ABSTRACT
Neuronal mechanisms underlying the directional sensitivity of the crayfish equilibrium system were studied in the brain by intracellular recording combined with mechanical statocyst hair deflection.
Five primary afferents were successfully characterized. Three of them showed a decrease in response to inward hair deflection. The remaining two showed the opposite directional response.
Directional sensitivity was found in six interneurones. Two of them were excited during inward hair deflection while the other four were excited during outward deflection. Both groups exhibited active inhibition during hair deflections in the opposite direction.
This ‘null-phase inhibition’ appeared to arise from the convergence of the two classes of afferents onto an interneurone with the opposite sign.
Three identified descending statocyst interneurones, S3, S6 and S7, were found to receive excitatory input from one statocyst and inhibitory input from the other.
The results thus indicated that the directional sensitivity of the crayfish equilibrium system was achieved by selective excitatory connections between the interneurone and the directionally arranged receptor and sharpened by inhibitory mechanisms.
Introduction
The neuronal mechanisms underlying the directional sensitivity of the equilibrium system of the crayfish Procambarus clarkii have been extensively studied (Takahata and Hisada, 1979, 1982a,b). Equilibrium responses are primarily controlled by a pair of specialized gravity-sensing organs, statocysts, that are located in the basal segments of the antennules. Sensory hairs are aligned in a crescent shape on the floor of the statocyst. The principles of operation of the statocyst were first described in the crayfish Astacus fluviatilis (Stein, 1975). In this species, all the hairs are polarized structurally towards the centre of the crescent.
Any tilt of the animal was thought to result in a specific pattern of deflection of the hairs distributed along the sensory crescent. Eye movements in response to selective stimulation of the statocyst hairs were observed, and showed indirectly that the relative magnitude of the excitation from a hair depended on the degree of coincidence between its polarization direction and the direction of shear, being maximal if both directions corresponded. In Procambarus clarkii, it was shown by extracellular recording from the statocyst nerve that the sensory neurones attached to the hairs had the same directional sensitivity, with maximal excitatory responses to hair deflection towards the centre of the crescent and maximal suppression for the opposite direction. Because of the crescent arrangement of the planes of polarization, the direction of hair deflection that elicited an excitatory response in the receptor was observed to vary with the location of the hair. Thus, it was suggested that a body tilt around a particular body axis would produce a regional distribution of excitation and suppression of receptors around the crescent (Takahata and Hisada, 1979). In each circumoesophageal connective of the crayfish, several statocyst interneurones have been found that receive input only from receptors located in a specific region of the sensory crescent and which show the same directional sensitivity (Takahata and Hisada, 1982a,b; Nakagawa and Hisada, 1989). These studies have suggested that the directional tuning of these statocyst interneurones is based on a selective connection onto them from a crescent-like distribution of polarized sensory hairs.
Selective connections to directionally arranged receptors have been shown to account for directionality of interneurones in other systems (e.g. giant interneurones of the cockroach, Westin et al. 1977; Walthall and Hartman, 1981). Different mechanisms of directional sensitivity have also been reported in other systems. In the crayfish telson, many hairs are dually innervated, with one afferent responding to headward bending and the other to tailward bending (Wiese, 1976). Some interneurones receive not only excitatory input from receptors with the same directional sensitivity but also indirect inhibitory input from ones with the opposite sensitivity. This ‘null-phase inhibition’ (Wiese et al. 1976) sharpens the system’s directional sensitivity. Central inhibitory interactions have also been reported to sharpen directional sensitivity in other vertebrate and invertebrate sensory systems (Precht, 1974; Edwards and Palka, 1974; Palka and Olberg, 1977; Matsumoto and Murphey, 1977; Levine and Murphey, 1980; Reichert et al. 1983). In this paper we show, on the basis of intracellular recording in the brain, that the directional sensitivity of the crayfish equilibrium system is attained by selective excitatory connections and is enhanced by such inhibitory mechanisms.
Materials and methods
Adult crayfish, Procambarus clarkii Girard, of either sex, measuring 8.5 –10.5 cm in length, were used. The preparations were essentially the same as those in a previous report (Nakagawa and Hisada, 1989). However, to facilitate intracellular microelectrode access to the brain, the animal was placed head-down in a chamber filled with crayfish saline (van Harreveld, 1936). The antennules were kept level in order to deliver direct mechanical stimulation to the statocyst hairs. Small groups of sensory hairs in the lateral region of the crescent were deflected towards or away from the centre by a miniature hook probe (Nakagawa and Hisada, 1989). To determine the effectiveness of the stimulation, we recorded the extracellular spike activity of both the optic nerve motor bundle and the circumoesophageal connective on the side contralateral to the stimulated statocyst. Glass microelectrodes filled with a 3 % solution of Lucifer Yellow were inserted into the deutocerebrum on both sides. After the directional responses of the statocyst-related neurones had been examined intracellularly, the dye was injected into them ionophoretically.
Results and Discussion
Here, we will identify two possible additional mechanisms that contribute to the directional sensitivity of the crayfish equilibrium system. The first is the bidirectional sensitivity of some interneurones arising from excitation of some afferents in response to deflection of statocyst hairs away from the centre of the crescent (Fig. 1). The neurones were judged to be afferents on the basis of the following observations. (1) Their action potentials arose directly from the baseline. No underlying postsynaptic potentials were visible. (2) No cell bodies were stained in the brain. (3) The neurones appeared to enter the brain nearly at the entrance of the statocyst nerve. So far, we have obtained successful intracellular recordings from five primary afferents. Three of these showed a decrease in response to hair deflection towards the crescent centre and an increase in response to a deflection in the opposite direction. The remaining two showed the opposite directional responses, like those reported in a previous study (Takahata and Hisada, 1979). Each of these two groups contained afferents which projected ipsilaterally or bilaterally (Fig. 1). The structural mechanisms underlying this novel directionality of the afferents are not clear. Two possibilities exist. (1) As in the telson, the statocyst sensory hairs are dually innervated, with one of the afferents responding to inward, and the other to outward, deflection. (2) Some statocyst sensory hairs are functionally polarized away from the centre of the crescent and are innervated by afferents which respond to an outward deflection. In Astacus each hair was shown to possess three sensory neurones in an ultrastructural study (Schöne and Steinbrecht, 1968). Patton (1969) reported that two or three qualitatively different receptors were driven simultaneously by movements of the same hair in Homarus americanus. Two of these responded to lateral deflection of the hair from its rest position, while the third was excited when the hair was returned to rest from a lateral deflection. This would tend to support the first possibility. In contrast, in Nephrops, several rows of statolith hairs were found in the lateral regions of the crescent with different polarizations (Newland and Neil, 1987). Also, sensory hairs oriented in opposite directions have been found in vertebrate vestibular sensory areas (Flock, 1964; Spoendlin, 1964). These observations provide precedents for the second possibility. A re-examination of the morphology of statocysts, including the inner hair row (Takahata and Hisada, 1979), in more detail is necessary to correlate this opposite directionality with statocyst morphology.
We have found six interneurones that are directionally sensitive and show opposite directional responses depending on the direction of hair deflection. Except for one projecting interneurone, all of them are local interneurones. Two of them were excited during inward deflection and actively inhibited by incoming IPSPs during outward deflection. The remaining four were actively inhibited during inward deflection and excited during outward deflection. Fig. 2 shows the morphology and typical responses of these interneurones. It should be noted that IPSPs or EPSPs could clearly be observed in response to outward deflection of the hairs. These potential changes cannot be attributed to the abolition of spontaneous afferent discharge. These findings suggest that: (1) statocyst sensory afferents are functionally endowed with two opposite directional sensitivities; (2) a given interneurone can be postsynaptic to both classes of afferents, but it is connected with opposite signs, with one afferent being excitatory and the other inhibitory; and (3) this convergence of the afferents of the two classes sharpens the directional sensitivity of the interneurone by the null-phase inhibition mechanism (Wiese et al. 1976) during hair deflection in the non-preferred direction.
The second mechanism is that several identified descending statocyst interneurones connect with opposite signs to the statocysts of the two sides. In previous papers, we have shown that these interneurones receive their major input from only one of the statocysts (Takahata and Hisada, 1982a,b;Nakagawa and Hisada, 1989). In this study, however, some descending statocyst interneurones were found to receive excitatory input from one statocyst and inhibitory input from the other, which we once thought to be the non-input statocyst. This is evident in the response of interneurone S7. We have demonstrated previously that this interneurone receives excitatory input from the statocyst ipsilateral to its axon (Nakagawa and Hisada, 1989). However, since the background discharge of this interneurone is very low, an inhibitory input, even if it existed, would not have been detected by measuring changes in the spike frequency. However, intracellular recordings from neurites in the brain clearly demonstrate inhibition of the neurone. Fig. 3 shows the response of S7 to inward deflection of the statocyst hairs on the side contralateral to the axon. The spike discharge was suppressed during the stimulation. The inhibition became clear when the background discharge frequency was increased (about 20spikes s−1) by intracellular injection of current. Interneurones S6 and S3 also showed a similar inhibition in response to inward deflection of the hairs of the contralateral and ipsilateral statocyst, respectively.
Spatial information has been shown to be transmitted in two different modes; by the pattern of excitation of the maculae and by the overall amount of excitation (Schöne, 1975). With respect to the former, the crayfish equilibrium receptor system was thought to be of relatively simple construction. This was based largely on the crescent-like distribution of inward-preferring polarization of sensory hairs and selective excitatory connections between receptors in specific regions and particular interneurones (Takahata and Hisada, 1979, 1982a,b). However, in this study, we have shown that additional mechanisms are involved in the equilibrium system. The first mechanism is the convergence of the two classes of afferents onto the interneurones that form the null-phase inhibitory connections. The second is the central inhibition of some identified descending statocyst interneurones derived from the differential connections from the statocysts of the two sides. Thus, several mechanisms which have been reported in other sensory systems also exist in the crayfish and serve to sharpen the directional sensitivity of the equilibrium system.
ACKNOWLEDGEMENTS
We are grateful to Dr H. Robinson for reading the manuscript and for constructive suggestions. This work was supported by Grants-in-aid from the Japanese Ministry of Education, Science and Culture to HN (no. 01790321) and to MH (nos 61480016 and 01480021).