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Central Projections of Primary Sensory Afferents to the Spinal Dorsal Horn in the Long-Tailed Stingray, Himantura fai
Abstract
The central projections of primary sensory afferents innervating the caudal region of the pectoral fin of the long-tailed stingray (Himantura fai) were labeled by applying the lipophilic carbocyanine dye DiI to the dorsal roots in fixed tissue. These observations were complemented by examination of hemotoxylin and eosin-stained paraffin sections of the dorsal root entry zone, and transmission electron microscopy of the dorsal horn. Transverse sections of the sensory nerve and dorsal root revealed two distinct myelinated axon sizes in the sensory nerve. Although the thick and thin axons do not appear to group together in the sensory nerves and dorsal root, they segregate into a dorsally directed bundle of thin fibers and a more horizontally directed bundle of thick fibers soon after entering the spinal cord. In DiI-labeled horizontal sections, fibers were observed to enter the spinal cord and diverge into rostrally and caudally directed trajectories. Branching varicose axons could be traced in the dorsal horn gray matter in the segment of entry and about half of the adjacent rostral and caudal segments. In transverse and sagittal sections, DiI-labeled afferents were seen to innervate the superficial and, to a lesser extent, deeper laminae of the dorsal horn, but not the ventral horn. Electron microscopy of unlabeled dorsal horn sections revealed a variety of synaptic morphologies including large presynaptic elements (some containing dense-core vesicles) making synaptic contacts with multiple processes in a glomerular arrangement; in this respect, the synaptic ultrastructure is broadly similar to that seen in the dorsal horn of rodents and other mammals.
... Thus far, most anatomical and electrophysiological studies have been conducted on teleost or bony fish with relatively few on elasmobranch (cartilaginous) fish. The very few published findings tend to be lacking in experimental detail (Leonard, 1985); however, a more recent study in the long-tailed stingray (Himantura fai) has confirmed some of the previous experiments in that there is a lack of unmyelinated C fibres, but small myelinated fibres are in abundance and could potentially be A-delta fibres (Kitchener et al., 2010). However, electrophysiological studies are needed to determine whether nociceptors occur in this group. ...
... Several tract tracing studies have demonstrated that the fish neuroanatomical pathways from peripheral areas to the brain are highly conserved. In the long-tailed stingray, dorsal horn distinguishable layers are apparent as seen in the equivalent of the spinal cord of mammals, and the synaptic ultrastructure is broadly similar to that of the dorsal horn of rodents and other mammals (Kitchener et al., 2010). The main tracts, including the spinothalamic and trigeminal, which convey pain from the body and face, respectively, are similarly organized (review in Sneddon, 2004), and within the teleost brain there are various connections to the thalamus and cortical areas (Rink and Wullimann, 2004) that innervate pain processing in mammals. ...
Recent developments in the study of pain in animals have demonstrated the potential for pain perception in a variety of wholly aquatic species such as molluscs, crustaceans and fish. This allows us to gain insight into how the ecological pressures and differential life history of living in a watery medium can yield novel data that inform the comparative physiology and evolution of pain. Nociception is the simple detection of potentially painful stimuli usually accompanied by a reflex withdrawal response, and nociceptors have been found in aquatic invertebrates such as the sea slug Aplysia. It would seem adaptive to have a warning system that allows animals to avoid lifethreatening injury, yet debate does still continue over the capacity for non-mammalian species to experience the discomfort or suffering that is a key component of pain rather than a nociceptive reflex. Contemporary studies over the last 10 years have demonstrated that bony fish possess nociceptors that are similar to those in mammals; that they demonstrate pain-related changes in physiology and behaviour that are reduced by painkillers; that they exhibit higher brain activity when painfully stimulated; and that pain is more important than showing fear or anti-predator behaviour in bony fish. The neurophysiological basis of nociception or pain in fish is demonstrably similar to that in mammals. Pain perception in invertebrates is more controversial as they lack the vertebrate brain, yet recent research evidence confirms that there are behavioural changes in response to potentially painful events. This review will assess the field of pain perception in aquatic species, focusing on fish and selected invertebrate groups to interpret how research findings can inform our understanding of the physiology and evolution of pain. Further, if we accept these animals may be capable of experiencing the negative experience of pain, then the wider implications of human use of these animals should be considered.
... There are two main types of nociceptive neuron in mammals, Aδ-fibres and C-fibres. Aδ-fibres, linked to sharp pains in us but also used for mechanoreception, have been found in elasmobranchs by at least three studies (Kitchener et al. 2010;Lacap 2022;Snow et al. 1993). Evidence of C-fibres, associated with lingering pains in us, was obtained for the first time by a study in 2022, but the C-fibres were specifically responding to nociceptive stimuli in the cranial region (Lacap 2022). ...
Martha Nussbaum’s Justice for Animals calls upon humanity to secure for all sentient beings the central capabilities they need to flourish. This essay review critically examines the ethical and scientific foundations of Nussbaum’s position. On the ethical side, we explore the tension between a robust defence of animal rights and political liberalism, which requires tolerance of a range of reasonable views. On the scientific side, we reflect on how our uncertainty regarding the distribution of sentience in the natural world leaves us uncertain about how many claimants of justice there are and about the relative strength of their claims. We bring out some further problems with Nussbaum’s views on animal killing and pest control. We also reflect on how animal welfare science might be done differently if guided by Nussbaum’s framework.
... Tract-tracing studies in fish have demonstrated that the nociceptive neuroanatomical pathways from the periphery to the brain are highly conserved and like those described above in mammals. Dorsal horn neuroanatomical strata and synaptic organization have been reported in certain species and considered to be analogous of those in mammals (Kitchener et al., 2010). In fish too, the spinothalamic and trigeminothalamic are the main pain pathways and are structured like in mammals (Sneddon, 2004). ...
In recent decades, the microbiological status of animal colonies, especially mice and rats, has been highly improved due to the amelioration of animal facilities, house caging systems, experimental procedures, and health monitoring programs. The microbiological status of experimental animals can dramatically influence the animal welfare, the robustness, and the reproducibility of research results. The Federation of European Laboratory Animal Science Associations (FELASA) has published several updated editions of recommendations for rodent health monitoring (1994, 1996, 2002, and 2014). These recommendations are intended to harmonize health monitoring programs and to standardize the microbiological quality of the animals to ensure reproducible results. Microorganisms can influence experimental results even if symptoms are not detected since many pathogens cause subclinical diseases. Subclinical disease may perturb biological parameters (behavior, growth rate, organ weight, immune response), and they may generate interferences with research. Symptom manifestations depend on many variables such as bacterial or viral infectious strains, the host immune status, the simultaneous presence of other pathogenic or opportunistic microorganisms, and the genetic background/mutation. Some pathogen of laboratory animals can also infect humans (zoonoses). For all these reasons, all institutions should establish a health monitoring program to guarantee the quality of the animals and the safety of personnel. It is important that a qualified person be responsible for health management (HM) of the facility since the decision whether a microorganism is acceptable or not in a given animal colony is a complex issue.
All the HM results of a microbiological unit (room, rack, isolator) are reported in the HM report.
... Tract-tracing studies in fish have demonstrated that the nociceptive neuroanatomical pathways from the periphery to the brain are highly conserved and like those described above in mammals. Dorsal horn neuroanatomical strata and synaptic organization have been reported in certain species and considered to be analogous of those in mammals (Kitchener et al., 2010). In fish too, the spinothalamic and trigeminothalamic are the main pain pathways and are structured like in mammals (Sneddon, 2004). ...
The aim of this chapter is to provide the reader with guidance and relevant sources related to implementation of animal welfare in husbandry practices, design, and daily operations of animal facility.
Specifically, the authors aim to address the following key requirements as indicated in the Directive 2010/63:
-To indicate how good welfare can promote good science, e.g., how the failure to attend to biological and behavioral needs may affect the outcome of procedures.
-To indicate how husbandry and care may influence experimental outcome and the number of animals needed, e.g., example where the place in the room influences the outcome, hence randomization.
-To describe the dietary requirements of the relevant animal species and explain how these can be met.
-To describe the importance of providing an enriched environment (appropriate to both the species and the science) including social housing and opportunities for exercise, resting, and sleeping.
-To describe suitable routines and husbandry practices for the maintenance, care, and welfare for a range of animals used in research, to include small laboratory species and large animal species where appropriate.
-To describe suitable environmental and housing conditions for laboratory animals, how conditions are monitored, and identify the consequences for the animal resulting from inappropriate environmental conditions.
-To recognize that changes to or disruption of circadian or photoperiod can effect animals.
-To describe the biological consequences of acclimatization, habituation, and training.
-To describe how to provide water and an appropriate diet for laboratory animals including the sourcing, storage, and presentation of suitable foodstuffs and water.
... Tract-tracing studies in fish have demonstrated that the nociceptive neuroanatomical pathways from the periphery to the brain are highly conserved and like those described above in mammals. Dorsal horn neuroanatomical strata and synaptic organization have been reported in certain species and considered to be analogous of those in mammals (Kitchener et al., 2010). In fish too, the spinothalamic and trigeminothalamic are the main pain pathways and are structured like in mammals (Sneddon, 2004). ...
This chapter introduces key aspects of morphophysiological features of the most used mammalian and fish species in biomedical research, highlighting the key aspects relevant to the choice of models. A brief overview on the evolution, anatomy, and physiology of pain pathways has been also reported due to the relevance in relation to the severity of procedures. The second part of the chapter is addressed to provide a general overview on breeding, genetics, genetic alterations, and strain differences (rodents and zebrafish) relevant for an accurate experimental design.
... This is intuitive since these fish may frequently encounter such low temperatures and it would not be adaptive to perceive them as noxious. In elasmobranchs unmyelinated C fibres are lacking but small myelinated fibres are in abundance and could be A-delta fibres (Cameron, Plenderleith, & Snow, 1990;Kitchener, Fuller, & Snow, 2010;Snow, Plenderleith, & Wright, 1993). However, electrophysiological studies are needed to determine whether nociceptors occur in this group. ...
The detection and assessment of pain in animals is crucial to improving their welfare in a variety of contexts in which humans are ethically or legally bound to do so. Thus clear standards to judge whether pain is likely to occur in any animal species is vital to inform whether to alleviate pain or to drive the refinement of procedures to reduce invasiveness, thereby minimizing pain. We define two key concepts that can be used to evaluate the potential for pain in both invertebrate and vertebrate taxa. First, responses to noxious, potentially painful events should affect neurobiology, physiology and behaviour in a different manner to innocuous stimuli and subsequent behaviour should be modified including avoidance learning and protective responses. Second, animals should show a change in motivational state after experiencing a painful event such that future behavioural decision making is altered and can be measured as a change in conditioned place preference, self-administration of analgesia, paying a cost to access analgesia or avoidance of painful stimuli and reduced performance in concurrent events. The extent to which vertebrate and selected invertebrate groups fulfil these criteria is discussed in light of the empirical evidence and where there are gaps in our knowledge we propose future studies are vital to improve our assessment of pain. This review highlights arguments regarding animal pain and defines criteria that demonstrate, beyond a reasonable doubt, whether animals of a given species experience pain.
Experiencing pain is one of the key drivers of deciding whether to protect an animal under legislation and guidelines. Over the last two decades empirical evidence for fish experiencing pain has grown and this chapter reviews the current state of our knowledge. Defining animal pain has been problematic but a definition based upon whether whole animal responses to pain differ from non-painful stimuli and whether the experience alters future behavioural decisions and motivation is adopted. Studies show that fish have a similar nociceptive system to mammals, that behaviour is adversely affected and that this is prevented by pain-relieving drugs demonstrating that fish respond to pain in a different manner to innocuous events. Further, fish are motivated to avoid areas where pain has been experienced and are consumed by the painful event such that they do not exhibit normal fear or antipredator responses. Taken together these results make a compelling case for pain in fish. However, this topic is still debated and the chapter discusses the opposing opinions. If we accept pain occurs in fish then the wider implications of the use of fish must be considered. It would be in the public’s interest to keep fish healthy for a myriad of reasons including disease-free fish production, preventing zoonoses, conservation and sustainability of fish stocks and valid experimental results from laboratory studies using fish models.
The study of diverse animal groups allows us to discern the evolution of the neurobiology of nociception. Nociception functions as an important alarm system alerting the individual to potential and actual tissue damage. All animals possess nociceptors, and, in some animal groups, it has been demonstrated that there are consistent physiological mechanisms underpinning the nociceptive system. This review considers the comparative biology of nociception and pain from an evolutionary perspective.
The ability to react to environmental change is crucial for the survival of an organism and an essential prerequisite is the capacity to detect and respond to aversive stimuli. The importance of having an inbuilt "detect and protect" system is illustrated by the fact that most animals have dedicated sensory afferents which respond to noxious stimuli called nociceptors. Should injury occur there is often sensitization, whereby increased nociceptor sensitivity and/or plasticity of nociceptor-related neural circuits acts as a protection mechanism for the afflicted body part. Studying nociception and nociceptors in different model organisms has demonstrated that there are similarities from invertebrates right through to humans. The development of technology to genetically manipulate organisms, especially mice, has led to an understanding of some of the key molecular players in nociceptor function. This review will focus on what is known about nociceptors throughout the Animalia kingdom and what similarities exist across phyla; especially at the molecular level of ion channels.
The present review examines the experimental evidence supporting the existence of central mechanisms able to modulate the synaptic effectiveness of sensory fibers ending in the spinal cord of vertebrates. The first section covers work on the mode of operation and the synaptic mechanisms of presynaptic inhibition, in particular of the presynaptic control involving axo-axonic synapses made by GABAergic interneurons with the terminal arborizations of the afferent fibers. This includes reviewing of the ionic mechanisms involved in the generation of primary afferent depolarization (PAD) by GABAergic synapses, the ultrastructural basis underlying the generation of PAD, the relationship between PAD and presynaptic inhibition, the conduction of action potentials in the terminal arborizations of the afferent fibers, and the modeling of the presynaptic inhibitory synapse. The second section of the review deals with the functional organization of presynaptic inhibition. This includes the segmental and descending presynaptic control of the synaptic effectiveness of group-I and group-II muscle afferents, the evidence dealing with the local character of PAD as well as the differential inhibition of PAD in selected collaterals of individual muscle-spindle afferents by cutaneous and descending inputs. This section also examines observations on the presynaptic modulation of large cutaneous afferents, including the modulation of the synaptic effectiveness of thin myelinated and unmyelinated cutaneous fibers and of visceral afferents, as well as the presynaptic control of the synaptic actions of interneurons and descending tract neurons. The third section deals with the changes in PAD occurring during sleep and fictive locomotion in higher vertebrates and with the changes of presynaptic inhibition in humans during the execution of a variety of voluntary movements. In the final section, we examine the non-synaptic presynaptic modulation of transmitter release, including the possibility that the intraspinal endings of primary afferents also release colocalized peptides in a similar way as in the periphery. The outcome of the studies presently reviewed is that intraspinal terminals of sensory fibers are not hard-wired conductors of the information generated in their peripheral sensory receptors, but dynamic systems that convey information that can be selectively addressed by central mechanisms to specific neuronal targets. This central control of information flow in peripheral afferents appears to play an important role in the generation of integrated movements and processing of sensory information, including nociceptive information.
The locomotory musculature of dogfish is innervated by the segmental spinal nerves. The sensory and motor innervation of the abdominal musculature was studied in a preparation consisting of a strip of the abdominal body wall innervated by the ventral rami of the spinal nerves.
Each ventral ramus consists of two separate nerve bundles which were found to be peripheral extensions of the dorsal and ventral spinal roots. Recordings from the sensory bundles showed that there are few sensory endings in the musculature and body wall of the dogfish. It was possible to differentiate between ephemeral responses produced by cutaneous free-nerve endings and prolonged discharges which were generated by more specialized sensory endings. In some details these endings were found to be unlike either muscle spindles or tendon organs. Further, skinning experiments suggested that these mechanoreceptors lay in the skin or the very outer layers of the myotome.
Histological searching, together with physiological isolation of units, suggested that these receptors were the corpuscular endings distributed sparsely amongst subcutaneous tissue. These endings are apparently the same as those described by Wunderer (1908) in the fins of elasmobranchs.
The combination of intracellular recording and staining techniques with electron microscopy offers a unique opportunity for correlating physiological and ultrastructural properties of single neurones. The spinal terminations of cutaneous primary afferent fibres have been studied by this method, and examination of the ultrastructural synaptology of their boutons sheds light on how sensory information may be processed at the first synapse in the spinal cord. The combination of intracellular staining techniques with other neuroanatomical tract-tracing methods can reveal some of the neuronal networks of the spinal cord and, particularly, the contributions made by primary afferent fibres to these networks.
The central projections of the electrosensory, mechanosensory, and octaval nerves of the Atlantic stingray were examined by transganglionic transport of horseradish peroxidase. Particular attention was paid to the relation of the projections to cell plates C1 and C2, and to a newly described cell plate, C3.
The electroreceptors in the stingray are found in three groups on the dorsal and ventral sides of the body. The electroreceptors are represented topographically on the ipsilateral dorsal nucleus. Those of the rostral part of the head and pectoral fins are represented on the rostroventral part of the nucleus, and those on the caudal part of the head and the pectoral fin on the dorsocaudal part of the nucleus.
Mechanosensory lateral line afferents terminate within the ipsilateral intermediate and caudal nuclei, and the lateral granular mass of the vestibulocerebellum. Anterior lateral line afferents also project to the magnocellular octaval nucleus. A topographic representation of the mechanosensory lateral line periphery is present on the intermediate and caudal nuclei. Mechanoreceptors on the trunk are represented laterally and those on the head medially. The terminal field of the anterior lateral line afferents on the intermediate nucleus surrounds cell plates C1 and C2. The anterior lateral line afferents also project to the medial part of the lateral granular mass, whereas the posterior lateral line afferents project to the lateral portion. Sparse projections of anterior lateral line afferents to the periventricular octaval nucleus were also observed.
The octaval nerve afferents terminate largely within the octaval column. Octaval nerve projections were also observed to the reticular formation, the periventricular octaval nucleus, the deep cerebellar nucleus, the vestibulocerebellum, particularly the lower lip and medial granular mass, and the intermediate nucleus. A sparse projection to cell plate C3 was found.
The relation between cell plates C1 and C2 and the anterior lateral line afferents suggests that these cell plates are related to processing lateral line information. While the relationship between cell plate C3 and the octaval afferents is not strong, the sparse octaval projection C3 receives suggests that it is relaying octaval information. © 1993 Wiley‐Liss, Inc.
Intracellular recordings were made from neurons of the trigeminal sensory ganglia of young adult sea lampreys. Receptive fields were mapped, and four classes of sensory cells were identified. Touch cells gave rapidly adapting responses to indentation of the skin. Pressure cells gave slowly adapting responses to indentation of the skin. Pit organ cells gave slowly adapting responses to mechanical stimulation of single lateral line pit organs. Nociceptive cells gave slowly adapting responses to destructive stimuli, such as puncture or burning of the skin. The axons of nociceptive cells had longer refractory periods and slower conduction velocities than did the axons of the other types of cell, indicating that nociceptive cells had smaller diameter axons.
We have exploited the segregation of motor and sensory axons into peripheral nerve sub-compartments to examine spinal reflex interactions in anaesthetized stingrays. Single, supra-maximal electrical stimuli delivered to segmental sensory nerves elicited compound action potentials in the motor nerves of the stimulated segment and in rostral and caudal segmental motor nerves. Compound action potentials elicited in segmental motor nerves by single stimuli delivered to sensory nerves were increased severalfold by prior stimulation of adjacent sensory nerves. This facilitation of the segmental reflex produced by intense conditioning stimuli decreased as it was applied to more remote segments, to approximately the same degree in up to seven segments in the rostral and caudal direction. In contrast, an asymmetric response was revealed when test and conditioning stimuli were delivered to different nerves, neither of which was of the same segment as the recorded motor nerve: in this configuration, conditioning volleys generally inhibited the responses of motoneurons to stimuli delivered to more caudally located sensory nerves. This suggests that circuitry subserving trans-segmental interactions between spinal afferents is present in stingrays and that interneuronal connections attenuate the influence that subsequent activity in caudal primary afferents can have on the motor elements.
This review emphasizes the properties of identified neurons and their synapses in the lamprey and the organization of motor systems, including muscle. Previous reviews of lamprey neurobiology have included the following topics: pituitary, pineal, nervous control of circulation, respiration, central nervous system, structure and function of sense organs, and Muller and Mauthner cells. Recent progress in several of these areas is also reviewed here.
1. The sizes and numbers of axons in peripheral nerves and spinal roots were investigated in the stingray, Dasyatis sabina. 2. The axons of the dorsal and ventral roots do not mingle in peripheral nerves of this animal as they do in higher vertebrates. Thus, it was usually possible to split the peripheral nerve into two portions, one containing only dorsal root axons, the other containing only ventral root axons. This feature was useful for the analysis of certain aspects of spinal cord organization. 3. The fact that dorsal and ventral root axons were segregated in peripheral nerves enabled us to demonstrate, without experimental surgery, that the central processes of the dorsal root ganglion cells and the proximal ventral root axons were 10-20% narrower, on the average, than the distal processes of the same dorsal root ganglion cells or the distal parts of the same ventral root axons. 4. The stingray is remarkable in having very few unmyelinated axons in the dorsal roots, ventral roots, or peripheral nerves. This paucity of unmyelinated axons distinguishes the Atlantic stingrays from all other vertebrates whose roots and nerves have been examined for unmyelinated fibers. 5. Similar findings were obtained for one spotted eagle ray (Aetobatus narinari) and two cow-nose rays (Rhinoptera bonasus).
1. Excitability changes in primary afferents and inhibitory interactions in evoked spinal cord activity were investigated in unanesthetized stingrays (Dasyatis subina) with high cervical spinal transections. 2. Primary afferent excitability increases following a conditioning stimulus to an adjacent segmental nerve were demonstrated with the Wall (31) technique. 3. Stimulation of A-alpha,beta and A-delta afferent fibers produced excitability increases in both A-alpha,beta and delta-fibers of the adjacent segment. 4. The excitability increase had a latency of about 10 ms, it peaked around 25 ms, and the change lasted more than 100 ms. 5. The central afferent volley in A-alpha,beta fibers and the N1- and late negative waves due to postsynaptic activity of dorsal horn interneurons were reduced by conditioning volleys in adjacent afferent nerves. The time course of the inhibition paralleled that of the excitability increases in afferent terminal arborizations, suggesting that the depression of postsynaptic activity is, at least in part, due to presynaptic inhibition. 6. Reduction of evoked discharges and excitatory postsynaptic potentials was observed in recordings from interneurons with a time course similar to that of the primary afferent depolarization (PAD). 7. Conditioning volleys in afferents of adjacent peripheral nerves produced facilitation or inhibition of segmental reflexes.
The central effects of selective stimulation of the sensory and motor components of peripheral nerves were investigated in the unanesthetized spinal cords of stingrays (Dasyatis sabina) with a spinal transection at a high cervical level. The large myelinated fibers in the sensory component of the peripheral nerve produce an afferent volley which is readily recorded from the dorsal horn. This is followed by a negative field potential, the N1-wave, which is largest in the deep parts of the dorsal horn. The small myelinated afferent fibers evoke a late negative potential, the LN-wave, which is largest in the dorsalmost part of the dorsal horn. The central volley in the small afferents is difficult to detect, although it may be demonstrated by anodally blocking the large afferents. Interneuronal activity can be evoked by large or small afferents or both. The excitation of interneurons undoubtedly underlies the N-waves. Antidromic field potentials are recorded in the ventral horn from motoneurons when the motor component of a peripheral nerve is stimulated. The antidromic invasion of motoneurons is facilitated by a conditioning volley in afferent fibers. The negative portion of the antidromic field potential shifts dorsolaterally with time, suggesting progressive invasion of the dorsolaterally oriented motoneuronal dendrites. Peripheral nerve stimulation evokes antidromic action potentials, monosynaptic and polysynaptic excitatory postsynaptic potentials, and polysynaptic inhibitory postsynaptic potentials which can be recorded intracellularly from motoneurons of the stingray spinal cord. Reflex discharges are easily detected in motor nerves following stimulation of sensory nerves. Most of the discharge is polysynaptic. Facilitation and posttetanic potentiation can be demonstrated.
An analysis of Nissl stained sections of the spinal cord taken from four species of elasmobranch showed that seven distinct cytoarchitectonic laminae are present. These laminae are compared with laminae described previously in the spinal cord of other vertebrates.
The distribution of immunoreactivity to serotonin, substance P, somatostatin, calcitonin gene-related peptide, neuropeptide Y, and bombesin was determined in the brown stingray (Dasyatis fluviorum), the eagle ray (Aetobatis narinari), the shovelnose ray (Rhinobatis battilum), and the black-tip shark (Carcharhinus melanopterus). In all species, dense immunoreactivity to most substances tested was found in the outer part of the substantia gelatinosa. Many fibres and varicosities immunoreactive to substance P, calcitonin gene-related peptide, and bombesin were found in this region and smaller numbers of fibres were found in the nucleus proprius. Immunoreactivity to somatostatin consisted of coarse fibre bundles that entered the dorsal horn at the nucleus proprius and radiated dorsally to the substantia gelatinosa. Axons and varicosities immunoreactive to serotonin and neuropeptide Y were found in all regions of the dorsal horn but were concentrated in the outer part of the substantia gelatinosa. The distribution of immunoreactivity to met-enkephalin in the shovelnose ray was concentrated in the lateral third of the substantia gelatinosa and to a lesser extent in the nucleus proprius.
The distribution of these substances is compared with that described in other vertebrates. Although the sensory information reaching the elasmobranch spinal cord is limited, compared with that of mammalian species, the distribution of these neuroactive factors in the dorsal horn of the two groups is strikingly similar.
The anterograde and retrograde transport of horseradish peroxidase was utilized to identify the motor nuclei and sensory connections of the IXth and Xth cranial nerves in the clearnose skate, Raja eglanteria. The majority of VIIth, IXth, and Xth nerve motoneurons form an ipsilateral dorsal visceromotor column, extending from the level of the posterior lateral line nerve root to the 3rd-4th ventral spinal roots. Within this column, the motor nucleus of IX (IXm) occurs rostral to vagal motoneurons (dorsal motor nucleus of X, Xmd). Vagal motoneurons are also located ventrolaterally in a ventral motor nucleus (Xmv), which extends from the level of the middle vagal rootlets to the 5th-6th ventral spinal roots. IXth and Xth nerve afferents terminate predominantly in the ipsilateral visceral sensory column (vS). Many vagal but few glossopharyngeal afferents form a solitary tract. A distinct nucleus of the solitary tract could not be identified; rather fibers terminate among cells scattered in the tract, adjacent vS, and nucleus of the commissura infima, where some vagal fibers decussate. Vagal and glossopharyngeal somatic sensory fibers were not found.
Horseradish peroxidase and cobaltous lysine tracers are used to determine the afferent and efferent projections of the vestibulolateral cerebellum (VLL) in the little skate, Raja erinacea. The skate VLL has separate divisions, pars medialis and pars lateralis, associated with vestibular and lateralis modalities, respectively. The pars medialis has a typical cerebellar structure with molecular and Purkinje cell layers and granular areas. In addition to known inputs from eighth nerve vestibular fibers and limited mechanosensory lateralis afferents, pars medialis afferents are from the ventral part of the descending octaval nucleus, the lateral funicular nucleus and nucleus of the medial longitudinal fasciculus. The pars lateralis and rostral anterior octaval nucleus may be additional afferent sources. Pars medialis efferents project to ventral descending and anterior octaval nuclei, as mossy fibers to the cerebellar corpus and as parallel fibers in the ventrolateral extreme of the molecular layer in the medial octavolateralis nucleus. The pars lateralis comprises granule and Golgi cells and is subdivided into a dorsal granular ridge (DGR) and lateral granular area (LG) that are the sources of parallel fibers in the molecular layers of the dorsal (electrosensory) and medial (mechanosensory) octavolateralis nuclei. Local injections of tracer reveal a systematic topography of pars lateralis parallel fiber projections and a mossy fiber projection to the corpus. Both DGR and LG receive direct spinal input but afferent sources to DGR and LG are otherwise distinct. While LG is known to receive mechanosensory lateralis afferents and limited eighth nerve fibers, DGR receives no direct cranial nerve input. Additional afferents to LG are predominantly from contralateral LG and the anterior octaval and lateral funicular nuclei. Additional DGR afferents are from three medullary nuclei beneath the cerebellar peduncle, nuclei F and K and paralemniscal nucleus, which also projects directly to the dorsal nucleus. Distinct inputs to DGR and LG suggest different contributions of VLL to medullary processing in electro- and mechanoreception.
More than 20 peptides have been identified in neurones of the brain, spinal cord and periphery. In several cases a peptide occurs together with a 'classical' transmitter in the same neurone. This raises new questions about the process of neurotransmission and may have important implications for our understanding and treatment of mental disorders.
The distribution and origin of four peptide neurotransmitter candidates of primary afferents (substance P, SP; somatostatin, SS; cholecystokinin, CCK; and vasoactive intestinal polypeptide, VIP) were studied in the stingray with peroxidase-antiperoxidase (PAP) immunohistochemistry. This elasmobranch has virtually no unmyelinated primary afferents, having instead only large and small myelinated afferents. SP-like immunoreactivity was distributed densely in the superficial aspect of the substantia gelatinosa (SG), particularly laterally, and was scattered in the nucleus proprius, the intermediate zone, and the ventral horn. The distributions of SS-, CCK-, and VIP-like immunoreactivities were similar to each other, but different from that of SP. Stained fibers appeared to issue from a prominent tract in the dorsolateral funiculus to form a plexus at the lateral margin of the nucleus proprius. The fibers spread dorsally and medially through the SG to terminate in a thin band at the superficial margin of the SG. Both SS and CCK were more dense in the lateral third of the SG, while VIP was more diffusely distributed within this structure. The remaining regions of the spinal gray matter contained immunoreactive fibers and terminals in variable densities. Many SS-positive cell bodies were observed in the ventral horn, in the deep dorsal horn, and in the ependymal layer. CCK-positive cells were observed in the medial ventral horn, and VIP-positive cells were observed subjacent to the SG and within the dorsolateral funiculus. After unilateral dorsal rhizotomies, SP-like immunoreactivity in the SG was depleted, while SP staining elsewhere and all SS, CCK, and VIP staining was indistinguishable from control. Thus all four peptides are present in the stingray spinal cord, although only SP appears to be a candidate primary afferent transmitter.
Modern views of agnathan phylogeny consider Petromyzoniformes and Myxiniformes to belong to distinct classes that diverged from a common ancestor at a remote period, perhaps in the lower Cambrian, greater than 600 million years ago. Both are more primitive than elasmobranchs, holocephalans and bony fishes. Myelin is well developed in elasmobranchs and other fishes but was reported to be lacking in the spinal cord of lampreys. In order to search further for possible early myelin in some part of the nervous system of one of the agnathan stems, or for further evidence that it first appeared in chondrichthians, we extended the sampling to many parts of the brain and cord of hagfish. Transmission electron microscopy was used as a nearly ideal criterion. We find no trace or forerunner of the spiral, multilaminate glial wrapping. Many axons are embedded within one or more glial cells, like unmyelinated fibers in other vertebrates, or lie contiguously in bundles without an obviously complete glial investment. True myelin must be presumed to have been invented within the vertebrates, in ancestors of the living cartilaginous fishes after the agnathans branched from the vertebrate stem.
The central projections of first‐order lateral line and octavus nerve afferents of the clearnose skate, Raja eglanteria , were, determined by nerve degeneration and horseradish peroxidase techniques. The octavolateralis area of the medulla, which receives these afferents, is organized into dorsal, intermediate, and ventral longitudinal columns of cells and neuropil. Fibers that innervate the electroreceptive sense organs enter the dorsal longitudinal column via the dorsal root of the anterior lateral line nerve and terminate within the dorsal nucleus. Mechanoreceptive fibers from neuromasts of the head and trunk are carried by the ventral root of the anterior lateral line nerve and posterior lateral line nerve, respectively. Both nerves enter the intermediate longitudinal column and terminate throughout the rostrocaudal extent of the intermediate nucleus. Fibers of the ventral root of the anterior lateral line nerve are confined to the medial portion of the intermediate nucleus and posterior lateral line nerve fibers to the lateral portion. In addition, ascending mechanoreceptive fibers from both head and trunk neuromasts project to the vestibulolateral lobe of the cerebellum.
Octavus nerve afferents enter the medulla and terminate primarily within the four octaval nuclei that comprise the ventral longitudinal column. Rostrocaudally, these nuclei are the anterior, magnocellular, descending, and posterior octaval nuclei. A few ascending axons continue beyond the anterior octaval nucleus and course to the vestibulolateral lobe of the cerebellum. Some descending axons emanate from the descending octaval nucleus and course to the reticular formation and intermediate nucleus.
Therefore, electroreceptive lateral line, mechanoreceptive lateral line, and octavus nerve afferents project ipsilaterally and terminate predominantly within separate medullary nuclei. The significance of octavus nerve projections to the intermediate nucleus and overlap of mechanoreceptive and octavus afferents within the vestibulolateral lobe of the cerebellum cannot be determined until it is known which fibers of the inner ear sense organs project to these areas.
Retrograde transport of horseradish peroxidase results in the labeling of large multipolar cells, both ipsilaterally and contralaterally, within a column of gray that lies dorsolateral to the reticular formation. These cells are interpreted as the cells of origin of the efferent components of the anterior and posterior lateral line nerves.
The dorsal octavolateral nucleus is the primary electrosensory nucleus in the elasmobranch medulla. We have studied the topographic organization of electrosensory afferent projections within the dorsal nucleus of the little skate, Raja erinacea , by anatomical (HRP) and physiological experiments.
The electrosensory organs (ampullae of Lorenzini) in skates are located in four groups on each side of the body, and each group is innervated by a separate ramus of the anterior lateral line nerve (ALLN). Transganglionic transport of HRP in individual rami demonstrated that electroreceptor afferents in each ramus project to a separate, nonoverlapping division of the central zone of the ipsilateral dorsal nucleus. These divisions, which are distinct areas separated by compact cell plates, are somatopically arranged. The volume of each division of the dorsal nucleus that is related to a single ramus is proportional to the number of ampullae innervated by the ramus, but not to the body surface area on which the receptors are distributed. Nearly one‐half of the nucleus is devoted to electrosensory inputs from the buccal and superficial ophthalmic ampullae concentrated in a small area on the ventral surface of the head rostral to the mouth. Multiple and single unit recordings demonstrated that adjacent cells in the nucleus have similar receptive fields on the body surface and revealed a detailed point‐to‐point somatotopy within the nucleus. With threshold stimuli most single units have ipsilateral receptive fields made up by excitatory inputs from 2–5 ampullary organs.
The somatotopy within the mechanosensory medial nucleus, also revealed by the HRP fills of individual ALLN rami, appears less rigid than that in the dorsal nucleus, as extensive overlap is present in the terminal fields of separate ALLN rami.
In order to assess the ability of sharks and rays to sense pain, the proportion of myelinated versus unmyelinated sensory fibres in the dorsal roots and the diameter spectrum of cells in the dorsal root ganglia of three species of elasmobranch fish were ascertained. Electron micrographs were used to count the numbers of myelinated and unmyelinated fibres in montages of whole dorsal roots of the long-tailed stingray (Himantura sp.), the shovelnose ray (Rhinobatus battilum), and small specimens of the black-tip shark (Carcharhinus melanopterus). The diameters of dorsal root ganglion cells in each species were measured by using the light microscope. Less than 1% of the dorsal root axons in the long-tailed stringray and a large specimen of the shovelnose were unmyelinated, whereas in smaller shovelnose rays and in the small black-tipped sharks, from 14% to 38% of axons were unmyelinated. Unmyelinated fibres differed from those in mammalian nerves in that there was a one-to-one association of the fibre with a Schwann cell. We conclude from these observations that myelination was incomplete in the black-tipped sharks and the smaller specimens of the shovelnose rays. The distribution of the diameter of cells of the dorsal root ganglia of these species was unimodal, resembling the diameter range that has been reported for the somata of myelinated fibres in the cat. We interpret these results as indications that sharks and rays lack the neural apparatus essential for the sensation of pain and we suggest that, to these life forms, the perception of pain might have little relevance to survival.
Enkephalin-like immunoreactivity (ENK-LI) was found throughout the spinal cord of the long-tailed ray Himantura fai. The densest ENK-LI was in the superficial portion of lamina A of the dorsal horn. Lamina B and the deeper parts of lamina A contained radially oriented, labelled fibres. Laminae C, D, and E contained many longitudinally oriented fascicles which were surrounded by a reticulum of transversely oriented, labelled fibres, some of which projected into the ventral and lateral funiculi. Labelled fibres were found in the dorsal commissure and around the central canal, but the later did not cross the midline. One-third of all enkephalinergic cells were found throughout laminae A and B, while two-thirds were located in the medial half of C, D, and E. Occasionally a labelled cell was located in the lateral funiculus. The ventral horn (laminae F and G) contained many enkephalinergic fibres but no labelled nuclei. A few dorsal column axons contained ENK-LI. In the lateral funiculus there were two groups of labelled axons, a superficial, dorsolateral group, and a deeper group, occupying a crescent-shaped region. The ventral funiculus also contained many labelled axons. The central projection of the dorsal root passed through the substantia gelatinosa and divided into rostrally and caudally projecting fascicles within lamina C. The root, and these fascicles, both lacked ENK-LI. In contrast, the fascicles in laminae D and E did contain enkephalinergic fibres. The origin of the various fibre systems and the role of enkephalin in the regulation of sensory processing and motor output are discussed.
Recorded action potentials in whole spinal nerves during mechanical stimulation of the skin of Himantura fai revealed that sequential nerves innervated sequential overlapping strips of the pectoral and pelvic fin skin. As found in previous studies, in which the dermatomes of the skate Raja clavata were measured behaviourally, approximately one-third of the rostral and caudal regions of each dermatome overlapped with the adjacent dermatomes. Consistent with dermatomal maps from non-mammalian vertebrates, but unlike the dermatomal maps obtained in mammals, there appears to be little difference in dermatome size when measured behaviourally or electrophysiologically. We suggest that neural mechanisms of the spinal cord, which appear to contribute to the discrepancy between behaviourally and electrophysiologically mapped dermatomes in mammals, are of negligible influence in stingrays.
Reorganization of the somatotopic map in the spinal dorsal horn may be elicited by a variety of deafferenting lesions, including transection of peripheral nerves or dorsal roots, or the application of neurotoxins. While such lesions give rise to a variety of neurochemical and morphological changes in the dorsal horn, collateral sprouting of intact primary afferents appears to be minimal. Recently, intraaxonal injection of neurobiotin has allowed visualization of the entire spinal arborization of single A beta primary afferent fibers in animals where the somatotopy of the relevant region of dorsal horn has also been mapped. In contrast to the somatotopic precision of the terminal fields of peripheral nerves suggested by transganglionic tracing, these studies have shown that afferents make connections many millimeters rostral and caudal to the region where their receptive field is represented in the somatotopic map. Intracellular recording from dorsal horn neurons has further shown that these long-ranging projections make functional, but weak, synaptic connections. Thus the functional somatotopic reorganization that follows nerve lesions in mature animals might be explained simply by an increased synaptic efficacy of these existing projections. In contrast to the negligible sprouting of intact A beta primary afferents, those undergoing axonal regeneration exhibit dense collateral sprouting into deafferented regions of the dorsal horn, particularly the superficial laminae, where the terminal arbors of many small (A delta and C) nociceptive afferent fibres degenerate following peripheral nerve lesions. The inappropriate connections made by these collateral sprouts may partly underlie the painful sequelae of nerve injury in man.
The present study documents that in the stingray Dasyatis sabina the numbers of (1) dorsal root ganglion cells, (2) dorsal root axons, (3) ventral root axons and (4) “motor” cells in the ventral horn increase steadily as the animals increase in size. This increase in axonal and neuronal numbers persists much further into adult life than is the case for other vertebrates that have been studied from this point of view. We hypothesize this steady increase in axonal numbers may be related to the ability of fish to regenerate parts of their central and/or peripheral nervous systems.
The neuronal organization of the spinal cord in red stingray was studied using the rapid Golgi method. The gray matter of the spinal cord was divided into seven laminae: RS-I, RS-II, RS-III, RS-IV, RS-V, RS-VI and RS-VII. RS-I is cell dense lamina which occupies the major part of the dorsal horn and corresponds to laminae I and II of the spinal cord of mammals, birds and reptiles. The neurons of the lamina I are interspersed with those of lamina II, without forming a discrete lamina. RS-II is located at the base of the dorsal horn and is considered to correspond to the nucleus proprius. RS-III and IV form the intermediate zone and are highly reticulated. A few neurons of various shapes and sizes are distributed among the numerous fibers. The nuclei such as the intermediolateral, intermediomedial or Clarke's nucleus cannot be identified in the intermediate zone. RS-V and VI constitute the ventral horn. RS-V occupies the major part of the ventral horn and contains motoneurons which are distributed diffusely, without forming any distinct cell groups. RS-VI is located in the ventromedial part of the ventral horn, contains commissural neurons and correspond to lamina VIII. RS-VII is a small area surrounding the central canal and corresponds to lamina X. Thus, while the major features of the spinal cord of the red stingray can be correlated with those of the spinal cord of mammals, birds and reptiles, the neuronal organization of the spinal cord of the red stingray remains in an undifferentiated state.
Trigeminal somatosensory receptors have not been characterised in teleost fish and studies in elasmobranchs have failed to identify nociceptors. The present study examined the trigeminal nerve of a teleost fish, the rainbow trout (Oncorhynchus mykiss) to determine what types of somatosensory receptors were present on the head of the trout specifically searching for nociceptors. Single unit recordings were made from receptive fields on the head of the fish innervated by the trigeminal nerve. Each receptive field was tested for sensitivity to mechanical, thermal and chemical stimulation. Five different receptor types were found: fast adapting receptors responding to mechanical stimulation; slowly adapting receptors responding to mechanical stimuli; polymodal nociceptors responding to mechanical, noxious thermal and chemical stimulation; mechanothermal nociceptors responding to mechanical stimulation and noxious heat; and mechanochemical receptors responsive to mechanical and chemical stimulation. Mechanical thresholds, receptive field diameter, conduction velocities and thermal thresholds of the receptors were determined and there was no significant difference between the receptor types in terms of these properties. Three shapes of action potential (AP) were recorded from these receptors: type 1 with no inflexion; type 2 with an inflexion on depolarisation; and type 3 with an inflexion on repolarisation. Conduction velocity, amplitude and duration of the APs, afterhypolarisation amplitude and duration, as well as the maximum rate of depolarisation were measured for each action potential type. No major differences were found when making comparisons within receptor type and between receptor types. The fish nociceptors had similar physiological properties to nociceptors found in higher vertebrates.
This chapter reviews the comparative anatomy of the spinal cord with particular attention to the lower forms. The spinal cord is generally considered as the lowest level and the most simply organized part of the central nervous system. Of all the parts of the central nervous system, the spinal cord or medulla spinalis preserves the early embryonic tube-like shape most clearly, and although secondary form changes occur in some groups, it may be said that this organ is generally of a cylindric appearance. The length of the spinal cord varies considerably. During the course of evolution, a relatively simply spinal nervous mechanism has been gradually overshadowed and superseded by a more complex secondary system. The gray matter of the spinal cord shows a progressive elaboration and an increasing segregation of separate cell masses in the series of vertebrates, but the primitive configuration: a central core of gray surrounded by an outer zone of white matter is principally maintained throughout the subphylum. The chapter gives some general remarks on the embryonic cord.
This study examined stimulus-response properties of somatosensory receptors on the head of rainbow trout, Oncorhynchus mykiss, using extracellular recording from single cells in the trigeminal ganglion. Of 121 receptors recorded from 39 fish, 17 were polymodal nociceptors, 22 were mechanothermal nociceptors, 18 were mechanochemical receptors, 33 were fast adapting mechanical receptors and 31 were slowly adapting mechanical receptors. Mechanical thresholds were higher in polymodal nociceptors than in either slowly adapting or fast adapting mechanical receptors, whereas thermal thresholds of mechanothermal nociceptors were higher than those of polymodal nociceptors. Polymodal nociceptors and mechanochemical receptors gave similar responses to topical applications of acid. All receptor types except mechanothermal nociceptors showed an increase in peak firing frequency with increased strength of mechanical stimulation, with evidence of response saturation at higher intensities. Mechanothermal, but not polymodal, nociceptors showed an increase in firing response to increased temperature. None out of 120 receptors tested gave any response to the temperature range +4 degrees C to -7 degrees C, indicating an absence of cold nociceptors. Attempts to evoke sensitization of receptors using chemical or heat stimuli were unsuccessful, with receptors showing either a return to control responses or irreversible damage. Comparisons are made between somatosensory receptors characterized here in a fish and those of higher vertebrates.