Cholinergic Mesopontine Signals Govern Locomotion and
Reward Through Dissociable Midbrain Pathways
Cheng Xiao
1
,
Jounhong Ryan Cho
2
,
Chunyi Zhou
1
,
Jennifer B. Treweek
1
,
Ken Chan
1
,
Sheri
L. McKinney
1
,
Bin Yang
1
, and
Viviana Gradinaru
1,*
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena,
California 91125, USA
2
Computation and Neural Systems, California Institute of Technology, Pasadena, California
91125, USA
Abstract
The mesopontine tegmentum, including the pedunculopontine and laterodorsal tegmental nuclei
(PPN and LDT), provides major cholinergic inputs to midbrain and regulates locomotion and
reward. To delineate the underlying projection-specific circuit mechanisms we employed
optogenetics to control mesopontine cholinergic neurons at somata and at divergent projections
within distinct midbrain areas. Bidirectional manipulation of PPN cholinergic cell bodies exerted
opposing effects on locomotor behavior and reinforcement learning. These motor and reward
effects were separable via limiting photostimulation to PPN cholinergic terminals in the ventral
substantia nigra pars compacta (vSNc) or to the ventral tegmental area (VTA), respectively. LDT
cholinergic neurons also form connections with vSNc and VTA neurons, however although photo-
excitation of LDT cholinergic terminals in the VTA caused positive reinforcement, LDT-to-vSNc
modulation did not alter locomotion or reward. Therefore, the selective targeting of projection-
specific mesopontine cholinergic pathways may offer increased benefit in treating movement and
addiction disorders.
Keywords
mesopontine tegmentum; pedunculopontine nucleus; laterodorsal tegmental nucleus; cholinergic
neuron; substantia nigra pars compacta; ventral tegmental area; locomotion; conditioned place
preference; optogenetics; retrograde tracing
*
To whom correspondence should be addressed: Viviana Gradinaru, Ph.D., Division of Biology and Biological Engineering, California
Institute of Technology, 1200 East California Blvd. MC 156-29, Pasadena, CA 91125, Phone: (626) 395-6813, viviana@caltech.edu.
Author Contributions:
C.X. and V.G. designed the project. C.X., R.C., C.Z., J.T., and V.G. planned experiments and analyzed data. C.X., R.C., C.Z., S.L.M.
and J.T. collected data. K.C. contributed to animal behavior tests and data illustration. B.Y. provided training in histology and image
processing. C.X. and V.G. wrote the manuscript with help from co-authors. Each author read and approved the final manuscript. VG
supervised all aspects of the project.
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Neuron
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Introduction
The pedunculopontine nucleus (PPN) is a heterogeneous brainstem structure that contains
cholinergic (ChAT), glutamatergic, and GABAergic neurons (
Benarroch, 2013
;
Jenkinson et
al., 2009
). Accumulating evidence suggests that PPN ChAT neurons play key roles in both
motor and non-motor behaviors (
Morita et al., 2014
). Parkinson’s disease patients with gait
disorders and postural instability display degeneration of the PPN ChAT neurons, and the
severity of balance deficits is correlated with a reduction in PPN ChAT-neuron numbers and
activity (
Bohnen et al., 2013
;
Bohnen et al., 2009
). In parkinsonian non-human primate
models, chemical lesion of PPN ChAT neurons is necessary and sufficient to impair gait and
balance (
Karachi et al., 2010
). In addition to affecting motor behavior, ChAT-neuron-
selective chemical lesions of the PPN also affect drug-seeking behavior (
Lanca et al., 2000
).
These studies suggest that PPN ChAT neurons are instrumental for normal function in
movement and reward reinforcement; however, they do not identify the downstream
effectors.
The PPN projects to multiple targets in the basal ganglia, midbrain, cerebellum, thalamus,
and the reticular formation (
Ballanger et al., 2009
;
Benarroch, 2013
;
Dautan et al., 2014
;
Jenkinson et al., 2009
;
Marani et al., 2008
). Although electrically and pharmacologically
stimulating PPN neurons
in vivo
leads to overall excitatory outcomes in downstream nuclei
(
Ballanger et al., 2009
;
Blaha et al., 1996
), the projections from the PPN to individual
downstream neurons are complex. Two notable targets of PPN ChAT circuitry, the substantia
nigra pars compacta (SNc) and the ventral tegmental area (VTA), are implicated in
locomotion and reward processing (
Bermudez and Schultz, 2014
;
Bromberg-Martin et al.,
2010
;
Ikemoto, 2007
;
Lerner et al., 2015
;
Roeper, 2013
). ChAT, glutamatergic, and
GABAergic neurons of the PPN form convergent connections onto SNc and VTA neurons,
but their synaptic contacts with individual neurons are often non-overlapping (
Futami et al.,
1995
;
Good and Lupica, 2009
;
Scarnati et al., 1986
). The multifaceted roles of SNc and
VTA neurons in exploratory activity, habituation, reinforcement, aversion (
Bromberg-Martin
et al., 2010
;
Friedman et al., 2014
;
Lerner et al., 2015
;
Roeper, 2013
;
Walsh et al., 2014
),
and the involvement of multiple nuclei in locomotion and reward (
Benarroch, 2013
;
Bromberg-Martin et al., 2010
;
Ikemoto, 2007
;
Jenkinson et al., 2009
;
Kravitz and Kreitzer,
2012
) adds experimental complexity to the assessment of how PPN ChAT tone in the VTA
and SNc patterns goal-directed behaviors. Whether selectively and temporarily enhancing
the tonic activity of PPN ChAT neurons, without affecting PPN glutamatergic and
GABAergic neurons, is sufficient to recruit SNc and VTA neurons and to regulate the
aforementioned behaviors is unknown. A strategy that enables specific modulation of ChAT
projections without perturbing glutamatergic and GABAergic projections can facilitate both
in vitro
and
in vivo
characterization of ChAT circuitry originated in the PPN.
To dissect out the behavioral effect of ChAT signaling along each PPN-to-midbrain circuit,
we have employed optogenetic tools (
Gradinaru et al., 2009
;
Gradinaru et al., 2010
;
Hausser,
2014
;
Walsh et al., 2014
) and ChAT-Cre rats (
Witten et al., 2011
). Electrical and chemical
interventions, which are commonly used to study the anatomy and physiology of the PPN,
do not offer the spatiotemporal or cell-type specificity required to selectively harness
specific PPN ChAT projections to the VTA and SNc, or to examine differences in PPN cell
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connectivity with SNc and VTA nuclei. Highlighting the requirement for precise spatial
targeting of neuronal manipulations, it is noteworthy that the dorsal and ventral tiers of the
SNc (dSNc and vSNc) project to distinct sub-regions of the striatum and may have
differential vulnerability to neurodegenerative assaults (
Hassan and Benarroch, 2015
). Thus
they merit investigation as separate subnuclei.
We found that optogenetic modulation of PPN ChAT somata altered both motor activity and
reward reinforcement, whereas targeted photo-stimulation of PPN ChAT terminals in the
vSNc or VTA granted separable control of these physiological processes. Importantly, these
results do not rule out cholinergic control of midbrain functions from non-PPN sources.
Indeed, as shown before (
Chen and Lodge, 2013
;
Lodge and Grace, 2006
;
Mena-Segovia et
al., 2008
) and confirmed here, ChAT neurons in the laterodorsal tegmental nucleus (LDT),
also formed functional connections with midbrain neurons. While photo-excitation of these
connections in the VTA regulated reward reinforcement, photoexcitation of LDT ChAT
terminals in the vSNc did not alter locomotion, which contrasts with the effects of PPN
originating cholinergic modulation. Therefore, ChAT projections from the PPN and the LDT
to the vSNc and VTA play distinct roles in regulating motor and reward behaviors, and could
be refined therapeutic targets for movement disorder and drug addiction.
Results
Previous studies (
Benarroch, 2013
;
Jenkinson et al., 2009
;
Mena-Segovia et al., 2008
;
Oakman et al., 1995
) have demonstrated connections between PPN neurons and SNc/VTA
neurons by neuronal tracing and electrophysiological techniques. As these techniques often
lacked cell specificity, ChAT connections were neither imaged in intact form nor were they
selectively stimulated without also perturbing glutamatergic and GABAergic connections.
We injected adeno-associated virus serotype 5 (AAV5) carrying Cre-dependent (DIO;
double-floxed inverse open reading frame) ChR2-eYFP into the PPN of ChAT-Cre rats to
selectively label ChAT neurons and their projections (Fig. 1A). To visualize the projections
to the SNc and VTA, we used PACT clearing (
Treweek et al., 2015
;
Yang et al., 2014
) for
facile and accurate mapping of dense, long-range fibers in 1 – 2 mm thick brain sections. We
observed ChR2-eYFP labeled axonal fibers from PPN ChAT neurons within both the dorsal
and ventral tiers of the SNc (dSNc and vSNc) as well as within the VTA (Fig. 1B, C).
We confirmed the functional properties of these connections by performing whole-cell
patch-clamp recordings from SNc and VTA neurons adjacent to ChR2-expressing PPN
ChAT axonal fibers in live midbrain slices of ChR2-injected rats (Fig. 1D). ChAT neurons
usually form connections with downstream neurons through non-synaptic volume
transmission (
Miwa et al., 2011
). Since ACh released in response to a short stimulation
epoch is rapidly hydrolyzed by acetylcholinesterase before reaching its receptors, long
stimulation epochs are necessary in order for ACh to accumulate and subsequently activate
ACh receptors. Therefore, to increase our probability of detecting existing functional ChAT
transmission, we applied 5 – 10 s continuous blue light stimulation (Fig. 1E–G). Under these
conditions, we detected inward currents in 33%, 24%, and 44% of vSNc, dSNc, and VTA
neurons, respectively (Fig. 1E–I). Meanwhile, blue light accelerated neuronal firing (Fig.
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1E–G, J). These data suggest that PPN ChAT neurons form excitatory connections with SNc
and VTA neurons.
Midbrain neurons contain both nicotinic and muscarinic ACh receptors (nAChRs and
mAChRs) (
Drenan and Lester, 2012
;
Miwa et al., 2011
;
Yeomans, 1995
), and either is
capable of mediating the response to light-induced ChAT release. We observed
photocurrents that were not significantly changed by atropine, a mAChR antagonist, but that
were significantly blocked by mecamylamine (MEC), a nAChR antagonist (Fig. 1K; Fig.
S1A, D); furthermore, MEC also blocked the enhancement of firing in response to photo-
excitation (Fig. 1E). These data suggest that nAChRs predominantly mediate the
connectivity between PPN ChAT neurons and SNc/VTA neurons.
Given that nAChRs modulate neurotransmitter release in midbrain (
Dani and Bertrand,
2007
), the nAChRs that mediate the photocurrents could be located on the recorded neurons
or on afferent terminals releasing other neurotransmitters, such as, glutamate or GABA. To
define where these nAChRs are located, we further characterized the pharmacological
properties of the photocurrents. We observed that bicuculline (10 μM, Bic), a GABA
A
receptor blocker, did not affect the photocurrents in midbrain neurons, but 10 μM MEC
blocked these currents (Fig. 1K; Fig. S1B, E). In some midbrain neurons (8/18), the
photocurrents were completely blocked by glutamate receptor antagonists (APV and
CNQX); whereas, in other neurons (10/18), the combined application of APV and CNQX
only partially reduced the currents, and MEC blocked the remaining currents (Fig. 1K; Fig.
S1C, F). These results suggest that nAChRs both in afferent glutamatergic terminals and in
midbrain neurons are important targets of endogenously released ACh from PPN.
The above optogenetic tracing data (Fig. 1B, C) support the hypothesis that the SNc and
VTA are downstream targets of PPN ChAT neurons. Since the SNc and VTA are involved in
locomotion and reward reinforcement (
Bromberg-Martin et al., 2010
;
Lerner et al., 2015
;
Maskos, 2008
;
Patterson et al., 2015
;
Roeper, 2013
), it follows that PPN ChAT activity may
impact locomotion and reward reinforcement. To test this hypothesis, we selectively and
reversibly manipulated opsin-expressing PPN ChAT neurons in freely behaving rats.
Similar to previous studies (
Jenkinson et al., 2009
), our data illustrate that ChAT neurons
reside throughout the PPN and account for almost half of all neurons in both the rostral and
caudal PPN (Fig. 2A, B; Fig. S2A). To tailor practical optogenetic stimulation paradigm (i.e.
duration and frequency) to modulate neurons within their spiking capability, we therefore
characterized the biophysical properties of PPN ChAT neurons using brain-slice patch-
clamp recordings (Fig. 2C–H). Most PPN neurons fired spontaneously at <10 Hz (Fig. S2B).
When increasing steps of depolarizing current were injected into PPN neurons, the firing
rates of ChAT neurons increased more than non-ChAT neurons (Fig. 2E, Tab S1), consistent
with our data showing that ChAT neurons had higher input resistance (Fig. 2F). The optimal
stimulation duration to evoke reliable single action potential for PPN neurons was 10 ms
(Fig. 2G, H; Fig. S2C, D; Tab S2). Thus, we applied 10 ms light pulses to evoke action
potentials in the following
ex vivo
and
in vivo
experiments involving optogenetic excitation.
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To achieve optogenetic control of PPN ChAT neurons we stereotaxically delivered AAV5-
DIO- ChR2-eYFP or Arch3.0-eYFP genes into the PPN of ChAT-Cre rats (
Chow et al.,
2010
;
Mattis et al., 2012
;
Witten et al., 2011
) (Fig. 3A–C, I–K; Fig. 4A). Whole-cell patch-
clamp recordings in PPN brain slices (Fig. S3A, D) confirmed functional expression levels
of virally transduced ChR2 and Arch3 in PPN neurons: blue and green light stimulation
evoked robust photocurrents in recorded cells (Fig. 3D, L; Fig. S3C, E, F) and effectively
modified the firing patterns (Fig. 3E, L). Opsin expression was specific to ChAT neurons
(Fig. 3C, K). Additionally, blue light pulses (10 ms) reliably evoked inward currents (Fig.
S3E) and action potentials (Fig. 3E–H) in PPN ChAT neurons expressing ChR2 (Fig. S3A).
To understand the behavioral contributions of PPN ChAT neurons, we monitored freely
moving rats for changes in locomotor activity in response to photo-modulation of opsin-
expressing PPN ChAT neurons. For photo-excitation, we applied 20 Hz stimuli (Fig. 4A;
Fig. S4A) because: 20 Hz is higher than the average spontaneous firing rates (<12 Hz) of
PPN neurons shown in our
in vitro
data (Fig. S2B; Tab S1) and in previous
in vivo
studies
(
Norton et al., 2011
), and 20 Hz is within the frequency range (20–25Hz) used for electrical
stimulation of the PPN and prior photo-excitation (at 20Hz) of PPN ChAT neurons that
effectively enhances movement (
Benarroch, 2013
;
Jenkinson et al., 2009
;
Plaha and Gill,
2005
;
Roseberry et al., 2016
;
Stefani et al., 2007
). To quantify locomotor behavior, we used
an open field test (see Methods). Exposure to ChR2-activating blue light (20 Hz, 10 ms
pulse width) robustly and reversibly increased locomotion (Fig. 4B, Fig. S4D). Moreover,
the enhancement of locomotion during photo-excitation was abolished by MEC (2 mg/kg,
i.p.) (Fig. S4G–I). These data indicate that increased PPN ChAT activity is sufficient to
promote locomotion by enhanced activation of nAChRs. To test whether spontaneous firing
in PPN ChAT neurons is necessary to maintain normal locomotion, we photo-inhibited
Arch3-expressing PPN ChAT neurons with continuous green light during open field tests.
Photo-inhibition significantly reduced locomotor activity, and this effect reversed upon light
termination (Fig. 4C, Fig. S4J). Neither blue nor green light stimulation altered locomotion
in control mCherry rats (Fig. 4B, C; Fig. S4B, D, J), eliminating the possibility that virus
transduction or light stimulation exerted non-specific actions on locomotion. During testing,
ChR2, Arch3 and corresponding mCherry cohorts exhibited similar levels of baseline
activity (Fig. S4E, K). These data provide a causal link between the activity of PPN ChAT
neurons and locomotion.
The PPN contains neurons that respond to either movement or reward signals, as shown by
prior
in vivo
single-unit recordings (
Lau et al., 2015
;
Norton et al., 2011
;
Okada and
Kobayashi, 2013
). Given that ChAT neurons are a dominant neuronal population in the PPN
(Fig. 2B), and that PPN neuronal activity has been linked to numerous physiological
functions, our optogenetic modulation of ChAT-neuron activity could affect non-motor
behaviors as well. To confirm the hypothesized role of PPN ChAT signaling in reward
processing, we used a conditioned place preference (CPP) paradigm (Fig. 4D, see
supplementary materials) to test whether photo-excitation and photo-inhibition of PPN
ChAT neurons during place conditioning would induce place preference and aversion,
respectively (Fig. 4E). On the first day, the rats preferred the dark compartment (350 – 550
s) to the white compartment (50 – 250 s) (Fig. S5A). Therefore, we applied a biased light-
conditioning paradigm on the following four consecutive days. Since lesioning PPN ChAT
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neurons reduces nicotine self-administration (
Lanca et al., 2000
), we hypothesized that
exciting and inhibiting PPN ChAT neurons would cause positive and negative reinforcement,
respectively. In a CPP paradigm, the time spent in the light-paired side should increase in
response to PPN ChAT photo-excitation but decrease with PPN ChAT photo-inhibition. To
avoid ceiling and floor effects for conditioning, we paired ChR2 rats with blue light pulses
in the non-preferred (white) compartment and Arch3 rats with green light in preferred (dark)
compartment (Fig. 4E). As controls, we paired mCherry rats with blue light pulses in the
white compartment and with green light in the dark compartment (Fig. 4E). After light-
conditioning, ChR2 rats exhibited a statistically significant increase in the time spent in the
compartment paired with photo-excitation (Fig. 4F, G), whereas Arch3 rats displayed a
significant reduction in the time spent in the compartment paired with photo-inhibition (Fig.
4H, I). Therefore, modulation of PPN ChAT somata is also sufficient to bidirectionally affect
reward processing. Note that four days of light-conditioning had only minor effects on
locomotion in the post-conditioning session (Fig. S5B, C).
Based on our findings that optogenetic modulation of PPN ChAT neurons modifies both
motor and reward behaviors, we reasoned that the downstream targets of ChAT projections
may be involved in conveying these behaviors. To understand the role of PPN projections to
the SNc and VTA in controlling locomotion and reward
in vivo
, we restricted optogenetic
modulation to ChAT axon terminals in the vSNc (Fig. 5A, B, Fig. S6A–C) and VTA (Fig.
6A, B; Fig. S7A–C). We chose to target vSNc-projecting and not dSNc-projecting fibers as
the smaller size of the vSNc and its anatomical segregation from the VTA and dSNc (Fig.
1B; Fig. 5A, B) facilitate its optogenetic isolation and recruitment.
In the open field test, photo-excitation of PPN-to-vSNc projections increased locomotion
(Fig. 5C, D; Fig. S6D). Photo-excitation of terminals could potentially cause back-
propagating action potentials (
Jennings et al., 2013
) in the vSNc projecting PPN ChAT
neurons and recruit downstream nuclei other than the vSNc, with the PPN-to-vSNc
connection playing only a minor role. To ensure that the vSNc was indeed a key contributor,
we photo-inhibited the projections of PPN ChAT neurons to the vSNc in rats injected with
AAV5-DIO-Arch3.0-eYFP in the PPN (Fig. 5A; Fig. S6C) and observed reduced
locomotion (Fig. 5E, F; Fig. S6E), confirming the critical role of this connection in motor
control. Photo-excitation of the PPN-to-vSNc projections had no effect on CPP (Fig. 5G, H).
In contrast to PPN-to-vSNc projections, photo-excitation of PPN-to-VTA projections did not
change locomotion in the open field test (Fig. 6C, D; Fig. S7A, B), but did attenuate place
aversion to the previously non-preferred compartment (Fig. 6E, F) in CPP. This effect could
be related to increased activity of VTA neurons (Fig. S7F–H), because direct stimulation of
VTA neurons is sufficient to cause reward reinforcement (
Tsai et al., 2009
). We next tested
whether photo-inhibition of PPN-to-VTA projections has opposite effects compared with
photo-excitation of the projections. Since Arch3.0 can have limitations for long epoch
terminal inhibition (
Mahn et al., 2016
), we virally transduced eNpHR3.0-eYFP into PPN
ChAT neurons and utilized green-light to inhibit the terminals of these neurons (Fig. 6A;
Fig. S7C–E). Conditioning eNpHR3.0-injected rats with photo-inhibition of PPN-to-VTA
projections reduced time spent in the light-paired compartment (Fig. 6G, H).
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Collectively, the above data indicate that targeted optogenetic modulation of PPN ChAT
terminals located in the vSNc and VTA bidirectionally affected locomotion and reward
reinforcement, respectively. In addition to the PPN, midbrain neurons also receive
cholinergic signaling from the LDT (
Hassan and Benarroch, 2015
;
Maskos, 2008
;
Mena-
Segovia et al., 2008
). Previous studies reveal that LDT ChAT neurons project preferentially
to the VTA and sparsely to the SNc (
Hassan and Benarroch, 2015
;
Mena-Segovia et al.,
2008
). However, it is still an open question whether the ChAT projections from the LDT to
the midbrain have similar functions to those from the PPN. To answer this question, we
performed photo-excitation of LDT ChAT terminals in both the VTA and vSNc. We
delivered AAV5-DIO-ChR2-eYFP to the LDT of ChAT-Cre rats; 66% (368/556) of ChAT
neurons contained ChR2 (Fig. 7A, B). In brain slices, photo-excitation of ChR2-eYFP
expressing LDT ChAT somata evoked inward currents and action potentials (Fig. 7C); when
applied to LDT ChAT terminals, blue light evoked inward currents in 22, 18, and 33% of
vSNc, dSNc and VTA neurons, respectively (Fig. 7D, E, F). These proportions were similar
to those of midbrain neurons responding to PPN ChAT terminal stimulation (Fig. 1H, Fig.
7F). We observed that APV+CNQX partially blocked the photocurrents in 5/10 neurons, but
did not change the photocurrents in 5/10 neurons. Meanwhile, Bic did not reduce
photocurrents in all 4 neurons we recorded. All of the remaining currents were eliminated by
MEC (Fig. 7G; Fig. S8C, D). These pharmacological properties are similar to the
photocurrents in response to photo-excitation of PPN ChAT terminals in midbrain except
that nAChRs on glutamatergic terminals had less contribution to these photocurrents (Fig.
7G vs. Fig. 1K).
The above electrophysiological data indicate that the LDT and PPN ChAT neurons may have
similar projections to the vSNc and VTA and therefore mediate similar behavioral outputs.
We performed photo-excitation of LDT ChAT terminals in the vSNc and VTA to test
whether the modulation leads to behavioral results similar to photo-excitation of PPN ChAT
terminals. In open field tests, photo-excitation of neither LDT-to-VTA nor LDT-to-vSNc
ChAT terminals altered locomotion (Fig. 7I, M), in contrast to the involvement of PPN-to-
vSNc projections in locomotion. In CPP assays, conditioning the rats with photo-excitation
of LDT-to-VTA ChAT projections (Fig. 7H; Fig. S8A) increased the time spent in the light-
paired compartment (Fig. 7J, K), while shining blue light on LDT-to-vSNc projections (Fig.
7L; Fig. S8B) to condition ChR2 and eYFP rats caused similar changes in their place
preference (Fig. 7N, O). The results suggest that the PPN and LDT sends similar ChAT
projections to the VTA regulating reward processing; in contrast to the PPN, LDT ChAT
signaling to the vSNc did not alter locomotion.
To map the PPN and LDT ChAT projections to the VTA and vSNc, we injected retrograde
herpes simplex viral (HSV) vectors carrying mCherry- and eYFP-Cre (
Antinone and Smith,
2010
;
Nieh et al., 2015
), respectively, into the vSNc and VTA (Fig. 8A). The virus is taken
up by terminals and transported along the axons to the somata, thereby labeling upstream
neurons in the PPN and LDT. In both the PPN and LDT, VTA and vSNc projecting ChAT
neurons and non-ChAT neurons were intermingled (Fig. 8B, C). The summarized data (Fig.
8D) show that more ChAT neurons projected to the vSNc (PPN: 58%; LDT: 29%) than to
the VTA (PPN: 20%; LDT: 25%), and 14% of ChAT neurons in both the PPN and LDT
projected to both the vSNc and VTA. Compared with the LDT (Fig. 8D), the PPN had
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significantly higher percentage of ChAT neurons projecting to either the vSNc, the VTA, or
both (64% vs. 40%), higher percentage of vSNc projecting neurons (58% vs. 29%), but
lower percentage of VTA projecting ChAT neurons (20% vs. 25%). The PPN and LDT had
similar percentage of ChAT neurons projecting to both the VTA and vSNc (~ 14%) (Fig. 8
D). Consistent with previous studies (
Lammel et al., 2012
;
Mena-Segovia et al., 2008
),
ChAT neurons are not the only neuron population in the PPN and LDT projecting to the
vSNc and VTA (Fig. 8B, C; Fig. S8E). Interestingly, a great majority of vSNc and a
minority of VTA projecting neurons were cholinergic (Fig. S8E).
To clarify whether the projections from these two groups of ChAT neurons are intermingled
in the vSNc and VTA, we delivered AAV5-DIO-eYFP in the PPN and AAV5-DIO-mCherry
in the LDT to trace these projections (Fig. S8F). We observed that the ChAT fibers were
adjacent to each other in both the vSNc and VTA (Fig. S8G). Interestingly, in the vSNc,
PPN ChAT fibers significantly outnumbered LDT fibers, which is consistent with the
retrograde tracing results and the differential roles observed in locomotion tests.
Discussion
The PPN contains heterogeneous cell populations with divergent brain-wide projections
(
Benarroch, 2013
;
Jenkinson et al., 2009
;
Mena-Segovia et al., 2008
;
Morita et al., 2014
). It
is implicated in a broad spectrum of behaviors, including locomotion, reward, and sleep
(
Arnulf et al., 2010
;
Benarroch, 2013
;
Maskos, 2008
). The link between the PPN and these
behaviors has mostly been obtained through neuromodulation with non-cell-specific
pharmacological tools, electrical stimulation, and chemical lesions. Optogenetics (
Gradinaru
et al., 2009
;
Gradinaru et al., 2010
;
Hausser, 2014
;
Walsh et al., 2014
) and chemogenetics
(
Sternson and Roth, 2014
;
Urban and Roth, 2015
) provide tools that enable dissection of
heterogeneous PPN circuits at the level of individual neuronal types. A few studies have
taken advantage of these techniques to selectively enhance the activity of PPN ChAT
neurons, from which it was observed that PPN ChAT neurons significantly modulate sleep
(
Van Dort et al., 2015
) and motor functions (
Pienaar et al., 2015
;
Roseberry et al., 2016
).
However, the behavioral effects of modulating PPN ChAT activity in opposing directions
had yet to be explored.
We applied optogenetics to selectively target and bidirectionally modulate PPN ChAT
neurons both at the somata and at the terminals, and further characterize the projections from
these neurons to midbrain areas. First, we employed techniques that enable intact
visualization of large tissue volume with high resolution (PACT (
Treweek et al., 2015
;
Yang
et al., 2014
), Fig. 1B–C) and imaged the projections of PPN ChAT neurons to midbrain
nuclei. Second, using retrograde HSV, we revealed that PPN ChAT neurons projecting to the
VTA and vSNc are intermingled, and some neurons project to both the VTA and vSNc
besides separate, parallel projections (Fig. 8B–D). Third, in acute brain slices, we applied
optogenetics to stimulate endogenous ACh release which evoked non-synaptic responses in
midbrain neurons through activating nAChRs in the recorded neurons and glutamate
terminals (Fig. 1D–K; Fig. S1). Although some PPN ChAT neurons have been reported to
contain glutamatergic and/or GABAergic marker proteins, it remains controversial whether
these neurotransmitters are co-released from the terminals (
Benarroch, 2013
;
Jenkinson et
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al., 2009
;
Mena-Segovia et al., 2008
;
Roseberry et al., 2016
;
Wang and Morales, 2009
). In
our experiments, the extensive current attenuation by MEC (Fig. 1E–G, J; Fig. S1) suggests
that ACh is the main underlying neurotransmitter and co-release of other neurotransmitters
(e.g. glutamate or GABA) plays a minor, if any, role in PPN ChAT terminals in the
midbrain, consistent with previous studies (
Roseberry et al., 2016
;
Wang and Morales,
2009
).
PPN neurons are known to modulate motor behaviors. Electrical and chemical stimulation of
the PPN enhance locomotion (
Jenkinson et al., 2004
), and nonspecific chemical lesion of the
PPN leads to hypokinesia (
Kojima et al., 1997
;
Masdeu et al., 1994
). Moreover, direct
in
vivo
electrophysiological recordings have shown that during voluntary movement (
Jenkinson
et al., 2009
), imaginary motion, and passive movement (
Tattersall et al., 2014
), PPN neurons
exhibit disparate changes (increased, decreased, or unchanged) in firing rates. These studies
suggest that subsets of PPN neurons may be involved in motor control. We observed that
photo-excitation of PPN ChAT neurons (20 Hz) caused hyperactivity (Fig. 4B) whereas
photo-inhibition caused hypokinesia (Fig. 4C). Furthermore, the hyperactivity evoked by
PPN ChAT-neuron stimulation was blocked by MEC (Fig. S4H,I), again suggesting either
that these neurons do not co-release glutamate or that any co-released glutamate plays only a
minor role in this behavior. These results demonstrate that bidirectional modulation of PPN
ChAT neurons is sufficient to regulate locomotion and may be a potential therapeutic target
for movement disorders involving both hypokinesia and involuntary movement.
In addition to locomotion, the PPN is also involved in reward processing: PPN neuronal
activity changes in response to reward signals (
Norton et al., 2011
), and pharmacological
inhibition or lesion of PPN neurons alters established addiction behavior, such as nicotine
self-administration (
Corrigall et al., 2002
;
Lanca et al., 2000
). However, a causal link to a
specific neuronal population in the PPN, whose modulation can affect reward behavior, has
been lacking. Our results illustrate that optogenetic activation of PPN ChAT neurons lends
positive emotional valence to previously aversive places (Fig. 4F–G). Conversely, inhibition
of PPN ChAT neurons produces place aversion (Fig. 4H, I). Thus, PPN ChAT signaling can
bidirectionally influence reinforcement learning.
A number of elegant studies have revealed complex roles for SNc and VTA neurons in
exploratory activity, habituation, reinforcement, aversion, etc (
Bromberg-Martin et al., 2010
;
Friedman et al., 2014
;
Lerner et al., 2015
;
Roeper, 2013
;
Walsh et al., 2014
). We observed
that stimulating ChAT terminals in the vSNc and VTA respectively modulate locomotion
and reward (Fig. 5), suggesting that vSNc and VTA neurons receiving PPN ChAT
innervation discriminately modulate locomotion and reward. Compared with the locomotor
and reward phenotypes during modulation of PPN ChAT-neuron somata, terminal
stimulation/inhibition tended to bring about smaller effects (Fig. 4B, C, G; Fig. 5C–F; Fig.
6F), suggesting that ChAT connections to the vSNc and VTA may only partially mediate the
roles of PPN ChAT neurons in motor control and reward processing.
Similar to the PPN ChAT neurons (Fig. 1H, K, Fig. S1), LDT ChAT neurons also innervate
midbrain neurons (Fig. 7D–F) through activating nAChRs on both midbrain neurons and
glutamatergic terminals (Fig. 7G; Fig. S8C–D). Optogenetic stimulation of VTA projecting
Xiao et al.
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LDT glutamatergic neurons has previously been found to produce place preference (
Lammel
et al., 2012
). Here, we observed that photoexcitation of LDT-to-VTA ChAT projections
produced similar effects. These dual contributions of glutamatergic and ChAT neurons to
conditioned reinforcement, as well as their physiological role in generating and maintaining
burst firing in VTA neurons (
Chen and Lodge, 2013
;
Lodge and Grace, 2006
), bolster the
role of the LDT in reward processing. By contrast, comparatively little is known about the
LDT’s contribution to locomotion. Our anatomical data showed that LDT ChAT neurons
also projected to the vSNc (Fig. 8C, D; Fig. S8G). However, the stimulation of these
projections
in vivo
did not affect locomotion and reward processing (Fig. 7M–O). Thus,
although the PPN and LDT are similar in neuronal composition, projection pathway, and
their contributions to reward processing, they differ in their modulation of locomotion (Fig.
8E). These differences and similarities align with their unique anatomical features. While the
percentages of VTA-projecting ChAT neurons are comparable between the PPN and the
LDT, noticeably more vSNc-projecting ChAT neurons reside in the PPN (Fig. 8D, E).
Brain-wide persistent augmentation of ChAT tone by genetically enhancing the sensitivity of
nicotinic acetylcholine receptors (nAChRs), the actuators of ChAT transmission, produces a
hyperactivity phenotype (
Drenan et al., 2008
) and facilitates nicotine reward-related
behaviors (
Tapper et al., 2004
). In contrast, the knock-out of major subtype of nAChRs
eliminates nicotine self-administration (
Picciotto et al., 1998
). These mouse genetic studies
suggest that ChAT tone plays critical roles in both locomotion and reward reinforcement.
Our results extend these studies by showing that temporally precise modulation of distinct
ChAT pathways, originating in the mesopontine tegmentum, can exert contrasting actions on
locomotion and reward (Fig. 8E). PPN ChAT neurons regulate locomotion and reward
through divergent pathways connecting to the vSNc and VTA, respectively. A parallel LDT
ChAT projection pathway modulates reward alone. Therefore, to refine the therapeutic
treatment of movement disorders or drug addiction, it may be advantageous to specifically
target discrete mesopontine cholinergic pathways (PPN-to-vSNc for enhancing motor
function, and PPN/LDT-to-VTA for curbing addiction).
Experimental procedures
We used Long Evans wild type and Choline acetyl-transferase (ChAT)-Cre rats with Long
Evans background (
Witten et al., 2011
). Animal husbandry and all experimental procedures
involving rats were approved by the Institutional Animal Care and Use Committee (IACUC)
and by the Office of Laboratory Animal Resources at the California Institute of Technology.
Surgeries for viral delivery, optical fiber implantation, and retrograde tracing
The rats were anesthetized with 2% isofluorane gas/carbogen mixture and stabilized on a
stereotaxic frame (Kopf Instruments). AAV5-Ef1
α
-DIO-ChR2-eYFP, AAV5-Ef1
α
-DIO-
Arch3.0-eYFP, AAV5-Ef1
α
-DIO-eNpHR3.0-eYFP, AAV5-Ef1
α
-DIO-mCherry, or AAV5-
Ef1
α
-DIO-eYFP (0.8 μL, viral concentration is 4X10
12
genome copies, UNC Vector Core)
was injected in the PPN or LDT of ChAT-Cre rats (3 months old), and custom-made optical
fiber guides (300 μm in diameter) were implanted above virus injection sites in the PPN, or
above their projection nuclei (i.e. the vSNc and the VTA). Following surgery, rats were
Xiao et al.
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single-housed on a 12 hrs light/dark cycle until behavioral testing (4–6 months old) or until
electrophysiological recordings (4–7 months old).
For retrograde tracing, we injected herpes simplex virus carrying eYFP- and mCherry-Cre
(MIT Viral Gene Transfer Core) in the VTA and the vSNc, respectively, and allowed the rats
to recover for 3 weeks before imaging eYFP and mCherry in PPN ChAT and non-ChAT
neurons.
Brain slice patch-clamp recordings
Electrophysiological recordings were performed on parasagittal brain slices, using the
protocol described with some modifications (
Xiao et al., 2015
;
Ye et al., 2006
) (see
supplementary materials).
In vivo single-unit recordings with optrodes
ChAT-Cre rats injected with AAV5-DIO-ChR2-eYFP into the PPN were anesthetized and
optrodes, made as previously described (
Gradinaru et al., 2007
), were inserted into the VTA
to track single-unit activity of VTA neurons before, during and after photoexcitation of
ChR2-expressing PPN ChAT terminals in VTA.
Immunohistochemistry
We performed immunohistochemistry with conventional histology to identify ChR2-eYFP-/
Arch3.0-eYFP-positive neurons and processes, ChAT neurons, and DA neurons. To visualize
optogenetically traced projections from PPN ChAT neurons to midbrain, we cleared 1–2 mm
brain sections from ChR2-injected rats with PACT (
Yang et al., 2014
), performed
immunohistochemistry to label ChR2-eYFP fibers and midbrain DA neurons (Fig. 1B, 5B),
immersed the brain sections in refractive index matching solution (RIMS) in 1–2 mm deep
square frames (iSpacer), and imaged the samples with a Zeiss LSM 780 confocal
microscope.
Behavioral Assays
The rats with optical fiber implants were subject to open field and conditioned place
preference (CPP) assays and their behaviors were acquired with a video camera controlled
by Ethovision XT 9 software (
Noldus et al., 2001
).
Data analysis
Statistical analysis was performed by SigmaPlot 11.0 (SPSS Inc.) and OpenEpi (
Dean et al.,
2010
). Values of p < 0.05 were considered significant.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank the entire Gradinaru lab for helpful discussions and Drs. David J. Anderson and Henry A. Lester for
helpful comments on the manuscript. This work was supported by grants to VG: NIH Director’s New Innovator
Xiao et al.
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IDP20D017782-01; NIH/NIA 1R01AG047664-01 (CX is a co-investigator); NIH BRAIN 1U01NS090577;
Heritage Medical Research Institute; Beckman Institute for Optogenetics and CLARITY; Pew Charitable Trust;
Michael J. Fox Foundation; Sloan Foundation. Work in the Gradinaru Laboratory at Caltech is also funded by the
following awards (to VG): NIH/NIMH 1R21MH103824-01; Kimmel Foundation; Human Frontiers in Science
Program; Mallinckrodt Foundation; Gordon and Betty Moore Foundation through Grant GBMF2809 to the Caltech
Programmable Molecular Technology Initiative; GSK Bioelectronics Research Grants; Hereditary Disease
Foundation; Caltech-GIST; Caltech-Amgen; Caltech-CEMI; Caltech-City of Hope Biomedical Initiative. C.X. is
partly supported by Michael J. Fox Foundation. J.B.T. acknowledges the Colvin Postdoctoral Fellowship. K.C. is
supported by the NIH Predoctoral Training in Biology and Chemistry (2T32GM007616-36).
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Highlights
•
Optogenetic characterization of mesopontine cholinergic cells inputs to
midbrain
•
Separable pedunculopontine cholinergic pathways govern locomotion and
reward
•
Laterodorsal tegmental cholinergic inputs to VTA modulates reward
•
Retrograde tracing reveals mesopontine cholinergic collateralization to VTA and
SNc
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Fig. 1. PPN ChAT neurons form functional connections with distinct midbrain areas
(A)
Stereotaxic microinjection of AAV5, carrying Ef1
α
-DIO-ChR2-eYFP, in the PPN of
ChAT-Cre rats.
(B)
Representative Images from a 0.8 mm thick parasagittal midbrain section
cleared with PACT and immune-stained for green fluorescent protein (ChR2-eYFP) and
tyrosine hydroxylase (TH).
(C)
Expanded views of the vSNc and dSNc from
(B)
. VTA
image was from a more medial section.
(D)
Brain-slice recordings were performed on
midbrain neurons. The neurons were selected by their location in the vSNc, dSNc, or VTA,
and their proximity to ChR2-positive fibers (arrows).
(E–G)
Blue light stimulation induced
inward currents in vSNc
,
dSNc, and VTA neurons (upper traces), and consequently
enhanced firing (lower traces in
E–G)
. Blue-light-evoked inward currents
(E, F)
and firing
increases
(E
) were blocked by 10 μM MEC.
(H)
Percentages of neurons in the vSNc (33%,
22/66), VTA (44%, 33/76), and dSNc (24.7%, 19/58) having responses to blue light. Black
and gray stacks indicate the percentages of neurons responding or not responding to light
stimulation, respectively.
(I)
Summary of light-evoked inward currents in vSNc (13.9±1.5
pA), dSNc (11.8±1.5 pA), and VTA neurons (10.2±1.4 pA) (one-way ANOVA: F
(2)
=1.59,
p
=0.22). Numbers of recorded neurons are shown in the blank bars.
(J)
Summary of light
enhancement of firing rates in vSNc (160±20% of baseline), dSNc (191±25% of baseline),
and VTA neurons (169±14% of baseline) (one-way ANOVA: F
(2)
= 0.58, p = 0.58).
(K)
Summary of the effects of 1 μM atropine (128±19%), 10 μM Bic (86±5.5)%, 50 μM APV
and 20 μM CNQX (APV+CNQX_1, partial blocking, 69±8.2%; APV+CNQX_2, complete
blocking, 5±3%) on photo-currents. Compared with control: *
p
<0.05; **
p
<0.01;
Compared with Drug only: ##: p<0.01.
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