Populations Of Primary Afferents Research Articles

Glutamate in primary afferents is required for itch transmission

Whether glutamate or itch-selective neurotransmitters are used to confer itch specificity is still under debate. We focused on an itch-selective population of primary afferents expressing MRGPRA3, which highly expresses Vglut2 and the neuropeptide neuromedin B (Nmb), to investigate this question. Optogenetic stimulation of MRGPRA3+ afferents triggers scratching and other itch-related avoidance behaviors. Using a combination of optogenetics, spinal cord slice recordings, Vglut2 conditional knockout mice, and behavior assays, we showed that glutamate is essential for MRGPRA3+ afferents to transmit itch. We further demonstrated that MRGPRA3+ afferents form monosynaptic connections with both NMBR+ and NMBR- neurons and that NMB and glutamate together can enhance the activity of NMBR+ spinal DH neurons. Moreover, Nmb in MRGPRA3+ afferents and NMBR+ DH neurons are required for chloroquine-induced scratching. Together, our results establish a new model in which glutamate is an essential neurotransmitter in primary afferents for itch transmission, whereas NMB signaling enhances its activities.

Kappa Opioid Receptor Distribution and Function in Primary Afferents

Primary afferents are known to be inhibited by kappa opioid receptor (KOR) signaling. However, the specific types of somatosensory neurons that express KOR remain unclear. Here, using a newly developed KOR-cre knockin allele, viral tracing, single-cell RT-PCR, and exvivo recordings, we show that KORis expressed in several populations of primary afferents: a subset of peptidergic sensory neurons, as well as low-threshold mechanoreceptors that form lanceolate or circumferential endings around hair follicles. We find that KOR acts centrally to inhibit excitatory neurotransmission from KOR-cre afferents in laminae I and III, and this effect is likely due to KOR-mediated inhibition of Ca2+ influx, which weobserved in sensory neurons from both mouse and human. In the periphery, KOR signaling inhibits neurogenic inflammation and nociceptor sensitization by inflammatory mediators. Finally, peripherally restricted KOR agonists selectively reduce pain and itch behaviors, as well as mechanical hypersensitivity associated with a surgical incision. These experiments provide a rationale for the use of peripherally restricted KOR agonists for therapeutic treatment.

Analgesia by Deletion of Spinal Neurokinin 1 Receptor Expressing Neurons Using a Bioengineered Substance P-Pseudomonas Exotoxin Conjugate.

Cell deletion approaches to pain directed at either the primary nociceptive afferents or second-order neurons are highly effective analgesic manipulations. Second-order spinal neurons expressing the neurokinin 1 (NK1) receptor are required for the perception of many types of pain. To delete NK1+ neurons for the purpose of pain control, we generated a toxin–peptide conjugate using DTNB-derivatized (Cys0) substance P (SP) and a N-terminally truncated Pseudomonas exotoxin (PE35) that retains the endosome-release and ADP-ribosylation enzymatic domains but with only one free sulfhydryl side chain for conjugation. This allowed generation of a one-to-one product linked by a disulfide bond (SP-PE35). In vitro, Chinese hamster ovary cells stably transfected with the NK1 receptor exhibited specific cytotoxicity when exposed to SP-PE35 (IC50 = 5 × 10−11 M), whereas the conjugate was nontoxic to NK2 and NK3 receptor-bearing cell lines. In vivo studies showed that, after infusion into the spinal subarachnoid space, the toxin was extremely effective in deleting NK1 receptor-expressing cells from the dorsal horn of the spinal cord. The specific cell deletion robustly attenuated thermal and mechanical pain sensations and inflammatory hyperalgesia but did not affect motoric capabilities. NK1 receptor cell deletion and antinociception occurred without obvious lesion of non–receptor-expressing cells or apparent reorganization of primary afferent innervation. These data demonstrate the extraordinary selectivity and broad-spectrum antinociceptive efficacy of this ligand-directed protein therapeutic acting via receptor-mediated endocytosis. The loss of multiple pain modalities including heat and mechanical pinch, transduced by different populations of primary afferents, shows that spinal NK1 receptor-expressing neurons are critical points of convergence in the nociceptive transmission circuit. We further suggest that therapeutic end points can be effectively and safely achieved when SP-PE35 is locally infused, thereby producing a regionally defined analgesia.

Resiniferatoxin: The Evolution of the "Molecular Scalpel" for Chronic Pain Relief.

Control of chronic pain is frequently inadequate or can be associated with debilitating side effects. Ablation of certain nociceptive neurons, while retaining all other sensory modalities and motor function, represents a new therapeutic approach to controlling severe pain while avoiding off-target side effects. transient receptor potential cation channel subfamily V member 1 (TRPV1) is a calcium permeable nonselective cation channel expressed on the peripheral and central terminals of small-diameter sensory neurons. Highly selective chemoablation of TRPV1-containing peripheral nerve endings, or the entire TRPV1-expressing neuron itself, can be used to control chronic pain. Administration of the potent TRPV1 agonist resiniferatoxin (RTX) to neuronal perikarya or nerve terminals induces calcium cytotoxicity and selective lesioning of the TRPV1-expressing nociceptive primary afferent population. This selective neuroablation has been coined “molecular neurosurgery” and has the advantage of sparing motor, proprioceptive, and other somatosensory functions that are so important for coordinated movement, performing activities of daily living, and maintaining quality of life. This review examines the mechanisms and preclinical data underlying the therapeutic use of RTX and examples of such use for the management of chronic pain in clinical veterinary and human pain states.

Identification of a Neuronal Receptor Controlling Anaphylaxis.

Allergic reactions can in severe cases induce a state of circulatory shock referred to as anaphylaxis. Histamine, the primary mediator of this condition, is released from immune cells, and, therefore, anaphylaxis has so far been considered an immune system disorder. However, we here show that the glutamatergic receptor mGluR7, expressed on a subpopulation of both peripheral and spinal cord neurons, controls histamine-induced communication through calcium-dependent autoinhibition with implications for anaphylaxis. Genetic ablation of mGluR7, and thus altered regulation of histamine-sensing neurons, caused an anaphylaxis-like state in mGluR7(-/-) mice, which could be reversed by antagonizing signaling between neurons and mast cells but not by antagonizing a central itch pathway. Our findings demonstrate the vital role of nervous system control by mGluR7 in anaphylaxis and open up possibilities for preventive strategies for this life-threatening condition.

De novo expression of neurokinin‐1 receptors by spinoparabrachial lamina I pyramidal neurons following a peripheral nerve lesion

Lamina I of the spinal dorsal horn is a major site of integration and transmission to higher centers of nociceptive information from the periphery. One important primary afferent population that transmits such information to the spinal cord expresses substance P (SP). These fibers terminate in contact with lamina I projection neurons that express the SP receptor, also known as the neurokinin-1 receptor (NK-1r). Three types of lamina I projection neurons have been described: multipolar, fusiform, and pyramidal. Most neurons of the first two types are thought to be nociceptive and express the NK-1r, whereas most pyramidal neurons are nonnociceptive and do not express the NK-1r. In this immunocytochemical and behavioral study, we induced a neuropathic pain-like condition in the rat by means of a polyethylene cuff placed around in the sciatic nerve. We document that this lesion led to a de novo expression of NK-1r on pyramidal neurons as well as a significant increase in SP-immunoreactive innervation onto these neurons. These phenotypic changes were evident at the time of onset of neuropathic pain-related behavior. Additionally, we show that, after a noxious stimulus (intradermal capsaicin injection), these NK-1r on pyramidal neurons were internalized, providing evidence that these neurons become responsive to peripheral noxious stimulation. We suggest that the changes following nerve lesion in the phenotype and innervation pattern of pyramidal neurons are of significance for neuropathic pain and/or limb temperature regulation.

Intact Aδ-fibers up-regulate transient receptor potential A1 and contribute to cold hypersensitivity in neuropathic rats

Mechanisms underlying cold hypersensitivity in neuropathic states are unclear. Recent data indicate both transient receptor potential (TRP) M8 and TRPA1 play a role. In relation to TRPA1, there are reported increases in mRNA. However, it is unknown whether TRPA1 mRNA is translated into functional receptors, whether these receptors are found on peripheral nociceptors and what population of primary afferents expresses the receptors. The present study provides several lines of evidence that TRPA1 receptors are expressed on intact primary sensory neurons and contribute to cold hypersensitivity following spinal nerve ligation (SNL). Immunohistochemical studies show that expression of TRPA1 is significantly increased in the ipsilateral compared with the contralateral L4 dorsal root ganglion (DRG). Using mustard oil (MO, selective TRPA1 agonist), Ca 2+ imaging demonstrates an increase in the percentage of MO-sensitive L4 DRG cells in SNL compared with sham and naive rats. The magnitude of the Ca 2+ response evoked by MO is also significantly larger in SNL compared with sham and naive rats. Behavioral studies demonstrate that SNL results in increased nocifensive behaviors to mechanical and cold stimulation that is not seen in sham or naive rats. Behavioral responses in sham rats are no different from naive rats. In vitro single fiber recordings demonstrate Aδ-fibers (intact L4 axons) in the nerve-injured hind paw have conduction velocities no different from naive rats. In contrast, compared with naive rats, mechanical thresholds of the Aδ-fibers in SNL rats are significantly decreased, the proportion of cold-sensitive and MO-sensitive Aδ-fibers is significantly increased and the response magnitude of Aδ-fibers to MO is significantly increased. MO-induced activity in Aδ-fibers is significantly reduced by Ruthenium Red (TRPA1 receptor antagonist). These results demonstrate that TRPA1 is expressed on peripheral nociceptors, and they are up-regulated on intact Aδ-fibers following nerve injury, contributing to cold hypersensitivity.

Retigabine-induced population primary afferent hyperpolarisation in vitro

Retigabine is a compound of potential interest in analgesia which acts as an M-channel opener to depress neuronal excitability. Here we study the effects of retigabine and its antagonist XE-991 on populations of primary afferents. Experiments were performed using a hemisected spinal cord preparation from rat pups maintained under in vitro conditions. Recording from dorsal roots were performed using tight fitting suction electrodes coupled to AC and DC amplifiers. The adjacent dorsal root was electrically stimulated at regular intervals. The effects of the modulators on basal potential, spontaneous potentials and dorsal root-dorsal root responses were studied. Superfusion of retigabine (10 μM) produced long lasting and robust hyperpolarisation of primary afferents which persisted during superfusion of picrotoxin (20 μM) and tetrodotoxin (0.5 μM). Other effects of retigabine were (1) increase in stimulation threshold; (2) increase in size of responses to suprathreshold stimuli; and (3) increase in amplitude and decrease in frequency of spontaneous dorsal root potentials. Superfusion of XE-991 had little effect on its own but blocked all the effects of retigabine. These results indicate the presence of functional M-currents in central terminals of primary afferents and in the interneurones that mediate dorsal root potentials. The depressant effects of retigabine on the excitability of these structures may contribute to its analgesic effects after pain-inducing treatments.

Cholera toxin B subunit and isolectin B4 label distinct populations of vagal afferents projecting to the nucleus tractus solitarii

The vagus nerve contains primary visceral afferents that convey information from cardiovascular, pulmonary, and gastrointestinal tissues to nucleus tractus solitarii (NTS). We tested the hypothesis that different anterograde tracers will label distinct populations of primary afferents. In somatic nerves, cholera toxin B subunit (CTb) and isolectin B4 (IB4) have been shown to label myelinated and unmyelinated fibers, respectively. We also examined the vesicular glutamate transporter 2 (VGlut2) content of these afferents. Male Sprague-Dawley rats (275–350 g) received injections of either CTb or IB4 (2% solution, 1–4 μl) into the vagus nerve under isoflurane anesthesia. Seven days later, rats were perfused and vibratome sections through NTS were examined with dual immunocytochemistry for either CTb (goat anti-CTb, List) or IB4 (goat anti-IB4, Vector) and VGlut2 (guinea pig anti-VGlut2, Chemicon), visualized using fluorescent secondary antibodies. CTb and IB4 produced distinct patterns of afferent labeling in NTS following vagal nerve injections. CTb produced both anterograde labeling in NTS and retrograde labeling of vagal motor neurons, while IB4 labeled only afferents. By confocal microscopy, we found VGlut2 in a subset of CTb-labeled vagal afferents, but VGlut2 was rarely found in IB4-labeled afferents. By electron microscopy we found that CTb afferents were both myelinated and unmyelinated, but IB4 afferents were only unmyelinated. These findings suggest that these tracers label largely distinct, but partially overlapping, populations of vagal afferents to NTS with distinct phenotypes. Support: NIH HL56301

Dorsal root ganglion neurons show increased expression of the calcium channel α2δ-1 subunit following partial sciatic nerve injury

Neuropathic pain is associated with changes in the electrophysiological and neurochemical properties of injured primary afferent neurons. A mRNA differential display study in rat L 4/5 dorsal root ganglia (DRGs) revealed upregulation of the calcium channel α2δ-1 subunit 2 weeks after partial sciatic nerve ligation (Seltzer model of neuropathic pain). The upregulated transcript appeared to represent previously unidentified sequence from the 3′-untranslated region of rat α2δ-1 mRNA. In situ hybridization using L 5 DRGs from sham operated rats showed that 73, 40 and 19% of small (<700 μm 2), medium (700–1100 μm 2) and large (>1100 μm 2) neuronal profiles, respectively, expressed α2δ-1 mRNA. Two weeks following nerve injury there was a significant ipsilateral increase, both in the percentage of DRG neurons expressing α2δ-1 mRNA and in the intensity of the hybridization signal. Comparison of this ipsilateral expression with that in sham animals, revealed that for small, medium and large neurons, respectively, the proportion of neurons labelled increased by 1.2-, 1.8- and 2.7-fold, while the hybridization signal in α2δ-1-labelled neurons increased by 2.8-, 2.5- and 3.7-fold. The most intensely labelled neuronal profiles in ipsilateral, sham and contralateral DRGs, were generally those with small cross-sectional areas. The α2δ-1 auxiliary subunit is known to modulate calcium channel function in heterologous expression systems via its association with the pore-forming α1 calcium channel subunit. Therefore the increased levels of this subunit in the populations of primary afferents described may, via modulation of calcium-dependent processes such as neurotransmitter release and neuronal excitability, influence the processing of sensory information.

Spatiotemporal processing of linear acceleration: primary afferent and central vestibular neuron responses.

Spatiotemporal convergence and two-dimensional (2-D) neural tuning have been proposed as a major neural mechanism in the signal processing of linear acceleration. To examine this hypothesis, we studied the firing properties of primary otolith afferents and central otolith neurons that respond exclusively to horizontal linear accelerations of the head (0.16-10 Hz) in alert rhesus monkeys. Unlike primary afferents, the majority of central otolith neurons exhibited 2-D spatial tuning to linear acceleration. As a result, central otolith dynamics vary as a function of movement direction. During movement along the maximum sensitivity direction, the dynamics of all central otolith neurons differed significantly from those observed for the primary afferent population. Specifically at low frequencies (</=0.5 Hz), the firing rate of the majority of central otolith neurons peaked in phase with linear velocity, in contrast to primary afferents that peaked in phase with linear acceleration. At least three different groups of central response dynamics were described according to the properties observed for motion along the maximum sensitivity direction. "High-pass" neurons exhibited increasing gains and phase values as a function of frequency. "Flat" neurons were characterized by relatively flat gains and constant phase lags (approximately 20-55 degrees ). A few neurons ("low-pass") were characterized by decreasing gain and phase as a function of frequency. The response dynamics of central otolith neurons suggest that the approximately 90 degrees phase lags observed at low frequencies are not the result of a neural integration but rather the effect of nonminimum phase behavior, which could arise at least partly through spatiotemporal convergence. Neither afferent nor central otolith neurons discriminated between gravitational and inertial components of linear acceleration. Thus response sensitivity was indistinguishable during 0.5-Hz pitch oscillations and fore-aft movements. The fact that otolith-only central neurons with "high-pass" filter properties exhibit semicircular canal-like dynamics during head tilts might have important consequences for the conclusions of previous studies of sensory convergence and sensorimotor transformations in central vestibular neurons.

A novel methodology for simultaneous assessment of the effects of 5-hydroxytryptamine on primary afferent polarisation and synaptic transmission in rat dorsal horn neurones in vitro

The rat hemisected spinal cord in vitro preparation was used to test simultaneously the effects of 5-hydroxytryptamine (5-HT) on primary afferent polarisation and synaptic transmission onto dorsal horn (DH) neurons. Primary afferent polarisation was measured from the cut end of a transected lumbar dorsal root (DR; L3–L6) using tight suction electrodes coupled to a D.C. amplifier. Conventional sharp microelectrode were used to record intracellularly the excitatory postsynaptic potential (EPSP) evoked by high intensity electrical stimulation (100 μA, 100 μs) of another DR contiguous to that used for the suction electrode recording. Superfusion of 5-HT (5–10 μM) caused primary afferent depolarisations (PAD) of 227.5 ± 26.5 μV (mean ± SEM) and 221 ± 32 μV, respectively, values comparable to the PAD caused by 10–100 μM γ-aminobutyric acid (GABA) superfusion. 5-HT-induced PAD was tetrodotoxin (TTX) resistant and non-additive to capsaicin-induced PAD suggesting a direct depolarising action of 5-HT on a population of primary afferents which may include a high proportion of unmyelinated fibres. Simultaneous intracellular recordings showed that 5-HT, in addition to generating PAD, depressed primary afferent-evoked synaptic transmission to DH neurons reflected by a significant reduction ( p < 0.05) in the amplitude and duration of the EPSP. In contrast, GABA, despite producing a PAD of similar amplitude, failed to depress synaptic transmission. These data suggest that PAD alone may be insufficient to account for the 5-HT-induced depression of synaptic transmission. This novel experimental approach offers a means to explore further the possible causal relationship between pre- and post-synaptic effects of 5-HT in the DH and its ability to modulate somatosensory processing and mociception.

A novel methodology for simultaneous assessment of the effects of 5-hydroxytryptamine on primary afferent polarisation and synaptic transmission in rat dorsal horn neurones in vitro

The rat hemisected spinal cord in vitro preparation was used to test simultaneously the effects of 5-hydroxytryptamine (5-HT) on primary afferent polarisation and synaptic transmission onto dorsal horn (DH) neurons. Primary afferent polarisation was measured from the cut end of a transected lumbar dorsal root (DR; L3–L6) using tight suction electrodes coupled to a D.C. amplifier. Conventional sharp microelectrode were used to record intracellularly the excitatory postsynaptic potential (EPSP) evoked by high intensity electrical stimulation (100 μA, 100 μs) of another DR contiguous to that used for the suction electrode recording. Superfusion of 5-HT (5–10 μM) caused primary afferent depolarisations (PAD) of 227.5 ± 26.5 μV (mean ± SEM) and 221 ± 32 μV, respectively, values comparable to the PAD caused by 10–100 μM γ-aminobutyric acid (GABA) superfusion. 5-HT-induced PAD was tetrodotoxin (TTX) resistant and non-additive to capsaicin-induced PAD suggesting a direct depolarising action of 5-HT on a population of primary afferents which may include a high proportion of unmyelinated fibres. Simultaneous intracellular recordings showed that 5-HT, in addition to generating PAD, depressed primary afferent-evoked synaptic transmission to DH neurons reflected by a significant reduction (p < 0.05) in the amplitude and duration of the EPSP. In contrast, GABA, despite producing a PAD of similar amplitude, failed to depress synaptic transmission. These data suggest that PAD alone may be insufficient to account for the 5-HT-induced depression of synaptic transmission. This novel experimental approach offers a means to explore further the possible causal relationship between pre- and post-synaptic effects of 5-HT in the DH and its ability to modulate somatosensory processing and mociception.

TrkA, CGRP and IB4 expression in retrogradely labelled cutaneous and visceral primary sensory neurones in the rat

The pattern of trkA expression in relation to other neurochemical markers (CGRP and IB4) was investigated in primary sensory neurones innervating either the skin or bladder. Retrograde tracing using the fluorescent marker Fast Blue was performed followed by histochemistry. A greater proportion of visceral afferents compared with cutaneous afferents were trkA-immunoreactive (75% and 43%, respectively). CGRP expression correlated with trkA expression in that it was higher in visceral afferents than cutaneous afferents (69% and 51%, respectively). IB4 expression was negatively correlated with trkA expression, being lower in visceral afferents compared with cutaneous afferents (29% and 43%, respectively). The results emphasise the heterogeneity of trkA expression (and hence nerve growth factor sensitivity) in afferents innervating different targets, and furthermore suggest that it is predominantly the CGRP-expressing population of primary afferents that express trkA.

Distribution of small-diameter primary afferents containing oligosaccharide residues recognized by monoclonal antibody LA4 in the dorsal horn of the cat

Small-diameter primary afferents expressing oligosaccharide residues recognized by the monoclonal antibody LA4 constitute a newly described primary afferent population in the cat. LA4-immunoreactive primary afferents are found mainly in lamina II (LII) at all levels of the cat spinal cord. However, within LII immunoreactive fibers are most concentrated in outer LII and practically absent from the inner third of the lamina. In addition at sacral levels, a lateral bundle of immunoreactive fibers enters laminae V–VII. The distribution pattern and the type of oligosaccharide expressed by LA4-immunoreactive fibers suggest that they may be homologous to primary afferents shown in the rat to contain fluoride-resistant acid phosphatase activity. However, in the rat spinal cord the whole of inner LII is the main projection area of LA4-immunoreactive fibers (Alvarez et al., J. Neurocytol., 18 (1989) 611–629). This difference in the location of primary afferents within LII may reflect a species difference in the physiology of LII inner.

Calcitonin gene-related peptide containing primary afferent fibers synapse on primate spinothalamic tract cells

Spinothalamic tract (STT) cells were identified by intracellular injection or retrograde labeling with horseradish peroxidase (HRP) in the primate spinal cord. Using immunohistochemical techniques, a population of primary afferents containing calcitonin gene-related peptide (CGRP) is demonstrated in synaptic contact with these neurons. Large glomerular type CGRP terminals with morphology considered typical of primary afferent fibers are occasionally observed in contact with STT profiles in laminae I and IIo; however, this morphological type targets STT profiles chiefly in the deeper dorsal horn (laminae III, IV and V). In contrast, the majority of CGRP terminals contacting STT profiles in the superficial dorsal horn (laminae I and IIo), are small, round or oblong shaped terminals. Thus, evidenced by these data, the absence of large, glomerular type terminals does not rule out primary afferent input.

Nerve section alters the interaction between C-fibre activity and intrathecal neuropeptides on the flexor reflex in rat

The hamstring flexor reflex evoked by C-fibre strength electric shocks to the sural nerve was recorded in unanaesthetised, decerebrate, spinalised rats with sciatic nerves intact or sectioned 14–21 days previously. The effect of a brief C-fibre strength conditioning stimulus (CS) train (20 s, 1 Hz) to the sural nerve or intrathecally applied neuropeptides substance P (SP), vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) on reflex excitability were quantified. The sural CS and all of the neuropeptides caused a brief (1–5 min) facilitation of the test reflex with no concomitant changes in the amplitude of the monosynaptic reflex. A synergistic facilitatory interaction between the CS and SP, but not between the CS and VIP was observed in rats with intact nerves. In nerve-sectioned animals no synergism was found between CS and SP, but a very strong synergistic facilitation was seen with CS and VIP. Since SP and VIP both coexist with CGRP in separate populations of primary afferents, the interaction between SP plus CGRP and VIP plus CGRP was tested in animals with intact and sectioned nerves. In animals with intact nerves, SP and CGRP had a strong synergistic effect, whereas VIP and CGRP had a weak facilitatory interaction. In animals with sectioned nerves, SP and CGRP did not interact, whereas VIP and CGRP had a stronger facilitatory interaction. Since it has been shown that there are changes in peptide levels in primary afferents and dorsal horn interneurons following nerve section, it is proposed that such alterations may underly some of the physiological changes in spinal cord function after damage to peripheral nerves.

Action of intrathecal capsaicin and its structural analogues on the content and release of spinal substance P: Selectivity of action and relationship to analgesia

Intrathecal injections of capsaicin (CAP) and 4 other homovanillic acid (HMV) derivatives related to the structure of CAP were carried out. Capsaicin, 1-nonenoylvanillylamide (NVA), HMV-dodecylamide (DCA) (but not HMV-cyclohexylamide (CHA) or HMV-hexadecylamide (HDC)) reduced the spinal content of substance P (SP), as measured by radioimmunoassay (RIA), and increased the tail-flick latency. Similar injection of kainic acid and piperine reduced levels of SP but failed to affect the tail-flick latency. None of the agents used affected spinal levels of cholecystokinin (CCK) or vasoactive intestinal peptide (VIP) as measured by RIA. In experiments using in vivo superfusion of the rat spinal cord, CAP, DCA and NVA were found to stimulate release of SP. Capsaicin had no effect on the levels of CCK or VIP immunoreactivity in the spinal superfusate. A tachyphylaxis to the effect of CAP and DCA on spinal SP release was demonstrated. Pretreatment with either agent blocked the releasing effect of the second. Pretreatment with an inactive analogue (HDC) had no effect on the subsequent activity of CAP. Kainic acid and piperine did not induce release of SP from the spinal cord. The relative selectivity of spinally administered capsaicinoids with regard to their effects on the content and release of peptides known to be contained in primary afferents and the presence of a similar structure-activity relationship for depletion and release of SP, desentization and antinociception suggest the presence of a specific receptor site associated with a specific population of primary afferents through which pain information may pass. Whether SP is an ‘afferent pain transmitter’ is not clear, but at the least, it appears to serve as a marker for a population of afferents acted upon by spinally administered capsaicinoids.

Light and electron microscopic autoradiographic study of the dorsal root projections to the cat dorsal horn

The distribution of terminals arising from dorsal root primary afferents was examined in the lumbar spinal cord of cats using light- and electron-microscopic autoradiography. Tritiated proline or leucine was injected into either the L6 or L7 dorsal root ganglion. The light-microscopic spinal cord distribution of radioactivity in the ganglia was independent of the type of amino acid used. Likewise, the length of the survival time after injection had no effect. The projections to the substantia gelatinosa and the marginal zone were consistently the densest. However, the topography of the dorsal horn distribution, relative the the segment of entry, varied significantly especially in the gelatinosa, depending upon the ganglia injected. Those to the substantia gelatinosa were largely limited to the segment of entry; those to the marginal zone and nucleus proprius extended many segments beyond the level of entry. At all levels the projection was exclusively ipsilateral to the side of injection. At the electron-microscopic level the distribution of radioactivity was determined in each of the three easily recognizable areas of the dorsal horn: the marginal zone, the substantia gelatinosa and the nucleus proprius. In each dorsal horn area the total terminal population was divided into four basic categories. Each of these areas was found to contain a characteristic distribution of these four terminal categories. The difference between areas arose, primarily, as a consequence of the dorsal to ventral decreasing frequency gradient of two types of terminal: those containing large, dense-cored vesicles and the increasing gradient of those containing flattened vesicles. The terminals with small pleomorphic vesicles and those with large round vesicles were frequently encountered in all three areas without a detectable frequency gradient. Similarly, the primary afferent terminal population, that is the subset of the total terminal population labelled after dorsal root ganglion injection, was also characteristic of the area, and each area was dominated by a different terminal type. In the marginal zone the terminals containing large dense-cored vesicles dominated. In the substantia gelatinosa the terminals with pleomorphic vesicles (which included the so-called ‘C’ type terminals) dominated. And the terminals containing large round vesicles dominated the primary afferent population in the nucleus proprius. The terminals containing flattened vesicles were never found to be specifically labeled in any of the areas examined.