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Peripheral targets of centrally located putative accessory neurons of MRO in the isopod Ligia exotica

Noriyuki Hama¹, Yoshinori Okada², Edit Pollák³, László Molnár³ and Akiyoshi Niida^2,*

¹ Animal Behavior and Intelligence, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
² Department of Biology, Faculty of Science, Okayama University, Okayama Tsushima 700-8530, Japan
³ Department of General Zoology and Neurobiology, University of Pécs and Adaptation Biological Research Group of the Hungarian Academy of Sciences, H-7601 Pécs, Ifjuság útja 6, Hungary

View larger version (33K): [in a new window]   Fig. 1. (A) Setup for ascertaining the inhibitory effect of accessory neurons on stretch-activated responses of TSR. An en passant suction electrode is used for antidromic stimulation of accessory neurons and for extracellular recording of stretch-activated responses which, at the same time, show the presence or absence of an inhibitory effect of putative accessory neurons. By pulling the tergite of the sixth thoracic segment, the receptor muscle of TSR-4 is stretched and stretch-activated responses occur as shown (arrow) in a representative trace composed of rapidly and slowly adapting responses. (B) Arrangement of electrodes for recording junctional potentials from two extensor muscles. Experiments were carried out to determine which muscle is innervated by neuron #1. To record junctional potentials of DEM1, the recording (R) and stimulating (S) electrodes were R1, R2, R4 and S. For DEM2, R1, R3, R4 and S were used. Each experiment was conducted in a separate animal. ANT, anterior; AX, axon of the receptor cell of the thoracic stretch receptor; D, dorsal; DEM1, medial deep extensor muscle; DEM2, lateral deep extensor muscle; EXT, extracellular recording; INT, intracellular recording; N3, third segmental nerve; npm, nerve to deep extensor muscle; nsm, nerve to superficial extensor muscle; POST, posterior; RC1, receptor cell of rapidly adapting stretch receptor; RC2, receptor cell of slowly adapting stretch receptor; RM, receptor muscle; ST, stretch stimulus; TG, thoracic ganglion; TS, thoracic segment; TSR, thoracic stretch receptor; V, ventral.

View larger version (89K): [in a new window]   Fig. 2. (A) Lucifer Yellow-filled putative accessory neurons and TSR axon in the fourth thoracic ganglion (TG4). Lucifer Yellow was applied to the cut end of the third segmental nerve of the third thoracic ganglion. Black fragments on the dorsal surface of the thoracic ganglion show melanophores. The dorsal side is up. Bar, 300µm. (B) Schematically depicted accessory neurons and TSR axon in TG5 based on backfilling with Lucifer Yellow. ANT, anterior; AXas, ascending axon of the receptor cell of the thoracic stretch receptor; #1-3, putative accessory neurons; TG, thoracic ganglion.

View larger version (70K): [in a new window]   Fig. 3. (A) Profile of intracellularly stained neuron #1. Neuron #1 has a large cell body and gives off dendritic branches in the next-anterior ganglion (double arrows in TG3). Its axon was traced near DEM2. Bar, 300µm. Based on this result, Lucifer Yellow was extracellularly applied to the distal cut end of npm (B). As a result, some motor neurons (arrowheads in the next-anterior ganglion, TG3) and neuron #1 are labeled (Bi). The photograph was taken under normal and ultraviolet light. (Bii) is rotated by 90° relative to (Bi). Bar, 500µm. N1/N2, first and second segmental nerve; N3, third segmental nerve; npm, nerve to deep extensor muscles; SR, stretch receptor; TG, thoracic ganglion.

View larger version (48K): [in a new window]   Fig. 7. Spatial arrangement of the superficial extensor muscle, the deep extensor muscle and the stretch receptor. They are depicted on one side bisected along the midline. The dorsal nerve (dnr) continues to the third segmental nerve. A pair of thoracic stretch receptors is located under the lateral deep extensor muscle (DEM2). The superficial extensor muscles (SEM1 and SEM2) lie under the medial deep extensor muscle (DEM1). ANT, anterior; npm, nerve to deep extensor muscles; nsm, nerve to superficial extensor muscle; POST, posterior; SR, stretch receptor.

View larger version (21K): [in a new window]   Fig. 4. Excitatory junctional potentials of DEM1 and DEM2 following electrical stimulation of nerve to deep extensor muscles (npm). Stimulation was gradually increased from A to C and from D to F. Two experiments (A–C and D–F) were performed in separate animals. (A–C) Recordings were taken simultaneously from neuron #1, DEM1) and N3. Junctional potentials in DEM1 (middle trace in B) were not obtained at a stimulus intensity that activated neuron #1, while a greater stimulus produced an excitatory junctional potential (middle trace in C). (D–F) Recordings were taken simultaneously from neuron #1, DEM2 and N3. A given stimulus intensity caused compound action potentials in N3 (bottom trace in E), but neither neuron #1 nor DEM2 are activated. However, a stimulus intensity that activates neuron #1 (top trace in F) brings about a junctional potential in DEM2 (middle trace in F).

View larger version (19K): [in a new window]   Fig. 5. Lack of effect of neuron #2 on the stretch-activated response. (A) Injection of depolarizing currents into neuron #2 produced regenerative action potentials (asterisks). (B) Such activation of neuron #2 had no effect on the stretch-activated response of TSR-4, where single and double asterisks indicate impulses conducted along the connective from depolarized neuron #2. If neuron #2 was an inhibitory accessory neuron, the stretch-activated response in B would be suppressed. Both the conducted impulses and the stretch-activated response were recorded through an en passant electrode at N3. Double arrows indicate stretch-activated impulses of TSR-4 derived from a rapidly adapting stretch receptor. The upward deflection below the trace shows a stretch stimulus at an amplitude of 1.5 mm.

View larger version (56K): [in a new window]   Fig. 6. Profile of intracellularly stained neuron #2. (A) The impaled neuron with the response properties shown in Fig. 5 was intracellularly labeled with Lucifer Yellow. The axon of neuron #2 labeled with Lucifer Yellow was directly traced to the superficial extensor muscle (Area-1), shown enlarged in Area-2. Note that there are no axonal branches to share with the stretch receptor. (B) The complete profile of another neuron #2 with a deformed -like dendritic structure. Bars, 500µm. N3, third segmental nerve; SEM1, medial superficial extensor muscle; SEM2, lateral superficial extensor muscle; SR, stretch receptor; TG, thoracic ganglion.

View larger version (33K): [in a new window]   Fig. 8. Inhibition of stretch-activated responses of the thoracic stretch receptor by a putative accessory neuron. (A) In accordance with the injection of depolarizing currents into the putative accessory neuron (asterisks in B), the stretch-activated response was suppressed (asterisks in A). Each arrow in trace A shows rapidly adapting impulses caused by a jerky stretch stimulus delivered manually, which appears as inflection points on the monitor trace below trace A. Paired arrows on the right hand side of the trace A indicate the impulse size of slowly adapting stretch receptor. (B) Electrical activities from the putative accessory neuron. Depolarizing current was injected into the putative accessory neuron, resulting in regenerative action potentials (asterisks). (C) Enlargement of the parts enclosed by rectangles (i) and (ii) in A. Stretch-activated EPSPs occur while the stretch stimulus is applied (i and ii) and disappear upon termination of the stretch stimulus. Summation of EPSPs (Ci) produces an action potential, which is shown in smaller amplitude than it really is (Bi). Upward deflections in monitor traces below traces A, B and C show stretch stimuli (amplitude 0.5 mm).

View larger version (129K): [in a new window]   Fig. 9. An example of an intracellularly stained inhibitory accessory neuron. This neuron showed the physiological properties given in Fig. 8 and was identified as a neuron #3, based on its profile. Parallel short vertical dendritic branches at the center of the ganglion (double arrows) are noted. This was revealed by intracellular staining of neuron #3. Bar, 500µm. The inset is an enlarged view of neuron #3 at the bottom. Bar, 40µm.

View larger version (154K): [in a new window]   Fig. 10. Stretch receptor axons overlapping on the vertical dendritic branches of neuron #3. (A) The axon of TSR-4 (Fig. 1A) reaches the root of N3 belonging to TG4, and bifurcates into ascending and descending axons in the connective. The descending axon overlaps on one vertical dendritic branch of neuron #3 in TG5 (thin arrow). The opposite arrowhead indicates another vertical dendritic branch of neuron #3. The circle shows the location of a neuron #3 covered with melanophores. Bar, 500µm. (B) Another example in which the descending stretch receptor axon overlaps on the vertical dendritic branch of neuron #3 (thin arrow) opposite that in A. In this case, the TSR axon was descending at the root of N3 in TG3. The arrowhead indicates another vertical dendritic branch of neuron #3. Bar, 300µm. AXas, ascending axon of TSR; AXde, descending axon of TSR; N3, third segmental nerve; TG, thoracic ganlion.

View larger version (21K): [in a new window]   Fig. 11. Inhibitory synaptic input from neuron #3 to the receptor cell of the stretch receptor. (A) Depolarizing currents applied to neuron #3 (middle trace) produce IPSPs in the rapidly adapting stretch receptor cell (RC1 in Fig. 1A) (bottom trace). Impulses of N3 (asterisks) that occurred with the application of depolarizing currents to neuron #3 and were conducted on the connective show one to one correspondence with hyperpolarizing potentials in a receptor cell (RC1) (arrowheads). (B) Demonstration of synaptic input to RC1. The amplitude of hyperpolarizing potential of RC1 mediated by the injection of current to neuron #3 changed depending on the magnitude of current injected into to RC1.