Abstract
Anxiety is one of the most common withdrawal symptoms of methamphetamine (METH) abuse, which further drives relapse to drugs. Interpeduncular nucleus (IPN) has been implicated in anxiety-like behaviors and addiction, yet its role in METH-abstinence-induced anxiety remains unknown. Here, we found that prolonged abstinence from METH enhanced anxiety-like behaviors in male mice, accompanied by more excited IPN GABAergic neurons, as indicated by the increased c-fos expression and the enhanced neuronal excitability by electrophysiological recording in the GABAergic neurons. Using the designer receptors exclusively activated by designer drugs method, specific inhibition of IPN GABAergic neurons rescued the aberrant neuronal excitation of IPN GABAergic neurons and efficiently reduced anxiety-like behaviors, whereas it did not induce depression-like behaviors in male mice after prolonged abstinence from METH. These findings reveal that IPN GABAergic neurons should be a promising brain target to alleviate late withdrawal symptoms from METH with few side effects.
SIGNIFICANCE STATEMENT Prolonged abstinence from METH triggers IPN GABAergic neurons and ultimately increases anxiety in male mice. Suppressing IPN GABAergic neurons rescues METH abstinence-induced aberrant neuronal excitation of IPN GABAergic neurons and efficiently reduces anxiety in mice.
Introduction
Methamphetamine (METH) is a highly addictive psychostimulant, and its high risk of relapse remains a big public health concern. Anxiety is a prominent withdrawal symptom of the METH withdrawal period (Hellem, 2016), which is generally believed to be a critical factor for driving relapse to METH (Glasner-Edwards et al., 2010). It is reported that the proportion of METH addicts with anxiety symptoms during withdrawal period was about 34.3% (Su et al., 2017), whereas laboratory animals exhibit increased anxiety-like behavior from prolonged abstinence (Nawata et al., 2019; Everett et al., 2020; Chen et al., 2022) but not early abstinence (Jacobskind et al., 2019) from METH. However, there is a lack of pharmacotherapy for METH-abstinence-induced anxiety, partially because of the psychiatric side effects such as depression caused by anxiolytic medicine (Chen et al., 2017). Therefore, understanding the precise neuroanatomical targets for METH-abstinence-induced anxiety is essential for developing clinical strategies and improving patient care.
Interpeduncular nucleus (IPN), consisting primarily of GABAergic neurons (Molas et al., 2017), has been implicated in anxiety-like behaviors and addiction (Antolin-Fontes et al., 2015; Wolfman et al., 2018; DeGroot et al., 2020; Elayouby et al., 2021). Recent studies have reported that IPN GABAergic neurons modulate somatic and affective symptoms of nicotine withdrawal (Avelar and George, 2022; Klenowski et al., 2022). Nicotine withdrawal-induced anxiety increases the activity of neurons in IPN (Zhao-Shea et al., 2015). Optogenetic silencing of IPN GABAergic neurons alleviates anxiety-like behaviors in nicotine-withdrawn mice (Klenowski et al., 2022). These studies highlight the critical role of IPN GABAergic neurons in regulating anxiety-like behaviors caused by drug withdrawal, yet the role of IPN in the development of METH abstinence-induced anxiety remains unknown.
In the present study, a male mice model of abstinence from METH was established. The anxiety-like behaviors and phenotypes of IPN activities were examined. Further, the role of specific modulating IPN GABAergic neurons in anxiety were explored in male mice from prolonged abstinence from METH.
Materials and Methods
Animals and treatments
C57BL/6 male mice (∼25 g, 8–10 weeks of age) were maintained on a 12 h reverse light/dark cycle with food and water available ad libitum. All mice were handled for 3 consecutive days before the experiment. Mice were assigned to receive a daily intraperitoneal injection of METH (M group, 3 mg/kg) or saline (S group, 0.2 ml) for 10 consecutive days, followed by a 1 d abstinence (early abstinence) or 14 d abstinence (prolonged abstinence) period (Shi et al., 2021). All procedures were conducted under the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at Nanjing University of Chinese Medicine.
Behavioral tests
The elevated plus maze (EPM), open-field Test (OFT), and marble buried test (MBT) were used to examine anxiety-like behaviors in mice. For the EPM (Fan et al., 2022), mice were released from the central platform individually and allowed to explore the maze freely for 5 min with a camera positioned overhead. The time spent, the distance traveled, and the number of entries in the open arms were recorded. For the OFT (Fan et al., 2022), mice were placed in the center of a square chamber (50 × 50 cm) to explore the whole chamber for 5 min. The time spent and the number of entries into the center zone (25 × 25 cm) were recorded. For the MBT (Nie et al., 2020), mice were placed individually in a clean cage with 5 cm of corncob bedding. Sixteen black marbles (14 mm diameter) were prearranged on top of the bedding in four evenly spaced rows of four marbles each. Mice were left undisturbed for 30 min, and the number of not buried marbles (more than half the surface of the marble uncovered with bedding) was counted every 5 min.
The tail suspension test (TST) and forced swim test (FST) were used to test depression-like behaviors in mice. Mice were individually suspended by the tail (TST) or placed into the water (FST) for 6 min. The immobility of each mouse during the last 4 min was recorded (Fan et al., 2022). All behavioral data were analyzed by TopScan software (Clever Systems Inc).
Immunofluorescence
The mice were perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) in PBS buffer. The brains were removed and postfixed in 4% PFA at 4°C overnight, then transferred to 30% (w/v) sucrose. Frozen coronal sections (30 µm) were cut on a cryostat (Leica). The sections were incubated with the primary antibodies overnight at 4°C, followed by the corresponding fluorophore-conjugated secondary antibodies for 1.5 h at room temperature. The primary antibodies used in the experiment were rabbit polyclonal anti-c-fos (1:3000; catalog #2250, Cell Signaling Technology; RRID:AB_2247211) and mouse polyclonal anti-GAD67 (1:800; catalog #MAB5406, Millipore; RRID:AB_2278725). The secondary antibodies used were Alexa Fluor 488-labeled donkey anti-mouse IgG (1:500; catalog #A-21202, Molecular Probes; RRID:AB_141607), Alexa Fluor 555-labeled donkey anti-rabbit (1:500; catalog #A32794, Thermo Fisher Scientific; RRID:AB_2762834), and Alexa Fluor 488-labeled donkey anti-rabbit (1:500; catalog #A32790, Thermo Fisher Scientific; RRID:AB_2762833). The images were captured by THUNDER imaging systems TCS SP8 and TCS SP8 STED 3 (Leica).
Slice preparation and electrophysiology
rAAV2/9-VGAT1-mCherry (catalog #PT-0325, BrainVTA) was injected into IPN before METH treatment. After EPM testing, mice were deeply anesthetized with isoflurane (RWD) and perfused with ice-cold cutting solution. Slice preparation was performed as previously described (Ge et al., 2021). Slices containing the IPN were cut at a 200 µm thickness using a vibratome in 4°C cutting solution. The slices were transferred to 37°C cutting solution for 9 min, then transferred to a holding solution to allow for recovery at room temperature for 1 h before recordings. During electrophysiological recordings, the brain slice was continuously perfused with oxygenated artificial CSF (aCSF) maintained at 30°C by a solution heater (TC-324C, Warner Instruments).
Loose-patch microelectrodes were filled with aCSF, and access resistance was maintained at 20–50 MΩ throughout the experiment. Recordings were performed under current-clamp mode with 0 holding current. Whole-cell current-clamp microelectrodes (3–5 MΩ) were filled with potassium internal solution. For measurements of neuronal excitability, current-step protocols were run (step, 10 pA; action potential range, 0–100 pA; input resistance range, 0−60 pA) at −70 mV. All signals were filtered at 4 kHz, amplified at 5× using a MultiClamp 700B amplifier (Molecular Devices) and digitized at 10 kHz with a Digidata 1440A analog-to-digital converter (Molecular Devices). All data were analyzed with Clampfit 10.6 software (Molecular Devices).
Designer receptors exclusively activated by designer drugs (DREADDs)
One hundred nanoliters of rAAV2/9-vGAT1-hM4Di-mCherry-WPRE-hGH-pA (Gi; catalog #PT-0488, BrainVTA) or control virus (Control; rAAV2/9-vGAT1-mCherry-WPRE-hGH-pA, catalog PT-0325, BrainVTA) was injected in the IPN before METH treatment. Thirty minutes before each behavioral test, mice received an intraperitoneal injection of clozapine-N-oxide (CNO, 2 mg/kg). Following the behavioral test, c-fos staining and electrophysiology experiments were performed in brain slices.
Statistical analysis
Statistical analysis was conducted using GraphPad Prism 9.0 software. The data are presented as the mean ± SD. Data of MBT, number of action potentials, and steady-state were analyzed by two-way repeated-measures ANOVAs followed by Bonferroni's post hoc test, and other data were analyzed by two-tailed unpaired t tests. Statistical significance was set as *p < 0.05, **p < 0.01.
Results
METH prolonged abstinence enhances anxiety-like behaviors and triggers the excitability of IPN GABAergic neurons in male mice
Male mice were assigned to receive an intraperitoneal injection of METH or saline once daily for 10 consecutive days, then underwent a 1 d abstinence (early abstinence) or 14 d abstinence (prolonged abstinence) period from METH at their home cage, forming the early abstinence from METH group (M') and saline early abstinence group (S') and prolonged abstinence from METH group (M) and saline prolonged abstinence group (S).
Anxiety-like behaviors were assessed via EPM, OFT, and MBT, and depression-like behaviors were assessed via TST and FST (Fig. 1a). During the prolonged abstinence period (Fig. 1a), in the EPM test (day 25), M group spent less time (t(26) = 4.533, p = 0.0001; Fig. 1b), traveled a shorter distance (t(26) = 3.372, p = 0.0023; Fig. 1c), and made fewer entries (t(26) = 5.431, p < 0.0001; Fig. 1d) into the open arms, whereas there was no difference between the two groups in the total distanced traveled during the EPM (t(26) = 1.215, p = 0.2354; Fig. 1e), when compared with the S group (Fig. 1f). In the OFT (day 26) and MBT (day 27) tests, neither the spent time (t(26) = 0.5509, p = 0.5864; Fig. 1g), the entries (t(26) = 0.7124, p = 0.4826; Fig. 1h) into the center area of the OFT (Fig. 1i), nor the numbers of remaining marbles during the MBT (F(6,156) = 0.5531, p = 0.7670; 0–30 min, p > 0.9999; Fig. 1j) were changed in the METH abstinence mice. In TST (day 28) and FST (day 29) tests, there was no statistically significant difference between M and S mice in the immobility during the TST (t(26) = 0.5484, p = 0.5881; Fig. 1k) and FST (t(26) = 0.8313, p = 0.4134; Fig. 1l).
Prolonged abstinence from METH enhances anxiety-like but not depression-like behaviors in male mice. a, Timeline of METH-prolonged-abstinence model and behavioral tests. b–f, EPM test (total n = 28). The time spent in the open arms (b), the distance traveled in the open arms (c), the number of entries into the open arms (d), the total distance traveled during the EPM (e), and representative traces of EPM exploration (f). g–i, OFT behaviors (total n = 28). The time spent in the center (g), number of entries into the center (h), and representative traces of open field exploration (i). j, MBT behaviors (total n = 28). k, TST behaviors (total n = 28). l, FST behaviors (total n = 28). N.S., no significance, p > 0.05; **p < 0.01 versus S mice.
Whereas during the early abstinence period (Fig. 2a), in the EPM test (day 11), there was no difference between the S' and M' groups in the time spent (t(15) = 0.9861, p = 0.3397; Fig. 2b), the distance traveled (t(15) = 0.04,955, p = 0.9611; Fig. 2c), and entries (t(15) = 0.2730, p = 0.7886; Fig. 2d) into the open arms (Fig. 2e). In the OFT (day 12), neither the spent time (t(15) = 1.111, p = 0.2839; Fig. 2f) nor the entries (t(15) = 1.346, p = 0.1982; Fig. 2g) into the center area were changed in METH early abstinence mice (Fig. 2h). These results confirmed that METH prolonged abstinence but not early abstinence enhanced anxiety-like behavior in male mice.
Early abstinence from METH did not alter anxiety-like behaviors in male mice. a, Timeline of METH early abstinence model and behavioral tests. b–e, EPM test (total n = 17). The time spent in the open arms (b), the distance traveled in the open arms (c), the number of entries into the open arms (d), and representative traces of EPM exploration (e). f–h, OFT behaviors (total n = 17). The time spent in the center (f), number of entries into the center (g), and representative traces of open field exploration (h). N.S., no significance, p > 0.05 versus S' mice.
Next, the activities of IPN GABAergic neurons were examined in M mice. The c-fos, GAD67, and CaMKII are used as markers for labeling neuronal activation, GABAergic neurons, and glutamatergic neurons, respectively. Compared with S mice, M mice showed enhanced activities of GABAergic neurons in the IPN, as indicated by increased numbers of c-Fos and GAD67 double-positive neurons (t(6) = 5.271, p = 0.0019, Fig. 3a). However, there was no difference between the two groups in the numbers of c-Fos and CaMKII double-positive neurons (t(6) = 1.061, p = 0.3297, Fig. 3b). As shown in Figure 3c, the neighboring ventral tegmental area (VTA) showed no difference in c-fos expression (t(10) = 0.5243, p = 0.6115) between S and M mice, indicating that prolonged METH abstinence did not change VTA activation in mice.
METH abstinence triggers the excitability of IPN GABAergic neurons in male mice. a, Immunostaining for c-fos/GAD67/DAPI in the IPN (total n = 8). White arrows indicate c-fos-positive and GAD67-positive neurons. Scale bar, 50 µm. b, Immunostaining for c-fos/CaMKII/DAPI in the IPN (total n = 8). White arrows indicate c-fos-positive and CaMKII-positive neurons. Scale bar, 50 µm. c, Immunostaining for c-fos/DAPI in the VTA (total n = 12). Scale bar, 50 µm. d, Timeline of virus injection, METH prolonged abstinence model, and behavioral tests. e, Schematic diagram of AAV2/9-vGAT1-mCherry injection into IPN (left) and representative images of electrophysiological recordings on IPN GABAergic neurons (right). Scale bar, 250 µm. f–h, Tonic firing of IPN GABAergic neurons under loose-patch configuration (total n = 27 cells from 10 mice). Frequency of spontaneous firing (f). Coefficient of variation (CV) of firing intervals (g). Sample traces of spontaneous firing (h). i–j, Action potentials (AP) of IPN GABAergic neurons under whole-cell current-clamp configuration (total n = 15 cells from 10 mic). Numbers of AP firing (i). Sample traces of AP firing (j). k, l, Voltage responses of IPN GABAergic neurons recorded under whole-cell current-clamp configuration in negative current injection (total n = 19 cells from 10 mice). The steady-state of IPN GABAergic neurons (k). Sample traces of voltage responses (l). N.S., no significance, p > 0.05, *p < 0.05, **p < 0.01 versus S group.
To further validate the functional alterations of IPN GABAergic neurons in M mice, electrophysiological recordings were conducted in brain slices transfected with virus (Fig. 3d). Virus AAV2/9-vGAT1-mCherry was injected into the IPN to label GABAergic neurons (Fig. 3e). For the loose-patch recordings, there was a higher frequency of spontaneous firing in IPN GABAergic neurons (t(25) = 3.059, p = 0.0052; Fig. 3f) and a lower level of the coefficient of variation (CV) of firing intervals (t(25) = 2.072, p = 0.0488; Fig. 3g) in M mice than in S mice (Fig. 3h). For the whole-cell recordings, there were higher evoked firing rates of IPN GABAergic neurons at 70–100 pA injecting currents (F(10,130) = 23.04, p < 0.0001; 10, 30–50 pA, p > 0.9999; 20 pA, p = 0.9624; 60 pA, p = 0.2170; 70 pA, p = 0.0326; 80 pA, p = 0.0040; 90 pA, p = 0.0007; 100 pA, p = 0.0006; Fig. 3i,j), and higher input resistance (F(6,102) = 4.032, p = 0.0012; 0, −50 pA, p > 0.9999; −10 pA, p = 0.6607; −20 pA, p = 0.0986; −30 pA, p = 0.0714; −40 pA, p = 0.1883; −60 pA, p = 0.1138; Fig. 3k,l) in M mice than in S mice.
Suppressing IPN GABAergic neurons alleviates METH-prolonged-abstinence-enhanced anxiety-like behaviors and rescues IPN abnormal activation
To examine the role of IPN GABAergic neurons in METH-prolonged-abstinence-enhanced anxiety, rAAV2/9-vGAT1-hM4Di-mCherry (Gi) or rAAV2/9-vGAT1-mCherry (Control) virus was injected in the IPN before METH treatment. Thirty minutes before each behavioral test, mice received an intraperitoneal injection of CNO to suppress IPN GABAergic neurons (Fig. 4a,b). In the EPM test (day 25), Gi mice spent more time (t(13) = 2.691, p = 0.0185; Fig. 4c), traveled a longer distance (t(13) = 2.151, p = 0.0509; Fig. 4d), and made more entries (t(13) = 2.837, p = 0.0140; Fig. 4e) into the open arms, but showed similar total distance traveled in the apparatus of EPM (t(13) = 0.3587, p = 0.7256; Fig. 4f) than that of Control mice (Fig. 4g). There was no difference on the time spent (t(13) = 0.4003, p = 0.6954; Fig. 4h) and the entries (t(13) = 0.6411, p = 0.5326; Fig. 4i) into the center area of the OFT (day 26; Fig. 4j), as well as the numbers of remaining marbles during the MBT (day 27; F(6,78) = 0.6385, p = 0.6991; 0–30 min, p > 0.9999; Fig. 4k) between Gi and Control mice. As for depression-like behaviors, there was no difference between the Gi and Control groups on immobility during the TST (day 28, t(13) = 1.099, p = 0.2918; Fig. 4l) and FST (day 29, t(13) = 0.5396, p = 0.5986; Fig. 4m).
Inhibition of IPN GABAergic neurons suppresses anxiety-like but not depression-like behaviors in male mice of METH prolonged abstinence. a, Timeline of DREADD treatment, METH abstinence model and behavioral tests. b, Schematic diagram of rAAV2/9-vGAT1-mCherry (Control) or rAAV2/9-vGAT1-hM4Di-mCherry (Gi) injection into IPN (left) and representative images of virus expression in the IPN. Scale bar, 2 mm (left), 250 µm (right). c–g, EPM test (total n = 15). The time spent in the open arms (c), the distance traveled in the open arms (d), the number of entries into the open arms (e), the total distance traveled during the EPM (f), and representative traces of EPM exploration (g). h–j, OFT behaviors (total n = 15). The time spent in the center (h), number of entries into the center (i), and representative traces of open field exploration (j). k, MBT behaviors (total n = 15). l, TST behaviors (total n = 15). m, FST behaviors (total n = 15). N.S., no significance, p > 0.05, *p < 0.05 versus Control mice.
Following the behavioral test, Gi and Control mice were harvested to detect the activity of IPN GABAergic neurons by c-fos staining and electrophysiology recording. As shown in Figure 5a, the number of c-fos and mCherry double-positive neurons in the IPN was at a lower level in Gi mice than in Control mice (t(4) = 10.65, p = 0.0004). As shown in Figure 5b, the virus had a few leakages into VTA. However, the neighboring VTA showed no difference in c-fos expression (t(4) = 0.5544, p = 0.6088) between Gi and controlled mice, indicating that the effect of inhibiting by designer receptors exclusively activated by designer drugs (DREADD) is specific to the IPN neurons without affecting VTA activation in mice.
Inhibition of IPN GABAergic neurons rescues abnormal IPN activation in male mice of METH prolonged abstinence. a, Immunostaining for c-fos/GAD67/DAPI in the IPN (total n = 6). White arrows indicate c-fos-positive and GAD67-positive neurons. Scale bar, 50 µm. b, Immunostaining for c-fos/DAPI in the VTA (total n = 6). Scale bar, 50 µm. ml, medial lemniscus. c–e, Tonic firing of IPN GABAergic neurons under loose-patch configuration (total n = 13 cells from 6 mice). Frequency of spontaneous firing (c). CV of firing intervals (d). Sample traces of spontaneous firing (e). f–g, Action potentials (AP) of IPN GABAergic neurons under whole-cell current-clamp configuration (total n = 17 cells from 6 mic). Numbers of AP firing (f). Sample traces of AP firing (g). h–i, Voltage responses of IPN GABAergic neurons recorded under whole-cell current-clamp configuration in negative current injection (total n = 16 cells from 10 mice). The steady-state of IPN GABAergic neurons (h). Sample traces of voltage responses (i). N.S., no significance, p > 0.05, **p < 0.01 versus Control mice.
For the loose-patch recordings, there was a lower frequency of spontaneous firing (t(11) = 3.533, p = 0.0047; Fig. 5c) and similar CVs of firing intervals (t(11) = 0.5217, p = 0.6122; Fig. 5d) in Gi mice than in Control mice (Fig. 5e). For the whole-cell recordings, there was no difference between Gi and Control groups in the evoked firing rates of IPN GABAergic neurons at 0–100 pA injecting currents (F(10,150) = 0.1691, p = 0.9980; 0–100 pA, p > 0.9999; Fig. 5f,g), whereas the input resistance of GABAergic neurons was at a lower level in Gi mice than in Control mice (F(6,84) = 6.193, p < 0.0001; 0 pA, p > 0.9999; −10 pA, p = 0.1102; −20 pA, p = 0.1564; −30 pA, p = 0.0986; −40 pA, p = 0.1776; −50 pA, p = 0.1731; −60 pA, p = 0.1028; Fig. 5h,i).
Discussion
The IPN is emerging as a viable target for the treatment of addiction as well as mood-associated disorders (McLaughlin et al., 2017). Recent studies have highlighted the role of IPN in nicotine-withdrawal symptoms (Elayouby et al., 2021; Jin and Drenan, 2022; Tapia et al., 2022). However, the contribution of IPN, especially the GABAergic neural assembles, to METH-abstinence-enhanced anxiety is still poorly understood. In the present study, we found that IPN GABAergic neurons were strongly activated, accompanied by an increase of anxiety-like behaviors in male mice after prolonged abstinence from METH. Specific inhibition of IPN GABAergic neurons efficiently reduced anxiety without inducing depression-like behaviors in METH-prolonged-abstinence mice and rescued the aberrant GABAergic neuronal excitation in their IPN. These findings demonstrate that the IPN GABAergic neural assemblies play a critical role in coding METH-prolonged-abstinence-induced anxiety symptoms (Fig. 6).
Schematic diagram of the present study. Prolonged abstinence from METH increases anxiety in male mice, along with more activated IPN GABAergic neurons, as indicated by more c-fos-positive GABAergic neurons and enhanced neuronal excitability by electrophysiological recording. With the DREADD method, specific suppressing IPN GABAergic neurons rescues the aberrant neuronal excitation of IPN GABAergic neurons and efficiently reduces the anxiety in male mice after METH abstinence.
The IPN serves as one of the key hubs in the system of dorsal diencephalic conduction (DDC; McLaughlin et al., 2017), and the DDC is well established in the process of anxiety (Okamoto and Aizawa, 2013). Anatomically, the IPN GABAergic neurons send projections to other emotion-related structures, such as the ventral hippocampus, lateral habenula, raphe nuclei, and laterodorsal tegmentum (Klemm, 2004; Antolin-Fontes et al., 2015; Ables et al., 2017; Lima et al., 2017; McLaughlin et al., 2017; Wolfman et al., 2018). In naive animals, silencing the dopaminergic projections from VTA to IPN deceased the exploration of mice in the open arms of EPM (DeGroot et al., 2020), whereas reducing neuropeptide signaling from medial habenula (MHb) to IPN pathway produced depression-like behaviors (Yoo et al., 2021). These findings indicate that IPN mediates both anxiety and depression, which might depend on different neural ensembles or different regulatory intensities on the same neural subgroups. In the present study, we found only GABAergic neurons but not glutamatergic neurons were activated by METH prolonged abstinence in the IPN. In parallel, selectively suppressing IPN GABAergic neurons sufficiently attenuated the anxiety-like behaviors without producing depression-like behaviors in METH-prolonged-abstinence mice, suggesting that the excitatory–inhibitory balance of IPN GABAergic neuronal ensembles might be specific for coding anxiety caused by drug abstinence. Thus, IPN GABAergic neurons might be promising anxiolytic targets for treating METH withdrawal symptoms.
As for the role of IPN in drug withdrawal anxiety, most current studies focus on animal models of nicotine withdrawal. Inconsistent with our findings, Zhao-Shea et al. (2013) found that optogenetic activation of IPN GABAergic neurons triggered somatic symptoms of nicotine withdrawal but did not influence anxiety disorder. Their follow-up study showed that pharmacological blockade of IPN corticotropin releasing factor receptor-1 or optogenetic silencing MHb–IPN inputs reduced nicotine withdrawal-induced anxiety (Zhao-Shea et al., 2015). Similar to our study, by GCaMP signals analysis in vivo, IPN GABAergic neuron activity was increased during the exploration of open arms in EPM of nicotine-withdrawn mice, and silencing IPN GABAergic neurons significantly attenuated anxiety from nicotine withdrawal (Klenowski et al., 2022). These discrepant findings may be because of the different IPN subregions, discrete IPN neuronal populations, or distinct IPN neural circuits that underly METH and nicotine withdrawal symptoms, respectively.
In summary, our study for the first time identified the critical role of IPN GABAergic neural assemblies in METH-abstinence-induced anxiety. Because modulating IPN GABAergic neurons sufficiently alleviate anxiety but do not produce depression in male mice after prolonged abstinence from METH, the IPN GABAergic neurons might emerge as a promising therapeutic target for treating METH withdrawal symptoms and facilitating addiction recovery with few side effects. Future studies need to comprehensively evaluate the role of IPN GABAergic neurons subpopulations, neural projections and underlying molecular mechanisms in METH-abstinence-induced anxiety.
Footnotes
This work was supported by National Natural Science Foundation of China Grants 81901353, 82071495, and 82271531; Natural Science Foundation of Jiangsu Province, China, Grants BK20201398 and BK20190805; and Open Project of Chinese Materia Medica First-Class Discipline of Nanjing University of Chinese Medicine Grant 2020YLXK004).
The authors declare no competing financial interests.
- Correspondence should be addressed to Xiaowei Guan at guanxw918{at}njucm.edu.cn or Feifei Ge at ffge{at}njucm.edu.cn
