Larval Neurons Research Articles

Persistent larval sensory neurones are required for the normal development of the adult sensory afferent projections in Drosophila.

We have tested the hypothesis that larval neurones guide growth of adult sensory axons in Drosophila. We show that ablation of larval sensory neurones causes defects in the central projections of adult sensory neurones. Spiralling axons and ectopic projections indicate failure in axon growth guidance. We show that larval sensory neurones are required for peripheral pathfinding, entry into the CNS and growth guidance within the CNS. Ablation of subsets of neurones shows that larval sensory neurones serve specific guidance roles. Dorsal neurones are required for axon guidance across the midline, whereas lateral neurones are required for posterior growth. We conclude that larval sensory neurones pioneer the assembly of sensory arrays in adults.

Developmental expression of ascidian neurotransmitter synthesis genes

To identify cholinergic neurons, we isolated a choline acetyltransferase (Ci-ChAT) gene from Ciona intestinalis by PCR methods. In the cloning process, we also obtained the gene encoding the vesicular acetylcholine transporter (Ci-vAChTP). These two genes shared the same 5'-UTR sequence as well as similar expression patterns. In both cases, the gene expression was first detected by whole-mount in situ hybridization in the anterior-dorsal region of the caudal nerve cord at the early tailbud stage. In the larva, the expression was seen in several cells of the visceral ganglion. These results suggest that ascidian larval motor neurons exist in the visceral ganglion.

Neuronal development in larval chiton Ischnochiton hakodadensis (Mollusca: Polyplacophora).

Chitons are the most primitive molluscs and, thus, a matter of considerable interest for understanding both basic principles of molluscan neurogenesis and phylogeny. The development of the nervous system in trochophores of the chiton Ischnochiton hakodadensis from hatching to metamorphosis is described in detail by using confocal laser scanning microscopy and antibodies raised against serotonin, FMRFamide, and acetylated alpha tubulin. The earliest nervous elements detected were peripheral neurons located in the frontal hemisphere of posthatching trochophores and projecting into the apical organ. Among them, two pairs of unique large lateral cells appear to pioneer the pathways of developing adult nervous system. Chitons possess an apical organ that contains the largest number of neurons among all molluscan larvae investigated so far. Besides, many pretrochal neurons are situated outside the apical organ. The prototroch is not innervated by larval neurons. The first neurons of the developing adult central nervous system (CNS) appear later in the cerebral ganglion and pedal cords. None of the neurons of the larval nervous system are retained in the adult CNS. They cease to express their transmitter content and disintegrate after settlement. Although the adult CNS of chitons resembles that of polychaetes, their general scenario of neuronal development resembles that of advanced molluscs and differs from annelids. Thus, our data demonstrate the conservative pattern of molluscan neurogenesis and suggest independent origin of molluscan and annelid trochophores.

Neuronal form in the central nervous system of the tadpole larva of the ascidian Ciona intestinalis.

The dorsal tubular central nervous system (CNS) of the ascidian tadpole larva is a diagnostic feature by which the chordate affinities of this group, as a whole, are recognized. We have used two methods to identify larval neurons of Ciona intestinalis. The first is serial electron microscopy (EM), as part of a dedicated study of the visceral ganglion (1), and the second is the transient transfection of neural plate progeny with green fluorescent protein (GFP) (2), to visualize the soma and its neurites of individual neurons in whole-mounted larvae of C. intestinalis. Our observations reveal that ascidian larval neurons are simple inform, with a single axonal neurite arising from a soma that is either monopolar or has only very few, relatively simple neurites arising from it, as part of a presumed dendritic arbor. Somata in the visceral ganglion giving rise to axons descending in the caudal nerve cord are presumed to be those of motor neurons.

Isolation of an early neural maker gene abundantly expressed in the nervous system of the ascidian, Halocynthia roretzi.

Ascidian tadpole larvae possess a primitive nervous system, which is a prospective prototype of the chordate nervous system. It is composed of relatively few cells but sufficient for complex larval behavior. Here we report on HrETR-1, a gene zygotically expressed in a large proportion of the developing neural cells of the ascidian, Halocynthia roretzi. HrETR-1 is an early neural marker which can be used for analyzing neural differentiation. HrETR-1 expression intensified in most neural cells of genes isolated to date, in both central and peripheral nervous systems including palps as early as the 110-cell stage. Using this gene as a probe, we characterized neural cells in the nervous system as well as confirming their origins. Also, we recognized three types of peripheral epidermal neurons which presumably correlate to the larval neurons previously reported for another ascidian. Among these, five bilateral neurons located in the anterior region of the trunk appeared to be derived from a8.26 blastomeres.

Remodeling of the femoral chordotonal organ during metamorphosis of the hawkmoth, Manduca sexta

During metamorphosis of the moth, Manduca sexta, the larval legs degenerate and are replaced by adult legs with a diverse array of new sensory organs. The majority of the larval sensory neurons degenerate but some hair sensilla and chordotonal organ sensory neurons survive metamorphosis (Consoulas [2000] J. Comp. Neurol. 419:154–174). In the present study nerve-tracing techniques, birth-date labeling (5-bromodeoxyuridine), and electrophysiology were used to describe the remodeling of the femoral chordotonal organ (FCO) in the prothoracic legs. The larval FCO is composed of two scoloparia, which are associated with separate apodemes. At the onset of metamorphosis, some of the 13 larval neurons degenerate, together with the larval FCO apodemes. The remaining larval FCO sensory neurons persist in the imaginal leg to become the precursors of the adult femoral and tibial chordotonal organs respectively. Early in the pupal stage, 45 to 60 new sensory neurons are generated de novo and become associated with 6 persistent larval neurons in the imaginal femur to compose the adult FCO. Two clusters of persistent and new neurons are enclosed into two scoloparia with short apodemes that eventually become fused. In both larval and adult stages, the FCO contains units that respond phasically and others that respond tonically to femorotibial movements. Nerve activity from the FCO neurons can be recorded continuously during the remodeling of the organ. A persistent leg flexor motoneuron receives inputs from the FCO sensory neurons in both larval and adult stages, offering the opportunity to investigate the remodeling of the neural circuits underlying the proprioceptive control of the femorotibial joint. J. Comp. Neurol. 426:391–405, 2000. © 2000 Wiley-Liss, Inc.

Larval Multidendrite Neurons Survive Metamorphosis and Participate in the Formation of Imaginal Sensory Axonal Pathways in the Notum of Drosophila

In each hemimesothorax of Drosophila, a cluster of five larval multidendrite neurons that survives metamorphosis is described. The cell bodies of these neurons, initially grouped together, spread out over the medial heminotum during early pupal stages and extend new dendrites. Growing axons from sensory bristle neurons first appear in a defined orientation specific for each macrochaete. They subsequently contact processes from the larval multidendrite neurons and then appear to follow the preestablished axon trajectories of the latter. Ablation of the multidendrite neurons during the larval stage causes bristle axons to adopt abnormal trajectories. We suggest that the persistent larval neurons participate in guiding axons of the bristles on the medial half of the notum to the posterior dorsal mesothoracic nerve leading to the central nervous system.

Reorganization of the nervous system during metamorphosis of a hydrozoan planula

Abstract. Laser scanning confocal microscopy is used to reveal the changes that occur in the RFamide‐positive nerve net as a free‐swimming, solid hydrozoan planula larva is transformed into a sessile, hollow, young polyp. Seven stages of development in Pennaria tiarella are described: planula competent to metamorphose, attaching planula, disc, pawn, crown, developing polyp, and developed primary polyp. The RFamide‐positive nervous system undergoes dramatic reorganization during metamorphosis: (1) larval neurons degenerate; (2) new neurons differentiate and reform a nerve net; and (3) the overall distribution pattern of the nervous system changes. This study confirms earlier observations on RFamide‐positive neurons of Hydractinia which also show the loss of these cells after the onset of metamorphosis.

Regulation of central neuron synaptic targeting by the Drosophila POU protein, Acj6.

Mutations in the Drosophila class IV POU domain gene, abnormal chemosensory jump 6 (acj6), have previously been shown to cause physiological deficits in odor sensitivity. However, loss of Acj6 function also has a severe detrimental effect upon coordinated larval and adult movement that cannot be explained by the simple loss in odorant detection. In addition to olfactory sensory neurons, Acj6 is expressed in a distinct subset of postmitotic interneurons in the central nervous system from late embryonic to adult stages. In the larval and adult brain, Acj6 is highly expressed in central brain, optic and antennal lobe neurons. Loss of Acj6 function in larval optic lobe neurons results in disorganized retinal axon targeting and synapse selection. Furthermore, the lamina neurons themselves exhibit disorganized synaptic arbors in the medulla of acj6 mutant pupal brains, suggesting that Acj6 may play a role in regulating synaptic connections or structure. To further test this hypothesis, we misexpressed two Acj6 isoforms in motor neurons where they are not normally found. The two Acj6 isoforms are produced from alternatively spliced acj6 transcripts, resulting in significant structural differences in the amino-terminal POU IV box. Acj6 misexpression caused marked alterations at the neuromuscular junction, with contrasting effects upon nerve terminal branching and synapse formation associated with specific Acj6 isoforms. Our results suggest that the class IV POU domain factor, Acj6, may play an important role in regulating synaptic target selection by central neurons and that the amino-terminal POU IV box is important for regulation of Acj6 activity.

Remodeling of the leg sensory system during metamorphosis of the hawkmoth, Manduca sexta.

The adult legs of the hawkmoth Manduca sexta are supplied by a diverse array of sensory organs and associated neurons (Kent and Griffin [1990] Cell Tissue Res. 259:209-223) that differ from those in the larval legs. In the present study, a combination of nerve-tracing techniques [biocytin, 1,1;-dioctadecyl-3,3,3;, 3;-tetramethyl-indocarbocyanine perchlorate (DiI)], birth date labeling (5-bromodeoxyuridine), confocal microscopy, and electrophysiology were used to describe the remodeling of the prothoracic leg sensory system. Four primary sensory branches carry the axons of all of the sensory neurons in the larval leg. At the onset of metamorphosis, the imaginal leg epidermis develops underneath the larval cuticle and encircles the sensory neurons, thus separating them from their target-organs. Most of the larval neurons degenerate during the larval-to-pupal transition and are replaced by new-adult sensory neurons that are born and differentiate in the pupa. Six sensory neurons that supply hair sensilla in the larval leg, together with 13 femoral and tibial chordotonal organ neurons, persist into the developing adult leg to serve similar functions. Early in the pupal stage, electrical activity can be recorded from these neurons despite the absence of target sensory structures. During the differentiation of the adult sensory system, the axons of the new-adult sensory neurons contact and fasciculate with the axons of the persistent neurons. Thus, five of the primary sensory branches of the adult leg are built on the preexisting larval sensory trajectories. Two sensory branches, however, are established de novo by the axons of specific adult sensory neurons.

Firing properties and electrotonic structure of Xenopus larval spinal neurons.

Whole cell voltage- and current-clamp measurements were done on intact Xenopus laevis larval spinal neurons at developmental stages 42-47. Firing patterns and electrotonic properties of putative interneurons from the dorsal and ventral medial regions of the spinal cord at myotome levels 4-6 were measured in isolated spinal cord preparations. Passive electrotonic parameters were determined with internal cesium sulfate solutions as well as in the presence of active potassium conductances. Step-clamp stimuli were combined with white-noise frequency domain measurements to determine both linear and nonlinear responses at different membrane potential levels. Comparison of analytic and compartmental dendritic models provided a way to determine the number of compartments needed to describe the dendritic structure. The electrotonic structure of putative interneurons was correlated with their firing behavior such that highly accommodating neurons (Type B) had relatively larger dendritic areas and lower electrotonic lengths compared with neurons that showed sustained action potential firing in response to a constant current (Type A). Type A neurons had a wide range of dendritic areas and potassium conductances that were activated at membrane potentials more negative than observed in Type B neurons. The differences in the potassium conductances were in part responsible for a much greater rectification in the steady-state current voltage (I-V curve) of the strongly accommodating neurons compared with repetitively firing cells. The average values of the passive electrotonic parameters found for Rall Type A and B neurons were c(soma) = 3.3 and 2.6 pF, g(soma) = 187 and 38 pS, L = 0.36 and 0.21, and A = 3.3 and 6.5 for soma capacitance, soma conductance, electrotonic length, and the ratio of the dendritic to somatic areas, respectively. Thus these experiments suggest that there is a correlation between the electrotonic structure and the excitability properties elicited from the somatic region.

Active dendritic membrane properties of Xenopus larval spinal neurons analyzed with a whole cell soma voltage clamp.

Voltage- and current-clamp measurements of inwardly directed currents were made from the somatic regions of Xenopus laevis spinal neurons. Current-voltage (I-V) curves determined under voltage clamp, but not current clamp, were able to indicate a negative slope conductance in neurons that showed strong accommodating action potential responses to a constant current stimulation. Voltage-clamp I-V curves from repetitive firing neurons did not have a net negative slope conductance and had identical I-V plots under current clamp. Frequency domain responses indicate negative slope conductances with different properties with or without tetrodotoxin, suggesting that both sodium and calcium currents are present in these spinal neurons. The currents obtained from a voltage clamp of the somatic region were analyzed in terms of spatially controlled soma membrane currents and additional currents from dendritic potential responses. Linearized frequency domain analysis in combination with both voltage- and current-clamp responses over a range of membrane potentials was essential for an accurate determination of consistent neuronal model behavior. In essence, the data obtained at resting or hyperpolarized membrane potentials in the frequency domain were used to determine the electrotonic structure, while both the frequency and time domain data at depolarized potentials were required to characterize the voltage-dependent channels. Finally, the dendritic and somatic membrane properties were used to reconstruct the action potential behavior and quantitatively predict the dependence of neuronal firing properties on electrotonic structure. The reconstructed action potentials reproduced the behavior of two broad distributions of interneurons characterized by their degree of accommodation. These studies suggest that in addition to the ionic conductances, electrotonic structure is correlated with the action potential behavior of larval neurons.

Sodium channels in central neurons of the tobacco budworm, Heliothis virescens: basic properties and modification by scorpion toxins

Voltage-activated sodium channels in central neurons of larval and adult Heliothis virescens were characterized using whole-cell patch clamp techniques. Macroscopic currents showing rapid activation and inactivation kinetics were uniformly sensitive to tetrodotoxin ( IC 50=1.9 nM). Currents began to activate at voltage steps to −45 mV and reached half maximal at −30 mV. Fast inactivation was evident at voltages as negative as −75 mV and reached half maximal at −50 mV. Full recovery from inactivation occurred within 1 to 2 ms. Currents in larval neurons exhibited similar properties to those of adult neurons, except for the rate of fast inactivation ( t 1), which was significantly slower in larval neurons. The biophysical properties of sodium channels remained unchanged for up to 3 days in culture. Two insecticidal neurotoxins, LqhαIT and AaIT, produced distinctly different modifications of H. virescens sodium channels. LqhαIT slowed channel inactivation, while AaIT specifically shifted voltage-dependent activation to more negative potentials. Overall, the results indicate that sodium channels in H. virescens neurons exhibit biophysical characteristics similar to those of vertebrates, yet possess pharmacological uniqueness with respect to scorpion toxin modification.

Remodeling of the femoral chordotonal organ during metamorphosis of the hawkmoth,Manduca sexta

During metamorphosis of the moth, Manduca sexta, the larval legs degenerate and are replaced by adult legs with a diverse array of new sensory organs. The majority of the larval sensory neurons degenerate but some hair sensilla and chordotonal organ sensory neurons survive metamorphosis (Consoulas [2000] J. Comp. Neurol. 419:154-174). In the present study nerve-tracing techniques, birth-date labeling (5-bromodeoxyuridine), and electrophysiology were used to describe the remodeling of the femoral chordotonal organ (FCO) in the prothoracic legs. The larval FCO is composed of two scoloparia, which are associated with separate apodemes. At the onset of metamorphosis, some of the 13 larval neurons degenerate, together with the larval FCO apodemes. The remaining larval FCO sensory neurons persist in the imaginal leg to become the precursors of the adult femoral and tibial chordotonal organs respectively. Early in the pupal stage, 45 to 60 new sensory neurons are generated de novo and become associated with 6 persistent larval neurons in the imaginal femur to compose the adult FCO. Two clusters of persistent and new neurons are enclosed into two scoloparia with short apodemes that eventually become fused. In both larval and adult stages, the FCO contains units that respond phasically and others that respond tonically to femorotibial movements. Nerve activity from the FCO neurons can be recorded continuously during the remodeling of the organ. A persistent leg flexor motoneuron receives inputs from the FCO sensory neurons in both larval and adult stages, offering the opportunity to investigate the remodeling of the neural circuits underlying the proprioceptive control of the femorotibial joint.

Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.

The mushroom bodies (MBs) are prominent structures in the Drosophila brain that are essential for olfactory learning and memory. Characterization of the development and projection patterns of individual MB neurons will be important for elucidating their functions. Using mosaic analysis with a repressible cell marker (Lee, T. and Luo, L. (1999) Neuron 22, 451-461), we have positively marked the axons and dendrites of multicellular and single-cell mushroom body clones at specific developmental stages. Systematic clonal analysis demonstrates that a single mushroom body neuroblast sequentially generates at least three types of morphologically distinct neurons. Neurons projecting into the (gamma) lobe of the adult MB are born first, prior to the mid-3rd instar larval stage. Neurons projecting into the alpha' and beta' lobes are born between the mid-3rd instar larval stage and puparium formation. Finally, neurons projecting into the alpha and beta lobes are born after puparium formation. Visualization of individual MB neurons has also revealed how different neurons acquire their characteristic axon projections. During the larval stage, axons of all MB neurons bifurcate into both the dorsal and medial lobes. Shortly after puparium formation, larval MB neurons are selectively pruned according to birthdays. Degeneration of axon branches makes early-born gamma neurons retain only their main processes in the peduncle, which then project into the adult gamma lobe without bifurcation. In contrast, the basic axon projections of the later-born (alpha'/beta') larval neurons are preserved during metamorphosis. This study illustrates the cellular organization of mushroom bodies and the development of different MB neurons at the single cell level. It allows for future studies on the molecular mechanisms of mushroom body development.

Altered outward K(+) currents in Drosophila larval neurons of memory mutants rutabaga and amnesiac.

K(+) currents in cultured Drosophila larval neurons have been classified into four categories according to their inactivation time constants, relative amplitude, and response to K(+) channel blockers 4-AP and tetraethylammonium. The percentage (65%) of neurons displaying K(+) currents which were reduced to 30% in amplitude by 5 mM cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP in both Drosophila memory mutants rutabaga (rut) and amnesiac (amn) was significantly larger than that (50%) in wild type. This initial characterization provides evidence for altered K(+) currents in both rut and amn mutants. Arachidonic acid, a specifical inhibitor of Kv4 family (shal) K(+) channels, was found to inhibit K(+) currents in cultured Drosophila neurons, suggesting the presence of shal channels in these neurons.

Genetic Dissection of Behavior: Modulation of Locomotion by Light in theDrosophila melanogasterLarva Requires Genetically Distinct Visual System Functions

The Drosophila larva modulates its pattern of locomotion when exposed to light. Modulation of locomotion can be measured as a reduction in the distance traveled and by a sharp change of direction when the light is turned on. When the light is turned off this change of direction, albeit significantly smaller than when the light is turned on, is still significantly larger than in the absence of light transition. Mutations that disrupt adult phototransduction disrupt a subset of these responses. In larvae carrying these mutations the magnitude of change of direction when the light is turned on is reduced to levels indistinguishable from that recorded when the light is turned off, but it is still significantly higher than in the absence of any light transition. Similar results were obtained when these responses were measured in strains where the larval photoreceptor neurons were ablated by mutations in the glass (gl) gene or by the targeted expression of the cell death gene head involution defective (hid). A mutation in the homeobox gene sine oculis (so) that ablates the larval visual system, or the targeted expression of the reaper (rpr) cell death gene, abolishes all responses to light detected as a change of direction. We propose the existence of an extraocular light perception that does not use the same phototransduction cascade as the adult photoreceptors. Our results indicate that this novel visual function depends on the blue-absorbing rhodopsin Rh1 and is specified by the so gene.

In vivo recording from identifiable neurons of the locomotor network in the developing zebrafish

The zebrafish is a popular model for developmental studies due to its accessibility by cellular, molecular and genetic approaches. As a complement to these other methods, we have devised an exposed hindbrain/spinal cord preparation in the curarized zebrafish embryo and larva that permits intracellular labeling and patch clamp recording from individually identified sensory neurons, motoneurons and interneurons in vivo. Regular bursts of synaptic potentials and action potentials were observed under whole-cell current clamp in embryonic motoneurons and in some identified interneurons. Larval neurons showed prolonged depolarizations with synaptically driven bursts of action potentials. Frequent spontaneous synaptic potentials were observed and synaptic currents were effectively space clamped. It is thus feasible to study in vivo the properties of identifiable neurons of the developing locomotor network in the zebrafish, including their synaptic activity, firing patterns and interconnections.

Metamorphic control of cyclic guanosine monophosphate expression in the nervous system of the tobacco hornworm, Manduca sexta.

During metamorphosis of Manduca sexta, defined sets of neurons show a dramatic accumulation of cyclic guanosine monophosphate (cGMP). Although many of these cells show low but detectable levels of cGMP during specific developmental windows, these levels are enhanced dramatically during dissection of the central nervous system (CNS). The ability of these neurons to show this induced cGMP expression depends on the developmental stage. Larvae do not show this capacity but it appears during the transition from the larval to the pupal stage. There are two different classes of response: the early expressing neurons start to show a cGMP response at the beginning of the prepupal stage while the late expressing cGMP neurons start at different times during the pupal-adult transition. The former set includes larval neurons that will likely be remodeled during metamorphosis, and a number of them are serotonergic. The late-expressing group also includes some larval cells, but most are adult-specific neurons. At least for one adult-specific cluster, the antennal lobe neurons, the cGMP expression parallels the maturation phase of these cells.

Drosophila EcR-B ecdysone receptor isoforms are required for larval molting and for neuron remodeling during metamorphosis.

During the metamorphic reorganization of the insect central nervous system, the steroid hormone 20-hydroxyecdysone induces a wide spectrum of cellular responses including neuronal proliferation, maturation, cell death and the remodeling of larval neurons into their adult forms. In Drosophila, expression of specific ecdysone receptor (EcR) isoforms has been correlated with particular responses, suggesting that different EcR isoforms may govern distinct steroid-induced responses in these cells. We have used imprecise excision of a P element to create EcR deletion mutants that remove the EcR-B promoter and therefore should lack EcR-B1 and EcR-B2 expression but retain EcR-A expression. Most of these EcR-B mutant animals show defects in larval molting, arresting at the boundaries between the three larval stages, while a smaller percentage of EcR-B mutants survive into the early stages of metamorphosis. Remodeling of larval neurons at metamorphosis begins with the pruning back of larval-specific dendrites and occurs as these cells are expressing high levels of EcR-B1 and little EcR-A. This pruning response is blocked in the EcR-B mutants despite the fact that adult-specific neurons, which normally express only EcR-A, can progress in their development. These observations support the hypothesis that different EcR isoforms control cell-type-specific responses during remodeling of the nervous system at metamorphosis.