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- Materials and methods
Although much information about metabotropic glutamate receptors (mGluRs) and their role in normal and pathologic brain function has been accumulated during the last decades, the role of group III mGluRs is still scarcely documented. Here, we examined mGluR4 knockout mice for types of behavior and synaptic plasticity that depend on either the hippocampus or the prefrontal cortex (PFC). We found improved spatial short- and long-term memory in the radial arm maze, which was accompanied by enhanced long-term potentiation (LTP) in hippocampal CA1 region. In contrast, LTP in the PFC was unchanged when compared with wild-type controls. Changes in paired-pulse facilitation that became overt in the presence of the GABAA antagonist picrotoxin indicated a function of mGluR4 in maintaining the excitation/inhibition balance, which is of crucial importance for information processing in the brain and the deterioration of these processes in neuropsychological disorders such as autism, epilepsy and schizophrenia.
Over the last decade, metabotropic glutamate receptors (mGluRs) have been intensively studied as potential targets for treating different diseases of the central nervous system (CNS), such as anxiety disorders, schizophrenia, Parkinson's disease, fragile X syndrome, Lewy body and Alzheimer's disease (Gross et al. 2012; Hopkins et al. 2009; Krystal et al. 2010; Lee et al. 1995; Luscher & Huber 2010; Niswender & Conn 2010; Osterweil et al. 2010; Price et al. 2010; Ribeiro et al. 2010; Sokol et al. 2011). The mGluRs belong to class C heptahelix G-protein-coupled receptors and are subdivided into three groups based on sequence homology, pharmacology and intracellular signaling mechanisms (Niswender & Conn 2010). Group I mGluRs (mGluR1 and mGluR5) are postsynaptically located and coupled to Gq/G11-like G-proteins, resulting in the release of inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol, and the activation of protein kinase C (PKC). Group II mGluRs (mGluR2 and mGluR3) and also group III mGluRs (mGluR4, 6, 7 and 8) are mainly presynaptically expressed and are Gi/o coupled leading to a decrease of cyclic AMP via inhibition of adenylyl cyclase. Once activated these receptors decrease postsynaptic activity by acting as presynaptic autoreceptors and heteroreceptors at glutamatergic and GABAergic terminals, respectively (Bordi & Ugolini 1999; Nakanishi 1994; Niswender & Conn 2010).
The mGluRs are not only involved in a variety of CNS disorders but have also been proven to play an important role in multiple forms of synaptic plasticity [e.g. (Anwyl 2009; Balschun et al. 1999a; Gladding et al. 2009; Huber et al. 2000; Klausnitzer et al. 2004; Luscher & Huber 2010; Moult et al. 2006)] and learning (Altinbilek & Manahan-Vaughan 2007, 2009; Balschun et al. 1999a; Callaerts-Vegh et al. 2006; Lyon et al. 2011; Riedel et al. 2003).
Disturbances in synaptic plasticity and learning, in turn, belong to the hallmarks of many psychiatric, neurodevelopmental, neurodegenerative and cognitive disorders [e.g. (Bear 2005; Goto et al. 2010; Marchetti & Marie 2011; Niswender & Conn 2010; Rowan et al. 2003; Wang et al. 2011)].
Group III mGluRs are the least understood among the mGluRs. Mainly owing to the lack of specific pharmacological tools, only a few studies addressed the function of this mGluR group in synaptic plasticity and learning. Thus, it was reported that intraventricular (i.c.v.) application of group III agonist l-2-amino-4-phosphonobutanoic acid (L-AP4) evoked robust long-term depression in the hippocampal CA1 region and the dentate gyrus (DG) (Manahan-Vaughan 2000; Naie et al. 2006). Long-term potentiation (LTP) in the CA1 region could be blocked by the group III antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) in vitro (Grover & Yan 1999) but not in vivo (Altinbilek & Manahan-Vaughan 2007).
In this study, we examined types of synaptic plasticity and learning in mGluR4 knockout (mGluR4−/−) mice that depend on either the hippocampus or the prefrontal cortex (PFC). The mGluR4−/− mice displayed a markedly enhanced LTP in the hippocampus but not in PFC. Furthermore, increased paired-pulse facilitation (PPF) upon application of GABAA antagonists suggests a function of mGluR4 in the regulation of the excitation/inhibition balance. These specific changes of short- and long-term synaptic plasticity were accompanied by improved spatial working (WM) and reference memory (RM) in mGluR4 mutant mice.
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- Materials and methods
Groups 2 and 3 mGluRs are both thought to act at glutamatergic and GABAergic terminals as inhibitory autoreceptors and heteroreceptors, respectively. Because of their negative coupling to adenylate cyclase signaling cascades (Niswender & Conn 2010), the primary consequence of their activation is a depression of glutamatergic and GABAergic transmission (Schoepp 2001). In so doing, they make a significant contribution to the tuning of transmission properties of various types of synapses (Ferraguti et al. 2005; Scanziani et al. 1997; Semyanov & Kullmann 2000; Shigemoto et al. 1996).
While the role of group 2 mGluRs in synaptic plasticity is documented by multiple studies, the investigation of group III mGluRs in these processes is only at its beginning. For example, i.c.v. application of the group III agonist L-AP4 evoked robust long-term depression in the hippocampal CA1 region and the DG that could be blocked by the group III antagonist (RS)-CPPG (Manahan-Vaughan 2000; Naie et al. 2006). The same compound had no effect on CA1-LTP (Altinbilek & Manahan-Vaughan 2007). Grover and Yan (1999) reported that NMDA receptor-independent LTP in the CA1 region in vitro could be blocked by the group III antagonist CPPG but not by (RS)-a-methylserine-O-phosphate. However, because the pharmacological effectors used in these studies do not distinguish between the subtypes of group III mGluRs, their genetic deletion is the method of choice to provide further insights into their functional role in synaptic plasticity. The mGluR4-deficient KO mice used in our study displayed a consistent strengthening of LTP in the CA1 region in vitro. This increase was observed irrespective of whether a robust potentiation was generated by repeated trains of tetanization or a decremental potentiation was induced by weak stimulation protocols. This seems to be in contradiction to a recent in vivo study by Altinbilek and Manahan-Vaughan (2007) in rats where application of the broad-band orthosteric group III antagonist CPPG had no effect on CA1-LTP. The difference to our results could be caused by: (1) species differences because rats and mice differ in the laminar distribution of mGluR4 in the hippocampus (Shigemoto et al. 1997) indicating an involvement in different functions. (2) A graded affinity of CPPG for the various group III mGluRs. Low concentrations of the compound are likely to bind to mGluR8 owing to the much higher affinity to this receptor when compared with mGluR4 (Naples & Hampson 2001). The mGluR8 is expressed in a subset of CA1 interneurons but not in pyramidal cells in this region, suggesting a role of mGluR8 in the generation of complex spike bursts (Ferraguti et al. 2005). (3) An incomplete inhibition of mGluR4 by the compound. In a recent study, Niswender et al. (2008) have shown that even at a high concentration as 1 mM, CPPG attained only about 60% inhibition of mGluR4 in vitro. Such high concentrations, however, cannot be applied in physiological experiments because CPPG has been described to inhibit group II mGluRs at higher concentrations.
Detailed analysis of the decay of responses during repeated TBS stimulation showed a slower synaptic fatigue in mGluR4−/− mice. This is indicative of a more sustained glutamate release and depolarization in mGluR4−/− mice during trains of TBS stimulation, thereby facilitating the induction of LTP.
Our findings of improved LTP after genetic abolishment of mGluR4-mediated presynaptic inhibition are reminiscent of a similar strengthening of potentiation after the application of group II antagonists (Behnisch et al. 1998). Interestingly, the improvement of potentiation after weak tetanization in mGluR4 mutant mice was stronger in response to a theta-burst protocol when compared with a weak, HFS of 40 pulses at 100 Hz. Both types of tetanization paradigms were shown to trigger different signal transduction cascades (Hoffman et al. 2002; Selcher et al. 2003).
Although the mean LTP values of mGluR4−/− mice in DG were consistently higher than controls, the genotype difference did not reach the level of significance. In this region, the paired-pulse measurements showed an interesting functional change in mGluR4 mutants, which became only overt when GABAA receptor-mediated inhibition was reduced by adding picrotoxin to the bath solution. This marked increase in paired-pulse response observed in picrotoxin-treated slices of KO mice is most likely owing to a changed interaction of the following mechanisms: presynaptically mediated PPF due to residual calcium that remains in the presynaptic terminal after the conditioning stimulus (Wu & Saggau 1994), a decrease in recurrent GABAA-mediated inhibition caused by picrotoxin (Fisher et al. 1997) and the lack of mGluR4-mediated control of glutamate and GABA release (Niswender & Conn 2010; Schoepp 2001). The latter seems to be particularly important when inhibition is weakened, as indicated by our data.
In further search of specific mechanisms underlying the increased excitability, we have to consider the fact that mGluR4 is not only localized in the presynaptic zones of principal cells but also of GABAergic interneurons (Corti et al. 2002; Kogo et al. 2004). This location is ideal to control GABA release by acting as heteroreceptor on GABAergic nerve terminals (Kogo et al. 2004; Somogyi et al. 2003). Probing further into the mechanisms, Rusakov et al. (2004) identified N-type Ca2+ channels as the principal target of l-(+)-2-amino-4-phosphonobutyric acid (L-AP4)-sensitive group III mGluRs at GABAergic terminals. The resulting depression of presynaptic Ca2+ influx mediates a reduction of GABA release in response to elevations of extracellular glutamate (Rusakov et al. 2004; Semyanov & Kullmann 2000). Thus, deletion of mGluR4 might have led in the DG to a disturbance of inhibition circuits owing to a change of presynaptic GABA release mechanisms and this had become overt at interpulse intervals of 10 and 20 milliseconds, respectively, after bath application of picrotoxin.
The paired-pulse ratio was not only changed in the DG but also in the CA1 region, but there without any use of GABAA antagonists. The mGluR4−/− mice showed increased PPF at 50-millisecond interpulse interval, indicating a lack of mGluR4-mediated tonic inhibition on glutamate release in mutant mice. On the background of different neuronal circuits and proportions of interneuronal subtypes in both regions (Freund & Buzsaki 1996), the increased PPF could be due to similar but not the same mechanisms as suggested above for the DG.
Although mGluR4 has been reported to reside in PFC (Corti et al. 2002; Gupta et al. 2005), we did not find any differences in basic excitability and LTP between mGluR4 mutant mice and their control wild-type littermates in basic excitability and LTP in this region. This does not exclude, of course, that other induction protocols for LTP would have resulted in discernible differences between the two genotypes, or that other types of synaptic plasticity could be more relevant to performance of the DMTS task.
Generally, there is not much known yet about the role of mGluRs in PFC synaptic plasticity. Activation of group II mGluRs was implicated in the induction of long-term depression in PFC (Huang & Hsu 2008; Otani et al. 2002) and mGluR7 was assigned a role in the regulation of NMDA receptors (Gu et al. 2012), which could point to a role of this group III mGluR subtype in plasticity.
The clear enhancement of LTP in the CA1 region of mGluR4−/− mice and the lack of significant effects of mGluR4 deletion on LTP in DG and PFC are difficult to explain because of the premature knowledge about this mGluR subtype (Niswender & Conn 2010; Niswender et al. 2008). Most likely, these differences are caused by different expression profiles of mGluR4 in these regions (Bradley et al. 1999; Shigemoto et al. 1997), which result in a region-specific participation of the receptor in diverse physiological functions such as the fine-tuning of glutamate and GABA release in response to the variety of activation patterns in the intact brain.
More importantly, however, we found a significant role of mGluR4 in LTP in the CA1 area, the principal hippocampal output region for spatial information that presumably further encodes information arriving from CA3 (Gigg 2006; Rolls 2010). Given the essential role of CA1 in NMDAR-dependent spatial learning and plasticity (Tsien et al. 1996), the improved RAM performance of mGluR4−/− mice is well in line with the enhanced LTP in the CA1 region.
One of the advantages of the RAM consists of allowing the distinction of both WM and RM components. Trial-specific WM allows the animal to recall arms entered previously in the current trial and to avoid re-entries. On the other hand, RM refers to stored representations and rules that are useful for all trials. In our study, better performance of mGluR4−/− mice was seen in short-term WM and long-term RM. The RAM learning has been shown to depend not only on the functional integrity of the hippocampus but also of the PFC (Kawabe et al. 1998; Olton & Papas 1979) and changes in WM were described to be positively correlated with LTP changes in PFC and hippocampal subregions (Cui et al. 2011; Dallerac et al. 2011, Holscher et al. 2007; Niewoehner et al. 2007). In contrast to the improved WM and RM, we did not observe changes in prefrontal LTP in vitro. However, there are several examples in the literature (and our own work) where changes in LTP were not accompanied by changes in memory performance and vice versa, or where LTP and memory changes were even negatively correlated (e.g. D'Hooge et al. 2005; Gerlai et al. 1998a; Jolas et al. 2002; Walther et al. 1998). This apparent discrepancy may be due to a number of reasons such as (1) a different sensitivity of LTP and learning performance to the molecular and physiological changes following a gene deletion or the insertion of a transgene, (2) the use of inappropriate or insensitive protocols, (3) the fact that learning represents the sum of putatively different effects of the manipulation in various brain regions, while the LTP effect is the synaptic readout of just one brain region and (4) learning but not LTP (in vitro) can be modulated by non-cognitive factors such as a higher motivational drive.
Interestingly, deletion of mGluR7, another group III mGluR subtype, resulted in partially opposite effects on RAM learning performance. The mGluR7−/− animals committed more WM errors than control mice in a four- and eight-arm RAM but the number of RM errors did not differ between genotypes (Callaerts-Vegh et al. 2006; Holscher et al. 2004). However, these effects were variable because in a configuration where only two of the arms of a four-arm maze were baited, there was no difference in WM errors. In a water maze study with mGluR4−/− mice, Gerlai et al. (1998a,b) found neither functional deficits in acquisition and retention (probe trial) of spatial memory nor in short-term memory (ITI = 90 min) and ‘medium-term’ memory (ITI = 17 h). They did see, however, a faster adaptation, i.e. an increased flexibility of mGluR4−/− mice after platform reversal, which is indicative of the ability of mGluR4−/− mice to show increased performance if demanded by specific circumstances of the task.
Repeated pharmacological blockade of group III mGluRs by the group III antagonist CPPG during RAM learning in rats has been described to result in an impairment of long-term RM while having no effect on short-term WM (Altinbilek & Manahan-Vaughan 2007). However, as outlined already above, because of the strong affinity of this compound for mGluR8 (Naples & Hampson 2001), it is not clear whether at the given dose an action on mGluR4 was involved in the effect.
In conclusion, increased LTP in the CA1 region as well as improved spatial short- and long-term memory after deletion of mGluR4 are in agreement with the presumed function of this mGluR subtype of exerting presynaptic inhibition. Changes in PPF in CA1 and DG point further to a role of mGluR4 in keeping the excitation/inhibition balance, which is one of the most critical variables in the processing of sensory information and of pivotal importance in neuropsychological disorders as autism, epilepsy and schizophrenia (Isaacson & Scanziani 2011; Yizhar et al. 2011).