Anatomical and physiological observations on the organization of mechanoreceptors and local interneurons in the central nervous system of the wandering spider Cupiennius salei

Summary In the wandering spider Cupiennius salei, the functional neuroanatomy of leg mechanosensory receptor neurons and interneurons associated with a single leg neumere was investigated by combined intracellular recording and Lucifer yellow ionophoresis. Trichobothria axons that selectively respond to air currents and to low-frequency airborne vibrations have arborizations restricted to ventral regions of the appropriate leg neuromere. Receptor afferents that respond selectively to substrateborne vibrations are distributed ventrally in the corresponding leg neuromere and extend into certain interganglionic tract neuropiles. Golgi impregnation and intracellular dye filling show that local interneurons originate in ventral sensory neuropiles of leg neuromeres and ascend dorsally to terminate amongst dendrites of motor neurons. Local interneurons generally show higher thresholds for vibration stimuli than do receptors. Local interneurons typically receive inputs from one or several types of receptors. Some respond to stimulation of a single leg, others respond to stimulation of several legs on the same side of the body. The functional morphology of the receptor afferents is correlated with known physiological characteristics of slit sensilla and trichobothria. Structure and activity of the local interneurons are compared with analogous interneurons in other arthropods.     

Homologues of serotonergic central pattern generator neurons in related nudibranch molluscs with divergent behaviors

Homologues of a neuron that contributes to a species-specific behavior were identified and characterized in species lacking that behavior. The nudibranch Tritonia diomedea swims by flexing its body dorsally and ventrally. The dorsal swim interneurons (DSIs) are components of the central pattern generator (CPG) underlying this rhythmic motor pattern and also activate crawling. Homologues of the DSIs were identified in six nudibranchs that do not exhibit dorsal–ventral swimming: Tochuina tetraquetra, Melibe leonina, Dendronotus iris, D. frondosus, Armina californica, and Triopha catalinae. Homology was based upon shared features that distinguish the DSIs from all other neurons: (1) serotonin immunoreactivity, (2) location in the Cerebral serotonergic posterior (CeSP) cluster, and (3) axon projection to the contralateral pedal ganglion. The DSI homologues, named CeSP-A neurons, share additional features with the DSIs: irregular basal firing, synchronous inputs, electrical coupling, and reciprocal inhibition. Unlike the DSIs, the CeSP-A neurons were not rhythmically active in response to nerve stimulation. The CeSP-A neurons in Tochuina and Triopha also excited homologues of the Tritonia Pd5 neuron, a crawling efferent. Thus, the CeSP-A neurons and the DSIs may be part of a conserved network related to crawling that may have been co-opted into a rhythmic swim CPG in Tritonia. This material is based upon work supported by the National Science Foundation, under Grant No. 0445768, and a GSU Research Program Enhancement grant to PSK.     

Regulation of afferent transmission in the feeding circuitry of Aplysia

Although feeding in Aplysia is mediated by a central pattern generator (CPG), the activity of this CPG is modified by afferent input. To determine how afferent activity produces the widespread changes in motor programs that are necessary if behavior is to be modified, we have studied two classes of feeding sensory neurons. We have shown that afferent-induced changes in activity are widespread because sensory neurons make a number of synaptic connections. For example, sensory neurons make monosynaptic excitatory connections with feeding motor neurons. Sensori-motor transmission is, however, regulated so that changes in the periphery do not disrupt ongoing activity. This results from the fact that sensory neurons are also electrically coupled to feeding interneurons. During motor programs sensory neurons are, therefore, rhythmically depolarized via central input. These changes in membrane potential profoundly affect sensori-motor transmission. For example, changes in membrane potential alter spike propagation in sensory neurons so that spikes are only actively transmitted to particular output regions when it is behaviorally appropriate. To summarize, afferent activity alters motor output because sensory neurons make direct contact with motor neurons. Sensori-motor transmission is, however, centrally regulated so that changes in the periphery alter motor programs in a phase-dependent manner.     

Pathfinding by sensory axons in Drosophila: substrates and choice points in early lch5 axon outgrowth

We have examined the pattern of axon growth from the lateral chordotonal (lch5) neurons in the body wall of the Drosophila embryo and identified cellular substrates and choice points involved in early axon pathfinding by these sensory neurons. At the first choice point (TP1), the lch5 growth cones contact the most distal cells of the spiracular branch (SB) of the trachea. The SB provides a substrate along which the axons extend internally to the level of the intersegmental nerve (ISN). In the absence of the SB, the lch5 axons often stall near TP1 or follow aberrant routes towards the CNS. At the second choice point (TP2), the lch5 growth cones make their first contact with other axons and turn ventrally toward the CNS, fasciculating specifically with the motor axons of the ISN.     

Glutamate drives the touch response through a rostral loop in the spinal cord of zebrafish embryos

Characterizing connectivity in the spinal cord of zebrafish embryos is not only prerequisite to understanding the development of locomotion, but is also necessary for maximizing the potential of genetic studies of circuit formation in this model system. During their first day of development, zebrafish embryos show two simple motor behaviors. First, they coil their trunks spontaneously, and a few hours later they start responding to touch with contralateral coils. These behaviors are contemporaneous until spontaneous coils become infrequent by 30 h. Glutamatergic neurons are distributed throughout the embryonic spinal cord, but their contribution to these early motor behaviors in immature zebrafish is still unclear. We demonstrate that the kinetics of spontaneous coiling and touch‐evoked responses show distinct developmental time courses and that the touch response is dependent on AMPA‐type glutamate receptor activation. Transection experiments suggest that the circuits required for touch‐evoked responses are confined to the spinal cord and that only the most rostral part of the spinal cord is sufficient for triggering the full response. This rostral sensory connection is presumably established via CoPA interneurons, as they project to the rostral spinal cord. Electrophysiological analysis demonstrates that these neurons receive short latency AMPA‐type glutamatergic inputs in response to ipsilateral tactile stimuli. We conclude that touch responses in early embryonic zebrafish arise only after glutamatergic synapses connect sensory neurons and interneurons to the contralateral motor network via a rostral loop. This helps define an elementary circuit that is modified by the addition of sensory inputs, resulting in behavioral transformation. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Neuronal control of heartbeat in the medicinal leech

Summary The leech heartbeat consists of a constriction-dilation rhythm of two lateral heart tubes extending over the length of the body. The beats of the segmental sections of these two tubes are coordinated in such a manner that the heart tube of one body side produces a frontward peristaltic wave while the heart tube on the other body side produces nearly concerted constrictions. This rhythm is metastable, in that left and right heart tubes alternate between peristaltic and concerted constriction modes, with a given mode lasting for tens or hundreds of beat cycles.The constriction-dilation cycles of the segmental heart tube sections are controlled by a set of rhythmically active motor neurons, the heart excitors, or HE cells. A bilateral pair of HE cells is located in all but the two frontmost and the two rearmost segmental ganglia of the ventral nerve cord. Each HE cell innervates via excitatory synapses the circular muscle fibers in the wall of the ipsilateral heart tube section. The activity cycle of the HE cells consists of an active phase, during which they are depolarized and produce a burst of impulses, and an inactive phase during which they are repolarized by a burst of inhibitory synaptic potentials. The intersegmentally coordinated activity cycles of the HE cell set are maintained in an isolated ventral nerve cord. Hence the generation of the heart excitor rhythm does not require sensory feedback.We are indepted to Amy Kelly and King-Wai Yau for advice on the use of the intracellular staining technique and to John Kretz for calling to our attention the existence of an afferent impulse burst rhythm emanating from denervated heart tube preparations. We thank Georgia Harper for excellent technical assistance. This research was supported by Grant GB 31933X from the National Science Foundation and NIH research grants GM17866 and Training Grant GM 01389 from the Institute of General Medical Sciences.     

Grainyhead-like 2 regulates neural tube closure and adhesion molecule expression during neural fold fusion

Defects in closure of embryonic tissues such as the neural tube, body wall, face and eye lead to severe birth defects. Cell adhesion is hypothesized to contribute to closure of the neural tube and body wall; however, potential molecular regulators of this process have not been identified. Here we identify an ENU-induced mutation in mice that reveals a molecular pathway of embryonic closure. Line2F homozygous mutant embryos fail to close the neural tube, body wall, face, and optic fissure, and they also display defects in lung and heart development. Using a new technology of genomic sequence capture and high-throughput sequencing of a 2.5 Mb region of the mouse genome, we discovered a mutation in the grainyhead-like 2 gene (Grhl2). Microarray analysis revealed Grhl2 affects the expression of a battery of genes involved in cell adhesion and E-cadherin protein is drastically reduced in tissues that require Grhl2 function. The tissue closure defects in Grhl2 mutants are similar to that of AP-2α null mutants and AP-2α has been shown to bind to the promoter of E-cadherin. Therefore, we tested for a possible interaction between these genes. However, we find that Grhl2 and AP-2α do not regulate each other's expression, E-cadherin expression is normal in AP-2α mutants during neural tube closure, and Grhl2;AP-2α trans-heterozygous embryos are morphologically normal. Taken together, our studies point to a complex regulation of neural tube fusion and highlight the importance of comparisons between these two models to understand more fully the molecular pathways of embryonic tissue closure.     

The organization of plurisegmental mechanosensitive interneurons in the central nervous system of the wandering spider Cupiennius salei

Summary In spiders the bulk of the central nervous system (CNS) consists of fused segmental ganglia traversed by longitudinal tracts, which have precise relationships with sensory neuropils and which contain the fibers of large plurisegmental interneurons. The responses of these interneurons to various mechanical stimuli were studied electrophysiologically, and their unilateral or bilateral structure was revealed by intracellular staining. Unilateral interneurons visit all the neuromeres on one side of the CNS. They receive mechanosensory input either from a single leg or from all ipsilateral legs via sensory neurons that invade leg neuromeres and project into specific longitudinal tracts. The anatomical organization of unilateral interneurons suggests that their axons impart their information to all ipsilateral leg neuromeres. Bilateral interneurons are of two kinds, symmetric and asymmetric neurons. The latter respond to stimulation of all legs on one side of the body, having their dendrites amongst sensory tracts of the same side of the CNS. Anatomical evidence suggests that their terminals invade all four contralateral leg neuromeres. Bilaterally symmetrical plurisegmental interneurons have dendritic arborizations in both halves of the fused ventral ganglia. They respond to the stimulation of any of the 8 legs. A third class of cells, the ascending neurons have unilateral or bilateral dendritic arborizations in the fused ventral ganglia and show blebbed axons in postero-ventral regions of the brain. Their response characteristics are similar to those of other plurisegmental interneurons. Descending neurons have opposite structural polarity, arising in the brain and terminating in segmental regions of the fused ventral ganglia. Descending neurons show strong responses to visual stimulation. Approximately 50% of all the recorded neurons respond exclusively to stimulation of a single type of mechanoreceptor (either tactile hairs, or trichobothria, or slit sensilla), while the rest respond to stimulation of a variety of sensilla. However, these functional differences are not obviously reflected by the anatomy. The functional significance of plurisegmental interneurons is discussed with respect to sensory convergence and the coordination of motor output to the legs. A comparison between the response properties of certain plurisegmental interneurons and their parent longitudinal tracts suggests that the tracts themselves do not reflect a modality-specific organization.Abbreviations BPI bilateral plurisegmental interneuron - CNS central nervous system - FVG fused ventral ganglia - LT longitudinal tract - PI plurisegmental interneuron - PSTH peristimulus timehistogram - UPI unilateral plurisegmental interneuron     

Fictive chewing activity in motor neurons and interneurons of the suboesophageal ganglion of Manduca sexta larvae

Alternating antiphasic rhythmic activity was observed in opener and closer mandibular motor neurons in the isolated suboesophageal ganglion of the larva of Manduca sexta (Lepidoptera: Sphingidae). This was interpreted provisionally as fictive chewing; the pattern is similar to that seen in semiintact animals but of lower frequency. Additionally, a variety of associated rhythmic activities were observed in suboesophageal interneurons. These could be classified into several different physiological types by their activity patterns in relation to the chewing cycle. Some of these neurons can modulate the rhythm when injected with current. It seems likely that they are part of or associated with a central pattern generator circuit for chewing.Abbreviations A anterior - CEC circumoesophageal connective - Cl-MN closer motor neuron - IN interneuron - MdN mandibular nerve - MN motor neuron - O-MN opener motor neuron     

Motor Axon Pathfinding

Motor neurons are functionally related, but represent a diverse collection of cells that show strict preferences for specific axon pathways during embryonic development. In this article, we describe the ligands and receptors that guide motor axons as they extend toward their peripheral muscle targets. Motor neurons share similar guidance molecules with many other neuronal types, thus one challenge in the field of axon guidance has been to understand how the vast complexity of brain connections can be established with a relatively small number of factors. In the context of motor guidance, we highlight some of the temporal and spatial mechanisms used to optimize the fidelity of pathfinding and increase the functional diversity of the signaling proteins.Motor neurons residing in the brain stem and spinal cord extend axons into the periphery and are the final relay cells for locomotor commands. These cells are among the longest projection neurons in the body and their axons follow stereotypical pathways during embryogenesis to synapse with muscle and sympathetic/parasympathetic targets. Cellular studies of motor axon navigation in developing chick and zebrafish embryos have shown that motor neurons located at different rostrocaudal positions show specific preferences for axonal pathways (see Landmesser 2001; Lewis and Eisen 2003 for reviews). This early cellular research laid the foundation for molecular studies of motor axon guidance by establishing the concept that motor neurons are in fact a diverse cell population. The molecular studies covered in this article have sought to identify genetic differences between motor neurons and to characterize the signaling pathways that underlie the specificity of motor axon targeting.     

Growth cones stall and collapse during axon outgrowth in Caenorhabditis elegans.

During nervous system development, neurons form synaptic contacts with distant target cells. These connections are formed by the extension of axonal processes along predetermined pathways. Axon outgrowth is directed by growth cones located at the tips of these neuronal processes. Although the behavior of growth cones has been well-characterized in vitro, it is difficult to observe growth cones in vivo. We have observed motor neuron growth cones migrating in living Caenorhabditis elegans larvae using time-lapse confocal microscopy. Specifically, we observed the VD motor neurons extend axons from the ventral to dorsal nerve cord during the L2 stage. The growth cones of these neurons are round and migrate rapidly across the epidermis if they are unobstructed. When they contact axons of the lateral nerve fascicles, growth cones stall and spread out along the fascicle to form anvil-shaped structures. After pausing for a few minutes, they extend lamellipodia beyond the fascicle and resume migration toward the dorsal nerve cord. Growth cones stall again when they contact the body wall muscles. These muscles are tightly attached to the epidermis by narrowly spaced circumferential attachment structures. Stalled growth cones extend fingers dorsally between these hypodermal attachment structures. When a single finger has projected through the body wall muscle quadrant, the growth cone located on the ventral side of the muscle collapses and a new growth cone forms at the dorsal tip of the predominating finger. Thus, we observe that complete growth cone collapse occurs in vivo and not just in culture assays. In contrast to studies indicating that collapse occurs upon contact with repulsive substrata, collapse of the VD growth cones may result from an intrinsic signal that serves to maintain growth cone primacy and conserve cellular material.     

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77–86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.     

Evidence that the Central Pattern Generator for Swimming in Tritonia Arose from a Non-Rhythmic Neuromodulatory Arousal System: Implications for the Evolution of Specialized Behavior

Comparisons of the nervous systems of closely related invertebratespecies show that identified neurons tend to be highly conservedeven though the behaviors in which they participate vary. Allopisthobranch molluscs examined have a similar set of serotonin-immunoreactiveneurons located medially in the cerebral ganglion. In a smallnumber of species, these neurons have been physiologically andmorphologically identified. In the nudibranch, Tritonia diomedea,three of the neurons (the dorsal swim interneurons, DSIs) havebeen shown to be members of the central pattern generator (CPG)underlying dorsal/ventral swimming. The DSIs act as intrinsicneuromodulators, altering cellular and synaptic properties withinthe swim CPG circuit. Putative homologues of the DSIs have beenidentified in a number of other opisthobranchs. In the notaspid,Pleurobranchaea californica, the apparent DSI homologues (As1–3)play a similar role in the escape swim and they also have widespreadactions on other systems such as feeding and ciliary locomotion.In the gymnosomatid, Clione limacina, the presumed homologousneurons (Cr-SP) are not part of the swimming pattern generator,which is located in the pedal ganglia, but act as extrinsicmodulators, responding to noxious stimuli and increasing thefrequency of the swim motor program. Putative homologous neuronsare also present in non-swimming species such as the anaspid,Aplysia californica, where at least one of the cerebral serotonergicneurons, CC3 (CB-1), evokes neuromodulatory actions in responseto noxious stimuli. Thus, the CPG circuit in Tritonia appearsto have evolved from the interconnections of neurons that arecommon to other opisthobranchs where they participate in arousalto noxious stimuli but are not rhythmically active.     

Different functions for homologous serotonergic interneurons and serotonin in species-specific rhythmic behaviours

Closely related species can exhibit different behaviours despite homologous neural substrates. The nudibranch molluscs Tritonia diomedea and Melibe leonina swim differently, yet their nervous systems contain homologous serotonergic neurons. In Tritonia, the dorsal swim interneurons (DSIs) are members of the swim central pattern generator (CPG) and their neurotransmitter serotonin is both necessary and sufficient to elicit a swim motor pattern. Here it is shown that the DSI homologues in Melibe, the cerebral serotonergic posterior-A neurons (CeSP-As), are extrinsic to the swim CPG, and that neither the CeSP-As nor their neurotransmitter serotonin is necessary for swim motor pattern initiation, which occurred when the CeSP-As were inactive. Furthermore, the serotonin antagonist methysergide blocked the effects of both the serotonin and CeSP-As but did not prevent the production of a swim motor pattern. However, the CeSP-As and serotonin could influence the Melibe swim circuit; depolarization of a cerebral serotonergic posterior-A was sufficient to initiate a swim motor pattern and hyperpolarization of a CeSP-A temporarily halted an ongoing swim motor pattern. Serotonin itself was sufficient to initiate a swim motor pattern or make an ongoing swim motor pattern more regular. Thus, evolution of species-specific behaviour involved alterations in the functions of identified homologous neurons and their neurotransmitter.     

GABA regulates the buccal motor output of Helisoma by two pharmacologically distinct actions

GABA was tested for its effects on patterned motor activity (PMA) underlying feeding. Using buccal motoneuron B19 to monitor PMA through intracellular recordings, GABA was found to exert effects at two levels. First, GABA stimulated rhythmic patterned activity resembling fictive feeding, which is under the control of the buccal CPG. In addition, GABA produced a direct inhibition of neuron B19. Both effects were observed when the buccal ganglia were studied in isolation from the rest of the central nervous system, suggesting local interactions with GABA receptors of buccal neurons. Furthermore, these two actions of GABA were found to be pharmacologically distinguishable. The direct hyperpolarization of neuron B19 was mimicked by muscimol, but not baclofen, and involved an increased chloride conductance, which was blocked by picrotoxin.Baclofen duplicated CPG activation by GABA. Picrotoxin had no effect on GABA- or baclofen-induced PMA.These results demonstrate that the Helisoma buccal ganglia have two GABA receptor types which resemble, pharmacologically, mammalian GABA_A and GABA_B receptors, and that GABA plays a key role in feeding patterned motor activity in Helisoma.Abbreviations CPG central pattern generator - GABA gammaamino butyric acid - HPLC high performance liquid chromatography - IPSP inhibitory postsynaptic potential - PMA patterned motor activity - SLRT supralateral radular tensor muscle