Synaptic interactions of substance P immunoreactive nerve terminals in the baro- and chemoreceptor reflexes of the cat

The neurochemical anatomy and synaptic interactions of morphologically identified chemoreceptor or baroreceptor afferents in the nucleus of the solitary tract (NTS) are poorly understood. A substantial body of physiological and light microscopic evidence suggests that substance P (SP) may be a neurotransmitter contained in first order sensory chemo- or baroreceptor afferents, however ultrastructural support of this hypothesis is lacking. In the present report we have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase. Medullary tissues including the commissural NTS (cNTS) were processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical detection of SP by dual labeling light and electron microscopic methods. At the light microscopic level, dense bilateral labeling with TMB was found in the tractus solitarius (TS) and cNTS, caudal to the obex. Rostral to the obex, significant ipsilateral TMB labeling was detected in the dorsal, dorso-lateral, and medial subnuclei of the NTS, as well as in the TS. Significant staining of SP immunoreactive processes was detected in most subnuclei of the NTS. The cNTS was examined by electron microscopy. Either HRP or SP were readily identified in single labeled unmyelinated axons, myelinated axons, and nerve terminals in the cNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the cNTS which were simultaneously identified as CSN primary afferents. These ultrastructural data support the hypothesis that SP immunoreactive first order neurons are involved in the origination of the chemo- and baroreceptor reflexes. Axo-axonic synapses were observed between CSN primary afferent terminals and: (a) unlabeled nerve terminals; (b) other CSN primary afferent terminals; and (c) terminals containing SP. Axo-axonic synapses were also observed between CSN primary afferents which contained SP, and other SP terminals. These observations may mediate the morphological bases for multiple forms of presynaptic inhibition in the cNTS, including those involved in cardiorespiratory integration. In conclusion, our results indicate that SP immunoreactive nerve terminals may be important in both the origination and the modulation of the chemo- and/or baroreceptor reflexes.     

Actions of capsaicin on mouse dorsal root ganglion cells in vitro

The effects of capsaicin were investigated on different populations of dorsal root ganglion cells in the in vitro mouse spinal cord-dorsal root ganglion preparation using intracellular electrodes. Dorsal root ganglion cells were characterised by the conduction velocity of their propagated action potential evoked by electrical stimulation of the dorsal root, and by the shape of their action potential. All cells with C-fiber characteristics (conduction velocity < 0.6 m/s; broad action potential with shoulder on the descending slope) were depolarised and generated action potentials when capsaicin (100-700 nM) was added to the bathing solution for 30 s. At these concentrations the membrane potential of DRG cells with myelinated fibers (conduction velocity > 2.0 m/s) was unaffected. Concentrations of capsaicin of 1.0-5.0 microM depolarised 50% of cells with conduction velocity > 10 m/s. During the depolarization of the membrane no action potentials were generated. In 50% of the capsaicin-sensitive neurons with conduction velocity faster than 10 m/s there was an initial hyperpolarization. Electrical stimulation of the dorsal root failed to evoke action potentials during the depolarization in 38% of the DRG cells with myelinated fibers and in all C-fibers tested within 10 min of the onset of the capsaicin effect. Passive depolarization of the membrane by intrasomal current injection mimicked the conduction block in neurons with large myelinated fibers. These observations confirm that capsaicin applied directly to the dorsal root ganglion affects, in a dose-dependent manner, both myelinated and unmyelinated primary afferents with a higher potency for C-neurons. Capsaicin evoked action potentials in C-neurons but not in neurons with myelinated fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential production of IL-10 by T cells and monocytes of HIV-infected individuals: association of IL-10 production with CD28-mediated immune responsiveness

The present study determines the proportions of unmyelinated cutaneous axons at the dermal-epidermal junction in glabrous skin and of myelinated and unmyelinated axons in the sural and medial plantar nerves that immunostain for subunits of the ionotropic glutamate receptors. Approximately 20% of the unmyelinated cutaneous axon profiles at the dermal-epidermal junction immunostain for either N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate receptor subunits. These findings are consistent with previous observations that NMDA and non-NMDA antagonists ameliorate nociceptive behaviors that result from noxious peripheral stimulation. In the sural nerve, where the large majority of myelinated fibers are sensory, approximately half of the myelinated axon profiles immunostain for the NMDA receptor 1 (R1) subunit, 28% immunostain for the glutamate receptor 1 (GluR1) AMPA subunit, and 11% for the GluR5,6,7 kainate subunits. Even higher proportions immunostain for these receptors in the medial plantar nerve, a mixed sensory and motor nerve. In the sural nerve, 20% of the unmyelinated axon profiles immunostain for NMDAR1 and only 7% label for GluR1 or GluR5,6,7. Because the sural nerve innervates hairy skin, these data suggest that glutamate will activate a higher proportion of unmyelinated axons in glabrous skin than in hairy skin. Measurements of fiber diameters indicate that all sizes of myelinated axon profiles, including Adelta and Abeta, are positively labeled for the ionotropic receptors. The presence of glutamate receptors on large-diameter myelinated axons suggests that these mechanosensitive receptors, presumably transducing touch and pressure, may also respond to local glutamate and thus be chemosensitive.     

Retrograde and transganglionic transport of horseradish peroxidase-conjugated cholera toxin B subunit, wheatgerm agglutinin and isolectin B4 from Griffonia simplicifolia I in primary afferent neurons innervating the rat urinary bladder

In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6-S1 spinal cord segments and in the T13-L2 and L6-S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit-horseradish peroxidase was injected. Cholera toxin B subunit-horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV-VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin-horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit-horseradish peroxidase. The isolectin B4 from Griffonia simplicifolia I-horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit-horseradish peroxidase than with wheat germ agglutinin- or isolectin B4-horseradish peroxidase. In the L6-S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with myelinated fibres). Double labelling with other neuronal markers showed that 71%, 43% and 36% of the cholera toxin B subunit-immunoreactive cells were calcitonin gene-related peptide-, isolectin B4-binding- and substance P-positive, respectively. A few cholera toxin B subunit cells showed galanin-immunoreactivity, but none were somatostatin-, vasoactive intestinal polypeptide-, or neuropeptide Y-immunoreactive or contained fluoride-resistant acid phosphatase. The results show that cholera toxin B subunit-horseradish peroxidase is a more effective retrograde and transganglionic tracer for pelvic primary afferents from the urinary bladder than wheat germ agglutinin-horseradish peroxidase and isolectin B4-horseradish peroxidase, but in contrast to somatic nerves, it is transported mainly by unmyelinated fibres in the visceral afferents.     

Sex differences in sensory and motor branches of the pudendal nerve of the rat

The morphology of the pudendal nerve was quantified in adult male and female rats. The sensory branch of the pudendal nerve was about three times as large in cross section in males as in females, and the motor branch was about five times as large. Electron microscopy was used to determine the ultrastructural bases of these gross size differences. Differences that were found included greater packing density of both myelinated and unmyelinated axons in females, larger myelinated and unmyelinated axons in males, larger myelin sheaths of sensory axons in males, more numerous myelinated axons in both branches of males, and more numerous unmyelinated axons in the sensory branch of males. There was also some indication that myelinated sensory axons were more likely to branch in the dorsal clitoral nerve of females than in the homologous nerve of males. Morphological differences in the structure of pudendal axons, their associated Schwann cells, and the extracellular matrix as well as differences in sensory and motor axonal number all have potential implications for the sexual differentiation of the central nervous system and behavior.     

The morphometric myelinated fiber composition of D11 as compared to L3, L4, and L5 ventral spinal roots of man

As compared to the diameter frequency distribution of myelinated fibers of L3, L4 and L5 ventral spinal roots (VSRs) which have two well developed peaks [large-diameter axons (AL) and intermediate-diameter axons (AI)] and a poorly developed or non-existent peak of small-diameter axons (AS), D11 VSRs contain small AL and AI peaks and a prominent AS peak. Because the ratio of AL to AI myelinated fibers of D11 VSRs is similar to that in L3, L4 and L5 VSRs and it is known that D11 VSRs contain many pre-ganglionic autonomic fibers it seems likely that AS is composed predominantly of preganglionic autonomic fibers. The mean diameter of the AS peak in D11 VSRs is approximately 2.4 micrometer. Average numbers of AS, AI and AL myelinated fibers for D11 VSRs are 3835, 738, and 1545 per root. These values should be useful as controls for morphometric studies of various neuropathies. The average diameter positions of the peak of AL, AI and AS of D11 VSRs as compared to those of L3, L4 and L5 are displaced to smaller diameter categoried which may reflect the smaller axon diameters of neurons which are shorter.     

Sprouting of primary afferent fibers after spinal cord transection in the rat

After spinal cord injury, hyper-reflexia can lead to episodic hypertension, muscle spasticity and urinary bladder dyssynergia. This condition may be caused by primary afferent fiber sprouting providing new input to partially denervated spinal interneurons, autonomic neurons and motor neurons. However, conflicting reports concerning afferent neurite sprouting after cord injury do not provide adequate information to associate sprouting with hyper-reflexia. Therefore, we studied the effect of mid-thoracic spinal cord transection on central projections of sensory neurons, quantified by area measurements. The area of myelinated afferent arbors, immunolabeled by cholera toxin B, was greater in laminae I-V in lumbar, but not thoracic cord, by one week after cord transection. Changes in small sensory neurons and their unmyelinated fibers, immunolabeled for calcitonin gene-related peptide, were assessed in the cord and in dorsal root ganglia. The area of calcitonin gene-related peptide-immunoreactive fibers in laminae III-V increased in all cord segments at two weeks after cord transection, but not at one week. Numbers of sensory neurons immunoreactive for calcitonin gene-related peptide were unchanged, suggesting that the increased area of immunoreactivity reflected sprouting rather than peptide up-regulation. Immunoreactive fibers in the lateral horn increased only above the lesion and in lumbar segments at two weeks after cord transection. They were not continuous with dorsal horn fibers, suggesting that they were not primary afferent fibers. Using the fluorescent tracer DiI to label afferent fibers, an increase in area could be seen in Clarke's nucleus caudal to the injury two weeks after transection. In conclusion, site- and time-dependent sprouting of myelinated and unmyelinated primary afferent fibers, and possibly interneurons, occurred after spinal cord transection. Afferent fiber sprouting did not reach autonomic or motor neurons directly, but may cause hyper-reflexia by increasing inputs to interneurons.     

Myelinated axons of ganglion cells in the retinae of teleosts

In the retina of the goldfish and the rainbow trout, the axons of ganglion cells belong to the unmyelinated or the myelinated types. The unmyelinated fibers are either arranged in bundles in direct contact with neighboring fibers or they are separated by intervening lamellae of oligodendroglial cytoplasm. The myelin sheaths of the myelinated fibers differ greatly in thickness. Most fibers show 3 to 5 myelin layers; single fiber elements, however, show 10 or even more layers.     

Effects of sciatic nerve crush on the L7 spinal roots and dorsal root ganglia in kittens

The number and size distribution of axons and neurons were examined in the L7 spinal roots and ganglia of kittens 14 to 220 days after early postnatal sciatic nerve crush. The results show that motoraxons in the ventral root as well as axons and perikarya of sensory neurons in the dorsal root remained growth-retarded throughout the examined period. This was most evident in the dorsal root. Both ventral and dorsal roots showed some loss of myelinated axons, but this was only half that previously observed after sciatic nerve resection. Whereas in the dorsal roots and dorsal root ganglia the loss seemed to be nonselective with respect to size, axons in the gamma range were primarily affected in the ventral roots. In the dorsal roots the proportion of unmyelinated axons was comparable with controls but in the ventral roots it was somewhat elevated. In most cases the loss of dorsal root ganglion neurons was relatively greater than the decrease of dorsal root axons.     

Unmyelinated nerve fiber analysis of the human abducent nerve

Unmyelinated nerve fibers of the abducent nerve have occasionally been observed with electron microscopes, but, to our knowledge, they have never been evaluated from the morphometric point of view. We analyzed the aging process of the unmyelinated nerve fibers in the human abducent nerve with the help of a new staining method suitable for morphometric research on the nervous system. We studied numbers and transverse areas of unmyelinated fibers of the abducent nerve in 10 cadavers. Our findings were that (1) these fibers were distributed diffusely, (2) their number decreased with age, and (3) the mean transverse area did not change with age. Most of the unmyelinated axons were thinner than the myelinated axons. These results may be important for analysis of clinical signs in relation to aging and ophthalmologic functions.     

Composition of the tectal and posterior commissures of the chick (Gallus domesticus)

In chick, the tectal and posterior commissures form a continuous band of axons lying in the dorsal aspect of the meso-diencephalon. In midline sagittal sections, two zones can be clearly defined. The rostral zone (RZ) contains about 290,000 axons of which 32% are myelinated. The caudal zone (CZ) contains about 911,000 fibers of which 12% are myelinated. The majority of unmyelinated fibers in RZ and CZ are between 0.15 and 0.40 micrometers in diameter. The majority of myelinated fibers in both RZ and CZ are less than 1 micrometer in diameter. It is likely that RZ corresponds to the posterior commissure while CZ corresponds to the tectal commissure.     

Unmyelinated nerve fibers in senile nerves and in late thalidomide neuropathy: a quantitative electron microscopic study (author's transl)

Sural nerve biopsies of four patients, aged 54--76 years, with a predominantly sensory type of neuropathy following high dosages of thalidomide were examined by light and electron microscopy. The present study includes a qualitative and quantitative evaluation of unmyelinated nerve fibers. Despite severe neuropathy, increased numbers of small unmyelinated axons per endoneurial area were noted in all patients. This numerical increase appeared to be independent of aging, since it was not seen in two senile controls, studied at the age of 83 and 88 years. The increase in the endoneurial density of unmyelinated axons, especially of small sized fibers, is likely to be related to regeneration following degeneration of unmyelinated axons although endoneurial shrinkage secondary to loss of large myelinated fibers could have caused an additional increase in the number of axons per endoneurial area. Axonal sprouting, despite degeneration of large numbers of myelinated and unmyelinated fibers, appears to be consistent with some of the characteristic clinical features of thalidomide neuropathy such as paresthesias, hyperesthesia for pain and temperature, and disturbances of autonomic functions. On the other hand, a variable number of empty Schwann cells (bands of Büngner) and pockets at the surface of many Schwann cells noted in the four patients with neuropathy were also seen in both senile controls with no signs of neuropathy. Thus, it is obvious that pockets and empty Schwann cells may be related to aging or other causes of slow axonal wasting with Schwann cell proliferation and are not necessarily associated with clinically manifest neuropathy.     

Innervation of posterior structures in the lumbar spine of the rat

OBJECTIVE: To determine the nature and distribution of afferent fibers to the interspinous tissues and facet joints of the lumbar spine in the rat. DESIGN: Dissection and photography of medial branch of the primary dorsal ramus; histological and electron microscopic examination of the medial branch; measurement of conduction velocities of fibers within the medial branch; recording of compound nerve activity in medial branch in response to mechanical and chemical stimulation of interspinous tissues and facet joints. RESULTS: In the rat, the medial branch of the primary dorsal ramus of lumbar spinal nerves is normally distributed to the facet joints and interspinous tissues one and two segmental levels caudad to its origin. This nerve contains unmyelinated and myelinated afferents with conduction velocities within the ranges of C fibers, and A-delta and A-beta fibers. The tissues served by this nerve are sensitive to mechanical and noxious chemical stimulation. CONCLUSIONS: There are many structural and functional similarities in the innervation of the lumbar spine in rats and humans. However, there are anatomical variants and, in rats, the medial branch of the primary dorsal ramus, which serves the interspinous tissues and facet joints, is distributed more caudally than in humans. This information should be taken into account in extrapolating experimental results from rats to the human situation.     

Myelination of S1 dorsal root axons in the cat

Samples of S1 dorsal root nerve fibers from cats of different pre- and postnatal ages were examined electron microscopically with regard to axon caliber and number of myelin lamellae. Each root was examined at four different cross-sectional levels. Two levels were situation close to the spinal cord entrance on each side of the peripheral (PNS) and central nervous system (CNS) border. The third and fourth levels were located more distally. The first compact myelin lamella was observed in the CNS part of the root in a 47-day-old fetus. In the 53-day-old fetus the degree of myelination was the same in the CNS as distal in the PNS part of the root. Surprisingly, all axons appeared unmyelinated close to the PNS-CNS border and remained so for a further 10-day period. After this time lag, this part of the root became myelinated and showed a rapid increase in myelin sheath thickness. Calculations of axonal growth, mesaxonal length, and myelin volume indicated a maturation process that progressed discontinuously. Myelination did not proceed in a strict somatofugal direction, but was a regionally differentiated process. The maximal myelin production, expressed as the increase in myelin volume per Schwann cell, was found during the second to fourth postnatal months, i.e., very late in development.     

The transplantation of syngeneic Schwann cells in medullary lesions: results, limitations and perspectives

After a central nervous system (CNS) injury, there is only an "abortive regeneration" of axons, while injured axons regenerate vividly in the peripheral nervous system (PNS). This difference is due, at least in part, to the existence in the periphery of Schwann cells and of growth promoting proteins they synthetize. One strategy to promote regrowth of central axons can be therefore, to modify (i.e. "peripheralize") the microenvironment by transplanting biologically active Schwann cells into the lesion site. In a rat model of traumatic paraplegia by inflation of a subdural microballoon, we performed syngeneic transplants of Schwann cells. These cells are cultured from adult dorsal root ganglia and can be kept in vitro for several months. They are transplanted in the injured spinal cord. The grafted Schwann cells are well integrated in the host tissue without detectable inflammatory reaction. Cystic cavitation and astrogliosis are reduced in grafted animals as compared to injured, non-grafted animals. The transplant is invaded by abundant, mainly unmyelinated axons which are immunoreactive for substance P, VIP or CGRP, i.e. transmitters known to be present in DRG afferents. Supraspinal afferents containing 5HT, TH or CCK accumulate at the rostral margin of the graft. Experimental procedures trying to stimulate the invasion of the graft by descending fibers, i.e. by inducing a chemoattraction are therefore of crucial importance for functional recovery.     

Kinetic characterisation and solubilisation of gamma-hydroxybutyrate receptors from rat brain

The effects of single electrical shocks to myelinated A and unmyelinated C afferent fibers of perineal and limb somatic nerves on the reflex discharges in pelvic parasympathetic (L6/S1) efferent nerves to the bladder were examined in anesthetized central nervous system (CNS)-intact and acute spinal rats. When the bladder was empty, stimulation of perineal somatic inputs to the L6 and S1 segments from the perineo-femoral branch of a pudendal nerve produced excitatory A- and C-reflex discharge components in postganglionic parasympathetic efferent nerve branches on the bladder surface. When the bladder was expanded and pelvic efferent neurons were rhythmically active, additional inhibitory A- and C-reflex components could be seen. After acute spinal transection, the same stimuli elicited excitatory A- and C-reflex discharges of similar latency as those observed before the spinal transection, but were of larger amplitude and longer duration; resting activity in the pelvic nerve was low, and no evoked inhibitory reflex components could be observed. Electrical stimulation of afferents in the tibial nerve had no effect when the bladder pressure was low, but when the bladder was distended, early and late components of reflex inhibition and excitation of parasympathetic activity were visible in CNS-intact rats; these reflex responses were abolished following spinalization.     

Fine structure and development of dorsal root ganglion neurons and Schwann cells in the newborn opossum Monodelphis domestica

The aim of these experiments was to determine the state of maturity of dorsal root ganglia and axons in opossums (Monodelphis domestica) at birth and to assess quantitatively changes that occur in early life. Counts made of dorsal root ganglion cells at cervical levels showed that the numbers were similar in newborn and adult animals, approximately 1,600 per ganglion. In cervical dorsal root ganglia of newborn animals, division of neuronal precursors cells had ceased. The number of axons in cervical dorsal roots was similar in newborn and adult animals (about 4,500). For each ganglion cell body, approximately three axons were counted in the dorsal root. At birth, dorsal roots contained several bundles about 30 microns in diameter consisting of small axons (0.05-2 microns in diameter). A few non-neural cells were identified as Schwann cell perikarya, each enclosing a number of neurites. Later, marked changes occurred in Schwann cells and in their relationship to axons in the roots. Thus, at 12 days, an increase occurred in the number of Schwann cells and fibroblasts, and the bundles had enlarged to about 80 microns with little increase in axon diameter (0.1-2 microns). By 18 days, the bundles were larger, and myelination had already started. At 23 days, the dorsal root contained more than 500 myelinated axons that could reach 5 microns in diameter. The adult dorsal root enclosed about 900 myelinated axons. Throughout this time, the relationship between the Schwann cells and axons changed. Together, these results indicate that the number of axons and cell bodies of sensory dorsal root ganglia in opossum do not show major changes after birth. In addition, these results set the stage for quantitative studies of regeneration of dorsal column fibers in injured neonatal opossum nervous system.     

Corticotropin releasing factor-like immunoreactivity in afferent projections to the sacral spinal cord of the cat

Distribution and origin of corticotropin releasing factor (CRF) in the thoraco-lumbar and sacral spinal cord of the cat has been studied using immunohistochemical method. CRF immunoreactive (CRF-IR) nerve fibers and terminals were most prominent in dorsal part of sacral spinal cord. In the sacral segments of the spinal cord, immunoreactivity for CRF was detected in a prominent bundle of axons and varicosities extending from Lissauer's tract (LT) along the lateral edge of the superficial dorsal horn (laminae I and II) to laminae V at the base of the dorsal horn. Individual CRF-IR fibers passed from the bundle in ventral medial and ventrolateral directions to the dorsal commissure and the sacral preganglionic nucleus (SPN), respectively. The bundle of CRF-IR axons closely resembled vasoactive intestinal polypeptide (VIP) containing fibers in LT and on the lateral edge of the dorsal horn. Sacral dorsal root transection eliminated both the CRF and VIP fiber staining in the dorsal horn. Spinal transection at the T12-T13 segmental level did not influence the CRF- or VIP-IR. Less intense CRF-IR was also present in fibers in: (1) the dorsal lateral funiculus adjacent to LT, (2) the superficial layers of the dorsal horn and intermediolateral nucleus at thoracolumbar spinal levels, (3) the ventral horn, including Onuf's nucleus, (4) the intermediate gray matter including the dorsal gray commissure, and (5) the SPN. The similarity in the distribution of CRF-IR and pelvic nerve afferent projections in the sacral spinal cord raises the possibility that CRF may be a transmitter in afferent neurons innervating the pelvic viscera.     

Reconstruction of trajectory of primary afferent collaterals in the dorsal horn of the cat spinal cord, using Golgi-stained serial sections

Using Golgi-stained serial sections obtained at the sacro-caudal levels of the cat spinal cord, it was possible to reconstruct the trajectory of primary afferents. They were classified into two groups: reliable primary afferents directly traced from the dorsal root and probable primary afferents traced from the dorsal funiculus or Lissauer's tract. The diameters of the reliable primary afferents vary from 0.88-1.88 mum. According to their courses, reliable primary afferents as well as probable primary afferents were classified into three groups: the first is distributed to both medial and lateral halves of the dorsal horn, the second to the medial half, and the third to the lateral half. Commissural fibers were also observed among the probable primary afferents. The rostro-caudal and medio-lateral extents of reliable primary afferents are found to be between 250 and 950 mum and 270 and 700 mum respectively, while those of the probable primary afferents were between 125 and 670 mum and 270 and 1,640 mum respectively. These primary afferent fibers are connected with at least two or more laminae of the dorsal horn gray matter.