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 12345. 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) 6789. Amongst these, small-diameter nociceptive neurons show a high density of GABA_B receptor expression 710. However, GABA_B receptors are also expressed postsynaptically on second order neurons and as well as at motor neuron synapses 1112. 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 1819. 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 2021. Indeed, GABA_B receptor agonists display muscle relaxant properties and are rather widely used in the control of spasticity 2223 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 GABA_B 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 GABA_B 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 GABA_B 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.

A large body of morphological, electrophysiological, behavioral, and pharmacological studies have implicated GABA_B receptors in the control of pain. However, owing to the broad expression of GABA_B receptors throughout the nervous system, spatially and temporally restricted manipulation of GABA_B receptor expression is needed to elucidate their role at different anatomical sites along the pain pathway. In this study, we generated mice lacking GABA_B(1) receptor specifically in the peripheral arm of the nociceptive pathway. These mice are well-suited for elucidating the relevance of GABA_B receptor-mediated presynaptic inhibition of neurotransmitter release from nociceptor terminals as well as a putative role for GABA_B 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 GABA_B 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-GABA_B(1)^-/- mice. 2. The magnitude and duration of chronic inflammatory pain and neuropathic pain was comparable between SNS-GABA_B(1)^-/- mice and control littermates. 3. Electrophysiological analyses of nociceptor activity revealed a higher basal excitability in Aδ-mechanoceptors in SNS-GABA_B(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 6789 as well as functional studies which show that GABA_B receptor activation on primary afferent terminals in the spinal cord reduces neurotransmitter release 434445. 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 28363738. 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 253949, 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 424352. 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 4345. 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 GABA_B 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.

In summary, this study clarifies a long-standing question in the field of GABAergic modulation of nociception, namely the contribution of presynaptic GABA_B receptors in primary afferent nociceptive neurons. The use of genetic tools to specifically delete GABA_B receptors in DRG neurons, while leaving their expression in the spinal cord and brain intact, revealed that GABA_B receptors in primary nociceptive neurons do not play a major role in the modulation of pain. Furthermore, our results suggest that anti-nociceptive effects evoked by GABA_B receptor agonists are not mediated by receptors in peripheral nociceptive neurons but by receptors in the central nervous system. Thus, our results suggest that it would be advantageous to focus on the central nervous system when harnessing the GABA_B receptor system for pain management.

CFA: complete Freud's adjuvant; DRG: dorsal root ganglia; GABA: γ-aminobutyric acid; PBS: phosphate buffered saline; PWL: paw withdrawal latency; SNI: spared nerve injury.

The authors declare that they have no competing interests.

VG performed a large portion of the experiments and analyzed data; NA, IT and SB performed experiments; BB provided the GABA_B(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.