Light and electron microscopy of contacts between primary afferent fibres and neurones with axons ascending the dorsal columns of the feline spinal cord

In addition to primary afferent fibres, the dorsal columns of the cat spinal cord contain ascending second-order axons which project to the dorsal column nuclei. The aim of the present study was to obtain morphological evidence that certain primary afferent axons form monosynaptic contacts with cells of origin of this postsynaptic dorsal column pathway. In ten adult cats, neurones with axons ascending the dorsal columns were retrogradely labelled with horseradish peroxidase using a pellet implantation method in the thoracic dorsal columns. In the lumbosacral regions of the same animals, primary afferent fibres were labelled intra-axonally with ionophoretic application of horseradish peroxidase. Tissue containing labelled axons was prepared for light and combined light and electron microscopy. Ultrastructural examination demonstrated that slowly adapting (Type I), hair follicle, Pacinian corpuscle and group Ia muscle spindle afferents formed monosynaptic contacts with labelled cells and light microscopical analysis suggested that they also received monosynaptic input from rapidly adapting (Krause) afferents. This evidence suggests that sensory information from large-diameter cutaneous and muscle spindle afferent fibres is conveyed disynaptically via the postsynaptic dorsal column pathway to the dorsal column nuclei. Some of the input to this pathway is probably modified in the spinal cord as the majority of primary afferent boutons forming monosynaptic contacts were postsynaptic to other axon terminals. The postsynaptic dorsal column system appears to constitute a major somatosensory pathway in the cat.     

Intracellular staining study of the feline cuneate nucleus. I. Terminal patterns of primary afferent fibers

The terminal arborizations of single identified cutaneous hair follicle and slowly adapting type I receptors and muscle (Ia) afferents have been studied in the cuneate nucleus of cats after intra-axonal injection of horseradish peroxidase. Penetrations were mainly at the middle and caudal levels of the nucleus--i.e., from obex to approximately 7 mm caudal to it. Following histochemical processing, the injected axons, along with their collateral branches and synaptic terminals, were visualized and examined with light and electron microscopy. Cutaneous afferents in middle cuneate (from obex to approximately 4 mm caudal to it) issued collateral branches, along the rostrocaudal axis of the nucleus, at intervals between 100 and 1,000 microns. The terminal field of each collateral's branches encompassed an area elongated largely rostrocaudally and virtually confined to the dorsal part of the middle cuneate. Although adjacent collaterals had nonoverlapping terminal arborizations, each one could give rise to separate foci of terminations. Muscle afferents differed, on the whole, from cutaneous afferents in the location and extent of collateral branching and terminal arborizations. However, because muscle fibers terminated primarily in the ventral region of the cuneate, but nevertheless exhibited sparser terminations in the dorsal part of the middle cuneate, there was some spatial overlap between zones of muscle and cutaneous projection. Synaptic boutons of cutaneous afferent fibers contained round clear vesicles, contacted dendritic profiles (sometimes more than one), and were postsynaptic to small boutons containing polymorphic vesicles. In contrast, boutons of muscle afferent fibers contacted somatic and dendritic profiles and were not postsynaptic to other boutons. The results are in general agreement with previous anatomical and electrophysiological work; however, the extent of the terminal field of single collateral branches may provide for a greater convergence of different receptor classes and of receptive fields on neurons in the middle cuneate than estimated by previous electrophysiological investigations.     

The morphology of hair follicle afferent fibre collaterals in the spinal cord of the cat

1. The enzyme horseradish peroxidase (HRP) was injected into single axons that innervated hair follicle receptors to study the morphology of their collaterals in the dorsal horn of the cord. The axons were impaled near the dorsal root entrance zone in the lumbosacral spinal cord of anaesthetized cats and HRP injected by passing current through the intra-axonal micro-electrode. The morphology was revealed by subsequent histochemistry.2. Thirteen hair-follicle afferent fibres were stained including six that innervated tylotrichs (type T hair follicle afferent units) and one that innervated guard hairs (type G unit). The remaining six axons were not classified according to hair type, but, on the basis of their axonal conduction velocities, would have been either type G or T.3. Eleven axons could be traced back into the dorsal roots. Eight of these, upon entering the cord, turned and ran towards the brain. They did not divide into rostral and caudal branches. Three of the eleven did divide and gave rise to both rostral and caudal branches.4. Sixty-three collaterals were given off the thirteen stained axons. All well-filled collaterals had a strikingly similar morphology. They descended through laminae I-III of the dorsal horn into the deeper parts of lamina IV or into lamina V, before turning and ascending back into superficial lamina IV and lamina III where they branched profusely to give rise to their terminal arborizations. Terminal boutons, most commonly of the ;en passant' type, were numerous in lamina III, but were also seen in the dorsal part of lamina IV and in ventral lamina II. None were observed in dorsal lamina II or near the junction of the grey and white matter (lamina I) or in lamina V.5. The terminal arborizations of collaterals from a single hair follicle afferent fibre were in line with one another in the longitudinal axis of the cord. In the better-stained preparations the terminal arborizations of adjacent collaterals from a single axon formed a continuous longitudinal column through the dorsal horn. There was a gradual shift of the column of arborizations from lateral to medial as the more rostral collaterals were given off.6. The hair-follicle afferent fibre collaterals are now identified as the ;collaterales grosses et profondes de la substance de Rolando' of Ramón y Cajal (1909) which give rise to the ;flame-shaped arbors' of Scheibel & Scheibel (1968).7. The importance of the longitudinal organization of the terminal arborizations for an understanding of the topographical properties of dorsal horn neurones is discussed.     

The relationship of dorsal root afferents to motoneuron somata and dendrites in the adult bullfrog: a light and electron microscopic study using horseradish peroxidase

The relationship of lumbar dorsal root afferents to lateral motor column motoneurons was studied using anterograde injury filling of dorsal roots and retrograde injury filling of ventral roots with horseradish peroxidase. At the light microscopic level, horseradish peroxidase labelled dorsal root axons were observed to separate into a medial division of large diameter axons which enter the dorsal funiculus and a lateral division of small diameter axons which form a compact bundle in the dorsolateral funiculus which may be homologous to the mammalian tract of Lissauer. Within the spinal gray, primary afferents terminate in two distinct regions. The more ventral of these terminal fields, which receives collaterals of primary afferent axons in the dorsal funiculus, overlaps the dendritic arborizations of the lateral motor column motoneurons. Some axons leave the ventral terminal field to enter the dorsal lateral motor column. Here they terminate on the primary dendrites and somata of lateral motor column motoneurons. At the electron microscopic level, labelled primary afferent terminals were seen to synapse upon lateral motor column motoneuron dendrites as well as upon the somata of dorsally positioned lateral motor column motoneurons. These terminals contain small spherical vesicles and occasional dense-cored vesicles. The synaptic specializations are characterized by a small amount of postsynaptic material. The lateral motor column may be divided into dorsal and ventral portions on the basis of the primary afferent distribution and this is in accord with functional, physiological and developmental data.     

Development of glomerular synaptic complexes and immunohistochemical differentiation in the superficial dorsal horn of the embryonic primate spinal cord

 Development of glomerular synapses in the superficial dorsal horn has been studied in the embryonic macaque spinal cord using light and electron microscopic techniques including Golgi impregnation, ³H-thymidine radioautography and pre-embedding immunohistochemistry of substance P (SP), calcitonin gene related peptide (CGRP), calbindin D-28 K (CB) and parvalbumin (PV). The study revealed that substantia gelatinosa cells of the primate dorsal horn are generated last, but unlike in rodents, synaptogenesis in this region starts at early embryonic (E) stages of the 165-day long gestation. Already by E30, both Type 1 (light) and 2 (dark) dorsal root axons and their growth cones are identifiable within the oval bundle of His, before they form synaptic contact with their final target cells. Subsequently they invade the dorsal horn and enter the bisecting interfaces formed by orderly programmed cell death. Each type of scalloped (sinusoid) central primary afferent terminal (i.e. DSA, RSV and LDCV) have well defined pre- and post-synaptic specializations already by E40. Among the neuropeptides studied, SP appears first at E67 and CGRP at E70 in the lateral position but within a few days both of them are spread to the entire superficial dorsal horn. Both SP and CGRP are present in the thin dorsal root axons and their growth cones, giving rise to scalloped and simple axon terminals. PV is transiently present in the entire length of the thick dorsal root afferents before becoming concentrated in the synaptic boutons. CB is displayed mainly in neurons of the lamina I and III. Dendrites of CB-immunoreactive cells establish synaptic connection with each type of dorsal root afferents, including glomerular synaptic complexes. These data reveal that the superficial dorsal horn in the primate spinal cord develops its characteristic synaptic complexes much earlier in gestation than in any other mammalian species studied. Furthermore, characteristic cytological features of the prospective glomerular complex emerge before establishment of the final synaptic contacts. Accepted: 20 July 1998     

Structure-function relationships in rat medullary and cervical dorsal horns. I. Trigeminal primary afferents

Intracellular recording and horseradish peroxidase (HRP) labeling were used to examine structure-function relationships in the medullary dorsal horn (MDH) and rostral cervical dorsal horn. In Nembutal-anesthetized rats, 78 trigeminal (V) primary afferent fibers were physiologically characterized and injected with HRP. Axons were sufficiently well stained to reconstruct all of their collaterals in the MDH. Many also extended into the cervical dorsal horn. Except for four axons, which responded best to noxious stimuli, all responded at short (mean = 0.50 ms) latencies to V ganglion shocks and to innocuous stimulation. Forty-five of our recovered fibers were associated with facial vibrissae and responded in either a rapidly adapting, slowly adapting type I, slowly adapting type IIa, or slowly adapting type IIb fashion. The adequate stimuli consisted of either slow deflection, high-velocity deflection, or a noxious pinch of the vibrissa follicle. The collaterals of all of the above-described mystacial vibrissa primary afferents proceeded directly to their region of arborization in a plane perpendicular to the lateral border of the medulla to collectively form a largely continuous, circumscribed terminal column. This longitudinally oriented column of terminal and en passant boutons angled from lamina V rostrally to lamina III caudally. In the magnocellular laminae of the MDH, all mystacial vibrissa primary afferents gave rise to similarly shaped arbors, regardless of their functional classification. While morphological variability was observed both within and between individual axons, variance between functional classes was no greater than that within a class. Moreover, number of collaterals, number of boutons, or bouton size did not distinguish functional classes. Nonmystacial vibrissa afferent arbors, with more caudal peripheral fields, had their primary arbor focus in C1 and C2 dorsal horn. These arbors had relatively little rostrocaudal overlap with mystacial vibrissa afferents, though they exhibited the same lamina V-to-III shift as they descended through the cervical cord. Unlike mystacial vibrissa afferents in the MDH, their collaterals followed a tortuous course and often occupied laminae II-V in one transverse section. The relative location of each vibrissa afferent's terminal field could be predicted by the particular vibrissa innervated. Dorsal vibrissae afferents had ventrolateral terminations and ventral vibrissae afferents terminated dorsomedially. Rostral vibrissae were represented in the rostral MDH, whereas caudal vibrissae were represented in the caudal MDH and rostral cervical dorsal horn.(ABSTRACT TRUNCATED AT 400 WORDS)     

Opioid neurons and pain modulation: an ultrastructural analysis of enkephalin in cat superficial dorsal horn

To clarify the circuitry through which opioid compounds modulate spinal and trigeminal nociceptive transmission, we have examined the synaptic associations formed by leucine-enkephalin-containing (enkephalin) neurons in the superficial dorsal horn of the cat. As described previously, punctate enkephalin immunoreactivity is concentrated in the marginal layer (lamina I) and in both the outer and inner layers of the substantia gelatinosa (lamina IIo and IIi). In colchicine treated cats, enkephalin perikarya are most numerous in lamina I and at the border between laminae I and II. Ultrastructural analysis reveals that enkephalin cells receive a diverse afferent input. The majority of afferent inputs are presynaptic to the enkephalin dendrites; few axosomatic synapses are seen. Among these presynaptic axonal profiles are unlabeled axons which resemble primary afferent terminals, including the characteristic central axonal varicosity. Enkephalin dendrites are also postsynaptic to enkephalin immunoreactive axons. Two types of enkephalin axonal profiles appear in the superficial dorsal horn. Class I profiles are only found in lamina I. These are large profiles which form few synapses; those synapses made are axodendritic. Class II enkephalin axons are smaller and are distributed in both layers I and II. While Class II axons most commonly form axo-dendritic synapses, they also form axo-axonic synapses with flat vesicle-containing profiles; the latter are generally presynaptic to the enkephalin terminals. Serial analysis further revealed that both the enkephalin and the flat vesicle-containing profile synapse onto a common dendrite. Although enkephalin axons frequently lie adjacent to round vesicle-containing profiles, anatomical evidence that opioid axons form synapses with this type of ending was not found. An additional type of enkephalin vesicle containing-profile is found in layer IIi; its morphological features do not clearly distinguish its axonal or dendritic origin. These endings are typically postsynaptic to unlabelled central endings, and provide minimal presynaptic input to other elements in the neuropil. Like some class II axons, these labelled profiles contain vesicles which cluster at the membrane immediately adjacent to unlabelled central axons. These results indicate that spinal enkephalin neurons receive a variety of synaptic inputs. These include inputs which may derive from primary afferent axons. Enkephalin neurons, in turn, influence nociceptive transmission predominantly through postsynaptic mechanisms. Finally, while we did not observe enkephalin terminals presynaptic in an axoaxonic relationship, the possibility that enkephalin neurons modulate the excitability of fine fiber nociceptive and nonnociceptive afferents via "nonsynaptic interactions" is discussed.     

The large synaptic complexes of the substantia gelatinosa

Summary A Golgi and electron microscope study, using also secondary degeneration after dorsal root transection and chronically isolated dorsal horn preparations, were undertaken with the objective to clarify the large (glomerulus-like) synaptic complexes in lamina II of the dorsal horn. The large sinusoid axon terminals forming the centers of these synaptic complexes are of intraspinal origin and are thought to arise from the hitherto unknown type of pyramid-shape nerve cells, situated at the border between laminae III and IV. The sinusoid axon terminals establish axo-dendritic synapses with substantia gelatinosa neurons and abundant axo-axonic synapses with smaller terminals that could be identified (at least partly) as endings of primary sensory afferents. The central sinuous axon terminals of the synaptic complexes are always presynaptic to the smaller axons and thus might be considered as a device for 1. presynaptic inhibition of impulse transmission from primary afferents to substantia gelatinosa neurons, and 2. as the anatomical basis for primary afferent depolarization.This study has been carried out and the paper has been prepared largely before the recent publications of the paper by Scheibel and Scheibel (1968), which accounts for some overlap between the Golgi informations presented.     

A quantitative study of cutaneous receptors and afferent fibres in the cat and rabbit

1. The discharge in myelinated afferent fibres innervating hairs in anaesthetized cats and rabbits, dissected from the saphenous nerve, was recorded during controlled movements of the hairs.

2. Three types of rapidly adapting afferent unit were found and they innervated three kinds of hair follicle—down hair, guard hair and tylotrich.

3. The down hair units had low thresholds (critical slopes) and some of the guard hairs had the highest thresholds and least sensitivity to displacement.

4. There was a good fit to a power function for the relation between velocity of displacement of a hair and the frequency of discharge in the corresponding afferent fibre.

5. It is concluded that the rapidly adapting hair follicle receptors can function as efficient exact movement detectors.

6. Tylotrich follicles were often associated with touch corpuscles, but there was independent innervation of the rapidly adapting tylotrich follicle receptors and the slowly adapting touch corpuscle receptors.

7. The conduction velocities of large populations of myelinated cutaneous axons innervating cutaneous mechanoreceptors were measured in cats and rabbits.

     

Anatomy of primary afferents and projection neurones in the rat spinal dorsal horn with particular emphasis on substance P and the neurokinin 1 receptor

The dorsal horn of the spinal cord plays an important role in transmitting information from nociceptive primary afferent neurones to the brain; however, our knowledge of its neuronal and synaptic organisation is still limited. Nociceptive afferents terminate mainly in laminae I and II and some of these contain substance P. Many projection neurones are located in lamina I and these send axons to various parts of the brain, including the caudal ventrolateral medulla (CVLM), parabrachial area, periaqueductal grey matter and thalamus. The neurokinin 1 (NK1) receptor on which substance P acts is expressed by certain neurones in the dorsal horn, including approximately 80 % of lamina I projection neurones. There is also a population of large NK1 receptor-immunoreactive neurones with cell bodies in laminae III and IV which project to the CVLM and parabrachial area. It has been shown that the lamina III/IV NK1 receptor-immunoreactive projection neurones are densely and selectively innervated by substance P-containing primary afferent neurones, and there is evidence that these afferents also target lamina I projection neurones with the receptor. Both types of neurone are innervated by descending serotoninergic axons from the medullary raphe nuclei. The lamina III/IV neurones also receive numerous synapses from axons of local inhibitory interneurones which contain GABA and neuropeptide Y, and again this input shows some specificity since post-synaptic dorsal column neurones which also have cell bodies in laminae III and IV receive few contacts from neuropeptide Y-containing axons. These observations indicate that there are specific patterns of synaptic connectivity within the spinal dorsal horn.     

Identification of dorsal root synaptic terminals on monkey ventral horn cells by electron microscopic autoradiography

Summary The projection of dorsal root fibres to the motor nucleus of the macaque monkey spinal cord has been examined utilizing light and electron microscopic autoradiography. Light microscopy demonstrates a very sparse labelling of primary afferent fibres in the ventral horn. Silver grains overlying radioactive sources are frequently clustered into small groups, often adjacent to dendritic profiles. Under the electron microscope, myelinated axons and a few large synaptic profiles containing rounded synaptic vesicles were overlain by numerous silver grains. These labelled profiles made synaptic contact with dendrites 1–3 m in diameter. The labelled profiles did not contact cell bodies or large proximal dendrites of ventral horn neurons. Frequently, small synaptic profiles containing flattened vesicles were presynaptic to the large labelled terminals and it is suggested that these axoaxonal synapses may mediate presynaptic inhibition of the primary afferent fibres. The relationship of the present findings to previously published physiological and anatomical studies is discussed.     

Prolonged GABAA-mediated inhibition following single hair afferent input to single spinal dorsal horn neurones in cats.

To study the central processing mechanisms of sensory input from low threshold afferents to the spinal cord, we examined the excitatory response of single lumbar dorsal horn neurones to stimulation of hairs in the receptive field using a mechanically driven probe, and to activation of single hair follicle afferents using an intracellular current pulse to the cell bodies in the dorsal root ganglion. Experiments were done on anaesthetized, paralysed cats, spinalized at the L1 lumbar level. Responses of spinal neurones to two types of hair afferent input were characteristically different. The excitatory response to input from a single group II hair afferent (A beta; innervating guard hair follicle receptors) was multimodal, characterized by a small early depolarization followed by a sharp, large component with a slow, prolonged decay phase, whereas the response to input from a single group III hair afferent (A delta; innervating down hair follicle receptors) was unimodal. The unitary EPSPs in response to activation of group III hair afferents had a slower rise time and longer decay time constant than those in response to activation of group II hair afferents. When the receptive field of the afferent was located in the centre of the receptive field of the dorsal horn neurone, the gain of the central response was greater for the input from a single group II afferent (> 1) than that for the input from a single group III afferent (< 1). In the case of single group II hair afferents, when pairs of single action potentials or pairs of trains of action potentials were generated at intervals of 20 ms to 3 s, the response in the dorsal horn neurone to the second volley was markedly depressed at intervals of less than 2 s, without any apparent inhibition of the on-going rate of firing. The response to the second volley in single group III afferents was less depressed. This inhibition of the response to the second of a paired volley in single group II hair afferents was attenuated by administration of bicuculline, but not strychnine or naloxone. This indicates that the inhibition involves a GABAA-receptor-mediated mechanism. Bicuculline did not affect the late component of the response to single group II hair afferent input, but unmasked a late component of the response to mechanical stimulation of hairs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of cutaneous and proprioceptive afferent projections in the chick spinal cord.

Muscle and cutaneous nerves were individually labeled with DiI in chick embryos to examine the development of sensory afferent arborizations in the spinal cord. Initially, cutaneous and muscle arbors were similar; both types first entered the spinal gray matter at stage 28-29 (embryonic day (E) 6). Differences in projections were first observed by late stage 34 (E8.5): muscle afferent collaterals extended almost unbranched to the level of motoneuronal dendrites while cutaneous afferents branched frequently and remained within the dorsal horn. Projections of putative small caliber axons into laminae 1 and 2, located laterally in the chick, did not develop until E13-14.     

内脏大神经初级传入神经元向中枢投射的光镜和电镜研究——CT-HRP跨神经节运送法

用猫20只,于内脏大神经注射CT—HRP,对初级传入在中枢内的投射进行了光、电镜观察。光镜下,内脏初级传入神经元的中枢投射纤维主要经Lissauer's束到脊髓后角边缘,部分可能终止于Ⅰ层,大部分分为内、外侧束包绕灰质后角边缘由浅部板层向深部板层进入Ⅴ、Ⅶ、Ⅹ层。内侧束可能有部分上升到薄束核。电镜观察,在Ⅰ、Ⅴ、Ⅶ、Ⅹ层看到标记的轴突末梢且可直接与交感节前神经元形成突触。本文还对内脏传入通路进行了讨论并认为交感传入纤维可能经脊髓后角神经元中继上传至孤束核。     

KCNQ4 K(+) channels tune mechanoreceptors for normal touch sensation in mouse and man

Mutations inactivating the potassium channel KCNQ4 (K(v)7.4) lead to deafness in humans and mice. In addition to its expression in mechanosensitive hair cells of the inner ear, KCNQ4 is found in the auditory pathway and in trigeminal nuclei that convey somatosensory information. We have now detected KCNQ4 in the peripheral nerve endings of cutaneous rapidly adapting hair follicle and Meissner corpuscle mechanoreceptors from mice and humans. Electrophysiological recordings from single afferents from Kcnq4(-/-) mice and mice carrying a KCNQ4 mutation found in DFNA2-type monogenic dominant human hearing loss showed elevated mechanosensitivity and altered frequency response of rapidly adapting, but not of slowly adapting nor of D-hair, mechanoreceptor neurons. Human subjects from independent DFNA2 pedigrees outperformed age-matched control subjects when tested for vibrotactile acuity at low frequencies. This work describes a gene mutation that modulates touch sensitivity in mice and humans and establishes KCNQ4 as a specific molecular marker for rapidly adapting Meissner and a subset of hair follicle afferents.     

Synaptic connexions between primary afferents and thoracic motoneurones in the frog

Summary Synaptic actions were evoked in thoracic motoneurones of the isolated frog spinal cord by 6th or 7th dorsal root volleys or impulses in single primary afferents. In a few cases the impaled motoneurones or the whole dorsal root were filled with horseradish peroxidase. Although HRP labelling has revealed that motoneurones have extensive dendritic arborizations extending into the dorsal horn, the zone in which thoracic dorsal root afferents terminate, there is no positive evidence for a direct link between them. Synaptic actions produced in thoracic motoneurones by dorsal root stimulation are mediated via interneurones and do not involve direct sensorimotor synapses. This conclusion is in agreement with results of simultaneous intracellular recording from motoneurones and individual dorsal root fibres afferent to them.Dr. A.I. Shapovalov deceased on September 28, 1983     

Cholecystokinin-like immunoreactivity in the dorsal horn of the spinal cord of the rat: a light and electron microscopic study

Cholecystokinin-like immunoreactivity was investigated with an indirect immunoperoxidase technique in the whole spinal cord with the light microscope and in the dorsal horn with the electron microscope. Intraparenchymal injections of colchicine were performed to allow the detection of cholecystokinin-like immunoreactive cell bodies. Rats treated at birth with capsaicin were also studied at the light microscope. Numerous cholecystokinin-like immunoreactive fibres and varicosities were found in the two superficial layers of the dorsal horn and in the intermedio-medial nucleus; cholecystokinin-like immunoreactive cell bodies were also present in these two regions. After neonatal capsaicin treatment, the number of cholecystokinin-like immunoreactive fibres and varicosities was strongly reduced in the dorsal horn. At the electron microscope level, cholecystokinin-like immunoreactivity was localized in numerous neurites often filled with vesicles (axon terminals and dendrites containing vesicles) and in few cell bodies and dendrites. The immunoreaction was found mainly associated with ribosomes, granular reticulum, neurotubules and vesicles. Large granular vesicles were filled with the reaction product whereas small and medium-sized vesicles showed a varying degree of immunoprecipitate around their membrane. In addition dense "granules" of precipitate were observed in numerous presynaptic neurites. Cholecystokinin-like immunoreactive axons were of small calibre and mostly unmyelinated. Cholecystokinin-like immunoreactive axon terminals made asymmetric synaptic contacts with generally unlabelled dendrites or dendritic spines. A single labelled nerve terminal could contact several different dendrites in structures resembling glomeruli. Few axo-somatic synapses but a relatively high number of axo-axonic contacts were seen. About half of these axo-axonic contacts involved pre- and postsynaptic profiles. Both light and electron microscopic observations led us to the conclusion that some of the cholecystokinin-like immunoreactive fibres of the dorsal horn originate in the spinal ganglia via capsaicin-sensitive C afferents; and some from intrinsic neurons, particularly islet cells. Other fibres may come from supraspinal centres, other local neurons or capsaicin-insensitive afferents from the spinal ganglia. The results are discussed with regard to data in the literature, particularly those concerned with the specificity of the cholecystokinin antibodies; it is hypothesized that several types of cholecystokinin-like immunoreactive peptides may be present in the dorsal horn, depending on their origin (supraspinal, intrinsic or peripheral).(ABSTRACT TRUNCATED AT 400 WORDS)     

The action of capsaicin on primary afferent central terminals in the superficial dorsal horn of newborn mice

Capsaicin was injected subcutaneously (50 mg/kg) into 10 mice on days 2 or 3 after birth, and 12 h, 3 and 5 days later the distribution and structure of degenerated primary afferent central axons or terminals (C-terminals) in the lumbar spinal dorsal horn were examined by electron microscopy. Degenerated terminal axons with dense or lamellar bodies or higher electron density were conspicuous 12 h after treatment with capsaicin. Severely degenerated unmyelinated axons, including dense or lamellar bodies engulfed by microglial cells, were numerous in the most superficial (marginal) layer, but rarely seen in the substantia gelatinosa. Two types of primary afferent central terminals in the substantia gelatinosa showed various extents of degeneration: small dark C-terminals (CI-terminals) with densely packed agranular synaptic vesicles, and large light ones (CII-terminals) with less dense agranular synaptic vesicles and a few granular synaptic vesicles. Thus, many central axon terminals of dorsal root ganglion (DRG) neurons that are sensitive to capsaicin enter the marginal layer and substantia gelatinosa. Degenerated primary afferent central axons or terminals markedly decreased in the superficial dorsal horn 3 and 5 days after capsaicin treatment, still, there were many degenerating DRG neurons at this time as shown by our previous study. Previously we also reported that fewer slightly degenerating unmyelinated dorsal root axons and small DRG neurons appear at 12 h and larger DRG neurons degenerate later than smaller ones after treatment with capsaicin. As a result, the discovery of many severely degenerated terminal axons in the superficial dorsal horn soon after treatment supports the idea that capsaicin first acts on the central terminals and that this is followed by damage to larger DRG neurons.     

Interactions between growing thalamocortical afferent axons and the neocortical primordium in normal and reeler mutant mice

The interactions between growing thalamocortical afferent axons and the neocortical primordium were examined during neocortical development of the mouse cerebrum, by labeling the afferents with the carbocyanine fluorescent dye, DiI, which was introduced into the dorsal thalamus of the fixed brains of control and reeler mutant mice. In the neocortical primordium of the control mouse, the labeled afferents running tangentially in the intermediate zone formed a dense plexus in the subplate, the layer below the cortical plate, as early as the 16th gestational day (E16). Small numbers of the afferents invaded the lower cortical plate at E16 and increasing numbers of labeled growing axons extended into the cortical plate at E17. At the 4th postnatal day (P4), labeled afferents grew radially up to the upper cortical plate and terminal arborizations of the afferents were evident in the forming layer IV. In contrast, in the E16 cerebrum of the reeler mutant mouse, in which the cortical layers are inverted, the labeled afferents traversed the neocortical primordium directly towards the superplate, the superficial layer above the cortical plate and the equivalent of the subplate in the control mouse. Thick bundles of labeled axons reached the superplate and made contact with the superplate neurons. At P4 in the reeler neocortex, the afferent axons that had reached the superplate began to change their direction of growth and turned towards the deeper layer. Electron-microscopic observations at E16 revealed that immature synapses were formed on the somata of the subplate neurons in the control mouse, and similar immature synapses were also formed on the superplate neurons of the reeler mutant. At E16 in the control, NGF receptor immunoreactivity was expressed in the intermediate zone, subplate and lower cortical plate, and the mode of expression corresponded to the distribution of thalamocortical afferents. At the same stage of the reeler mutant, expression of NGF receptor immunoreactivity was confined to the afferent axons that had grown through the neocortical primordium towards the superplate. In the control at E17, highly polysialylated NCAM (NCAM-H), a homophilic cell adhesion molecule, was expressed in the subplate, marginal zone and afferent axons. In the reeler mutant at the same stage, this adhesion molecule was expressed in both the superplate and the bundles of the afferent axons. These findings suggest that the subplate and the superplate, which are composed of neurons generated at the earliest stage, attract growing thalamocortical afferent axons specifically by a chemotropic mechanism through the expression of NGF receptor. Furthermore, growth cones of the afferent axons may make contact with the subplate or superplate neurons specifically through the homophilic adhesive activity of NCAM-H expressed on them. On the basis of such mechanisms, the subplate or the superplate could play a role as a tentative target for the thalamocortical afferents prior to arrival at their final targets, i.e., the layer IV cortical neurons in the control and the equivalent neurons in the reeler mutant.     

The physiology and anatomy of long ranging afferent fibres within the spinal cord.

The caudal extent of the terminal arborizations of dorsal root afferents was determined in adult cats. The method used micro-electrode stimulation within the dorsal horn and the recording on a distant dorsal root filament of the antidromic action potentials evoked by the stimulation of axons within the spinal cord. 2. It was found that all filaments examined in the L2, 3 and 4 dorsal roots contained axons which projected at least as far as the S1 segment. The axons descended in or near the dorsal columns and from there penetrated into the grey matter. 3. The course of single fibres was followed to their apparent terminals. Thresholds, latencies and relative and absolute refractory periods were measured for single axons. These measurements confirmed that continuous axons ran from dorsal roots to distant segments and that the action potentials recorded were not dorsal root reflexes. 4. The majority of fibres with long range central arborizations were shown to have normal receptive fields in the dermatome of their parent dorsal root. They were not aberrant fibres leaving the spinal cord. 5. The long range afferents exist in substantial numbers since fifteen of eighty axons isolated by micro-electrode recording in the L2 dorsal root sent their axons as far as the S1 segment. The presence of these afferents from five segments away does not fit the data published on the inhibitory and excitatory receptive fields or dorsal horn cells which appear adequately explained by afferents arriving over nearby dorsal roots up to two segments away.