Magnocellular nuclei of the basal forebrain project to neocortex, brain stem, and olfactory bulb. Review of some functional correlates

Brain Research, 93 (1975) 385-398 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

385

Research Reports

M A G N O C E L L U L A R N U C L E I OF T H E BASAL F O R E B R A I N PROJECT TO N E O C O R T E X , BRAIN STEM, A N D O L F A C T O R Y BULB. REVIEW OF SOME FUNCTIONAL CORRELATES

IVAN DIVAC

Laboratory of Behavioral Physiology, Institute of Neurophysiology, University of Copenhagen, Copenhagen DK-2100 (Denmark) (Accepted March 3rd, 1975)

SUMMARY

Horseradish peroxidase was injected into the neocortex of squirrel monkeys, rats, tree shrews and one opossum, in the brain stem of one squirrel monkey and rats, and in the olfactory bulb, the corpus vitreum or the vascular system of rats. Following the cortical, brain stem and bulbar injections labeled cells were found (predominantly ipsilaterally) in the magnocellular nuclei of the basal forebrain: nucleus of the diagonal band, the magnocellular preoptic nucleus and nucleus basalis. These nuclei may, therefore, be classified together hodologically as well as cytologically and histochemically. The number of labeled cells was proportional to the size of the injected region. It is uncertain whether the same cells project to all target regions. Large labeled cells were found scattered among pallidal and entopeduncular neurons in rats with cortical or brain stem injections. These neurons may be the equivalent to the nucleus basalis in other species.

INTRODUCTION

The basal forebrain has been implicated in sleep s4, reward sS, thirst 4s, learning 44 and attention 4a. Its functions are, however, not understood partly because the region contains several cell groups and pathways and merges without distinct borders with a number of prosencephalic formationsa2,6a, 67. A conspicuous component of the basal forebrain is a chain of large neurons extending from the septum to the posterior

386 end of nucleus lentiformis. These neurons are differently grouped by different anatomists, e.g., some authors describe only the nucleus of the diagonal band 17,38, others divide them into diagonal band, magnocellular preoptic, and basal nuclei 6v (see descriptions and synonyms in refs. 19, 63, 67, 71 and an illustration in ref. 16). This chain of magnocellular nuclei of the basal forebrain (MNBF) has been described in species ranging from 'primitive' opossum to chimpanzee and man 9,11,17,19,2a,27,2s, 3s.4a,46,66,v3,92. A study of neurogenesis of the mouse brain indicated that the genesis of the neurons in MNBF begins early and at approximately the same time throughout the chain, but continues for a longer time in the nucleus of the diagonal band than in the preoptic region 10. In primates, all MNBF neurons show similar enzymatic composition, indicating vigorous phosphate, glycolytic, and aerobic oxidative metabolism; they contain no secretory granulesZS, 49. The dendrites of the cells in the region of the nucleus of the diagonal band are oriented predominantly radially in the frontal plane, with axons bifurcating in one ascending and one descending branch 52. High concentrations of the specific cholinesterase in the cell bodies and their processes 26,32,a6,37,49,s° are taken to indicate their cholinergic character. This is supported by detection of choline acetyltransferase in M N B F 66a (P.L. McGeer, personal communication). The MNBF neurons seem to have widespread afferent and efferent connections. The basal forebrain region was found to receive projections from the hippocampus ss, the prefrontal cortex 4°,62, the olfactory tubercle 22, the symmetrical region on the contralateral side 72, the pontine taste area 64,65, the median and dorsal raphe nuc!ei 7, locus coeruleus 6s and some other poorly identified formations in the brain stem 47 which possibly correspond to the supramammillary nuclei and the ventral tegmental areaS, 7z. Which of these projections terminate on MNBF neurons remains uncertain. MNBF in turn appear to project to the olfactory bulb 71, hippocampus 13'39'42' 51,79, the mediodorsal nucleus 20, the lateral habenular nucleus 69,71, several hypothalamic and brain stem nuclei 54,61, and the lateral neocortex 19,a6,3v,s°. Krnjevid and Silver36, 37 and Shute and Lewis s° probably misidentified M N B F cells as belonging to the lentiform nucleus. Their histochemical studies indicated that axons originating in the anterior part of the MNBF chain distribute to the medial portions of the cortex via the fornix, the 'subcallosal band' and cingulum 36,42, whereas the fibers from the posterior part reach the lateral cortex after piercing the putamen 36,aT,s°. The acetylcholinesterase-containing fibers terminate in deep (5th and 6th) and superficial (lst) cortical layers a6. Most of the anatomical results need confirmation or additional details, e.g., the cortical projections suggested electrophysiologically78,sl as well as by histochemical 36,37,s0 and retrograde degeneration methods 19 were not confirmed in studies with silver impregnation methods 54,71. However, negative results obtained with silver impregnation tehcniques do not exclude the presence of projections zl. Furthermore, when a lesion is made in a cytologically heterogenous area traversed by axons originating elsewhere, silver techniques can neither identify the cell bodies corresponding to the degenerating axons terminating in different target areas, nor distinguish between fibers originating in the destroyed area and those passing through it.

387 The method based on retrograde axonal transport of horseradish peroxidase (HRP) 3~ may overcome these difficulties and was therefore used in the present reinvestigation of the ascending and descending connections of the basal forebrain. METHODS

Three adult squirrel monkeys (Saimiri sciureus), 27 Long-Evans rats (Rattus norwegicus) (21-100 days old), two adult tree shrews (Tupaia glis) and one adult opossum (Didelphis virginiana) were used in the study. Two of the monkeys received multiple injections of H R P solution, spaced about 2 mm apart, into the neocortex of one hemisphere. In the third monkey, two stereotaxic injections were made into the mesencephalon (A: + 2.5; L: 2.5; Hi: --2.0; H~: ÷ 1.0). In 4 rats most of the cortex was injected; 13 rats were given more limited cortical injections involving the medial (n = 2), mediofrontal (n ~ 5), dorsolateral frontal (n = 2), suprarhinal (n = 3), or occipital (n ~- 1) area; 3 rats were injected in the olfactory bulb; two in the corpus vitreum of one eye; and two into the brain stem (stereotaxic coordinates: A: 0.0; L: 1.4; H: 8.0 from the surface of the skull which was in the horizontal plane). The remaining 3 rats, serving as controls, received 100-1000 mg/kg of H R P intravenously. In one of the control animals 5 mg of dissolved HRP was in addition poured over the pia of one hemisphere. The tree shrews received injections into the frontal cortex. In the opossum the prefrontal cortex was injected on one side and the sensorimotor cortex on the other. The injected volumes of 50 ~ H R P (Sigma type VI), dissolved in physiological saline, varied from 0.1 to 0.8 #1 for one point, with rates of 0.06-0.6 #l/min. The monkeys were operated under 25 mg/kg, and the opossum under 35 mg/kg, pentobarbital injected intraperitoneally. The rats were anesthetized with 3.3 ml/kg Equithesin. Tree shrews received 50 mg/kg pentobarbital i.p. 30 min after injections of a mixture of 50 mg/kg chlorpromazine and 4 mg/kg atropine. The animals were re-anesthetized 24-48 h after surgery and transcardially perfused with a solution of 1 ~ formaldehyde and 1.25 ~ glutaraldehyde (TAAB, electron microscopic purity) in 0.1 M phosphate buffer (pH: 7.4). The brains were left in situ for 2-6 h, removed from the skull, stored overnight in the same fixative at 4 °C, and transferred into a solution of 3 0 ~ sucrose in 0.1 M phosphate buffer (pH: 7.4). They were stored in this solution until the brains sank. Frozen sections were cut from the brains in the coronal stereotaxic plane at 40/zm. Every 7th section of the monkey brains and every 4th section for the rats and the opossum were processed largely according to a standard protocol ag. The sections were incubated at room temperature for 15 rain in a solution containing 0 . 0 5 ~ diaminobenzidine tetrahydrochloride and 5 ~ sucrose in 0.1 M phosphate buffer (pH : 7.4), and for an additional 25 min after hydrogen peroxide was added to the solution, making a concentration of 0.01 ~. Following incubation, the sections were transferred to a 5 sucrose-buffer solution, then to distilled water, and finally to 0.75 ~ gelatin dissolved in 4 0 ~ alcohol. From the gelatin solution the sections were mounted on slides previously covered with chromalum gelatin 21, dried, dehydrated, exposed to xylene to remove lipids, and lightly counterstained with cresyl violet.

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389 RESULTS

Injections to the neocortex Injections designed to infiltrate the neocortex occasionally spread to the claustrum and neostriatum (in one monkey and 11 rats) or to the hippocampus (in 4 rats; Figs. 1-3). In the second monkey, 6 rats, both tree shrews, and the opossum, the injections were smaller and restricted to the neocortex. In all except two rat brains, cells containing fine brown granules were found in the basal forebrain region. The number of labeled cells seemed proportional to the size of the injected neocortical area, but since all sections were not preserved, precise cell counts could not be made. In 4 of the rats with injections restricted to the neocortex (Fig. ld), a few labeled cells were found in the basal forebrain region. However, in two rats with similar injections (Fig. lc), the basal forebrain region in the preserved slides did not contain any labeled ceils. Assuming that only some of the cells of the population projecting to the neocortex are labeled in animals with small cortical injections, the results of the monkey with the larger cortical injection (Figs. la and 2), and one rat with a comparable injection (Figs. lb and 3) will be described in detail. M N B F in the monkey with the small injection restricted to the neocortex contained a smaller number of labeled cells. For the rat, a picture similar to Fig. 3 could be obtained by plotting on composite diagrams all the labeled cells seen in all the brains with injections restricted to the neocortex. In both illustrated brains (Figs. 2 and 3) a chain of labeled cells extended through the basal forebrain predominantly on the side of the injection. From the medial septum, this chain of large cells runs posteriorly and ventrolaterally to the region situated between the anterior commissure and the ventral surface of the brain (substantia innominata). In the monkey, some large cells were densely grouped, and others were more scattered. At the caudal end of this chain labeled ceils clustered around the posterolateral end of the anterior commissure and the dorsolateral border of the optic tract close to the central and medial amygdala nuclei, and, in largely separate bands, surrounded the lateral, ventral and medial surface of the lateral segment of the globus pallidus (Fig. 2). This arrangement was not seen in the rat, where the labeled ceils showed a less orderly distribution. In the posterior sections of the rat brain, the labeled neurons did not concentrate around the anterior commissure and aligned themselves along the dorsomedial border of the optic tract and the ventrolateral border of the internal capsule, approaching and mixing with the neurons of the interpeduncular nucleus and globus pallidus (Fig. 3). In both monkeys and rats, an occasional labeled cell was found in the same nuclei on the contralateral side;

Fig. 1. Sections with maximal spread of HRP in respective brains, a: the squirrel monkey with the larger cortical injection, b: the rat with the largest cortical injection, c: a rat in which no labeled neurons were found in MNBF, although such cells were found in the thalamus, d: a rat with a few labeled cells in MNBF. e: the squirrel monkey with mesencephalic injection, f: a rat with brain stem injection, g: the rat with the larger injection confined to the olfactory bulb. h: a rat with injection into corpus vitreum. Scale: a and e, 10 mm; b, c, d, and f, 5 mm; g and h, 1 mm.

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Fig. 2. Charting of the brain of the squirrel monkey (Fig. la) with the larger cortical injection. The labeled neurons in MNBF are represented by dots. The arrows point at some less conspicuous cells. The numbers indicate approximate distances from the interaural line (compare with the atlas by Emmers and Akerta6). The striped areas indicate spread of HRP.

such neurons were seen only in anterior sections (Figs. 2 and 3). N o small cells in the basal forebrain region 67 (Fig. 4) were labeled in these or any other brains in the present series. The M N B F neurons appeared to be more strongly labeled in younger rats. The available material did not permit a precise analysis o f relations between the positions o f the injected cortical area and the labeled cells in the basal forebrain region.

Injections to the brain stem In two rats with injections extending bilaterally f r o m the posterior commissure to the medulla oblongata (Fig. lb), the label accumulated in a large n u m b e r o f M N B F neurons o f both hemispheres (Fig. 4e). The distribution o f the labeled neurons appeared the same as in the rats with cortical injections, spanning the distance between the medial septum and the lentiform nucleus. In the monkey, H R P was injected into one side o f the mesencephalon (Fig. le). Labeled cells were scattered throughout the basal forebrain, most densely at the level o f A = 14.5 (compare with Fig. 2). Most o f these cells were situated on the side o f the injection; only two were found on the contralateral side. N o n e of the grouped large cells which were labeled in the monkeys with neocortical injections (Figs. 2 and 4a) contained the label in this animal. Injections to the olfactory bulb The injections were incomplete but confined to the olfactory bulb o f one rat (Fig. le); in this specimen the label was f o u n d in the cells o f M N B F only in the ven-

391

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Fig. 3. Plotting of the labeled cells in MNBF in a rat brain with large injection on standard diagrams3a. The labeled cells are represented by crosses. The dotted areas indicate spread of HRP in this animal.

tromedial portion of the septum (Fig. 4f), i.e., in the horizontal limb of the nucleus of diagnonal band and posteriorly to the level 6790 (compare with Fig. 3). In another rat, H R P infiltrated the whole bulb and spread to the frontal pole; in this case also the dorsomedial septum contained labeled neurons. In the third rat, where H R P was restricted to the central portion of the bulb, no labeled ceils were found in the basal forebrain.

Injections to the corpus vitreum The retinas of the injected eyes were filled with the oxidation product (Fig. lh), but no labeled cells were seen in any part of the basal forebrain. Intravenous injections No labeled cells were found in the basal forebrain in any of the 3 animals.

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Fig. 4. Labeled cells in the MNBF. a: the cells at the level a - - 14.5 of the monkey illustrated in F i g s . l a a n d 2. b: the cells lying between the external and internal segment of the globus pallidus, level a -- 8.5, in the same animal, c: the cells at the level a - 8380 of the rat illustrated in Fig. 3. d: the dorsalmost cells at the level a - - 5150 in the same animal, e: the cells approximately at the level a - - 8620 of the rat with injection illustrated in Fig. lf. f: the cells approximately at the level a - 7190 of the rat with injection illustrated in Fig. l g . a, b, c a n d d have the same orientation as on corresponding sections in Figs. 2 a n d 3.

DISCUSSION

Large neurons of the basal forebrain contained the characteristic brown granular form of the labelaa, 39 both in the present series of animals with HRP injected into the neocortex and in a group of rhesus monkeys with similar injections to the parietal cortex (Divac et al., in preparation). The labeling was not dependent on involvement of subcortical formations. Thus, M N B F seem to project directly to the neocortex in 5 species from 4 orders. The brain stem injections in rats accidentally infiltrated the cortex of one hemisphere (Fig. lf). This cortical involvement can hardly, however, account for the large number and bilateral distribution of labeled M N B F neurons in these animals since none of the cortical injections of a similar size (Fig. ld) produced comparable labeling. It may therefore be concluded that M N B F project also to the brain stem. In addition, projections from M N B F (with the exception of the

393 vertical limb of nucleus of the diagonal band and nucleus basalis) to the olfactory bulb are confirmed. All of these connections were indicated in earlier studies based on different anatomical methods19,36,aT,54,71, s0. Further work is required to provide information about the detailed topology of these connections and other possible efferents from MNBF (see Introduction). The presence of granules in the perikarya of the basal forebrain neurons cannot be an artefact of accumulation of any substance other then H R P (e.g., lipofuscin) since (a) these neurons were not labeled in rats with systemic or intraocular injections, (b) very few neurons on the side contralateral to the injections contained the label, and (c) these cells were even more strongly labeled in the 21-day-old rats than in the older animals. If all M N B F perikarya were labeled in animals with injections to either cortex or the brain stem, and at least some of the perikarya in the animals injected to the brain stem or the neocortex, respectively, it could be concluded that some, if not all, neurons of M N B F project to both targets. Since, however, the present material contained neither complete labeling of M N B F nor complete injections to the cortex or the brain stem, it is not possible to determine whether MNBF neurons send axons to both targets or, alternatively, each target is supplied by separate fibers. Millhouse 5z found in Golgi-impregnated material that axons of neurons of the nucleus of the diagonal band divide into ascending and descending branches, an observation supporting the second alternative. A definite solution of this problem must await the development of a complementary somatopetally transported tracer. If indeed various brain regions receive axon collaterals from the same basal forebrain neurons, some similarities in the effects of electrical stimulation in formations as widely separated as the caudate nucleus 34, internal capsule and globus pallidus 1~, the medial thalamic region 3, and the olfactory bulb z, could be explained by antidromic-orthodromic invasion of axons originating in the basal forebrain (see also refs. 55, 58). The caudalmost nucleus of MNBF, nucleus basalis (of Meynert), was found in several species, but not in the rat 19. Since the present data suggest that some M N B F neurons in rats do reach globus pallidus and entopeduncular nucleus, it is possible that they represent an equivalent of nucleus basalis. These neurons mix with the pallidal and entopeduncular cells in a less orderly way in rodents than in primates. The spatial relation of the nucleus basalis and nucleus lentiformis requires reexamination of the connections of both globus pallidus and putamen. In the rats with intraocular injections no labeling was found in the basal forebrain. Thus, the present experiments have failed to provide evidence that the acetylcholinesterase-containing fibers in the rat optic nerve, thought to be centrifugal 41, originate in MNBF. The cortical projections described here provide a morphological substrate for the ventral of the two 'diffuse' prosencephalic corticopetal systems postulated on the basis of electrophysiological 7s, biochemical 86, histochemical s0, and Golgi 77 studies. The dorsal system, originating in the intralaminar thalamic nuclei 57, has already been confirmed by anatomical methods29,3~,59, 74. The two systems differ in at least two ways; the large neurons of the basal forebrain contain larger amounts of acetyl-

394 cholinesterase than the intralaminar nuclei3Z,36,49,s0, and the thalamic axons, but not the fibers of the basal forebrain neurons, pass through the reticular nucleus of the thalamus on the way to the cortex 76. The present data suggest that the corticopetal projection of the basal forebrain is not diffuse: each area apparently receives axons from a small number of M N B F neurons. The physiological 'diffuseness' may result from the organization of the local circuitry, or be an artefact of the technique of electrical macrostimulation, or both. The full functional significance of the widespread projections of MNBF remains to be determined. The presently available data, based on techniques of macrostimulation, ablation, or unit recording in awake animals, must be treated with caution because of the difficulty of identifying the involved elements of this highly heterogenous regionS~,67. Some findings indicate a role of the basal forebrain in synchronization of electrocortical activity and slow-wave sleeplS,24,~0,60,83,s4,s9,9° (see also reviews refs. 5, 81). This region may be under the influence of the raphe nuclei 7 which also seem to be involved in slow-wave sleep 30. Application of serotonin, the presumed transmitter of the raphe neurons 12, in the basal forebrain induces electrocorticographic synchronization and onset of behavioral sleep 91. Unit recordings have indicated that neurons surrounding globus pallidus in the squirrel monkey sS, rhesus monkey 14, and rat 44, are involved in drinking and eating, and the cells in the lateral preoptic area in rats increase firing in response to systemic injections of hypertonic solutions 4s. These are probably the same units which 'learned' not to fire in response to an unconditioned stimulus in a classical conditioning paradigm 44. Whether the same neurons are involved in reward, reinforcement, or attention processes 43 remains to be seen. It is possible that M N B F play a role in all these phenomena by means of a common, probably cholinergic s9 neurophysiological mechanism: electroencephalographic synchronization accompanies sleep, but is also found in connection with reinforcement6, vS. MNBF with their connections are further candidates for the morphological substrate of the controversial behavioral phenomena of self-stimulation and aphagia. Thus, interruption of the projection from the pontine taste area to MNBF 64,65 rather than damage to the lateral hypothalamus 1, the nigrostriatal pathway 87, or to the trigeminal lemniscus 94, may be responsible for the aphagia. This suggestion is supported by the results of unit recordings in MNBF 14,43,~4,85 and by the occurrence of the aphagia in animals with lesions of the brain stem posterior to substantia nigra 94 or of the basal forebrain anterior to the thalamus 56. Some results indicate that electrical stimulation of the same pathway may be responsible for the self-stimulation behavior v0. Pathological changes were found in perikarya of nucleus basalis of schizophrenic patients 4. This problem may be worth a reexamination in the light of sleep disturbancesSZ, 93 and failure to respond to common reinforcers 53 in some schizophrenic patients.

395 ACKNOWLEDGEMENTS This research was supported in part by N.I.H. G r a n t s NS 09211 awarded to D. N. P a n d y a , NS 06209, H a r o l d Goodglass, Principal Investigator, a n d a t w i n n i n g g r a n t from E u r o p e a n T r a i n i n g P r o g r a m m e for Research in Brain a n d Behavior. The largest a m o u n t o f work was completed at the H a r v a r d Neurological U n i t , B o s t o n City Hospital during leave of absence. The o p o s s u m was injected in c o l l a b o r a t i o n with T h o m a s J. Tobias a n d the tree shrews in c o l l a b o r a t i o n with R i c h a r d E. Passingham. T. L. K e m p e r , J. H. LaVail, P. Rakic, a n d R. G. E. W i k m a r k , offered helpful c o m m e n t s during p r e p a r a t i o n of the manuscript.

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