Distribution of ‘non-specific’ cholinesterase histochemical staining in the dorsal thalamus: a comparative study in rodents

Brain Research, 522 (1990) 315-321 Elsevier

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BRES 24155

Distribution of 'non-specific' cholinesterase histochemical staining in the dorsal thalamus: a comparative study in rodents Norman H. Liu, Benjamin P. Yu, Kathy A. Gallardo and Richard T. Robertson Department of Anatomy and Neurobiology, College of Medicine, University of California, lrvine, CA 92717 (U.S.A.) (Accepted 13 March 1990)

Key words: Anterior nuclei; Cholinesterase; Comparative; Rodent; Thalamus

Histochemical studies in rat dorsal thalamus demonstrate that 'non-specific' cholinesterase (ChE) enzyme activity is characteristic of neurons of the anterior dorsal (AD) and reuniens (Re) nuclei and in a cell group found as part of the central lateral (CL) and lateral dorsal (LD) nuclei. Extra-somatal ChE staining also is seen in the anterior ventral (AV) nucleus. Parallel histochemical studies in other rodents reveal slight ChE activity in neurons of the mouse AD and LD, but not in other thalamic nuclei. The dorsal thalami of hamsters, gerbils and guinea pigs show no detectable cellular staining of ChE, although low levels of extra-somatal ChE appear in AV and the internal medullary lamina. These data indicate that 'non-specific' cholinesterase activity is not found commonly in neurons of the dorsal thalamus and prominent ChE staining may be unique to the laboratory rat.

Several recent studies have described patterns of cholinesterase histochemical staining in the brain. In addition to patterns of 'specific' acetylcholinesterase (ACHE) activity 1'8"11-13'17'1s'2°-22,evidence has indicated that some brain regions display significant levels of 'non-specific' cholinesterase (ChE) activity 3"4"6"8-1°A4-

16,19.

Many of these histochemical investigations have been done in the laboratory rat. Within dorsal thalamus of the rat, ChE-positive n e u r o n s have been found within the anterodorsal ( A D ) , anteroventral (AV), and reuniens (Re) nuclei, and in a region that corresponds to portions of the centrolateral (CL) and laterodorsal (LD) nuclei 14-16. Despite considerable attention and provocative theories 2-4'6'9'1°'14-16"19"21,the function(s) of ChE in brain remains u n k n o w n . The localization of ChE within particular neural systems may, however, provide some clues to its function. In this light, it is interesting that ChE-positive n e u r o n s in dorsal thalamus of the rat appear to send axonai projections to medial iimbic cortex 5"14"23, suggesting a possible function of ChE in limbic system function. If ChE is playing a fundamentally important role in limbic system function, then we could expect to find evidence of ChE histochemical staining of these neurons in a variety of species is. This study was u n d e r t a k e n to determine whether thalamic neurons in a variety of species of rodents display C h E activity. Experiments were performed on adult animals of 5 species of rodents including 5 S p r a g u e - D a w l e y rats

weighing 255-372 g, 6 gerbils weighing 101-139 g, 6 mice weighing 19-32 g, 3 guinea pigs weighing 628-670 g, and 5 hamsters weighing 102-111 g. A n i m a l s were anesthetized with chloral hydrate (350 mg/kg) supplemented with sodium pentobarbital (10 mg/kg) and perfused through the heart with normal saline followed by 10% buffered formalin. The brains were removed and fixed overnight in a 10% f o r m a l i n - 2 0 % sucrose mixture. Brains were cut TABLE I

Presence of ChE staining in selected nuclei of dorsal thalamus of rodents Intensity of ChE reaction product is indicated as intense (++ +), moderate (++), light (+), or absent (-).

Thalamic nucleus

Rat

A. Axonal staining AD + AV +++ CL ++ LD ++ Re + Pt + + B.

Somatal staining AD +++ AV ++ CL ++ LD ++ Re +++ Pt +

Mouse

Hamster

Gerbil

Guinea pig

++ + .

+ + -

+ -

+ + -

.

.

.

+

-

-

-

+ + . + .

-

-

-

-

-

-

-

+

.

.

.

.

.

. -

Correspondence: R.T. Robertson, Department of Anatomy and Neurobiology, College of Medicine, University of California, Irvine, CA 92717, U.S.A. 0(XI6-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

316 2 v

ti

Fig. 1. A - H . (For legend, see next page.)

317

m

d

Fig. 1. Photomicrographs of transverse sections through anterior thalamus of rodents, stained histochemically for ChE (left photographs) or AChE (right photographs). AD, anterior dorsal n.; AV, anterior ventral n,; AM, anterior medial n.; Pt, parataenial n. A,B: rat. C,D: mouse. E,F: gerbil. G,H: hamster, I,J: guinea pig. Bar in A = 1.0 mm for all photographs. transversely on freezing microtome at section thickness of 64 ~m. In most experiments, 3 complete series of transverse sections were collected, extending from the striatum through the midbrain. Two of these series of sections were processed for cholinesterase histochemistry, following the procedure outlined by Koelle and Friedenwald 1'7. This method employs a sodium acetate-buffered aqueous medium (pH 5.3), copper-glycine, and one of two substrates. Series 1 was used to study the distribution of ACHE, using 1.0 x 10 -4 M acetylthiocholine as the substrate, while ChE was inhibited by 1.0-1.5 x 10-4 M tetraisopropyl pyrophosphoramide (iso-OMPA). Series 2 was used to study the distribution of ChE, using 1.0-1.5 × 10 -4 M propionylthiocholine as the substrate while AChE was inhibited by 1,5-bis-(4-allyl-dimethylammoniumphenyl)petan-3-one dibromide (BW284c51). Sections for both series were incubated for 21-24 h at room temperature; the histochemical reaction product was developed by immersing the free sections in an aqueous solution of 1% ammonium sulfide for 25-35 s. In some cases, 1.5 × 10 -4 M butyrylthiocholine was used as the substrate in ChE histochemistry, and in some cases the BW284c51 was omitted. A third series of sections was processed for a standard Nissl stain. Sections were examined under the light microscope using bright field illumination. Density of histochemical reaction product was used as an indicator of level ChE activity22. ChE histochemical staining within forebrain tissue of rodents is seen in certain neuron cell bodies and their proximal dendrites or in some forebrain fiber tracts. The intensity and distribution of ChE staining varies considerably between species, as illustrated in Table I and Figs. 1-3. The distribution of AChE histochemical staining, in comparison, appears remarkably similar among different species. Photomicrographs presented in Figs. 1-3 illustrate the

pattern of ChE histochemical staining in transverse sections through rostral and middle portions of the dorsal thalamus of the 5 species studied. The distribution of AChE staining is also shown for comparison. In each region of the dorsal thalamus, ChE staining was most intense in the rat, with markedly reduced staining in each of the other species. For example, Fig. 1A,B presents examples of ChE (Fig. 1A) and A C h E (Fig. 1B) staining in the anterior thalamus of rat. Note the distinct ChE staining of virtually all neuron cell bodies in the anterior dorsal (AD) nucleus. A few neuron cell bodies in the anterior ventral (AV) nucleus stain for ChE, but most staining in AV appears associated with axons rather than cell bodies. Note also that the pattern of AChE staining in the rat anterior thalamus is similar to the pattern of ChE staining. Other photomicrographs in Fig. 1 illustrate that ChE staining in A D is essentially absent in most of the other rodent species. A few labeled cells are seen only in mouse AD (Fig. 3B). ChE staining in AV of other rodents is much reduced, relative to the rat, with light staining apparently associated with axons. AV displays a few ChE labeled neurons. Other regions in anterior thalamus that display ChE staining include the parataenial (Pt) nucleus of the rat and mouse, but not the other rodent species. Photomicrographs in Figs. 2 and 3 illustrate the distributions of ChE and AChE histochemical reaction products in transverse sections through mid-thalamus. Note in the rat that ChE staining occurs in a band of cells that are found within the lateral portion of the central lateral (CL) nucleus and the ventral part of the lateral dorsal (LD) nucleus. In the same sections, clear ChE staining also can be seen in the midline reuniens (Re) nucleus. Note also that the distribution of ChE staining is distinctly different from the distribution of AChE staining (cf. Fig. 2A,B). The mouse shows moderate staining for ChE in the

318

Fig. 2. A-H. (For legend, see next page.)

319

Fig. 2. I,J. Photomicrographs of transverse sections through middle thalamus of rodents, stained histochemically for ChE (left photographs) or AChE (right photographs). CL, central lateral n.; LD, lateral dorsal n.; Re, reuniens n. A,B: rat. C,D: mouse. E,F: gerbil, G,H: hamster. I,J: guinea pig. Bar in A = 1.0 mm for all photographs.

CL, but this staining is not associated with neural somata and a p p e a r s to be axonal. A s shown in Fig. 2D and at higher magnification in Fig. 3D, the mouse displays scattered C h E positive neurons in the dorsal lateral part of L D ; this group of ChE-positive neurons extends caudally to the level of the lateral geniculate body.

Gerbil, guinea pig and h a m s t e r show very slight, if any, C h E staining in C L and L D . The staining in C L in these species does not involve n e u r o n cell bodies but appears associated with axons. While the rat displays rather p r o m i n e n t C h E staining of neurons in Re, all o t h e r of the species studied did not.

Fig. 3. Photomicrographs of transverse sections through thalamus of rodents, stained histochemically for ChE. Abbreviations as in Figs. 1 and 2. A: rat anterior thalamus. B: mouse anterior thalamus; note labeled cells in A D . C: rat lateral thalamus. D: mouse lateral thalamus; note labeled cells in dorsal LD. Bar in A = 250/~m for all photographs.

320 Thus, the present data reveal only very slight levels of ChE in thalami of rodents other than the rat. Indeed, ChE activity in gerbil, hamster and guinea pig generally is found only in regions of the thalamus that display prominent levels of A C h E activity, and this ChE activity is not found within neuronal somata but appears in associated axons. Only the mouse shows ChE-positive neurons and these lightly stained neuron somata are found in AV, A D and in LD. The absence of ChE staining in the dorsal thalamus of species other than rat was unexpected, but further experiments indicated that this absence of ChE activity is likely not due to experimental artifact 1. First, patterns of A C h E in these animals were robust and similar to the pattern seen in rat, indicating that problems relating to perfusion, fixation or other aspects of the procedure likely did not contribute to absence of staining. Second, use of butyrylthiocholine as a substrate rather than propionylthiocholine did not reveal ChE staining. Although we and others a4'21 have demonstrated that propionylthiocholine is the preferred substrate in studies of C h E in rat, this would not necessarily hold for other species. Third, even deletion of the BW284c51 inhibitor of A C h E did not reveal ChE histochemical reaction product in these species. Most of the previous studies of cholinesterase activity in the brain have focused on A C h E (EC 3.1.1.7) activity 1'sA1-13"17"18'2°-z2,primarily because of the impor-

denced by it commonly being referred to as 'specific' or 'true' cholinesterase, while ChE (EC 3.1.1.8) has been referred to as 'non-specific' cholinesterase or 'pseudocholinesterase '3'4,9'14-16'21. Recent demonstrations that significant levels of ChE can be found in neurons in certain regions of the brain challenged the prevalent belief that ChE is a 'non-specific' cholinesterase 3'4' 6,9,10,14-16,19. Importantly, ChE in rat thalamus is found

tant role of A C h E in catabolism of the neurotransmitter acetylcholine. The focus of attention on A C h E is evi-

Supported in part by NSF Grant 87-08515. We thank Jennifer Bruce and Kerry Lee for their participation in initial experiments.

1 Butcher, L.L., Acetylcholinesterase histochemistry. In A. Bj0rklund and T. HOkfelt (Eds.), Handbook of Chemical

brain, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 6413-6417. 10 Layer, P.G., Alber, R. and Sporns, O., Quantitative development and molecular forms of acetyl- and butyryi-cholinesterase during morphogenesis and synaptogenesis of chick brain and retina, J. Neurochem., 49 (1987) 175-182. 11 Parent, A. and Butcher, L.L., Organization and morphologies of acetylcholinesterase containing neurons in the thalamus and hypothalamus of the rat, J. Comp. Neurol., 170 (1976) 205-226. 12 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 13 Robertson, R.T., A morphogenic role for transiently expressed acetylcholinesterase in developing thalamocortical systems?, Neurosci. Lett., 75 (1987) 259-264. 14 Robertson, R.T. and Gorenstein, C., 'Non-specific' cholinesterase-containing neurons of the dorsal thalamus project to medial limbic cortex, Brain Research, 404 (1987) 282-292. 15 Robertson, R.T., Lieu, C.L., Lee, K. and Gorenstein, C., Distribution of 'non-specific' cholinesterase-containing neurons in the dorsal thalamus of the rat, Brain Research, 368 (1986) 116-124. 16 Robertson, R.T. and Mostamand, E, Development of nonspecific cholinesterase containing neurons in the dorsal thalamus of the rat, Dev. Brain Res., 41 (1988) 43-60. 17 Robertson, R.T., Poon, H.K., Duran, M.R. and Yu, J., Neonatal enucleations reduce number, size, and acetylcholinesterase staining in neurons of the dorsal lateral geniculate nucleus, Dev. Brain Res., 47 (1989) 209-225. 18 Robertson, R.T., Poon, H.K., Mirrafati, S.J. and Yu, J., Transient patterns of acetylcholinesterase activity in developing

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Elsevier, Amsterdam, 1983, pp. 1-49. 2 Clitherow, J.W., Mitchard, M. and Harper, N.J., The possible biological function of pseudocholinesterase, Nature (Lond.), 199 (1963) 1000-1001. 3 Gorenstein, C., Bundman, M.C., Bruce, J.L. and Rotter, A., Neuronal localization of pseudocholinesterase in the rat cerebellum: sagittal bands of Purkinje cells in the nodulus and uvula, Brain Research, 418 (1987) 68-75. 4 Graybiel, A.M. and Ragsdale, C.W. Jr., Pseudocholinesterase staining in the primary visual pathway of the macaque monkey, Nature (Lond.), 299 (1982) 439-442. 5 Herkenham, M., The connections of the nucleus reuniens thalami; evidence for a direct thalamo-hippocampal pathway in the rat, J. Comp. Neurol., 177 (1978) 589-610. 6 Horn, K.M. and Carey, R.G., Origin of butyrylcholinesterase in the lateral geniculate nucleus of tree shrew, Brain Research, 448 (1988) 386-390. 7 Koelle, G.B. and Friedenwald, J.S., A histochemical method for Localizingcholinesterase activity, Proc. Soc. Exp. Biol. Med., 70 (1949) 617-622. 8 Kostovic, I. and Goldman-Rakic, P.S., Transient cholinesterase staining in the mediodorsal nucleus of the thalamus and its connections in the developing human and monkey brain, J. Cornp. Neurol., 219 (1983) 431-447. 9 Layer, P.G., Comparative localization of acetylcholinesterase and pseudocholinesterase during morphogenesis of the chicken

not just in regions that also contain A C h E activity, but appears distinctly sequestered in particular thalamic regions. Specifically, the demonstration that ChE is found associated with thalamocortical neurons that send their axons to medial limbic cortex suggested that this 'non-specific' cholinesterase may, indeed, have some specific role relating to limbic system function 14-16. However, if ChE was performing a particular neuronal function, parsimony would predict that the distribution of ChE is conserved across species, particularly across species within a particular phylogenetic order such as the rodents TM. The present results, demonstrating that only the rat displays intense levels of C h E activity in the dorsal thalamus, do not support the hypothesis that C h E activity is a c o m m o n characteristic of rodent thalamus. These results leave unanswered the issue of whether ChE in the rat thalamus is a fundamentally important or just a spurious phenomenon.

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21 Silver, A., The Biology of Cholinesterases, North Holland Press, Amsterdam, 1974. 22 Storm-Mathisen, J., Quantitative histochemistry of acetylcholinesterase in rat hippocampal region correlated to histochemical staining, J. Neurochem., 17 (1970) 739-750. 23 Thompson, S.M. and Robertson, R.T., Sensory projections to the limbic system. I. Thalamocortical projections to medial limbic cortex in the rat, J. Comp. Neurol., 265 (1987) 175-188.