Distribution of noradrenaline immunoreactivity in the forebrain and midbrain of the lizardGekko gecko

THE JOURNAL OF COMPARATIVE NEUROLOGY 285:453-466 (1989)

Distribution of Noradrenaline Immunoreactivity in the Forebrain

and Midbrain of the Lizard G e k b g e c b

WILHELMUS J.A.J. SMEETS AND HARRY W.M. STEINBUSCH Departments of Anatomy and Embryology(W.J.A.J.S.) and Pharmacology (H.W.M.S.),

Vrije Universiteit, Amsterdam, The Netherlands

ABSTRACT The distribution of noradrenaline (NA) immunoreactivity in the fore-

brain and midbrain of the lizard Gekko gecko was studied by means of recently developed antibodies against NA. Noradrenaline-containing cell bodies are found in the hypothalamic periventricular organ and ependymal wall of the infundibular recess of the diencephalon. They are also present in the locus coeruleus and the nucleus of the solitary tract of the brainstem. Noradrena- line-immunoreactive (NAi) fibers and varicosities are widely, but not uniformly, distributed throughout the forebrain and midbrain. In the telencephalon, dense plexuses of NAi fibers are found in the bed nucleus of the medial forebrain bundle, the vertical limb of the nucleus of the diagonal band of Broca, and the caudoventral part of the septa1 region. The diencephalon, the periventricular preoptic area, the supraoptic nucleus, and, in particular, the medial habenular nucleus are densely innervated by NAi fibers, whereas in the midbrain NAi plexuses are found in the ventral tegmental area, the substantia nigra and its dorsolateral extension (RA8), and in an area ventral to the nucleus interpeduncularis, pars ventralis. Moderately dense plexuses of NAi fibers are found in the small-celled medial cortex, the dorsal cortex, and the midbrain tectum. The remaining forebrain and midbrain areas are generally not or only sparsely innervated by NAi fibers.

The distribution of NAi cell bodies and fibers resembles the pattern revealed with antibodies against dopamine-P-hydroxylase (DBH). A remarkable exception is that the cells in the hypothalamic periventricular organ and ependymal wall of the infundibular recess are immunonegative for DBH. Pos- sible explanations for this discrepancy are discussed. The present study on the distribution of NA immunoreactivity in the brain of Gekko gecko combined with the results of a previous report on the distribution of dopamine in the same species (Smeets et al., '86b) offer the opportunity to differentiate between the two catecholamines in the brain of this vertebrate.

Key words: locus coeruleus, dopamine-8-hydroxylase, organon periventriculare hypothalami, cortex, substantia nigra, reptiles

Since the first demonstrations of noradrenaline in the peripheral (Von Euler, '51) and the central nervous system (Vogt, '54), a vast number of studies have been devoted to unraveling the noradrenergic system in the brains of vertebrates. With the original formaldehyde-induced fluorescence (FIF) technique (Falck, '62; Falck et al., '62) or its subsequent modifications (for review, see Hokfelt et al., '84), it is, however, difficult to discriminate between the various catecholamines. In mammalian species, the development of antibodies against the catecholamine synthesizing enzymes

has made it possible to determine the distribution of cell bodies and fibers of each catecholaminergic system (see, e.g., Armstrong et al., '82; Hokfelt et al., '84; Jaeger et al., '84; Miachon et al., '84; Ciriello et al., '86; Reiner and Vincent,

Accepted March 28,1989. Address reprint requests to Wilhelmus J.A.J. Smeets, Ph.D., Department

of Anatomy and Embryology, Vrije Universiteit, P.O. Box 7161, 1007 MC Amsterdam, The Netherlands.

0 1989 ALAN R. LISS, INC.

454

'86; Vincent, '88). By contrast, in reptiles only studies with the nondiscriminative tyrosine hydroxylase (TH) antibodies have been fruitful (Wolters et al., '84; Brauth, '88). Attempts to distinguish between specific catecholamines with antisera against dopamine-P-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) had no success (Wolters et al., '84). New impetus to the research of catecholaminergic systems in vertebrates was given by the development of antibodies against the transmitters themselves. At present, antisera raised to bovine serum albumin (BSA) conjugated noradrenaline (Verhofstad et al., '80, '82; Steinbusch et al., '81, '88) and antibodies to dopamine like- wise conjugated to BSA (Geffard et al., '84) enable us to demonstrate specifically and in great detail these two catecholaminergic systems.

As part of a research program to elucidate the organiza- tion of the reptilian forebrain, we currently attempt to chemically characterize the forebrain and midbrain of the lizard Gekko gecko. In previous studies, the distribution of dopamine and serotonin, visualized with specific antibodies, has been reported (Smeets et al., '86b; Smeets and Stein- busch, '88). The present study deals, for the first time in reptiles, with the noradrenergic contribution to the catecholaminergic innervation of the gekkonid forebrain and midbrain. The main goal, therefore, is to differentiate between the two catecholamines with respect to their innervation of forebrain and midbrain structures in Gekko.

In addition to material stained with antibodies to noradrenaline, series stained with antisera against dopamine- P-hydroxylase, the enzyme that converts dopamine to noradrenaline, are available. A remarkable difference between the two immunohistochemical procedures, as observed in the hypothalamic periventricular organ, demonstrates that both methods are invaluable for a good insight into the noradrenergic system.

W.J.A.J. SMEETS AND H.W.M. STEINBUSCH

Ace Alh Ame Am1 Apol Bmfb ca Cgld cg1v CgP Chab Cxd Cxla Cxlp Cxml Cxms Dlh D11 Dls Dm DVR flm GP Habl Habm IPV lfb lfbd lfbv Lte LtP MP M t

MATERIALS AND METHODS For the present study 8 adult Tokay gekkos, Gekko

gecko, of both sexes were anesthetized with Nembutal and perfused transcardially. The animals were processed immu- nohistochemically with antibodies against either noradrenaline (NA, n = 2) or dopamine-0-hydroxylase (DBH, n = 6).

Immunohistochemical procedure with antibodies to noradrenaline

After perfusion with 50 ml ice-cold (4"C), oxygen- enriched Ca2+-free Tyrode's solution (Lo& et al., '80) for 2 minutes, immediately followed by 200-300 ml of a mixture of ice-cold, 4% paraformaldehyde, 0.570 glutaraldehyde, and 0.2% picric acid in 0.1 M sodium phosphate buffer (pH 7.4), the brains were removed from the skull. They were postfixed for 2 hours in ice-cold 0.1 M phosphate buffer containing 4% paraformaldehyde and 1% Na2S205 (pH 7.4). Subsequently, the brains were kept for at least one night in 0.1 M phosphate buffer with 30% sucrose and 1% NazS2O5. To reduce double bonds, they were processed in Tris-Buff- ered-Saline (TBS) containing 1% Na2S20, and 10 mM NaBH, for 30 minutes at room temperature. The brains were then cut at 40 ym on a freezing microtome and the sections were collected in TBS containing 1% Na2S205. The sections were processed according to an immunocytochemical procedure consisting of the following incubations: goat antiserum to NA, diluted 1:60.000 in TBS containing 0.5 % Triton X-100 (TBS-T) for 48 hours; rinsed three times in TBS for 10 minutes, and subsequent incubation with don- key antigoat serum (Nordic), diluted 1 : l O O in TBS-T for 60 minutes. After rinsing three times for 15 minutes in TBS, the sections were incubated with goat peroxidase antiperoxidase serum (Nordic), diluted 1:600 for 2 hours. They were then rinsed again three times for 15 minutes in TBS.

nucleus accumbens area lateralis hypothalami nucleus externus amygdalae nucleus lateralis amygdalae area preoptica lateralis bed nucleus of the medial forebrain bundle commissura anterior corpus geniculatum laterale, pars dorsalis corpus geniculatum laterale, pars ventralis corpus geniculatum pretectale commisura habenulae cortex dorsalis cortex lateralis, pars anterior cortex lateralis, pars posterior cortex medialis, large-celled part cortex medialis, small-celled part nucleus dorsolateralis hypothalami nucleus dorsolateralis thalami, large-celled part nucleus dorsolateralis thalami, small-celled part nucleus dorsomedialis thalami dorsal ventricular ridge fasciculus longitudinalis medialis globus pallidus nucleus lateralis habenulae nucleus medialis habenulae nucleus interpeduncularis, pars ventralis lateral forebrain bundle lateral forebrain bundle, dorsal peduncle lateral forebrain bundle, ventral peduncle nucleus lentiformis thalami, pars extensa nucleus lentiformis thalami, pars plicata nucleus medialis posterior nucleus medialis thalami

Abbreviations

nlll NdB Nolfa Nsa Nsd Nsi Nsl Nsm Nsph oph Ph PPO Pth Rot Rub Sn s o Sped Str tect topt Torc tsh tub olf V Vltd Vltv Vmh Vmt VTA I11 IIId IIIV

nervus oculomotorius nucleus of the diagonal band of Broca nucleus olfactorius anterior nucleus septalis anterior nucleus septalis dorsalis nucleus septalis impar nucleus septalis lateralis nucleus septalis medialis nucleus sphericus organon periventriculare hypothalami nucleus periventricularis hypothalami periventricular preoptic area pallial thickening nucleus rotundus nucleus ruber substantia nigra nucleus supraopticus nucleus suprapeduncularis striatum tectum tractus opticus nucleus centralis of the torus semicircularis tractus septohypothalamicus tuberculum olfactorium ventricle nucleus ventrolateralis thalami, pars dorsalis nucleus ventrolateralis thalami, pars ventralis nucleus ventromedialis hypothalami nucleus ventromedialis thalami ventral tegmental area nucleus nervi oculomotorii nucleus nervi oculomotorii, pars dorsalis nucleus nervi oculomotorii, pars ventralis

NORADRENALINE IMMUNOREACTIVITY IN GEKKONID FOREBRAIN AND MIDBRAIN

Finally, after incubation in 0.05 % 3,3-diaminobenzidine (Sigma) with 0.01% HzOz in TBS for 10-15 minutes and rinsing, the sections were mounted on glass slides (mounting medium: 0.2-0.3 5% gelatin in Tris-buffer) and, after drying overnight, coverslipped. Some sections were counterstained with cresyl violet. Details about the production and characterization of the noradrenaline-antiserum have been described elsewhere (see Steinbusch and Tilders, '87; Stein- busch et al., '88).

Immunohistochemical procedure with antibodies to DBH

After perfusion with 150 ml, ice-cold (4"C), Ringer's solution followed by 4% paraformaldehyde, 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), the brains were removed from the skull. They were postfixed in ice-cold phosphate buffer containing 4% paraformaldehyde for 60 minutes and, after rinsing with phosphate buffer, kept overnight in the same solution with 30% sucrose. The next day the brains were cut at 40 pm on a freezing microtome and the sections were collected in phosphate buffer. After rinsing three times for 15 minutes in TBS, the sections were processed according to an immunocytochemical procedure that is identical to that used for NA-immunohistochemistry, with the following modifications: (1) the first antibody is a rabbit antiserum to DBH (kindly provided by Dr. M. Gold- stein, New York), diluted 1:300, with an incubation time of 16 hours, (2) the second antibody is swine antirabbit serum (Nordic), diluted 150, and (3) the third antibody is rabbit peroxidase antiperoxidase serum (Dakopatts), diluted 1:800.

Evaluation and presentation of the results The distribution of noradrenaline immunoreactivity was

charted in a representative series of transverse sections through the forebrain and midbrain of Gekko gecko. The levels of the sections as well as the nomenclature used are essentially the same as those described in a previous paper (Smeets et al., '86a). The mapping of the noradrenergic cell bodies and fibers is mainly based on sections that were stained with antibodies against noradrenaline, since staining of fibers is most distinct in material processed with the NA antiserum. The most obvious example is the noradrenergic, sympathetic innervation of the walls of cerebral blood vessels, particularly in the dorsal ventricular ridge (Fig. 1). In material stained with the DBH-antiserum such immunoreactive plexuses could not be observed. For clarity, the NAi plexuses in close apposition to blood vessels have been omit- ted in the chartings of Figure 2. Noradrenergic cell bodies, on the contrary, stain generally more darkly with the DBH antiserum.

RESULTS Noradrenaline immunoreactive cell bodies The most rostra1 group of NAi cell bodies in the forebrain

of the lizard Gekko gecko is found in the hypothalamic periventricular organ in the diencephalon. The cells are arranged in several layers and appear to be in direct contact with the cerebrospinal fluid (CSF) of the third ventricle (Figs. 2G, 3). Slightly more caudally, another small group of NA cells is observed in the ependymal layer of the infundibular recess (not illustrated). Also, these cells seem to contact directly the CSF. Surprisingly, the NAi cells in both the

455

Fig. 1. Photomicrograph from a transverse section through the forebrain of the lizard Gekko gecko showing the noradrenergic, sympathetic innervation of a blood vessel in the dorsal ventricular ridge as visualized with antibodies against noradrenaline. Bar = 0.1 mm.

periventricular organ and the ependymal layer of the infundibular recess do not stain with the DBH antiserum (Figs. 3, 4).

Although it is outside the scope of this study, it should be mentioned that the majority of the NAi cell bodies in the gekkonid brain are located in the presumed locus coeruleus at isthmic levels and in the nucleus of the solitary tract at the level of the obex (Figs. 5,6). The NAi cell bodies of the locus coeruleus occupy a rather large area and possess generally three of four processes that do not show a preferential orientation. The NAi cells at the level of the obex are, in general, bipolar and their processes are oriented in a mediolateral direction.

Noradrenaline immunoreactive fibers In the olfactory bulb, the noradrenergic fibers are pre-

dominantly confined to the internal granular and plexiform layers. Some NAi fibers are observed in the mitral cell layer, whereas other surround the glomeruli. Numerous NAi fibers with almost no varicosities are found in the olfactory peduncle.

In the telencephalon proper, NAi varicose fibers can be observed in cortical as well as in subcortical areas. On both sides of the cellular layer of the smaI1-celled medial cortex, a

456

plexus of NAi fibers is present; those in the superficial layer have the highest density (Figs. 2A-E, 7 ) . The cell bodies of the large-celled medial cortex are not in close contact with NA fibers. However, the most distal part of both their apical and basal dendrites may receive an input from NAi fibers that course dorsally as well as ventrally to the longitudinal association bundle encompassing the cellular layer (Fig. 2C- E). As in the small-celled medial cortex, the NAi fibers in the dorsal cortex form a plexus superficial and deep to the cellular layer with the highest density in the superficial layer (Figs. 2B-F, 8). Whereas the NAi fibers have a rather homogeneous distribution throughout the small-celled medial cortex, they are less uniformly distributed in the dorsal cortex both in mediolateral and rostrocaudal directions. NAi fibers terminate predominantly in its medial and inter- mediate portions, whereas from rostra1 to caudal the density of NAi fibers increases (Fig. 2B-F). The lateral cortex receives a relatively weak noradrenergic input. Throughout its rostrocaudal extent, NAi fibers are found in the molecular layer (Fig. 2B-E). In addition, noradrenergic fibers are observed in the cellular layer, particularly in the ventral part of the anterior lateral cortex.

In the subcortical telencephalic areas, dense NAi plexuses are found in the bed nucleus of the medial forebrain bundle, the vertical limb of the nucleus of the diagonal band of Broca, and the caudoventral part of the septal area (Figs. 2C-E, 9-11). A very weak noradrenergic input reaches the remaining parts of the septal area, the nucleus accumbens, the striatum, and the olfactory tubercle, whereas the external, lateral, and spherical nuclei of the amygdaloid complex receive a weak to moderate NA innervation (Fig. 2B-F). The dorsal ventricular ridge is almost devoid of NAi fibers, except for its dorsomedial portion where numerous varicose fibers are observed in the superficial cell plate (Fig. 2C-E, 12).

Diencephalic cell masses that show a dense noradrenergic innervation are the periventricular preoptic area, the supraoptic nucleus, and the medial habenular nucleus (Figs. 2E-F, 13). In addition, numerous NAi fibers are observed in an area ventromedial to the lateral forebrain bundle that partly matches the region occupied by the medial forebrain bundle (Fig. 2E,F). A moderate to dense plexus of NAi varicose fibers is present in the subependymal layer of both the thalamus and the hypothalamus, in the periventricular hypothalamic nucleus, and in the lateral hypothalamic area (Figs. 2F,G, 13). Other diencephalic areas receive only a sparse noradrenergic innervation.

In the midbrain, a very dense NAi plexus is observed in the ventromedial part of the ventral tegmental area (Figs. 2H, 14). Caudally, the plexus continues into an area ventral to the nucleus interpeduncularis, pars ventralis (Fig. 21). Another conspicuous NAi plexus is found in an area that matches the region containing the dopaminergic neurons of the substantia nigra (Figs. 21,15). A moderate to dense NAi plexus is further observed in the retrorubral (RA8) dopaminergic cell group at caudal midbrain levels. The remaining parts of the midbrain tegmentum contain only a few dif- fusely arranged NAi fibers, except for the periventricular layers including the torus semicircularis, where numerous NAi fibers run parallel to the ventricular surface. The tec-

W.J.A.J. SMEETS AND H.W.M. STEINBUSCH

Fig. 2. Drawings of a series of transverse sections through the forebrain and midbrain of the lizard Gekko gecko showing the distribution of NA immunoreactive cells (large dots) and fibers (interrupted lines).

NORADRENALINE IMMUNOREACTIVITY IN GEKKONID FOREBRAIN AND MIDBRAIN 457

Figure 2 continued

458 W.J.A.J. SMEETS AND H.W.M. STEINBUSCH

Imm

Figure 2 continued


Fig. 3. Photomicrograph from a transverse section through the diencephalon of the lizard Gekkogecko showing the CSF-contacting NAi cell bodies in the periventricular organ as visualized with antibodies against NA. Bar = 0.1 mm.

Fig. 4. Photomicrograph from a transverse section at a level comparable to that shown in Figure 3, but stained with antibodies against dopamine-0-hydroxylase. Note the absence of immunoreactive cells in the periventricular organ (between arrows). Bar = 0.1 mm.

tum shows a moderate to dense plexus of NAi fibers in all tectal layers with the exception of layer 6 (nomenclature of Senn, '79), which contains only a few NAi fibers (Figs. 2H,I, 16). The NAi fibers in the periventricular layers are continuous with those in the periventricular layers of the tegmentum.

DISCUSSION Since the present study has revealed some unexpected

discrepancies between the results obtained with antibodies against noradrenaline and against its synthetic enzyme dopamine-P-hydroxylase, we first comment on these techniques. Next we try to differentiate between the noradrenergic and dopaminergic innervation of neuronal structures in the gekkonid forebrain and midbrain. Finally, by com- bining data of hodological studies with those obtained by the immunohistochemical studies, the presumed sites of origin of the projections of the two catecholaminergic systems to forebrain structures are determined.

Technical considerations The observed distribution of NAi cell bodies and varicose

fibers throughout the forebrain and midbrain of Gekho

gecko, as visualized with specific antibodies against NA, was identical in both specimens (1 male, 1 female) siudied. The specificity of the antibodies has been demonstrated previ- ously by Steinbusch et al. ('87, '88). Moreover, additional brain material of Gekko stained with antibodies against DA, TH, DBH, and PNMT supports the assumption that the NA antiserum effectively detects NA neuronal structures. For example, PNMT immunoreactive cell bodies at caudal brainstem levels do not stain with antibodies to NA. On the contrary, cell bodies in the locus coeruleus stain with antibodies against NA and DBH, but not with antiserum against PNMT. A detailed analysis of the catecholaminergic systems in reptiles, as revealed with antibodies against the transmitters themselves as well as against the various enzymes involved in the catecholamine synthesis, will be pub- lished separately (Smeets and Steinbusch, '89).

When we confine ourselves to the data obtained with antisera against NA and DBH, several conclusions can be drawn. First, there is a remarkable similarity between the results obtained with the two antisera when it is taken into account that neuronal structures that stain with PNMT have to be subtracted from those stained with anti-DBH. Second, in previous studies it was found that the hypothalamic periventricular organ of Gekko contains both dopa-


Figures 5 and 6

NORADRENALINE IMMUNOREACTIVITY IN GEKKONID FOREBRAIN AND MIDBRAIN

Figs. 7, 8. Photomicrographs from transverse sections through the telencephalon of the lizard Gekko gecko showing the distribution of noradrenergic fibers in the small-celled medial cortex (Fig. 7) and the dorsal cortex (Fig. 8) as visualized with antibodies against NA. Bar = 0.1 mm.

minergic and serotoninergic neurons (Smeets et al., '86b; Smeets and Steinbusch, '88). The present study reveals that this organ may also contain noradrenergic CSF-contacting cells. This appears to be the first time that such a rostra1 location of NAi cell bodies in the brain of vertebrates has been observed. Possible explanations for this are that: (1) with the FIF technique only a discrimination between green (catecholaminergic) and yellow (serotoninergic) fluorescent cell bodies can be made (for review, see Parent et al., '84), and (2) that antibodies against T H fail to demonstrate catecholaminergic neurons in the periventricular organ of reptiles and birds (Reiner et al., '83; Wolters et al., '84; Kiss and PBczely, '87; Brauth, '88; Smeets, '88), whereas conflicting results have been reported for teleosts (cf. Hornby et al., '87;

Fig. 5. Photomicrograph from a transverse section through the isth- ma1 region of the lizard Gekko gecko showing the DBH immunoreactive cells in the presumed locus coeruleus complex. Arrow points to the meningeal surface. Bar = 0.1 mm.

Fig. 6. Photomicrograph from a transverse section immediately caudal to the obex in Gekko gecko showing noradrenergic cell bodies in the nucleus of the solitary tract as visualized with antibodies against NA. Note the mediolateral orientation of the cell processes. Bar = 0.1 mm.

46 1

Meek et al., '89). In the present study it was found that antibodies against DBH also fail to stain cells in the gekkonid periventricular organ.

Thus it emerges from the above results that the cell bodies in the organ contain either dopamine or noradrenaline, but lack the enzymes involved in the biosynthesis of these catecholamines. This implies that the DA- and NA immunoreactivity in these cells is the result of a local uptake mechanism. The second possibility is that the cells in the periventricular organ contain the biosynthetic enzymes in concentrations below the detection level of the antibodies. This can be investigated by administration of the precursors tyrosine or L-dopa to the specimen. If the enzymes are present, they will be activated and produce a large increase in the concentrations of both the enzyme and the neurotransmitter. Still another possibility, i.e., influence of seasonal factors and temperature (Marschall, 'SO), can be ruled out, since in one and the same specimen of Gekko cells in the periventricular organ stain with antibodies to DA, but not with antibodies to T H (Smeets, '88). When sections stained with antisera against NA or DBH are compared, it is obvious that our results support the notion by Hokfelt et al. ('84) that, in general, cell bodies are better visualized with enzyme immunocytochemistry, whereas axons and terminals are best demonstrated with antibodies against the neurotransmitters themselves.


Figures 9-12


Comparison of the noradrenergic and dopaminergic system

The present study on the distribution of NAi immunoreactivity in the brain of Gekko gecko combined with the results of a previous account on the distribution of DA immunoreactivity (Smeets et al., '86b) offers, for the first time, the opportunity to differentiate between both catecholamines in a vertebrate brain. In the following section, we compare the distribution of DAi- and NAi-fibers in the gekkonid forebrain and midbrain in order to determine whether brain regions are innervated by both types of fibers or preferentially by only one of them.

Telencephalon In the small-celled medial cortex, both DA- and NA-

immunoreactive fibers may contact the proximal parts of the apical as well as the basal dendrites. However, the distal part of the apical dendrites appears to receive almost exclu- sively a NA input. The NA innervation of the large-celled part of the medial cortex matches that of DAi fibers, which suggests that only the most distal parts of the apical dendrites are contacted by DAi and NAi fibers. Although a considerable overlap of DAi- and NAi-fibers occurs in the dorsal cortex, there is some indication that DAi fibers reach primarily the lateral portion of this cortical region, whereas NAi fibers terminate predominantly in its medial and inter- mediate portions. Throughout its rostrocaudal extent, the molecular layer of the lateral cortex receives a noradrenergic input. On the contrary, a dopaminergic innervation prevails only in the cellular layer of the ventral portion of the anterior lateral cortex.

With respect to subcortical telencephalic areas, pericellu- lar baskets in the septal region are observed only in material stained with the antiserum against DA, suggesting that these presumed axosomatic contacts are dopaminergic. In contrast, there is considerable overlap between NAi- and DAi fibers in the caudoventral part of the septum at the level of the anterior commissure. Both catecholamines are also observed throughout the nucleus of the diagonal band of Broca. However, at caudal levels the noradrenergic innervation seems to prevail in the vertical limb of this band. The dopaminergic input to the nucleus accumbens and striatum exceeds by far that of NA. This finding is not in agreement with the conclusion reached by Baumgarten and Braak ('68) that the fluorescence in the striatum of the lizards Lacerta muralis and Lacerta uiridis, as observed with the FIF technique, is mainly due to the presence of noradrenaline. How- ever, our finding in Gekko is in accordance with the results of biochemical studies in turtles (Juorio, '69; Parent, '79) where dopamine is reported to occur in the striatum at more than twice the concentration of noradrenaline. A similar predominance of DA over NA occurs in the dorsal ventricular ridge (DVR). Whereas both the core and the superficial cell plate of the DVR contain a moderate to dense DAi plexus, only the dorsomedial portion of the superficial cell plate receives a rather dense noradrenergic input. There is

Figs. 9-12. Photomicrographs from transverse sections through the forebrain of the lizard Gekko gecko showing noradrenergic fibers and varicosities in the bed nucleus of the medial forebrain bundle (Fig. 9), the vertical limb of the nucleus of the diagonal band of Broca (Fig. lo), the caudoventral septal region (Fig. ll), and the dorsomedial portion of the dorsal ventricular ridge (Fig. 12) as demonstrated with antibodies against NA. Bar = 0.1 mm.

also an overlap of DAi- with NAi-varicose fibers and terminals in the lateral and external amygdaloid nuclei and in the dorsal half of the nucleus sphericus, although, in general, the dopaminergic input appears to be stronger.

Diencephalon and midbrain In the diencephalon, the subependymal layer, the peri-

ventricular preoptic area, the periventricular hypothalamic nucleus, the lateral hypothalamic area, and the dorsomedial thalamic nucleus contain both DAi- and NAi varicose fibers and terminals. Differentially innervated neuronal structures are the supraoptic nucleus, the medial habenular nucleus, the lateral geniculate body, and the pretectum. The first two structures display a predominantly NAi plexus, whereas the latter two are primarily innervated by DAi fibers.

Comparing the innervation of the gekkonid tectum by the two catecholamines, several conclusions can be drawn: (1) the midbrain tectum receives a strong input of both dopaminergic and noradrenergic fibers, (2) compared to NAi fibers, DAi varicose fibers show a remarkably laminar orga- nization, (3) an equally dense innervation of DAi and NAi fibers is observed in tectal layers 2-5 (nomenclature by Senn, '79), and (4) the tectal layers 9 and 11 receive a predominantly dopaminergic input, whereas the layers 7 and 8 and the most superficial layers (12-14) are primarily innervated by NAi fibers. The DAi-and NAi fibers in the periventricular tectal layers (2-5) are continuous with those in the periventricular tegmental layers. A further notable result of the present study is the demonstration of a strong noradrenergic projection to the substantia nigra and the ventral tegmental area.

Sites of origin of DA and NA fibers Information on the sites of origin of the DA- and NA

innervation of neuronal structures in the forebrain and midbrain of Gekko can be obtained by comparing the location of DAi and NAi cells with data on connectivity. I t is plausible that the dopaminergic input to the dorsal cortex originates from DAi cells located in the periventricular hypothalamic nucleus, in the lateral hypothalamic area, and in the ventral tegmental area (Bruce and Butler, '84; ten Donkelaar and de Boer-van Huizen, '881, whereas the noradrenergic input most probably comes from the locus coeruleus and, perhaps, the nucleus of the solitary tract (see ten Donkelaar and de Boer-van Huizen, '88, their Fig. 5). The lateral cortex may receive its dopaminergic input from DAi cells in the most caudal part of the periventricular hypothalamic nucleus and in the ventral tegmental area (Bruce and Butler, '84). The sites of origin of its noradrenergic input are still unknown. The only possible source of dopaminergic and noradrenergic projection fibers to the small-celled medial cortex seems to be the periventricular hypothalamic organ (Bruce and Butler, '84).

Regarding the subcortical telencephalic areas, hodological evidence has been presented that the DVR receives its dopaminergic input from cells in the ventral tegmental area and the substantia nigra, whereas its noradrenergic innervation originates from the locus coeruleus (ten Donkelaar and de Boer-van Huizen, '88). However, it can not presently be excluded that DAi or NAi cells in the nucleus of the solitary tract also project to the DVR (cf. Fig. 1 of ten Donkelaar and de Boer-van Huizen, '88). The dopaminergic innervation of the striatum and the nucleus accumbens has its origin in the substantia nigra and the ventral tegmentaI area (Parent,


Figures 13-16

465 NORADRENALINE IMMUNOREACTIVITY IN GEKKONID FOREBRAIN AND MIDBRAIN

'79). Little is known about the cells of origin of the catecholaminergic innervation of diencephalic structures, although the study by Hoogland ('82) suggests that the dopaminergic input to the dorsomedial thalamic nucleus originates from the periventricular hypothalamic nucleus, whereas the locus coeruleus provides the noradrenergic innervation of this cell mass. Finally, the dopaminergic and noradrenergic inputs to the midbrain tectum probably arise from the substantia nigra and the locus coeruleus, respectively, although the posterodorsal nucleus may also contribute (ten Donkelaar et al., '87). I t is evident that other techniques such as combined retrograde labeling and immunofluorescence or neurotoxic specific depletion studies are needed to confirm these ideas.

Concluding remarks This study reveals the location of noradrenergic cell bod-

ies and their terminal fields in the forebrain and the midbrain of the lizard Gekko gecko with specific antibodies against noradrenaline. The results obtained with these antibodies are largely comparable to those with antibodies against DBH after subtracting structures labeled with antibodies against PNMT. It has also become clear that, at least in Gekko, noradrenergic fibers are, in general, not stained with antibodies against TH. This is particularly well illustrated by comparing the staining patterns for the three different antibodies (DA, NA, TH) in the small-celled medial cortex and the midbrain tectum (Smeets et al., '86b; Smeets, '88; present study). However, both antibodies against the catecholamines themselves and antibodies against their biosynthetic enzymes are needed to get a full understanding of the catecholaminergic systems. For example, antibodies to DA and NA have made possible a very detailed mapping of cell bodies and fibers. On the contrary, antibodies to T H and DBH have demonstrated that in the forebrain of Gekko cell bodies stain for TH, but not for DA, NA, or DBH. This leaves the possibility open that DOPA by itself is a putative neurotransmitter (Smeets, '88; Smeets and Steinbusch, '89).

Finally, the distribution of NA immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko resembles in many respects that reported for mammals (for review see, e.g., Moore and Card, '84). However, if our results obtained with these antibodies in Gekko are considered decisive, it is to be expected that their application to the brains of mammalian species will considerably enlarge our knowledge of their noradrenergic systems.

ACKNOWLEDGMENTS The authors are much indebted to Dr. A.H.M. Lohman

for critically reading the manuscript, to Mr. J.G.M. Bol, Mr. P.H. Goede, and Mr. A.J. Jonker for technical assistance, to Mr. D. de Jong for preparing the photomicrographs, and to Mrs. N. Nauta-Kaat and Mrs. R. Stoevelaar-de Zeeuw for typing the manuscript.

Figs. 13-16. Photomicrographs from transverse sections through the diencephalon and midbrain of the lizard Gekko gecko showing noradrenergic fibers and varicosities in the medial habenular nucleus (Fig. 13), the ventral tegmental area (Fig. 14), the substantia nigra (Fig. 15), and the various layers of the midbrain tectum (Fig. 16) as demonstrated with antibodies against NA. Bar = 0.1 mm.

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Distribution of noradrenaline immunoreactivity in the forebrain and midbrain of the lizardGekko gecko