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68 Alcohol andtheCerebellum
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the brain. In particular, although studies of isolated brain tissue have identied numerous targets and neural impacts of
higher concentrations of EtOH ([EtOH]>20mM), in many
brain regions, impacts of low concentrations of EtOH
([EtOH] ≤ 10 mM) are minimal or lacking. Thus, it is
unlikely that low [EtOH] has such widespread action in the
brain and on behavior by direct actions. Instead, it is likely
that low [EtOH] has more localized actions that are then
transmitted more widely, and the cerebellum is clearly
involved in this process.
In vivo studies of humans and animals indicate that low
[EtOH] alters cerebellar processing and associated behaviors
(Volkow et al. 2008; Gallaher et al. 1996; Schuckit 1985;
Schuckit etal. 2003). Importantly, studies of cerebellar brain
slices have shown that 9–10 mM EtOH can powerfully
enhance or suppress (depending on genotype) GABAA
receptor (GABAAR)-mediated inhibition of rat cerebellar
GCs (Carta etal. 2004; Kaplan etal. 2013), which are the
primary integrators of afferent information to the cerebellar
cortex (Fig.68.1). Such actions are effectively transmitted to
modulate excitation of PCs (Kaplan etal. 2016a), which are
the sole output of the cerebellar cortex to the cerebellar
nuclei (Fig.68.1). The Inferior Olivary neurons that supply
the climbing ber input to, and powerfully excite output PCs
are also inhibited by 10mM EtOH (Welsh etal. 2011), as is
the N-methyl-D-aspartate receptor (NMDAR)-mediated
component of their synaptic input to PCs and associated
plasticity (He etal. 2013), providing another clear pathway
for cerebellar sensitivity to low [EtOH]. Finally, 10 mM
EtOH also increases mixed GABAergic/glycinergic inhibition of Unipolar Brush cells, the only other non-GC excitatory interneuron in the GC layer (not shown in Fig.68.1,
given restricted expression in specic cerebellar regions)
(Richardson and Rossi 2017). Importantly, in rodent studies
of EtOH-induced motor impairment, specic modulation of
cerebellar processing with locally infused modulatory drugs
(including nicotine) can effectively reduce or even eliminate
motor impairing effects of systemic EtOH, verifying that
EtOH-induced motor incoordination is primarily specically
mediated by actions in the cerebellum (Dar 2015). Given that
low [EtOH] clearly modulates cerebellar output, and enough
to inuence known cerebellar-dependent behaviors, it is reasonable to consider such altered output will then similarly
indirectly inuence all other brain regions that the cerebellum communicates with (Fig. 68.1), which includes most
regions that are activated invivo by low [EtOH], as described
above. Thus, in addition to mediating motor discoordination,
the observed widespread changes in brain activity and associated behavioral responses to consumption of a unit or two
of EtOH (leading to BACs of ~10mM) are likely driven in
part by altered cerebellar output.
As the concentration of EtOH increases above 10mM,
but still in the survivable recreational/clinical range
(20–50mM), progressively at least some aspect of most of
the cells and synapses within the cerebellar cortex are
affected, including enhancement of molecular layer interneuron inhibition of PCs (25mM EtOH) (Ming etal. 2006),
direct excitation of PCs (50mM EtOH) (Ming etal. 2006),
and direct inhibition of input layer GCs (35mM) (Lewohl
etal. 1999). Thus, starting at 10mM, progressively increasing components of the cerebellar cortical circuit are affected
by EtOH, with almost all components affected by survivable
BACs (50mM). Such high sensitivity, and progressive incorporation of cerebellar components underlies increasingly
impaired motor performance starting at 1–2 units of
EtOH.And, as suggested above, the cerebellum likely contributes, via projections throughout the brain, to many behavioral aspects of EtOH consumption.
68.2.2 The Cerebellum Is aCommon
andDominant Site ofBrain Damage
inPeople withAlcohol Use Disorder
(AUD)
Brain damage is commonly observed in humans with AUD,
and in addition to the specic deleterious behavioral impacts
of such brain damage, such as impaired memory formation
and ataxia, alterations in various cognitive functions, such as
impaired judgement, likely contribute to the maintenance of
AUD (Bates etal. 2013; Segobin etal. 2014; Le Berre etal.
2013), creating a devastating vicious cycle. The cerebellum
is a common and prominent site of AUD-associated brain
damage, as reected by overall shrinkage (Fig.68.2) as well
as PC loss (Sullivan etal. 2003), although damage occurs
preferentially in subsections of the cerebellum, notably the
anterior vermis (Sullivan etal. 2003). Moreover, there is considerable heterogeneity across individuals in the extent of
cerebellar damage. In this regard, a major ongoing quandary
is whether AUD-associated brain damage generally and cerebellar damage specically is primarily due to chronic excessive EtOH exposure per se, due to malnutrition (in particular
thiamine deciency) which often accompanies AUD, or
both. Anatomical studies indicate that cerebellar damage is
more common and severe in patients that also experienced
thiamine deciency, and the cerebellum is notably sensitive
to damage by thiamine deciency, but it is difcult to disentangle the presence of thiamine deciency from increased
EtOH exposure levels in the uncontrolled clinical scenario.
However, detailed statistical analysis of various behavioral
patterns in patients with AUD suggest that brain structures
subserving motor control and executive function can be doubly dissociated in terms of etiology from memory impairments, with motor control/executive function impairments
being most correlated with the level of EtOH exposure but
not thiamine deciency, and memory impairment being most
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Fig. 68.2 Representative MRI images of the brains from a control subject (left) and a subject with AUD (right), showing shrunken cerebellum
in subject with AUD.The cerebellum is circled in both images. Adapted
correlated with the level of EtOH exposure and thiamine
deciency (Fama etal. 2019). Thus, it is likely that much of
the reported cerebellar damage in AUD is in fact due to levels
of EtOH exposure, which is in keeping with its unusual sensitivity to EtOH, as described above. This conclusion is supported by animal studies in which prominent cerebellar
damage occurs upon forced exposure to EtOH despite maintaining optimal nutrition (Jaatinen and Rintala 2008). Adding
to the face validity of such animal models, the extent of cerebellar damage varies across subregions (Jaatinen and
Rintala 2008).
The statistical analysis of behavioral correlates of regional
brain damage also clearly associates both motor and cognitive impairment with EtOH-induced cerebellar damage
(Sullivan etal. 2020; Pinner etal. 2020), making it likely that
cerebellar damage contributes to poor decision making that
sustains AUD (Bates et al. 2013; Segobin et al. 2014; Le
Berre etal. 2013).
The mechanisms by which EtOH damages the cerebellum
are not particularly well understood, but likely involve excitotoxicity, oxidative damage and inammatory responses
(Jaatinen and Rintala 2008). The concept of EtOH exposure
causing excitotoxicity is on its face curious, given that in
general EtOH dampens excitation, both through enhancing
GABAergic inhibition and suppressing glutamatergic excitation. However, this apparent paradox can be reconciled by
considering that excitotoxicity likely occurs during withdrawal from chronic EtOH, when the brain’s homeostatic
adaptations to EtOH dampening of excitability leads to a
rebound hyperexcitable state (Jaatinen and Rintala 2008).
with permission from original images provided by Dr. Edith Sullivan,
used in Sullivan etal. (2003) ACER, 27:301
68.2.3 The Cerebellum Is aDominant Site
ofBrain Damage Underlying Fetal
Alcohol Spectrum Disorder (FASD)
Fetal alcohol spectrum disorder (FASD) is a spectrum of
lifelong physical and neurological symptoms resulting from
fetal exposure to EtOH.The range and degree of damage and
consequent symptoms vary depending on the duration and
level of exposure to EtOH (Sullivan etal. 2020; Pinner etal.
2020). The developing cerebellum is particularly sensitive to
EtOH, and its damage and consequent malfunction is a key
substrate of many of the neurological manifestations of
FASD, including reduced balance, ne motor skill, broad
motor coordination, and likely via its non-motor roles, language, emotional regulation and cognitive function (Sullivan
etal. 2020; Pinner etal. 2020; Gill and Sillitoe 2019). Similar
to the adult cerebellum, damage to the developing cerebellum manifests as overall shrinkage and involves damage to
PCs (Nirgudkar etal. 2016). Moreover, the damage is not
uniform, but rather some sub-regions, particularly the anterior vermis are more commonly damaged (Nirgudkar etal.
2016), and this may have to do with molecular and physio-
logical differences across sub-regions, despite the otherwise
uniform cellular makeup of the cerebellar cortex (Pinner
etal. 2020; Gill and Sillitoe 2019).
In addition to spatial heterogeneity in EtOH-induced cerebellar damage, the developing cerebellum has critical developmental time points when susceptibility to damage is
greatest. Since it is difcult to accurately ascertain when and
to what degree a human fetus is exposed to EtOH, such infor-
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mation comes largely from rodent models, in which evidence
indicates that the developing cerebellum has two distinct
phases of sensitivity to the damaging effects of EtOH exposure, equivalent to the second and third trimester of gestation
in humans (Nirgudkar etal. 2016) (and references therein).
In particular, EtOH exposure during rodent gestational age
10–19 (neurodevelopmentally equivalent to the second trimester in humans) results in cerebellar shrinkage without
notable PC loss. In contrast, EtOH exposure in newborn
pups, in particular, postnatal days 4–9 (neurodevelopmentally equivalent to the third trimester in humans), results in
loss of PCs without dramatic cerebellar shrinkage. Thus, the
overall phenotype of humans with FASD (cerebellar shrinkage and PC loss) likely reects exposure during the second
and third trimesters respectively.
Similar to the adult cerebellum, EtOH-induced damage is
induced in animal models with a controlled diet, suggesting
at least some of the damage is induced by EtOH itself,
although damage is likely exacerbated by concomitant thiamine deciency. Likewise, although specic mechanisms of
damage are not yet well understood, EtOH-induced fetal cerebellar damage likely involves some combination of excitotoxicity, oxidative damage and inammatory responses
(Jaatinen and Rintala 2008). Also, like the adult cerebellum,
EtOH-induced excitotoxic damage may occur during the
hyperexcitable withdrawal phase of chronic alcohol abuse
(Jaatinen and Rintala 2008). In the context of excitotoxicity
being a likely contributing factor in EtOH-induced cerebellar
damage during this transient period of development, but also
in adult cerebellum (Sect. 68.2.2), it is noteworthy that PCs,
which are damaged in both contexts, have an atypical temporal expression pattern of functional NMDARs, exhibiting a
transient expression in newborn rodents, which disappear at
about postnatal day 8, and then re-emerge in fully adult PCs
(Piochon et al. 2007). Given the well-known role of Ca2+
inux through NMDARs in triggering excitotoxic neuronal
damage, it is possible that the developmental pattern of PC
NMDARs contributes to the developmental prole of their
damage in the context of FASD and AUD.
68.3 The Cerebellum andGenetic Risk
forDeveloping AUD
AUD (FH−). And preclinical animal studies suggest that
genetic differences in cerebellar response to EtOH inuence
how much EtOH a given rodent genotype will consume.
68.3.1 Cerebellar Anatomy andPhysiology
inHumans withGenetic Risk forAUD
MRI studies indicate that the cerebellum of FH+ offspring is
signicantly larger than in trait-matched FH− offspring, due
primarily to increased grey matter, potentially due to reduced
synaptic pruning during development (Hill 2010; Hill etal.
2007, 2011, 2016). Importantly, this difference is separable
from effects related to prenatal exposure to EtOH, which
also affects the size of the cerebellum, but in the opposite
direction and in distinct lobes (Sharma and Hill 2017). While
it is not clear how such anatomical differences inuence predilection for AUD, increased cerebellar volume in FH+ individuals is associated with an allelic variation in the GABAAR
α2 subunit (Hill et al. 2011), which when knocked out in
mice results in reduced EtOH consumption in females
(Boehm etal. 2004). In addition to these anatomical differences, there are also considerable individual differences in
cerebellar processing and communication with other brain
regions that correlate with FH status. In functional MRI studies, relative to FH− individuals, alcohol naïve FH+ offspring
show less functional connectivity between the cerebellum
and two brain regions known to be involved in addictive
behaviors, the prefrontal cortex (PFC) and nucleus accumbens (Cservenka etal. 2014; Herting etal. 2011). Further,
FH+ individuals show reduced cerebellar activity during cognitive tasks (risky decision making and spatial working
memory), despite not exhibiting any decits in task performance (Cservenka and Nagel 2012; Mackiewicz Seghete
etal. 2013). Thus, in addition to exhibiting reduced communication with addiction associated brain regions, alcohol
naïve, FH+ individuals exhibit altered cerebellar processing
of behavioral tasks that likely play a role in addiction.
Collectively, while specic mechanisms remain unclear,
genetic risk for developing an AUD is associated with altered
cerebellar anatomy, communication with known reward centers of the brain, and altered activity during behaviors likely
to inuence development of AUD.
Adoption and twin studies suggest that predilection for
developing AUD is 50–60% genetically determined (Hasin
etal. 2007; Hill 2010), and both clinical and preclinical stud-
ies suggest that the cerebellum plays a signicant role in
mediating such genetic predilection. In particular, clinical
studies of humans with a family history of AUD (FH+), and
thus elevated risk for developing AUD, indicate that cerebellar anatomy, physiology and sensitivity to EtOH in FH+ individuals differs from individuals without a family history of
68.3.2 A Low Level ofResponse toAlcoholInduced Cerebellar-Dependent Motor
Impairment Is aRisk Factor
forExcessive Alcohol Consumption
inRodent Models andAUD inHumans
As with many addictive drugs, an endophenotypic component to genetic risk for developing an AUD is increased sen-
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1-2g/kg/day
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sitivity to the rewarding aspects of EtOH and reduced
sensitivity to the aversive aspects of EtOH. One wellestablished cerebellar-associated behavioral measure of sensitivity to EtOH in humans is alcohol-induced static ataxia,
which manifests as body sway, resulting from impaired vestibular and ocular feedback control of balance. Such studies
consistently nd that a low level of sensitivity to EtOHinduced body sway is heritably associated with FH+ family
history status and predictive of development of AUD
(Schuckit et al. 2005, 2011). A similar covariation also
occurs in preclinical rodent models. In particular, sensitivity
to EtOH-induced ataxia shows an inverse relationship with
EtOH consumption in several inbred strains of mice (e.g.
DBA/2 J (D2) and C57BL/6 J (B6) mice), and lines of
rodents selected for differences in alcohol consumption (e.g.
Alko, Alcohol/Alko, Non-Alcohol rats, and alcoholpreferring (P)/alcohol non-preferring (NP) rats) (Rossi and
Richardson 2018). Thus, there is substantial evidence that in
both humans and rodents, genetically determined insensitivity to the motor impairing effects of EtOH is a risk factor for
AUD in humans and excessive EtOH consumption in rodents.
Golgi cell to GC synapse
-
Cl
-
Cl
Cl
Cl
-
-
Cl
-
SDR granule cell
response to EtOH
68.3.3 Genetic Dierences inCerebellar
Cellular Responses toAlcohol Correlate
with, andlikely Contribute totheLevel
ofAlcohol Consumption inAnimal
Models
As introduced above, a primary component of the cerebellum’s sensitivity to EtOH is via EtOH actions at the Golgi
cell to GC inhibitory GABAergic synapse. Inhibition at this
synapse is mediated by two forms of GABAA receptor
(GABAAR)-mediated inhibition: traditional phasic spontaneous inhibitory postsynaptic currents (sIPSCs) generated by
postsynaptic GABAARs responding to the vesicular release
of GABA (Fig. 68.3a), and a powerful form of tonic
inhibitory current generated by high afnity, nondesensitizing extrasynaptic GABAARs responding to the
ambient extracellular concentration of GABA (Fig.68.3a)
(Hamann etal. 2002). Early studies of the cerebellum in low
EtOH consuming Sprague Dawley rats (SDRs) demonstrated
that low [EtOH] (starting at ~10mM) enhances Golgi cell
inhibition of GCs (Carta etal. 2004), via both an increase in
SDR
D2 mouse
52mM 52mM
EtOH
B6 mousePrairieVole
52mM52mM
-
Cl
GABA
δ
β
x
α
4,
6
α
4
,6
β
x
A
Rcurrent
e f
D2
SDR
1-2 1-2 1-4 11-22
Typical g/kg/day EtOH consumed
Phasic
γ
2
α
β
1
x
α1β
x
-
Cl
GABAAR IPSC
Tonic
Fig. 68.3 (a) Top: Schematic diagram showing GABAARs in the syn-
aptic cleft (blue) and outside of the synaptic cleft (green). Bottom:
Phasic IPSCs (left) are mediated by synaptic GABAARs (as evidenced
by their sensitivity to the GABAAR antagonist, GABAzine) that are rapidly activated by the high concentrations of vesicular GABA released
into the synaptic cleft. Tonic GABAAR-mediated currents (right)
blocked by GABAzine. Note: because GABA released into the synaptic cleft diffuses out of the cleft where it can activate extrasynaptic
GABAARs, the magnitude of the tonic GABAAR current increases or
decreases in parallel with changes in vesicle release rate, either from the
presynaptic neuron or from neighboring synapses not directly connected to the recorded cell. (b) Example recording (left) showing that
EtOH (52mM) increases sIPSC frequency and tonic GABAAR current
magnitude in a GC in a slice of cerebellum from a low EtOH consuming Sprague Dawley rat (SDR). (c and d) Example recordings showing
that EtOH (52mM) enhances the tonic GABAAR current in low EtOH
B6
NHP
~12g/kg
~12
consuming rodent genotypes (SDRs and D2 mice; c), but suppresses the
tonic GABAAR current in high EtOH consuming rodent genotypes
(Prairie Voles and B6J mice; c). (e) Plot of mean EtOH-induced change
in magnitude of GC tonic GABAAR current across mammalian genotypes with divergent EtOH consumption phenotypes. Note, EtOH consumption values are rough estimates of average amount consumed
across a 24h period for each mammalian genotype, without consideration for consumption pattern across the day. (f) Example recordings
and bar chart of mean responses to varying doses of EtOH in SDRs and
B6J mice showing that opposite action of EtOH is preserved at low to
high [EtOH]. (g) Mean amount of EtOH consumed by B6J mice during
a 2hr., 2 bottle choice (water and 10% EtOH) session, under control
conditions and after a local injection of the GABAAR agonist, THIP,
into lobe 3 of the cerebellum. Images adapted with permission from
Mohr etal. 2013 and Kaplan etal. 2013, 2016a, b
PV
PV
g
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the frequency of sIPSCs and an increase in the magnitude of
the tonic GABAAR current (Fig. 68.3b), which in GCs is
mediated by extrasynaptic α6 and δ subunit-containing
GABAARs (Fig.68.3a) (Hamann etal. 2002). The primary
mechanism for both components is EtOH-induced increased
action potential ring by Golgi cells, with the resultant
increase in vesicular GABA release increasing both sIPSC
frequency and tonic GABAAR current magnitude, due to the
associated elevation of ambient extracellular [GABA] (Carta
etal. 2004; Kaplan etal. 2013).
The notably high sensitivity of the Golgi cell to GC
GABAAR system to EtOH, combined with the clear role of
this response in mediating at least one behavioral response to
EtOH (motor impairment) makes it a potential mediator of
genetic differences in response to low [EtOH] that inuences
initial subjective reactions to consumption of EtOH, and thus
predilection for developing an AUD (as in Sect. 68.3.2).
Indeed, more recent studies of different rodent genotypes
with divergent EtOH consumption phenotypes determined
that a general pattern exists, in which the impact of EtOH on
GC tonic GABAAR currents varies in polarity and magnitude
in parallel with the EtOH consumption phenotype of the
mammal, with high and low EtOH consumption associated
with suppression and enhancement of tonic GABAAR currents respectively (Fig.68.3b–e). This relationship persists
across the dose-response range of EtOH concentrations
tested, including 9mM (Fig.68.3f), indicating that EtOH has
opposite actions on GC tonic GABAAR currents in high and
low EtOH-consuming genotypes at all levels of consumption. Importantly, even at 10mM, EtOH suppression of the
GC tonic GABAAR current observed in high EtOH consuming genotypes is strong enough to increase excitation of output PCs (Kaplan et al. 2016a), providing a plausible
mechanism by which EtOH could affect activity in the VTA
reward center of the brain, via recently discovered direct
excitatory connections (Carta etal. 2019).
Further studies suggest that the striking geneticallydetermined correlation between GC tonic GABAAR current
response to EtOH and consumption phenotype plays a causative role. Specically, the GABAAR agonist THIP, which
has an order of magnitude higher afnity for δ-subunit containing GABAARs, was focally injected into the cerebellum
of B6J mice in vivo. This would effectively increase GC
tonic GABAAR currents similar to what EtOH does in low
EtOH- consuming genotypes (Kaplan et al. 2013). Such
injections effectively reduced EtOH consumption by normally high EtOH-consuming B6J mice (Fig.68.3g), without
affecting water consumption or overall locomotion (Kaplan
et al. 2016a). While more selective manipulations of the
tonic GABAAR current will need to be tested, and ideally it
should be determined if EtOH consumption can be increased
or decreased based on the direction of manipulation, these
studies suggest that either EtOH-induced suppression of GC
tonic GABAAR currents promotes EtOH consumption, or
EtOH-induced enhancement of GC tonic GABAAR currents
deters consumption or both.
The molecular mechanisms that mediate the response
polarity of GC tonic GABAAR currents to EtOH have been
partially determined. Specically, enhancement of tonic
GABAAR currents is mediated by EtOH inhibition of neuronal nitric oxide synthase (nNOS), which excites Golgi cells
enough to increase their action potential ring (Kaplan etal.
2013). Genetic control of this process appears to be imple-
mented by expression of nNOS, with low levels of expression in high EtOH-consuming B6J mice and PVs, high levels
of expression in low EtOH-consuming SDRs and D2 mice,
and intermediate and variable levels of expression across
individual non-human primates (NHPs) which also show
variable levels of EtOH consumption (Kaplan et al. 2013,
2016b; Mohr etal. 2013). Conversely, the suppression of GC
tonic GABAAR currents observed in B6J mice and PVs is
mediated solely by postsynaptic actions on the GABAARs,
which is genetically determined by the level of postsynaptic
PKC activity. In particular, PKC activity appears to prevent
EtOH from suppressing GC tonic GABAAR currents (Kaplan
etal. 2013).
Collectively, the data indicate that there are two genetically controlled molecular switches (presynaptic nNOS
expression and postsynaptic PKC activity), and that the balance of the two processes dictates the polarity and magnitude
of the effect of EtOH on GC tonic GABAAR currents. High
post synaptic PKC activity and high nNOS expression results
in EtOH enhancing GC tonic GABAAR currents, and low
post synaptic PKC activity and low nNOS expression results
in EtOH suppressing tonic GABAAR currents. Importantly,
the resultant direction of EtOH effects correlates with and
inuences EtOH consumption phenotype, wherein suppression and enhancement are associated with high and low
EtOH consumption, respectively.
68.4 Summary
The cerebellum is uniquely sensitive to EtOH, responding
physiologically and behaviorally to concentrations of EtOH
achieved by an adult consuming 1–2 units of alcohol
(10mM), and having nearly its entire cortical circuit affected
by higher, but survivable, concentrations (<50mM). Through
such responses, the cerebellum contributes to many of the
behavioral/psychological responses to recreational and abusive consumption of EtOH, ranging from motor incoordination to cognitive and rewarding aspects of EtOH. Its
heightened sensitivity also makes it susceptible to damage
by chronic excessive alcohol consumption by humans with
AUD, and upon fetal exposure, thereby contributing to
symptoms of FASD. Finally, genetic variation in cerebellar
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anatomy, physiology and divergent responsivity to EtOH
likely contribute to the risk for developing AUD, which may
be further exacerbated by EtOH-induced cerebellar damage
which reduces cognitive skills critical for abstaining from
ongoing abusive EtOH consumption.
Acknowledgments This work was supported by the National Institute
on Alcohol Abuse and Alcoholism Grants R01AA-012439 and
R01AA- 026078, and Washington State University Alcohol and Drug
Abuse Research Program (ADARP) grants to D.J.R.
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Moonwalker Mouse
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MohamedF.Ibrahim andEstherB.E.Becker
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Abstract
The Moonwalker (Mwk) mouse is a dominant ataxic
mouse model of inherited cerebellar ataxia caused by a
gain-of-function mutation in the gene encoding the
TRPC3 cation channel. Mwk mice display overt ataxia,
impaired Purkinje cell development, altered Purkinje cell
excitability and loss of TRPC3-expressing neurons in the
cerebellum. Recently, dysregulated mGluR1-TRPC3 signalling has been implicated in multiple human spinocerebellar ataxias. Here, we discuss the behavioural,
morphological, and functional changes in Mwk mice with
an emphasis on their relevance to the human spinocerebellar ataxias.
Keywords
Spinocerebellar ataxia · Cerebellum · Purkinje Cell ·
TRPC3 · mGluR1 · IP3R1 · Calcium signalling
69.1 Introduction
The Moonwalker (Mwk) mouse is a dominant ataxic mouse
model caused by a gain-of-function mutation in the transient receptor potential channel 3 (TRPC3) and was rst
described by Becker and colleagues (Becker etal. 2009).
The mutant is named after its shufing gait with retropulsion and displays decits in motor coordination and balance. Notably, a functionally similar gain-of-function
mutation in the human TRPC3 gene underlies spinocerebellar ataxia type 41 (SCA41) (Fogel etal. 2015). TRPC3
and its upstream activator, the metabotropic glutamate
receptor type 1 (mGluR1), are critical for normal Purkinje
M. F. Ibrahim · E. B. E. Becker (*)
Kavli Institute for Nanoscience Discovery, Nufeld Department of
Clinical Neurosciences, University of Oxford, Oxford, UK
e-mail: mohamed.ibrahim@ndcn.ox.ac.uk;
esther.becker@ndcn.ox.ac.uk
cell (PC) development and function (Hartmann etal. 2008).
Interestingly, abnormal mGluR1-TRPC3 signalling is
observed in several pre-clinical ataxic mouse models
(Power etal. 2016a; Kano and Watanabe 2017), suggesting
a common disease mechanism. This makes the Mwk mouse
a valuable model to gain insights into the pathogenic
mGluR1-TRPC3 signalling mechanisms underlying multiple genetic subtypes of cerebellar ataxia. This chapter provides an overview of the behavioural, cellular, and
functional changes in the Mwk cerebellum and discusses
the relevance of TRPC3 dysfunction for human spinocerebellar ataxia.
69.2 Mwk Mice Harbour aMutation
intheTRPC3 Channel
The Mwk mouse was generated through a large-scale
N-ethyl-N-nitrosourea (ENU) mutagenesis screen and harbours a single non-synonymous point mutation (A-to-G) in
exon 7 of the Trpc3 gene, resulting in a threonine-to-ala-
nine amino acid change (T561A; UniProtKB entry
Q9QZC1) in the TRPC3 channel (Becker et al. 2009)
(Fig. 69.1). TRPC3 belongs to the canonical subfamily
(TRPC1–7) of the large transient receptor (TRP) superfamily. TRPCs are calcium- permeable, non-selective cation
channels that are expressed in multiple cell types including
neurons and are involved in numerous physiological functions (Ramsey etal. 2006). TRPC3, TRPC6, and TRPC7
are unique in being activated by the lipid second messenger
diacylglycerol (DAG).
Structurally, TRPC channels resemble voltage-gated
potassium channels and possess similar functional domains
including a transmembrane domain (TMD) comprising six
transmembrane helices (S1–S6) in between the N-terminal
domain (NTD) containing four ankyrin repeats and the
coiled C-terminal domain (CTD) (Fig. 69.1). The poreforming domain between S5 and S6 is conserved across the
TRPC family (Fan etal. 2018; Tang etal. 2018). Compared
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
D. L. Gruol et al. (eds.), Essentials of Cerebellum and Cerebellar Disorders, https://doi.org/10.1007/978-3-031-15070-8_69
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Fig. 69.1 Schematic
representation of the domain
structure and topology of
TRPC3. Disease-causing
gain-of-function point
mutations in the S4–S5 linker
region (Mwk) and the TRP
domain (SCA41) are
indicated. Mwk Moonwalker
mouse; NTD N-terminal
domain; CTD C-terminal
domain
M. F. Ibrahim and E. B. E. Becker
to other TRP channels, TRPC3 displays several unique features including an unusually long S3 and two lipid-binding
sites in key regions implicated in TRPC3 channel gating;
one in the pre-S1 domain and the other in the S4–S5 linker
region (Fan etal. 2018). In addition, in TRPC3 the highly
conserved TRP domain is arranged at a right angle to S6in
contrast to the obtuse angles seen in other TRP channels.
This arrangement brings the TRP domain closer to both the
lipid-binding site in pre-S1 and the S4–S5 linker region,
suggesting a unique gating mechanism in TRPC3 (Fan
et al. 2018). Interestingly, both the Mwk and the human
SCA41 mutation are found within this key region of TRPC3
and result in an increased channel function; the Mwk mutation lies within the cytoplasmic S4–S5 linker region of
TRPC3 (Becker etal. 2009), whereas the SCA41 mutation
resides within the TRP domain (Fogel et al. 2015)
(Fig.69.1).
TRPC3 is strongly expressed in the nervous system
with the highest expression of TRPC3 in the cerebellum
(Hartmann etal. 2008; Wu etal. 2019). Within the cerebellum, TRPC3 is mainly found in the somatodendritic compartment of PCs (Hartmann etal. 2008), with an expression
pattern somewhat complementary to zebrin, where the
highest expression of TRPC3 is found in the faster ring,
zebrin- negative (Z-) PCs (Wu etal. 2019). TRPC3 is also
expressed in mGluR1a-expressing excitatory type II unipolar brush cells (UBCs), which are mainly located in lobules IX and X (Sekerková etal. 2013). Compared to other
brain regions, the predominant TRPC3 isoform in the cerebellum is the short isoform TRPC3c, which has an
increased channel conductance (Kim et al. 2012;
Cederholm etal. 2019).
69.3 Mwk Mice Display Overt Cerebellar
Ataxia
Mwk mutants have growth decits but a normal life span
(Becker etal. 2009). Mutant mice exhibit a complex behavioural phenotype including both neurological and nonneurological symptoms. Consistent with the importance of
TRPC3 for cerebellar functioning, the main neurological
feature in the Mwk mouse is cerebellar ataxia, starting as
early as 3 weeks of age (Becker et al. 2009). Specically,
Mwk mice display a wide shufing gait with a nonuniform
alternate left-right step pattern. Placed on a smooth surface,
mutants often walk backwards (retropulsion), an observation
that led to the naming of the mutant. Furthermore, Mwk
mutants are greatly impaired in their ability to maintain balance in the static rod test (Becker etal. 2009) (Fig. 69.3a).
Mwk mice also display other neurological features including
decreased pre-pulse inhibition and increased startle reex,
and non-neurological phenotypes such as corneal vascularization and abnormalities in metabolism that include altered
triglyceride, cholesterol, iron, and glucose levels (Becker
2020). The results of extensive phenotyping of the Mwk
mouse are accessible via the International Mouse Phenotyping
Consortium Web Portal (www.mousephenotype.org).
69.4 Loss ofTRPC3-Expressing Neurons
intheMwk Cerebellum
In the Mwk cerebellum, a slow but progressive loss of PCs
is observed, starting at 4months of age. The death of PCs is
more evident in the anterior cerebellum of the vermis and
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pronounced in the lateral hemispheres (Becker etal. 2009),
consistent with TRPC3’s expression pattern. Notably, anterior cerebellar atrophy is also observed in the SCA41
patient cerebellum (Fogel etal. 2015). In addition to the
death of PCs, type II UBCs are almost completely lost in
the Mwk cerebellum by 1month of age (Sekerková etal.
2013) (Fig.69.3b). It is conceivable that cerebellar neurons
might be particularly vulnerable to gain-of-function mutations in TRPC3 as they predominantly express the shorter
and more active TRPC3c isoform (Kim et al. 2012;
Cederholm et al. 2019). As a consequence of increased
TRPC3 function, excessive calcium load is likely to cause
neuronal death (Fig. 69.2). Indeed, biochemical evidence
for activated calcium signalling was found in the Mwk cerebellum (Dulneva et al. 2015) and in cell-based assays
investigating the functional consequences of TRPC3 mutations (Fogel etal. 2015; Hanson etal. 2015). Death by calcium overload might also explain the differences in the
timing of PC versus UBC loss. The particularly high calcium buffering capacity of PCs (Fierro and Llano 1996)
due to the presence of high concentrations of calcium-binding proteins likely renders these neurons more resilient to
TRPC3-mediated calcium overload compared to type II
UBCs (Sekerková etal. 2013).
Fig. 69.2 mGluR1-TRPC3 signalling is key to Purkinje cell function
and altered in cerebellar ataxia. Left: mGluR1 activation induces slow
excitatory postsynaptic currents (sEPSC) mediated by TRPC3 and activates phospholipase C (PLC) by coupling to Gα protein and produces
inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 binds to
IP3R1-gated endoplasmic receptor (ER) calcium stores resulting in Ca2+
release. In addition, mGluR1 contributes to PC intracellular Ca2+ rise by
interacting with CaV2.1 (P/Q-type) and CaV3.1 (T-type) voltage-gated
calcium channels. Protein kinase γ (PKCγ) is activated both by DAG
and Ca2+ and inhibits TRPC3. Human mutations in the genes encoding
mGluR1 and downstream signalling result in spinocerebellar ataxia
(SCA) types 44, 41, 14, and 15 and 29. Right: The Mwk gain-offunction mutation in TRPC3 alters TRPC3 gating resulting in enhanced
sEPSCs and disrupted PC ring. Mutant TRPC3 also causes excess calcium inux affecting cell function and ultimately leading to cell death.
Both abnormal PC ring and cell death contribute to cerebellar ataxia
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