Brain Research, 131 (1977) 303-312 © Elsevier/North-HollandBiomedicalPress

303

DOPAMINE-SENSITIVE ADENYLATE CYCLASE WITHIN LAMINAE OF THE OLFACTORY TUBERCLE

NElL R. KRIEGER,JOHN S. KAUER, GORDON M. SHEPHERD and PAUL GREENGARD Departments of Pharmacology and Physiology, and the Neurosurgical Research Laboratories, Yale University School of Medicine, New Haven, Conn. 06510 (U.S.A.)

(Accepted December 3rd, 1976)

SUMMARY There are three histological laminae within the rat olfactory tubercle: plexiform, pyramidal and polymorphic. We have assayed dopamine-sensitive adenylate cyclase in homogenates of frozen sections cut parallel to these laminae. Consecutive sections were cut of alternating thickness, 16 #m and 100 #m. The 16 #m sections were stained with toluidine blue to ascertain the depth and orientation of each section. The 100 #m sections were homogenized and assayed for dopamine-sensitive adenylate cyclase. Substantial levels of dopamine-sensitive adenylate cyclase were found within all three laminae. The results suggest that the enzyme occurs in cell processes, including dendrites, of the plexiform layer, and they are consistent with the localization of dopamine-sensitive adenylate cyclase in the pyramidal cells.

INTRODUCTION It has been postulated that a receptor for the neurotransmitter dopamine may be dopamine-sensitive adenylate cyclaselL Within the rat brain, both dopamine and dopamine-sensitive adenylate cyclase are present in high amounts in caudate nucleus, nucleus accumbens, and olfactory tubercleS,6,12,17,18; these are sites of projections of dopaminergic pathways from the brain stem 7,22. In view of the potential importance of dopamine-sensitive adenylate cyclase, it was decided to determine its neuronal localization. For this purpose the tubercle is advantageous because of its clearly laminated organizationa,4 and its accessibility at the brain surface (see Fig. 1A). There are three well-defined histological laminae within the rat olfactory tubercle: a superficial plexiform layer, a thin dense layer of pyramidal cell bodies, and a deep layer of scattered pyramidal and polymorphic cell bodies (see Fig. 1B). We have assayed dopamine-sensitive adenylate cyclase in homogenates of frozen sections cut tangential to these layers. The topographical arrangement of the layers, including

304 dense clusters of small cell bodies (islands of Calleja), is seen especially clearly in these tangential sections. The laminar distribution of dopamine-sensitive adenylate cyclase is described and the implications for neuronal localization are discussed.

METHODS Male Sprague-Dawley rats, weighing approximately 300 g, were killed by decapitation. The hemisected brain was rapidly frozen in powdered dry ice while resting on its ventral surface on a plate of teflon. The brain's own weight flattened the tubercle and improved the alignment of successive layers with the plane of section. The tissue was mounted in a cryostat and sectioned at -15 °C. Tangential sections which included the entire tubercle were approximately 18 sq. mm in area. The curvature of successive layers (see Fig. 1B) meant that such sections usually included material from more than one layer. We were able to obtain tangential sections of more uniform laminar composition by paring the frozen tubercle down to a pedestal of 4 sq. mm cross-section before sectioning it (see Fig. 1A). The pedestal remained attached to the hemisected brain, in the experiments described here, the pedestal was located in the posterior medial region of the tubercle where the layers are most uniform (see Fig. 2). It remains to be determined whether the distribution of dopamine-sensitive adenylate cyclase observed in this portion of the tubercle is representative of that in the other regions. Consecutive tangential sections were cut of alternating thickness, 16/~m and 100 #m. The 16 #m sections were stained with toluidine blue to establish the pattern of cells at each depth, and the 100 #m sections were used for measurement of enzyme activity. Homogenization of tissue, incubation of dopamine-sensitive adenylate cyclase, and cyclic AMP determinations were carried out by methods similar to those described by Kebabian et al.lL Frozen tissue sections were homogenized in 0.5 ml of 2 m M Tris-(hydroxymethyl)aminomethane-maleate buffer (pH 7.4)-2 m M ethylene° glycol-bis(fl-aminoethylether)-N,N'-tetraacetic acid (EGTA). The standard assay mixture (final vol. 0.5 ml) for measurement of adenylate cyclase activity contained (in mmole/liter): Tris-(hydroxymethyl)aminomethane-maleate, 80, pH 7.4; ATP, 0.5; MgSO4, 2.0; isobutylmethylxanthine, 1; EGTA, 0.6; GTP, 0.01. The enzyme (0.05 ml of tissue homogenate) was preincubated with all components of the standard assay system except ATP, plus any added test substances, for 20 min at 0 °C; the reaction was initiated by the addition of ATP and carried out for 10 min at 30 °C. The reaction was terminated by boiling. Aliquots of each homogenate were assayed in triplicate in the presence and absence of dopamine and test substances. The cyclic AMP values were determined by the procedure of Brown et al.L Cyclic AMP standards from 0.1 to 0.8 pmoles were used. Protein was determined by the method of Lowry et al. 14. Consecutive 100 #m (4 sq. mm) sections were found to contain approximately constant (40/~g) amounts of protein. Under the experimental conditions used, dopamine-sensitive adenylate cyclase activity was linear with respect to time and protein concentration.

305

A

C, OLE TUBER,

A - PLEXIFORM B" PYRAMIDAL C- POLYMO RPHIC

B

Fig. 1. A: outline drawing of sagittal section of rat brain. Pedestal of olfactory tubercle is indicated; dotted lines show tissue trimmed away (see text). Tangential sections through pedestal are indicated by A, B, C (see Fig. 2). OLF. BULB, olfactory bulb; AON, anterior olfactory nucleus; ACC, accumbens nucleus; OLF. TUBER, olfactory tubercle. B: frontal section through olfactory tubercle, showing histological layers. Toluidine blue. Magnification × 30.

306 A

Fig. 2. A and B.

307 C

Fig. 2. Tangential sections through the three main laminae of the olfactory tubercle. A: plexiform layer. B: pyramidal layer. C: polymorphic layer. Weigert stain. A, x 50; B, × 37, C, x 28. Regions outlined by rectangles indicate approximate portions of sections occupied by tissue pedestal (see text). Arrows indicate islands of Calleja, as discussed in text. RESULTS

Anatomy The histology of the rat olfactory tubercle is shown in the frontal section of Fig. 1B. The outermost plexiform layer contains few cell bodies. The middle layer consists of a dense sheet of small and medium-size pyramidal cell bodies (15-30/tm diameter). In the deepest layer, one encounters, superficially, scattered medium-size pyramidal cells (15-25 #m diameter); deeper, scattered polymorphic cells ( 1 0 4 0 #m diameter) predominate. The tangential sections in Fig. 2 show cell patterns from the three laminae. The most superficial section (A) contains exclusively the outer plexiform layer, together with the overlying pia. The middle section (B) contains predominantly pyramidal cells, localized in the dense layer of cell bodies and the zone just deep to it. Because of the

308 curvature of the tubercle, there is also a rim of plexiform layer. The deepest section (C) passes through the deep polymorphic cell layer. The approximate borders of the pedestals of tissue that were assayed are indicated by the rectangular outline. Certain topographical aspects of the laminae may be noted. Fig. 2B shows that the lateral border of the pyramidal cell body layer has the form of serrations or fingers. These extend in a general orientation laterally and anteriorly toward the lateral olfactory tract (see also Fig. 2C). Scattered through the layers are dense groups of small cell bodies (5-10 #m diameter): the islands of Calleja. In the tangential sections we could distinguish three types of island. One was located superficially within the plexiform layer, at or near the tips of the fingers of pyramidal cell bodies (arrows in Fig. 2B). The second was composed of one or more large groups of cells in the medial part of the tubercle at a level just deep to the pyramidal cell body layer (not illustrated). The third type consisted of small groups of cells scattered within the deeper layer; some were located within the fingers (i.e. just deep to the pyramidal layer) (see arrow in Fig. 2C). In serial sections it was found that most of these small islands were interconnected to each other and to the larger medial islands by groups of cells arranged in thin strands. These arrangements can be followed much more clearly in tangential than in frontal sections. The types of islands are consistent with classical descriptions1,3, 4. c

.o 120 •t . ¢J



¢1) to

"

100

'~.

80

a "'

60

IX: 0 LL

D.. =E < ¢0 ._1 0>.0

4O

2O

Plexiform I 100

>j:yromidal I 200

I 300

TUBERCLE

P o l y m o r p h i e >1

I 400

I 500

DEPTH

I 600

I 700

I 800

(microns)

Fig. 3. Dopamine-sensitive adenylate cyclase activity as a function of depth in the olfactory tubercle. The activities plotted are averages from 5 independent experiments with no dopamine (O ©) or 100/~M dopamine ( • • ) . The depths of the midpoints of the assayed sections are plotted on the abscissa. The approximate locations of the successive layers of the tubercle are indicated above the abscissa. To show the combined data from several experiments, the adenylate cyclase activities at each depth, both in the absence and presence of dopamine, were normalized within each experiment. For this purpose, the activities observed in the absence of dopamine were multiplied by a constant such that their sum was 250 arbitrary units for each tubercle. The corresponding activities in the presence of dopamine were then multiplied by the same constant. These normalized values are called relative specific activities.

309

Biochemistry In Fig. 3 dopamine-sensitive adenylate cyclase activity in homogenates of consecutive sections is plotted as a function of the depth in microns within the tubercle. Adenylate cyclase activities are shown in the presence and in the absence of 100 #M dopamine. The figure shows the combined data from 5 tubercles in 5 independent experiments. In each experiment, each homogenate was assayed in triplicate, both in the absence and presence of dopamine, and the enzyme activity of each section expressed as relative specific activity (see legend). The data represent the mean i S.E.M. of the relative specific activities in these 5 experiments, calculated at each depth. Using regression analysis, straight lines can be calculated for the two conditions shown. For the condition with dopamine, the calculated line has a slope of--0.050 (units/min/section)/(Fm) with a standard deviation of 0.037. For the condition without dopamine, the slope of the line is --0.020 (units/min/section)/(#m) with a standard deviation of 0.012. Neither of these slopes is significantly different from zero. The specific activity of dopamine-sensitive adenylate cyclase in homogenates of these 100 #m sections was approximately 50 pmoles cyclic AMP/mg protein/min in the absence of dopamine and 100 pmoles/mg protein/min in the presence of 100/~M dopamine. In homogenates of the whole tubercle we observed an activity of 30 pmoles/mg protein/min in the absence and 100 pmole/mg protein/min in the presence of 100 #M dopamine. Fig. 4 illustrates the effect of the dopamine antagonist trifluoperazine and of the /%adrenergic antagonist propranolol on the activation of the enzyme by dopamine. 12 o o

~e

10

.=_ E _=

~Y %%%

o E

8

¢3 IJJ

6

nO IJ_ n :E

/'%, + DOPAMINE

DOPAMINE+

"~% "~_

TRIFLUOPERAZINE

4

¢J --

_J ¢.) >-

2

-DOPAMINE

I

I

I

I

I

I

I

I

I00

200

300

400

500

600

"tO0

800

TUBERCLE

DEPTH

(microns)

Fig. 4. Dopamine-sensitive adenylate cyclase activity as a function of depth in the olfactory tubercle with no dopamine (O O), 100 # M dopamine (O O), 100 # M dopamine plus 10 FM propranolol ([] . . . . []), and 100/~M dopamine plus 2/~M trifluoperazine ( A . . . . A). Data are from a single tubercle (see text), and represent the mean -k S.E.M. of triplicate determinations.

310 Dopamine stimulated the enzyme activity by 2- to 3-fold at every depth. Trifluoperazine (2 #M) reduced this stimulation by 70-90 ~o at various depths in the tubercle. This antipsychotic drug is known to inhibit the activation by dopamine of dopaminesensitive adenylate cyclase in homogenates of whole olfactory tubercle and caudate nucleus 5A7. Control experiments on sections of the tubercle showed that trifluoperazine did not affect adenylate cyclase activity in the absence of added dopamine. In contrast to the inhibitory action of trifluoperazine on dopamine-sensitive adenylate cyclase activity, the enzyme was unaffected by the presence of 10/~M propranolol. The failure of propranolol to block the stimulation by dopamine indicates that the dopamine is not acting through a fl-receptor. DISCUSSION Substantial levels of dopamine-sensitive adenylate cyclase were found at all depths of the tubercle. Thus, elements which might contain the dopamine-sensitive adenylate cyclase occur in all three laminae. In terms of neuronal localization we can deduce that the location of dopamine-sensitive adenylate cyclase is not limited to the neuronal cell bodies since the plexiform layer contains mainly processes, yet it shows as much or more activity than the other layers (see Figs. 3 and 4). The processes of the plexiform layer include the apical dendrites of the pyramidal cells 19, and extrinsic axons and terminals from the olfactory bulb, from the olfactory cortex 19, and from dopaminergic cells in the brain stem 22. There are also superficial islands of Calleja 1,3,4, and axons and dendrites ascending from cells in deeper layers. The hypothesis that pyramidal cells contain dopamine-sensitive adenylate cyclase is consistent with the observed distribution of activity. Their cell bodies are found in both the dense (pyramidal) layer and the deeper layer, and their dendrites are known from Golgi studies to be arborized both superficially and centrally 19. This distribution of dendrites could account for the substantial levels of dopamine-sensitive adenylate cyclase activity seen in all three of the laminae of the olfactory tubercle. However, we cannot rule out contributions from dendrites of other cell types, or even from the dopaminergic axon terminals. With regard to the islands of Calleja in the plexiform layer, several of our sections lacked these cells while still showing strong dopamine-sensitive adenylate cyclase activity. The possible contribution of glia at different depths in the tubercle is unknown. These results and interpretations are consistent with evidence concerning the localization of the dopaminergic input which originates in the ventral tegmentum. The data of Fuxe show a rich fluorescence for the dopamine axon terminals in the plexiform and pyramidal layers with decreases in the deeper layers 7,8. Further studies will be needed to quantitate the dopamine content in the different layers. Similarly, stains for degenerating axon terminals after lesions of the ventral tegmentum in the brainstem show the greatest localization of silver grains in the plexiform and pyramidal cell body layersl°,lL Significantly, the islands of Calleja tend not to fluoresce for dopamine 7 nor show the presence of degenerating terminals 1°. It may be noted that, in the caudate nucleus, there is evidence that the

311 dopaminergic pathway (from the substantia nigra) terminates on interneurons which in turn feed onto the o u t p u t cells (cf. refs. 13, 16). Our results suggest that the dopaminergic fibers to the tubercle (from the ventral tegmentum) may terminate directly on pyramidal cells. It is possible that these are the main output cells in the tubercle 2°,21, although they m a y also have short projections to other populations within this cortex. The present findings, considered together with previous anatomical studies in the olfactory tubercle 7,s,10,15, suggest that the dopamine pathway terminates not only in relation to cell bodies but also their dendrites. Further work on neuronal localization o f dopamine-sensitive adenylate cyclase should aid in understanding the role o f this enzyme system in dopaminergic transmission 9 as well as the role of dopaminergic systems in the clinical disorders in which they have been implicated. ACKNOWLEDGEMENTS This work was supported in part by fellowships f r o m the Scottish Rite F o u n d a t i o n for Research in Schizophrenia and the National Institute of Mental Health (MH-01241) ( N R K ) ; Neurology Training G r a n t (NS-05477) (JSK); Spinal Cord T r a u m a Research G r a n t (NS-10174); Research G r a n t NS-07609 ( G M S ) ; and Research Grants MH-17387 and NS-08440 (PG). We thank Miss K. Beardsley for technical assistance,

REFERENCES l Beccari, N, II lobo paraolfattorio nei mammiferi, Arch. Ital. Anat. Embriol., 9 (1910) 173-220. 2 Brown, B. L., Albano, J. D. M., Ekins, R. P. and Sgherzi, A. M., A simple and sensitive saturation assay method for the measurement of adenosine 3':5'-cyclic monophosphate, Biochem. J., 121 (1971) 561-562. 3 Cajal, S. R., Studies on the Cerebral Cortex, (Translated by Lisbeth M. Kraft), Lloyd-Luke, London, 1955, pp. 70-75. 4 Calleja, C., La Region Olfactoria Del Cerebro, Madrid, 1893. 5 Clement-Cormier, Y. C., Kebabian, J. W., Petzold, G. L. and Greengard, P., Dopamine-sensitive adenylate cylcase in mammalian brain: A possible site of action of antipsychotic drugs, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1113-1117. 6 Clement-Cormier, Y. C., Parrish, R. G., Petzold, G. L., Kebabian, J. W. and Greengard, P., Characterization of a dopamine-sensitive adenylate cyclase in the rat caudate nucleus, J. Neurochem., 25 (1975) 143-149. 7 Fuxe, K., The distribution of monoamine terminals in the central nervous system, Acta physiol. scand., 64 (1965) Suppl. 247, 37-85. 8 Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system, Z. Zellforsch., 65 (1965) 573-596. 9 Greengard, P., Possible role for cyclic nudeotides and phosphorylated membrane proteins in postsynaptic actions of neurotransmitters, Nature (Lond.), 260 (1976) 101-108. l0 Hedreen, J. C. and Chalmers, J. P., Neuronal degeneration in rat brain induced by 6-hydroxydopamine: A histological and biochemical study, Brain Research, 47 (1972) 1-36. 1 i Heimer, L. and Wilson, R., The subcortical projections of the allocortex: similarities in the neural associations of the hippocampus, the piriform cortex, and the neocortex. In M. Santini (Ed.), Golgi Centennial Symposium Proceedings, Raven Press, New York, 1975, pp. 177-193. 12 Kebabian, J. W., Petzold, G. L. and Greengard, P., Dopamine-sensitive adenylate cylcase in caudate nucleus of rat brain and its similarity to the 'dopamine receptor', Proc. nat. Acad. Sci. (Wash.), 69 (1972) 2145-2149.

312 13 Kemp, J. M. and Powell, T. P., The structure of the caudate nucleus of the cat, Phil. trans. B, 262 (1971) 383-401. 14 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 15 Maler, L., Fibiger, H. C. and McGeer, P. L., Demonstration of the nigrostriatal projection by silver staining after nigral injections of 6-hydroxy-dopamine, Exp. Neurol., 40 (1973) 505-515. 16 McGeer, P. L., Grenwall, D. S. and McGeer, E. G., Influence of non-cholinergic drugs on rat striatal acetylcholine levels, Brain Research, 80 (1974) 211-217. 17 Miller, R.J.,Horn, A.S.andlversen, L.L.,Theactionofneuroleptic drugs on dopamine-stimulated adenosine cyclic 3',5'-monophosphate production in rat neostriatum and limbic forebrain, Molec. PharmacoL, 10 (1974) 759-766. 18 Mishra, R. K., Gardner, E. L., Katzman, R. and Makman, M. H., Enhancement of dopaminestimulated adenylate cyclase activity in rat caudate after lesions in substantia nigra: Evidence for denervation supersensitivity, Proc. nat. Acad. Sci., (Wash.), 71 (1974) 3883-3887. 19 Price, J. L., An autoradiographic study of complementary laminar patterns of termination of afferent fibers of the olfactory cortex, J. comp. Neurol., 150 (1975) 87-108. 20 Scott, J. W. and Leonard, C., The olfactory connections of the lateral hypothalamus in the rat, mouse and hamster, J. comp. Neurol., 141 (1970) 331-344. 21 Scott, J. W. and Chafin, B. R., Origin of olfactory projections to lateral hypothalamus and nuclei gemini of the rat, Brain Research, 88 (1975) 64-68. 22 Ungerstedt, U., Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand. Suppl. 367 (1971) 1--48.

Dopamine-sensitive adenylate cyclase within laminae of the olfactory tubercle.

Brain Research, 131 (1977) 303-312 © Elsevier/North-HollandBiomedicalPress 303 DOPAMINE-SENSITIVE ADENYLATE CYCLASE WITHIN LAMINAE OF THE OLFACTORY...
4MB Sizes 0 Downloads 0 Views