TAK1 inhibition leads to RIPK1-dependent apoptosis in immune-activated cancers.

Poor survival and lack of treatment response in glioblastoma (GBM) is attributed to the persistence of glioma stem cells (GSCs). To identify novel therapeutic approaches, we performed CRISPR/Cas9 knockout screens and discovered TGFß activated kinase (TAK1) as a selective survival factor in a significant fraction of GSCs. Loss of TAK1 kinase activity results in RIPK1-dependent apoptosis via Caspase-8/FADD complex activation, dependent on autocrine TNFα ligand production and constitutive TNFR signaling. We identify a transcriptional signature associated with immune activation and the mesenchymal GBM subtype to be a characteristic of cancer cells sensitive to TAK1 perturbation and employ this signature to accurately predict sensitivity to the TAK1 kinase inhibitor HS-276. In addition, exposure to pro-inflammatory cytokines IFNγ and TNFα can sensitize resistant GSCs to TAK1 inhibition. Our findings reveal dependency on TAK1 kinase activity as a novel vulnerability in immune-activated cancers, including mesenchymal GBMs that can be exploited therapeutically.

EGFR amplification and EGFRvIII predict and participate in TAT-Cx43266-283 antitumor response in preclinical glioblastoma models.

BACKGROUND: Glioblastoma (GBM) commonly displays epidermal growth factor receptor (EGFR) alterations (mainly amplification and EGFRvIII) and TAT-Cx43266-283 is a Src-inhibitory peptide with antitumor properties in preclinical GBM models. Given the link between EGFR and Src, the aim of this study was to explore the role of EGFR in the antitumor effects of TAT-Cx43266-283. METHODS: The effect of TAT-Cx43266-283, temozolomide (TMZ) and erlotinib (EGFR inhibitor) was studied in patient-derived GBM stem cells (GSCs) and murine neural stem cells (NSCs) with and without EGFR alterations, in vitro and in vivo. EGFR alterations were analyzed by Western blot (WB) and Fluorescence In Situ Hybridization (FISH) in these cells, and compared with Src activity and survival in GBM samples from TCGA. RESULTS: The effect of TAT-Cx43266-283 correlated with EGFR alterations in a set of patient-derived GSCs and was stronger than that exerted by TMZ and erlotinib. In fact, TAT-Cx43266-283 only affected NSCs with EGFR alterations, but not healthy NSCs. EGFR alterations correlated with Src activity and poor survival in GBM patients. Finally, tumors generated from NSCs with EGFR alterations, showed a decrease in growth, invasiveness and vascularization after treatment with TAT-Cx43266-283, which enhanced the survival of immunocompetent mice. CONCLUSION: Clinically relevant EGFR alterations are predictors of TAT-Cx43266-283 response and part of its mechanism of action, even in TMZ- and erlotinib-resistant GSCs. TAT-Cx43266-283 targets NSCs with GBM-driver mutations, including EGFR alterations, in an immunocompetent GBM model in vivo, suggesting a promising effect on GBM recurrence. Together, this study represents an important step towards the clinical application of TAT-Cx43266-283.

Elevated FOXG1 in glioblastoma stem cells cooperates with Wnt/ß-catenin to induce exit from quiescence.

Glioblastoma (GBM) stem cells (GSCs) display phenotypic and molecular features reminiscent of normal neural stem cells and exhibit a spectrum of cell cycle states (dormant, quiescent, proliferative). However, mechanisms controlling the transition from quiescence to proliferation in both neural stem cells (NSCs) and GSCs are poorly understood. Elevated expression of the forebrain transcription factor FOXG1 is often observed in GBMs. Here, using small-molecule modulators and genetic perturbations, we identify a synergistic interaction between FOXG1 and Wnt/ß-catenin signaling. Increased FOXG1 enhances Wnt-driven transcriptional targets, enabling highly efficient cell cycle re-entry from quiescence; however, neither FOXG1 nor Wnt is essential in rapidly proliferating cells. We demonstrate that FOXG1 overexpression supports gliomagenesis in vivo and that additional ß-catenin induction drives accelerated tumor growth. These data indicate that elevated FOXG1 cooperates with Wnt signaling to support the transition from quiescence to proliferation in GSCs.

Oncogene expression from extrachromosomal DNA is driven by copy number amplification and does not require spatial clustering in glioblastoma stem cells.

Extrachromosomal DNA (ecDNA) are frequently observed in human cancers and are responsible for high levels of oncogene expression. In glioblastoma (GBM), ecDNA copy number correlates with poor prognosis. It is hypothesized that their copy number, size, and chromatin accessibility facilitate clustering of ecDNA and colocalization with transcriptional hubs, and that this underpins their elevated transcriptional activity. Here, we use super-resolution imaging and quantitative image analysis to evaluate GBM stem cells harbouring distinct ecDNA species (EGFR, CDK4, PDGFRA). We find no evidence that ecDNA routinely cluster with one another or closely interact with transcriptional hubs. Cells with EGFR-containing ecDNA have increased EGFR transcriptional output, but transcription per gene copy is similar in ecDNA compared to the endogenous chromosomal locus. These data suggest that it is the increased copy number of oncogene-harbouring ecDNA that primarily drives high levels of oncogene transcription, rather than specific interactions of ecDNA with each other or with high concentrations of the transcriptional machinery.

Combination of BMI1 and MAPK/ERK inhibitors is effective in medulloblastoma.

BACKGROUND: Epigenetic changes play a key role in the pathogenesis of medulloblastoma (MB), the most common malignant pediatric brain tumor. METHODS: We explore the therapeutic potential of BMI1 and MAPK/ERK inhibition in BMI1High;CHD7Low MB cells and in a preclinical xenograft model. RESULTS: We identify a synergistic vulnerability of BMI1High;CHD7Low MB cells to a combination treatment with BMI1 and MAPK/ERK inhibitors. Mechanistically, CHD7-dependent binding of BMI1 to MAPK-regulated genes underpins the CHD7-BMI1-MAPK regulatory axis responsible of the antitumour effect of the inhibitors in vitro and in a preclinical mouse model. Increased ERK1 and ERK2 phosphorylation activity is found in BMI1High;CHD7Low G4 MB patients, raising the possibility that they could be amenable to a similar therapy. CONCLUSIONS: The molecular dissection of the CHD7-BMI1-MAPK regulatory axis in BMI1High;CHD7Low MB identifies this signature as a proxy to predict MAPK functional activation, which can be effectively drugged in preclinical models, and paves the way for further exploration of combined BMI1 and MAPK targeting in G4 MB patients.

On-chip perivascular niche supporting stemness of patient-derived glioma cells in a serum-free, flowable culture.

Glioblastoma multiforme (GBM) is the most common and the most aggressive type of primary brain malignancy. Glioblastoma stem-like cells (GSCs) can migrate in vascular niches within or away from the tumour mass, increasing tumour resistance to treatments and contributing to relapses. To study individual GSC migration and their interactions with the perivasculature of the tumour microenvironment, there is a need to develop a human organotypic in vitro model. Herein, we demonstrated a perivascular niche-on-a-chip, in a serum-free condition with gravity-driven flow, that supported the stemness of patient-derived GSCs and foetal neural stem cells grown in a three-dimensional environment (3D). Endothelial cells from three organ origins, (i) human brain microvascular endothelial cells (hCMEC/D3), (ii) human umbilical vein endothelial cells (HUVECs) and, (iii) human lung microvascular endothelial cells (HMVEC-L) formed rounded microvessels within the extracellular-matrix integrated microfluidic chip. By optimising cell extraction protocols, systematic studies were performed to evaluate the effects of serum-free media, 3D cell cultures, and the application of gravity-driven flow on the characteristics of endothelial cells and their co-culture with GSCs. Our results showed the maintenance of adherent and tight junction markers of hCMEC/D3 in the serum-free culture and that gravity-driven flow was essential to support adequate viability of both the microvessel and the GSCs in co-culture (>80% viability at day 3). Endpoint biological assays showed upregulation of neovascularization-related genes (e.g., angiopoietins, vascular endothelial growth factor receptors) in endothelial cells co-cultured with GSCs in contrast to the neural stem cell reference that showed insignificant changes. The on-chip platform further permitted live-cell imaging of GSC - microvessel interaction, enabling quantitative analysis of GSC polarization and migration. Overall, our comparative genotypic (i.e. qPCR) and phenotypic (i.e. vessel permeability and GSC migration) studies showed that organotypic (brain cancer cells-brain endothelial microvessel) interactions differed from those within non-tissue specific vascular niches of human origin. The development and optimization of this on-chip perivascular niche, in a serum-free flowable culture, could provide the next level of complexity of an in vitro system to study the influence of glioma stem cells on brain endothelium.

Glioblastomas acquire myeloid-affiliated transcriptional programs via epigenetic immunoediting to elicit immune evasion.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.

The white matter is a pro-differentiative niche for glioblastoma.

Glioblastomas are hierarchically organised tumours driven by glioma stem cells that retain partial differentiation potential. Glioma stem cells are maintained in specialised microenvironments, but whether, or how, they undergo lineage progression outside of these niches remains unclear. Here we identify the white matter as a differentiative niche for glioblastomas with oligodendrocyte lineage competency. Tumour cells in contact with white matter acquire pre-oligodendrocyte fate, resulting in decreased proliferation and invasion. Differentiation is a response to white matter injury, which is caused by tumour infiltration itself in a tumoursuppressive feedback loop. Mechanistically, tumour cell differentiation is driven by selective white matter upregulation of SOX10, a master regulator of normal oligodendrogenesis. SOX10 overexpression or treatment with myelination-promoting agents that upregulate endogenous SOX10, mimic this response, leading to niche-independent pre-oligodendrocyte differentiation and tumour suppression in vivo. Thus, glioblastoma recapitulates an injury response and exploiting this latent programme may offer treatment opportunities for a subset of patients.

Inositol treatment inhibits medulloblastoma through suppression of epigenetic-driven metabolic adaptation.

Deregulation of chromatin modifiers plays an essential role in the pathogenesis of medulloblastoma, the most common paediatric malignant brain tumour. Here, we identify a BMI1-dependent sensitivity to deregulation of inositol metabolism in a proportion of medulloblastoma. We demonstrate mTOR pathway activation and metabolic adaptation specifically in medulloblastoma of the molecular subgroup G4 characterised by a BMI1High;CHD7Low signature and show this can be counteracted by IP6 treatment. Finally, we demonstrate that IP6 synergises with cisplatin to enhance its cytotoxicity in vitro and extends survival in a pre-clinical BMI1High;CHD7Low xenograft model.

Regional identity of human neural stem cells determines oncogenic responses to histone H3.3 mutants.

Point mutations within the histone H3.3 are frequent in aggressive childhood brain tumors known as pediatric high-grade gliomas (pHGGs). Intriguingly, distinct mutations arise in discrete anatomical regions: H3.3-G34R within the forebrain and H3.3-K27M preferentially within the hindbrain. The reasons for this contrasting etiology are unknown. By engineering human fetal neural stem cell cultures from distinct brain regions, we demonstrate here that cell-intrinsic regional identity provides differential responsiveness to each mutant that mirrors the origins of pHGGs. Focusing on H3.3-G34R, we find that the oncohistone supports proliferation of forebrain cells while inducing a cytostatic response in the hindbrain. Mechanistically, H3.3-G34R does not impose widespread transcriptional or epigenetic changes but instead impairs recruitment of ZMYND11, a transcriptional repressor of highly expressed genes. We therefore propose that H3.3-G34R promotes tumorigenesis by focally stabilizing the expression of key progenitor genes, thereby locking initiating forebrain cells into their pre-existing immature state.

Experimental models and tools to tackle glioblastoma.

Glioblastoma multiforme (GBM) is one of the deadliest human cancers. Despite increasing knowledge of the genetic and epigenetic changes that underlie tumour initiation and growth, the prognosis for GBM patients remains dismal. Genome analysis has failed to lead to success in the clinic. Fresh approaches are needed that can stimulate new discoveries across all levels: cell-intrinsic mechanisms (transcriptional/epigenetic and metabolic), cell-cell signalling, niche and microenvironment, systemic signals, immune regulation, and tissue-level physical forces. GBMs are inherently extremely challenging: tumour detection occurs too late, and cells infiltrate widely, hiding in quiescent states behind the blood-brain barrier. The complexity of the brain tissue also provides varied and complex microenvironments that direct cancer cell fates. Phenotypic heterogeneity is therefore superimposed onto pervasive genetic heterogeneity. Despite this bleak outlook, there are reasons for optimism. A myriad of complementary, and increasingly sophisticated, experimental approaches can now be used across the research pipeline, from simple reductionist models devised to delineate molecular and cellular mechanisms, to complex animal models required for preclinical testing of new therapeutic approaches. No single model can cover the breadth of unresolved questions. This Review therefore aims to guide investigators in choosing the right model for their question. We also discuss the recent convergence of two key technologies: human stem cell and cancer stem cell culture, as well as CRISPR/Cas tools for precise genome manipulations. New functional genetic approaches in tailored models will likely fuel new discoveries, new target identification and new therapeutic strategies to tackle GBM.

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein.

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

Dietary energy requirements in relatively healthy maintenance hemodialysis patients estimated from long-term metabolic studies.

BACKGROUND: Studies that examined dietary energy requirements (DERs) of patients undergoing maintenance hemodialysis (MHD) have shown mixed results. Many studies reported normal DERs, but some described increased energy needs. DERs in MHD patients have been estimated primarily from indirect calorimetry and from nitrogen balance studies. The present study measured DERs in MHD patients on the basis of their dietary energy intake and changes in body composition. OBJECTIVE: This study assessed DERs in MHD patients who received a constant energy intake while changes in their body composition were measured. DESIGN: Seven male and 6 female sedentary, clinically stable MHD patients received a constant mean (±SD) energy intake for 92.2 ± 7.9 d while residing in a metabolic research ward. Changes in fat and fat-free mass, measured by dual-energy X-ray absorptiometry, were converted to calorie equivalents and added to energy intake to calculate energy requirements. RESULTS: The average DER was 31 ± 3 kcal · kg(-1) · d(-1) calculated from energy intake and change in fat and fat-free calories, which was 28 ± 197 kcal/d over the 92 d of the study. DERs of MHD patients correlated strongly with their body weight (r = 0.81, P = 0.002) and less closely with their measured resting energy expenditure expressed as kcal/d (r = 0.69, P = 0.01). Although the average observed DER in MHD patients was similar to published estimated values for normal sedentary individuals of similar age and sex, there was wide variability in DER among individual patients (range: 26-36 kcal · kg(-1) · d(-1)). CONCLUSIONS: Average DERs of sedentary, clinically stable patients receiving MHD are similar to those of sedentary normal individuals. Our data do not support the theory that MHD patients have increased DERs. Due to the high variability in DERs, careful monitoring of the nutritional status of individual MHD patients is essential. This trial was registered at clinicaltrials.gov as NCT02194114.

The effects of prolonged administration of norepinephrine reuptake inhibitors on long-term potentiation in dentate gyrus, and on tests of spatial and object recognition memory in rats.

Phasic norepinephrine (NE) release events are involved in arousal, novelty detection and in plasticity processes underlying learning and memory in mammalian systems. Although the effects of phasic NE release events on plasticity and memory are prevalently documented, it is less understood what effects chronic NE reuptake inhibition and sustained increases in noradrenergic tone, might have on plasticity and cognitive processes in rodent models of learning and memory. This study investigates the effects of chronic NE reuptake inhibition on hippocampal plasticity and memory in rats. Rats were administered NE reuptake inhibitors (NRIs) desipramine (DMI; 0, 3, or 7.5mg/kg/day) or nortriptyline (NTP; 0, 10 or 20mg/kg/day) in drinking water. Long-term potentiation (LTP; 200 Hz) of the perforant path-dentate gyrus evoked potential was examined in urethane anesthetized rats after 30-32 days of DMI treatment. Short- (4-h) and long-term (24-h) spatial memory was tested in separate rats administered 0 or 7.5mg/kg/day DMI (25-30 days) using a two-trial spatial memory test. Additionally, the effects of chronically administered DMI and NTP were tested in rats using a two-trial, Object Recognition Test (ORT) at 2- and 24-h after 45 and 60 days of drug administration. Rats administered 3 or 7.5mg/kg/day DMI had attenuated LTP of the EPSP slope but not the population spike at the perforant path-dentate gyrus synapse. Short- and long-term memory for objects is differentially disrupted in rats after prolonged administration of DMI and NTP. Rats that were administered 7.5mg/kg/day DMI showed decreased memory for a two-trial spatial task when tested at 4-h. In the novel ORT, rats receiving 0 or 7.5mg/kg/day DMI showed a preference for the arm containing a Novel object when tested at both 2- and 24-h demonstrating both short- and long-term memory retention of the Familiar object. Rats that received either dose of NTP or 3mg/kg/day DMI showed impaired memory at 2-h, however this impairment was largely reversed at 24-h. Animals in the high-dose NTP (20mg/kg/day) group were impaired at both short- and long-term intervals. Activity levels, used as an index of location memory during the ORT, demonstrated that rats receiving DMI were again impaired at retaining memory for location. DMI dose-dependently disrupts LTP in the dentate gyrus of anesthetized rats and also disrupts memory for tests of spatial memory when administered for long periods.

Convergence of cMyc and ß-catenin on Tcf7l1 enables endoderm specification.

The molecular machinery that directs formation of definitive endoderm from pluripotent stem cells is not well understood. Wnt/ß-catenin and Nodal signalling have been implicated, but the requirements for lineage specification remain incompletely defined. Here, we demonstrate a potent effect of inhibiting glycogen synthase kinase 3 (GSK3) on definitive endoderm production. We find that downstream of GSK3 inhibition, elevated cMyc and ß-catenin act in parallel to reduce transcription and DNA binding, respectively, of the transcriptional repressor Tcf7l1. Tcf7l1 represses FoxA2, a pioneer factor for endoderm specification. Deletion of Tcf7l1 is sufficient to allow upregulation of FoxA2 in the presence of Activin. In wild-type cells, cMyc contributes by reducing Tcf7l1 mRNA, while ß-catenin acts on Tcf7l1 protein. GSK3 inhibition is further required for consolidation of endodermal fate via upregulation of Sox17, highlighting sequential roles for Wnt signalling. The identification of a cMyc/ß-catenin-Tcf7l1-FoxA2 axis reveals a de-repression mechanism underlying endoderm induction that may be recapitulated in other developmental and patho-logical contexts.

Self-Reported Interview-Assisted Diet Records Underreport Energy Intake in Maintenance Hemodialysis Patients.

OBJECTIVES: Studies suggest that maintenance hemodialysis (MHD) patients report dietary energy intakes (EIs) that are lower than what is actually ingested. Data supporting this conclusion have several important limitations. The present study introduces a novel approach of assessing underreporting of EI in MHD patients. DESIGN: Comparisons of EI of free-living MHD patients determined from food records to their measured energy needs. SETTING: Metabolic research ward. SUBJECTS: Thirteen clinically stable MHD patients with unchanging weights whose EI was assessed by dietitian interview-assisted 3-day food records. INTERVENTION: EI was compared with (1) patients' resting energy expenditure (REE), measured by indirect calorimetry, and estimated total energy expenditure (TEE) and (2) patients' dietary energy requirements (DER) measured while patients underwent nitrogen balance studies and consumed a constant energy diet in a research ward for a mean duration of 89.5 days. DER was calculated as the actual EI during the research study corrected for changes in body fat and lean body mass measured by Dual X-Ray Absorptiometry. MAIN OUTCOME MEASURE: Underreporting of EI was determined by an EI:REE ratio <1.27 and an EI:TEE ratio or EI:DEE ratio <1.0. RESULTS: Seven of the 13 MHD patients studied were male. Patient's ages were 47.7 ± standard deviation 9.7 years; body mass index averaged 25.4 ± 2.8 kg/m2, and dialysis vintage was 53.3 ± 37.1 months. The EI:REE ratio (1.03 ± 0.23) was significantly less than the cutoff value for underreporting of 1.27 (P = .001); 12 of 13 patients had EI:REE ratios <1.27. The mean EI:TEE ratio was significantly less than the cutoff value of 1.0 (0.73 ± 0.17, P < .0001), and 12 MHD patients had EI:TEE ratios <1.0. The EI:DER ratio was also <1.0 (0.83 ± 0.25, P = .012), and 10 MHD had EI:DER ratios <1.0. CONCLUSIONS: Dietitian interview-assisted diet records by MHD patients substantially underestimate the patient's dietary EI.

A lateralized odor learning model in neonatal rats for dissecting neural circuitry underpinning memory formation.

Rat pups during a critical postnatal period (≤ 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

Unlearning: NMDA receptor-mediated metaplasticity in the anterior piriform cortex following early odor preference training in rats.

Here we demonstrate metaplastic effect of a change in NMDA receptor (NMDAR) number in the anterior piriform cortex (aPC) in rat induced by a 10 min pairing of peppermint odor + stroking, which significantly modifies later learning and memory. Using isolated synaptoneurosomes, we found NR1 receptor downregulation 3 h after training and upregulation at 24 h. Consistent with the NR1 pattern, the NMDAR-mediated EPSP was smaller at 3 h and larger at 24 h. Subunit composition was unchanged. Whereas LTP was reduced at both times by training, LTD was facilitated only at 3 h. Behaviorally, pups, given a pairing of peppermint + stroking 3 h after an initial peppermint + stroking training, lost the normally acquired peppermint preference 24 h later. To probe the pathway specificity of this unlearning effect, pups were trained first with peppermint and then, at 3 h, given a second training with peppermint or vanillin. Pups given peppermint training at both times lost the learned peppermint preference. Pups given vanillin retraining at 3 h had normal peppermint preference. Downregulating NR1 with siRNA prevented odor preference learning. Finally, the NMDAR antagonist MK-801 blocked the LTD facilitation seen 3 h after training, and giving MK-801 before the second peppermint training trial eliminated the loss of peppermint odor preference. A training-associated reduction in NMDARs facilitates LTD 3 h later; training at the time of LTD facilitation reverses an LTP-dependent odor preference. Experience-dependent, pathway-specific metaplastic effects in a cortical structure have broad implications for the optimal spacing of learning experiences.

A role for the anterior piriform cortex in early odor preference learning: evidence for multiple olfactory learning structures in the rat pup.

cFos activation in the anterior piriform cortex (aPC) occurs in early odor preference learning in rat pups (Roth and Sullivan 2005). Here we provide evidence that the pairing of odor as a conditioned stimulus and ß-adrenergic activation in the aPC as an unconditioned stimulus generates early odor preference learning. ß-Adrenergic blockade in the aPC prevents normal preference learning. Enhancement of aPC cAMP response element-binding protein (CREB) phosphorylation in trained hemispheres is consistent with a role for this cascade in early odor preference learning in the aPC. In vitro experiments suggested theta-burst-mediated long-term potentiation (LTP) at the lateral olfactory tract (LOT) to aPC synapse depends on N-methyl-D-aspartate (NMDA) receptors and can be significantly enhanced by ß-adrenoceptor activation, which causes increased glutamate release from LOT synapses during LTP induction. NMDA receptors in aPC are also shown to be critical for the acquisition, but not expression, of odor preference learning, as would be predicted if they mediate initial ß-adrenoceptor-promoted aPC plasticity. Ex vivo experiments 3 and 24 h after odor preference training reveal an enhanced LOT-aPC field excitatory postsynaptic potential (EPSP). At 3 h both presynaptic and postsynaptic potentiations support EPSP enhancement while at 24 h only postsynaptic potentiation is seen. LOT-LTP in aPC is excluded by odor preference training. Taken together with earlier work on the role of the olfactory bulb in early odor preference learning, these outcomes suggest early odor preference learning is normally supported by and requires multiple plastic changes at least at two levels of olfactory circuitry.