The subnuclear organization of acetylcholinesterase-containing neurons in the interpeduncular nucleus of rats

Brain Reseurch

Bulktin,

Vol.

13, pp. 503-508,

1984. 0 Ankho

International

Inc. Printed

in

the U.S.A.

0361-9230184 $3.00 + .OO

The Subnuclear Organization of Acetylcholinesterase-Containing Neurons in the Interpeduncular Nucleus of Rats BARRY

FASS*’

AND GEOFFREY

S. HAMILLt

“Department

of Neurosurgery, University of Virginia School of Medicine Charlottesville, VA 22908 ?Department of Anatomy, The Milton S. Hershey Medical School The Pennsylvania State University, Hershey, PA 17033 Received

4 June 1984

FASS, B. AND G. S. HAMILL. The suhnucleur orgunizufion of acetylcholinesterase-contuining neurons in the internucleus of ruts. BRAIN RES BULL 13(4) 503-508, 1984.-The interpeduncular nucleus (IPN) is a heterogeneous structure comprised of seven subnuclei which differ with regard to their cytoarchitecture, synaptology, connectivity, and content of neuropeptides and biogenic amines. In the present study, we used Butcher’s pharmacohistochemical regimen to assess the subnuclear distribution and staining intensity of acetylcholinesterase (AChE)-containing neurons in the IPN. Although AChE-positive somata were present in every subnucleus, their staining intensity differed within and between the subnuclei. The most intensely stained somata were found in the apical and central subnuclei; however, they comprised only IO-25% of the total population of AChE-positive somata in these subnuclei. Heavily stained somata were observed in the apical, central, and lateral subnuclei; moderately stained somata in the central, lateral, intermediate, and rostra1 subnuclei; and lightly stained somata in the lateral, intermediate, rostral, dorsal lateral, and rostra1 lateral subnuclei. The present findings indicate that AChE-containing neurons are differentially distributed between subnuclei of the IPN.

peduncular

lnterpeduncular nucleus Diisopropylfluorophosphate

Subnuclear organization

Acetylcholinesterase

interpeduncular nucleus (IPN) historically has been considered a homogeneous structure. However, recent evidence indicates that the IPN is organized into subnuclei which are distinguishable on the basis of their cytoarchitecture and synaptology [12]. A review of the existing nomenclature for these subnuclei has attempted to consolidate and standardize the divergent terminology into one scheme which recognizes seven subnuclei [18]. Four subnuclei are bilateral (the lateral, intermediate, rostra1 lateral, and dorsal lateral subnuclei), and three are on the midline (the rostra], central, and apical subnuclei). These subnuclei have been further differentiated on the basis of their content of putative neurotransmitters and distribution of afferents from the nucleus of the diagonal band [11,13]. Since the IPN contains one of the highest concentrations of cholinergic enzymes in the brain (e.g., [22,23]), we became interested in whether such enzymes are distributed differentially across the subnuclei. The presence of histochemically demonstrable acetylcholinesterase (AChE) often has been interpreted as evidence for acetylcholine activity (e.g., [19]), especially in those neurons which stain intensely for AChE after peripheral injections of diisopropylfluorophosphate (e.g., [4, 5, 6, 10, 171). Recent studies of

pharmacohistochemistry

AChE localization within the neuropil of IPN revealed a heterogeneous pattern of staining between subnuclei; the rostra], apical, and lateral subnuclei exhibited the most intense staining [ 12,261. However, these studies did not investigate the distribution of AChEcontaining cell bodies within subnuclei. Butcher and Woolf [6] reported the presence of AChE-positive somata in IPN, but did not analyze their subnuclear organization. Consequently, we utilized Butcher’s pharmacohistochemical regimen [4, 5,6] in the present study to map the distribution of AChE-containing cell bodies in the subnuclei of IPN.

THE

METHOD

AChE Pharmacohistochemistty

Four intact male Sprague-Dawley rats (250-350 g; Dominion Labs) served as subjects. Each rat was pretreated with atropine methyl sulfate (4.0 mg/kg, IP), to reduce the severity of autonomic symptoms. Then the subjects were injected intramuscularly with diisopropylfluorophosphate (DFP; Sigma Chemical Co.; dissolved in peanut oil, 6.67 mg/ml) at a dose of 1.8 mgikg. After a survival interval of 5.5-10 hr, the rats were anesthetized and perfused intracardially with 10%

*Requests for reprints should be addressed to either author. B. Fass now is at the Department of Psychology, Clark University, MA 01610.

so3

Worcester,

504

PONS ---y_

D.

,,

_-

_//”

FIG. 1. Camera lucida tracings of the IPN at four coronal levels, from rostral (A) to caudal (D). Dashed lines indicate the boundaries ofeach subnucleus. Abbreviations: RL, rostra1 lateral; I, intermediate; I,. lateral. Redrawn from Hamill and Lenn [ 121

neutral buffered formalin. Their brains postfixed overnight in 30% sucrose-formalin,

were removed, and cut in the

coronal plane on a freezing microtome. AChE histochemistry was performed according to the method of Naik 1211, using promethazine (Wyeth Labs) as an inhibitor of nonspecific cholinesterase (after [20]). Every other section through the IPN was incubated in substrate medium for 48 hr and then reacted in 1% sodium sulfide. The sections subsequently were mounted, defatted, and coverslipped with Eukitt (Calibrated Instruments, Inc.). Control material was processed similarly, except that (1) ethopropazine hydrochloride (Sigma) was used in place of promethazine as the inhibitor, or (2) butyrylthiocholine iodide (Sigma) was used instead of acetylthiocholine iodide as the substrate. The first control yielded a pattern of staining virtually identical to that obtained with promethazine, whereas the second yielded virtually no staining. Quulitative

Analyses

The distribution and staining intensity of AChE-positive cell bodies were assessed for each subnucleus, at four rostrocaudal levels of the IPN (illustrated diagrammatically in Fig. 1). Two independent observers used the following scale to rate the staining intensity: intense, heavy, moderate, light, and weak. This rating scale was calibrated by defining “intense” as the intensity of staining found in the red nucleus,

oculomotor nucleus, and substantia nigra pars compacta: other investigators (e.g., [6]) also have rated~neurons in these nuclei as “intense.” Since our two sets of ratings were highly correlated, a composite rating was determined for the AChE-positive somata within each subnucleus.

Morphometric measurements of AChE-positive cell bodies within each subnucleus were obtained with the aid of a Leitz microscope, video camera, and Zeiss Videoplan” image-analyzing system. A 100x oil objective was used in evaluating the mean area, AChE-stained somata.

perimeter,

and diameter

of the

RESUI.TS

The optimal survival interval for revealing AChE-positive somata in the IPN was 8 hr. At 5.5 and 10 hr after DFP staining was the intensity of neuropil treatment, moderate-to-heavy and it obscured less intensely stained somata. Even at 8 hr, however, the neuropil of the rostra1 and lateral subnuclei was moderately stained, thereby distinguishing the borders of these subnuclei from the remaining subnuclei (they exhibited virtually no neuropil staining; Figs. 2 and 3A). AChEstained cell bodies were present in all sub-

AChE-CONTAINING

NEURONS

IN IPN

FIG. 2. Acetylcholinesterase (AChE) staining in the IPN (level C of Fig. 1) after pretreatment with diisopr~pyIfluorophospbate an d 8 hr survival. The neuropil of rostra1 (R) and lateral (L) subnuclei is distinguished by intense and heavy staining for AChE, respectively. Little staining of the neuropil is present in the intermediate (I) and central (C) subnuclei. The majority of AChE-positive somata in these subnuclei are tightly or moderately stained, although a small proportion of heavily stained somata are present in the lateral subnucleus {see text). Calibration bar= f@l hrn.

TABLE

1

Rostrd Subnucleus

~~STRI~UTIGN AND STAINING ~~ARA~E~I~I~S OF AChE-C(~NTAIN~NGNEURONS IN SUBNUCLEI OF THE ~TE~PE~UNCULAR NUCLEUS Subnuclei Apical Central Lateral lntermediate Rostra1 Dorsal Lateral Rostra1 Lateral

lntense

Heavy

* *

* * *

Moderate

* 8 * *

Light

* * * * *

nuclei. However, there were no histochemicaliy detectable AChE-containing dendrites or processes in the IPN. The distribution and staining properties of the AChE-positive cell bodies are summarized in Table I, by subnucleus (except for the dorsal lateral and rostra1 lateral subnuclei, which contained only lightly stained somata and were otherwise unremarkable).

As illustrated in Fig. 2, light-to”m~erate~y stained somata are dist~but~d homogeneously throughout the rostrocaudal and medioiater~ extent of the rostral subnucleus. Approximately 10% of these AChE-positive somata were stained lightly, while the remainder stained moderately. Morphometric analyses of the AChE-stained somata in the rostra1 subnucleus revealed that their mean area was 32.9 f-Lmz, perimeter 21.6 grn, and diameter 6.9 pm (n=20 somata),

Apical Subtzucleus Some of the most intensely stained AChE-positive somata in the IPN were found in the apical subnucleus (Fig. 3); they were stained at least as intensely as the most intensely stained neurons in the oeuiumotor nucleus, red nucleus, and substantia nigra pars compacta. These intensely stained somata were homogeneously distributed but comprised only approximately 25% of the AChE-positive somata in this subnucleus. The remaining somata were heavily (about 25%) or moderately (about 50%) stained. The mean area for AChE-positive somata in the apicai subnucleus was 88.5 pm2, perimeter 36.7 pm, and diameter 11.7 pm (n=40),

AChE-CONTAINING

NEURONS

IN IPN

Central Subnucleus The distribution of AChE-positive cell bodies in the central subnucleus is shown in Figs. 2 and 3A. AChE-positive somata appeared to be distributed in horizontal laminations at rostra1 levels (corresponding to level C of the IPN; Fig. 1) of the central subnucleus (Fig. 2). More caudally (level D of the IPN; Fig. l), however, AChE-positive somata were organized into columns along the lateral margins of the subnucleus (adjacent to the intermediate subnucleus). These somata extended dorsally and medially (just ventral to the apical subnucleus), thereby forming a horseshoe shape (Fig. 3A). This pattern was consistent across the coronally-cut brains. Most of these AChE-positive cells were stained moderately, although a small proportion (approximately 10%) stained heavily. By contrast, the central region of the subnucleus at this rostrocaudal level was virtually devoid of AChE-stained somata. The morphometric measurements for these somata suggest that there might be two cell types: a larger one, with a mean area of 142 pm2, mean perimeter 45.1 pm, and mean diameter 15.0 pm (n=29); and a smaller one, with a mean area of 57.8 pm*, mean perimeter 28.8 pm, and mean diameter 9.5 pm (n=36). Neither type was restricted to a specific region of the subnucleus, and neither exhibited one unique intensity of staining. Lateral Subnucleus Light-to-moderately stained cell bodies were distributed homogeneously throughout the lateral subnucleus (Figs. 2 and 3A), which also demonstrated a characteristic neuropil staining. Occasional heavily stained somata were observed in the lateral subnucleus, although there was no discernible pattern to their distribution. The AChE-positive somata in this subnucleus were among the largest we measured in the IPN; their mean area was 105.3 pm*, mean perimeter 39.6 pm, and mean diameter 13.3 pm (n=30). Intermediate

Subnucleus

Light-to-moderately stained cell bodies were observed in the rostra1 portion of this subnucleus (cf, Figs. 2 and 3A). Their mean area was 92.6 pm*, mean perimeter 36.8 pm, and mean diameter 12.0 pm (n=34). DISCUSSION

The present findings suggest that AChE-containing neurons are distributed differentially across subnuclei of the IPN. While every subnucleus was found to contain AChEpositive cell bodies, the most intensely stained ones were restricted (for the most part) to the apical and central subnuclei. Such intensely stained somata comprised only a small proportion of the total population of AChE-positive cells in these subnuclei (roughly lO-25%). The other subnuclei contained mostly light-to-moderately stained cell bodies, with a small percentage (approximately lO-25%) which stained heavily. Thus, the subnuclei of the IPN may be further dif-

507 ferentiated on the basis of their intrinsic organization of AChE-containing neurons. Previous studies using the pharmacohistochemical regimen for AChE have yielded negative results on the distribution and staining properties of AChE-containing neurons in the IPN. For example, Butcher and Woolf [6] reported the presence of only lightly-stained somata in the IPN. In a more recent report by Satoh, Armstrong and Fibiger [27], the IPN is represented as being devoid of “intensely stained AChE cells.” Fibiger [IO], citing pharmacohistochemical [lo] and immunohistochemical [16] data, additionally has stated that the IPN does not contain cholinergic neurons. However, these studies may have been restricted to one subnucleus or level of IPN. We found, by contrast, a small population of cells in the apical and central subnuclei which stained at least as intensely for AChE as neurons in the oculomotor nucleus, red nucleus, and substantia nigra pars compacta. Based upon the general view that very intense AChE staining/high AChE activity is a necessary (but not sufficient) criterion for identifying a neuron as cholinergic [6,171, one might suppose that these neurons could be cholinergic. In some regions of the brain, however, there are neurons which stain intensely for AChE but probably do not use acetylcholine as a transmitter (e.g., [9]). Recent immunocytochemical studies of the distribution of choline acetyltransferase (ChAT) have revealed a lack of ChATpositive cells in the IPN [l, 9, 14, 251. Since these studies may have been restricted to one subnucleus or level of the IPN, it will be important to reexamine in detail the distribution of ChAT within each IPN subnucleus. It is not known whether the intensely stained neurons in the apical and central subnuclei might be the cells-of-origin of IPN efferents. HRP studies have shown that the hippocampal formation receives an afferent projection from the IPN [2, 24, 291. Although the subnuclear distribution of the cells which project to the hippocampus has not been examined systematically, it appears that they might be contained within the apical and central subnuclei. Riley and Moore [24], using Ives’ [15] nomenclature, reported that retrogradely labeled cells were present in the dorsal magnocellular division of the IPN (apical subnucleus; after [ 181) following intrahippocampal injections of HRP. Wyss et al. [29], using the nomenclature of Berman [3], found labeled cells of medium size (130 pm*) in the apical and outer posterior divisions of the IPN (apical subnucleus; after [18]). Therefore, the intensely stained AChE-containing cells which we found in the apical and central subnuclei might contribute to the pathway from the IPN to the hippocampus. The neurochemical and neuroanatomical significance of the less intensely staining cell bodies in the IPN is not clear, at present. They conceivably could be cholinoceptive; i.e., the targets of a presumably cholinergic input to the IPN originating from the nucleus of the diagonal band or the habenula [11,28]. Alternatively, such cells might be related to peptidergic systems. There now is evidence that AChE participates in activities unrelated to acetylcholine, such as

FACING PAGE FIG. 3. A. Acetylcholinesterase (AChE) staining in the IPN (level D of Fig. 1) after pretreatment with diisopropylfluorophosphate and 8 hr survival. Intensely staining cells are present throughout the apical (A) subnucleus (arrows). Moderately and heavily stained AChEcells are organized into columns adjacent to the lateral margins of the central (C) subnucleus. Such cells extend dorsallv and mediallv iust ventral to the apical subnucleus, thereby forming a characteristic horseshoe pattern. The central region of the central subnucleus appearsV&tually devoid of AChE-positive cells. Calibration bar= 100 pm. B. Intensely stained cells in the apical subnucleus. Arrows correspond to those in Fig. 3A. Calibration bar=50 pm.

the hydrolysis of substance P and enkephalins [7,8]. Recent findings suggest a correspondence between the subnuclear distribution of AChE and putative peptide neurotransmitters in the IPN. For example, Rotter and Jacobowitz [26] reported that immunohistochemical staining for substance P coincides with neuropil staining for AChE in regions of the IPN corresponding to the lateral and rostra1 subnuclei. Hamill et ul. [13] found immunofluorescent processes reactive for substance P and leu-enkephalin within the rostra1 and lateral subnuclei, and these subnuclei exhibit considerable neuropil staining for AChE even after DFP treatment. Thus, it will be important to determine the extent to which AChE and neuropeptides are colocalized within IPN subnuclei. ln conclusion, the present study demonstrates that

AChE-containing cells are distributed differentially acro\~ the subnuclei of the IPN. We propose thnr although she majority of neurons in the IPN most likely do not use awry1 choline as a transmitter, a small group of neurons restricted to the apical and central subnuclei is rich in AC’hE :md might contribute to the IPN’s inputs to the hippocampus. Elucid:ition of the chemical neuroanatomy of these cells :cwait\ further investigation.

This study wab supported by NSF grant BNS76-17750 awarded to Oswald Steward. We are very grateful to Dry. 0. Steward folsupport, E. Rubel for use of the image analy\i\ system. and ‘I Reeves for technical assistance. We also thank Mr. F. 1.. Snavel\ for assistance in preparing the figures.

REFERENCES I. Armstrong, D. A., C. B. Saper, A. I. Levey, B. H. Wainer and R. D. Terry. Distribution of cholinergic neurons in the rat brain: demonstrated by the immunohistochemical localization of choline acetyltransferase. J Camp Neurnl 216: 53-68, 1983. 2. Baisden, R. H., D. B. Hoover and R. J. Cowie. Retrograde demonstration of hippocampal afferents from the interpeduncular and reuniens nuclei. Neurosci Left 13: 105-109, 1979. 3. Berman, A. L. The Brain Stem of the Cut. A Cytoarchitec,tclnii. Atlas With Slereotaxic Coordinates. Madison: University of Wisconsin Press, 1968. 4. Butcher, L. L. Acetylcholinesterase histochemistry. in: Handbook of’ Chemical Neuroanatomy, vol I: Methods in Chemical Neuroanatomy, edited by A. Bjiirklund and T. HBkfelt. Amsterdam: Elsevier Science Publishers, 1983, pp. l-49. 5. Butcher, L. L., K. Talbot and L. Bilezikjian. Acetylcholinesterase neurons in dopamine-containing regions of the brain. .I Neural Trunsm 37: 127-153, 1975. _ 6. Butcher, L. L. and N. J. Woolf. Cholinergic

and serotonergic organization. role in intercellular communication processes, and interactive mechanisms. In: Chemical Transmission in the Bruin, edited by R. M. Buijs, P. Pevet and D. F. Swaab. Amsterdam: Elsevier Biomedical Press, 1982, pp. 3-40. 7. Chubb, I. W., A. J. Hodgson and G. H. White. Acetylcholinesterase hydrolyzes substance P. Neuroscience 5: 2065-2072,

systems in the brain and spinal cord-anatomical

1980. 8.

Chubb, I. W., E. Ranieri, G. H. White and A. J. Hodgson. The

enkephalins are amongst the peptides hydrolyzed by purified acetylcholinesterase. Neuroscience 10: 1369-1377, 1983. 9. Eckenstein, F. and M. V. Sofroniew. Identification of central cholinergic neurons containing both choline acetyltransferase and acetylcholinesterase and of central neurons containing only acetylcholinesterase. J Neurosci 3: 22862291, 1983. 10. Fibieer. H. C. The organization and some projections of cholynergic neurons of the mammalian forebrain. Brain Res Ret, 4: 327-388,

1982.

11 Hamill, G. S. and B. Fass. Differential distribution of diagonal band afferents to subnuclei of the interpeduncular nucleus in rats. Neurosci Left 48: 43-48, 1984. 12 Hamill, G. S. and N. J. Lenn. The subnuclear organization of the rat interpeduncular nucleus: a light and electron microscopic study. J Comp Neural 222: 396408, 1984. 13 Hamill, G. S., J. A. Olschowka, N. J. Lenn and D. M. Jacobowitz. The subnuclear organization of substance P. cholecystokinin, vasoactive intestinal peptide, somatostatin, leu-enkephalin, dopamine-/3-hydroxylase and serotonin in the rat interpeduncular nucleus. J Comp Neural 226: 580-596, 1984. 14. Houser, C. R., G. D. Crawford, R. P. Barber, P. M. Salvaterra and J. E. Vaughn. Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase. Bruin Res 266: 97-121,

1983.

1.5. Ives, W. R. The interpeduncular nuclear complex uf selected rodents. J Camp Nrurol 141: 77-94. 1971. 16. Kimura, H.. P. L. McGeer, J. H. Peng and E. G. McGecr. The central cholinergic system studied by choline acetyltransferase immunohistochemistry in the cat. .I Camp Nettrol 200: 151-201. 1981. 17. Lehmann, J. and H. C. Fibiger. Acetylcholinesterase and the cholinergic neuron. Lifi, Sci 25: 1939-1947, 1980. 18. Lenn, N. J. and G. S. Hamill. Subdivisions of the interpeduncular nucleus: A proposed nomenclature. Broirl Ret B/r// 13: 20?? 204, 1984. 19. Lewis, P. R. and C. C. D. Shute. The cholinergic limbic system: projections to the hippocampal formation, medial cortex, nuclei of the ascending cholinergic reticular system, and the subfornical organ and supra-optic crest. Brain 90: 521-540, 1967. 20. Lynch, G., D. A. Matthews, S. Mosko, T. Parks and C. c’oman. Induced acetylcholinesterase-rich layer in rat dentale gyrus following entorhinal lesions. Bruin Res 42: 3 I l-3 16, 1972. 21. Naik, N. T. Technical variations in Koelle’s histochemical method for demonstrating cholinesterase activity. p .I Mic,ro\c Method.% 104: 89-100, 1963. 22. Palkovits, M. and D. M. Jacobowitz. Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. J Camp Neural 157: 29-42, 1974. 23. Palkovits. M.. J. M. Saavedra. R. M. Kobayashi and M. Brownstein. Choline acetyltransferase content of limbic nuclei of the rat. Bruin Re3 79: 443-450, 1974. 24. Riley, J. N. and R. Y. Moore. Diencephalic and bramstem aiferents to the hippocampal formation of the rat. Rvuin Res R///l 6: 437-444, 1981. 25. Ross, M. E., D. H. Park, G. Teitelman, V. M. Picket, D. J. Rein and T. H. Joh. lmmunohistochemical localization of choline acetyltransferase using a monoclonal antibody: a radioautographic method. Nerrroscience IO: 907-922. 1983. 26. Rotter, A. and D. M. Jacobowitz. Localization of substance P. alphaacetylcholinesterase, muscarinic receptors and bungarotoxin binding sites in the rat intepeduncular nucleus. Brrrin Res Bull 12: 83-94. 1984. 27. Satoh, K., D. M. Armstrong and H. C. Fibiger. A comparison 01

the distribution of central cholinergic neurons as demonstrated by acetylcholinesterase pharmacohistochemistry and choline acetyltransferase immunohistochemistry. Brain RP\ Bu11 II: 693-720, 1983. 28. Villani, L.. A. Contestabile

and F. Fonnum. Autoradiographic labeling of the cholinergic habenulo-interpeduncular prqjection. Ncurosci Lctt 42: 261-266, 1983. 29. Wyss, J. M., L. W. Swanson and W. M. Cowan. A study oi subcortical afferents to the hippocampal formation in the rat. Neuroscience

4: 463-476.

1979.