Background
Metabotropic GABA receptors, namely GABA
B receptors, mediate the slow and prolonged physiological effects of the inhibitory neurotransmitter, GABA. They play an important role in the modulation of synaptic transmission. Contribution of pre- as well as post-synaptic GABA
B receptors in the modulation of long-term plasticity phenomena in brain regions, such as the hippocampus and amygdala, has been described [
1‐
5]. GABA
B receptors are also highly concentrated in the superficial dorsal horn, predominantly on afferent terminals of sensory neurons located in the dorsal root ganglia (DRG) [
6‐
9]. Amongst these, small-diameter nociceptive neurons show a high density of GABA
B receptor expression [
7,
10]. However, GABA
B receptors are also expressed postsynaptically on second order neurons and as well as at motor neuron synapses [
11,
12]. The expression of GABA
B receptor subunits is enhanced in lumbal spinal cord and dorsal root ganglion following chronic nociceptive activation in models of axotomy and chemogenic pain [
13].
Multiple lines of evidence support an antinociceptive role for GABA
B receptors in animal models of acute and chronic pain. baclofen, a GABA
B receptor agonist, exhibits antinociceptive effects in model of peripheral nerve injury and chronic inflammation [
14]. baclofen also attenuates pain-related behaviors evoked by the formalin injection in rats and also reduces allodynia-like behavioral symptoms in disease models of chronic pain inducing monoarthritis [
15], ischemic injury to the spinal cord [
16], carrageenan-produced inflammation [
17] or trigeminal neuralgia [
18,
19]. In the view of extensive literature in animal models of acute and chronic pain, it is rather surprising that the clinical administration of GABA
B receptor agonists as analgesics has been restricted to trigeminal neuralgia and post-herpetic neuralgia [
20,
21]. Indeed, GABA
B receptor agonists display muscle relaxant properties and are rather widely used in the control of spasticity [
22,
23] and have been implicated in dystonia [
24]. Because evoked pain behaviors in animal studies mostly rely upon a motor behavioral response, the motor deficits caused by GABA
B receptor modulation occlude an unequivocal interpretation of behavioral responses. Another important caveat is that currently available GABA
B ligands suffer from lack of selectivity with respect to the locus of action within the different components of the spinal sensory-motor circuit. Thus, a complete delineation of the sensory antinociceptive actions from the motor inhibitory actions of GABA
B receptors is not possible using the conventional approach of ligand delivery in animals.
We reasoned that the application of genetic tools to manipulate GABAB receptor expression in a site-specific manner may enable delineating their specific role in the modulation of nociception and chronic pain. We generated mice lacking the GABAB receptors specifically in nociceptive neurons of the dorsal root ganglia. Detailed behavioral and electrophysiological analyses in the paradigms of basal and pathological nociception revealed that although GABAB receptors localized in first order nociceptive neurons have the capacity to modulate sensitization phenomena, their contribution towards the modulation of pain at the level of the whole living organism is not pronounced. Furthermore, pharmacological experiments showed that baclofen-induced antinociception is mechanistically-independent of GABA receptors in the first order nociceptive neurons.
Discussion
A large body of morphological, electrophysiological, behavioral, and pharmacological studies have implicated GABAB receptors in the control of pain. However, owing to the broad expression of GABAB receptors throughout the nervous system, spatially and temporally restricted manipulation of GABAB receptor expression is needed to elucidate their role at different anatomical sites along the pain pathway. In this study, we generated mice lacking GABAB(1) receptor specifically in the peripheral arm of the nociceptive pathway. These mice are well-suited for elucidating the relevance of GABAB receptor-mediated presynaptic inhibition of neurotransmitter release from nociceptor terminals as well as a putative role for GABAB receptors in peripheral nociceptive terminals in physiological and pathophysiological states. We analyzed mice with respect to the excitability of nociceptors and its manifestations in several models of pain, including unilateral hindpaw inflammation, chemogenic activation of nociceptors and peripheral neuropathy. Surprisingly, our detailed analyses revealed very few phenotypic differences between mice lacking GABAB receptors in nociceptors and control mice. Briefly, our main findings were: 1. Chemogenic pain evoked by formalin and early nociceptive hypersensitivity were slightly prolonged in SNS-GABAB(1)
-/- mice. 2. The magnitude and duration of chronic inflammatory pain and neuropathic pain was comparable between SNS-GABAB(1)
-/- mice and control littermates. 3. Electrophysiological analyses of nociceptor activity revealed a higher basal excitability in Aδ-mechanoceptors in SNS-GABAB(1)
-/- mice; however, this did not translate into clear functional changes with respect to nociceptive behavior.
Our findings are surprising in the view of previous studies reporting GABA
B receptor expression in primary afferent terminals [
6‐
9] as well as functional studies which show that GABA
B receptor activation on primary afferent terminals in the spinal cord reduces neurotransmitter release [
43‐
45]. Although it is clear that GABA
B receptors are densely expressed in peripheral nociceptive neurons, the literature on the regulation of GABA
B receptor expression in pathological pain states is somewhat mixed. For example, some studies reported an increase in GABA
B receptor expression in the spinal dorsal horn and peripheral nociceptors in inflammatory pain states [
13]. In contrast, Engle et al. [
46] found that spinal nerve ligation does not alter the expression or function of GABA
B(1) and GABA
B(2) in the spinal cord and dorsal root ganglia of rats and also does not lead to changes in GABA
B receptor binding affinity in inflammatory and neuropathic states. Furthermore, findings in a model of diabetic neuropathy suggest reduced function of presynaptic GABA
B receptors at primary afferent terminals, but not those on GABAergic and glycinergic interneurons, in the spinal cord [
45]. Interestingly, a series of experiments with novel ligands at GABA
B receptors have also suggested a functional contribution of GABA
B(1) expressed in peripheral nociceptive neurons; e.g. α conotoxins and Rg1A peptides derived from the venom of marine Conus snails, which are currently in development for the treatment of neuropathic pain, have been shown to inhibit native calcium channel currents by the virtue of activation of GABA
B receptors in first order neurons [
47]. Thus, considerable support implicates a role for GABA
B receptors expressed in peripheral nociceptive neurons in the endogenous modulation of nociception and pathological pain.
In this study, we deleted the primary ligand-binding subunit of metabotropic GABA receptors, GABA
B(1), specifically in peripheral nociceptive neurons leaving their expression in the spinal cord and brain intact. Numerous studies in cell lines as well as native tissues have demonstrated that a loss of GABA
B(1) leads to a complete lack of ligand binding and a total loss of function of native GABA
B receptors [
28,
36‐
38]. Therefore, based upon our findings, we infer that a conditional loss of GABA
B receptor function in peripheral nociceptive neurons
in vivo does not lead to significant changes in nociception and the development of pathological pain.
It is interesting to note that we have found a phenotype in firing properties of Aδ peripheral afferents, but not in C-afferents in SNS-GABA
B(1)
-/- mice compared to GABA
B(1)
fl/fl. This might result from higher expression of GABA
B(1) in Aδ- as compared to C-fibers. As noted previously GABA
B receptor mRNA has been shown to be expressed in all DRG neurons [
7]. However, studies on differential expression of the protein in different types of DRG neuron are lacking due to antibody specificity issues. Other possible explanation of the phenotype would be a more important role for GABA
B(1) in Aδ-fibers in comparison to C-fibers. This hypothesis is supported by work of Sengupta et al., who observed a more prominent blockade of Aδ-fiber, than C-afferent fiber, activity upon application of systemic baclofen in pelvic nerve afferent fibers responding to isobaric colorectal distension [
48].
It cannot be ruled out that compensatory changes, such as an increase in inhibition via other inhibitory transmitters and receptors, come into place to reinstate inhibition in pathological states. However, this is unlikely given that loss of GABA
B(1) beginning at very early developmental stages, such as in classical knockout mice, does not lead to compensation of GABA
B-mediated inhibition with respect to pain; classical GABA
B(1) knockout mice demonstrate a prominent hyperalgesic phenotype [
39]. Analyses in classical GABA
B(1) knockout mice have confirmed that a loss of the GABA
B(1) subunit is paralleled by a loss of all biochemical and electrophysiological GABA
B(1) responses [
25,
39,
49], demonstrating that GABA
B(1) is an essential component of pre- and postsynaptic GABA
B receptors. Directly comparing the phenotypes of global GABA
B(1) receptor knockout mice and nociceptor-specific GABA
B(1) knockout mice therefore leads to the inference that although GABA
B receptors in the nervous system are important in the control of pain, these functions are likely mediated by receptors expressed in the central nervous system rather than those expressed in peripheral nociceptive neurons. However, it deserves to be noted that classical GABA
B(1) null mutants also exhibit morphological and molecular changes in the constitution of peripheral myelin and demonstrate gate abnormalities, as revealed by very recent studies [
50], thereby raising the possibility that these alterations in the periphery may have contributed to the sensory phenotype in GABA
B(1)-deficient mice. These abnormalities would not be expected in nociceptor-specific null mutants studied here.
Interestingly, we have found a slight phenotype in the second phase of formalin response in SNS-GABA
B(1)
-/- compared to GABA
B(1)
fl/fl mice. There is evidence that the second phase of the formalin response depends not only on central, spinal mechanisms but also on the neural activity generated during the first phase and continuing firing activity during the second phase [
51]. Therefore, the phenotype in phase IIb of the formalin response could be caused by exaggerated activation of primary afferents due to the lack of GABA
B mediated inhibition in the first phase of the formalin test.
Experimental studies with the classical GABA
B receptor agonist, baclofen, have implicated a therapeutic role for GABA
B receptors in the inhibition of nociceptive hypersensitivity. However, baclofen has only found limited clinical utility in the treatment of pain. We found that systemically administered baclofen can reduce nociceptive hypersensitivity, e.g. evoked by formalin, consistent with previous reports [
42,
43,
52]. However, analysis of nociceptor-specific GABA
B(1) receptor mutants revealed that this anti-nociceptive activity of baclofen occurs independently of GABA
B(1) expression in peripheral nociceptive neurons. Indeed, there is considerable evidence supporting a spinal action of baclofen in inhibiting pain; in particular, administration of baclofen attenuates mechanical allodynia in a rat spinal cord injury model, whereas a GABA
B receptor antagonist, phaclofen, shows opposite effects [
53]. Furthermore, GABA
B receptors expressed in dorsal horn neurons have been shown to participate in the modulation of secondary hyperalgesia in monoarthritic rats, which is reduced by intrathecal injection of baclofen [
16]. However, some studies have also suggested a presynaptic locus of action of baclofen. For example, electrophysiological studies have suggested inhibition of neurotransmitter release from presynaptic terminals via baclofen-mediated activation of GABA
B receptors [
43,
45]. However, the consequences of baclofen-induced inhibition of presynaptic neurotransmitter release from nociceptive afferents in the spinal cord are somewhat tampered by the consequential reduction of GABAergic and glycinergic synaptic transmission onto substantia gelatinosa neurons, which are typically also activated by the glutamatergic inputs coming in via peripheral afferents [
54].
Indeed, GABA
B receptors are also widely distributed in a variety of brain regions which play an important role in the modulation of pain, e.g. the rostral agranular insular cortex, a cortical area which is constantly activated by painful stimuli [
55]. Furthermore, it has been shown that a local increase of GABA
B concentrations in higher brain centres results in lasting bilateral analgesia [
56]. Thus, the locus of baclofen action remains unclear.
Our analyses suggest that baclofen-induced inhibition of anti-nociception, particularly at doses which do not cause motor impairment, is not mediated by GABAB receptors on presynaptic nerve terminals. A detailed analysis of baclofen-induced anti-nociception is considerably hindered by the marked motor impairment caused by baclofen at higher doses. We observed that intraplantar injection of baclofen in the hind paw did not lead to anti-nociception at low doses (data not shown); doses which evoked anti-nociception upon intraplantar administration were accompanied by a marked impairment of motor function and paralysis. Based upon these pieces of evidence, we conclude that peripheral nociceptive neurons are not the primary locus of baclofen action in the modulation of pain.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
VG performed a large portion of the experiments and analyzed data; NA, IT and SB performed experiments; BB provided the GABAB(1)
fl511/fl511 mice; RK designed and supervised the study and helped with the writing of the manuscript; MK performed a large portion of experiments, analyzed data and wrote the manuscript. All authors read and approved the final manuscript.