Segmentation of the central nervous system in leech.

Central nervous system (CNS) in leech comprises segmentally iterated progeny derived from five embryonic lineages (M, N, O, P and Q). Segmentation of the leech CNS is characterized by the formation of a series of transverse fissures that subdivide initially continuous columns of segmental founder cells in the N lineage into distinct ganglionic primordia. We have examined the relationship between the N lineage cells that separate to form the fissures and lateral ectodermal and mesodermal derivatives by differentially labeling cells with intracellular lineage tracers and antibodies. Although subsets of both lateral ectoderm and muscle fibers contact N lineage cells at or near the time of fissure formation, ablation experiments suggest that these contacts are not required for initiating fissure formation. It appears, therefore, that this aspect of segmentation occurs autonomously within the N lineage. To support this idea, we present evidence that fundamental differences exist between alternating ganglionic precursor cells (nf and ns primary blast cells) within the N lineage. Specifically, ablation of an nf primary blast cell sometimes resulted in the fusion of ipsilateral hemi-ganglia, while ablation of an ns primary blast cell often caused a 'slippage' of blast cells posterior to the lesion. Also, differences in cell behavior were observed in biochemically arrested nf and ns primary blast cells. Collectively, these results lead to a model of segmentation in the leech CNS that is based upon differences in cell adhesion and/or cell motility between the alternating nf and ns primary blast cells. We note that the segmentation processes described here occur well prior to the expression of the leech engrailed-class gene in the N lineage.

Identification of a neurogenic sublineage required for CNS segmentation in an Annelid.

In embryos of leeches (phylum Annelida), metameric structures arise sequentially from a germinal plate comprising the descendants of five pairs of embryonic stem cells called teloblasts. It has been shown that transverse stripes of cells expressing ht-en (a homolog of engrailed, a Drosophila segment polarity gene), arise in the germinal plate prior to the appearance of segmental ganglia and that, in the main neurogenic lineage (derived from the N teloblasts), the stripe of cells expressing ht-en demarcates the boundary between prospective segmental ganglia. Previous lineage-tracing experiments had suggested that the clones of nf and ns primary blast cells in the N lineage are confined to within segmental borders. This conclusion was called into question by the observation that the cells expressing ht-en do not appear to be at the very posterior edge of the nf clone, from which they arise. To resolve this issue, we have injected individual primary blast cells with fluorescent lineage tracers; we find that cells in the nf clone actually straddle two adjacent ganglia. Moreover, using photoablation techniques, we find that the nf clone is required for proper morphogenesis of the segmentally iterated central nervous system (CNS).

Leech neurogenesis. I. Positional commitment of neural precursor cells.

This paper reports analyses of the differentiation and distribution of identified peripheral neurons and central 5-HT-containing neurons in embryos of the glossiphoniid leech Theromyzon rude that have been deprived of one of the bilaterally paired major ectodermal cell lines called the n bandlets. Cells descended from a lone surviving n bandlet were abnormally distributed across both sides of the ventral midline. Nevertheless, they produced the complement of identified neurons that they would have produced in a normal embryo. Neurons produced by cells that crossed the midline occupied the normal positions of their absent homologs, as demonstrated by morphometric analysis of normal and n-bandlet-deprived ganglia. Ablations of ectodermal cell lines other than the n bandlets (o and p, or q) allowed the formation of normal distributions of neurons descended from the n bandlets. These results are interpreted as showing that neural precursor cells are committed to occupy particular positions before reaching those positions and probably use positional cues of predominantly nonectodermal origin to recognize those positions. Together, the results reported here and in the accompanying paper (S. Torrence, M. Law, and D. Stuart, 1989, Dev. Biol. 136, 40-60) suggest that ectodermal cells that are committed to give rise to specific neurons use cues provided by the mesoderm to find positions appropriate to their fates.

Leech neurogenesis. II. Mesodermal control of neuronal patterns.

This paper reports analyses of the effects of eliminating mesoderm from one or both sides of embryos of the glossiphoniid leech Theromyzon rude on the differentiation and distribution of ectodermal cells, especially identified peripheral neurons and central 5-hydroxytryptamine (5-HT)-containing neurons arising from the bilateral pair of cell lines called the n bandlets (n-kinship cells). In mesoderm-deprived regions, no segmental hemiganglia formed, and identified neurons were not organized into recognizable patterns, although 5-HT neurons underwent neurochemical differentiation and grew axons. In unilaterally mesoderm-deprived embryos, segmental hemiganglia were formed in a midbody experimental zone, and cells that had abnormally crossed the ventral midline from the deprived side gave rise to identified neurons that were incorporated as supernumeraries into the normal organization of hemiganglia on the nondeprived side. In a posterior experimental zone, ganglionic morphology was disrupted on both sides. We conclude that precursor cells are committed to specific neuronal fates regardless of whether they occupy normal positions and that mesodermal tissues provide positional cues necessary for such precursor cells to find positions appropriate to their fates.

Cell lineage, cell death, and the developmental origin of identified serotonin- and dopamine-containing neurons in the leech.

The nervous system of the glossiphoniid leech includes segmentally iterated neurons that contain serotonin (5-HT) and dopamine. These have been investigated in Helobdella triserialis, Theromyzon rude, and Haementeria ghilianii. Five types of 5-HT neurons are identified by immunocytochemistry in the abdominal ganglia of the ventral nerve cord: the bilaterally paired Retzius, anteromedial, ventrolateral and dorsolateral neurons, and the unpaired posteromedial (pm) neuron. Three types of bilaterally paired dopamine neurons are identified by glyoxylic acid-induced fluorescence in the segmental body wall: MD, LD1, and LD2. Each left or right half of the segmental complement of the leech nervous system is known to develop from 6 distinct ectodermal primary blast cells (ns, nf, o, p, qs, and qf). To identify the blast cells of origin of the 5-HT and dopamine neurons, fluorescent cell lineage tracers were injected into the various precursors of the blast cells in early (stage 6) embryos. The embryos were then raised until their 5-HT and dopamine neurons could be scored (stage 11) for the presence or absence of lineage tracer. We find that the Retzius, anteromedial, and posteromedial 5-HT neurons are derived from the ns blast cell, while the ventrolateral and dorsolateral 5-HT neurons are derived from the nf blast cell. The unpaired pm 5-HT neuron arises as one of a bilateral pair of neurons, of which one later dies. Whether the left or right pm neuron survives in any given ganglion is the consequence of some form of competitive interaction between cells derived from the left and right n primary blast cells, possibly between the left and right pm neurons themselves. We find that, of the dopamine neurons, the LD1 neuron is derived from the o blast cell, the LD2 neurons from the p blast cell, and the MD neuron from one of the 2 kinds of q blast cells. These results show that the 5-HT and dopamine neurons arise from 5 different primary blast cells in a highly determinate manner, and they support the view that cells of a similar phenotype need not be closely related in the developmental cell lineage tree.

Development of neurotransmitter metabolism in embryos of the leech Haementeria ghilianii.

We have investigated the development of neurotransmitter metabolism in embryos of the glossiphoniid leech Haementeria ghilianii. The neurotransmitter content of dissected embryonic tissues was measured by means of radioenzymatic assays, while the presence of neurotransmitters in individual identified neurons was detected by means of immunocytochemical and monoamine histofluorescence techniques. The capacity for synthesis of neurotransmitters was measured by incubating dissected embryonic tissues in radiolabeled neurotransmitter precursors. A specific neurotransmitter uptake system present in some neurons was detected by means of an autoradiographic technique. At an early stage of development of the nervous system, when most neurons are just beginning process outgrowth, the nerve cord acquires the capacity to synthesize ACh, 5-HT, and GABA from their immediate precursors, and contains ACh. Moreover, 5-HT-immunoreactive neurons and neurons that are capable of GABA uptake can be identified. Dopamine-containing neurons are first detected by their histofluorescence at a slightly later stage, after process outgrowth is under way. As development continues, the content of and capacity for synthesis of these neurotransmitters increase, as does the number of neurons capable of GABA uptake. During the earlier stages of development, ACh content exceeds 5-HT content, which in turn exceeds dopamine content. By the end of embryogenesis, however, 5-HT and dopamine contents have greatly increased relative to ACh content, with 5-HT content exceeding ACh content by a factor of 2. Of the neurotransmitters thus far studied, 5-HT is present in the highest amount in the juvenile and adult nerve cord. Our results indicate that in the development of the leech nervous system neurotransmitter metabolism is one of the first neuronal characters to differentiate and that the subsequent levels of the different neurotransmitters are differentially regulated.

Gangliogenesis in leech embryos: migration of neural precursor cells.

In the metameric CNS of leeches, identified neurons occupy highly stereotyped positions in each segmental ganglion. Although many of the neural precursor cells arise near their definitive positions, some arise outside the prospective domain of the segmental ganglia and thus must migrate into the CNS. Here, we report the results of an analysis of the role of cell migration in gangliogenesis in the leech Theromyzon rude. Segmental ganglia of the ventral nerve cord arise as laterally thickened sheets of tissue lying astride the ventral midline. Particular identified circular and longitudinal muscle fibers, visualized by indirect immunofluorescence using a monoclonal antibody against leech muscle, outline the presumptive ganglionic territories even before the ganglionic rudiments become morphologically distinct and serve as anatomical landmarks to which the cell movements are related. Cell lineage tracers microinjected into precursor blastomeres are used to visualize migratory cells. Small groups of neural precursor cells that arise outside the prospective ganglionic territories migrate with stereotyped timing along stereotyped pathways to reach their definitive positions, and each group of migratory cells gives rise to a stereotyped subset of the cells in a ganglion. No segmental or regional differences are observed in any aspect of cell migration studied here, supporting the view that segmental differences in the architecture of the leech CNS arise only after the initial condensation of the ganglionic rudiments.

Neuronal sites of action of a neurosecretory peptide, egg-laying hormone, in Aplysia californica.

1. Egg-laying hormone (ELH) is a polypeptide of about 4,500 mol wt synthesized in the bag cell neurons of the abdominal ganglion of Aplysia. We studied the effects of ELH on the neuronal activity of the attached head ganglia (buccal, cerebral, pleural, and pedal), on the isolated buccal ganglia, as well as on feeding in intact Aplysia. 2. Starved animals (n = 7) injected with crude extract containing ELH stopped eating algae at 17 +/- 4 min and their eggs first appeared at 29 +/- 4 min after injection at 20 degrees C. This cessation of eating is significant when compared to the seven controls (P less than 0.01). These data clearly indicate that a suppression of feeding activity occurs before the appearance of eggs. 3. ELH applied to the paired buccal ganglia in vitro activates a pair of neurons into a tonic pacemaker mode (approximately 1 spike/s). This activation also occurs in a high-magnesium, zero calcium solution that blocks chemical synapses. The time for the full appearance of this activity in vitro correlates well with the time for suppression of feeding in vivo. Each of these neurons has an ipsilateral axon in buccal nerve 3. The neuron has been identified by intracellular recording. 4. ELH increases the rate of firing of a second pair of buccal neurons, each with an ipsilateral axon in the cerebrobuccal connective. 5. ELH, when applied to the attached head ganglia, causes large bursts of neuronal activity in pedal nerves to the foot and increased activity in the nerve to the penis; the relevant neurons remain to be identified. 6. These in vitro effects were produced by ELH partially purified from bag cell cluster homogenates using ammonium sulfate precipitation followed by anion exchange and gel filtration chromatography or by ELH released from activated bag cells in isolated abdominal ganglia and then purified by gel filtration. The isolated buccal ganglia effects have been confirmed with fully purified ELH. 7. The ELH effects on the in vitro nervous system support the hypothesis that ELH in vivo acts directly on the nervous system to suppress feeding activity, controlled by the buccal and cerebral ganglia. ELH may also produce characteristic movements of the head during egg laying, controlled probably by the pedal and cerebral ganglia.

Neurosecretion of egg-laying hormone and other peptides from electrically active bag cell neurons of Aplysia.

1. Radiolabeled peptides released from an isolated cluster of bag cell neurons, during an after discharge, were compared with the polypeptide egg-laying hormone (ELH), 4,500 daltons, pI 9.0-9.3, as purified from homogenates of bag cell clusters. A peptide, labeled with methionine, leucine, and arginine, which is selectively released from after discharging bag cell clusters comigrates with marker ELH, purified from cluster homogenates, on P-6 gel filtration columns and subsequent isoelectric focusing gels. At least three other presumed peptides of unknown function are also released, including one of 5,000-6,000 mol wt, pI 4.5-5.0. 2. When bag cells afterdischarge in vitro, bioactive material is released that will induce egg laying when injected into an Aplysia. This released bioactive material also comigrates with bioactive material from cluster homogenates on P-6 columns. 3. These experiments demonstrate that ELH (4,500 mol wt, pI 9.0-9.3), as purified from bag cell cluster homogenates, is also the major form secreted from bag cells that induces egg laying. This purified ELH can now be used to study the physiological effects of a secreted neurohormone and their relationship to behavior.

Purification and primary structure of the neuropeptide egg-laying hormone of Aplysia californica.

Egg-laying hormone (ELH), a neuropeptide synthesized by the bag cell neurons, induces egg laying and its correlated behavior in Aplysia californica. In the present study, ELH has been purified to homogeneity and its primary structure has been determined. We find this molecule to have 36 amino acid residues with a M(r) of 4385 and a calculated isoelectric point of 9.7. Direct microsequence analysis revealed a single amino acid sequence that is in agreement with the amino acid composition determined after acid hydrolysis of ELH: H-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu-Gln- Ile-Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-OH. Enzyme data indicate that the COOH-terminal lysine may be modified but its exact nature remains to be determined. There is no similarity between the amino acid sequence of ELH and that of presently known vertebrate neuropeptides. The two-step purification procedure, starting with a homogenate of bag cell clusters, consisted of cation exchange chromatography on SP C25 (Sephadex) followed by gel filtration on Bio-Gel P-6. Our purification results in a 100-fold enrichment of ELH from bag cell homogenates and a 36% recovery of purified radiolabeled marker ELH. Analysis of purified ELH radiolabeled with [(35)S]methionine or [(3)H]leucine on isoelectric focusing gels and on 8 M urea/sodium dodecyl sulfate gels showed only a single peak containing 90% of the radiolabel. Radiolabeled ELH migrated with a pI of 9.0-9.2 and an apparent M(r) of 3500-5700. ELH retained egg-laying bioactivity when eluted from this segment of the gel. We find that 2.5 nmol of pure ELH consistently induces egg laying at 20 degrees C.

Differentiated short latency response increases after conditioning in inferior colliculus neurons of alert rat.

The responses of 35 inferior colliculus multiple units (MUs) to tone onset were measured in 10 freely moving rats before and after differential behavioral conditioning. MU response changes were found in the 16.8 msec after tone onset (includes 2 msec air travel time) after learning. Responses to CS+ onset increased in 12 of the 35 individual MUs, and in the group of MUs as a whole, after conditioning. The CS+--CS- difference increased in 17 of the 35 individual MUs, and in the group of MUs, after conditioning. The response differentiation was significant in the 3.6-6.4 msec interval after the tone reached the animal's ears, the time at which neuronal responses were first evident. Since the inferior colliculus increases were differentiated between CS+ and CS-, they could not be explained by sensitization caused by changed middle ear contractions or by alterations in orientation to the speakers.

Trial sequence of changed unit activity in auditory system of alert rat during conditioned response acquisition and extinction.

1. The activity of units in the auditory system of alert, freely moving rats was studies during the acquisition and extinction of a tone-signaled, appetitive classically conditioned response. Responses of neurons in inferior colliculus (N = 28), medial geniculate (N = 32), posterior nucleus of thalamus (N = 28), pretectal region (N = 19), and cortex (N = 100) were studies in 74 rats across 10-trial blocks. 2. During behavioral acquisition, neurons in posterior nucleus of thalamus were the first to show response increments to CS+ onset. They were followed by neurons in cortex, pretectal region, medial geniculate, inferior colliculus and by movement behavior. 3. Prestimulus background rates during acquisition showed significant decrements in cortical neurons. These background decrements began to be evidenced in the trial series before the response increases in posterior nucleus. These data strengthened the suggestion of a previous study that posterior nucleus responses could be dependent on tonic modulation from cortex. 4. Extinction appeared to be largely a reverse of acquisition. Cortex and behavior showed response decrements first in the trial series. They were followed by medial geniculate, pretectal region, posterior nucleus, and inferior colliculus neurons. 5. The hypothesis was advanced that the auditory lemniscal adjunct afferent system may play a primary role in the early phases of auditory conditioned-response acquisition.