Development of catecholamine systems in the brain of the lizardGallotia galloti

THE JOURNAL OF COMPARATIVE NEUROLOGY 350~41-62 (1994)

Development of Catecholamine Systems in the Brain of the Lizard GaZZotia galloti

LORETA MEDINA, LUIS PUELLES, AND WILHELMUS J.A.J. SMEETS Department of Microbiology and Cell Biology, University of La Laguna, Tenerife, Spain

and Department of Anatomy and Neurobiology, University of Tennessee, Memphis, Tennessee (L.M.); Department of Morphological Sciences, University of Murcia, Murcia, Spain (L.P.); and Graduate School Neurosciences Amsterdam, Research Institute

Neurosciences Vrije Universiteit, Department of Anatomy and Embryology, Amsterdam, The Netherlands (W.J.A.J.S.).

ABSTRACT For a better insight into general and derived traits of developmental aspects of catecholamin-

ergic (CA) systems in amniotes, we have studied the development of these systems in the brain of a lizard, Gullotia gulloti, with tyrosine hydroxylase (TH)- and dopamine (DA) immunohisto- chemical techniques. Two main groups of TH-immunoreactive (THi) perikarya appear very early in development: one group in the midbrain which gives rise to the future ventral tegmental area, substantia nigra and retrorubral cell groups, and another group in the tuberomammillary hypothalamus. Somewhat later in development, THiDA-immunoreactive cells are observed in the thalamus, rostrodorsal hypothalamus and spinal cord, and, with another delay, in the suprachiasmatic nucleus, the periventricular organ, and the pretectal posterodorsal nucleus. CA cell groups that appear rather late in development include the cells in the olfactory bulb, the locus coeruleus and the caudal brainstem. As expected, the development of immunoreactive fibers stays behind that of the cell bodies, but reaches the adult-like pattern just prior to hatching.

The present study revealed considerable variation in the relation between the state of cytodifferentiation and first expression of THiDA immunoreactivity between CA cell groups. Catecholamine cells in the midbrain and tuberomammillary hypothalamus are still migrating, immature (absence of dendrites) and express only TH immunoreactivity at the time of first detection. Cells which appear at later developmental stages lie already further away from the ventricle, possess two or more dendritic processes, and generally express both TH- and DA immunoreactivity. o 1994 Wiley-Liss, Inc.

Key words: tyrosine hydroxylase, dopamine, embryonic brain, reptiles, comparative neuroanatomy

The adult brains of most vertebrates have several fea- tures in common with respect to the distribution of catechol- amine (CAI containing cell bodies. For example, in all vertebrates, CA cell bodies are observed in the olfactory bulbs, hypothalamus, midbrain tegmentum, isthmic region and caudal brainstem (for review, see Smeets and Reiner, 1994). Developmental studies of avian and mammalian brains have shown that CA cell bodies in many of these locations are already present at early embryonic stages (Tennyson et al., 1973; Lauder and Bloom, 1974; Specht et al., 1981a; Yurkewicz et al., 1981; Guglielmone and Pan- zica, 1985; Verney et al., 1991; Puelles and Medina, 1994). Moreover, evidence has been obtained that the cell groups do not arise at the same time: CA cell bodies appear first in the hypothalamus and the isthmic region and, subse- quently, in the midbrain tegmentum and other brain regions. A similar sequence of appearance is observed in the

developing brain of anamniotes (Ekstrom et al., 1992,1994; Manso et al., 1993; Gonzdez and Smeets, 1994). However, in teleost fish CA cells were never observed in the midbrain tegmentum of either the developing or adult brain (Ek- strom et al., 1992, 1994; Manso et al., 19931, whereas in anuran amphibians a distinct dopaminergic (DA) midbrain cell group could be distinguished, but relatively late in development (Gonzalez and Smeets, 1994). In the latter class of vertebrates, the midbrain cell group seems to separate from the hypothalamic CA cell group, which is in sharp contrast with birds and mammals, where the ventral tegmental areaisubstantia nigra DA cell group extends

Accepted June 9,1994 Address reprint requests to LoretaMedina, Ph.D., Department of Anatomy

and Neurobiology, University of Tennessee, Memphis, 875 Monroe Avenue, Memphis, TN 38163.

o 1994 WILEY-LISS, INC.

42 L. MEDINA ET AL.

from the isthmic region to the diencephalon (Puelles and Medina, 1994).

For a better understanding of the evolution and the ontogeny of the CA systems of vertebrates, it seems to be opportune to study these systems in reptiles, a class of vertebrates that occupies a crucial position in phylogeny. In the present study, therefore, we have analyzed the develop- ment of the CA systems of a lizard, Gallotia galloti, by means of antibodies against the enzyme tyrosine hydroxy- lase (TH) and dopamine. The use of both antibodies enables us to differentiate between the dopamine system and the noradrenalineladrenaline systems. Gallotia galloti is an endemic lacertid lizard from the Canary Islands (Spain), that has been selected as an experimental model because of the similarity of its CA systems to those of other reptilian species (Smeets, 1994), and also because many data are available on its development (e.g., Trujillo, 1982; Yanes et al., 1987, 1989; Medina, 1991) including a timetable of development on basis of external morphology (Ramos Stef- fens, 1980).

MATERIALS AND METHODS Twenty-four embryos of the lizard, Gallotia galloti (Rep-

tilia, Lacertidae), ranging from developmental stage 32 632) to hatching, were used for the present study (Table 1). Embryos were removed from the egg, immersed in Ringer’s solution, and carefully staged according to the timetables of development by Ramos Steffens (1980) and Dufaure and Hubert (1961). The embryonic brains were fixed by immersion in 0.1 M phosphate buffer (pH 7.4) containing 5% glutaraldehyde and 1% NazSz06. After 15 to 30 minutes (depending on the size of the brain), the brains were immersed in phosphate-buffered 30% sucrose contain- ing 1% NazS2O5. Subsequently, the brains were cut on a cryostat at 50 pm in a sagittal, corrected horizontal (paral- lel to the optic tract), or transverse plane. Sections were serially collected in 0.05 M Tris-buffered saline (TBS, pH 7.6) containing 1% Na2SzO5. One series of sections was processed for tyrosine hydroxylase (TH) immunohistochem- istry, whereas the other series was used for dopamine (DA) immunohistochemistry .

In addition, seven adult specimens of G. galloti were deeply anesthetized with ethyl ether and perfused transcar- dially with saline solution followed by a phosphate-buffered solution of either 4% paraformaldehyde (n = 4) or 5% glutaraldehyde and 1% NazSz05 (n = 3) . The brains were cryoprotected and sectioned on a cryostat (sagittal, cor- rected horizontal and transverse sections, 40-pm-thick). Sections were then processed for either TH or DA immuno-

TABLE 1. Number and Developmental Stages of Embryos of Gallotia galloti Used for the Present Study

Developmentalstage (S) 32 33 34 35 36 37 38 39 40 hatching Number of animals 1 2 1 4 3 3 2 2 2 4

histochemistry following the procedures as previously de- scribed for other reptiles (Smeets and Steinbusch, 1990). In brief, for DA immunohistochemistry, sections were incu- bated in: (1) rabbit anti-dopamine antiserum (generously provided by Dr. R.M. Buijs, Netherlands Institute for Brain Research, Amsterdam), diluted 1:3,000 for 16 hours; (2) swine anti-rabbit antiserum (Nordic), diluted 1 5 0 for 1 hour; and ( 3 ) rabbit peroxidase anti-peroxidase complex (Dakopatts), diluted 1:BOO for 1 hour. For TH immunohis- tochemistry, sections were incubated in: (1) mouse anti- tyrosine hydroxylase antiserum (Incstar), diluted 1:2,000 for 16 hours; (2) goat anti-mouse antiserum (Nordic), diluted 1 : l O O for 1 hour; and (3) mouse peroxidase anti- peroxidase complex (Dakopatts), diluted 1500 for 1 hour.

Control sections were processed omitting the primary antisera. No specific labeling of somata or fibers was found in these sections. Additional series of embryonic brains stained with cresyl violet or histochemically for acetylcholin- esterase were available, facilitating the interpretation of the location of immunoreactive elements.

RESULTS The distribution of TH-immunoreactive (THi) and dopa-

mine-immunoreactive (DAi) cell bodies and fibers in the adult brain of the lizard Gallotia galloti is largely the same as that reported for another lizard species, i.e., Gekko gecko (Smeets et al., 1986; Smeets, 1994). Only minor differences exist between the two species in the dopaminergic innerva- tion of the superficial tectal layers and in the retinorecipi- ent thalamic nuclei (Medina and Smeets, 1992).

Embryonic development of the brain As is true for most reptiles, lacertid lizards are oviparous.

Their embryonic development lasts for approximately 45 days at an average temperature of 25°C. On the basis of morphological criteria, the embryonic period of the lizard Lacerta muralis has been subdivided into 40 developmental stages (Dufaure and Hubert, 1961). For another lizard, i.e., Gallotia galloti, Ramos Steffens (1980) has described the developmental stages that correspond to stages 22 to 40 of L. muralis. A summary of the development of G. galloti is shown in Table 2. In brief, when the female lays eggs, embryos are at developmental stages that range from 24 to

BG

Cxl d DVR

cc

eP1 gl Gld Ht Hr i Lc m ob

basal ganglia central canal lateral cortex diencephalon dorsal ventricular ridge external plexiform layer glomerular layer dorsal geniculate nucleus tuberomammillary hypothalamus rostrodorsal periventricular hypothalamus isthmus locus coeruleus mesencephalon olfactory bulb

Abbreviations

oc OPh Pd POA r SCN SN Sol SP Sph t TeO VTA

optic chiasm periventricular hypothalamic organ posterodorsal nucleus preoptic area rhombencephalon suprachiasmatic nucleus substantia nigra nucleus of the solitary tract spinal cord nucleus sphericus telencephalon optic tectum ventral tegmental area

DEVELOPMENT OF CATECHOLAMINE SYSTEMS IN A REPTILE

28. At that moment, the embryo (mainly the head and upper part of the body) is bent over itself, and the trunk is open at its ventral side (Ramos Steffens, 1980). Rudiments of the heart and the intestine are present in the embryo, and limb buds start to develop at stage 28. The number of somites varies from 17 pairs (stage 24) to 36 pairs (stage 28), and one to four branchial arches are recognized. In the head, the neuropore is closed, while eye and ear vesicles are present. The neural tube is still at an early proliferation phase, with most cells accumulated in a very thick ventricu- lar zone. Major subdivisions of prosencephalon, mesencepha- lon and rhombencephalon are already recognized, with the mesencephalon lying at the level of the cephalic flexure. The amniotic membrane totally covers the embryo and a periem- bryonic vascular system is present.

The first developmental stage at which lightly labeled THi cell bodies are found is stage 32 (S32). For clarity, a description of the major events in the development of the body and head of G. galloti for each developmental stage preceeds the description of the distribution of THi and DAi cells and fibers at the corresponding stage. The results are summarized in Figures 1 and 2, in which the THi and DAi perikarya and fibers are depicted in schematics of sagittal sections of the brain at each developmental stage. In addition, on the right side of each sagittal section, a drawing of G. galloti of a corresponding stage (after Ramos Steffens, 1980) is shown.

Developmental stages 32 and 33 (S32, S33). At S32, the trunk of the embryo only remains open through a narrow umbilicus, which connects the embryo to the yolk sac and embryonic membranes (Fig. 1A). Rudiments of the liver and of the excretory and reproductive systems are already present. Four limbs are visible and each of them presents a flattening at the end which will become feet at later stages. The branchial arches are in regression and early jaw prominences are formed around the stomodeum. The olfac- tory vesicle is formed, and pigmentation of the eyes has started. In the brain, the prosencephalon is clearly divided into telencephalon and diencephalon. The majority of cells are located in the ventricular zone, but a number of neuroblasts and immature neurons are already observed in the mantle layer. In the mantle layer of some regions, clusters of cells are present which represent the primordia of nuclei such as the pretectal geniculate nucleus (Medina, 1991) and the ventral geniculate nucleus (Trujillo, 1982). In the mantle layer of the basal part of the telencephalon, cell bodies begin to form the basal ganglia, but it is not possible to distinguish between striatum and pallidum at this early stage manes et al., 1989). The boundary between the early basal ganglia and the dorsal ventricular ridge (DVR) is also unclear at S32 (Yanes et al., 1989).

At this stage, a few weakly THi cell bodies are found in the midbrain tegmentum dorsal to the cephalic flexure and close to the midline (Fig. 1A). One stage later, at S33, both the number of THi cell bodies in the midbrain and the intensity of their immunoreactivity have increased substan- tially (Figs. lA, 3A,B). The cell bodies display still some immature features, such as the absence of dendrites and the presence of a long, thick process which is directed either rostrally, laterally, or dorsally (Fig. 3A). A few THi neuro- blasts possess a long process that is directed caudally. A small THi fiber bundle arises from the midbrain cell group and reaches, following the longitudinal axis of the brain, as far rostrally as the basal region of the caudal diencephalon (Fig. 3A).

TABLE 2. Summary of the Embryonic Development of the Lizard Gallotia~uZloti (see text for more details)

43

~~ ~~

524-528 Rudiments of heart and intestine are present. 17-36 somite pairs. 14 branchial arches. Limb buds start to develop. Eye and ear vesicles are present. Neuropore is closed. Neural tube is divided in prosencephalon, mesencephalon and rhombencephalon. Most cells of neural tube are accumulated in avery thick ventricular zone.

Rudiments of liver and excretory system are present. Four limbs are visible. BranchiaJ arches are in regression. Early jaw prominences are observed. Olfactory vesicle is formed. Pigmentation starts in eyes. The brain is divided into telencephalon, diencephalon, mesencephalon and

Most cells of the brain are located close to the ventricle; some cells are in the

S32-S33

rhombencephalon.

mantle and start to form the primordia of the basal ganglia, the thalamic ven- tral geniculate nucleus or the pretectal geniculate nucleus.

s34-535 Toes differentiate in the limbs. All branchial arches have disappeared. Jaw prominences have merged. Vascularization starts in the brain. The mantle of the brain becomes thicker and contains many neuroblasts and

immature neurons; primordia of many nuclei are observed, such as the oculo- motor nuclei, the ventral tegmental area, and the suhstantia nigra in the mes- encephalon; the ventrolateral and ventral geniculate nuclei of the ventral thala- mus; the periventricnlar and ventromedial hypothalamic nuclei; the striatum and the dorsal ventricular ridge in the telencephalon.

Synaptogenesis starts in the ventral thalamus.

Reproductive system shows sexual dimorphism. Claws start to develop in the toes. The abdominal skin contains scales. Most grisea of the brain are outlined, including the locus coeruleus, the magno-

cellular and parvocellular isthmic nuclei, the nucleus rotundus of the dorsal thalamus, the periventricular organ in the hypothalamus, and the nucleus accumbens in the telencephalon.

S36-S37

S38 Pigmentation starts to differentiate in the skin. In the brain, most cell groups are formed, and many neurons are undergoing

cytodifferentiation (dendritic and axonal branching and synaptogenesis). Synaptogenesis starts in the striatum.

Gonads become internalized in females and the embryo acquires an adult-like

Brain neurons undergo final steps of cytodifferentiation (formation of dendritic

S39-S40

aspect.

arbor, acquisition of spines, completion of synatogenesis).

At late S33, the THi perikarya of the midbrain tegmen- tum are even more numerous and strongly immunoreactive than observed at early S33 (Fig. 3B). Some THi cell bodies lie more laterally in the tegmentum. The ascending THi fibers can now be traced further rostrally as far as the hypothalamus. At late S33, a few weakly THi perikarya are observed at tuberomammillary hypothalamic levels, where they are located close to the midline. These cells are either round without any process or monopolar with a single, very thick process.

Developmental stage 34 (5'34). At S34, toes start to differentiate in the limbs of the embryo and all branchial arches have disappeared. In the head, jaw prominences have merged, and a rudimentary palate appears. The tongue is already observed in the mouth. In the brain, more neuroblasts and immature neurons are located in the mantle, which has become thicker than in previous stages, particularly in the rhombencephalic and mesencephalic tegmentum, the pretectum, the ventral thalamus, and the tuberomammillary hypothalamus.

At this developmental stage, the THi cell group in the midbrain tegmentum largely resembles the corresponding group as observed at late S33. In a sagittal section, most THi perikarya are located in the caudal part of the mesence-

44

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L. MEDINA ET AL.

m

DEVELOPMENT OF CATECHOLAMINE SYSTEMS IN A REPTILE 45

phalic tegmentum, where a sharp boundary is found be- tween the mesencephalic and isthmic tegmental regions (Figs. lB, 4). Only a few THi perikarya are found caudal to this limit or crossing it. Other THi cell bodies lie rostral to the main mesencephalic group, between and parallel to the ascending THi fibers which, at this stage, reach the caudal telencephalon (Fig. 1B).

At S34, the THi cell group in the tuberomammillary hypothalamus has increased considerably both in the num- ber of cells and their immunoreactivity (Fig. 4). Some of the perikarya show the beginning of dendritic arborization, which gives them a more mature appearance. Others are still monopolar neuroblasts. A number of THi perikarya are located close to the infundibular region (Fig. 4).

Developmental stage 35 (5'35). At S35, individual toes connected by a membrane are distinguished in the limbs of the embryo. The reproductive system is more developed, but still not sexually differentiated. In the head, a rudimen- tary eardrum is present. Vascularization starts in the brain at S35 (Trujillo, 1982; Yanes et al., 1989). The mantle of the brain is considerably thicker than in previous stages, and primordia of several nuclei are distinguishable in it, such as the oculomotor nuclei, the ventral tegmental area and the substantia nigra in the mesencephalon, the ventral genicu- late nucleus, the ventrolateral thalamic nucleus and the area triangularis in the ventral thalamus, and the periven- tricular and ventromedial nuclei in the hypothalamus. In the telencephalon, the limit between the striatum and DVR becomes clear. Synaptogenesis starts in the thalamus at S35 (Trujillo, 1982).

Compared to previous stages, the THi cell group of the midbrain tegmentum has undergone dramatic changes. These are: (1) a remarkable increase in the number of immunoreactive cells; (2) the majority of the cell bodies located in the midbrain tegmentum are now not only THi, but also DAi; (3) the cells show a stronger TH immunoreac- tivity; and (4) the presence of first and second order dendrites, which gives the cells a more mature appearance (Figs. lB, 5A-C). Another notable change is that the midbrain cell group is now clearly subdivided into a lateral and a medial cell cluster (Fig. 5A,B). The cells in the lateral group will most likely give rise to the future substantia nigra (SN), while most of the cells in the medial group will probably constitute the ventral tegmental area (VTA) DA cell group.

With respect to the hypothalamus, the THi perikarya of the tuberomammillary hypothalamic cell group (Ht) are now located more laterally (Fig. 6). Most of these cell bodies lie perpendicular to the ventricle, are monopolar or bipolar neuroblasts and are also DAi. A bundle of immunoreactive axons originating from the tuberomammillary cell group

Fig. 1. A-C: Schematic drawings of sagittal sections of the brain of the lizard, Gallotia galloti, at different developmental stages (S32- S36), showing the distribution of catecholamine (CAI neuronal ele- ments in the embryonic brain for each stage. Empty circles represent cell bodies that are tyrosine hydroxylase immunoreactive (THi), but dopamine (DA)-immunonegative (DAi). Filled circles represent cell bodies that are both THi and DAi. Triangles represent cell bodies that are DAi, but TH-immunonegative. The distribution of immunoreactive fibers at each stage is indicated by fine lines and dots, and the developing CA fiber bundles are represented by a single line that ends in an asterisk, which represents a growth cone. Drawings of the whole embryo of G. galloti, at different developmental stages (Ramos Steffens, 1980), are presented on the right side of the brains. Bar = 0.5 mm for brain; 1 mm for embryo.

can be traced caudally, following the longitudinal axis of the brain (Figs. lB, 6A). At S35, another THi cell group appears at rostrodorsal hypothalamic levels (Hr), consisting of a small number of cells which lie close to the midline and have an immature appearance (Fig. 1B).

Developmental stage 36 (S.36). At S36, the reproductive system of the embryo starts to show sexual dimorphism, and the abdominal skin contains scales. In the brain, several new nuclei which were not clearly observed in previous stages are outlined, such as the locus coeruleus, the magnocellular and parvocellular isthmic nuclei, the central nucleus of the torus semicircularis, the nucleus rotundus of the dorsal thalamus, the periventricular organ in the hypothalamus, and the nucleus accumbens in the telencephalon. At this stage, strongly THiiDAi perikarya are observed, for the first time, ventral to the central canal of the spinal cord (Figs. lC, 7). These cell bodies contact the ventricle by means of a short, thick process. Axons arising from these spinal cell bodies can be traced rostrally and caudally.

Compared to the previous stage, there are several fea- tures of the midbrain THiiDAi cell groups at S36 that deserve comment. First, the subdivision into a medial (VTA) and a lateral (SN) cell group has become more distinct. Moreover, a few THi cell bodies are found in a more dorsal position, close to the periaqueductal gray. Some other THi cells are, at this stage, observed in the isthmic tegmentum, adjacent to the midline (Fig. 1C). These cell bodies most likely constitute a caudal continua- tion of the VTA. Other THi cell bodies are observed rostral to the main group of CA cells of the midbrain (Fig. 1C). The latter cells are located in the basal region of the caudal diencephalon, and constitute a rostral continuation of the VTA.

In the hypothalamus, the THiiDAi cell bodies in the tuberomammillary region lie lateral to the periventricular hypothalamic organ, in which neither THi nor DAi cell bodies are observed yet. Most of the immunoreactive cell bodies of the tuberomammillary hypothalamic group are located in the future lateral hypothalamic area, whereas a few cells lie in a ventrocaudal part of the periventricular hypothalamic nucleus. In general, the tuberomammillary cells have a triangular or bipolar shape and already possess second order dendrites (Fig. 6C). The number of THi perikarya observed in the rostrodorsal periventricular hypo- thalamic group at S36 has increased considerably (Fig. 1C). The majority of these rostrodorsal cells consists of monopo- lar neuroblasts that lie parallel to the ventricle, extending from dorsocaudal to rostroventral levels of the periventricu- lar hypothalamus (Figs. lC, 8A,B). A number of THi cell bodies located in the dorsocaudalmost portion of this hypothalamic group are arranged radial to the ventricle, and some of them occupy more lateral positions (Fig. 8A).

Developmental stage 37 (S37). At S37, the sexual dimor- phism of the reproductive system is more evident than at S36. In the limbs of the embryo, the membrane between toes has withdrawn, and claws start to develop. The eyelids are partially closed. Most of the grisea in the brain are outlined, except for the superficial layers of the tectum and the cortex, which are still forming.

With respect to the pattern of immunoreactivity, there are several events that are noteworthy. First, the appear- ance of three new CA cell groups, viz. the periventricular hypothalamic organ, the pretectal posterodorsal nucleus and the suprachiasmatic nucleus (Fig. 2A). Whereas, in

A

-- I / s37

B m

S38

S39-40(PRENATAL STAGES)

Fig. 2. A-C: Schematic drawings of sagittal sections of the brain of Gallotia galloti at developmental stages S37-S40, showing the distribution of CA neuronal elements at these stages. For further explanation, see Figure 1.

DEVELOPMENT OF CATECHOLAMINE SYSTEMS IN A REPTILE 47

general, developing CA cells first display TH immunoreac- tivity, the cells in the hypothalamic periventricular organ are strongly immunoreactive for DA but never show TH immunoreactivity either in the developing or in the adult lizard brain (Fig. 9). All DAi cells in the latter organ seem to contact the cerebrospinal fluid of the ventricle. The other two new groups of CA cell bodies, i.e., the pretectal pos- terodorsal nucleus and the suprachiasmatic nucleus, con- tain a few weakly THi perikarya (Fig. 2A). The cell bodies of the posterodorsal nucleus and the suprachiasmatic nucleus are immunonegative for the DA antiserum.

The CA cell bodies of the midbrain/isthmic group and the hypothalamus show a higher degree of maturation than at S36 (Figs. 8C, 9). This is specially noticeable for the cell bodies of the rostrodorsal periventricular hypothalamic group, which show first and second order dendritic pro- cesses and are also DAi. In the midbrain, a few TH- immunopositive, but DA-immunonegative cell bodies are observed in the periaqueductal gray, close to the ventricle.

Immunoreactive fibers are observed in the regions where the CA cell bodies are located (Fig. 2A). Further, lightly labeled fibers are observed in the basal ganglia of the telencephalon, although varicosities cannot be distin- guished.

Developmental stage 38 (S38). At S38, scales and a light, diffuse pigmentation start to differentiate in the skin of the dorsal part of the embryo. In the brain, most cell groups are formed and many neurons are undergoing cytodifferentiation, such as dendritic and axonal branching, and synaptogenesis. Synaptogenesis starts in the striatum at S38 vanes et al., 19891, although in more caudal areas like the thalamus this process started at S35 and goes on through subsequent stages (Trujillo, 1982).

At S38, weakly THi cell bodies are, for the first time, observed in the locus coeruleus (Lc; Fig. 2B). The number of THi cells and their immunoreactivity in the pretectal posterodorsal nucleus and the suprachiasmatic nucleus is considerably increased, but they still do not express DA immunoreactivity .

Strongly immunoreactive plexuses of fibers and varicosi- ties are observed in the VTA, SN and hypothalamus. Moderate immunoreactivity is observed in the basal ganglia of the telencephalon, but varicosities cannot be recognized yet.

During the prenatal stages, the gonads of females become internalized, and scales and pigmentation differentiate in the skin of the head and body, so that the embryo acquires an adult-like aspect. At the same time, the lamination of the superficial tectal layers of the brain achieves the adult state (Medina, 1991). The neurons of the brain undergo the final steps of their cytodifferentiation (formation of the dendritic arbor, acquisition of dendritic and somatic spines, completion of synaptogenesis), and the brain acquires an adult-like appear- ance (Figs. 2C, 10-12).

At S39, THi cell bodies are observed, for the first time, in the olfactory bulb and the caudal brainstem (nucleus of the solitary tract and ventrolateral tegmentum; Figs. 2C, 11). During these stages, THi fibers become apparent through- out the brain, and varicosities are already recognized at 540. The number of CA fibers and varicosities increases dramatically at late S40, giving the brain an adult-like CA innervation pattern just prior to hatching (Fig. 12). Moder- ate to dense plexuses of fibers and varicosities are observed in the nucleus accumbens, striatum, olfactory tubercle, septum, dorsal ventricular ridge, nucleus sphericus, poste-

Prenatal deuelopmental stages (S39, S40).

rior lateral cortex, dorsal geniculate nucleus, midbrain tectum, as well as in all regions that contain CA cell bodies.

DISCUSSION In the present account, a survey has been presented of

the development of CA systems in the brain of the lizard Gallotia galloti as demonstrated by means of TH- and DA immunohistochemical methods. There are several aspects of this development that deserve comment. In the following sections, we discuss first the spatiotemporal sequence of appearance of CA cell bodies in Gallotia and compare it with the sequences observed in birds and mammals. Subse- quently, the development of catecholamine systems in vertebrates will be discussed taking a segmental point of view. Finally, the transient expression of CA cell bodies and the relationship between developing CA fiber tracts and other developmental events in the brain of Gallotia will be dealt with.

Spatiotemporal sequence of appearance of CA cell bodies in GaZZotia

The sequence of appearance of CA cell bodies in the developing brain of the lizard Gallotia galloti is represented in Figure 13. Three different categories of CA cell bodies can be distinguished in the embryonic brain of Gallotia on the basis of their degree of cytodifferentiation at the moment when they first become immunoreactive. The first category consists of cell bodies that become THi at a very early moment of their cytodifferentiation. When these cell bodies become immunoreactive, they lie still close to the ventricle, have a round, monopolar or bipolar shape (with thick varicose processes), and are DA-immunonegative. To this category belong the CA cell bodies of the midbrain tegmentum, the tuberomammillary hypothalamus and the rostrodorsal periventricular hypothalamus. At subsequent stages, CA cell bodies in these groups are gradually found in more lateral positions in the mantle, and show signs of maturation (they become DAi and acquire second and third order dendrites). This suggests that these cell bodies be- come THi prior to their migration into the mantle and before their maturation.

A second category is formed by CA cell bodies that become THi at a relatively late moment of their cytodiffer- entiation. In general, when these cells become immunoreac- tive, they lie already further away from the ventricle, in a position that corresponds to the one occupied in the adult brain, and they possess two or more dendritic processes. These features suggest that these cell bodies become immu- noreactive after having finished their migration into the mantle, and after initial maturing. The CA cell bodies of the olfactory bulb, preoptic region, pretectal posterodorsal nucleus, locus coeruleus, and nucleus of the solitary tract are representatives of this category.

The third category includes CA cell bodies that remain attached to the ventricle during and after their cytodifferen- tiation. CA perikarya of the periventricular hypothalamic organ (only DAi) and spinal cord (THi/DAi) belong to this category. Since these cell bodies do not develop a dendritic arbor, it is difficult to know at which moment of their cytodifferentiation they are when they first become immu- noreactive. The observation of THiiDAi growing axons arising from the CSF-contacting CA cell bodies in the spinal cord at S36 indicates that these neurons express TH- and DA immunoreactivity at early stages of their cytodifferentiation.

48 L. MEDINA ET AL.

Fig. 3. A Photomicrograph of a sagittal section of the brain of Gullotzu at S33, showing THi perikarya located in the midbrain tegmentum (large arrow), just above the cephalic flexure. The ventricle is observed in the dorsal part of the photograph (V. Note that THi perikarya are located adjacent to the limit between midbrain (m) and isthmus (i; limit is indicated with small arrows), and from here they spread rostrally in the midbrain. The immunoreactive cells observed in the mesencephalon have a fuzzy appearance and are lightly labeled. Very intensely labeled blood cells are also observed in the photomicro-

graph (medium-sized arrows). B Photomicrograph of a corrected horizontal section (parallel to the optic tract) of the brain at late S33, showing THi cell bodies in the midbrain tegmentum, at both sides of the midline (large arrows). The ventricle is observed in the dorsal part of the photograph (V). Very intensely labeled blood cells are also observed in the photomicrograph (medium-sized arrows). Photomicrographs A and B have been taken using differential interference contrast (Nomar- ski optics). Bar = 100 pm.

DEVELOPMENT OF CATECHOLAMINE SYSTEMS IN A REPTILE 49

Fig. 4. Photomicrograph of a sagittal section of the brain at S34, showing THi perikarya located in the midbrain tegmentum (m) and the tuberomammillary hypothalamus (Ht). Note the difference between the immunoreactive cells observed in the hypothalamus, which are clearly outlined and intensely labeled, and the immunoreactive cells observed in the mesencephalon, which have a fuzzy appearance and are lightly labeled. Most of the THi perikarya of the midbrain are located adjacent to the limit between mesencephalon (m) and isthmus (i; limit indicated with small arrows). A few THi cell bodies are displaced caudal to the limit, in the isthmic tegmentum (curved arrow). A fiber bundle originates in the THi cell group of the midbrain and courses rostrally

(empty arrow). In the hypothalamus, some THi cell bodies are located in the infundibular region (large arrow). Note the high degree of curvature of the cephalic flexure (arrowhead). This high curvature causes the angle of the longitudinal (rostrocaudal) axis of the brain to change continuously. The reader can get a better idea of the orientation of this photograph by comparing it with the schematics of sagittal sections of the embryonic brain shown in Figure 1. Very intensely labeled blood cells are also observed in the photomicrograph (medium- sized arrows). Photomicrograph has been taken using differential interference contrast (Nomarski optics). Bar = 100 km.

Figure 5

DEVELOPMENT OF CATECHOLAMINE SYSTEMS IN A REPTILE 51

Spatiotemporal sequence of appearance of CA cell bodies in other vertebrates

As in Gallotia, CA cell bodies have been detected in embryonic brains of mammals and birds at early stages of their cytodifferentiation (Specht et al., 1981a; Guglielmone and Panzica, 1984,1985; Ugrumov et al., 1989; Di Porzio et al., 1990; Shults et al., 1990; Verney et al., 1991; Puelles and Medina, 1994). However, in mammals and birds these early CA cell bodies are located not only in the hypothala- mus and midbrain tegmentum, but also in the locus coeruleus and medulla (see Table 3). A developmental study of CA systems in the chick (Puelles and Medina, 1994) has revealed that CA cell bodies first appear in the tuberomam- millary hypothalamus (5.5-6 days of incubation), and, a few days later, in the substantia nigra, locus coeruleus and medulla (8-9 days of incubation; Table 3). This sequence of appearance is different from that in Gallotia, in which THi cell bodies are observed first in the midbrain tegmentum (S32-S33), and, shortly thereafter, in the tuberomammil- lary hypothalamus (late S33-S34; Table 3). In contrast to rat and chicken, a remarkable delay in appearance was found for CA cells in the locus coeruleus and medulla (S38-S39) of the lizard Gallotia (Table 3). The observation that the locus coeruleus of Gallotia is cytologically apparent by S36, but that neurons in the locus coeruleus do not become THi until S38, indicates that there is a considerable delay between the time of birth of these cells and their first expression of the enzyme TH. This remarkable delay in the appearance of CA neurons in the locus coeruleus of the lizard Gallotia is also in contrast with teleost fishes and amphibians, in which the locus coeruleus is one of the first structures of the brain containing CA cells during develop- ment (Ekstrom et al., 1992, 1994; Manso et al., 1993; Gonz6lez and Smeets, 1994). Interestingly, the develop- ment of the CA systems of the sheep shows a spatiotempo- ral sequence of appearance of CA cells very similar to that of Gallotia, with the midbrain tegmetum and the tuberomam- millary hypothalamus being the earliest groups expressing CA, and with a delay of the locus coeruleus (Tillet and Thibault, 1987). Further studies in other reptilian species will be necessary in order to know whether the delay in the expression of CA in the locus coeruleus during development is a rule that applies to the development of CA systems of all reptiles.

As in Gallotia, in the chicken embryo, dopamine is detected in the cell bodies of the paraventricular organ several days later than in other hypothalamic areas (Gugliel-

Fig. 5. Photomicrographs of transverse sections of the brain at S35, showing the THi cell bodies at rostral (A) and caudal levels (B) of the midbrain tegmentum, at both sides of the midline. Note that two different subgroups can be distinguished in the midbrain (m): one located close to the midline (future VTA), and another located in a lateral position (future SN). At caudaI levels (B), some THi neurobIasts located close to the midline are displaced into the isthmus (i; large arrow), whereas some other THi cell bodies are observed across the midline (curved arrow). The ventricle is observed in the dorsal part of the photograph (V). Very intensely labeled blood cells are also observed in the photomicrographs (small arrows). C: Photomicrograph of a sagittal section of the brain at S35, showing THi perikarya located in the substantia nigra. The majority of the cells lie within the midbrain, adjacent to the limit (indicated by small arrows) between the mesen- cephalon (m) and isthmus (i). Note the THi neuroblast which is caudally displaced from the main group of immunoreactive cells (empty arrow). Blood cells are indicated by medium-sized arrows in A-C. Photomicrographs have been taken using interference contrast (Nomar- ski optics). Bars = 100 pm in Aand B, 50 pm in C .

mone and Panzica, 1984; Puelles and Medina, 1994). An interesting finding of the present study is that the cell bodies of the hypothalamic periventricular organ never express TH immunoreactivity during any developmental stage, and that they become strongly DAi at S37 without any apparent presence of TH, an enzyme that is needed for the synthesis of dopamine. In adult specimens of all non- mammalian vertebrates studied so far, cell bodies of the periventricular organ are DAi, but TH-immunonegative (Smeets, 1988b; Ekstrom et al., 1990; Smeets and GonzB- lez, 1990; Smeets and Steinbusch, 1990; Gonzalez and Smeets, 1991). Evidence has been obtained that the cell bodies of the periventricular organ do not synthesize dopa- mine, but instead accumulate it from the CSF (Smeets et al., 1991). In this respect, it is interesting to note that just one stage before the first detection of DA immunoreactivity in the cell bodies of the periventricular organ (S37), CSF- contacting cell bodies located ventral to the central canal of the spinal cord become strongly THi and DAi (S36). This raises the possibility that dopamine may be released into the ventricle by CSF-contacting cell bodies of the spinal cord, and accumulated from the CSF by the cell bodies of the periventricular organ of the hypothalamus.

In the lizard brain, the last CA cell group that becomes THi during development is that in the olfactory bulb (S39). This finding agrees with the data on the development of CA systems in rats, in which THi cells in the olfactory bulb are also detected at very late, prenatal stages (Specht et al., 1981b).

Development of CA fiber tracts in relation to other developmental events

In the present study, we observed growing THi/DAi s o n s in the embryonic brain of the lizard Gallotia. Catechol- amines and TH have previously been detected also in growing axons of other vertebrates (Tennyson et al., 1973; Specht et al., 1981a; Voorn et al., 1988; Pindzola et al., 1990; Reisert et al., 1990). In fact, they are not only present in the growing axonal shaft, but also in the axonal growth cones (Specht et al., 1981a; present study), suggesting that they play a role in finding the migration pathway or influencing the development of the target. In support of this notion, recent experiments in vitro have shown that stimu- lation of the D2 dopamine receptor results in both neurite branching and outgrowth (Todd, 1992), whereas stimula- tion of the D1 receptor inhibits growth cone motility and neurite outgrowth (Lankford et al., 1987, 1988).

The outgrowth of three different CA fiber bundles could be observed in the present study: (1) ascending THi fibers arising from the CA cell group of the midbrainiisthmic tegmentum; (2) descending THi fibers from the CA cell group of the tuberomammillary hypothalamus; and (3) ascending and descending THi/DAi fibers arising from the CA cell bodies of the spinal cord. These CA fiber bundles were observed in the embryonic brain from S33 to S36, and after growing, they constitute or contribute to some impor- tant CA pathways of the brain, such as the nigrostriatal pathway which provides an important CA innervation to the basal ganglia (see below). The CA cell groups that become positive at a later moment during their differentia- tion, such as the posterodorsal pretectal nucleus, the locus coeruleus, and the nucleus of the solitary tract, also contrib- ute to some of the CA fiber tracts and innervation that are present in the adult brain (e.g., the ascending noradrener- gic fiber bundle arising from the locus coeruleus, which provides the forebrain with a wide innervation). However, since cell bodies in these groups are already considerably

52 L. MEDINA ET AL.

Fig. 6. A,B: Photomicrographs of a corrected horizontal section at S35, showing THi cells in the tuberomammillary hypothalamic cell group (Ht). At this stage, many cells are monopolar or bipolar neuro- blasts, and have one or two thick processes with growth cones (small arrows in B, which is a higher magnification of A). Note the THi fiber bundle that originates in this cell group and courses caudally (large

arrows). C: Photomicrograph of THi cells in the tuberomammillary hypothalamic cell group at S36. Note that the cells have started maturing, and that they already show some dendritic branches. Photo- micrographs A-C have been taken using interference contrast (Nomar- ski optics). Bars = 50 bm in A, 25 km in B and C.

matured before becoming immunoreactive, their main con- nections are probably established by that time.

The THi fiber bundle originated in the CA cell group of the

midbrainiisthmic tegmentum starts growing at S32-33, and reaches the caudal telencephalon at ,334 (Table 4). However, it appears that these CA fibers do not innervate the lateral striatum until S37-S38; the adult-like innerva-

Development of the nigrostriatal DA pathway.

DEVELOPMENT OF CATECHOLAMINE SYSTEMS IN A REPTILE 53

Fig. 7. Photomicrograph of a sagittal section of the brain at S36, showing THi cell bodies located in the spinal cord, ventral to the central canal (cc). Axons originating in these cells decussate in a commissure ventral to them (arrow). Photomicrograph has been taken using interference contrast (Nomarski optics). Bar = 50 wm.

tion of the striatum and nucleus accumbens (with clear varicosities) is not observed until prenatal stage S40, just prior to hatching. These results are in agreement with those of Yanes et al. (1989) on the development of the striatum in the lizard Gallotia galloti (Table 4). According to the latter authors, striatal neurons (born from S31 to S35) mature between S38 and S40, while synaptogenesis (mainly axodendritic synapses) starts at S38. The same authors also noted that axodendritic synapses acquire an adult-like morphology by S40, coinciding with the moment when we observed THi varicosities in the striatum (Table 4). The early arrival of the THi axons in the striatum suggests that they have an influence on the maturation of the striatal neurons. This idea has also been proposed for mammals, where the CA innervation of the rostra1 striatum follows a lateroventral to dorsomedial gradient which coin- cides with the maturation gradient of the striatal neurons (Tennyson et al., 1973; Specht et al., 1981b; Bayer, 1984; Voorn et al., 1988; Table 4). The molecular mechanisms by which dopaminergic axons may influence the development of the striatum are not known yet, but in any case they must be mediated by specific receptors. In line with this, there is recent evidence that during the development of the rat striatum, dopamine receptors are detected soon after the arrival of THi fibers (Foster et al., 1987; De Vries et al., 1992), and that the postsynaptic D1 dopamine receptors become functionally active much earlier than the postsynap- tic D2 dopamine receptors (De Vries et al., 1992). This

differential developmental profile of the D1 and DZ dopa- mine receptors may play an important role in the develop- ment of the striatum, considering that stimulation of D1 or D2 receptor results in different effects on neurite out- growth.

Development of CA systems: A segmental point of view

During development, the neural tube becomes subdivided into transverse segmental domains, called neuromeres, which represent regions of increased cell proliferation (Bergquist and KSllBn, 1954; Vaage, 1969; Keyser, 1972; Puelles et al., 1987; Lumsden and Keynes, 1989; Noden, 1991). These domains are bounded by lines of clonal restriction (Fraser et al., 1990; Figdor and Stern, 1993) and by slowly proliferating boundary cells that restrict interseg- mental gap-junctional cell communication (Lumsden, 1990; Guthrie et al., 1991; Martinez et al., 1992). In brief, the following segments have been described in the brain of diverse vertebrates (Figs. 14, 15): (1) 7-8 rhombencephalic segments or rhombomeres (rhl-rh8); (2) a large isthmocer- ebellar segment (sometimes referred to as rhl); (3) 1 mesencephalic segment or mesomere; and (4) 6 prosence- phalic segments (pl-p6; for review, see Bulfone et al., 1993; Puelles and Rubenstein, 1993; Puelles and Medina, 1994). In support of the existence of these segments, several homeobox genes (which produce important regulatory fac-

54 L. MEDINA ET AL.

Fig. 8. Photomicrographs of corrected horizontal (parallel to the optic tract) sections of the brain at either S36 (A,B) or 537 (C), showing THi perikarya in the rostrodorsal hypothalamic cell group (Hr). Many of these perikarya are located adjacent to the ventricle (V; large filled arrows). Note that at S36 (A,B) many of these THi cells are monopolar neuroblasts (arrows in B) with a long, thick process arranged parallel to the ventricle (shown in B, which is a higher magnification of A). At S37

(C), the cells in the corresponding cell group have already a more mature aspect. In the dorsocaudalmost part ofthe rostrodorsal hypotha- lamic group, THi perikarya are located further away from the ventricle and are arranged perpendicular to the latter (empty arrow). Photomicro- graphs A-C have been taken using interference contrast (Nomarski optics). Bars = 100 pm in A and C , 50 pm in B.

Fig. 9. Photomicrographs of corrected horizontal (parallel to the optic tract), adjacent sections of the brain at 537 stained for TH (A) or DA (B), showing immunoreactive perikarya in the tuberomamrnillary

hypothalamic cell group (Ht) and in the periventricular organ (oph). Photomicrographs A and B have been taken using interference contrast (Nomarski optics). Bar = 100 pm.

DEVELOPMENT OF CATECHOLAMINE SYSTEMS IN A REPTILE 55

Fig. 10. A,B: High power photomicrographs of immature THi cell bodies (large arrows) of the midbrain cell group at S35, as viewed in sagittal sections. Note the presence of a growth cone on the process of one of the cells (small arrow in B). C,D: Low (C) and high (D) power photomicrographs of a sagittal section of the brain at S39, showing

highly matured THi cells in the midbrain (VTA, SN, AS) and in the isthmus (arrow). Photomicrographs A-D have been taken using inter- ference contrast (Nomarski optics). Bars = 25 pm in A, B, and D, 100 pm in C.

tors affecting the expression of other genes) show, during development of the neural tube, a segmented pattern of expression which coincides with the morphologically de- fined segmental boundaries (Keynes and Lumsden, 1990; Noden, 1991; Price et al., 1991; Bulfone et al., 1993; Figdor and Stern, 1993; Puelles and Rubenstein, 1993).

Several lines of evidence indicate that interneuromeric boundaries may act as barriers to longitudinal cell move- ment (Noden, 1991). Although this may be a general rule for the development of the brain, many exceptions to it are found during development, one example being the facial motoneurons in mammals and reptiles. In these animals, the facial motoneurons are born in rhombomeres 4-5, and migrate into the more caudal rhombomeres 6-7 (Medina et

al., 1993). In the context of the development of CA systems, a number of studies including this one have shown the expression of CA by cell bodies at a very early time in their cytodifferentiation, probably before the completion of their migration (references and Discussion above). In the lizard Gallotia, these early CA cell bodies are located in the midbrain tegmentum, the tuberomammillary hypothala- mus (mostly in basal part of p4) and the rostrodorsal periventricular hypothalamus and nearby ventral thalamus (alar parts of p3-p5; Figs. 14, 15). As discussed above, there is some morphological evidence that CA cell bodies of these groups in the lizard Gatlotia may migrate from a periven- tricular into a more lateral position. This migration may be considerable for the CA cells of the midbrain tegmentum

56 L. MEDINA ET AL.

Fig. 11. A Photomicrograph of a horizontal section through the olfactory bulb of Gallotia galloti at ,339, showing THi cell bodies in the glomerular layer (gl) and the external plexiform layer (epl). In this photograph, rostral is to the left. B-D: Photomicrographs of sagittal sections of the brain at S39, showing THi cell bodies in the pretectal

posterodorsal nucleus (B), locus coeruleus (0, and nucleus of the solitary tract (D). In these photographs, rostral is to the left, and dorsal is to the top. Photomicrographs A-D have been taken using phase- contrast brightfield. Bars = 50 Frn in A, B, and D, 100 krn in C.

that are going to constitute the lateral parts of the substan- tia nigra, a migration that has also been proposed for the substantia nigra of mammals (Levitt and Rakic, 1982; Marchand and Poirier, 1983). In contrast, this migration should be very short for the CA cells of the rostrodorsal periventricular hypothalamus which are going to constitute part of the periventricular hypothalamic nucleus, and for the CA cells of the midbrain tegmentum which are going to constitute the VTA. In addition to these standard migra- tions into the mantle within the same segment, our mate- rial has provided morphological evidence of immature CA cells of the midbrain tegmentum that may be migrating into the isthmic tegmentum (see Figs. 4,5B). Morphological evidence for a migration across the limit between mesen-

cephalon and isthmus has been found also in chick embryos (Puelles and Medina, 1994). Nevertheless, we must be cautious with interpreting migration on the basis of morpho- logical observations of immunoreactive cell bodies in the brain, and other kinds of experiments will be needed before any conclusion about migration can be made definitively.

In our material, we have observed THi/DAi cell bodies located in the basal region of p l at late and prenatal developmental stages, as well as in adults. These neurons are caudally continuous with the main group of CA cell bodies of the midbrain tegmentum, and they constitute a rostral part of the VTA in the lizard Gallotia, as in birds and mammals (Puelles and Medina, 1994). The origin of the CA neurons of the basal region of p l is unclear, and no clear

DEVELOPMENT OF CATECHOLAMINE SYSTEMS IN A REPTILE 57

Fig. 12. Photomicrographs of sagittal sections of the brain at S40, at lateral (A) or medial (B) levels, showing the distribution of the different CA cell groups in the midbrain (VTA, SN), tuberomammillary hypothalamus (Ht), rostrodorsal periventricular hypothalamus (Hr),

and suprachiasmatic nucleus (SCN). Note the immunoreactive fiber bundle that arises in the tuberomammillary hypothalamus and courses caudally (arrows). Photomicrographs A and B have been taken using brightfield. Bar = 200 pm.

58 L. MEDINA E T AL.

olfactory bulb

suprachiasmatic nucleus

rostrodorsal hypothalamus

tuberomammilar hypothalamus

periventricular organ

pretectal posterodorsal nucleus

midbrain tegmentum

locus coeruleus

solitary tracvarea postrema

spinal cord/central canal

Developmental stages

Fig. 13. Schematic showing the sequence of appearance of THi (hatched bar) and DAi (solid bar) perikarya in the different structures of the brain of GaZZotia galloti during ontogeny (developmental stages 32 to hatching). The first THi cell bodies appear in the midbrain tegmentum at S32433. Shortly thereafter, THi perikarya appear in the tuberomammillary hypothalamus, and later in the rostrodorsal periventricular hypothalamus. These cell bodies become DAi during subsequent developmental stages. At 536, cell bodies located ventral to

TABLE 3. Comparison of Spatiotemporal Sequence of Appearance of CA Cells in the Main CA Cell Groups of Different Vertebrates

the central canal of the spinal cord become strongly THilDAi, and one stage later, at S37, cell bodies of the periventricular hypothalamic organ become DAi, although they are TH-immunonegative. During intermedi- ate-late (S37, S38) and prenatal (S39, S40) developmental stages, THi cell bodies appear in the olfactory bulb, suprachiasmatic nucleus, the posterodorsal pretectal nucleus, the locus coeruleus, and the nucleus of the solitary tract.

TABLE 4. Relation Between Development of the CA Nigrostriatal Pathway and Striatal Cytodifferentiation

G. galloti Chicken’ Rat2 Rat G. galloti

Lc S38 E S E 9 E12.5 VTAiSN S32-533 E8-E9 E12.5 Ht s33.5-s34 E5.5-E6 E12.5 Hr + POA s35-s37 E1.5-E8 E12.5-E14.5 OB s39 E l 8 E21

‘Puelles and Medina (1994). 2Specht et al. (1981a, b); Ugrumov et al. (1989).

evidence for a migration from the midbrain into p l has been obtained in Gallotia or birds (Puelles and Medina, 1994). With respect to the rostroventral periventricular hypotha- lamic cell group, it is interesting to note that at S36 the most dorsocaudal cells of the group are arranged radial to the ventricle, in contrast to the rest of them which are arranged parallel to it. Using neuromeric terms, the dorso- caudal cells of this group are probably located in the alar part of p3 and may correspond to the zona incerta of mammals (Hokfelt et al., 19841, whereas the rest of the CA cells of this group are probably located in the alar parts of

Neurogenesis VTAiSN ? CA cells in VTAiSN S32-533 Neurogenesis striatum S31-S3Fi3 Maturation of striatal neurons S38-S403 Arrival of CA fibers in striatum 5 3 4 4 3 5 CA innervation of striatum s37-s39 Appearance of varicosities S40

Ell-El6.5l E12.52 E12-Post4 E13-Post4 E15-ElG ElI-PostS Post5

‘Altman and Eayer (1981). ZSpecht et al. (1981a); Burgunder andYoung111 (1990); Solberget al. (1993) 3Yanes et a1 (1989). 4Bayer (1964). Voorn et al. (1988)

p4 and p5 and correspond to “rostral” parts of A14 of mammals (Hokfelt et al., 1984). Our results provide some morphological evidence of some CA neuroblasts of p4-p5 that may migrate rostrally during development. No evi- dence for such a migration has been obtained in mammals or birds (see Puelles and Medina, 1994), and from a segmental viewpoint, the probability for an intersegmental migration is very low, although not impossible (see above).

DEVELOPMENT OF CATECHOLAMINE SYSTEMS IN A REPTILE 59

s33

Fig. 14. Schematic drawings of a sagittal section of the embryonic brain of the lizard GaZZotia at early (S33, S35) and intermediate (S37) developmentat stages, showing the distribution of the CA celI groups with respect to the brain segmental (6 prosomeres, pl-p6; 1 mesomere,

m; isthmus, i; 8 rhombomeres, rhl-rh8; see text for details) and longitudinal (alar and basal plates) domains. Shaded areas represent CA cell groups observed in the embryonic brain.

60 L. MEDINA ET AL.

Fig. 15. Schematic drawing of a sagittal section of the brain of the lizard GuZZotia at a perinatal stage, showing the distribution of the CA cell groups with respect to the brain segmental (6 prosomeres, pl-p6; 1 mesomere, m; isthmus. i: 8 rhombomeres. rhl-rh8; see text for details) and longitudinal (alar and basal plates) domains.

Transient expression of CA cell bodies in the brain of Gattotia

CA cell bodies are not present in the central gray of the adult brain of Gullotiu. This indicates that the THi peri- karya observed in the central gray of the embryonic brain from S37 until prenatal stages represent transient CA neurons. The presence of transient CA neurons in the developing brain has been reported also in mammals (e.g., Cochard et al., 1978, 1979; Teitelman et al., 1979, 1981; Specht et al., 1981b; Jaeger and Joh, 1983; Jonakait et al., 1984; Berger et al., 1985; Verney et al., 1988). In fact, the transient expression of a CA phenotype occurs in a variety of cells of diverse embryonic origin. Therefore, it is now thought that this transient expression is not just a feature related to cells that die after aberrant development, but may represent a more complex feature of cell bodies that transiently express detectable levels of TH and retain this ability during the entire life (Jonakait et al., 1984). From a phylogenetic point of view, our finding of transient CA cell bodies in the central gray of Gullotia is of interest, since CA cell bodies are observed in the central gray of some adult reptiles such as the lizards Vurunus and Anolis (Wolters et al., 1984; Smeets, 1994) and crocodiles (Medina, unpub- lished observations), but not in other lizards (Gullotiu, Gekko), turtles and snakes (Smeets et al., 1986, 1987; Smeets, 1988a, 1994). Because dopaminergic neurons are present in the central gray of several species of birds (Guglielmone and Panzica, 1984; Bailhache and Balthaz- art, 1993; Puelles and Medina, 1994; Reiner et al., 1994) andin rats (dorsalA10, Hokfelt et al., 1984), it is most likely that the presence of such cells is a general or primitive feature of CA systems in amniotes.

ACKNOWLEDGMENTS We thank the U.D.I. of Cell Biology of the University of

La Laguna (Tenerife, Spain) for generously providing us with the lizard embryos for this study. We also thank E.J. Karle for her valuable help with the illustrations, Dr. R.M. Buijs for generously providing the dopamine antiserum, and Dr. A. Reiner (Department of Anatomy and Neurobiol- ogy, University of Tennessee, Memphis) for partially sup- porting this work (NIH Grant NS-19620) and his helpful suggestions for improving the manuscript. This work was also partially supported by the Spanish DGICYT PB87-0688- CO1-C02 and PB90-0296-COl-CO2 (L.P.), and by postdoc- toral fellowships from the Spanish Ministerio de Educacion y Ciencia (L.M.).

LITERATURE CITED Altman, J., and S.A. Bayer (1981) Development of the brain stem in the rat.

V. Thymidine-radiographic study of the time of origin of neurons in the midbrain tegmentum. J. Comp. Neurol. 198:677-716.

Bailhache, T., and J. Balthazart (1993) The catecholaminergic system of the quail brain: Immunocytochemical studies of dopamine-p-hydroxylase and tyrosine hydroxylase. J. Comp. Neurol. 329t230-256.

Bayer, S.A. (1984) Neurogenesis in the rat striatum. Int. J. Devl. Neurosci. 2163-175.

Berger, B., C. Verney, P. Gaspar, andA. Febvret (1985) Transient expression of tyrosine-hydroxylase immunoreactivity in some neurons of the rat neocortex during postnatal development. Dev. Brain Res. 23~141-144.

Bergquist, H., and B. m l 6 n (1954) Notes on the early histogenesis and morphogenesis of the central nervous system in vertebrates. J. Comp. Neurol. 100:627-660.

Bulfone, A,, L. Puelles, M.H. Porteus, M.A. Frohman, G.R. Martin, and J.L.R. Rubenstein (1993) Spatially restricted expression of Dlx-1, Dk-2 (Tes-11, Gbx-2 and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J. Neurosci. 13:3155-3172.

DEVELOPMENT OF CATECHOLAMINE SYSTEMS IN

Burgunder, J.M., and W.S. Young 111 (1990) Ontogeny of tyrosine hydroxy- lase and choleocystokinin gene expression in the rat mesencephalon. Dev. Brain Res. 5285-93.

Cochard, P., M. Goldstein, and I.B. Black (1978) Ontogenetic appearance and disappearance of tyrosine hydroxylase and catecholamines in the rat embryo. Proc. Nat. Acad. Sci. USA 75:2986-2990.

Cochard, P., M. Goldstein, and I.B. Black (1979) Initial development of the noradrenergic phenotype in autonomic neuroblasts of the rat embryo in viva. Dev. Biol. 71:lOO-114.

De Vries, T.J., A.H. Mulder, and A.N.M. Schoffelmeer (1992) Differential ontogeny of functional dopamine and muscarinic receptors mediating presynaptic inhibition of neurotransmitter release and postsynaptic regulation of adenylate cyclase activity in rat striatum. Dev. Brain Res. 66:9 1-96.

Di Porzio, U., A. Zuddas, D.B. Cosenza-Murphy, and J.L. Barker (1990) Early appearance of tyrosine hydroxylase immunoreactive cells in the mesencephalon of mouse embryos. Int. J. Dev. Neurosci. 8:523-532.

Dufaure, J.P., and J. Hubert (1961) Table de developpement du lezard vivipare (Lacerta uiuipara Jacquin). Archiv. Anat. Microsc. Morphol. Exp. 50t309-327.

Ekstrom, P., T. Honkanen, and H.W.M. Steinbusch (1990) Distribution of dopamine-immunoreactive neuronal perikarya and fibers in the brain of a teleost, Gastwostens acuteatus L. Comparison with tyrosine hydroxy- lase- and dopamine-b-hydroxylase-immunoreactive neurons. J. Chem. Neuroanat. 3233-260.

Ekstrom, P., T. Honkanen, and B. Borg (1992) Development of tyrosine hydroxylase-, dopamine- and dopamine P-hydroxylase-immunoreactive neurons in a teleost, the three-spined stickleback. J. Chem. Neuroanat. 5481-501.

Ekstrom, P., T. Honkanen, and B. Borg (1994) Development of catechol- amine neurons in the CNS of teleosts. In W.J.A.J. Smeets and A. Reiner (eds): Phylogeny and Development of Catecholamine Systems in the CNS of Vertebrates. Cambridge: Cambridge University Press, pp. 325- 342.

Figdor, M.C., and C.D. Stern (1993) Segmental organization of embryonic diencephalon. Nature 3633330-634.

Foster, G.A., M. Schultzberg, T. Hokfelt, M. Goldstein, H.C. Hemmings, C.C. Ouimet, S.I. Walaas, and P. Greengard (1987) Development of a dopamine- and cyclic adenosine 3’ :5’-monophosphate-regrtlated phospho- protein (DARPP-32) in the prenatal rat central nervous system, and its relationship to the arrival of presumptive dopaminergic innervation. J. Neurosci. 7:1994-2018.

Fraser, S., R. Keynes, and A. Lumsden (1990) Segmentation in the chick embryo hindbrain is defined by cell lineage restrictions. Nature 344:431- 435.

Gonzalez, A., and W.J.A.J. Smeets (1991) Comparative analysis of dopamine and tyrosine hydroxylase immunoreactivities in the brain of two amphib- ians, the anuran Rana ridibunda and the urodele Pleurodeles waltlii. J. Comp. Neurol. 303:457477.

Gonzaez, A., and W.J.A.J. Smeets (1994) Ontogeny of catecholamine systems in the CNS of anuran amphibians. An immunohistochemical study with antibodies against tyrosine hydroxylase and dopamine. J. Comp. Neurol. 346:63-79.

Guglielmone, R., and G.C. Panzica (1984) Typology, distribution and development of the catecholamine-containing neurons in the chicken brain. Cell Tissue Res. 23757-79.

Guglielmone, R., and G.C. Panzica (1985) Early appearance of catecholamin- ergic neurons in the central nervous system of precocial and altricial avian species. A fluorescence-histochemical study. Cell Tissue Res. 240:381-384.

Guthrie, S.C., M. Butcher, and A. Lumsden (1991) Patterns of cell division and interkinetic nuclear migration in the chick embryo hindbrain. J. Neurobiol. 22:742-754.

Hokfelt, T., R. Martensson, A. Bjorklund, S. Kleinau, and M. Goldstein (1 984) Distributional maps of tyrosine-hydroxylase-immunoreactive neu- rons in the rat brain. In A. Bjorklund and T. Hokfelt (eds): Handbook of Chemical Neuroanatomy. Vol. 2: Classical Transmitters in the CNS, Part I. Amsterdam: Elsevier, pp. 277-379.

Jaeger, C.B., and T.J. Joh (1983) Transient expression of tyrosine hydroxy- lase in some neurons of the developing inferior colliculus of the rat. Dev. Brain Res. 11.128-132.

Jonakait, G.M., K.A. Markey, M. Goldstein, and I.B. Black (1984) Transient expression of selected catecholaminergic traits in cranial sensory and dorsal root ganglia of the embryonic rat. Dev. Biol. 101:51-60.

Keynes, R., and A. Lumsden (1990) Segmentation and the origin of regional diversity in the vertebrate central nervous system. Neuron 2: 1-19.

A REPTILE 61

Keyser, A. (1972) The development of the diencephalon of the Chinese hamster. An investigation of the validity of the criteria of subdivision of the brain. Acta Anat. Suppl. 59:l-178.

Lankford, K., F.G. DeMello, and W.L. Klein (1987) A transient embryonic dopamine receptor inhibits growth cone motility and neurite outgrowth in a subset of avian retina neurons. Neurosci. Lett. 75169-174.

Lankford, K., F.G. DeMello, and W.L. Klein (1988) D1-type dopamine receptors inhibit growth cone motility in cultured retina neurons: Evidence that neurotransmitters act as morphogenic growth regulators in the developing central nervous system. Proc. Nat. Acad. Sci. USA 85:45674571.

Lauder, J.M., and F.E. Bloom (1974) Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat. J. Comp. Neurol. 155:469482.

Levitt, P., and P. Rakic (1982) The time of genesis, embryonic origin and differentiation of the brain stem monoamine neurons in the Rhesus monkey. Dev. Brain Res. 4t35-57.

Lumsden, A. (1990) The cellular basis of segmentation in the developing hindbrain. Nature 13:329-335.

Lumsden, A., and R. Keynes (1989) Segmental patterns of neuronal development in the chick hindbrain. Nature 337:424-428.

Manso, M.J., M. Becerra, P. Molist, 1. Rodriguez-Moldes, and R. Anadon (1993) Distribution and development of catecholaminergic neurons in the brain of the brown trout. A tyrosine hydroxylase immunohistochemi- cal study. J. Hirnforsch. 34239-260.

Marchand, R., and L.J. Poirier (1983) Isthmic origin of neurons of the rat substantia nigra. Neuroscience 9:373-381.

Martinez, S., E. Geijo, M.V. Sanchez-Vives, L. Puelles, and R. Gallego (1992) Reduced junctional permeability at interrhombomeric boundaries. Devel- opment 116:1069-1076.

Medina, L. (1991) Estudio Ontogenetico e Inmunohistoquimico de 10s Centros Visuales Primarios de Reptiles. Ph.D. Dissertation, Universidad de La Laguna, Spain.

Medina, L., and W.J.A.J. Smeets (1992) Cholinergic, monoaminergic and peptidergic innervation of the primary visual centers in the brain of the lizards Gekkogecko and Gallotiagalloti. Brain Behav. Evol. 40:157-181.

Medina, L., W.J.A.J. Smeets, P.V. Hoogland, and L. PueUes (1993) Distribu- tion of choline acetyltransferase immunoreactivity in the brain of the lizard Gallotiagalloti. J. Comp. Neurol. 331.261-285.

Noden, D.M. (1991) Vertebrate craniofacial development: The relation between ontogenetic process and morphological outcome. Brain Behav. Evol. 38: 190-225.

Pindzola, R.R., R.H. Ho, and G.F. Martin (1990) Development of catechol- aminergic projections to the spinal cord in the north american opossum, Didelphis uirginiana. J. Comp. Neurol. 294t399-417.

Price, M., M. Lemaistre, M. Pischetola, R. Di Lauro, and D. Duboule (1991) A mouse gene related to Distal-less shows a restricted expression in the developing forebrain. Nature 351:748-751.

Puelles, L., and L. Medina (1994) Development of neurons expressing tyrosine hydroxylase and dopamine in the chicken brain. In W.J.A.J. Smeets and A. Reiner (eds): Phylogeny and Development of Catechol- amine Systems in the CNS of Vertebrates. Cambridge: Cambridge University Press, pp. 381-404.

Puelles, L., and J.L.R. Rubenstein (1993) Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. TINS 16t472-479.

Puelles, L., J.A. Amat, and M. Martinez-de-la-Torre (1987) Segment-related, mosaic neurogenetic pattern in the forebrain and mesencephalon of early chick embryos: I. Topography of AChE-positive neuroblast up to stage HH18. J. Comp. Neurol. 266.247-268.

Ramos Steffens, A. (1980) Tabla de desarrollo embrionario de Lacerta galloti galloti (period0 de organogenesis), y aspectos de su reproduccion. Universidad de La Laguna, Spain.

Reiner, A., E.J. Karle, K.D. Anderson, and L. Medina (1994) Catecholaminer- gic perikarya and fibers in the avian nervous system. In W.J.A.J. Smeets and A. Reiner (eds): Phylogeny and Development of Catecholamine Systems in the CNS of Vertebrates. Cambridge: Cambridge University Press, pp. 135-181.

Reisert, I., R. Schuster, R. Zienecker, and C. Pilgrim (1990) Prenatal development of mesencephalic and diencephalic dopaminergic systems in the male and female rat. Dev. Brain Res. 53t222-229.

Shults, C.W., R. Hashimoto, R.M. Brady, and F.H. Gage (1990) Dopaminer- gic cells align along radial glia in the developing mesencephalon of the rat. Neuroscience 38r42 7-436.

62

Smeets, W.J.A.J. (1988a) Distribution of dopamine immunoreactivity in the forebrain and midbrain of the snake Python regzus: A study with antibodies against dopamine. J. Comp. Neurol. 271t115-129.

Smeets, W.J.A.J. (198813) The monoaminergic systems of reptiles investi- gated with specific antibodies against serotonin, dopamine, and nor- adrenaline. In W.K. Schwerdtfeger and W.J.A.J. Smeets (eds): The Forebrain of Reptiles. Current Concepts of Structure and Function. Basel: Karger, pp. 97-109.

Smeets, W.J.A.J. (1994) Catecholamine systems in the CNS of reptiles: Structure and functional correlations. In W.J.A.J. Smeets and A. Reiner (eds): Phylogeny and Development of Catecholamine Systems in the CNS of Vertebrates. Cambridge: Cambridge University Press, pp. 103- 133.

Smeets, W.J.A.J., and A. Gonzaez (1990) Are putative dopamine-accumulat- ing cell bodies in the hypothalaniic periventricular organ a primitive brain character of non-mammalian vertebrates? Neurosci. Lett. 114:248- 252.

Smeets, W.J.A.J., and A. Reiner (1994) Catecholamines in the CNS of vertebrates: Current concepts of evolution and functional significance. In W.J.A.J. Smeets and A. Reiner (eds): Phylogeny and Development of Catecholamine Systems in the CNS of Vertebrates. Cambridge: Cam- bridge University Press, pp. 463481.

Smeets, W.J.A.J., and H.W.M. Steinbusch (1990) New insights into the reptilian catecholaminergic systems as revealed by antibodies against the neurotransmitters and their synthetic enzymes. J. Chem. Neuroanat. 3:25-43.

Smeets, W.J.A.J., P.V. Hoogland, and P. Voorn (1986) The distribution of dopamine immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko: An immunohistochemical study with antibodies against dopamine. J. Comp. Neurol. 253:46-60.

Smeets, W.J.A.J., A.J. Jonker, and P.V. Hoogland (1987) Distribution of dopamine in the forebrain and midbrain of the red-eared turtle, Pseud- emys scriptu eleguns, reinvestigated using antibodies against dopamine. Brain Behav. Evol. 30:121-142.

Smeets, W.J.A.J., M.N. Kidjan, andA.J. Jonker (1991) a-MPT does not affect dopamine levels in the periventricular organ of lizards. NeuroReport 2:369-372.

Solberg, Y., W.F. Silverman, and Y. Pollack (1993) Prenatal ontogeny of tyrosine hydroxylase gene expression in the rat ventral mesencephalon. Dev. Brain Res. 739-97.

Specht, L.A., V.M. Pickel, T.H. Joh, and D.J. Reis (1981a) Light-microscopic immunocytochemical localization of tyrosine hydroxylase in prenatal rat brain. I. Early ontogeny. J. Comp. Neurol. 199t233-253.

Specht, L.A., V.M. Pickel, T.H. Joh, and D.J. Reis (1981b) Light-microscopic immunocytochemical localization of tyrosine hydroxylase in prenatal rat brain. 11. Late ontogeny. J. Comp. Neurol. 199:255-276.

Teitelman, G., H. Baker, T.H. Joh, and D.J. Reis (1979) Appearance of catecholamine-synthesizing enzymes during development of rat sympa- thetic nervous system: Possible role of tissue environment. Proc. Nat. Acad. Sci. USA 76.309-13.

L. MEDINA ET AL.

Teitelman, G., M.D. Gershon, T.P. Rothman, T.H. Joh, and D.J. Reis (1981) Proliferation and distribution of cells that transiently express a catechol- aminergic phenotype during development in mice and rats. Dev. Biol. 86:348-355.

Tennyson, V., C. Mytilineou, and R.E. Barrett (1973) Fluorescence and electron microscopic studies of the early development of the substantia nigra and area ventralis tegmenti in the fetal rabbit. J. Comp. Neurol.

Tillet, Y., and J. Thihault (1987) Early ontogeny of catecholaminergic structures in the sheep brain. Anat. Embryol. 177:173-181.

Todd, R.D. (1992) Neural development is regulated by classical neurotrans- mitters: Dopamine D2 receptor stimulation enhances neurite outgrowth. Biol. Psychiatry 31394-807.

Trujillo, C.M. (1982) Ontogenesis de 10s Nucleos Talamicos en Gallotia galloti (Reptil, Lacertidae): Estudio Estructural y Ultrastructural. Ph.D. Dissertation, Universidad de La Laguna, Spain.

Ugrumov, M.V., J. Taxi, A. Tixier-Vidal, J. Thibault, and M.S. Mitskevich (1989) Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. An atlas of neuronal cell bodies. Neuroscience 29:135-156.

Vaage, S. (1969) Segmentation of the primitive neural tube in chick embryos. A morphological, histochemical and autoradiographical investigation. Adv. Anat. Embryol. Cell Biol. 41:l-88.

Verney, C., P. Gaspar, A. Febvret, and B. Berger (1988) Transient tyrosine hydroxylase-like immunoreactive neurons contain somatostatin and substance P in the developing amygdala and bed nucleus of the stria terminalis of the rat. Dev. Brain Res. 42t45-58.

Verney, C., N. Zecevic, B. Nikolic, C. Alvarez, and B. Berger (1991) Early evidence of catecholaminergic cell groups in 5- and 6-week-old human embryos using tyrosine hydroxylase and dopamine-P-hydroxylase immu- nohistochemistry. Neurosci. Lett. 131:121-124.

Voorn, P., A. Kalsbeek, B. Jorritsma-Byham, and H.J. Groenewegen (1988) The pre- and postnatal development of the dopaminergic cell groups in the ventral mesencephalon and the dopaminergic innervation of the striatum of the rat. Neuroscience 25:857-887.

Wolters, J.G., H.J. Ten Donkelaar, and A.A.J. Verhofstad (1984) Distribu- tion of catecholamines in the brain stem and spinal cord of the lizard Varanus exanthematicus: An immunohistochemical study based on the used of antibodies to tyrosine hydroxylase. Neuroscience 13t469493.

Yanes, C.M., M.A. Perez-Batista, J.M. Martin-Trujillo, M. Monzon, and A. Marrero (1987) Anterior dorsal ventricular ridge in the lizard: Embry- onic development. J. Morphol. 194~55-64.

Yanes, C., M.A. Perez-Batista, J.M. Martin-Trujillo, M. Monzon, and A. Rodriguez (1989) Development of the ventral striatum in the lizard Gallotia galloti. J. Anat. 164~93-100.

Yurkewicz, L., M. Marchi, J.M. Lauder, and E. Giacobini (1981) Develop- ment and aging of noradrenergic cell bodies and axon terminals in the chicken. J. Neurosci. Res. 6:621-641.

149:233-258.