146 Brain Research, 290 (1984) 146-151 Elsevier

Cholinergic and non-cholinergic septo-hippocampal projections: a double-label horseradish peroxidase-acetylcholinesterase study in the rabbit

RONALD H. BAISDEN l, MICHAEL L. WOODRUFF 1 and DONALD B. HOOVER 2

Departments of lAnatomy and 2pharmacology, Quillen-Dishner College of Medicine, East Tennessee State University, Johnson City, TN37614 (U.S.A.)

(Accepted September 13th, 1983)

Key words: hippocampus - - septal area - - diagonal band - - cholinergic - - horseradish peroxidase

The existence of a massive cholinergic projection from cells in the medial septal nucleus (MS) and nucleus of the diagonal band (DB) to the hippocampal formation has been recognized for some time. However, the actual percentages of cholinergic and non-cho- linergic neurons in the MS and DB which project to the hippocampus have not been reported. A procedure which combines horserad- ish peroxidase (HRP) and acetylcholinesterase (ACHE) histochemistry in the same tissue was used to determine these percentages in the rabbit. Less than 50% of the neurons in the MS and DB which were labeled with reaction product following an HRP injection into the dorsal hippocampus also stained for ACHE. Moreover, 70% of all neurons containing HRP reaction product were located in the DB, but neurons in the DB could not be differentiated from those in the MS on the basis of size or morphology. These data are taken to indicate that much of the MS-DB hippocampal projection is not cholinergic. Substance P is suggested as another possible transmitter within this anatomical system.

The septal p ro jec t ion to the h ippocampus is known

to originate from cells located in the medial septal nu-

clei and nuclei of the diagonal band8A 1. Ear ly work

emphasized the cholinergic nature of this projec-

tion4, 5 to the extent that the possibil i ty of a significant

non-cholinergic sep to-h ippocampal pro jec t ion was,

with two exceptions7,10, largely ignored.

Lynch et al. 7 compared the dis tr ibut ion of ACHE-

stained neuron cell bodies in the septa l -diagonal

band region of some rats to the dis tr ibut ion of cells

containing horseradish peroxidase (HRP) react ion

product in o ther rats. Based upon this approach,

these investigators concluded that there was essen-

tially complete over lap in distr ibution be tween the

locations in the medial septal nucleus (MS) and nu-

cleus of the diagonal band (DB) of AChE-s ta in ing

neuron somata (and, therefore , p resumably ace-

tylcholine-containing cells) and somata labe led with

H R P react ion product subsequent to H R P injections

into dorsal h ippocampus of the rat. Lynch et al. also

emphasized that the dis tr ibut ion of terminals arising

from cells in the MS-DB region, as demons t ra ted by

anter0grade hodological techniques, were found

only in areas where A C h E his tochemistry p roduced

staining. The coextensive nature of both ACHE-

stained cell bodies and terminals , with cell bodies and

terminals identif ied with techniques to demons t ra te

connectivity, could be taken to suggest that the sep-

toh ippocampal system is ent i rely cholinergic.

Lynch et al. suggest this conclusion only in a tenta-

tive manner , while point ing out that Mesulam et

al. 10, who employed a double- label p rocedure com-

bining H R P and A C h E staining in the same tissue,

demonst ra ted , following in t rah ippocampal H R P in-

jection in the monkey , that not all neurons in the MS

and DB containing H R P inject ion product also

stained for ACHE. This observat ion indicates that at

least par t of the sep to-h ippocampal pro jec t ion is not

cholinergic. However , the exact propor t ion of non-

cholinergic neurons projec t ing to the h ippocampus

from the septal and diagonal band regions was not

identif ied in this study.

The purpose of the present exper iment was to ex-

pand the observat ions of Lynch et al. and Mesulam et

Correspondence: R. H. Baisden, Department of Anatomy, Quillen-Dishner College of Medicine, East Tennessee State University, Johnson City, TN 37614, U.S.A.

0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B .V.

al. A double-labeling technique first used by Warr 15

in his study of the olivocochlear system, was em-

ployed in rabbits to compare the relative numbers of

AChE-neurons which project to the hippocampus to

the number of non-AChE neurons projecting to the hippocampus. In addition, several rabbit brains were

prepared using the Golgi-Kopsch technique and the

shape, size, and dendritic configuration of neurons in

the MS and DB areas were described and compared

/

Fig. 1. Summary diagram through the septum and diagonal band demonstrating relative amounts of cells which labeled with HRP and either did (stars) or did not (circles) stain for ACHE. Circles or stars do not reflect an actual count of cells, but rather the number of stars and circles reflect relative pro- portions as obtained from all cells analyzed.

147

to the HRP- and AChE-stained neurons. This tech-

nique was employed to provide a better description

of cell type than can be produced by examination of

HRP-reacted material only and has been used pre-

viously to study the morphology of thalamic neurons

sending afferents to the hippocampus 2.

Seventeen male New Zealand albino rabbits,

weighing between 3.5 and 4.2 kg were anesthetized

with an intramuscular injection of a mixture of ket-

amine hydrochloride (100 mg) and acepromazine (10

mg) followed 15 min later by an intravenous injection

of sodium pentobarbital (50 mg). After surgical anes-

thesia was achieved, the rabbit's scalp was shaved

and the animal was placed in a Kopf stereotaxic in-

strument such that lambda was 1.5 mm lower than

bregma. A midline incision was made and the mus-

cles and fascia underlying the scalp were retracted to

expose the skull. A burr hole was produced in the

skull 4 mm posterior to bregma and 6 mm lateral to

the midline. In twelve of the rabbits the tip of a 10 ~1

Hamilton syringe was lowered through this hole into

the dorsal hippocampus (5 mm ventral to the surface

of the brain) and 0.5/~1 of a 50% HRP (Boehringer,

Grade 1) 0.9% saline solution was injected. In the re-

maining 5 rabbits the HRP solution was injected into

the dorsolateral neocortex overlying the hippocam-

pus. Six of the rabbits given intrahippocampal injec-

tions of HRP and all of the rabbits given neocortical

injections were killed with an overdose of sodium

pentobarbital approximately 48 h following the injec-

tion and perfused through the heart with 0.9% saline

followed by 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed, sectioned

frozen at 50/~m and processed according to the cobalt intensification-diamino benezidine procedure 1. The

labeled cells found in this series of experiments were compared to cells in the same region of three addi-

tional rabbit brains prepared using a Golgi-Kopsch stain12.

The remaining 6 HRP-injected rabbits were also

killed by overdose of sodium pentobarbital approxi-

mately 48 h after the injection. These rabbits were perfused through the heart with a 0.1 M phosphate

buffer (pH = 7.4) followed by a solution containing 1% paraformaldehyde and 1.25% glutaraldehyde in

the same phosphate buffer. The brains were stored

overnight in phosphate buffer containing 6% sucrose

148

and sectioned frozen at 50 ~tm. The sections were processed using the combined A C h E - H R P proce- dure of Mesulamg.

A total of 875 labeled cells were classified from the 6 rabbit brains processed using the combined

A C h E - H R P procedure. Although H R P reaction

product was found predominantly ipsilateral to the injection, HRP-labeled somata were also found in

the contralateral MS and DB. Sixty-eight percent (594) of the cells projecting to the hippocampus were

found in the nucleus of the diagonal band. Labeled

neurons observed in the DB were found in essentially

equal numbers in both its vertical and horizontal limbs along the entire rostro-caudal extent of the

structure (Fig. 1). Fifty-four percent of these neu-

rons (323 cell bodies) contained only H R P reaction

product and did not stain for ACHE, while 46% (271

cell bodies) stained for both enzymes. Thirty-one

percent (281 neuron perikarya) were found in the

medial septal nucleus and 59% (166) of these con- tained H R P reaction product only, while 41% (115)

of the labeled MS cells contained both enzymes. Less

than 1% of the total population of labeled cells was

found in the septal raphe. All of these cells contained only the H R P reaction product.

Two hundred five cells in the MS and 117 cells in

the DB were analyzed from the 6 rabbit brains proc-

essed using the cobalt intensification H R P proce-

dure. These neuron perikarya were outlined using a drawing tube attached to an Olympus Model BH mi-

croscope. Measurement of the diameter of each cell body was made along the long axis of the cell. The

mean diameter of the neuron cell bodies labeled in

the MS was 35.04ktm (S.E. = 0.87). The mean diam- eter labeled cells in the DB was 36.32/~m (S.E. =

1.24). The results of a t-test indicated that the diame-

ters of the labeled cells in these two areas did not dif- fer statistically (t = 0.86; d.f. = 321).

Fig. 2. A and B: HRP injection sites in the dorsal hippocampus of two rabbits used in this study; bar = 1 mm. C-F: single- and double- labeled neuron cell bodies of the diagonal band nucleus (C and E) and the medial septal nucleus (F) after hippocampal injection of HRP and combined HRP-AChE histochemical processing; bar = 50/~m.

149

Fig. 3. Multipolar neurons of the diagonal band (A-C) and medial septal nuclei (D and E); bar --- 50/~m. A distal dendritic segment of a neuron of the diagonal band (F) showing spinous processes; bar = 50 ¢tm.

The outlines of neuron cell bodies filled with HRP

subsequent to intrahippocampal injections of the

tracer were compared to neurons stained in other

preparations with the Golgi-Kopsch procedure. Gol-

gi-stained neurons which matched neurons filled with

HRP reaction product were found in both the MS and

DB. For these matched neurons, no observable dif-

ferences were found in the morphology of cells con-

tained within the medial septal nucleus as compared

to the nucleus of diagonal band. All neurons de-

scribed are of the isodendritic type with long, ta-

pering dendrites. A few fusiform-shaped cells can be

observed near either the midline (MS), or the ventral

surface of the brain, (DB), however, most cell bodies

are radiate and tend to have round to pyramidal

shaped perikarya. Only the most proximal portion of

the axons could be observed. Therefore, no

statement can be made regarding axonal orientation.

Three to five dendrites arise from the soma. These

dendrites are thick proximally, tapering as they ex-

tend from the cell body. Branches can be observed

from both the primary and secondary portions of the

dendrites. Dendritic spines are present, however,

many cells exhibit very smooth dendrites. When

present the spines are most dense on the more distal

dendritic segments.

Examination of the sections taken from the brains

of the rabbits given HRP injections into the dorsolat-

eral neocortex overlying the hippocampus revealed a

total of 37 labeled cell bodies. All of these were lo-

cated in the lateral two-thirds of the horizontal limb

of the diagonal band. None of the sections from the

control rabbits were processed with the combined

H R P - A C h E procedure. For this reason, no com-

ment can be made concerning the possible

neurotransmitter content of the HRP-containing

150

neurons. However, these brains do provide adequate control for neocortical uptake of HRP in the rabbits

given intrahippocampal HRP injections. The distri- bution and number of labeled neurons after control neocortical HRP injections indicates that neocortical uptake of the HRP directed into the hippocampus in

the experimental preparations could not account for any significant number of the labeled neurons ob- served in the MS and DB in these rabbits.

In summary, the results of the present study indi- cate that slightly less than 70% of the cells labeled in

the DB and MS after injection of HRP into the dorsal hippocampus of the rabbit are contained within the DB. The double-labeling procedure reveals that, re- gardless of the location of the neuron, less than 50% of the HRP-labeled cells also contain ACHE. Cells giving rise to axons reaching the hippocampus have diameters ranging from 11 to 80pm. DB neurons and MS neurons cannot be distinguished as separate pop- ulations on the basis of size alone, nor can they be dis- tinguished on the basis of morphology as revealed in the Golgi-Kopsch preparations. Both the DB and MS contain isodendritic, multipolar neurons with a variety of perikaryal shapes ranging from fusiform to round. Dendritic spines are found on many cells in both regions and tend to be most dense along the dis- tal portions of the dendrites.

These observations are in agreement with the non- quantitative report of Mesulam et al. 10, who also em-

ployed the H R P - A C h E double-labeling procedure, observing that an ' intermediate' number of neuron cell bodies were labeled only with HRP reaction product after an injection of HRP into the dorsal hip-

pocampus of rhesus monkeys. Our results indicate that at least one half of the projections from the MS and DB to the hippocampus in the rabbit do not con- tain ACHE. Presumably these neurons do not employ acetylcholine as a neurotransmitter, since, although a neuron may contain AChE but not be cholinergic or cholinoceptive, true cholinergic neurons always have

high levels of AChE 3. Indeed, if our data err, they would, therefore, err in over-estimating the number of true cholinergic DB-MS hippocampal efferent neurons.

Although it would appear that more than half of the hippocampally projecting neurons of the DB and MS do not contain ACHE, this conclusion should be accepted cautiously. The injections were placed only

into the dorsal hippocampus. It may be that a larger percentage of double-labeled neurons would have been found in the DB-MS if the ventral hippocampus had received the injection. This possibility should be tested directly. However, a part of the DB-MS pro- jections to ventral hippocampus were very likely la-

beled by HRP in the present experiment as the HRP injections into dorsal hippocampus were sufficiently large to permit diffusion and incorporation of the substance by fibers of passage originating in the DB- MS areas and terminating in the ventral hippocam- pus. For this reason it seems unlikely that the propor- tion of double-labeled to single-labeled DB-MS neu- rons would change greatly following HRP injections limited to ventral hippocampus.

Even if the data reported from the present experi- ments do not permit exact generalization of percent- ages of cholinergic and non-cholinergic DB-MS pro- jections to the entire hippocampus it yet appears jus- tified to conclude that a substantial part of this pro- jection is indeed non-cholinergic. However, these data do not indicate what transmitter is used. Sub- stance P is one very likely candidate. Substance P- containing cell bodies have been found in the DB and MS 6, and Vincent and McGeer TM have demonstrated

that electrolytic lesions of the septal area reduce both the activity of choline acetyltransferase and the amount of substance P in the hippocampus. Other potential transmitters or modulators may, of course, also be contained within the neurons of the DB and MS.

The results of the present experiment do not sup- port the contention of Lynch et al. 7 that the ACHE-

containing neurons of the MS demonstrate AChE staining characteristics which are distinct from those of the DB. Lynch et al. report that the majority of the AChE-staining neuron perikarya contained within the MS are small, stain only lightly, and exhibit few visible dendrites, while the AChE-stained cells of the

DB are larger and more intensely stained with ob- vious branching dendrities. A variety of neurons could be characterized in the present study in terms of size, shape, dendritic arborization and AChE staining density, but these were not observed to be organized into groupings such as those described by Lynch et al. Several differences between the two ex- periments might account for these discrepancies in results. Rabbits served as subjects in the present

151

study, while Lynch et al. employed rats and species

differences might exist in organization of the MS and

DB. The use of the double-label procedure might

also account for the observed differences, although

the basis for such an effect is not apparent. Finally,

Lynch and his colleagues treated their rats with diiso-

propyldifluorophosphate (DFP) to inhibit ACHE,

allowing 24 h for the enzyme to recover before killing

the animals. Since A C h E is produced in the soma and

transported distally into axons and dendrities, this

technique eliminates staining of the neuropil and al-

lows easier observation of the cell bodies of ACHE-

positive cells. It may be, however, that the apparent

differences in neuron staining density for A C h E and

dendritic arborization patterns observed by Lynch et

al. were due to differential regenerat ion and trans-

port of the enzyme, rather than actual differences in

morphology of the neurons.

Analysis of the HRP-reacted brains in the present

study indicated that neurons of many sizes project to

the hippocampus from the DB and MS. The smallest

cells found to be labeled with HRP reaction product

after the hippocampal injection had a diameter of 11

~m. These cells are of the size which, based upon

analysis of Golgi-stained material from rabbits,

T6mb61 and Petsche 13 proposed to be septal inter-

neurons. Comparison of the HRP-reacted and Golgi-

stained brains in the present study indicated that

these small cells also have the same shape as the

small neurons observed by T6mb61 and Petsche.

These observations suggest that at least some of the

small neurons found within the MS and DB are not

interneurons, but project to the hippocampus.

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