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We studied the development of the primary olfactory system of a teleost, the brown trout, with the aims of clarifying whether the caudal projection pertains to the olfactory or to the terminal nerve system, of identifying the brain regions receiving this projection, and of investigating its possible functional significance. As olfactory markers (OMs) we used two polyclonal antibodies (to substance P and to alpha-melanocyte-stimulating hormone) that were found to label the olfactory projection strongly after preadsortion of the antibody with the corresponding antigen (OMs), and as a terminal nerve marker we used an antiserum to FMRF-amide peptide. OM labelling was observed in both perikarya and axons of olfactory neurons. In adults, olfactory neurons projected not only to olfactory glomeruli in the olfactory bulb but also, as has been reported previously, to more caudal targets in the forebrain through the medial olfactory tract. Our results show that these targets include the ventral and commissural nuclei of the area ventralis telencephali, the periventricular preoptic region, and the organum vasculosum laminae terminalis. Glomeruli were not observed before hatching, and the extrabulbar olfactory projections appear late in development. Extensive periventricular preoptic olfactory plexuses and olfactory innervation of the organum vasculosum laminae terminalis did not appear until adulthood. The cells of the ganglion nervus terminalis, which form ganglionic groups along the olfactory nerves, were not stained with these olfactory markers at any developmental stage studied, nor was the medial olfactory tract FMRP-amide peptide immunoreactive. Our results thus confirm the existence of primary olfactory projections to extrabulbar targets in trout. The target regions identified in this study are implicated in sexual behaviour: We discuss the related possibility that, in teleosts, these extrabulbar olfactory projections (rather than projections of the terminal nerve, as is widely held) are the primary mediators of neuroendocrine response to pheromones.

In our initial effort to study the ontogeny of the gastropod nervous system, we used histological techniques to examine the post-embryonic development of cells which exhibit serotoninlike immunoreactivity in Lymnaea (Croll and Chiasson, J. Comp. Neurol. 230:122-142, '89). The present study complements that report by examining the embryonic development of these neurons. The first serotoninlike immunoreactive (SLIR) cells to be detected in the embryos are the paired C4 neurons of the cerebral ganglia. These cells are faintly visible at about 37-38% of embryonic development and have already produced axons which traverse the cerebral commissure. By about 2-3% later the axon tips reach the pedal ganglia and appose the next SLIR cells to appear, the EPe1 neurons. Over the next 30% of development four more pairs of cerebral neurons are added adjacent to the C4 neurons and over ten cells are added to each of the pedal ganglia. At about 70% of development SLIR fibers are first detected in the parietal and visceral ganglia forming the abdominal ring. Around this time the somata of the C1 neurons also first appear in the cerebral ganglia together with their prominent axons projecting to the buccal ganglia. The last 30% of development is marked by a massive addition of SLIR cells (up to 60) in each pedal ganglion. The early appearance of the first SLIR cells suggests that they may be among the first nerve cells to differentiate and that they may play central roles in the formation of the CNS. We hypothesize that most of the animal's neural circuitry is laid down during embryogenesis by a stereotypic ontogenetic program with post-embryonic neurogenesis subserving mostly compensatory and modulatory purposes.

In order to give rise to the enteric nervous system (ENS), cells migrating from the neural crest must find the bowel and cease migrating at appropriate locations within the gut. Previous studies of the development of the ENS in a mutant mouse have led to the hypothesis that laminin in the enteric mesenchyme may act as a signal to crest-derived cells to cease migrating and extend neurites (or glial processes). Implied in this hypothesis is the idea that crest-derived cells, as a prelude to their participation in ganglion formation, acquire a neurally related laminin receptor, which they do not express at pre-enteric stages of migration. As a partial test of this hypothesis, single and double label immunocytochemistry at light and electron microscopic (EM) levels were used to study the expression of cell surface laminin binding proteins by crest-derived cells in the process of migrating to or within the developing chick gut. Two antibodies (called 3070 and alpha-110) raised against neuronal cell surface laminin binding proteins were employed for this purpose. Laminin binding protein immunoreactivity was found to be expressed within the bowel and ganglion of Remak by a subset of crest-derived cells (identified immunocytochemically with NC-1/HNK-1 antibodies) and by all of those developing as neurons (identified immunocytochemically with antibodies to neurofilament-associated proteins). Laminin binding protein immunoreactivity was also found to be expressed in fixed neural structures elsewhere in the embryos, including cranial and spinal roots, nerves, and ganglia. In contrast, laminin binding protein immunoreactivity was not expressed by migrating crest-derived cells in the vicinity of the vagal or sacral regions of the neuraxis (from which the precursors of the ENS take origin); nor was it expressed by juxta-pharyngeal vagal crest-derived cells migrating to the foregut through the caudal branchial arches or by the caudal stream of sacral crest-derived cells approaching the hindgut. EM immunocytochemistry confirmed that laminin binding protein immunoreactivity in the bowel was located on the surfaces of crest-derived cells, and was exhibited both by those cells that could only be distinguished from their neighbors by their NC-1/HNK-1 immunoreactivity and by cells developing as neurons or glia. EM immunocytochemistry also revealed that the surfaces of crest-derived cells migrating through the enteric mesenchyme were contacted by many small osmiophilic "puffs" of laminin-immunoreactive extracellular material. These puffs coincided in location with membrane sites that expressed the immunoreactivity of the laminin binding protein. These observations are consistent with the hypothesis that laminin plays a role in the formation of enteric ganglia.

Vestibular axons form synapses on a restricted area of the lateral dendrite of the Mauthner cell, a large, identified brainstem neuron found in fish and amphibians. The differentiation of the vestibular nerve, medullary neuropil, and Mauthner cell of the axolotl (Ambystoma mexicanum) was studied to understand better the means by which this synaptic specificity arises. The Mauthner cell first extends a medial process and then a lateral dendrite. The latter initially elongates as a simple process and later sends out branches. As the lateral dendrite grows, vestibular axons enter the brainstem to form one of the earliest of several discrete axon fascicles that course longitudinally through the neuropil. The fascicles, many of which are identifiable on the basis of their location and axonal morphology, are the precursors of the longitudinal tracts of the mature salamander. The lateral dendrite grows dorsally over the orthogonally oriented fascicles, making contact with each at a characteristic time and place. The first afferents to form synapses do so on the soma and proximal lateral dendrite; subsequent afferent groups terminate more distally. Axons within a given fascicle form synapses with the Mauthner cell in a discrete and initially homogeneous domain. As dendritic branches form and the organization of the longitudinal fascicles becomes more complex, the homogeneity of axons terminating on a given region of the Mauthner cell surface is lost, but no major rearrangement or migration of terminals is apparent. These observations are consistent with both active recognition and passive spatiotemporal models of synaptic site specificity.

We have examined the formation of myenteric ganglia in the developing avian enteric nervous system. The monoclonal antibody HNK-1 was used to identify neural-crest-derived cells in whole mounts of fore- and midgut of chick embryos. We find that the crest-derived cells extend processes to their neighbors and form a complex network in the wall of the gut. Formation of this network is an unusual behavior of crest-derived cells and suggests the gut microenvironment is critical to this behavior. This cellular network disappears after ablation of the vagal neural crest, indicating the HNK-1-stained cellular network arises from crest-derived cells. The network is found in the gut wall before the vagal nerve fibers are present. This network is first found in the primordium of the proventriculus, distal to the evagination of the lung buds, and progresses just proximal to the yolk stalk at embryonic day (E) 3.5 and almost to the ileocecal junction at E5.5. The number of cells and the complexity of the network decrease in a rostral-caudal direction down the length of the gut at these stages. The leading edge of the network consists of cells serially arranged in longitudinally running strands. The organization of the network changes with increasing embryonic age; we have focused on network changes in the proventriculus. In the primordium of the proventriculus at E3.5, the network consists of a cluster of one or two adjacent crest-derived cells, which extend processes to a number of neighboring crest-derived cells. At E5.5 large increases in the number of cells per cluster and in the length of cellular connectives between clusters are apparent. At E6.5 a crude meshwork of clusters is seen. At E10.5 the arrangement of cell clusters resembles the pattern of ganglia found in the adult myenteric plexus. This network may provide the environmental cues for the differentiation of enteric neurons and a framework for the pattern of ganglia found in the adult enteric nervous system.

The primordium of the compound eye of the moth Manduca sexta was removed by surgery within 12 hours after the molt to the pupa. When the operated animals were examined as pharate adults just prior to emergence from the pupal cuticle, no eye tissue was present on the operated side. Histological examination of the brain at successive developmental stages showed that the volume of laminar neuropil on the operated side increased very little after deafferentation while the volume of the control-side laminar neuropil increased more than 20-fold. The impairment of development of the lamina was accompanied by a reduction in the synthesis and storage by the optic lobe of two neurotransmitter candidates, acetylcholine and 5-hydroxytryptamine. The endogenous content of 5-hydroxytryptamine was also reduced in these preparations. Excision of a section of the stemmatal nerve, which connects the primordium of the compound eye to the brain, also arrested the development of the lamina, but in this case the compound eye itself developed apparently normally without making contact with the brain. This finding supports the hypothesis that the centripetal growth of photoreceptor-cell axons normally is contact-guided. Results of neurochemical experiments on retinal tissue argue against the idea that acetylcholine, 5-hydroxytryptamine, or gamma-aminobutyric acid is a likely photoreceptor-cell neurotransmitter. From these and our previous studies of the metamorphosing brain in Manduca we conclude that different classes of neurons in the same central nervous system can exhibit widely different degrees of dependency on their normal innervation for their survival and differentiation.

The role of afferent and efferent connections in the differentiation of optic lobe interneurons was investigated by using laser ablations of neuronal precursors in the brain of Musca domestica and analysis of two eye mutants of the same species. The first mutant, split eye, had no connections between the retina and the optic lobes. In this case the optic lobes were drastically reduced in volume and the neural organization within the neuropil regions was altered. The other mutant, spindle, had reduced retinae that innervated reduced optic lobes with a normal-appearing orderly arrangement of neurons. In addition disordered neuropil, composed of identified visual interneurons, was found that had no afferent innervation. Three main types of alterations resulting from laser ablations were analyzed. These ablations removed entire neuropil regions or parts of these: (1) removal of the first optic neuropil region (the lamina) resulting in receptor axons projecting directly to the second neuropil (the medulla) and sprouting of medulla neurons toward the receptor layer; (2) removal of one part of the third optic neuropil (the lobula plate) and severe alteration of the other part (the lobula) resulting in sprouting of lobula neurons into the medulla neuropil; and (3) removal of the entire optic lobe resulting in reduction of the volume of the lateral midbrain and photoreceptor axons forming a tangle beneath the retina. Our findings confirm that afferent retinal input is essential for normal differentiation and maintenance of many optic lobe interneurons. Furthermore, it was seen that a normal columnar organization of the neuropils and the dendritic patterns of visual interneurons are dependent on afferent inputs. A common response to removal of inputs was a reorganization of axonal and dendritic projections.

The differentiation of serotonin-immunoreactive (5-HTi) neurons in the optic lobes of fleshflies was studied during in situ development and in in vivo cultures. All 5-HTi neurons with cell bodies in the imaginal optic lobes differentiate during postembryonic (pupal) development. These are local anaxonal neurons. In addition there are two large 5-HTi bilateral neurons that connect all optic lobe neuropil regions on both sides of the brain and have their cell bodies in the midbrain proper. Deafferentation of optic lobes cultured in vivo leads to drastic reduction in optic lobe volume and increased cell death. All the 5-HTi neurons differentiate after deafferentation but their morphology changes. The neuropil receiving the photoreceptor inputs, the lamina, degenerates but a disorganized "pseudolamina" is formed by the processes of the two large 5-HTi neurons. The layering of the optic lobe neuropils cannot be distinguished and 5-HTi processes form novel projectional patterns. Hence, the 5-HTi neurons do not require afferent inputs from the retina for their differentiation and survival, but the effect on other optic lobe interneurons is reflected in the morphological plasticity of the 5-HTi neurons.

Although identified neurons are defined as constant and unique from animal to animal within the same species, we might expect to find phenotype variability in the number, morphology, and physiology of identified neurons if we examined them in enough sexually reproduced animals of the same species. Morphological variability and axonal abnormalities were previously encountered in a study of the large ocellar interneurons in 50 grasshoppers. Both the somata and arborizations in the brain of the ocellar interneurons from one or more ocelli can be stained easily and repeatedly with cobalt, so that one can routinely examine the morphology of large numbers of cells in large numbers of animals. In the present study, the large ocellar interneurons were examined in three types of animals: (i) animals from two different breeding populations and their offspring, (ii) the offspring within clutches from single mated pairs of known phenotypes, and (iii) isogenic animals from the parthenogenetic clones of single unmated females. The soma location, axonal pathway, location of arborizations, and fine branching patterns of 14 of the large ocellar interneurons were examined in 430 grasshoppers. The large ocellar interneurons were examined in 11 clones raised in nearly constant and identical conditions. In most of the clones, the morphology of interneurons L5L and L5R was normal; all of the 82 animals examined from clones 4, 7, 9, 10, 12, 13, 16, and 17 were normal. Clones 8 and 2 had high percentages of animals with axonal abnormalities of interneuron L5: 16 out of 24 animals (66%) in clone 8 and 37 out of 42 animals (88%) in clone 2. All of the ocellar interneurons except for L5 were normal in all of these animals. Thus, the abnormal development of at least one identified interneuron, L5, can occur with a high degree of genetic control and a high degree of specificity. The axonal abnormality of interneuron L5 was not the same in each of the 37 animals from clone 2, although common mistakes and patterns were detected.

The G, B1, and B2 neurons are three prominent interneurons located in adjacent segmental ganglia in the central nervous system of locusts. Previous studies on the adult nervous system have shown that each of these cells has its own distinctive morphology and responsiveness to auditory input. Previous studies on the embryonic nervous system have described the lineage and development of one of these cells, the G neuron, in the mesothoracic (T2) segment. In this paper it is shown that the G, B1, and B2 neurons are segmental homologues in that they arise from equivalent lineages during embryogenesis in the T2, T3, and A1 segments, respectively. Each cell arises (along with its identified sibling neuron) from the division of the second ganglion mother cell of neuroblast 7-4. The segment-specific morphology of the G homologues was determined in the T3 and A1 segments between 60-70% of embryonic development, and their identity was established as the adult B1 and B2 neurons by comparing the distinctive cell-specific features of their morphology between embryo and adult. Although all three neurons display striking morphological differences, they all share certain structural features in common, including the location of their primary axons and neurites in specific tracts in the neuropil. By recording intracellularly from the main neurites of the G, B1, and B2 neurons, clear differences were found in the synaptic inputs each of the neurons receives and the synaptic outputs each makes. For example, G and B2, but not B1, receive direct monosynaptic input from the descending contralateral movement detector (DCMD) interneurons and from auditory afferents; B1, but not B2, connects directly to G; and B2, but not B1 or G, connects directly to flight motoneurons. The main conclusion from these observations is that lineally equivalent neurons in different segments can develop similar primary structures but quite different secondary morphologies and synaptic connections. How these segment-specific differences arise during embryogenesis remains unknown.

We have examined the morphology and neuronal elements of the cerebral neuroendocrine system in the larval, pupal, and adult stages of the moth Manduca sexta with a variety of neuroanatomical techniques. The larval brain contains several discrete groups of neurosecretory and non-neurosecretory cells that project to the associated neurohemal organs (the corpora cardiaca-allata complex, or CC-CA) and to a variety of more peripheral structures. A previously undescribed set of cells in the subesophageal ganglion have also been found that project out the neurosecretory nerves. During metamorphosis, the cerebral neuroendocrine system undergoes a dramatic structural reorganization, including the reduction or loss of many larval nerves and a repositioning of the cell groups and their dendritic fields. Despite these changes, most of its central elements are retained. In addition, by the completion of adult development a new cluster of cells can be found on either side of the dorsal midline of the brain. We have also determined the relative contributions of the different cell groups to the moth neuroendocrine system by intracellular iontophoresis of dye into individual cells. Within the dorsal protocerebrum, five separate morphological types of cells can be recognized, each with a distinctive pattern of dendritic arborization in the brain and terminal neurohemal processes that project to the CC, the CA, the aorta, or to a combination of these regions. The large intrinsic cells of the CC have also been filled, revealing an unusual set of morphological features in these peripheral neurosecretory cells.

Neuropeptide-FMRFamide-like immunoreactivity was characterized in the fruit fly, Drosophila melanogaster. In the adult central nervous system, a stereotypic pattern of immunoreactive cell bodies and immunoreactive nerve processes and varicosities was observed, indicating a neurochemical role for FMRFamide-like substance(s) in Drosophila. Localization of immunoreactivity in the central nervous system of early larval stage revealed that the majority of the prominent FMRFamide-like immunoreactive neurons were already differentiated. The FMRFamide-like immunoreactive neurons remain immunoreactive throughout postembryonic stage and persist in the adult central nervous system. In the larva, in addition to the central nervous system, FMRFamide-like immunoreactivity was localized in the fibers innervating the ring gland, in the ganglion innervating the gut and in the gastric caeca.

The object of this study was to describe the embryonic and postembryonic morphogenesis of a grasshopper interneuron in order to determine how, and when, this cell comes to assume its mature form. DCMD is an intensively investigated interneuron whose morphology, input and output physiology, and role in behavior are relatively well-known in the adult. We examined the morphology of DCMD in the brain at each stage of its development with silver-intensified cobalt-fills. It arises at 40 +/- 4% of embryogenesis and is probably one of the early progeny from its stem cell. In the ensuing 40% of development, its brain arborization grows quite directly into its mature form. Branches appear first and are always longest and densest in the brain region where the adult arborization is found. Thus, the adult form arises by initially directed growth and not by secondary selection of branches from a diffuse or overgrown arborization. Restricted secondary branch loss of lateral filopodia and probably of a few early branches does occur. Embryonic and postembryonic development of the cell are distinctly different. Embryogenesis is the period of morphological differentiation as indicated by the growth and shaping of the brain and also thoracic (axonal) arborizations, the appearance of cytological specializations, and the logarithmic growth of the neurite and soma. The brain arborization has its mature form, although not size, by the completion of embryogenesis. Postembryonic development is a period of substantial, but primarily allometric, growth. The soma and neurite grow linearly (with time), and the arborization grows in proportion to brain size.

In order to map the expression of receptors for nerve growth factor (NGF) during brain and cranial ganglia development, iodinated NGF (125I beta NGF) was used as a probe in an autoradiographical analysis performed between embryonic day 3 (E3) and posthatching day 3 (P3) of chicken development. Heavy autoradiographic labelling was observed at the classical NGF target sites, the proximal cranial sensory ganglia and the sympathetic superior cervical ganglion, throughout development and after hatching. In contrast, only weak labelling could be detected during a restricted time span in the vestibulocochlear (E4-E8) and the distal cranial sensory ganglia (E4-E10), the neurons of which originate from the otic and epibranchial placodes. Specific 125I beta NGF binding was also observed in various brain regions during early brain development. NGF receptor expression there followed a characteristic pattern. The neuroepithelial layer displayed very low levels of specific 125I beta NGF binding, while strong 125I beta NGF labelling was found in the mantle layer. Brainstem somatomotor nuclei, visceromotor columns, brainstem alar plate, cerebellar anlage, tectum, and basal forebrain (epithalamus, striatum) were found to be transiently labelled by 125I beta NGF in early development (E4-E12). Non-nervous tissues such as parts of the otic vesicle epithelium and skeletal muscle anlagen of the head were also labelled. These results, showing specific binding of 125I beta NGF to cranial cells of different origin (neural tube, neural crest, placode, and possibly mesoderm) strengthen the concept that NGF may have diverse functions in growth and differentiation of various tissues and cell types.

The synaptic neuropil of the olfactory (antennal) lobe of the moth Manduca sexta is subdivided into histologically conspicuous structures called glomeruli that are typical of olfactory systems in vertebrates and invertebrates. Each glomerulus consists of the highly branched neuritic arbors of both primary olfactory axons and antennal-lobe neurons, bounded by a nearly complete envelope of glial cells. We have studied events occurring during the first half of metamorphic adult development. The first signs of organization of the neuropil into glomeruli are changes in glial cells. Prior to the ingrowth of olfactory axons from the antenna, glial cells form a continuous border around the neuropil. When olfactory axons begin to reach the lobe, glial cells embark on a stereotyped series of changes: the border becomes disrupted, glial cells begin to proliferate and extend processes into the outer regions of the neuropil, and some glial cells migrate toward the center of the neuropil. Shortly thereafter, glomeruli emerge from the neuropil, delineated by glial cells. If, however, afferent axons are prevented from ever reaching the antennal lobe, glomeruli never develop and the glial cells remain almost entirely restricted to a thick layer bordering the neuropil. Thus sensory axons have a direct influence not only on neuronal but also on glial differentiation. Our results lead us to suggest that the glial cells may be in a position to act as intermediaries in developmental interactions between sensory axons and antennal-lobe neurons.

The fate of descending brain-thoracic neurons in the metamorphosing supraoesophageal ganglion of the mealworm beetle, Tenebrio molitor, is described. Comparison of the descending neurons of the larval, various pupal, and adult stages outlines a high degree of topological invariance in the structure of descending interneurons; i.e., the basic organization of the imaginal set of descending neurons is anticipated by the structure of the larval neurons. Single descending neurons of analogous clusters of larval and imaginal neurons outline a virtual identical structural organization in both the larval and imaginal brain. There is a quantitative increase from approximately 70 to approximately 120 brain-thoracic interneurons during metamorphosis. This increase does not cause complex transformations in the structural organization of the descending interneurons. Experiments with the DNA-inhibitor hydroxyurea prove that the described topological invariant structure of the set of descending neurons is based on the persistence of individually descending neurons. There is evidence that the whole set of larval interneurons persists throughout the beetles' metamorphosis. The essential characteristics of the later imaginal set of descending neurons are qualitatively and quantitatively established within the first 10% of pupation. Structural invariance of the set of descending neurons is discussed with regard to the significance of cellular interaction for the mechanisms of metamorphic reorganization of nervous tissue.

In hirudinid leeches, the segmental ganglia associated with the sexual organs contain several hundred more neurons than other midbody ganglia. To determine whether this difference arises by differential cell addition or by differential cell death, cell counts were made in several segmental ganglia during the course of embryonic and postembryonic development. The results show that all ganglia behave equally in early development. In each case, at least 10-20% more cells than will make up the adult complement of about 400 neurons is generated, and by about 20 days of embryonic development cell loss brings the number down to about 400 cells. By about 30 days, when animals emerge from their cocoons, additional cells have begun to appear in the sex ganglia. The number of extra cells continue to increase gradually over the next several months, until the adult number of 600-700 neurons is attained. These observations indicate that at least some segmental differences in the size of neuronal populations are due to differential cell proliferation and that these differences can arise quite late in the maturation of an animal.

Posthatching growth in the pond snail Lymnaea stagnalis involves approximately a 20-fold increase in the linear dimensions of the ganglia composing the central nervous system. Developmental change within the population of neurons exhibiting serotoninlike immunoreactivity (SLIR) was examined in order to explain this growth in cellular terms. The study indicates that at least two factors contribute to the growth of the nervous system. First, SLIR cells approximately double in number from the 200-250 cells in hatchlings to the complement found in animals approaching sexual maturity. Much of this increase in cell number occurred within identifiable discrete clusters of neurons with different clusters adding cells at different rates and at different times. The number of SLIR cells also increased in more diffuse populations, particularly along the medial aspects of the paired pedal and the right parietal ganglion. No identified cells were added postembryonically. In addition to the increases in neuron numbers, posthatching development in Lymnaea also involves the growth of individual cells. All cells examined showed continuous somatic growth during posthatching development, but different identified cells and different cell clusters were characterized by different rates of relative growth. Together, the results highlight the complexity of postembryonic development in the snail by indicating the temporal and spatial specificity for both cell addition and cell growth within the nervous system.