mGluR1/TRPC3-mediated Synaptic Transmission and Calcium Signaling in Mammalian Central Neurons

Metabotropic glutamate receptors type 1 (mGluR1s) are required for a normal function of the mammalian brain. They are particularly important for synaptic signaling and plasticity in the cerebellum. Unlike ionotropic glutamate receptors that mediate rapid synaptic transmission, mGluR1s produce in cerebellar Purkinje cells a complex postsynaptic response consisting of two distinct signal components, namely a local dendritic calcium signal and a slow excitatory postsynaptic potential. The basic mechanisms underlying these synaptic responses were clarified in recent years. First, the work of several groups established that the dendritic calcium signal results from IP₃ receptor-mediated calcium release from internal stores. Second, it was recently found that mGluR1-mediated slow excitatory postsynaptic potentials are mediated by the transient receptor potential channel TRPC3. This surprising finding established TRPC3 as a novel postsynaptic channel for glutamatergic synaptic transmission.Glutamate is the predominant neurotransmitter used by excitatory synapses in the mammalian brain (Hayashi 1952; Curtis et al. 1959). At postsynaptic sites, glutamate binds to two different classes of receptors, namely the ionotropic glutamate receptors (iGluRs) and the metabotropic glutamate receptors (mGluRs) (Sladeczek et al. 1985; Nicoletti et al. 1986; Sugiyama et al. 1987). The iGluRs represent ligand-gated nonselective cation channels that underlie excitatory postsynaptic currents (EPSCs). Based on their subunit composition, gating, and permeability properties, they are subdivided into three groups named after specific agonists: AMPA- (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), NMDA receptors (N-methyl D-aspartate receptors) and kainate receptors (Alexander et al. 2009). The other class of glutamate receptors, the mGluRs, consists of receptors that are coupled to G proteins and act through distinct downstream signaling cascades. They are structurally different from iGluRs and characterized by the presence of seven transmembrane domains (Houamed et al. 1991; Masu et al. 1991). The mGluRs exist as homodimers that do not by themselves form an ion-permeable pore in the membrane (Ozawa et al. 1998). To date, eight different genes (and more splice variants) encoding mGluRs have been identified and form the mGluR1 through mGluR8 subtypes (Alexander et al. 2009). Based on the amino acid sequence homology, downstream signal transduction pathways, and pharmacological properties, each of the subtypes was assigned to one of three groups. Group I receptors consist of mGluR1 and mGluR5 that positively couple to the phospholipase C (PLC). The receptors mGluR2 and mGluR3 constitute group II, whereas the remaining mGluRs, namely mGluR4, mGluR6, mGluR7, and mGluR8, belong to group III. Both groups II and III inhibit the adenylyl cyclase and thereby reduce the concentration of cAMP in the cytosol.Of all different subtypes, mGluR1 is the most abundantly expressed mGluR in the mammalian central nervous system. In the brain, mGluR1 is highly expressed in the olfactory bulb, dentate gyrus, and cerebellum (Lein et al. 2007). The highest expression level of mGluR1 in the brain is found in Purkinje cells, the principal neurons of the cerebellar cortex (Shigemoto et al. 1992; Lein et al. 2007). Together with the AMPA receptors, mGluR1s are part of the excitatory synapses formed between parallel fibers and Purkinje cells (Fig. 1A). Each Purkinje cell is innervated by 100,000–200,000 parallel fibers (Ito 2006) that are axons of the cerebellar granule cells, the most abundant type of neuron in the brain. A second type of excitatory input to Purkinje cells is represented by the climbing fibers that originate in the inferior olive in the brain stem (Ito 2006). The two excitatory synaptic inputs to Purkinje cells are important determinants for the main functions of the cerebellum, including the real-time control of movement precision, error-correction, and control of posture as well as the procedural learning of complex movement sequences and conditioned responses.Open in a separate windowFigure 1.Parallel fiber-evoked mGluR1-dependent signals. (A) Diagram showing the parallel fiber synaptic input to Purkinje cell dendrites. (B) Microelectrode recording of glutamatergic postsynaptic potentials from a Purkinje cell in an acute slice of adult rat cerebellum. Short trains of stimuli to the parallel fibers (5–6 at 50 Hz) caused summation of the early AMPA receptor-dependent EPSPs (leading to spike firing) and a slow, delayed, depolarizing potential (slow EPSP), which was reversibly inhibited by antagonist of mGluRs (+)-MCPG (1mM). (C) Confocal image of a patch-clamped Purkinje cell in a cerebellar slice of an adult mouse. The patch-clamp pipette and the glass capillary used for electrical stimulation of parallel fibers are depicted schematically. The site of stimulation is shown at higher magnification in D. (D) Left: Parallel fiber-evoked (five pulses at 200 Hz, in 10 mM CNQX) synaptic responses consisting of a dendritic mGluR1-dependent Ca²⁺ transient (ΔF/F, top) and an early rapid and a slow excitatory postsynaptic current (EPSC, bottom). Block of the mGluR1-dependent components by the group I-specific mGluR-antagonist CPCCOEt (200 µM) is shown as indicated. Right: Pseudocolor image of the synaptic Ca²⁺ signal. (B, Reprinted with modifications, with permission, from Batchelor and Gaithwaite 1997 [Nature Publishing Group].)It is expected that mGluR1 is involved in many of these cerebellar functions. This view is supported by the observation that mGluR1-deficient knockout mice show severe impairments in motor coordination. In particular, the gait of these mice is strongly affected as well as their ability for motor learning and general coordination (Aiba et al. 1994). The phenotype of the general mGluR1-knockout mice is rescued by the insertion of the gene encoding mGluR1 exclusively into cerebellar Purkinje cells (Ichise et al. 2000) and blockade of mGluR1 expression only in Purkinje cells of adult mice leads to impaired motor coordination (Nakao et al. 2007). These findings established mGluR1 in Purkinje cell as synaptic receptors that are indispensable for a normal cerebellar function.Synaptic transmission involving mGluR1s is found at both parallel fiber-Purkinje cell synapses (Batchelor and Garthwaite 1993; Batchelor et al. 1994) as well as at climbing fiber-Purkinje cell synapses (Dzubay and Otis 2002). Most of our knowledge on the mGluR1 was gained from the analysis of the parallel fiber synapses. The parallel fiber synapse is quite unique in the central nervous system regarding its endowment with neurotransmitter receptors. In contrast to most other glutamatergic synapses in the mammalian brain, it lacks functional NMDA receptors (Shin and Linden 2005). The entire synaptic transmission at these synapses relies on AMPA receptors and on mGluR1 (Takechi et al. 1998). Although AMPA receptors are effectively activated even with single shock stimuli (Konnerth et al. 1990; Llano et al. 1991b), activation of mGluRs requires repetitive stimulation (Batchelor and Garthwaite 1993; Batchelor et al. 1994; Batchelor and Garthwaite 1997; Takechi et al. 1998). A possible explanation for the need of repetitive stimulation may relate to the observation that mGluR1s are found mostly at the periphery of the subsynaptic region (Nusser et al. 1994). At these sites outside the synaptic cleft, glutamate levels that are sufficiently high for receptor activation may be reached only with repetitive stimulation.At parallel fiber-Purkinje cell synapses, repetitive stimulation produces an initial AMPA receptor postsynaptic signal component, followed by a more prolonged mGluR1 component (Fig. 1). Figure 1B shows a current clamp recording of this response consisting of an early burst of action potentials, followed by a prolonged depolarization known as a “slow excitatory postsynaptic potential” (slow EPSP) (Batchelor and Garthwaite 1993; Batchelor et al. 1994; Batchelor and Garthwaite 1997). Voltage-clamp recordings allow a clear separation of the initial rapid, AMPA receptor mediated excitatory postsynaptic current (EPSC) and the mGluR1-mediated slow EPSC (Fig. 1D) (Takechi et al. 1998; Hartmann et al. 2008). In addition of inducing the slow EPSPs, mGluR1s mediate a large and highly localized dendritic calcium transient in cerebellar Purkinje cells (Fig. 1D) (Llano et al. 1991a; Finch and Augustine 1998; Takechi et al. 1998).