Open Access
How to translate text using browser tools
27 January 2004 Effects of Nitric Oxide on Proprioceptive Signaling
Hansjürgen Schuppe, Makoto Araki, Hitoshi Aonuma, Toshiki Nagayama, Philip L. Newland
Author Affiliations +

We have analysed the effects of the neuromodulator nitric oxide (NO) on proprioceptive information processing by ascending intersegmental interneurons that form part of the local circuits within the terminal abdominal ganglion of the crayfish. NO modulates the synaptic inputs to ascending interneurons, enhancing the amplitude of class I interneurons and reducing the amplitude of class II interneurons. Repetitive proprioceptive stimulation leads to rapid depression in a specific set of identified interneurons but not in others. Bath application of a nitric oxide scavenger, PTIO, causes a significant decrease in the rate of depression of the interneurons showing a rapid depression, independent of interneuron class, but has no effect on the dynamic responses of the interneurons that show little initial depression. These results indicate that NO exerts multiple effects at the very first stage of synaptic integration in local circuits.


The tailfan of decapod crustaceans is richly innervated by many sensory structures that point to the central role it plays in the production and control of movement, from ballistic swimming to slower equilibrium movements. The surface of the tailfan is covered with many exteroceptive hairs (Wiese, 1976), and spanning the joints of the different segments of the tailfan of crayfish are numerous internal proprioceptors that play a crucial role in controlling the movements of its different parts (Field et al., 1990). Signals from these proprioceptors are processed in local circuits within the terminal abdominal ganglion, that include spiking and nonspiking local interneurons, ascending interneurons and motor neurons (Nagayama et al., 1994; Newland et al., 2000). The ascending interneurons serve a number of functions; first, they integrate sensory signals, second, they distribute these inputs to other segments of the animal, thus placing movements into appropriate behavioural context, and third, they act as premotor elements controlling sets of motor neurons in the terminal abdominal ganglion (Nagayama, 1997, 2002).

These local circuits, and ascending interneurons in particular, are subject to continuous modulation through the actions of many neuromodulators, including the gaseous neuromodulator nitric oxide (NO) (Bicker et al., 1996; Garthwaite and Boulton, 1995). Imaging studies using the NO-specific indicator 4,5-diaminofluoroscein (Schuppe et al., 2002), and histochemical studies (Schuppe et al., 2001a) have described the distribution of putative nitric oxide synthase containing neurons in the terminal abdominal ganglion. Moreover, double labelling studies have shown that many of the 65 pairs of ascending interneurons in the terminal abdominal ganglion synthesise NO (Schuppe et al., 2001b). Recent pharmacological studies have shown that sensory inputs to the ascending interneurons, activated by means of electrical stimulation, are subject to modulation by NO. Ascending interneurons whose inputs are enhanced by NO have been defined as class I interneurons, whereas ascending interneurons whose inputs are depressed by NO have been defined as class II interneurons (Aonuma and Newland, 2001). Since previous studies used electrical stimulation to evoke excitatory postsynaptic potentials (EPSPs) in the ascending interneurons, it is not clear whether such NO-induced modulation might occur during sensory activation caused by uropod movement, what modality of inputs are modulated or whether NO has any dynamic effects at the synapse. The aim of this study was, therefore, to analyse the effects of NO on sensory signals evoked by movement stimuli applied to the strand of an identified proprioceptor in the tailfan at velocities encountered during escape swimming movements.


Adult crayfish, Pacifastacus leniusculus (Dana), obtained locally from a commercial supplier (Riversdale Farm, Dorset, UK), were prepared for experiments using methods described in detail elsewhere (Aonuma et al., 1999; Schuppe and Newland, 2004). Briefly, an animal was decapitated, the abdomen removed and pinned ventral-side-uppermost in a small Sylgard lined chamber that was constantly perfused with saline at 14ml/min. The terminal abdominal ganglion was exposed, as well as the protopodite-endopodite chordotonal organ (PEnCO) that monitors movements of the endopodite about the protopodite (Field et al., 1990) (Fig. 1A). The strand of the PEnCO was displaced by means of a small pin attached to a Ling vibrator, the movements of which were controlled by computer. The responses of the sensory neurons innervating the PEnCO were monitored using an oil hook electrode placed on nerve 3 as it entered the ganglion.

Fig. 1

A. Photograph showing the ventral surface of the left uropod of the crayfish showing the protopodite (Pro), endopodite (Endo) and exopodite (Exo). Insert shows higher power view of the PEnCO that is innervated by sensory neurons in nerve 3 (N3). B. Rampform displacement of the strand of the PEnCO, equivalent to an opening movement about the joint, evoked a compound EPSP in an ascending interneuron.


Intracellular recordings were made from the neuropilar processes of the ascending interneurons with glass microelectrodes filled with 3% Lucifer Yellow CH (Sigma Chemical Co.) and with resistances of 60–120MΩ. All recordings were stored on computer using a Cambridge Electronic Design 1401 analogue-to-digital interface for subsequent off-line analysis using the software Spike 2. After physiological and pharmacological analysis interneurons were stained by iontophoretic injection of Lucifer Yellow using 5–10 nA hyperpolarizing current pulses of 500 ms duration at 1 Hz for 5–10 min. Ganglia were dissected from the animals, fixed in 10% formalin, dehydrated in an ascending alcohol series and cleared with methyl salicylate before being imaged on a Nikon E-800 fluorescent microscope fitted with a Sony microMax CCD camera using Metamorph software (Universal Imaging Corporation). Interneurons were identified according to published morphological criteria (Nagayama et al., 1993, 1994).

The recording chamber was perfused for at least 15min prior to drug application that lasted for 5 min, and the preparation washed again for up to 30 min in normal saline. All drugs were obtained from Sigma Chemical Co., and dissolved in normal saline to the required concentration 5 min before use (L-arginine; L-NAME, Nω-nitro-L-arginine methyl ester; SNAP, S-nitroso-N-acetylpenicillamine and PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide). Results are based on successful recordings from 35 ascending interneurons.


Effects of NO on proprioceptive inputs to ascending interneurons

The PEnCO spans the joint between the protopodite and endopodite of the tailfan (Fig. 1A) and is innervated by sensory neurons that respond to the position, direction and velocity of movement of the endopodite relative to the protopodite (Field et al., 1990). Displacement of the strand of the PEnCO evoked compound EPSPs in ascending interneurons of both classes (Fig. 1B), the rise time and amplitude of which were dependent on the amplitude and velocity of applied movement (Aonuma et al., 1999). To analyse the effects of NO on these synaptic inputs we manipulated both endogenous and exogenous NO levels using different pharmacological agents.

L-arginine acts as a substrate for NO synthesis and is normally regulated to low levels within cells. Increasing the available substrate for NO synthesis leads to increases in NO synthesis in the terminal abdominal ganglion of crayfish (Schuppe et al., 2002). Bath application of 1 mM L-arginine for 5 min increased significantly the amplitude of proprioceptive inputs in class I ascending interneurons by 50.6±13.6% (mean±standard error of the mean, S.E.M., n=8 from 2 animals, p<0.05, Student's t test) (Fig. 2A). By contrast, 1 mM L-arginine caused a significant decrease in the amplitude of class II interneurons (Fig. 2A) by 19.1±02.6% (n=8 from 2 animals, p<0.05). In some class II interneurons the action potentials evoked by proprioceptive stimulation were abolished by L-arginine application (Fig. 2A).

Fig. 2

Effects of the NO precursor L-arginine, and the NOS inhibitor, L-NAME, on EPSP amplitude. A. Bath application of 1 mM L-arginine caused an increase in the amplitude of class I interneurons and a reduction in amplitude of class II interneurons during ramp stimulation of the PEnCO (bottom trace). Three or four superimposed sweeps are shown for each example. After washing for 15 min responses returned close to control levels. B. Bath application of 10 mM L-NAME caused a decrease in the responses of class I interneurons but an increase in amplitude of responses of class II interneurons. Dotted lines in this and subsequent figures indicate the resting membrane potential level.


Imaging studies of NO levels in the terminal ganglion have also shown that bath application of an inhibitor of the NO Modulation of Ascending Interneurons 3 enzyme nitric oxide synthase, L-NAME, results in a reduction of endogenous NO levels (Nakatsubo et al., 1998; Schuppe et al., 2002). Bath application of 10 mM L-NAME, in contrast to L-arginine, significantly reduced the amplitude of EPSPs in class I interneurons (25.9±3.4%, n=4 from 1 animal, p<0.05) but increased those in class II interneurons by 29.3±5.9% (n=12 from 3 animals, p<0.05) (Fig. 2B). These increases in class II interneurons were sufficiently large to give rise to action potentials during stimulation of the PEnCO. PTIO, that acts as a NO scavenger, has been shown directly to reduce NO levels in the central nervous system (Fujie et al., 2000) and bath application of 500 μM PTIO reduced the amplitude of EPSPs in class I by 36.3±12.3% (n=18 from 3 animals, p<0.05) while it increased the amplitude of those in class II interneurons by 33±13.7% (n=18 from 3 animals, p<0.05)(Fig. 3A).

Fig. 3

Effects of the NO scavenger PTIO and NO donor SNAP on EPSP amplitude. A. Signal averages (of 30 responses to PEnCO stimulation) showing that bath application of 500 μM PTIO decreased EPSP amplitude in class I interneurons but increased EPSP amplitude in class II interneurons. B. Bath application of 200 μm SNAP increased the amplitude of EPSPs in class I interneurons and deceased the amplitude of potentials in class II interneurons.


Direct bath application of the NO donor SNAP significantly increases NO levels in the terminal ganglion (Schuppe et al., 2002), and bath application of 500 μM SNAP led to an increase in the amplitude of EPSPs in class I interneurons by 47.5±17.9% (n=4 from 1 animal) and a decrease in class II interneurons by 28.6±3.9% (n=18 from 3 animals, p<0.05) (Fig. 3B).

Effects of NO on the depression of synaptic inputs of ascending interneurons

Recent studies have shown that NO can influence the rate of depression of excitatory junction potentials in the flexor muscles in the crayfish evoked by giant motor neuron activation (Aonuma et al., 2000). We investigated whether similar changes in depression rate is likely for sensory inputs at the very first stages in processing in local circuits, by the ascending interneurons. We analysed the rates of depression of 15 ascending interneurons to repeated proprioceptive stimulation (Fig. 4A) in which 6 ramp stimuli were delivered at 0.33Hz and repeated each minute. The relative rate of depression between the first and sixth stimulus was calculated (the sixth divided by the first). Nine out of the 15 interneurons showed a marked rapid depression (0.46±0.06, n=9) with amplitudes decreasing by up to 90% in some interneurons, while the remaining six interneurons showed little depression in their responses (0.96±0.038, n=6). These two groups of interneuron showed significantly different rates of depression (p<0.05, Student's t test) but included interneurons of both classes in each group. Bath application of the NO scavenger PTIO (500 μM) for 5 min caused a significant (p<0.05) reduction in the rate of depression in those interneurons that showed an initial rapid depression (Fig. 4B), irrespective of interneuron class. On the other hand, PTIO had no significant effect on the responses of those interneurons that showed little initial depression (p>0.05).

Fig. 4

Depression of responses of ascending interneurons to repeated proprioceptive stimulation. A. Responses of an interneuron to a group of 6 ramp stimuli showing rapid depression. B. Bath application of 500 μM PTIO reduced the rate of depression in interneurons showing initial rapid depression, but has no significant effect on interneurons that showed little initial depression. * indicates significant difference (t = −2.9, p<0.05, Student's t test).



Our results have shown that consistent, but contrasting, changes in response of the 2 classes of ascending interneurons were caused by drugs that increased NO levels (such as L-arginine and SNAP) and those that reduced NO levels (L-NAME and PTIO), with changes being the mirror image for class I and class II interneurons (Aonuma and Newland, 2001). It is not yet clear whether these changes are related to the input properties of these interneurons, such as whether the connections between sensory neurons and interneurons are mediated through electrical or chemical synapses, that are known to occur in the terminal ganglion (Nagayama et al., 1997). Nevertheless, the results we have obtained parallel the changes that occur in these same classes of interneuron during electrical stimulation of the entire sensory nerve bundle. The results we describe here, however, demonstrate that the inputs from a small number of sensory neurons (in this case approximately 12 sensory neurons innervate the PEnCO, Field et al., 1990) of a specific modality are modulated by NO and moreover, and crucially, that these inputs occur at velocities that are likely to occur during normal movements of the crayfish (Field et al., 1990), unlike the resulting inputs from electrical stimulation which evoke a large barrage of input. What is also clear is that changing NO levels can have profound changes on the excitability of the ascending interneurons so that some that respond at only subthreshold levels under control conditions can give rise to action potentials when exposed to NO, and conversely those inputs that give rise to action potentials at control levels are reduced to subthreshold levels after changes in NO levels. NO can therefore change the overall balance of inputs from the PEnCO to these ascending interneurons with the ultimate result that changes in the outputs of the local circuits are likely to occur.

NO regulates synaptic depression in ascending interneurons

Our results indicate that NO plays a key role in the dynamic responses of the interneurons by enhancing the rate at which they depress. Similar results have also been demonstrated by Aonuma et al. (2000) between motor neurons and flexor muscles in the crayfish. It has been suggested that such depression at the neuromuscular junction results from a reduction in intracellular Ca2+ concentration (Czternasty and Bruner, 1980; Czternasty et al., 1980). In vertebrates it has been shown that NO can decrease intracellular Ca2+ levels by eleavating cGMP (Rashatwar et al., 1987). It is possible that such a NO/cGMP pathway underlies the changes in rates of depression in the ascending interneurons, especially since NO is thought to act via soluble guanylate cyclase and cGMP in local circuits (Aonuma and Newland, 2002). Further studies are however necessary to demonstrate this directly.

We have recently shown that NO does not influence the pattern of activity of proprioceptive sensory spikes when applied in the periphery (Schuppe and Newland, 2004) suggesting that the effects we describe here must all occur at a central level. These results also point to NO having multiple effects at the synapse. For example, NO has been shown to decrease the amplitude of presynaptic afferent depolarisation in the terminals of PEnCO afferents, thereby increasing the effectiveness of the sensory signal. This increase in efficacy could account for the changes that occur in the responses of class I interneurons but could not alone account for the responses of class II interneurons, or indeed, the changes in the rate of depression that occur in the ascending interneurons irrespective of class. Aonuma and Newland (2001) hypothesised that NO may be synthesised in specific ascending interneurons as a result of increased mechanosensory input and that this NO could then act retrogradely to reduce transmitter release, thus accounting for the effects of NO on class II interneurons. Neither of these possible actions of NO could account for the changes in the rate of depression in both classes of interneuron and point to NO have multiple actions at this very early synapse in local circuits and possibly acting through a number of mechanisms.

The role of NO in local circuits

The sensory neurons that innervate the PEnCO make monosynaptic, chemical connections with specific spiking and non-spiking local interneurons as well as many ascending interneurons in the local circuits of the terminal abdominal ganglion (Newland and Nagayama, 1993). In addition they also form electrical connections with specific ascending interneurons and the lateral giant interneurons that form part of the pathway involved in tail-flip production (Newland et al., 1997). We have recently demonstrated that the synaptic inputs to spiking local interneurons, evoked by electrical stimulation of the sensory nerve, are also modulated by NO (Aonuma and Newland, 2002) as with many ascending interneurons (Aonuma and Newland, 2001), implying that NO modulation occurs at many levels, on many interneuron types and on a number of sensory modalities in these circuits. Given the role of proprioceptive inputs in controlling many different movements and behaviours it is likely that the modulation of their signals is likely to have profound changes on sensory inputs to local circuits, the processing of sensory information and the intersegmental and local outputs of the local circuits. The consequences of this modulation, at the level of the outputs of these circuits on the uropod motor neurons, has yet to be analysed in detail, but it is clear that if we want to understand the action of NO in detail on these circuits we should now analyse the effects of NO on the outputs and also determine under what conditions NO levels are modulated in vivo.


This work was supported by an award from the Yamada Science Foundation (to TN and PLN) whose support we gratefully acknowledge.



H. Aonuma and P. L. Newland . 2001. Opposing actions of nitric oxide on synaptic inputs of identified interneurones in the central nervous system of the crayfish. J Exp Biol 204:1319–1332. Google Scholar


H. Aonuma and P. L. Newland . 2002. Synaptic inputs onto spiking local interneurons in crayfish are depressed by nitric oxide. J Neurobiol 52:144–155. Google Scholar


H. Aonuma, P. L. Newland, and T. Nagayam . 1999. Processing of proprioceptive signals by ascending interneurones in the terminal abdominal ganglion of the crayfish. J Exp Biol 202:2975–2984. Google Scholar


H. Aonuma, T. Nagayama, and M. Takahata . 2000. Modulatory effect of nitric oxide on synaptic depression in the crayfish neuromuscular system. J Exp Biol 203:3595–3602. Google Scholar


G. Bicker, O. Schmachtenberg, and J. De Vente . 1996. The nitric oxide/cyclic GMP messenger system in olfactory pathways of the locust brain. Eur J Neurosci 8:2635–2643. Google Scholar


G. Czternasty and J. Bruner . 1980. On the mechanism of a long-lasting neuromuscular depression in crayfish. Comp Biochem Physiol 66:143–148. Google Scholar


G. Czternasty, J. Bruner, and C. Galeano . 1980. Role of long-lasting neuromuscular depression in muscle activity in crayfish. Comp Biochem Physiol 66:137–142. Google Scholar


L. H. Field, P. L. Newland, and M. Hisada . 1990. Physiology and structure of three new uropod proprioceptors in the crayfish Procambarus clarkii. J Exp Biol 154:179–200. Google Scholar


S. Fujie, H. Aonuma, I. Ito, A. Gelperin, and E. Ito . 2002. The nitric oxide/cyclic GMP pathway in the olfactory processing system of the terrestrial slug Limax marginatus. Zool Sci 19:15–26. Google Scholar


J. Garthwaite and C. L. Boulton . 1995. Nitric oxide signaling in the central nervous system. Ann Rev Physiol 57:683–706. Google Scholar


T. Nagayama 1997. Organization of exteroceptive inputs onto non-spiking local interneurons in the crayfish terminal abdominal ganglion. J Exp Zool 279:29–42. Google Scholar


T. Nagayama 2002. Synaptic organization of local circuit neurons in the terminal abdominal ganglion of crayfish. In “The Crustacean Nervous System”. Ed by K. Wiese , editor. Springer-Verlag. Berlin. pp. 591–600. Google Scholar


T. Nagayama and P. L. Newland . 1993. A sensory map based on velocity threshold of sensory neurons from a chordotonal organ in the tailfan of the crayfish. J Comp Physiol A 172:7–15. Google Scholar


T. Nagayama, H. Namba, and H. Aonuma . 1994. Morphological and physiological bases of crayfish local circuit neurones. Histol Histopathol 9:791–805. Google Scholar


T. Nagayama, Y. Isogai, M. Sato, and M. Hisada . 1993. Intersegmental ascending interneurons controlling uropod movements of the crayfish Procambarus clarkii. J Comp Neurol 332:155–174. Google Scholar


T. Nagayama, H. Aonuma, and P. L. Newland . 1997. Convergent chemical and electrical synaptic input from proprioceptive afferents onto an identified interneurone in the crayfish. J Neurophysiol 77:2826–2830. Google Scholar


N. Nakatsubo, H. Kojima, K. Kikuchi, Y. Hirata, D. Maeda, Y. Imai, T. Irimura, and T. Nagano . 1998. Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS Lett 427:263–266. Google Scholar


P. L. Newland and T. Nagayama . 1993. Parallel processing of proprioceptive information in the terminal abdominal ganglion of the crayfish. J Comp Physiol 172:389–400. Google Scholar


P. L. Newland, H. Aonuma, and T. Nagayama . 1997. Monosynaptic excitation of lateral giant fibres by proprioceptive afferents in the crayfish. J Comp Physiol 181:103–109. Google Scholar


P. L. Newland, H. Aonuma, and T. Nagayama . 2000. The role of proprioceptive signals in the crayfish escape circuit. Zool Sci 17:1185–1195. Google Scholar


S. S. Rashatwar, T. L. Cornwell, and T. M. Lincoln . 1987. Effects of 8-bromo-cGMP on Ca2+ levels in vascular smooth muscle cells: possible regulation of Ca2+-ATPase by cGMP-dependent protein kinase. Proc Nat Acad Sci USA 84:5685–5689. Google Scholar


H. Schuppe and P. L. Newland . 2004. Nitric oxide modulates presynaptic afferent depolarisation of mechanosensory neurons. J Neurobiol in press. Google Scholar


H. Schuppe, H. Aonuma, and P. L. Newland . 2001a. NADPH-diaphorase histochemistry in the terminal abdominal ganglion of the crayfish. Cell Tissue Res 303:289–299. Google Scholar


H. Schuppe, H. Aonuma, and P. L. Newland . 2001b. Distribution of NADPH-diaphorase positive ascending interneurons in the crayfish terminal abdominal ganglion. Cell Tissue Res 305:135–146. Google Scholar


H. Schuppe, M. Cuttle, J. E. Chad, and P. L. Newland . 2002. 4,5-diaminofluoroscein imaging of nitric oxide synthesis in crayfish terminal ganglia. J Neurobiol 53:361–369. Google Scholar


K. Wiese 1976. Mechanoreceptors for near-field water displacement in crayfish. J Neurophysiol 39:283–319. Google Scholar
Hansjürgen Schuppe, Makoto Araki, Hitoshi Aonuma, Toshiki Nagayama, and Philip L. Newland "Effects of Nitric Oxide on Proprioceptive Signaling," Zoological Science 21(1), 1-5, (27 January 2004).[1:EONOOP]2.0.CO;2
Received: 12 August 2003; Accepted: 1 September 2003; Published: 27 January 2004
local circuits
Back to Top