S14.5: Phylogenetic principles in avian brain organisation

Anatomy-Cell Biology, Faculty of Medicine, University of Giessen, Aulweg 123, 35392 Giessen, Germany, fax 49 641 99 47009, Blaehser@anatomie.med.uni-giessen.de

The cytoarchitectural organisation realised in the avian brain, mostly in the forebrain, is difficult to decipher. In an attempt to relay weakly structured neuronal groups to functional, mainly visceral and sensory circuitries, the spatial distribution of neuropeptide-producing systems was studied. Because of the confusing organisational aspect even of these systems in the avian brain, a comparative onto- and phylogenetic research was undertaken.

In the course of these immunocytochemical studies we were among the very first to use serially cut whole brain sections of agnathan, elasmobranch fish, amphibian, reptile and avian brains. By means of these complete series of brain sections, oriented in the horizontal, sagittal and frontal planes, the spatial distribution of numerous neuropeptide-producing (npp) systems could be described including the morphological equivalents of the peptide's double function as neuromodulators and as neurohormones (Blähser 1982; 1983; 1984; 1988a; Blähser & Dubois 1980; Blähser et al. 1978; Goosens et al. 1977; Kuenzel & Blähser 1991; 1994). Phylogenetically directed comparisons revealed an evolutionary topographical constancy of the neuropeptide-reacting structures, including the presence of mixed fibre networks in the areas of contact (Blähser 1983; 1985; 1987a; 1987b; 1988b; 1989; 1992; 1994; 1995; Blähser et al. 1989; Blähser & Fellmann 1985; Blähser & Kuenzel 1991; Blähser & Ueck 1983; Neubert 1998). The demonstration of densely packed fibre networks belonging to different neuropeptide systems and occurring mainly in areas of visceral control showed the uselessness of experimental interventions into these areas with the aim to influence or eliminate only one of the participating systems.

The ontogenetic studies performed on amphibian and avian brains gave no indication of brain development in view of understanding the fine organisation of the adult organ, due to the late appearance of immunoreactivity of neuropeptides (Blähser & Heinrichs 1982; Heinrichs 1984; Lang 1991; Luhnenschloss 1987; 1988).

The results of the comparative studies showed that each peptide system repeats in all vertebrate classes a characteristic basic pattern as an integral component of the brain's overall organisation: constancy in location and number of neuropeptide perikaryal groups; constancy of extra- and intracerebral projection areas; constancy in location and composition of the areas of contact; constancy in the spatial relationship between some neuropeptide systems and visceral and sensory circuitries.

In the following we will focus on results related to know functional cicuitries as well as indications concerning selected areas of avian brain development.

Studies were performed mostly by light microscopical immunocytochemical techniques. Brains of Hagfish, Myxine glutinosa; Brook Lamprey, Lampetra planeri; Marbled Electric Ray, Torpedo marmorata; African Clawed Toad, Xenopus laevis; Frog, Rana temporaria; Axolotl; Ambystoma mexicanum; Ceylon Caecilian, Ichthyophis glutinosus; Common Cobra, Naja n. naja; Ringed Snake, Natrix natrix; Ganges Softshell Turtle, Tryonix gangeticus; Indian Lizard, Calotes versicolor; Tokay Gecko, Gekko gecko; Japanese Quail, Coturnix coturnix japonica; Domestic Chicken, Gallus gallus forma domestica; Pigeon, Columba livia; White-Crowned Sparrow, Zonotrichia leucophrys gambelii; Peking Duck, Anas platyrhinchos were submitted to fixation by perfusion or immersions, embedded in paraffin or in gelatin and sectioned serially in horizontal, frontal and sagittal planes. For topographical orientation, sections were stained according to Kluver & Barrera (1953). For immunocytochemical reactions, antisera directed against numerous neuropeptides were raised in rabbits by means of synthetic antigens designed corresponding to the mammalian molecular form. The antisera were tested by enzyme-linked immuno-assays (ELISA; Blähser et al. 1989) in order to determine the specificity of the antiserum as well as the intramolecular location of the most heavily reacting epitope. The test of appropriateness in view of phylogenetic studies was performed on brain sections. The immunocytochemical reactions were performed by use of the PAP-technique of Sternberger (1979) and the ABC-technique of Hsu et al. (1981). (Details concerning all technical and methodological aspects can be obtained from the corresponding author).

The polyclonal antisera directed against the mammalian molecular form of numerous neuropeptides always reacted specifically in the brains studied. Only neurotensin-antiserum was not bound in the Hagfish brain.

The results of the comparative immunocytochemical studies revealed the existence of some fundamental morphological characteristics of the neuropeptide-producing systems:

(1) Immunoreactive perikarya and their fibre projections mostly form a widespread but continuous system, demonstrating that each neuropeptide is represented by its own peculiar distribution pattern of structural components. The entity of all structures reacting with a specific antiserum is morphologically defined as a neuropeptide system.

(2) All neuropeptide systems, except ACTH, project within the brain as well as into neurohemal areas, demonstrating thereby the morphological equivalents of their double function as neuromodulatory and neurohormal agents.

(3) Location and arrangement of peptide-producing cell groups are independent of the 'classical' nuclei revealed by nissl and fibre stains.

(4) Fibres from different peptide systems project into well-delimited central nervous areas, forming overlapping fibre networks contacting local, non-immunoreactive perikarya: areas of contact or of integration. These areas are mainly related to systems of visceral control (e.g. caudal brainstem, medio-basal hypothalamus) or to limbic circuitries (septal and preoptic region). They occur independently of local fibre concentrations due to recognised spatial arrangements, as seen, e.g. in the bottleneck of the median eminence or in association to larger fibre bundles. The number of the areas of contact, their spatial relationship to landmark structures (ventricles, commissures, etc.), and the respective composition of participating peptide systems are always identical.

(5) Independent of the final location of peptide-producing perikaryal groups in the adult brain, the target areas of the fibre projections are identical in all vertebrate brains. Due to this conservation, the final perikaryal location indicates the local development of the concerned brain region.

(6) The fusion of originally separated peptide-producing cell groups (e.g. the diencephalic NPY immunoreactive cells) and the segregation of primarily undivided cell populations into numerous sub-groups (e.g. the diencephalic AVT-reacting cells) depend on the structural development of the concerned region.

The bifunctional role of neuropeptides as neuromodulatory and neurohormonal agents implies a seemingly confusing aspect, namely, of their spatial distribution in the avian brain. Phylogenetic studies on less complex vertebrate brains have helped to elucidate this problem in some points and have demonstrated a nearly unchanged location of immunoreactive (ir) structures from cyclostomes to mammals (Blähser 1983). Hence, it may be inferred that the presence of neuropeptides in vertebrates remains related mainly to phylogenetically old mechanisms. However, it should be bone in mind that the final functional results of neuropeptide actions depend on the evolutionary changes of their intracerebral target neurons.

The neuropeptide-producing cell combines the properties of electrical conductivity and the secretion of transmitters and peptides. It can be assumed that peptidergic neurons represent the ancestral form of neurons, appearing for the first time in a coelenterate and possessing from the very beginning all of the above-cited properties (Lentz 1968). At this early stage of evolution, the nervous function is identical with neurosecretion. Remnants of this very first stage are preserved even in birds by cerebrospinal-fluid contacting neurons.

The conservation of very ancestral morphological aspects of neuropeptide systems in the avian brain is reflected functionally by their association to basic visceral and sensory circuitries, and by their sparcity in pallial, and pallium-derived areas. The lack of a consistent projection of peptide-ir fibres into, e.g. the pallial centre of vocal control demonstrates this assumption. In contrast to this sparseness of peptides, the pallial centres of dorsally positioned (high) control are targets of dense projections from transmitter systems, appearing later than neuropeptides during brain evolution. However, at lower levels of control, e.g. the thalamic relay centres integrating somatosensory and visual information into the ascending pathways of vocal control, the afferent fibres belonging to several neuropeptide systems, mainly Neuropeptide Y (NPY), Somatostatin (SOM), Neurotensin (NT), Corticotropin-releasing factor (better: Corticoliberin; CRF) and Vasoactive-intestinal peptide (VIP) are consistent and comparable in all vertebrate brains studied. The same holds true for the intercollicular nucleus involved in vocal production via its influence on the motor nuclei of the hypoglossal and the glossopharyngeal nerves controlling the activity of larynx and syrinx muscles, as well as for the parabrachial nucleus involved in respiratory activity; these nuclei are reached by fibres ir for Arginine-Vasotocin (AVT), Substance P (SP), VIP and Methionine-Enkephalin (ME) (Kolsch et al. 1997; see also Wild 1997).

Another example for the strong association of neuropeptides to centres of visceral control is represented by the dorsal vagal complex, a very consistent area of contact in all vertebrate classes. The complex is composed of a viscero-motor nucleus, Nc. motorius dorsalis n. vagi, a viscero-sensory portion, the Nc. tractus solitarii and a neurohemal zone, the Area postrema. The neuropeptides studied, ME and Leucine Enkephalin (LE), SP, VIP, NT and AVT each demonstrate a distinct spatial distribution related to the subnuclear organisation of the complex. The viscero-motor portion of the complex, innervating glands and smooth muscle of the upper gastro-intestinal tract, receive mainly dense NT- and SP-immunoreactive (ir) projections, whereas just the ventral-most subnucleus is densely labelled for all peptides investigated. The viscero-sensory part, Nc. tractus solitarii, receives dense NT-, SP- and VIP-ir projections into its gastro-intestinal-related area, receives SP-ir fibres into its pulmonary-associated area and receives NT, SP, VIP and M-Enk fibres into its lateral tier related to cardiovascular and pulmonary control. The Area postrema, involved in baroreceptive and gastro-intestinal reflex circuits, receives strong VIP- and Enk-ir fibre projections (for details concerning the nuclear subdivision of the complex and the area-related neuropeptide afferences, see Neubert 1998). Comparative studies resulted in the demonstration of very consistent SP-ir afferents into the vagal complex area also in Hagfish, Frog, Tokay Gecko, Indian Lizard and Ganges Softshell Turtle (Neubert et al. 1996).

At a superior level of integration in the ventromedial and lateral hypothalamus of all vertebrates studied, excluding the tuberal region, another area of contact characterised by the presence of densely overlapping fibre networks belonging mainly to the neuropeptide systems VIP, NT, CRF, NPY, SP, SOM and ME is relayed to non-ir nuclei involved in the control of reproductive and food intake behaviour. These centres are, among others, components of a visceral forebrain system, also related to pulmonary and cardiovascular functions (birds: Kuenzel & Blähser 1993; rat: Romijn et al. 1997).

Phylogenetically conserved projections into centres of main sensory control and integration are particular evident in well-delimited layers of the optic tectum, being reached by fibres ir for NPY, SP, NT and VIP (VIP in the chicken: Blähser et al. 1991; Kuenzel & Blähser 1994). Some nuclei relayed to different optic circuitries receive also peptidergic inputs: The ventral portion of the lateral geniculate nucleus contains dense NPY-ir fibre nets and the pretectal nucleus, connected to visual-motor pathways, receives NT-ir afferents. Structures related to olfaction gain their sensory information via the terminal and olfactory nerves. Two peptidergic systems are strongly related to nearly all levels of the concerned sensory pathways: SP and gonadotropin releasing hormone (GnRH). Sp-ir fibres are mainly related to primary and secondary olfactory pathways with a predominance of the vomeronasal portions. The GnRH system demonstrates a strong association to centres integrating olfactory information, including areas functionally related to the limbic system. In the blind gymnophionians this association emphasises the involvement of the neuropeptide in mechanisms of adaptation to environmental stimuli. In birds it is at least initially somewhat astonishing. The association is especially evident in the domestic fowl to which a sense of smell is largely denied. However, when the definition of olfaction is not restricted to conscious smelling but includes the unconscious reception of olfactory information, the association points to a similar involvement as in apodans: the connection of olfactory inputs to endocrine, optic, behavioural and vegetative centres (Blähser 1984).

One of the most fascinating aspects of neuropeptide producing (npp) structures in the vertebrate brain is the possibility to deduce from the final location of perikaryal groups or of projection areas to principles of local brain development in different classes and species. The results of comparative studies have led to the concept of topographical displacement (Blähser 1992). The proliferation of the cellular matrix including the presumptive npp cells starts from the periventricular cell layer. The rate of proliferation and the directions of migration result from a multitude of influences, among others, the 'bauplan' of the species and the structural and volume development of the adjacent brain regions. However, some topographical aspects of npp groups never change: the projection areas are constant and a topographically displaced subgroup remains always interconnected by fibre projections to the non-displaced portion of the group. These relationships enable one to deduce the direction of the proliferation from the final location of the cells. The location of the main part of a npp in brains or in brain regions with a restricted ventriculo-fugal migration, as seen in Lampreys, indicates that the crucial point of origin lies in the rostral division of the third ventricle/preoptic recess.

It is assumed that presence or absence of npp cells within telencephalic areas depends on location and extent of the matrix material involved in the very first formation of the lateral telencephalic vesicles containing or lacking presumptive npp material as well as on the extent and direction of proliferating cellular matrix of the lateral ventricular walls (such differences have been discussed by Wicht & Nieuwenhuys (1998) for the development of telencephalic hemispheres in Lampreys and Hagfishes). In this way, the overall presence of NPY-ir cells within the amphibian pallium and the absence or scarcity of such cells in the reptilian and the avian brain can be explained. A proliferation of the cellular matrix originating in the baso-preoptic region into caudo-lateral telencephalic areas seemingly occurs in some reptiles but not in the avian brain. Both brain types are morphologically characterised by the development of what is called the dorsal ventricular ridge (DVR). The origin of the components of this formation is still being discussed (Dubbeldam 1998). The presence of NT-ir perikarya and of VIP-ir fibre networks within the DVR of the Ganges Softshell Turtle and The Tokay Gecko points to the origin of the neuronal material from the matrix located rostral to the third ventricle. The npp cells maintain a fibre-based connection with the ir cells retained in original location and project at short distances into olfaction-associated areas. The short-distance projection is comparable to what can be observed in the brain of the Indian Lizard and in birds. In these animals no NT- or VIP-ir cells are present within the DVR; their NT cells remain in a preoptic position and perform, among others, a short-distance projection into the telencephalon medium. This lack of a rostro-laterally directed proliferation from the preoptic area into the DVR may depend, among others, on the time of the developing hypopallial wall, on the volume of the striatum and the extensive mediobasal telencephalic evagination in birds.

The fusion of originally separated npp cell groups can be demonstrated by means of diencephalic NPY-ir cells. In the brain of the Brook Lamprey, Hagfish and Frog a dorso-lateral NPY-ir group projects into vision-related di- and mesencephalic areas, whereas a ventromedial group lies in close spatial relationship to the paraventricular nucleus. In the Frog a primordial thalamic round nucleus separates both groups. In reptiles the space-demanding volumetric increase of the round nucleus results in an approach of the two groups. In the avian brain with its dramatically increased volume of the round nucleus, both NPY-ir groups fuse. However, due to the conservation of the original projection areas, they can be individually defined. In contrast to such a fusion, an originally compact cell group can be segregated by a volumetric increase of fibre tracts, e.g. the forebrain bundles. In this way, the AVT-ir paraventricular nucleus is subdivided into numerous subunits and several portions of the total cellular population are displaced dorsolaterally (details in: Blähser 1992).

Independent of all changes occurring during evolution, the avian brain reflects morphological principles realized in all vertebrate brains. The comparative study of the primitive neuropeptide-producing structures provides a morphological basis for a better understanding of function-related topographical changes during brain evolution. The importance of conserved fibre-based connections between the neuropeptide-producing systems and 'classical' neuronal groups lies in the maintenance of basic information channels, represented by the neuropeptide systems and providing access to the targeted non-ir neuronal centres for structural development in order to augment the complexity of brain function.

The investigations were supported by several Research Grants from the Deutsche Forschungsgemeinschaft to S.B.. The authors are greatly indebted to Sabine Tasch for excellent technical assistance.

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