Vta Glutamate Neuron Activity Drives Positive Reinforcement Absent Dopamine Co Release Vivien Zell Thomas Steinkellner Nick G Hollon Shelley M Warlow Elizabeth Souter Lauren Faget Avery full chapter pdf docx

VTA Glutamate Neuron Activity Drives
Positive Reinforcement Absent

Dopamine Co-release Vivien Zell &
Thomas Steinkellner & Nick G. Hollon &
Shelley M. Warlow & Elizabeth Souter &
Lauren Faget & Avery C. Hunker & Xin
Jin & Larry S. Zweifel & Thomas S.
Hnasko
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VTA Glutamate Neuron Activity Drives Positive

Reinforcement Absent Dopamine Co-release
Graphical Abstract Authors
Vivien Zell, Thomas Steinkellner,
Nick G. Hollon, ..., Xin Jin,
Larry S. Zweifel, Thomas S. Hnasko
Correspondence
thnasko@health.ucsd.edu
In Brief
Activation of VTA glutamate neurons
leads to dopamine co-release in nucleus
accumbens. Zell et al. genetically block
this dopamine signal to show that VTA
glutamate projections to nucleus
accumbens can reinforce behaviors
independently. These findings establish a
parallel dopamine-independent
mesolimbic pathway capable of
supporting positive reinforcement.
Highlights
d Activation of VTA glutamate neurons can reinforce behavior
d Activation of VTA glutamate neurons also leads to dopamine

(DA) release
d We generated two distinct models to abolish this coincident

DA signal
d VTA glutamate neuron activity can serve as a reinforcer

independent from DA
Zell et al., 2020, Neuron 107, 1–10

September 9, 2020 Published by Elsevier Inc.
https://doi.org/10.1016/j.neuron.2020.06.011 ll
Please cite this article in press as: Zell et al., VTA Glutamate Neuron Activity Drives Positive Reinforcement Absent Dopamine Co-release, Neuron
(2020), https://doi.org/10.1016/j.neuron.2020.06.011
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VTA Glutamate Neuron Activity Drives Positive
Reinforcement Absent Dopamine Co-release
Vivien Zell,1 Thomas Steinkellner,1 Nick G. Hollon,2 Shelley M. Warlow,1 Elizabeth Souter,1 Lauren Faget,1
Avery C. Hunker,3 Xin Jin,2 Larry S. Zweifel,3 and Thomas S. Hnasko1,4,5,*
1Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA
2Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
3Department of Pharmacology, University of Washington, Seattle, WA 98195, USA
4Research Service VA San Diego Healthcare System, San Diego, CA 92161, USA
5Lead Contact
*Correspondence: thnasko@health.ucsd.edu
https://doi.org/10.1016/j.neuron.2020.06.011
SUMMARY
Like ventral tegmental area (VTA) dopamine (DA) neurons, VTA glutamate neuron activity can support positive
reinforcement. However, a subset of VTA neurons co-release DA and glutamate, and DA release might be
responsible for behavioral reinforcement induced by VTA glutamate neuron activity. To test this, we used op-
togenetics to stimulate VTA glutamate neurons in which tyrosine hydroxylase (TH), and thus DA biosynthesis,
was conditionally ablated using either floxed Th mice or viral-based CRISPR/Cas9. Both approaches led to
loss of TH expression in VTA glutamate neurons and loss of DA release from their distal terminals in nucleus
accumbens (NAc). Despite loss of the DA signal, optogenetic activation of VTA glutamate cell bodies or axon
terminals in NAc was sufficient to support reinforcement. These results suggest that glutamate release from
VTA is sufficient to promote reinforcement independent of concomitant DA co-release, establishing a non-DA
mechanism by which VTA activity can support reward-seeking behaviors.
INTRODUCTION et al., 2014, 2018). Optogenetic activation of VTA glutamate cell

bodies or terminals in NAc can support self-stimulation (Wang
The ventral tegmental area (VTA) has a pivotal role in the control et al., 2015; Yoo et al., 2016), but their activation has also been
of motivated behaviors. Dopamine (DA) transmission from VTA shown to drive apparent avoidance in some behavioral assays
to nucleus accumbens (NAc) and other limbic structures contrib- (Qi et al., 2016; Yoo et al., 2016). This avoidance behavior might
utes to the processes underlying motivated behavior and behav- be a paradoxical consequence resulting from a preference for
ioral reinforcement and is a key structure for the initiation of drug short duration trains of VTA glutamate neuron stimulation (Yoo
addiction (Ikemoto and Bonci, 2014). DA release from VTA neu- et al., 2016). Alternatively, glutamate release from VTA terminals
rons is sufficient to support positive reinforcement, reward in NAc might drive avoidance through preferential activation of
learning, and to invigorate reward seeking; thus, DA release is NAc interneurons, even though DA co-release from a subset of
often conceptualized as a primary reward signal (Berridge these neurons contributes to reward (Qi et al., 2016). Thus, it re-
et al., 2009; Fields et al., 2007; Keiflin and Janak, 2015). Howev- mains unclear whether the reward signals supporting self-stimu-
er, increasing evidence has established important functional and lation of VTA glutamate neurons are mediated by glutamate per
physiological roles for non-DA neurons in VTA, including those se, or by the concomitant co-release of DA.
that release glutamate or GABA (Bariselli et al., 2016; Pupe Here, we tested whether VTA glutamate projections to NAc
and Wallén-Mackenzie, 2015). Further complexity is added by drive reward independently of DA co-release. We generated con-
the fact that some VTA neurons co-release multiple neurotrans- ditional knockout (cKO) mice in which Tyrosine hydroxylase (Th)
mitters (Hnasko and Edwards, 2012; Trudeau et al., 2014). was specifically knocked out from VGLUT2-expressing neurons
Approximately 36% of NAc-projecting VTA neurons express to disrupt DA synthesis. Optogenetic stimulation of VTA gluta-
the type-2 vesicular glutamate transporter (VGLUT2) (Yamaguchi mate neurons or their axon terminals led to robust DA release in
et al., 2011), and electrophysiological data demonstrate that NAc of controls but not cKO mice. Further, while cKOs displayed
essentially all medium spiny neurons (MSNs) in medial NAc shell only a minor reduction in basal locomotor activity, loss of DA co-
receive glutamatergic input from VTA (Hnasko et al., 2012; Min- release did not abolish or otherwise impact self-stimulation or
gote et al., 2015, 2019; Stuber et al., 2010; Yoo et al., 2016) as other measures of behavioral reinforcement. To overcome poten-
do striatal cholinergic interneurons (Cai and Ford, 2018; Chuhma tial compensatory adaptations associated with the loss of Th from
Neuron 107, 1–10, September 9, 2020 Published by Elsevier Inc. 1

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Figure 1. Disruption of TH Expression and DA Release from VTA Glutamate Neurons

(A) Genetic disruption of floxed Th gene in Slc17a6Cre (VGLUT2-Cre) mice.
(B) Coronal sections through VTA labeled with antibodies against TH and dopamine transporter (DAT), plus RNAscope probes targeting VGLUT2 transcripts.
Neurons co-expressing TH and VGLUT2 (arrows) in controls were virtually absent in cKO; scale, 500 mm (top) and 100 mm (bottom); t test, ****p < 0.0001 (n = 3
mice/group).
(legend continued on next page)
2 Neuron 107, 1–10, September 9, 2020

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VTA glutamate neurons during development, we also imple- medial VTA of control and cKO mice (Figure 1C). Approximately
mented a virus-based CRISPR/Cas9 approach to disrupt Th 15% of mCherry+ VTA glutamate cell bodies co-labeled with TH
expression and DA release from VTA glutamate neuron in adult in control mice, but co-expressing cells were virtually absent in
mice, to similar effect. Together, our results indicate that VTA cKOs, further confirming the conditional loss of TH from
glutamate neuron activity can support robust behavioral rein- VGLUT2+ VTA neurons (Figure 1D).
forcement independent of their ability to recruit DA release. To test whether the loss of TH from VTA glutamate neurons
was sufficient to abolish evoked DA release, we used fast-scan
RESULTS cyclic voltammetry (FSCV) in brain slices through the medial
NAc shell (Figure 1E). Optogenetic stimulation of VTA glutamate
Conditional Ablation of DA Synthesis in VTA Glutamate terminals in the NAc produced a robust frequency-dependent in-
Neurons crease in evoked DA in controls but not the cKO (Figure 1F).
To test the contribution of DA co-transmission to VTA glutamate However, bath application of DA (10 mM, 10 min) rescued
neuron function, we generated mice with conditional deletion ChR2-evoked DA transients in cKOs (Figure 1G), demonstrating
of Th (Darvas and Palmiter, 2010) selectively in VGLUT2-Cre- that VTA glutamate terminals retain the capacity to release DA
expressing neurons (Figure 1A). Brain sections from cKO when substrate is available.
(Slc17a6+/Cre; Thlox/lox), and heterozygous control littermates (con- Importantly, we also demonstrate that DA release is absent
trol: Slc17a6+/cre; Th+/lox) were used to confirm the loss of TH in following optogenetic stimulation of VTA glutamate neurons
VTA glutamate neurons. Using a combination of in situ hybridiza- in vivo. In this experiment, ChR2 was expressed in VTA gluta-
tion and immunohistochemistry, we found that 14% of neurons mate neurons, optic fibers were implanted in VTA, and carbon fi-
expressing VGLUT2 mRNA were also TH+ in control mice, bers were implanted in medial NAc (Figures 1H and S4). Optoge-
whereas TH+ VGLUT2 neurons were essentially absent in the netic stimulation (1 s) elicited frequency-dependent DA release in
VTA of cKO mice (Figure 1B). Although numerous studies demon- control but not cKO mice (Figure 1I). Together, these results
strate that 10%–20% of VTA DA neurons express VGLUT2 in the demonstrate the functional loss of NAc DA release in cKO
adult, a much higher proportion transiently express VGLUT2 dur- mice upon optogenetic stimulation of VTA glutamate neurons.
ing development (Bérubé-Carrière et al., 2009; Kouwenhoven
et al., 2019; Mendez et al., 2008; Steinkellner et al., 2018). As a VTA Glutamate Transmission Is Similar at Both D1
consequence, we observed 80% decrease in the number of and D2 MSNs and Is Intact in cKO
TH-immunoreactive cell bodies in both VTA and SNc of cKOs (Fig- To test whether glutamate release was altered in cKO mice, we
ures S1A and S1B). We also observed a corresponding decrease measured excitatory postsynaptic currents (oEPSCs) evoked by
in TH-immunoreactive DA axon terminals in the NAc, and caudate/ optogenetic stimulation of VTA glutamate terminals in NAc (Fig-
putamen (CPu) (Figures S1C and S1D). In contrast, DA transporter ure S3A). We found 6,7-dinitroquinoxaline-2,3(1H,4H)-dione
(DAT) immunoreactivity was unaltered, indicating that DA neuronal (DNQX)-sensitive oEPSCs of similar amplitude in cKO and control
architecture was intact in cKO mice. Despite the reduced TH mice (Figure S3B), suggesting that, unlike DA release, glutamate
expression, we observed only a modest reduction in baseline transmission was intact. We also measured paired-pulse ratios
locomotor activity in cKO mice, and locomotor responses to (PPRs) and found substantial paired-pulse depression in both ge-
L-DOPA, amphetamine, or the D1R agonist SKF81297 were notypes, though depression was reduced in the cKO (Figure S3C),
similar between cKO and control mice, suggesting intact psycho- which could be due to reduced DA auto-receptor activation at
motor function (Figure S2). these excitatory pre-synaptic terminals (Adrover et al., 2014;
Chuhma et al., 2009). The onset delay and deactivation time con-
Disruption of DA Transmission from VTA Glutamate stant (t) of oEPSCs did not differ across genotypes (Figure S3D).
Neurons D1- and D2-type MSNs comprise >95% of NAc neurons but
To control VTA glutamate neuron activity, we infused an adeno- have distinct functional roles (Cole et al., 2018; Kravitz et al.,
associated virus (AAV) engineered for Cre-dependent expres- 2012; Lobo and Nestler, 2011; Thibeault et al., 2019). To test
sion of Channelrhodopsin-2:mCherry (ChR2:mCherry) into the whether VTA glutamate neurons might preferentially impact
(C) Strategy for selective expression of ChR2:mCherry in VGLUT2 VTA neurons.

(D) Coronal sections show colocalization of TH and ChR2:mCherry is nearly absent in the cKO; scale, 100 mm (top) and 50 mm (bottom); t test, ***p = 0.0003 (n = 3
mice/group).
(E) Schematic of ex vivo FSCV to measure DA evoked from VTA glutamate terminals.
(F) Photostimulation elicited frequency-dependent DA transients in the medial NAc shell in control but not cKO; RM two-way ANOVA, main effect of frequency,
F(4, 28) = 11.0, p < 0.0001; genotype, F(1, 7) = 22.5, p = 0.002 and interaction, F(4,28) = 9.6, p < 0.0001. Sidak’s multiple comparisons test ****p < 0.0001, **p < 0.01
(control n = 4, cKO n = 5 mice). Right insets show example color plots following 20 Hz photostimulation. Voltammogram scale: 2 nA; 0.3 V.
(G) Bath application of DA (10 mM, 10 min) rescued opto-triggered DA transients in cKO; RM one-way ANOVA, main effect of treatment, F(16, 64) = 17.5, p < 0.0001
(n = 5 mice). Bonferroni’s multiple comparisons test ****p < 0.0001. Right insets show example color plots following 20 Hz photostimulation at 0 (top) and 30
(bottom) min after DA bath application/wash. Voltammogram scale: 2 nA; 0.3 V.
(H) Schematic of in vivo FSCV to measure evoked DA from VTA glutamate terminals.
(I) Photostimulation of VTA glutamate cell bodies elicited frequency-dependent DA signals in NAc; Friedman test, main effect of frequency in controls, p < 0.0001
(n = 4 mice) but not cKO mice, p = 0.43 (n = 5). Voltammogram scale: 1 nA; 0.3 V.
Data are represented as mean ± SEM. See also Figures S1–S4.
Neuron 107, 1–10, September 9, 2020 3

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these sub-populations, we used D1R-Cre 3 VGLUT2-Cre or We next tested mice in the RTPP procedure where they were
A2a-Cre 3 VGLUT2-Cre double-transgenic mice to achieve con- provided free access to two compartments: entrance into active
ditional expression of (1) ChR2:EYFP in VTA VGLUT2 neurons side A-triggered 40-Hz optogenetic stimulation that discontin-
and (2) mCherry in D1 or D2 (A2a) subtype-defined NAc MSNs ued upon entry into side B (Figure 3A). When targeting VTA gluta-
(Figure S3E). To our knowledge, there is no VGLUT2 expressing mate fibers in NAc, both genotypes spent less time in the active
neurons in the NAc nor D1R or A2a expressing neurons in VTA, chamber and switched their side preference to the inactive
cf. Allen Brain Atlas: Slc17a6 (73818754), Drd1 (352), and chamber when side B was switched to active; mCherry-express-
Adora2a (72109410) (Lein et al., 2007). We observed oEPSCs ing mice showed no significant preference or avoidance (Fig-
at both MSN types, and no significant difference was detected ure 3B). Both genotypes also showed an increased approach
between D1R+ versus putative D1R– nor A2a+ versus putative rate into the active side compared to mCherry-expressing
A2a– neurons (Figure S3F). PPR, delay, and t were also un- mice (Figure 3C), and there was no significant difference be-
changed across MSN subtypes (Figures S3G and S3H). tween controls and cKO mice that lack the evoked DA signal.
Together these results show that VTA glutamate neurons do Mice with implants dorsal to VTA were used to target the cell
not make preferential contact onto D1-type compared to D2- bodies (Figure 3D) and displayed similar patterns of active side
type projecting MSNs. avoidance (Figure 3E), with increased approach rates over time
(Figure 3F), and no effect of genotype on these behaviors.
Self-stimulation of VTA Glutamate Neurons in the Together, these results demonstrate that VTA glutamate neuron
Absence of DA Co-release activation can drive self-stimulation and approach behavior in
Previous work from our lab demonstrated that optogenetic activa- the absence of concomitant DA co-release, suggesting VTA
tion of VTA glutamate cell bodies or their terminals in NAc can sup- glutamate neurons represent a DA-independent mesolimbic
port intracranial self-stimulation (ICSS) (Wang et al., 2015; Yoo reward circuit.
et al., 2016). Curiously, mice that self-stimulated these neurons
in an operant task showed a counter-intuitive frequency-depen- CRISPR/Cas9 Disruption of DA Transmission from VTA
dent avoidance for their stimulation in a real-time place preference Glutamate Neurons
(RTPP) assay. However, this apparent avoidance was accompa- While the above data suggest that VTA glutamate neurons can
nied by an increased rate of entries into the active side and a pref- convey a reinforcement signal that does not depend on
erence for short duration trains of VTA glutamate neuron stimula- increased DA release, the disruption of DA synthesis and release
tion (Yoo et al., 2016). While these data imply distinct functional included neurons that do not express VGLUT2 in the adult due to
roles for VTA DA and glutamate neurons in positive reinforcement, transient developmental expression of VGLUT2 in a wider popu-
optogenetic activation of VTA glutamate neurons also increases lation of midbrain DA neurons (Figure S1). Further, loss of TH
DA signaling in NAc (Figure 1) through transmitter co-release from early development might cause compensatory adaptations
and/or local excitatory synapses onto VTA DA neurons (Dobi in cKO mice. To bypass these issues, we turned to a recently
et al., 2010; Qi et al., 2016; Yoo et al., 2016). Therefore, to test developed viral-based CRISPR/Cas9 approach to disrupt DA
the contribution of DA to behavioral reinforcement driven by VTA synthesis and release selectively from adult VTA neurons that ex-
glutamate neurons, we performed ICSS and RTPP using cKO press VGLUT2. This approach relied on a single AAV vector for
mice that lack the ability to synthesize and co-release DA. Cre-dependent expression of Staphylococcus aureus Cas9
ChR2:mCherry or mCherry was expressed in VTA glutamate (SaCas9) and U6 promoter-driven expression of a single-guide
neurons in control and cKO mice, and optic fibers were bilaterally RNA to induce indel mutations in the Th gene (sgTh) (Hunker
implanted dorsal to medial NAc shell to target axon terminals. et al., 2020). This AAV vector (or sgCTRL) was infused together
Mice were first tested on a 2-hole ICSS assay where nosepokes with AAV-DIO-ChR2:mCherry into VTA of VGLUT2:Cre mice
on hole ‘‘A’’ (active) triggered a laser to deliver a 1 s 40-Hz stim- (Figure 4A). Immunohistochemical analysis showed 19% of
ulus, while nosepokes on hole ‘‘B’’ (inactive) were without effect Cas9-expressing (VGLUT2) VTA neurons expressed TH in the
(Figure 2A). This frequency and pattern of stimulation was sgCTRL group, but only 2% expressed TH in the sgTh group
selected because prior works show that mice can discriminate (Figure 4B). Using ex vivo FSCV, we found substantially reduced
and show preference for it and because VTA glutamate neurons ChR2-evoked DA release in the sgTh- compared to sgCTRL-in-
can fire at similar frequency in response to salient stimuli (Root jected brains, demonstrating successful disruption of DA release
et al., 2018a; Wang et al., 2015; Yoo et al., 2016). Across the first from VTA glutamate neurons (Figures 4C and 4D). Exogenous DA
4 test sessions mice displayed strong preference for the active application rescued evoked DA transients from the sgTh brains,
hole with no significant differences detected between genotypes confirming that VTA glutamate terminals retained the ability to
(Figures 2B). Over the subsequent 4 sessions holes ‘‘A’’ and ‘‘B’’ recycle and release DA when substrate was available (Figure 4E).
were swapped, and both genotypes switched their preference to The sgCTRL and sgTh animals were also tested for self-stim-
engage with the active hole; mCherry-expressing control mice ulation of VTA glutamate neurons using the 2-hole ICSS task. In
did not show a preference or a switch and performed few nose- accordance with our results obtained in cKO mice, sgCTRL and
pokes (Figures 2B and 2C). We also tested a separate cohort of sgTh mice displayed an equivalent preference for the nosepoke
animals with optic fibers implanted dorsal to VTA glutamate cell hole coupled to optogenetic stimulation of VTA glutamate termi-
bodies rather than NAc terminals (Figure 2D); these animals also nals in NAc and switched preference when we switched the
displayed a robust preference for the active nosepoke with no active nosepoke (Figure 4F). We observed similar results
difference between genotype (Figures 2E and 2F). when the active nosepoke hole was coupled to optogenetic

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Figure 2. Self-stimulation of VTA Glutamate Neurons in cKO Mice

(A) Approach for self-stimulation of VTA glutamate terminals in NAc.
(B) ChR2:mCherry-expressing control and cKO mice displayed equivalent preference for the active nosepoke hole (days 1–4, hole A) and followed the active side
after a switch (days 5–8, hole B); RM two-way ANOVA, main effect of day, F(7, 175) = 18.6, p < 0.0001 and interaction, F(14,175) = 7.2, p < 0.0001 (n = 10 control,
n = 10 cKO, n = 8 mCherry). Tukey-Kramer’s multiple comparisons test ***p < 0.001, **p < 0.01, *p < 0.05.
(C) ChR2:mCherry-expressing control and cKO increase the number of photostimulations earned across sessions compared to mCherry-expressing mice; RM
two-way ANOVA, main effect of genotype, F(2, 25) = 5.2, p = 0.013 and interaction, F(14, 175) = 1.8, p = 0.043. Sidak’s multiple comparisons test ***p < 0.001, **p <
0.01, *p < 0.05.
(D) Approach for self-stimulation of VTA glutamate cell bodies.
(E) Control and cKO mice developed an equivalent preference for the active nosepoke hole; RM two-way ANOVA, main effect of day, F(3,57) = 15.9, p < 0.0001.
(F) Control and cKO mice earned an equivalent number of photostimulations per session across days; RM two-way ANOVA, main effect of day, F(3,57) = 27.2, p <
0.0001 (n = 11 control, n = 10 cKO mice).
Data are represented as mean ± SEM. See also Figure S4.
stimulation of glutamate cell bodies in VTA (Figure 4G). These 2019; Stuber et al., 2010). In addition, VTA glutamate neurons
data provide additional independent evidence that VTA gluta- make local synapses onto VTA DA neurons, representing
mate neuron activity can support positive reinforcement absent another mechanism by which they might elicit increased DA
concomitant increases in DA signaling. signaling (Dobi et al., 2010; Qi et al., 2016; Yoo et al., 2016). In
this report, we used conditional ablation of TH in glutamate neu-
DISCUSSION rons to test the contribution of DA transmission for reinforcement
behaviors evoked by VTA glutamate neuron activity. We found
Prior work has demonstrated that, like VTA DA neurons, optoge- that, while VTA glutamate neuron stimulation evokes DA release,
netic activation of VTA glutamate neurons can support approach this DA signal was not necessary for the activation of VTA gluta-
behaviors (Wang et al., 2015; Yoo et al., 2016). However, the mate neurons to support self-stimulation and approach behav-
extent to which VTA glutamate neurons rely on DA signaling to iors. We thus propose that glutamate and DA projections from
drive reward behaviors remained unclear. Indeed, a subset of VTA to NAc represent partially overlapping but functionally
VTA glutamate neurons co-express DA markers and co-release distinct parallel pathways that can independently and distinc-
DA directly (Kawano et al., 2006; Lavin et al., 2005; Mingote et al., tively support positive reinforcement.

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Figure 3. Behavior in Real-Time Place Preference Assay Is Unchanged by the Loss of DA Co-release in cKO Mice
(A) Schematic of the real-time place preference assay (RTPP) with stimulation of VTA glutamate terminals in NAc.
(B) ChR2:mCherry-expressing control and cKO mice spent less time in the active side compared to mCherry-expressing mice; RM two-way ANOVA, main effect
of day, F(6, 150) = 23.8, p < 0.0001 and interaction, F(12,150) = 3.7, p < 0.0001 (n = 10 control mice, n = 10 cKO, n = 8 mCherry). Tukey-Kramer’s multiple comparisons
test ***p < 0.001, **p < 0.01, *p < 0.05.
(C) ChR2:mCherry-expressing control and cKO displayed an increase in approach rate into the active compartment; RM two-way ANOVA, main effect of ge-
notype, F(2, 25) = 17.1, p < 0.0001 and interaction F(25,150) = 7.3, p < 0.0001. Tukey-Kramer’s multiple comparisons test ****p < 0.0001, ***p < 0.001.
(D) Schematic of RTPP with stimulation of VTA glutamate cell bodies.
(E) Control and cKO mice spent less time in the side paired with laser; RM two-way ANOVA, main effect of day, F(6,78) = 14.6, p < 0.0001.
(F) Control and cKO displayed a concomitant increase in approach rate across sessions; RM two-way ANOVA, main effect of day, F(6,78) = 6.6, p < 0.0001 (n = 11
control, n = 10 cKO mice). Data are represented as mean ± SEM. See also Figure S4.
We consistently found that 10%–20% of VTA neurons that ex- of DA co-release from VTA glutamate neurons in our models.
press VGLUT2 in adult mouse VTA also co-express the DA Importantly, our data also suggest that any DA release that might
marker TH. This is in line with other recent reports and demon- be evoked through feedforward mechanisms or through recy-
strates that there is a larger population of non-DA glutamate neu- cling of extracellular DA released by other neurons is abrogated
rons concentrated in medial VTA of mice (Hnasko et al., 2012; in the cKO mice that lacked evoked DA release in vivo. Despite
Yamaguchi et al., 2015). We provide new evidence that both the loss of DA signals in our cKO and sgTh animals, optogenetic
in vivo and ex vivo optogenetic stimulation of glutamate cell activation of VTA glutamate neurons supported reinforcement
bodies in VTA or their terminals can directly drive DA release in and approach behaviors in our ICSS and RTPP assays that
the medial NAc. Dual TH/VGLUT2-expressing neurons were were indistinguishable from mice with intact DA/glutamate co-
virtually absent in our cKO and sgTh animals, as was optoge- transmission. Similar results were obtained whether optogenetic
netic-evoked DA release, demonstrating successful disruption stimulation was delivered at the level of VTA glutamate cell

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Figure 4. Self-stimulation of VTA Glutamate Terminals in NAc following CRISPR/Cas9 Disruption of TH and DA Co-release
(A) Schematic of AAV vectors and approach for CRISPR/Cas9 conditional deletion of Th from VTA glutamate neurons with ChR2:mCherry expression.
(B) Coronal sections show co-localization of TH in Cas9-expressing neurons in sgCTRL mice is absent in sgTh mice; scale, 100 mm (top), 50 mm (bottom); t test,
**p = 0.0025 (n = 4 mice).
(C) Schematic of ex vivo FSCV approach.
(D) Photostimulation evoked DA transients from VTA glutamate terminals in medial NAc shell of sgCTRL but not sgTh mice; RM two-way ANOVA, main effect of
frequency, F(4, 24) = 12.9, p < 0.0001; treatment F(1, 6) = 7.1, p = 0.038 and interaction, F(4,24) = 8.1, p = 0.0003, Sidak’s multiple comparisons test **p < 0.01, *p <
0.05 (n = 4 mice per group). Right insets show example color plots following 20 Hz photostimulation.
(E) Time course of opto-triggered DA transient sizes after bath application/wash; RM one-way ANOVA, main effect of treatment, F(16, 48) = 15.5, p < 0.0001.
Bonferroni’s multiple comparisons test ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 (n = 4 mice). Right insets show example color plots following 20 Hz
photostimulation at 0 min (top) and 30 min (bottom) after DA bath application/wash. Voltammogram scale: 2 nA; 0.4 V.
(F) Left: approach for self-stimulation of VTA glutamate terminals in NAc. Right: mice injected with either sgTh or sgCTRL develop an equivalent preference for the
active nosepoke hole; RM two-way ANOVA, main effect of day, F(7,126) = 5.7, p < 0.0001 (n = 7 sgTh, n = 8 sgCTRL, n = 6 mCherry mice).
(legend continued on next page)

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bodies, or their terminals in NAc. Thus, a concomitant increase in the cKO. This might indicate a lower release probability in the
DA signaling does not appear necessary for behavioral reinforce- cKO, reduced D2-autoreceptor inhibition, or reduced glutamate
ment driven by VTA glutamate neuron activity. quantal content as a consequence of reduced vesicular synergy
Consistent with our previous observations, we found that the (Adrover et al., 2014; Aguilar et al., 2017; Hnasko and Edwards,
same animals that readily self-stimulated VTA glutamate neu- 2012; Hnasko et al., 2010; Silm et al., 2019).
rons displayed counter-intuitive avoidance behavior in the Previous studies showed that VTA glutamate synapses might
RTPP assay (Yoo et al., 2016). However, the reduction in time preferentially impact striatal interneurons (Cai and Ford, 2018;
spent in the active chamber was accompanied by an increased Chuhma et al., 2014, 2018; Qi et al., 2016). However, VTA gluta-
approach rate to that same active chamber, and both of these mate neurons also directly synapse onto and can alter in vivo firing
findings were also observed in the cKO. This pattern of behavior rate of NAc projection neurons (Mingote et al., 2015; Stuber et al.,
increases the number of short duration stimuli and probably re- 2010; Tecuapetla et al., 2010; Wang et al., 2017). Yet it had not
flects mice adjusting their behavior over time to optimize their been determined whether D1 or D2 neurons, which play different
preferred pattern of stimulation. Indeed, using an instrumental functional roles, might be differentially connected to glutamate
assay, mice displayed strong preference for brief stimulation neurons from VTA. Although more studies will be necessary to
of VTA glutamate neurons, and by comparison longer stimula- assess how VTA inputs onto distinct NAc cell types influence their
tion of VTA DA neurons was preferred (Yoo et al., 2016). We activity and effect on behavior in vivo, our data comparing oEPSC
therefore hypothesize that both VTA DA and glutamate neuron properties suggest no clear bias in the connectivity of VTA gluta-
activity can drive positive reinforcement but that they do so in mate synapses on to MSN cell type.
distinctive ways and that reward driven by VTA glutamate Previous attempts at understanding the relative contributions
neuron activity does not depend on accompanying increases of dual DA/glutamate co-transmission focused primarily on
in DA release. isolating the DA component. Mingote and colleagues achieved
An alternative hypothesis is that different subpopulations of VTA a partial reduction in glutamate release from midbrain DA neu-
glutamate neurons differentially drive reward and aversion through rons with intact DA transmission and observed a decrease in
different mechanisms, similar to different projection-defined VTA amphetamine sensitization and a potentiation of latent inhibition,
DA sub-populations (de Jong et al., 2019). Indeed, initial investiga- implying a role for glutamate co-release in motivational salience
tions of VTA glutamate neuron activity in vivo shows a diversity of and context discrimination (Mingote et al., 2017). Conditional
responses to salient rewarding and aversive stimuli (Montardy deletion of VGLUT2 from DAT-expressing DA neurons abolished
et al., 2019; Root et al., 2018b). Thus, one population of VTA gluta- evoked oEPSCs in NAc and other terminal structures (Soden
mate neurons could drive approach through intra-VTA excitation et al., 2016; Stuber et al., 2010; Tritsch et al., 2012) and led to
of DA neurons, while another population may drive avoidance reduced psychostimulant-induced locomotor activity and
via projections to NAc or other distal structures. In this case, reward-seeking behaviors (Alsiö et al., 2011; Birgner et al.,
perhaps different patterns of stimulation differentially recruit sub- 2010; Hnasko et al., 2010; Wallén-Mackenzie et al., 2010), while
populations of VTA glutamate neurons resulting in complex se- self-stimulation of DA neurons was unaffected (Wang et al.,
quences of approach and avoidance. However, our present find- 2017). Loss of VGLUT2 also sensitized DA neurons to neuro-
ings argue against this mechanism, as mice that lack DA release toxins, suggesting a role for VGLUT2 in selective DA neuron
evoked by VTA glutamate neuron stimulation continue to self- vulnerability (Shen et al., 2018; Steinkellner et al., 2018).
stimulate VTA glutamate cell bodies or terminals in NAc. Further, Here, we report the first results that isolate the glutamate
our finding that mice that self-stimulate in an operant assay can component of VTA projection neurons using conditional genetic
show apparent place avoidance in the RTPP assay caution against manipulations. We found that optogenetic stimulation of VTA
interpreting widely used measures of avoidance behaviors as glutamate neurons is sufficient to drive positive reinforcement
conclusively indicative of aversion. Instead, we suggest that a and that this appears to be independent of concurrent DA
reduced approach rate or evidence of a negative affective reaction release. Glutamate neurons therefore represent an alternate
(e.g., conditioned avoidance or innate fear responses) might be VTA pathway with unique features by which mesolimbic projec-
necessary to conclude a stimulus is aversive. In any case, future tions could integrate reward and motivational signals to control
studies will be necessary to reconcile the heterogeneity in VTA reward-seeking behaviors.
glutamate responses found in vivo with their functional roles in
reward-related or other behaviors.
STAR+METHODS
We also examined the impact of ablated DA synthesis and
release from VTA VGLUT2 neurons on glutamate neurotransmis-
Detailed methods are provided in the online version of this paper
sion in the NAc using electrophysiology. Light-triggered DNQX-
and include the following:
sensitive oEPSCs were recorded in both control and cKO
mice, with no difference in their characteristics. However, we d KEY RESOURCES TABLE
observed a significant reduction in paired-pulse depression in d RESOURCE AVAILABILITY
(G) Left: approach for self-stimulation of VTA glutamate cell bodies. Right: mice injected with either sgTh or sgCTRL develop an equivalent preference for the
active nosepoke hole; RM two-way ANOVA, main effect of day, F(7, 84) = 43.1, p < 0.0001 and interaction, F(7, 84) = 2.7, p = 0.015 (n = 6 sgTh, n = 8 sgCTRL mice).
Sidak’s multiple comparisons test *p < 0.05.
Data are represented as mean ± SEM. See also Figure S4.

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B Lead Contact Bariselli, S., Glangetas, C., Tzanoulinou, S., and Bellone, C. (2016). Ventral
B Materials Availability tegmental area subcircuits process rewarding and aversive experiences.
B Data and Code Availability J. Neurochem. 139, 1071–1080.
d EXPERIMENTAL MODEL AND SUBJECT DETAILS Berridge, K.C., Robinson, T.E., and Aldridge, J.W. (2009). Dissecting compo-
B Animals nents of reward: ‘liking’, ‘wanting’, and learning. Curr. Opin. Pharmacol.
9, 65–73.
d METHOD DETAILS
B Stereotactic surgery Bérubé-Carrière, N., Riad, M., Dal Bo, G., Lévesque, D., Trudeau, L.É., and
Descarries, L. (2009). The dual dopamine-glutamate phenotype of growing
B Immunohistochemistry
mesencephalic neurons regresses in mature rat brain. J. Comp. Neurol. 517,
B In situ hybridization followed by immunohistochemistry 873–891.
B Electrophysiological recordings from adult brain slices
Birgner, C., Nordenankar, K., Lundblad, M., Mendez, J.A., Smith, C., le
B Fast-scan cyclic voltammetry recordings Grevès, M., Galter, D., Olson, L., Fredriksson, A., Trudeau, L.E., et al. (2010).
B Behavioral studies VGLUT2 in dopamine neurons is required for psychostimulant-induced behav-
d QUANTIFICATION AND STATISTICAL ANALYSIS ioral activation. Proc. Natl. Acad. Sci. USA 107, 389–394.
Cai, Y., and Ford, C.P. (2018). Dopamine Cells Differentially Regulate Striatal
SUPPLEMENTAL INFORMATION Cholinergic Transmission across Regions through Corelease of Dopamine
and Glutamate. Cell Rep. 25, 3148–3157.
Supplemental Information can be found online at https://doi.org/10.1016/j. Chuhma, N., Choi, W.Y., Mingote, S., and Rayport, S. (2009). Dopamine
neuron.2020.06.011. neuron glutamate cotransmission: frequency-dependent modulation in the
mesoventromedial projection. Neuroscience 164, 1068–1083.
ACKNOWLEDGMENTS Chuhma, N., Mingote, S., Moore, H., and Rayport, S. (2014). Dopamine neu-
rons control striatal cholinergic neurons via regionally heterogeneous dopa-
We thank Dr. Martin Darvas for providing TH floxed mice and Dr. Nicholas Spit- mine and glutamate signaling. Neuron 81, 901–912.
zer for guidance on combining RNAscope with immunohistochemistry. This
Chuhma, N., Mingote, S., Yetnikoff, L., Kalmbach, A., Ma, T., Ztaou, S., Sienna,
work has been supported by the NIH (R01DA036612, K99AG059834,
A.C., Tepler, S., Poulin, J.F., Ansorge, M., et al. (2018). Dopamine neuron
K99MH119312, and K99DA046514), VA (I01 BX003759), a NIDA-INSERM
glutamate cotransmission evokes a delayed excitation in lateral dorsal striatal
postdoctoral Drug Abuse Research Fellowship, a Schrӧdinger postdoctoral
cholinergic interneurons. eLife 7. Published online October 8, 2018. https://
fellowship from the Austrian Science Fund (J3656-B24), and a Jonas Salk
doi.org/10.7554/eLife.39786.
Fellowship.
Clark, J.J., Sandberg, S.G., Wanat, M.J., Gan, J.O., Horne, E.A., Hart, A.S.,
Akers, C.A., Parker, J.G., Willuhn, I., Martinez, V., et al. (2010). Chronic micro-
AUTHOR CONTRIBUTIONS
sensors for longitudinal, subsecond dopamine detection in behaving animals.
V.Z. and T.S.H. conceived the project and designed experiments. N.G.H. con- Nat. Methods 7, 126–129.
ducted and analyzed the in vivo FSCV recordings with support from X.J. V.Z. Cole, S.L., Robinson, M.J.F., and Berridge, K.C. (2018). Optogenetic self-stim-
conducted and analyzed the electrophysiological recordings. V.Z. conducted ulation in the nucleus accumbens: D1 reward versus D2 ambivalence. PLoS
and analyzed the behavioral and optogenetic studies with the help of E.S., L.F., ONE 13, e0207694.
and S.W. V.Z. conducted and analyzed ex vivo FSCV recordings with support
Darvas, M., and Palmiter, R.D. (2010). Restricting dopaminergic signaling to
from N.G.H. T.S. conducted and analyzed ISH/IHC experiments and behav-
either dorsolateral or medial striatum facilitates cognition. J. Neurosci. 30,
ioral pharmacology. A.C.H. and L.Z. provided AAV CRISPR/Cas9 constructs.
1158–1165.
V.Z. and T.S.H. supervised all aspects of the work. V.Z. and T.S.H. wrote the
paper with editorial input from all authors. de Jong, J.W., Afjei, S.A., Pollak Dorocic, I., Peck, J.R., Liu, C., Kim, C.K., Tian,
L., Deisseroth, K., and Lammel, S. (2019). A Neural Circuit Mechanism for
Encoding Aversive Stimuli in the Mesolimbic Dopamine System. Neuron
DECLARATION OF INTERESTS
101, 133–151.
The authors declare no competing interests. Dobi, A., Margolis, E.B., Wang, H.-L., Harvey, B.K., and Morales, M. (2010).
Glutamatergic and nonglutamatergic neurons of the ventral tegmental area
Received: February 11, 2019 establish local synaptic contacts with dopaminergic and nondopaminergic
Revised: April 21, 2020 neurons. J. Neurosci. 30, 218–229.
Accepted: June 7, 2020
Fields, H.L., Hjelmstad, G.O., Margolis, E.B., and Nicola, S.M. (2007). Ventral
Published: June 30, 2020
tegmental area neurons in learned appetitive behavior and positive reinforce-
ment. Annu. Rev. Neurosci. 30, 289–316.
REFERENCES
Heien, M.L.A.V., Khan, A.S., Ariansen, J.L., Cheer, J.F., Phillips, P.E.M.,
Wassum, K.M., and Wightman, R.M. (2005). Real-time measurement of dopa-
Adrover, M.F., Shin, J.H., and Alvarez, V.A. (2014). Glutamate and dopamine
mine fluctuations after cocaine in the brain of behaving rats. Proc. Natl. Acad.
transmission from midbrain dopamine neurons share similar release proper-
Sci. USA 102, 10023–10028.
ties but are differentially affected by cocaine. J. Neurosci. 34, 3183–3192.
Aguilar, J.I., Dunn, M., Mingote, S., Karam, C.S., Farino, Z.J., Sonders, M.S., Heusner, C.L., Beutler, L.R., Houser, C.R., and Palmiter, R.D. (2008). Deletion
Choi, S.J., Grygoruk, A., Zhang, Y., Cela, C., et al. (2017). Neuronal of GAD67 in dopamine receptor-1 expressing cells causes specific motor def-
Depolarization Drives Increased Dopamine Synaptic Vesicle Loading via icits. Genesis 46, 357–367.
VGLUT. Neuron 95, 1074–1088. Hnasko, T.S., and Edwards, R.H. (2012). Neurotransmitter corelease: mecha-
Alsiö, J., Nordenankar, K., Arvidsson, E., Birgner, C., Mahmoudi, S., Halbout, nism and physiological role. Annu. Rev. Physiol. 74, 225–243.
B., Smith, C., Fortin, G.M., Olson, L., Descarries, L., et al. (2011). Enhanced su- Hnasko, T.S., Chuhma, N., Zhang, H., Goh, G.Y., Sulzer, D., Palmiter, R.D.,
crose and cocaine self-administration and cue-induced drug seeking after loss Rayport, S., and Edwards, R.H. (2010). Vesicular glutamate transport pro-
of VGLUT2 in midbrain dopamine neurons in mice. J. Neurosci. 31, motes dopamine storage and glutamate corelease in vivo. Neuron 65,
12593–12603. 643–656.

ll
Report
Hnasko, T.S., Hjelmstad, G.O., Fields, H.L., and Edwards, R.H. (2012). Ventral Qi, J., Zhang, S., Wang, H.L., Barker, D.J., Miranda-Barrientos, J., and
tegmental area glutamate neurons: electrophysiological properties and pro- Morales, M. (2016). VTA glutamatergic inputs to nucleus accumbens drive
jections. J. Neurosci. 32, 15076–15085. aversion by acting on GABAergic interneurons. Nat. Neurosci. 19, 725–733.
Howard, C.D., Li, H., Geddes, C.E., and Jin, X. (2017). Dynamic Nigrostriatal Root, D.H., Zhang, S., Barker, D.J., Miranda-Barrientos, J., Liu, B., Wang,
Dopamine Biases Action Selection. Neuron 93, 1436–1450. H.-L., and Morales, M. (2018a). Selective Brain Distribution and Distinctive
Synaptic Architecture of Dual Glutamatergic-GABAergic Neurons. Cell Rep.
Hunker, A.C., Soden, M.E., Krayushkina, D., Heymann, G., Awatramani, R.,
23, 3465–3479.
and Zweifel, L.S. (2020). Conditional Single Vector CRISPR/SaCas9 Viruses
for Efficient Mutagenesis in the Adult Mouse Nervous System. Cell Rep. 30, Root, D.H., Estrin, D.J., and Morales, M. (2018b). Aversion or Salience
4303–4316. Signaling by Ventral Tegmental Area Glutamate Neurons. iScience 2, 51–62.
Ikemoto, S., and Bonci, A. (2014). Neurocircuitry of drug reward. Shen, H., Marino, R.A.M., McDevitt, R.A., Bi, G.-H., Chen, K., Madeo, G., Lee,
Neuropharmacology 76 (Pt B), 329–341. P.-T., Liang, Y., De Biase, L.M., Su, T.-P., et al. (2018). Genetic deletion of ve-
sicular glutamate transporter in dopamine neurons increases vulnerability to
Kawano, M., Kawasaki, A., Sakata-Haga, H., Fukui, Y., Kawano, H., Nogami,
MPTP-induced neurotoxicity in mice. Proc. Natl. Acad. Sci. USA 115,
H., and Hisano, S. (2006). Particular subpopulations of midbrain and hypotha-
E11532–E11541.
lamic dopamine neurons express vesicular glutamate transporter 2 in the rat
Silm, K., Yang, J., Marcott, P.F., Asensio, C.S., Eriksen, J., Guthrie, D.A.,
brain. J. Comp. Neurol. 498, 581–592.
Newman, A.H., Ford, C.P., and Edwards, R.H. (2019). Synaptic Vesicle
Keiflin, R., and Janak, P.H. (2015). Dopamine Prediction Errors in Reward Recycling Pathway Determines Neurotransmitter Content and Release
Learning and Addiction: From Theory to Neural Circuitry. Neuron 88, 247–263. Properties. Neuron 102, 786–800.
Keithley, R.B., and Wightman, R.M. (2011). Assessing principal component Soden, M.E., Miller, S.M., Burgeno, L.M., Phillips, P.E.M., Hnasko, T.S., and
regression prediction of neurochemicals detected with fast-scan cyclic vol- Zweifel, L.S. (2016). Genetic Isolation of Hypothalamic Neurons that
tammetry. ACS Chem. Neurosci. 2, 514–525. Regulate Context-Specific Male Social Behavior. Cell Rep. 16, 304–313.
Keithley, R.B., Heien, M.L., and Wightman, R.M. (2009). Multivariate concen- Steinkellner, T., Zell, V., Farino, Z.J., Sonders, M.S., Villeneuve, M., Freyberg,
tration determination using principal component regression with residual anal- R.J., Przedborski, S., Lu, W., Freyberg, Z., and Hnasko, T.S. (2018). Role for
ysis. Trends Analyt. Chem. 28, 1127–1136. VGLUT2 in selective vulnerability of midbrain dopamine neurons. J. Clin.
Kouwenhoven, W.M., Fortin, G., Penttinen, A.-M., Florence, C., Delignat- Invest. 128, 774–788.
Lavaud, B., Bourque, M.-J., Trimbuch, T., Luppi, M.P., Poulin, J.-F., Stuber, G.D., Hnasko, T.S., Britt, J.P., Edwards, R.H., and Bonci, A. (2010).
Rosenmund, C., et al. (2019). Vglut2 expression in dopamine neurons contrib- Dopaminergic terminals in the nucleus accumbens but not the dorsal striatum
utes to post-lesional striatal reinnervation. bioRxiv, 12.23.887323. corelease glutamate. J. Neurosci. 30, 8229–8233.
Kravitz, A.V., Tye, L.D., and Kreitzer, A.C. (2012). Distinct roles for direct and Tecuapetla, F., Patel, J.C., Xenias, H., English, D., Tadros, I., Shah, F., Berlin,
indirect pathway striatal neurons in reinforcement. Nat. Neurosci. 15, 816–818. J., Deisseroth, K., Rice, M.E., Tepper, J.M., and Koos, T. (2010). Glutamatergic
signaling by mesolimbic dopamine neurons in the nucleus accumbens.
Lavin, A., Nogueira, L., Lapish, C.C., Wightman, R.M., Phillips, P.E., and
J. Neurosci. 30, 7105–7110.
Seamans, J.K. (2005). Mesocortical dopamine neurons operate in distinct tem-
poral domains using multimodal signaling. J. Neurosci. 25, 5013–5023. Thibeault, K.C., Kutlu, M.G., Sanders, C., and Calipari, E.S. (2019). Cell-type
and projection-specific dopaminergic encoding of aversive stimuli in addic-
Lein, E.S., Hawrylycz, M.J., Ao, N., Ayres, M., Bensinger, A., Bernard, A., Boe,
tion. Brain Res. 1713, 1–15.
A.F., Boguski, M.S., Brockway, K.S., Byrnes, E.J., et al. (2007). Genome-wide
atlas of gene expression in the adult mouse brain. Nature 445, 168–176. Tritsch, N.X., Ding, J.B., and Sabatini, B.L. (2012). Dopaminergic neurons
inhibit striatal output through non-canonical release of GABA. Nature 490,
Lobo, M.K., and Nestler, E.J. (2011). The striatal balancing act in drug addic-
262–266.
tion: distinct roles of direct and indirect pathway medium spiny neurons.
Trudeau, L.E., Hnasko, T.S., Wallén-Mackenzie, A., Morales, M., Rayport, S.,
Front. Neuroanat. 5, 41.
and Sulzer, D. (2014). The multilingual nature of dopamine neurons. Prog. Brain
Mendez, J.A., Bourque, M.-J., Dal Bo, G., Bourdeau, M.L., Danik, M., Williams, Res. 211, 141–164.
S., Lacaille, J.-C., and Trudeau, L.-E. (2008). Developmental and target-
Wallén-Mackenzie, A., Wootz, H., and Englund, H. (2010). Genetic inactivation
dependent regulation of vesicular glutamate transporter expression by dopa-
of the vesicular glutamate transporter 2 (VGLUT2) in the mouse: what have we
mine neurons. J. Neurosci. 28, 6309–6318.
learnt about functional glutamatergic neurotransmission? Ups. J. Med. Sci.
Mingote, S., Chuhma, N., Kusnoor, S.V., Field, B., Deutch, A.Y., and Rayport, 115, 11–20.
S. (2015). Functional Connectome Analysis of Dopamine Neuron
Wang, H.L., Qi, J., Zhang, S., Wang, H., and Morales, M. (2015). Rewarding ef-
Glutamatergic Connections in Forebrain Regions. J. Neurosci. 35,
fects of optical stimulation of ventral tegmental area glutamatergic neurons.
16259–16271.
J. Neurosci. 35, 15948–15954.
Mingote, S., Chuhma, N., Kalmbach, A., Thomsen, G.M., Wang, Y., Mihali, A., Wang, D.V., Viereckel, T., Zell, V., Konradsson-Geuken, Å., Broker, C.J.,
Sferrazza, C., Zucker-Scharff, I., Siena, A.C., Welch, M.G., et al. (2017). Talishinsky, A., Yoo, J.H., Galinato, M.H., Arvidsson, E., Kesner, A.J., et al.
Dopamine neuron dependent behaviors mediated by glutamate cotransmis- (2017). Disrupting Glutamate Co-transmission Does Not Affect Acquisition of
sion. eLife 6. Published online July 13, 2017. https://doi.org/10.7554/ Conditioned Behavior Reinforced by Dopamine Neuron Activation. Cell Rep.
eLife.27566. 18, 2584–2591.
Mingote, S., Amsellem, A., Kempf, A., Rayport, S., and Chuhma, N. (2019). Yamaguchi, T., Wang, H.-L., Li, X., Ng, T.H., and Morales, M. (2011).
Dopamine-glutamate neuron projections to the nucleus accumbens medial Mesocorticolimbic glutamatergic pathway. J. Neurosci. 31, 8476–8490.
shell and behavioral switching. Neurochem. Int. 129, 104482.
Yamaguchi, T., Qi, J., Wang, H.L., Zhang, S., and Morales, M. (2015).
Montardy, Q., Zhou, Z., Lei, Z., Liu, X., Zeng, P., Chen, C., Liu, Y., Sanz-Leon, Glutamatergic and dopaminergic neurons in the mouse ventral tegmental
P., Huang, K., and Wang, L. (2019). Characterization of glutamatergic VTA neu- area. Eur. J. Neurosci. 41, 760–772.
ral population responses to aversive and rewarding conditioning in freely-mov- Yoo, J.H., Zell, V., Gutierrez-Reed, N., Wu, J., Ressler, R., Shenasa, M.A.,
ing mice. Sci. Bull. (Beijing) 64, 1167–1178. Johnson, A.B., Fife, K.H., Faget, L., and Hnasko, T.S. (2016). Ventral tegmental
Pupe, S., and Wallén-Mackenzie, Å. (2015). Cre-driven optogenetics in the area glutamate neurons co-release GABA and promote positive reinforce-
heterogeneous genetic panorama of the VTA. Trends Neurosci. 38, 375–386. ment. Nat. Commun. 7, 13697.

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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Sheep Anti-tyrosine hydroxylase (TH) Pel-Freeze P60101-0
Rabbit Anti-tyrosine hydroxylase (TH) Millipore AB152
Rat Anti-dopamine transporter (DAT) Millipore MAB369
Rabbit Anti-DsRed Clontech 632496
Rabbit Anti-HA Sigma H6908
Donkey Anti-Sheep Alexa Fluor 647 Jackson Immuno Research 713-605-147
Donkey Anti-Rat Alexa Fluor 488-conjugated Jackson Immuno Research 712-545-150
Donkey Anti-Rabbit Fluor 594-conjugated Jackson Immuno Research 711-585-152
Donkey Anti-Rabbit Fluor 647-conjugated Jackson Immuno Research 711-605-152
Bacterial and Virus Strains
rAAV1-EF1a-DIO-hChR2(H134R)-mCherry UNC virus vector core N/A
rAAV5-EF1a-DIO-mCherry UNC virus vector core N/A
rAAV5-EF1a-DIO-hChR2(H134R)-EYFP UNC virus vector core N/A
AAV1-FLEX-SaCas9-sgTh Hunker et al., 2020 N/A
Chemicals, Peptides, and Recombinant Proteins
6,7-Dinitroquinoxaline-2,3(1H,4H)-dione (DNQX) Sigma-Aldrich D0540
Dopamine hydrochloride Alfa Aesar A11136
3,4-Dihydroxy-L-phenylalanine (L-DOPA) Sigma-Aldrich D9628
Benserazide hydrochloride Sigma-Aldrich B7283
D-amphetamine hemisulfate Sigma-Aldrich A5880
SKF81297 hydrobromide Tocris 1447
Critical Commercial Assays
RNAscope Multiplex Fluorescent Kit Advanced Cell Diagnostics 320850
Mm-Slc17a6-C1 (VGLUT2 Antisense probe) Advanced Cell Diagnostics 319171
Experimental Models: Organisms/Strains
Mouse: Slc17a6tm2(cre)Lowl The Jackson Laboratory 016963
Mouse: Thlox/lox Darvas and Palmiter, 2010 N/A
Mouse: Tg(Adora2a-cre)K139Gsat/Mmcd The Jackson Laboratory 036158
Mouse: Drd1a+/Cr Heusner et al., 2008 N/A
RESOURCE AVAILABILITY
Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Thomas
S. Hnasko (thnasko@health.ucsd.edu)
Materials Availability
d This study did not generate new plasmids.
d This study did not generate new mouse lines.
d This study did not generate new unique reagents.
Data and Code Availability

d This study did not generate any code.
d This study did not generate any particular type of dataset.
Neuron 107, 1–10.e1–e4, September 9, 2020 e1

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EXPERIMENTAL MODEL AND SUBJECT DETAILS
Animals
Mice were bred at the University of California, San Diego (UCSD), group housed (max 5 mice/cage), and maintained on a 12h light–
dark cycle with food and water available ad libitum unless noted. Initial breeders for Slc17a6tm2(cre)Lowl and Tg(Adora2a-cre)
K139Gsat/Mmcd breeders were obtained from The Jackson Laboratory (stock no: 016963 and 036158, respectively). Drd1a+/Cre
(Heusner et al., 2008) breeders were obtained from the lab of Larry Zweifel (University of Washington) and Thlox/lox mice were provided
by the lab of Martin Darvas (University of Washington). All mice were maintained fully backcrossed on to C57BL/6. Control and Th
cKO mice were generated by breeding homozygous Thlox/lox mice to double heterozygous Th+/lox; Slc17a6+/Cre mice and heterozy-
gous littermates were used as controls. Drd1+/Cre or Adora2a-Cre mice were bred to Slc17a6Cre/Cre. Mice were 3-8 months old for
behavioral experiments, in vivo FSCV and immunohistochemistry and 8-12 weeks old for slice electrophysiology and ex vivo
FSCV experiments. Male and female mice were included in all experiments, and all experiments performed in accordance with pro-
tocols approved by the UCSD Institutional Animal Care and Use Committee.
METHOD DETAILS
Stereotactic surgery
Mice (> 4-week-old) were anaesthetized with isoflurane, placed in a stereotaxic frame (Kopf), and 300 nL of AAV1-EF1a-DIO-
ChR2(H134R):mCherry or AAV5-EF1a-DIO-mCherry (UNC gene therapy center virus vector core) infused into the VTA of heterozy-
gote (Th+/lox; Slc17a6+/Cre) and TH cKO (Thlox/lox; Slc17a6+/Cre) mice (ml: ± 0.35, ap: 3.4, dv: 4.4 mm relative to Bregma) at 100
nl.min-1 (WPI UltraMicroPump) using custom made 30-gauge stainless steel (Plastics One, VA) injectors. For CRISPR/Cas9 exper-
iments VGLUT2:Cre mice were injected bilaterally with either AAV1-FLEX-SaCas9-U6-sgTh or a control vector targeting human Th at
a region of low complementarity AAV1-FLEX-SaCas9-U6-sgTh(human) (sgCTRL) (Hunker et al., 2020). Cas9 vectors were mixed 2:1
with AAV1-EF1a-DIO-ChR2(H134R):mCherry and a total of 300 nL injected per side. For the electrophysiology recordings from
defined D1 or D2 type neurons, Drd1+/cre; Slc17a6+/Cre or Adora2a-Cre; Slc17a6+/Cre mice were infused with 2 different AAVs unilat-
erally: first with AAV5-EF1a-DIO-mCherry in the medial NAc shell (ml: 0.5, ap: +1.1, dv: 4.5) plus AAV5-EF1a-DIO-hChR2(H134R)-
EYFP in VTA (ml: 0.35, ap: 3.4, dv: 4.4). For each infusion, the injection tip was left in place for 10 min and then slowly retracted.
Mice used in behavioral experiments, were also implanted with a 200-mm-core optic fiber (Newdoon Inc.) inserted either unilaterally in
VTA (ml: ± 0.5, ap: 3.4, dv: 4.0) or bilaterally in NAc following a 10 mediolateral angle (ml: ± 1.13, ap: +1.4, dv: 3.81) following
viral infusion. Fibers were stabilized using dental cement (Lang dental) secured by at least two skull screws. Animals were treated with
the analgesic Carprofen (Pfizer, 5mg.kg-1 s.c.) before and after surgery. Mice were monitored daily and allowed to recover from sur-
gery for > 3 weeks before subsequent assay.
Immunohistochemistry
Mice were deeply anaesthetized with pentobarbital (200 mg.kg-1 i.p.; Virbac) and transcardially perfused with 10–20 mL of phos-
phate-buffered saline (PBS) followed by 60-70 mL of 4% paraformaldehyde (PFA) at a rate of 6 ml.min-1. Brains were extracted,
post-fixed in 4% PFA at 4 C overnight and cryoprotected in 30% sucrose in PBS for 48–72h at 4 C. Brains were snap frozen in chilled
isopentane and stored at 80 C. Sections (30 mm) were cut using a cryostat (CM3050S, Leica) and collected in PBS containing
0.01% sodium azide. For immunostaining, brain sections were blocked with 5% normal donkey serum in PBS containing 0.2% Triton
X-100 (block) for 1 h at room temperature. Sections were then incubated with one or more of the following primary antibodies (rabbit
anti-TH, 1:2,000, Millipore AB152; sheep anti-TH, 1:1,000, Pel-Freez P60101-0; rabbit anti-DsRed, 1:2000, Clontech 632496; rat anti-
DAT 1:1000, Millipore MAB369; mouse anti-TH 1:1000, Millipore MAB318; rabbit anti-HA, 1:1000, H6908, Sigma - SaCas9 has a
hemagglutinin (HA)-epitope tag on the C terminus) in blocking buffer overnight at 4 C. Sections were rinsed 3x15 min with PBS
and incubated in appropriate secondary antibodies (Jackson ImmunoResearch) conjugated to Alexa 488, Alexa 594 or Alexa 647
fluorescent dyes (5 mg.ml-1) for 2 h at room temperature. Sections were washed 3x15 min with PBS, mounted onto glass slides
and coverslipped with Fluoromount-G mounting medium (Southern Biotech) + DAPI (Roche, 0.5 mg.ml-1).
Images were acquired using widefield epifluorescence (Zeiss AxioObserver). For the colocalization of TH with mCherry, images of
VTA were acquired using a 20X objective with identical acquisition settings across slides. For fluorescence densitometry, images of
the ventral and dorsal striatum were acquired and four to six striatal sections were analyzed per animal. Briefly, regions of interest in
the striatum were delineated and pixel densities were quantified using ImageJ. Background staining was quantified by measurement
of pixel intensities in the corpus callosum and subtracted from striatal regions for normalization. For cell counting, sections covering
the rostrocaudal extent of the VTA were collected and stained for TH and mCherry (Figure 1D) or HA (to visualize SaCas9) and TH
(Figure 4B). Three to four sections covering the VTA were counted per animal by an experimenter blind to the treatment. For densi-
tometry quantification, four striatal sections per animal were analyzed using ImageJ. Regions of interest in the dorsal and ventral
striatum were delineated and pixel densities were quantified using ImageJ. Background staining was quantified by measurement
of pixel density in the dorsomedial cortex and subtracted from striatal regions for normalization.
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In situ hybridization followed by immunohistochemistry

Mice were anesthetized with pentobarbital (200 mg.kg-1, i.p.) and transcardially perfused with 4% PFA. Brains were removed, post-
fixed in 4% PFA overnight, and cryoprotected in 30% sucrose in PBS for 48-72h. Brains were cut serially (16 mm) on a cryostat
(CM3050S, Leica) and mounted directly onto Superfrost glass slides (Fisher). Sections were stored at –80 C before starting the RNA-
scope assay (Advanced Cell Diagnostics). Briefly, slides were baked at 60 C for 30 min, rehydrated in PBS followed by boiling in
Target Retrieval Solution (ACD) for 5 min. Slides were washed twice in distilled water and once in 100% ethanol before incubation
with protease III (ACD) at 40 C for 30 min. RNA hybridization was performed according to the Fluorescent Multiplex Kit (ACD) using
an antisense probe against Slc17a6/VGLUT2 (ACD 319171-C1). After the last wash step, slides were rinsed two times in PBS,
blocked in 5% normal donkey serum/0.2% Triton X-100 in PBS for 1h at room temperature and incubated with primary antibodies
(rabbit Anti-TH 1:500; AB152, Chemicon; rat-Anti-DAT 1:500; MAB369, Chemicon) overnight at 4 C in blocking buffer. Next day,
slides were washed three times in PBS for 15 min and incubated with secondary antibodies (1:400; Alexa Fluor 488 and 647; Jackson
ImmunoReseach) for two hours at room temperature. Finally, slides were washed three times in PBS for 15 min and coverslipped
using DAPI-containing Fluoromount-G. Images were taken at 20x magnification using a Zeiss Axio Observer Epifluorescence micro-
scope. For cell counting quantification, sections covering the rostrocaudal extent of the VTA were collected and stained for TH, DAT
and VGLUT2. For RNAscope quantification of VGLUT2-positive neurons, cells were deemed positive above a threshold of 4 puncta.
3-4 sections covering the VTA were counted per animal by an experimenter blind to the treatment.
Electrophysiological recordings from adult brain slices

Adult mice (7–12 wks) were deeply anaesthetized with pentobarbital (100 mg.kg-1 i.p.; Virbac) and transcardially perfused with 10 mL
ice-cold sucrose-artificial cerebrospinal fluid (ACSF) containing (in mM): 75 sucrose, 87 NaCl, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25
NaH2PO4, 25 NaHCO3 and continuously bubbled with carbogen (95% O2–5% CO2). Brains were extracted, and 200-mm coronal
slices were cut in sucrose-ACSF using a Leica Vibratome (vt1200). Slices were transferred to a perfusion chamber containing
ACSF at 31 C (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.4 NaH2PO4, 25 NaHCO3, 11 glucose, continuously bubbled in carb-
ogen. After > 45 min recovery, slices were transferred to a recording chamber continuously perfused with ACSF (2–3 ml.min-1) main-
tained at 29–31 C using an in-line heater. Patch pipettes (3.5–5.5 MU) were pulled from borosilicate glass (King Precision Glass) and
filled with internal recording solution containing (in mM): 120 CsCH3SO3, 20 HEPES, 0.4 EGTA, 2.8 NaCl, 5 TEA, 2.5 Mg-ATP, 0.25
Na-GTP, at pH 7.25 and 285 ± 5 mOsm. mCherry-labeled VGLUT2 VTA terminals as well as D1R- and A2a-expressing NAc neurons
were visualized by epifluorescence and visually guided patch recordings were made using infrared-differential interference contrast
(IR-DIC) illumination (Axiocam MRm, Examiner.A1, Zeiss). ChR2 was activated by flashing blue light (5-ms) through the light path of
the microscope using a light-emitting diode (UHP-LED460, Prizmatix) under computer control. Excitatory postsynaptic currents
(EPSCs) were recorded in whole-cell voltage clamp (Multiclamp 700B amplifier, Axon Instruments), filtered at 2 KHz, digitized at
10 KHz (Axon Digidata 1550, Axon Instruments), and collected online using pClamp 10 software (Molecular Device). Series resistance
and capacitance were electronically compensated before recordings. Estimated liquid-junction potential was 12mV and left uncor-
rected. Series resistance and/or leak current were monitored during recordings and cells that showed > 25% change during record-
ings were considered unstable and discarded. Neurons were held in voltage-clamp at 60mV to record AMPAR EPSCs in whole-cell
configuration and single-pulse (5-ms) photostimuli were applied every 55 s and 10 photo-evoked currents were averaged per neuron
per condition. DMSO stock solution of DNQX (10 mM, Sigma) was diluted 1,000-fold in ACSF and bath applied at 10 mM. Current
sizes were calculated by using peak amplitude from baseline. Decay time constants (t) were calculated by fitting an exponential func-
tion to each averaged current trace using the following formula: f(t) = e-t/t+C.
Fast-scan cyclic voltammetry recordings

In vivo FSCV
VGLUT2-Cre-expressing control and cKO mice were prepared as described above with Cre-dependent ChR2:mCherry virus infusion
and optic fiber implantation targeting the VTA unilaterally. Following > 6 weeks recovery, mice were anesthetized with isoflurane
(induced at 3%, maintained at 0.5%–1.5% throughout recordings) and placed in a stereotaxic frame (Kopf). A Ag/AgCl reference
electrode was implanted in the contralateral hemisphere, and a carbon-fiber microelectrode (Clark et al., 2010) was lowered into
the medial NAc shell (ml: ± 0.4, ap: 1.34, dv: 3.55 mm). A PC-based system running TarheelCV software written in LabView (National
Instruments) was used for voltammetric waveform application and data acquisition. The potential at the carbon-fiber electrode was
held at 0.4 V versus the Ag/AgCl reference, ramped to +1.3 V and back to 0.4 V at 400 V.s-1. This voltammetric waveform initially
was applied at 60 Hz for 15 min and then at 10 Hz for the duration of the recording. Optogenetic stimulation was delivered to the VTA
through the optic fiber coupled to a 473-nm laser (LaserGlow). Stimulation trains (1 s, 10 mW, 10 ms pulse width) at frequencies of 5,
10, 20, or 50 Hz were delivered every 2 min in interleaved, ascending order, with four repetitions of each frequency. This full stimu-
lation protocol was repeated at 3-5 recording sites per animal 200 mm apart in the dorsoventral axis. This multisite procedure
increased chances of appropriate recording site within the medial NAc shell, then confirmed using post hoc histology. The last
recording site was marked with an electrolytic lesion (70 mA, 20 s). When multiple recording sites were located within the medial shell
for a given animal, a single site was included in the final group analyses, based on minimized noise throughout the stimulation pro-
tocol. Dopamine signal was isolated from the background-subtracted voltammetric signal using chemometric analysis (Heien et al.,
2005) with custom code for principle component regression (Keithley et al., 2009) written in MATLAB (MathWorks). Training sets for
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this analysis included standard cyclic voltammograms of dopamine and pH changes obtained from optogenetic stimulation in DAT-
cre mice expressing ChR2 in the VTA, as well as electrode-specific background drift from the current recordings (Howard et al., 2017;
Keithley and Wightman, 2011). Dopamine concentration was estimated based on average post-implantation electrode sensitivity
(Clark et al., 2010). Nissl staining was used to visualize lesion sites created following FSCV measurements. Briefly, 30 mm frozen vi-
bratome sections were mounted on slides, air-dried and placed into 1:1 ethanol/chloroform overnight. Slides were then rehydrated
through ethanol dilutions to distilled water, stained in 0.1% cresyl violet solution for 10 minutes. Slides were then rinsed with water
and differentiated in 95% ethanol for 30 minutes and dehydrated in 100% ethanol for 2 3 5 minutes before being cleared in xylene 2 3
5 minutes and coverslipped.
In vitro FSCV
For recording electrodes, cylindrical carbon-fiber electrodes were prepared with 7 mm diameter fibers (100 mm of exposed fiber,
GoodFellow) inserted into a glass pipette (1.0 mm, A-M systems) and pulled to seal the pipette around the carbon-fiber. The electrode
was held at 0.4 V versus Ag/AgCl and a triangular voltage ramp ( 0.4 to +1.3 at 400 V.s-1) was delivered at 10 Hz. DA transients were
evoked in the medial NAc shell by optogenetic stimulations (473 nm, 1 s, 5 ms pulse-width) at various frequencies (5-50 Hz) through
the light path of the microscope. Data were collected and analyzed using TarheelCV software. The amplitude of DA transients was
measured from the oxidation peak region and 3 responses were averaged. For positive control recovery experiments DA was pre-
pared fresh daily and bath-applied at 10 mM for 10 min followed by bath application of ACSF (10 min, wash) prior to repeated DA
measurement at the same recording site.
Behavioral studies
Behavioral pharmacology
Horizontal locomotor activity was measured in square plastic chambers (17 3 8.9 cm) using an automated video tracking system
(ANY-maze, Stoelting Co.). The following drugs (all Sigma-Aldrich unless indicated) were injected: 50 mg.kg-1 L-DOPA and
12.5 mg.kg-1 benserazide (i.p.) in 0.25% (wt/ vol) ascorbate in PBS, d-amphetamine hemisulfate (5 mg.kg-1, i.p.) in saline,
SKF81297 (0.2 mg.kg-1, i.p.; Tocris #1447) in saline. All drugs were injected at 10 ml.kg-1 except for L-DOPA/benserazide, which
was injected at 33 ml.kg-1.
2-nosepoke self-stimulation
Mice were placed on a restricted feeding schedule: food restriction consisted of removing food the evening before the first day and ad
libitum access was then restricted to a 3-h daily period following the assay. At the beginning of the session, ferrules were connected
to a 50-mm optical patch cable connected to an optical commutator (Doric Lenses) and mice were placed in operant chambers (Med
Associates) controlled by MedPC IV software. The start of the session was signaled by a brief tone (2 kHz, 1 s), illumination of over-
head house light, and LED cue lights over the nosepoke holes; sessions lasted 45 min. The chamber contained two photobeam-
equipped nosepoke holes which were each baited at the start of each session with a sucrose pellet (Bio-Serv, F0071). Beam-breaks
on the active nosepoke led to a 0.5 s tone, the LED cue lights over the nosepokes turned off for the duration of the photostimulus, and
the activation of a TTL-controlled DPSS laser (473 nm, Shanghai or OEM laser) set to deliver pulses at 10 mW (80 mW.mm-2 at 200-m
fiber tip) at 40 Hz (1 s, 10-ms pulse width) controlled by an Arduino stimulus generator. Laser power was measured using a digital
power meter (Thorlabs PM100D/S121C). Nosepokes that occurred during ongoing photostimulation were recorded but were without
effect; inactive nosepokes led to identical tone and cue light effects but did not trigger the laser.
Real-time place preference
On a baseline (pre-test) day, mice were placed on the border between two adjoining (20 3 20 cm) homogeneous gray compartments
and the amount of time spent in each compartment was recorded using video tracking software (ANY-maze). On the subsequent day,
one side was designated active, where entries triggered photostimulation (473 nm, 10 mW, 40 Hz, 10-ms pulse width) using the lasers
as described above but controlled by an ANY-maze interface (San Diego Instruments). Sessions lasted for 30 min and the amount of
time spent in each compartment and the number of crossings was recorded.
QUANTIFICATION AND STATISTICAL ANALYSIS
To evaluate statistical significance, data from Figures 1B, 1D, 4B, S1B, S1D, S2A–S2C, S3B, S3D, S3F, and S3H were subjected to
Student’s t tests (KyPlot). Data from Figures 1F, 1G, 2B, 2C, 2E, 2F, 3B, 3C, 3E, 3F, 4D–4G, S2A–S2C, S3C, and S3G were subjected
to RM one- or two-way ANOVAs followed by Sidak or Tukey post hoc analysis (GraphPad Prism v6). Friedman nonparametric test
was used for non-Gaussian-assumed data from Figure 1I. In all the figures data are presented as means ± SEM unless noted and
statistical significance was set at p < 0.05.
e4 Neuron 107, 1–10.e1–e4, September 9, 2020

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The Project Gutenberg eBook of Navy boys to the
rescue
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eBook.
Title: Navy boys to the rescue

or, Answering the wireless call for help
Author: Halsey Davidson
Illustrator: Clare Angell
Release date: January 15, 2024 [eBook #72723]
Language: English
Original publication: United States: George Sully and Company,

1919
Credits: Roger Frank and the Online Distributed Proofreading

Team at https://www.pgdp.net (This file was produced
from images made available by the Library of
Congress)
*** START OF THE PROJECT GUTENBERG EBOOK NAVY BOYS

TO THE RESCUE ***
NAVY BOYS TO THE RESCUE
With her smoke trailing behind her and the guns barking
in rapid succession, the Colodia raced toward the scene.
OR
ANSWERING THE WIRELESS CALL FOR HELP
BY
HALSEY DAVIDSON
Author of “Navy Boys After a Submarine,” “Navy
Boys Chasing a Sea Raider,” etc.
NEW YORK
GEORGE SULLY AND COMPANY
BOOKS FOR BOYS
NAVY BOYS SERIES

By Halsey Davidson
12mo, cloth, illustrated.
NAVY BOYS AFTER A SUBMARINE

Or Protecting the Giant Convoy
NAVY BOYS CHASING A SEA RAIDER
Or Landing a Million Dollar Prize
NAVY BOYS BEHIND THE BIG GUNS
Or Sinking the German U-Boats
Or Answering the Wireless Call for Help
NAVY BOYS AT THE BIG SURRENDER
Or Rounding Up the German Fleet
GEORGE SULLY & COMPANY

Publishers New York
Copyright, 1919, by
GEORGE SULLY & COMPANY
Navy Boys to the Rescue
Printed in U. S. A.
CONTENTS
I The Friendly Grip
II The Hun in His Fury
III The Missing Man
IV The Paper Chase
V The Trickster
VI Work Ahead
VII On the Grey Waters
VIII The Yankee Way
IX “Schmardie”
X The Terror of the Seas
XI Action
XII Wireless Whispers
XIII The Super-submersible
XIV The Mirage
XV Combing the Sea
XVI Stations
XVII The Spitfire
XVIII “Ghost Talk” Again
XIX A Difference of Opinion
XX Too Late Again
XXI The Mystery Message
XXII The Wireless Call for Help
XXIII The Sea Pigeon in Sight
XXIV The Blind Chase
XXV A New Convoy

CHAPTER I—THE FRIENDLY GRIP
“And yonder’s a snap-dragon; and that’s a buttercup. That is
feverfew growing over there; and there’s foxglove right there in that
swampy place. Those are cowslip blossoms—the English cowslip is
different from ours.”
“Whew!” blew Phil Morgan, unpuckering his lips and breaking off
the haunting little air he had been whistling. “I wouldn’t believe you
knew so much about the flora of this strange land, Frenchy.”
“Oi, oi! Is it Flora he’s bragging about? Then Frenchy’s got a new
girl!”
“Sounds to me,” mumbled Al Torrance, who lay along the flower-
bestrewed bank with his hat over his eyes, “that he was discussing
the fauna of the country—with his snap-dragons, and fox’s gloves,
and cows slipping.”
“Ignoramus that you are!” scoffed Michael Donahue, otherwise
“Frenchy.” “I am talkin’ to Whistler. He knows something and
appreciates the profundity of me learnin’.”
“Ye-as,” drawled Torrance, otherwise “Torry,” as their leader began
droning away, his lips puckered again. “He knows just enough to
whistle the same awful tune for an hour. What is it, anyway, Phil?”
“The tune the old cow died on, I guess,” suggested Ikey
Rosenmeyer.
“It’s a tune Phoebe was playing on the piano a good deal the last
time we were home,” said Whistler with some gravity. “Wish I’d hear
from the folks again. I am worried about Phoebe.”
He spoke of his eldest sister, who during the last few months had
not been well. Although, like many brothers and sisters, Philip
Morgan, by his chums usually called Whistler, and Phoebe had their
differences, now when far from home, “the folks” seem nearer and
dearer than ever in his mind.
Philip Morgan lay with his chums on a bank beside a tiny trickle of
water called a brook in that shire, although it was nothing more than
a rill. They were high up on “the downs,” overlooking a port in which
the American destroyer Colodia lay at anchor amid a multitude of
naval vessels of three nations.
Over the sea a thick haze, on land the yellow sunshine, so
welcome when it is seen in England that it seems more beautiful
than elsewhere. The boys had forty-three hours’ shore leave, and for
that brief space of time they desired, as most sailors do, to get just
as far away in spirit and in surroundings from the ship as possible.
They had tramped into the country the day before, spent the night
in four wonderful beds in an old inn that might have harbored some
of Sir Francis Drake’s men at the time of the Armada, and were now
due at the wharf in a few hours.
Life aboard a destroyer or an American submarine chaser in
foreign service is not very pleasant if it is exciting. The space for
sleeping, for instance, on these fast vessels is scarcely greater than
that assigned to the crews of submarines. As Ikey Rosenmeyer, who
possessed a riotous imagination, had said at the inn:
“Oi, oi! sleeping in a real bed again is better than bein’ at home in
Ireland, and Frenchy says that’s heaven ’cause his mother came
from there. Why, it is better’n heaven! You could spread out your
legs and wiggle all your toes without havin’ the master-at-arms down
on you like a thousand of brick.”
Frenchy, in a dreamy and poetic mood, not infrequent when the
romantic Irish blood in his veins was stirred, was gazing off over the
sea at the fogbank.
“Think of it,” he murmured, “How many hundred an’ thousan’ of
ships have sailed out of this harbor into just such a fogbank as that
—”
“And never came back,” interrupted Torry. “Some tough old gobs,
the ancient British seamen, boy.”
“‘Tough’ is right,” chuckled Frenchy, his poetic feelings exploded.
“And they haven’t got over it yet. They’ve got old-timers in the British
Navy now that can remember when the cat was used on the men’s
backs, reg’lar.”
“And every British sailor had tar on his breeches—that’s why they
used to call ’em ‘brave British tars,’” scoffed Torry. “Can it! These
English chaps are all right. They aren’t much different from us
garbies.”
“Is that so?” exclaimed Ikey, whose sharp eyes allowed little to
escape them. “What kind of a deep-sea crab do you call this comin’
down the road right now, I want to know?”
Phil Morgan paid no attention to what his mates were talking
about. The peaceful English landscape charmed his eye.
Down the gently sloping road which, after a mile or so, led into the
Upper Town, as it was called in distinction from the port, or Lower
Town, the stone cottages—some almost hidden by vines—stood
sentinel-wise along the way.
One rather larger house was a schoolhouse. Nothing at all like the
schoolhouses in America in appearance. But Phil Morgan knew it
was a schoolhouse, and that the school was in session, for he had
seen the children filing in not long before and their voices had been
raised in song just before Frenchy had begun to note the different
flowers.
The excited chatter of the other boys finally aroused Morgan from
his contemplation of the peaceful scene. In the other direction,
toward which his mates were looking, the outlook was not so
peaceful. At least, not at one particular spot in the hedge-bordered
road. It did not need a sailor’s weather eye to see that the situation
was “squally.”
The “deep-sea crab,” the presence of which Ikey had announced,
proved on further examination to be two individuals, not one. But
they were closely attached to one another and the way they “wee-
wawed,” as Torry said, from one side of the road to the other,
certainly would lead to the supposition that intoxication was the
cause of such tacking from hedge to hedge.
“And one of ’em’s one of our own garbies,” declared Frenchy. “Isn’t
that a shame?”
“But look at that big feller, will you?” gasped Ikey. “Why, he must
weigh a ton!”
“You’re stretching that a bit, Ikey,” admonished Whistler, breaking
off in his tune to speak. “But he is a whale of a man.”
“Biggest garby I ever saw,” breathed Torry, amazed.
It was the big fellow only, it proved, who was partly intoxicated. He
was a British sailor. His companion was both perfectly sober and
perfectly mad. His face was aflame as he and his unwelcome
companion approached the four Navy Boys.
The big fellow gripped him by the collar of his blouse, and it was
utterly impossible for the Yankee lad to get away from “the friendly
grip.”
“Talk about this ‘hands across the sea’ stuff,” murmured Torrance.
“Here’s a case where it is going too far. We’ll have to rescue a
brother garby, won’t we?”
“Believe me, that’s a reg’lar mamma’s boy Johnny Bull has got his
grip on, too,” chuckled Frenchy.
“Hush up, you fellows,” advised Phil Morgan, with sudden interest.
“I believe I know that fellow.”
“Not Goliath yonder?” cried Ikey Rosenmeyer. “I didn’t know you
sailed with such craft.”
“The other chap,” Morgan explained.
“If he’s a friend,” began Torrance, commencing to roll back his
sleeves suggestively.
“Sit down!” advised the older boy, sharply. “We’d look nice piling
onto that big fellow, wouldn’t we?”
“And the whole of us couldn’t handle him,” murmured Frenchy.
“You never know till you try,” said the optimistic Torrance.
“This is a case for strategy,” stated Morgan. “Now, don’t any of you
fellows lose your heads.” Then he hailed the two tacking along the
road:
“Ahoy! Hey, you!”
The American lad who was held in durance by the British sailor
looked up and showed something besides the red flag of annoyance
in his countenance.
“I say, you fellows!” he cried. “Help me out of this, will you?”
At this the huge British seaman for the first time appeared to see
the four boys on the bank beside the road.
“My heye!” he bellowed, standing still, but wagging his head from
side to side in a perfectly ridiculous way. “My heye! ’Ere’s a ’ole
bloomin’ ship load of ’em. Ahoy, me ’earties, let the heagle scream!”
and he led off in a mighty cheer that awoke the echoes of the
heretofore peaceful countryside.
Frenchy and Ikey, in great glee, sprang up and cheered with him.
But the expression in the countenance of the giant’s captive caused
the two older Navy Boys to smother their amusement.
“That’s the way he’s been going on for four hours—and more,”
groaned the captive. “Why! he hung on to my collar all the time we
were eating dinner up there at that inn. Made the barmaid cut up his
victuals for him. Paid her a shilling for doing it.”
“Say, is her name Flora?” Ikey asked, at once interested. “Is that
the girl Frenchy was just talking about?”
But Torrance quenched him with a hand on his mouth. The
situation of the Yankee youth in that giant’s hands seemed more
serious than they supposed. The grip of the big hand never relaxed.
“’Ere we are, all together, me ’earties,” rumbled the giant. “Hi’m
glad to know yuh. Hi’m Willum Johnson, ’im that ’ad a barrow hin the
Old Kent Road before the war. Hand jolly well knowed Hi was to the
perlice,” confessed the man frankly.
“Hit allus took six bobbies to take me hin, lads. Hand now one o’
the bloomin’ hofficers makes me walk a chalkline, haboard ship. Hi
tell yuh, ain’t this war terrible?”
“That’s what it is,” admitted Frenchy, staring at the man with wide-
open eyes.
“Come over here and sit down—and tell us all about it,” Whistler
Morgan said, beckoning.
“Hi’ll go yuh!” declared the giant seaman. “Hand so wull me friend
—one o’ the nicest little Yankees Hi ever come across.”
The strange Yankee sailor was too much disturbed by his situation
to look very closely at Phil and his comrades. The viselike grip of the
semi-intoxicated giant on his collar was the principal thing in the
victim’s mind.
Almost as soon as the British seaman sprawled on the grassy
bank his head began to nod and his eyes to close.
“He’s going off,” whispered Al Torrance.
“You’d think he would,” returned the victim of the over-friendly
seaman, in the same tone, “if you could have seen him eat and
drink. You never saw such an appetite! He had everybody at that inn
standing around and gaping at us.”
It was evident that the young sailor felt his position deeply. He was
a nice looking fellow, very neat in his dress, and with delicate
features.
“How did you come to fall in with him in the first place?” Al asked,
as the giant began to snore.
“Why,” explained the stranger, “I started to walk down to the port
because it was so pleasant. He was sitting outside the place where I
stopped for tea and muffins after I’d walked a way. I had no idea he
was so—so far gone. But he must have been drinking for days,”
casting a disgusted glance at his close companion whose hamlike
hand never relaxed. “He learned where I was going, and he at once
got a grip on my collar. He hasn’t let go since—I never saw such a
man!” concluded the stranger morosely.
“His hand will drop off when he gets sound asleep,” Whistler said
comfortingly. “Then we’ll sneak.”
“Don’t you believe it!” whispered the other in vast disgust. “He fell
asleep after dinner, but his fingers are just clamped on to my collar.
When I tried to wriggle away, he awoke. See!”
He tried to pull away from the friendly grip. At once the British
seaman half aroused; but his fingers never relaxed.
“William Johnson his my nyme—

Seaman’s my hav-o-cation!
Hi’m hin this war for a penny-bun—
Hand so is hall my nation!
Hoo-roo!” mumbled the gigantic sailor, and fell asleep again.

“Now, what do you know about that?” demanded the victim of
brotherly love. “And me—Well, I’m due aboard the Colodia to-day.”
“The Colodia!” exclaimed the four Navy Boys in chorus.
None of them wore a designating mark, for they had on their white
service caps. But the Colodia was the Yankee destroyer to which
Morgan, Torrance, Donahue and Rosenmeyer belonged. The four
gazed on the stranger with increased interest at his statement.
“Say,” Whistler asked, “aren’t you George Belding? Didn’t you and
your folks come up to Seacove from New York five or six years ago
and spend the summer in the old Habershaw House? I’m Phil
Morgan. We lived right next to the Habershaw House.”
“My goodness!” exclaimed the strange youth, sticking out his hand
to grab Whistler’s. “And your father, Dr. Morgan, and mine went to
college together. That’s what brought us up to Seacove. Sure! My
mother wasn’t well. We all got fat and sassy up there. I declare I’m
glad to see you, Phil Morgan!”
“Me long lost brother!” whispered Frenchy to the others. “Have you
still got the strawberry mark upon your arm?”
But Al Torrance was quite as serious as Whistler and the newly-
introduced George Belding.
“Say, fellows,” Al said, “if he’s going to be one of us on the old
destroyer, we’ve got to help him out of this mess.”
“Go ahead! How?” demanded Ikey.
Al produced a pocketknife which he opened quickly. It had a long
and sharp blade. He approached the snoring giant on the bank.
“Oi Oi!” gasped Ikey Rosenmeyer. “Never mind! Don’t kill him in
cold blood. Remember, Torry, it’s Germans we’re fightin’, not these
Britishers.”
“What are you going to do?” demanded Belding.
“Cut your collar away,” said Torrance. “That’s about all you can do.
If he wants to hang on to the collar, let him.”
“It’ll spoil his shirt,” objected Ikey.
“Sh! Go ahead,” murmured Belding. “It’s a good idea. I couldn’t get
at my own knife and do it, with him hanging on to me so tightly.”
“Take care, Al,” advised Whistler. “And you other fellows stand
aside. Be ready to run when George is free.”
His advice was good. The giant seaman still snored, but it would
not take much to rouse him.
The five boys were now so much interested in the attempt to get
Belding free that they took no heed of anything else. So they were all
shocked when a chorus of steam whistles and sirens suddenly broke
forth from the port below them. A gun boomed on the admiral’s ship.
Pandemonium was let loose without warning.
“Oh, my aunt!” groaned George Belding, “what is that?”
“Willum Johnson” awoke with a start and a grunt, and, sitting up on
the bank, demanded of everybody in general, “’Oo’s shootin’ hof the
bloomin’ gun?”
But Whistler and Torry had whirled to look out to sea. They had
heard a similar alarm before. Out of the blue-gray fogbank over the
sea, and high, high up toward the hazy sky, whirled a black object,
no bigger at first than a bird. But how rapidly it approached the port,
and how quickly its outline became perfectly clear!
“A Zep, boys!” cried Al Torrance. “There’s a raid on! That’s a
German machine, sure’s you are a foot high!”
“Are you sure?” murmured Belding, who had been dragged quickly
to his feet by the giant.
“Hit’s the bloomin’ ’Uns—no fear it ain’t!” ejaculated the big British
seaman. “Ah! There goes the a-he-rial guns.”
Splotches of white smoke sprang up from several shoulders of the
hill that overlooked the port. The watchful coast-defense men were
not unprepared; but the enemy airship, rapidly growing bigger in the
boys’ eyes, winged its way nearer to the land, boldly ignoring the
shells sent up to meet it.
“She’s going to drop her bombs right over the town!” gasped
Whistler, grabbing Belding, who was nearest.
CHAPTER II—THE HUN IN HIS FURY
Wheeling up from behind them on the higher shoulder of the hill, an
airplane spiraled into the upper ether, in an attempt to get above the
huge machine that had, two minutes before, appeared out of the sea
fog. But this attempt to balk the Hun, like those of the anti-aircraft
guns in their emplacements about the port, promised little success.
The fog had made the close approach of the huge Zeppelin
possible, and now the rumble of the motors of the enemy machine
could be heard clearly by the four Navy Boys on the hillside and their
two companions.
“Oh, cracky!” gasped Al Torrance. “She’s coming!”
“And right this way!” gulped Ikey Rosenmeyer. “If she drops a
bomb—”
“Good-night!” completed Frenchy in a sepulchral tone.
“Let’s get under cover!” cried George Belding, striving again to get
away from the “friendly grip” of the British sailor, Willum Johnson.
“Hold on!” commanded Whistler Morgan. “No use losing our heads
over this.”
“If one of those bombs lands near us we’ll likely lose more than
our heads,” grumbled Torry.
“Wait! If we run like a bunch of scared rabbits, we are likely to run
right into danger rather than away from it.”
“Those horns down there say ‘Find a cellar!’” whispered Frenchy.
“Oi, oi!” added Ikey. “There ain’t no cellars up here on this hill yet.”
“Keep cool,” repeated Whistler. The other boys were used to
listening to him, and to following his advice. He was a cautious as
well as a courageous lad, and his chums were usually safe in
following Philip Morgan’s lead.
These four boys, all hailing from the New England coast town of
Seacove, had begun their first “hitch,” as an enlistment is called, in
the United States Navy as apprentice seamen, several months
before America got into the Great War, and some months before the
oldest of the four was eighteen.
They had now spent more than a year and a half in the service,
and their experiences had been many and varied. After their initial
training at Saugarack, the big Naval training camp, the four chums,
with others of their friends and camp associates, had been sent
aboard the torpedo boat destroyer, Colodia, one of the newest,
largest, and fastest of her type in the United States Navy.
The Colodia’s first two cruises were full of excitement and
adventure for the four Navy Boys, especially for Philip Morgan; for he
fell overboard from the destroyer and was picked up by the German
submarine U-812, and his experiences thereon and escape
therefrom, are narrated in the first volume of this series, entitled:
“Navy Boys After the Submarines; Or, Protecting the Giant Convoy.”
The second of the series, “Navy Boys Chasing a Sea Raider; or
Landing a Million Dollar Prize,” relates the experiences of these four
friends on a longer and even more adventurous cruise of the
Colodia. Under the command of Ensign MacMasters, the Navy Boys
as members of a prize crew, took the captured Graf von Posen into
Norfolk; and their experiences on the captured raider made a
dramatic and exciting story of the day-by-day work of the boys of the
Navy.
Through their kind friend Mr. Alonzo Minnette, who was holding a
volunteer position at Washington in the Navy Department, the four
chums obtained a chance to cruise with the superdreadnaught
Kennebunk, a brand new and one of the largest of the modern
American fighting machines launched during the first months of the
war. The Colodia having gone across the Atlantic while the boys
were with the captured raider, they with Ensign MacMasters were
very glad to join the crew of the huge superdreadnaught in the
interim.
The third volume of the series, “Navy Boys Behind the Big Guns;
Or, Sinking the German U-Boats,” took our heroes into perils and
adventures which they will long remember, for they included work in
the gun turrets of the Kennebunk, a wreck that threatened the lives
of all four chums, a mix-up with German spies, and finally a record
trip across the Atlantic by which the huge superdreadnaught arrived
at the rendezvous in time to take part in a naval engagement which
put a part of the Hun navy to flight.
Now the four friends were back on the Colodia which was doing
patrol duty off the English and French coasts, and convoying troop
and food ships through the submarine and mine zones. The base of
the squadron of which the destroyer was a member was at this
English port, on the hillside above which Philip Morgan, Alfred
Torrance, Michael Donahue and Ikey Rosenmeyer have been
introduced just as they met the American sailor lad, George Belding,
and his doubtful friend, the giant ex-coster, “Willum” Johnson.
“Keep cool,” Whistler urged again, as the Zeppelin sailed inland.
“There is no use running——”
His further speech was smothered by a terrific explosion from the
port below. A lurid burst of flame, stronger than the sunlight, shot into
the air where a wharf and warehouse had been. Smoke followed,
instantly hiding the mark the bomb from the Zeppelin had found.
This daylight raid was the boldest the Germans had attempted.
The enemy must have supposed the fog was over the land as well
as the sea, or he would never have risked the attack.
Again a nerve-racking explosion following a flash of light that
seared their eyeballs, and the middle of the town—the market place
—was shrouded by thick smoke.
“The dirty ’ounds!” bawled the British seaman, suddenly finding his
voice. “The dirty ’ounds! They’re killin’ women an’ kids down there!
Lemme get my bloomin’ ’ands on ’em!”
He dropped George Belding’s collar at last and would have started
in a clumsy run down the hill. It was Whistler who stopped him, with
a two-handed grip on the Englishman’s collar now.
“What good would you be down there, man?” the American youth
demanded. “You’d only get yours, too, maybe. Those bombs are
falling two or three thousand feet.”
“Argh!” growled Willum Johnson, shaking his huge fists in the air,
his face raised to the coming Zeppelin. The growl was animal-like,
not human. “Argh! Lemme get ’em——”
A third bomb exploded. A big house below them, half way down
the hillside, disappeared. It was as though a monstrous sponge had
been wiped across that spot and erased the building!
“Oh! Look out! Look out!” sobbed Frenchy, and covered his eyes
with his hands.
His chum Ikey shook beside him, but could not close his eyes to
the horror.
The Zeppelin was curving around, evidently determined to make
for the sea and the fogbank again. Beneath it, on either side, even
above it, the bursts of white smoke betrayed the explosion of aerial
shells the defense guns were firing at the enemy machine. And all
the time the single British airplane on duty was climbing skyward.
“If that thing can only get above the Zep.!” murmured Al Torrance.
Suddenly the airplane darted toward the sea, in a sharp slant
upward. Bravely the pilot sought to cut off the Zeppelin’s escape into
the fogbank out of which she had burst five minutes before.
Guns from the Huns’ airship began to bark. They were firing on the
British plane. The latter’s guns made no reply as she continued to
mount into the upper air.
The course of the Hun machine was changed again. In
approaching the hills surrounding the port the Zeppelin was brought
much nearer to the earth.
The ship was indeed a monster! Swung landward to escape the
mounting airplane, the Zeppelin, its motors thundering, came closer
and closer to the spot where the American sailor boys were
standing.
“Bli’me!” roared the apparently fast-sobering Britisher. “They are
goin’ to drop one o’ them blarsted buns on our bloomin’ ’eads!”
“‘Buns’ is good,” groaned Al. “Here she comes!”
It seemed as though the great airship was directly above them.
The boys actually saw the bomb released and fall!
There was no possible mistake on the part of the brutal crew and
commander of the Zeppelin. They knew very well the bomb would
fall upon no warship in the harbor, or any possible storage place of
munitions. Up here on the hillside were nothing but little dwellings
and—the schoolhouse!
As though it were aimed at that house of instruction, the great
shell fell and burst! If teacher and pupils had descended into the
cellar at the first alarm of the horns and guns, it would scarcely have
availed to save them. The shot was too direct.
One moment the green-tiled, freshly whitened walls of the
schoolhouse stood out plainly against the yellow and green

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VTA Glutamate Neuron Activity Drives

Positive Reinforcement Absent

Elsevier Weekblad - Week 26 - 2022 Gebruiker

Jock Seeks Geek: The Holidates Series Book #26 Jill

The New York Review of Books – N. 09, May 26 2022

Calculate with Confidence, 8e (Oct 26,

Green Micro and Nanocomposites Thomas S.

Coal Geology 3rd Edition Larry Thomas

The Movement Thomas C. Holt [Thomas C. Holt]

UNDERSTANDING HEALTH POLICY : a clinical approach. 8th

VTA Glutamate Neuron Activity Drives Positive

d Activation of VTA glutamate neurons also leads to dopamine

d We generated two distinct models to abolish this coincident

d VTA glutamate neuron activity can serve as a reinforcer

Zell et al., 2020, Neuron 107, 1–10

INTRODUCTION et al., 2014, 2018). Optogenetic activation of VTA glutamate cell

Neuron 107, 1–10, September 9, 2020 Published by Elsevier Inc. 1

Figure 1. Disruption of TH Expression and DA Release from VTA Glutamate Neurons

(legend continued on next page)

2 Neuron 107, 1–10, September 9, 2020

(C) Strategy for selective expression of ChR2:mCherry in VGLUT2 VTA neurons.

Neuron 107, 1–10, September 9, 2020 3

4 Neuron 107, 1–10, September 9, 2020

Figure 2. Self-stimulation of VTA Glutamate Neurons in cKO Mice

Neuron 107, 1–10, September 9, 2020 5

6 Neuron 107, 1–10, September 9, 2020

(legend continued on next page)

Neuron 107, 1–10, September 9, 2020 7

8 Neuron 107, 1–10, September 9, 2020

Neuron 107, 1–10, September 9, 2020 9

10 Neuron 107, 1–10, September 9, 2020

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Data and Code Availability

Neuron 107, 1–10.e1–e4, September 9, 2020 e1

EXPERIMENTAL MODEL AND SUBJECT DETAILS

e2 Neuron 107, 1–10.e1–e4, September 9, 2020

In situ hybridization followed by immunohistochemistry

Electrophysiological recordings from adult brain slices

Fast-scan cyclic voltammetry recordings

Neuron 107, 1–10.e1–e4, September 9, 2020 e3

QUANTIFICATION AND STATISTICAL ANALYSIS

e4 Neuron 107, 1–10.e1–e4, September 9, 2020

Title: Navy boys to the rescue

Author: Halsey Davidson

Illustrator: Clare Angell

Release date: January 15, 2024 [eBook #72723]

Original publication: United States: George Sully and Company,

Credits: Roger Frank and the Online Distributed Proofreading

*** START OF THE PROJECT GUTENBERG EBOOK NAVY BOYS

NAVY BOYS SERIES

NAVY BOYS AFTER A SUBMARINE

GEORGE SULLY & COMPANY

NAVY BOYS TO THE RESCUE

“William Johnson his my nyme—

Hoo-roo!” mumbled the gigantic sailor, and fell asleep again.

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