Morphine coordinates SST and PV interneurons in the prelimbic cortex to disinhibit pyramidal neurons and enhance reward
Morphine coordinates SST and PV interneurons in the prelimbic cortex to disinhibit pyramidal neurons and enhance reward
Changyou Jiang 0
● Xueying Wang 0
● Qiumin Le Ping Zheng 0
● Feifei Wang 0
● Lan Ma 0
0 Department of Neurology, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, School of Basic Medical Sciences, Institutes of Brain Science and Zhongshan Hospital, Fudan University , 200032 Shanghai , China
Opioids, such as morphine, are clinic analgesics which induce euphoria. Morphine exposure modifies the excitability and functional interactions between neurons, while the underlying cellular and molecular mechanisms, especially how morphine assembles heterogeneous interneurons (INs) in prelimbic cortex (PrL) to mediate disinhibition and reward, are not clear. Using approaches of optogenetics, electrophysiology, and cell type-specific RNA-seq, we show that morphine attenuates the inhibitory synaptic transmission from parvalbumin+ (PV)-INs onto pyramidal neurons in PrL via μ-opioid receptor (MOR) in PV-INs. Meanwhile, morphine enhances the inhibitory inputs from somatostatin+ (SST)-INs onto PV-INs, and thus disinhibits pyramidal neurons via δ-opioid receptor (DOR)-dependent Rac1 upregulation in SST-INs. We show that MOR in PV-INs is required for morphine-induced behavioral sensitization, while DOR as well as Rac1 activity in SST-INs is required for morphine-induced conditioned place preference and hyper-locomotion. These results reveal that SST- and PVINs, functioning in PrL as a disinhibitory architecture, are coordinated by morphine via different opioid receptors to disinhibit pyramidal neurons and enhance reward.
Addictive drug-induced long-lasting modifications in the
brain are associated with the neuronal plasticity in reward
circuits, which depends on dopamine neurons in the ventral
tegmental area (VTA) and their downstream targets,
including nucleus accumbens (NAc), anterior cingulate
cortex, medial prefrontal cortex (mPFC), etc. [
responds diversely to reward-predictive cues and is critical
for motivated behaviors . Prelimbic subregion of mPFC
(prelimbic cortex, PrL) has been implicated in regulation
and innervation of addictive processes [
]. Both VTA
and NAc are main recipients of the glutamatergic inputs
from the PrL, indicating this nuclei is crucial for reward
processing and the expression of drug-induced sensitization
3, 9, 10
The information processing of cortical circuits depends
on the functional interactions between the excitatory and
inhibitory connectivity. Diverse types of GABAergic
interneurons (INs) can receive, integrate, and encode
information to stringently control the projective outputs
]. Somatostatin (SST) and parvalbumin (PV) INs are two
major subtypes of inhibitory neurons in PrL of rodent and
human cortex, and they target distal dendritic and
perisomatic regions of postsynaptic excitatory neurons,
respectively, to exert their distinct inhibitory effects on excitatory
]. Functional connections between different
subtypes of INs are observed and thus INs collaboratively
regulate information integration in neural network [
The plasticity of PV-INs changes after fear conditioning and
environment enrichment, and the dysfunction of PV-INs is
found in schizophrenia mouse model [
of SST-INs in cortex is found in the Alzheimer’s mouse
]. These data indicate that the plasticity of distinct
INs in cortical circuits participates in the process of
cognition and psychiatric disorders.
Opioids, such as morphine, are clinical effective
analgesics, but they also induce euphoria and adaptive
changes of reward circuits [
]. Morphine acts through
Gprotein coupled opioid receptors to modulate presynaptic
and postsynaptic ion channels [
] and disinhibit the
inhibitory control to modulate pain and reward [
Recent studies indicate that morphine exposure causes
complex modifications of anatomical and functional
connections in reward circuits, especially between excitatory
]. However, the effect and the biological basis
of opioids on the connectivity among the excitatory and the
inhibitory neurons in PrL, are not fully understood.
In this study, we combined morphological tracing,
electrophysiology, and optogenetic methods, investigated
morphine-induced alterations of inhibitory inputs from
SST-INs and PV-INs onto local pyramidal neurons. Our
results reveal a disinhibitory architecture consisting of
SSTand PV-INs in the PrL, which is coordinated by morphine
via different subtypes of opioid receptors to disinhibit
pyramidal neurons, thus enhance morphine reward and
Morphine attenuates the inhibitory transmission to
pyramidal neurons from PV-INs, but not SST-INs in
PrL via a MOR-dependent pathway
To investigate morphine-induced disinhibition of pyramidal
neurons mediated by different INs in PrL, PV-INs or
SSTINs expressing hChR2-H134R were activated by serial laser
pulses, and the responsive inhibitory postsynaptic currents
(IPSCs) were measured in nearby pyramidal neurons
(Fig. 1a, b). The responsive probability was comparable
upon PV-INs or SST-INs activation (Fig. 1c, d). All
lightevoked IPSCs could be abolished by 20 mM bicuculline
(Fig. 1e, g). Morphine treatment (10 mg/kg, i.p.)
significantly reduced the peak amplitude and the half-width of
responsive IPSCs upon optogenetic activation of PV-INs,
while did not change the 10–90% rise time or synaptic
latency (Fig. 1e, f, and Supplementary Fig. 1a). Optogenetic
activation of SST-INs in PrL evoked much weaker IPSCs in
the nearby pyramidal neurons than activation of PV-INs,
while the responsive probability and the peak amplitude of
responsive IPSCs in pyramidal neurons were not changed
by morphine exposure (Fig. 1g, h).
To investigate whether μ-opioid receptor (MOR) in the
INs mediates the disinhibitory effect of morphine on PrL
pyramidal neurons, we co-infected
AAV-DIO-(hChR2H134R)-mCherry with AAV-Flex-MOR-shRNA-EGFP or
AAV-Flex-Scramble-shRNA-EGFP in the PrL of PV-Cre or
SST-Cre mice (Fig. 1a). The downregulation of MOR in
PV- and SST-INs was evaluated by immunostaining
(Supplementary Fig. 2a–c). Laser evoked action potentials (APs)
in PV-INs and SST-INs were not different between the
Scramble or MOR-shRNA expressing cells (Supplementary
Fig. 2d, e). Selective expression of MOR-shRNA in PV-INs
abolished the inhibition by morphine on responsive IPSC
amplitude in pyramidal neuron to laser stimulation of
PVINs (Fig. 1e, f). However, conditional knockdown of MOR
in SST-INs had no effect on the response probability or the
properties of the responsive IPSCs in pyramidal neurons to
laser activation of SST-INs (Fig. 1d, g, h, and
Supplementary Fig. 1b).
These results show that there is a broad connectivity
between pyramidal neurons and PV- or SST-INs in the PrL,
and PV-INs are able to evoke larger IPSCs in nearby
pyramidal neurons than SST-INs. Morphine decreases the
strength of synaptic inputs from PV-INs, but not SST-INs
onto pyramidal neurons via a MOR-dependent pathway.
Morphine increases neurite complexity of SST-INs and inhibitory transmission onto PV-INs in PrL
Addictive drugs are shown to regulate the density of
dendritic spines and the electrophysiological activity of neurons
in the mPFC [
]. To evaluate morphine-induced
morphological changes in PrL INs, we used reporter mice
(SST-Cre::EYFP and PV-Cre::EYFP) and injected a
fluorescent dye into SST-INs and PV-INs for morphological
tracing (Supplementary Fig. 3a). The results showed that 12
h after a single or five consecutive morphine injections,
the neurite complexity (Sholl intersections) and total
neurite length of SST-INs were significantly increased
compared with the saline control group (Supplementary
Fig. 3b, c). However, no significant difference in neurite
complexity (Sholl intersections) and total neurite length in
PV-INs were detected after morphine exposure
(Supplementary Fig. 3d, e).
To examine synaptic transmission in these INs in PrL,
we performed whole-cell patch-clamp recordings in PV-INs
or SST-INs 12 h after saline or morphine exposure
(Supplementary Fig. 4a). The amplitude of miniature excitatory
postsynaptic currents (mEPSCs) recorded from SST-INs
and PV-INs were both moderately decreased, while no
difference in the frequency of mEPSCs was observed after
morphine exposure (Supplementary Fig. 4b–e). Morphine
exposure did not affect the amplitude or frequency of the
miniature IPSCs (mIPSCs) in SST-INs (Fig. 1i, k, m), but
increased both the frequency and the amplitude of mIPSCs
in PV-INs (Fig. 1j, l, n), indicating that morphine enhances
inhibitory inputs onto PV-INs in PrL. In addition, morphine
increased the number of induced spikes in SST-INs
(Fig. 1o, p), while decreased the number of induced spikes
in PV-INs (Fig. 1q, r). The intrinsic electrophysiological
characteristics of SST-INs and PV-INs did not change after
morphine treatment (Supplementary Table 1).
These results suggest that morphine differentially
regulates neurite complexity, inhibitory synaptic
transmission, and membrane excitability of SST-INs and PV-INs
Fig. 1 Morphine decreases the strength of inhibitory transmission from
PV-INs to pyramidal neurons in PrL via MOR, and increases
inhibitory synaptic transmission to PV-INs. a Schematic diagram indicating
where AAV-Flex-MOR-shRNA-EGFP and
AAV-DIO-hChR2(H134R)mCherry were injected into PrL of PV-Cre or SST-Cre mice. b
Representative confocal images showing the pyramidal neurons (PYR,
Lucifer yellow) in PrL after whole-cell recordings upon optogenetic
stimulation of PV- or SST-interneurons co-expressing
hChR2mCherry and shRNA-EGFP. Percentage of pyramidal neurons
responsive to light-evoked activation of PV-INs (c: n = 30 cells/5 mice
in saline/Scramble group, 33 cells/6 mice in morphine/Scramble
group, 35 cells/6 mice in morphine/MOR-shRNA group; χ2 test) or
SST-INs (d: n = 24 cells/4 mice in saline/Scramble group, 27 cells/4
mice in morphine/Scramble group, n = 23 cells/4 mice in morphine/
MOR-shRNA group; χ2 test) 1 h after saline or 10 mg/kg morphine
treatment. e–h Representative traces and responsive IPSC amplitudes
onto pyramidal neurons from PV-INs (e, f: n = 27 cells/5 mice in
saline/Scramble group, 29 cells/6 mice in morphine/Scramble group,
30 cells/6 mice in morphine/MOR-shRNA group; One-way ANOVA
by the Bonferroni’s post-hoc test, F(2,83) = 5.508, P = 0.0057) or
SSTINs (g, h: n = 23 cells/4 mice in saline/Scramble group, 23 cells/4
mice in morphine/Scramble group, 22 cells/4 mice in
morphine/MORshRNA group; One-way ANOVA by the Bonferroni’s post-hoc test,
F(2,65) = 1.505, P = 0.2297) 1 h after saline or morphine (10 mg/kg,
i.p.) treatment in PrL slices expressing Scramble or MOR-shRNA.
EYFP+ cells in PrL were recorded in acute slice from SST-Cre::EYFP
or PV-Cre::EYFP mice 12 h after saline or morphine (10 mg/kg, i.p.)
injection. Representative traces (i, j), cumulative probability
distribution and average amplitude (k, l), and frequency (m, n) of mIPSCs
recorded from SST-INs (n = 22–27 cells/4 mice in each group) and
PV-INs (n = 36–40 cells/7–8 mice in each group; Mann–Whitney U
test for the average and two-sample Kolmogorov–Smirnov test for
cumulative probability). Representative AP traces and number of
induced spikes in SST-INs (o, p) or PV-INs (q, r) (o, p: n = 35 cells/7
mice; current: F(25,1700) = 191, P < 0.0001, treatment: F(1,68) = 15.84,
P = 0.0002, interaction: F(25,1700) = 9.079, P < 0.0001; q, r: n = 29–30
cells/6 mice; current: F(25,1425) = 192.9, P < 0.0001, treatment: F(1,57)
= 7.959, P = 0.0066, interaction: F(25,1425) = 2.822, P < 0.0001;
twoway RM ANOVA by the Bonferroni’s post-hoc test) in PrL after saline
or morphine injection. Data are presented as mean ± S.E.M; *P < 0.05,
**P < 0.01, ***P < 0.001, and ****P < 0.0001
Morphine enhances the inhibitory synaptic transmission from SST-INs to fast-spiking (FS) PVINs in PrL
Accumulating evidence indicates that the disinhibitory
microcircuits in the cortex involve interactions among
different subtypes of INs [
]. We thus investigated
whether morphine regulates the strength of synaptic inputs
from SST-INs onto PV-INs. We took advantage of
LhX6EGFP transgenic mice, in which the majority of INs derived
from the medial ganglionic eminence are labeled with
], and bred this transgenic line with SST::
tdTomato line (LhX6-EGFP/SST-tdTomato alleles) to
distinguish SST-INs (tdTomato+) from other types of INs
(EGFP+/tdTomato−) (Fig. 2a).
Immunostaining result showed that 56.62% of EGFP
+/tdTomato− INs is PV positive (Fig. 2b). Since FS is the
most prominent electrical property of PV-INs, we recorded
EGFP+/tdTomato− INs with FS character. To examine
synaptic transmission from SST-INs onto FS PV-INs,
AAVDIO-(hChR2-H134R)-mCherry was infected in the PrL of
LhX6-EGFP/SST-Cre mice, and EGFP+/tdTomato− FS-INs
nearby tdTomato+ SST-INs were recorded upon
optogenetic activation of SST-INs (Fig. 2c, d). The connection
probability did not change after morphine exposure
(Fig. 2e). The responsive amplitude, but not the 10–90%
rise time or half-width of light-evoked IPSCs in FS-INs,
was increased 12 h after morphine exposure (Fig. 2f–h),
suggesting that morphine exerts long-lasting effect on the
inhibitory synaptic transmission from SST-INs to FS-INs.
We next assessed the presynaptic release probability by
analysis of paired-pulse ratio (PPR) and the coefficient of
variation in FS PV-INs upon optogenetic stimulation of
SST-INs. Morphine significantly reduced PPR ratio at the
second and third stimulations (Fig. 2i, j). The coefficient of
variation of responsive IPSCs did not significantly change
(Fig. 2k). Combined with result of the increased mIPSCs
frequency in PV-INs after morphine exposure, these data
suggest that morphine increases the presynaptic release
probability of SST-INs to PV-INs, and indicate that
morphine-enhanced inhibitory GABAergic transmission
from SST-INs onto PV-INs involves presynaptic
Cell type-specific RNA-seq reveals that morphine
upregulates Rac1 pathway specifically in SST-INs,
but not PV-INs of PrL
Given the observed difference of morphine-induced
neuronal plasticity between SST-INs and PV-INs, the
neuronalspecific molecular mechanisms between these two subtypes
of neurons were assessed. SST-Cre and PV-Cre mice were
crossed to RPL22-HA reporter mice to produce mice that
express HA-tagged ribosomal protein (ribotag) specifically
in SST- or PV-INs (Fig. 3a). Transcripts associated with
ribosomes (in the process of de novo protein synthesis)
were isolated from PrL 12 h after saline or morphine
injection and sequenced (Fig. 3b). Analysis of the
ribosome-associated transcripts in these two groups showed
that Pvalb and Sst were respectively enriched in PV-INs and
SST-INs (Supplementary Fig. 5a), indicating the successful
enrichment of interneuron subtype-specific transcripts.
Morphine injection induced more transcriptional alterations
in SST-INs, compared with PV-INs (translational change in
1558 vs. 328 genes; Fig. 3c and Supplementary Fig. 5b).
To infer potential intracellular pathways altered by
morphine, ClueGO was used for regulatory network construction
of these two subtypes of INs. The analysis indicates that
SST-INs exhibited significant differences in genes involved
in the pathways of cAMP and insulin signaling, addiction,
and neurotrophic factor signal transduction, etc., which have
group; χ2 test). Responsive IPSC amplitudes (f), the 10–90% rise time
(g) and the half-width (h) of responsive IPSCs in FS PV-INs after
saline or morphine exposure (n = 25–26 cells/five mice in each group;
Mann–Whitney U test). i Representative traces of responsive
amplitudes of IPSC responses in SST-INs to FS PV-INs connections. Light
interval: 100 ms. Paired-pulse ratio (PPR) (j) and the coefficient of
variation (k) of FS PV-INs in the PrL of mice injected with saline or
morphine (n = 20 cells/five mice in each group; j two-way RM
ANOVA by the Bonferroni’s post-hoc test. Number: F(3,114) = 266.2,
P < 0.0001, treatment: F(1,38) = 8.944, P = 0.0049, interaction: F(3,114)
= 5.394, P = 0.0017; k Unpaired Student’s t test). Data are presented
as mean ± SEM; n.s. not significant; *P < 0.05 and ***P < 0.001
been associated with learning and memory, cognition, and
neural plasticity. Consistent with the neurite complexity of
SST-INs revealed by morphological study, Rac1, a member
of the Rho family of GTPases and an important regulator of
actin cytoskeleton and structural plasticity [
located on the hub section of the morphine-regulated
signaling network in SST-INs (Fig. 3d). The changed
ribosomeassociated transcripts in PV-INs were enriched in the
network including the insulin signaling, cell differential
pathways, and protein–protein interaction, etc.
(Supplementary Fig. 5c). qRT-PCR of ribosome-associated transcripts
showed that the expression of the Rac1/Cdc42 guanine
nucleotide exchange factor 6 (Arhgef6), and the immediate
early genes such as Arc were upregulated in SST-INs
(Supplementary Fig. 5d), but not in PV-INs (Supplementary
Fig. 5e). These results indicate that morphine-induced
morphological alterations in SST-INs couples with the changes
of Rac1-related signaling pathways.
Fig. 3 Morphine upregulates the expression of Rac1 and Arhgef6 in
SST-INs via a DOR-dependent mechanism. Cell type-specific
RNAseq reveals morphine upregulates Rac1 pathway in SST-INs. a
Breeding scheme of SST-Cre::RPL22-HA mice and
PV-Cre::RPL22HA mice. b Schematic procedure showing ribotag
immunoprecipitation (IP) and RNA-seq of the ribosome-associated transcripts in
PVINs or SST-INs in PrL after saline or 10 mg/kg morphine injection. c
The heat map of hierarchical clustering of normalized level of
ribotagisolated transcripts in SST-INs (five mice/group). Each row
corresponds to a single gene. d Representation of the morphine-regulated
signaling network enrichment analysis including all modules and
contributing genes in SST-INs. e–f Single-molecule fluorescence ISH
for Rac1 or Arhgef6 transcript in SST-INs 1 h or 12 h after saline or
morphine (10 mg/kg, i.p.) injection. Quantification of the fluorescent
intensity of Rac1 or Arhgef6 transcripts in SST-INs. Three mice/group:
Rac1: Sal, 247 cells, Mor 1 h, 150 cells, Mor 12 h, 192 cells; Arhgef6:
Sal, 448 cells, Mor 1 h, 355 cells, Mor 12 h, 294 cells. g, h smFISH for
Rac1 or Arhgef6 in SST-INs expressing DOR-shRNA, MOR-shRNA, or
Scramble-shRNA 12 h after morphine (10 mg/kg, i.p.) injection.
Quantification of the fluorescent intensity of Rac1 or Arhgef6
transcripts in SST-INs expressing shRNA. 4 mice/group; Rac1: Scramble,
172 cells, DOR-shRNA, 185 cells, 154 cells; Arhgef6: Scramble,
125 cells, DOR-shRNA, 258 cells, MOR-shRNA, 106 cells.
Oneway ANOVA by the Bonferroni’s post-hoc test for intensity (e: F(2,586)
= 12.12, P < 0.0001; f: F(2,1094) = 12.45, P < 0.0001; g: F(2,508) =
52.05, P < 0.0001; h: F(2,486) = 13.57, P < 0.0001). Two-sample
Kolmogorov–Smirnov test for cumulative frequency. Data are
presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and
****P < 0.0001
Morphine upregulates the expression of Rac1 and
Arhgef6 in SST-INs via DOR
Since the plasticity-related gene Rac1 in SST-INs was
identified as a hub gene in morphine-regulated signaling
pathways, and the expression of Arhgef6 was markedly
upregulated in SST-INs after morphine exposure
(Supplementary Fig. 5d), we performed single-molecule RNA
in situ hybridization (ISH) to analyze the level of Rac1
and Arhgef6 transcripts in SST-INs. The results of
RNAscope ISH showed that the intensity of Rac1 and
Arhgef6 transcripts in SST-INs was upregulated 1 h after
morphine injection and maintained at high level 12 h after
the injection (Supplementary Fig. 6 a, b and Fig. 3e, f).
Morphine binds preferentially to MOR, while
prolonged stimulation of neurons with morphine, both
in vitro and in vivo, markedly increases recruitment of
intracellular δ-opioid receptor (DOR) to the cell surface
]. To explore the potential role of MOR and DOR in
morphine-induced upregulation of Rac1 and Arhgef6
mRNAs in SST-INs, we infected
AAV-Flex-DOR-shRNAEGFP or AAV-Flex-MOR-shRNA-EGFP into PrL of
SST-Cre mice. Cre-dependent DOR downregulation in
SST-INs and PV-INs was verified by RNAscope ISH
(Supplementary Fig. 7). We found that 12 h after
morphine exposure, the fluorescent intensity of Rac1 and
Arhgef6 transcripts was decreased in SST-INs expressing
DOR-shRNA, whereas not changed in SST-INs
expressing MOR-shRNA (Supplementary Fig. 6c, d and Fig. 3g,
h). These results suggest that morphine enhances the
expression of Rac1 and Arhgef6 in SST-INs via DOR, but
DOR and Rac1 in SST-INs mediate the enhancement of inhibitory transmission from SST-INs onto PV-INs by morphine and the disinhibition of pyramidal neurons in PrL
We further assessed whether morphine-enhanced
inhibitory inputs onto PV-INs were mediated by DOR and Rac1
in SST-INs. We infected Cre-dependent DOR-shRNA and
hChR2-mCherry viruses into the PrL of
LhX6-EGFP/SSTCre mice, and performed whole-cell recordings in FS
PVINs or pyramidal neurons nearby the opto-activated
SSTINs 12 h after morphine exposure (Fig. 4a, e). Knockdown
of DOR did not affect the light-evoked responsive
probability in FS PV-INs (Fig. 4c), but decreased the
responsive amplitude in FS PV-INs (Fig. 4b, d). In
addition, knockdown of DOR did not affect the
lightevoked responsive probability and the responsive
amplitude of IPSCs in pyramidal neurons (Fig. 4f–h). These
results suggest that knockdown of DOR in SST-INs
decreased the strength of the inhibitory inputs from
SSTINs to FS PV-INs.
We also constructed the
AAV-EF1α-DIO-Rac1-DNmcherry which expresses the dominant negative mutant of
Rac1 (Rac1-DN) in a Cre-dependent manner. The neurite
complexity and total neurite length of SST-INs or PV-INs
were significantly decreased by the expression of
Rac1DN in PrL (Supplementary Fig. 8). Moreover, we infected
Cre-dependent Rac1-DN and hChR2-mCherry viruses into
the PrL of LhX6-EGFP/SST-Cre mice, and performed
whole cell recordings in FS PV-INs or pyramidal neurons
(Fig. 4i, m). Expressing Rac1-DN in SST-INs did not
affect the light-evoked responsive probability in FS
PVINs (Fig. 4k), while attenuated the light-evoked
responsive amplitude in PV-INs (Fig. 4j, l). However, expressing
Rac1-DN in SST-INs decreased the light-evoked
responsive probability (Fig. 4o), while did not affect the
responsive amplitude in pyramidal neurons (Fig. 4n, p). In
addition, pyramidal neurons nearby the SST-INs were
significantly disinhibited by morphine, reflected by the
increased current pulses, while the increased current
pulses were abolished by expressing Rac1-DN in SST-INs
(Fig. 4q, r). These data indicate that morphine increases
the strength of the inhibitory transmission from SST-INs
onto PV-INs via DOR and Rac1 signaling in SST-INs,
and may thus attenuate the inhibitory effect of PV-INs
onto pyramidal neurons in PrL.
Distinct opioid receptor pathways in PrL SST-INs and
PV-INs mediate morphine-conditioned place preference (CPP) and behavioral sensitization
Prefrontal cortex has been implicated in the reward
processing and the development of addictive-drug-induced
behavioral sensitization [
]. To investigate the effect of
MOR and DOR signaling pathways in specific INs on the
rewarding properties and locomotor-activating effects of
morphine, SST-Cre and PV-Cre mice were infected with
AAV-Flex-DOR-shRNAEGFP or AAV-Flex-Scramble-shRNA-EGFP in PrL
(Fig. 5a). Results showed that knockdown of DOR in
SSTINs significantly inhibited morphine-induced CPP and
hyper-locomotion, while knockdown of MOR in SST-INs
had no such effects (Fig. 5b, c). We then injected
AAV-EF1α-DIOmCherry into the PrL of SST-Cre and PV-Cre mice
(Fig. 5d). Expressing Rac1-DN in SST-INs abolished
morphine-induced CPP and hyper-locomotion (Fig. 5e, f),
while expressing Rac1-DN in PV-INs had no effect
(Supplementary Fig. 9). These results indicate that DOR-Rac1
pathway in the SST-INs, but not in the PV-INs, is involved
in the reward properties and locomotor-activating effects of
Knockdown of MOR in PV-INs had no effect on
morphine-induced CPP, but decreased locomotor activity
90 min after the initial morphine injection (Fig. 5h, i) and
behavioral sensitization after repeated morphine exposures
(Supplementary Fig. 10), suggesting that MOR of PV-INs
in PrL is involved in both the initiation and expression of
behavioral sensitization to morphine. Knockdown of DOR
in PV-INs had no effect on morphine-induced CPP and
hyper-locomotion (Fig. 5h, i).
Taken together, these data reveal that DOR-Rac1
pathway in SST-INs is required for morphine-induced CPP and
hyper-locomotion, while MOR pathway in PV-INs is
involved in behavioral sensitization, and indicate that
morphine, via distinct opioid receptors, coordinates an
architecture consisting of SST-INs and PV-INs to disinhibit
pyramidal neuron in PrL and enhance reward.
GABAergic INs, as a minority of the cortical neuronal
population in the forebrain, are crucial in fine-tuning
cortical microcircuits. The interactions of GABAergic INs with
excitatory glutamatergic neurons maintain balanced
electrical activity and normal cortical functions [
activates presynaptic GABAergic neurons in VTA and
disinhibits dopaminergic neurons, increasing dopamine
release and inducing reward [
]. However, the molecular
targets and the inhibitory architecture recruited by morphine
to promote reward and behavioral sensitization are unclear.
PV-INs includes FS basket and chandelier cells. FS-INs
are the largest population of INs in the neocortex. They
regulate action potential firing and form complex structural
contacts between themselves to promote synchronization of
electrical activity [
14, 43, 44
]. In contrast to PV-INs,
SSTINs are dendritic targeting and they mediate double-synapse
inhibition on nearby pyramidal neurons [
]. We examined
the amplitude of responsive IPSC from PV-INs and
SSTINs onto pyramidal neurons by optogenetic stimulation, and
observed that as compared with SST-INs, PV-INs showed
stronger predominant inhibitory inputs onto pyramidal
neurons (Fig. 1). SST-INs showed an increased intrinsic
membrane excitability, while PV-INs showed a decreased
intrinsic membrane excitability upon morphine exposure.
Consistently, SST-INs exhibited more translational and
morphological changes upon morphine exposure (Fig. 3 and
Supplementary Fig. 3), indicating that morphine-induced
alterations are interneuron subtype-specific and likely via
differential modulatory mechanisms.
SST-INs not only innervate pyramidal neurons but also
strongly innervate other types of INs in the cortex
33, 34, 46
]. The increased frequency of mIPSC in PV-INs
indicates an increased inhibitory input. Utilizing
LhX6Fig. 5 Distinct opioid receptors of SST-INs and PV-INs in the PrL
coordinate morphine-induced CPP and behavioral sensitization. a
Schematic of the PrL area where the AAV-Flex-DOR-shRNA-EGFP,
AAV-Flex-MOR-shRNA-EGFP or AAV-Flex-Scramble-shRNA-EGFP
was injected in SST-Cre or PV-Cre mice.The effect of downregulating
DOR or MOR in SST-INs on morphine-induced CPP (b: n = 18 mice
in Scramble group, 15 mice in MOR-shRNA group, 11 mice in
DORshRNA group; Paired Student’s t test, Scramble, P = 0.0041,
MORshRNA, P = 0.0095, DOR-shRNA, P = 0.6107) and hyper-locomotion
(c: n = 24 mice in Scramble group, 12 mice in MOR-shRNA group, 13
mice in DOR-shRNA group; two-way RM ANOVA by the
Bonferroni’s post-hoc test. Scramble vs. DOR-shRNA, time: F(11,385) = 37.74,
P < 0.0001, virus: F(1,35) = 8.463, P = 0.0063, interaction, F(11,385) =
5.586, P < 0.0001; Scramble vs. MOR-shRNA, time: F(11,374) = 29.51,
P < 0.0001, virus: F(1,34) = 0.0693, P = 0.7940, interaction: F(11,374) =
1.427, P = 0.1584). d Schematic of the PrL area where
AAV-DIORac1-DN-mCherry or AAV-DIO-mCherry was injected in SST-Cre or
PV-Cre mice. The effect of downregulating Rac1 activity in SST-INs
on morphine-induced CPP (e: n = 11 mice in mCherry group, 8 mice
in Rac1-DN group; paired Student’s t test, mCherry, P = 0.0086,
Rac1-DN, P = 0.9184) and hyper-locomotion (f: n = 8 mice in
mCherry group, 12 mice in Rac1-DN group; two-way RM ANOVA
by the Bonferroni’s post-hoc test. Time: F(11,198) = 8.23, P < 0.0001,
virus: F(1,18) = 2.957, P = 0.1026, interaction, F(11,198) = 4.973, P =
0.00029). g Experimental schedule for morphine-induced CPP. The
effect of down-regulating DOR or MOR in PV-INs on
morphineinduced CPP (h: n = 13 mice in Scramble group, 11 mice in
MORshRNA group, 14 mice in DOR-shRNA group; paired Student’s t-test,
Scramble, P = 0.0108, MOR-shRNA, P = 0.0049, DOR-shRNA, P =
0.0073) and hyper-locomotion (i: n = 20 mice in Scramble group, n =
10 mice in MOR-shRNA group, 12 mice in DOR-shRNA group;
twoway RM ANOVA by the Bonferroni’s post-hoc test. Scramble vs.
MOR-shRNA, time: F(11,308) = 27.42, P < 0.0001, virus: F(1,28) =
0.6785, P = 0.4171, interaction: F(11,308) = 2.641, P = 0.0031;
Scramble vs. DOR-shRNA, time: F(11,330) = 29.33, P < 0.0001, virus: F
(1,30) = 0.8693, P = 0.3586, interaction: F(11,330) = 1.119, P = 0.3449).
Data are presented as mean ± SEM; n.s. not significant; *P < 0.05, **P
< 0.01, ***P < 0.001, and ****P < 0.0001. j A model depicting the
disinhibitory architecture in PrL and coordination by morphine.
SSTINs (orange) innervate distal dendrites, while PV-INs (green) mainly
target soma of pyramidal neurons (PYR), to exert distinct inhibitory
effect on PYR in physiological state. Morphine attenuates the
inhibitory input from PV-INs onto PYR via MOR (blue), while upregulates
the Rac1 in SST-INs via DOR (gray) to enhance its inhibitory effect
onto PV-INs. This architecture specifically coordinated by morphine
via different opioid receptors disinhibits pyramidal neurons in PrL, and
thus enhances reward
EGFP/SST-tdTomato mice, we identified PV-INs by
EGFP+/tdTomato− labeling and FS features to study the
synaptic transmission from SST-INs to FS PV-INs. Our
data suggest that morphine exposure increases the inhibitory
transmission from SST-INs to FS PV-INs in PrL and this is
dependent on a presynaptic mechanism. We postulate that
after morphine stimulation, SST-INs extend their neurites to
form extensive reciprocal connections and stronger
inhibition to adjacent FS PV-INs, and this is accompanied with
reduced FS PV-INs firing, leading to disinhibition of nearby
pyramidal neurons and behavioral changes.
Morphine exhibits affinity to MOR, DOR, and KOR
subtypes, but has higher affinity to MOR and thus
preferentially binds to MOR [
]. The results from
MORand DOR-knockout mice indicate that MOR is essential for
both of analgesia and tolerance of morphine [
DOR is required for the development of sensitization and
tolerance to the locomotor-activating effects of morphine
]. Previous histological data showed that MORs are
expressed prominently in PV-INs, whereas DORs are
expressed prominently in SST, neuropeptide Y, and
corticotrophin releasing factor (CRF) INs, as well as in
pyramidal neurons in hippocampus [
]. It is interesting to
know how morphine remodels the circuits to promote
reward by activating signaling pathways mediated by
different opioid receptors in different INs.
In this study, we focus on MOR and DOR signaling and
the inhibitory transmission from PV- and SST-INs onto
pyramidal neurons in PrL. Our results reveal that morphine
exerts its disinhibition function on pyramidal neurons via
neuronal subtype- and opioid receptor-specific signaling
pathways. The activation of MOR-signaling pathway in
PVINs by morphine attenuates the inhibitory inputs to
pyramidal neurons directly, while activation of DOR signaling
pathway in SST-INs by morphine enhances the inhibitory
inputs to PV-INs and thus further disinhibits pyramidal
neurons nearby (Fig. 5j). Our results showed that morphine
specifically increases the neurite complexity and
upregulates Rac1 and Arhgef6 in SST-INs, while knockdown of
DOR in SST-INs decreased the mRNA level of Rac1 and
Arhgef6. Knockdown of Rac1 in SST-INs abolished
morphine-induced strengthening of the inhibitory inputs to
FS PV-INs and the increase of activity of the pyramidal
neuron. These data indicate that the Rac1 is downstream of
opioid receptor in SST-INs, and it mediates the inhibition of
SST-INs on nearby PV-INs.
MOR in PV-INs and DOR in SST-INs in PrL mediate
morphine-induced CPP and behavioral sensitization,
respectively, indicating that morphine-induced reward
processing and behavioral sensitization require the activation of
neuronal-specific opioid receptors in PrL. Our results
suggest that the acute effect of morphine to disinhibit pyramidal
neurons is via attenuating the inhibitory synaptic
transmission from PV-INs to pyramidal neurons, while the
longlasting disinhibition effect of morphine (12 h after morphine
exposure) on pyramidal neurons is through enhancing
the strength of the inhibitory transmission from SST-INs
onto PV-INs. Since morphine has higher affinity for MOR
than DOR, we hypothesize that morphine initially activates
the MORs in the PV-INs, directly inhibits PV-INs
producing acute disinhibition of pyramidal neurons and
behavioral sensitization, and excessive morphine activates
DORs in SST-INs, thus inducing prolonged inhibition on
PV-INs via upregulation of Rac1, blocking the inhibitory
inputs from PV-INs to the PrL pyramidal neurons and
Research on the structural and functional connectivity
between inhibitory INs and pyramidal neurons is
important for understanding on how inhibitory architecture in
PrL gates neuronal network excitability. The circuitry for
behavioral sensitization includes glutamatergic
projections from the mPFC to the NAc [
], and the
prefrontal glutamate release into NAc mediates drug-seeking
behaviors . The output circuits of PrL guide
conditioned reward seeking through divergent PFC → NAc and
PFC → PVT encoding [
]. mPFC → NAc population
dynamics predict individual reward seeking or
suppression decision [
]. These results indicate that
glutamatergic projection in PrL, which is modulated precisely by
local INs, is required for behavioral sensitization and
reward. Our data indicate that morphine remodels
SSTand PV-interneuron plasticity, which in turn induces
behavioral changes via distinct molecular pathways in the
two types of INs. The MOR and DOR signaling pathways
in the INs play important regulatory roles in behavioral
sensitization and reward processing.
Materials and methods
SST-Cre mice (013044), PV-Cre mice (012358), Rosa 26
reporter mice (006148), Ai14 reporter mice (007914), and
Ribotag mice (011029) were purchased from The Jackson
Laboratory (CA, USA). LhX6-EGFP mice (000246-MU)
were purchased from Mutant Mouse Resource & Research
Centers (MMRRC). These mice were bred to C57BL/6 J
for more than 6 generations. SST-Cre::EYFP or PV-Cre::
EYFP alleles were generated by crossing SST-Cre or
PVCre mice with Rosa 26 reporter mice;
SST-Cre::RPL22HA or PV-Cre:: RPL22-HA alleles were generated by
crossing SST-Cre or PV-Cre mice with Ribotag mice;
SST-Cre::tdTomato alleles were generated by crossing
SST-Cre mice with Ai14 mice; LhX6-EGFP/SST-Cre::
tdTomato mice were generated by crossing LhX6-EGFP
mice with SST-Cre::tdTomato mice;
LhX6-EGFP/SSTCre were generated by crossing LhX6-EGFP mice with
SST-Cre mice. 6–10-week-old male offsprings were used
in the experiments, and randomly assigned to groups.
Mice used for the experiments were housed in groups on a
12 h light/dark cycle (light on from 8 a.m. to 8 p.m.) with
access to food and water ad libitum. All experiment
procedures were strictly in accordance with the National
Institutes of Health Guide for the Care and Use of
Laboratory Animals, and were approved by Animal Care
and Use Committee of the animal facility at Fudan
Fragment encoding Rac1 dominant negative mutant
(Rac1DN, T17N) [
] was subcloned into
pAAV-EF1α-DIOmCherry using AscI/NheI restriction sites to yield
pAAVEF1α-DIO-Rac1-DN-mcherry. For Cre-dependent
expression of shRNAs in cells and transgenic mice, the shRNAs
coding sequence targeting mouse MOR
(5′-CGGCTAATACAGTGGATCGAA-3′) or DOR
(5′GTGCTATGGCCTCATGCTACT-3′) were cloned into the
pAAV-CMV-Flex-MIR30shRNA-EGFP vector (Obio
Technology, Shanghai, China) using EcoRI/XhoI restriction
AAV9-FlexMOR-shRNA-EGFP, AAV9-Flex-DOR-shRNA-EGFP or
AAV9-Flex-Scramble-shRNA-EGFP viruses were packaged
by Obio Technology (Shanghai, China).
AAV9-EF1α-DIOmCherry and AAV9-EF1α-DIO-hChR2(H134R)-mCherry
were purchased from Taitool Bioscience (Shanghai, China).
Mice were anesthetized with 2% isoflurane and placed in a
stereotactic instrument (Stoelting, Kiel, WI, USA).
Microinjections were performed using 33-gauge needle connected
to a 10 μl Hamilton syringe. The intended stereotaxic
coordinates for PrL were: AP + 2.0 mm; ML ± 0.3 mm
(with an angle of 14° from the middle to the lateral); DV —
2.0 mm. Each site was injected with 0.5 μl of purified and
concentrated AAV (1012 IU/ml) with a slow injection rate
(0.1 μl/min). All mice were given at least 3 weeks to recover
before behavioral experiments or electrophysiological
recordings, and the efficiency of viral infection and the
shRNA knockdown was verified by immunostaining. The
histology slides were examined blindly to check the
expression of EGFP or mCherry in PrL. Only the mice with
virus infection in correct place were chosen for further
Brain slice preparation and electrophysiological recording
Coronal sections (300 μm) containing PrL were prepared as
previously described [
]. Briefly, the mice were
anesthetized by isoflurane and then transcardially perfused with cold
artificial cerebrospinal fluid [ACSF; 92 mM
N-methyl-Dglucamine, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM
NaHCO3, 20 mM HEPES, 25 mM D-glucose, 2 mM
thiourea, 5 mM Na-ascorbate, 3 mM Na-pyruvate, 0.5 mM
CaCl2, and 10 mM MgCl2]. Brains were quickly removed,
sliced with the vibratome (Thermo Scientific, MA, USA) and
incubated in protective ACSF saturated with 95% O2, 5%
CO2, and the slices were used within 6 h after preparation.
Individual neurons were identified under a BX51WI
microscope (Olympus, Tokyo, Japan) equipped with Rolera Bolt
CCD camera (QImaging, Surrey, BC, Canada). Whole-cell
voltage clamp recordings were performed in oxygenated
ACSF (124 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4,
24 mM NaHCO3, 5 mM HEPES, 12.5 mM glucose, 2.4 mM
CaCl2, and 1.2 mM MgCl2) at 31~32 °C with an EPC-10
amplifier and Patchmaster software (HEKA Elektronik,
Lambrecht/Pfalz, Germany). The pipette resistance was in
the range of 8–10 MΩ. Current clamp recordings were
filtered at 2.9 kHz and sampled at 5 kHz.
For light-evoked postsynaptic currents whole-cell
recordings, ChR2 was excited with a 473 nm LED blue
light source (Xcite-110) delivered through the
epifluorescence pathway and fed into a 60× water-immersion
objective lens (Olympus BX51). Photo-stimulation (2–6
mW/mm2, 1–2 ms duration) was controlled by a TTL input
(HEKA Instruments). Pyramidal neurons or FS INs were
clamped at −70 mV. For recordings, the pipettes were filled
with intracellular solution (60 mM K-gluconate, 66 mM
KCl, 2 mM MgCl2, 10 mM HEPES, 0.2 mM EGTA, 4.5
mM MgATP, 0.5 mM Na3GTP, 10 mM
Na-phosphocreatine, 0.25% neurobiotin, pH 7.25, 300 mOsm). D-APV
(100 mM) and CNQX (20 mM) were added to block
excitatory currents. A train of light pulses (10 Hz) was delivered
to the presynaptic cells, and the postsynaptic responses were
recorded 20–30 repeated trails at 15 or 20 s interval. Data
were analyzed off-line with Clampfit 10.3 (Molecular
Devices, Union City, CA, USA) or Mini Analysis Program
(Synaptosoft, Fort Lee, NJ, USA). The peak amplitude was
calculated by subtracting the baseline. The synaptic latency
was determined as the duration from current onset time to
peak time. The rise time of evoked IPSCs was assessed
from the 10 to 90% rising phase, and the half-width of
evoked IPSCs was defined as the duration at the half
amplitude. For the paired-pulse ratio calculation, the
averaged peak amplitude of the first IPSC was defined as the
basal level of synaptic strength. The variable coefficient was
assessed from the amplitudes of each sweep.
We used modified intracellular solution (127.5 mM
cesium methanesulfonate, 7.5 mM CsCl, 10 mM HEPES,
2.5 mM MgCl2, 4 mM Na2ATP, 0.4 mM Na3GTP, 10 mM
sodium phosphocreatine, 0.6 mM EGTA, pH 7.25, 290
mOsm) to adjust the reversal potential of the γ-aminobutyric
acid-A receptor (GABAaR) response. mEPSC events were
recorded in the presence of 2 μM TTX and GABAaR
blocker (bicuculline methiodide, 10 μM) (Tocris
Bioscience, Bristol, UK), at a holding potential of −60 mV.
mIPSC events were recorded in the presence of 2 μM TTX,
NMDA receptor blocker (10 μM D-APV), and AMPA
receptor blocker (CNQX, 20 μM), at a holding potential of
+10 mV. The intracellular solution (130 mM K-gluconate,
6 mM KCl, 2 mM MgCl2, 10 mM HEPES, 2.5 mM
ATPMg, 0.5 mM GTP-Na2, 10 mM creatine phosphate, 0.6 mM
EGTA, pH 7.25, 290 mOsm) was used for recording action
potential. After achieving whole cell configuration, a
current-step protocol (from −200 to +200 pA, with 10 pA
increment) was run and repeated. Recordings with Rs >
30 MΩ were excluded from statistical analysis. Data were
filtered at 300 Hz and were analyzed by Mini Analysis
Morphological reconstruction and quantitation
For neuronal reconstruction and morphological analysis,
SST and PV INs in PrL were randomly selected, patched,
and filled with 2% lucifer yellow (L0259, Sigma-Aldrich, St
Louis, WA, USA) for at least 10 min. After 10 min
additional diffusion, slices were fixed in 4% PFA in 0.1 M
phosphate-buffered saline (PBS) overnight. Sections were
blocked in 10% serum and 0.1% Triton-X in PBS, and
incubated with an anti-Lucifer Yellow antibody (Invitrogen,
#A-575c, Carlsbad, CA, USA) overnight at 4 °C. Z-series
images were taken at 2 μm interval using an Olympus
FV1000 confocal laser scanning microscope with a
60× objective (Olympus). Full cell 3-dimensional
reconstructions and analysis were made by Neurolucida
(MicroBrightField, Williston, VT, USA).
Mice were anesthetized with isoflurane and perfused with
saline followed by 4% paraformaldehyde in 0.1 M PBS.
The brains were removed, fixed in 4% paraformaldehyde
overnight and subjected to dehydration in 30% sucrose at
4 °C for 72 h before slicing 30 μm per slice. Slices were
incubated with diluted antibodies in blocking solution
containing 0.2% Triton X-100 (Sigma-Aldrich) and 3%
goat serum (Jackson ImmunoResearch, West Grove, PA,
USA) at 4 °C overnight. The primary antibodies used were:
anti-SST (Santa Cruz, #sc-47706, Dallas, TX, USA),
antiPV (Merck Millipore, #MAB1572, Darmstadt, Germany)
and anti-MOR (Abcam, #ab10275, Cambridge, MA, USA).
Slices were rinsed in 0.1 M PBS then incubated in Cy3
antimouse, Alexa 647 anti-rat or Cy3 anti-rabbit IgG antibodies
(Jackson ImmunoResearch) for 1 h at room temperature,
then mounted after rinsing with 0.1 M PBS. Images were
acquired on a Nikon A1 microscope (Tokyo, Japan) using
20× air or 60× oil objective lens. The observer analyzing the
expression of MOR in SST or PV INs was blinded to the
The frozen brain tissue was sliced into 10 μm coronal
sections and mounted onto Colorfrost Plus slides
(ThermoFisher, Waltham, MA, USA). Slices were
incubated with hydrogen peroxide 10 min RT, target-retrieval
solution and Protease III using RNAscope® 2.5 Universal
Pretreatment Reagents (Advanced Cell Diagnostics,
#322380, Newark, CA, USA). smFISH for all genes
examined, Rac1 (#517461), Arhgef6 (#574371), EGFP
(#400281-C3), Sst (#404631-C2), Pvalb (#421931-C2), and
Oprd1 (#427371-C3) were performed hybridization for 2 h.
After hybridization, we used the RNAscope® Multiplex
Fluorescent Detection Kit v2 (#323110) to amplify signal
and mounted. Images were acquired with a Nikon A1
microscope using 20× objective. IOD in SST+ or PV+
neurons was analyzed by Image-Pro Plus 6.0 (Media
Cybernetics, Rockville, MD, US). The observer analyzing
the expression of DOR, MOR, Rac1, and Arhgef6 in SST, or
PV INs was blinded to the group allocation.
Purification of ribosome-associated mRNA was performed
as described previously with slight modification [
were decapitated, and the brains were removed
immediately. The PrL were dissected in ice-cold PBS. The brain
tissue was homogenized in 1 ml Supplemented
Hybridization Buffer (25 mM Tris pH 7.0, 25 mM Tris pH 8.0,
12 mM MgCl2, 100 mM KCl, 1% Triton X-100) containing
1 mM DTT, 1 × protease inhibitors (Roche, Upper Bavaria,
Germany), 200 U/ml RNase inhibitor (Promega, Madison,
WI, USA), 100 μg/ml cycloheximide (Cayman, Ann Arbor,
MI, USA), and 1 mg/ml heparin (Sigma-Aldrich). The
supernatant was incubated with 10 μg anti-HA antibody
(Sigma-Aldrich, #H6908) and 100 μl Dynabeads Protein G
(Invitrogen) for 12 h. Purified mRNA was eluted from the
Dynabeads using TRIzol LS (Invitrogen) according to the
manufacturer’s instructions with the inclusion of a DNase
digestion step. The Agilent RNA 6000 Pico Kit (Agilent,
Santa Clara, CA, USA) and Agilent 2100 bioanalyzer were
used to evaluate the quality of purified mRNA. Samples
with RIN number > 7 were used.
mRNA was enriched using NEB Next Poly(A) mRNA
Magnetic Isolation Module (NEB, E7490S, Ipswich, MA,
USA). Library was prepared with NEB Next Ultra RNA
Library Prep Kit (E7530S) and sequenced on a HiSeq 4000
(Illumina) by Novogene Technology Co. Ltd (Beijing,
China). Raw reads were quality checked and trimmed with
FASTX-toolkit to remove adapter contamination and
lowquality reads (quality score < 28). The clipped reads were
aligned to mouse reference sequence (GRCm38/mm10)
using HISAT2. Mapped reads for each transcript were
counted using HTseq and differential expression analysis
was performed with DESeq2. Genes with more than twofold
expression changes, and were significantly different (P <
0.05) were selected for further analysis. ClueGo [
used for signaling pathway and network construction.
Reverse transcription, and quantitative real-time PCR (qRT-PCR)
Reverse transcription was completed using the PrimeScript
RT reagent Kit (RR037A, Takara Biotechnology, Dalian,
China). The cDNA was subjected to qRT-PCR using SYBR
Premix Ex Taq (RR420A, Takara) and Eppendorf
Mastercycler PCR System (Eppendorf, Hamburg, Germany).
The primers are listed in Supplementary Table 2.
An activity monitor system (43.2 cm length × 43.2 cm
width × 30.5 cm height, Med-Associates, St. Albans, VT,
USA) was used to detect morphine-induced locomotor
activities and behavioral sensitization. Each mouse was
placed in the center of the open field and allowed to explore
freely for 30 min (baseline). After given an intraperitoneal
injection of morphine (10 mg/kg) (Shenyang 1st
Pharmaceutical Company, Shenyang, China), the mice were
confined to the open field for 120 min. To evaluate
morphineinduced behavioral sensitization, mice were placed in
chamber for 10 min, then injected with morphine
(10 mg/kg, i.p.) and placed in chamber for 1 h. The total
distance traveled was recorded.
Conditioned place preference
Morphine-induced CPP was performed using a two
chamber (15 × 15 × 20 cm) apparatus with distinct tactile
environments to maximize contextual differences. A
manual guillotine door (15 × 20 cm) separated the two
chambers. The observer was blinded to the group
allocation. On the first day, mice were allowed to freely explore
the entire apparatus for 15 min (pretest). The mice staying
in one chamber for more than 10 min were excluded from
the experiment. From the second to the sixth days, mice
were daily given an intraperitoneal injection of morphine
(10 mg/kg, i.p.) and confined to one of the chambers
(drugpaired) for 30 min, and 6 h later, they received an i.p.
injection of saline (equivalent volume to that of morphine)
and confined to the other chamber for 30 min
(conditioning). On the seventh day, mice were allowed to freely
explore the entire apparatus for 15 min (test). The time
spent in each chamber was recorded during the pretest and
test sessions. CPP score was defined as the time (in
seconds) spent in morphine-paired chamber minus the time
spent in saline-paired chamber.
Data were analyzed with SPSS 20 software (IBM, Armonk,
NY, USA). Sample size estimation was conducted on alpha
value of 0.05 and desired power of 0.80. Comparisons between
groups were made by unpaired or paired two-tailed student’s t
test, Mann–Whitney U test, χ2 test, one-way ANOVA, or
twoway ANOVA. Two-sample Kolmogorov–Smirnov test was
used for analyzing the cumulative distribution. Results of
locomotion and neuronal excitability were analyzed by
twoway repeat-measure (RM) ANOVA followed by the
Bonferroni’s post-hoc test. Statistical significance was represented as
*P < 0.05; **P < 0.01; ***P < 0.001, and ****P < 0.0001.
All data are presented as mean ± SEM.
All data needed to evaluate the conclusions in the paper are
present in the paper and/or the supplementary materials.
Additional data related to this paper may be requested from
the authors. Raw and processed NGS data are deposited in
the National Center for Biotechnology Information
BioProject database under accession number (PRJNA508422).
Acknowledgements We thank Dr. Yongchun Yu (Fudan University)
for helpful discussions.
Funding This work was supported by grants from the Natural Science
Foundation of China (31430033 and 91632307 to LM, and 31671042
and 31871021 to FW), the Ministry of Science and Technology
(2015CB553501 to LM), National Key R&D Program of China
(2018YFC1004500 to QL), Shanghai Municipal Science and
Technology Major Project (2018SHZDZX01 to LM and FW) and ZJLab.
Author contributions FW and LM designed and supervised the study.
CJ, FW and LM planned the experiments and analyzed the data. CJ
carried out the electrophysiology experiments. CJ, FW and GH carried
out the immunohistochemical experiments and RNAscope ISH. QL
and CL carried out the ribo-tag purification and bioinformatics
analysis. XW and PL contributed to the acquisition of the behavioral data.
XW carried out the morphological reconstruction and quantitation.
ZW constructed the AAV vectors. CJ drafted the paper. FW, PZ and
LM revised the paper.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
Publisher’s note: Springer Nature remains neutral with regard to
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1. Saunders A , Oldenburg IA , Berezovskii VK , Johnson CA , Kingery ND , Elliott HL , et al. A direct GABAergic output from the basal ganglia to frontal cortex . Nature . 2015 ; 521 : 85 - 9 .
2. Nestler EJ , Hope BT , Widnell KL . Drug addiction: a model for the molecular basis of neural plasticity . Neuron . 1993 ; 11 : 995 - 1006 .
3. Steketee JD , Kalivas PW . Drug wanting: behavioral sensitization and relapse to drug-seeking behavior . Pharmacol Rev . 2011 ; 63 : 348 - 65 .
4. Vertes RP. Differential projections of the infralimbic and prelimbic cortex in the rat . Synapse . 2004 ; 51 : 32 - 58 .
5. Sun W , Rebec GV . Repeated cocaine self-administration alters processing of cocaine-related information in rat prefrontal cortex . J Neurosci . 2006 ; 26 : 8004 - 8 .
6. Marquis JP , Killcross S , Haddon JE . Inactivation of the prelimbic, but not infralimbic, prefrontal cortex impairs the contextual control of response conflict in rats . Eur J Neurosci . 2007 ; 25 : 559 - 66 .
7. Hearing M , Kotecki L , Marron Fernandez de Velasco E , FajardoSerrano A , Chung HJ , Lujan R , et al. Repeated cocaine weakens GABA(B)-Girk signaling in layer 5/6 pyramidal neurons in the prelimbic cortex . Neuron 2013 ; 80 : 159 - 70 .
8. Kalivas PW , Volkow ND . New medications for drug addiction hiding in glutamatergic neuroplasticity . Mol Psychiatry . 2011 ; 16 : 974 - 86 .
9. Kalivas PW . The glutamate homeostasis hypothesis of addiction . Nat Rev Neurosci . 2009 ; 10 : 561 - 72 .
10. Li Y , Hu XT , Berney TG , Vartanian AJ , Stine CD , Wolf ME , et al. Both glutamate receptor antagonists and prefrontal cortex lesions prevent induction of cocaine sensitization and associated neuroadaptations . Synapse . 1999 ; 34 : 169 - 80 .
11. Hattori R , Kuchibhotla KV , Froemke RC , Komiyama T. Functions and dysfunctions of neocortical inhibitory neuron subtypes . Nat Neurosci . 2017 ; 20 : 1199 - 208 .
12. Kawaguchi Y , Kubota Y. GABAergic cell subtypes and their synaptic connections in rat frontal cortex . Cereb Cortex . 1997 ; 7 : 476 - 86 .
13. Naka A , Adesnik H . Inhibitory circuits in cortical layer 5 . Front Neural Circuits . 2016 ; 10 : 35 .
14. Tremblay R , Lee S , Rudy B. GABAergic interneurons in the neocortex: from cellular properties to circuits . Neuron . 2016 ; 91 : 260 - 92 .
15. Zhu Q , Ke W , He Q , Wang X , Zheng R , Li T , et al. Laminar distribution of neurochemically-identified interneurons and cellular co-expression of molecular markers in epileptic human cortex . Neurosci Bull . 2018 ; 34 : 992 - 1006 .
16. Chen SX , Kim AN , Peters AJ , Komiyama T . Subtype-specific plasticity of inhibitory circuits in motor cortex during motor learning . Nat Neurosci . 2015 ; 18 : 1109 - 15 .
17. Kvitsiani D , Ranade S , Hangya B , Taniguchi H , Huang JZ , Kepecs A . Distinct behavioural and network correlates of two interneuron types in prefrontal cortex . Nature . 2013 ; 498 : 363 - 6 .
18. Li Z , Luan W , Chen Y , Chen M , Dong Y , Lai B , et al. Chronic morphine treatment switches the effect of dopamine on excitatory synaptic transmission from inhibition to excitation in pyramidal cells of the basolateral amygdala . J Neurosci . 2011 ; 31 : 17527 - 36 .
19. Donato F , Rompani SB , Caroni P . Parvalbumin-expressing basket-cell network plasticity induced by experience regulates adult learning . Nature . 2013 ; 504 : 272 - 6 .
20. Lin H , Hsu FC , Baumann BH , Coulter DA , Anderson SA , Lynch DR . Cortical parvalbumin GABAergic deficits with alpha7 nicotinic acetylcholine receptor deletion: implications for schizophrenia . Mol Cell Neurosci . 2014 ; 61 : 163 - 75 .
21. Schmid LC , Mittag M , Poll S , Steffen J , Wagner J , Geis HR , et al. Dysfunction of somatostatin-positive interneurons associated with memory deficits in an Alzheimer's Disease Model . Neuron . 2016 ; 92 : 114 - 25 .
22. Le Merrer J , Becker JA , Befort K , Kieffer BL . Reward processing by the opioid system in the brain . Physiol Rev . 2009 ; 89 : 1379 - 412 .
23. Tedford HW , Zamponi GW . Direct G protein modulation of Cav2 calcium channels . Pharmacol Rev . 2006 ; 58 : 837 - 62 .
24. Lüscher C , Slesinger PA. Emerging roles for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease . Nat Rev Neurosci . 2010 ; 11 : 301 - 15 .
25. Nockemann D , Rouault M , Labuz D , Hublitz P , McKnelly K , Reis FC , et al. The K(+) channel GIRK2 is both necessary and sufficient for peripheral opioid-mediated analgesia . EMBO Mol Med . 2013 ; 5 : 1263 - 77 .
26. Endres-Becker J , Heppenstall PA , Mousa SA , Labuz D , Oksche A , Schafer M , et al. Mu-opioid receptor activation modulates transient receptor potential vanilloid 1 (TRPV1) currents in sensory neurons in a model of inflammatory pain . Mol Pharm . 2007 ; 71 : 12 - 8 .
27. Zhang Z , Tao W , Hou YY , Wang W , Lu YG , Pan ZZ . Persistent pain facilitates response to morphine reward by downregulation of central amygdala GABAergic function . Neuropsychopharmacology . 2014 ; 39 : 2263 - 71 .
28. Baimel C , Borgland SL . Orexin signaling in the VTA gates morphine-induced synaptic plasticity . J Neurosci . 2015 ; 35 : 7295 - 303 .
29. Kim J , Ham S , Hong H , Moon C , Im HI . Brain reward circuits in morphine addiction . Mol Cells . 2016 ; 39 : 645 - 53 .
30. Russo SJ , Dietz DM , Dumitriu D , Morrison JH , Malenka RC , Nestler EJ . The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens . Trends Neurosci . 2010 ; 33 : 267 - 76 .
31. Somogyi P , Katona L , Klausberger T , Lasztoczi B , Viney TJ . Temporal redistribution of inhibition over neuronal subcellular domains underlies state-dependent rhythmic change of excitability in the hippocampus . Philos Trans R Soc Lond B Biol Sci . 2014 ; 369 : 20120518 .
32. Ma Y , Hu H , Agmon A . Short-term plasticity of unitary inhibitory-to-inhibitory synapses depends on the presynaptic interneuron subtype . J Neurosci . 2012 ; 32 : 983 - 8 .
33. Pfeffer CK , Xue M , He M , Huang ZJ , Scanziani M. Inhibition of inhibition in visual cortex: the logic of connections between molecularly distinct interneurons . Nat Neurosci . 2013 ; 16 : 1068 - 76 .
34. Xu H , Jeong HY , Tremblay R , Rudy B . Neocortical somatostatinexpressing GABAergic interneurons disinhibit the thalamorecipient layer 4 . Neuron . 2013 ; 77 : 155 - 67 .
35. Tuncdemir SN , Wamsley B , Stam FJ , Osakada F , Goulding M , Callaway EM , et al. Early somatostatin interneuron connectivity mediates the maturation of deep layer cortical circuits . Neuron . 2016 ; 89 : 521 - 35 .
36. Guan W , Cao JW , Liu LY , Zhao ZH , Fu Y , Yu YC . Eye opening differentially modulates inhibitory synaptic transmission in the developing visual cortex . Elife . 2017 ; 6 : e32337 .
37. Hedrick NG , Harward SC , Hall CE , Murakoshi H , McNamara JO , Yasuda R . Rho GTPase complementation underlies BDNF-dependent homo- and heterosynaptic plasticity . Nature . 2016 ; 538 : 104 - 08 .
38. Tashiro A , Minden A , Yuste R . Regulation of dendritic spine morphology by the rho family of small GTPases: antagonistic roles of Rac and Rho . Cereb Cortex . 2000 ; 10 : 927 - 38 .
39. Hayashi-Takagi A , Yagishita S , Nakamura M , Shirai F , Wu YI , Loshbaugh AL , et al. Labelling and optical erasure of synaptic memory traces in the motor cortex . Nature . 2015 ; 525 : 333 - 8 .
40. Cahill CM , Morinville A , Lee MC , Vincent JP , Collier B , Beaudet A . Prolonged morphine treatment targets delta opioid receptors to neuronal plasma membranes and enhances delta-mediated antinociception . J Neurosci . 2001 ; 21 : 7598 - 607 .
41. Kepecs A , Fishell G . Interneuron cell types are fit to function . Nature . 2014 ; 505 : 318 - 26 .
42. Johnson SW , North RA . Opioids excite dopamine neurons by hyperpolarization of local interneurons . J Neurosci . 1992 ; 12 : 483 - 8 .
43. Courtin J , Chaudun F , Rozeske RR , Karalis N , Gonzalez-Campo C , Wurtz H , et al. Prefrontal parvalbumin interneurons shape neuronal activity to drive fear expression . Nature . 2014 ; 505 : 92 - 6 .
44. Hu H , Gan J , Jonas P. Interneurons . Fast-spiking, parvalbumin(+) GABAergic interneurons: from cellular design to microcircuit function . Science . 2014 ; 345 : 1255263 .
45. Pi HJ , Hangya B , Kvitsiani D , Sanders JI , Huang ZJ , Kepecs A . Cortical interneurons that specialize in disinhibitory control . Nature . 2013 ; 503 : 521 - 4 .
46. Xu H , Liu L , Tian Y , Wang J , Li J , Zheng J , et al. A disinhibitory microcircuit mediates conditioned social fear in the prefrontal cortex . Neuron . 2019 ; 102 : 668 - 82 e5.
47. Yamada H , Shimoyama N , Sora I , Uhl GR , Fukuda Y , Moriya H , et al. Morphine can produce analgesia via spinal kappa opioid receptors in the absence of mu opioid receptors . Brain Res . 2006 ; 1083 : 61 - 9 .
48. Sora I , Takahashi N , Funada M , Ujike H , Revay RS , Donovan DM , et al. Opiate receptor knockout mice define mu receptor roles in endogenous nociceptive responses and morphine-induced analgesia . Proc Natl Acad Sci USA . 1997 ; 94 : 1544 - 9 .
49. Chefer VI , Shippenberg TS . Augmentation of morphine-induced sensitization but reduction in morphine tolerance and reward in delta-opioid receptor knockout mice . Neuropsychopharmacology . 2009 ; 34 : 887 - 98 .
50. Milner TA , Burstein SR , Marrone GF , Khalid S , Gonzalez AD , Williams TJ , et al. Stress differentially alters mu opioid receptor density and trafficking in parvalbumin-containing interneurons in the female and male rat hippocampus . Synapse . 2013 ; 67 : 757 - 72 .
51. Pierce RC , Kalivas PW . A circuitry model of the expression of behavioral sensitization to amphetamine-like psychostimulants . Brain Res Brain Res Rev . 1997 ; 25 : 192 - 216 .
52. McFarland K , Lapish CC , Kalivas PW . Prefrontal glutamate release into the core of the nucleus accumbens mediates cocaineinduced reinstatement of drug-seeking behavior . J Neurosci . 2003 ; 23 : 3531 - 7 .
53. Otis JM , Namboodiri VM , Matan AM , Voets ES , Mohorn EP , Kosyk O , et al. Prefrontal cortex output circuits guide reward seeking through divergent cue encoding . Nature . 2017 ; 543 : 103 - 07 .
54. Kim CK , Ye L , Jennings JH , Pichamoorthy N , Tang DD , Yoo AW , et al. Molecular and circuit-dynamical identification of topdown neural mechanisms for restraint of reward seeking . Cell . 2017 ; 170 : 1013 - 27 e14.
55. Gao Q , Yao W , Wang J , Yang T , Liu C , Tao Y , et al. Post-training activation of Rac1 in the basolateral amygdala is required for the formation of both short-term and long-term auditory fear memory . Front Mol Neurosci . 2015 ; 8 : 65 .
56. Zhao S , Ting JT , Atallah HE , Qiu L , Tan J , Gloss B , et al. Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function . Nat Methods . 2011 ; 8 : 745 - 52 .
57. Sanz E , Yang L , Su T , Morris DR , McKnight GS , Amieux PS . Cell-type-specific isolation of ribosome-associated mRNA from complex tissues . Proc Natl Acad Sci USA . 2009 ; 106 : 13939 - 44 .
58. Bindea G , Mlecnik B , Hackl H , Charoentong P , Tosolini M , Kirilovsky A , et al. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks . Bioinformatics . 2009 ; 25 : 1091 - 3 .