979 by ANKHO International, Inc.

It is not sufficient to refer to data on diffusions of dyes, local anesthetics, or radioactively labeled chemicals (Albert & Madryga, 1980; Routtenberg, 1972), because compounds with different chemical and physical properties have widely different diffusion properties. This is elegantly illustrated by the work of Herz and Teschemacher (1971) on the analgesic effects of a series of opiates in rabbits after intraventricular injection. From their findings it could be concluded that a more lipophilic opiate such as fentanyl spreads rapidly through brain tissue and also quickly leaves the brain. Thus, for intracerebral studies where limited spread and an extended duration of action is wished, one should select a hydrophilic compound (e.g., Britt & Wise, 1983).

Another reason not to rely on observed spread of a labeled compound is the uncertainty about the detection threshold for visualization in comparison to the threshold for a pharmacological effect. If an effect is mediated by concentrations that cannot be visualized, such diffusion data are inconclusive. Therefore the safest way to exclude mediation of an effect by areas neighboring the injection site is to actually inject the drug into the surrounding areas or into the cerebral ventricle and to demonstrate that (1) higher doses are needed, (2) a smaller effect is obtained, or (3) a longer latency for an effect is observed. This worked reasonably well for morphine and the A₉-A₁₀ site for self-stimulation enhancement (Broekkamp et al., 1979a): Animals were tested for self-stimulation each hour for a 15-minute period following the microinjection of morphine into the A₉-A₁₀ area or surrounding structures. Despite the high variance a coherent picture emerged. The latency for the occurrence of an increase to at least 150% of the baseline was correlated significantly with the distance from the center of the dopamine-cell conglomerate A₉-A₁₀ (see Figure 3). The slope of the regression line indicated a diffusion speed for morphine through brain tissue of 0.9 mm/hour. This figure was also obtained by Herz and Teschemacher (1971), who derived it from the latency for analgesia after intraventricular injections.
 

Figure 3: Scatter diagram illustrating the latency for an increase in self-stimulation by local injections of 2 x 1 mg morphine HCl and the distance (in mm) between the infused site and a point located in the middle of the A9-A10 area. Reprinted with permission from Broekkamp, 1976.

The other crucial question in relation to the local morphine effect is whether nonspecific effects related to a high local concentration are involved. Controls with local anesthetics or excitant substances can provide useful information, but the best way to address this problem is to investigate the pharmacological properties of the effect with other agonists and/or antagonists. The pharmacological characteristics of an effect from local application should not have unexplainable aspects different from the known characteristics of effects mediated by similar receptors. For example, in order to meet this requirement for pharmacological consistency of an opiate effect, one should be able to obtain a similar effect with an endorphin or an enkephalin. Indeed the stabilized enkephalin analog D-Ala²-Met⁵-enkephalinamide also enhances self-stimulation when injected bilaterally into the morphine sensitive site (Broekkamp, et al., 1979a). This is useful information because, from a chemical point of view, the compound is entirely different from morphine and will have different nonspecific effects. Support for pharmacological consistency is also provided by the observation that the local effect of morphine can be antagonized by systemic treatment with naloxone (Broekkamp et al., 1979a). Several other behaviors have been shown to be influenced by injections of opiates into the dopamine cell area. Stereotypy, circling, and locomotor activity increase after b -endorphin or D-Ala-Met-enkephalinamide infusions into this area (Broekkamp, Phillips, & Cools, 1979b; Iwamoto & Way, 1977; Joyce, Koob, Strecker, Iversen, & Bloom, 1981). Similar microinjections induce place preference in a conditioning paradigm (Phillips & LePiane, 1980, 1982), and self-administration is supported by microinjections into the ventral tegmental area (Bozarth & Wise, 1981). All these effects were antagonized by an opiate antagonist. Reports on effects with unmodified leucine or methionine enkephalins into the A₉-A₁₀ area are lacking. Enkephalin doses as high as 50 to 200 mg into the ventricle were required to depress lever pressing for food by rats (Belluzzi & Stein, 1977). It is relevant to point out here that local injections with natural transmitters provide special problems. This will be discussed separately after this section.

From the data reviewed above, it emerges that the opiate effects in the A₉-A₁₀ area are well established and have adequate controls. Results obtained with self-stimulation behavior and additional data with locomotor activity made it easier to establish the importance of this area for the mediation of opiate reward with techniques such as place-preference conditioning or local self-administration (Bozarth & Wise, 1981, 1983; Phillips & LePiane, 1980; Wise & Bozarth, 1981).

We should remain alert for the possibility that other sites contribute to the reinforcement by opiates in man. The behavior depressant effect in man and animals has not been investigated explicitly for its role in opiate reward. A direct test for reinforcement related to opiate depression will be possible now that more precise information is available on the site in the brain where opiates depress behavior of rats. Behavioral depression or akinesia was measured by placing the experimental animals in abnormal postures and observing the duration of maintenance of such postures (for details of the method, see Dunstan, Broekkamp, & Lloyd, 1981). With local injections it was found that a site identical to or near the nucleus raphe pontis mediates akinesia induced by morphine (Broekkamp, LePichon, & Lloyd, 1984). A comparison of bilateral injections confirmed that the sensitive site is in the midsagittal plane (see Figure 4). More work on the pharmacological properties of this effect is needed in order to rule out nonspecific drug effects.
 

Figure 4: Coronal section illustrating infusion sites for comparing akinesia response to local morphine after bilateral (2 x 5 mg/0.5 ml) or midline infusion (10 mg/ ml). Reprinted with permission from Broekkamp, LePichon, & Lloyd, 1984. Copyright 1984 by Elsevier Scientific Publishers.

Not many reports are available describing effects with natural transmitters after intracerebral injections, and when effects are demonstrated, they invariably occur at doses in the microgram range (Garrigou, Broekkamp & Lloyd, 1981; Leibowitz, 1978; Leibowitz & Brown, 1980). This is a much higher concentration than endogenously present. Part of the reason is that natural compounds are vulnerable to endogenous enzymes which makes it difficult for them to diffuse through membranes and reach the receptor unmodified. For example, dopamine has an effect in the nucleus accumbens only in rats pretreated with the monoamine oxidase inhibitor nialamide (Pijnenburg & van Rossum, 1973). Despite the evidence for rapid metabolism, critics often still feel that the concentrations required are too high to be of physiological relevance. In considering this problem it is important to realize that the endogenous transmitter is packaged in a high concentration in synaptic vesicles which are discharged directly over the postsynaptic receptors. In view of this it is more likely that when "physiological" transmitter concentrations in locally applied solutions have any effect, this is mediated by presynaptic receptors inhibiting the release of the endogenous transmitter rather than by a direct postsynaptic effect.

Systemic dexamphetamine increases self-stimulation. This is evident not only by lever pressing rate enhancement for brain stimulation but also on measures more directly related to the reward value of brain stimulation (Atrens, Von Vietinghoff-Reisch, Der-Karabetian, & Masliyah, 1974; Liebman & Butcher, 1974; Phillips & Fibiger, 1973; Zarevics & Setler, 1979). This effect of amphetamine has been linked to an effect in the nucleus accumbens and nucleus caudatus (Broekkamp, Pijnenburg, Cools, & van Rossum, 1975). Control experiments ruled out the possibility that the anterior hypothalamus was involved or that the ventricular system transported the locally applied drug elsewhere. The latency for the self-stimulation enhancement was less than one minute. In contrast to the akinesia-inducing effect of morphine, amphetamine was most effective after bilateral injections. Pharmacological consistency is supported by experiments showing that the dopamine-receptor blocker haloperidol has opposite effects when applied into the same area (Broekkamp & van Rossum, 1975). In addition, there is an obvious substrate for the amphetamine effect available in the form of a rich innervation of dopamine containing terminals in the nucleus accumbens and caudate nucleus. The relevance for amphetamine reward has been clearly demonstrated by local self-administration experiments wherein the nucleus accumbens proved to be a sensitive brain area (Hoebel et al., 1983).

The technique of combining brain stimulation and local injections can also be used in research on brain systems involved in the addiction potential of benzodiazepines. For this type of drug, it is better to use aversive brain stimulation which is strongly influenced by benzodiazepines (Bovier, Broekkamp, & Lloyd, 1982). It is probably also the aversion antagonizing effect of benzodiazepines which makes them drugs of potential abuse. A soluble and potent benzodiazepine is available for local infusions (Pieri et al., 1981; Shibata, Kataoka, Gomita, & Ueki, 1982), and the newly developed benzodiazepine antagonists allow control for pharmacological specificity of local effects (Bonetti et al., 1982; Czernik et al., 1982).

The technique of local injections combined with brain self-stimulation at a steady rate enables the collection of information concerning the sensitive brain areas possibly involved in mediating reinforcing effects of drugs of abuse. In this chapter the sensitive sites for self-stimulation effects of morphine and dexamphetamine are discussed. It is illustrated that with the local injection technique and self-stimulation attention can be paid to the following issues:

• Is there a dose dependent effect?


• What is the duration of action and latency of onset?


• Should the area be influenced in both hemispheres or is unilateral injection sufficient?


• Can the effect of a low dose be linked to a particular brain site? Have injections into surrounding brain tissue a smaller effect or an effect after a longer latency?


• Is there a pharmacological consistency? This can be studied by using other agonists and antagonists for the receptor under investigation.


• Is there a physiological consistency? This can be studied by using transmitter metabolism inhibitors or transmitter reuptake inhibitors.

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