Toshiki Nagayama

Local spikeless interaction of motoneuron dendrites in the crayfishProcambarus clarkii girard

Summary1.Spikeless communication between dendrites of crayfish motoneurons was demonstrated by intracellular current injection.2.Subthreshold depolarization of one motoneuron (Add MN) innervating the adductor exopodite, one of the closer muscles of the uropod, increased the spontaneous discharge rate of another motoneuron (Red MN No. 1) innervating another closer muscle, reductor exopodite.3.Hyperpolarization of the Add MN caused a decrease in the spike frequency of the Red MN No. 1.4.The effects are graded and dependent on the membrane potential changes in the current-injected cells.5.Spikeless communication was also observed between opener motoneurons. Such communication was observed only between synergistic motoneurons and not between antagonistic ones.6.Regions in the Add MN in which current injection effectively changed the Red MNs activity were always distant from the spike-initiating region, and confined to the distal dendritic branches.7.The amplitude of e.p.s.p.s recorded from the Red MN No. 1 in response to antidromic spikes of the Add MN was increased by hyperpolarization and decreased by depolarization at the site of recording. The spikeless, graded interaction appears to be mediated by chemical transmission via a monosynaptic connection.8.It is concluded that the crayfish uropod motoneurons function not only as simple output elements but also as complex integrative elements by forming local circuits at restricted dendritic regions.

Functional characteristics of local non-spiking interneurons as the pre-motor elements in crayfish

SummaryControl of the crayfish uropod motoneurons by the unilateral-type local non-spiking interneurons (LNSNs) in the terminal (sixth) abdominal ganglion was studied with intracellular recording and current injection.1.Current injected into LNSNs affected the spike activity of uropod motoneurons in a graded manner. The effects depended on both the duration and the intensity of current pulses.2.Simultaneous intracellular recordings from an LNSN and an opener motoneuron showed that LNSNs could change the motoneuron membrane potential by their own membrane potential change without generating spikes.3.In fifty-nine cases, we penetrated LNSNs which affected uropod motoneurons bidirectionally. Hyperpolarization of 23 LNSNs decreased and that of other 36 LNSNs increased the spontaneous discharge rate of an identified reductor exopodite motoneuron (Red MN) No. 1. Depolarization had the opposite effect in either case.4.It is suggested that at least one chemical synapse should be intercalated between the LNSN and the motoneuron. although whether their connection is monosynaptic or not still remains open to future study.5.Twenty-four penetrated LNSNs co-actively controlled the synergistic set of motoneurons. Other 23 LNSNs also controlled the antagonistic set in addition to the synergistic set of motoneurons in a reciprocal way, and other 21 LNSNs in a co-activating way.6.The possible role of unilateral-type LNSNs in the motor control, especially in the non-rhythmical, episodic movement such as uropod steering, is discussed.

Physiological and morphological characterization of anaxonic non-spiking interneurons in the crayfish motor control system

Spike activation of the motoneurons innervating uropod muscles in crayfish is controlled by anaxonic interneurons located within the terminal (the 6th abdominal) ganglion. These neurons do not generate spikes either spontaneously at the resting potential level or in response to current injection of either polarity. Yet the change in the membrane potential of these non-spiking interneurons caused an increase or decrease in the discharge frequency of motoneuron spikes, depending upon the direction of the membrane potential change. These non-spiking interneurons within the terminal ganglion presumably integrate various descending command signals and select the adequate information to be gated to the motoneurons.

Cholinergic transmission at mechanosensory afferents in the crayfish terminal abdominal ganglion

Electrical stimulation of mechanosensory afferents innervating hairs on the surface of the exopodite in crayfish Procambarus clarkii (Girard) elicited reciprocal activation of the antagonistic set of uropod motor neurones. The closer motor neurones were excited while the opener motor neurones were inhibited. This reciprocal pattern of activity in the uropod motor neurones was also produced by bath application of acetylcholine (ACh) and the cholinergic agonist, carbamylcholine (carbachol). The closing pattern of activity in the uropod motor neurones produced by sensory stimulation was completely eliminated by bath application of the ACh blocker, d-tubocurarine, though the spontaneous activity of the motor neurones was not affected significantly. Bath application of the acetylcholinesterase inhibitor, neostigmine, increased the amplitude and extended the time course of excitatory postsynaptic potentials (EPSPs) of ascending interneurones elicited by sensory stimulation. These results strongly suggest that synaptic transmission from mechanosensory afferents innervating hairs on the surface of the tailfan is cholinergic.Bath application of the cholinergic antagonists, dtubocurarine (vertebrate nicotinic antagonist) and atropine (muscarinic antagonist) reversibly reduced the amplitude of EPSPs in many identified ascending and spiking local interneurones during sensory stimulation. Bath application of the cholinergic agonists, nicotine (nicotinic agonist) and oxotremorine (muscarinic agonist) also reduced EPSP amplitude. Nicotine caused a rapid depolarization of membrane potential with, in some cases, spikes in the interneurones. In the presence of nicotine, interneurones showed almost no response to the sensory stimulation, probably owing to desensitization of postsynaptic receptors. On the other hand, no remarkable changes in membrane potential of interneurones were observed after oxotremorine application. These results suggest that ACh released from the mechanosensory afferents depolarizes interneurones by acting on receptors similar to vertebrate nicotinic receptors.

The organization of exteroceptive information from the uropod to ascending interneurones of the crayfish

The organization of exteroceptive inputs to identified ascending interneurones of the crayfish, Procambarus clarkii (Girard), has been analyzed by stimulation of hairs on the uropod and simultaneous intracellular recordings from ascending interneurones. The spikes of single afferent neurones which innervated hairs on the distal ventral surface of the exopodite were consistently followed by a depolarizing synaptic potential in many identified ascending interneurones with a constant and short central delay of 0.7–1.5 ms. The amplitude of the potentials depended on the membrane potential of the ascending interneurones. Each afferent neurone made divergent outputs onto several ascending interneurones and each ascending interneurone received convergent inputs from several afferent neurones. Certain ascending interneurones made inhibitory or excitatory connections with other ascending interneurones. These central interactions were always one-way, and the spikes from one ascending interneurone consistently evoked excitatory or inhibitory post-synaptic potentials in other interneurones which followed with a constant and short latency of 0.7–1.0 ms. The inhibitory postsynaptic potential was reversed by injection of steady hyperpolarizing current.

Monosynaptic excitation of lateral giant fibres by proprioceptive afferents in the crayfish

Abstract Giant interneurones mediate a characteristic `tail flip escape response of the crayfish, Procambarus clarkii, which move it rapidly away from the source of stimulation. We have analysed the synaptic connections of proprioceptive sensory neurones with one type of giant interneurone, the lateral giant. Spikes in sensory neurones innervating an exopodite-endopodite chordotonal organ in the tailfan, which monitors the position and movements of the exopodite, are followed at a short and constant latency by excitatory postsynaptic potentials in a lateral giant interneurone (LG) recorded in the terminal abdominal ganglion. These potentials are unaffected by manipulation of the membrane potential of LG, by bath application of saline with a low calcium concentration, or by one containing the nicotinic antagonist, curare. The potentials evoked in LG by chordotonal organ stimulation are thus thought to be monosynaptic and electrically mediated. This is the first demonstration that LG receives input from sensory receptors other than exteroceptors in the terminal abdominal ganglion.

Parallel processing of proprioceptive information in the terminal abdominal ganglion of the crayfish

The processing of proprioceptive information from the exopodite-endopodite chordotonal organ in the tailfan of the crayfish Procambarus clarkii (Girard) is described. The chordotonal organ monitors relative movements of the exopodite about the endopodite. Displacement of the chordotonal strand elicits a burst of sensory spikes in root 3 of the terminal ganglion which are followed at a short and constant latency by excitatory postsynaptic potentials in interneurones. The afferents make excitatory monosynaptic connections with spiking and nonspiking local interneurones and intersegmental interneurones. No direct connections with motor neurones were found.Individual afferents make divergent patterns of connection onto different classes of interneurone. In turn, interneurones receive convergent inputs from some, but not all, chordotonal afferents. Ascending and spiking local interneurones receive inputs from afferents with velocity thresholds from 2–400°/s, while nonspiking interneurones receive inputs only from afferents with high velocity thresholds (200–400°/s).The reflex effects of chordotonal organ stimulation upon a number of uropod motor neurones are weak. Repetitive stimulation of the chordotonal organ at 850°/s produces a small reduction in the firing frequency of the reductor motor neurone. Injecting depolarizing current into ascending or non-spiking local interneurones that receive direct chordotonal input produces a similar inhibition.

Distribution of GABAergic premotor nonspiking local interneurones in the terminal abdominal ganglion of the crayfish

The inhibitory neurotransmitter of premotor nonspiking local interneurones in the crayfish terminal abdominal ganglion was investigated physiologically and immunocytochemically. Depolarization of a nonspiking interneurone evoked a hyperpolarization in a uropod motor neurone. The amplitude of hyperpolarization in the motor neurone was gradually decreased under low‐calcium/high‐magnesium saline. Local pressure injection of γ‐aminobutyric acid (GABA) into the neuropil caused a similar hyperpolarization of the motor neurone. These physiological studies suggested a GABAergic inhibitory interaction between nonspiking interneurones and the motor neurones. Premotor nonspiking interneurones are classified into two subgroups of posterolateral (PL) and anterolateral (AL) interneurones, and AL interneurones are further divided into three subtypes. A combination of intracellular staining from nonspiking local interneurones with Lucifer yellow and immunocytochemical staining with an antiserum directed against GABA revealed that all the PL interneurones sampled in this study showed GABA‐like immunoreactivity. A population of cell bodies (n = 6–11) with a small diameter (15–30 μm) packed together forming a cluster showed GABA‐like immunoreactivity, and the cell bodies of most PL interneurones were found in this cluster. To compare the number and the pattern of main branches of PL interneurones, cells were classified into three identifiable sets of interneurones, called PL‐1, PL‐2, and PL‐3. By contrast, about one‐half of AL interneurones, especially the third subtype of AL interneurones, which have cell bodies located ventrolaterally in the ganglion, did not show GABA‐like immunoreactivity. Furthermore, the position of cell bodies of GABA‐immunoreactive AL interneurones was scattered compared to that of PL interneurones. J. Comp. Neurol. 389: 139–148,1997.

A sensory map based on velocity threshold of sensory neurones from a chordotonal organ in the tailfan of the crayfish

The central projections of sensory neurones innervating a strand chordotonal organ (CO) in the tailfan of the crayfish, Procambarus clarkii (Girard) have been investigated. The CO monitors movement of the exopodite of the tailfan relative to the endopodite. Intracellular recording and staining were used to characterise the response of the sensory neurones to applied stretches of the chordotonal organ and to reveal their morphology. Two gross morphological types of afferents were found: those that terminated in the terminal (6th) abdominal ganglion on the side ipsilateral to the sensory receptor, and those that had branches in the terminal ganglion and an intersegmental axon that ascended rostrally. Afferents responded to position, velocity and direction of imposed CO displacement. Afferents with particular physiological properties had similar morphologies in different crayfish. Irrespective of their directional responses, afferents had central projection areas dependent upon their velocity thresholds. Many afferents responded only during movement of the CO, and those with the lowest velocity thresholds (2°/s) had branches that projected most anteriorly, while those with progressively higher velocity thresholds (up to 200°/s) projected progressively more posteriorly. Afferents that responded to low velocity ramp movements and spiked tonically projected to more posterior areas of the ganglion than those that responded only to movements.

Decrease in excitability of LG following habituation of the crayfish escape reaction

Crayfish escapes from threatening stimuli to the abdomen by tailflipping upwards and forwards. This lateral giant (LG)-mediated escape reaction habituates readily upon repetitive sensory stimulation. Using an isolated abdominal nerve cord preparation, we have analyzed the change in LG activity by applying additional sensory stimulation after different periods following habituation to characterize the retention of LG habituation. Results show that the LG mediated response habituates more quickly, but the retention time is shorter, as repetitive sensory stimulation is applied at progressively shorter inter-stimulus time intervals. The spike response of LG recovers quickly, within several minutes after habituation, but they fail to spike when an additional stimulus is applied after specific long periods following habituation. The critical period of the delay for this decrease in excitability of LG is dependent on the inter-stimulus time interval of the initial repetitive stimulus. As the inter-stimulus interval became longer, the delay needed for decrease in excitability became shorter. Furthermore, the local injection of 10−6xa0molxa0l−1 octopamine into the neuropil just following habituation promotes the achievement of decrease in excitability. No effects were observed when 10−6xa0molxa0l−1 serotonin and tyramine were injected. These results suggested octopamine promotes decrease in excitability of LG following habituation.