J Comp Physiol (1983) 152:529-534 Journal of Comparative Physiology. A �9 Springer-Verlag 1983

The sensory systems of embryos of the newt:

Alan Roberts and J.D.W. Clarke* Department of Zoology, The University, Bristol BS8 lUG, England

Accepted April 18, 1983

Triturus vulgaris

Summary. We have investigated the sensory sys- tems of Triturus vulgaris embryos after they be- come capable of swimming. Reducing the light in- tensity produces no short latency response but leads to an increase in spontaneous movements not dependent on the lateral eyes. The pineal eye is implicated, and recordings show that it is excited by dimming. Mechanical stimulation of trunk skin evokes movements. The innervation of trunk skin by free nerve-endings from Rohon-Beard cells is demonstrated using horseradish peroxidase stain- ing. Recordings from Rohon-Beard cells show their receptive fields. They respond to deformation of the skin. The embryos appear to lack a propa- gated skin impulse.

Introduction

The sensory systems of late stages of amphibian embryos are very simple in organization when compared to those of any adult vertebrate. How- ever, they have to provide sufficient information to control the limited behaviour of the embryos. There is therefore an innervation of the skin by mechanoreceptive 'free' nerve endings (Roberts and Blight 1975; Roberts and Hayes 1977; Roberts I980), a system for monitoring noxious stimuli to the skin which is based on the skin itself being excitable (Roberts and Stirling 1971; Roberts 1971; Roberts and Smyth 1974; Sato et al. 1981; Fan Shifan and Dai Rong• 1962; Sun Yian and Dai Rongxi 1982), and a visual system based on the pineal eye which is excited by dimming (Ro- berts 1978; Foster and Roberts 1982). Most of

* Present address: Department of Anatomy, King's College, The Strand, London WC2R 2LS, England

these sensory systems have only been studied in Xenopus laevis so we have made a broad study of them in the common newt (Triturus vulgaris). In our study the properties of the sensory systems have been closely related to the motor behaviour of the newt embryos which we describe elsewhere (Soffe et al. 1983). While possessing broadly simi- lar sensory anatomy and physiology, each system in the newt operates in some way differently to the corresponding system in J(enopus. An account of the physiology of the innervation of the head skin by trigeminal ganglion cells in Triturus helveti- cus has already been presented (Roberts 1980).

Materials and methods

Embryos of Triturus vulgaris developed from eggs laid in aquaria and where necessary were removed from their egg mem- branes with fine forceps. They were staged using Gallien and Bidaud's tables for Triturus helvetieus (1959). All methods have been described previously (Foster and Roberts 1982; Roberts 1980; Roberts and Clarke !982; Roberts and Hayes 1977; Hayes and Roberts 1983). All recordings were made in frog saline (in mmol/l: NaC1, 115; KC1, 2.5; CaC12, 5; NaHCO3, 2.4; pH 7.2).

The light source was calibrated in I~Einsteins m-2"S- 1. One Einstein = one Mole of light = 6 x 1023 photons .m- 2.s - t. One lux=0.016 gE-m 2-s 1.

Results

Responses to dimming

Unlike Xenopus embryos (Roberts 1978; Foster and Roberts 1982)Triturus embryos show no clear, short latency movements when the light is dimmed. However, dimming (from 1,500 to 5 ~ E . m Z . s -1 ) leads to an increase in the number of spontaneous movements mainly in the first minute after dim- ming (Fig. 1). Despite this lack of a short latency response in normal embryos, dimming can evoke

530 A. Roberts and J.D.W. Clarke: Newt embryo sensory systems

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dark Fig. 1 A, B. Increase in movements following dimming in stage 34-36 embryos still in their egg membranes. A 10 embryos were observed during alternating 3 min light and dark periods. Number of embryos moving increased markedly during the 1st min in the dark. B After 10 min in the light, 14 embryos were observed for 90 s in the dark. The maximum number move 20 s after the light was dimmed. (In this casemovements were not recorded during the light phase.)

motor root activity in embryos which are immobi-, lized in 10-4 mol/1 tubocurarine. These responses occur 500 ms or more after light intensity reduc- tion (Soffe et al. 1983).

The pineal eye was implicated by observations on one embryo which developed no lateral eyes. In this animal dimming the light still increased the number of spontaneous movements and on some occasions led immediately to swimming as in the Xenopus embryo. Extracellular recordings were therefore made from the Triturus embryo pineal. A small (60 ~tm tip opening) suction electrode placed on the pineal vesicle showed low levels of spontaneous background firing which increased briefly on dimming (Fig. 2). The increase in firing depended on the degree of dimming. After dim- ming firing adapted rapidly in the first 5 s (Fig. 2) and then returned to the background level within about 2 min. There was no evidence of sustained firing related to the steady level of illumination. In one embryo the lateral eyes were removed prior to recording from the pineal. Similar responses to dimming were still observed.

Responses to mechanical stimulation of trunk skin

Like Xenopus embryos, those of Triturus will move when stroked or poked quickly with a fine hair

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Fig. 2A, B. Recordings from units in the pineal vesicte of a stage 34 embryo showing responses to different degrees of dim- ming. A Direct recordings of responses to dimming from 1,500 gE.m-Z.s -1 t o the levels indicated (5.3, ll0, 540 and 1,000 gE.m-l .s -1 respectively). Dimming started at start of each trace (arrow). B Plots of instantaneous spike frequency against time after dimming for the records shown in A

(about 5 gm in diameter) which causes a local in- dentation of the skin. However, Triturus embryos will also move in response to less localized and slower distortion of the skin caused by: pressing with the side of a fine hair, pressing or stroking

A. Roberts and J.D.W. Clarke: Newt embryo sensory systems 531

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Fig. 3A-C. Responses to skin stimulation in units recorded from the dorsal spinal cord with suction electrodes (se). For each embryo a camera lucida drawing was made of the rostral part of the trunk viewed from the side (area between arrows on the drawing of a whole embryo). Receptive fields were then marked on these drawings and arrows used to indicate where the skin was stroked with a hair. In the recordings, arrows indicate when strokes were given and lines when the skin was indented normal to its surface. A Unit with receptive field over myotomes which responded to (i) touch with a hair; (ii) stroke with hair (e.g. arrow on receptive field), (iii) repeated strokes, and (iv) indentation of the skin (at the dot) with a 250 gm glass ball. B Unit (a) with receptive field on fin was recorded with electrode at se2. It gave few spikes to quick strokes with the hair. (i) Short strokes gave one spike, (ii) strokes along the field gave more. Unit (b) with a ventral field was recorded with electrode at se l . It responded (i) to touch but also (ii) to slow indentation with the side of the hair. C 4 units recorded at se2 showing receptive fields and responses to single or repeated strokes (e.g. Units (a) and b)). Unit (c) had large spikes but the records show that stroking within its receptive field also evoked smaller spikes from a second unit whose receptive field overlapped unit (c). Unit (d) has a large field and the records show responses to long strokes which later excite unit (b) (vertical gain half that in (bi) and (bii)). Unit (e), recorded at se l , has a large receptive field which overlaps extensively into the fields of units (c) and (d). Time calibration in B applies to all records. Arrows on receptive fields indicate locations of strokes for recordings illustrated

with a 250 I~m diameter smooth glass ball, gently squeezing the tail or belly with forceps. They also move in response to stronger stimuli: pinch with forceps, 1 ms current pulse to the skin, or damage to the skin. The types of movements made in re-

sponse to these stimuli are described by Soffe et al. (1983). As in Xenopus, pressing on the head during swimming can cause swimming to stop (Roberts 1980).

If Triturus embryos are immobilized in

532

10 -4 mmol/1 tubocurarine and the dorsal spinal cord is exposed locally, then a fine suction elec- trode can be placed on the dorsal midline of the spinal cord to record from sensory Rohon-Beard cells (Roberts and Hayes 1977). In Xenopus this method of recording has been corroborated by in- tracellular recordings from Rohon-Beard cells (Clarke, Hayes, Hunt and Roberts, in prep.). Units recorded from the mid-dorsal spinal cord in Tritur- us respond to mechanical stimulation of localized receptive fields on the trunk skin (Fig. 3). The re- ceptive fields ranged widely in area (0.023 mm 2 to 0.208 ram2), and in shape. Sensitivity to stimula- tion was uneven within receptive fields there often being small particularly sensitive patches. Overlap between receptive fields was not seen often but could be considerable (e.g. between units (c), (d) and (e) in Fig. 3 C). In one case illustrated, overlap was clear physiologically as stimulation led to co- excitation of two units with different spike ampli- tudes (Fig. 3 Cc).

Local indentation of the skin surface with a fine hair either poked into or stroked across the receptive field was an effective stimulus for all re- corded units (Fig. 3). While some units were like Xenopus t runk skin units (Roberts and Hayes 1977) in all their properties (e.g. Fig. 3, Ba), most differed in being able to respond well to repeated stimulation (Figs. 3A and C). They differed also in responding to slower and less local distortion of the skin, produced by pressing with the side of a hair or by pressing or rubbing with a 250 gm diameter glass ball (Figs. 3Aiv and Bbii). To be effective these forms of stimulation needed to in- dent the skin clearly, by about 40 to 60 gin. Using the glass ball mounted on a loudspeaker cone re- sponses to sinusoidal indentation were obtained with cycle periods of 90 ms or less. Ramp indenta- tions could evoke spikes if rise times were 400 ms or less.

Innervation of trunk skin

As in Xenopus (Roberts and Hayes 1977; Clarke, Hayes, Hunt and Roberts, in prep.) the trunk skin of Triturus embryos is innervated by Rohon-Beard cells whose somas form a double row along the dorsal midline of the spinal cord. When stained with Horseradish peroxidase (HRP) the peripheral neurites of these Rohon-Beard cells can be fol- lowed in serial sections from the spinal cord to the skin. We have also examined wholemounts of skin which contain Rohon-Beard neurites stained with HRP. In favourable cases these can be fol- lowed to their terminations within the trunk skin (Fig. 4). After reaching the skin, neurites lie on the basal lamina under the skin. In this position

A. Roberts and J.D.W. Clarke: Newt embryo sensory systems

'branches ' separate from the main neurite. How- ever, with the light microscope it is impossible to distinguish single neurites from pairs or small bun- dles of neurites. Consequently 'branches ' could be two neurites separating from one another (see also Roberts and Hayes 1977). Despite this uncertainty, it is likely that all the 'branches ' shown in Fig. 4 are in fact from a single main neurite since the area covered by the 'branches ' corresponds closely in size and shape to the kinds of receptive fields found physiologically (e.g. Fig. 3Cb). From their position under the ski n neurites penetrate between the inner skin cells and run between the two cell layers of the skin. Here, further 'branching' occurs and, approaching the ends of the neurites, small bulges, or varicosities become common. Only very rarely do neurites lie between superficial skin cells.

Absence of propagated skin impulse

A broad range of tests have failed to produce any evidence for a propagated skin impulse in Triturus vulgaris embryos between stages 33 and 37. If the spinal cord and myotomes are cut through in the mid-trunk region stimulation of the tail does not evoke movements rostral to the cut (Wintrebert 1904; Roberts 1971). Electrical recordings were made from the skin with i intracellular microelec- trodes, microelectrodes inserted through the skin (Roberts 1971) and suction electrodes applied to the outside of the skin. No propagated events were recorded in response to mechanical or electrical stimulation of the skin.

Discussion

The sensory systems that we have described here and previously (Roberts 1980) in Triturus embryos between stages 33 and 36 (Gallien and Bidaud 1959 and see Soffe et al. 1983) seem sufficient to account for the behavioural responses seen to dimming the lights and mechanical stimulation. The acoustico- lateralis system and lateral eyes do not yet appear to be functional and we have not investigated re- sponses to chemical stimuli. The aim of this study was to compare the embryonic pineal eye and so- matosensory systems of a urodele Triturus, with those of the anuran Xenopus. In general there are strong parallels between the two groups.

The pineal eye in Triturus is a small flattened vesicle on the dorsal midline between the fore- and mid-brain (Kelly 1971). As in Xenopus, units re- corded from the pineal in Triturus show low spon- taneous activity which is markedly increased on dimming. We assume that activity is recorded from pineal ganglion cells. The effects o f dimming on behaviour in Triturus are weaker than in Xenopus

A. Roberts and J.D.W. Clarke: Newt embryo sensory systems 533

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Fig. 4. Rohon-Beard cell neurites innervating ventral flank skin in a stage 33/34 embryo. Camera lucida drawing made from HRP stained whole-mount of skin from the region shown in the drawing of the whole embryo. A large neurite was followed below the skin (u) from the level of the myotomes. It did not branch until it had passed the ventral row of pigment cells; all its branches are therefore shown in the drawing. Branches mainly lie between the inner and outer skin cell layers. One short section of neurite (between arrows) lies between cells of the outer layer of skin cells, whose outlines are shown as dotted fines where they were clear

but for about 60 s after dimming embryos show raised activity. Lateral eyes are not necessary for this, and we conclude that the pineal eye produces central excitation fol lowing dimming as in Xenopus embryos (Roberts 1978; Foster and Roberts 1982).

The somatosensory system in Triturus is based on an innervation of the skin o f the whole body surface by unmyelinated nerve fibres forming 'free' nerve endings within the skin. We have de- scribed the innervation o f trunk skin by R o h o n -

534 A. Roberts and J.D.W. Clarke: Newt embryo sensory systems

Beard cells. The pattern of innervation of the head skin by trigeminal ganglion cells is very similar, resembling that seen in more densely innervated regions of trunk skin (Roberts, unpublished). In head skin, HRP staining shows that beaded neu- rites lie mainly between the inner and outer skin cell layers where they can branch extensively. As in trunk skin few neurites are seen between the superficial cells in head skin. Physiologically the whole skin surface is sensitive to light touch through its innervation by trigeminal ganglion cells and by Rohon-Beard cells, which sense local, ra- pid, transient deformations of the skin surface. However, while trunk skin is innervated by a single physiological and anatomical type of sensory neu- rone, the Rohon-Beard cell (this conclusion as- sumes that extra-medullary cells, (Hughes 1957; Roberts and Clarke 1982) are a subclass of Rohon- Beard cells and have similar physiology), head skin is also innervated by a second physiological class of trigeminal ganglion cell in both Triturus and Xenopus (Roberts 1980). This class responds to slower and more general deformation of the skin with a more vigorous and less rapidly adapting discharge. Stimuli exciting this class of receptor are normally effective in stopping ongoing swim- ming in both Xenopus and Triturus embryos. Thus, when they swim into objects they tend to stop.

What differences are there in the somatosen- sory systems of Xenopus and Triturus? Many Tri- turus Rohon-Beard cells are less phasic than those in Xenopus. They will respond repeatedly to more diffuse and slower skin deformations, as will the corresponding rapid-transient detectors of the head skin (Roberts 1980). This correlates with behaviour since Triturus embryos will swim when gentle pressure is applied to the trunk skin while Xenopus embryos wili not. The physiological dif- ference between Triturus and Xenopus is not ob- viously related to differences in the neuroanatomy of the neurites innervating the trunk skin. Rohon- Beard neurites in the skin of Triturus and Xenopus (Roberts and Hayes 1977; Clarke, Hayes, Hunt and Roberts, in prep.) are similar to each other and to rapid-transient neurites in Xenopus head skin (Hayes and Roberts 1983).

A second difference between Triturus and Xen- opus is the apparent absence of a propagated skin impulse (Roberts and Stirling 1971; Roberts 1971). This is surprising, since skin impulses have been recorded in other urodeles (Cynops Fan Shifan and Dai Rongxi 1962; Sun Yian and Dai Rongxi 1982; Sato etal. 1981: Ambystoma; Blight, personal communication). If absent, this means that Tri- turus has no sensory system specifically monitoring

stronger, possibly noxious stimuli. We do not yet understand the significance of these differences in sensory capability between embryos which might otherwise be expected to have similar require- ments.

Acknowledgements. We thank the SERC and MRC for their support, K. Williams for photographic assistance, Dr. B.P. Hayes for the drawing in Fig. 4, and Dr. S.R. Soffe for help at all stages.

References

Fan Shifan, Dai Rongxi (1962) Electric activity of embryonic epithelium in Urodeles. Kexuw Tongboa 10:38-9

Foster RG, Roberts A (1982) The pineal eye in Xenopus laevis embryos and larvae:photoreceptors with a direct excitatory effect on behaviour. J Comp Physiol 145: 413~419

Gallien L, Bidaud O (1959) Table chronologique du developpe- ment chez Triturus helveticus Razoumowski. Bull Soc Zool Fr 84:22-32

Hayes BP, Roberts A (1983) The anatomy of two functional types of 'free' nerve ending in the head skin of Xenopus embryos. Proc R Soc Lond B 218:61-76

Hughes AFW (1957) The development of the primary sensory system in Xenopus laevis. J Anat 91:323-338

Kelly DE (1971) Development aspects of amphibian pineal sys- tems. In: Wolstenholme GE, Knight J (eds) The pineal gland. Churchill and Livingston, Edinburgh London

Roberts A (1971) The role of propagated skin impulses in the sensory system of young tadpoles. Z Vergl Pbysiol 75 : 388-401

Roberts A (1978) Pineal eye and behaviour in Xenopus tadpoles. Nature 273 : 774

Roberts A (1980) The function and role of two types of mecha- noreceptive ' free' nerve endings in the head skin of amphi- bian embryos. J Comp Physiol 135:341-348

Roberts A, Blight AR (1975) Anatomy, physiology and behav- ioural role of sensory nerve endings in the cement gland of embryonic Xenopus. Proc R Soc Lond B 192:111-127

Roberts A, Clarke JDW (1982) The neuroanatomy of an am- phibian embryo spinal cord. Phil Trans R Soc Lond B 296:195-212

Roberts A, Hayes BP (1977) The anatomy and function of 'free' nerve endings in an amphibian skin sensory system. Proc R Soc Lond B 196:415-429

Roberts A, Smyth D (1974) The development of a dual touch sensory system in embryos of the amphibian Xenopus laevis. J Comp Physiol 88:3142

Roberts A, Stirling CA (1971) The properties and propagation of a cardiac-like impulse in the skin of young tadpoles. Z Vergl Physiol 71:295-310

Sato E, Adachi S, Ito S (1981) The genesis and transmission of epidermal potentials in an amphibian embryo. Dev Biol 88 : 137-146

Soffe SR, Clarke JDW, Roberts A (1983) Swimming and other centrally generated motor patterns in newt embryos. J Comp Physiol 152:535-544

Sun Yian, Dai Rongxi (1982) Further studies on the electrical activity of epithelial cells in embryos of Cynops orientalis. Acta Biol Exp Sinica 15:351-359

Wintrebert P (1904) Sur l'existence d'une irritabilit6 excito- motrice primitive independante de voies nerveuses chez les embryons cili~es des batraciens. CR Soc Biol Paris 57: 645-647