Anatomy and activity patterns in a multifunctional motor neuron and its surrounding circuits

Dorsal Excitor motor neuron DE-3 in the medicinal leech plays three very different dynamical roles in three different behaviors. Without rewiring its anatomical connectivity, how can a motor neuron dynamically switch roles to play appropriate roles in various behaviors? We previously used voltage-sensitive dye imaging to record from DE-3 and most other neurons in the leech segmental ganglion during (fictive) swimming, crawling, and local-bend escape (Tomina and Wagenaar, 2017). Here, we repeated that experiment, then re-imaged the same ganglion using serial blockface electron microscopy and traced DE-3’s processes. Further, we traced back the processes of DE-3’s presynaptic partners to their respective somata. This allowed us to analyze the relationship between circuit anatomy and the activity patterns it sustains. We found that input synapses important for all the behaviors were widely distributed over DE-3’s branches, yet that functional clusters were different during (fictive) swimming vs. crawling.

Anatomy and activity patterns in a multifunctional motor neuron and its surrounding circuits

Dorsal Excitor motor neuron DE-3 in the medicinal leech plays three very different dynamical roles in three different behaviors. Without rewiring its anatomical connectivity, how can a motor neuron dynamically switch roles to play appropriate roles in various behaviors? We previously used voltage-sensitive dye imaging to record from DE-3 and most other neurons in the leech segmental ganglion during (fictive) swimming, crawling, and local-bend escape (Tomina and Wagenaar, 2017). Here, we repeated that experiment, then re-imaged the same ganglion using serial blockface electron microscopy and traced all of DE-3’s processes. Further, we traced back the processes of all of DE-3’s presynaptic partners to their respective somata. This allowed us to analyze the relationship between circuit anatomy and the activity patterns it sustains. We found that input synapses important for all of the behaviors were widely distributed over DE-3’s branches, yet that functional clusters were different during (fictive) swimming vs. crawling.

Local and Intersegmental Interactions of Coordinating Neurons and Local Circuits in the Swimmeret System

During forward swimming, periodic movements of swimmerets on different segments of the crayfish abdomen progress from back to front with the same period. Information encoded as bursts of spikes by coordinating neurons in each segmental ganglion is necessary for this coherent organization. This information is conducted to targets in other ganglia. When an individual coordinating neuron is stimulated at different phases in the system's cycle of activity, the timing of motor output from other ganglia may be altered. In models of this coordinating circuit, we assumed that each coordinating neuron encodes information about the state of the local pattern-generating circuit in its home ganglion but is not part of that local circuit. We tested this assumption by stimulating individual coordinating neurons of two kinds—ASCE and DSC—at different phases under two conditions: with the target ganglion functional, and with the target ganglion silenced. Blocking a DSC neuron's target ganglion did not alter its negligible influence on the output from its home ganglion; the phase-response curves (PRC) remained flat. Blocking an ASCE neuron's target ganglion significantly affected its influence on the output from its home ganglion. We had predicted that ASCE's modest phase-dependent influence would disappear with the target silenced, but instead the amplitude of the PRCs increased significantly. Thus we have two different results: DSC neurons conformed to prediction based on the models’ assumptions, but ASCE neurons showed an unexpected property, one that is partially masked when the bidirectional flow of information between neighboring ganglia is operating normally.

Development of sensory processes during limb regeneration in adult crayfish.

The capacity of the crayfish Procambarus clarkii to regenerate its walking legs provides a system for studying the mechanisms of neural regeneration and repair. A set number of excitatory and inhibitory motor neurons innervate all the limb musculature throughout the normal development and regeneration of a limb. The cell bodies of the motor neurons reside within the segmental ganglion and, upon loss of the limb, their axons regrow from their severed distal ends. The cell bodies of the sensory neurons, in contrast, are located close to their sensory endings within the limb, and they are therefore lost, along with the limb, upon autotomy, leaving the severed, distal axonal stumps of the sensory neurons within the ganglionic root. During the regeneration of a limb, new sensory neurons develop within the limb, and their axons must then grow into the ganglionic root to make the appropriate connections for the new limb to become functional. Evidence is presented in the present paper that the sensory axonal stumps do not degenerate before the new sensory neurons appear within the root as the limb regenerates. These results also indicate a progressive advance of growth cones, presumably sensory in origin, towards the neuropil within the ganglion over time.

Photoinactivation of the giant neuropil glial cells in the leech Hirudo medicinalis: effects on neuronal activity and synaptic transmission

1. We studied the effects of photoinactivation of neuropil glial (NG) cells of the leech Hirudo medicinalis on neuronal activity and synaptic transmission. Each segmental ganglion contains two of these giant glial cells, which are electrically and dye coupled. 2. One of the two NG cells in an isolated segmental ganglion was filled with the dye Lucifer yellow (LY). Subsequent irradiation of the ganglion with laser light (440 nm) to photolyze LY caused irreversible depolarization of both NG cells. The NG cells that were filled with LY depolarized from -73 +/- 1.1 (SE) mV to -22 +/- 2.4 mV within 25 +/- 2.8 min of continuous irradiation (n = 22). The other NG cell, which was not directly filled with LY, depolarized with some delay. 3. Photoinactivation of the NG cells caused an irreversible depolarization of Retzius neurons and noxious (N) sensory cells by a mean of 14 mV (n = 36) and 9 mV (n = 24), respectively. In addition, the input resistance was reduced by 54% in Retzius cells and by 34% in N cells. Spikes could not be evoked in Retzius cells after the inactivation of the NG cells, either by intracellular current injection or by electrical nerve stimulation. Similarly, anterior pagoda neurons, annulus erector neurons, and the excitor neurons of the ventrolateral circular muscles became inexcitable. However, N cells, heart interneurons, and most of the heart motor neurons, touch cells, and pressure cells could still generate spontaneous or evoked action potentials. 4. Photoinactivation of the NG cells impaired the electrical connection between the two Retzius neurons. The electrical coupling was completely eliminated in six of eight cell pairs and reduced by 66% in two others. 5. Photoinactivation of the NG cells in the 3rd and 4th segmental ganglion caused a complete block of the chemical synapse between reciprocal inhibitory heart interneurons in these ganglia; the bursting rhythm either stopped or changed to a tonic activity, whereas inhibitory postsynaptic potentials could not be recorded in either heart interneuron anymore. 6. Laser irradiation alone had no effect on neuronal activity and synaptic transmission. Addition of glutathione (10 mM) and ascorbic acid (10 mM) to the saline to bind extracellular radicals that might be produced by the irradiation did not suppress the effects caused by photoinactivation of NG cells. 7. Elevation of bath K+ concentration to 12 mM, acidification of the saline to pH 5.5, and alkalinization to pH 8.5 for 6 min each did not mimick the effects on membrane properties of Retzius cells as produced by inactivation of NG cells. The results suggest some role of glial cells in the maintenance of neuronal activity and electrical and chemical synaptic transmission.

Properties of the nociceptive neurons of the leech segmental ganglion

1. The electrical responses of nociceptive (N) lateral and N medial neurons of the leech segmental ganglion to mechanical, chemical, and thermal stimulation of the skin were studied in a superfused ganglion-body wall preparation. 2. Mechanical indentation of the skin > 10 mN evoked in both types of cells a sustained discharge of impulses; afterdischarge was often observed with suprathreshold stimulations. 3. Application to the cutaneous receptive area of 10-100 mM acetic acid or of NaCI crystals and solutions also elicited a firing response in N medial and N lateral cells. In contrast, capsaicin applied to the skin (3.3 x 10(-5) to 3.3 x 10(-2) M) excited N lateral but not N medial neurons. Likewise, impulse discharges were obtained when capsaicin was applied to the cell bodies of N lateral but not of N medial neurons. 4. In both types of N neurons, heating of the skin above 39 degrees C evoked a discharge of impulses whose frequency was roughly proportional to temperature values. 5. Application of repeated suprathreshold heating cycles at 10-min intervals enhanced the impulse frequency of the response (sensitization). Shorter time intervals between heating cycles depressed the response to heat. Sensitization could not be obtained by equivalent soma depolarizations obtained by intracellular current injection. 6. Impulse discharges evoked by irritant agents were also augmented by previous application of noxious heat. 7. N lateral neurons fired in response to low-pH solutions and capsaicin directly applied onto the ganglion. N medial neurons responded inconsistently to acid and were insensitive to capsaicin. Action potentials evoked in N lateral cells by capsaicin had a slow rise, a prominent hump, and a prolonged afterhyperpolarization. 8. It is concluded that N neurons of the leech segmental ganglion respond to different modalities of noxious stimuli applied to their peripheral receptive fields and develop sensitization after repeated noxious stimulation. These properties are typical of mammalian polymodal nociceptors; thus N neurons may be a simple model for analysis of membrane mechanisms associated with polymodality of nociceptive neurons.

A separate local pattern-generating circuit controls the movements of each swimmeret in crayfish

1. Within an abdominal segment, the motor output from the segmental ganglion to the swimmerets consists of coordinated bursts of impulses in the separate pools of motor neurons innervating the left and right limbs. This coordinated motor pattern features alternating (out-of-phase) bursts of impulses in the power-stroke (PS) and return-stroke (RS) motor axons that innervate each swimmeret. PS bursts on both sides of each segment occur simultaneously (in-phase), and so RS bursts on both sides are also in-phase. 2. With all intersegmental connections interrupted, isolated abdominal ganglia were able to sustain the normal swimmeret motor pattern of alternating PS/RS activity that was bilaterally in-phase. 3. After an isolated ganglion was surgically bisected down the midline, the isolated hemiganglia that resulted could produce stable, coordinated alternation of PS and RS bursts. 4. The neuropeptide proctolin could induce rhythmic oscillations of membrane potential in swimmeret neurons when spiking was blocked by tetrodotoxin (TTX). For neurons within the same hemiganglion, these oscillations retained the same phase relations they displayed in controls, but the oscillations of neurons in different hemiganglia became uncoordinated. 5. Synaptic transmission between swimmeret neurons in the same hemiganglion persisted in the presence of TTX. Swimmeret interneurons that could activate the pattern-generating circuitry under control conditions could induce membrane-potential oscillations in swimmeret neurons of the same hemiganglion when TTX was present. 6. We conclude that a separate hemisegmental pattern-generating circuit controls the rhythmic PS and RS movements of each swimmeret. Each circuit is located in the same hemiganglion as the population of motor neurons that innervates the local swimmeret. Graded transmission is sufficient to coordinate the timing of oscillatory activity within the hemisegmental circuitry. These hemisegmental circuits are coupled by intersegmental and bilateral coordinating pathways that are dependent on sodium action potentials for their operation.

THE MULTISEGMENTAL MOTOR SUPPLY TO TRANSVERSE MUSCLES DIFFERS IN A CRICKET AND A BUSHCRICKET

Most abdominal sternites of the cricket Gryllus bimaculatus and the bushcricket Decticus albifrons are bridged by a transverse muscle (TM) which supports expiratory movements. In the cricket, ventilatory contractions are controlled both within each segment, by a bilateral pair of excitatory motoneurones in the abdominal ganglion supplying the left and right halves of the TM independently, and intersegmentally, by peripheral collaterals of homologous motoneurones from adjacent segments. The axons of these motoneurones run in the ipsilateral paramedian nerve. This unique divergence of excitatory motoneurones to different muscles also results in massive convergence of excitatory inputs from different ganglia, especially on the TMs of the middle abdominal segments. TM contraction rates are increased by this intersegmentally divergent and convergent motor supply, especially in the middle abdominal segments. In bushcrickets, each transverse muscle in segments 3–7 is innervated bilaterally by four pairs of neurones: (i) two pairs of contralateral excitatory motoneurones with axons that diverge, supplying two adacent muscles; (ii) one pair of contralateral excitatory neurones found in the second anterior ganglion and (iii) a pair of median inhibitory neurones in the segmental ganglion. Transverse muscles 2 and 8 receive reduced innervation. The excitatory motoneurones generate slow excitatory postsynaptic potentials (EPSPs), which must sum to cause muscle contractions. During ventilation, contralateral paired transverse motoneurones fire at similar frequencies, thus sychronizing the contractions of the left and right halves of the muscle so that the whole muscle acts as a single unit.

On the Function of a Locust Flight Steering Muscle and its Inhibitory Innervation

1. In tethered flying locusts, the pleuroalar (or pleuroaxillary) muscle of the forewing (M85) was stimulated via its efferent nerve. The effect on the angular setting of the wing was observed using photogrammetry. Maximal tetanic contraction of the muscle reduced downstroke pronation and upstroke supination by more than 25°. A more physiological stimulus regime resulted in angular changes of about 7°, which is near the range observed during steering manoeuvres. This confirms that the pleuroalar muscle plays an important role in adjusting the wing's aerodynamic angle of attack, as proposed in anatomical studies by Pfau (1978). 2. Unit a of the pleuroalar muscle was found to be innervated by the common inhibitor neurone 1 (CI) of the segmental ganglion. IJPs with amplitudes between 2 and 10mV were elicited by action potentials in CI. 3. A basic tonus was observed in the pleuroalar muscle in the absence of activity in excitatory motoneurones. CI input reduced this basic contracture but did not affect EJPs or muscle twitches elicited by excitatory input. 4. Activity of the common inhibitor was recorded intracellularly and with nerve electrodes in tethered flying locusts. Tonic discharges were observed with spike frequencies ranging from 5 to 35 Hz, 25 Hz being a typical value. 5. EMG recordings from the two units of the pleuroalar muscle showed that only unit a was active during most horizontal flight sequences. While its discharge was modulated in response to imposed roll movements, unit b was recruited primarily during ipsilateral roll. These results indicate functional specialization between pleuroalar muscle units a and b and suggest that the inhibitory innervation of unit a functions in the fine adjustment of wing pronation.

Leg Coordination in the Stick Insect Carausius Morosus: Effects of Cutting Thoracic Connectives

Behavioral studies of stick insects have identified six mechanisms which coordinate leg stepping. All six are active between ipsilateral leg pairs. As a first step towards locating the neurons mediating these interactions, the present study describes the effects of cutting one of the paired thoracic connectives. After the operation the following changes in step coordination occurred. The ipsilateral leg immediately caudal to the severed connective generally showed weak stepping. In free-walking animals it often remained near its posterior extreme position and dragged along the substratum. During supported walking, rhythmic stepping was common, but the swing phase of this leg was longer and both temporal and spatial coordination were disturbed. When the leg made a pause it usually stopped in the air near the end of its swing movement. During steady walking, the operation interrupted information from the adjacent forward leg normally used to guide the end-point of the swing or to signal errors in leg placement and elicit a correctivetreading-on-tarsus reflex. It also interrupted position information affecting the start of the swing. For the leg rostral to the cut, the inhibition during the swing of the posterior leg and the excitation when the latter started its retraction were both interrupted. These results indicate that all six ipsilateral coordination mechanisms are primarily mediated by the ipsilateral connective. In addition, the data show that contralateral coordination within the segmental ganglion is strongest for the front legs, weaker for the rear legs, and not discernible for the middle legs.