Molecular nutrition & food research
May, S;Greenow, KR;Higgins, AT;Derrick, AV;Taylor, E;Pan, P;Konstantinou, M;Nixon, C;Wooley, TE;Sansom, OJ;Wang, LS;Parry, L;
PMID: 36045438 | DOI: 10.1002/mnfr.202200234
Black raspberries (BRBs) have colorectal cancer (CRC) chemo-preventative effects. As CRC originates from an intestinal stem cell (ISC) this study has investigated the impact of BRBs on normal and mutant ISCs.Mice with an inducible Apcfl mutation in either the ISC (Lgr5CreERT2 ) or intestinal crypt (AhCre/VillinCreERT2 ) are fed a control or 10% BRB-supplemented diet. This study uses immunohistochemistry, gene expression analysis, and organoid culture to evaluate the effect of BRBs on intestinal homeostasis. RNAscope is performed for ISC markers on CRC adjacent normal colonic tissue pre and post BRB intervention from patients. 10% BRB diet has no overt effect on murine intestinal homeostasis, despite a reduced stem cell number. Following Apc ISC deletion, BRB diet extends lifespan and reduces tumor area. In the AhCre model, BRB diet attenuates the "crypt-progenitor" phenotype and reduces ISC marker gene expression. In ex vivo culture BRBs reduce the self-renewal capacity of murine and human Apc deficient organoids. Finally, the study observes a reduction in ISC marker gene expression in adjacent normal crypts following introduction of BRBs to the human bowel.BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment and further supports the use of BRBs in CRC prevention.
Ziskin JL, Dunlap D, Yaylaoglu M, Fodor IK, Forrest WF, Patel R, Ge N, Hutchins GG, Pine JK, Quirke P, Koeppen H, Jubb AM (2013).
PMID: 22637696 | DOI: 10.1136/gutjnl-2011-301195.
OBJECTIVE:
Wnt/Tcf, Lgr5, Ascl2 and/or Bmi1 signalling is believed to define the mouse intestinal stem cell niche(s) from which adenomas arise. The aim of this study was to determine the relevance of these putative intestinal stem cell markers to human colorectal cancer.
DESIGN:
19 putative intestinal stem cell markers, including Ascl2 and Lgr5, were identified from published data and an evaluation of a human colorectal gene expression database. Associations between these genes were assessed by isotopic in situ hybridisation (ISH) in 57 colorectal adenocarcinomas. Multiplex fluorescent ISH and chromogenic non-isotopic ISH were performed to confirm expression patterns. The prognostic significance of Lgr5 was assessed in 891 colorectal adenocarcinomas.
RESULTS:
Ascl2 and Lgr5 were expressed in 85% and 74% of cancers respectively, and expression was positively correlated (p=0.003). Expression of Bmi1 was observed in 47% of cancers but was very weak in 98% of cases with expression. Both Ascl2 and/or Lgr5 were positively correlated with the majority of genes in the signature but neither was correlated with Cdk6, Gpx2, Olfm4 or Tnfrsf19. Lgr5 did not have prognostic significance.
CONCLUSION:
These data suggest that 74-85% of colorectal cancers express a Lgr5/Ascl2 associated signature and support the hypothesis that they derive from Lgr5(+)/Ascl2(+) crypt stem cells, not Bmi1(+) stem cells. However, Olfm4 was not found to be a useful marker of Lgr5(+) cells in normal colon or tumours. In this large series, Lgr5 expression is not associated with increased tumour aggressiveness, as might be expected from a cancer stem cell marker.
Cloft, S;Miska, K;Jenkins, M;Proszkowiec-Weglarz, M;Kahl, S;Wong, E;
| DOI: 10.1016/j.psj.2023.102537
Infection with the protozoan parasite Eimeria can cause the economically devastating disease coccidiosis, which is characterized by gross tissue damage and inflammation resulting in blunted villi and altered intestinal homeostasis. Male broiler chickens at 21 d of age were given a single challenge with Eimeria acervulina. Temporal changes in intestinal morphology and gene expression were investigated at 0, 3, 5, 7, 10, and 14 d post-infection (dpi). There were increased crypt depths for chickens infected with E. acervulina starting at 3 dpi and continuing to 14 dpi. At 5 and 7 dpi, infected chickens had decreased Mucin2 (Muc2), and Avian beta defensin (AvBD) 6 mRNA at 5 and 7 dpi and decreased AvBD10 mRNA at 7 dpi compared to uninfected chickens. Liver-enriched antimicrobial peptide 2 (LEAP2) mRNA was decreased at 3, 5, 7, and 14 dpi compared to uninfected chickens. After 7 dpi, there was increased Collagen 3a1 and Notch 1 mRNA compared to uninfected chickens. Marker of proliferation Ki67 mRNA was increased in infected chickens from 3 to 10 dpi. In addition, the presence of E. acervulina was visualized by in situ hybridization (ISH) with an E. acervulina sporozoite surface antigen (Ea-SAG) probe. In E. acervulina infected chickens, Ea-SAG mRNA was only detectable on 5 and 7 dpi by both ISH and qPCR. To further investigate the site of E. acervulina infection, Ea-SAG and Muc2 probes were examined on serial sections. The Muc2 ISH signal was decreased in regions where the Ea-SAG ISH signal was present, suggesting that the decrease in Muc2 by qPCR may be caused by the loss of Muc2 in the localized regions where the E. acervulina had invaded the tissue. Eimeria acervulina appears to manipulate host cells by decreasing their defensive capabilities and thereby allows the infection to propagate freely. Following infection, the intestinal cells upregulate genes that may support regeneration of damaged intestinal tissue.
Biological Psychiatry Global Open Science
Jiang, S;Zhang, H;Eiden, L;
| DOI: 10.1016/j.bpsgos.2023.04.001
Background The neuropeptide PACAP is a master regulator of central and peripheral stress responses, yet it is not clear how PACAP projections throughout the brain execute endocrine and behavioral stress responses. Methods We used AAV neuronal tracing, an acute restraint stress (ARS) paradigm, and intersectional genetics, in C57Bl6 mice, to identify PACAP-containing circuits controlling stress-induced behavior and endocrine activation. Results PACAP deletion from forebrain excitatory neurons, including a projection directly from medial prefrontal cortex (mPFC) to hypothalamus, impairs c-fos activation and CRH mRNA elevation in PVN after 2 hr of restraint, without affecting ARS-induced hypophagia, or c-fos elevation in non-hypothalamic brain. Elimination of PACAP within projections from lateral parabrachial nucleus to extended amygdala (EA), on the other hand, attenuates ARS-induced hypophagia, along with EA fos induction, without affecting ARS-induced CRH mRNA elevation in PVN. PACAP projections to EA terminate at PKCδ neurons in both central amygdala (CeA) and oval nuclei of bed nucleus of stria terminalis (BNSTov). Silencing of PKCδ neurons in CeA, but not in BNSTov, attenuates ARS-induced hypophagia. Experiments were carried out in mice of both sexes with n>5 per group. Conclusions A frontocortical descending PACAP projection controls PVN CRH mRNA production, to maintain hypothalamo-pituitary adrenal (HPA) axis activation, and regulate the endocrine response to stress. An ascending PACAPergic projection from eLPBn to PKCδ neurons in central amygdala regulates behavioral responses to stress. Defining two separate limbs of the acute stress response provides broader insight into the specific brain circuitry engaged by the psychogenic stress response.
Frontiers in molecular neuroscience
Kim, JJ;Sapio, MR;Vazquez, FA;Maric, D;Loydpierson, AJ;Ma, W;Zarate, CA;Iadarola, MJ;Mannes, AJ;
PMID: 35706427 | DOI: 10.3389/fnmol.2022.892345
Ketamine, an N-methyl-D-aspartate (NMDA)-receptor antagonist, is a recently revitalized treatment for pain and depression, yet its actions at the molecular level remain incompletely defined. In this molecular-pharmacological investigation in the rat, we used short- and longer-term infusions of high dose ketamine to stimulate neuronal transcription processes. We hypothesized that a progressively stronger modulation of neuronal gene networks would occur over time in cortical and limbic pathways. A continuous intravenous administration paradigm for ketamine was developed in rat consisting of short (1 h) and long duration (10 h, and 10 h + 24 h recovery) infusions of anesthetic concentrations to activate or inhibit gene transcription in a pharmacokinetically controlled fashion. Transcription was measured by RNA-Seq in three brain regions: frontal cortex, hippocampus, and amygdala. Cellular level gene localization was performed with multiplex fluorescent in situ hybridization. Induction of a shared transcriptional regulatory network occurred within 1 h in all three brain regions consisting of (a) genes involved in stimulus-transcription factor coupling that are induced during altered synaptic activity (immediate early genes, IEGs, such as c-Fos, 9-12 significant genes per brain region, p < 0.01 per gene) and (b) the Nrf2 oxidative stress-antioxidant response pathway downstream from glutamate signaling (Nuclear Factor Erythroid-Derived 2-Like 2) containing 12-25 increasing genes (p < 0.01) per brain region. By 10 h of infusion, the acute results were further reinforced and consisted of more and stronger gene alterations reflecting a sustained and accentuated ketamine modulation of regional excitation and plasticity. At the cellular level, in situ hybridization localized up-regulation of the plasticity-associated gene Bdnf, and the transcription factors Nr4a1 and Fos, in cortical layers III and V. After 24 h recovery, we observed overshoot of transcriptional processes rather than a smooth return to homeostasis suggesting an oscillation of plasticity occurs during the transition to a new phase of neuronal regulation. These data elucidate critical molecular regulatory actions during and downstream of ketamine administration that may contribute to the unique drug actions of this anesthetic agent. These molecular investigations point to pathways linked to therapeutically useful attributes of ketamine.
Caprioli D, Venniro M, Zhang M, Bossert JM, Warren BL, Hope BT, Shaham Y.
PMID: 27980115 | DOI: 10.1523/JNEUROSCI.3091-16.2016
We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we studied the role of dorsolateral and dorsomedial striatum (DLS, DMS) in this incubation.We trained rats to self-administer palatable food pellets (6 days, 6-h/d) and methamphetamine (12 days, 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/day) for 19 days. We used in situ hybridization to measure co-labeling of the activity marker Fos with Drd1 and Drd2 in DMS and DLS after the tests. Based on the in situ hybridization co-labeling results, we tested the causal role of DMS D1- and D2-family 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 days of voluntary abstinence than after 1 day (incubation of methamphetamine craving). The 'incubated' response was associated with increased Fos expression in DMS but not DLS; Fos was co-labeled 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 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.
Cloft, S;Uni, Z;Wong, E;
| DOI: 10.1016/j.psj.2023.102495
Mature small intestines have crypts populated by stem cells which produce replacement cells to maintain the absorptive villus surface area. The embryonic crypt is rudimentary and cells along the villi are capable of proliferation. By 7 d post-hatch the crypts are developed and are the primary sites of proliferation. Research characterizing the proliferative expansion of the small intestine during the peri-hatch period is lacking. The objective of this study was to profile the changes of genes that are markers of stem cells and proliferation: Olfactomedin 4 (Olfm4), Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and marker of proliferation Ki67 from embryonic day 17 to 7 d post-hatch using quantitative PCR and in situ hybridization (ISH). The expression of the stem cell marker genes differed. Olfm4 mRNA increased while Lgr5 mRNA decreased post-hatch. Ki67 mRNA decreased post-hatch in the duodenum and was generally the greatest in the ileum. The ISH was consistent with the quantitative PCR results. Olfm4 mRNA was only seen in the crypts and increased with morphological development of the crypts. In contrast Lgr5 mRNA was expressed in the crypt and the villi in the embryonic periods but became restricted to the intestinal crypt during the post-hatch period. Ki67 mRNA was expressed throughout the intestine pre-hatch, but then expression became restricted to the crypt and the center of the villi. The ontogeny of Olfm4, Lgr5 and Ki67 expressing cells show that proliferation in the peri-hatch intestine changes from along the entire villi to being restricted within the crypts.
Pfarr S, Schaaf L, Reinert JK, Paul E, Herrmannsdörfer F, Roßmanith M, Kuner T, Hansson AC, Spanagel R, Körber C, Sommer WH.
PMID: - | DOI: Fos Bcl11b Rgs8
Cue-reward associations form distinct memories that can drive appetitive behaviors and are involved in craving for both drugs and natural rewards. Distinct sets of neurons, so called neuronal ensembles, in the infralimbic area (IL) of the medial prefrontal cortex play a key role in alcohol seeking. Whether this ensemble is specific for alcohol or controls reward seeking in general remains unclear. Here, we compared IL ensembles formed upon recall of drug (alcohol) or natural reward (saccharin) memories in male Wistar rats. Using an experimental framework that allows identification of two distinct reward-associated ensembles within the same animal, we found that cue-induced seeking of either alcohol or saccharin activated ensembles of similar size and organization, whereby these ensembles consist of largely overlapping neuronal populations. Thus, the IL seems to act as a general integration hub for reward seeking behavior, but also contains subsets of neurons that encode for the different rewards.
SIGNIFICANCE STATEMENT
Cue-reward associations form distinct memories that can act as drivers of appetitive behaviors and are involved in craving for natural rewards as well as for drugs. Distinct sets of neurons, so called neuronal ensembles, in the infralimbic area of the medial prefrontal cortex play a key role in cue-triggered reward seeking. However, it is unclear whether these ensembles act as broadly tuned controllers of approach behavior or represent the learned associations between specific cues and rewards. Using an experimental framework that allows identification of two distinct reward-associated ensembles within the same animal we find largely overlapping neuronal populations. Repeated activation by two distinct events could reflect the linking of the two memory traces within the same neuron.
Greenwood, MP;Greenwood, M;Bárez-López, S;Hawkins, JW;Short, K;Tatovic, D;Murphy, D;
PMID: 36773648 | DOI: 10.1016/j.molmet.2023.101692
The excessive release of the antidiuretic hormone vasopressin is implicated in many diseases including cardiovascular disease, diabetes, obesity, and metabolic syndrome. Once thought to be elevated as a consequence of diseases, data now supports a more causative role. We have previously identified CREB3L1 as a transcription factor that co-ordinates vasopressin synthesis and release in the hypothalamus. The objective here was to identify mechanisms orchestrated by CREB3L1 that co-ordinate vasopressin release.We mined Creb3l1 knockdown SON RNA-seq data to identify downstream target genes. We proceeded to investigate the expression of these genes and associated pathways in the supraoptic nucleus of the hypothalamus in response to physiological and pharmacological stimulation. We used viruses to selectively knockdown gene expression in the supraoptic nucleus and assessed physiological and metabolic parameters. We adopted a phosphoproteomics strategy to investigate mechanisms that facilitate hormone release by the pituitary gland.We discovered glucagon like peptide 1 receptor (Glp1r) as a downstream target gene and found increased expression in stimulated vasopressin neurones. Selective knockdown of supraoptic nucleus Glp1rs resulted in decreased food intake and body weight. Treatment with GLP-1R agonist liraglutide decreased vasopressin synthesis and release. Quantitative phosphoproteomics of the pituitary neurointermediate lobe revealed that liraglutide initiates hyperphosphorylation of presynapse active zone proteins that control vasopressin exocytosis.In summary, we show that GLP-1R signalling inhibits the vasopressin system. Our data advises that hydration status may influence the pharmacodynamics of GLP-1R agonists so should be considered in current therapeutic strategies.
Liu, J;Wu, R;Seaman, R;Manz, KM;Johnson, B;Vu, J;Huang, Y;Zhang, Y;Robison, AJ;Neve, R;Grueter, BA;Dietz, D;Li, JX;
PMID: 35079125 | DOI: 10.1038/s41380-022-01448-3
Relapse remains a major challenge to the treatment of cocaine addiction. Recent studies suggested that the trace amine-associated receptor 1 (TAAR1) could be a promising target to treat cocaine addiction and relapse; however, the underlying mechanism remains unclear. Here, we aimed to investigate the neural mechanism underlying the role of TAAR1 in the drug priming-induced reinstatement of cocaine-seeking behavior in rats, an animal model of cocaine relapse. We focused on the shell subregion of nucleus accumbens (NAc), a key brain region of the brain reward system. We found that activation of TAAR1 by systemic and intra-NAc shell administration of the selective TAAR1 agonist RO5166017 attenuated drug-induced reinstatement of cocaine-seeking and prevented drug priming-induced CaMKIIα activity in the NAc shell. Activation of TAAR1 dampened the CaMKIIα/GluR1 signaling pathway in the NAc shell and reduced AMPAR-EPSCs on the NAc slice. Microinjection of the selective TAAR1 antagonist EPPTB into the NAc shell enhanced drug-induced reinstatement as well as potentiated CaMKIIα activity in the NAc shell. Furthermore, viral-mediated expression of CaMKIIα in the NAc shell prevented the behavioral effects of TAAR1 activation. Taken together, our findings indicate that TAAR1 regulates drug-induced reinstatement of cocaine-seeking by negatively regulating CaMKIIα activity in the NAc. Our findings elucidate a novel mechanism of TAAR1 in regulating drug-induced reinstatement of cocaine-seeking and further suggests that TAAR1 is a promising target for the treatment of cocaine relapse.
Centanni SW, Morris BD, Luchsinger JR, Bedse G, Fetterly TL, Patel S, Winder DG.
PMID: 30390064 | DOI: 10.1038/s41386-018-0257-8
Negative affect is a core symptom domain associated with an array of neurological and psychiatric disorders and is only partially targeted by current therapies, highlighting the need for better, more targeted treatment options. This study focuses on negative affective symptoms associated with prolonged alcohol abstinence, one of the leading causes of relapse. Using a mouse model of chronic alcohol consumption followed by forced abstinence (CDFA), prolonged alcohol abstinence increased c-fos expression and spontaneous glutamatergic neurotransmission in the dorsal bed nucleus of the stria terminalis (dBNST), a region heavily implicated in negative affect in both humans and rodents. Further, pharmacologically enhancing eCBs with JZL184 prevents abstinence-induced increases in dBNST neuronal activity, underscoring the therapeutic potential of drugs targeting the brain's eCB system. Next, we used a channelrhodopsin-assisted mapping strategy to identify excitatory inputs to the dBNST that could contribute to CDFA-induced negative affect. We identified the insular cortex (insula), a region involved in regulating interoception, as a dense, functional, endocannabinoid-sensitive input to the dBNST. Using a chemogenetic strategy to locally mimic eCB signaling, we demonstrate that the insula strongly influences CDFA behavioral and BNST neuronal activity. Lastly, we used viral anterograde transsynaptic expression in combination with a Gq-DREADD to selectively recruit dBNST neurons receiving insula projections. Chemogenetic recruitment of these neurons mimicked behavioral and c-fos responses observed in CDFA. Collectively, this study supports a role for the insula-BNST neural circuit in negative affective disturbances and highlights the therapeutic potential of the endocannabinoid system for treating negative affective disorders.
Behavioural brain research
Van Savage, J;Avegno, EM;
PMID: 37352979 | DOI: 10.1016/j.bbr.2023.114553
Designer receptors exclusively activated by designer drugs (DREADDs) are a promising tool for analyzing neural circuitry, and improved DREADD-selective ligands continue to be developed. Relative to clozapine-N-oxide (CNO), JHU37160 is a selective DREADD agonist recently shown to exhibit higher blood brain barrier penetrance and DREADD selectivity in vivo; however, relatively few studies have characterized the behavioral effects of systemic JHU37160 administration in animals. Here, we report a dose-dependent anxiogenic effect of systemic JHU37160 in male Wistar and Long-Evans rats, regardless of DREADD expression, with no impact on locomotor behavior. These results suggest that high dose (1 mg/kg) JHU37160 should be avoided when performing chemogenetic experiments designed to evaluate circuit manipulation on anxiety-like behavior in rats.
