Activity Modes in Thalamocortical Relay Neurons are Modulated by Gq/G11 Family G-proteins – Serotonergic and Glutamatergic Signaling

In thalamocortical relay (TC) neurons, G-protein-coupled receptors play an important part in the control of activity modes. A conditional Gαq knockout on the background of a constitutive Gα11 knockout (Gαq/Gα11−/−) was used to determine the contribution of Gq/G11 family G-proteins to metabotropic serotonin (5-HT) and glutamate (Glu) function in the dorsal part of the lateral geniculate nucleus (dLGN). In control mice, current clamp recordings showed that α-m-5-HT induced a depolarization of Vrest which was sufficient to suppress burst firing. This depolarization was concentration-dependent (100 μM: +6 ± 1 mV, n = 10; 200 μM: +10 ± 1 mV, n = 7) and had a conditioning effect on the activation of other Gαq-mediated pathways. The depolarization was significantly reduced in Gαq/Gα11−/− (100 μM: 3 ± 1 mV, n = 11; 200 μM: 5 ± 1 mV, n = 6) and was apparently insufficient to suppress burst firing. Activating Gαq-coupled muscarinic receptors affected the magnitude of α-m-5-HT-induced effects in a reciprocal manner. Furthermore, the depolarizing effect of mGluR1 agonists was significantly reduced in Gαq/Gα11−/− mice. Immunohistochemical stainings revealed binding of 5-HT2CR- and mGluR1α-, but not of 5-HT2AR-specific antibodies in the dLGN of Gαq/Gα11−/− mice. In conclusion, these findings demonstrate that transmitters of ascending brainstem fibers and corticofugal fibers both signal via a central element in the form of Gq/G11-mediated pathways to control activity modes in the TC system.

Recordings were only performed on recombined TC neurons. We distinguished these from non-recombined GABAergic interneurons based on our previously established physiological and morphological criteria (Broicher et al., 2008a). All cells had a resting membrane potential negative of −60 mV, the access resistance was in the range of 5-15 MΩ and series resistance compensation of 30% or more was routinely applied. A liquid junction potential of 8 ± 2 mV (n = 10) was measured and taken into account.

5-ht recePtor signaling and coMPetition between different recePtor classes in gα q /gα 11 −/− Mice
In order to mimic an arousal by brainstem and cortical inputs to the thalamus, we have chosen the paradigm described in the following. A hyperpolarized membrane potential negative to −70 mV is necessary to achieve burst firing of TC neurons in response to a depolarizing current step in vivo (Steriade, 1991), and thus, cells were held at about −71 mV by DC current injection. Rather small sleep-related variations of the membrane potential (∼10 mV) found in TC neurons are sufficient to mediate a switch between burst and tonic firing in vivo (Hirsch et al., 1983). The experiments described below tested to what extent the knockout of Gα q has affected the ability of TC neurons to perform this switch. Acetylcholine plays a major role in the modulation of thalamic states of activity and the function of this transmitter depends on Gα q -coupled muscarinic receptors. However, it is unknown which G-proteins are targeted by the other brainstem neurotransmitters. Thus, we tested neurotransmitter candidates expected to be connected to membrane potential of TC neurons by 6 ± 1 mV (n = 10) and 10 ± 1 mV (n = 7), respectively ( Figure 1B). We also tested the effect of a more specific agonist for 5-HT 2C receptors (CP 809101) on the membrane potential in current clamp recordings (Siuciak et al., 2007). This agonist produced comparable depolarizations (10 μM: 8.3 ± 2.0 mV, n = 5; 100 μM: 17.5 ± 1.4 mV, n = 4). This suggests that 5-HT 2C receptors in the dLGN could be responsible for a large portion of the observed effect. The response to α-m-5-HT was significantly reduced in Gα q /Gα 11 −/− (100 μM: 3 ± 1 mV, n = 11, p < 0.001; 200 μM: 5 ± 1 mV, n = 6, p < 0.01). In the presence of 200 μM α-m-5-HT, depolarizing current steps elicited tonic firing in Gα 11 −/− (f i = 69 ± 14 Hz, n = 7; data not shown). During application of 100 μM α-m-5-HT in Gα q /Gα 11 −/− burst firing was either preserved (f i = 126 ± 6 Hz, n = 4) or the LTS was crowned by a single action potential (n = 7, Figure 1A, lower right trace). For the other two recording conditions (100 μM in controls, 200 μM in Gα q /Gα 11 −/− ) depolarizing current steps either evoked an intermediate response with slow bursting (f i ≈ 100 Hz) followed by 1-4 tonic action potentials (not shown) or passive membrane responses ( Figure 1A, upper right trace). See also Sherman (1996). G q /G 11 -mediated signaling pathways. Because of the known coupling of 5-HT 2 receptors to G q /G 11 family G-proteins (Roth et al., 1998), we applied the 5-HT 2 receptor agonist α-m-5-HT and monitored changes in TC neurons under current clamp conditions. A shift from burst to tonic firing relies on the initial membrane potential. In order to provide comparable conditions between neurons with a different resting membrane potential and to ensure robust bursting with two or more action potentials riding on top of a low-threshold Ca 2+ spike (LTS), all TC neurons investigated here were set to a membrane potential of −70.7 ± 0.2 mV (n = 70) by DC offset. The resting membrane potential was −69.4 ± 0.9 mV and the applied DC offset current was −10 ± 2.4 pA (n = 70). Of 70 neurons investigated under these conditions, only 1 had a resting membrane potential positive to −65 mV (−63 mV) and almost all (68 of 70 TC neurons) were able to generate a LTS. Cells with a membrane potential positive to −65 mV and cells that could not generate a LTS were excluded from further analysis.
Step depolarization resulted in high-frequency burst firing in both mice genotypes (Gα 11  As shown in Figure 1D, the variability of a combined effect of the two agonists, namely α-m-5-HT and muscarine is rather small (open circles). Moreover, the variation in the effect of α-m-5-HT is comparable to the variation in the effect of muscarine. Still, when applied together, the combined effect does not exceed a given value. Or, in other words, both agonist effects vary, depending on how occupied the system is by the presence of the respective other agonist. This suggests that both mechanisms of activation involve a common and limiting mechanism.
In summary, these data demonstrate that serotonergic signaling depends on G q -type G-proteins, and suggest that the same pool of G-proteins can be accessed by different transmitter pathways.

exPression of 5-ht and mglu recePtor subtyPes in dlgn
To provide further evidence that Gα q -coupled receptors are functionally expressed in TC neurons we performed immunohistochemical stainings by antibody labeling.
Application of 5-HT 2A R-specific antibodies did not lead to staining by binding of the secondary antibody ( Figure 4B, middle image). Strong staining was observed upon application of 5-HT 2C R- (Figure 4C, middle image) and mGluR1α-specific antibodies ( Figure 4D, middle image). We co-stained slices with the neuronspecific nucleus marker NeuN (Figures 4B-D left images; merged images to the right). This revealed that 5-HT 2C R and mGluR1α were not somatically expressed in neurons of the LGN. We verified this by examining slices that were co-stained with a marker for microtubule associated protein 2 (MAP2) at a higher magnification. The images revealed that neither the staining for 5-HT 2C (not shown) nor for mGluR1α clearly marked the outline of the soma ( Figure S1 in Supplementary Material). We could not observe any differences between stainings in the dLGN of control and Gα q / Gα 11 −/− mice ( Figure S2 in Supplementary Material). The densitometric analysis of 5-HT 2C R-specific fluorescence revealed significant differences between control (mean fluorescence intensity = 32 ± 1 a.u.; n = 3 independent slices) and Gα q / Gα 11 −/− mice (mean fluorescence intensity = 41 ± 1 a.u.; n = 3; data not shown). No differences were found for mGluR1α-specific saturating response. In both mouse strains, the t-ACPD-dependent depolarization was strong enough to induce a shift from burst to tonic firing of action potentials, which is also reflected in the firing frequency (Gα 11 −/− , 50 μM: f i = 32 ± 2 Hz, n = 7; Gα 11 −/− , 100 μM: f i = 44 ± 11 Hz, n = 8; Gα q /Gα 11 −/− , 50 μM: f i = 38 ± 3 Hz, n = 7; Figures 2A,B). In Gα q /Gα 11 −/− at 100 μM t-ACPD four out of six cells revealed an intermediate response with a slow burst (f i = 96 ± 9 Hz, n = 4) followed by 2-8 tonic action potentials (data not shown). Only one cell showed tonic firing (∼27 Hz), and the remaining cell was lost in the course of the experiment.
It could be argued that the application of a neuromodulator could lead to changes in interneuron firing (Sherman, 2004;Munsch et al., 2005), which in turn could lead to changes in RMP unrelated to the activation of Gα q -coupled receptors of the recorded cell. To test for this, we applied 50 μM DHPG in the presence of 0.5 μM TTX, eliminating all action potential firing and excluding presynaptic effects. Under these conditions, the observed depolarization was very similar to control conditions (12 ± 3 mV, n = 3).

5-HT receptor-dependent signaling
Waking, but not REM sleep, is accompanied by increased serotonergic activity in the thalamus (Steriade et al., 1997). The role of 5-HT in the thalamus seems complex and is not yet fully understood: Cellular effects may be direct or indirect and show regional differences. In vitro studies demonstrated that 5-HT causes a small depolarization and a shift in voltage dependency of the hyperpolarization activated cation current, I h . The latter is achieved via G S -proteins and cAMP production (McCormick and Pape, 1990;Lee and McCormick, 1996). Moreover, inhibition of an I SO component occurred, resembling the current mediated by TASK channels (S. G. Meuth and T. Budde, unpublished results). In consequence, oscillatory burst activity is suppressed. An indirect modulation of I h via G q -proteins may include IP 3 -induced Ca 2+ release from intracellular stores and subsequent activation of a Ca 2+ -dependent adenylyl cyclase (Lüthi and McCormick, 1998). The findings of the present study indicate that a significant part of the 5-HT-induced depolarization is mediated by G q /G 11 -coupled receptors. All receptors of the 5-HT 2 subclass are coupled to G q /G 11 -proteins. These in turn, activate PLC-β. 5-HT 2A and 5-HT 2C are widely distributed in the fluorescence (control: mean fluorescence intensity = 44 ± 1 a.u.; Gα q /Gα 11 −/− : mean fluorescence intensity = 42 ± 1 a.u.; n = 3 independent slices each; data not shown). These findings point to an attempt to compensate the loss of 5-HT 2C receptor function by an increased number of receptors in Gα q /Gα 11 −/− mice.

discussion
The results of the present study can be summarized as follows: (1) The action of 5-HT and glutamate in TC neurons depends on G q -coupled intracellular signaling cascades. The effects of α-m-5-HT, t-ACPD, and DHPG under current clamp conditions were significantly reduced in Gα q /Gα 11 . As a consequence, the stimulation of metabotropic 5-HTR and GluR could not induce a full shift from burst to tonic firing of action potentials in Gα q /Gα 11 −/− mice.
(2) The amplitude of membrane potential depolarization by muscarine depends on the degree of prior utilization of the G q -dependent pathway. (3) It is concluded that G q /G 11 family G-proteins play a central role in the state-dependent control of thalamic activity modes.

gα q /gα 11 -dePendent signaling in the thalaMus
The flow of sensory information from the sensory organs to the cerebral cortex via the thalamus is highly regulated by inputs from the ascending activating brainstem system, releasing ACh, 5-HT, and noradrenalin (Steriade et al., 1997). These neuromodulators exert their influence onto the state of the thalamus by altering specific ion channels. This is achieved through the activation of were unchanged in dLGN tissue (Wettschureck et al., 2006;Broicher et al., 2008b) and a lack of strong plastic compensation has been noted before in TC neurons of mice deficient for HCN2 (Ludwig et al., 2003) and TASK-1 (Meuth et al., 2006). Nevertheless, the densitometric analysis of immunohistochemical staining in the present study indicated an increased expression of 5-HT 2C R in Gα q /Gα 11 −/− mice, thereby possibly showing a futile attempt to compensate the loss of receptor function by an increased number of membrane receptors.
(3) G-protein signaling outside the canonical seven transmembrane domain receptors and G-protein independent pathways of these receptors may exist. Recent evidence indicates that G-proteins play important roles in receptor tyrosine kinase signaling and may be activated by accessory proteins (Marty and Ye, 2010;Sato and Ishikawa, 2010). Furthermore, increasing evidence indicates that ERK, JAK/STATs, Src-family tyrosine kinases, β-arrestins, and PDZ domain-containing proteins directly relay signals from seven transmembrane domain receptors, independent of G-proteins (Sun et al., 2007). Future studies have to show the coupling of metabotropic ACh, 5-HT, and Glu receptors to parallel pathways, activated by βγ-subunits, by G-proteins not belonging to the G q /G 11 -family, and/or by G-protein-independent mechanisms. (4) Gα q may be incompletely eliminated. The CaMKII-Cre mouse line has been shown to express Cre in forebrain principal neurons, but not in forebrain interneurons (Mantamadiotis et al., 2002;Marsicano et al., 2003). With respect to recombination efficiency within the population of forebrain principal neurons it is generally assumed that recombination is complete (Marsicano et al., 2003). Within a particular forebrain principal neuron, Cre expression always results in a complete inactivation of Gα q , since the CaMKII promoter chosen to drive Cre expression is very strong. We are therefore positive that we can rule out any partial Gα q inactivation (i.e., only one of the two Gα q alleles is recombined) in Cre-expressing principal neurons.

exPression of 5-ht and mglu recePtors
Immunohistochemical stainings provided evidence that Gα qcoupled receptors are functionally expressed in TC neurons. However, the application of 5-HT 2A R-specific antibodies failed to stain the tissue. Why this is the case remains unclear. The strong staining that was observed upon application of 5-HT 2C R-and mGluR1α-specific antibodies was not reduced in Gα q /Gα 11 −/− mice, suggesting that the deletion of G q does not lead to a down regulation of its upstream receptors (compare Figure 4 and Figure S2 in Supplementary Material). Co-staining slices with the neuronspecific nucleus marker NeuN revealed that 5-HT 2C R and mGluR1α were not somatically expressed in neurons of the LGN. This was confirmed in a co-staining with MAP2.
The finding that a specific agonist for 5-HT 2C receptors had the same effect on the membrane potential in current clamp recordings than α-m-5-HT in control mice suggests that 5-HT 2C receptors in the dLGN could be the functionally dominant isoform. brain and are present in the rodent dLGN (Li et al., 2004). Thus, the strong reduction of the effect of α-m-5-HT in Gα q /Gα 11 −/− is in good agreement with a 5-HT 2 expression in dLGN and is possibly connected to the modulation of TASK channels.
In the course of this study, we made the interesting observation that muscarinic and serotonergic receptors seem to compete for the same G q -protein-coupled signaling pathway. The effect of activation of one receptor class is strongly and negatively correlated to the strength of prior activation of the other receptor class, thereby suggesting convergence onto the same -limited -pool of G q -protein-coupled signaling pathways. This view is supported by results obtained from cat and guinea pig TC neurons that suggested acetylcholine-and noradrenalin-induced slow depolarizations to occur through the activation of the same second-messenger system (McCormick, 1992b).

mGluR-dependent signaling
Group I mGluRs consist of mGluR1 and mGluR5 that are positively coupled to PLC-β. Several types of mGluRs are expressed in the dLGN of different species, with retinal (mGluR5) and cortical (mGluR1) inputs accessing specific subtypes (Godwin et al., 1996b;Lourenco Neto et al., 2000). Application of t-ACPD and selective mGluR1 agonists depolarizes TC neurons and switches their activity mode from burst to tonic firing, thereby mediating TC transmission (McCormick and von Krosigk, 1992;Godwin et al., 1996a;Salt, 2002). The results of the present study are in line with these findings. However, the remaining t-ACPD effect in Gα q / Gα 11 −/− indicates that mGluRs not coupled to G q /G 11 contribute to the response. This conclusion is corroborated by the finding that mGluR3, mGluR4, and mGluR7 are expressed in rodent dLGN (Lourenco Neto et al., 2000). During postnatal development, specific changes in the subcellular location of mGluRs have been observed (Liu et al., 1998) which are the basis for the topographical association to different input systems (Godwin et al., 1996b;Turner and Salt, 2000). residual effects of neurotransMitters in gα q /gα 11 −/− Mice While Gα q and Gα 11 have very similar effector-coupling properties and may substitute each other (Offermanns, 1999), the remaining effect of receptor agonists in Gα q /Gα 11 −/− may result from different mechanisms: (1) Other G-protein families may compensate for the lack of Gα q in deficient mice. Findings from hippocampal neurons of Gα o -deficient mice have shown that in the absence of Gα o , ion channels will be regulated by other G-proteins with different properties (Greif et al., 2000). In particular Gα 15 and Gα 16 can link a variety of predominantly Gα qcoupled receptors to the PLCβ pathway (Offermanns and Simon, 1995). (2) Receptors, intracellular signaling proteins, and effectors of the Gα q -dependent pathways may be up-regulated. However, the Gα q /Gα 11 −/− mice develop a severe epileptic phenotype with a reduced life span, which argues against an effective compensation mechanism (Wettschureck et al., 2006;Broicher et al., 2008b). Indeed, mRNA expression of effector channels (TASK channels, HCN channels) and all mAChR subtypes