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Spinal Pharmacology of Nociceptive Transmission
SUMMARY
The activity evoked in primary afferent fibers by high-intensity stimuli or tissue injury leads to a pattern of evoked spinal activity and then spinifugal outflow, which in turn activates supraspinal linkages. This review considers the multiple transmitter systems in terms of their agonists and the respective receptors that subserve these afferent–spinal, spinal–brain stem, and brain stem–forebrain projections. A variety of local and long loop connections exist at every neuraxial level to modulate (either enhance or attenuate) the evoked afferent traffic. As important as the afferent linkages are, systems that regulate their processing through these rostrally projecting elements are equally important. Accordingly, the pharmacology of these modulatory systems is also reviewed. Importantly, the behavioral relevance of these anatomically and pharmacologically defined systems to pain processing is addressed by merging techniques that locally deliver agonists and antagonists with measurement of the pain behavior evoked in well-defined behavioral models. The resulting picture is one of complex interactions between systems activated by noxious stimuli and the modulatory processes that alter the message and behavior generated by these stimuli.
Introduction
Acute thermal or mechanical stimuli—or chemicals released from damaged tissue—applied acutely to the skin, muscle, or viscera in the absence of prior conditioning or training evoke a constellation of well-defined behavior and characteristic changes in autonomic function that are defined as nociception. As , “Stimuli become adequate as excitants of pain when they are of such intensity as threatens damage to the skin.” The composition of the behavioral sequelae to such stimuli in the unanesthetized, intact animal varies with the state of arousal, species, and age but will typically include signs of agitation, vocalization, and coordinated efforts to escape (e.g., withdrawal of the limb) or to attenuate the magnitude of the stimulus (e.g., licking or shaking the stimulated limb). The more intense the acute stimulus, the greater the pain indices (e.g., decreased response latency or increased magnitude of responding). With frank tissue injury or inflammation, the organism will often display evidence of ongoing pain behavior even after the injuring stimulus has been removed, and the same stimulus may now elicit an enhanced magnitude of pain behavior. The state corresponding to this facilitated behavioral response is referred to as “hyperalgesia.” Pragmatically, if the hyperalgesia includes an exaggerated response produced by a frankly non-noxious stimulus (e.g., light brushing of the skin), we may further define this second component as allodynia. Our aim is to understand the pharmacology of the systems that mediate these behaviorally relevant phenomena. Such systems may be considered in terms of the overall organization of the encoding substrates.
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The forward flow of information of excitatory input through the dorsal horn to cortical centers is regulated at every level by local and long loop circuits, which by actions pre- and post-synaptic to the afferent pathway modulate the excitability of the synapse (see Chapter 5 , Chapter 6 , Chapter 8 ) such that the response to a given afferent input may be either augmented or reduced.
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Finally, it is understood that the pain experience is organized by substrates that define the affective component associated with stimuli that affect physical integrity. Functional imaging studies have revealed that brain regions such as the limbic cortex, which is not believed to contribute directly to somatosensory encoding, receive information affiliated with the pain experience and that activity in these regions often parallels the behavioral correlates of the stimulus conditions (see Chapter 7 ).
The present chapter seeks to provide an overview of the pharmacology associated with the several components of this afferent trafficking, with an emphasis on the effects of such agents on the pain behavior of the organism.
Thus, combined study of the behavioral states induced by specific and well-defined nociceptive stimuli with specific effort to assess receptor pharmacology within terminal regions of the anatomical tracts through which this information projects allows us to define the behavioral relevance of these systems to nociceptive processing. Such focal pharmacological manipulation in the intact and unanesthetized animal is achieved through the delivery of drugs in a reliable, delimited manner to specific regions of the central nervous system (CNS). In the brain, placement of intracerebroventricular cannulae permits assessment of central action but affords little anatomic specificity of the site of action. However, stereotaxic placement of microinjection cannulae combined with small injection volumes and iontophoretic administration of agents helps define local CNS pharmacology. Spinal drug delivery using chronic catheters or acute injections has permitted examination of the pharmacology of spinal systems that alter nociceptive transmission ( ). Factors governing the degree of localization of drug action after intracerebral or intrathecal delivery have been reviewed intensively elsewhere.
Excitatory Transmitters in the Afferent Components of Nociceptive Processing
The following sections consider the pharmacology of the systems that subserve the rostral flow of information generated by small afferent input into the dorsal horn and the subsequent projections via crossed and uncrossed tracts into the brain stem and diencephalon.
Primary Afferents: Transmitters and Receptor Systems
Physiology of the First-Order Synapse
Several properties characterize the nature of the interaction between primary afferent fibers and second-order neurons.
Post-synaptic Effect
Single-unit recording has indicated that primary afferent stimulation results in powerful excitation of dorsal horn neurons. Dating from the earliest systematic studies ( ), there has been no evidence that primary afferents induce monosynaptic inhibition in the dorsal horn (see, for example, reviews of dorsal horn function: ). This property suggests that the putative afferent transmitters should largely be characterized by their ability to evoke excitatory post-synaptic potentials (EPSPs) in second-order dorsal horn neurons.
Multiple Neurotransmitters in a Terminal
Stimulation of nerve filaments at intensities that activate small, slowly conducting afferents typically reveals the existence of at least two populations of EPSPs that are believed to be monosynaptic: (1) fast and of brief duration and (2) delayed and of extended duration ( ). Although the presence of different EPSPs on the same membrane may reflect monosynaptic input from two different families of axons and/or the presence of interneurons contributing to the slow EPSP, such multiple EPSP morphologies in fact also reflect the presence of several distinct classes of neurotransmitters released from a given terminal acting on the dorsal horn neuron, including excitatory amino acids ( ; ; ), purines ( ), and peptides ( ). Release of multiple transmitters from a single terminal at a single synapse is supported by electron microscopy, which frequently shows the presence of morphologically distinct (small clear-core versus large dense-core) populations of vesicles within the same terminal bouton (see ). These differences are consistent with the broader appreciation in neurobiology that such morphologically distinct vesicles reflect the co-containment of distinct classes of releasable neurotransmitters within the same terminal ( ). Examination of the distribution of glutamate indicates, for example, that it is probably contained in small open-core vesicles whereas large dense-core vesicles are believed to contain peptides (see ).
The association of peptides with dense-core vesicles and amino acids with clear-core vesicles has practical consequence when it comes to the depolarization/secretion properties of these transmitter classes. Dense-core vesicles typically reside farther from the synaptic density than clear-core vesicles do. The intracellular Ca 2+ required to couple local depolarization to vesicular release arises from voltage-dependent Ca 2+ channels within the synaptic density. Thus, in general, greater depolarization (associated with a higher firing frequency as observed after tissue injury) is required for the intracellular Ca 2+ concentration to reach the mobilization threshold in the vicinity of the dense-core vesicles ( ). This association supports the notion that peptide release is comparatively enhanced with persistent activation.
Stimulus Intensity and Afferent Release
As reviewed elsewhere in this volume, an important characteristic of the primary afferent–encoding process is that the magnitude of the generator potential and the frequency of the action potential are largely a function of peripheral stimulus intensity. At the spinal terminal, larger generator potentials lead to the progressive opening of more voltage-sensitive calcium channels that serve to mobilize vesicles for release of transmitter. Accordingly, transmitter release and post-synaptic depolarization will typically be a function of action potential frequency. Importantly, as reviewed below, coupling between afferent traffic and release can be significantly increased or decreased by local modulatory factors that regulate excitation–secretion coupling (e.g., as in opening of the voltage-sensitive calcium channel, mobilization of synaptic proteins) or terminal depolarization.
Primary Afferent Terminal Calcium Channels
Depolarization of the primary afferent terminal leads to the opening of voltage-gated calcium channels (VGCCs). A variety of VGCCs have been identified as defined by their activation characteristics, structural subunit composition, and pharmacology ( ). Several are present in the dorsal root ganglion (DRG) and primary afferent fiber central terminals ( ). Activation of these channels, presynaptic to the primary afferent, serves a number of critical functions: (1) they generate depolarizing membrane current at the terminal, (2) they initiate release of transmitter by promoting the activation of membrane docking proteins such as SNAP 25 and VAMP ( ), (3) they initiate phosphorylation of membrane proteins (e.g., N -methyl- d -aspartate [NMDA] and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] receptors, which can enhance channel efficacy; ), or (4) they activate cytosolic and membrane enzymes (e.g., phospholipase A 2 [PLA 2 ]; ). Blockade of several species of calcium channels, notably those for the N-, T-, and L-type channels, potently diminishes post-synaptic depolarization. Interestingly, direct assessment of peptide release via substance P (SP) receptor internalization has shown that N- but not T- or L-type blockers ( ) prevent the evoked release of SP from small nociceptive afferents ( ). It should be noted that post-synaptic calcium channels are also important. Post-synaptic currents, initiated by afferent input, are also reduced by N-type channel blockers, but much less so by P/Q-type and L-type channel blockers.
Afferent Transmitters and Their Receptors
The role of distinctive populations of terminals remains to be determined, but the physiological properties of coupling of the respective receptors suggest distinct mechanisms of afferent encoding. An essential characteristic of these agents is their ability to be released into the extracellular milieu following depolarization of the primary afferent terminals. Thus, in vivo, activation of C-fiber afferents elicits the release of SP ( ), calcitonin gene–related peptide (CGRP; ), vasoactive intestinal polypeptide (VIP; ), somatostatin (SST; ), and glutamate ( ). At present, analysis of laminae I and II of the dorsal horn (regions where small afferents are known to terminate, see Chapter 5 ) and small DRG cells (considered to be the cells of origin of small unmyelinated and finely myelinated afferent axons) has revealed the presence of a large number of possible transmitter candidates.
As noted above, multiple neurotransmitters are commonly present within any given terminal, frequently the excitatory amino acid glutamate and a peptide such as SP. These neurotransmitters are summarized in Figures 28-1 and 28-2 . Given the ability of glutamate, acting through receptor-gated Na + or Ca 2+ channels, to produce rapid EPSPs and the ability of peptides to decrease K + conductance and yield slow, long-lasting EPSPs, co-containment allows a single terminal to evoke multiphasic post-synaptic events. Distinct populations of afferent fibers can be defined on the basis of their peptide contents ( ). For example, histochemical analysis of lumbar DRG cells has typically revealed that 50% contain CGRP and 30% contain SP; 96% of the CGRP-positive cells also showed SP immunoreactivity ( ). Populations of C fibers have been identified as peptidergic (containing, for example, SP and CGRP) and as non-peptidergic (identified by binding of the plant lectin isolectin B4 [IB4]) ( ). A significant number of large Aβ fibers (up to 20%) are also nociceptive (see for review), but little is known about their specific pharmacology, and thus they will be noted but not specifically considered.
Amino Acids
Transmitter System
Glutamate is found in 65–80% of DRG and trigeminal ganglion neurons ( ). Although aspartate was at first considered to be an afferent neurotransmitter, there is no functional evidence for this and only glutamate will be considered. Many sensory neurons exhibiting glutamate immunoreactivity have small perikarya that link them to small primary afferent fibers. Electromicrographic studies using afferent transport markers have shown glutamate to be present in the dorsal horn terminals of large fractions of both myelinated and unmyelinated axons ( ). Specific activation of small afferents with capsaicin evokes the release of glutamate from primary afferent neurons ( ). Recovery of glutamate in microdialysates of the dorsal spinal cord in vivo is increased several-fold after the injection of noxious chemicals into the periphery ( ), thus providing additional support for the hypothesis that glutamate is released from afferent nociceptors, although other cellular sources of excitatory amino acids are not excluded by these studies. These findings are consistent with the observation of vesicular glutamate transporters in Aβ, Aδ, and C fibers ( ). Subtypes of glutamate transporters are located predominantly, perhaps exclusively, on specific cell types; for example, excitatory amino acid carrier 1 (EAAC1) is found on dorsal horn and DRG neurons and axonal terminals, whereas glutamate aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are found on astrocytes and microglia in the spinal cord. Astrocytic transporters are thought to be important in the newly appreciated tripartite synapse, where they transport excess glutamate into astrocytes, which is then converted into glutamine by the enzyme phosphate-activated glutaminase. Glutamine is released into the synapse, where it is picked up by axon terminals and converted back into glutamate by the resident mitochondria. Under basal conditions, transporter inhibition results in increased levels of extracellular glutamate, spontaneous pain behavior, and evoked hypersensitivity. The latter two phenomena are reversed by glutamate receptor antagonists ( ). Decreased dorsal horn expression of GLT-1 and GLAST is observed following partial sciatic nerve ligation ( ), chronic constriction injury (CCI; ), and paclitaxel neuropathy ( ), thus suggesting that injury induces the loss of astrocytic transporters and the resultant glutamate-mediated excitotoxicity. Interestingly, reported that administration of ceftriaxone, an agent that up-regulates GLT-1 expression, reverses both the loss of GLT-1 and the pain behavior seen after a variety of injury states. These data conflict with those seen after intraplantar injection of formalin or complete Freund’s adjuvant, where glutamate transporter blockade or knockdown is reported to enhance pain behavior ( ).
Receptors
The post-synaptic excitatory effects of spinal excitatory amino acids are reflected by their potent ability to initiate pain behavior in animals after spinal delivery. These effects are mediated by specific interactions with a variety of glutamate receptors that are broadly divided into ionotropic and metabotropic subtypes.
The ionotropic glutamate AMPA, kainate, and NMDA receptors will be considered first. Receptors in each class are constituted from multiple subunits from different gene families to form transmembrane glutamate-activated pores. Details of assembly are provided elsewhere ( ). Intrathecal injections of glutamate receptor agonists have emphasized the importance of both NMDA and non-NMDA sites on dorsal horn neurons in producing powerful algogenic behavior ( , , ). Equally important is the fact that presynaptic ionotropic autoreceptors, found on primary afferent terminals, regulate the release of glutamate (see Fig. 28-3 ).
AMPA Receptors
Together with kainite receptors, AMPA receptors form a division of ionotropic receptors referred to as non-NMDA. Tetrameric AMPA receptors are glutamate-activated ionophores, which when activated, lead to a transient increase in the conductance of small cations (sodium) that results in depolarization. They are composed of two subunit dimers (GluA1–4) and are present in high concentration in the dorsal horn on non–primary afferent neuronal membranes and on ventral horn motor neurons and Renshaw cells ( ). Receptor subunits have multiple phosphorylation sites that individually contribute to receptor trafficking, movement into synapses, channel conductance, and open time. Non-neuronal cells are also immunopositive for AMPA receptors. Dorsal horn AMPA receptors show a decrease after rhizotomy ( ), consistent with the finding that more than one-third of putative nociceptive DRG neurons are immunopositive for AMPA receptors ( ). Furthermore, electrophysiological studies show activity mediated by presynaptic AMPA receptors at spinal afferent terminals ( ). Activation of these autoreceptors has been reported to inhibit release of glutamate ( ). A population of AMPA receptors are also Ca 2+ permeable, a property endowed by the absence of GluA2 subunits ( ). Such calcium-permeable AMPA receptors are present on lamina I neurons, some of which are neurokinin 1 (NK1) receptor positive, and on outer lamina II neurons ( ). A second population of gephyrin-coated lamina I neurons project to the midbrain and thalamus and contain GluA4 rather than GluA1 subunits ( ); these neurons lack NK1 receptors ( ). Other NK1-positive neurons in deeper dorsal horn laminae also lack GluR1 subunits ( ).
AMPA Physiology
Work with AMPA antagonists has emphasized that fast synaptic transmission between primary afferent fibers and both superficial and deep dorsal horn neurons is primarily driven by AMPA receptors ( ; ; ; ; ); kainite and NMDA receptors contribute only a small component of the early EPSP. Accordingly, iontophoretically applied AMPA antagonists block the acute excitation in dorsal horn neurons initiated by all classes of afferent fibers. Thus, selective AMPA antagonists are effective in blocking the responses of dorsal horn neurons to acute noxious mechanical and thermal stimuli in normal animals ( ). Studies of the calcium-permeable AMPA site in the spinal cord ex vivo have shown that activation of these receptors leads to increased calcium flux and serves to strengthen the synaptic transmission mediated by AMPA receptors ( ). Blocking spinal calcium-permeable AMPA sites with intrathecal Joro spider toxin facilitates C, but not A fiber–evoked responses of dorsal horn neurons ( ). Importantly, expression of calcium-permeable AMPA receptors on membranes of NK1-positive ( ) and NK1-negative lamina I neurons ( ) is regulated by ongoing afferent traffic and increases as a result of tissue inflammation. Afferent evoked activity, mediated via activation of either NMDA or tumor necrosis factor (TNF) receptors in the dorsal horn, enhances AMPA receptor trafficking ( , Tao 2010).
AMPA-Mediated Behavior
Intrathecal injection of AMPA antagonists produces a frank block of the behavioral response to acute aversive stimuli, such as on the hot plate or tail flick test, as well as facilitated states induced by tissue injury ( ). Importantly, at doses that are slightly higher, hindlimb dysfunction occurs after intrathecal delivery, a finding emphasizing the effect on ventral horn function and the probable block of excitatory input from large proprioceptive afferents. Thus, although behavioral analysis suggests that AMPA antagonists alter nociceptive input, their functional profile emphasizes the broad spectrum of end points blocked after the intrathecal delivery of such antagonists. Clinical trials with AMPA receptor antagonists indicated modest anti-hyperalgesia, especially against dynamic allodynia and cold pain, and agents were generally ineffective in reversing spontaneous pain ( ; ).
Animals genetically engineered to have fewer calcium-permeable AMPA receptors have reduced inflammation-induced pain behavior, whereas animals with decreased GluA2 subunits have prolonged and increased inflammatory hyperalgesia ( ). It should also be noted that intrathecal jorotoxin and philanthotoxin, blockers of the calcium-permeable AMPA site, blocked thermal injury–induced mechanical allodynia, carrageenan-evoked thermal hyperalgesia, and mechanical allodynia and had minimal effect on acute thermal escape latencies ( ). Additionally, although AMPA receptor antagonists prevent the development of both primary and secondary hyperalgesia following surgical incision, antagonists specific to the calcium-permeable site selectively block only secondary hyperalgesia ( ). Lack of jorotoxin blockade of primary hyperalgesia is an example of differences between calcium-permeable AMPA and NMDA receptor blockade, thus implying that the second messengers downstream of Ca 2+ entry in these two systems trigger distinct second-messenger pathways ( ).
Kainate Receptors
Kainate receptors are tetramers of subunits, each with distinct physiological and pharmacological properties ( ). The subunits GluR5–7 can form low-affinity receptors but develop higher affinity when paired with either KA1 or KA2. When activated, kainate receptors become permeable to monovalent cations (Na + , K + ), although variants are reported that are also permeable to Ca 2+ ( ). Persistent desensitization can occur at low agonist concentrations ( ). Autoradiography shows dense kainate binding in laminae I and II and less dense binding in deeper laminae ( ). Immunohistochemistry shows kainate subunit labeling on perikarya in laminae I–III ( ). Immunostaining also co-localizes with IB4 and cholera toxin subunit B and is significantly reduced by rhizotomy ( ). Presynaptic afferent localization is confirmed by identification of kainate subunits on DRG cells labeled with IB4, vanilloid receptor 1, and P2X 3 receptors, but not with SP ( ) ( ). Both pre- and post-synaptic kainate receptors may play a role in transmission at spinal primary afferent synapses. Presynaptically, kainate subunits are present on primary afferent terminals, where they may serve as autoreceptors ( ) and increase ( ) or decrease ( ) release of glutamate from primary afferents.
Kainate Physiology
Kainate receptor block has revealed an AMPA/NMDA-independent slow potential that was most pronounced for stimulation intensities sufficient to activate high-threshold Aδ and C fibers ( ). In addition, kainate receptors are found on inhibitory dorsal horn neurons, and approximately one-third of terminals in the superficial dorsal horn are positive for GABAergic markers and co-stain for kainate receptors ( ). Activation of these receptors may lead to increased release of γ-aminobutyric acid (GABA); paradoxically this may induce an ultimate decrease in GABA inhibition through negative feedback at GABA B autoreceptors ( ).
Kainate-Mediated Behavior
Intrathecal kainate receptor–preferring antagonists displayed antinociceptive action in the acute tail flick, hot plate, formalin, and mechanical pain threshold tests, as well as nerve injury hyperpathia ( ).
NMDA Receptor
The NMDA receptor is a glutamate-activated calcium ionophore that is constructed from four subunits: two NR1 subunits and two from the NR2 family—the latter have a great deal more variability than the NR1 subunits ( ). There are binding sites for glutamate and an allosteric site for glycine.
NMDA Physiology
Antagonism of the NMDA receptor has been shown to have little effect on acute post-synaptic excitation in the absence of conditioning input ( ) because of Mg 2+ blockade under basal membrane voltage conditions.
NMDA-Mediated Behavior
Blockade of spinal NMDA receptors by intrathecal delivery does not alter the acute thermal or mechanical thresholds ( ). Accordingly, the details of this receptor will not be further considered here. As reviewed below, however, this receptor does play an important role in augmenting afferent-evoked excitation in the presence of conditioning stimulation.
Metabotropic Receptors
Metabotropic glutamate receptors (mGluRs) are G protein–coupled receptors that are divided into three principal groups based on their intracellular signaling cascades. In group I, mGluR1 and mGluR5 stimulate phospholipase C (PLC), thereby leading to mobilization of intracellular Ca 2+ , activation of protein kinase C (PKC), and phosphoinositide hydrolysis; groups II (mGluR2 and 3) and III (mGluR4 and 6–8) are negatively coupled to adenylate cyclase. At the spinal level, delivery of group I agonists enhances basal glutamate release, and group II and III agonists diminish evoked glutamate release ( ). These results suggest that group I mGluRs may be pro-nociceptive by enhancing the spinal release of glutamate whereas group II and III mGluRs may be antinociceptive by suppressing the spinal release of glutamate. This supposition is strengthened by the finding that group I mGluR agonists increase phosphorylation of the spinal NMDA NR2B subunit ( ) and activate the mitogen-activated protein kinases (MAPKs) extracellular signal–regulated kinases 1 and 2 (ERK1 and ERK2) ( ). In parallel, spinal mGluR1 and mGluR5 antagonists reduce the hyperalgesia and receptor phosphorylation engendered by paw inflammation ( ).
Group II agonists produce reductions in basal ( ) or stimulated ( ) glutamate levels in the caudate and striatum, respectively. In DRGs, more than half the neurons, many of them presumptive nociceptors, are positive for group II (mGluR2/3) receptors. Activation of these receptors is without effect in naïve animals but reduces both pain behavior and single-fiber activation in the sensitized state ( ). These effects may be mediated via modulation of transient receptor potential vanilloid 1 (TRPV1) receptors and tetrodotoxin (TTX)-resistant Na + channels. Systemic treatment with group II agonists reduces pain behavior in both nerve injury and inflammation models. This is thought to be due in great part to presynaptic inhibition of A (including Aδ) fiber input into superficial dorsal horn neurons ( ).
Group III mGluR agonists reduce release of glutamate in the nucleus accumbens ( ) and hippocampus ( ). Conversely, local application of the group I agonists dihydroxyphenylglycine (DHPG) ( ) or (RS)-2-chloro-5-hydroxyphenylglycine ( ) increases and local application of the group I antagonist 2-methyl-6-(phenylethynyl) pyridine (MPEP) ( ) decreases glutamate levels in the parietal cortex or striatum in vivo.
Neurokinins
Transmitter System
SP was the first peptide identified as being specific for small sensory afferents and remains the best characterized. It, along with several sequence-similar peptides (e.g., neurokinin A [NKA]), are widely distributed among small IB4-negative DRG neurons whose central terminals synapse in spinal laminae I and inner II ( ) (see Fig. 28-2 ). Based on axons identified by conduction velocity, about half of C fibers and 20% of Aδ fibers contain SP ( ). In addition, populations of bulbospinal-projecting neurons also contain and probably release SP ( ). Spinal cord release of SP is secondary to direct stimulation of central C-fiber terminals by capsaicin ( ), by acute activation of C fibers ( ), and by noxious mechanical ( ) and cold ( ) stimuli. Using antibody-coated microelectrodes, SP and NKA were found to be released in the superficial dorsal horn in response to noxious thermal, mechanical, and chemical stimuli ( ; ). Using NK1 receptor internalization as an index of synaptic activity, peripheral noxious stimuli were found to initiate a stimulation intensity–dependent release of SP ( ).
Receptor
Several classes of NK receptors have been identified ( ). These G protein–coupled receptors stimulate PLC, thereby leading to breakdown of phosphoinositol and elevation of intracellular calcium levels. As with other G protein–coupled receptors, when this receptor is occupied, it undergoes internalization ( ). NK1 receptors are densely distributed on superficial dorsal horn neurons, many of which project to the brain stem (rostroventral medulla) and diencephalon (nucleus parabrachialis) ( ) and to a lesser degree to deeper dorsal horn neurons ( ). NK3 receptors are also found superficially in the dorsal horn ( ).
Physiology
Spinal delivery of neurokinins, particularly SP, has been shown to (1) evoke activity in nociceptive dorsal horn neurons ( ), (2) produce mild agitation ( ), and (3) induce potent hyperalgesia ( ) in unanesthetized animals. At the several tachykinin receptors it appears that NK1 and perhaps NK2 receptors are of most importance in nociception ( ). Spinal NK1 receptor antagonists reduce the afterdischarge in dorsal horn neurons evoked by acute noxious stimulation ( ).
Behavior
Behavioral studies in animal models have emphasized that intrathecal neurokinin antagonists fail to alter acute nociceptive behavior (e.g., hot plate test) but do diminish the hyperalgesia induced by persistent stimuli, such as in the phase 2 formalin test ( ), carrageenan-evoked thermal hyperalgesia ( ), and visceral nociception ( ). Convergent results have been reported in rats with reduced expression of NK1 protein because of intrathecal injection of antisense oligonucleotides ( ) and in mice lacking the NK1 receptor ( ). NK3-preferring antagonists depress spinal wind-up ( ) and central sensitization of a spinal withdrawal reflex ( ) and reduce hyperalgesia in arthritic models ( ).
Calcitonin Gene–Related Peptide
Transmitter System
CGRP-like immunoreactivity is expressed in approximately 45–70% of lumbar DRG neurons ( ). Based on identification of axonal conduction velocity, the majority of CGRP-containing neurons were classified as nociceptive (e.g., CGRP in 46% of C fibers, in 33% of Aδ fibers, and in 17% of Aβ fibers) ( ). CGRP is released from the spinal terminals of primary afferent neurons by high-intensity mechanical and thermal stimuli, as well as by local injection of irritants ( ).
Receptors
The effects of CGRP are believed to be mediated by the calcitonin-like receptor, that is, a G s -coupled seven-transmembrane–spanning receptor ( ).
Physiology
Application of CGRP induces spinal facilitation of dorsal horn responses that were blocked by putative CGRP antagonism ( ). Iontophoretic application of CGRP potentiates the depolarizing effects of SP ( ).
Behavior
Intrathecal delivery of partial CGRP sequences believed to be antagonistic resulted in a reduction in the hyperalgesia induced by intradermal capsaicin ( ) and carrageenan ( ). Spinal delivery of a CGRP antagonist increased thermal escape latency with and without tissue inflammation ( ). In addition, CGRP antagonism diminished the writhing response induced by phenylbenzoquinone ( ) and the thermal hyperalgesia and tactile allodynia otherwise observed after cord hemisection ( ).
Somatostatin
Transmitter System
SST immunoreactivity is limited to populations of small cells in DRGs and small dorsal horn neurons ( ). SST has also been identified in populations of bulbospinal-projecting cells ( ). Early work showed that SST is released from the spinal cord by capsaicin ( ). Subsequent work indicated differential release of SST in the spinal cord in response to noxious thermal but not noxious mechanical stimuli ( ).
Receptors
SST and its analogues act through a family of G protein–coupled receptors (SST1–5) that are widely distributed in the brain and periphery. SST1, 2, and 5 inhibit the opening of voltage-sensitive calcium channels ( ). Binding and parallel immunohistochemistry showed SST receptor subtypes 1, 2, and 3 in laminae I–III and in the ventral horn ( ). Some of this immunoreactivity is probably present on interneurons and on terminals of sensory afferents. Immunoreactivity for the SST3 receptor is also present on a large percentage of DRG neurons and motoneurons ( ).
Physiology
STT has been shown to inhibit spinal dorsal horn neuronal firing in response to noxious stimuli ( ) through a decrease in post-synaptic membrane excitability by activation of inwardly rectifying K + conductance ( ). Other work has emphasized a biphasic concentration-dependent activation of neurons and long-lasting depression suggesting toxicity ( ). After intrathecal application, SST increased the hindpaw electromyographic reflex ( ) and facilitated thermal nociception ( ).
Behavior
Considerable controversy exists regarding the effects of spinal SST and its analogues. Early work suggested that it was antinociceptive. However, other reports indicated that antinociception was observed at doses that resulted in pronounced motor dysfunction ( ; ; ). It is probable that important differences are related to the nature of the multiple receptors being activated by the several agonists. The spinal pharmacology of these excitatory and inhibitory receptor-mediated effects has not been fully studied to date.
Vasoactive Intestinal Polypeptide/Pituitary Adenylate Cyclase–Activating Peptide
Transmitter System
VIP and pituitary adenylate cyclase–activating peptide (PACAP) are both structurally related members of the glucagon/secretin superfamily ( ). VIP-positive neurons are numerous in primary afferent neurons of the thoracic and, in particular, the sacral spinal nerves, as well as in cranial nerves that innervate viscera ( ). VIP protein and mRNA expression are localized primarily in small to medium-sized DRG neurons ( ). Afferent stimulation, but not spinal capsaicin, releases VIP from the spinal cord ( ).
Receptors
VIP binding is concentrated in spinal laminae I and II ( ). PACAP has also been identified in small afferents, which unlike VIP, are capsaicin sensitive. Capsaicin results in the release and depletion of PACAP in the spinal cord ( ). VIP and PACAP are both recognized by a family of three receptors. Cloning reveals them to be G protein–coupled, adenylate cyclase–activating receptors ( ). Message for each of the three receptors is present in the spinal dorsal horn, particularly in laminae II–IV ( ).
Physiology/Behavior
Iontophoretic VIP and PACAP evoke the excitation of dorsal horn neurons ( ; ). Intrathecal VIP initiates the facilitation of spinal flexor reflexes, but spinal delivery of a VIP antagonist was without effect on this reflex ( ). Application of PACAP or a putative PACAP agonist (maxadilan) resulted in long-lasting spinal depolarization ( ) and hyperalgesia ( ). Conversely, application of a putative PACAP antagonist was found to induce a slow depolarizing response and reduce stimulation-evoked activation in spinal cord slices. Others have reported PACAP-induced inhibition of the C fiber–evoked flexor reflex ( ), block of the tail flick ( ), and a reduction in formalin-induced flinching ( ). Accordingly, whether PACAP is nociceptive or antinociceptive is controversial and doubtless depends on the specific receptors and systems examined ( ).
Galanin
Transmitter System
Galanin is expressed in DRGs and the spinal dorsal horn ( ). In the dorsal horn, galanin is primarily located in small GABAergic and enkephalinergic cells ( ). In the DRG, neither the fiber caliber associated with galanin-positive neurons ( ) nor the stimuli to which they respond have been characterized. Galanin staining density in the superficial dorsal horn decreases with C- but not with A-fiber stimulation, probably indicating release ( ). The physiological stimuli that evoke spinal galanin release in normal animals have not been defined. However, the peptide does not appear to be released in response to noxious thermal or mechanical stimulation ( ).
Receptors
Three receptors have been cloned for galanin (Gal 1–3 ) and belong to the superfamily of G protein–coupled receptors ( ). Activation of either the Gal 1 or Gal 3 receptor produces hyperpolarization via G i/o , whereas Gal 2 receptor activation leads to stimulation of G q/11 , thereby producing mobilization of calcium ( ). All three receptor transcripts are present in the DRG and spinal cord ( ). Gal 1 receptor mRNA is present in lamina II local neurons ( ).
Physiology
Early work indicated that intrathecal galanin facilitates the flexor reflex in response to noxious stimulation at low doses and inhibits it at higher doses ( ). It is now known that intrathecal Gal 1 receptor (Gal 1–29 ) but not Gal 2 receptor (Gal 2–13 )–preferring agonists inhibit spinal SP release, as assessed by NK1 receptor internalization evoked by paw compression. Spinal release of prostaglandin E 2 (PGE 2 ) evoked by intrathecal SP was blocked by both Gal 1 and Gal 2 receptor–preferring agonists. These data were taken to support both a pre- and post-synaptic action for Gal 1 receptor and a post-synaptic action for Gal 2 receptor at the level of the spinal dorsal horn ( ).
Behavior
Intrathecal low doses of galanin produce a significant reduction in the mechanical threshold ( ), whereas higher doses are reported to produce vocalization ( ). Based on Gal 1 versus Gal 2 receptor–preferring agonists, this enhanced sensitivity is believed to be mediated by the Gal 2 receptor. Spinal Gal 1–29 but not Gal 2–11 markedly inhibited the flinching behavior induced by paw formalin, whereas both agents blocked the hyperalgesia induced by intrathecal SP ( ).
Adenosine Triphosphate
Transmitter System
Adenosine triphosphate (ATP) is believed to be released, in part, from primary afferent terminals ( ). In culture, ATP is released from DRG axons following electrical stimulation ( ).
Receptors
Given the multiple subunits, at least 10 functional R -homomeric and heteromeric P2X receptors have been identified ( ). P2X receptors are expressed at a variety of sites on neurons and non-neuronal cells ( ). These effects are antagonized by the local application of antagonists. An important effect on the primary afferent terminal has also been postulated based on the ability of P2X agonists to initiate afferent transmitter release (see below). Current thinking points to an important role of such afferent-evoked ATP release in activating adjacent glia ( ). Further discussion on purines in pain transmission and the results of manipulating its effects on behavior are considered below.
Brain-Derived Nerve Growth Factor
Brain-derived neurotrophic factor (BDNF) is synthesized by small DRG neurons, transported to spinal terminals ( ), and released via capsaicin or electrical stimulation of the dorsal roots ( ). Importantly, this release is maximized by high-frequency burst stimulation and diminished by NMDA receptor antagonism. The role of spinal BDNF after release is not known, although it may serve as a modulator of synaptic transmission ( ). The complexity of its actions is suggested by the observation that although intrathecal BDNF diminishes the formalin flinching response ( ), NMDA-evoked responses are enhanced following up-regulation of BDNF in DRGs, and this enhanced excitability is reduced by BDNF-binding receptor protein ( ).
Mix of Post-synaptic Effects
An important element evident from this component of the review is that the excitatory effects of primary afferent fibers are mediated by multiple transmitters (e.g., amino acids and several peptides) and by multiple receptors for a given transmitter, as with glutamate. Current evidence suggests that high-intensity afferent input initiates the concurrent release of multiple transmitters. Not surprisingly, the post-synaptic consequences are extremely complex. In this instance, concurrent spinal injection of SP and glutamate produces a significant mutual augmentation of the algogenic effect as compared with the injection of either alone ( ; see also ). Similar results have been noted after iontophoretic delivery of SP and glutamate onto dorsal horn neurons ( ). Conversely, a noxious thermal, mechanical, or subcutaneous irritant (formalin) activates a complex profile of activation of large and small afferents that serves to activate spinal c-Fos or a neuronal marker such as Zif/268. It has been shown that activation of c-Fos by thermal stimuli is reduced by an NMDA or AMPA antagonist whereas Zif/268 expression is unaltered. Following formalin application, c-Fos and Zif/268 expression was reduced by NMDA but not by AMPA antagonism alone ( ). It is clear that at the level of the first synapse there is a very high degree of pharmacologically defined, behaviorally relevant encoding.
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