Are Type 1 metabotropic glutamate receptors a viable therapeutic target for the treatment of cerebellar ataxia?

Abstract

The cerebellum is a key brain structure for accurate coordination of sensory and motor function. Compared with other brain regions, the cerebellum expresses a particularly high level of Type 1 metabotropic glutamate receptors (mGluR1). In this review we aim to explore the significance of these receptors for cerebellar synapse function and their potential for treating cerebellar ataxia, a poorly treated degenerative motor disorder that is often hereditary. We find a significant and historical literature showing pivotal mechanisms linking mGluR1 activity with healthy cerebellar synaptic function and motor coordination. This is best illustrated by the impaired motor behaviour in mGluR1 knockout mice that bears strong resemblance to human ataxias. More recent literature also indicates that an imbalance of mGluR1 signalling is as critical as its removal. Too much, as well as too little, mGluR1 activity contributes to ataxia in several clinically relevant mouse models, and perhaps also in humans. Given the availability and ongoing refinement of selective pharmacological tools to either reduce (negative allosteric modulation) or boost (positive allosteric modulation) mGluR1 activity, our findings suggest that pharmacological manipulation of these receptors should be explored as an exciting new approach for the treatment of a variety of human cerebellar ataxias.

Abbreviations

BG

Bergmann glia

CF

climbing fibre

EAAT

excitatory amino acid transporter

LTD

long‐term depression

mGluR

metabotropic glutamate receptor

NAM

negative allosteric modulator

PAM

positive allosteric modulator

PF

parallel fibre

PN

Purkinje neuron

PSD

postsynaptic density

Background and rationale

The metabotropic glutamate receptor (mGluR) family of G‐protein‐coupled receptors bind glutamate and act through intracellular chemical messenger signalling cascades to activate a wide variety of downstream effectors (Conn & Pin, 1997). The eight different mGluRs are divided into three different types, and of these the Type1 mGluRs, mGluR1 and mGluR5, are both functionally coupled to phospholipase C (PLC) and influence inositol trisphosphate (InsP ₃) and calcium (Ca²⁺) signalling. These Type 1 mGluRs are the focus of this review since their high levels of expression in the cerebellum and their acknowledged links to synaptic plasticity and motor learning (Ito, 2002; Gao et al. 2012) make them an attractive area of study in the context of cerebellar ataxia.

The Purkinje neurons (PNs) provide the sole output from the cerebellar cortex and their firing behaviour provides a fundamental component of the sensorimotor ‘error correction signal’ that is critical for motor learning and coordination. The PNs sit at the centre of a synaptic microcircuitry that translates cerebellar inputs to change the gain of their firing output (Fig. 1). In both rodents and humans, these fundamentally important neurons express very high levels of mGluR1a and ‐b (long and short splice variants, respectively) at their glutamatergic AMPA receptor‐dependent excitatory synapses (Berthele et al. 1999; Mateos et al. 2000). mGluR1 expression at PN synapses is located outside the postsynaptic density (PSD) and only activated when glutamate levels spill over from the active zone of the synapse. mGluR1 recruitment occurs during high frequency activation of parallel fibres (PFs) (Batchelor & Garthwaite, 1997; Tempia et al. 2001) and climbing fibre (CF) activation (Dzubay & Otis, 2002). Mature PNs do not express mGluR5 (Casabona et al. 1997) under normal conditions (but see ‘Loss‐ and gain‐of‐function of Type 1 mGluR signalling in clinically relevant mouse models of cerebellar ataxias’ section).

Figure 1. Microcircuitry of the cerebellar cortex .

The Purkinje neurons (PNs) provide the output from the cerebellar cortex modified by extrinsic excitatory synaptic inputs from mossy fibres (via granule cells, GCs) and climbing fibres (CFs). Synaptic activity can be directly modified by postsynaptic mGluR1‐dependent mechanisms to weaken the excitatory parallel fibre (PF) synapse (LTD). PN firing output is also further indirectly modified by PF activation of interneurons (basket cells, stellate cells) and Bergmann glia (BG). Golgi and unipolar brush cell activity influences GCs, so indirectly influencing PF input to PNs. The schematic diagram summarises Type 1 mGluR expression amongst the cells in the microcircuit, suggesting that there are a variety of opportunities for Type 1 mGluR pharmacological modulation in the circuit to directly or indirectly modify PN output. Note that mature PNs express the highest level of mGluR1 and do not express mGluR5. ML, molecular layer; GCL, granule cell layer; BG, Bergmann glia.

PN output is also influenced by the activity of inhibitory interneurons within the microcircuitry. These neurons also express Type 1 mGluRs (both mGluR1 and ‐5) but at much lower levels than PNs; although still functionally important, this expression is not discussed further here (but summarised in Fig. 1).

Activation of mGluR1 following glutamatergic activity at healthy PN synapses leads to a number of diverse downstream postsynaptic signalling mechanisms (Fig. 2 A). Together these influence Ca²⁺ levels in the postsynaptic spines (and dendrites) triggering signalling cascades that lead to a permanent weakening of the synapses, called long‐term depression (LTD), and memory formation (Wang et al. 2014). Note that several other types of synaptic plasticity exist in the cerebellar microcircuit, often called distributed circuit plasticity (Hansel et al. 2001; Gao et al. 2012; D'Angelo et al. 2016), including mGluR1‐dependent long‐term potentiation (LTP) at the PF–PN synapse in vivo (Chu et al. 2014).

Figure 2. Type 1 mGluR signalling at healthy and ataxic cerebellar PF synapses .

A summary of Type 1 mGluR signalling interactions at healthy (A) and ataxic (B) cerebellar PF synapses. Note the loss of key elements between A and B in a variety of clinically relevant ataxic mouse models and as a consequence of human mutations that cause ataxia. Spiral represents diverse downstream signalling pathways involving PKC and tyrosine‐dependent kinases and phosphatases where activity culminates in the removal of AMPA‐R from the postsynaptic membrane in LTD. It is not known if defects in these components occur in ataxias. Increased line thicknesses in B versus A indicates increased efficiency of coupling between mGluR5 and InsP ₃ mobilisation (compared with mGluR1) or increased Na⁺, Ca²⁺ or K⁺ ion fluxes.

The functional importance of PN mGluR1‐mediated signalling at PF and CF synapses is highlighted in mGluR1 knockout mice (Aiba et al. 1994; Conquet et al. 1994) by diminished postsynaptic LTD at PF–PN synapses, together with abnormal multiple, as opposed to single, CF innervation of PNs (Kano et al. 1997), movement ataxia and impaired motor learning (eye blink conditioning). Remarkably, rescue of these impairments is possible using PN‐specific expression of mGluR1 in the knockout mice (Ichise et al. 2000). The latter finding suggests that PN synapses are the most important site of mGluR1 action, in line with their high levels of mGluR1 expression. More recently, specific reintroduction of mGluR1a to the knockout mouse rescued all cerebellar deficits whilst reintroduction of the shorter mGluR1b variant rescued the PN TRPC3 currents and motor coordination but failed to rescue multiple CF innvervation, impaired Ca²⁺ handling and motor learning (Ohtani et al. 2014). This more recent study highlights the specific importance of mGluR1 splice variants and their relative functional contributions to downstream signalling.

The development of selective and potent pharmacology to specifically modify mGluR1 activity (independent of mGluR5) has further confirmed the importance of mGluR1 for motor learning and lever pressing tasks including selectivity of action at other sites in the brain (Lavreysen et al. 2004; Hodgson et al. 2011). These potent but more specific positive and negative allosteric modulators (PAMs and NAMs) of Type 1 mGluRs (Gasparini et al. 2002) have the potential to reveal how synaptic mGluR signalling mechanisms influence cerebellar function in vivo.

The acknowledged importance of Type 1 mGluRs at cerebellar synapses and the resurgent interest in their actions based upon improved pharmacology now makes it timely to gather together the recent and historical literature on mGluR1 signalling at cerebellar synapses. We are also motivated by recent exciting literature showing that both loss and gain of mGluR1 function contribute to ataxia in a number of clinically relevant mouse models and in humans. In this way we hope to explore if Type 1 mGluR pharmacology can be a viable approach for the treatment of human cerebellar ataxias.

mGluR1 signalling at healthy PN synapses

Glutamate released at both CF and PF synapses activates mGluR1 and its G‐protein complex so that Gα activates phospholipase C (PLC) to catalyse the breakdown of phosphatidyl‐inositol‐bisphosphate (PIP₂) to InsP ₃ and diacyl‐glycerol (DAG) and the release of Ca²⁺ from intracellular stores via activation of InsP ₃ receptors (Blackstone et al. 1989). At the PFs, mobilisation of PN Ca²⁺ from stores is restricted to single postsynaptic spines or small spino‐dendritic domains depending on the strength of stimulation (Finch & Augustine, 1998; Takechi et al. 1998). Subsequent activation of a variety of downstream kinases and phosphatases (see Fig. 2) drives the phosphorylation and removal of AMPA receptor from the synapse giving rise to mGluR1‐dependent synaptic weakening (or LTD); these downstream pathways are very briefly summarised in Fig. 2 A and not further discussed.

Interactions with K⁺‐dependent outward currents

Flash photolysis of InsP ₃ to mimic mGluR1 activation also mobilises PN Ca²⁺ in spines and dendrites and is accompanied by an outward K⁺ current that slowly hyperpolarises the PN (Khodakhah & Ogden, 1993; Canepari & Ogden, 2006). Conversely, mGluR1‐dependent local Ca²⁺ also inactivates the A‐type Kv4.3 K⁺ conductance in the outer PN dendrites to enhance, or unlock, CF dendritic Ca²⁺ spike initiation and so directly alter PN output (Otsu et al. 2014).

Activation of Na⁺‐ and Ca²⁺‐permeable inward currents

More specifically at synapses, applications of mGluR1 agonists or short bursts of high frequency synaptic activity evoke a slow, transient inward cationic current called an mGluR1‐mediated long‐lasting EPSP/C (Batchelor & Garthwaite, 1997) and mediated by Ca²⁺ and Na⁺ entry (Knopfel et al. 2000; Canepari et al. 2004). These currents are mediated by the non‐specific cation channel TRPC3 (Hartmann et al. 2008) and although Ca²⁺ entry via this route is much less than the Ca²⁺ released from intracellular stores (Canepari et al. 2004) the current is critical for LTD induction (Chae et al. 2012). Importantly, TRPC3 is inactivated by PKCɣ, a PN synapse enriched Ca²⁺‐dependent protein kinase C (PKC) isoform (Kwan et al. 2006) as part of a negative feedback mechanism to curtail Ca²⁺ and Na⁺ entry.

mGluR1‐mediated endocannabinoid retrograde signalling

An important consequence of PF‐mediated mGluR1 activation (G protein activation, PLCβ, calcium rises) in PNs is an endocannabinoid‐mediated reduction in presynaptic PF glutamate release (Maejima et al. 2001; Brown et al. 2003; Maejima et al. 2005; Marcaggi, 2015). This mechanism, which is also required for LTD, uses the endocannabinoid 2‐arachidonoylglycerol produced by the PN that is then released back onto PFs expressing the CB1 receptor (Maejima et al. 2005; Carey et al. 2011). This mGluR1‐dependent retrograde signalling could provide a negative feedback loop to prevent overactivity of PNs during barrages of high‐frequency PF input (Kano et al. 2008).

mGluR1 interaction with glutamate transporters

Inhibition of excitatory amino acid transporters (EAATs) in Bergmann glia (BG; EAAT1) or PNs (EAAT4) also significantly enhances mGluR1‐dependent EPSP/Cs (Reichelt & Knopfel, 2002; Power & Empson, 2014) by slowing glutamate clearance. Similarly, inhibiting these transporters facilitates mGluR1‐dependent PF LTD (Brasnjo & Otis, 2001). Furthermore, in PNs from EAAT4‐positive parasaggital zones, PF mGluR1 currents and LTD are curtailed (Wadiche & Jahr, 2005) presumably because EAAT4 removes glutamate so efficiently from the synaptic cleft that glutamate cannot reach the more remotely located mGluR1s.

mGluR1 interaction with AMPA receptors

AMPA receptor (AMPA‐R) activation inhibits the mGluR1 current through a tyrosine kinase pathway, perhaps even within the same synapse. The implication is that mGluR1‐mediated EPSP/Cs will be most powerful at ‘silent’ AMPA‐R‐negative synapses (Auger & Ogden, 2010).

mGluR1 interaction with GluRδ2

GluRδ2 is an atypical ionotropic glutamate receptor expressed at high levels in PNs (Lomeli et al. 1993). Cerebellin (Cbln1) is a natural ligand for GluRδ2 (Matsuda et al. 2010) and together they interact with neurexins for critical stabilisation of cerebellar synapses during development (Uemura et al. 2010). More recently, mGluR1 has been shown to trigger GluRδ2 gating (Ady et al. 2014). Furthermore, when GluRδ2 is deleted, cerebellar LTD is impaired and normal elimination of CFs that takes place during development is lost (Uemura et al. 2007) (as in mGluR1 knockout mice). GluRδ2 also functionally interacts with mGluR1, PKCɣ and TRPC3 in cerebellar tissue to the extent that PF‐evoked mGluR1‐dependent slow EPSCs are slowed in the absence of GluRδ2 (Kato et al. 2012).

Loss‐ and gain‐of‐function of Type 1 mGluR signalling in clinically relevant mouse models of cerebellar ataxias

Given the pivotal role of mGluR1 at cerebellar synapses it is perhaps not surprising that the literature reveals mGluR1‐associated synaptic dysfunction in a number of mouse models of human cerebellar ataxias (Fig. 2 B).

mGluR1 loss‐of‐function

There are approximately 40 types of spinocerebellar ataxia (SCA), most caused by autosomal dominant mutations in a wide variety of proteins. Many SCAs are well capitulated in mouse models expressing the mutant genes.

In the SCA1 154Q and the SCA1 82Q mouse models (Burright et al. 1995; Watase et al. 2002), the mice express 154 or 82 CAG (Q) repeats in the human ataxin‐1 gene. The repeats cause the ataxin‐1 protein (ATXN‐1) to misfold and aggregate leading to toxicity and the formation of inclusion bodies specifically in PN soma. Misfolded ATXN‐1 in the SCA1 mouse interrupts the normal physiological function of the retinoid‐related orphan receptor (RORα) probably by disrupting transcription of downstream effectors (Serra et al. 2006). In SCA1 82Q the inclusion bodies contain high levels of sequestered mGluR1, GluRδ2 and PKCɣ (Skinner et al. 2001) and mGluR1 expression on PN dendrites is reduced and disordered, respectively, in the 154Q (Notartomaso et al. 2013) and 82Q (Zu et al. 2004) mice. Despite this reduction, Type 1 mGluR‐mediated slow Ca²⁺ signals are easier to evoke in PN dendrites from SCA1 82Q mice (Inoue et al. 2001). We can speculate that this occurs via an increased availability of glutamate (see above) as expression of EAAT4 in PNs (Serra et al. 2004) and EAAT1 in BG (Cvetanovic, 2015) is downregulated in this model of SCA1. We might also speculate that down‐regulation of mGluR1 expression is a necessary compensation to prevent excessive cytosolic Ca²⁺ and so permit PN survival, particularly as SERCA3 is also down‐regulated (Serra et al. 2004) and could lead to slowed uptake of Ca²⁺ into the endoplasmic reticulum (ER). More speculatively, loss of mGluR1 may disrupt the functional interaction between mGluR1 and Kv4.3 A‐type potassium channels seen in healthy synapses (Otsu et al. 2014). Indeed increased Kv4.3 channel expression reduces phasic PN firing in the early, pre‐symptomatic stages of SCA1 82Q (Hourez et al. 2011).

In the SCA1 154Q mouse, decreased mGluR1 (mRNA and protein) expression is accompanied by increased mGluR5 expression in the cerebellum (Notartomaso et al. 2013). The mGluR5 expression appears to be in PNs although mature PNs do not normally express detectable levels of mGluR5. However, since mGluR5 stimulates PLC with greater efficiency than mGluR1 (Casabona et al. 1997), the switch to mGluR5 could represent a gain‐of‐function to rescue Ca²⁺ signalling, perhaps to prevent PN death.

The spontaneously ataxic staggerer mice exhibit an autosomal recessive mutation in the RORα^sg gene and so bear similarity to the SCA1 mouse model above. As above, the PNs from staggerer mice in the advanced stages of ataxia also lack functional PF–PN mGluR1‐mediated synaptic currents and diminished expression of mGluR1 in PNs (Mitsumura et al. 2011).

Similarly in a mouse model of human SCA5 where a human β‐III spectrin mutation underlies the development of ataxia (Perkins et al. 2010), disrupted mGluR1 expression in PN dendrites is associated with a functional loss of mGluR1‐mediated responses and altered PF function (Armbrust et al. 2014). This is despite loss of EAAT4 expression from PNs and EAAT1 (GLAST) from BG in this model, which would be expected to make more glutamate available to mGluR1 (Perkins et al. 2010).

Recently, in SCA3 where CAG repeats disrupt the Atx‐3 gene and where RORα signalling is also disrupted (as in SCA1 above), mGluR1‐dependent endocannabinoid‐mediated retrograde suppression of PF function is completely lost (Konno et al. 2014).

mGluR1 gain‐of‐function

Whilst diminished expression and loss‐of‐function of mGluR1 in some ataxias is consistent with the ataxic phenotype of the mGluR1 knockout mice, some more recent literature indicates that a gain‐of‐function of mGluR1 signalling also leads to ataxia.

In a mouse model of SCA28, PNs exhibit defective mitochondrial Ca²⁺ handling leading to excessive cytosolic Ca²⁺ levels, but interestingly the ataxic phenotype is rescued by reducing mGluR1 expression in the SCA28 mice (Maltecca et al. 2015).

Hyperactive mGluR1‐mediated TRPC3 currents are seen in the PNs of spontaneously ataxic moonwalker mice (Becker et al. 2009) suggesting increased Na⁺ and Ca²⁺ influx (see above). The moonwalker mutation causes a threonine to alanine switch that modifies TRPC3 gating in PNs so that the channel opens under conditions of lower glutamate and mGluR1 activation. More recently, complete loss of unipolar brush cells in moonwalker, perhaps caused by overactive TRPC3 currents and Ca²⁺ overload, could help explain the severity of this model of ataxia (Sekerkova et al. 2013). Similarly, in a mouse model of SCA14, mutant PKCγ fails to inactivate TRPC3, so mGluR1‐mediated inward currents in PNs are larger than normal. The SCA14 mice also fail to exhibit LTD, and CF synapse elimination falters during development (Shuvaev et al. 2011); a similar phenotype is also associated with mGluR1 loss‐of‐function in mGluR1 knockout mice (see above).

In the hotfoot‐4J (GluRδ2 ^4Hojo) mouse, mutations in the GRID2 gene lead to a truncated GluRδ2 that is retained in the ER and fails to reach the plasma membrane (Matsuda & Yuzaki, 2002); this functional loss of GluRδ2 leads to increased extrasynaptic mGluR1 and TRPC3 expression and prolonged mGluR1‐mediated slow EPSCs in PNs (Kato et al. 2012). Ultimately the ataxia in hotfoot is likely to be caused by defective PF and CF synapse function. However, prolonged mGluR1 signalling could also contribute to the hotfoot phenotype and we can speculate that it might even drive the synaptic defects. Another well‐known mouse model of spontaneous ataxia, lurcher, is also associated with mutations in GRID2. This gene seems to be a hotspot for mutations as over 20 have been identified in this gene and many involve partial deletion of the GluRδ2 protein. In lurcher the mutation causes an alanine to threonine switch that transforms GluRδ2 into a leaky channel (Zuo et al. 1997). As a result PNs are depolarised leading to oxidative stress, underdevelopment of PN dendrites (Soha & Herrup, 1995) and increased apoptosis (Zuo et al. 1997; Selimi et al. 2000). Whether mGluR1 signalling is also disrupted in lurcher is not known.

Loss‐ and gain‐of‐function of Type 1 mGluR signalling in human ataxias

Recent clinical findings provide direct evidence that altered mGluR1 function causes human cerebellar ataxia.

In cerebellar autoimmune ataxias, antibodies to Homer‐3 (Hoftberger et al. 2013) and mGluR1 (Sillevis Smitt et al. 2000) are present in patient sera. The latter antibodies prevented LTD in a mouse cerebellar slice suggesting that this autoimmune ataxia represents a loss‐of‐mGluR1 function (Coesmans et al. 2003). Importantly administration of immunoglobulins reverses the ataxia.

More recently, a mutation in the region encoding mGluR1 was linked to an early‐onset autosomal‐recessive congenital cerebellar ataxia (Guergueltcheva et al. 2012) although the functional nature of this mutation and its impact (loss‐ or gain‐of‐function) is not known. Early in 2015 a rare case of adult onset ataxia was also identified as a gain‐of‐function mutation in TRPC3. This p.Arg762His mutation, although different from the mutation in moonwalker mice, is predicted to influence channel gating in a similar manner. Significantly, expression of the p.Arg762His mutant TRPC3 increases cell death in functional assays (Fogel et al. 2015).

Very recently, several other families with autosomal recessive cerebellar ataxias have been identified as having GRID2 mutations suggesting that GRID2 may be a fragile area of the genome in both mouse (hotfoot) and humans (Van Schil et al. 2015). How these GRID2 mutations exert their effect in the human cerebellum is not known, but we can speculate that similar mGluR1 gain(s)‐of‐function observed in hotfoot mice may contribute, along with severe defects in PF and CF function. However, the greater severity of cerebellar atrophy in humans compared with mice suggests that GRID2 may have an expanded role(s) in humans (Hills et al. 2013).

Recent successful treatment of ataxia using specific Type 1 mGluR interventions

The somewhat conserved nature of the cerebellar ataxia phenotype caused by mGluR1 loss‐ or gain‐of‐function in humans and mouse nevertheless gives strong potential for translation of mGluR1 based treatments. We are not there yet, but two recent studies that manipulated mGluR1 successfully restored motor deficits in two mouse models of SCA1 and SCA28.

The mGluR1 PAM alleviated the ataxia symptoms in severely ataxic 30‐week‐old SCA1 154Q mice when mGluR1 expression drops precipitously. In contrast, the mGluR1 NAM (albeit at very high doses) worsened the motor symptoms in the later stages of this model. Significantly, a single administration of the mGluR1 and mGluR5 PAM alleviated the ataxia consistent with a functional mGluR1–mGluR5 switch (see ‘mGluR loss‐of‐function’ section) (Notartomaso et al. 2013).

More recently in the SCA28 mouse model, genetic manipulation to reduce mGluR1 expression and signalling in PNs restored their cytosolic Ca²⁺ to more manageable levels and improved the ataxic behaviour of the mice (Maltecca et al. 2015).

These two quite distinct studies in two very different models of human cerebellar ataxia provide strong motivation for testing Type 1 mGluR1 and ‐5 PAMs and NAMs in other preclinical mouse models of ataxia. Possible candidates include tackling the early stages of the ataxic, gain‐of‐function moonwalker and hotfoot mouse with an mGluR1 NAM, or the mid to late stages of the SCA1 82Q and 154Q mouse and staggerer with an mGluR1, or even an mGluR5 PAM.

Closing remarks

Finding therapeutic targets for the treatment of ataxia remains critically important for those at risk, especially in families where known dominant and recessive mutations might occur. The recent success of pharmacological approaches to tackle mGluR1 malfunction in a variety of mouse preclinical ataxia models is incredibly exciting, although we should be mindful that both loss‐ and gain‐of‐function are likely to be relevant, perhaps even at different stages of ataxia progression. The rather conserved nature of mGluR1 malfunction in human ataxias and mouse preclinical models, and the availability of potent and specific allosteric modulators also make continued preclinical research and mechanistic determinations very worthwhile. Armed with greater knowledge, we think manipulation of Type 1 mGluR holds considerable promise for translation to a viable treatment for human ataxias.

Additional information

Competing interests

The authors state that they have no competing interests.

Author contributions

All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

A University of Otago PhD Scholarship to E.M.P.

Biographies

Emmet Power has a long‐held interest in motor systems and completed his MSc in Neuroscience at University College Cork where he worked on microRNA expression in the developing spinal cord with Kieran McDermott. Emmet left Ireland for an adventure in ‘Middle Earth’, the South Island of New Zealand, and recently completed his PhD on cerebellar ataxia with Ruth Empson in the Department of Physiology at Otago. He is currently a post‐doc in her lab.

Natalya English is a Neuroscience Honours student in Ruth Empson's lab and loves reconstructing Purkinje neurons on the confocal.

Ruth Empson is an Associate Professor at Otago having immigrated to New Zealand in 2007 from the UK where she was at the School of Biological Sciences, Royal Holloway. Their research questions address circuitry in the cerebellum and motor cortex – and anything in between – using dynamic imaging (calcium and voltage) electrophysiology, behaviour and immunohistochemistry.