Second messengers, the substantia gelatinosa and injury-induced persistent pain

PAIN Pain Supplement

6 (1999) X-S 12

Second messengers, the substantia gelatinosa and injury-induced persistent pain Karla R. Petersen-Zeitz”’ b, Allan I. Basbauma* b.* “Departmenr.~ of Anatomy and Physiology. hW.M. Keck Foundation

Center,for

University of Cal(forrtia San Francisco.

Integrati\se Neuroscience,

Uniwr,ri@ of California

San Francisco,

San Francisco.

Box 0452.

CA 94143,

USA

San Francisco,

CA 94143,

USA

Abstract Although there is now unequivocal evidence that the circuitry within the substantia gelatinosa is a major contributor to the transmission and control of nociceptive messages, this was not known 35 years ago, when Pat Wall first focussed attention on this region. In addition to being the target of neurochemically distinct nociceptors, this region contains a heterogeneous population of excitatory and inhibitory interneurons. This review focuses on the contribution of second messenger systems that are found in the substantia gelatinosa. In particular the review highlights their critical contribution to the development of persistent pain conditions in the setting of tissue and nerve injury. Several of the studies used animals with deletions of genes that encode major second messenger molecules, including protein kinase A, C and nitric oxide synthase. Our laboratory has shown that mice with a deletion of the gene that encodes the gamma isoform of protein kinase C (which is almost exclusively expressed in a population of interneurons of the inner part of the substantia gelatinosa) have completely normal acute pain responses. However, the allodynia that characteristically develops after injury does not occur in these mice, particularly when it is generated by partial sciatic nerve injury. By contrast, deletion of genes that encode protein kinase A subunits only show deficits in the development of tissue inflammation-induced pain. These differences highlight the selectivity that characterizes the contribution of different second messenger molecules. Because of the restricted distribution of these molecules, it is likely that they are activated by different populations of primary afferent nociceptor and under very different conditions of injury. Understanding the circuitry within the substantia gelatinosa is thus critical to elucidating the mechanisms through which these second messenger molecules contribute to the development of

persistent pain in the setting of injury. 0 1999 International Association for the Study of Pain. Published by Elsevier Science B.V. Kqwordst

Substantia

gelatinosa;

Second messenger

systems; Persistent pain conditions

1. Introduction Our understanding of the mechanisms that underlie the transmission of nociceptive messages has changed considerably in the last 35 years, i.e. since the publication of the Gate Control Theory of Pain. For many of the intervening years, the emphasis was on the use of tract tracing and electrophysiological studies to identify the anatomy and physiology of the pathways through which information about acute noxious stimuli is transmitted from the spinal cord to the brainstem and thalamus. With a view to understanding the basis of pain conditions that were more clinically relevant, attention then turned to the long term consequences of injury, i.e. of maintained C-fiber input. Pat Wall’s studies were instrumental in focusing attention

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on the mechanisms that regulate the flow of nociceptive information (Hillman and Wall, 1969) and on altered states of nociceptive processing that occur in the setting of injury (Cook et al., 1986), including increased spontaneous activity, lowered threshold, etc. In fact, a major rationale for proposing the Gate Control Theory (Melzack and Wall, 1965) was the recognition that clinically relevant pains cannot be explained by activity in a single class of afferent fiber. Rather, the activity of segmental and supraspinal modulatory systems are also critical determinants of the ultimate perception that is generated in the setting of injury. We could identify a host of areas that were influenced by Pat Wall’s remarkable insights into how the nervous system functions. In our view, however, Pat’s most important contribution to our present day understanding of the mechanisms through which nociceptive messages are processed derives from his early studies of the workings of the substantia gelatinosa. Most people who have read the Science paper describing Gate Control Theory will

for the Study of Pain. Published by Elsevier Science B.V

recall Melzack and Wall’s critical discussion of the importance of the balance of large and small fibers to nociceptive processing. Readers remember the argument that pain is not just a function of the activity generated in C-fibers and they can readily discuss the emphasis that was placed on importance of large-fiber mediated inhibitory controls and on descending modulation. Perhaps because of our laboratory’s use of immunocytochemical and neurochemical approaches to dissect the circuits in the dorsal horn, it is our opinion that the focus of that paper on the workings of the substantia gelatinosa was particularly memorable. Indeed, based merely on its appearance in simple histological sections, Pat suggested that the substantia gelatinosa has a unique neurochemistry. How prescient can you get‘? Remember that 1965 was several years before immunocytochemistry was applied to the nervous system and before the demonstration that peptides are abundant in superficial dorsal horn of the spinal of the cord. The original ideas about the function of the substantia gelatinosa derived from Pat’s electrophysiological studies of the effects of small and large-fiber stimulation on the properties of dorsal horn neurons and on the propagation of dorsal root potentials (Wall, 1962; Mendell and Wall, 1964; Wall. 1967). Particular importance was placed on the contribution of presynaptic controls of primary afferent terminals via interneurons of the substantia gelatinosa (Wall, 1964). Today that concept is taken for granted, but it was remarkably controversial in the sixties, in part because of the paucity of anatomical evidence for presynaptic controls. At the conclusion of a talk describing his studies of the presynaptic controls that operate in the generation of positive and negative dorsal root potentials (Wall. 1964), Pat was confronted by Sir John Eccles who noted that ‘George Gray has informed me (i.e. Eccles) that he finds axo-axonic synapses to be very rare in the substantia gelatinosa’. Pat responded as only he can that ‘It is open season and the hunting is good with the electron microscope. You choose only to see synapses where the bubbles appear. but I can’t believe that there is only one type of anatomical synapse in the whole animal kingdom’. We’re not sure whether the bubbles referred to boutons or to vesicles, but Pat was obviously correct. Few people today would deny the importance of presynaptic controls. Today when we don’t see the ‘bubbles’ in the electron microscope, we turn to the possibility of volume conduction and so-called non-synaptic modes of transmission (Zoli et al., 1998). Pat’s mind was always open to the unconventional explanation and more often than not his novel interpretation was correct. There are probably also very few scientists in the field who realize that Pat Wall (in collaborative studies with Lennert Heimer) was the first to establish that the substantia gelatinosa is in fact targeted by primary afferents (Heimer and Wall, 1968). Previous studies argued that the afferents only target regions ventral to the substantia gelatinosa (Ralston, 1968). Although some of the anatomy upon which Pat’s ideas were generated have been modified (nota-

bly the extent to which large diameter afferents directly interact with substantia gelatinosa neurons), the importance of this region in the transmission of nociceptive messages has been documented repeatedly. Indeed. with every paper that appears. the neurochemical complexity of the substantia gelatinosa increases. A recent review (Snider and McMahon, 1998) highlighted the fact that in addition to containing a heterogeneous population of intemeurons (see below). two completely distinct populations of primary afferent nociceptor target the inner and outer parts of this region. The outer part of the substantia gelatinosa receives inputs from small diameter afferents that are rich in peptides (notably substance P and CGRP); the inner part of the substantia gelatinosa is targeted by afferents that contain few peptides. Rather these afferents express a unique purinergic (P2X:) receptor and a unique cell surface glycoprotein that can be labeled with the lectin IB4. The peptide and nc;n-peptide populations of afferents are also distinguished by their sensitivity in the adult, to different neurotrophins, nerve growth factor and glial-derived neurotrophic factor. respectively. Pat could not have imagined how chemically complex this region was, but his insights into the importance of the region have been confirmed many times over. Interest in the neurochemical complexity of the substantia gelatinosa, of course, is sustained because we assume that when we understand the functional correlates of this neurochemistry, we will have a much better insight into the causes of persistent pain. It is also hoped that these insights will lead to the development of new therapeutic approaches to treatment. Because of the long-standing interest that Pat has in the organization and function of this region of the spinal cord and because of his particular interest in the mechanisms through which long term changes are produced in the spinal cord, (remember that his student. Lorne Mendell. discovered wind-up (Mendell, 1966)). we dedicate the following review to him. Specifically. we will address recent studies that have identified second messenger systems that are activated by C-fiber inputs and that contribute to the persistent pain conditions produced by tissue ot nerve injury.

2. Cellular mechanisms

of spinal cord plasticity

As in other systems where long-term changes have been examined (e.g. hippocampus and cerebellum (Malenka and Nicoll, 1993)). the majority of studies that have addressed long term changes in nociceptive processing have addressed the effects induced via a glutamate action at the NMDA receptor (Wilcox, 1991). This follows, in part because of the implication of NMDA receptors in long term potentiation, because of the availability of antagonists to this receptor and because the major excitatory neurotransmitter of the C-fiber is glutamate. Indeed. there is considerable evidence that a glutamate action at the NMDA receptor underlies the induction and maintenance, not only of wind-up (Dicken-

K.R. Petersen-Zeit;

A.I. Bnshautn /Pain

son, 1990), but also of the central sensitization that is produced by intense electrical or chemical stimulation of C-fibers (Woolf and Thompson, 1991). Thus, injuryinduced hyperalgesia and allodynia associated with tissue or nerve injury (Davar et al., 1991; Coderre and Melzack, 1992; Mao et al., 1992b; Ren et al., 1992; Yamamoto and Yaksh, 1992; Tal and Bennett, 1993; Chaplan et al., 1997) and the electrophysiological correlates of these (Dickenson and Sullivan, 1987; Dougherty et al., 1993) are inhibited by systemic or intrathecal administration of NMDA receptor antagonists. Providing further evidence for the importance of presynaptic controls (particularly of the unconventional type that Pat championed in the early sixties), our own laboratory has provided a somewhat novel perspective on the mechanisms through which this NMDA-mediated facilitation of the transmission of nociceptive messages is generated. In the course of our immunocytochemical analysis of the distribution of the NMDARl subunit of the receptor, we noted that the receptor is expressed in both pre- and postsynaptic elements in the superficial dorsal horn (Liu et al., 1994). We next used double label studies and confirmed that the receptor is synthesized in dorsal root ganglion cells, transported to the central terminals of primary afferent terminals and localized to the vesicle release site of glutamatergic terminals. Our subsequent studies provided evidence that the presynaptic receptor is functional and that via an autoreceptor on the primary afferent, noxious inputs can generate enhanced release of primary afferent excitatory drive to postsynaptic neurons (Liu et al., 1997). On the other hand, neither activation of the pre nor the postsynaptic NMDA receptor is sufficient to sustain the long-term changes that occur as a result of persistent injury. Because the NMDA receptor is highly permeable to calcium, a number of calcium-dependent second messengers pathways, including protein kinase C (PKC), nitric oxide (NO), and alpha calcium-calmodulin kinase II (aCaMKII), are activated when Ca2+ is gated by the NMDA receptor (Bliss and Collingridge, 1993; Mao et al., 199.5; Mayer and Westbrook, 1987). These second messenger cascades, in turn establish long-term changes in neurons (Bliss and Collingridge, 1993; Coderre et al., 1993). In many respects the types of changes that have been observed in the development of long term potentiation in the hippocampus have been observed in the spinal cord, when inputs are generated in the setting of intense stimulation or persistent injury (Malinow et al., 1989; Coderre, 1992; Abeliovich et al., 1993; Meller et al., 1994; Mao et al., 1995; Lin et al., 1996). The problem with many of these studies is that the antagonists that have been used to implicate, for example PKC, are not at all selective for the multiple isoforms of this enzyme, or even for the particular class of enzyme. We, therefore, turned to a study of mice in which the genes that encode different second messenger molecules have been eliminated.

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3. Cellular mechanisms kinase A

Sl

of spinal cord plasticity:

protein

Our first studies examined the contribution of the gene that encodes the RIP regulatory subunit of protein kinase A, which is a CAMP-dependent second messenger that is distributed in the peripheral and central nervous system (Malmberg et al., 1997a,b). Compared to their wild type littermates, we found that acute pain indices did not differ in mice that carry a null mutation in this gene. However, tissue injury-evoked persistent pain behavior, inflammation of the hindpaw and ipsilateral dorsal horn Fos expression were significantly reduced in the mutant mice, as was plasma extravasation induced by intradermal injection of capsaicin into the paw. Consistent with studies of Levine and colleagues (Taiwo et al., 1989) who implicated CAMPdependent mechanisms in the sensitization of primary afferent C-fibers, we also found that the enhanced thermal sensitivity observed in wild-type mice after intraplantar or intrathecal (spinal) administration of prostaglandin E2 was significantly reduced in the mutant mice. In contrast, indices of pain behavior produced by partial sciatic nerve section (Malmberg and Basbaum, 1998), namely thermal and mechanical allodynia, were not altered in the mutant mice. Based on these studies, we concluded that the RIP subunit of PKA is necessary for the full expression of tissue injuryevoked (nociceptive) pain but does not appear to be required for nerve injury-evoked (neuropathic) pain. Because the RIP subunit is only present in the nervous system, including small diameter trkA receptor-positive dorsal root ganglion cells, we suggested that in inflammatory conditions, the RIP subunit of PKA is specifically required for nociceptive processing in the central and peripheral terminals of small-diameter primary afferent fibers. On the other hand, although the animals showed a deficit in the response to intrathecally administered PGE?, these studies did not specifically point to the substantia gelatinosa as the locus of the central changes that influence the primary afferent terminal. In fact, we are still ignorant as to the cells of origin of the endogenous PGE, that can influence the terminal; it may be neuronal or glial-derived.

4. Cellular mechanisms kinase C gamma

of spinal cord plasticity:

protein

Our subsequent studies of the development of persistent pain conditions pointed directly to second messenger systems produced by substantia gelatinosa neurons. In these studies. we evaluated mice with a deletion of the gene that encodes the gamma subunit of protein kinase C (PKCy; (Malmberg et al., 1997a,b)). Importantly, this isoform of PKC (a calcium-dependent enzyme) is not present until after birth, so that developmental abnormalities or compensatory responses to its loss are less likely to have occurred. Furthermore, unlike other PKC isoforms, PKCy is

only expressed in the CNS. As for PKA, mice with deletion of PKCy were completely normal in tests of acute pain. This was true for noxious thermal and noxious mechanical stimuli. However. the PKCy null mice almost completely failed to develop a neuropathic pain syndrome (thermal and mechanical allodynia) after partial sciatic nerve section. Although PKCy is expressed in numerous places in the brain (particularly in the hippocampus and cerebellum) its expression in the spinal cord is restricted to a population of interneurons in the inner part of the substantia gelatinosa. When viewed in sagittal sections (Martin et al., 1999), the neurons have the characteristic morphology of the islet cells (Gobel, 1978). whose dendrites and axons arborize within the inner part of the substantia gelatinosa. In other words, the circuitry through which these interneurons are likely to influence the transmission of nociceptive messages must occur within the inner part of the substantia gelatinosa. Furthermore, based on our subsequent double label studies, we showed that markers of inhibitory interneurons rarely were found in the PKCy-containing interneurons (Martin et al., 1999). It follows that the great majority of the PKCy-containing intemeurons must be excitatory. Based on these observations, we believe that the failure to develop nerve injury induced thermal and mechanical allodynia reflects alterations in a circuit that involves the non-peptide population of C-fiber (which selectively targets the inner substantia gelatinosa), a PKCy-containing excitatory interneuron and a nociresponsive projection neuron. Given that the dendrites of overlying projection neurons of lamina I rarely penetrate the substantia gelatinosa (Gobel. 1978; Beal, 1979; Light et al., 1979). we further suggest that the circuit controlled via the PKCy-containing interneuron involves the lamina V neuron. In fact, our recent electrophysiological studies in the spinal cord of mice with a deletion of PKCy indicate that neurons of lamina V do not develop long term sensitization in the setting of injury, even though the response to acute noxious stimulation is comparable to that of the wild type mice (Martin and Basbaum, 1998). As noted above. one of the propositions upon which the Gate Control Theory of Pain is based is that there is a critical interaction of activity generated via inputs in small (A delta and C) and large fibers. Any disruption of the balance in activity in these different afferent groups could result in an abnormal pain condition. Of course Gate Control emphasized the ‘closing’ function of the large fibers. via their activation of inhibitory interneurons in the substantia gelatinosa. Often overlooked, however, is the fact the Gate Control circuit also included a direct excitatory input from the large fibers to the ‘transmission’ neuron. That connection made sense because of the evidence for the existence of wide-dynamic range neurons. which receive a convergent nociceptive and non-nociceptive input. Recent studies. of course, have provided evidence that under pathological conditions, the input transmitted via A beta (i.e. large diameter afferents) is a major contributor to the mechanical

allodynia that occurs (Campbell et al., 1988; Andersen et al., 1995). Furthermore, thtre is now considerable evidence that in the setting of injury large diameter afferents can sprout and eventually make contact with neurons with which they do not normally interact (Doubell et al.. 1997). One interesting possibility is that the PKCy-containing interneurons come under the influence of sprouted large diameter afferents, thus contributing to the allodynia observed in the setting of nerve injury. Calcium-dependent protein kinases have not only been strongly implicated in spinal cord mechanisms of sensitization, leading to allodynic and hyperalgesic conditions (Coderre. 1992; Mao et al., 1992a,b, 1993; Yashpal et al.. 1995; Peng et al., 1997) but they have also been implicated in the development of tolerance to the analgesic effects of morphine (Mayer et al., 1995). Most interestingly, based on studies of Mao and colleagues, it has been hypothesized that activation of PKC in neurons of the superficial dorsal horn contributes both to the development of sensitization in the setting of injury and to the development of morphine (Mao et al., 1995). For example, chronic administration of morphine not only produces tolerance but it also increases the level of PKC, notably within laminae I and II. The latter was concluded from the finding of an increase in membrane bound PKC under morphine tolerant conditions. The increase in membrane bound PKC was inferred from an increase in phorbol ester binding and was presumed to result from a translocation of the enzyme from the cytosol to the membrane during tolerance development. In light of the evidence for a contribution of NMDA receptor mechanisms to the development of tolerance (Trujillo and Akil. 1991: Elliott et al.. 1994) and because PKC has been shown to regulate the NMDA receptor (its phosphorylation reduces the magnesium block (Chen and Huang, 1992)), the authors hypothesized that the development of tolerance reflects a PKC-mediated increase in opioid regulation of the NMDA receptor. Numerous studies remain to be performed to confirm those observations, however, they provide further evidence for the critical importance of the substantia gelatinosa to the development of long term changes in nociceptive processing, in this case under conditions in which drugs that normally block the transmission of nociceptive messages, namely opioids, are administered.

5. Cellular mechanisms oxide

of spinal cord plasticity:

nitric

The fact that the PKCy appears to be concentrated in excitatory interneurons of the substantia gelatinosa provides some clues to the contribution of the excitatory population of interneuron, but we are left with the contribution of the far larger group of inhibitory interneurons. Perhaps Gate Control Theory’s most controversial aspect relates to the complex function that it assigned to the interneurons of the substantia gelatinosa. Although the Gate Control Theory model only

included a single inhibitory substantia gelatinosa interneuron, it was obvious that this was a black box formulation. The inhibitory interneuron included in the ‘Gate’ diagram was merely the final interneuron in a complex circuit located within the substantia gelatinosa. Because primary afferents are exclusively excitatory (or were certainly assumed to be so in 1965) the hypothesis that the C fibers inhibited the ‘inhibitory’ substantia gelatinosa neuron required that there were multiple intervening neurons involved. Activation of cascades of inhibitory interneurons leading to disinhibition was certainly a possibility and indeed recent studies have provided considerable evidence that loss of GABAergic inhibitory control in the substantia gelatinosa may underlie the development of persistent pain conditions (Hao et al., 1992). But this loss of GABAergic control would more likely account for the loss of large fiber-mediated inhibition, not for the enhancement produced via the activation of C fibers. Studies of the distribution and contribution of nitric oxide to nociceptive processing in the setting of injury, however, provide evidence that the GABAergic inhibitory interneurons are far more complex than previously imagined. Based on studies of hippocampal synaptic plasticity, it has been demonstrated that nitric oxide (NO), which is produced by the calcium/calmodulin dependent enzyme nitric oxide synthase (NOS), contributes to NMDA receptor-dependent long term potentiation (Schuman and Madison, 1991). Subsequent studies of its function in the context of nociceptive processing provided evidence for a comparable function in the spinal cord. For example, inhibitors of NOS do not alter the processing of acute nociceptive messages, but significantly reduce the hyperalgesia associated with persistent injury (Meller et al., 1992; Malmberg and Yaksh, 1993; Meller and Gebhart, 1993). Although it was not surprising to find that the enzyme is concentrated in the superficial dorsal horn (Bredt et al., 1991) and to show that the enzyme increases in a chronic arthritis model (Wu et al., 1998) it was quite surprising to find that the enzyme is almost exclusively found to colocalize with markers of GABAergic inhibitory intemeurons (Spike et al., 1993). How then can the same neuron mediate a facilitatory and an inhibitory control of nociceptive processing? Although the precise molecular mechanisms by which NO mediates injury-induced changes in spinal cord processing are not understood, some insight has been gained by further analysis of the neurochemistry of the substantia gelatinosa. Specifically, it has been suggested that NO signaling occurs primarily through the activation of soluble isoforms of guanylyl cyclase that increase intracellular cGMP levels to activate cGMP-dependent protein kinases. The cGMPdependent protein kinase, cGKI, however is not expressed in neurons intrinsic to the dorsal horn, but rather, is concentrated in small- and medium-diameter DRG neurons (Qian et al., 1996). This suggests that in order to mediate synaptic plasticity NO must either act through different cellular mechanisms in spinal cord neurons or that it must act upon neurons outside of the cells in which it is synthesized.

Because NO is a small, gaseous molecule, it is has been proposed that NO diffuses out of neurons in the substantia gelatinosa to act in a retrograde fashion on primary afferent neurons and that this activity underlies changes in nociceptive processing associated with injury (Meller and Gebhart. 1993). Importantly, cGMP analogs as well as guanylyl cyclase inhibitors have been demonstrated to have effects similar to those seen with activators and inhibitors of the NOS pathway (Meller et al., 1992; Garry et al., 1994). Thus the same substantia gelatinosa interneuron may exert presynaptic inhibitory as well as facilitatory controls upon primary afferents. Of course, the controls exerted via NOS would not have an anatomical substrate that can be observed in the electron microscope. This would clearly be one more example of presynaptic controls that cannot be identified when one only focuses on the ‘bubbles’ in the electron microscope.

6. Cellular mechanisms &aMKII

of spinal cord plasticity:

The studies described above provide strong evidence that the chemistry of the intemeurons of the substantia gelatinosa is key to understanding how long term changes are induced by injury and how these changes contribute to the development of persistent pain conditions. More recently, our attention has turned to other second messenger systems that are likely to contribute to the hyperexcitability that characterizes the injury state. The alpha isoform of calcium-calmodulin dependent protein kinase II (crCaMKII) is a neural-specific enzyme that has been implicated in prolonged changes in synaptic function (Hanson and Schulman, 1992). Among the proteins targeted by cuCaMKII are the AMPA and NMDA receptors and synapsin I (Benfenati et al., 1992; Kitamura et al.. 1993; McGladeMcCulloh et al., 1993; Omkumar et al., 1996; Barria et al., 1997). Abundantly expressed in postsynaptic densities, this enzyme is a serine-threonine kinase that is activated by the binding of calcium-bound calmodulin to a domain near its C-terminal end. This relieves the protein’s catalytic domain of autoinhibition and permits the enzyme to autophosphorylate a key threonine residue (Miller et al., 1988; Thiel et al., 1988; Lou and Schulman, 1989). Interestingly, Thr286 phosphorylation enables cuCaMKI1 to switch into an ‘on’ state that persists when calcium levels return to baseline (Saitoh and Schwartz, 1985; Miller and Kennedy, 1986; Miller et al., 1988). This calcium-independent activity of cvCaMKI1 has given rise to the hypothesis that the enzyme serves to store synaptic memories for postsynaptic neurons (Lisman, 1994). Indeed, aCaMKII does appear to be necessary for the development of prolonged synaptic changes, notably NMDA receptor-dependent long-term potentiation (LTP) at the CA3-CA1 synapse in the hippocampus (Malenka et al., 1989; Malinow et al., 1989; Silva et al., 1992). Importantly, the action of this enzyme is not limited

to the hippocampus. (wCaMKI1 has also been implicated in activity-dependent plasticity in adult mouse visual cortex and in whisker barrel cortex (Glazewski et al.. 1996; Kirkwood et al., 1997). Based on preliminary studies of the distribution of the enzyme in the spinal cord, we hypothesize that aCaMKI1 also contributes to the development of persistent changes in nociceptive processing at the spinal cord level. In fact, there is evidence that intracellular application of autophosphorylated cvCaMKI1 can enhance excitatory synaptic transmission of substantia gelatinosa neurons (Kolaj et al.. 1994). Although in situ studies found only moderate levels of aCaMKI1 in spinal cord (Benson et al., 1992) our immunocytochemical analysis found very high concentrations in superficial dorsal horn (Basbaum and Kennedy, 1986). Of particular interest, we recently found that a very high proportion of PKCy-containing interneurons of the inner part of the substantia gelatinosa cocontain uCaMKII. This observation is particularly interesting because there is evidence that the two second messenger systems interact in the generation of long term potentiation in hippocampal pyramidal neurons. For example, it has been demonstrated that the LTP produced by injection of calcium-calmodulin (which activates aCaMKI1) is blocked by inhibitors of PKC (Wang and Kelly, 1995). Given the colocalization of aCaMKI1 with PKCy in the substantia gelatinosa, we are very interested in determining whether these two molecules interact in the development of central sensitization.

7. Summary We have come a long way from the simple circuit that Gate Control Theory described. Gate Control Theory emphasized how large and small fibers close and open a gate, respectively, by turning a key that was inherent in the workings of a substantia gelatinosa interneuron. We now know that there are numerous ways to enhance nociceptive transmission. Clearly, the Gate can be opened and closed by a slew of keys, not all of which have been identified. The presence of many keys, of course, means that there are multiple substantia gelatinosa circuits through which the transmission of nociceptive messages can be reduced or enhanced. Among Pat’s incredible contributions to the growth of pain research was his recognition that the substantia gelatinosa is the depository of the keys. We do not know how many more keys are stored there, but we will continue to seek them out.

Acknowledgements This work was supported by: NS 14627. KRP is supported by a fellowship from the National Science Foundation.

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