Lai, Y;Liu, W;Lee, T;Kuo, C;Liu, Y;Huang, C;Chen, Y;Chen, I;Wu, S;Wang, S;Lee, P;Liu, C;Lo, J;Chang, Y;Kuo, H;Hsieh, C;Li, C;Liu, P;
| DOI: 10.1016/j.phymed.2022.154597
Background Retinoblastoma, the most common pediatric intraocular malignancy, can develop during embryogenesis, with most children being diagnosed at 3-4 years of age. Multimodal therapies are typically associated with high levels of cytotoxicity and side effects. Therefore, the development of novel treatments with minimal side effects is crucial. Magnolol has a significant anti-tumor effect on various cancers. However, its antitumor effect on retinoblastoma remains unclear. Purpose The study aimed to determine the effects of magnolol on the regulation of EMT, migration, invasion, and cancer progression in retinoblastoma and the modulation of miR-200c-3p expression and the Wnt/ zinc finger E-box binding homeobox 1 (ZEB1)/E-cadherin axis in vivo and in vitro. Methods The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to evaluate magnolol-induced cell toxicity in the Y79 retinoblastoma cell line. Flow cytometry and immunostaining assays were performed to investigate the magnolol-regulated mitochondrial membrane potential and the intracellular and mitochondrial reactive oxygen species levels in Y79 retinoblastoma cells. Orthotopic and subcutaneous xenograft experiments were performed in eight-week-old male null mice to study retinoblastoma progression and metastasis. In situ hybridization and quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays were performed to evaluate the level of the anti-cancer miRNA miR-200c-3p. The mRNA and protein levels of E-cadherin, β-catenin, α-smooth muscle actin (α-SMA), fibronectin-1, and ZEB1 were analyzed using RT-qPCR, immunoblot, immunocytochemistry, and immunohistochemistry assays in vitro and in vivo. Results Magnolol increased E-cadherin levels and reduced the activation of the EMT signaling pathway, EMT, tumor growth, metastasis, and cancer progression in the Y79 retinoblastoma cell line as well as in the orthotopic and subcutaneous xenograft animal models. Furthermore, magnolol increased the expression of miR-200c-3p. Our results demonstrate that miRNA-200c-3p inhibits EMT progression through the Wnt16/β-catenin/ZEB1/E-cadherin axis, and the ZEB1 silencing response shows that miR-200c-3p regulates ZEB1-mediated EMT in retinoblastoma. Conclusion Magnolol has an antitumor effect by increasing E-cadherin and miRNA-200c-3p expression to regulate ZEB1-mediated EMT and cancer progression in retinoblastoma. The anti-tumor effect of magnolol by increasing E-cadherin and miRNA-200c-3p expression to regulate ZEB1-mediated EMT and cancer progression in retinoblastoma has been elucidated for the first time.
Ziminski J, Hessler S, Margetts-Smith G, Sieburg MC, Crombag HS, Koya E.
PMID: 28213443 | DOI: 10.1523/JNEUROSCI.3766-16.2017
Cues that predict the availability of food rewards influence motivational states and elicit food-seeking behaviors. If a cue no longer predicts food availability, animals may adapt accordingly by inhibiting food seeking responses. Sparsely activated sets of neurons, coined neuronal ensembles, have been shown to encode the strength of reward-cue associations. While alterations in intrinsic excitability have been shown to underlie many learning and memory processes, little is known about these properties specifically on cue-activated neuronal ensembles. We examined the activation patterns of cue-activated orbitofrontal cortex (OFC) and nucleus accumbens (NAc) shell ensembles using wild-type and Fos-GFP mice following appetitive conditioning with sucrose and extinction learning. We also investigated the neuronal excitability of recently activated, GFP+ neurons in these brain areas using whole-cell electrophysiology in brain slices. Exposure to a sucrose cue elicited activation of neurons in both the NAc shell and OFC. In the NAc shell, but not the OFC, these activated GFP+ neurons were more excitable than surrounding GFP- neurons. Following extinction, the number of neurons activated in both areas was reduced and activated ensembles in neither area exhibited altered excitability. These data suggest that learning-induced alterations in the intrinsic excitability of neuronal ensembles is regulated dynamically across different brain areas. Furthermore, we show that changes in associative strength modulate the excitability profile of activated ensembles in the NAc shell.SIGNIFICANCE STATEMENTSparsely distributed sets of neurons called 'neuronal ensembles' encode learned associations about food and cues predictive of its availability. Widespread changes in neuronal excitability have been observed in limbic brain areas after associative learning, but little is known about the excitability changes that occur specifically on neuronal ensembles that encode appetitive associations. Here we reveal that sucrose cue exposure recruited a more excitable ensemble in the nucleus accumbens, but not orbitofrontal cortex compared to their surrounding neurons. This excitability difference was not observed when the cue's salience was diminished following extinction learning. These novel data provide evidence that the intrinsic excitability of appetitive memory-encoding ensembles is differentially regulated across brain areas and dynamically adapts to changes in associative strength.
Shin S, Pribiag H, Lilascharoen V, Knowland D, Wang XY, Lim BK.
PMID: 29276054 | DOI: 10.1016/j.neuron.2017.11.040
Early life stress (ELS) in the form of child abuse/neglect is associated with an increased risk of developing social dysfunction in adulthood. Little is known, however, about the neural substrates or the neuromodulatory signaling that govern ELS-induced social dysfunction. Here, we show that ELS-induced downregulation of dopamine receptor 3 (Drd3) signaling and its corresponding effects on neural activity in the lateral septum (LS) are both necessary and sufficient to cause social abnormalities in adulthood. Using in vivo Ca2+ imaging, we found that Drd3-expressing-LS (Drd3LS) neurons in animals exposed to ELS show blunted activity in response to social stimuli. In addition, optogenetic activation of Drd3LS neurons rescues ELS-induced social impairments. Furthermore, pharmacological treatment with a Drd3 agonist, which increases Drd3LS neuronal activity, normalizes the social dysfunctions of ELS mice. Thus, we identify Drd3 in the LS as a critical mediator and potential therapeutic target for the social abnormalities caused by ELS.
Chen, G;Xu, J;Luo, H;Luo, X;Singh, SK;Ramirez, JJ;James, ML;Mathew, JP;Berger, M;Eroglu, C;Ji, RR;
PMID: 36256481 | DOI: 10.1172/jci.insight.161028
Hevin/Sparcl1 is an astrocyte-secreted protein and regulates synapse formation. Here we show that astrocytic hevin signaling plays a critical role in maintaining chronic pain. Compared to wild-type mice, hevin-null mice exhibited normal mechanical and heat sensitivity but reduced inflammatory pain. Interestingly, hevin-null mice have faster recovery than wild-type mice from neuropathic pain after nerve injury. Intrathecal injection of wild-type hevin was sufficient to induce persistent mechanical allodynia in naïve mice. In hevin-null mice with nerve injury, AAV-mediated re-expression of hevin in GFAP-expressing spinal cord astrocytes could reinstate neuropathic pain. Mechanistically, hevin is crucial for spinal cord NMDA receptor (NMDAR) signaling. Hevin potentiated NMDA currents mediated by the GluN2B-containing NMDARs. Furthermore, intrathecal injection of a neutralizing antibody against hevin alleviated acute and persistent inflammatory pain, postoperative pain, and neuropathic pain. Secreted hevin was detected in mouse cerebrospinal fluid (CSF) and nerve injury significantly increased CSF hevin abundance. Finally, neurosurgery caused rapid and substantial increases in SPARCL1/HEVIN levels in human CSF. Collectively, our findings support a critical role of hevin and astrocytes in the maintenance of chronic pain. Neutralizing of secreted hevin with monoclonal antibody may provide a new therapeutic strategy for treating acute and chronic pain and NMDAR-medicated neurodegeneration.
Cutando, L;Puighermanal, E;Castell, L;Tarot, P;Belle, M;Bertaso, F;Arango-Lievano, M;Ango, F;Rubinstein, M;Quintana, A;Chédotal, A;Mameli, M;Valjent, E;
PMID: 35710984 | DOI: 10.1038/s41593-022-01092-8
The cerebellum, a primary brain structure involved in the control of sensorimotor tasks, also contributes to higher cognitive functions including reward, emotion and social interaction. Although the regulation of these behaviors has been largely ascribed to the monoaminergic system in limbic regions, the contribution of cerebellar dopamine signaling in the modulation of these functions remains largely unknown. By combining cell-type-specific transcriptomics, histological analyses, three-dimensional imaging and patch-clamp recordings, we demonstrate that cerebellar dopamine D2 receptors (D2Rs) in mice are preferentially expressed in Purkinje cells (PCs) and regulate synaptic efficacy onto PCs. Moreover, we found that changes in D2R levels in PCs of male mice during adulthood alter sociability and preference for social novelty without affecting motor functions. Altogether, these findings demonstrate novel roles for D2R in PC function and causally link cerebellar D2R levels of expression to social behaviors.
Liu, JJ;Tsien, RW;Pang, ZP;
PMID: 34980924 | DOI: 10.1038/s41593-021-00984-5
Hypothalamic melanin-concentrating hormone (MCH) polypeptide contributes to regulating energy homeostasis, sleep and memory, although the mechanistic bases of its effects are unknown. In this study, in mice, we uncovered the physiological mechanism underlying the functional role of MCH signaling in projections to the dorsolateral septum (dLS), a region involved in routing hippocampal firing rhythms and encoding spatial memory based on such rhythms. Firing activity within the dLS in response to dorsal CA3 (dCA3) excitation is limited by strong feed-forward inhibition (FFI). We found that MCH synchronizes dLS neuronal firing with its dCA3 inputs by enhancing GABA release, which subsequently reduces the FFI and augments dCA3 excitatory input strength, both via pre-synaptic mechanisms. At the functional level, our data reveal a role for MCH signaling in the dLS in facilitating spatial memory. These findings support a model in which peptidergic signaling within the dLS modulates dorsal hippocampal output and supports memory encoding.
Rytova V, Ganella DE, Hawkes D, Bathgate RAD, Ma S and Gundlach AL
PMID: 30891856 | DOI: 10.1002/hipo.23089
Anxiety disorders are highly prevalent in modern society and better treatments are required. Key brain areas and signaling systems underlying anxiety include prefrontal cortex, hippocampus, and amygdala, and monoaminergic and peptidergic systems, respectively. Hindbrain GABAergic projection neurons that express the peptide, relaxin-3, broadly innervate the forebrain, particularly the septum and hippocampus, and relaxin-3 acts via a Gi/o -protein-coupled receptor known as the relaxin-family peptide 3 receptor (RXFP3). Thus, relaxin-3/RXFP3 signaling is implicated in modulation of arousal, motivation, mood, memory, and anxiety. Ventral hippocampus (vHip) is central to affective and cognitive processing and displays a high density of relaxin-3-positive nerve fibers and RXFP3 binding sites, but the identity of target neurons and associated effects on behavior are unknown. Therefore, in adult, male rats, we assessed the neurochemical nature of hippocampal RXFP3 mRNA-expressing neurons and anxiety-like and social behavior following chronic RXFP3 activation in vHip by viral vector expression of an RXFP3-selective agonist peptide, R3/I5. RXFP3 mRNA detected by fluorescent in situ hybridization was topographically distributed across the hippocampus in somatostatin- and parvalbumin-mRNA expressing GABA neurons. Chronic RXFP3 activation in vHip increased anxiety-like behavior in the light-dark box and elevated-plus maze, but not the large open-field test, and reduced social interaction with a conspecific stranger. Our data reveal disruptive effects of persistent RXFP3 signaling on hippocampal GABA networks important in anxiety; and identify a potential therapeutic target for anxiety disorders that warrants further investigation in relevant preclinical models.
Biological Psychiatry Global Open Science
Guerri, L;Dobbs, L;da Silva e Silva, D;Meyers, A;Ge, A;Lecaj, L;Djakuduel, C;Islek, D;Hipolito, D;Martinez, A;Shen, P;Marietta, C;Garamszegi, S;Capobianco, E;Jiang, Z;Schwandt, M;Mash, D;Alvarez, V;Goldman, D;
| DOI: 10.1016/j.bpsgos.2022.08.010
Background A salient effect of addictive drugs is to hijack the dopamine reward system, an evolutionarily conserved driver of goal-directed behavior and learning. Reduced dopamine type-II receptor (D2R) availability in the striatum is an important pathophysiological mechanism for addiction that is both consequential and causal for other molecular, cellular, and neuronal network differences etiologic for this disorder. Here, we sought to identify gene expression changes attributable to innate low expression of the Drd2 gene in the striatum and specific to striatal indirect medium spiny neurons (iMSNs). Methods Cre-conditional, Translating Ribosome Affinity Purification (TRAP) was used to purify and analyze the translatome (ribosome-bound mRNA) of iMSNs from mice with low/heterozygous or wild-type Drd2 expression in iMSNs. Complementary electrophysiological recordings and gene expression analysis of postmortem brain tissue from human cocaine users were performed. Results Innate low expression of Drd2 in iMSNs led to differential expression of genes involved in GABA and cAMP signaling, neural growth, lipid metabolism, neural excitability, and inflammation. Creb1 was identified as a likely upstream regulator, among others. In human brain, expression of FXYD2, a modulatory subunit of the Na/K pump, was negatively correlated with DRD2 mRNA expression. In iMSN-TRAP-Drd2HET mice, increased Cartpt and reduced S100a10 (p11) expression recapitulated previous observations in cocaine paradigms. Electrophysiology experiments supported a higher GABA tone in iMSN-Drd2HET mice. Conclusion This study provides strong molecular evidence that in addiction inhibition by the indirect pathway is constitutively enhanced through neural growth and increased GABA signaling.
Gu, X;Zhang, YZ;O'Malley, JJ;De Preter, CC;Penzo, M;Hoon, MA;
PMID: 36894654 | DOI: 10.1038/s41593-023-01268-w
Supraspinal brain regions modify nociceptive signals in response to various stressors including stimuli that elevate pain thresholds. The medulla oblongata has previously been implicated in this type of pain control, but the neurons and molecular circuits involved have remained elusive. Here we identify catecholaminergic neurons in the caudal ventrolateral medulla that are activated by noxious stimuli in mice. Upon activation, these neurons produce bilateral feed-forward inhibition that attenuates nociceptive responses through a pathway involving the locus coeruleus and norepinephrine in the spinal cord. This pathway is sufficient to attenuate injury-induced heat allodynia and is required for counter-stimulus induced analgesia to noxious heat. Our findings define a component of the pain modulatory system that regulates nociceptive responses.
Caprioli D, Venniro M, Zhang M, Bossert JM, Warren BL, Hope BT, Shaham Y.
PMID: 28123032 | DOI: 10.1523/JNEUROSCI.3091-16.2017
Abstract
We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we studied the role of dorsolateral striatum (DLS) and dorsomedial striatum (DMS) in this incubation. We trained rats to self-administer palatable food pellets (6 d, 6 h/d) and methamphetamine (12 d, 6 h/d). We then assessed relapse to methamphetamine seeking under extinction conditions after 1 and 21 abstinence days. Between tests, the rats underwent voluntary abstinence (using a discrete choice procedure between methamphetamine and food; 20 trials/d) for 19 d. We used in situ hybridization to measure the colabeling of the activity marker Fos with Drd1 and Drd2 in DMS and DLS after the tests. Based on the in situ hybridization colabeling results, we tested the causal role of DMS D1 and D2family receptors, and DMS neuronal ensembles in "incubated" methamphetamine seeking, using selective dopamine receptor antagonists (SCH39166 or raclopride) and the Daun02 chemogenetic inactivation procedure, respectively. Methamphetamine seeking was higher after 21 d of voluntary abstinence than after 1 d (incubation of methamphetamine craving). The incubated response was associated with increased Fos expression in DMS but not in DLS; Fos was colabeled with both Drd1 and Drd2 DMS injections of SCH39166 or raclopride selectively decreased methamphetamine seeking after 21 abstinence days. In Fos-lacZ transgenic rats, selective inactivation of relapse test-activated Fos neurons in DMS on abstinence day 18 decreased incubated methamphetamine seeking on day 21. Results demonstrate a role of DMS dopamine D1 and D2 receptors in the incubation of methamphetamine craving after voluntary abstinence and that DMS neuronal ensembles mediate this incubation.
SIGNIFICANCE STATEMENT:
In human addicts, abstinence is often self-imposed and relapse can be triggered by exposure to drug-associated cues that induce drug craving. We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we used classical pharmacology, in situ hybridization, immunohistochemistry, and the Daun02 inactivation procedure to demonstrate a critical role of dorsomedial striatum neuronal ensembles in this new form of incubation of drug craving.
Bernanke, A;Burnette, E;Murphy, J;Hernandez, N;Zimmerman, S;Walker, QD;Wander, R;Sette, S;Reavis, Z;Francis, R;Armstrong, C;Risher, ML;Kuhn, C;
PMID: 34898621 | DOI: 10.1371/journal.pone.0260577
Females are more affected by psychiatric illnesses including eating disorders, depression, and post-traumatic stress disorder than males. However, the neural mechanisms mediating these sex differences are poorly understood. Animal models can be useful in exploring such neural mechanisms. Conditioned taste aversion (CTA) is a behavioral task that assesses how animals process the competition between associated reinforcing and aversive stimuli in subsequent task performance, a process critical to healthy behavior in many domains. The purpose of the present study was to identify sex differences in this behavior and associated neural responses. We hypothesized that females would value the rewarding stimulus (Boost ) relative to the aversive stimulus (LiCl) more than males in performing CTA. We evaluated behavior (Boost intake, LiCl-induced behaviors, ultrasonic vocalizations (USVs), CTA performance) and Fos activation in relevant brain regions after the acute stimuli [acute Boost (AB), acute LiCl (AL)] and the context-only task control (COT), Boost only task (BOT) and Boost -LiCl task (BLT). Acutely, females drank more Boost than males but showed similar aversive behaviors after LiCl. Females and males performed CTA similarly. Both sexes produced 55 kHz USVs anticipating BOT and inhibited these calls in the BLT. However, more females emitted both 22 kHz and 55 kHz USVs in the BLT than males: the latter correlated with less CTA. Estrous cycle stage also influenced 55 kHz USVs. Fos responses were similar in males and females after AB or AL. Females engaged the gustatory cortex and ventral tegmental area (VTA) more than males during the BOT and males engaged the amygdala more than females in both the BOT and BLT. Network analysis of correlated Fos responses across brain regions identified two unique networks characterizing the BOT and BLT, in both of which the VTA played a central role. In situ hybridization with RNAscope identified a population of D1-receptor expressing cells in the CeA that responded to Boost and D2 receptor-expressing cells that responded to LiCl. The present study suggests that males and females differentially process the affective valence of a stimulus to produce the same goal-directed behavior.
Matsushima, A;Pineda, SS;Crittenden, JR;Lee, H;Galani, K;Mantero, J;Tombaugh, G;Kellis, M;Heiman, M;Graybiel, AM;
PMID: 36650127 | DOI: 10.1038/s41467-022-35752-x
Striatal projection neurons (SPNs), which progressively degenerate in human patients with Huntington's disease (HD), are classified along two axes: the canonical direct-indirect pathway division and the striosome-matrix compartmentation. It is well established that the indirect-pathway SPNs are susceptible to neurodegeneration and transcriptomic disturbances, but less is known about how the striosome-matrix axis is compromised in HD in relation to the canonical axis. Here we show, using single-nucleus RNA-sequencing data from male Grade 1 HD patient post-mortem brain samples and male zQ175 and R6/2 mouse models, that the two axes are multiplexed and differentially compromised in HD. In human HD, striosomal indirect-pathway SPNs are the most depleted SPN population. In mouse HD models, the transcriptomic distinctiveness of striosome-matrix SPNs is diminished more than that of direct-indirect pathway SPNs. Furthermore, the loss of striosome-matrix distinction is more prominent within indirect-pathway SPNs. These results open the possibility that the canonical direct-indirect pathway and striosome-matrix compartments are differentially compromised in late and early stages of disease progression, respectively, differentially contributing to the symptoms, thus calling for distinct therapeutic strategies.
