Brain Research BuNerin,
Vol. 8,
pp. 727-749,
1982. Printed in the
U.S.A.
Cholinergic Projections from the Basal Forebrain to Frontal, Parietal, Temporal, Occipital5 and Cingulate Cortices: A Combined Fluorescent Tracer and Acetylcholinesterase Analysis’ VOLKER
Department
of Psychology
BIGL, NANCY
J. WOOLF
and Brain Research Institute, Received
AND LARRY
L. BUTCHER2
University of California, Los Angeles,
CA 90024
13 February 1982
BIGL, V., N. J. WOOLF AND L. L. BUTCHER. Cholinergic projections from the basal forebrain to frontal, parietal, temporal, occipital, and cingulate cortices: A combined jluorescent tracer and acetylcholinesterase analysis. BRAIN RES. BULL. 8(6) 727-749, 1982.-The morphologies, intercellular organization, and cortical projection patterns of putative cholinergic neurons in the basal forebrain of the rat were examined by use of fluorescent tracer histology in combination with the pharmacohistochemical regimen for acetylcholinesterase (AChE). Intensely staining AChE-containing cells projecting to frontal sensorimotor (Area IO), parietal (Area 2), and temporal (Area 41) cortices were found ipsilaterahy in nucleus preopticus magnocellularis, in nucleus basalis, and in association with the substantia innominata, the ansa lenticularis, and the lateral hypothalamic area; an essentially rostrocaudal topography was observed for these projections. AChE-containing pathways to cingulate (Area 29) and visual (Area 17) cortices derived from ipsilateral somata associated with the vertical and horizontal limbs of the diagonal band, nucleus preopticus magnocellularis, rostral portions of nucleus basalis, and the substantia innominata. Neurons innervating Area 29 were generally located more rostrally than those giving rise to AChE atferents to Area 17. The vast majority of cells appeared to innervate relatively discrete areas of the cortex. Evidence for collateralization was found only in neurons projecting to visual and cingulate cortices, and these represented only 3.2% of the cells providing AChE afferents to Areas 17 and 29. The basal forebrain AChE projection cells were typically large (>25 pm in maximum cell body extent), and their somata were predominantly oval, with lesser proportions being fusiform or triangular. Many were organized in clusters, particularly in nucleus basalis. Basal forebrain Cortex Cholinergic afferents
Acetylcholinesterase
THE basal forebrain of the rat contains an appreciable number of projection neurons that stain intensely for acetylcholinesterase (AChE, EC 3.1.1.7) and show rapid recovery
of enzyme activity following systemic administration of the irreversible inhibitor bis(l-methylethyl)phosphorofluoridate (di-isopropylfluorophosphate: DFP) [9, 39; 51, 521. These cells, referred to collectively in this report as the basal forebrain AChE projection system (BFAPS), are typically large (>25 pm in maximum soma extent) and comprise a relatively contiguous, and at certain loci continuous, constellation of neurons associated at various levels with the medial septal nucleus, the lateral preoptic area, the ventral pallidumkubstantia innominata region, the vertical and horizontal limbs
Retrogradely
transported
fluorescent
labels
of the diagonal band and nucleus preopticus magnocelhrlaris, the pallidally-associated nucleus basalis, the ansa lenticularis, and lateral aspects of the lateral hypothalamic area [9, 17, 18, 19, 20, 25, 26, 39, 44, 51, 52, 61, 67, 69, 70, 741. Considerable anatomic, biochemical, and histochemical data, considered together, suggest that somata demonstrating strong AChE activity in the above-delimited brain regions are cholinergic and that many are the source of cholinergic atferent fibers to various telencephalic, and possibly also brainstem, structures [9, 13, 17, 19, 29, 32, 33, 34, 35, 36, 39, 41, 44,47, 50, 54, 57, 61, 69, 70, 72, 73, 741. Fiber systems deriving.from the basal forebrain AChE complex detailed in the preceding paragraph include (a) pro-
‘The data contained within this paper were presented at the 89th Annual Convention of the American Psychological Association; Los Angeles, CA; August 25, 1981, and a preliminary report was published in the Abstracts of the First World Congress of IBRO; Lausanne, Switzerland; March 31-Apri16, 1982 (Neuroscience Suppl. 7: S26S27, 1982). *To whom correspondence and reprint requests should be mailed at the following address: Department of Psychology; University of California; 405 Hilgard Avenue; Los Angeles, CA 90024.
Copyright 8 1982 ANKHO
International Inc.-O361-9230/82/060727-23%03.00/O
BIGL, WOOLF AND BUTCHER
728 LIST OF NEUROANATOMIC
a ad al
nucleus accumbens anterior dorsal nucleus of the thalarnus nucleus of the ansa lenticularis anterior ventral nucleus of the thalamus basolateral nucleus of the amygdala anterior commissure internal capsule central nucleus of the amygdala ventral hippocampal commissure claustrum optic chiasm caudate-putamen complex entopeduncular nucleus timbria medial forebrain bundle globus pallidus anterior hypothalamic area lateral hypothalamic area Islands of Caheja lateral nucleus of the amygdala nucleus basalis nucleus of the lateral olfactory tract lateral preoptic area
Z CA CA1 ce CFV Cl co
CP eP
FH FMP gP ha hl IC la nb 01
PO1
wm medial preoptic area poma re sf si sm & st tam td td, td, td, TO TOL tr ts tuo tv VP
zi
jections from the medial setpal nucleus and diagonal band to the hippocampal formation and medial limbic cortex [41, 43, 45, 691, (b) pathways from nucleus preopticus magnocellularis, called by some authors the nucleus of the horizontal limb of the diagonal band (e.g., [24]), to olfactory bulbs and entorhinal cortex [69,70], (c) fibers from nucleus basalis and other large neurons described as belonging to substantia innominata to most, if not all, areas of the neocortex [17, 19, 32, 39, 44, 50, 57, 691, and (d) projections from the medial septal nucleus, nuclei of the diagonal band, lateral preoptic area, ventral pallidum/substantia innominata region, and nucleus preopticus magnocellularis to the amygdala [19, 47, 741. Of these putative cholinergic pathways, the cortical projection component of the BFAPS is perhaps least understood at present. Little is known, for example, about its topography, although a crude, predominantly ipsilateral, reverse rostro-caudal organization has been suggested in previous biochemical and histochemical studies [45,69]. Furthermore, it has not been established if individual neurons in the BFAPS project discretely or diffusely to different cortical areas, and there is a paucity of data concerning the morphologies and intemeuronal organization of those cells. In the present study, we have attempted to determine some of the cortical projection patterns of the BFAPS by infusing different retrogradely transported fluorescent compounds into various regions of the cerebral cortex and, following microscopic evaluation of single and double labelled neurons, processing the same tissue section for AChE according to the pharmacohistochemical regimen developed in this laboratory [4, 5, 81. This latter procedure permits visualization of AChE-containing neuronal somata and their proximal processes to an extent impossible to achieve with other protocols for the enzyme [4]. METHOD
Experimental
Animnls
Twenty-seven
male
and female
Sprague-Dawley
ABBREVIATIONS
rats
nucleus preopticus magnocelhilaris nucleus reuniens nucleus septahs timbrialis substantia innominata medial septal nucleus supraoptic nucleus rhinal sulcus interstitial nucleus of the stria terrninahs anterior medial nucleus of the thalamus diagonal band diagonal band, pars angularis diagonal band, horizontal limb diagonal band, vertical limb optic tract lateral olfactory tract reticular nucleus of the thalamus nucleus triangularis septi olfactory tubercle ventral nucleus of the thalamus ventral pallidurn zona incerta
(Simonsen Laboratories; Gilroy, CA) were used. The animals weighed 180-320 g at the time of experimentation and were housed under conditions of constant temperature (22°C) and relative humidity (50%). They were maintained on a 12 hr light-dark schedule of illumination; injections and animal euthanasia were performed during the light phase of the cycle (6:00-18:OO hr). Surgical and Intracerebral
Rats were anesthetized
infusion Procedures
with chloral hydrate,
350 mg/kg
IP. Their heads were shaved and then mounted by use of ear
plugs within a Kopf stereotaxic apparatus (David Kopf Instruments; Tujunga, CA). Two different solutions of fluorescent tracers were prepared immediately prior to intracortical infusion. One was a 30% solution of Evans Blue (Matheson, Coleman and Bell; Norwood, OH) and the other was a solution consisting of 2.5% 4’,6-diamidino-2-phenylindole HCl (DAPI; Sigma Chemical Co.; St. Louis, MO) and 10% primuline (Matheson, Coleman and Bell; Not-wood, OH); the vehicle for both tracer preparations was distilled, deionized water. A 1 ~1 syringe with permanently attached cannula (outer diameter, 0.5 mm; Hamilton Co.; Reno, NV) was used to inject a total volume of 0.05, 0.1, 0.15, 0.2 or 0.3 ~1 of tracer solution during a 10 min period; larger volumes were used for larger cortical regions. The cannula was allowed to remain in place for 4 min following the termination of the injection period before being withdrawn slowly. In each rat, one fluorescent tracer solution was infused into one cortical area and, immediately thereafter, the other solution was injected into another region of the cortex. All possible injection combinations of any two cortical areas were prepared, with infusions being made into the following Krieg-categorized [38] areas according to the indicated anterior-posterior (AP), lateral (L), and ventral (V, from the cortical surface) coordinates, based on a Bregma zero, from the stereotaxic atlas in Skinner [62]: Area 10 of the frontal-sensorimotor cortex (AP,
AChE PROJECTIONS
TO CORTEX
+5.5; L, 2.5; V, 0.6), Area 2 of the parietal cortex (AP, +O.S; L, 5.5, V, 0.4), Area 41 of the temporal cortex (AP, -4.0; L, 7.3; V, 0.5), Area 17 of the occipital cortex (AP, -5.5; L, 3.2; V, 0.3), and Area 29 of the cingulate cortex (AP, -3.5; L, 0.3; V, 0.8). Following surgery the animals were placed on a heating pad maintained at 30°C until they recovered from anesthesia. They were then housed individually in stainless steel cages until they were sacrificed 48 hr after intracortical introduction of Evans Blue and DAPUprimuline. This time interval has been found to produce optimal labelling of neurons in the BFAPS. Histochemical
and Histologic Procedures
All rats intracortically infused with Evans Blue and DAPUprimuline were injected intramuscularly with 1.8 mg/kg DFP (Calbiochem, Inc. ; La Jolla, CA) 4 hr prior to euthanasia. The 4 hr survival time was chosen in order to visualize AChE optimally in basal forebrain somata (see [S]). Animals experiencing respiratory difftculty during the survival period after DFP were administered 10 mg/kg atropine methyl bromide (Sigma Chemical Co.; St. Louis, MO). In preliminary experiments it was found that DFP did not influence the transport of Evans Blue or DAPI/primuline. Furthermore, atropine methyl bromide did not interfere with Evans Blue and DAPVprimuline transport or with AChE staining after DFP. The rats were anesthetized with 350 mgikg chloral hydrate IP and, subsequently, were sacrificed by cardiac perfusion with 120 ml cold (4°C) 0.9% saline followed by 120 ml cold 10% buffered Formalin (pH, 7 The brains were removed and placed into cold buffered .reutral Formalin for 48 hr before being transferred to a cold 30% sucrose solution for an additional 48 hr. The brains were blocked in the coronal plane of Kiinig and Klippel [37], cut into 46 mm slabs, frozen with solid CO2 on an aluminum specimen holder, and cut on a freezing microtome at 40 pm intervals. The resulting tissue sections were collected in cold 0.9% saline and immediately thereafter were mounted on glass slides coated with pig gelatin or chrome alum. After the brain sections dried at room temperature (22”C), they were rinsed in distilled deionized water, air dried again, and coverslipped under mineral oil (Nujol@; Plough, Inc.; Memphis, TN). The slides thus prepared were examined within 48 hr with a Zeiss RA fluorescence microscope. Transmitted illumination was used. The red fluorescence of Evans Blue was visualized optimally with an excitation system consisting of a combination of Zeiss LP 520 and KP 560 filters; the barrier filter was a Zeiss LP 590. A Zeiss BG 365 excitation filter and LP 435 barrier filter were used to visualize the predominantly light blue fluorescence of the DAPUprimuline tracers. The entire basal forebrain AChE complex was inspected and regions containing labelled cells were photographed with a Zeiss camera and Ektachrome@ film (400 ASA; Kodak; Rochester, NY). Following the recording of the location on the microscope stage of the regions photographed, the slides were taken from the stage and their coverslips removed manually. The slides were blotted on absorbent paper, immersed in xylene for 1 min to remove the mineral oil coverslipping medium, blotted again to remove excess xylene, and allowed to air dry. The mounted brain sections were then rinsed in 0.9% saline for 2 min and subsequently processed for AChE according to the procedure of Karnovsky and Roots [31] as modified by Butcher et al. [6]. Three to six glass slides were
729 placed for 30 min into Coplin jars (volume, 65 ml) containing 50 ml of 30 PM N,N’-bis(l-methylethyl)pyrophosphorodiamidic anhydride (iso-OMPA; K and K Laboratories; Plainview, NY) to inhibit butyrylcholinesterase. The iso-OMPA solution was then poured out and replaced with 50 ml of the AChE reaction mixture containing 25 mg acetylthiocholine iodide, 32.5 ml of 0.2 M Tris-maleate buffer (pH, 5.7), 2.5 ml of 0.1 M sodium citrate, 5.0 ml of 0.03 M cupric sulfate, 5.0 ml of 0.005 M potassium ferricyanide, and 5.0 ml of distilled deionized water. Slides remained in the AChE incubation medium at 22°C for 4-12 hr, depending on the length of time necessary to visualize the AChE reaction product most clearly in the basal forebrain neurons. Additi ma1 control experiments for the specificity of the AChE rLaction wers performed as described in Butcher and Hodge 1121. After the histochemical reaction for AChE was complete, slides were removed from the Coplin jars, rinsed in distilled deionized water, air dried, immersed in xylene, and coverslipped under Permount@ (Fisher Scientific Co.; Fairlawn, NJ). The same regions of the basal forebrain examined for Evans Blue and DAPI/primuline labelled cells were then analyzed with bright-field or dark-field illumination for the presence of AChEcontaining neurons. Some brain sections were additionally stained with cresyl violet as described in Woolf and Butcher [73]. In addition to the histochemical and histologic material prepared specifically for the current study, we had available to us for analysis an extensive library of AChE slides generated during the past 11 years according to procedures detailed in Butcher and Bilezikjian [5], Butcher et al. [8], Butcher and Hodge [12], and Butcher [4]. This material was used for basic studies on the delineation of the BFAPS and for assessment of cellular shapes and dimensions (for methods, see [73]). RESULTS
The BFAPS: Nomenclature
and Cellular Characteristics
The rostrocaudal extent of the BFAPS, as defined in this paper, is displayed in Figs. l-8. The terminology used derives from several sources and was selected to provide communality with currently accepted and commonly employed nomenclature. Because of the complexity of this region of the brain, however, it is likely that entirely different appellative schema will evolve as more detailed anatomic and histochemical studies are conducted. Additional structures may be added also. For most portions of the BFAPS we have adopted essentially the terminologies of Parent and his associates [51, 52, 531 and of Heimer [24]. Like Heimer [24], we refer to the rostral extension of substantia innominata as the ventral pallidum. Parent and O’Reilly-Fromentin [53] note in the cat that most AChEcontaining cells in the lateral preoptic area stain weakly or moderately for the enzyme. We have also observed this profile in the rat (Figs. 2-3), but at the rostromedial interface of the ventral pallidumsubstantia innominata region with the lateral preoptic area a few large, intensely-staining AChE somata, probably displaced substantia innominata neurons, appear to invade what has been traditionally called the lateral preoptic area (Fig. 2). These heavily-staining AChE cells in the lateral preoptic area, therefore, are also considered to be a part of the BFAPS, although they appear to project preferentially to the amygdala 1741.
BIGL, WOOLF AND BUTCHER
FIG. 1. THIS PAGE AND FOLLOWING: FIGS. 1-8. Rostrocaudal extent of the Basal Forebrain AChE Projection System. Pharmacohistochemical regimen for AChE 14-81 in which the DFP-sacrifice interval was C8 hr. Unlabelled arrows in Figs. 3, 4, and 8 point to intensely staining AChE-containing somata in globus pallidus proper (Figs. 3-4) or internal capsule (Fig. 8). For further explanation, see text. Scale, 400 pm.
AChE PROJECTIONS TO CORTEX
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BIGL, WOOLF AND BUTCHER
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AChE PROJECTIONS
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AChE PROJECTIONS
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AChE PROJECTIONS TO CORTEX
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738
BIGL. WOOLF AND BUTCHER TABLE PERIKARYAL
SHAPES AND SIZES OF INTENSELY
1
STAINING AChE NEURONS IN FIVE REGIONS OF THE BASAL FOREBRAIN AChE PROJECTION SYSTEM Number
and Percent Having Indicated Soma Shape
Number of Cells Analyzed
Oval
A. Medial Septa! Nucleus
48
41 (85%)
5 (11%)
B. Lateral PreopticVentral Pallidumi Substantia Innominata Region
38
19 (50%)
C. Diagonal Band Nuclei
82
D. Nucleus Preopticus Magnocellularis E. Nucleus Basalis
Subdivision
Fusiform
Triangular
Maximum Soma Dimension f&m) Complex
Mean + SD*
Range
2 (4%)
0
25.3 t 4.4i
t8-37
11 (29%)
7 (18%)
1 (3%)
28.1 f 4.84
18-38
37 (45%)
29 (35%)
14 (17%)
2 (3%)
27.5 f 4.65
18-40
70
33 (47%)
23 (33%)
14 (20%)
0
26.8 + 4.41
18-40
75
35 (47%)
32 (43%>)
8 (10%)
0
27.8 r 5.63
18-43
*A one-way analysis of variance revealed that significant differences existed in maximum soma dimension among the different regions, F(4,308)=2.66, ~~0.05. A posteriori analyses with the Newman-Keuls procedure [71] showed the following comparisons to be significantly different @<0.05): A-B, A-C, and A-E. No other comparisons reached significance +>O.OS).
The term nucleus basalis is essentially as used by Parent and coworkers [51,52]. In addition to the primary localiza-
tion of these cells ventral and medial to globus pallidus (Figs. 3-X), some intensely-staining AChE neurons of nucleus basalis are found occasionally within pallidum proper (Figs. 3-4) and within the internal capsule (e.g., Fig. 8). Unlike Parent et al. [53], however, we do not emphasize to the same extent the relationship of nucleus basalis cells to the globus pallidus. Indeed, somata in globus pallidus proper are smaller and stain less intensely for AChE than those in nucleus basalis (Figs. 3-8; cf. [S&53]). Pallidal cells are similar in size and AChE staining intensity, however, to neurons in the entopeduncular nucleus (Fig. 8; [50,53]), which strengthens the point of view that the entopeduncular nucleus is the rodent homolo~e of the internal segment of the globus pallidus in primates (see [52]). The “cellular bridge” between nucleus basalis and the lateral hypothalamic area (al in Fig. 8), although noted but unnamed by Parent et al. [52], appears to be associated with
what Kiinig and Klippel [37] have called the ansa lenticularis, and, for want of a better term at this time, we will retain that nomenclature. All other appellations are standard and can be found in the atlases of Kanig and Klippel[37] and Sherwood and Timiras [60]. Most AChE-cont~ning somata in the BFAPS stain intensely for the choline& degradative enzyme at short intervals (4-8 hr) following DFP administration (Figs. 1-8). The majority of these somata are oval, with lesser proportions being fusiform or triangular (Table 1). Cell bodies having complex shapes (no readily classifiable geometry) are infrequently seen (Table 1). The mean maximum soma extents range from 25.3 Frn in the medial septal nucleus to 28.1 pm in the lateral preoptic-ventral pallidumlsubstantia innominata region, with cells in the medial septal nucleus being significantly smaller than those in most other subdivisions of
the BFAPS (Table 1). Although the perikaryal dimensions we observed are highly compatible with those found by Ribak and Kramer [57] in the cat and by Nagai et ai. [47] in the rat, they are somewhat less than those reported by Parent and O’Reilly-Fromentin ([S3], cat) and by Lehmann et af. ([39], rat). This variability may be attributable to technical difficulties attendant with accurately measuring soma sizes in neurons such as those of the BFAPS having isodendritic processes. Because such dendrites taper gradually from the cell body [ 10,561, determination of where the soma ends and the process begins is rendered difficult (Figs. 1-9). In addition, in many regions of the BFAPS there is a tendency for individual neurons to form clusters (Figs. l-9), a feature particularly prominent in nucleus basalis (Figs. 3-9) where certain somata appear to be inte~oven by a confluence of cellular processes (Fig. 9). In the present report, therefore, we have used conservative criteria for the determination of cell body dimensions: relatively isolated somata were measured, and the border of the perikaryon was considered to exist at the first indication of dendritic tapering. Knjection Sites and Spread of Fluorescent
Tracers
Fluorescent compounds infused into each cortical area did not exceed the boundaries indicated in Fig. 10 for any of the expe~ment~ animals. For a given rat, however, the spread of tracer was somewhat less than that delimited (Fig. 10). The number of labelled cells in the basal forebrain did not vary significantly as a function of tracer used; approximately the same numbers of labelled somata were observed after Evans Blue infusion into a given cortical region as after injection of DAPVprimuline into that same area. Examples of basal forebrain neurons labelled after cortical Evans Blue or DAPUprimuline administration are shown in Fig. 11. The intraneuronal presence of these fluorescent tracers did not
AChE PROJECTIONS
TO CORTEX
FIG. 9. Interneuronal organization of intensely staining AChE-containing neurons in nucleus basalis. iharmacohistochemica regimen for ACM H-81 in which the DFP-sacrifice interval was 6 hr. Transverse sections. Dark-field illumination. Frames B and D are higher power portrayals of frames A and C, respectively. Scale in C is 2OOpm and applies also to A. Scale in D is 100 pm and is applicable to 3 also.
739
740
BIGL. WOOLF AND BUTCHER
appear to interfere appreciably with subsequent demonstration of AChE activity (Fig. 11, compare H with F and G). Location of AChE-Containing Neurons in the Basal Forebrain Labelled with Evans Blue or DAPIIPrimuline Following Cortical Infusions Frontal cortex injections. AChE-containing cells labelled with Evans Blue or DAPVprimuline after Area 10 infusions were found ipsilateral to the injection site primarily in the middle third of nucleus basalis at levels between the posterior anterior commissure rostrally and posterior portions of the optic chiasm caudally (Fig. 12). Retrogradely labelled AChE neurons were also observed in nucleus preopticus magnocelhrlaris and in association with substantia innominata and lateral aspects of the lateral hypothalamic area. There was no evidence for AChE-containing somata that were double-labelled with fluorescent tracers. This last finding indicates that cells in the BFAPS pro’ecting to Area 10 of the frontal cortex do not provide afIJerents to other cortical regions, at least those investigated in the current study, a result in agreement with the conclusion of Divac [ 131 that the “. . . corticopetal projection of the basal forebrain is not diffuse: each area apparently receives axons from a small number of MNBF [magnocellular nuclei of the basal forebrain] neurons” (p. 394). Parietal cortex injections. AChE-containing neurons labelled after fluorescent tracer infusions into Area 2 of the parietal cortex were found ipsilateral to the injection site and almost exclusively in nucleus basalis along the medial border of the globus pallidus, although a few labelled cells were seen in substantia innominata and the lateral hypothalamic area (Fig. 13). These somata were generally located more caudally than those labelled after Area 10 injections (cf., Fig. 12). Like neurons projecting to frontal cortex, cells projecting to Area 2 were not double-labelled and, therefore, did not appear to innervate the other cortical regions examined in this investigation. Temporal cortex injections. Area 41 infusions of fluorescent tracers resulted in ipsilateral labelling of AChEcontaining neurons of nucleus basalis at levels generally caudal to those observed after Area. 10 and 2 injections (Fig. 14) cf., Figs. 12-13). Labelled AChE cells were also found in the internal capsule and in association with ansa lenticularis (Fig. 14). No double-labelled AChE-containing somata were observed. Occipital and cingulate cortex injections. The topography of the projections from the BFAPS to occipital (Area 17) and cingulate (Area 29) cortices are considered together because only after these injections was evidence found for AChEcontaining neurons labelled with both Evans Blue and DAPI/ primuline. Double-labelled AChE cells were observed in
FIG. 10. Visible extents of diffusion (stippled areas) of fluorescent tracers infused into different regions of the cerebral cortex. Numbers refer to cortical areas classified according to Krieg [38]. Template diagrams are redrawn from Skinner 1621.
FACING PAGE: FIG. 11. Examples of labelled neurons in various regions of the Basal Forebrain AChE Projection System following infusion of fluorescent tracers into different areas of the cerebral cortex. Frame A: Nucleus basalis cell in the internal capsule labelled with DAPFprimuline following tracer infusion into Area 2 of the parietal cortex. Frame B: Neuron in the horizontal limb of the diagonal band labelled with Evans Blue following tracer injection into Area 17 of the visual cortex. Frame C: Nucleus basalis cells labelled with DAPUprimuline following tracer infusion into Area 10 of the frontal cortex. Frame D: Nucleus basalis neurons labelled with Evans Blue following tracer infusion into Area 41 of the temporal cortex. Frame E: Cells in nucleus preopticus magnocellularis labelled with Evans Blue following tracer infusion into Area 29 of the cingulate cortex. Frames F-H: Evans Blue (F), DAPFprimuline (G), and AChE (H) demonstrated on the same tissue section. Shown are neurons associated with the horizontal limb of the diagonal band following Evans Blue infusion into Area 29 of the cingulate cortex (F) and DAPUprimuline injection into Area 17 of the visual cortex (G). Tracer-containing cells also demonstrated AChE activity (H). EB, Evans Blue labelled cells; D, DAPYprimuline labelled cell. Scale, 70 pm.
AChE PROJECTIONS TO CORTEX
FIG. 11.
741
742
BIGL, WOOLF AND BUTCHER
FIG. 13. Location of intensely staining AChE-containing neurons labelled with Evans Blue or DAPUprimuline following tracer infusion into Area 2 of the parietal cortex (asterisks). The level of each section is indicated on the left according to coordinates in Kijnig and Klippel [37].
FIG. 12. Location of intensely staining AChE-containing neurons labelled with Evans Blue or DAPI/primuline following tracer infusion into Area 10 of the frontal cortex (asterisks). The level of each section is indicated on the left according to coordinates in Kiinig and Klippel [37].
ticus magnocehularis, in substantia innominata, and in nucleus basalis at rostra1 levels (Fig. 15). AChE somata giving rise to afferent fibers to Area 29 were observed ipsilaterahy in all subdivisions of the diagonal band and in nucleus preopticus magnocellularis (Fig. 15).
DISCUSSION
the flexure (pars angularis, Fig. 1) of the diagonal band and its horizontal limb (Fig. 15). Neurons containing AChE and projecting to both Areas 17 and 29 were not numerous, however, and most cells projected to either one or the other cortical region (Fig. 15). In fact, double-labelled somata accounted for only 3.2% of the total number of AChE neurons containing tracer after occipital and cingulate cortex infu-
sions . AChE-containing cells projecting to Area 17 were found ipsilaterahy primarily in association with pars angularis and the horizontal limb of the diagonal band, in nucleus preop-
Use of Retrogradely Transported Fluorescent Combination with AChE Histochemistry
Tracers in
Previous studies concerned with assessing the cortical projections of AChE-containing neurons in the basal forebrain, demonstrated in pharmacologically unmanipulated animals or according to the pharmacohistochemical regimen, have employed horseradish peroxidase as the retrograde tracer [25,39,44,45,50,573. As pointed out by us previously [73], however, fluorescent labels offer certain advantages compared to horseradish peroxidase when these markers are used in combination with AChE histochemistry. First, the
AChE PROJECTIONS
TO CORTEX
chemical reactions necessary to demonstrate horseradish peroxidase frequently render AChE staining weak and sometimes indistinct [39], possibly increasing the probability of false negatives in double- and triple-staining experiments (see [64]). This problem is not as apparent with retrogradely transported fluorescent labels because they require no chemical processing for their visualization. Second, AChE loci are often difficult to detect in neurons also containing horseradish peroxidase because of the dark and intense color of the horseradish peroxidase reaction product. In our procedure, fluorescent tracers and AChE are demonstrated sequentially on the same tissue section under different illumination conditions. The presence of fluorescent compounds in the same neurons containing AChE, therefore, does not interfere significantly, if at all, with cholinesterase visualization. Third, fluorescent labels may be the most sensitive tools currently available for the demonstration of neuronal pathways. Sawchenko and Swanson [59] found that considerably more cells were labelled in the paraventricular nucleus after spinal cord injections of True Blue or bisbenzimide than after spinal infusions of horseradish peroxidase. Finally, combined fluorescent tracer and AChE histochemistry procedures are relatively simple and can be executed comparatively rapidly.
Organization
of AChE-Containing
Somata in the BFAPS
The suggestion has been made that the corticahyprojecting portion of the BFAPS represents, in part at least, a rostra1 extension of the brainstem reticular formation, a prominent difference being that the component neurons project directly to the cortex without a relay in the thalamus [44,56]. As such, some investigators have conjectured that the BFAPS projects diffusely upon the cortex (e.g., [69], a viewpoint apparently reinforced by results from numerous pharmacologic experiments on cortical acetylcholine efflux as a function of administration of cholinergic agonists and antagonists (see [12]) and by the findings that electrical stimulation of the mesencephalic reticular formation enhances acetylcholine release from all areas of the cortex [55,65]. The results of the present experiments, however, do not support the hypothesis that neurons of the BFAPS diffusely innervate the cortex, if it is meant by the word “diffuse” that a given neuron can supply two or more distinct regions of the brain by axon collateralization. Although cells projecting to different cortical areas were intermingled, each neuron of the BFAPS, with few exceptions, innervated a relatively discrete portion of the cortex. The somata of the BFAPS appear, therefore, to be mosaically organized, with each element (i.e., neuron) of the mosaic projecting to a particular cortical target. It must be emphasized, however, that this organizational schema does not rule out the possibility that the system as a whole acts “diffusely.” As indicated in this paper, many somata of the BFAPS apparently are interwoven by the confluence of their cellular processes (dendritic bundles?). If individual neurons in the BFAPS communicate with neighboring or more distant cells by dendritic release of transmitter (e.g., see [9,73]), possibly acetylcholine, then stimulation of only one such neuron could affect several other cells projecting to different cortical areas. In this way, the neuronal mosaic of the BFAPS could provide the morphologic substrate for what might be interpreted holistically to be a “diffuse” projection system.
FIG. 14. Location of intensely staining AChE-containing neurons labelled with Evans Blue or DAPUurimuline followinz tracer infusion into Area 41 of the temporal cortex (asterisks).-The’level of eacgsection is indicated on the left according to coordinates in Kiinig and Klippel [37].
Topographic Organization of Projections from the Basal Forebrain to the Cerebral Cortex Selectedprevious investigations. Although species differences are apparent and although we concentrated specifically on AChE projections, our results on the cortical projections of neurons in the basal forebrain of the rat are reasonably compatible with previous observations, particularly those of Divac [13] and of Henderson [25], deriving from the use of horseradish peroxidase as the retrograde tracer (Table 2). Most of these studies examined the projections of basal forebrain neurons to only one or two cortical areas (Table 2), however, and none assessed the degree of collateralization of those cells. Unlike Lehmann et al. [39], we did not find evidence that posterior portions of nucleus basalis projected to visual cortex (cf., results in the monkey, Table 2). The data presented by these authors [39] were sketchy, however, and it is possible that the injection site was not completely charac-
744
BIGL, WOOLF AND BUTCHER
FIG. 15. Location of intensely staining AChE-containing neurons labehed with Evans Blue or DAPI/primuline following tracer infusion into Area 17 of the visual cortex (solid black circles) and Area 29 of the cingulate cortex (solid black triangles). Cells labelled with both Evans Blue and DAPUprimuline are indicated by white triangles on black circles (arrows in sections A8380, A7020, and A6790). The level of each section is indicated on the left according to coordinates in Konig and Khppel [37].
terized in their preliminary study. Similarly, Divac [ 141 observed labelling of somata in medial septum, the vertical and horizontal limbs of the diagonal band, and in substantia innominata following True Blue or bisbenzimide infusions into “anteromedial prefrontal cortex.” Our Area 10 injections, which we assume would correspond in part to Divac’s [14] “anteromedial prefrontal cortex,” did not entirely reproduce his pattern of labelling, pa~icul~ly in rostral portions of the BFAPS. It is possible, however, that Divac’s [ 141 injections involved areas of cingulate cortex adjacent to Area 10, as well as Area 10 itself. If so, then we would predict the labelling of cells in the BFAPS that he reported. AChE projections to cortex. According to Papez [49], mammals are I‘. . . distinguished by the presence of a welldeveloped bilateral forebrain. This is divisible into (1) the primitive olfactory brain and (2) dorsal to it the non-olfactory cerebrum. The cerebrum of each side is as a rule clearly separated from the olfactory brain by the rhinal fissure” (p. 3). Krieg [38], in extensive studies of the rat cerebral cortex, proposed that the pallium could be divided into three parts: “Dorsaily, where the hemisphere is attached to the
thalamus, is the archipallium or hippocampus. Ventrally, is the paleopallium or pyriform region . , . . The third portion is the neop~li~ or somatic cortex . . . . What distinguishes the cortex of the mammal from that of any other class is the strong projection from the thalamus of the visual, somesthetic and auditory system . . . . The projection is a direct one. The location on the hemisphere of these three primary projections is predicated by the position in the thalamus that these nuclei happen to have. The geniculate nuclei, as representing special senses are further lateral than the more generalized somesthetic sense” (p. 254). He further observed “Between the neocortex and the hippocamthat pus . . . there is an interesting development. . . . This is the area which developed into the cingulate region which shows a different pattern of differentiation from the other areas . . . . It is highly significant, perhaps, that in their early differentiation the areas which develop from the cingulum parallel in position the areas which they lie opposite. Thus, area 24 corresponds to areas 10 and 6,23 is next to 4 , . . and 29 connects with 17 and 18” (p. 257). In the current study we examined AChE projections to
AChE PROJECTIONS TO CORTEX
745
TABLE 2 TOPOGRAPHY OF PROJECTIONS FROM THE BASAL FOREBRAIN TO THE CEREBRAL CORTEX REVEALED BY INTRACORTICAL INFUSION OF HORSERADISH PEROXIDASE
cortical Injection Site
Location of Labelled Somata*
Rat
Frontal, parietal, and medial frontal cortex
td, nb, medullary laminae between capsula intema and gp, poma
Rat
Dorsal neocortex
No label in nb, but in .,td if cingulate cortex involved
Temporal cortex
nb
Frontal and anterodorsal cortex
nb ventral to gp, concentrated in posterior half of gp
Visual cortex
Posterior parts of nb
Authors
Animal
Divac [13]
Burton and Fitzgerald
[31 Lehmann et al.[39]
Rat
Henderson r241
Rat
Visual cortex
td
Jayaraman
Cat
Temporal cortex
Intrapallidal part of nb
Parent et al.[50]
Cat
Auditory cortex
Medial and caudoventral putamen, gp, si
Ribak and Kramer [57]
Cat
Anterior and posterior sigmoid gyri
Vertical and horizontal limbs of td; anterior nb; si; hl
Kievet and Kuypers [33,341
Rhesus Monkey
Pre- and postcentral gyri, parietal lobe
sm, td, anterior parts of nb, medullary laminae of gp
Visual cortex
Caudal parts of nb
[281
Wintield et al. [72]
Monkey
Visual cortex
Caudal parts of nb
Jones et al.
Rhesus, Squirrel Monkey
Pre- and postcentral gyri
Anterior parts of nb near td, medullary laminae between gp and putamen, si and pallidal part of nb
Rhesus Monkey
Areas 4 and 6
Septum, nb of substantia innominata, medullary laminae between gp and putamen
Visual cortex
nb lateral to TO, anterior nb between gp and putamen
Cingulate, frontal, parietal, and temporal cortices
nb of substantia innominata
~291
Mesulam and Van Hoesen
[Ml Ogren and Hendrickson 1481 Mesulam et al. [45]
Rhesus Monkey
*For meaning of symbols, see list of neuroanatomic
abbreviations.
BIGL, WOOLF AND BUTCHER
746
TABLE 3 SOME
NORMAL
AND
PATHOLOGIC BEHAVIORS AND PHYSIOLOGIC ACETYLCHOLINE HAS BEEN IMPLICATED
IN WHICH
Selected Experimental Evidence
Biologic process A. Temperature
FUNCTIONS
Regulation
B. Ingestive Behavior I. Drinking
2. Feeding
Injection of carbachol or acetylcholine and eserine into the hypothalamus produces hyperthermia in monkeys [46].
Infusion of carbachol into the hypothalamus, preoptic area, septum, and hippocampus elicits drinking in rats [40]. Int~hy~thal~ic injection of carbachol elicits feeding in rabbits 1631.
C. Aggression
“Rage” is produced in into the anteromedial applied to the lateral mouse killing by rats
D. Learning and Memory
Cerebroventricular infusion of hemicholinium-3 increases number of trials to criterion in a conditioned avoidance task [%I; physostigmine produces retrograde amnesia [22]; scopolamine impairs recall in humans [16].
E. Sleep and Wakefulness
Depletion creases release chrony
F.
DFP and pilocarpine produce analgesia in
Nociception
G. Normal Aging
cats by infusion of carbachol hypothalamus [Z]; neostigmine hypothalamus increases [If.
of acetylcholine with hemicholinium-3 deamount of REM sleep [15]; acetylcholine from cortex is greatest during EEG desynof wakefulness and REM sleep 1271. Kits
[30].
Cell loss occurs in humans in the nucleus of the substantia innominata, sublenticular part 1221.
H. Disease 1. Parkinsonism
Atropine ameliorates tremor [2 I J.
2. Huntington’s Chorea
Physostigmine and choline decrease involuntary movements [ 111; benztropine exacerbates those movements [68].
3. Schizophrenia
Physostigmine
4. Mania
Predominantly euphoric mat&s become less manic after physostigmine [ 111.
5. Alzheimer’s Disease and Senile Dementia of the Alzheimer type
Choline acetyltransferase and AChE are reduced in the cerebral cortex [66].
cortical areas that Krieg [38] would call neocortex (Areas 10,2,41, and X7)and one area (Area 29) of “interesting development” representing a portion of what subsequent authors have referred to as “medial limbic cortex” (e.g., [69]), or mesocortex [753. Within the context of this term~ology, some general statements can be made about the organization of the AChE innervation of the neocortex and posterior portions of medial limbic cortex. First, nucleus basalis and somata associated with caudal substantia iunominata, the ansa lenticularis, and the lateral hypothalamic
four
and arecholine ameliorate symptoms [42].
area provide AChE afferents preferenti~ly to neocortex. This is further evidenced by the finding that lesions in by the finding that lesions in these regions produce a loss of biochemically and histochemically assessed AChE primarily in neocortex that parallels decrements of ChAT in those same cortical areas [32, 39, 691. Second, more rostral portions of the BFAPS preferen:i&y knervate cingulate, retrosplenial, entorhinal and probably other related cortical areas, as well as the amygdala, olfactory bulbs, and hippocampus [47, 69, 741. It is conceivable, therefore, that the BFAPS
AChE PROJECTIONS
TO CORTEX
747
consists of two different but not necessarily mutually exclusive components: (a) a rostral, predominantly medial, portion comprised of the medial septal nucleus, nuclei of the diagonal band, cells in the rostra1 lateral preoptic area and the ventral pallidum/rostral substantia innominata region, and nucleus preopticus magnocellularis that provides AChE afferents primarily to limbic structures in the telencephalon and (b) a caudal, essentially lateral, segment consisting of somata in caudal substantia innominata, nucleus basalis, ansa lenticularis, and lateral hypothalamic area that are the source of afferent AChE fibers preferentially innervating neocortical regions. If the above-delimited parcellation of the BFAPS is valid, then certain other gene~i~tions can be advanced. First, neurons of the caudal BFAPS do not show a tendency to collateralize. Second, more medially-located neocortical areas (Areas 10, 17) are innervated by more rostral portions of the caudal BFAPS and more laterally-located neocortical regions (Areas 2, 41) are supplied by more posteriorlysituated cells of the caudal BFAPS. If Area 17, the so-called primary visual area [75], is omitted from consideration, then the caudal BFAPS projects essentially in a rostrocaudal manner upon the frontal, parietal, and temporal cortices. Inclusion of Area 17, however, allows no simple topography to be fo~ulated. A subst~ti~ number of cells in what we have tentatively called the rostral BFAPS project to that region of the occipital cortex, suggesting possible preferential influences of the limbic system on the processing of visual information. Third, only cells in the “limbic” BFAPS display a tendency to collateralize, and these are few in number. It must be remembered, however, that only Area 29 was examined in relation to collateral projections to Areas 10, 2,41, and 17. If Krieg 1381is correct that cingulate cortex develops in register with adjacent regions of the neocortex (vide supru), then it would be predicted that neurons innervating anterior cingulate cortex might also collaterahze to supply AChE fibers to more rostral portions of neocortex (e.g., cells innervating Area 24 of the cingulate cortex might also innervate Area 10 of the frontal cortex). Furthermore, these neurons would probably lie more posteriorly in the BFAPS. Such conjectures await experimental ionization, however.
Functions of Cholinergic Projections to the Cerebral Cortex Central choline& systems have been implicated in a variety of physiologic and behavioral processes, both normal and pathologic (Table 3; for a more complete treatment, see [12]). Whether or not these involve AChE-containing neurons in the BFAPS is open to speculation, but some of the functions appear to involve areas of the nervous system where appreciable numbers of intensely staining AChE somata or their terminal fields are found, including the basal forebrain, cortex, and hippocampus (Table 3). Particularly significant in this regard is the finding that Alzheimer’s disease and senile dementia of the Alzheimer type are correlated with major involvement of cholinergic mechanisms. In addition to the usual neuropathologic profile of neuritic plaques, neurofibrillary tangles, and granulovacular degeneration seen in several brain regions of Alzheimer patients, but especially in the hippocampus and cortex, both cortical and hippocampal choline acetyltransferase and AChE are si~~c~tly reduced with little or no alteration in dopamine, norepinephrine, S-hydroxytryptamine, GABA, or enzymes associated with their metabolism [66]. One explanation for these observations is that, in Alzheimer’s disease and in senile dementia of the Alzheimer type, there is preferential degeneration of cholinergic neurons projecting to the cortex and hippocampus. Indeed, over 40 years ago, Hassler [23] reported that even in normal aging there was significant loss of neurons in the sublenticular part of the nucleus of the substantia innominata.
This research was supported by USPHS grants NS-10928 and AGO1745 to L.L.B. V.B.‘s stav in the United States was made possible by a travel grant from the Ministry of Health of the German Democratic Republic. N.J.W. is an ARCS scholar. Carol Bergdoll and Howard Tribe are thanked for their skillful assistance in preparing the maffusc~pt and figures.
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