Extracellular Signal-Regulated Kinases Mediate an Autoregulation of GABAB-Receptor-Activated Whole-Cell Current in Locus Coeruleus Neurons.

The norepinephrine-releasing neurons in the locus coeruleus (LC) are well known to regulate wakefulness/arousal. They display active firing during wakefulness and a decreased discharge rate during sleep. We have previously reported that LC neurons express large numbers of GABAB receptors (GABABRs) located at peri-/extrasynaptic sites and are subject to tonic inhibition due to the continuous activation of GABABRs by ambient GABA, which is significantly higher during sleep than during wakefulness. In this study, we further showed using western blot analysis that the activation of GABABRs with baclofen could increase the level of phosphorylated extracellular signal-regulated kinase 1 (ERK1) in LC tissue. Recordings from LC neurons in brain slices showed that the inhibition of ERK1/2 with U0126 and FR180204 accelerated the decay of whole-cell membrane current induced by prolonged baclofen application. In addition, the inhibition of ERK1/2 also increased spontaneous firing and reduced tonic inhibition of LC neurons after prolonged exposure to baclofen. These results suggest a new role of GABABRs in mediating ERK1-dependent autoregulation of the stability of GABABR-activated whole-cell current, in addition to its well-known effect on gated potassium channels, to cause a tonic current in LC neurons.

receptor is formed from the heterodimerization of the GABA B1 and GABA B2 receptor subunits, with the former constituting the GABA binding site and the latter being coupled to the Gproteins, comprising α i/o , β and γ subunits [11][12][13] . The binding of GABA to the GABA B1 receptor activates the coupled G protein to gate the pre-and postsynaptic ion channels described above via the β and γ subunits 8,10 . Despite the well-understood functional roles of the β and γ subunits, much remains to be learned about the role of receptor-induced lowering of cAMP levels by the α i/o subunit.
Electron microscopic studies have revealed that the subcellular distribution of GABA B Rs is mostly at peri-/ extrasynaptic loci [4][5][6][7] , implying that, similar to GABA A Rs, these extrasynaptic GABA B Rs can mediate a tonic form of signaling by detecting ambient GABA. Indeed, it has been shown that ambient GABA can tonically induce a low level of presynaptic and postsynaptic GABA B R activation to provide the control of transmitter release at the hippocampus and calyx of Held synapses and the control of the excitability of pyramidal neurons in the medial prefrontal cortex and noradrenergic (NAergic) neurons in the locus coeruleus (LC) 7,9,[14][15][16] . The physiological roles of GABA B R-mediated tonic inhibition have begun to emerge. Recently, it has been shown that tonic inhibition of LC NAergic neurons (hereafter referred to as LC neurons) could be an important player in the regulation of brain function states 7,17 . LC neurons have global NAergic projections to the forebrain and play important roles in the control of behaviors through the regulation of vigilance 18,19 . Furthermore, GABAergic transmission in the LC has been implied to be a mechanism underlying the effect of some anesthetics on consciousness 17,[20][21][22][23][24] . It has been shown that LC neurons and NAergic A7 neurons in the pons express a large amount of GABA B Rs and are subject to GABA B R-mediated tonic inhibition in brain slice preparations and in vivo 7,9,17 . Moreover, the suppression of the tonic inhibition of LC neurons could accelerate the regain of consciousness from isoflurane-induced deep anesthesia 17 . Tonic inhibition would require the activity of a substantial number of GABA B Rs on the membrane for a long period. Nevertheless, this would appear to conflict with the features of most GPCRs, including GABA B Rs, such that the receptor will undergo rapid desensitization upon activation by the ligand 25 . In this study, we report that, in LC neurons, the activation of GABA B Rs also activates extracellular signal-regulated kinase 1 (ERK 1 ) signaling pathways, which is consistent with previous studies in the hippocampus and cerebellum [26][27][28] . We further show that the activation of ERK 1 signaling pathways by GABA B Rs could prevent a rapid decline in the GABA B R-activated whole-cell membrane current and help stabilize tonic inhibition.

Results
Activation of GABA B Rs increases peRK levels in the Lc. We first examined whether the activation of GABA B Rs could also increase phosphorylated-ERK 1/2 (pERK 1/2 ) levels in the LC, as previously reported in hippocampal and cerebellar tissue [26][27][28] . We examined pERK 1/2 levels in LC tissue punched from slices ( Fig. 1A) bathed in 50 μM baclofen, a GABA B R agonist, and the vehicle, artificial cerebrospinal fluid (aCSF) containing synaptic blockers (see Materials and Methods), using western blot analysis. As pERK 1/2 levels were reported to peak at 10 min and start to decline at 20 min of baclofen stimulation in cultured cerebellar granule cells 27 , 15 min of baclofen stimulation was used in this study. In comparison to the tissues from the vehicle-bathed slices, the pERK 1 level increased by 29.5 ± 8.2% in the LC tissues from the baclofen-bathed slices (Fig. 1B1) (p = 0.006; n = 9, Student's paired t-test). There was no increase in the pERK 2 level (p = 0.183; n = 9, Student's paired t-test). We also compared pERK 1/2 levels between LC tissues punched from slices bathed in baclofen and in baclofen plus 10 μM CGP54626, a GABA B R antagonist. Compared to LC tissues from the baclofen-bathed slices, the inhibition of GABA B Rs with CGP54626 reduced the pERK 1 level by 25.9 ± 4.1% (Fig. 1B2) (p = 0.003, n = 6, Student's paired t-test), showing that the increase in the pERK 1 level by baclofen stimulation was specific to GABA B R activation. Interestingly, compared to LC tissues from the baclofen-bathed slices, the pERK 2 level also significantly decreased by 31.3 ± 6.1% in CGP54626-bathed slices (p = 0.018, n = 6, Student's paired t-test). As the ambient GABA in the pontine area can continuously activate GABA B Rs to exert tonic inhibition of LC neurons 7,9 , it could be that there was a basal pERK 2 level produced by continuous GABA B R activation. Accordingly, tonic GABA B R activation left less room for a further increase in the pERK 2 level by baclofen stimulation, and the inhibition of GABA B Rs could result in a significant reduction.

characterization of i GABABR in Lc neurons.
To confirm that the increase in pERK 1 levels upon GABA B R activation occurred in LC neurons and to explore the possible physiological role of the elevated ERK activity, we performed whole-cell patch recording from LC neurons and tested the effects of ERK blockers on the whole-cell current induced by GABA B R activation. All recordings described hereafter were performed with the addition of synaptic blockers to the bath medium to avoid secondary effects via fast synaptic transmissions. We adapted previously described criteria for identifying NAergic neurons in the dorsal pontine area 7,29,30 to validate that the recorded neurons were LC neurons. The criteria were as follows: (1) the recorded neuron should be immunoreactive to anti-tyrosine hydroxylase (TH) antibody ( Fig. 2A); (2) the recorded neuron should be able to spontaneously fire APs followed by prominent afterhyperpolarization; and (3) the recorded neuron should display a delay in AP generation upon the injection of depolarizing current pulses, with V m held at ~ −70 mV (Fig. 2B). A very interesting observation from the whole-cell recordings of LC neurons was the appearance of spontaneous oscillation at ~ 0.2 Hz of the membrane voltage in current-clamp recordings (Fig. 2C) or of the membrane current in voltage-clamp recordings (Fig. 2D). The oscillating events displayed some similar features to those of spontaneous APs, such as being biphasic and generated at a rate similar to spontaneous APs. Since LC neurons are electrically coupled to gap junctions 31,32 , these events could be due to flow through the gap junctions of currents underlying the APs generated from other LC neurons in the proximity and electrically coupled to the recorded neuron 7 . This argument is further supported by the results showing that these events were blocked by CBX, a gap junction blocker (Fig. 2D). We refer to the events recorded using the voltage clamp as I Osc .
www.nature.com/scientificreports www.nature.com/scientificreports/ Bath application of 100 μM baclofen induced an outward current that was blocked by subsequent application of 10 μM CGP54626 (Fig. 3A), showing that the current was mediated by GABA B Rs. This observation is consistent with our previous reports 7, 9 , further demonstrating that the current was generated by the opening of The images show two sagittal brainstem slices from an animal. The LC in the left slice was punched (A1) for western blot analysis, and the right slice was used for comparison (A2). IHC with anti-TH antibody was performed for the two slices, as shown in the insets showing merged fluorescence images of anti-TH (red) and DAPI (blue) staining of the dashed rectangular areas at high magnification. A comparison of the two slices shows that the punched area contained mostly TH-ir tissue. (B) Images show representative western blot analysis results for pERK 1/2 in LC tissue punched from slices bathed in vehicle or baclofen (B1) and from slices bathed in baclofen or baclofen plus CGP54626 (B2). The plot in the right panels summarizes the results. Each paired circle and line indicates the result of a single experiment; bars and capped lines denote the mean and SEM, respectively. The asterisks denote significant differences compared to the control at p < 0.05 (*) and p < 0.01 (**); ns denotes no significance compared to the control. GIRK channels downstream of the activation of GABA B Rs by baclofen. Hereafter, we refer to the current as the GABA B R-mediated current (I GABABR ). Interestingly, the induction of I GABABR was associated with the suppression of I Osc activity, and the activity reappeared upon subsequent application of CGP54626 to counteract the effect of baclofen ( Fig. 3A-C).
The I GABABR underwent partial decline upon prolonged (15 minutes) exposure to baclofen in the control condition. As seen in Fig. 3A, upon the application of baclofen, I GABABR was quickly induced and peaked with a mean amplitude of 98.1 ± 20.4 pA (n = 11; Fig. 3D), followed by a gradual decline to approximately half the peak amplitude. We quantified the decline in I GABABR by measuring τ 50 , defined as the time required for the I GABABR to decline from its peak amplitude to half that value, and it was 356 ± 49 s in the control condition (Fig. 3E). The subsequent coapplication of CGP54626 15 min after baclofen application suppressed I GABABR to a level below baseline (the membrane current before baclofen application; see green dotted lines and asterisk in Fig. 3A), showing a basal tone of GABA B R activation in LC neurons. The membrane current underlying the basal tone of GABA B R activation is referred to as the tonic current (I Tonic ), measured as the difference between the membrane currents  www.nature.com/scientificreports www.nature.com/scientificreports/ at baseline and upon CGP54626 application (see asterisks in Fig. 3A). The I Tonic in the control was −9.4 ± 2.4 pA (n = 11) (Fig. 3F). Consistent with the observation of the basal tone of GABA B R activation, we also observed a significantly higher frequency of I Osc during CGP54626 application than at baseline.The frequency of I Osc at baseline and during the CGP54626 application was 0.15 ± 0.05 Hz and 0.38 ± 0.03 Hz, respectively (n = 11; p = 0.001, post hoc Bonferroni test after two-way repeated-measures ANOVA) (Fig. 3Aa, Fig. 3G).
inhibition of eRK 1/2 accelerates the decline of i GABABR Lc neurons. We next tested the effects of two selective ERK blockers, U0126 and FR180204, on I GABABR in LC neurons. We first examined whether there were possible effects of the two ERK 1/2 inhibitors on LC neurons by recording the basal spontaneous firing rate (SFR). The cell-attached configuration of the patch recording was used to avoid interference in the ion composition of the cytoplasm by the pipette solution. We found that the application of 20 μM U0126 for 30 min significantly decreased the SFR from 0.51 ± 0.13 to 0.33 ± 0.09 Hz (Fig. 4A,B) (n = 6 cells, p = 0.028, paired Wilcoxon-sign rank test). In contrast, the application of 20 μΜ FR180204 for 30 min significantly increased the SFR from 0.47 ± 0.09 to 0.73 ± 0.08 Hz (Fig. 4A,C) (n = 6 cells, p = 0.006, Student's paired t-test), and the application of the vehicle (0.1% DMSO) did not have an effect on the SFR (Fig. 4A,D) (baseline: 0.54 ± 0.14 Hz, DMSO: 0.55 ± 0.14 Hz, n = 8 cells, p = 0.8, Student's paired t-test). The differential effects of the two drugs on the SFR might be ascribed to the fact that FR180204 directly targets ERK 1/2 , while U0126 targets the mitogen-activated protein kinase that acts upstream of ERK 1/2 . The results also suggest that LC neurons might have basal ERK 1/2 activity, which could regulate various types of ion channels involved in the regulation of the membrane potential of LC neurons. To minimize the nonspecific effects, we pretreated the slices for 2 hrs and continuously perfused them throughout the recording with U0126 or FR180204 so that a stable baseline could be obtained before www.nature.com/scientificreports www.nature.com/scientificreports/ the application of baclofen and CGP54626.In slices pretreated and perfused with U0126 or FR180204, the peak amplitude of I GABABR showed no difference in U0126-(n = 9 cells) and FR180204-(n = 10 cells) treated slices compared to the peak amplitude of I GABABR measured in control slices (Kruskal-Wallis test, p = 0.644 among the comparisons between control and ERK blocker groups) (Fig. 3A-D). In contrast, both drugs significantly accelerated the decline in I GABABR compared with the control condition. The τ 50 measured from I GABABR recorded in the U0126-treated and FR180204-treated slices was 163 ± 21 s and 150 ± 21 s, respectively; both were significantly shorter than the measurements obtained using the control slice (Kruskal-Wallis test, p = 0.001 among the comparison between control and ERK blocker groups; p = 0.009 for control vs. U0126 and p = 0.002 for control vs. FR180204 using post hoc Dunn's multiple comparisons test) (Fig. 3A-C,E). The I Tonic revealed by the subsequent application of CGP54626 in slices pretreated with U0126 was −7.6 ± 4.0 pA; it was 0.9 ± 7.0 pA in slices pretreated with FR180204; no difference was observed in either case compared with the control (two-way repeated-measures ANOVA, sphericity assumed, F(1, 27) = 3.672, p = 0.066 for CGP effects; sphericity assumed, F (2, 27) = 1.337, p = 0.279 for the comparison among ERK blockers) (Fig. 3A−C,F).
Compared with baseline, the application of CGP54626 also increased the frequency of I Osc in U0126-and FR180204-treated slices, with the extent of the increase being significantly less in FR180204-treated slices than that in the control slices (two-way repeated-measures ANOVA, sphericity assumed, F(1, 26) = 55.106, p = 0.000 for CGP effects; sphericity assumed, F (2, 26) = 6.255, p = 0.006 for the comparison among ERK blockers). Upon CGP54626 application, the frequency of I Osc significantly increased from 0.24 ± 0.02 to 0.33 ± 0.3 Hz in U0126-treated slices (n = 9 cells, p = 0.025, post hoc Bonferroni test); however, the extent of the increase showed no difference compared to the control (p = 1, post hoc Bonferroni test). In FR180204-tretaed slices, the frequency of I Osc significantly increased from 0.48 ± 0.06 to 0.57 ± 0.08 Hz (n = 9 cells, p = 0.032, post hoc Bonferroni test)and the extent of the increase was significantly less than that in the control (p = 0.001, post hoc Bonferroni test) (Fig. 3G). Again, the significant reduction in the extent of the increase in SFR upon CGP54626 application observed in FR180204-tretaed slices but not in U0126-treatred slices might be ascribed to the fact that FR180204 specifically targets ERK 1/2 , while U0126 targets the mitogen-activated protein kinase that acts upstream of ERK 1/2 . eRK 1/2 activated by GABA B Rs is essential to sustain tonic inhibition of Lc neurons. An interpretation of the above observations could be that the activation of GABA B Rs in LC neurons not only opened GIRK channels but also triggered ERK-dependent autoregulation of receptors to prevent the quick desensitization of GABA B Rs upon prolonged exposure to the agonist. To test this possibility, we examined the effects on tonic inhibition of LC neurons 7 with prolonged exposure of GABA B Rs to baclofen at a saturating concentration with the inhibition of ERK activity. If the above interpretation is correct, the inhibition of ERK-dependent autoregulation would result in a reduced functionality of GABA B Rs, as indexed by a reduction in the tonic inhibition of LC neurons after prolonged agonist exposure. We found that a precise assessment of tonic inhibition by directly measuring GABA B R-mediated I Tonic was difficult due to the high noise level imposed by the high I Osc activity. This phenomenon might account for the lack of a significant difference in I Tonic in the U0126-or FR180204-treated slices compared to the control (Fig. 3F).Accordingly, we examined the SFR of LC neurons to assess GABA B R-mediated tonic inhibition.
To examine the effects on tonic inhibition of LC neurons with prolonged exposure of GABA B Rs to baclofen with the inhibition of ERK activity, we perfused the slices with 100 μM baclofen, the minimum dosage for producing the saturation of GABA B R functionality in LC neurons 7 , for 15 min after obtaining a stable cell-attached recording from an LC neuron. The slices were then washed with 20 μM baclofen for an additional 15 min, followed by the co-administration of 20 μM baclofen and CGP54626 (Fig. 5A). Based on the dose-dependent relationship of I GABABR induced by baclofen 7 , we estimated that 20 μM baclofen would produce 70% of the maximum GABA B R activation in LC neurons. Therefore, bathing the slices in 20 μM ambient baclofen could largely amplify tonic inhibition for easier observation. As seen (Fig. 5A,B), after a 15-minute period of pre-exposure to the agonist at a saturating concentration, a significant increase in the SFR upon CGP54626 application was observed in LC neurons bathed in 20 μM baclofen in the control slices (n = 10 cells, p = 0.000, Student's paired t-test), the U0126-treated slices (n = 7 cells, p = 0.012, Student's paired t-test) and in the FR180204-treated slices (n = 7 cells, p = 0.001, Student's paired t-test). The GABA B R-mediated tonic inhibition under the condition was defined as: where SFR Bac and SFR Bac+CGP54626 are the SFRs recorded in 20 μM baclofen and 20 μM baclofen plus CGP54626, respectively. The calculated tonic inhibition was 147.3 ± 39.4% in the control (n = 10), which was significantly stronger than that in U0126-treated slices of 34.8 ± 8.9% (n = 7) and in FR180204-treated slices of 16.6 ± 3.2% (n = 7) (One-way ANOVA, p = 0.008 among the comparison between control and ERK blocker groups; p = 0.045 for control vs. U0126 and p = 0.013 for control vs. FR180204 using post hoc Bonferroni test) (Fig. 5C). eRK 1/2 activated by GABA B R does not have an effect on pGABA B2 R. Finally, we examined the possible regulatory site of ERK 1/2 regulation of GABA B R functionality upon receptor activation. To this end, we compared the level of phosphorylated GABA B2 receptor subunit at the serine 783 (S783) site between LC tissue treated with baclofen and that treated with baclofen plus FR180204 using western blot analysis, as this regulatory site is shown to enhance GABA B R activation of GIRK 33 . If ERK 1/2 regulated GABA B R functionality by phosphorylating the S783 site of the GABA B2 receptor subunit, the pGABA B2 receptor level should be significantly higher in baclofen-treated LC tissue than in the tissue treated with baclofen plus FR180204. However, we did not find a difference between the two groups in the level of phosphorylation of the GABA B2 receptor at the S783 site. Compared to the control (LC tissues from the baclofen-bathed slices), the pGABA B2 R level was 96.3 ± 6.9% of the Scientific RepoRtS | (2020) 10:7869 | https://doi.org/10.1038/s41598-020-64292-x www.nature.com/scientificreports www.nature.com/scientificreports/ control in LC tissues from the slice bathed in baclofen plus FR180204 (Fig. 6) (n = 6, p = 0.651, Student's paired t-test).

Discussion
In this study, we provide biochemical and electrophysiological evidence showing that GABA B Rs can mediate the autoregulation of GABA B R-activated whole-cell current through the activation of ERK 1 in LC neurons. Since ambient GABA in the LC is significantly higher during rapid eyemovement (REM)/non-REM sleep than during wakefulness 20,22 , ERK 1 -dependent autoregulation of GABA B R functionality could be a mechanism enabling the receptors to be continuously activated by the ambient GABA without undergoing severe desensitization, thereby providing a stable, tonic inhibition of LC neurons.
In many brain regions, including the LC, electron microscopic studies have shown that GABA B Rs are located predominantly close to neurotransmitter release sites at presynaptic terminals; in contrast, they are mainly located at peri-/extrasynaptic but not at synaptic active zones in postsynaptic neurons [4][5][6][7] . These locations of GABA B Rs imply that most of the receptors are not directly activated by synaptically released GABA in the synaptic cleft but by ambient GABA in peri-/extrasynaptic spaces. The concentration of ambient GABA critically depends on GABA spillover from the synaptic cleft, and the amount of GABA spillover is determined by the frequency and pattern of AP arrival at axonal terminals. It is also determined by the operation of GABA transporters located in the neuron and glia cell membrane [34][35][36][37][38][39] . GABA transporters not only serve in the reuptake of GABA in the synaptic cleft but also operate in a reverse mode that actually causes release but not reuptake of the neurotransmitter, a so-called nonvesicular release process, which would occur when high-frequency and repeated APs arrive at axonal terminals [40][41][42] . Accumulating reports have shown that ambient GABA generated by the abovementioned neuronal and glial activity could sufficiently and tonically activate peri-/extrasynaptic GABA B Rs at both presynaptic and postsynaptic sites, thereby exerting tonic inhibition for the control of transmitter release from www.nature.com/scientificreports www.nature.com/scientificreports/ presynaptic terminals and the spiking activity in postsynaptic neurons 7,9,[14][15][16][34][35][36]39 . The regulation of ambient GABA by AP-dependent vesicular and nonvesicular release could modulate tonic inhibition of neurons that express peri-/extrasynaptic GABA A Rs and/or GABA B Rs, and the processes could be linked to the long-term regulation of brain function. LC neurons are well known to be involved in the regulation of the wakefulness-sleep cycle; they fire APs in a brain state-dependent manner, displaying active firing during wakefulness, a decreased discharge rate during non-REM sleep and silence during REM sleep 18,43 . Obviously, a mechanism that provides prolonged and stable inhibition of LC neurons could promote REM/non-REM sleep.
In addition to ambient GABA, the operation of GABA B R-mediated tonic inhibition would require a substantial and steady amount of GABA B Rs on the membrane. Nevertheless, many GPCRs undergo desensitization, a process in which a receptor reduces its response after prolonged exposure to the agonist, which helps to attenuate or terminate signal transduction to protect the target cells from overstimulation 25 . It is now well known that desensitization involves the phosphorylation of GPCRs by G protein-coupled receptor kinases (GRKs), which lead to the uncoupling of the receptors from G proteins. This process is followed by the internalization of GPCRs through the recruitment of arrestins that trigger clathrin-mediated endocytosis of the phosphorylated receptor 44 . Interestingly, a growing amount of evidence suggests that GABA B Rs, unlike many other GPCRs, are not the substrate for GRKs other than GRK4/5 and do not conform to the above-described agonist-induced internalization 45 . Nevertheless, GABA B Rs exhibit significant rates of constitutive endocytosis via clathrin-dependent and dynamin-dependent mechanisms under basal conditions, followed by the recycling of large numbers of GABA B Rs back to the plasma membrane to maintain steady-state cell surface numbers [45][46][47][48] . These processes perhaps reflect the long cell surface half-lives of GABA B Rs and make them suitable for mediating a stable and persistent effect such as tonic inhibition. For example, unlike neurokinin receptors, which undergo almost complete desensitization after a few seconds of exposure to substance-P in NAergic A7 neurons 29 , GABA B R desensitization progresses at a much slower rate in NAergic A7 neurons 9 as well as in LC neurons. The regulation of the surface stability of the GABA B R number usually involves the phosphorylation of the receptor molecule per se. It has been shown that the phosphorylation of S892 of the GABA B2 receptor by protein kinase-A promotes cell surface stability of the GABA B R number 46 ; in cultured hippocampal neurons, S867 in GABA B1 receptors is phosphorylated by Ca 2+ / calmodulin-dependent protein kinases downstream of NMDA receptor activation, which induces the internalization of GABA B Rs. The phosphorylation of the receptor molecule also regulates GABA B R signaling by altering the efficacy of receptor-effector coupling. The phosphorylation of S783 of theGABA B2 receptor by 5′AMP-dependent protein kinase (AMPK) has been shown to enhance GABA B R activation of GIRKs 33 . In addition to the promotion of cell surface stability of the receptor number, the phosphorylation of S892 of the GABA B2 receptor by protein kinase-A facilitates receptor-effector coupling 49 and slows KCTD12-induced desensitization of the GIRK current 50 . In addition to the receptor molecules, the phosphorylation of effectors, such as N-type Ca 2+ channels, by tyrosine kinase can interact with regulators of G protein signaling (RGS) to form a regulatory complex that provides additional mechanism for regulation in the GABA B R-mediated currents 51 . Another mechanism reported to be involved in the regulation of GABA B R desensitization includesthe agonist-induced association of the receptors with GRK4/5, which results in GABA B R desensitization in a phosphorylation-independent manner. The interaction of GABA B1 and GABA B2 receptor subunits with NEM-sensitive fusion protein (NSF) primes the receptors for phosphorylation by protein kinase C (PKC) to uncouple them from G proteins. The association of GABA B Rs with various types of auxiliary proteins, such as potassium channel tetramerization domain-containing proteins (KCTDs), via the C-terminus of GABA B2 receptors can render the receptor complex competent for desensitization.
Our western blot analysis showed that baclofen could increase pERK 1 levels in LC tissue, and this effect was not observed in LC tissue treated with CGP54626 to block GABA B Rs. These observations show that the www.nature.com/scientificreports www.nature.com/scientificreports/ baclofen-induced increase in pERK 1 levels in the LC was GABA B R-dependent, consistent with the results of previous reports in the hippocampus and cerebellum [26][27][28] . In addition to the well-known effect on gating GIRKs, the results of these previous and present studies demonstrate an additional role of GABA B R activation in activating ERK in hippocampal CA1 pyramidal neurons, cerebellar granule neurons and LC neurons. By performing whole-cell recordings directly from LC neurons, we further observed that the inhibition of ERK 1/2 with two selective blockers produced consistent effects on the activity induced by baclofen application in LC neurons. These results confirmed that the elevation of pERK 1 levels by GABA B R activation occurred in LC neurons, although we could not completely rule out the possibility that the effects on LC neuron activity were secondary. We did not validate the link between GABA B R activation and the increased pERK 1 level. However, it has been reported that the increase in pERK 1/2 levels by GABA B R activation relies on a pertussis-toxin-sensitive G i/o heterotrimeric protein-dependent pathway by releasing G βγ in cultured cerebellar granule neurons 27 . Interestingly, this signaling pathway for ERK 1/2 phosphorylation seems to be unique to GABA B Rs, as the activation of G i/o -coupled α 2 -adrenoceptors or G i/o -coupled 5HT 1A receptors in the adult mouse CA1 region did not result in ERK 1/2 phosphorylation 26 . As shown in our electrophysiological experiments, the inhibition of ERK 1/2 activity with two selective blockers accelerated the decay of I GABABR , and it is likely that the signaling mechanisms of GABA B R trafficking and/or desensitization may involve an ERK-dependent intermediate step that has not yet been identified. Therefore, the inhibition of the ERK-dependent intermediate step accelerated the decline of I GABABR . Our western blot analysis showed that the activation of ERK 1 downstream of GABA B R activation did not have an effect on the level of phosphorylation of the GABA B2 receptor at the serine 783 site. Activated ERK 1 could regulate GABA B R functionality at other serine sites in GABA B1 and/orGABA B2 receptors, as previously described. Furthermore, it could be involved in the interaction between RGS and phosphorylated GIRKs by tyrosine kinase 51,52 , as it has been recently suggested that the GABA B R activates tyrosine kinase and downstream ERK 1/2 53 . Apparently, the mechanisms underlying the regulation of GABA B R desensitization by ERK 1 remain to be clarified.
In conclusion, here, we report that the activation of GABA B Rs with baclofen increases the phosphorylation of ERK 1 in LC tissue and that the blockade of ERK 1/2 activity with two selective blockers, U0126 and FR180204, accelerates the decay of I GABABR induced by prolonged baclofen application in LC neurons. Furthermore, an increase in the spontaneous firing rate and a decrease in the tonic inhibition of LC neurons occurs after prolonged GABA B R activation by baclofen at a saturating concentration. These results suggest a new functional role of Gαi/ο upon GABA B R activation in mediating an ERK 1 -dependent autoregulation of the stability of GABA B R-activated whole-cell current in LC neurons, in addition to the well-known action of gating GIRKs with G β,γ subunits. We argue that activated ERK 1 signaling might help maintain a dynamic balance between the desensitization and recycling of receptors back to the membrane to sustain GABA B R-mediated tonic inhibition of LC neurons. However, it should be noted that postnatal day 8-10 rats were used in this study, and there is a possible weakness that the same phenomena may not be fully present in adulthood.

Materials and Methods
Animals. The use of animals in this study was approved by the Institutional Animal Care and Use Committee for National Taiwan University (permission #NTU103-EL-00076) and the Institutional Animal Care and Use Committee for Chung Shan Medical University(permission #1423), the guidelines of which comply with the "Codes for Experimental Use of Animals" of the Council of Agriculture of Taiwan based on the Animal Protection Law of Taiwan. Every effort was made to minimize the number of animals used and their suffering. Sprague-Dawley rat pups of both sexes were used and sacrificed for slice preparation on postnatal days 8-10 for the electrophysiological experiments and postnatal days 14-16 for the western blot analysis. preparation of brainstem slices and electrophysiology. The animals were anesthetized with 5% isoflurane in O 2 and decapitated, followed by rapid exposure of the brain and chilling with ice-cold aCSF consisting of the following (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO 4 , 26.2 NaHCO 3 , 1 NaH 2 PO 4 , 2.5 CaCl 2 , and 11 glucose, oxygenated with 95% O 2 and 5% CO 2 , pH 7.4. Sagittal brainstem slices (300 μm) containing the LC were prepared using a vibroslicer (D.S.K. Super Microslicer Zero 1, Dosaka EM, Kyoto, Japan), maintained in a moist air-liquid (aCSF) interface chamber and allowed to recover for at least 90 minutes before being transferred to an immersion-type chamber mounted on an upright microscope (BX51WI, Olympus Optical Co., Ltd., Tokyo, Japan) for recording. Throughout the recording period, they were perfused at 2-3 ml/min with oxygenated aCSF containing synaptic blockers. The synaptic blockers contained 5 mM kynurenic acid, 100 μM picrotoxin and 1 μM strychnine to block glutamatergic, GABAergic and glycinergic synaptic transmission, respectively. The baseline described in the control slices refers to the recordings made under these conditions before administering baclofen and CGP54626. For recordings made in the U0126-treated slices and FR180204-treated slices, baseline refers to recording in the aCSF containing synaptic blockers, 0.1% DMSO and U0126 or FR180204.
Neurons were viewed using Nomarski optics. Patch pipettes were pulled from borosilicate glass tubing (1.5 mm outer diameter, 0.32 mm wall thickness; Warner Instruments Corp., Hamden, CT, USA) and had a resistance of approximately 3-5 MΩ when filled with the pipette solutions. To record the GABA B R-mediated current, the pipette solution consisted of the following (in mM): 131 K-gluconate, 20 KCl, 10 HEPES, 2 EGTA, 8 NaCl, 2 ATP, and 0.3 GTP; pH adjusted to 7.2 with KOH. Recordings were performed at 29-31 °C in the whole-cell or cell-attached configuration with a patch amplifier (Multiclamp 700 B; Axon Instruments Inc., Union City, CA, USA). For current-clamp recordings of whole-cell configuration, the bridge was balanced, and the recordings were accepted only if the recorded neuron had a membrane potential (V m ) of at least −45 mV without applying a holding current and if the APs were able to overshoot 0 mV. For voltage-clamp recordings, the V m was clamped to −70 mV unless specified. The serial resistance was monitored throughout the recording, and the data were discarded if the values varied by more than 20% of the original value, which was usually less than 20 MΩ. In a series of experiments, the cell-attached configuration was used. In the recording, the patch amplifier was set in