- Open Access
Conditional gene deletion reveals functional redundancy of GABAB receptors in peripheral nociceptors in vivo
Molecular Painvolume 5, Article number: 68 (2009)
γ-aminobutyric acid (GABA) is an important inhibitory neurotransmitter which mainly mediates its effects on neurons via ionotropic (GABAA) and metabotropic (GABAB) receptors. GABAB receptors are widely expressed in the central and the peripheral nervous system. Although there is evidence for a key function of GABAB receptors in the modulation of pain, the relative contribution of peripherally- versus centrally-expressed GABAB receptors is unclear.
In order to elucidate the functional relevance of GABAB receptors expressed in peripheral nociceptive neurons in pain modulation we generated and analyzed conditional mouse mutants lacking functional GABAB(1) subunit specifically in nociceptors, preserving expression in the spinal cord and brain (SNS-GABAB(1) -/- mice). Lack of the GABAB(1) subunit precludes the assembly of functional GABAB receptor. We analyzed SNS-GABAB(1) -/- mice and their control littermates in several models of acute and neuropathic pain. Electrophysiological studies on peripheral afferents revealed higher firing frequencies in SNS-GABAB(1) -/- mice compared to corresponding control littermates. However no differences were seen in basal nociceptive sensitivity between these groups. The development of neuropathic and chronic inflammatory pain was similar across the two genotypes. The duration of nocifensive responses evoked by intraplantar formalin injection was prolonged in the SNS-GABAB(1) -/- animals as compared to their control littermates. Pharmacological experiments revealed that systemic baclofen-induced inhibition of formalin-induced nociceptive behaviors was not dependent upon GABAB(1) expression in nociceptors.
This study addressed contribution of GABAB receptors expressed on primary afferent nociceptive fibers to the modulation of pain. We observed that neither the development of acute and chronic pain nor the analgesic effects of a systematically-delivered GABAB agonist was significantly changed upon a specific deletion of GABAB receptors from peripheral nociceptive neurons in vivo. This lets us conclude that GABAB receptors in the peripheral nervous system play a less important role than those in the central nervous system in the regulation of pain.
Metabotropic GABA receptors, namely GABAB 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 GABAB receptors in the modulation of long-term plasticity phenomena in brain regions, such as the hippocampus and amygdala, has been described [1–5]. GABAB 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 GABAB receptor expression [7, 10]. However, GABAB receptors are also expressed postsynaptically on second order neurons and as well as at motor neuron synapses [11, 12]. The expression of GABAB receptor subunits is enhanced in lumbal spinal cord and dorsal root ganglion following chronic nociceptive activation in models of axotomy and chemogenic pain .
Multiple lines of evidence support an antinociceptive role for GABAB receptors in animal models of acute and chronic pain. baclofen, a GABAB receptor agonist, exhibits antinociceptive effects in model of peripheral nerve injury and chronic inflammation . 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 , ischemic injury to the spinal cord , carrageenan-produced inflammation  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 GABAB receptor agonists as analgesics has been restricted to trigeminal neuralgia and post-herpetic neuralgia [20, 21]. Indeed, GABAB receptor agonists display muscle relaxant properties and are rather widely used in the control of spasticity [22, 23] and have been implicated in dystonia . Because evoked pain behaviors in animal studies mostly rely upon a motor behavioral response, the motor deficits caused by GABAB receptor modulation occlude an unequivocal interpretation of behavioral responses. Another important caveat is that currently available GABAB 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 GABAB 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.
Materials and methods
Homozygous mice carrying the GABAB1 flox allele (GABAB(1) flanked by loxP sites, GABAB(1) fl511/fl511) have been described previously in details  GABAB(1) fl511/fl511 mice were crossed with SNS-Cre mice  to obtain GABAB1-LoxPSNS-Cre+ mice (referred to as SNS-GABAB(1) -/- mice in this manuscript) and GABAB(1) fl/fl mice (control littermates). Genotyping was done on mouse genomic tail DNA using primers: for sense strand 5'-ATCTCTTCCTTGGCTGGGTCTTTGCTTCGCTCG-3' and for anti-sense 5'-GGGTTATTGAATATGATCGGAATTCCTCGACT-3' to detect GABAB1 flox allele, and for sense strand 5'-GAAAGCAGCCATGTCCAATTTACTGACCGTAC-3' and for anti-sense strand 5'-GCGCGCCTGAAGATATAGAAGA-3' to detect SNS-Cre transgene expression. Both, SNS-Cre and GABAB(1) fl511/fl511 mice were backcrossed individually into their background for more than 8 generations before being crossed with each other. Littermates were used in all experiments to control for background effects.
Western blots were performed with lysates of mouse DRG, spinal cord and brain with antibodies recognizing murine GABAB(1) (AB1531, Chemicon) and alpha-tubulin (Sigma Aldrich) according to standard protocols .
In Situ Hybridisation
For generation of riboprobes, 1.7 kb-long GABAB(1)-specific probes were generated and in situ hybridisation using nonradioactive Dig-UTP-labelled antisense and sense probes was performed on cryostat sections of DRG (16 μm) as described in details previously .
Nociceptive tests and mouse models of pain
All animal use procedures were in accordance with ethical guidelines imposed by the local governing body (Regierungspräsidium Karlsruhe, Germany). All behavioral measurements were done in awake, unrestrained, age-matched, female or male mice which were more than 3 months of age. Complete Freund's adjuvant (CFA, Sigma Aldrich) was injected unilaterally in the intraplantar surface of the mouse hindpaw (20 μl) as described in details previously . Analysis of latency of paw withdrawal in response to noxious heat was done as described in details  (Ugo Basile Inc.). Mechanical sensitivity was tested in the same group of animals using von Frey hairs.
The 'spared nerve injury' (SNI) model for neuropathic pain was performed as described in details previously . Two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) were ligated and cut leaving the remaining third branch (sural nerve) intact. Mechanical allodynia following SNI was measured as paw withdrawal latency to dynamic von Frey stimulation (Ugo Basile, maximum force 5 g, ramp 10 sec) . Response to thermal stimulus (52°C) in the SNI model was done via a hot plate latency test. The latency to paw licking, paw withdrawal or jumping after placing the animal to the hot plate was measured. Cold allodynia following SNI was assessed by counting the number of responses (flinching, licking, jumping, and shaking) on a 5°C cold plate during an observation period of 90 seconds. Additionally, cold allodynia was determined by measuring the duration of paw licking, lifting and flinching in response to plantar application of a drop of acetone. The nociceptive behavior was observed for 90 sec starting immediately after acetone application.
Formalin (1%, 20 μl) was injected into the plantar surface of the hindpaw and the duration of nocifensive behaviors (lifting, licking, or flinching) was measured in 5 min bins for 60 min after formalin injection as described previously . baclofen (2 mg/kg of body weight; Lioresal intrathecal, Novartis) or sterile PBS for control animals were injected intraperitonially 30 min before 1% formalin administration.
Afferent recordings in skin-nerve preparation
A total of 19 mice (10 GABAB(1) fl/fl and 9 SNS-GABAB(1) -/-) were used in the electrophysiological investigations. An in vitro skin nerve preparation was used to study the properties of mechanosensitive C- and Aδ-afferent fibers which innervate the skin in the inflamed area. Experiments were performed on the dissected skin of control animals and animals sacrificed 4 hours following CFA inoculation into the hindpaw (30 μl). Animals were killed by CO2 inhalation, the saphenous nerve was dissected with the skin of the dorsal hind-paw attached and mounted in an organ bath "inside-up" to expose the dermis. The preparation was perfused with an oxygen-saturated modified synthetic interstitial fluid solution containing (in mM) 123 NaCl, 3.5 KCl, 0.7 MgSO4, 1.5 NaH2PO3, 1.7 NaH2PO4, 2.0 CaCl2, 9.5 sodium gluconate, 5.5 glucose, 7.5 sucrose, 10 Hepes at temperature of 32 ± 1°C and pH 7.4 ± 0.05. Fine filaments were teased from the desheathed nerve placed in separate chamber and placed on a recording electrode.
Nerve fibers were classified according to their conduction velocities, von Frey thresholds, and firing properties. Electrical stimulation of the nerve fiber was employed to calculate conduction velocities of individual nerve fibers. Fibers which conducted < 1 m/s and fibers conducting between 1-10 m/s were considered to be unmyelinated C-fibers and myelinated Aδ-fibers, respectively. Some of the fibers of the velocities around 1 m/s where not included into the analyses if further detailed classification according to the firing properties and threshold was not possible.
Once the receptive field was identified using the glass rod a computer-controlled linear stepping motor (Nanomotor Kleindiek Nanotechnik)  was used to apply standardized mechanical stimuli. Each fiber was tested with series of displacement mechanical stimuli ranging from 6 to 384 μm for both control and CFA injected animals. Electrophysiological data were collected with Powerlab 4.0 system and analyzed off-line with the spike histogram extension of the software.
Data analysis & statistics
All data are presented as mean ± standard error of the mean (S.E.M.). Analysis of variance (ANOVA) for random measures was performed followed by post-hoc Fisher's test to determine statistically significant differences. p < 0.05 was considered significant.
Conditional and specific deletion of GABAB(1) in nociceptors
We utilized the Cre-loxP system to generate mice conditionally lacking GABAB receptors specifically in primary nociceptors. We have previously described the generation of BAC transgenic mice expressing the Cre-recombinase under the influence of promoter elements of the mouse Scn10a gene encoding the NaV1.8 sodium channel. This line enables gene deletion specifically in c- and Aδ-nociceptors in the dorsal root ganglia while preserving gene expression in the central nervous system and other tissues [26, 34]. Mice carrying floxed GABAB1 allele  were crossed with SNS-Cre+ mice to generate homozygous mice in which the Cre-mediated excision of GABA B(1) exon will assure the absence of GABAB1 gene product in pre-synaptic order nociceptive neurons and their peripheral and central terminals. These include all of the known C-terminal variants of GABAB(1), such as GABAB(1a), which contributes largely to the GABAB(1) expression in nociceptors, GABAB(1b), and the additional, newly described GABAB(1e), that can mediate dominant-negative effects on GABAB receptor heteromerization and is indeed expressed in pain pathways . Based on our understanding of GABAB receptor heteromerization, lack of the GABAB(1) subunit precludes the assembly of functional GABAB receptor [28, 36–38]. Previous studies have shown that the deletion of GABAB(1) subunit is sufficient to cause the loss of pre- and post-synaptic GABAB responses [25, 39].
Western blot analysis showed that in SNS-GABAB(1) -/- mice expression of GABAB(1) protein in the dorsal root ganglia (DRG) is largely reduced, whereas expression in the brain and spinal cord remains unchanged (Fig. 1A and 1B). In situ mRNA hybridisation using GABAB(1)-specific riboprobes revealed that only a small proportion of neurons expressing mRNA for GABAB(1) belongs to the group of large diameter neurons. A majority of GABAB(1) expressing DRG neurons are small-diameter neurons. SNS-GABAB(1) -/- mice showed a selective deletion of GABAB(1) in small diameter neurons of the DRG whereas expression in large-diameter neurons was preserved (Fig. 1C).
Electrophysiological analyses of peripheral nerve activity in SNS-GABAB(1) -/- mice
To clarify the specific contribution of GABAB receptors on peripheral terminals, we performed electrophysiological recordings on peripheral polymodal C-fiber nociceptors and Aδ nociceptors, which were identified on the basis of stimulation and conduction properties in a hindpaw skin-nerve preparation . The skin was dissected from both genotypes from either naive mice or 4 hours after injection of 30 μl CFA into the hindpaw.
Recordings were made from C- and Aδ-fibers because these neurons are targeted by the SNS-Cre-mediated deletion of GABAB(1). Stimulus-response functions of C-fibers from naive SNS-GABAB(1) -/- mice and GABAB(1) fl/fl mice demonstrated no significant changes between the responsiveness of this population to mechanical stimulation (Fig. 2A). At 4 h following CFA-induced hind paw inflammation, the excitability of mechanoreceptive C-fibers increased significantly in GABAB(1) fl/fl mice (Fig. 2B; p < 0.005, ANOVA followed by posthoc Fisher's test; typical examples of traces are shown below the quantitative summary in Fig. 2B). Mechanoreceptive C-fibers of SNS-GABAB(1) -/- mice also demonstrated increased excitability following CFA-induced inflammation; however, statistical significance was only reached at higher forces of mechanical stimulation. Surprisingly, analysis of the basal activity of mechanoreceptive AM-fibers (Aδ-fibers) revealed that the basal excitability is significantly increased in mice lacking GABAB(1) in peripheral nociceptors (Fig. 2D) whereas GABAB(1) fl/fl mice demonstrated strong hyperexcitability to the entire range of intensities of mechanical stimuli employed following CFA-induced inflammation (Fig. 2E). Aδ-fibers in SNS-GABAB(1) -/- mice failed to demonstrate any further increases in excitability following CFA (Fig. 2F). The magnitude of responses shown by SNS-GABAB(1) -/- mice to various mechanical forces in the naive state was equivalent to those shown by the GABAB(1) fl/fl mice in the CFA-induced sensitized state and no further sensitization was then seen in SNS-GABAB(1) -/- mice following CFA-induced inflammation.
Development of acute nociceptive hypersensitivity and its pharmacological modulation by baclofen
To determine how GABAB receptor affects behavioral correlates of rapid sensitization in pain pathways, we performed the plantar formalin test on SNS-GABAB(1) -/- mutant mice and their wild-type controls . Intraplantar injection of formalin in rodents evokes nocifensive behaviors such as licking, shaking, and lifting of the injected paw in a biphasic manner. Phase I of the formalin response (0-10 min after injection) is caused by persistent activation and acute sensitization of nociceptors, whereas the phase II response (10-60 min after injection) result from continual activation of nociceptors and a sensitization of central synapses via mechanisms which are triggered by repetitive stimulation during the first phase . GABAB(1) mutant mice failed to reveal changes in the total cumulative duration of phase I and phase II responses to formalin injection (1%, 20 μl) (Fig. 3B). However, the latter part of the phase II formalin response (so called phase IIb, ) was significantly prolonged in SNS-GABAB(1) -/- (n = 5) when compared to GABAB(1) fl/fl animals (n = 5) (Fig. 3A).
Previous studies on formalin-induced nocifensive behavior showed that intraperitoneal or intrathecal injection of GABA-B receptor agonist, baclofen induces antinociception in both phases of the formalin test [41, 42]. Consistent with previous studies, baclofen (2 mg/kg of body weight), administered intraperitoneally 30 min prior to the injection of formalin showed antinociceptive effect on phase I and II (Fig. 3B, C; p < 0.05) behavior. However, baclofen was equally efficient in depressing nociceptive behavior on SNS-GABAB(1) -/- (n = 5) and GABAB(1) fl/fl (n = 5) mice (Fig. 3B, C; p < 0.05) suggesting that the effect of baclofen is independent on expression of GABAB receptors in peripheral nervous system.
Development of chronic inflammatory pain in SNS-GABB(1) -/- mice
Development of somatic inflammatory pain and hyperalgesia was assessed in GABAB(1) fl/fl mice and SNS-GABAB(1) -/-mice at 12 h, 1, 2, 4, 6 and 8 days following unilateral hindpaw inflammation induced by intraplantar injection of complete Freud's adjuvant (CFA; 20 μl). In CFA-injected animals paw withdrawal latency (PWL) to noxious radiant heat decreased significantly for up to 4 days (Fig. 4A). CFA-induced thermal hyperalgesia was comparable in SNS-GABAB(1) -/- (n = 6) and GABAB(1) fl/fl (n = 6) mice (Fig. 4B). Magnitudes of mechanical hyperalgesia that developed after CFA injection was tested using von Frey filaments on SNS-GABAB(1) -/- mice, and their respective control littermates (Fig. 4C-F). Upon CFA injection, no significant variance in the magnitudes of both allodynia (defined as responses to 0.16-0.4 g force) as well as mechanical hyperalgesia (defined as responses to 0.6 - 4 g force) was observed in SNS-GABAB(1) -/- (n = 6) mice compared to GABAB(1) fl/fl mice (n = 6) (Fig. 4C-E). No significant difference in the relative drop in response thresholds that is defined as minimum force required to elicit 40% response frequency over the basal state was observed between genotypes (Fig. 4F). These results imply that a loss of GABAB receptors in nociceptive DRG neurons does not produce a major effect on nociceptive behavior and is not involved in the modulation of chronic inflammatory pain.
Development of neuropathic pain in SNS-GABAB(1) -/- mice
To assess whether peripheral GABAB receptors play a role in nociceptive hypersensitivity which develops following a peripheral nerve lesion, we employed the spared nerve injury model (SNI) of neuropathic pain . At any of the time points studied following injury (3, 7, 9, 16 and 23 days), no differences were found with respect to hyperalgesia and allodynia between SNS-GABAB(1) -/- mice and GABAB(1) fl/fl mice. Following SNI, profound mechanical allodynia was recorded by measuring paw withdrawal latency using a dynamic aesthesiometer. Mechanical allodynia developed to a similar extent in SNS-GABAB(1) -/- and GABAB(1) fl/fl mice (Fig. 5A). Thermal hyperalgesia to heat developed to a comparable extent in GABAB(1) -/- mice and GABAB(1) fl/fl mice (Fig. 5B). Similarly, cold allodynia (responses to 5°C) developed equally in SNS-GABAB(1) -/- mice and GABAB(1) fl/fl mice following SNI (Fig. 5C, D). From these data, we infer that GABAB receptors expressed by Aδ- and C-nociceptive peripheral neurons are not involved in regulation of neuropathic pain.
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 GABAB receptor expression in primary afferent terminals [6–9] as well as functional studies which show that GABAB receptor activation on primary afferent terminals in the spinal cord reduces neurotransmitter release [43–45]. Although it is clear that GABAB receptors are densely expressed in peripheral nociceptive neurons, the literature on the regulation of GABAB receptor expression in pathological pain states is somewhat mixed. For example, some studies reported an increase in GABAB receptor expression in the spinal dorsal horn and peripheral nociceptors in inflammatory pain states . In contrast, Engle et al.  found that spinal nerve ligation does not alter the expression or function of GABAB(1) and GABAB(2) in the spinal cord and dorsal root ganglia of rats and also does not lead to changes in GABAB receptor binding affinity in inflammatory and neuropathic states. Furthermore, findings in a model of diabetic neuropathy suggest reduced function of presynaptic GABAB receptors at primary afferent terminals, but not those on GABAergic and glycinergic interneurons, in the spinal cord . Interestingly, a series of experiments with novel ligands at GABAB receptors have also suggested a functional contribution of GABAB(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 GABAB receptors in first order neurons . Thus, considerable support implicates a role for GABAB 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, GABAB(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 GABAB(1) leads to a complete lack of ligand binding and a total loss of function of native GABAB receptors [28, 36–38]. Therefore, based upon our findings, we infer that a conditional loss of GABAB 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-GABAB(1) -/- mice compared to GABAB(1) fl/fl. This might result from higher expression of GABAB(1) in Aδ- as compared to C-fibers. As noted previously GABAB receptor mRNA has been shown to be expressed in all DRG neurons . 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 GABAB(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 .
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 GABAB(1) beginning at very early developmental stages, such as in classical knockout mice, does not lead to compensation of GABAB-mediated inhibition with respect to pain; classical GABAB(1) knockout mice demonstrate a prominent hyperalgesic phenotype . Analyses in classical GABAB(1) knockout mice have confirmed that a loss of the GABAB(1) subunit is paralleled by a loss of all biochemical and electrophysiological GABAB(1) responses [25, 39, 49], demonstrating that GABAB(1) is an essential component of pre- and postsynaptic GABAB receptors. Directly comparing the phenotypes of global GABAB(1) receptor knockout mice and nociceptor-specific GABAB(1) knockout mice therefore leads to the inference that although GABAB 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 GABAB(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 , thereby raising the possibility that these alterations in the periphery may have contributed to the sensory phenotype in GABAB(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-GABAB(1) -/- compared to GABAB(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 . Therefore, the phenotype in phase IIb of the formalin response could be caused by exaggerated activation of primary afferents due to the lack of GABAB mediated inhibition in the first phase of the formalin test.
Experimental studies with the classical GABAB receptor agonist, baclofen, have implicated a therapeutic role for GABAB 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 GABAB(1) receptor mutants revealed that this anti-nociceptive activity of baclofen occurs independently of GABAB(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 GABAB receptor antagonist, phaclofen, shows opposite effects . Furthermore, GABAB 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 . 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 GABAB 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 .
Indeed, GABAB 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 . Furthermore, it has been shown that a local increase of GABAB concentrations in higher brain centres results in lasting bilateral analgesia . 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.
In summary, this study clarifies a long-standing question in the field of GABAergic modulation of nociception, namely the contribution of presynaptic GABAB receptors in primary afferent nociceptive neurons. The use of genetic tools to specifically delete GABAB receptors in DRG neurons, while leaving their expression in the spinal cord and brain intact, revealed that GABAB 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 GABAB 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 GABAB receptor system for pain management.
complete Freud's adjuvant
dorsal root ganglia
phosphate buffered saline
paw withdrawal latency
spared nerve injury.
Huang CC, Hsu KS: Local protein synthesis and GABAB receptors regulate the reversibility of long-term potentiation at murine hippocampal mossy fibre-CA3 synapses. J Physiol 2004, 561: 91–108. 10.1113/jphysiol.2004.072546
Remondes M, Schuman EM: Molecular mechanisms contributing to long-lasting synaptic plasticity at the temporoammonic-CA1 synapse. Learn Mem 2003, 10: 247–252. 10.1101/lm.59103
Pan BX, Dong Y, Ito W, Yanagawa Y, Shigemoto R, Morozov A: Selective gating of glutamatergic inputs to excitatory neurons of amygdala by presynaptic GABAb receptor. Neuron 2009, 61: 917–929. 10.1016/j.neuron.2009.01.029
Vigot R, Barbieri S, Bräuner-Osborne H, Turecek R, Shigemoto R, Zhang YP, Luján R, Jacobson LH, Biermann B, Fritschy JM, Vacher CM, Müller M, Sansig G, Guetg N, Cryan JF, Kaupmann K, Gassmann M, Oertner TG, Bettler B: Differential compartmentalization and distinct functions of GABAB receptor variants. Neuron 2006, 50: 589–601. 10.1016/j.neuron.2006.04.014
Shaban H, Humeau Y, Herry C, Cassasus G, Shigemoto R, Ciocchi S, Barbieri S, Putten H, Kaupmann K, Bettler B, Lüthi A: Generalization of amygdala LTP and conditioned fear in the absence of presynaptic inhibition. Nat Neurosci 2006, 9: 1028–1035. 10.1038/nn1732
Price GW, Kelly JS, Bowery NG: The location of GABAB receptor binding sites in mammalian spinal cord. Synapse 1987, 1: 530–5838. 10.1002/syn.890010605
Towers S, Princivalle A, Billinton A, Edmunds M, Bettler B, Urban L, Castro-Lopes J, Bowery NG: GABAB receptor protein and mRNA distribution in rat spinal cord and dorsal root ganglia. Eur J Neurosci 2000, 12: 3201–3210. 10.1046/j.1460-9568.2000.00237.x
Yang K, Wang D, Li YQ: Distribution and depression of the GABA B receptor in the spinal dorsal horn of adult rat. Brain Res Bull 2001, 55: 479–485. 10.1016/S0361-9230(01)00546-9
Castro AR, Pinto M, Lima D, Tavares I: Nociceptive spinal neurons expressing NK1 and GABA B receptors are located in lamina I. Brain Res 2004, 1003: 77–85. 10.1016/j.brainres.2003.12.027
Charles KJ, Evans ML, Robbins MJ, Calver AR, Leslie RA, Pangalos MN: Comparative immunohistochemical localisation of GABA(B1a), GABA(B1b) and GABA(B2) subunits in rat brain, spinal cord and dorsal root ganglion. Neuroscience 2001, 106: 447–467. 10.1016/S0306-4522(01)00296-2
Fritschy JM, Meskenaite V, Weinmann O, Honer M, Benke D, Mohler H: GABAB-receptor splice variants GB1a and GB1b in rat brain: developmental regulation, cellular distribution and extrasynaptic localization. Eur J Neurosci 1999, 11: 761–768. 10.1046/j.1460-9568.1999.00481.x
Calver AR, Medhurst AD, Robbins MJ, Charles KJ, Evans ML, Harrison DC, Stammers M, Hughes SA, Hervieu G, Couve A, Moss SJ, Middlemiss DN, Pangalos MN: The expression of GABA B1 and GABA B2 receptor subunits in the CNS differs from that in peripheral tissues. Neuroscience 2000, 100: 155–170. 10.1016/S0306-4522(00)00262-1
McCarson KE, Enna SJ: Nociceptive regulation of GABA(B) receptor gene expression in rat spinal cord. Neuropharmacology 1999, 38: 1767–1773. 10.1016/S0028-3908(99)00121-5
Smith GD, Harrison SM, Birch PJ, Elliott PJ, Malcangio M, Bowery NG: Increased sensitivity to the antinociceptive activity of (+/-)-baclofen in an animal model of chronic neuropathic, but not chronic inflammatory hyperalgesia. Neuropharmacology 1994, 33: 1103–1108. 10.1016/0028-3908(94)90149-X
Castro AR, Pinto M, Lima D, Tavares I: Secondary hyperalgesia in the monoarthritic rat is mediated by GABAB and NK1 receptors of spinal dorsal horn neurons: a behavior and c-fos study. Neuroscience 2006, 141: 2087–2095. 10.1016/j.neuroscience.2006.05.048
Hao JX, Xu XJ, Yu YX, Seiger A, Wiesenfeld-Hallin Z: Baclofen reverses the hypersensitivity of dorsal horn wide dynamic range neurons to mechanical stimulation after transient spinal cord ischemia; implications for a tonic GABAergic inhibitory control of myelinated fiber input. J Neurophysiol 1992, 68: 392–396.
Buritova J, Chapman V, Honoré P, Besson JM: The contribution of GABAB receptor-mediated events to inflammatory pain processing: carrageenan oedema and associated spinal c-Fos expression in the rat. Neuroscience 1996, 73: 487–496. 10.1016/0306-4522(96)00071-1
Takemura M, Shimada T, Shigenaga Y: GABA B receptor-mediated effects on expression of c-Fos in rat trigeminal nucleus following high- and low-intensity afferent stimulation. Neuroscience 2001, 103: 1051–1058. 10.1016/S0306-4522(01)00031-8
Takeda M, Tanimoto T, Ikeda M, Kadoi J, Matsumoto S: Activaton of GABAB receptor inhibits the excitability of rat small diameter trigeminal root ganglion neurons. Neuroscience 2004, 123: 491–505. 10.1016/j.neuroscience.2003.09.022
Hosny A, Simopoulos T, Collins B: Response of intractable post herpetic neuralgia to intrathecal baclofen. Pain Physician 2004, 7: 345–347.
Cheshire WP: Trigeminal neuralgia: for one nerve a multitude of treatments. Expert Rev Neurother 2007, 7: 1565–1579. 10.1586/14737126.96.36.1995
Kakinohana O, Hefferan MP, Nakamura S, Kakinohana M, Galik J, Tomori Z, Marsala J, Yaksh TL, Marsala M: Development of GABA-sensitive spasticity and rigidity in rats after transient spinal cord ischemia: a qualitative and quantitative electrophysiological and histopathological study. Neuroscience 2006, 141: 1569–1583. 10.1016/j.neuroscience.2006.04.083
Slonimski M, Abram SE, Zuniga RE: Intrathecal baclofen in pain management. Reg Anesth Pain Med 2004, 29: 269–276.
van Hilten JJ, Hoff JI, Thang MC, van de Meerakker MM, Voormolen JH, Delhaas EM: Clinimetric issues of screening for responsiveness to intrathecal baclofen in dystonia. Neural Transm 1999, 106: 931–941. 10.1007/s007020050213
Haller C, Casanova E, Müller M, Vacher CM, Vigot R, Doll T, Barbieri S, Gassmann M, Bettler B: Floxed allele for conditional inactivation of the GABAB(1) gene. Genesis 2004, 40: 125–130. 10.1002/gene.20073
Agarwal N, Offermanns S, Kuner R: Conditional gene deletion in primary nociceptive neurons of trigeminal ganglia and dorsal root ganglia. Genesis 2004, 38: 122–129. 10.1002/gene.20010
Tappe A, Klugmann M, Luo C, Hirlinger D, Agarwal N, Benrath J, Ehrengruber MU, During MJ, Kuner R: Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain. Nat Med 2006, 12: 677–816. 10.1038/nm1406
Kuner R, Köhr G, Grünewald S, Eisenhardt G, Bach A, Kornau HC: Role of heteromer formation in GABAB receptor function. Science 1999, 283: 74–77. 10.1126/science.283.5398.74
Hartmann B, Ahmadi S, Heppenstall PA, Lewin GR, Schott C, Borchardt T, Seeburg PH, Zeilhofer HU, Sprengel R, Kuner R: The AMPA receptor subunits GluR-A and GluR-B reciprocally modulate spinal synaptic plasticity and inflammatory pain. Neuron 2004, 44: 637–650. 10.1016/j.neuron.2004.10.029
Decosterd I, Woolf CJ: Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000, 87: 149–158. 10.1016/S0304-3959(00)00276-1
Schmidtko A, Luo C, Gao W, Geisslinger G, Kuner R, Tegeder I: Genetic deletion of synapsin II reduces neuropathic pain due to reduced glutamate but increased GABA in the spinal cord dorsal horn. Pain 2008, 139: 632–643. 10.1016/j.pain.2008.06.018
Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K: The formalin test: an evaluation of the method. Pain 1992, 51: 5–17. 10.1016/0304-3959(92)90003-T
Wetzel C, Hu J, Riethmacher D, Benckendorff A, Harder L, Eilers A, Moshourab R, Kozlenkov A, Labuz D, Caspani O, Erdmann B, Machelska H, Heppenstall PA, Lewin GR: A stomatin-domain protein essential for touch sensation in the mouse. Nature 2007, 445: 206–209. 10.1038/nature05394
Agarwal N, Pacher P, Tegeder I, Amaya F, Constantin CE, Brenner GJ, Rubino T, Michalski CW, Marsicano G, Monory K, Mackie K, Marian C, Batkai S, Parolaro D, Fischer MJ, Reeh P, Kunos G, Kress M, Lutz B, Woolf CJ, Kuner R: Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors. Nat Neurosci 2007, 10: 870–879. 10.1038/nn1916
Schwarz DA, Barry G, Eliasof SD, Petroski RE, Conlon PJ, Maki RA: Characterization of gamma-aminobutyric acid receptor GABAB(1e), a GABAB(1) splice variant encoding a truncated receptor. J Biol Chem 2000, 275: 32174–32181. 10.1074/jbc.M005333200
Jones KA, Borowsky B, Tamm JA, Craig DA, Durkin MM, Dai M, Yao WJ, Johnson M, Gunwaldsen C, Huang LY, Tang C, Shen Q, Salon JA, Morse K, Laz T, Smith KE, Nagarathnam D, Noble SA, Branchek TA, Gerald C: GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2. Nature 1998, 396: 674–679. 10.1038/25348
Kaupmann K, Malitschek B, Schuler V, Heid J, Froestl W, Beck P, Mosbacher J, Bischoff S, Kulik A, Shigemoto R, Karschin A, Bettler B: GABA(B)-receptor subtypes assemble into functional heteromeric complexes. Nature 1998, 396: 683–687. 10.1038/25360
White JH, Wise A, Main MJ, Green A, Fraser NJ, Disney GH, Barnes AA, Emson P, Foord SM, Marshall FH: Heterodimerization is required for the formation of a functional GABA(B) receptor. Nature 1998, 396: 679–682. 10.1038/25354
Schuler V, Lüscher C, Blanchet C, Klix N, Sansig G, Klebs K, Schmutz M, Heid J, Gentry C, Urban L, Fox A, Spooren W, Jaton AL, Vigouret J, Pozza M, Kelly PH, Mosbacher J, Froestl W, Käslin E, Korn R, Bischoff S, Kaupmann K, Putten H, Bettler B: Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABA(B) responses in mice lacking GABA(B(1)). Neuron 2001, 31: 47–58. 10.1016/S0896-6273(01)00345-2
Tegeder I, Niederberger E, Vetter G, Brautigam L, Geisslinger G: Effects of selective COX-1 and -2 inhibition on formalin-evoked nociception behavior and prostaglandin E2 release in the spinal cord. J Neurochem 2001, 79: 777–786. 10.1046/j.1471-4159.2001.00613.x
Shafizadeh M, Semnanian S, Zarrindast MR, Hashemi B: Involvement of GABAB receptors in the antinociception induced by baclofen in the formalin test. Gen Pharmacol 1997, 28: 611–615.
Dirig DM, Yaksh TL: Intrathecal baclofen and muscimol, but not midazolam, are antinociceptive using the rat-formalin model. J Pharmacol Exp Ther 1995, 275: 219–227.
Ataka T, Kumamoto E, Shimoji K, Yoshimura M: Baclofen inhibits more effectively C-afferent than Adelta-afferent glutamatergic transmission in substantia gelatinosa neurons of adult rat spinal cord slices. Pain 2000, 86: 273–282. 10.1016/S0304-3959(00)00255-4
Iyadomi M, Iyadomi I, Kumamoto E, Tomokuni K, Yoshimura M: Presynaptic inhibition by baclofen of miniature EPSCs and IPSCs in substantia gelatinosa neurons of the adult rat spinal dorsal horn. Pain 2000, 85: 385–393. 10.1016/S0304-3959(99)00285-7
Wang XL, Zhang HM, Chen SR, Pan HL: Altered synaptic input and GABAB receptor function in spinal superficial dorsal horn neurons in rats with diabetic neuropathy. J Physiol 2007, 579: 849–861. 10.1113/jphysiol.2006.126102
Engle MP, Gassman M, Sykes KT, Bettler B, Hammond DL: Spinal nerve ligation does not alter the expression or function of GABA(B) receptors in spinal cord and dorsal root ganglia of the rat. Neuroscience 2006, 138: 1277–1287. 10.1016/j.neuroscience.2005.11.064
Callaghan B, Haythornthwaite A, Berecki G, Clark RJ, Craik DJ, Adams DJ: Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABAB receptor activation. J Neurosci 2008, 28: 10943–10951. 10.1523/JNEUROSCI.3594-08.2008
Sengupta JN, Medda BK, Shaker R: Effect of GABA(B) receptor agonist on distension-sensitive pelvic nerve afferent fibers innervating rat colon. Am J Physiol Gastrointest Liver Physiol 2002, 283: G1343–1351.
Kaupmann K, Cryan JF, Wellendorph P, Mombereau C, Sansig G, Klebs K, Schmutz M, Froestl W, Putten H, Mosbacher J, Bräuner-Osborne H, Waldmeier P, Bettler B: Specific gamma-hydroxybutyrate-binding sites but loss of pharmacological effects of gamma-hydroxybutyrate in GABA(B)(1)-deficient mice. Eur J Neurosci 2003, 18: 2722–2730. 10.1111/j.1460-9568.2003.03013.x
Magnaghi V, Ballabio M, Camozzi F, Colleoni M, Consoli A, Gassmann M, Lauria G, Motta M, Procacci P, Trovato AE, Bettler B: Altered peripheral myelination in mice lacking GABAB receptors. Mol Cell Neurosci 2008, 37: 599–609. 10.1016/j.mcn.2007.12.009
Puig S, Sorkin LS: Formalin-evoked activity in identified primary afferent fibers: systemic lidocaine suppresses phase-2 activity. Pain 1996, 64: 345–355. 10.1016/0304-3959(95)00121-2
Sabetkasai M, Khansefid N, Yahyavi SH, Zarrindast MR: Baclofen and antidepressant-induced antinociception in formalin test: possible GABA(B) mechanism involvement. Psychopharmacology 1999, 142: 426–431. 10.1007/s002130050909
Gwak YS, Tan HY, Nam TS, Paik KS, Hulsebosch CE, Leem JW: Activation of spinal GABA receptors attenuates chronic central neuropathic pain after spinal cord injury. J Neurotrauma 2006, 23: 1111–1124. 10.1089/neu.2006.23.1111
Choi IS, Cho JH, Jeong SG, Hong JS, Kim SJ, Kim J, Lee MG, Choi BJ, Jang IS: GABA(B) receptor-mediated presynaptic inhibition of glycinergic transmission onto substantia gelatinosa neurons in the rat spinal cord. Pain 2008, 138: 330–342. 10.1016/j.pain.2008.01.005
Ohara PT, Granato A, Moallem TM, Wang BR, Tillet Y, Jasmin L: Dopaminergic input to GABAergic neurons in the rostral agranular insular cortex of the rat. J Neurocytol 2003, 32: 131–141. 10.1023/B:NEUR.0000005598.09647.7f
Jasmin L, Rabkin SD, Granato A, Boudah A, Ohara PT: Analgesia and hyperalgesia from GABA-mediated modulation of the cerebral cortex. Nature 2003, 424: 316–320. 10.1038/nature01808
The authors thank H.-J. Wrede and D. Baumgartl-Ahlert for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (KU1438/3-1 and KU1438/4-1 to RK), the Frontier Excellence Program of the University of Heidelberg (to RK), the Swiss Science Foundation (3100A0-117816 to BB) and the European Community's Seventh Framework Programme (FP7/2007-2013) under Grant Agreement 201714 (to BB). MK is a recipient of a Postdoctoral Fellowship from the Medical Faculty of the University of Heidelberg
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 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.