International Journal of

Molecular Sciences

Review
The Optogenetic Revolution in
Cerebellar Investigations
Francesca Prestori 1 , Ileana Montagna 1 , Egidio D’Angelo 1,2 and Lisa Mapelli 1, *
1 Department of Brain and Behavioral Sciences, University of Pavia, 27100 Pavia, Italy;
[email protected] (F.P.); [email protected] (I.M.); [email protected] (E.D.)
2 IRCCS Mondino Foundation, 27100 Pavia, Italy
* Correspondence: [email protected]




Received: 16 March 2020; Accepted: 1 April 2020; Published: 3 April 2020


Abstract: The cerebellum is most renowned for its role in sensorimotor control and coordination, but a
growing number of anatomical and physiological studies are demonstrating its deep involvement
in cognitive and emotional functions. Recently, the development and refinement of optogenetic
techniques boosted research in the cerebellar field and, impressively, revolutionized the methodological
approach and endowed the investigations with entirely new capabilities. This translated into a
significant improvement in the data acquired for sensorimotor tests, allowing one to correlate
single-cell activity with motor behavior to the extent of determining the role of single neuronal types
and single connection pathways in controlling precise aspects of movement kinematics. These levels of
specificity in correlating neuronal activity to behavior could not be achieved in the past, when electrical
and pharmacological stimulations were the only available experimental tools. The application of
optogenetics to the investigation of the cerebellar role in higher-order and cognitive functions, which
involves a high degree of connectivity with multiple brain areas, has been even more significant. It is
possible that, in this field, optogenetics has changed the game, and the number of investigations
using optogenetics to study the cerebellar role in non-sensorimotor functions in awake animals is
growing. The main issues addressed by these studies are the cerebellar role in epilepsy (through
connections to the hippocampus and the temporal lobe), schizophrenia and cognition, working
memory for decision making, and social behavior. It is also worth noting that optogenetics opened a
new perspective for cerebellar neurostimulation in patients (e.g., for epilepsy treatment and stroke
rehabilitation), promising unprecedented specificity in the targeted pathways that could be either
activated or inhibited.

Keywords: cerebellum; optogenetics; sensorimotor system; non-sensorimotor functions

1. Introduction
The first reports on cerebellar structure and the first hypothesis on function pointed out its high
degree of connectivity with the rest of the brain and the possible relationship with higher-order
functions [1]. However, since the nineteenth century, with the work of Flourens (who provided the first
descriptions of the motor syndrome now called ataxia [2]), the cerebellum has been investigated for its
role in motor functions, while the role of its extensive connectivity to other brain areas was overlooked.
The shift in the cerebellar paradigm is only relatively recent and can be traced back to Schmahmann’s
first reports of the Cerebellar Cognitive Affective Syndrome ([3] and the introduction of the Dysmetria
of Thought concept in 1998 [4]. Since then, several studies have reported cerebellar abnormalities
or lesions at the core of cognitive dysfunctions [5–7] as well as higher order function impairments
associated with motor syndromes involving the cerebellum [8,9]. While the research on the cerebellar
role in sensorimotor integration can rely on a well-defined anatomy and easily measurable motor

Int. J. Mol. Sci. 2020, 21, 2494; doi:10.3390/ijms21072494 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 2494 2 of 24

outputs, investigating the cerebellar role in non-sensorimotor functions proved challenging. To find
the role of the cerebellum in these cases, one needs to take into account cerebellar connectivity with
other brain regions, such as the hippocampus or the neocortex, that are not usually direct. Moreover,
while considering cognitive functions, the involvement of brain cortical associative areas makes the
identification of the specific role of the cerebellum hard. Over the last decade, the revolution of technical
approaches in neurophysiological investigations has provided new tools and granted unprecedented
resolution in determining the effects of neuronal types and single pathways in complex behaviors.
In particular, optogenetics proved to be a game-changing tool that neuroscience needed in order to
achieve stimulation specificity, in vitro and in vivo, that was not foreseeable only twenty years ago.
This technical revolution affected cerebellar research too, allowing huge steps in understanding the
cerebellar contribution to both motor and non-motor functions. This review will focus on the use of
optogenetics in the cerebellum to analyze behavior, to different extents. Concerning the section about
the role of cerebellum in the sensorimotor system, the focus will be on the unprecedented level of
detail that optogenetics allow to achieve in terms of specific neuronal types or pathways involved in
precise aspects of movement. The non-sensorimotor section will summarize the main findings of the
last decade, where the use of optogenetics allowed us to tackle the role of cerebellum in functions
and behaviors usually associated with other brain areas. Importantly, this optogenetic revolution in
cerebellar investigations might be key in achieving cerebellar stimulation (or inhibition) as a clinical
treatment for a broad spectrum of disorders.
In this review, we will provide a focused and critical summary of recent advances in cerebellar
optogenetics and its applications to study neuronal circuits in physiological or pathological conditions,
highlighting the advantages of photostimulation techniques, as well as emerging questions and
future perspectives.

2. Brief Overview of the Cerebellar Anatomy and Microcircuits Organization

The cerebellum is part of the hindbrain, located above the brainstem, and is characterized by two
lateral hemispheres and a medial region called the vermis. The whole cerebellum is organized in the
deep cerebellar nuclei (DCN), enclosed in the white matter, and in the three layers characterizing the
cerebellar cortex. Throughout the whole cortex, the network organization is repeated, with very few
differences among regions. In essence, the first set of inputs is conveyed by the mossy fibers, making
excitatory synaptic contacts with granule cells and Golgi cells in the granular layer (the inner cortical
layer of the cerebellum). Granule cells are excitatory neurons that make synaptic contacts (through the
ascending axon or the parallel fibers) with the Golgi cells, molecular layer interneurons, and Purkinje
cell (PC) dendrites (in the molecular layer, the external layer of the cerebellar cortex; in between,
PCs soma originate the Purkinje cell layer). Interestingly, granule cells are the only excitatory neurons
in the cerebellar cortex (except for unipolar brush cells in specific regions), giving rise to an inhibitory
organization in feedback and feedforward loops that endow the cortical processing with peculiar
features (for a detailed review of this topic see [10–12]). Another set of inputs comes from the climbing
fibers, originating in the inferior olive. Climbing fibers directly contact PCs, which provide the sole
output of the cerebellar cortex. PCs axons make inhibitory synaptic contacts with DCN neurons.
The DCN are composed of three sets of nuclei (from medial to lateral): the fastigial, the interpositus
(that includes the globose and emboliform nuclei in primates), and the dentate nuclei. Both mossy and
climbing fibers send collaterals to the DCN before entering the cerebellar cortex. Therefore, the DCN
can compare and integrate the input signals conveyed to the cerebellum and the result of cortical
processing of those same inputs. Several studies report that this anatomical organization of cerebellar
microcircuits can be divided into modules, operating in loops with the regions of origin of their inputs,
being the inferior olive or the rest of the brain [13]. The cerebellar role in both sensorimotor and
non-sensorimotor functions likely relies on the brain regions involved in these loops. This is also
evident from the recent advances in cerebellar development studies, though this topic is beyond the
scope of this review (see for details on this subject [14,15]).
Int. J. Mol. Sci. 2020, 21, 2494 3 of 24

3. Pros and Cons of Optogenetics

The obvious advantage of optogenetics is the possibility to modify neuronal activity in a cell-specific
or region-specific manner. This is usually achieved using promoters for proteins expressed in specific
neuronal subtypes or by localizing the viral injection to confined areas. The second main advantage is
the possibility to activate or inhibit the target neurons. Previously, neuronal inhibition was achieved
by local lesions, pharmacological tools, or decreasing the temperature in the brain region of interest.
All of these approaches have evident disadvantages. Electrical lesions are usually not precisely
localized and are not reversible. Optogenetic inhibition of neuronal activity can be achieved with
millisecond precision and brief duration, if needed. The pharmacological tool lacks temporal precision,
and the reversibility might not be complete. The drop in local temperature is usually achieved by
perfusing cold solutions, lacking both temporal and spatial precisions. The high spatial and temporal
resolutions of optogenetic modulation of neuronal activity, together with the specificity of cell types
and pathways, led to a definitive advance in the study of neuronal activity and connectivity on the
basis of complex behaviors and pathological conditions. This was even more evident in the last few
years, where technological improvements made miniaturized devices available, allowing us to modify
the activity of selected neuronal types and specific connections during a wide range of behaviors and
behavioral tasks, providing causal relationships between the optogenetically targeted neurons and the
observed behavior.
Though the pros of optogenetics are impressive, some technical issues need to be taken into
account. First of all, the need to inject viral constructs when genetically modified animals are not
available. Different viral batches may come with different titer and, in every case, the dilution and
the volume of the injections must be calibrated each time. This is true even when using the same
batch but changing injection location, since different brain areas show different infection levels with
the same construct. Moreover, the specificity of the cell type is allowed only where the target cells
have unique markers, not common to other cells in the surrounding areas. This condition is not
always achievable. Finally, the amount of opsins expressed on neuronal membranes might differ from
trial to trial, making the net effect of light-driven neuronal activation/inhibition difficult to reproduce
consistently. Nevertheless, the pros of optogenetics use in neuroscience mostly overcomes the cons,
and its unique features need to be taken into account when designing experiments and interpreting
results. Before proceeding, another critical issue is worth pointing out. Optogenetics is indeed a
revolutionary method to excite or inhibit neurons, since it involves the opening of ion channels on
their membranes, generating ion fluxes to modify membrane voltage. This condition is very similar to
the physiological processes involved during neuronal activity, but it is essential to keep in mind that
optogenetic stimulation is very different from the physiological condition. It is not possible to have
control over the amount of currents induced in a single neuron or to affect only those neurons that are
physiologically activated together by a common pathway. Every neuron expressing the opsins will
react when illuminated, therefore activating or inhibiting entire regions. This condition is quantitatively
different from physiological activation in terms of current amplitude in single neurons and the number
of neurons affected. Though this specification was necessary, a stimulation method acting directly on
neurons and mimicking the exact physiological activation is not available at the moment.
Indeed, optogenetics has moved cerebellar research ahead by allowing us to disentangle
intertwined pathways at a spatiotemporal precision unmatched by other techniques. In particular,
optogenetic tools allowed for (1) the genetic specificity and anatomical strategies controlling the
electrical activity of selected cerebellar neurons, (2) the targeting of projections between cerebellum
and other brain regions by delivering light to opsin-expressing axons, (3) the link to data obtained from
in vitro/in vivo electrophysiological recordings and targeted cerebellar neurons during behavioral
tasks and (4) closed-loop interventions in which optical cerebellar stimulation is guided by real-time
readouts of ongoing activity [16].
In the following sections, the use of optogenetic tools to dissect the cerebellar role in behavior is
divided for investigations of the sensorimotor and non-sensorimotor functions.
Int. J. Mol. Sci. 2020, 21, 2494 4 of 24

4. Sensorimotor Functions
The renowned function of the cerebellum is the integration of sensorimotor information. Despite
the first reports on this topic dating back almost two centuries ago, the underlying physiological
mechanisms remain incompletely defined still at the neuronal and microcircuit levels. The recent
development of optogenetics boosted neurophysiological research, providing a suitable tool to
investigate the impact of specific neurons or pathways on behavior. Indeed, optogenetics is now
primarily employed to stimulate specific components of the cerebellar circuit in order to investigate
the cerebellar contribution to perception and motor control. The cerebellum is involved in loops
with the cerebral cortex, including motor and sensory areas. Inputs coming to the cerebellum send
collaterals to the DCN before entering the cerebellar cortex. The DCN are in the position to integrate
the “raw” signal sent to the cerebellum with the results of the cortical processing of the same input.
In turn, the DCN convey the integrated signal back to the brain regions of origin. These sensorimotor
cortico-cerebellar loops play a crucial role in the fine control of voluntary movements [17,18].
The following sections briefly summarize the main findings made possible by optogenetics in the
investigation of the cerebellar role in sensorimotor control and learning.

4.1. Sensorimotor Integration and Voluntary Movement

Cerebellar contribution to sensorimotor integration is particularly evident in the rodent whisker
system. Cortical sensory and motor information converge at the cellular level in the cerebellar cortex
(as the lateral part of Crus I), which forms a closed functional loop with the whisker motor cortex. Indeed,
optogenetic stimulation of this cortical area in awake CD1-L7-ChR2-YFP mice resulted in alterations
of ongoing movements and touch events against surrounding objects [17]. In particular, selective
photostimulation of PCs of the crus I area receiving motor inputs produced inhibition of the DCN that
resulted in a post-inhibitory activation of the cerebellar-thalamo-cortical pathway, yielding motor cortex
activation. Lesions at the cerebro-cerebellar loop level in rodents impaired whisking-linked behavior,
though not affecting the ability to move the whiskers and touch surrounding objects [17]. Interestingly,
the same study reported that optogenetic perturbation of cerebellar processing led to suppression of
coherent activities in the gamma band between motor and sensory cortexes. Investigating cerebellar
integration of sensory and motor information might, therefore, be essential not only for the study of
movement control, but also for a deeper understanding of the sensory process itself.

4.2. Associative Learning (Eyeblink Conditioning)

The cerebellum is known to be involved in eyeblink conditioning (EBC), which is often used in
investigations involving the cerebellum in physiological and pathological conditions. In EBC, a neutral
conditioned stimulus (CS, either visual or auditory) anticipates an aversive unconditioned stimulus
(US), usually an air puff delivered to the eye or a peri-orbital shock, see [19,20], that causes the animal
to close an eyelid out of protection. After multiple presentations of CS and US with the same temporal
pattern, the animals learn to associate the CS with the following US, resulting in the closing of the
eyelid when the CS (neutral stimulus) is delivered, anticipating the aversive stimulation (US). CS and
US signals are conveyed to the cerebellum through distinct input pathways, the mossy fibers and the
climbing fibers, respectively [21]. Both these inputs project to the cerebellar cortex and send collaterals
to the DCN, where learning is thought to take place [19,21,22]. So far, investigations on EBC mechanisms
have focused mainly on the role of the cerebellar cortex and, in particular, of long-term depression and
long-term potentiation at the fiber parallel to the PC synapse [23–28], intrinsic plasticity of PCs [25,29],
and their dependence on the external feedback provided by the climbing fibers [30,31]. Indeed, it has
been shown that electrical stimulation of mossy fibers is able to substitute the sensory CS to drive
eyelid conditioning in rabbits [32]. Optogenetic stimulation has the advantage of avoiding tissue
damage provoked by current injection and allows for the precise activation of neuronal subpopulations
or specific pathways. For these reasons, optogenetics was applied in several studies investigating
Int. J. Mol. Sci. 2020, 21, 2494 5 of 24

cerebellar role in associative learning. A recent study extensively applied this technique in order
to investigate the impact of behavioral states on associative learning, the pathways and neuronal
types involved [33]. The CS was mimicked as optogenetic activation of cerebellar mossy fibers, using
Thy1-ChR2/EYFP transgenic mice expressing channelrhodopsin 2 (ChR2) in cerebellar mossy fibers
(MF-ChR2 mice). In particular, the authors show that locomotion is able to enhance associative
learning (using delay EBC as a model), exploiting optogenetics to selectively activate mossy fibers
terminals (mimicking the CS) in a specific region of the cerebellar cortex involved in eyelid movements.
Their results showed that locomotor contribution to eyelid closure during the task is likely to rely
on mechanisms involving the mossy fiber pathway (CS) and downstream. Optogenetics was further
used to address the contribution of cerebellar neuronal types, showing that the processing of the input
downstream of the mossy fibers is deeply involved in the associative learning process and that the
different neuronal types impact differently on the timing of the response (with milliseconds precision).
It is evident that optogenetics was key to obtain this kind of factorization of the process underlying
associative learning in the cerebellum.
The cerebellar circuit underlying EBC mechanisms was further characterized, addressing the role
of the internal feedback between the DCN and the cerebellar cortex within the same modules [34].
Projection neurons in the DCN send excitatory fibers that enter the granular layer to originate the
so-called nucleocortical mossy fibers. The terminals of these fibers make direct synaptic contact
with granule cells and Golgi cells, and provide indirect inhibition to PCs through the parallel
fibers—molecular layer interneurons pathway. Gao and colleagues obtained optogenetic control of
nucleocortical mossy fibers activity by injecting the AAV-hSyn-ChR2-eYFP in the interposed nuclei
(Figure 1). At the same time, this procedure allowed them to label mossy fibers rosettes in the
granular layer originating from nucleocortical projections. Therefore, an optical fiber implanted in
the cerebellar cortex of mice used for EBC tests was sufficient to modulate nucleocortical mossy
fiber activity, while the YFP labelling was used to unravel structural modifications of these terminals
after learning. This protocol allowed them to characterize cortical activity during the behavioral
test (using extracellular recordings mainly from PCs) and the synaptic strength of the connections to
granule cells and Golgi cells (using patch-clamp recordings in vitro). These complex experimental
settings allowed them to unravel a specific role of nucleocortical projections during EBC in enhancing
PCs inhibition, most likely preferentially activating granule cells, in which parallel fibers activate
molecular layer interneurons more than PCs (Figure 1B). In untrained animals, the optogenetic
activation of nucleocortical mossy fibers did not induce the conditioned eyeblink response, while in
mice where associative learning already took place, optogenetics was able to modify the strength and
the latency of the response. The authors concluded that the nucleocortical pathway acts as a gain
amplifier in the learned EBC response, while it cannot be sufficient to generate this form of associative
learning. These observations might be generalized, suggesting the role of nucleocortical cerebellar
connections to be components of an internal amplification loop that might be involved in mechanisms
underlying associative motor learning [30,31,35]. Here, optogenetics proved to be indispensable to
select nucleocortical fibers among the mossy fiber bundle that enters the granular layer. Electrical
stimulation of the interposed nuclei could have achieved a similar effect in the cortex, but it would
have activated all the downstream connections outside the cerebellum (a condition that is clearly
detrimental when working with awake animals).
Int. J. Mol. Sci. 2020, 21, 2494 6 of 24
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Figure
Figure1.1.Role
RoleofofExcitatory
ExcitatoryCerebellar
CerebellarNucleocortical
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ininthe
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Mossy afferents
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can directly cell (PC) activity
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GrC-MLI-PC increases
processing. The the
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in PCs, indicating a preferential
inhibitory/excitatory (I/E) ratio in enhancement of theafeedforward
PCs, indicating preferentialinhibitory
enhancement GrC-MLI-PC pathway.
of the feedforward
inhibitory GrC-MLI-PC pathway.
Recently, a new approach to EBC studies combined optogenetics and pharmacological tools in
awake and freely
Recently, moving
a new rats [36].
approach Again,
to EBC the CS
studies was substituted
combined withand
optogenetics the pharmacological
optogenetic activation of
tools in
mossy fibers
awake (in this
and freely case, following
moving rats [36]. the injection
Again, of was
the CS the construct
substitutedpAAVwith2/9-hSyn-ChR2-mCherry
the optogenetic activationin the
of
pontine nuclei). The pharmacological approach was used to block both excitatory and
mossy fibers (in this case, following the injection of the construct pAAV 2/9-hSyn-ChR2-mCherry in inhibitory inputs
to the
the pontine
pontine nuclei,
nuclei). in order
The to “isolate”approach
pharmacological the learning
wasprocess
used toinblock
the cerebellum. These
both excitatory experiments
and inhibitory
showedtothat
inputs thethe cerebellum
pontine in itself
nuclei, is sufficient
in order to generate
to “isolate” associative
the learning learning
process in thein cerebellum.
the form of simple
These
EBC, though extra-cerebellar inputs are required to facilitate such learning [36].
experiments showed that the cerebellum in itself is sufficient to generate associative learning in the
form of simple EBC, though extra-cerebellar inputs are required to facilitate such learning [36].
4.3. Eye Movements in Monkeys
4.3. EyeTheMovements
efficacy of in Monkeys stimulation in rodents opened the possibility of a potential application
optogenetic
of this technique
The efficacyinofa wide variety stimulation
optogenetic of animal models. In monkeys,
in rodents openedPCs the are known toofplay
possibility a crucial
a potential
role in the execution of accurate movements, although their impact is still poorly
application of this technique in a wide variety of animal models. In monkeys, PCs are known to understood, due to
play
athecrucial
lack ofrole
techniques for selective
in the execution of manipulation of their spiking
accurate movements, althoughactivity. A recentisinvestigation
their impact still poorly
applied optogenetics to modify PC firing, using an AAV-L7-ChR2 construct
understood, due to the lack of techniques for selective manipulation of their spiking that isactivity.
specificAfor this
recent
neuronal type,applied
investigation in rhesus macaquesto
optogenetics [37]. The efficacy
modify PC firing,of using
the expression in this animal
an AAV-L7-ChR2 model
construct was
that is
properly confirmed using immunohistochemical analysis and neurophysiological recordings.
specific for this neuronal type, in rhesus macaques [37]. The efficacy of the expression in this animal To test
the impact
model wasofproperly
photoactivation of PCs
confirmed on behavior,
using an optical fiber analysis
immunohistochemical was implanted in the oculomotor
and neurophysiological
vermis to deliver
recordings. To testlight pulses (453
the impact nm) after saccade
of photoactivation initiation
of PCs on randomly
on behavior, an opticalinterleaved
fiber wastrials (50%).
implanted
in the oculomotor vermis to deliver light pulses (453 nm) after saccade initiation on randomly
interleaved trials (50%). Interestingly, the optical stimulation failed to evoke saccades, although it
Int. J. Mol. Sci. 2020, 21, 2494 7 of 24

Interestingly, the optical stimulation failed to evoke saccades, although it was observed to provoke
saccade dysmetria. This observation confirmed, for the first time, the feasibility of genetic manipulation
techniques in primates. It is worth noting that previous studies reported the ability to evoke saccades
in primates using electrical stimulation to activate PCs in the oculomotor vermis [38–41], showing that
the effects of electrical stimulation are stronger than optical ones, at least on primate behavior [42–46].
This might be explained by considering that optical stimulation exerts its effects on nearby neurons [47],
which is specific for PCs, and ChR2-expressing axons generate only low frequency spiking when
activated [48,49]. Nevertheless, optogenetics proved to be a valuable tool for studies in monkeys,
not only in rodents. Indeed, an even more recent study used a similar approach to investigate the
role of PC firing irregularity in cerebellar functions of rhesus monkeys [50]. It has been suggested,
both for mice and monkeys, that the irregularity of interspike intervals in PCs might play a role in
the information transfer from the cerebellar cortex to DCN. Payne and colleagues used oculomotor
behavior to test this hypothesis. Interestingly, while mean PCs firing varied accordingly to mean
eye velocity, a strong correlation between these terms was not found observing moment-to-moment
variations, which was therefore not related to spike irregularity. The optogenetic stimulation of PCs
was able to independently control spike rate and irregularity, eliciting eye movements related to a linear
rate code with 3–5ms temporal precision. These results are remarkable and show that the cerebellum
exerts its control on movements using a firing code that works at impressively high rates, while a
possible role of spike irregularity was not evident.

4.4. Movement Kinematics

The study of the role of specific neuronal types and pathways in the kinematics of movement
execution is likely one of the fields that benefits more from the use of optogenetic tools. For example,
it was recently shown that the transient suppression of spontaneous activity in a population of PCs
was able to control movement kinematics in terms of modulation of size, speed, and timing [51].
The suppression of PC firing acts as a powerful mechanism that alters the precise control of movement,
presumably due to disinhibition of target neurons in the DCN. Before the development of optogenetics,
the precise and selective inactivation of PCs on behaviorally relevant timescales in vivo was impossible.
In particular, in this study, the PC inhibition was achieved by activation of molecular layer interneurons
(both stellate and basket cells), using a transgenic mouse line that expresses ChR2 in 85% of molecular
layer interneurons, called nNos-BAC mice [52]. The transient suppression of PC activity in the eyeblink
microzone resulted in the closure of the ipsilateral eyelid. In contrast, the same stimulation pattern in
nearby locations of paravermal lobules IV-VI generated other orofacial movements such as mouth
opening, cheek/lip lifting, and forward thrusting of the vibrissa. A change in the duration of the
light pulse caused a corresponding change in the duration of eyelid closure, without affecting the
blink speed. Both the intensity and the latency of the light pulse influenced the time needed to
obtain the complete eyelid closure. These findings confirm previous observations that indicate the
cerebellar cortex as playing a crucial role for adaptively controlling the timing of classically conditioned
eyelid movements [19,53,54]. DCN neurons receiving inhibitory inputs from the PCs of the same
module coherently showed excitatory responses that were modulated by the photostimulation intensity
delivered to the cortex. Moreover, a strong linear correlation was found between the DCN neuron
firing rate and both the size and speed of eyelid movements. This relationship suggests that PC
firing and disinhibition-driven increases in DCN activity play a prominent role in the control of
movement kinematics.
Another study used optogenetics to silence molecular layer interneurons by selectively expressing
Archaerhodopsin 3.0 (Arch 3.0) using a Nos1-Cre transgenic mouse line and viral-mediated gene transfer
(rAAV2-EF1a-DIO-eArch3.0-eYFP), while recording PC activity from the soma and the dendrites [55].
This experimental setting allowed the researchers to investigate PC activity in detail during self-paced
locomotion. The optogenetic silencing of molecular layer interneurons was used to test whether the
perturbation of excitation/inhibition balance in PCs affected self-paced locomotion. Indeed, optical
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in magnitude and duration observed were similar to those generated by direct optogenetic activation
stimulation
of PCs usinginducedChr2 [56], consistent
suggesting changes in locomotorofbehavior
the involvement the samein the 70% of mice
downstream tested, leading
motor-related pathways. to a
slowdown or complete halt in locomotion. Changes in magnitude
Together, these findings suggest that the regulation of PCs excitation/inhibition balanceand duration observed were similar
to those generated
provides a sensitive by direct optogenetic
modulatory activationofof cerebellar
mechanism PCs using Chr2 output [56], suggesting
that the involvement
is necessary for correct
of the same downstream
movement performance. motor-related pathways.
Together, these findings
Another approach focusedsuggest that the regulation
the attention of PCs excitation/inhibition
on the cerebellar output, the DCN, and balance provides a
their modulation
sensitive modulatory[57].
during locomotion mechanism
Different of studies
cerebellar output
have shown thatthat
is necessary
movement, for correct
both asmovement
initiation andperformance.
learning,
Another approach focused the attention on the cerebellar
is accompanied by an increase in DCN neuron firing, either by disinhibition or direct output, the DCN, and their modulation
excitation
during locomotion
[17,51,58–64]. As [57].
well Different
as DCN studies
silencing, haveinactivation
shown that movement,
or ablation both has as initiation
been related andto learning,
prolonged is
accompanied by an increase in DCN neuron firing, either by disinhibition
reaction times or disruption of movements in behaving animals [65–70]. Sarnaik and colleagues or direct excitation [17,51,58–64].
As well as DCN
performed silencing,
extracellular inactivation
recordings from orPCsablation
and DCN has been related
neurons (in to
theprolonged
interpositus reaction
nucleus) times or
while
disruption
monitoring of hind
movements in behavinginanimals
paw movement awake,[65–70]. Sarnaik andmice
head-restrained colleagues
running performed
on a freelyextracellular
rotating
recordings from PCs and DCN neurons (in the interpositus nucleus)
treadmill (Figure 2). Optogenetics were used to modulate PC activity in mice offspring of Ai27D while monitoring hind pawx
movement in awake, head-restrained mice running on
Pcp2-cre crosses expressing ChR2 in PCs with the use of the viral constructsa freely rotating treadmill (Figure 2). Optogenetics
were used to modulate PC activity in mice offspring of Ai27D
AAV9.CBA.Flex.ChR2(H134R)-mCherry.WPRE.SV40 or x Pcp2-cre crosses expressing ChR2
AAV9.EF1a.DIO.hChR2(H134R)-
in PCs with the use of the viral constructs AAV9.CBA.Flex.ChR2(H134R)-mCherry.WPRE.SV40
eYFP.WPRE.hGH [57]. Photostimulation was used to modify the rate and temporal pattern of the or
AAV9.EF1a.DIO.hChR2(H134R)-eYFP.WPRE.hGH
inhibition of DCN neurons, at different times of[57]. Photostimulation
the stride cycle during was used to modify
locomotion on the thetreadmill.
rate and
temporal pattern of the inhibition of DCN neurons, at different times of the
The fast timing kinetics of perturbations pemitted by optogenetics was used to find clear correlations stride cycle during locomotion
on the treadmill.
between DCN activityThe fastperturbation
timing kinetics andofalterations
perturbations pemitted
of the observed by optogenetics
movements was used to find
(as “slips”). The
clear
directcorrelations
stimulation between
of PCsDCN activitymice
in awake perturbation
is then and alterations
sufficient of the observed
to provoke movements
slips during voluntary (as
“slips”). The direct stimulation of PCs in awake mice is then sufficient
locomotion, in a way that is related to DCN inhibition mediated by PCs. A correlation was found to provoke slips during voluntary
locomotion, in a way that
between locomotion is relateddisruptions
transient to DCN inhibition
and the mediated
modulationby PCs. ofADCNcorrelation wasrather
activity, found between
than the
locomotion transient disruptions and the modulation of DCN
absolute firing rate. These results suggest that the role of DCN in locomotion goes beyond activity, rather than the absolute firing
the
rate. These results suggest that the role of DCN in locomotion goes
transmission of incoming inputs to downstream regions, providing further details on how the beyond the transmission of incoming
inputs to downstream
cerebellar output actively regions, providing
regulates further details
locomotion. on how
While DCN theinactivation
cerebellar output actively disrupted
consistently regulates
locomotion. While DCN inactivation consistently disrupted ongoing
ongoing movements, dampening the DCN activity still had the effect of causing irregularities movements, dampening the DCN of
activity still had the
movements during locomotion.effect of causing irregularities of movements during locomotion.

Figure2.2.Modulation
Figure Modulationof ofPCs
PCsandandDCNDCNduring
duringvoluntary
voluntarylocomotion.
locomotion.Left,
Left,in
inPcp2-cre
Pcp2-cremice,
mice,aamixture
mixture
of
ofviruses
viruses(AAV2/9)
(AAV2/9)was
wasinjected
injectedto to
express ChR2
express ChR2with a fluorescent
with marker
a fluorescent conjugated
marker withwith
conjugated eYFPeYFP
in the
in
interpositus nucleus.
the interpositus Right, Right,
nucleus. schematics of the setup
schematics of thewith a head-fixed
setup mouse running
with a head-fixed mouse onrunning
the cylindrical
on the
treadmill.
cylindricalPaw movements
treadmill. are recordedare
Paw movements with an infrared
recorded withcamera. The patch
an infrared pipette
camera. The contained an
patch pipette
electrode wire and an optical fiber.
contained an electrode wire and an optical fiber.

4.5.
4.5. Movement
Movement Disorders
Disorders
The
Thecerebellum
cerebellumisisnotoriously
notoriouslyinvolved
involvedin inaanumber
numberof ofmotor
motordisorders,
disorders,involving
involvingalterations
alterationsofof
cerebellar
cerebellar processing or of cerebellar connections to other brain regions. Optogenetics isisevidently
processing or of cerebellar connections to other brain regions. Optogenetics evidently
helpful
helpfulininthese
thesecases
casesin
inorder
orderto tounderstand
understandthe thespecificity
specificityof
ofthe
thealterations
alterationsat
atthe
thelevel
levelof
ofneuronal
neuronal
types
typesororpathways
pathwaysinvolved.
involved.ForForexample,
example, a recent study
a recent described
study thethe
described cerebellar rolerole
cerebellar in modulating the
in modulating
activity of basal ganglia neurons in the striatum [71]. Basal ganglia and cerebellum are
the activity of basal ganglia neurons in the striatum [71]. Basal ganglia and cerebellum are brainbrain structures
fundamental for the initiation
structures fundamental and
for the correct execution
initiation and correct of execution
voluntaryofmovement
voluntary[72,73], and impairment
movement [72,73], and
in their function
impairment is responsible
in their function isforresponsible
several motorfor disorders, suchdisorders,
several motor as Parkinson’s
suchdisease and ataxia
as Parkinson’s [74].
disease
and ataxia [74]. The optogenetic activation of the cerebellum (using AAV2-Syn-ChR2(H134R)-YFP
Int. J. Mol. Sci. 2020, 21, 2494 9 of 24

The optogenetic activation of the cerebellum (using AAV2-Syn-ChR2(H134R)-YFP injected in the

dentate nucleus) proved capable of altering striatal neuron activity in half the cases of freely moving
mice [71]. This effect had a short latency (around 10ms), though it involved an indirect pathway through
the thalamus and affected cortical-driven plasticity in this region. Beyond this, Chen and colleagues
characterized the cerebellar influence on basal ganglia activity in a mouse model of cerebellar-induced
dystonia (obtained with ouabain infusion, to mimic rapid onset dystonia Parkinsonism). In this
model, cerebellar-induced dystonic postures were correlated with abnormal neuronal activity in the
dorsolateral striatum. Interestingly, the alteration in cerebellar output was transmitted through the
thalamic station (intralaminar nuclei) and altered basal ganglia activity. Silencing this thalamic nucleus
using optogenetics (AAV2/5-CAG-ArchT-GFP) alleviated the dystonic symptoms.
The cerebellar role in motor diseases involving brain circuits were recently investigated for Parkinson’s
disease, revealing a reorganization of cerebellar–cerebral circuit in a mouse model of this disease, causing
a lesion of midbrain dopaminergic neurons [75]. Interestingly, though bearing alterations at the cerebellar
level, this mouse model did not show any motor symptoms usually associated to cerebellar dysfunction,
suggesting normal coupling to descending pathways. Indeed, the alterations in the cerebellum were
described at the level of populations of PCs showing slow and irregular firing, compared to controls,
likely explaining the increased activity of DCN neurons [75]. Cerebellar stimulation elicited weaker
electrocortical responses in the motor cortex of lesioned animals, in agreement with the hypothesis
of a decrease in cerebellar modulation of motor cortex activity in Parkinson’s disease [76–78]. These
observations suggest a putative role of the cerebellum in Parkinson’s disease tremor [79,80].
Though the details of cerebellar coordination of multi-joint movements are still debated, new
clues were derived from an investigation of these mechanisms in the ataxic and dystonic mouse (the
tottering mouse). The olivo-cerebellar system is thought to play a major role, through climbing fibers
generating complex spikes in PCs. The inferior olive displays oscillatory dynamics that have been
proposed to take part in controlling the motor output [81,82]. The tottering mouse is characterized
by reduced levels of complex spikes synchronicity, delayed compared to the initiation of movement,
likely affecting the motor deficits. A recent study exploited this model and optogenetics to investigate
the role of synchronicity of PC activation in microzones [56]. This study exploits the fast kinetics
of photostimulation and the possibility to select its spatial extension. The authors reported that
synchronous activation of PC ensembles could facilitate both the initiation and coordination of
movements, showing that simple spikes in PCs play a major role. The mechanisms highlighted in this
study are likely the same as those involved in multi-joint movement coordination.
On a related note, the cerebellum was reported to play a role in post-stroke rehabilitation. This effect
is likely to be mediated by the connections from the cerebellum to various cerebral cortex areas. A recent
paper addressed this issue in a mouse model with induced photothrombotic stroke in the sensorimotor
cortex [83]. Indeed, it was reported that the stimulation of the dentate nucleus in rodents positively
affected post-stroke motor recovery [84]. Zhang and colleagues demonstrated that the effectiveness
of environmental enrichment, known to promote rehabilitation [85], is facilitated when paired with
cerebellar stimulation. To obtain the stimulation specificity, they photo-activated the DCN, previously
injected with rAAV-hsyn-hChR2-mcherry-WPRE-PA. Notably, this investigation also demonstrated that
the cerebellum is necessary to mediate the positive effects of environmental enrichment on rehabilitation,
by inactivating the DCN using optogenetics (rAAV-hsyn-eNPHR3.0-mcherry-WPRE-PA). It is likely
that the cerebellar role in post-stroke rehabilitation relies on cerebellar contribution to cortical plasticity,
an issue still debated, but sustained by accumulating evidence [86–89]. This paper is a good example
of the advancements made possible by optogenetics: obtaining stimulation precision, exploring the
possibility to evaluate the effects of activation and inhibition of the same neurons, and achieving
temporal accuracy. As the authors themselves stated, future investigations will address the role of
single cerebellar neuronal types in these mechanisms.
Int. J. Mol. Sci. 2020, 21, 2494 10 of 24

5. Non-Sensorimotor Functions
While the cerebellar role in sensorimotor functions and motor coordination is well established,
the nature of its impact on cognition and emotion remains more difficult to address. The connections
involved are usually indirect and the convergence of more inputs to associative areas makes it
incredibly difficult to detect the specific role of the cerebellum among the contributions of other brain
areas. To further complicate the picture, the behavioral counterpart to the cerebellar involvement in
non-sensorimotor functions is difficult to retrieve. The understanding of the cerebellar involvement in
non-sensorimotor functions was first prompted by the development of functional magnetic resonance
imaging (fMRI), during the past 25 years. Recent anatomical, structural and functional evidence
has revealed that cerebellar activation is associated with addiction, social cognition and emotional
processing [90–93]. Furthermore, cerebellar lesions are implicated in cognitive disorders and abnormal
social behavior such as in autism spectrum disorders (ASD), cognitive affective syndrome, schizophrenia
and epilepsy [93–100]. Notably, ASD patients report both motor dysfunctions together with non-motor
symptoms, suggesting that cerebellar impairment might indeed contribute to both [95,100,101], likely
depending on the connected brain areas [102]. In non-human primates, tract-tracing investigations
have demonstrated that topographically distinct regions (called output channels) of the DCN project to
different cortical areas: with dorsal parts sending efferent fibers to the motor cortex and ventral parts
to the prefrontal and parietal cortexes, which are generally involved in cognitive and higher-order
executive functions [103,104]. Recently, neuroimaging studies have reported a similar connectivity
topography in human DCN [105,106]. The prefrontal cortex and its extensive connections with other
cortical, subcortical and brain stem areas have extensively been investigated. These studies provided
evidence for two different pathways through which DCN communicate with the prefrontal cortex.
The main pathway involves glutamatergic neurons located in the DCN, which connect to primary
thalamic nuclei such as ventrolateral, ventromedial and, additionally, centrolateral nuclei [103,107,108].
The connectivity among the DCN and thalamic nuclei is yet to be fully characterized. It is unclear
whether the cerebellum projects to the mediodorsal thalamic nuclei, which are known to provide the
main thalamic inputs to the prefrontal cortex [109,110]. Recently, reciprocal connectivity between the
prefrontal cortex and ventral thalamic nuclei (specifically ventromedial) has been shown [109,111,112].
The second pathway involves DCN projections to the ventral tegmental area (VTA) which, in turn,
send dopaminergic fibers to the prefrontal cortex [113]. A growing body of evidence has reported
that VTA dopaminergic projections in the prefrontal cortex, in addition to influencing stress-related
function and working memory [114,115], mediate many of the higher-order cognitive functions,
including reward, motivation, attention and behavioral flexibility [116–119]. Notably, alterations in
dopaminergic neurotransmission in the prefrontal cortex has been shown in a number of patients
diagnosed with schizophrenia and autism [120–122]. Recently, it has been shown that the electrical
stimulation of DCN was able to indirectly evoke the release of dopamine in the prefrontal cortex
(dentate-reticulotegmental-peduncolopontine-VTA-prefrontal cortex pathway) [123,124], suggesting a
cerebellar contribution to reward driven behaviors.
The following sections summarize the recent findings in the main non-sensorimotor functions
addressed using optogenetics (see Table 1).
Int. J. Mol. Sci. 2020, 21, 2494 11 of 24

Table 1. Viral constructs used in optogenetic manipulation of non-motor behavior.

Expression Cerebellar
Serotype Promoter Opsin Behavior Behavioral Outcomes
(Cell type) Region
AAV1 hSyn Neuron-specific ChR2 DCN Reward Increased place preference [125]
Social
AAV5 CAG All cells ArchT DCN Altered social preference [125]
behavior
Reduction in hippocampal seizure
Cortex
Pcp2-Cre * Purkinje cells ChR2 Epilepsy duration and seizure-induced
(simplex)
inhibition [126]
DCN Reduction in hippocampal seizure
AAV9 VGluT-Cre * Glutamatergic ChR2 Epilepsy
(fastigial) duration [127]
DCN Reduction in thalamocortical
AAV2 hSyn Neuron-specific ChR2 Epilepsy
(dentate) oscillations [128]
Cortex Working Reduction in performance accuracy
AAV2/9 Pcp2-Cre * Purkinje cells ChR2
(Crus I) memory [129]
Cortex Postural Reduction in the extent of blood
Lentivirus L7 Purkinje cells eNpHR3.0
(uvula) alterations pressure recovery [130]
DCN
AAV CamKII Glutamatergic ChR2 Schizophrenia Increase in prefrontal activity [131]
(dentate)
Transgenic lines that have been used in mouse studies are denoted by an asterisk (*).

5.1. Reward and Social Behavior

A recent report exploited optogenetic specificity to demonstrate the involvement of the cerebellum
in non-motor functions through a direct pathway. This is particularly interesting, as cerebellar impact
on non-sensorimotor functions is usually considered indirect, passing mainly through the thalamic relay.
In this work, Carta and colleagues reported the presence, the physiological nature and the functional
significance of the direct connectivity between DCN and the VTA in mice [125]. In particular,
the adeno-associated virus carrying the ChR2 (AAV1-hSyn-ChR2-YFP) or the archaerhodopsin
(AAV5-CAG-ArchT-GFP) was injected into the DCN (Figure 3). The optogenetic modulation of
DCN axons activity was achieved by illuminating the infected fibers directly in the VTA in acute slices.
This allowed them to rule out the spurious activation of other fibers and it provided a unique tool
to determine the monosynaptic origin of the responses detected in the VTA. This same investigation
showed that DCN axons form glutamatergic synapses with both dopaminergic and non-dopaminergic
neurons in the VTA and that their optogenetic activation directly increased the activity of VTA neurons,
both ex vivo and in vivo. While the experiments in slices allowed to characterize the high efficacy of the
transmission at these connections, behavioral tests were key to determine the functional significance
of this pathway and the effects of cerebellar activity on VTA-dependent behavior involving reward
and sociability. Interestingly, the optogenetic activation of DCN axons in the VTA was sufficient to
cause long-term place preference, while their inhibition completely blocked social preference in the
three-chamber sociability test [125]. Though this test is known to also involve the prefrontal cortex [132],
the use of optogenetics to activate or inhibit DCN axons in the VTA allowed the researchers to prove a
causal relationship between cerebellar activity and VTA-driven motivated behavior. This experimental
setting provided the demonstration of a direct, functional, glutamatergic pathway from the DCN to
the VTA, actively contributing to motivated behavior, further expanding the view of cerebellar role in
non-motor functions [93].
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 12 of 25

contributing to motivated
Int. J. Mol. Sci. 2020, 21, 2494 behavior, further expanding the view of cerebellar role in non-motor
12 of 24
functions [93].

Figure 3.3. Cerebellar

Cerebellar influence
influence on on
reward andand
reward sociability. (A) Left,
sociability. (A)schematic viral injection
Left, schematic of AAV1-
viral injection of
hSyn-ChR2-eYFP targeting
AAV1-hSyn-ChR2-eYFP the deep
targeting the cerebellar nucleinuclei
deep cerebellar (DCN). Optical
(DCN). fibersfibers
Optical werewere
implanted
implantedintointo
the
the ventral
ventral tegmental
tegmental areaarea (VTA),
(VTA), bilaterally.
bilaterally. Right,
Right, mice
mice were
were allowedtotoexplore
allowed exploreaasquare
square chamber,
chamber,
divided in quadrants, one of which was associated to a reward. Whenever the mouse entered in the
reward quadrant, cerebellar axons innervating the VTA were optically optically stimulated.
stimulated. For the time the
spent in
mouse spent in the
the reward
reward quadrant,
quadrant, the thestimulus
stimulus(20 (20HzHzfor
for33sec)
s) was
wasrepeated
repeatedevery
every10 s. (B)
10s. Left,
(B) Left,
AAV5-hSyn-ArchT-GFP
AAV5-hSyn-ArchT-GFPwas wasinjected
injectedin the DCN
in the in order
DCN to selectively
in order inhibit inhibit
to selectively cerebellar axons through
cerebellar axons
bilateral bilateral
through optical fibers implantation
optical into the into
fibers implantation VTA.theRight,
VTA.Social
Right,preference was examined
Social preference using a
was examined
three-chambered
using social task.
a three-chambered socialThetask.
testedThe mouse
testedwas
mouseallowed to approach
was allowed a “stimulus”
to approach mouse confined
a “stimulus” mouse
to one side
confined tochamber
one side(social
chamber side) or a novel
(social object
side) or placed
a novel on the
object otheron
placed side
the(non-social
other side side). On theside).
(non-social first
daythe
On (training day),
first day the mouse
(training day),wastheallowed
mouseto freely
was explore
allowed to all threeexplore
freely chambers. On the
all three subsequent
chambers. On day,
the
the cerebellar axons innervating the VTA were optically inhibited for the time
subsequent day, the cerebellar axons innervating the VTA were optically inhibited for the time thethe mouse stayed by the
social side. The optogenetic stimulation was immediately ceased when the
mouse stayed by the social side. The optogenetic stimulation was immediately ceased when the mouse exited the social side.
mouse exited the social side.
5.2. Working Memory and Decision-Making
5.2. Working
WorkingMemory
memory and
is Decision-Making
a fundamental part of the decision-making process. These are fundamental
cognitive functions involving distributed interacting network of different brain areas, with the
Working memory is a fundamental part of the decision-making process. These are fundamental
prefrontal cortex at the core [133]. The cerebellar contribution to working memory has been reported
cognitive functions involving distributed interacting network of different brain areas, with the
by several studies in humans. Cerebellar damages or dysfunctions often lead to working memory
prefrontal cortex at the core [133]. The cerebellar contribution to working memory has been reported
impairments [3,134–136]. In order to investigate this aspect of cerebellar function in behaving mice,
by several studies in humans. Cerebellar damages or dysfunctions often lead to working memory
Deverett and colleagues exploited optogenetics to test whether direct, precise and transient disruption
impairments [3,134–136]. In order to investigate this aspect of cerebellar function in behaving mice,
of cerebellar activity modulated the accumulation of sensory information in working memory [129].
Deverett and colleagues exploited optogenetics to test whether direct, precise and transient
Mice expressing ChR2 in PCs were obtained by crossing mice with genotypes Pcp2-Cre for PC specificity
disruption of cerebellar activity modulated the accumulation of sensory information in working
and Ai27D for ChR2. The authors showed the optogenetic manipulation of PC-impaired decision
memory [129]. Mice expressing ChR2 in PCs were obtained by crossing mice with genotypes Pcp2-
making by reducing the ability to effectively retain past information in working memory (Figure 4).
Cre for PC specificity and Ai27D for ChR2. The authors showed the optogenetic manipulation of PC-
In particular, they tested the accumulation of sensory information using a behavioral task for head-fixed
impaired decision making by reducing the ability to effectively retain past information in working
mice. In each trial, the animal received air-puffs directed both on the left and right whisker pads,
memory (Figure 4). In particular, they tested the accumulation of sensory information using a
simultaneously, for seconds (sensory evidence presentation). After a delay, an auditive cue signaled
behavioral task for head-fixed mice. In each trial, the animal received air-puffs directed both on the
that the mouse could retrieve a water reward, either by licking a left- or right-placed port. The task was
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 13 of 25

left
Int. J.and
Mol. right whisker
Sci. 2020, 21, 2494 pads, simultaneously, for seconds (sensory evidence presentation). After a
13 of 24
delay, an auditive cue signaled that the mouse could retrieve a water reward, either by licking a left-
or right-placed port. The task was successful, and the reward delivered, when the licking direction
successful,
was the sameand theas thereward delivered,
whisker pad when
whichthe licking direction
received was the(see
more air-puffs sameFigure
as the whisker
4 for a pad which
schematic
received more air-puffs (see Figure 4 for a schematic representation
representation of this task). ChR2-expressing PCs were stimulated during the sensory evidence of this task). ChR2-expressing
PCs were stimulated
presentation, preceding during the sensory
the decision, and ledevidence presentation,
to reductions precedingaccuracy.
in performance the decision, and
In this ledthe
case, to
reductions in performance accuracy. In this case, the use of optogenetics
use of optogenetics allowed the researchers to narrow down the specificity of the effect on behavior allowed the researchers to
narrow
to down
a single the specificity
neuronal type, theof the that
PCs, effect on behavior
provide to output
the sole a singleofneuronal type, the
the cerebellar PCs,While
cortex. that provide
the use
the sole output of the cerebellar cortex. While the use of mutant
of mutant mice could have helped identifying an important role of these neurons, optogeneticsmice could have helped identifying
an important
provided role of these
the advantage toneurons, optogenetics
modify neuronal provided
activity the advantage
in a transient to modify
way. This allowedneuronal activity
them to modify
in a transient
neuronal way.inThis
activity onlyallowed
the temporalthem towindowmodify significant
neuronal activity
for the in only the temporal
accumulation of the window
sensory
evidence in working memory. The transient and reversible nature of the optogenetictransient
significant for the accumulation of the sensory evidence in working memory. The and
interference
reversible nature of the optogenetic interference with neuronal activity
with neuronal activity allowed to rule out possible motor impairments leading to failing the test and allowed to rule out possible
motor impairments
reinforcing the concept leading to failing
that the the test
cerebellar cortexandisreinforcing
indeed involvedthe concept that evidence
in sensory the cerebellar cortex
retainment
is indeed involved in sensory evidence retainment for working memory.
for working memory. These results may help account for the many clinical findings linking cerebellar These results may help
account for the many clinical findings linking cerebellar activity
activity to working memory and decision making in humans [103,136–138]. Since working memory to working memory and decision
making
is in humans
considered dependent[103,136–138]. Since working
on the activity of forebrainmemory is considered
regions [139,140], itdependent on the
is likely that the activity
cerebellarof
forebrain regions [139,140], it is likely that the cerebellar role
role described above is mediated by indirect cerebellar–neocortical projections. Furtherdescribed above is mediated by indirect
cerebellar–neocortical
investigations on the projections.
subject would Further investigations
definitely on theoptogenetics
benefit from subject would usedefinitely
to dissectbenefit from
cerebellar
optogenetics
pathways. use to dissect cerebellar pathways.

Figure 4. Impaired
Figure 4. Impaired decision-making
decision-making by by disruption
disruption of of cerebellar activity during sensory evidence
accumulation.
accumulation. Left,
Left, mice
mice ofof genotypes
genotypes Pcp2-Cre
Pcp2-Cre forfor PC
PC specificity
specificity and
and Ai27D
Ai27D for
for ChR2
ChR2 were
were used.
used.
ChR2-expressing PCs were stimulated using light delivered through optical fibers
ChR2-expressing PCs were stimulated using light delivered through optical fibers implanted bilaterally implanted
bilaterally
over crus I over
of thecrus I of the cerebellum.
cerebellum. Right,ofschematic
Right, schematic of the evidence-accumulation
the evidence-accumulation decision-makingdecision-
task.
making task. two
In each trial, In each trial,oftwo
streams streamstimed
randomly of randomly
air puffstimed
were air puffs were
delivered to thedelivered to thewhiskers.
left and right left and
right whiskers.
After an 800-msAfter
delay,an 800-ms
mice delay,
licked mice
one of twolicked one of
lick ports two lick the
indicating ports indicating
side with more thecumulative
side with more
puffs
cumulative puffs to
to receive a water receive a water reward.
reward.

5.3. Schizophrenia
5.3. Schizophrenia and
and Cognition
Cognition
Cerebellar projections
Cerebellar projections to to the
the frontal
frontal cortexes,
cortexes, passing
passing through
through thethe thalamus
thalamus and
and the
the VTA,
VTA, are
are
considered the
considered the base
baseofofitsitsrole
roleinincognitive
cognitive performances
performances andand related
related diseases,
diseases, suchsuch as autism
as autism and
and schizophrenia [102]. The cerebellum, thalamus and frontal cortex have
schizophrenia [102]. The cerebellum, thalamus and frontal cortex have been found to be involved been found to be
in
involved in schizophrenia,
schizophrenia, leadingoftoa adistributed
leading to a theory theory of network
a distributed network
impairment at impairment at the
the core of this core
disease.
of this disease.the
Interestingly, Interestingly, the electrical
electrical stimulation of stimulation of the
the cerebellar cerebellar
vermis usingvermis using MRI-guided
MRI-guided transcranial
transcranial magnetic stimulation (TMS) proved to be a successful treatment
magnetic stimulation (TMS) proved to be a successful treatment in patients with in patients with refractory
refractory
schizophrenia [141–143].
schizophrenia [141–143]. ThisThisevidence
evidenceisisofof particular
particular relevance
relevance since
since only
only fewfew treatment
treatment options
options are
are available for this neurological disorder. Parker and colleagues proposed a novel hypothesis
Int. J. Mol. Sci. 2020, 21, 2494 14 of 24

that cerebellar stimulation could improve cognitive symptoms of schizophrenia by normalizing

prefrontal cortex activity, suggesting an experimental design combining optogenetics, pharmacology,
and electrophysiology tools in awake-behaving animals to investigate the cerebellar role in cognitive
processes [144,145]. The injection of optogenetic constructs in the DCN allowed to specifically activate
the cerebellar inputs to the VTA and different thalamic nuclei. The schizophrenia condition is mimicked
by pharmacologically disrupting D1-mediated dopamine signaling in the prefrontal cortex in rodents,
to reproduce the effect of reduced expression of D1-like dopamine receptors observed in the same brain
area in human patients, considered at the core of schizophrenia dysfunctions as deficits in internal
timing behavior [131,146–148]. In these animals, optogenetic stimulation of ChR2-expressing DCN
axons in the thalamic ventrolateral nuclei rescued the performance in an interval timing cognitive task,
found impaired by disrupting D1 dopamine signaling [131]. Moreover, the prefrontal cortex neurons
showed an increase in the firing rate following optogenetic stimulation of DCN projections in the
thalamus. These data support the idea that the cerebellar circuit can compensate for dysfunctional
prefrontal cortex activity in schizophrenia-mimicking conditions [131]. The use of optogenetics in
this experimental settings allowed for the matching of the two formerly separated observations that
D1-like dopamine receptors in the prefrontal cortex are involved in timing processes [145] and the
general knowledge that the cerebellum is a timing-machine operating in the sub-millisecond time
scale [149–151], likely contributing to the internal timing task.

5.4. Temporal Lobe Epilepsy and Absence Seizure

Temporal lobe epilepsy (TLE) is the most diffused focal epilepsy in adults. TLE patients are often
unresponsive to anti-epileptic drugs and not suitable for surgical intervention. In this type of epilepsy,
seizures often start at the hippocampal level. Nevertheless, a direct intervention on hippocampal
neurons activity proved inefficient to block the seizures. Over the last few decades, electrophysiological
and neuroimaging studies have provided evidence for a cerebellar role in influencing cerebral epileptic
activity. For example, seizures can arise directly from cerebellar structures [152,153] and changes in
cerebellar blood flow, and electroencephalogram and neuronal activity have been reported during
seizures [154–157]. Despite this evidence, experimental investigations using animal models of TLE
to unravel the role of the cerebellum in the epileptic phenomenon have produced mixed results.
The lack of specificity inherent with electrical stimulation was most likely responsible for these
unclear conclusions. A recent study using mice lacking cerebellar long-term depression showed that
information processing in hippocampal place cells is disrupted [158], and suggested the cerebellum as a
new target for intervention in TLE. Thus, Krook-Magnuson and colleagues tested whether optogenetic
intervention targeting the cerebellum could provide seizure control in TLE, exploiting the specificity
achievable with this technique [126]. In particular, the unilateral intrahippocampal kainate mouse
model of TLE was used. ChR2 and NpHr (light-driven chloride pump halorhodopsin) were used in
different experiments to test the effects of activation or inhibition of parvalbumin-expressing neurons,
including PCs, in the cerebellar cortex. In this case, the specificity of optogenetics was not confined to a
single neuronal type, since also molecular layer interneurons might express parvalbumin. Nevertheless,
the authors could draw noticeable conclusions on the effect of activating or silencing cerebellar cortical
neurons and these results prompted further research. In particular, they achieved the puzzling result
that the seizure duration in the hippocampus was somehow reduced by both the activation and
inhibition of parvalbumin-expressing neurons in the cerebellar cortex, suggesting that the direction
of modulation was not critical. The authors comment that these apparent contradictory results can
arise from the fact that parvalbumin is also expressed in molecular layer interneurons, and that the
net effect on DCN is therefore difficult to infer. This is in agreement with a recent report using a
pan-neuronal promoter for ChR2 expression in the cerebellar molecular layer while recording from
the DCN, showing the mixed effect of light activation likely depending on the prevalent effect of
direct activation of PCs or inhibition through molecular layer interneurons [159]. Krook-Magnuson
and colleagues further exploited optogenetic specificity by replicating their key findings in a mouse
Int. J. Mol. Sci. 2020, 21, 2494 15 of 24

line expressing ChR2 selectively in PCs. Their results showed that selective excitation of PCs was
not only capable of reducing hippocampal seizure duration but also of inhibiting seizure induction
(increasing the time between seizures) [126]. In conclusion, the use of optogenetic approaches was key
to demonstrate that the cerebellum can be an effective target to inhibit hippocampal seizures. Moreover,
the complex response observed to optogenetic manipulation of PCs offers one possible explanation
for the fact that the direction of modulation (excitation/inhibition) was not critical for hippocampal
seizure inhibition. During optogenetic excitation of PCs, DCN show decreased firing rates [63,160,161].
However, PC excitation is subsequently followed by a pause in PC activity [126], in agreement with
the burst–pause behavior typical of these neurons [162], and a corresponding increase in firing in the
DCN [161]. Therefore, both optogenetic inhibition and excitation of PCs could result in excitation
of the DCN and thereby the cessation of hippocampal seizures. To verify this hypothesis, Streng
and Krook-Magnuson optogenetically stimulated glutamatergic neurons in the DCN (mainly in the
fastigial nucleus) during hippocampal seizures in a mouse model of TLE [127]. Indeed, optogenetic
inhibition of fastigial nuclei neurons had no effect on hippocampal seizures while excitation robustly
attenuated them. These results strongly suggest that DCN excitation was responsible for the decrease
in hippocampal seizures observed while modifying the activity in the cerebellar cortex. Addressing
DCN activity might therefore be key to successful intervention on TLE.
The role of the cerebellum in epileptic phenomena was also proved by recent work on cerebellar
contributions to absence epilepsy [128]. Absence epilepsy is characterized by sudden periods of
impaired consciousness which typically last up to ten seconds, associated to behavioral arrest.
The electrophysiological counterpart was found in thalamocortical oscillations due to excessive
activation of the cerebral cortex, originating the so-called generalized spike-and-wave discharges
(GSWDs). Kros and colleagues tested whether altering the output of the cerebellum might affect
thalamocortical oscillations in the tottering mouse, characterized by a missense mutation of the Cacna1a
gene leading to a loss-of-function of calcium channels, and considered an established model for absence
epilepsy. To modify DCN activity, the virus containing the ChR2 (AAV2-hSyn-ChR2(H134R)-EYFP)
was injected into the DCN. The specificity of the optogenetic approach was, in this case, obtained
using localized injections. This experimental setting allowed the researchers to determine the effect of
cerebellar activation on GSWDs, recorded through electrodes implanted in the primary motor and
sensory cortexes. The results showed that optical activation of DCN neurons significantly reduced
or even stopped GSWDs within 150ms from the onset of stimulation (both bilateral and unilateral).
The authors showed that the optogenetic activation of DCN neurons was able to alter the spontaneous
activity of these neurons, often involved in oscillatory cycles. Interestingly, the precise temporal
efficiency of optogenetic stimulation was ideal to explore the temporal window of the GSWD most
responsive to DCN activation, showing that the most effective results were obtained when stimulation
was applied during the “wave” phase of GSWD, that is, when cortical neurons are normally silent.
In this paper, optogenetic stimulation was irreplaceable for both the specificity of DCN activation and
for the temporal precision involved in these kinds of investigations.

5.5. Control of Blood Pressure

The cerebellar lobule IX of the vestibulocerebellum (also called the uvula) is known to be
extensively connected to brainstem regions involved in vestibular signal processing and, interestingly,
cardiovascular regulation. Thus, the uvula could take part in the reflex mechanisms that intervene
during postural alterations to homeostatically regulate blood pressure. Indeed, previous investigations
have shown that lesions of this cerebellar region are able to impair the baroreceptor reflex and
cardiovascular responses during postural alterations [163,164]. PCs in the uvula project, indirectly,
to the solitary tract nucleus, which receives primary afferent fibers from the baroreceptors. Though this
pathway was the best candidate to explain the experimental evidence, no clear causal connection could
be detected between PC activity and cardiovascular responses to postural alterations. Tsubota
and colleagues [130] addressed this issue by infecting rat cerebellar uvula with a lentivirus
Int. J. Mol. Sci. 2020, 21, 2494 16 of 24

carrying the eNpHR, selectively expressed in PCs due to the specificity of the L7 promoter
(Lenti-sL7-eNpHR-EYFP-WPRE). By inhibiting PCs at different times during the postural alterations
(consisting in 30◦ head-up or head-down tilts), this study clearly showed that PC activity in the uvula
is involved in the regulation of blood pressure in response to postural alterations (in particular during
head-down tilts) and also in recovery to baseline levels. Previous studies showing the uvula role in these
mechanisms were performed by lesioning the cerebellar areas involved [164]. The optogenetic approach
provides several advantages, as the cell-type specificity, the transient nature of neuronal silencing,
the temporal modulation of the inhibition (that lasted from 1s to several seconds in Tsubota’s study)
and the possibility to avoid neural compensations as consequences to the lesion. The investigation
summarized in this section is conducted on anesthetized rats, unlike all the other reports taken into
consideration in this review. Nevertheless, the study by Tsubota and colleagues was considered
here due to the notable advances provided by the use of optogenetics and to the peculiarity of the
topic addressed. Beyond its role in sensorimotor and non-sensorimotor functions, the cerebellum
is involved in homeostatic reflexes as the baroreceptor one, controlling blood pressure through PC
activity. Optogenetics is helping us to unravel these less investigated mechanisms.

6. Clinical Aspects
Cerebellar stimulation as a clinical tool to reduce patients’ symptoms of motor and non-motor
diseases is gaining more and more attention as of late. For a comprehensive review of this topic,
see [165]. In the context of this review, it is interesting to point out that optogenetic stimulation of the
cerebellum could provide a suitable tool to overcome some of the issues raised by the use of Deep
Brain Stimulation, Transcranial Direct Current Stimulation, and Transcranial Magnetic Stimulation.
First of all, optogenetics can guarantee specificity of the target zone. Where specific markers are
available, even single neuronal types might be selectively targeted. This would avoid the unspecific
and unforeseeable effects of stimulating the cerebellar cortex, for example. Secondly, optogenetics
provide the possibility to either activate or inhibit neuronal activity. At the moment, inhibition can
only be achieved through lesioning or surgical ablation of brain tissue. Thirdly, optogenetics is ideal to
provide neuronal activation/inhibition in precise and sharp temporal windows, that can be modulated
down to the milliseconds time scale. Interestingly, this aspect of optogenetics is particularly suitable to
be paired to systems involving brain–computer interface (BCI) technology. An example is reported in
one of the papers cited in the previous section on absence epilepsy [128]. Kros and colleagues found
that optogenetic intervention on the DCN in the tottering mouse was more efficient in the “wave”
phase of the GSWD. The authors suggested that the use of a BCI in this case might be useful in order to
provide stimulation to the DCN in the most effective way, and they indeed provided a proof of principle
of the validity of this procedure in their experimental conditions. The future of brain stimulation will
most likely involve the cerebellum on various motor and non-motor pathologies, and will be radically
improved wherever the implementation of optogenetics will be successful. Indeed, recent constructs
show reduced vector-associated cytotoxicity, suggesting that a clinical application in substitution to
deep brain stimulation might be feasible soon. At the moment, optogenetics is applied only to vision
restoration [166], but its overcoming of previous technical issues [167] might open several clinical
applications in the field of cerebellum-related pathologies, also.

Author Contributions: Writing-original draft, F.P., I.M., L.M.; visualization, F.P.; writing-review and editing, L.M.;
Funding acquisition, E.D. and L.M. All authors have read and agreed to the published version of the manuscript.
Funding: This work has received funding from: the European Union’s Horizon 2020 Framework Programme for
Research and Innovation under the Specific Grant Agreement No. 785907 (Human Brain Project SGA2) to ED;
Blue-Sky Research Grant of the University of Pavia (Università degli Studi di Pavia; BSR77992) to LM.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the
decision to publish the results.
Int. J. Mol. Sci. 2020, 21, 2494 17 of 24

Abbreviations
AAV Adeno-associated viruses
Arch Archaerhodopsin
ASD Autism Spectrum Disorders
ChR2 Channelrhodopsin-2
CS Conditioned stimulus
DCN Deep Cerebellar Nuclei
EBC Eyeblink conditioning
eNPHR3.0 Enhanced Natronomonas halorhodopsin
GSWDs Generalized spike-and-wave discharges
NPHR Halorhodopsin
PC Purkinje cell
TLE Temporal lobe epilepsy
TMS Transcranial magnetic resonance
US Unconditioned stimulus
VTA Ventral Tegmental Area
YFP Yellow Fluorescent Protein

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