A map of taste neuron projections in the Drosophila CNS

We provide a map of the projections of taste neurons in the CNS of Drosophila. Using a collection of 67 GAL4 drivers representing the entire repertoire of Gr taste receptors, we systematically map the projections of neurons expressing these drivers in the thoracico-abdominal ganglion and the suboesophageal ganglion (SOG). We define 9 categories of projections in the thoracico-abdominal ganglia and 10 categories in the SOG. The projection patterns are modular, and can be interpreted as combinations of discrete pattern elements. The elements can be interpreted in terms of the taste organ from which the projections originate, the structures from which they originate, and the quality of taste information that they represent. The extensive diversity in projection patterns provides an anatomical basis for functional diversity in responses elicited by different taste stimuli.     

Sensory neurons and peripheral pathways in Drosophila embryos

Summary The thoracic and abdominal segments of the Drosophila embryo contain 373 neurons innervating external sensory structures and 162 neurons innervating chordotonal organs. These neurons are arranged in ventral, lateral and dorsal clusters within each segment, in a highly invariant pattern. Two fascicles are formed in each segment as the sensory axons grow ventrally towards the CNS and meet motor axons growing dorsally from the CNS. In all but the last segment, the anterior fascicle is contributed by the dorsal and lateral neurons, while the posterior one is formed by the ventral neurons. Five distinct segmental patterns are described, corresponding to (1) the prothorax, (2) the other two thoracic segments, (3) the first seven abdominal segments, (4) the eighth and (5) the ninth (and possibly the tenth) abdominal segments.The publisher regrets that two companion papers unfortunately were published out of sequence. The present paper should have preceded the paper entitled The sense organs in the Drosophila larva and their relation to the embryonic pattern of sensory neurons, which appeared in Volume 195, Number 4 of the journal (pp 222–228)     

Calcium spikes in a leech nonspiking neuron

The NS neurons are nonspiking cells, present as pairs in each midbody ganglion of the leech nervous system, which display a very extensive arborization. They were shown to regulate the coactivation of motoneurons. Here we have investigated the electrophysiological properties of these neurons under the hypothesis that transmission along the extensive neurites requires the aid of voltage-dependent conductances. The results indicate that NS neurons respond to electrical stimulation with a spike-like event, which was not an all-or-none but rather a graded phenomenon that depended on the intensity and duration of the electrical stimulus. The spike-like response was activated at a membrane potential of approximately −50 mV; its amplitude was a logarithmic function of the extracellular Ca²⁺ concentration and was unaffected by a broad range of changes in the extracellular Na⁺ concentration; intracellular application of tetraethylammonium (TEA) caused a large increase in its amplitude and duration. These data indicate that NS neurons bear voltage-dependent low-threshold Ca²⁺ and TEA-sensitive K⁺ conductances that could contribute to shaping synaptic signals, or transmission along the extensive neuritic tree.     

Axon extension and retraction by leech neurons: severing early projections to peripheral targets prevents normal retraction of other projections

During leech embryogenesis, interactions between homologous neurons in neighboring segments lead to the selective retraction of longitudinal axonal projections by midbody AP and AE neurons, which maintain lateral axonal projections to the periphery. Results of experiments reported here show that disconnecting the lateral projections from the periphery rescues the projections normally fated to retract. We propose that these neurons normally progress through two states during early development, one in which they are insensitive to interactions with their homologs (state A) and a second in which they are sensitive (state B). Establishment of lateral connections with their targets triggers the switch from state A to state B; cutting these projections puts neurons back to state A.     

Tract formation and axon fasciculation of molecularly distinct peripheral neuron subpopulations during leech embryogenesis.

In leech, the central projections of peripheral sensory neurons segregate into specific axonal tracts, which are distinguished by differential expression of surface antigens recognized by the monoclonal antibodies Lan3-2 and Lan4-2. Lan3-2 recognizes an epitope expressed on axons that segregate into three distinct axon fascicles. In contrast, the Lan4-2-positive axons selectively project into only one of the Lan3-2-positive axon tracts. These observations provide evidence for a hierarchy of guidance cues mediating specific pathway formation in this system. Since the Lan3-2 antibody has been shown to perturb this process and since, as shown here, the Lan3-2 and Lan4-2 antigens are closely molecularly interrelated, these antibodies may help define molecules and epitopes mediating neuronal recognition and axonal guidance.     

Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation. There are four main DA neuron classes (I-IV)(1). They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation(2-10). The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology(11-13) because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses. DA neuron activity modifies the output of a larval locomotion central pattern generator(14-16). The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses(14,16-20). Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)(21). These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field(7,22,23). Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping(7,22,23), as well as the wiring of a simple circuit modulating larval locomotion(14-17). We present here a practical guide to generate and analyze genetic mosaics(24) marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)(1,10,25) and Flp-out(22,26,27) techniques (summarized in Fig. 1).     

Modulation of the pattern of axonal projections of a leech motor neuron by ablation or transplantation of its target

The sixth segmental ganglion in the ventral nerve cord of the leech H. medicinalis contains a bilateral pair of rostral penile evertor motor neurons (RPEs) that in the adult innervate the male genitalia. During embryogenesis, the RPEs extend numerous extraganglionic projections. Only two of these innervate the target and are normally retained in the adult, while the others retract. Early, but not late, removal of the male genitalia results in the indefinite retention and continued growth of projections that would normally retract. Any of these projections can innervate targets transplanted to ectopic locations. We conclude that an RPE motor neuron requires a signal, provided by its interaction with the target organ during a critical period, in order to stop extending axons, stabilize those axons that contact the target, and retract those that do not.     

Calmodulin and son of sevenless dependent signaling pathways regulate midline crossing of axons in the Drosophila CNS

The establishment of axon trajectories is ultimately determined by the integration of intracellular signaling pathways. Here, a genetic approach in Drosophila has demonstrated that both Calmodulin and Son of sevenless signaling pathways are used to regulate which axons cross the midline. A loss in either signaling pathway leads to abnormal projection of axons across the midline and these increase with roundabout or slit mutations. When both Calmodulin and Son of sevenless are disrupted, the midline crossing of axons mimics that seen in roundabout mutants, although Roundabout remains expressed on crossing axons. Calmodulin and Son of sevenless also regulate axon crossing in a commissureless mutant. These data suggest that Calmodulin and Son of sevenless signaling pathways function to interpret midline repulsive cues which prevent axons crossing the midline.     

Photosensory input pathways in the medicinal leech

Summary The medicinal leech,Hirudo medicinalis possesses two types of photosensory organs: five bilateral pairs of eyes embedded in two longitudinal rows in the dorsal surface of the head, and seven bilateral pairs of sensilla situated in both the dorsal and the ventral surface of each of the 21 body segments. The photoreceptor cells of each eye or sensillum project their axons centrally via a characteristic cephalic or segmental nerve which carries the photosensory input to the brain or to the segmental ganglion. In response to a pulse of light the photoreceptors produce a train of impulses whose frequency first rises to anearly peak and then declines to asteady state plateau at which it remains until the end of the pulse. The amplitude of the early peak response and the level of the steady state plateau rise linearly with the log of the light pulse intensity, but the dynamic range of the early peak response is much narrower than that of the plateau. Both ocular and sensillar photoreceptors adapt to the intensity of interpulse background illumination; the ocular receptors adapt so completely that their level of background activity is nearly independent of the background light intensity, whereas the ventral sensillar photoreceptors adapt incompletely, so that their background activity rises with the background light intensity. Ocular and sensillar photoreceptors make their maximal response to green light at a wavelength of about 540 nm. They are almost insensitive to red and violet light at both extremes of the visible spectrum. The photosensory response of a single eye is directionally selective, whereas that of a single sensillum has much less directional selectivity. Several higher order sensory neurons were identified in the segmental ganglion that receive photosensory input from the sensilla. One of these neurons has the sensillum in the ipsilateral dorso-medial body wall of the same segment as its receptive field and another neuron the bilateral set of ventral sensilla in the body wall of the next posterior segment.We are indebted to Frank S. Werblin for valuable advice and discussions. We thank Kenneth L. Carlock for designing and constructing much of the special electronic equipment used in this study. We also thank Alexander Petruncola for his helpful suggestions regarding the computational analysis of the experimental results and for writing the computer programs used in the processing of the data.This research was supported by Grant No. GB 31933X from the National Science Foundation, and NIH research grant No. GM 17866 and Training Grant No. GM 00829 from the Institute for General Medical Sciences.     

Localization of the myomodulin-like immunoreactivity in the leech CNS

The distribution of myomodulinlike immunoreactivity in the leech CNS was determined using an antiserum raised against Aplysia myomodulin. Segmental ganglia contained approximately 60 immunoreactive neurons. In addition, numerous fibers containing immunoreactive varicosities were found throughout the neuropil. Using a combination of Lucifer Yellow injections and immunocytochemistry, we identified neurons including the anterior Pagodas (AP), annulus erector (AE), motor neurons, Leydig, longitudinal muscle motoneurons (L), S cells, and coupling interneurons, all of which are active during the touch-elicited shortening reflex. FMRF-amide-like immunoreactivity in three of these cells (L, AP, and AE) was previously demonstrated. Specific staining for myomodulin was abolished by preadsorption of the antiserum with synthetic myomodulin, but not with FMRF-amide. These results suggest a potential role for myomodulin in both intrinsic and extrinsic modulation of the leech touch-elicited shortening reflex. Further, it is possible that several neurons mediating this reflex contain multiple neuromodulatory peptides. © 1996 John Wiley & Sons, Inc.     

Leydig neuron activity modulates heartbeat in the medicinal leech

1. Leydig neurons fire spontaneously at low rates (less than 4 Hz), but their activity increases with mechanical stimulation or electrical stimulation of mechanosensory neurons. These conditions also cause acceleration of bursting in heart motor neurons. 2. The firing rate of Leydig cells was found to regulate heart rate in chains of isolated ganglia. When Leydig neurons were made to fire action potentials at relatively high frequencies (ca. 5-10 Hz), however, heart motor neurons ceased bursting and were either silenced or fired erratically. 3. Firing of Leydig neurons at high rates caused bilateral heart interneurons of ganglia 3 or 4 to fire tonically rather than in their normal alternating bursts Tonic firing of these heart interneurons accounts for the prolonged barrages of ipsps recorded in heart motor neurons and the disruption of their normal cyclic activity. 4. Preventing spontaneous activity of Leydig neurons with injected currents in isolated ganglia caused deceleration of the heartbeat rhythm but did not halt oscillation. 5. Electrical stimulation of peripheral nerve roots with Leydig neuron activity suppressed in isolated ganglia caused acceleration of heart rate.     

Developmental interdeterminacy in embryos of the leech Helobdella triserialis

In embryonic development of the leech Helobdella triserialis, each of the four paired positionally identifiable, ectodermal teloblasts (N, O, P, and Q) generates a bandlet of blast cell progeny that merges with ipsilateral bandlets into a germinal band. Left and right germinal bands coalesce into the germinal plate which gives rise to the segmental tissues of the leech and wherein the progeny of each teloblast generate a characteristic pattern of epidermal and neuronal cells. Experiments reported here show that the positionally identified O teloblast sometimes generates the P pattern and vice versa. The reversal of these teloblasts' generative identities was shown to correspond to the formation of chiasmata by their blast cell bandlets, so that the positions of their bandlets in the germinal band are reversed as well. Thus it is the position of the bandlet in the germinal band, rather than the position of the parent teloblast, which correlates with the fate of o and p blast cells. Moreover, two types of ablation experiments have shown that, in the absence of generative P teloblast progeny, those cells which would normally generate the O pattern take on a new fate and give rise to the P pattern in the nervous system, both at the gross pattern level in the segmental ganglia, and at the level of identified neurons in the peripheral nervous system. If related, these phenomena suggest that the O and P teloblasts, which derive from the symmetric cleavage of the OP proteloblasts, have a common developmental pluripotency. And in that case, the fates of their progeny are determined hierarchically on the basis of relative position in the nascent germinal band, with P-type fate being preferred.     

Disruption of peripheral target contact influences the development of identified central dendritic branches in a leech motor neuron in vivo

Retrograde signaling from target tissues has been shown to influence many aspects of neuronal development in a number of developmental systems. In these experiments using embryonic leeches (Hirudo medicinalis), we examined how depriving a neuron of contact with its peripheral target affects the development of the cell's central arborization. We focused our attention on the motor neuron cell 3, which normally stimulates dorsal longitudinal muscle fibers to contract. At different locations in the periphery and in embryos of several different stages, we cut the nerve containing the growing axon of cell 3. This surgery led to dramatic overgrowth of cell 3's central dendritic branches, which normally accept synaptic contacts from other neurons, including the inhibitory motor neuron cell 1. When cell 3's peripheral axon was cut relatively early in development, its overgrown central branches eventually retracted. However, cells that were disrupted later in development retained their overextended branches into adulthood. In addition, if the axon was cut close to the ganglion early in development, depriving the cell of contact with any dorsal tissues, the central branches failed to retract and were instead retained into adulthood. Unlike cell 3, the central branches of cell 1, which has the same peripheral target muscles as cell 3, remained unchanged following all axotomy protocols. These results suggest that in at least some neurons contact with peripheral targets can influence development of the central processes that normally mediate synaptic contacts.     

Caspase-dependent apoptotic pathways in CNS injury

Recent studies have suggested a role for neuronal apoptosis in cell loss following acute CNS injury as well as in chronic neurodegeneration. Caspases are a family of cysteine requiring aspartate proteases with sequence similarity to Ced-3 protein of Caenorhabditis elegans. These proteases have been found to contribute significantly to the morphological and biochemical manifestations of apoptotic cell death. Caspases are translated as inactive zymogens and become active after specific cleavage. Of the 14 identified caspases, caspase-3 appears to be the major effector of neuronal apoptosis induced by a variety of stimuli. A role for caspase-3 in injury-induced neuronal cell death has been established using semispecific peptide caspase inhibitors. This article reviews the current literature relating to pathways regulating caspase activation in apoptosis associated with acute and chronic neurodegeneration, and suggests that identification of critical upstream caspase regulatory mechanisms may permit more effective treatment of such disorders.     

Processing of sensory signals by a non-spiking neuron in the leech

The non-spiking neurons 151 are present as bilateral pairs in each midbody ganglion of the leech nervous system and they are electrically coupled to several motorneurons. Intracellular recordings were used to investigate how these neurons process input from the mechanosensory P neurons in isolated ganglia. Induction of spike trains (15 Hz) in single P cells evoked responses that combined depolarizing and hyperpolarizing phases in cells 151. The phasic depolarizations, transmitted through spiking interneurons, reversed at around -20 mV. The hyperpolarization had two components, both reversing at around -65 mV, and which were inhibited by strychnine (10 micromol l(-1)). The faster component was transmitted through spiking interneurons and the slower component through a direct P-151 interaction. Short trains (<400 ms) of P cell spikes (15 Hz) evoked the phasic depolarizations superimposed on the hyperpolarization, while long spike trains (>500 ms) produced a succession of depolarizations that masked the hyperpolarizing phase. The amplitude and duration of the hyperpolarization reached their maximum at the initial spikes in a train, while the depolarizations persisted throughout the duration of the stimulus train. Both phases of the response were relatively unaffected by the spike frequency (5-25 Hz). The non-spiking neurons 151 processed the sensory signals in the temporal rather than in the amplitude domain.     

The role of matrix molecules in regeneration of leech CNS.

Extracellular matrix (ECM) molecules extracted from the leech central nervous system (CNS) provide substrates that induce extensive growth of processes of identified leech nerve cells in culture. Two ECM molecules, laminin and tenascin, have been identified. The laminin-like molecule has been purified and shown to be a cross-shaped molecule similar to vertebrate laminin with subunits of 340, 220, 180, and 160 kD. Purified laminin as a substrate induces rapid outgrowth of Retzius (R) and Anterior Pagoda (AP) cells in culture. The tenascin molecule has been partially purified. In electronmicrographs, leech tenascin, like vertebrate tenascin, has six arms of equal size joined in a central globule. Highly enriched fractions of leech tenascin induce rapid and extensive outgrowth of Retzius and AP cells in culture. Substrate molecules not only induce outgrowth of processes but also affect the growth patterns of individual nerve cells. Neurites are straight with few branches in laminin, but curved with profuse branches on tenascin. During regeneration of the CNS in the animal, laminin appears at new sites associated with growth cones. The appearance of laminin correlates with the accumulation of microglial cells. Thus, ECM molecules with growth-promoting activity for leech nerve cells in vitro appear to be involved in inducing regeneration and allowing the neurites to reconnect with former targets.     

Posttranslational processing of alpha-tubulin during axoplasmic transport in CNS axons

Tubulin proteins in mouse retinal ganglion cell (RGC) neurons were analyzed to determine whether they undergo posttranslational processing during axoplasmic transport. Alpha- and beta-tubulin comprised heterogeneous proteins in the primary optic pathway (optic nerve and optic tract) when examined by two-dimensional (2D) PAGE. In addition, however, alpha-tubulin exhibited regional heterogeneity when consecutive 1.1-mm segments of the optic pathway were analyzed separately. In proximal segments, alpha-tubulin consisted of two predominant proteins separable by isoelectric point and several less abundant species. In more distal segments, these predominant proteins decreased progressively and the alpha-tubulin region of the gel was represented by less abundant multiple forms only; beta-tubulin region of the gel was represented by less abundant multiple forms only; beta- tubulin was the same in all segments. After intravitreal injection of [3H]proline to mice, radiolabeled alpha- and beta-tubulin heteroproteins were conveyed together at a rate of 0.1-0.2 mm/d in the slowest phase of axoplasmic transport. At 45 d postinjection, the distribution of radiolabeled heterogeneous forms a alpha- and beta- tubulin in consecutive segments of optic pathway resembled the distribution of unlabeled proteins by 2D PAGE, indicating that regional heterogeneity of tubulin arises during axonal transport. Peptide mapping studies demonstrated that the progressive alteration of alpha- tubulin revealed by PAGE analysis cannot be explained by contamination of the alpha-tubulin region by other proteins on gels. The results are consistent with the posttranslational processing of alpha-tubulin during axoplasmic transport. These observations, along with the accompanying report (J. Cell Biol., 1982, 94:150-158), provide additional evidence that CNS axons may be regionally specialized.