Mercury stable isotope signatures of world coal deposits and historical coal combustion emissions.

Catecholamine Innervation of the Basal Forebrain IV. TOPOGRAPHY OF THE DOPAMINE PROJECTION TO THE BASAL FOREBRAIN AND NEOSTRIATUM JAMES H. FALLON AND ROBERT Y. MOORE Department ofiveurosciences, University of California at Sun Diego, La Jolla, California 92093 U.S.A.

ABSTRACT In this study the location of dopamine (DA) neuron perikarya in the rostra1 mesencephalon of the r a t was determined using the glyoxylic acid fluorescence histochemical technique. Subsequently the topography of the projection of these mesencephalic neurons on the basal forebrain and striatum was analyzed using the anterograde transport-autoradiographic tracing method and the retrograde transport-horseradish peroxidase (HRP) technique. The results of these anatomical studies were correlated with the biochemical and histochemical studies presented in previous reports (Moore, '78; Fallon and Moore, '78; Fallon e t al., '78) to provide the following conclusions. The topography of the DA neuron projection on the basal forebrain and neostriatum is organized in three planes, dorsal-ventral, medial-lateral and anterior-posterior. DA cells are found almost exclusively in the substantia nigra (SN) and ventral tegmental area (VTA). Ventral cells of the SN and VTA project to the dorsal structures of the basal forebrain such a s the septum, nucleus accumbens and neostriatum. The latter includes some DA cells located ventrally in the pars reticulata of the SN. Dorsal cells project to ventral structures. The medial-lateral topography is organized such that the medial sectors of the SN-VTA area project to the medial parts of nuclei in the basal forebrain and neostriatum whereas lateral sectors of the SN-VTA area project to the lateral parts of nuclei in the basal forebrain and neostriatum. An anterior-posterior topography also is evident such that anterior parts of the SN-VTA project anteriorly whereas the posterior SN-VTA projects more posteriorly in these areas. These observations are consistent with the view t h a t the DA neurons of the SN-VTA complex form a single nuclear group with a highly topographically organized projection innervating not only deep nuclei of the telencephalon but allocortical structures as well. In previous reports (Moore, '78; Fallon and Moore, '78; Fallon e t al., '78) the catecholamine (CAI innervation of the basal forebrain was described. These studies indicated that, whereas the projection of the brainstem and locus coeruleus norepinephrine (NE) neuron systems to the basal forebrain do not exhibit a n evident topography, the projection of the mesencephalic dopamine (DA) neurons to the basal forebrain is topographically organized. The general view of the organization of the projection of DA systems on the forebrain (cf. Ungerstedt, '71; Moore and Kromer, '77; Lindvall and Bjorklund, '77; Moore and Bloom, '78, for reviews) has been t h a t three major DAforebrain systems exist; a nigrostriatal sysJ. COMP. NEUR. (1978)180: 545-580.

tem arising from the Dahlstrom and Fuxe ('64) cell groups A8 and A9, a mesolimbic system arising from cell group A10 and a mesocortical system arising from cell groups A10 and possibly A9. In the present study the precise topography of the projection of the mesencephalic DA neuron systems was examined using three methods. First, the location of the DA-containing cell bodies of these neuronal groups was determined using the Vibratome-glyoxylic acid (GA) modification of the FalckHillarp fluorescence histochemical technique (Lindvall and Bjorklund, '74a). Second, the general pattern of projection of the cell groups was analyzed using the anterograde trans-

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port-autoradiographictracing method (Cowan et al., '72) and, third, the horseradish peroxidase (HRP)-retrograde transport method was employed to provide a precise delineation of the topography of the projections. These studies taken together with results obtained in the previous biochemical and histochemical studies (Fallon and Moore, '76a,b, '78; Moore, '78; Fallon e t al., '78) provide correlative evidence demonstrating the topography of the projection of mesencephalic DA neurons upon the cerebral cortex, basal forebrain and striatum. MATERIALS AND METHODS

The animals used in this study were approximat,ely 100 female albino rats, 100-300 g, of the Sprague-Dawley strain (Hilltop Farms, Scottsdale, Pennsylvania). All surgical procedures were carried out under deep ether or pentobarbital (40 mg/kg) anesthesia with the animal in a stereotaxic apparatus (Kopf Instruments, Tujunga, California). The methodology for the histochemical and

anatomical studies is presented in detail in previous reports (Moore, '78, Fallon and Moore, '78; Fallon et al., '78) with the following exceptions.

Location of DA cell bodies The location of the DA-containing cell bodies of the substantia nigra (SN) and ventral tegmental area (VTA), which includes the DA cell groups AS, A9 and A10 of Dahlstrom and Fuxe ('641, was determined in ten rats using the Vibratome-GA modification of the Falck-Hillarp technique (Lindvall et al., '73; Lindvall and Bjorklund, '74a). Coronal sections of the mesencephalon and diencephalon, 15-25p m thick, were cut in the coronal plane. Following the examination of the GA-induced fluorescence of the DA cell bodies, the sections were stained with cresyl violet in order to correlate the position of the DA-containing cell bodies with standard cytoarchitectonic analysis. Drawings were made of sections from each brain showing the loca-

A b breuiations A, Nucleus accumbens AA, Anterior amygdaloid area ABN, Medial basal nucleus of amygdala AC. Central nucleus of amygdala ACO, Cortical nucleus of amygdala ALA, Anterior lateral nucleus of amygdala ALP, Posterior lateral nucleus of amygdala AM, Medial nucleus of amygdala CA, Anterior commissure CAA, Anterior commissure, pars anterior CAI, Internal capsule CC, Crus cerebri CG, Central gray CE, Entorhinal cortex CL, Claustrum CO, Optic chiasm CP, Caudate-putamen CP,, Ventral part of caudate-putamen CPF, Piriform cortex CPF,,, Layer I1 of piriform cortex F, Fornix DR, Dorsal raphe FMT, Mammilothalamic tract FR, Fasciculus retroflexus H, , Forel's field H1 H,, Forel's field H2 HI, Hippocampus IC, Inferior colliculus IPN, Interpeduncular nucleus L, Medial lemniscus LGN. Lateral geniculate nucleus LC, Locus coeruleus LG, Lamina glomerulosa of the olfactory bulb LGE, Lamina granularis externa of the olfactory bulb LGI, Lamina granularis interna of the olfactory bulb MFB, Medial forebrain bundle

MG, Medial geniculate nucleus MM, Mammillary nuclei MI, Intercalated nuclei of amygdala MTN, Medial terminal nucleus of the accessory optic tract OAD, Pars dorsalis of the anterior olfactory nucleus OAL, Pars lateralis of the anterior olfactory nucleus OAP, Pars posterior of the anterior olfactory nucleus OL, Nucleus of the lateral olfactory tract OT, Olfactory tubercle PC, Posterior commissure PF, Frontal pole of cortex P,, Nucleus paranigralis PN, Pontine nuclei POL, Lateral preoptic area POM, Medial preoptic area Nucleus parabrachialis pigmentosus ed nucleus RET, Mesencephalic reticular formation RF, Reticular formation SC, Superior colliculus SN,, Pars compacta of the substantia nigra SNL, Pars lateralis of the substantia nigra SN,, Pars reticulata of the substantia nigra SR, Rhinal sulcus SRC, Suprarhinal or perirhinal cortex ST, Interstitial nucleus of the stria terminalis (bed nucleus) STT, Stria terminalis TO, Optic tract TOL, Lateral olfactory tract TV, Ventral thalamic nucleus VAFB, Ansa peduncularis-ventral amygdaloid bundle VTA, Ventral tegmental area (Tsai) Y, Area y of medial piriform cortex 21, Zona incerta

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TOPOGRAPHY OF DOPAMINE CELL PROJECTIONS

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A

Fig. 1 Drawings of coronal sections that correspond to the same levels represented in figure 2. See Abbreviations for identification of nuclear areas and fiber tracts.

tion of DA neurons and these were used to make a composite drawing to show the location and relative density of DA neurons in relation to other structures in a series of representative coronal levels from caudal diencephalon to caudal midbrain.

Anterograde transport studies Injections of a mixture of 3H-proline and 3H-leucine (4-40 pCi in 0.03-0.05 pl) were made into the SN-VTA region of 25 rats. The

procedures used for the autoradiographic studies have been described in detail previously (Moore, '78).

Retrograde transport studies Injections of HRP were made into different areas of the cerebral cortex, basal forebrain or neostriatum of 65 rats and the brains examined for retrograde and anterograde transport of HRP. The details of this procedure are described in a previous report (Moore, '78).

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JAMES H. FALLON AND ROBERT Y. MOORE RESULTS

Location of DA cell bodies The location of the DA cell bodies in the ventral tegmentum is illustrated in figures 1 and 2 and figure 18f in relation to a variety of landmarks at each of the coronal levels shown. It is evident that the DA cell bodies (fig. 2) form a continuum and, on this basis, a separation into sub-nuclei is not warranted. I t is helpful, on the other hand, to refer t o components of the overall nuclear group as being within the ventral tegmental area (VTA) or substantia nigra (SN), recognizing that there is no clear boundary between them. I t is also important to note that, whereas many cells in the VTA and SN are DA neurons, there are numerous cells in both areas, and particularly the VTA, that do not contain DA. VTA The VTA, the A10 cell group of Dahlstrom and Fuxe, ('641, has DA neuron cell-bodies that are continuous laterally with the SN and dorsally with the periventricular DA cells. Within the VTA, the DA cells form a component of the ventral tegmental area of Tsai, nucleus linearis and two other nuclear areas described by Taber ('61) in the cat as nucleus parabrachialis pigmentosus and nucleus paranigralis. Nucleus parabrachialis pigmentosus is situated ventromedial to the red nucleus (figs. 1, 2d-f) whereas the nucleus paranigralis is triangular in shape and lies just medial to the caudal half of the SN, ventral to the nucleus parabrachialis and dorsolateral to the interpeduncular nucleus (figs. 1,2D-F). In the present study, the analysis of projections of DA neurons suggested that the VTA could be viewed as consisting of four confluent regions. The medial VTA DA neurons are referred to as medial VTA whereas the lateral neurons, extending laterally t o the red nucleus, medial lemniscus, medial terminal nucleus of the accessory optic system (MTN: figs. lC,2) and SN are referred to as the lateral VTA. Superimposed over this medial-lateral subdivision is a dorsal-ventral subdivision. The cells situated just dorsal to the interpeduncular nucleus which include the DA neurons within nucleus paranigralis and ventral portion of the ventral tegmental area of Tsai are referred to as the ventral VTA, while cells lying dorsal to these are referred to as the dorsal VTA. The DA neurons of the SN include a dense

layer of cells in the pars compacta (cell group A9 of Dahlstrom and Fuxe, '64). The dorsalmost cells of the pars compacta have dendritic processes that appear oriented primarily mediolaterally (fig. 18f), while the ventral DA cells have dendrites oriented both in the mediolateral and in the ventrolateral direction, the latter often extending into the pars reticulata. Scattered DA cells are situated dorsally among the fibers of the medial lemniscus and the area just ventral to the red nucleus (figs. 1,2F), laterally in the pars lateralis of the substantia nigra (figs. 1,2C-E) to the ventral border of the paralemniscal nucleus and, ventrally, a number of DA cells are evident throughout the pars reticulata of the SN (figs. 1,2: B-F).Rostrally, some DA cells are present in the medial zona incerta and the prerubral field of Fore1 (figs. 1,2A).Caudally, scattered DA cells are found in the ventrolateral midbrain tegmentum dorsal to the medial lemniscus and lateral to the red nucleus (figs. 1,2F-H).These caudolateral cells, referred to as cell group A8 by Dahlstrom and Fuxe ('64) are continuous with DA neurons of the SN and constitute a caudal extension of the nigral group. Some DA cells can be found within and along the border of the cerebral peduncle (figs. 1,2F,G). The SN, like the VTA can be viewed on the basis of projections as consisting of confluent sectors of DA neurons. In the medial-lateral direction, three subdivisions can be designated, a medial SN, middle SN and lateral SN. In the dorso-ventral plane the SN is subdivided into a dorsal SN (cells near the red nucleus, cells in the medial lemniscus and dorsal cells of the pars compacta and pars lateralis of the SN), and a ventral SN (cells in ventral pars compacta and ventral pars lateralis of SN, cells in pars reticulata of the SN). The A8 cell group of Dahlstrom and Fuxe ('64) will be referred to as the caudal SN. These subdivisions are useful in describing the topography of the projections from the SN and VTA to the cerebral cortex, basal forebrain and striatum.

Anterograde transport studies Autoradiographic studies with injections of 3H-leucine and 3H-proline were made in various sectors of the SN-VTA in order to determine the topography of the ventral mesencephalic projections to the forebrain and especially to determine the pattern of the projections within each terminal area. It is important to note that this analysis is limited


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Fig. 2 Drawings of coronal sections through the SN-VTA. Black dots represent CA cell bodies seen in glyoxylic acidtreated sections of the rat brain. The location of nuclei (outlined by dotted lines) and fiber tracts (solid lines) is also illustrated.

by the fact that the injection sites label a number of neurons and only gross topographical relationships can be determined and that the injections were made into areas that contain both DA and non-DA cells. Thus, the anterograde labeling represents both DA neuron projections and non-DA neuron projections. In addition, there may be preferential uptake and transport of the amino acids within each, or both, of the cell populations. A further problem is the distinction of labeled preterminal fibers and terminal field labeling. Preterminal fibers are interpreted as such when in a clear fiber pathway such as the internal capsule or when the labeling is in linear

arrays. The interpretation of terminal field labeling is based on the location of the labeling and its appearance, usually as scattered silver grains. Despite these limitations the autoradiographic technique is a powerful anatomical tool, particularly when correlated with the results of other techniques. Injection sites Labeled injection sites in the ventral tegmentum of 25 rats are evident in nearly all sectors of the SN-VTA. These include the medial VTA (fig. 17b), lateral VTA, medial SN, middle SN or lateral SN. A t least two injection sites were in these sectors a t either the

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Fig. 3 Representation of coronal sections from olfactory bulb levels (A) to posterior meaencephalon and ventral tegmentum (M),with accompanying abbreviations which identify nuclear areas and fiber tracts. These levels correlate with figures 4-6. See Abbreviations. Solid black lines in piriform cortex and olfactory tubercle represent cell dense layer (11) below superficial layer (I).


Fig. 4 Illustration of a case in which 3H-leucine/prolinewas injected bilaterally into the ventral tegmenturn, including the VTA and interpeduncular nucleus. The shaded area in L-M represents the injection site. Large black dots represent label over fibers of passage and small black dots represent anterograde label over a terminal area. All the projections to the surrounding tegmentum, hypothalamus and thalamus are not il. lustrated. The identification of nuclear areas and fiber pathways is illustrated in figure 3.

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Fig. 5 Illustration of a case in which 'H leucine/proline was injected into the ventral tegmentum, including the lateral VTA and medial SN. The shaded area in J-M represents the injection site. Large black dots represent fibers of passage and small black dots represent anterograde label over a terminal area. All the projections to the surrounding tegmentum, hypothalamus and thalamus are not illustrated.


Fig. 6 Drawings of coronal sections through brain for a case where 3H prolinefleucine was injected into the middle section of the SN (K,L,M) and surrounding tegmentum. Large black dots represent labeled axons and small black dots represent labeling found over terminal areas. For identification of nuclei and fiber tracts see figure 3.

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anterior or posterior half of the SN-VTA. Therefore, in the medial-lateral and anteriorposterior plane there are ten overlapping but distinct components of the SN-VTA in which labeled injection sites are localized. Three examples of the injection sites, with the resulting anterograde labeling are illustrated in figures 4-6. The nuclei and fiber tracts that correspond to the sections in figures 4-6 are illustrated in figure 3. All slides including those containing the injection sites were exposed in the dark for 30 days. The long exposure times used for the injection sites result in more dense and intensive labeling over the injection sites than if the more standard 7-day exposure time was used. The longer exposure times, therefore, probably result in a more conservative estimate of the extent of the injection site, defined here as the area containing dense cellular labeling. The injection site in the VTA, illustrated in figures 4K-M, was centered in the caudal twothirds of the ipsilateral and contralateral VTA and interpeduncular nucleus with minimal extension to the medial edge of the medial lemniscus and SN. The injection site in the medial SN, illustrated in figures 5J-M, was centered in the medial S N with some extension into the medial one-third of the medial lemniscus, lateral VTA, ventral red nucleus, lateral interpeduncular nucleus and lateral posterior mammillary nucleus. The injection site in the middle SN, illustrated in figures 6K-M was centered in the middle SN with some extension into the medial lemniscus, ventral mesencephalic reticular formation and cerebral peduncle. Fiber pathways Following autoradiographic injections into the VTA (fig. 4), some labeled axons course short distances into both components of the SN. Longer, ascending axons traverse the ventral medial tegmentum to the medial forebrain bundle (fig. 4D). Labeled fibers leave the medial forebrain bundle in the ansa peduncularis-ventral amygdaloid bundle (figs. 4E-G) to innervate the amygdala and other cortical areas. The anterior basal forebrain is innervated by rostra1 extension of the medial forebrain bundle (figs. 4C-F). Laterally directed fibers, some within the medial edge of the internal capsule enter the striatum (figs. 4C-G). Fibers enter the olfactory tubercle, nucleus accumbens, anterior olfactory nucleus and ol-

factory bulb (fig. 4A) directly from the medial forebrain bundle, Some fibers traverse the diagonal band to the septum (fig. 4D). Some of these continue dorsally and rostrally around the genu of the corpus callosum to enter the cingulum and innervate cingulate cortex. Other medial forebrain bundle fibers continue rostrally into the external capsule to innervate frontal cortex (figs. 4A,B). Following autoradiographic injections into lateral VTA extending into the medial S N (fig. 51, fibers ascend in the lateral hypothalamus, medial forebrain bundle and medial internal capsule (figs. 5E-I).The distribution of ascending fibers is similar to that following VTA injections except that there is a more laterally situated component. The fiber pathways that enter the striatum are more extensive with numerous fascicles evident in the globus pallidus (figs. 5E,F). Following autoradiographic injections into the middle SN (fig. 6) and lateral SN (not illustrated), ascending axons distribute somewhat more laterally in the medial forebrain bundle and internal capsule. No labeled axons are present over the stria terminalis and less label is present over the ansa peduncularisventral amygdaloid bundle (figs. 6E-G), however, some label is still present above the optic tracts (figs. 6G-H) and lateral sectors of the olfactory tubercle and nucleus accumbens (figs. 6C-E). In summary, the ascending fiber pathways to the cortex, olfactory structures and striatum are very similar following autoradiographic injections into the VTA and SN with the major difference being that medial injections (VTA, medial SN) result in label over the medial components of the medial forebrain bundle and internal capsule while lateral injections (middle SN, lateral SN) result in more lateral labeling of these pathways. Terminal fields Anterograde label was distributed over the cortex, olfactory structures and striatum following injections into different sectors of the VTA or SN. The terminal areas described below include isocortical and allocortical structures (amygdala, septum, piriform cortex, suprarhinal cortex, entorhinal cortex, cingulate cortex, frontal cortex, olfactory tubercle, anterior olfactory nuclei and olfactory bulb) and striatum (nucleus accumbens, globus pallidus, neostriatum). Projections to areas not known to receive a DA innervation


will not be described on the assumption they arise from the non-DA neurons in the VTA-SN region. In this regard it should be noted that some non-DA projections to regions receiving a DA neuron innervation may be shown. The value of the study lies in the extent to which it correlates with other data. Amygdala

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plex with the medial SN providing the major input. Suprarhinal cortex Sparse to moderately dense label can be seen over suprarhinal cortex following injections into the S N or VTA. The label is densest following injections into the medial SN (fig. 5) and is greatest over the deeper cortical layers (fig. 19d). In addition some label is present over more dorsal neocortical areas (e.g., figs. 5C-F). Thus, like the piriform cortex, the innervation of the suprarhinal cortex appears to originate over a wide region of the SN-VTA complex, with the medial SN and lateral VTA providing the major input.

Injections into VTA (fig. 4) and, particularly the medial SN (fig. 5) result in moderately dense labeling over the amygdala, particularly the central nucleus, lateral basal nucleus and intercalated cell groups (fig. 17c). Less dense labeling is evident over the anterior amygdaloid areas and lateral posterior nucleus. As the injection site is placed more lat- Entorhinal cortex erally in the middle SN (fig. 6), considerably The innervation of the entorhinal cortex apless labeling is found in the amygdala, but some continues to be present over the central pears to be from more restricted areas of the nucleus. Injections into the lateral SN do not ventral tegmentum. Injections into the VTA result in appreciable labeling of any compo- (fig. 4) result in sparse to moderate label over the deep cortical layers (figs. 4J-L, 19c). More nent of the amygdaloid complex. lateral injections (fig. 5) result in sparse labeling and lateral injections (fig. 6) result in Septum no labeling of entorhinal cortex. Thus, it apInjections that include VTA (figs. 4, 5) re- pears that the VTA provides most, if not all, of sult in considerable labeling over the septum, the SN-VTA complex innervation of the entoespecially over a band that corresponds to the rhinal cortex. medial part of the lateral septa1 nucleus (fig. 4D). As the injection site is placed more later- Cingulate cortex ally (fig. 5) some label is present over the latInjections into the VTA or SN (figs. 4-61 reeral septum (fig. 5D) but the label is more dif- sult in anterograde label over the cingulate fuse. cortex, particularly the anterior one-third. VTA injections tend to result in label over the Piriform cortex cingulum (figs. 4D,E) whereas more lateral The anterograde labeling over the piriform injections result in more label over the supercortex and claustrum is sparse following ficial layers of the cingulate cortex (figs. injections into the VTA or SN (figs. 4-61. 5,6E). Following a medial VTA injection the labeling is present more medially in piriform Frontal cortex cortex (fig. 4), whereas following a lateral Injections into the VTA and medial S N VTA and medial SN injection the labeling is (figs. 4, 5) result in a modest labeling of fronpresent more laterally (fig. 5). The label is tal cortex, especially in the deeper layers. present in all cortical layers with deep layers Whereas VTA injections result in more medial receiving the densest innervation. Some label labeling (fig. 4A), SN injections result in addican be seen over layer I1 and rarely over layer tional labeling of lateral and ventral compoI (figs. 4: I, 5 : I). The labeling following VTA nents (figs. 5,6A,B). Thus, the majority of the and medial SN injections indicates that there innervation to anterior-medial frontal cortex is a medial-lateral topography in the projec- arises from the VTA but the medial and midtion (figs. 4, 5). Other injections not shown dle SN projects to more lateral-ventral cortex. here indicate that there is an anterior-posterior topography present in the SN and VTA Olfactory bulb The innervation of the olfactory bulb is projection to the piriform cortex. Thus the projection to the piriform cortex appears to sparse to moderately dense and arises from arise from a large area of the SN-VTA com- the VTA and medial SN. Label is evident over

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all Layers of the bulb, especially the internal plexiform layer (figs. 4, 5A) and is concentrated over the ventral-medial part of the posterior half of the olfactory bulb. Little, if any, label is present over the glomeruli. No topography in the projection is evident. Anterior olfactory nuclei The innervation of the anterior olfactory nuclei is sparse to moderately dense and is also topographically organized. Following injections into the VTA label can be found over the ventromedial nuclei (figs. 4A,B) whereas injections in the medial SN result in label over more lateral and dorsal areas (figs. 5A,B). Very sparse label is found over the most dorsal-lateral sectors following injections into the middle SN (fig. 6A). Olfactory tubercle The projection from the SN and VTA to the olfactory tubercle is clearly topographically organized such t h a t the VTA projects to the medial sectors of the tubercle (figs. 4C-E) and the medial-middle SN projects to more lateral areas of the tubercle (figs. 5,6C-E). The labeling is dense following VTA and medial SN injections, especially over layers I1 and I. Layer I11 is characterized by a moderately dense labeling. Nucleus accumbens The innervation to the nucleus accumbens is topographically organized. Injections into the VTA result in dense label over the medial half of the nucleus (figs. 4C, 19a), injections into the medial SN result in moderately dense label over the lateral and ventral parts of nucleus (figs. 5D,E, 19b), injections into the middle SN result in sparse to moderate label over the lateral edge of the nucleus (figs. 6C,D) and injections into the lateral SN do not result in label over the nucleus. Globus pallidus The innervation to the globus pallidus arises exclusively from the SN. Numerous fibers of passage are evident following SN injections. In addition, sparse terminal field labeling is evident over the globus pallidus (figs. 5, 6E). Neostriatum The innervation of the neostriatum is dense, topographically organized and arises from both VTA and SN. VTA injections result in la-

bel over anterior and medial sectors of the neostriatum (figs. 4C-G). Medial SN injections result in label over the medial and middle neostriatum (figs. 5C-I). Injections involving both the middle and lateral SN result in labeling of the middle, lateral and posterior neostriatum (figs. 6C-I). Lateral SN injections result in label over the most lateral and posterior neostriatum. Injections into the lateral and caudal SN (A8 of Dahlstrom and Fuxe, ’64) result in label over the ventral and posterior putamen (fundus striata). If the SN-VTA projection to the neostriatum is considered in toto, the projection to the “rim” areas of the neostriatum is more dense than more central or “core” areas. Therefore, it would appear from these studies t h a t the neostriatal innervation arises from the VTA and SN with the more anterior and medial VTA and SN projecting anteriorly, medially and dorsally and the more lateral and caudal SN projecting laterally, posteriorly and ventrally. Thus, autoradiographic studies have provided data concerning the topography of the projection from the mesencephalic neurons to the cerebral cortex, basal forebrain and striatum. The technique is limited in its usefulness for this type of analysis, however, because of the overlap of injection sites, the difficulty in producing small, highly localized injections into a nuclear group with a restricted dorsalventral dimension. In addition the injection sites include other tegmental and hypothalamic areas t h a t do not contain DA cell bodies (e.g., interpeduncular nucleus, mammillary nuclei, red nucleus) which may give rise to projections that make interpretation of terminal field labeling difficult. Only areas shown in previous studies (Moore, ‘78 Fallon et al., ’78; Fallon and Moore, ’78; Lindvall e t al., ’74; Hokfelt e t al., ’74; Thierry e t al., ‘74; Berger e t al., ’76) to receive a mesencephalic DA neuron projection have been described. As noted above this is based on the assumption that terminal field labeling in these areas following injections into DA neuron groups predominantly represents projections of DA neurons. Further confirmation for this is obtained from the HRP-retrograde transport studies to be described below. The data obtained with the retrograde transport technique allow for a precise determination of the topography of the mesencephalic DA neuron projection on the forebrain. This is possible, first, because the projection is from a relatively restricted neuronal group to much more extensive, diver-


gent terminal fields and, second, because the DA neuronal group has been precisely identified using the GA fluorescence histochemical method.

Retrograde transport studies The cells labeled by retrograde transport of HRP following injections into known DA terminal areas conformed in location, size and appearance to the DA neurons described in the GA fluorescence histochemical study of mesencephalic DA cell groups. Injections of HRP were made into a large number of cortical, olfactory and striatal areas. A variety of stereotaxic approaches were used, including vertical to horizontal injection angles in order to minimize the possibility that HRP uptake along the needle tract would produce confounding results. Listed below are areas into which HRP was injected and a description of the location of retrogradely labeled cells found in the SN-VTA area after such injections. Although cells in the locus coeruleus (NE system) and raphe (serotonin system) were consistently retrogradely labeled with HRP following injections of HRP into basal forebrain structures, it is beyond the scope of this report t o describe these results in detail. Amygdala Injections of HRP into the amygdala result in retrograde labeling of cells in the VTA and SN. Since the majority of the DA innervation of the amygdala is concentrated in the dorsal or middle areas (central nucleus, intercalated nuclei and lateral basal nucleus), (Fallon et al., '781, it was difficult to obtain retrograde labeling of cells in the SN-VTA unless the HRP injection was located in the dorsal two-thirds of the amygdala. The dorsal amygdala lies adjacent to the ventral neostriatum which is also densely innervated by DA terminals. For this reason the distinction between SN-VTA neurons projecting t o the amygdala and those projecting to the neostriatum is difficult because HRP spread into the adjacent neostriatum often occurs. Thus, two populations of cells in the SN-VTA are frequently retrogradely labeled as a result of HRP injections into the dorsal amygdala. It is possible to discriminate the two projections, however, as injections of HRP confined to the neostriatum result in retrograde labeling of cells in the ventral portion of the SN pars compacta and SN pars reticulata whereas HRP injections confined almost entirely to the amygdala re-

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sult in retrograde labeling of cells in the dorsal part of the VTA and SN pars compacta adjacent to and within the medial lemniscus. Injections involving both neostriatum and amygdala result in very dense anterograde HRP labeling of axons and terminals located immediately ventral to the retrogradely labeled cells in the ventral SN pars compacta (fig. 17d). In addition, labeled cells are found a t a distance from those associated with the anterograde HRP transport (figs. 17d-f). Injections of HRP largely restricted to the amygdala result in little, if any, anterograde HRP label in the SN. Examples of these phenomena are illustrated in figure 7. Injection of HRP into the anterior portion of the lateral amygdala and ventral putamen (fig. 7A) results in retrograde labeling of ventral cells in the SN (fig. 7A: 1-31 and another population of dorsal cells in the lateral VTA (fig. 7A: 2). Adjacent to the labeled SN pars compacta cells is a dense anterograde label in the SN, pars reticulata. The retrograde labeling of cells in the dorsal and lateral VTA results from the HRP injection into the lateral amygdala and the retrograde labeling of cells in the lateral SN results from the ventral putamen component of the HRP injection. When the injection of HRP is made into more caudal and medial sectors of the amygdala (fig. 7B), the retrograde labeling of dorsal cells in the VTA and medial S N is shifted more caudally (cf. figs. 7A: 3, 7B: 3). In addition, the retrograde and anterograde labeling in the lateral SN is shifted caudally, (cf. figs. 7A: 3, 4, 7B: 3, 4). With some HRP injections into the amygdala, retrogradely labeled cells are located in the prerubral field and around the mammilothalamic tract, an area known to contain DA cells (Bjorklund and Nobin, '73). From the analysis of 25 injections of HRP into the amygdala, using different injection angles into all components of the amygdala, i t is concluded that the dorsal cells of the lateral VTA, medial SN and middle SN project topographically to the amygdala with the VTA projecting slightly more medially and the medial and middle SN projecting more laterally to the amygdala. In addition, the anterior sectors of the SN-VTA project to the anterior amygdala and the posterior SN-VTA projects to the posterior amygdala. There is also a possibility that the lateral SN projects to the amygdala but, if this projection does exist, it is a unique and separate projection that parallels the more medial SN-VTA projection. The

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Fig. 7 Illustration of two cases of HRP injections (left) into the posterior basal telencephalon and the resulting retrograde cell labeling (large black dots) and anterograde terminal labeling in SN, (small black dots) in ventral tegmenturn a t right. Injection a t top (A) was centered in anterior portion of posterior lateral nucleus of amygdala. Spread of HRP into ventral caudal putamen resulted in retrograde label of lateral SN, cells with additional anterograde label in adjacent SN, (small black arrows). The retrogradely labeled cells in the more medial SN and lateral VTA (large black arrows) was due to HRP injection into amygdala and possibly piriform cortex. Injection a t bottom (B) was placed more medially and posteriorly in amygdala than case A. Note t h a t retrograde cell labeling in medial SN and lateral VTA tends to be more medial and caudal than in A (compare figs. A: 2 and B: 2 as well as figs. A: 3 and B: 3). Also note that the retrogradely labeled cells in lateral VTA and medial SN a r e situated dorsally in both cases A and B. These topographical relationships indicate a projection pattern from SK and VTA to basal forebrain as being medial to medial, anterior to anterior and dorsal t o ventral.

lateral S N could possibly project to the central nucleus of the amygdala, a connection t h a t would be reciprocal to central amygdalofugal projections to the lateral S N (Bunney and Aghajanian, '76). However, a direct projection from the lateral S N to the amygdala is not supported by the results of autoradiography

tracing (see above) or biochemical studies (Fallon et al., '78). Septum Injections of HRP into the septum result in retrograde labeling of cells in the VTA as illustrated in figures 8A,B and, in some in-


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Fig. 8 Drawings of coronal sections through the brain after injections of HRP into the lateral (A) or medial (B) septum (left side of figure). The ventral tegmental cells that were labeled after these injections are represented by black dots. In comparing the two case8 it should be noted tha t lateral septal injections (A) resulted in more retrograde labeling of cells in the ventral VTA (A: 2, 3) while medial septal region injections (B) resulted in more retrograde labeling of cells around the mammilothalamic tract, in the vicinity of the caudal diencephalic-DA cell group (B: 1, arrow).

stances, in the caudal diencephalic cell group (fig. 8).The cells labeled in the VTA are in the ventral area, just above the interpeduncular nucleus (fig. 8A: 2, 3). Injections centered in the medial septum (fig. 8B) result in retrograde labeling of cells both in the VTA and around the mammillothalamic tract (fig. 8B: 1) whereas injections restricted to the lateral septum result in retrograde labeling of cells only in the VTA. This indicates that the ventral cells in the VTA project more laterally in the septal nuclei whereas cells in the caudal diencephalon, around the mammillothalamic

tract, project to a more medial septal region. The data also indicate a rostro-caudal topography to the VTA-septa1 projection. Piriform cortex Injections of HRP into the piriform cortex result in retrograde labeling of cells in the lateral VTA and a wide area of the SN. As with the amygdala, it is difficult to make injections restricted t o piriform cortex which do not spread into the ventral neostriatum or lateral amygdala. Injections centered in the piriform cortex consistently lead to retrograde labeling

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Fig. 9 HRP injections into the piriform cortex, claustrum, anterior amygdaloid area and ventral-lateral caudate-putamen of two rats. When compared with more localized injections into t h e ventral-lateral caudate-putamen (fig. 11B), these cases resulted in more labeling found in the dorsal cells of the lateral VTA, medial and middle SN and less retrograde labeling in the caudal SN area (A: 4, B: 4). These results and the results of figure 11B taken together, suggest t h a t the caudal SN projects to the ventral-lateral caudate-putamen, while the dorsal cells of the lateral VTA and SN project to the adjacent basal forebrain.

of dorsal cells in the lateral VTA (fig. 9A: 3 and fig. 9B: 2 ) and wide expanses of the SN (figs. 9A,B). With spread of the HRP into the ventral neostriatum (fundus striata), some cells in the lateral caudal SN were labeled (figs. 9A: 4,and 9B: 41. Thus, the innervation of the piriform cortex appears to arise from approximately the same areas of the SN-VTA that project to the amygdala.

tubercle result in retrograde labeling of the dorsal cells in the middle and lateral VTA and medial SN. Injections of HRP into the lateral half of the olfactory tubercle result in retrograde labeling of cells in the dorsal VTA. Injections of HRP t h a t were restricted to the olfactory bulb or anterior olfactory nuclei did not result in retrograde labeling of SN-VTA cells.

Olfactory nuclei Large injections of HRP into the olfactory

Nucleus accumbens I t was not possible to obtain HRP injections


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Fig. 10 HRP injections centered in the anteromedial caudate (A) or nucleus accumbens and medial-ventral caudate (B).Anteromedial caudate injection (A) resulted in retrograde labeling (large black dots) in the pars reticulata of the SN. Injection of HRP into the nucleus accumbens and medial-ventral caudate (B)resulted in a similar pattern of retrograde and anterograde labeling in the ventral SN-VTA but also included a slightly more dorsal component in the lateral VTA and medial SN (B:2, 3).

restricted to the nucleus accumbens without some spread into the adjacent areas of the neostriatum or lateral septa1 area. An HRP injection centered in the nucleus accumbens, but with some spread of HRP into the adjacent neostriatum (fig. IOB) results in retrograde labeling of cells in the ventral-lateral VTA and medial SN (fig. 18d). These injections, as well as neostriatal injections, also result in anterograde labeling of axons and presumed terminals in the pars reticulata of the SN. The location of the anterograde label also reflects a topographic organization of the pro-

jection in that it is always located just ventrolateral to the retrogradely labeled cells in the ventral SN. Neostriatum The HRP studies as well as the autoradiographic studies reveal a precise topographic pattern for the projections of the SN-VTA to the neostriatum. The projection from the neostriatum to the SN also is organized in a reciprocal topographic fashion. This latter conclusion is obtained from analysis of the pattern of the anterograde transport of HRP

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Fig. 11 HRP injection sites centered in the dorsal-lateral caudate-putamen (A) or ventro-lateral caudateputamen (B). After both injections, retrograde labeling was found in the ventral cells middle SN, with accompanying anterograde HRP label in the adjacent pars reticulata (A: 1, 2, 3 and B: 1, 2, 3). After ventro-lateral injections (B), however, additional retrograde label was found in the caudal SN (arrow in B: 4) and dorsal sector of the medial SN (B: 3).

to the S N after neostriatal injections. The anteromedial sector of the neostriatum receives projections from the most ventral cells of the lateral VTA and medial SN (fig. 10A). When the HRP injection site is shifted laterally and slightly caudally (fig. 11A), labeled cells are found ventrally in the middle SN (figs. 18a,b) including some cells in the pars reticulata (fig. 18e). The pars reticulata in this area also contains anterograde label. In cases in which the injection is located ventrally in the neostriatum the labeled cells are located lateral and caudal in the S N (fig. 11B).

The additional labeling of dorsal cells in the medial SN (fig. 11B: 3) reflects spread of HRP into the anterior amygdaloid area and piriform cortex (fig. 18c). This conclusion is based on results obtained when the injection site is centered more ventrally in the piriform cortex, claustrum and anterior amygdaloid area (figs. 9A, B). These more ventral HRP injections result in more labeling of dorsal cells of the lateral VTA (fig. 9A: 3) as well as the middle and medial SN without anterograde label in the adjacent pars reticulata (figs. 9A: 1,3,4 9B: 1-3).Since some HRP did spread into the


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Fig. 12 HRP injections with two different electrode angles into the caudal putamen, dorsal amygdala and suprarhinal cortex (A) and a similar injection site sparing the dopamine-containing axons in the amygdala (B). These results suggest t h a t t he lateral and caudal SN (B: 3) projects to the caudal putamen while dorsal cells of the more medial areas of the SN and VTA project to the amygdala (A). The spread of HRP into the temporal cortex in (B) accounts for retrograde HRP label of non-DA cells in the medial geniculate nucleus (B: 2, 3).

ventral caudal putamen in these two cases, some caudal S N cells are labeled (figs. 9A: 2,4 9B: 4). Further cases substantiate these observations. In these cases, and if the HRP injection sites are placed more caudally in the neostriatum (figs. 12A,B), the retrograde and anterograde label in the SN is shifted to the lateral SN. When the HRP spread to the amygdala is minimal (fig. 12B) the only cells that are retrogradely labeled in the SN are lateral SN cells (fig. 12B: 3). If the injection site spread into the central and posterior lat-

eral nucleus of the amygdala, additional labeling is found in the dorsal cells of the VTA and medial SN (fig. 12A: 1-31. DISCUSSION

This study concludes a series intended to provide as detailed an account of the CA innervation of the basal forebrain as possible using currently available methodology. The first three studies (Moore, '78; Fallon et al., '78; Fallon and Moore, '78) were directed to two aspects of this general problem. The first was

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to assess the relative density of the CA innervation of the basal forebrain areas under study using regional analysis of N E and DA content and to identify the nuclei of origin of the innervation by determining the effects of restricted lesions on regional NE and DA content. I t is apparent that biochemical assays provide only a very gross estimate of innervation density but the assays have proven useful as one method for determining the nuclei of origin of the innervation when combined with lesions. The major limitations to this methodology are as follows. First, the variabilities of the analytical methodology and t h a t of the dissections makes it difficult to distinguish differences less than 20%. Consequently, a small contribution to the CA innervation of a n area from a given nucleus probably would not be appreciated. Second, the lesions destroy large areas of a nucleus and this methodology does not permit conclusions concerning a precise topography of projection. Third, the loss of CA in an area analyzed could be throughout the area or in a restricted part of it and this would not be apparent in the data. The GA fluorescence histochemical method, in contrast, not only is extremely sensitive but allows a very precise, detailed analysis of the organization of the CA innervation of the areas studied. This correlates well with the assay data in the respect that areas with dense innervation evident histochemically have high CA content by biochemical assay. The histochemical method is limited in that it is difficult to trace a pathway in normal material and that it may be difficult to appreciate a very limited CA innervation, such as the N E innervation to neostriatum and olfactory tubercle, in the presence of a very dense innervation, in these instances a DA innervation. The method does have a n advantage over the original Falck-Hillarp method, however, - that i t permits differential identification of N E and DA axons on morphological grounds in most instances (Lindvall and Bjorklund, '74a,b). The use of lesions with the fluorescence histochemical method can provide some information (Moore, '78) but this is limited particularly in the case of diffuse projections, and it was apparent early in the course of these studies t h a t the autoradiographic tracing method and the HRP-retrograde transport method would give much more reliable and precise information on the topography of projections. This is particularly true for the DA projections, the subject of the present study.

We have not observed any discrete topography in the projections of the N E cell groups. The biochemical and histochemical data indicate t h a t the NE neuron innervation of the basal forebrain with few exceptions such a s the interstitial nucleus of the stria terminalis, is relatively homogeneous and distributed in a diffuse, fairly even, plexiform manner in nearly all areas studied. Most of the NE neuron innervation of the basal forebrain arises from locus coeruleus with a much smaller contribution from caudal brainstem NE cell groups restricted to septum, olfactory tubercle and amygdala. Following HRP injections into basal forebrain areas we routinely observed labeling of locus coeruleus neurons (e.g., from septum, Moore, '78) but without clear evidence for any topography of labeling within the nucleus. No labeling of caudal brainstem neurons was noted but these NE neurons are scattered and this could easily be missed. The striking finding of the present study is the precise topography of the projections of the mesencephalic DA neurons. When the observations from the biochemical, histochemical, autoradiographic and retrograde transport studies are considered together, it is possible to delineate the organization of both the NE and DA projections to basal forebrain (Moore, '78; Fallon e t al., '78; Fallon and Moore, '78), to identify the cell groups giving rise to the projections and, in large part, the exact topography of mesencephalic DA neuron projections to the entire telencephalon. The latter point will be discussed in greater detail below. The study of the connections of DA-containing cells of the midbrain recently has a t tracted many investigators, in large part because of the discovery that loss of the DA neurons of the nigrostriatal pathway plays a n important role in the pathogenesis of Parkinson's disease (Hornykiewicz, '66; '73). Subsequently, the existence of major DA projections to the basal forebrain and striatum has been demonstrated experimentally by a number of techniques. The DA projection systems conventionally have been divided into three major groups, a nigrostriatal system (Anden e t al., '64; Poirier and Sourkes, '65; Bedard et al., '69; Hokfelt and Ungerstedt, '69; Moore et al., '71; Ungerstedt, '71; Carpenter and Peter, '72; Golden, '72; Hattori et al., '73; Maler et al., '73; Shimizu and Ohnishi, '73; Lindvall and Bjorklund, '74b; Nauta et al., '74; Sotelo and Riche, '74; Butcher et al., '76; Kitai et al., '76; Usunoff et al., '761, a mesolimbic system


CELLS

PROJECTION VTA

Fig. 13 Schematic drawing of the location of DA-containing cell bodies of the SN and VTA a s viewed from a dorsal perspective (top left) and two representative drawings of coronal sections (A, B top right) through the S N and VTA. The generalized scheme of projections of the SN-VTA cells is shown in drawing a t bottom of figure. For explanation see DISCUSSION. Abbreuiations: R, red nucleus; MG, medial geniculate nucleus; M,, posterior mamrnillary nucleus; L, medial lemniscus; MT, medial terminal nucleus of accessory optic system (accounts for “clear” area in top left drawing); P, pontine nuclei; IPN, interpeduncular nucleus; VTA, ventral tegmental area; SN,, pars cornpacta of the substantia nigra; SN,, pars reticulata of the substantia nigra.

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and mesocortical system (Anden e t al., '66; Ungerstedt, '71; Thierry et al., '73; Hokfelt e t al., '74; Lindvall et al., '74a; Berger et al., '76). From these studies cited above, the nigrostriatal DA pathway has been thought to arise largely from pars compacta of the S N (A9 group of Dahlstrom and Fuxe, '64) whereas the mesolimbic and mesocortical DA systems were thought to arise from the VTA (A10 group of Dahlstrom and Fuxe, '641.The results of the present study and the previous reports in this series (Fallon et al., '78; Fallon and Moore, '78; Moore, '78) do not support this strict parcellation of the DA cell groups and projection systems but rather suggest that the DA cell groups form a continuum, with both the VTA and SN providing DA neuron projections to the majority of the telencephalic terminal fields. In this respect, our results are in agreement with observations obtained by others (Moore e t al., '71; Fuxe e t al., '74; Lindvall e t al., '74; Avendano e t al., '76; Domesick e t al., '76). The data from our studies add to the previous observations by proposing t h a t the projection of the SN-VTA system of DA neurons has a discrete topography which includes not only medial-lateral and anteriorposterior components, but also contains a n important dorsal-ventral component which accounts for, a t least in part, the separation of the cortical and striatal projection systems. This separation is not exclusive since one cell may contribute fibers to both systems, for example some DA fibers were found to bifurcate with one axonal branch innervating the neostriatum and one axonal branch innervating adjacent amygdala (Fallon et al., '78). An alternative explanation of this divergence would be to consider the central nucleus a s a component of the neostriatum. A clearer example of this dorsal-ventral separation of the SN-VTA projection is that the dorsal cells of the SN and VTA project to ventral basal forebrain structures (e.g., amygdala, olfactory tubercle) while ventral cells of the VTA and SN project to more dorsal basal forebrain and striatal structures (e.g., septum, neostriatum). It should be noted, however, t h a t we cannot state on the basis of the evidence currently available the extent to which one DA neuron may project to more than one component of the overall topographic organization. That is, a ventral VTA cell for example, could project to both septum and rostral medial neostriatum. While this does not seem likely

on conceptual grounds, it cannot be excluded by the available data. This dorsal-ventral separation is illustrated in the highly schematized diagram of a coronal section through the SN-VTA in figure 13. The location of the DA cell bodies in the SNVTA is illustrated in figure 13 from a dorsal view (left side of fig. 13) and two representative coronal sections (right side of fig. 13). The general projection patterns of the SN-VTA cells is shown a t the bottom of figure 13. The dorsal-ventral organization of the SN and VTA projection systems includes a ventral zone in the pars reticulata, most of the cells of which are not DA-containing and which project to the tectum (Graybiel and Sciascia, '75; Faull and Mehler, '76; Hopkins and Niessen, '76). Cells throughout the pars reticulata, especially in the rostro-lateral aspect of the nucleus, project to the thalamus (Rinvik, '75; Carpenter e t al., '76; Clavier et al., '761, but this is not a DA neuron projection. Cells in the ventral portion of the SN and lateral VTA, including some cells in the pars reticulata, project to the neostriatum. Some ventral cells in the VTA also project to the septum. The dorsal cells of the S N and VTA prcject to the other areas of the limbic forebrain, including the nucleus accumbens, olfactory tubercle, amygdala and the piriform cortex. The separation and topographical relationships of the projections of the dorsal cells, ventral cells and cortical projections are summarized in figures 14, 15 and 16 (see figures and legends). The topography of the cortical projections are in general agreement with previous studies (Fuxe et al., '74; Lindvall et al., '74; Avendano et al., '76) but do not support the findings of Avendano et al. ('76) in the cat that the cortical projections are bilateral. The anterior-posterior component of the topography of the nigrostriatal pathway is in agreement with results obtained in the cat (Bedard e t al., '69; Usunoff et al., '76) and rhesus monkey (Carpenter and Peter, '72). The finding t h a t the VTA contributes DA fibers to the anterior and medial strip of the caudate agrees with recent observations in the r a t (Simon e t al., '76). This VTA-neostriatal projection may reflect the heterogeneity of the DA innervation in the neostriatum with different transmitter-receptor properties associated with the rostral neostriatum (VTA projection) and the caudal neostriatum (SN projection), as reported by Tassin et al. C76). Olson e t al. ('72) reported


DORSAL CELLS

7

567

VENTRAL FOREBRAIN

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/r posterior $.

project ion

VTA

Fig. 14 Schematic representation of the topography of t he projection of the dorsal cell of the SN-VTA to th e ventral structures of the basal forebrain. Three drawings a t left represent dorsal view of SN-VTA as explained in figure 13. The width of the arrows reflect the density of the DA innervation to the basal forebrain structures a t right. The stippling in the drawings represents the VTA area and the resulting projection while th e cross hatching represents the SN area and its projection zones. The overall topography is medial to medial, lateral to lateral. Some evidence exists also for a n anterior to anterior and posterior to posterior topography. The origin of the DA projection to the olfactory bulb is in doubt but probably originates from the VTA area. There is also a possibility t hat the lateral SN provides a n additional input to the central nucleus of the amygdala (arrow to question mark) and t he caudal diencephalic DA groups may also project to the amygdala.

two different types of DA innervation in the neostriatum and limbic structures evident in the rat DA system during development. These two types include a diffuse-type of DA termi-

nals and “island”-like DA terminals. The “island” type of DA innervation develops first and the two types of terminals have a differential response to pharmacological agents which

568


VENTRAL CELLS

DORSAL SUBCORT.

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4% Q)

E

Fig. 15 Schematic representation of the projection of the more ventral cells of the SN and VTA to the dorsal forebrain structures. The projection from the VTA to the lateral septum is shown but the projection from the caudal diencephalic DA group to the septum is not represented. The SN-VTA projection to the nucleus accumbens has a clear medial to medial and lateral to lateral topography. The nigrostriatal projection arises from t h e lateral VTA to the anteromedial caudate while the SN projection, represented by three zones, is to the more lateral, caudal and ventral caudate-putamen. The inset a t the lower right of the figure is a n illustration of a coronal section through the brain a t t h e level of t h e tic mark (caudate-putamen, at right). Note t h a t the VTA projects medially in the caudate-putamen while the SN projects more laterally and ventrally. The caudal S N (A8 of Dahlstrom and Fuxe, '64) projects to the area of the ventral putamen (fundus striata) and possibly to the dorsal anterior amygdaloid area.


569

CORTEX

NT. CINGULATE

-U 4-q$

!i

Fig. 16 Schematic representation of the projection of the VTA and SN to the cortex. The anterior and posterior VTA projects to the anterior medial frontal cortex and entorhinal cortex respectively. Some contribution from the most medial SN just adjacent to the VTA to the entorhinal cortex is also possible. The projection of these more polar cortices from the VTA is contrasted with the projection from more lateral areas of the VTA and SN to the anterior cingulate and piriform-suprarhinal cortices. The topography within these systems is not established. Dorsal cells account for the majority of the projections, especially to more ventral cortical areas.

is topographical within the neostriatum (01son et al., ’72).Since DA terminals from the VTA are known to give rise to “island”-like terminals (Lindvall et al., ’74b;Fallon et al.,

’781, these different terminal morphologies in the DA innervation of the neostriatum and limbic structures may reflect the different origins of these pathways in the VTA and SN.

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The most consistent feature of the projection of caudal nigral DA cells is to t h e fundus striata at t h e level of lateral limb of t h e anterior commissure. Ungerstedt ('71) first suggested that this cell group (A8 of Dahlstrom and Fuxe, '74) might project to the neostriatum and Nauta ('76) has also discussed t h e unique projection of these cells to t h e ventral striatum. Special note is required of t h e projections of diencephalic and caudal mesencephalic DA cell groups. The caudal diencephalic cell group (Bjorklund and Nobin, '73) which was not thought to project to the basal forebrain (Fuxe e t al., '74) has been shown to contribute ascending axons in t h e medial forebrain bundle (Ungerstedt, "71). Evidence was presented in the present study t h a t some cells in t h e appropriate area of the caudal diencephalon (but not proven to be DA cells) probably project to t h e septum a n d amygdala. A projection of diencephalic DA cells to telencephalon has not been observed with the possible exception of a very small component of the incerto-hypothalamic system of Bjorklund et al. ('75). I t has been suggested recently t h a t the mesencephalic DA cell groups project to hypothalamus (Kizer et al., '76) but we have obtained no evidence in this study to support such a projection and this is in accord with t h e observation of Weiner e t al. ('72) t h a t complete hypothalamic deafferentation does not alter hypothalamic DA levels. In addition, fluorescence histochemical studies suggest t h a t most hypothalamic DA innervation is associated either with the periventricular DA system (Lindvall and Bjorklund, '74b) or the tuberohypophysial system (Bjorklund et al., '73). In summary, t h e DA neurons of t h e SN and VTA are formed as a single unit ontogenetically (Olson and Seiger, '72) and constitute a continuous group in t h e adult. The projections of the SN-VTA can be broken down into two components, a striatal projection and a cortical projection. The striatal projection arises from the ventral tier of DA cells in the S N and VTA with a clear medial-lateral and anteriorposterior topography. The cortical projections arise from the dorsal tier of DA cells in t h e SN and VTA, with t h e exception of t h e septum which receives projections from t h e ventral cells in the VTA. I n most allocortical areas there is a medial-lateral topography. The neocortical and transitional cortical projections arise from the S N and VTA and are topographically organized such t h a t polar cortices

(frontal cortex, entorhinal cortex) are innervated by VTA projections while lateral and medial cortices (piriform cortex, suprarhinal cortex, cingulate cortex) are innervated by broad expanses of t h e SN and VTA. These results do not support t h e concept of a strict distinction between a mesolimbic and mesocortical DA system arising from t h e VTA and a nigrostriatal DA system arising from t h e SN. The results of the present study do support the concept that precise topography of projection is a distinctive feature of t h e DA systems in general. A typical example is t h e tubero-hypophysial DA system (Bjorklund et al., '73). The organization of t h e DA systems is in marked contrast to other monoamine systems such as the norepinephrine (cf. Moore and Kromer, '77, for review), epinephrine (Hokfelt et al., '74) and serotonin (Anden et a]., '66; Fuxe e t al., '68; Conrad e t al., '74; Moore e t al., '78) systems which are generally characterized by a much more widespread distribution of cell bodies and more diffuse projections. The DA systems a r e characterized by distinct localization of neuronal perikarya, a topographically organized projection system and, in all likelihood, a distinct function determined by the relationships of a specific DA system to other, well-defined functional systems with which i t interacts. ACKNOWLEDGMENTS

This work was supported by USPHS Grants NS-12080 a n d NS-05187 from the National Institutes of Health. We are grateful to Mr. Bart Zeigler for his skilled technical assistance in the preparation of t h e photographic material. LITERATURE CITED Anden, N.-E., A. Carlsson, A. Dahlstrom, K. Fuxe, N.-A.Hillarp and K. Larsson 1964 Demonstration and mapping out of nigro-neostriatal dopamine neurons. Life Sci., 3: 523-530. Anden, N.-E., A. Dahlstrom, K. Fuxe, K. Larsson, L. Olson and U. Ungerstedt 1966 Ascending monoamine neurons to the telencephalon and diencephalon. Acta physiol. scand., 67: 313-326. Avendano, C.,F.Reinoso-Suarez and A. Llamas 1976 Projections to gyrus sigmoideus from the substantia nigra in the cat, as revealed by the horseradish peroxidase retrograde transport technique. Neurosci. Letters, 2: 61-65. Bedard, P., L. Larochelle, A. Parent and L. J. Poirier 1969 The nigrostriatal pathway: A correlative study based on neuroanatomical criteria in the cat and the monkey. Exp. Neur., 25: 365-377. Berger, B., A. M. Thierry, J . P. Tassin and M. A. Myre 1976 Dopaminergic innervation of the rat prefrontal cortex: a fluorescence histochemical study. Brain Res., 106: 133-145. Bjorklund, A., 0. Lindvall and A. Nobin 1975 Evidence of

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Fallon, J. H., andR. Y. Moore 1978 Catecholamine innervation of the basal forebrain. 111. Olfactory bulb, anterior olfactory nuclei, olfactory tubercle and piriform cortex. J. Comp. Neur., 180: 533-544. Faull, R. L. M., and W.R. Mehler 1976 Studies of the fiber connections of the substantia nigra in the rat using the method of retrograde transport of horseradish peroxidase. Neuroscience Abs., 2: 62. Fuxe, K., T. Hokfelt, 0. Johansson, G. Jonsson, P. Lidbrink and A. Ljundiihl 1974 The origin of the dopamine nerve terminals in limbic and frontal cortex. Evidence for meaocortico dopamine neurons. Brain Res., 82: 349-355. Fuxe, K., T.Hokfelt and U. Ungerstedt 1968 Localization of indolealkylamines in CNS. Adv. Pharmacol., 6:

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Golden, G. S. 1972 Embryologic demonstration of a nigro-striatal projection in the mouse. Brain Res., 44:

278-282. Graybiel, A. M., T. R. Sciascia 1975 Origin and distribution of nigrotectal fibers in the cat. Neuroscience Abs., 1: 174. Hattori, T.,H. C. Fibiger, P. L. McGeer and L. Maler 1973 Analysis of the fine structure of the dopaminergic nigrostriatal projection by electron microscopic autoradiography. Exp. Neur., 41: 599-611. Hokfelt, T., K. Fuxe, M. Goldstein and 0. Johansson 1974 Immunohistochernical evidence for t h e existence of adrenaline neurons in the r a t brain. Brain Res., 66:

235-251. Hokfelt, T., and U. Ungerstedt 1969 Electron and fluorescence microscopical studies on the nucleus caudatus putamen of the r a t after unilateral lesions of ascending nigro-neostriatal dopamine neurons. Acta physiol. scand., 76: 415-426.

Hopkins, D. A., and L. W.Niessen 1976 Substantia nigra projections to the reticular formation, superior colliculus and central gray in the rat, cat and monkey. Neurosci. Letters, 2: 253-259. Hornykiewicz, 0. 1966 Dopamine (3-hydroxytryptamine) and brain function. Pharmacol. Rev., 18: 925-964. 1973 Parkinson’s disease: From brain homogenate to treatment. Fed. Proc., 32: 183-190. Kitai, S. T., J. D. Kocsis and J. Wood 1976 Origin and characteristics of t h e cortico-caudate afferents: a n anatomical and electrophysiological study. Brain Res., 118: 137 141. Kizer, J. S., M. Palkovits and M. J. Brownstein 1976 The projections of the A8, A9 and A10 dopaminergic cell emibodies: Evidence for a nigral-hypothalamic-median nence pathway. Brain Res., 108: 363-370. Lindvall, O.,and A. Bjorklund 1974a The glyoxylic acid fluorescence histochemical method: a detailed account of the methodology for the visualization of central catecholamine neurons. Histochem., 39: 97-127. 1974b The organization of the ascending catecholamine neuron systems in the r a t brain. Acta phyaiol. scand., Suppl. 412: 1-48. 1977 Organization of catecholamine neurons in the rat central nervous system. In: Handbook of Psychopharmacology. L. Iversen, I. Iversen and S.H. Snyder, eds. Plenum Press, New York, in press. Lindvall, O., A. Bjorklund and T. Hokfelt 1973 Application of the glyoxylic acid method to vibratorne sections for the improved visualization of central catecholamine neurons. Histochemie, 35: 31-38. Lindvall, O., A. Bjorklund, R. Y. Moore and U. Stenevi 1974 Mesencephalic dopamine neurons projecting to neocortex. Brain Res., 81: 325-331. Maler, L., H. C. Fibiger and P. L. McGee 1973 Demonstration of the nigrostriatal projection by silver staining after nigral injections of 6-hydroxydopamine. Exp. Neuro., 40: 505-515. Moore, R.Y. 1978 The catecholamine innervation of the basal forebrain. I. The Septa1 area. J. Comp. Neur., 177: ~

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Note Added in Proof: Since this paper was submitted for publication it has been shown that Substance P fibers innervating the SN originate from the rostra1 neostriatum whereas GABA fibers originate from the caudal neostriatum (Gale et al., '77, Brain Res.,136: 371-3751, The Substance P fibers appear to preferentially innervate medial sectors of the SN-VTA (fig. 13 of Cuello and Kanazawa, '78; J. Comp. Neur., 178: 129156). These observations, in conjunction with those of the present study represent a further demonstration of the components within the reciprocal connections between the substantia nigra and neostriatum with identified neurotransmitters and a topographical organization.

PLATES

PLATE 1 EXPLANATION OF FIGURES

17a-f Photomicrographs of coronal sections of the brain processed for autoradiography ( a d or HRP retrograde transport (d-f). b-f are photomicrographs taken with a darkfield condensor. (a) Injection site of 'H prolineileucine into the ventral tegmentum. The injection site was in the anterior-medial ventral tegmentum, including the medial and middle SN and lateral VTA with some spread into the lateral mammillary nuclei, crus cerebri, medial lemniscus and ventral Forel's fields. This is the most extensive injection site of any brain included in the study. (b) Anterograde transport in the medial forebrain bundle, medial cerebral peduncle and above the optic tracts after 3H prolinefleucine into the medial ventral tegmentum. Marker bar, 200 pm, (c) Anterograde transport into one of the intercalated nuclei of the amygdala (MI) and less dense label in the central nucleus (AC) after 3H proline/leuc~ne injection into the medial ventral tegmentum. Marker bar, 200 Fm. (d) Middle and lateral SN area. HRP was injected into the dorsal amygdala with some spread into the caudal-ventral putamen. The anterograde HRP transport into the lateral SNR, associated with the retrogradely labeled ventral cells of the lateral SN,, were mainly the result of the HRP injection into the putamen while the more heavily labeled dorsal cells of the middle SN were t h e result of the HRP injection into the amygdala. Marker bar, 200 wm. A higher power photograph of the area outlined in the white box of (d) is shown in (el. The retrogradely labeled cells in (e) (arrows) have dendrites with a more medial-lateral orientation than the ventral cells of the SN,, whose dendrites a r e oriented in a more ventro-lateral orientation. Thus, the ventral SN, cells have dendrites in t h e area of the SNR which receives striatal efferents from the same area to which the SN, cells project. These topographical connections are the reciprocal nigro-striatal-nigral loops. Marker bar, 100 pm. (f) Retrogradely labeled cells in the dorsal portion of the lateral VTA and medial SN after a n HRP injection into the amygdala. Some retrogradely labeled cells were found in the medial lemniscus (L). Note the paucity of anterograde HRP labeling. Marker bar, 200 pm.

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TOPOGRAPHY O F DOPAMINE CELL PROJECTIONS J a m e s H. Fallon a n d Robert Y. Moore

PLATE 1

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16a-f Photomicrographs of coronal sections through the SN and VTA following injections of HRP into areas of the basal forebrain and neostriatum (a-f). (a) section through the middle-anterior SN following injection of HRP into the anterior-lateral caudate-putamen. The retrogradely labeled ventral cells in the SN, lie adjacent to t h e anterograde label in SNR. Arrow (a) designates an axon adjacent to anterograde labeling in SNR. (b) A higher magnification of section shown in (a). (c) Retrograde labeling of two populations of cells in the SN and VTA following injection of HRP in the antero-lateral caudate-putamen with localized spread into the temporal, suprarhinal and piriform cortex. The labeled cells in the mid-lateral SN, was associated with anterograde labeling in the SNR (left side of c). This labeling was due to the striatal injection of HRP, while the retrogradely labeled cells in the medial SN-lateral VTA area was probably due to the cortical component of the HRP injection (d). Retrogradely labeled cells in the ventral part of the medial SN-lateral VTA following injection of HRP into the nucleus accumbens and adjacent caudate-putamen (fig. 6B). (e) Retrogradely labeled cells in the mid-caudal SN following large injection of HRP into the dorsal caudate-putamen. Note t h a t in addition to the labeled cells in the SN,, there is additional labeling of cells in the SNR. The labeled cells in the SNR lie within the area receiving anterograde HRP labeling. Marker bars, 200 gm. (f) Photomicrograph of section processed for GA-induced fluorescence demonstrating DA-containing cells in the SN. A DA neuron in the dorsal tier is designated by an arrow. L denotes medial lemniscus. Marker bar, 50 gm.

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TOPOGRAPHY OF DOPAMINE CELL PROJECTIONS James H. Fallon and Robert Y. Moore

PLATE 2

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19a-e Photomicrographs of coronal sections processed for autoradiography following injections of 3H proline/ leucine into the ventral tegmentum. (a) and (b) show anterograde label in the posterior nucleus accumbens. In (a) the injection site was placed in the VTA. The anterograde label was concentrated in the dorsal and medial nucleus accumbens. In (b) the injection was placed in the medial SN. The anterograde label was found to spread more laterally in the nucleus accumbens than was found after more medially placed injections into the VTA (cf. a). (c) Anterograde label in ventral-anterior entorhinal cortex (CE) following injections of 3H proline/leucine into the caudal part of the VTA. In addition to the sparse to moderate label over widespread areas of the entorhinal cortex, some small “islands” of moderately dense label could be found over layers I1 and I11 of ventral CE (arrow). These may correspond to the “islands” of DA innervation seen in sections processed for glyoxylic acid-induced fluorescence. (d) and (e) are photomicrographs of anterograde label seen over the suprarhinal (SRC) and piriform (CPF) cortices following injections of 3H proline/leucine into the lateral VTA-medial S N area. Note t h a t the anterograde labeling over the SRC is greater than over the CPF. The anterograde label in SRC extends to the superficial cortical layers (e). Marker bars, 200 fim.

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TOPOGRAPHY OF DOPAMINE CELL PROJECTIONS James H. Fallon and Robert Y. Moore

PLATE 3

ME DIAL

579

Environmental effects of increased coal utilization: ecological effects of gaseous emissions from coal combustion.

Grindability and combustion behavior of coal and torrefied biomass blends.

Health effects of coal mining and combustion: carcinogens and cofactors.

Presence of stable coal radicals in autopsied coal miners' lungs and its possible correlation to coal workers' pneumoconiosis.

Coal-packed methane biofilter for mitigation of green house gas emissions from coal mine ventilation air.

Natural attenuation of coal combustion waste in river sediments.

Estimate of sulfur, arsenic, mercury, fluorine emissions due to spontaneous combustion of coal gangue: An important part of Chinese emission inventories.

Semi-coke briquettes: towards reducing emissions of primary PM2.5, particulate carbon, and carbon monoxide from household coal combustion in China.

Reducing NOx Emissions for a 600 MWe Down-Fired Pulverized-Coal Utility Boiler by Applying a Novel Combustion System.

Coal workers' pneumoconiosis: a historical perspective on its pathogenesis.

The secondary release of mercury in coal fly ash-based flue-gas mercury removal technology.

Thermochemical and trace element behavior of coal gangue, agricultural biomass and their blends during co-combustion.

Patterns of coal workers' pneumoconiosis in Appalachian former coal miners.

Temporal measurements and kinetics of selenium release during coal combustion and gasification in a fluidized bed.

Coal-mining and mortality.

Coal dust and compensation.

Coal dust and compensation.

Coal dust and compensation.

Investigation on thermal and trace element characteristics during co-combustion biomass with coal gangue.

Coal-mining and mortality.

Adverse effects of coal combustion related fine particulate matter (PM2.5) on nematode Caenorhabditis elegans.

Analysis of the leaching behavior of elements from coal combustion residues for better management.

pH-dependent leaching of constituents of potential concern from concrete materials containing coal combustion fly ash.

Mouse skin tumorigenicity studies of indoor coal and wood combustion emissions from homes of residents in Xuan Wei, China with high lung cancer mortality.