Discharge characteristics of fine medial articular afferents at rest and during passive movements of inflamed knee joints

After induction of an experimental knee joint inflammation, the activity of single Group III and IV afferent units in the medial articular nerve of the cat was recorded at rest and during passive movements. the properties of these units were compared to those sampled from normal knee joints. The proportion of units displaying resting discharges was higher in the inflamed group. The frequency of discharges was also higher. The receptive fields were larger than those in the control units. Passive movements in the normal working range of the joint activated many more units in the inflamed joint than in the control sample. We conclude that joint inflammation sensitizes articular nociceptors to be active not only at rest, but also during normally innocuous joint movements.     

Somatic and visceral primary afferents in the lower thoracic dorsal root ganglia of the cat

Anterograde transport of horseradish peroxidase (HRP) through somatic and visceral nerves was used to estimate the proportions of somatic and visceral dorsal root ganglion (DRG) cells of the lower thoracic ganglia of the cat. A concentrated solution of HRP was applied for at least 5 hours to the central end of the right greater splanchnic nerve and of the left T9-intercostal nerve of adult cats. Some animals remained under chloralose anaesthesia for the duration of the HRP transport time (up to 53 hours) whereas longer HRP application and transport times (4-5 days) were allowed in animals that recovered from barbiturate anaesthesia. Visceral DRG cells were found in approximately equal numbers in all ganglia examined (T7-T11). Population estimates were obtained for the T8 and T9 ganglia where visceral DRG cells were found to be 6.2% (T8) and 5.2% (T9) of the total cell population. In contrast, somatic DRG cells were found in large numbers in the ganglia examined (T8 and T9) where they amounted to over 90% of the cell population. Measurement of cross-sectional areas and estimates of cell diameters of the DRG cells showed greater proportions of large somatic cells (diameter greater than 40 micron) than of large visceral cells. Similar distributions of cell size were found for both somatic and visceral DRG cells with diameters less than 40 micron. These results show that the proportion of visceral afferent fibres in the dorsal roots that mediate the spinal cord projection of the splanchnic nerve is very small. Since viscerosomatic convergence in the thoracic spinal cord is very extensive, the present results suggest considerable divergence of the visceral afferent input to the central nervous system.     

Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord.

Subunits of glutamate receptors participate in the regulation of sensory transmission at primary afferent synapses in the superficial laminae of dorsal horn (DH). We report here on the distribution of kainate receptors (GluR5/6/7) in these laminae by using light microscope (LM) and electron microscope (EM) immunocytochemistry. Standard (4%) paraformaldehyde fixation resulted in immunostaining for GluR5/6/7 in perikarya and fine processes in lamina II, especially its inner part (IIi). Preembedding EM revealed immunostaining of dendrites, perikarya, and occasional terminals, presumed to be from primary afferent fibers, at the center of glomerular arrangements. In rats perfused with 0.5% paraformaldehyde, LM showed a more punctate staining, mainly in the ventral part of lamina IIi and lamina III, than in material fixed with 4% paraformaldehyde. Approximately two-thirds of GluR5/6/7 puncta were also immunostained with synaptophysin, suggesting that in material fixed with 0.5% paraformaldehyde, a large fraction of these are synaptic terminals. Double immunostained puncta disappear 4 days after dorsal rhizotomy, suggesting that most of GluR5/6/7-immunopositive terminals are from primary afferent fibers. EM material fixed with 0.5% paraformaldehyde confirmed the expression of GluR5/6/7 in numerous synaptic endings with morphology of primary afferents. To determine the type of primary afferent terminals that express GluR5/6/7, two neuroanatomic tracers were injected in the sciatic nerves. The lectin from Bandeiraea simplicifolia (IB4) is selectively taken up by unmyelinated primary afferent fibers that terminate in the outer part of lamina II (IIo) and dorsal part of lamina IIi, whereas the B subunit of the cholera toxin (CTB) is selectively taken up by a broader class of primary afferents which, in superficial DH, terminate mainly in laminae I, ventral part of IIi, and III. Approximately 20% of GluR5/6/7-immunoreactive puncta colocalized with IB4, whereas approximately 40% of GluR5/6/7-immunoreactive puncta colocalized with CTB. The present study shows that (1) GluR5/6/7 does not have a clear and consistent spatial relation with postsynaptic sites, (2) a large number of primary afferents express GluR5/6/7, and (3) these are not limited to one functional class. Thus, modulation by glutamate of primary afferent terminals by means of kainate receptors in the superficial laminae of DH may predominantly involve presynaptic mechanisms.     

Cholecystokinin in Mammalian Primary Sensory Neurons and Spinal Cord: In Situ Hybridization Studies in Rat and Monkey

The peptide cholecystokinin (CCK) has been suggested to be involved in nociception, but its exact localization at the level of the spinal cord and in spinal ganglia has been a controversial issue. Therefore the distribution of messenger RNA (mRNA) for CCK was studied by in situ hybridization using oligonucleotide probes on sections of adult rat lumbar dorsal root ganglia following unilateral section of the sciatic nerve and on sections of untreated monkey trigeminal ganglia, spinal cord and spinal ganglia from all levels. For comparison, calcitonin gene-related peptide (CGRP) mRNA was also studied in the monkey tissue using the same techniques. Peripheral sectioning of the sciatic nerve in the rat resulted in the appearance of detectable CCK mRNA in up to 30% of remaining ipsilateral L4 and L5 dorsal root ganglion neurons 3 weeks after surgery, with a distinct but more limited appearance also in the contralateral ganglia. No cells, or only single cells, could be seen in normal control rat ganglia. In contrast, in the normal monkey, ∼20% of dorsal root ganglion neurons, regardless of spinal level, and 10% of trigeminal ganglia neurons expressed mRNA for CCK. CGRP mRNA was expressed at detectable levels in ∼80% of these monkey dorsal root ganglion neurons. In the monkey spinal cord, CCK mRNA was detected in the dorsal horn and in motoneurons, whereas CGRP mRNA was only seen in motoneurons. The present results suggest that CCK peptides can be involved in sensory processing in the dorsal horn of the spinal cord in normal monkeys and in rats after peripheral nerve injury, adding one more possible excitatory peptide to the group of mediators in the dorsal horn.     

Adjuvant-induced inflammation of rat paw is associated with altered calcitonin gene-related peptide immunoreactivity within cell bodies and peripheral endings of primary afferent neurons

Local inflammation is associated with profound changes in the biochemistry and physiology of primary afferent nerve fibers and the central neurons responding to their signals. In some tissues, the neural changes accompanying inflammation include sprouting and cytochemical changes that are delayed several days after the initial injury. In the present stydiy, we have analyzed the effect of complete Freund's adjuvant (CFA)-induced inflammation in the rat paw on clacitonin gene-related peptide (CGRP) immunoreactivity (IR) in dorsal root ganglia and within tissue of the inflamed paw. We quantified the CGRP-IR within the L₁, L₄, and L₆ ganglia, and in ankle, midpaw, joint and toe tissues. Analysis of the processed tissue revealed a significant increase in the percentage of CGRP-positive cells within L₄ dorsal root ganglia ipsilateral to an inflamed hindpaw six days after administration of CFA. There was a parallel increase in the number and staining density of detectable CGRP-immunoreactive fibers in periarticular and perivascular tissues of the inflamed digits and inflamed ankle. The other tissues of the paw, including epidermis and the regions surrounding the abceses, did not have detectable changes in CGRP-immunoreactive fibers, despite tissue swelling and dystrophic changes in the foot that included loss of mast cell staining. These data demonstrate that local inflammation of the rat paw has delayed influences in the peripheral nervous system, in addition to a number of previoulsy characterized acute effects. The alterations of CGRP-IR were focused around specific tissue types, such as joints and subdermal blood vessels, and absent from others, such as epidermis or in the areas surrounding abscesses. This suggests production of local factors within reactive tissues that selectively interact with nerve fibers to induce changes in CGRP-IR within the fibers. © 1994 Wiley-Liss, Inc.     

Chemical characterization of substance P-like immunoreactivity in primary afferent neurones

Substance P-like immunoreactivity (SPLI) in rat dorsal root ganglia and dorsal spinal cord was characterized by high-performance liquid chromatography followed by radioimmunoassay. In spinal cord and ganglia, respectively, 87% and 64% of SPLI eluted with authentic SP. The remainder of the SPLI in ganglia eluted as a single peak which did not represent the sulphoxide of SP or any of its C-terminal fragments.     

Identification of neuropeptides in pelvic and pudendal nerve afferent pathways to the sacral spinal cord of the cat

The distribution of several neuropeptides, including vasoactive intestinal polypeptide (VIP), substance P, somatostatin, leucine enkephalin, methionine enkephalin, and cholecystokinin, in sacral afferent pathways of the cat was examined by immunohistochemical techniques. Certain peptides (substance P, somatostatin, and leucine enkephalin) could be demonstrated in normal dorsal root ganglion cells; however, topical administration or injections of colchicine solution into ganglia 36-56 hours prior to removal markedly increased the number of cells labeled and the intensity of staining. Other peptides (VIP, cholecystokinin, and methionine enkephalin) were only detected in significant numbers of cells following intraganglionic injections of colchicine. The distribution of peptides in dorsal root ganglion cells projecting to the pelvic nerve (visceral) and the pudendal nerve (somatic) was examined by retrograde dye labeling combined with immunohistochemistry. Fluorescent dyes were applied to the cut ends of the nerves 2 weeks prior to removal. A considerably higher percentage of pelvic nerve afferent neurons than pudendal nerve afferent neurons exhibited peptide immunoreactivity; e.g., VIP (42% vs. 10%), cholecystokinin (29% vs. 12%), substance P (24% vs. 21%), leucine enkephalin (30% vs. 24%), and methionine enkephalin (10% vs. 3%). Somatostatin was present in only a small percentage of either type of afferent neuron (0.3-2%). The total percentage of peptide-containing pelvic afferent neurons exceeded 100% (137%), suggesting that more than one peptide is present in some visceral afferent neurons. This has been confirmed in preliminary experiments. The peptide-containing cells were in general less than 40 micron in average diameter; however, a significant percentage of substance P and cholecystokinin neurons ranged from 40 to 60 micron in average diameter. VIP cells had the smallest average diameter (30 micron) whereas somatostatin cells had the largest average diameter (36 micron). Statistical analysis of cell sizes revealed that substance P cells projecting to the pelvic nerve were smaller than substance P cells sending axons into the pudendal nerve. On the other hand, VIP cells in the two afferent pathways were not significantly different in size. Sacral visceral and somatic afferent neurons contain a wide spectrum of neuropeptides, some of which (e.g., VIP and cholecystokinin) seem to be preferentially distributed in the visceral afferent systems.(ABSTRACT TRUNCATED AT 400 WORDS)     

Peripheral axotomy induces only very limited sprouting of coarse myelinated afferents into inner lamina II of rat spinal cord

Peripheral axotomy-induced sprouting of thick myelinated afferents (A-fibers) from laminae III-IV into laminae I-II of the spinal cord is a well-established hypothesis for the structural basis of neuropathic pain. However, we show here that the cholera toxin B subunit (CTB), a neuronal tracer used to demonstrate the sprouting of A-fibers in several earlier studies, also labels unmyelinated afferents (C-fibers) in lamina II and thin myelinated afferents in lamina I, when applied after peripheral nerve transection. The lamina II afferents also contained vasoactive intestinal polypeptide and galanin, two neuropeptides mainly expressed in small dorsal root ganglion (DRG) neurons and C-fibers. In an attempt to label large DRG neurons and A-fibers selectively, CTB was applied four days before axotomy (pre-injury-labelling), and sprouting was monitored after axotomy. We found that only a small number of A-fibers sprouted into inner lamina II, a region normally innervated by C-fibers, but not into outer lamina II or lamina I. Such sprouts made synaptic contact with dendrites in inner lamina II. Neuropeptide Y (NPY) was found in these sprouts in inner lamina II, an area very rich in Y1 receptor-positive processes. These results suggest that axotomy-induced sprouting from deeper to superficial layers is much less pronounced than previously assumed, in fact it is only marginal. This limited reorganization involves large NPY immunoreactive DRG neurons sprouting into the Y1 receptor-rich inner lamina II. Even if quantitatively small, it cannot be excluded that this represents a functional circuitry involved in neuropathic pain.     

Neurokinin A-like immunoreactivity in articular afferents of the cat.

Dorsal root ganglion cells with axons innervating the cat's knee joint via the medial articular nerve were retrogradely labelled with Fast blue. Neurokinin A-like immunoreactivity was found in 4.5 +/- 1.1% (mean +/- S.D. of 5 nerves and 695 cells) of the articular afferents. Colchicine treatment of the ganglia increased the percentage of immunopositive cells to 8.5 +/- 0.7% (mean +/- S.D. of 6 nerves and 554 cells) after 3-22 h. The diameter distribution of the immunopositive somata ranged from 20 to 50 microns with a maximum at 26-30 microns. Comparing the proportions of neurokinin A-immunopositive cells with those of substance P, it can be calculated on the basis of mRNA encoding that neurokinin A is synthetized in about half of the substance P-containing primary articular afferents.     

Presynaptic and postsynaptic subcellular localization of substance P receptor immunoreactivity in the neostriatum of the rat and rhesus monkey (Macaca mulatta)

The substance P receptor (SPR) gene is expressed at high levels in basal ganglia, but the paucity of information about localization of the encoded receptor protein has limited our understanding of this peptide's involvement in cellular and subcellular mechanisms in this region. Morphological evidence in the rodent striatum indicates that SPRs are expressed in postsynaptic neuronal elements, while pharmacological studies suggest the existence of presynaptic SPRs in this structure. We have examined the issue of subcellular distribution of this receptor protein in rat and primate neostriatal tissue, employing an antiserum raised against SPR. Electron microscopic analysis revealed that SPR immunoreactivity is present in presynaptic and postsynaptic neuronal elements in both species. In agreement with earlier studies, SPR immunoreactivity was found predominantly in perikarya and dendrites of a small subset of striatal neurons, the large and medium-sized aspiny interneurons. In addition, a small but significant proportion of the immunoreaction product was localized in presynaptic profiles, both in axons and axon terminals. The majority of SPR immunoreactive boutons formed asymmetric synapses with dendrites and dendritic spines. The association of SPRs with asymmetric synapses provides a morphological substrate for peptidergic modulation of excitatory neurotransmission of extrastriatal origin. A minor proportion of immunolabeled axons established symmetric synaptic junctions with unlabeled dendrites. The presence of SPRs in these synapses suggests a presynaptic peptidergic modulation of intrinsic striatal transmitter systems. The observations in this study also indicate that SPR mediates a complex combination of postsynaptic and presynaptic effects on acetylcholine release in the mammalian striatum. © 1996 Wiley-Liss, Inc.     

Origins and distribution of cerebrovascular nerve fibers showing calcitonin gene-related peptide-like immunoreactivity in the major cerebral artery of the dog

The origins and overall distribution of perivascular nerve fibers showing calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the major cerebral arteries were investigated immunohistochemically in the dog by using whole-mount preparations of the arterial trees around the circle of Willis. Perivascular nerve fibers with CGRP-LI were seen most abundantly in the basilar artery, vertebral artery, common anterior cerebral artery, proximal part of the anterior cerebral artery, and terminal part of the internal carotid artery. They were far less numerous in the middle cerebral artery, posterior cerebral artery, superior cerebellar artery, and distal part of the anterior cerebral artery. Neuronal cell bodies with CGRP-LI were observed in the trigeminal, nodose, superior cervical, and dorsal root ganglia. CGRP-LI fibers in the large pial arteries in the circle of Willis were eliminated ipsilaterally after unilateral transection of the ophthalmic division of the trigeminal nerve, and slightly reduced in number ipsilaterally after unilateral transection of the maxillary division of the trigeminal nerve. They did not show any noticeable changes after unilateral transection of the mandibular division of the trigeminal nerve. On the other hand, CGRP-LI fibers in the basilar and vertebral arteries did not show any appreciable changes after unilateral transection of the trigeminal nerve, but they were eliminated after bilateral ganglionectomy of the dorsal root ganglia of the first, second, and third cervical nerves. After ganglionectomy of the ciliary, pterygopalatine, otic, nodose, or superior cervical ganglion, no changes were observed in perivascular nerve fibers with CGRP-LI in the major cerebral arteries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and neurochemical identification of pancreatic afferents in the mouse

Dysfunction of primary afferents innervating the pancreas has been shown to contribute to the development of painful symptoms during acute and chronic pancreatitis. To investigate the distribution and neurochemical phenotype of pancreatic afferents, Alexa Fluor-conjugated cholera toxin B (CTB) was injected into the pancreatic head (CTB-488) and tail (CTB-555) of adult male mice to label neurons retrogradely in both the dorsal root ganglia (DRG) and nodose ganglia (NG). The NG and DRG (T5-T13) were processed for fluorescent immunohistochemistry and visualized by using confocal microscopy. Spinal pancreatic afferents were observed from T5 to T13, with the greatest contribution coming from T9-T12. The pancreatic afferents were equally distributed between right and left spinal ganglia; however, the innervation from the left NG was significantly greater than from the right. For both spinal and vagal afferents there was significantly greater innervation of the pancreatic head relative to the tail. The total number of retrogradely labeled afferents in the nodose was very similar to the total number of DRG afferents. The neurochemical phenotype of DRG neurons was dominated by transient receptor potential vanilloid 1 (TRPV1)-positive neurons (75%), GDNF family receptor alpha-3 (GFRalpha3)-positive neurons (67%), and calcitonin gene-related peptide (CGRP)-positive neurons(65%) neurons. In the NG, TRPV1-, GFRalpha3-, and CGRP-positive neurons constituted only 35%, 1%, and 15% of labeled afferents, respectively. The disparity in peptide and receptor expression between pancreatic afferents in the NG and DRG suggests that even though they contribute a similar number of primary afferents to the pancreas, these two populations may differ in regard to their nociceptive properties and growth factor dependency.     

Age alters the ability of substance P to sensitize joint nociceptors in Guinea pigs

Pain perception is altered during senescence and it is thought that this could in part be due to changes in peripheral pain sensing processes. The present study examined the effect of substance P (SP) (10⁻¹⁰−10⁻⁸ mol; 0.1 mL bolus close intraarterial) on knee joint afferent mechanosensitivity in young (2- to 5-mo-old) and aged (17- to 36-mo-old) Dunkin-Hartley guinea pigs. Single unit electrophysiological recordings were made from knee joint primary afferent nerves in response to normal (nonnoxious) and painful (noxious) rotation of the joint. In young and old animals, local application of SP had a sensitizing effect on joint afferents in response to movements made in the normal working range of the knee. With noxious hyper-rotation of the joint, SP was able to increase afferent firing rate in young but not in old animals. These data demonstrate a lack of SP-mediated sensitization of joint nociceptors during senescence and suggest a peripheral deficiency in joint nociception with respect to age.     

The distribution and origin of a novel brain peptide, neuropeptide Y, in the spinal cord of several mammals

The distribution of neuropeptide Y [NPY]-immunoreactive material was examined in the spinal cord and dorsal root ganglia of rat, guinea-pig, cat, marmoset, and horse. Considerable concentrations of NPY and similar distribution patterns of immunoreactive nerve fibres were found in the spinal cord of all species investigated. The dorsal root ganglia of the cat and the horse contained numerous immunoreactive nerve fibres, but in these species, as in the other three studied [rat, guinea-pig, marmoset], no positively stained cell bodies were found. Neuropeptide Y-immunoreactive nerves were observed at all levels of the spinal cord, being most concentrated in the dorsal horn. In the rat, guinea-pig, and marmoset, there was a marked increase of NPY-immunoreactive fibres in the lumbosacral regions of the spinal cord, and this was reflected by a considerable increase of extractable NPY. Estimations of NPY-immunoreactive material in the various regions of the rat spinal cord were as follows: cervical, 13.8 +/- 1.0; thoracic, 21.1 +/- 2.5; lumbar, 16.3 +/- 2.9; sacral, 92.4 +/- 8.5 pmol/gm wet weight of tissue +/- SEM. In the ventral portion of the guinea-pig spinal cord they were as follows: cervical, 7.1 +/- 1.2; thoracic, 8.2 +/- 3.6; lumbar, 22.6 +/- 7.0; sacral, 36.7 +/- 9.5 pmol/gm wet weight of tissue +/- SEM. Analysis of spinal cord extracts by reverse phase high performance liquid chromatography [HPLC] demonstrated that NPY-immunoreactive material elutes in the position of pure NPY standard. No changes in the concentration and distribution of the NPY-like material in the rat spinal cord were observed following a variety of surgical and pharmacological manipulations, including cervical rhizotomy, sciatic nerve section and ligation, and local application of capsaicin [50 mM] to one sciatic nerve. It is therefore suggested that most of the NPY-immunoreactive material in the spinal cord is derived either from intrinsic nerve cell bodies or from supraspinal tracts.     

Noradrenergic inhibition of the release of substance P from the primary afferents in the rabbit spinal dorsal horn

To investigate pre-synaptic influence of the descending noradrenergic system on the primary afferents containing substance P (SP), effects of noradrenergic manipulations on the in situ release of immunoreactive SP (iSP) from the dorsal horn were examined in the thalamic rabbit. Local application of noradrenaline (10 microM) to the dorsal horn produced complete inhibition of the noxious mechanical stimuli-evoked release of iSP. This effect was reversed by yohimbine (the more selective alpha 2-blocker, 10 microM) and partially antagonized by prazosin (the more selective alpha 1-blocker, 10 microM). The resting release of iSP was not affected by noradrenaline. The noxious mechanical stimuli-evoked release of iSP was significantly increased by acute spinal transection and local application of yohimbine (10 microM) alone to the dorsal horn. Prazosin (10 microM) slightly increased the evoked iSP release, and a beta-blocker metoprolol did not affect it. These results suggest that the nociceptive primary afferents containing SP are tonically inhibited by the descending noradrenergic system linked to alpha-adrenoceptors, and that such alpha-adrenoceptors located on the primary afferent terminals may be one of the sites of action of noradrenaline spinal analgesia. In contrast to the evoked iSP release, the resting iSP release was increased only by acute spinal transection and not by yohimbine, prazosin and metoprolol. All these observations suggest that tonic inhibition in propriospinal neurons containing SP is mediated by a non-noradrenergic system.     

Somatostatin-like immunoreactivity in the midbrain of the cat

Somatostatin (SS) immunoreactivity was localized in cat brain sections with an immunoperoxidase technique. Cell bodies in the midbrain containing SS immunoreactivity were found in the superficial and intermediate gray layers of the superior colliculus, the interpeduncular nucleus, the raphe, the inferior colliculus and nucleus of its brachium, the nucleus of the optic tract, and the lateral tegmental field. Additional positive neurons were seen in the parabigeminal nucleus and in the dorsal periaqueductal gray in kitten material. Immunoreactive fibers were observed in the periaqueductal gray and in the midbrain tegmentum, with particularly dense labeling just dorsal to the substantia nigra and in the parabrachial nuclei. This is the first report of the distribution of SS immunoreactivity in the midbrain of the cat. It is concluded that somatostatin has a distribution compatible with a role as a major neurotransmitter/neuromodulator within certain midbrain nuclei, especially the interpeduncular nucleus and the superior colliculus.     

Central distribution of trigeminal and upper cervical primary afferents in the rat studied by anterograde transport of horseradish peroxidase conjugated to wheat germ agglutinin

Injections of WGA-HRP were made in the rat trigeminal ganglion and C1-3 dorsal root ganglia (DRGs) to study the central projection patterns and their relations to each other. Trigeminal ganglion injections resulted in heavy terminal labeling in all trigeminal sensory nuclei. Prominent labeling was also observed in the solitary tract nucleus and in the medial parts of the dorsal horn at C1-3 levels, but labeling could be followed caudally to the C7 segment. Contralateral trigeminal projections were found in the nucleus caudalis and in the dorsal horn at C1-3 levels. The C1 DRG was found to be inconstant in the rat. When it was present, small amounts of terminal labeling were found in the external cuneate nucleus (ECN) and the central cervical nucleus (CCN). No dorsal horn projections were seen from the C1 DRG. Injections in the C2 DRG resulted in heavy labeling in the ECN, nucleus X, CCN, and dorsal horn, where it was mainly located in lateral areas. Labeling could be followed caudally to the Th 7 segment. C2 DRG projections also appeared in the cuneate nucleus (Cun), in all the trigeminal sensory nuclei, and in the spinal, medial, and lateral vestibular nuclei. A small C2 DRG projection was observed in the ventral cochlear nucleus. C3 DRG injections resulted in heavy labeling in both medial middle and lateral parts of the dorsal horn, in the ECN, and in nucleus X, whereas the labeling in the CCN was somewhat weaker. Smaller projections were seen to trigeminal nuclei, Cun, and the column of Clarke. Comparisons of the central projection fields of trigeminal and upper cervical primary afferents indicated a somatotopic organization but with a certain degree of overlap.     

The projection of the medial and posterior articular nerves of the cat's knee to the spinal cord

We studied the spinal projections of the medial and posterior articular nerves (MAN and PAN) of the knee joint in the cat with the aid of the transganglionic transport of horseradish peroxidase. The afferent fibers of the MAN entered the spinal cord via the lumbar dorsal roots L5 and L6 and those of the PAN entered via the dorsal roots L6 and L7. Within the dorsal root ganglia, most labeled neurons had small to medium diameters. A relatively higher number of medium-size cell bodies were labeled from the PAN than from the MAN. In the spinal cord labeled MAN afferent fibers and terminations were most dense in the L5 and L6 segments, and those of the PAN were most dense in L6 and L7, that is, in the respective segments of entry. Labeled afferent fibers from both nerves projected rostrally at least as far as L1 and caudally as far as S2. Labeled fibers were found in Lissauer's tract as well as in the dorsal column immediately adjacent to the dorsal horn. In the spinal gray matter, both nerves had two main projection fields, one in the cap of the dorsal horn in lamina I, the other in the deep dorsal horn in laminae V-VI and the dorsal part of lamina VII. Both nerves, but particularly the PAN, projected to the medial portion of Clarke's column. No projection was found to laminae II, III, and IV of the dorsal horn or to the ventral horn. Since these findings parallel observations on hindlimb muscle afferent fibers, the present data support the existence of a common pattern for the central distribution of deep somatic afferent fibers.     

Co-localization of endomorphin-2 and substance P in primary afferent nociceptors and effects of injury: a light and electron microscopic study in the rat

Endomorphin-2 (EM2) is a tetrapeptide with remarkable affinity and selectivity for the mu-opioid receptor. In the present study, we used double-fluorescence and electron microscopic immunocytochemistry to identify subsets of EM2-expressing neurons in dorsal root ganglia and spinal cord dorsal horn of adult rats. Within the lumbar dorsal root ganglia, we found EM2 immunoreactivity mainly in small-to-medium size neurons, most of which co-expressed the neuropeptide substance P (SP). In adult rat L4 dorsal root ganglia, 23.9% of neuronal profiles contained EM2 immunoreactivity and ranged in size from 15 to 36 microM in diameter (mean 24.3 +/- 4.3 microM). Double-labelling experiments with cytochemical markers of dorsal root ganglia neurons showed that approximately 95% of EM2-immunoreactive cell bodies also label with SP antisera, 83% co-express vanilloid receptor subtype 1/capsaicin receptor, and 17% label with isolectin B4, a marker of non-peptide nociceptors. Importantly, EM2 immunostaining persisted in mice with a deletion of the preprotachykinin-A gene that encodes SP. In the lumbar spinal cord dorsal horn, EM2 expression was concentrated in presumptive primary afferent terminals in laminae I and outer II. At the ultrastructural level, electron microscopic double-labelling showed co-localization of EM2 and SP in dense core vesicles of lumbar superficial dorsal horn synaptic terminals. Finally, 2 weeks after sciatic nerve axotomy we observed a greater than 50% reduction in EM2 immunoreactivity in the superficial dorsal horn. We suggest that the very strong anatomical relationship between primary afferent nociceptors that express SP and EM2 underlies an EM2 regulation of SP release via mu-opioid autoreceptors.