Kenneth E. McCarson

The role of GABA in the mediation and perception of pain.

A great deal of effort has been expended in attempting to define the role of GABA in mediating the transmission and perception of pain. Pursuit of this question has been stimulated by the fact that GABAergic neurons are widely distributed throughout the central nervous system, including regions of the spinal cord dorsal horn known to be important for transmitting pain impulses to the brain. In addition, GABA neurons and receptors are found in supraspinal sites known to coordinate the perception and response to painful stimuli and this neurotransmitter system has been shown to regulate control of sensory information processing in the spinal cord. The discovery that GABA receptor agonists display antinociceptive properties in a variety of animal models of pain has provided an impetus for developing such agents for this purpose. It has been shown that GABA receptor agonists, as well as inhibitors of GABA uptake or metabolism, are clinically effective in treating this symptom. However, even with an enhanced understanding of the relationship between GABAergic transmission and pain, it has proven difficult to exploit these findings in designing novel analgesics that can be employed for the routine management of pain. Work in this area has revealed a host of reasons why GABAergic drugs have, to date, been of limited utility in the management of pain. Chief among these are the side effects associated with such agents, in particular sedation. These limitations are likely due to the simultaneous activation of GABA receptors throughout the neuraxis, most of which are not involved in the transmission or perception of pain. This makes it difficult to fully exploit the antinociceptive properties of GABAergic drugs before untoward effects intervene. The discovery of molecularly and pharmacologically distinct GABAA receptors may open the way to developing subtype selective agents that target those receptors most intimately involved in the transmission and perception of pain. The more limited repertoire of GABAB receptor subunits makes it more difficult to develop subtype selective agents for this site. Nonetheless, a GABAB agonist, CGP 35024, has been identified that induces antinociceptive responses at doses well below those that cause sedation (Patel et al., 2001). It has also been reported that, unlike baclofen, tolerance to antinociceptive responses is not observed with CGP 44532, a more potent GABAB receptor agonist (Enna et al., 1998). While the reasons for these differences in responses to members of the same class remain unknown, these findings suggest it may be possible to design a GABAB agonist with a superior clinical profile than existing agents. Besides the challenges associated with identifying subtype selective GABAA and GABAB receptor agonists, the development of GABA analgesics has been hindered by the fact that the responsiveness of these receptor systems appear to vary with the type and duration of pain being treated and the mode of drug administration. Further studies are necessary to more precisely define the types of pain most amenable to treatment with GABAergic drugs. Inasmuch as the antinociceptive responses to these agents in laboratory animals are mediated, at least in part, through activation or inhibition of other neurotransmitter and neuromodulator systems, it is conceivable that GABA agonists will be most efficacious as analgesics when administered in combination with other agents. The results of anatomical, biochemical, molecular, and pharmacological studies support the notion that generalized activation of GABA receptor systems dampens the response to painful stimuli. The data leave little doubt that, under certain circumstances, stimulation of neuroanatomically discreet GABA receptor sites could be of benefit in the management of pain. Continued research in this area is warranted given the limited choices, and clinical difficulties, associated with conventional analgesics.

D3 dopamine receptors in rat spinal cord: implications for sensory and motor function

Quantitative autoradiography was used to determine the distribution of D(3) receptors in rat spinal cord and compare it with the distribution of D(1)-like and D(2) (and D(4)) receptors. [(3)H]PD 128907-labeled D(3) sites were observed in roughly 6-fold lower density than [(3)H]spiperone-labeled D(2) (D(4)) sites and 60-fold lower density than [(3)H]SCH 23390-labeled D(1)-like sites. Highest densities of D(3) binding were observed in the superficial layers of the dorsal horn at cervical and lumbar levels followed by the pars centralis and dorsal horn. Lowest densities of D(3) sites were detected in the ventral horn. These observations suggest that spinal D(3) receptors may play a role in sensory and/or motor function or contribute to the pharmacological effects of dopaminergic drugs.

Hippocampal neurokinin-1 receptor and brain-derived neurotrophic factor gene expression is decreased in rat models of pain and stress

Acute or chronic stress can alter hippocampal structure, cause neuronal damage, and decrease hippocampal levels of the neurotrophin brain-derived neurotrophic factor (BDNF). The tachykinin substance P and its neurokinin-1 (NK-1) receptor may play a critical role in neuronal systems that process nociceptive stimuli; their importance in stress-activated systems has recently been demonstrated by the antidepressant-like actions of NK-1 receptor antagonists. However, the functional similarities between neurokinin receptors in the hippocampus and those in sensory systems are poorly understood, as is the significance of hippocampal NK-1 receptor in the context of chronic pain. Therefore, we investigated the effects of immobilization stress or inflammatory stimuli on NK-1 receptor and BDNF gene expression in the rat hippocampus. Rats received an acute or chronic immobilization stress, or an acute (formalin) or chronic (complete Freunds adjuvant) inflammatory stimulus to the right hind paw. Subsequently hippocampal volume and specific gravity were measured and NK-1 receptor and BDNF mRNA levels quantified using ribonuclease protection assays. Results showed that either stress or pain down-regulates expression of both NK-1 receptor and BDNF genes in the hippocampus. Hippocampal volume was increased by either pain or stress; this may be due to edema (decreased specific gravity). Thus, BDNF and NK-1 receptor gene plasticity may reflect sensory activation or responses to neuronal injury. These data may provide useful markers of hippocampal activation during chronic pain, and suggest similarities in the mechanisms underlying chronic pain and depression.

Constitutive μ-Opioid Receptor Activity Leads to Long-Term Endogenous Analgesia and Dependence

Pain and Dependence The properties and functions of µ-opioid receptors have been studied intensively with respect to the binding of endogenous or exogenous ligands. However, much less is known about the constitutive, ligand-independent, activation of opioid receptors. Working in mice, Corder et al. (p. 1394) observed the prolonged constitutive activation of µ-opioid receptors in the spinal dorsal horn after transient peripheral inflammation. The results suggest that constitutive activation of µ-opioid receptors depresses nociception—the perception of pain—for long periods of time and induces cellular and physical dependence on endogenous opioid signaling. Transient inflammation can lead to prolonged activation of pain-relieving opioid receptors in the spinal cord. Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced μ-opioid receptor (MOR) constitutive activity (MORCA) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3′,5′-monophosphate overshoot and hyperalgesia) that required N-methyl-d-aspartate receptor activation of adenylyl cyclase type 1. Thus, MORCA initiates both analgesic signaling and a compensatory opponent process that generates endogenous opioid dependence. Tonic MORCA suppression of withdrawal hyperalgesia may prevent the transition from acute to chronic pain.

Decreased brain docosahexaenoic acid content produces neurobiological effects associated with depression: Interactions with reproductive status in female rats

Decreased tissue levels of docosahexaenoic acid (DHA; 22:6n-3) are implicated in the etiologies of non-puerperal and postpartum depression. With the aim of determining neurobiological sequelae of decreased brain DHA content, this study examined the effects of a loss of brain DHA content and concurrent reproductive status in adult female Long-Evans rats. An alpha-linolenic acid-deficient diet and breeding protocols were used to produce virgin and parous female rats with cortical phospholipid DHA levels 23-26% lower than virgin and parous rats fed a control diet containing adequate alpha-linolenic acid. Parous dams were tested/euthanized at weaning (postnatal day 20) of the second litter; virgin females, during diestrus. Decreased brain DHA was associated with decreased hippocampal BDNF gene expression and increased relative corticosterone response to an intense stressor, regardless of reproductive status. In virgin females with decreased brain DHA, serotonin content and turnover in frontal cortex were decreased compared to virgin females with normal brain DHA. In parous dams with decreased brain DHA, the density of 5-HT(1A) receptors in the hippocampus was increased, corticosterone response to an intense stressor was increased, and the latency to immobility in the forced swim test was decreased compared to parous dams with normal DHA. These findings demonstrate neurobiological alterations attributable to decreased brain DHA or an interaction of parous status and brain DHA level. Furthermore, the data are consistent with findings in depressed humans, and thus support a role for DHA as a factor in the etiologies of depressive illnesses, particularly postpartum depression.

Nociceptive regulation of GABAB receptor gene expression in rat spinal cord

Activation of gamma-aminobutyric acid (GABA) neurotransmission evokes antinociceptive responses in laboratory animals. The recent cloning of GABA(B) receptor gene products makes it possible to examine the regulation of this receptor system as it relates to the mediation of pain-related sensory information. Inasmuch as acute and chronic pain alter the expression of a number of nociception-related receptors, and because such changes are important components in the regulation of pain, the present study was undertaken to determine whether GABA(B) receptor gene expression is altered in sensory systems following a peripheral nociceptive stimulus. Solution hybridization-nuclease protection assays conducted 24 h after formalin injection into the right hindpaw of the rat revealed a significant bilateral increase in GABA(B) R1 and R2 receptor expression in the dorsal lumbar spinal cord, and a significant increase in GABA(B) R1 receptor mRNA in the ipsilateral lumbar dorsal root ganglion. These findings indicate an activity-dependent, differential regulation of GABA(B) R1 and R2 receptor gene expression in spinal sensory systems in response to chemogenic nociceptive activation, suggesting that GABA(B) receptor plasticity may play an important role in regulating the mediation, and perception, of chronic pain.

Neurokinin-1 (NK-1) receptor and brain-derived neurotrophic factor (BDNF) gene expression is differentially modulated in the rat spinal dorsal horn and hippocampus during inflammatory pain.

Persistent pain produces complex alterations in sensory pathways of the central nervous system (CNS) through activation of various nociceptive mechanisms. However, the effects of pain on higher brain centers, particularly the influence of the stressful component of pain on the limbic system, are poorly understood. Neurokinin-1 (NK-1) receptors and brain-derived neurotrophic factor (BDNF), known neuromediators of hyperalgesia and spinal central sensitization, have also been implicated in the plasticity and neurodegeneration occurring in the hippocampal formation during exposures to various stressors. Results of this study showed that injections of complete Freunds adjuvant (CFA) into the hind paw increased NK-1 receptor and BDNF mRNA levels in the ipsilateral dorsal horn, supporting an important role for these nociceptive mediators in the amplification of ascending pain signaling. An opposite effect was observed in the hippocampus, where CFA down-regulated NK-1 receptor and BDNF gene expression, phenomena previously observed in immobilization models of stress and depression. Western blot analyses demonstrated that in the spinal cord, CFA also increased levels of phosphorylated cAMP response element-binding protein (CREB), while in the hippocampus the activation of this transcription factor was significantly reduced, further suggesting that tissue specific transcription of either NK-1 or BDNF genes may be partially regulated by common intracellular transduction mechanisms mediated through activation of CREB. These findings suggest that persistent nociception induces differential regional regulation of NK-1 receptor and BDNF gene expression and CREB activation in the CNS, potentially reflecting varied roles of these neuromodulators in the spinal cord during persistent sensory activation vs. modulation of the higher brain structures such as the hippocampus.

Vitamin D Deficiency Promotes Skeletal Muscle Hypersensitivity and Sensory Hyperinnervation

Musculoskeletal pain affects nearly half of all adults, most of whom are vitamin D deficient. Previous findings demonstrated that putative nociceptors (“pain-sensing” nerves) express vitamin D receptors (VDRs), suggesting responsiveness to 1,25-dihydroxyvitamin D. In the present study, rats receiving vitamin D-deficient diets for 2–4 weeks showed mechanical deep muscle hypersensitivity, but not cutaneous hypersensitivity. Muscle hypersensitivity was accompanied by balance deficits and occurred before onset of overt muscle or bone pathology. Hypersensitivity was not due to hypocalcemia and was actually accelerated by increased dietary calcium. Morphometry of skeletal muscle innervation showed increased numbers of presumptive nociceptor axons (peripherin-positive axons containing calcitonin gene-related peptide), without changes in sympathetic or skeletal muscle motor innervation. Similarly, there was no change in epidermal innervation. In culture, sensory neurons displayed enriched VDR expression in growth cones, and sprouting was regulated by VDR-mediated rapid response signaling pathways, while sympathetic outgrowth was not affected by different concentrations of 1,25-dihydroxyvitamin D. These findings indicate that vitamin D deficiency can lead to selective alterations in target innervation, resulting in presumptive nociceptor hyperinnervation of skeletal muscle, which in turn is likely to contribute to muscular hypersensitivity and pain.

Central and peripheral expression of neurokinin-1 and neurokinin-3 receptor and substance P-encoding messenger RNAS: Peripheral regulation during formalin-induced inflammation and lack of neurokinin receptor expression in primary afferent sensory neurons

The neurokinin-1 receptor and its tachykinin neuropeptide ligand substance P are associated with the mediation of nociception. Substance P released from primary afferent sensory neurons activates neurokinin receptors on both central and peripheral targets that mediate specific aspects of central sensitization and inflammatory function; however, an autoreceptor function for the neurokinin-1 receptor remains highly controversial. Activation of the neurokinin-1 receptor by substance P during chronic nociception increases neurokinin-1 receptor gene expression in the spinal cord. Similarly, neurokinin-3 receptors on peripheral or target tissues or neurons could play an important role in the sensitization of sensory neurons. Therefore, this study (i) mapped the steady-state levels of substance P-encoding preprotachykinin, neurokinin-1 and neurokinin-3 receptor messenger RNAs in central and peripheral tissues including sensory ganglia, and (ii) investigated whether formalin-evoked nociception altered the quantity or location of neurokinin-1 or neurokinin-3 receptor messenger RNAs in the sensory ganglia or inflamed peripheral targets for substance P. Solution hybridization-nuclease protection assays quantified neurokinin receptor messenger RNA levels in central and peripheral tissues from normal and formalin-inflamed rats. High concentrations of the neurokinin-1 receptor were found in whole brain, spinal cord, and peripheral target organs innervated by substance P-containing neurons. Measurable levels of neurokinin-3 receptor messenger RNA were found only in brain, spinal cord and urinary bladder. Results also show that neither neurokinin-1 nor neurokinin-3 receptor messenger RNAs were detectable in primary afferent sensory neurons in the dorsal root ganglia of normal or formalin-inflamed rats. Neurokinin-1 receptor messenger RNA levels were, however, significantly increased in hindpaw tissues inflamed by formalin for 6 h. These results indicate that the plasticity of neurokinin-1 receptor gene expression in non-neuronal peripheral cells could regulate sensitivity to substance P in a manner similar to that in the spinal cord dorsal horn. Altered neurokinin-1 receptor gene expression provides a useful marker of long-term nociceptive activation and may mediate peripheral mechanisms of hyperalgesia and cellular sensitization during inflammation. Importantly, inflammation does not induce a phenotypic change in afferent sensory neurons providing neurokinin receptor targets for the direct sensitization of these neurons by substance P.

The formalin-induced expression of tachykinin peptide and neurokinin receptor messenger RNAs in rat sensory ganglia and spinal cord is modulated by opiate preadministration

Tachykinin peptides such as substance P and neurokinin B have been widely studied as mediators of pain transmission. The expression of neurokinin-1 and neurokinin-3 receptor messenger RNAs in the spinal cord is increased following intense nociception. The opiate ligands morphine and naltrexone alter behavioral responses to formalin-induced pain and alter evoked substance P release. This study investigated whether these opiates similarly alter the expression of substance P-, neurokinin B-, neurokinin-1 receptor- and neurokinin-3 receptor-encoding messenger RNAs in spinal systems following formalin-induced nociception. Expression levels of various messenger RNAs were quantitated using solution hybridization-nuclease protection assays. Six hours after hindpaw treatment, the levels of substance P-encoding preprotachykinin messenger RNA in the lumbar dorsal root ganglia and neurokinin B, neurokinin-1 receptor and neurokinin-3 receptor messenger RNAs in the lumbar dorsal horn were increased by approximately two-fold as compared to sham-treated controls. Pretreatment with naltrexone resulted in a further increase in the nociception-induced substance P messenger RNA expression in the dorsal root ganglia; preprotachykinin messenger RNA expression was not affected by morphine. Nociception-induced neurokinin-1 receptor messenger RNA expression in the dorsal horn was blocked by morphine, but was not affected by naltrexone. Both morphine and naltrexone blocked the formalin-induced increases in neurokinin B and neurokinin-3 receptor messenger RNA levels. Increased neurokinin B messenger RNA expression may reflect increased neurokinin B turnover in spinal interneurons activated by nociception. Neurokinin-3 receptor messenger RNA expression levels varied closely with, and thus may be regulated by, the levels of neurokinin B messenger RNA in the same regions. The results of this study indicate that pretreatment with opiate ligands modulates the expression of tachykinin peptide and neurokinin receptor encoding mRNAs in spinal systems following a peripheral chemogenic inflammatory stimulus. Thus, endogenous opioid systems may be involved in activity-induced changes in the expression of spinal tachykinin peptides and neurokinin receptors.