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Control of Brain Serotonin and Norepinephrine by Specific Neural Systems
Control of Brain Serotonin and Norepinephrine by Specific Neural Systems ALFRED HELLERAND ROBERTY. MOORE Department of Pharmacology, University of Chicago, Chicago, Illinois and
Departments of Pediatrics, Medicine (Neurology) and Anatomy, University of Chicago, Chicago, Illinois
Int r o d uction Despite the anatomic complexity and relative inaccessibility of the central nervous system, substantial experimental advances have been made in understanding its function. One classical approach to this has been the delineation of altered functional states resulting from destruction of specific groups of neurons. This technique has been applied extensively in neuropsychology and neurophysiology but has not been utilized as widely in studies of central neurochemistry. I n the experiments to be discussed here, the technique of selective destruction of discrete groups of central neurons was employed t o study the monoamines, 5-HT and NE. The experiments were initiated to identify neuronal systems in brain responsible for the presence and metabolism of these substances as one step in the elucidation of their functional role in this organ. Although the validity of this approach has not as yet been completely established for brain, it is based on the precedent of denervation experiments which have proved so useful in demonstrating the neural origin of transmitter substances in the peripheral nervous system.
Central Nervous System Lesions Affecting Brain Monoamines AND
THE PERIPHERAL MODELOF DENERVATION ITSAPPLICATION TO THE CENTRAL NERVOUS SYSTEM
The use of denervation has proved a powerful experimental tool in the analysis of the neural origin of transmitter substances in the periphery. It is well known that destruction of peripheral autonomic nerves will alter the content of acetylcholine or catecholamines in tissues innervated by the affected nerves. Section and degeneration of preganglionic sympathetic fibers causes a marked reduction in the acetylcholine content of the superior cervical ganglion (Brown and Feldberg, 1936; MacIntosh, 1938) and tissue 191
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ALFRED HELLER AND ROBERT Y . MOORE
catecholamine levels show a similar decrease following postganglionic sympathetic denervation (Cannon and Lissak, 1939; von Euler and Purkhold, 1951). These findings have generally been considered to provide substantive evidence for the neural origin of acetylcholine in ganglia and catecholamines in peripheral tissues innervated by the sympathetic nervous system. The use of denervation in the periphery has been particularly useful since the effects would appear to be restricted to structures innervated by the neurons destroyed. Section of preganglionic sympathetic fibers does not reduce catecholamine levels in tissues receiving postganglionic sympathetic innervation from the denervated ganglia (Rehn, 1958; Fischer and Snyder, 1965). These considerations suggest that selective denervation by the use of discrete lesions would represent a feasible approach to the determination of the neural elements responsible for the presence of monoamines in the central nervous system, although it should be recognized from the outset that the peripheral model of denervation need not be wholly applicable to the central nervous system. Possibly the most obvious dissimilarity is the fact that while peripheral nerves regenerate, the destruction of central neurons is not followed by a functional regeneration. Selective denervation in the central nervous system may be produced by a number of techniques. One of the most common approaches is the use of stereotaxic placement of electrodes into specific brain areas and passage of direct current of appropriate amperage and duration to produce the extent of electrolytic lesion desired. Such lesions can be quite restricted and their anatomic size and location determined by histological techniques. I n addition, the use of silver impregnation methods allows a precise analysis of the anatomic itreas denervated by any particular lesion (see the section on Interpretation of Lesion Effects on Brain Monoamines). It is possible, therefore, to employ the peripheral denervation model in the study of the central nervous system. Specific central neurons can be destroyed, the areas they innervate can be identified and, by appropriate tissue analysis, the neurochemical consequences of such lesions can be determined. THE MEDIALFOREBRAIN BUNDLEAND BRAINSEROTONIN The applicability of denervation as an experimental approach to the identification of the anatomic elements responsible for the presence of monoamines in the brain was first demonstrated by the finding that selective central nervous system lesions in the rat could reduce brain 5-HT levels (Heller et al., 1962). Electrolytic lesions were placed in a series of mesencephalic, diencephalic, and telencephalic areas and whole brain 5-HT levels determined 35 days postoperatively. Table I presents the percent change in brain 5-HT produced by these lesions and sham-operated controls as corn-
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TABLE I EFFECT OF CENTRALNERVOUS SYSTEM LESIONS ON RATBRAIN SEROTONIN@ Experimental lesion
Difference from normal
Sham operated Medial forebrain bundle Ventral midbrain tegmentum Dorsomedial tegmentum Septa1 area Medial hypothalamus Hippocampus Cortex Caudate Lateral pontine tegmentum ~~
+ -
3% 36%’
- 15%’ - 14Y0b - 12%b - 7% - 5% - 2%
+
0% 7% ~~
Data taken from Heller et al. (1962). Differences statistically significant; p < .01. All other values not significant; p > .05. a
pared to normal unoperated animals. Four of the lesions used produced significant reductions in brain 5-HT. Lesions in the septa1 region, dorsomedial tegmentum, and ventral midbrain tegmentum reduced brain 5-HT levels by 12-15y0. Lateral hypothalamic lesions had a more marked effect. producing a 36% reduction in this amine. The changes observed with other lesions or sham operation were not significant. Consideration of the anatomic elements whose destruction resulted in a significant decrease in whole brain 5-HT led to the conclusion that the integrity of one particular neural system was necessary for the maintenance of normal 5-HT levels in brain. The most pronounced effect (- 36%) was produced by lesions of the lateral hypothalamus. This portion of hypothalamus contains few cells and a complex tract, the medial forebrain bundle, which interconnects the hypothalamus with the basal telencephalon and midbrain (see the section on the Medial Forebrain Bundle). Septal, dorsomedial tegmental, and ventral tegmental lesions significantly reduced brain 5-HT and destroyed areas contributing to and receiving significant numbers of fibers from the medial forebrain bundle. I n contrast, the five other lesions which were without effect on monoamine level did not involve medial forebrain bundle components. Lesions of the medial hypothalamus destroyed midline hypothalamic nuclei but spared the medial forebrain bundle. Caudate nucleus lesions, transecting many internal capsule fibers, and lateral pontine tegmentum destruction, which severed a considerable number of specific sensory system fibers, were also without effect on brain 5-HT levels, Hippocampal lesions had no effect and this is of particular interest since this area contributes some fibers to the
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medial forebrain bundle in the preoptic area. The neocortex, as will be discussed in some detail below, neither contributes nor receives direct innervation from the medial forebrain bundle. Although the lesions we used were fairly restricted, Adler et at. (1965) have reported that frontal or posterior cortical ablation has no effect on brainstem 5-HT or NE. Thus, each lesion which significantly reduced brain 5-HT, in contrast to those which did not, either transected the medial forebrain bundle or ablated areas contributing fibers to that tract in the lateral hypothalamus. On the basis of analogy to peripheral denervation experiments, it was suggested that the loss of brain 5-HT might be a consequence of degeneration of 5-HTproducing fibers within the medial forebrain bundle (Heller et al., 1962).
SELECTIVE EFFECTSOF CENTRALNERVOUS SYSTEM LESIONSON BRAIN MONOAMINES Destruction of the medial forebrain bundle in the rat was also found to produce a reduction in whole brain levels of NE as did lesions of the dorsomedial tegmentum (Heller and Harvey, 1963). A comparison of the effects of other anatomically verified lesions on rat brain 5-HT and NE revealed a particularly interesting characteristic of such lesion effects, namely that destruction of selective areas could produce amine-specific effects (Heller and Moore, 1965). A summary of these findings is shown in Table 11. Medial TABLE I1 SELECTIVITY OF EFFECT OF CENTRALNERVOUS SYSTEM LESIONS ON BRAIN AND NOR EPINEPHRINE^ SEROTONIN Difference from sham level Experimental lesion
Medial forebrain bundle Dorsomedial tegmentum Ventrolateral tegmentum Central gray
6-HT
NE
- 33%b - 28YOb
- 26%b - 24%b
-
3%
- 18%b
-
32%b
- 7%
Data taken from Heller and Moore (1965). Differences statisticelly significant; p < .01. All other values not significant; p > .06. a b
forebrain bundle and dorsomedial tegmental lesions produce significant decreases in both 5-HT and NE. The two other tegmental lesions demonstrate lesion selectivity. Ventrolateral tegmentum destruction reduces NE without
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195
affecting 5-HT, whereas central gray lesions lower only 5-HT. Lateral tegmental nuclei would appear, therefore, to be necessary for maintenance of brain NE whereas normal 5-HT levels are dependent upon the integrity of more dorsal and medial tegmental nuclei. Although whole brain levels of NE were not significantly affected by septa1 lesions, recent regional studies have shown that this lesion produces some reduction in cortical NE. This latter point is of significance since it reveals that a lesion may produce a neurochemical effect in a restricted area of brain which is obscured by whole brain amine determinations. Hippocampal lesions, for example, do not reduce whole brain NE but Donoso (1966) has reported that such lesions affect hypothalamic NE. I n general, lesions whose effects can be detected by whole brain determinations must produce either fairly widespread changes or else massive decreases in monoamines in a restricted area. As in the case of effects on 5-HT, reductions in NE can be related in the case of each lesion with such an effect to destruction of either the medial forebrain bundle itself or areas contributing axons to this tract. The results would indicate that the integrity of the medial forebrain bundle is necessary for the maintenance of normal brain levels of both 5-HT and NE and these findings represent presumptive evidence favoring a neural origin for these monoamines in brain.
Regional Localization of the Effects of Central Nervous System Lesions on Brain Monoamines THEMEDIAL FOREBRAIN BUNDLE-ANATOMICAL
CONSIDERATIONS
The experiments outlined above (Heller et al., 1962; Heller and Moore, 1965) implicated the medial forebrain bundle as a neuronal system of primary importance in the maintenance of brain monoamine levels. The question which arises, however, is whether the effects of the lesions are secondary to section and degeneration of monoamine-producing neurons within the medial forebrain bundle in a manner analogous to the loss of peripheral transmitters following appropriate denervation. As noted previously, the loss of transmitter substances in the periphery is restricted to the structure denervated. If there is a similarity between the peripheral and central effects of denervation and the effects of brain lesions are a function of degeneration of monoamine-producing neurons, then the lesion effects should be restricted to the regional areas innervated by the neurons destroyed. To test this possibility directly, a comparison was made between the areas innervated by the medial forebrain bundle and the areas showing a reduction in monoamine content following its destruction.
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Although considerable information already was available on the connections of the medial forebrain bundle (for reviews, cf. Ingram, 1940; Guillery, 1957; Nauta, 1958; Wolf and Sutin, 1966; Moore and Heller, 1967), we elected to trace the exact distribution of the axons transected by our medial forebrain bundle lesions. Studies were carried out on both cats and rats in which lesions were placed in the identical manner to those used in the amine experiments. Animals were killed a t several postoperative time periods (rats, 2-12 days; cats, 5-14 days) and series of sections from the brains were stained by the Nauta-Gygax technique (Nauta, 1957) for the selective impregnation of degenerating axons. The results of these studies have been presented in some detail (Moore et al., 1965a; Moore and Heller, 1967) and will only be summarized here. I n addition, the descending connections of the medial forebrain bundle will not be considered since they do not bear directly on the amine data. From a lesion in the lateral hypothalamus, ascending medial forebrain bundle fibers traverse the lateral hypothalamic and lateral preoptic areas. Large numbers of preterminal fibers are present among the degenerating fibers of passage and these presumably represent sites of termination of degenerating axons. The telencephalic sites where preterminal axons are found are the septa1 area and the anterior amygdaloid area. The number of degenerating fibers traced to these areas, though, is relatively few and the vast majority of transected axons appear to terminate in the lateral hypothalamic and lateral preoptic areas. No degenerating axons could be traced to the striatum, hippocampus, or any area of the neocortex except in unusual cases when the lesion encroached extensively on the internal capsule or the subthalamic area. It should be emphasized that such cases were unusual and in very few instances was there a significant projection to the caudate-putamen complex (in the rat) or to neocortex.
OF MONOAMINE DECREASES FOLLOWING MEDIAL REGIONALLOCALIZATION FOREBRAIN BUNDLELESIONS
I n contrast to the restricted distribution within the telencephalon of the axons transected in these studies, levels of both 5-HT and NE were decreased throughout the telencephalon by medial forebrain bundle lesions (Table 111). I n both the cat and rat all of the major telencephalic subdivisions, cortex, striatum, and rhinencephalon (septum, amygdala, and hippocampus) demonstrate significant falls in 5-HT and NE levels even though only the septum and amygdala are directly innervated. The effect is restricted to telencephalon; diencephalic and brainstem amine content is unaffected.
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CONTROL O F BRAIN 5-HT AND NE BY SPECIFIC NEURAL SYSTEMS
TABLE I11 REGIONAL EFFECTSOF MEDIAL FOREBRAIN BUNDLELESIONSON SEROTONIN, NOREPINEPHRINE, A N D 5-EYDROXYTRYPTOPHAN/DOPA DECARBOXYLASE IN THE CATAND RAT BRAIN^
Area analyzed
5-HT Cat
~
5-HTPldopa decarboxylase
NE Rat
Cat
Rat
Cat
Rat
~~~~
Telencephalonb Frontal cortex Parietal cortex Occipital cortex Septa1 areac Striatum A m ygdalac Hippocampus DiencephaIonC Midbrainc Pons medulla
-
42% -66%
-
17%
- 52% -
+
59% 4%
49% - 51% - 54% - 68% -
-
I I
- 63% -51% - 67%
- 63% -71% -
10%
- 62% 35% - 41% 1
-
-
-
35%
-
47%
-
45%
-50% -
4%
- 46% - 64% - 41% -
31%
-
a Data taken from Moore et al. (1965a), Heller et al. (1966a,b), Moore and Heller (1967), and unpublished observations. I n each case the data are expressed in percent difference from a control value (see original publications for control used). A single percent difference given for more than one area (bracketed) means that those areas were analyzed as a single sample in that experiment. 6 The percent difference for each telencephalic sample was statistically significant; p < .05. Other differences, save for cat midbrain 5-HTP decarboxylase, were not statistically significant; p z .05. Areas directly innervated by the medial forebrain bundle.
OTHERLESIONS-ANATOMICAL AND NEUROCHEMICAL CONSIDERATIONS Preliminary anatomical and regional neurochemical studies have been carried out in the rat on the septal lesion, the dorsomedial tegmental lesion, and the ventrolateral tegmental lesion. As noted above, these have differing effects on whole brain monoamine levels. Studies of the distribution of axons transected by these lesions were performed using the Nauta technique. Lesions in the septal area produce two primary groups of degenerating axons. Ascending fibers either enter adjacent frontal cortical areas or run in the fornix t o terminate in the hippocampus. Descending axons enter the stria medullaris, postcommissural fornix, and medial forebrain bundle. The
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terminal distribution of these projections is well known (Nauta, 1956; Raisnian, 1966) and will not be described further. With the tegmental lesions, ascending projections will be considered exclusively, since only these are pertinent to the regional distribution of the amine changes produced b;y the lesions. Both the ventrolateral and dorsomedial lesions result in a cimilar pattern of ascending degenerating axons. A dorsal group traverses the central reticular formation to enter the subthalamus and intralaminar thalamic nuclei. This group predominates following the ventrolateral tegmental lesion. A second, ventral group of axons is also present after each lesion and these fibers traverse the ventral tegmental area to enter the medial forebrain bundle. This group predominates following the dorsomedial tegmental lesion, particularly when the lesion is quite dorsally and medially situated. It should be noted, however, that both of these lesions destroy axons which project into the medial forebrain bundle. Some axons continue through this tract into the septum but this projection is quite sparse with the ventrolateral tegmental lesion. No fibers from either lesion could be traced to the amygdala, striatum, hippocampus, or neocortex. The effects of the septa1 and ventrolateral tegmental lesions on regional 5-HT and NE levels are shown in Table IV. Septal lesions produce small but significant decreases in both 5-HT and NE in the cortex and a large decrease TABLE 117 REGIONAL EFFECTS OF SEPTALAND VENTROLATERAL TEGMENTAL LESIONS ON SEROTONIN A N D NOREPINEPHRINE IN THE RATBRAIN= ~~
Ventrolateral tegmental lesion
Septal lesion Area analyzed
NE
5-HT Telencephalon Cortex Septum Striatum Amygdala Hippocampus Diencephalon Brainstem
- 15%’ -
c
+ 20%b
+
2% - 40%” 14% + 9%
+
-
26%b -
c
+ 20%
- 10% - 9%
+ 10%
+
2%
5-HT
NE
2% 5% + 4% - 3% - 12% + 5%
- 43700 - 53%b - 31%b - 44x0 - 43x0
-
-
c
-
47 -
%b c
Unpublished observations. Differences statistically significant; p < .05. All other values not significant; p > .05. c Area including lesions not analyzed. Septal lesions verified by gross inspection; ventrolateral tegmental lesions verified histologically. a
b
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in 5-HT in the hippocampus. Each of these areas is directly innervated by the area destroyed. The increase in striatal amines cannot be accounted for a t present. I n contrast to the septa1 lesion, ventrolateral tegmental lesions produce large decreases in NE alone in all telencephalic areas and in the diencephalon. Although only preliminary experiments are available, it appears a t present that the falls in amines resulting from dorsomedial tegmental lesions will have a regional distribution identical to that of the ventrolateral tegmental lesion. Thus, as in the case of the medial forebrain bundle lesion, tegmental lesions result in amine falls in widespread areas that are distant to the sites of termination of the axons destroyed.
Interpretation of Lesion Effects on Brain Monoamines The discrepancy between the distribution of the amine falls and the projection of transected medial forebrain bundle fibers which was apparent in our initial regional studies in the cat (Moore and Heller, 1964; Moore et al., 1965a; Heller et al., 1964, 1966a) brought up an obvious difficulty in the interpretation of the results. Three alternatives were available. First, we could dismiss the neurochemical effects of the medial forebrain bundle lesions on the grounds that they were the result of some nonspecific change in neuronal metabolism. This seemed unlikely for several reasons. The effect appears in restricted areas (i.e., the telencephalon) and not throughout the brain. It occurs only on the lesion side of the brain when unilateral lesions are employed (Harvey et al., 1963) and the change in amine levels appears to be a permanent effect, lasting to at least 120 days after operation. Lastly, the effect on the amines is dependent upon exact placement of the lesion. Ablation of areas immediately adjacent to the critical area is without effect. Second, we could adhere to our original hypothesis that the effects of the lesions on brain monoamine levels were the direct result of section and subsequent degeneration of amine-producing neurons (Heller et al., 1962). I n subsequent experiments confirming our observations on the effects of lesions on brain monoamines using the histochemical technique of Falck and Hillarp (Falck et al., 1962; Falck, 1962), the loss of “monoaminergic” neural elements has been interpreted in this manner (Anden et al., 1966; Hillarp et al., 1966).As in our experiments, the loss of both 5-HT and NE was found in diencephalon and telencephalon following midbrain tegmental and medial forebrain bundle lesions. On the basis of their observation of histochemically demonstrable neuronal perikarya within the brainstem, the apparent absence of such cells in the diencephalon and telencephalon, and the loss of fluorescent telencephalic terminals with a concomitant increase in brainstem cell-body monoamine content, these workers have proposed the existence of a system
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of monoamine-producing neurons. The cell bodies of this system are diagrammed (Anden et al., 1966, p. 323) as lying in the brainstem with axons ascending from them to traverse the medial forebrain bundle to innervate the entire telencephalon, including the neocortex. The neurochemical effects of lesions involving the medial forebrain bundle or regions providing ascending projections into i t would, according to this scheme, be a consequence of section and degeneration of ascending axons of monoamine-producing brainstem cells which directly innervate the telencephalon. As noted above, however, such a n interpretation is a t variance with present knowledge of medial forebrain bundle projections. The basic problem, we believe, comes with respect to the lack of histochemical demonstration of monoamine cell bodies above the diencephalon. Dahlstrom and Fuxe (1965, p. 18) have noted that “cell bodies of the specific monoamine neurons in brain show very low to low levels of their respective amines,” but these can apparently be more easily visualized by pretreatment of animals with pharmacologic agents. This would suggest that some cell bodies may contain too little monoamine to be detectable by current methods. Nickerson (1966, p. 802) has quite succinctly summarized this question with respect to the sensitivity of the fluorescent histochemical method by asking, L L H absent ~ ~is absent?” With these considerations in mind i t would seem premature, in our opinion, to rule out the possibility that monoamine-containing cell bodies exist within the telencephalon or t o discard available anatomic information on the connections of the medial forebrain bundle. If the proposal that the effects of lesions on monoamines in all areas of brain is a direct result of section and degeneration of monoamine-producing neurons is accepted, it would have two consequences. It would require an extension of current concepts of the connections of the medial forebrain bundle t o include projections t o the striatum, the hippocampus, and all regions of the neocortex, and it would impugn the validity of all techniques which have been used to date to study the connections of the medial forebrain bundle. We have recently brought together many of the anatomical arguments which bear on this problem (Moore and Heller, 1967). The major question is whether the Nauta method is sufficiently reliable and sensitive to demonstrate all of the connections of the medial forebrain bundle. There is nothing to suggest that i t is not. I n our material we routinely resolve degenerating axons which have been measured to be 0.5 p diameter. This is well within the size range which should include most, if not all, central axons and terminals. And, taking into account the welhecognized swelling which occurs during the process of axonal degeneration, we may well be demonstrating axons whose normal diameter is in the range of 0.2 p. Studies of medial forebrain bundle connections have used several modifications of the basic Nauta method and given essentially the same results (Gudlery, 1957;
CONTROL OF BRAIN 5-HT AND N E BY SPECIFIC NEURAL SYSTEMS
201
Wolf and Sutin, 1966; Moore and Heller, 1967). Furthermore, there is good evidence that the Nauta method does impregnate degenerating axon terminals (Guillery and Ralston, 1964), and, in initial experiments using a new technique for the demonstration of axon terminals (Fink and Heimer, 1967), we can find no indication for a much wider distribution of medial forebrain bundle axons than was heretofore suspected. It must be admitted, however, that the medial forebrain bundle does contain some small axons which are normally beyond the resolution of the light microscope. There is nothing to suggest, though, that the axons of their terminals would not be within the range of resolution of the light microscope if properly demonstrated by silverstaining methods. Indeed, in a recent review of the use of electron microscopy as a tool in tracing degenerating axons, Alksne et al. (1966, p. 28) state “that for the demonstration of the terminal area of a nervous pathway one will always find one or more of the available silver methods, used with much caution, and often after much modification, to be suitable”. Thus, in evaluation of our studies and those of others, there are no substantial grounds for assuming that a significant area of termination of the medial forebrain bundle would have been overlooked. For these reasons we do not believe that i t would be valid, a t present, to view the medial forebrain bundle as a system of monoamine-producing neurons innervating the entire telencephalon. Based upon these anatomical considerations, we came to accept and propose the third alternative that seemed available to u‘s. That is, if the effect of the lesion is not due either to a nonspecific alteration of neuronal metabolism or to degeneration of monoamine-producing cells, then it must represent a change in the control of neuronal metabolism which is mediated across one or more intermediate neurons which may or may not be similarly affected. We have termed such changes, occurring one or more synapses from the neurons destroyed, “trans-synaptic’’ neurochemical effects of central lesions.
Biochemical Mechanisms Underlying Lesion Effects
As a n approach to understanding the biochemical basis of lesion effects, we have conducted a series of investigations of the effect of such lesions on brain levels of the enzymes involved in monoamine biosynthesis. These studies have involved the placement of specific lesions, followed by in vitro determination of brain level of enzyme. Medial forebrain bundle lesions which decrease telencephalic 5-HT and NE also reduce telencephalic decarboxylase activity using either 5-HTP or dopa as the substrate (Heller et al., 1965; Moore et al., 1966). A detailed regional analysis of the anatomic pattern of enzyme loss in the cat revealed that 5-HTP decarboxylase was reduced by 35-59y0 in all regional areas showing a loss in monoamines (Heller et al.,
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ALFRED HELLER AND ROBERT Y. MOORE
1966b, Table 111).Similar regional losses in dopa decarboxylase have subsequently been found following medial forebrain bundle lesions in the rat. There is, therefore, a corresponding loss of a n enzyme activity necessary for the synthesis of both 5-HT and NE in all areas of brain which show reduced monoamine levels following medial forebrain bundle lesions. It may be noted that this effect on enzyme activity occurs, as does the amine effect, in several areas one or more synapses removed from the transected axons of the medial forebrain bundle and is, in this sense, transynaptic. We have suggested, on the basis of the correspondence between regional amine and enzyme loss, that the effect of medial forebrain bundle lesions on telencephalic monoamine levels is due, a t least in part, to a defect in monoamine biosynthesis a t the decarboxylase step. Reduction of deoarboxylase activity of the magnitude found need not lead to a decrease in in vivo biosynthesis. To relate the effects on decarboxylase activity to the effects on amine levels and in vivo biosynthesis, one must consider what changes may occur a t the cellular level following the lesion. The loss of enzyme may be total a t some neuronal sites of synthesis and the resultant loss in absolute number of synthetic sites available might well lead to a decrease in monoamine biosynthesis regardless of the levels of other enzymes or considerations of what steps might be rate-limiting in the biosynthetic pathway. I n this respect, however, we have recently found that the telencephalic level of tyrosine hydroxylase is also reduced (- 44%, Table V) by medial forebrain TABLE V DIFFERENTIAL NEUROCHEMICAL EFFECTS OF HYPOTHALAMIC AND TEGMENTAL ON TELENCEPHALIC MONOAMINESAND ENZYMES IN THE RAT^ LESIONS Difference in telencephalic level from sham control
Experimental lesion
5-HT
NE
Medial forebrain bundle Ventroleteral tegmentum
- 66%’ - 12%
- 51%b - 76%’
Dopa decarboxylase
- 28%b
-
5%
Tyrosine hydroxylaae
- 44Y0b - 30Y0b
Unpublished observations. Differences statistically significant; p < .01. All other values not significant; p > .05. 0
b
bundle lesions and effects on this enzyme would most probably be reflected in reductions in NE level. It would seem likely then, that the effects of medial forebrain bundle lesions on monoamines are, a t least in part, due to a reduction in synthetic enzymes. The finding that incorporation of dopa-5H to
CONTROL OF BRAIN
5-HT AND
NE BY SPECIFlC NEURAL SYSTEMS
203
catecholamines in vivo is reduced in those areas of cat brain which show a loss of monoamines and decarboxylase activity following medial forebrain bundle lesions (Moore et al., 1965b) indicates that the loss of enzyme activity observed by in vitro measurements is of functional significance with respect to in vivo monoamine biosynthesis. Although the loss of monoamines is probably secondary to an enzymic defect, this relationship does not provide information about the state of neurons in which these losses have occurred. Particularly in areas such as neocortex in which the loss of amines and enzymes following medial forebrain bundle lesions is transynaptic, it is not clear whether the neurons affected undergo some form of transneuronal degeneration or remain intact but suffer a selective deficit in enzymes involved in monoamine biosynthesis. Following lesions of the ventrolateral tegmentum which produce a selective reduction in telencephalic NE there is a decrease in telencephalic tyrosine hydroxylase (- 30%; Table V), but no effect on dopa decarboxylase activity. This finding reveals that central nervous system lesions, depending on their anatomic locus, may be specific in their effects on the level of enzyme activity necessary for one of the steps in a biosynthetic sequence without altering others. The maintenance of normal levels of individual neuronal enzymes involved in monoamine biosynthesis appears to be it function of the integrity of specific neural systems which may be identified by the use of central nervous system lesions. If the loss of NE following ventrolateral tegmental lesions is secondary to degeneration of NE-producing neurons, it would be difficult to visualize this occurring without a loss in all biosynthetic enzymes involved in NE formation, including dopa decarboxylase. The results obtained would indicate that the decrease in telencephalic NE produced by ventrolateral tegmental lesions is secondary to it selective reduction in tyrosine hydroxylase without neuronal degeneration.
Conclusions The reduction in brain monoamines resulting from central lesions represents, for the most part, a series of complex phenomena which are not entirely explicable on the basis of a simple model of section and degeneration of monoamine-producing neurons. The results obtained do permit, however, some general statements to be made about the interpretation of lesion effects and about the organization of neural mechanisms controlling various aspects of brain monoamine metabolism. Each area whose destruction was demonstrated to lower brain levels of either 5-HT or NE involves neural elements either within or directly related to the neurons of the medial forebrain bundle. Section of this tract itself markedly alters levels of both amines throughout the telencephalon as well
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as reducing telencephalic tyrosine hydroxylase and dopa decarboxylase activity. Lesions placed more caudally in the midbrain tegmentum may, depending upon their exact location, alter either 5-HT, NE, or both. Such lesions also appear to be selective with respect t o the enzymes involved and changes are observed in the diencephalon as well as the telencephalon. The effects of the lesions, thus, are always anatomically rostral to the region destroyed but are apparently transmitted via the medial forebrain bundle. It is not evident why the medial forebrain bundle is critical for the maintenance of brain monoamines although its importance as the primary source of hypothalamic afferent and efferent connections is well recognized (Bleier et al., 1966).The anatomic course of the polysynaptic projections of the medial forebrain bundle which may influence areas rostral to the hypothalamus is unknown a t present. Septa1 lesions do reduce hippocampal 5-HT and result in some minimal reduction in cortical 5-HT and NE which may be either polysynaptic in character or a function of loss of monoamine neurons innervating these areas. It seems unlikely, however, that the majority of neurons controlling telencephalic monoamines pass through the septum since destruction of this regional area does not produce changes of the magnitude seen with lateral hypothalamic lesions except in the case of hippocampal 5-HT. Rather, it would appear more likely that above the hypothalamus the system becomes too diffuse to be markedly affected by a discrete lesion. Whatever the elements involved in this system and their course to the sites of termination, i t is considered a system made up of sets of neurons with its influences mediated transynaptically. At first glance the invocation of such a n anatomically ill-defined system for neural control within the diencephalon and telencephalon would appear unwarranted, but it is not without precedent. Ascending influences maintaining behavioral and electrophysiological evidences of arousal are localized in the rostral midbrain tegmentum but spread diffusely beyond that region (Magoun, 1963). As in the case of the system influencing monoamines, these effects are mediated through polysynaptic connections, since no known simple projection system will account for their distribution. The concept of transynaptic control of neuronal monoamine metabolism raises several important questions. It necessitates consideration of the status of the neurons affected. Since transneuronal degeneration is an uncommon event in &becentral nervous system outside of the primary sensory projections, we would expect the cells with reduced amine levels to be morphologically intact. If this is the case, then the effects of the lesions on cellular chemical phenomena could be viewed as a consequence of an altered functional status. Each central neuron receives input over much of the surface of its soma and dendrites and may be influenced by vast numbers of presynaptic elements. The neurochemical integrity of any given cell can be interpreted
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205
as a function of this afferent “pool” of innervation. Alteration of this afferent pool either by direct denervation or by distant effects which change the pattern of input may result in some alteration of the function of the affected neuron. If important influences are removed permanently as in the instance of destructive lesions (e.g., in the medial forebrain bundle), the morphologically intact neuron could respond by a reduction in enzyme content and thereby, in amine level. The proposed model is not inconsistent with what we now know about the neurochemical effects of a t least some central lesions. Regardless of the nature of these effects a t the cellular level, their widespread distribution has implications on a physiological or behavioral level. For example, the reinforcing properties of stimulation within the tegmentum and hypothalamus (Olds, 1962) need not be directly attributable to events a t the site stimulated but may reflect events at a distance. It has been shown recently, in this regard, that stimulation of the tegmentum may cause release of 5-HT within the telencephalon (Aghajanian et al., 1967). Similarly, the syndrome of adipsia and aphagia resulting from lateral hypothalamic lesions (Teitlebaum and Epstein, 1962) could represent distant changes in the functional activity of widespread areas on the basis of transsynaptic changes such as those described here. The lesion technique has proved a useful approach to the study of neuronal systems controlling brain monoamines. The maintenance of 5-HT and NE in brain appears to be a function of the integrity of a specific system of hypothalamic neural elements, the medial forebrain bundle. The complexity of the relation of this system to the control of central monoamine metabolism is a reflection of the complex physiologic and anatomic organization of the brain. ACKNOWLEDGMENT The work reported here was supported by research grants MH-04954 and NB-05002 from the National Institutes of Health. A. Heller is supported by a Career Research Development Fellowship ( K 3-MH-21, 850) from the National Institute of Mental Health. R. Y. Moore is supported by Career Research Development Fellowship (K 3-NB7, 389) from the National Institute of Neurological Diseases and Blindness and is a John and Mary R. Markle Scholar in Medical Science.
REFERENCES Adler, M. W., Kuriyama, K., and Maynert, E. W. (1965). Life Sci. 4, 141. Aghajanian, G . K., Rosecrans, J. A., and Sheard, M. H. (1967). Science 156, 402. Alksne, J . F., Blackstad, T. W., Walberg, F., and White, L. E., Jr. (1966). Ergeb. Ancit. Entwicklungsgesch. 39, 1 . Anden, N.-E., Dahlstrom, A., Fuxe, K., Larsson, K., Olson, L., and Ungerstedt, U. (1966). Actn Physiol. Scand. 67, 313.
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Bleier, R., Bard, P., and Woods, J. W. (1966). J.Comp. Neurol. 128, 255. Brown, G. L., and Feldberg, W. (1936). J . Physiol. ( L o r ~ d o n86, ) 290. Cannon, W. B., and Lissak, K. (1939). A m . J . Physiol. 125, 765. Dahlstrom, A., and Fuxe, K. (1965). Acta Physiol. Scnnd. Suppl. 247. Donoso, A. 0 . (1966). Experientia 22, 191. Falck, B. (1962). Acta Physiol. Scaitd. Suppl. 197. Falck, B., Hillarp, N.-A., Thieme, G., and Torp, A. (1962). J. Histochem. Cytochem. 7, 323. Fink, R. P., and Heimer, L. (1967). Brain Res. 4, 369. Fischer, J. E., and Snyder, S. (1965). J . Pharmacol. Exptl. Thernp. 150, 190. Guillery, R. W. (1957). J . Anat. 91, 91. Guillery, R. W., and Ralston, H. J. (1964). Science 143, 1331. Harvey, J. A., Heller, A., and Moore, R. Y . (1963). J . Pharmacol. Exptl. Therap. 140, 103. Heller, A., and Harvey, J. A. (1963). Phnrmncologist 5 ( 2 ) , 264. Heller, A., and Moore, R. Y . (1965). J. Pharmacol. Exptl. Therap. 150, 1. Heller, A., Harvey, J. A., and Moore, R. Y . (1962). Biochem. Pharmacol. 11, 859. Heller, A., Seiden, L. S., and Moore, R. Y . (1964). Pharmncologzst 6, 196. Heller, A., Seiden, L. S., Porcher, W., and Moore, R. Y . (1965). Science 147, 887. Heller, A., Seiden, L. S., and Moore, R. Y . (1966a). Ztztern. J . Neurophnrmocol. 5, 91. Heller, A., Seiden, L. S., Porcher, W., and Moore, R. Y . (1966b). J . Neurochem. 13, 967 Hillarp, N.-A., Fuxe, K., and Dahlstrom, A. (1966). Pknrmrzcol. Rev. 18, 727. Ingram, W. R. (1940). Res. Publ. Assoc. Res. Nervous Mentnl Disense 28, 105. MacIntosh, F. C. (1938). Arch. Zntern. Physiol. 47, 321. Magoun, H. W. (1963). “The Waking Brain”. Thomas, Springfield, Illinois. Moore, R. Y . ,and Heller, A. (1964). Trans. A m . Neurol. Assoc. 89, 143. Moore, R. Y . , and Heller, A. (1967). J . Phamncol. Exptl. Therap. 156, 12. Moore, R. Y . , Wong, S. L. R., and Heller, A. (1965a). Arch. Neurol. 13, 346. Moore, R. Y . ,Margolis, R. U., and Heller, A. (1965b). Federntion Proc. 24, 195. Moore, R. Y . , Bhatnagar, R., and Heller, A. (1966). Intern. J . Neurophnmncol. 5, 287. Nauta, W. J. H. (1956). J . Comp. Neurol. 104, 247. Nauta, W. J. H. (1957). Z?z “New Research Techniques of Neuroanatomy” (W. F. Windle, ed.), p. 17. Thomas, Springfield, Illinois. Nauta, W. J. H. (1958). Brnilt 81, 319. Nickerson, M. (1966). Pharmncol. Rev. 18, 801. Olds, J. (1962). Physiol. Rev. 42, 554. Raisman, G. (1966). Brain 89, 317. Rehn, N. 0. (1958). Actn PhysioZ.Scnd. 42, 309. Teitlebaum, P., and Epstein, A. (1962). Psychol. Rev. 69, 74. von Euler, U. S., and Purkhold, A. (1951). Acta Physiol. Scnnd. 24, 218. Wolf, G., and Sutin, J. (1966). J. Comp. A’eurol. 127, 137.
Departments of Pediatrics, Medicine (Neurology) and Anatomy, University of Chicago, Chicago, Illinois
Int r o d uction Despite the anatomic complexity and relative inaccessibility of the central nervous system, substantial experimental advances have been made in understanding its function. One classical approach to this has been the delineation of altered functional states resulting from destruction of specific groups of neurons. This technique has been applied extensively in neuropsychology and neurophysiology but has not been utilized as widely in studies of central neurochemistry. I n the experiments to be discussed here, the technique of selective destruction of discrete groups of central neurons was employed t o study the monoamines, 5-HT and NE. The experiments were initiated to identify neuronal systems in brain responsible for the presence and metabolism of these substances as one step in the elucidation of their functional role in this organ. Although the validity of this approach has not as yet been completely established for brain, it is based on the precedent of denervation experiments which have proved so useful in demonstrating the neural origin of transmitter substances in the peripheral nervous system.
Central Nervous System Lesions Affecting Brain Monoamines AND
THE PERIPHERAL MODELOF DENERVATION ITSAPPLICATION TO THE CENTRAL NERVOUS SYSTEM
The use of denervation has proved a powerful experimental tool in the analysis of the neural origin of transmitter substances in the periphery. It is well known that destruction of peripheral autonomic nerves will alter the content of acetylcholine or catecholamines in tissues innervated by the affected nerves. Section and degeneration of preganglionic sympathetic fibers causes a marked reduction in the acetylcholine content of the superior cervical ganglion (Brown and Feldberg, 1936; MacIntosh, 1938) and tissue 191
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ALFRED HELLER AND ROBERT Y . MOORE
catecholamine levels show a similar decrease following postganglionic sympathetic denervation (Cannon and Lissak, 1939; von Euler and Purkhold, 1951). These findings have generally been considered to provide substantive evidence for the neural origin of acetylcholine in ganglia and catecholamines in peripheral tissues innervated by the sympathetic nervous system. The use of denervation in the periphery has been particularly useful since the effects would appear to be restricted to structures innervated by the neurons destroyed. Section of preganglionic sympathetic fibers does not reduce catecholamine levels in tissues receiving postganglionic sympathetic innervation from the denervated ganglia (Rehn, 1958; Fischer and Snyder, 1965). These considerations suggest that selective denervation by the use of discrete lesions would represent a feasible approach to the determination of the neural elements responsible for the presence of monoamines in the central nervous system, although it should be recognized from the outset that the peripheral model of denervation need not be wholly applicable to the central nervous system. Possibly the most obvious dissimilarity is the fact that while peripheral nerves regenerate, the destruction of central neurons is not followed by a functional regeneration. Selective denervation in the central nervous system may be produced by a number of techniques. One of the most common approaches is the use of stereotaxic placement of electrodes into specific brain areas and passage of direct current of appropriate amperage and duration to produce the extent of electrolytic lesion desired. Such lesions can be quite restricted and their anatomic size and location determined by histological techniques. I n addition, the use of silver impregnation methods allows a precise analysis of the anatomic itreas denervated by any particular lesion (see the section on Interpretation of Lesion Effects on Brain Monoamines). It is possible, therefore, to employ the peripheral denervation model in the study of the central nervous system. Specific central neurons can be destroyed, the areas they innervate can be identified and, by appropriate tissue analysis, the neurochemical consequences of such lesions can be determined. THE MEDIALFOREBRAIN BUNDLEAND BRAINSEROTONIN The applicability of denervation as an experimental approach to the identification of the anatomic elements responsible for the presence of monoamines in the brain was first demonstrated by the finding that selective central nervous system lesions in the rat could reduce brain 5-HT levels (Heller et al., 1962). Electrolytic lesions were placed in a series of mesencephalic, diencephalic, and telencephalic areas and whole brain 5-HT levels determined 35 days postoperatively. Table I presents the percent change in brain 5-HT produced by these lesions and sham-operated controls as corn-
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193
TABLE I EFFECT OF CENTRALNERVOUS SYSTEM LESIONS ON RATBRAIN SEROTONIN@ Experimental lesion
Difference from normal
Sham operated Medial forebrain bundle Ventral midbrain tegmentum Dorsomedial tegmentum Septa1 area Medial hypothalamus Hippocampus Cortex Caudate Lateral pontine tegmentum ~~
+ -
3% 36%’
- 15%’ - 14Y0b - 12%b - 7% - 5% - 2%
+
0% 7% ~~
Data taken from Heller et al. (1962). Differences statistically significant; p < .01. All other values not significant; p > .05. a
pared to normal unoperated animals. Four of the lesions used produced significant reductions in brain 5-HT. Lesions in the septa1 region, dorsomedial tegmentum, and ventral midbrain tegmentum reduced brain 5-HT levels by 12-15y0. Lateral hypothalamic lesions had a more marked effect. producing a 36% reduction in this amine. The changes observed with other lesions or sham operation were not significant. Consideration of the anatomic elements whose destruction resulted in a significant decrease in whole brain 5-HT led to the conclusion that the integrity of one particular neural system was necessary for the maintenance of normal 5-HT levels in brain. The most pronounced effect (- 36%) was produced by lesions of the lateral hypothalamus. This portion of hypothalamus contains few cells and a complex tract, the medial forebrain bundle, which interconnects the hypothalamus with the basal telencephalon and midbrain (see the section on the Medial Forebrain Bundle). Septal, dorsomedial tegmental, and ventral tegmental lesions significantly reduced brain 5-HT and destroyed areas contributing to and receiving significant numbers of fibers from the medial forebrain bundle. I n contrast, the five other lesions which were without effect on monoamine level did not involve medial forebrain bundle components. Lesions of the medial hypothalamus destroyed midline hypothalamic nuclei but spared the medial forebrain bundle. Caudate nucleus lesions, transecting many internal capsule fibers, and lateral pontine tegmentum destruction, which severed a considerable number of specific sensory system fibers, were also without effect on brain 5-HT levels, Hippocampal lesions had no effect and this is of particular interest since this area contributes some fibers to the
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ALFRED RELLER AND ROBERT Y. MOORE
medial forebrain bundle in the preoptic area. The neocortex, as will be discussed in some detail below, neither contributes nor receives direct innervation from the medial forebrain bundle. Although the lesions we used were fairly restricted, Adler et at. (1965) have reported that frontal or posterior cortical ablation has no effect on brainstem 5-HT or NE. Thus, each lesion which significantly reduced brain 5-HT, in contrast to those which did not, either transected the medial forebrain bundle or ablated areas contributing fibers to that tract in the lateral hypothalamus. On the basis of analogy to peripheral denervation experiments, it was suggested that the loss of brain 5-HT might be a consequence of degeneration of 5-HTproducing fibers within the medial forebrain bundle (Heller et al., 1962).
SELECTIVE EFFECTSOF CENTRALNERVOUS SYSTEM LESIONSON BRAIN MONOAMINES Destruction of the medial forebrain bundle in the rat was also found to produce a reduction in whole brain levels of NE as did lesions of the dorsomedial tegmentum (Heller and Harvey, 1963). A comparison of the effects of other anatomically verified lesions on rat brain 5-HT and NE revealed a particularly interesting characteristic of such lesion effects, namely that destruction of selective areas could produce amine-specific effects (Heller and Moore, 1965). A summary of these findings is shown in Table 11. Medial TABLE I1 SELECTIVITY OF EFFECT OF CENTRALNERVOUS SYSTEM LESIONS ON BRAIN AND NOR EPINEPHRINE^ SEROTONIN Difference from sham level Experimental lesion
Medial forebrain bundle Dorsomedial tegmentum Ventrolateral tegmentum Central gray
6-HT
NE
- 33%b - 28YOb
- 26%b - 24%b
-
3%
- 18%b
-
32%b
- 7%
Data taken from Heller and Moore (1965). Differences statisticelly significant; p < .01. All other values not significant; p > .06. a b
forebrain bundle and dorsomedial tegmental lesions produce significant decreases in both 5-HT and NE. The two other tegmental lesions demonstrate lesion selectivity. Ventrolateral tegmentum destruction reduces NE without
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195
affecting 5-HT, whereas central gray lesions lower only 5-HT. Lateral tegmental nuclei would appear, therefore, to be necessary for maintenance of brain NE whereas normal 5-HT levels are dependent upon the integrity of more dorsal and medial tegmental nuclei. Although whole brain levels of NE were not significantly affected by septa1 lesions, recent regional studies have shown that this lesion produces some reduction in cortical NE. This latter point is of significance since it reveals that a lesion may produce a neurochemical effect in a restricted area of brain which is obscured by whole brain amine determinations. Hippocampal lesions, for example, do not reduce whole brain NE but Donoso (1966) has reported that such lesions affect hypothalamic NE. I n general, lesions whose effects can be detected by whole brain determinations must produce either fairly widespread changes or else massive decreases in monoamines in a restricted area. As in the case of effects on 5-HT, reductions in NE can be related in the case of each lesion with such an effect to destruction of either the medial forebrain bundle itself or areas contributing axons to this tract. The results would indicate that the integrity of the medial forebrain bundle is necessary for the maintenance of normal brain levels of both 5-HT and NE and these findings represent presumptive evidence favoring a neural origin for these monoamines in brain.
Regional Localization of the Effects of Central Nervous System Lesions on Brain Monoamines THEMEDIAL FOREBRAIN BUNDLE-ANATOMICAL
CONSIDERATIONS
The experiments outlined above (Heller et al., 1962; Heller and Moore, 1965) implicated the medial forebrain bundle as a neuronal system of primary importance in the maintenance of brain monoamine levels. The question which arises, however, is whether the effects of the lesions are secondary to section and degeneration of monoamine-producing neurons within the medial forebrain bundle in a manner analogous to the loss of peripheral transmitters following appropriate denervation. As noted previously, the loss of transmitter substances in the periphery is restricted to the structure denervated. If there is a similarity between the peripheral and central effects of denervation and the effects of brain lesions are a function of degeneration of monoamine-producing neurons, then the lesion effects should be restricted to the regional areas innervated by the neurons destroyed. To test this possibility directly, a comparison was made between the areas innervated by the medial forebrain bundle and the areas showing a reduction in monoamine content following its destruction.
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Although considerable information already was available on the connections of the medial forebrain bundle (for reviews, cf. Ingram, 1940; Guillery, 1957; Nauta, 1958; Wolf and Sutin, 1966; Moore and Heller, 1967), we elected to trace the exact distribution of the axons transected by our medial forebrain bundle lesions. Studies were carried out on both cats and rats in which lesions were placed in the identical manner to those used in the amine experiments. Animals were killed a t several postoperative time periods (rats, 2-12 days; cats, 5-14 days) and series of sections from the brains were stained by the Nauta-Gygax technique (Nauta, 1957) for the selective impregnation of degenerating axons. The results of these studies have been presented in some detail (Moore et al., 1965a; Moore and Heller, 1967) and will only be summarized here. I n addition, the descending connections of the medial forebrain bundle will not be considered since they do not bear directly on the amine data. From a lesion in the lateral hypothalamus, ascending medial forebrain bundle fibers traverse the lateral hypothalamic and lateral preoptic areas. Large numbers of preterminal fibers are present among the degenerating fibers of passage and these presumably represent sites of termination of degenerating axons. The telencephalic sites where preterminal axons are found are the septa1 area and the anterior amygdaloid area. The number of degenerating fibers traced to these areas, though, is relatively few and the vast majority of transected axons appear to terminate in the lateral hypothalamic and lateral preoptic areas. No degenerating axons could be traced to the striatum, hippocampus, or any area of the neocortex except in unusual cases when the lesion encroached extensively on the internal capsule or the subthalamic area. It should be emphasized that such cases were unusual and in very few instances was there a significant projection to the caudate-putamen complex (in the rat) or to neocortex.
OF MONOAMINE DECREASES FOLLOWING MEDIAL REGIONALLOCALIZATION FOREBRAIN BUNDLELESIONS
I n contrast to the restricted distribution within the telencephalon of the axons transected in these studies, levels of both 5-HT and NE were decreased throughout the telencephalon by medial forebrain bundle lesions (Table 111). I n both the cat and rat all of the major telencephalic subdivisions, cortex, striatum, and rhinencephalon (septum, amygdala, and hippocampus) demonstrate significant falls in 5-HT and NE levels even though only the septum and amygdala are directly innervated. The effect is restricted to telencephalon; diencephalic and brainstem amine content is unaffected.
197
CONTROL O F BRAIN 5-HT AND NE BY SPECIFIC NEURAL SYSTEMS
TABLE I11 REGIONAL EFFECTSOF MEDIAL FOREBRAIN BUNDLELESIONSON SEROTONIN, NOREPINEPHRINE, A N D 5-EYDROXYTRYPTOPHAN/DOPA DECARBOXYLASE IN THE CATAND RAT BRAIN^
Area analyzed
5-HT Cat
~
5-HTPldopa decarboxylase
NE Rat
Cat
Rat
Cat
Rat
~~~~
Telencephalonb Frontal cortex Parietal cortex Occipital cortex Septa1 areac Striatum A m ygdalac Hippocampus DiencephaIonC Midbrainc Pons medulla
-
42% -66%
-
17%
- 52% -
+
59% 4%
49% - 51% - 54% - 68% -
-
I I
- 63% -51% - 67%
- 63% -71% -
10%
- 62% 35% - 41% 1
-
-
-
35%
-
47%
-
45%
-50% -
4%
- 46% - 64% - 41% -
31%
-
a Data taken from Moore et al. (1965a), Heller et al. (1966a,b), Moore and Heller (1967), and unpublished observations. I n each case the data are expressed in percent difference from a control value (see original publications for control used). A single percent difference given for more than one area (bracketed) means that those areas were analyzed as a single sample in that experiment. 6 The percent difference for each telencephalic sample was statistically significant; p < .05. Other differences, save for cat midbrain 5-HTP decarboxylase, were not statistically significant; p z .05. Areas directly innervated by the medial forebrain bundle.
OTHERLESIONS-ANATOMICAL AND NEUROCHEMICAL CONSIDERATIONS Preliminary anatomical and regional neurochemical studies have been carried out in the rat on the septal lesion, the dorsomedial tegmental lesion, and the ventrolateral tegmental lesion. As noted above, these have differing effects on whole brain monoamine levels. Studies of the distribution of axons transected by these lesions were performed using the Nauta technique. Lesions in the septal area produce two primary groups of degenerating axons. Ascending fibers either enter adjacent frontal cortical areas or run in the fornix t o terminate in the hippocampus. Descending axons enter the stria medullaris, postcommissural fornix, and medial forebrain bundle. The
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ALFRED HELLER AND ROBERT Y. MOORE
terminal distribution of these projections is well known (Nauta, 1956; Raisnian, 1966) and will not be described further. With the tegmental lesions, ascending projections will be considered exclusively, since only these are pertinent to the regional distribution of the amine changes produced b;y the lesions. Both the ventrolateral and dorsomedial lesions result in a cimilar pattern of ascending degenerating axons. A dorsal group traverses the central reticular formation to enter the subthalamus and intralaminar thalamic nuclei. This group predominates following the ventrolateral tegmental lesion. A second, ventral group of axons is also present after each lesion and these fibers traverse the ventral tegmental area to enter the medial forebrain bundle. This group predominates following the dorsomedial tegmental lesion, particularly when the lesion is quite dorsally and medially situated. It should be noted, however, that both of these lesions destroy axons which project into the medial forebrain bundle. Some axons continue through this tract into the septum but this projection is quite sparse with the ventrolateral tegmental lesion. No fibers from either lesion could be traced to the amygdala, striatum, hippocampus, or neocortex. The effects of the septa1 and ventrolateral tegmental lesions on regional 5-HT and NE levels are shown in Table IV. Septal lesions produce small but significant decreases in both 5-HT and NE in the cortex and a large decrease TABLE 117 REGIONAL EFFECTS OF SEPTALAND VENTROLATERAL TEGMENTAL LESIONS ON SEROTONIN A N D NOREPINEPHRINE IN THE RATBRAIN= ~~
Ventrolateral tegmental lesion
Septal lesion Area analyzed
NE
5-HT Telencephalon Cortex Septum Striatum Amygdala Hippocampus Diencephalon Brainstem
- 15%’ -
c
+ 20%b
+
2% - 40%” 14% + 9%
+
-
26%b -
c
+ 20%
- 10% - 9%
+ 10%
+
2%
5-HT
NE
2% 5% + 4% - 3% - 12% + 5%
- 43700 - 53%b - 31%b - 44x0 - 43x0
-
-
c
-
47 -
%b c
Unpublished observations. Differences statistically significant; p < .05. All other values not significant; p > .05. c Area including lesions not analyzed. Septal lesions verified by gross inspection; ventrolateral tegmental lesions verified histologically. a
b
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199
in 5-HT in the hippocampus. Each of these areas is directly innervated by the area destroyed. The increase in striatal amines cannot be accounted for a t present. I n contrast to the septa1 lesion, ventrolateral tegmental lesions produce large decreases in NE alone in all telencephalic areas and in the diencephalon. Although only preliminary experiments are available, it appears a t present that the falls in amines resulting from dorsomedial tegmental lesions will have a regional distribution identical to that of the ventrolateral tegmental lesion. Thus, as in the case of the medial forebrain bundle lesion, tegmental lesions result in amine falls in widespread areas that are distant to the sites of termination of the axons destroyed.
Interpretation of Lesion Effects on Brain Monoamines The discrepancy between the distribution of the amine falls and the projection of transected medial forebrain bundle fibers which was apparent in our initial regional studies in the cat (Moore and Heller, 1964; Moore et al., 1965a; Heller et al., 1964, 1966a) brought up an obvious difficulty in the interpretation of the results. Three alternatives were available. First, we could dismiss the neurochemical effects of the medial forebrain bundle lesions on the grounds that they were the result of some nonspecific change in neuronal metabolism. This seemed unlikely for several reasons. The effect appears in restricted areas (i.e., the telencephalon) and not throughout the brain. It occurs only on the lesion side of the brain when unilateral lesions are employed (Harvey et al., 1963) and the change in amine levels appears to be a permanent effect, lasting to at least 120 days after operation. Lastly, the effect on the amines is dependent upon exact placement of the lesion. Ablation of areas immediately adjacent to the critical area is without effect. Second, we could adhere to our original hypothesis that the effects of the lesions on brain monoamine levels were the direct result of section and subsequent degeneration of amine-producing neurons (Heller et al., 1962). I n subsequent experiments confirming our observations on the effects of lesions on brain monoamines using the histochemical technique of Falck and Hillarp (Falck et al., 1962; Falck, 1962), the loss of “monoaminergic” neural elements has been interpreted in this manner (Anden et al., 1966; Hillarp et al., 1966).As in our experiments, the loss of both 5-HT and NE was found in diencephalon and telencephalon following midbrain tegmental and medial forebrain bundle lesions. On the basis of their observation of histochemically demonstrable neuronal perikarya within the brainstem, the apparent absence of such cells in the diencephalon and telencephalon, and the loss of fluorescent telencephalic terminals with a concomitant increase in brainstem cell-body monoamine content, these workers have proposed the existence of a system
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of monoamine-producing neurons. The cell bodies of this system are diagrammed (Anden et al., 1966, p. 323) as lying in the brainstem with axons ascending from them to traverse the medial forebrain bundle to innervate the entire telencephalon, including the neocortex. The neurochemical effects of lesions involving the medial forebrain bundle or regions providing ascending projections into i t would, according to this scheme, be a consequence of section and degeneration of ascending axons of monoamine-producing brainstem cells which directly innervate the telencephalon. As noted above, however, such a n interpretation is a t variance with present knowledge of medial forebrain bundle projections. The basic problem, we believe, comes with respect to the lack of histochemical demonstration of monoamine cell bodies above the diencephalon. Dahlstrom and Fuxe (1965, p. 18) have noted that “cell bodies of the specific monoamine neurons in brain show very low to low levels of their respective amines,” but these can apparently be more easily visualized by pretreatment of animals with pharmacologic agents. This would suggest that some cell bodies may contain too little monoamine to be detectable by current methods. Nickerson (1966, p. 802) has quite succinctly summarized this question with respect to the sensitivity of the fluorescent histochemical method by asking, L L H absent ~ ~is absent?” With these considerations in mind i t would seem premature, in our opinion, to rule out the possibility that monoamine-containing cell bodies exist within the telencephalon or t o discard available anatomic information on the connections of the medial forebrain bundle. If the proposal that the effects of lesions on monoamines in all areas of brain is a direct result of section and degeneration of monoamine-producing neurons is accepted, it would have two consequences. It would require an extension of current concepts of the connections of the medial forebrain bundle t o include projections t o the striatum, the hippocampus, and all regions of the neocortex, and it would impugn the validity of all techniques which have been used to date to study the connections of the medial forebrain bundle. We have recently brought together many of the anatomical arguments which bear on this problem (Moore and Heller, 1967). The major question is whether the Nauta method is sufficiently reliable and sensitive to demonstrate all of the connections of the medial forebrain bundle. There is nothing to suggest that i t is not. I n our material we routinely resolve degenerating axons which have been measured to be 0.5 p diameter. This is well within the size range which should include most, if not all, central axons and terminals. And, taking into account the welhecognized swelling which occurs during the process of axonal degeneration, we may well be demonstrating axons whose normal diameter is in the range of 0.2 p. Studies of medial forebrain bundle connections have used several modifications of the basic Nauta method and given essentially the same results (Gudlery, 1957;
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201
Wolf and Sutin, 1966; Moore and Heller, 1967). Furthermore, there is good evidence that the Nauta method does impregnate degenerating axon terminals (Guillery and Ralston, 1964), and, in initial experiments using a new technique for the demonstration of axon terminals (Fink and Heimer, 1967), we can find no indication for a much wider distribution of medial forebrain bundle axons than was heretofore suspected. It must be admitted, however, that the medial forebrain bundle does contain some small axons which are normally beyond the resolution of the light microscope. There is nothing to suggest, though, that the axons of their terminals would not be within the range of resolution of the light microscope if properly demonstrated by silverstaining methods. Indeed, in a recent review of the use of electron microscopy as a tool in tracing degenerating axons, Alksne et al. (1966, p. 28) state “that for the demonstration of the terminal area of a nervous pathway one will always find one or more of the available silver methods, used with much caution, and often after much modification, to be suitable”. Thus, in evaluation of our studies and those of others, there are no substantial grounds for assuming that a significant area of termination of the medial forebrain bundle would have been overlooked. For these reasons we do not believe that i t would be valid, a t present, to view the medial forebrain bundle as a system of monoamine-producing neurons innervating the entire telencephalon. Based upon these anatomical considerations, we came to accept and propose the third alternative that seemed available to u‘s. That is, if the effect of the lesion is not due either to a nonspecific alteration of neuronal metabolism or to degeneration of monoamine-producing cells, then it must represent a change in the control of neuronal metabolism which is mediated across one or more intermediate neurons which may or may not be similarly affected. We have termed such changes, occurring one or more synapses from the neurons destroyed, “trans-synaptic’’ neurochemical effects of central lesions.
Biochemical Mechanisms Underlying Lesion Effects
As a n approach to understanding the biochemical basis of lesion effects, we have conducted a series of investigations of the effect of such lesions on brain levels of the enzymes involved in monoamine biosynthesis. These studies have involved the placement of specific lesions, followed by in vitro determination of brain level of enzyme. Medial forebrain bundle lesions which decrease telencephalic 5-HT and NE also reduce telencephalic decarboxylase activity using either 5-HTP or dopa as the substrate (Heller et al., 1965; Moore et al., 1966). A detailed regional analysis of the anatomic pattern of enzyme loss in the cat revealed that 5-HTP decarboxylase was reduced by 35-59y0 in all regional areas showing a loss in monoamines (Heller et al.,
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1966b, Table 111).Similar regional losses in dopa decarboxylase have subsequently been found following medial forebrain bundle lesions in the rat. There is, therefore, a corresponding loss of a n enzyme activity necessary for the synthesis of both 5-HT and NE in all areas of brain which show reduced monoamine levels following medial forebrain bundle lesions. It may be noted that this effect on enzyme activity occurs, as does the amine effect, in several areas one or more synapses removed from the transected axons of the medial forebrain bundle and is, in this sense, transynaptic. We have suggested, on the basis of the correspondence between regional amine and enzyme loss, that the effect of medial forebrain bundle lesions on telencephalic monoamine levels is due, a t least in part, to a defect in monoamine biosynthesis a t the decarboxylase step. Reduction of deoarboxylase activity of the magnitude found need not lead to a decrease in in vivo biosynthesis. To relate the effects on decarboxylase activity to the effects on amine levels and in vivo biosynthesis, one must consider what changes may occur a t the cellular level following the lesion. The loss of enzyme may be total a t some neuronal sites of synthesis and the resultant loss in absolute number of synthetic sites available might well lead to a decrease in monoamine biosynthesis regardless of the levels of other enzymes or considerations of what steps might be rate-limiting in the biosynthetic pathway. I n this respect, however, we have recently found that the telencephalic level of tyrosine hydroxylase is also reduced (- 44%, Table V) by medial forebrain TABLE V DIFFERENTIAL NEUROCHEMICAL EFFECTS OF HYPOTHALAMIC AND TEGMENTAL ON TELENCEPHALIC MONOAMINESAND ENZYMES IN THE RAT^ LESIONS Difference in telencephalic level from sham control
Experimental lesion
5-HT
NE
Medial forebrain bundle Ventroleteral tegmentum
- 66%’ - 12%
- 51%b - 76%’
Dopa decarboxylase
- 28%b
-
5%
Tyrosine hydroxylaae
- 44Y0b - 30Y0b
Unpublished observations. Differences statistically significant; p < .01. All other values not significant; p > .05. 0
b
bundle lesions and effects on this enzyme would most probably be reflected in reductions in NE level. It would seem likely then, that the effects of medial forebrain bundle lesions on monoamines are, a t least in part, due to a reduction in synthetic enzymes. The finding that incorporation of dopa-5H to
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catecholamines in vivo is reduced in those areas of cat brain which show a loss of monoamines and decarboxylase activity following medial forebrain bundle lesions (Moore et al., 1965b) indicates that the loss of enzyme activity observed by in vitro measurements is of functional significance with respect to in vivo monoamine biosynthesis. Although the loss of monoamines is probably secondary to an enzymic defect, this relationship does not provide information about the state of neurons in which these losses have occurred. Particularly in areas such as neocortex in which the loss of amines and enzymes following medial forebrain bundle lesions is transynaptic, it is not clear whether the neurons affected undergo some form of transneuronal degeneration or remain intact but suffer a selective deficit in enzymes involved in monoamine biosynthesis. Following lesions of the ventrolateral tegmentum which produce a selective reduction in telencephalic NE there is a decrease in telencephalic tyrosine hydroxylase (- 30%; Table V), but no effect on dopa decarboxylase activity. This finding reveals that central nervous system lesions, depending on their anatomic locus, may be specific in their effects on the level of enzyme activity necessary for one of the steps in a biosynthetic sequence without altering others. The maintenance of normal levels of individual neuronal enzymes involved in monoamine biosynthesis appears to be it function of the integrity of specific neural systems which may be identified by the use of central nervous system lesions. If the loss of NE following ventrolateral tegmental lesions is secondary to degeneration of NE-producing neurons, it would be difficult to visualize this occurring without a loss in all biosynthetic enzymes involved in NE formation, including dopa decarboxylase. The results obtained would indicate that the decrease in telencephalic NE produced by ventrolateral tegmental lesions is secondary to it selective reduction in tyrosine hydroxylase without neuronal degeneration.
Conclusions The reduction in brain monoamines resulting from central lesions represents, for the most part, a series of complex phenomena which are not entirely explicable on the basis of a simple model of section and degeneration of monoamine-producing neurons. The results obtained do permit, however, some general statements to be made about the interpretation of lesion effects and about the organization of neural mechanisms controlling various aspects of brain monoamine metabolism. Each area whose destruction was demonstrated to lower brain levels of either 5-HT or NE involves neural elements either within or directly related to the neurons of the medial forebrain bundle. Section of this tract itself markedly alters levels of both amines throughout the telencephalon as well
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as reducing telencephalic tyrosine hydroxylase and dopa decarboxylase activity. Lesions placed more caudally in the midbrain tegmentum may, depending upon their exact location, alter either 5-HT, NE, or both. Such lesions also appear to be selective with respect t o the enzymes involved and changes are observed in the diencephalon as well as the telencephalon. The effects of the lesions, thus, are always anatomically rostral to the region destroyed but are apparently transmitted via the medial forebrain bundle. It is not evident why the medial forebrain bundle is critical for the maintenance of brain monoamines although its importance as the primary source of hypothalamic afferent and efferent connections is well recognized (Bleier et al., 1966).The anatomic course of the polysynaptic projections of the medial forebrain bundle which may influence areas rostral to the hypothalamus is unknown a t present. Septa1 lesions do reduce hippocampal 5-HT and result in some minimal reduction in cortical 5-HT and NE which may be either polysynaptic in character or a function of loss of monoamine neurons innervating these areas. It seems unlikely, however, that the majority of neurons controlling telencephalic monoamines pass through the septum since destruction of this regional area does not produce changes of the magnitude seen with lateral hypothalamic lesions except in the case of hippocampal 5-HT. Rather, it would appear more likely that above the hypothalamus the system becomes too diffuse to be markedly affected by a discrete lesion. Whatever the elements involved in this system and their course to the sites of termination, i t is considered a system made up of sets of neurons with its influences mediated transynaptically. At first glance the invocation of such a n anatomically ill-defined system for neural control within the diencephalon and telencephalon would appear unwarranted, but it is not without precedent. Ascending influences maintaining behavioral and electrophysiological evidences of arousal are localized in the rostral midbrain tegmentum but spread diffusely beyond that region (Magoun, 1963). As in the case of the system influencing monoamines, these effects are mediated through polysynaptic connections, since no known simple projection system will account for their distribution. The concept of transynaptic control of neuronal monoamine metabolism raises several important questions. It necessitates consideration of the status of the neurons affected. Since transneuronal degeneration is an uncommon event in &becentral nervous system outside of the primary sensory projections, we would expect the cells with reduced amine levels to be morphologically intact. If this is the case, then the effects of the lesions on cellular chemical phenomena could be viewed as a consequence of an altered functional status. Each central neuron receives input over much of the surface of its soma and dendrites and may be influenced by vast numbers of presynaptic elements. The neurochemical integrity of any given cell can be interpreted
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as a function of this afferent “pool” of innervation. Alteration of this afferent pool either by direct denervation or by distant effects which change the pattern of input may result in some alteration of the function of the affected neuron. If important influences are removed permanently as in the instance of destructive lesions (e.g., in the medial forebrain bundle), the morphologically intact neuron could respond by a reduction in enzyme content and thereby, in amine level. The proposed model is not inconsistent with what we now know about the neurochemical effects of a t least some central lesions. Regardless of the nature of these effects a t the cellular level, their widespread distribution has implications on a physiological or behavioral level. For example, the reinforcing properties of stimulation within the tegmentum and hypothalamus (Olds, 1962) need not be directly attributable to events a t the site stimulated but may reflect events at a distance. It has been shown recently, in this regard, that stimulation of the tegmentum may cause release of 5-HT within the telencephalon (Aghajanian et al., 1967). Similarly, the syndrome of adipsia and aphagia resulting from lateral hypothalamic lesions (Teitlebaum and Epstein, 1962) could represent distant changes in the functional activity of widespread areas on the basis of transsynaptic changes such as those described here. The lesion technique has proved a useful approach to the study of neuronal systems controlling brain monoamines. The maintenance of 5-HT and NE in brain appears to be a function of the integrity of a specific system of hypothalamic neural elements, the medial forebrain bundle. The complexity of the relation of this system to the control of central monoamine metabolism is a reflection of the complex physiologic and anatomic organization of the brain. ACKNOWLEDGMENT The work reported here was supported by research grants MH-04954 and NB-05002 from the National Institutes of Health. A. Heller is supported by a Career Research Development Fellowship ( K 3-MH-21, 850) from the National Institute of Mental Health. R. Y. Moore is supported by Career Research Development Fellowship (K 3-NB7, 389) from the National Institute of Neurological Diseases and Blindness and is a John and Mary R. Markle Scholar in Medical Science.
REFERENCES Adler, M. W., Kuriyama, K., and Maynert, E. W. (1965). Life Sci. 4, 141. Aghajanian, G . K., Rosecrans, J. A., and Sheard, M. H. (1967). Science 156, 402. Alksne, J . F., Blackstad, T. W., Walberg, F., and White, L. E., Jr. (1966). Ergeb. Ancit. Entwicklungsgesch. 39, 1 . Anden, N.-E., Dahlstrom, A., Fuxe, K., Larsson, K., Olson, L., and Ungerstedt, U. (1966). Actn Physiol. Scand. 67, 313.
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Bleier, R., Bard, P., and Woods, J. W. (1966). J.Comp. Neurol. 128, 255. Brown, G. L., and Feldberg, W. (1936). J . Physiol. ( L o r ~ d o n86, ) 290. Cannon, W. B., and Lissak, K. (1939). A m . J . Physiol. 125, 765. Dahlstrom, A., and Fuxe, K. (1965). Acta Physiol. Scnnd. Suppl. 247. Donoso, A. 0 . (1966). Experientia 22, 191. Falck, B. (1962). Acta Physiol. Scaitd. Suppl. 197. Falck, B., Hillarp, N.-A., Thieme, G., and Torp, A. (1962). J. Histochem. Cytochem. 7, 323. Fink, R. P., and Heimer, L. (1967). Brain Res. 4, 369. Fischer, J. E., and Snyder, S. (1965). J . Pharmacol. Exptl. Thernp. 150, 190. Guillery, R. W. (1957). J . Anat. 91, 91. Guillery, R. W., and Ralston, H. J. (1964). Science 143, 1331. Harvey, J. A., Heller, A., and Moore, R. Y . (1963). J . Pharmacol. Exptl. Therap. 140, 103. Heller, A., and Harvey, J. A. (1963). Phnrmncologist 5 ( 2 ) , 264. Heller, A., and Moore, R. Y . (1965). J. Pharmacol. Exptl. Therap. 150, 1. Heller, A., Harvey, J. A., and Moore, R. Y . (1962). Biochem. Pharmacol. 11, 859. Heller, A., Seiden, L. S., and Moore, R. Y . (1964). Pharmncologzst 6, 196. Heller, A., Seiden, L. S., Porcher, W., and Moore, R. Y . (1965). Science 147, 887. Heller, A., Seiden, L. S., and Moore, R. Y . (1966a). Ztztern. J . Neurophnrmocol. 5, 91. Heller, A., Seiden, L. S., Porcher, W., and Moore, R. Y . (1966b). J . Neurochem. 13, 967 Hillarp, N.-A., Fuxe, K., and Dahlstrom, A. (1966). Pknrmrzcol. Rev. 18, 727. Ingram, W. R. (1940). Res. Publ. Assoc. Res. Nervous Mentnl Disense 28, 105. MacIntosh, F. C. (1938). Arch. Zntern. Physiol. 47, 321. Magoun, H. W. (1963). “The Waking Brain”. Thomas, Springfield, Illinois. Moore, R. Y . ,and Heller, A. (1964). Trans. A m . Neurol. Assoc. 89, 143. Moore, R. Y . , and Heller, A. (1967). J . Phamncol. Exptl. Therap. 156, 12. Moore, R. Y . , Wong, S. L. R., and Heller, A. (1965a). Arch. Neurol. 13, 346. Moore, R. Y . ,Margolis, R. U., and Heller, A. (1965b). Federntion Proc. 24, 195. Moore, R. Y . , Bhatnagar, R., and Heller, A. (1966). Intern. J . Neurophnmncol. 5, 287. Nauta, W. J. H. (1956). J . Comp. Neurol. 104, 247. Nauta, W. J. H. (1957). Z?z “New Research Techniques of Neuroanatomy” (W. F. Windle, ed.), p. 17. Thomas, Springfield, Illinois. Nauta, W. J. H. (1958). Brnilt 81, 319. Nickerson, M. (1966). Pharmncol. Rev. 18, 801. Olds, J. (1962). Physiol. Rev. 42, 554. Raisman, G. (1966). Brain 89, 317. Rehn, N. 0. (1958). Actn PhysioZ.Scnd. 42, 309. Teitlebaum, P., and Epstein, A. (1962). Psychol. Rev. 69, 74. von Euler, U. S., and Purkhold, A. (1951). Acta Physiol. Scnnd. 24, 218. Wolf, G., and Sutin, J. (1966). J. Comp. A’eurol. 127, 137.