Immunohistochemical localization of neuropeptide FF-like in the brain of the turtle: relation to catecholaminergic structures.

A previous study in the lizard Gekko gecko has revealed that neuropeptide FF (NPFF, a neuropeptide involved in nociception, cardiovascular regulation, and endocrine function) is widely distributed throughout the brain and spinal cord. Although the distribution of NPFF immunoreactivity shares many features with that found in other vertebrates, it was noted that Gekko shared more features with anamniotes in terms of number of cell groups, more elaborate networks of fibers, and lack of colocalization with catecholamines, than with mammals. To assess the primitive or derived character of these features, NPFF and tyrosine hydroxylase (TH) antibodies have been applied to the brain and spinal cord of the turtle, Pseudemys scripta elegans, which belongs to a different radiation of reptiles. As in Gekko, major NPFF-ir cell groups were found in the diagonal band nucleus of Broca and in the hypothalamus, whereas additional cells were identified in the anterior olfactory nucleus, lateral and dorsal cortices, dorsal ventricular ridge, and the intergeniculate leaflet formation. Notable differences are the presence of NPFF-ir cells in the medial cortex and striatum of Pseudemys, which are lacking in Gekko. On the other hand, no NPFF-ir cells could be detected in the septal region and dorsal horn of the spinal cord in Pseudemys. Double staining with NPFF and TH antibodies revealed an intimate relationship between NPFF-ir and TH-ir structures but colocalization could not be established. In conclusion, the distribution of NPFF in the brain of Pseudemys has corroborated previous results in Gekko, but also revealed some notable species differences.

Origin and development of descending catecholaminergic pathways to the spinal cord in amphibians.

The origin and development of the supraspinal catecholaminergic (CA) innervation of the spinal cord was studied in representative species of the three amphibian orders (Anura: Xenopus laevis and Rana perezi; Urodela: Pleurodeles waltl; Gymnophiona: Dermophis mexicanus). Using retrograde dextran amine tracing in combination with tyrosine hydroxylase (TH)-immunohistochemistry, we showed that only four brain centers contribute to the CA innervation of the adult spinal cord: (1) the ventrolateral component of the posterior tubercle, (2) the periventricular nucleus of the zona incerta, (3) the locus coeruleus, and (4) the nucleus of the solitary tract (except for gymnophionans). The pattern observed is largely similar in all amphibian species studied. The development of the CA innervation of the spinal cord was studied with in vitro double labeling methods in Xenopus laevis tadpoles. At stage 40/41, the first CA neurons projecting to the spinal cord were found to originate in the posterior tubercle. At stage 43, spinal projections were found from the periventricular nucleus of the zona incerta and the locus coeruleus, whereas spinal projections from the nucleus of the solitary tract were not observed before stage 53. These results demonstrate a temporal sequence in the appearance of the CA cell groups projecting to the anuran spinal cord, organized along a rostrocaudal gradient.

Vasotocin and mesotocin in the brains of amphibians: state of the art.

Immunohistochemical studies during the last decade have revealed elaborate systems of vasotocinergic (AVT) and mesotocinergic (MST) neuronal elements in the brain of a variety of amphibians including anurans, urodeles, and gymnophionans. Apart from a well-developed hypothalamo-hypophysial system, the antibodies demonstrated the existence of extrahypothalamic AVT- and MST-immunoreactive cell groups as well as extensive extrahypothalamic networks of immunoreactive fibers. The wide distribution of AVT- and MST-immunoreactive fibers throughout the brains of amphibians suggests that the two neuropeptidergic systems are involved not only in hypothalamo-hypophysial interactions, but also in a variety of other brain functions. Moreover, there is now evidence that sex-related differences occur in amphibians as previously shown for amniotes. It should be noted, however, that substantial variation occurs in the relative densities of AVT- and MST-immunoreactive fibers and number of cells between species, even within a single order of amphibians. Similar observations have been made in other classes of vertebrates and prompt us, therefore, to critically evaluate conclusions with respect to specific functions of AVT and MST in the central nervous system of vertebrates.

Immunohistochemical localization of DARPP-32 in the brain of the lizard, Gekko gecko: co-occurrence with tyrosine hydroxylase.

To assess the relationship between dopaminergic neuronal structures and dopaminoceptive structures in a reptile, single and double immunohistochemical procedures with antibodies directed against DARPP-32 (dopamine- and cAMP-regulated phosphoprotein with an apparent molecular mass of 32,000 daltons),a phosphoprotein related to the dopamine D(1)-receptor, and tyrosine hydroxylase (TH) were applied to the brain of the lizard, Gekko gecko. The DARPP-32 antibody yielded a well-differentiated pattern of staining in the brain of Gekko. In general, areas that are densely innervated by TH-immunoreactive, putative dopaminergic fibers, such as the nucleus accumbens, striatum, dorsal ventricular ridge, and amygdaloid complex, display strong immunoreactivity for DARPP-32 in somata and neuropil. Distinct cellular DARPP-32 immunoreactivity was also found in the lateral cortex, ventral hypothalamus, habenula, central nucleus of the torus semicircularis, midbrain tectum, parvicellular isthmic nucleus, raphe nuclei, caudal rhombencephalic tegmentum, and spinal cord. Striatal projections to the midbrain and their target, i.e., the substantia nigra pars reticulata, were found to be strongly immunoreactive. Double immunofluorescence staining revealed that dopaminergic cells generally do not stain for DARPP-32, except for cells in the ventral hypothalamus and at caudal rhombencephalic levels. In conclusion, the distribution of DARPP-32 in the brain of the lizard Gekko gecko largely resembles the pattern observed in birds and mammals, at least as far as basal ganglia structures are concerned. On the other hand, there are several specific features of DARPP-32 distribution in the gekkonid brain that deserve further attention, such as cellular colocalization of DARPP-32 and TH immunoreactivity in hypothalamic and caudal rhombencephalic areas, and cellular DARPP-32 immunoreactivity in the tectum and central nucleus of the torus semicircularis of the midbrain, the superior and inferior raphe nuclei, and the spinal cord.

Descending supraspinal pathways in amphibians. II. Distribution and origin of the catecholaminergic innervation of the spinal cord.

Immunohistochemical studies with antibodies against tyrosine hydroxylase, dopamine, and noradrenaline have revealed that the spinal cord of anuran, urodele, and gymnophionan (apodan) amphibians is abundantly innervated by catecholaminergic (CA) fibers and terminals. Because intraspinal cells occur in all three orders of amphibians CA, it is unclear to what extent the CA innervation of the spinal cord is of supraspinal origin. In a previous study, we showed that many cell groups throughout the forebrain and brainstem project to the spinal cord of two anurans (the green frog, Rana perezi, and the clawed toad, Xenopus laevis), a urodele (the Iberian ribbed newt, Pleurodeles waltl), and a gymnophionan (the Mexican caecilian, Dermophis mexicanus). To determine the exact site of origin of the supraspinal CA innervation of the amphibian spinal cord, retrograde tracing techniques were combined with immunohistochemistry for tyrosine hydroxylase in the same sections. The double-labeling experiments demonstrated that four brain centers provide CA innervation to the amphibian spinal cord: 1.) the ventrolateral component of the posterior tubercle in the mammillary region, 2.) the periventricular nucleus of the zona incerta in the ventral thalamus, 3.) the locus coeruleus, and 4.) the nucleus of the solitary tract. This pattern holds for all three orders of amphibians, except for the CA projection from the nucleus of the solitary tract in gymnophionans. There are differences in the strength of the projections (based on the number of double-labeled cells), but in general, spinal functions in amphibians are controlled by CA innervation from brain centers that can easily be compared with their counterparts in amniotes. The organization of the CA input to the spinal cord of amphibians is largely similar to that described for mammals. Nevertheless, by using a segmental approach of the CNS, a remarkable difference was observed with respect to the diencephalic CA projections.

Catecholamine systems in the brain of vertebrates: new perspectives through a comparative approach.

A comparative analysis of catecholaminergic systems in the brain and spinal cord of vertebrates forces to reconsider several aspects of the organization of catecholamine systems. Evidence has been provided for the existence of extensive, putatively catecholaminergic cell groups in the spinal cord, the pretectum, the habenular region, and cortical and subcortical telencephalic areas. Moreover, putatively dopamine- and noradrenaline-accumulating cells have been demonstrated in the hypothalamic periventricular organ of almost every non-mammalian vertebrate studied. In contrast with the classical idea that the evolution of catecholamine systems is marked by an increase in complexity going from anamniotes to amniotes, it is now evident that the brains of anamniotes contain catecholaminergic cell groups, of which the counterparts in amniotes have lost the capacity to produce catecholamines. Moreover, a segmental approach in studying the organization of catecholaminergic systems is advocated. Such an approach has recently led to the conclusion that the chemoarchitecture and connections of the basal ganglia of anamniote and amniote tetrapods are largely comparable. This review has also brought together data about the distribution of receptors and catecholaminergic fibers as well as data about developmental aspects. From these data it has become clear that there is a good match between catecholaminergic fibers and receptors, but, at many places, volume transmission seems to play an important role. Finally, although the available data are still limited, striking differences are observed in the spatiotemporal sequence of appearance of catecholaminergic cell groups, in particular those in the retina and olfactory bulb.

Evolution of the basal ganglia: new perspectives through a comparative approach.

The basal ganglia (BG) have received much attention during the last 3 decades mainly because of their clinical relevance. Our understanding of their structure, organisation and function in terms of chemoarchitecture, compartmentalisation, connections and receptor localisation has increased equally. Most of the research has been focused on the mammalian BG, but a considerable number of studies have been carried out in nonmammalian vertebrates, in particular reptiles and birds. The BG of the latter 2 classes of vertebrates, which together with mammals constitute the amniotic vertebrates, have been thoroughly studied by means of tract-tracing and immunohistochemical techniques. The terminology used for amniotic BG structures has frequently been adopted to indicate putative corresponding structures in the brain of anamniotes, i.e. amphibians and fishes, but data for such a comparison were, until recently, almost totally lacking. It has been proposed several times that the occurrence of well developed BG structures probably constitutes a landmark in the anamniote-amniote transition. However, our recent studies of connections, chemoarchitecture and development of the basal forebrain of amphibians have revealed that tetrapod vertebrates share a common pattern of BG organisation. This pattern includes the existence of dorsal and ventral striatopallidal systems, reciprocal connections between the striatopallidal complex and the diencephalic and mesencephalic basal plate (striatonigral and nigrostriatal projections), and descending pathways from the striatopallidal system to the midbrain tectum and reticular formation. The connectional similarities are paralleled by similarities in the distribution of chemical markers of striatal and pallidal structures such as dopamine, substance P and enkephalin, as well as by similarities in development and expression of homeobox genes. On the other hand, a major evolutionary trend is the progressive involvement of the cortex in the processing of the thalamic sensory information relayed to the BG of tetrapods. By using the comparative approach, new insights have been gained with respect to certain features of the BG of vertebrates in general, such as the segmental organisation of the midbrain dopaminergic cell groups, the occurrence of large numbers of dopaminergic cell bodies within the telencephalon itself and the variability in, among others, connectivity and chemoarchitecture. However, the intriguing question whether the basal forebrain organisation of nontetrapods differs essentially from that observed in tetrapods still needs to be answered.

Integration and segregation of limbic cortico-striatal loops at the thalamic level: an experimental tracing study in rats.

The frontal lobe and the basal ganglia are involved in a number of parallel, functionally segregated circuits. Information is thought to pass from distinct parts of the (pre)frontal cortex, via the striatum, the pallidum/substantia nigra and the thalamus, back to the premotor/prefrontal cortices. Currently, different views exist as to whether these circuits are to be considered as open or closed loops, as well as to the degree of interconnection between different circuits. The main goal of the present study is to answer some of these questions for the limbic corticostriatal circuits. The latter circuits involve the nucleus accumbens, the ventral pallidum/dorsomedial substantia nigra pars reticulata, the medial parts of the mediodorsal and ventromedial thalamic nuclei and the prefrontal cortex. Within the nucleus accumbens, a core and a shell region are recognized on the basis of anatomical and functional criteria. The shell of the nucleus accumbens projects predominantly to the mediodorsal, the midline and the reticular thalamic nuclei via the ventral pallidum, whereas the core reaches primarily the medial part of the ventromedial thalamic nucleus, the intralaminar and mediodorsal thalamic nuclei via a relay in the dorsomedial substantia nigra pars reticulata. By means of double labeling experiments with injections of anterograde tracers in both the ventral pallidum and the substantia nigra of rats, we were able to demonstrate that circuits involving the shell and the core of the nucleus accumbens remain largely segregated at the level of the thalamus. Only restricted areas of overlap of ventral pallidal and reticular nigral projections occur in the mediodorsal and ventromedial thalamic nuclei, which allows for a limited degree of integration, at the thalamic level, of information passing through the two circuits.

Evidences for shared features in the organization of the basal ganglia in tetrapods: studies in amphibians.

In a series of recent studies, the organization of the basal ganglia of amphibians, more in particular their connectivity and chemoarchitecture, has been thoroughly analyzed. The pattern of organization found for the amphibian basal ganglia includes dorsal and ventral striatopallidal systems, reciprocal connections between the striatopallidal complex and structures derived from the diencephalic and mesencephalic parts of the basal plate (striatonigral and nigrostriatal projections), and descending pathways from the striatopallidal system to the midbrain tectum and the reticular formation of the brain stem. A comparative analysis of the organization of the basal ganglia in tetrapods strongly supports the notion that a primitive pattern was most likely present in ancestral tetrapods, and that many features can still be recognized in extant amphibians and amniotes.

Cholinergic and catecholaminergic neurons relay striatal information to the optic tectum in amphibians.

In the amphibians Rana perezi and Xenopus laevis, the involvement of cholinergic and catecholaminergic neurons in the relay of basal ganglia inputs to the tectum was investigated. Tract-tracing experiments, in which anterograde tracers were applied to the basal ganglia and retrograde tracers to the optic tectum, were combined with immunohistochemistry for choline acetyltransferase and tyrosine hydroxylase. The results of these experiments suggest that dopaminergic neurons of the suprachiasmatic nucleus and pretectal region, noradrenergic cells of the locus coeruleus and the cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei mediate at least part of the basal ganglia input to the tectum in anurans.

Evolution of the basal ganglia in tetrapods: a new perspective based on recent studies in amphibians.

It has been postulated frequently that the fundamental organization of the basal ganglia (BG) in vertebrates arose with the appearance of amniotes during evolution. An alternative hypothesis, however, is that such a condition was already present in early anamniotic tetrapods and, therefore, characterizes the acquisition of the tetrapod phenotype rather than the anamniotic-amniotic transition. Re-examination of the BG organization in tetrapods in the light of recent findings in amphibians strongly supports the notion that elementary BG structures were present in the brain of ancestral tetrapods and that they were organized according to a general plan shared today by all extant tetrapods.

Basal ganglia organization in amphibians: evidence for a common pattern in tetrapods.

The results of recent studies investigating the connections and chemoarchitecture of the basal forebrain of amphibians provide strong evidence that tetrapod vertebrates share a common pattern of basal ganglia organization. This pattern includes the existence of dorsal and ventral striatopallidal systems, reciprocal connections between the striatopallidal complex and the diencephalic and mesencephalic basal plate (striato-nigral and nigro-striatal projections), and descending pathways from the striatopallidal system to the midbrain tectum and reticular formation. The connectional similarities are parallelled by similarities in the distribution of chemical markers of striatal and pallidal structures such as dopamine, substance P and enkephalin. Moreover, studies of development and expression of homeobox genes have given further support to the notion that both amniotic and anamniotic tetrapods have a common pattern of basal ganglia organization. A new nomenclature of basal forebrain structures in amphibians is proposed which reflects our current understanding of basal ganglia organization in this class of vertebrates.

Basal ganglia organization in amphibians: chemoarchitecture.

Recent studies dealing with the investigation of the afferent and efferent connections of the basal ganglia of amphibians have revealed many similarities with basal ganglia structures of amniotes. In a further step, the chemoarchitecture of basal ganglia of the frog Rana perezi has been investigated. For use as main markers of amphibian basal ganglia structures, antibodies against tyrosine hydroxylase, substance P, and enkephalin were selected. Moreover, the distributions of nitric oxide synthase (nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry), calretinin, dopamine-beta-hydroxylase, choline acetyltransferase, mesotocin, vasotocin, somatostatin, neuropeptide Y, neuropeptide FF, and serotonin were studied to corroborate a comparison with both basal ganglia and amygdaloid structures of amniotes. On the basis of connections and chemoarchitecture, a striatum proper, nucleus accumbens, dorsal and ventral pallidum, bed nucleus of the stria terminalis, and amygdaloid complex have been identified. Accordingly, a new terminology is proposed that is in line with our current understanding of basal ganglia organization in amphibians.

Basal ganglia organization in amphibians: development of striatal and nucleus accumbens connections with emphasis on the catecholaminergic inputs.

To broaden our insight into the organization of the basal ganglia of amphibians, the development of the connections of the striatum and the nucleus accumbens was studied by means of tract-tracing techniques based on the transport of biotinylated dextran amines. In a number of experiments, these techniques were combined with tyrosine hydroxylase immunohistochemistry to identify the sources of catecholaminergic inputs to the striatum and the nucleus accumbens. Already at late embryonic stages, the basal telencephalon receives inputs from cells located in the amygdala, the thalamus, the suprachiasmatic nucleus, the raphe nucleus, and the rhombencephalic reticular formation. At these stages, the rostral part of the posterior tubercle seems to be the only source of the dopaminergic input to the basal telencephalon. During premetamorphosis, not only a differentiation between connections of the striatum and the nucleus accumbens could be made, but new sources of inputs were also detected in the mesencephalic and isthmic tegmentum, the parabrachial nucleus, and the nucleus of the solitary tract. Double-labeling experiments revealed that, at these stages, in addition to the posterior tubercle, cells within the mesencephalic tegmentum, the locus coeruleus, and the solitary tract nucleus contribute to the catecholaminergic innervation of the basal forebrain. During prometamorphic stages, a gradual increase occurs in the number of cells that project to the basal telencephalon. At the beginning of the metamorphic climax, the organization of the basal ganglia afferents largely resembles the pattern observed in juveniles and adults. Remarkably, during larval stages, the cells that contribute to the dopaminergic innervation of the basal forebrain show a rostrocaudal gradient in time of appearance. Moreover, the dopaminergic fibers reach the striatum earlier than the nucleus accumbens, and they precede markedly the development of the efferent connections of both brain structures. These developmental aspects are easily correlated with the situation in amniotes; therefore, the notion that amphibians share an essentially similar pattern of basal ganglia organization with other tetrapods is further strengthened.

Distribution of choline acetyltransferase immunoreactivity in the brain of anuran (Rana perezi, Xenopus laevis) and urodele (Pleurodeles waltl) amphibians.

Because our knowledge of cholinergic systems in the brains of amphibians is limited, the present study aimed to provide detailed information on the distribution of cholinergic cell bodies and fibers as revealed by immunohistochemistry with antibodies directed against the enzyme choline acetyltransferase (ChAT). To determine general and derived features of the cholinergic systems within the class of Amphibia, both anuran (Rana perezi, Xenopus laevis) and urodele (Pleurodeles waltl) amphibians were studied. Distinct groups of ChAT-immunoreactive cell bodies were observed in the basal telencephalon, hypothalamus, habenula, isthmic nucleus, isthmic reticular formation, cranial nerve motor nuclei, and spinal cord. Prominent plexuses of cholinergic fibers were found in the olfactory bulb, pallium, basal telencephalon, ventral thalamus, tectum, and nucleus interpeduncularis. Comparison of these results with those obtained in other vertebrates, including a segmental approach to correlate cell populations, reveals that the cholinergic systems in amphibians share many features with amniotes. Thus, cholinergic pedunculopontine and laterodorsal tegmental nuclei could be identified in the amphibian brain. The finding of weakly immunoreactive cells in the striatum of Rana, which is in contrast with the condition found in Xenopus, Pleurodeles, and other anamniotes studied so far, has revived the notion that basal ganglia organization is more preserved during evolution than previously thought.

Afferent connections of the nucleus accumbens of the snake, Elaphe guttata, studied by means of in vitro and in vivo tracing techniques in combination with TH immunohistochemistry.

The aim of the present study was to determine the afferent connections of the nucleus accumbens in snakes, in particular its catecholaminergic input. For that purpose, in vitro and in vivo applications of retrograde tracers in the nucleus accumbens of Elaphe guttata were combined with tyrosine hydroxylase (TH) immunohistochemistry. Both techniques revealed telencephalic inputs to the nucleus accumbens originating from the diagonal band of Broca, ventral pallidum, amygdaloid complex, and dorsal cortex. Major diencephalic inputs arise from the dorsomedial thalamic nucleus and the hypothalamus. In the brainstem, a few retrogradely labeled cells were observed in the raphe nucleus and the locus coeruleus. Considerably more cells were found in the midbrain tegmentum. Within the confines of the locus coeruleus and, in particular, the midbrain tegmentum, retrogradely labeled cells stained also for TH suggesting that those areas constitute the major catecholaminergic input to the nucleus accumbens of snakes. The experimental approach used in the present study, in particular the in vitro technique, seems to be very suited for studying the development of basal ganglia organization of reptiles in the near future.

Basal ganglia organization in amphibians: efferent connections of the striatum and the nucleus accumbens.

As a further step in unraveling the organization of the basal ganglia of amphibians, the efferent connections of the striatum and the nucleus accumbens have been studied in the brains of the anurans, Rana perezi and Xenopus laevis, and the urodele, Pleurodeles waltl, by using biotinylated or fluorescent dextran amines as anterograde tracers. A common pattern of efferent connections was observed in both groups of amphibians, but those in the anurans were more elaborate. Striatal efferent fibers were found to reach the lateral and medial amygdala, the anterior and posterior entopeduncular nuclei, several thalamic nuclei, the dorsomedial posterior tubercle, the pretectum, the optic tectum, the torus semicircularis, the pontomesencephalic reticular formation, and the caudal brainstem. Efferent fibers of the nucleus accumbens project to the medial amygdala, the preoptic area, the ventral hypothalamic nucleus, the dorsomedial posterior tubercle, the medial tegmental area, the pontomesencephalic reticular formation, and the raphe. In addition, the study has revealed the existence of intrinsic connections within the ventral telencephalic wall, suggesting a possible further compartmentalization of the amphibian basal forebrain. In conclusion, the results of the present study corroborate the notion that the basal ganglia of amphibians share many features with their presumed homologues in amniotes.