High-mobility group protein B1 (HMGB1) has been implicated in numerous tumour types where expression regulates tumour cell growth and survival. We hypothesised that high HMGB1 expression in ovarian tumours would predict poor patient survival. Using tissue microarrays of primary ovarian cancers combined with a comprehensive database of clinicopathological variables, the expression of HMGB1 was assessed by immunohistochemistry in two independent cohorts (n=194 and n=360) using a monoclonal antibody specific for HMGB1. Kaplan-Meier analysis showed an association of HMGB1 expression with progression free survival in the primary cohort (p=0.023). In the validation cohort, expression was associated with overall survival (p=0.002). Low expression of HMGB1 was protective and in a multivariate model HMGB1 expression was shown to be an independent predictor of poor survival in ovarian cancer (p=0.006). The role of HMGB1 in cancer is complex. As high levels of HMGB1 expression are likely to render ovarian cancer cells resistant to chemotherapy, therapies targeting the HMGB1 axis may be appropriate in the treatment of ovarian cancer patients.

Cholinergic interneurons (CINs) provide the main source of acetylcholine to all striatal regions, and strongly modulate dopaminergic actions through complex regulation of pre- and post-synaptic acetylcholine receptors. Although striatal CINs have a well-defined electrophysiological profile, their biochemical properties are poorly understood, likely due to their low proportion within the striatum (2-3%). We report a strong and sustained phosphorylation of ribosomal protein S6 on its serine 240 and 244 residues (p-Ser²⁴⁰⁻²⁴⁴-S6rp), a protein integrant of the ribosomal machinery related to the mammalian target of the rapamycin complex 1 (mTORC1) pathway, which we found to be principally expressed in striatal CINs in basal conditions. We explored the functional relevance of this cellular event by pharmacologically inducing various sustained physiological activity states in CINs and assessing the effect on the levels of S6rp phosphorylation. Cell-attached electrophysiological recordings from CINs in a striatal slice preparation showed an inhibitory effect of tetrodotoxin (TTX) on action potential firing paralleled by a decrease in the p-Ser²⁴⁰⁻²⁴⁴-S6rp signal as detected by immunofluorescence after prolonged incubation. On the other hand, elevation in extracellular potassium concentration and the addition of apamin generated an increased firing rate and a burst-firing activity in CINs, respectively, and both stimulatory conditions significantly increased Ser²⁴⁰⁻²⁴⁴-S6rp phosphorylation above basal levels when incubated for one hour. Apamin generated a particularly large increase in phosphorylation that was sensitive to rapamycin. Taken together, our results demonstrate for the first time a link between the state of neuronal activity and a biochemical signaling event in striatal CINs, and suggest that immunofluorescence can be used to estimate the cellular activity of CINs under different pharmacological and/or behavioral conditions.

In mammalian species, the capacity for goal-directed action relies on a process of cognitive-emotional integration, which melds the causal and incentive learning processes that link action-goal associations with the current value of the goal [1]. Recent evidence suggests that such integration depends on a cortical-limbic-striatal circuit centered on the posterior dorsomedial striatum (pDMS) [2]. Learning-related plasticity has been described at both classes of principal neuron in the pDMS, the direct (dSPNs) and indirect (iSPNs) pathway spiny projection neurons [3-5], and is thought to depend on inputs from prelimbic cortex (PL) [6] and the basolateral amygdala (BLA) [7]. Nevertheless, the relative contribution of these structures to the cellular changes associated with goal-directed learning has not been assessed, nor is it known whether any plasticity specific to the PL and BLA inputs to the pDMS is localized to dSPNs, iSPNs, or both cell types. Here, by combining instrumental conditioning with circuit-specific manipulations and ex vivo optogenetics in mice, we discovered that the PL and not the BLA input to pDMS is pivotal for goal-directed learning and that plasticity in the PL-pDMS pathway was bilateral and specific to dSPNs in the pDMS. Subsequent experiments revealed the BLA is critically but indirectly involved in striatal plasticity via its input to the PL; inactivation of the BLA projection to PL blocked goal-directed learning and prevented learning-related plasticity at dSPNs in pDMS.

Pavlovian conditioning enables predictive stimuli to control action performance and action selection. The present experiments used sensory-specific satiety to examine the role of outcome value in these two forms of control. Experiment 1 employed a general Pavlovian-instrumental transfer design to show that a stimulus predicting a food outcome energizes the performance of an instrumental action earning another food outcome. This energizing effect was removed when the stimulus-predicted outcome or a novel outcome was devalued by sensory-specific satiety. Experiments 2 and 3 employed a specific Pavlovian-instrumental transfer design to demonstrate that a stimulus predicting a particular food outcome promotes the selection of an instrumental action earning the same, but not a different, food outcome. Remarkably, this effect was maintained when all or just one of the stimulus-predicted outcomes were devalued by sensory-specific satiety. These results indicate that satiety alone removes the expression of general PIT. By contrast, satiety or outcome-specific devaluation does not regulate the expression of specific PIT, which is insensitive to changes in outcome value. This dissociation is consistent with the view that general and specific PIT are two separate phenomena driven by distinct psychological mechanisms.

Evidence suggests that, in Pavlovian conditioning, associations form between conditioned stimuli and multiple components of the unconditioned stimulus (US). It is common, for example, to regard USs as composed of sensory and affective components, the latter being either appetitive (e.g., food or water) or aversive (e.g., shock or illness) and, therefore, to suppose different USs of the same affective class activate a common affective system. Furthermore, evidence is growing for the suggestion that, in competitive learning situations, competition between predictive stimuli is primarily for association with the affective system activated by the US. Thus, a conditioned stimulus (CS) previously paired with one US will block conditioning to another CS when both are presented together and paired with a different US of the same affective class, a phenomenon called transreinforcer blocking. Importantly, similar effects have been reported when steps are taken to turn the pretrained CS into a conditioned inhibitor, which activates the opposing affective state to the excitor that it inhibits. Thus, an appetitive inhibitor can block conditioning to a second CS when they are presented together and paired with foot shock. Here we show that the same is true of an aversive inhibitor. In two experiments conducted in rats, we found evidence that an aversive inhibitor blocked conditioning to a second CS when presented in a compound and paired with food. Such findings demonstrate that affective processes and their opponency organize appetitive-aversive interactions and establish the valences on which they are based, consistent with incentive theories of Pavlovian conditioning.

Multidisciplinary evidence suggests that the control of voluntary action arbitrates between two major forms of behavioral processing: cognitively guided (or goal directed) and autonomously guided (or habitual). Brain-state irregularities affecting the striatum-such as aging-commonly shift control toward the latter, although the responsible neural mechanisms remain unknown. Combining instrumental conditioning with cell-specific mapping and chemogenetics in striatal neurons, we explored strategies that invigorate goal-directed capacity in aged mice. We found that, under conditions favoring goal-directed control, aged animals resiliently expressed autonomously guided behavior, a response that was underpinned by a characteristic one-to-one functional engagement of the two main neuronal populations in the striatum-D1- and D2-dopamine receptor-expressing spiny projection neurons (SPNs). Chemogenetically induced desensitization of D2-SPN signaling in aged transgenic mice recapitulated the striatal plasticity state observed in young mice, an effect that shifted behavior toward vigorous, goal-directed action. Our findings contribute to the understanding of the neural bases of behavioral control and propose neural system interventions that enhance cognitive functioning in habit-prone brains.

Accumulation of the protein tau characterises Alzheimer's disease and other tauopathies, including familial forms of frontotemporal dementia (FTD) that carry pathogenic tau mutations. Another hallmark feature of these diseases is the accumulation of dysfunctional mitochondria. Although disease-associated tau is known to impair several aspects of mitochondrial function, it is still unclear whether it also directly impinges on mitochondrial quality control, specifically Parkin-dependent mitophagy. Using the mito-QC mitophagy reporter, we found that both human wild-type (hTau) and FTD mutant tau (hP301L) inhibited mitophagy in neuroblastoma cells, by reducing mitochondrial translocation of Parkin. In the Caenorhabditis elegans nervous system, hTau expression reduced mitophagy, whereas hP301L expression completely inhibited it. These effects were not due to changes in the mitochondrial membrane potential or the cytoskeleton, as tau specifically impaired Parkin recruitment to defective mitochondria by sequestering it in the cytosol. This sequestration was mediated by aberrant interactions of Parkin with the projection domain of tau. As mitochondria are dysfunctional in neurodegenerative conditions, these data suggest a vicious cycle, with tau also inhibiting the degradation of damaged mitochondria.

The present experiments examined whether extinction of a stimulus predicting food affects the ability of that stimulus to energize instrumental performance to obtain food. We first used a general Pavlovian instrumental transfer (PIT) paradigm in which rats were first given Pavlovian conditioning with a stimulus predicting one type of food outcome and were then trained to lever press for a different food outcome. We found that the Pavlovian stimulus enhanced performance of the lever press response and that this enhancement was preserved after extinction of that stimulus (Experiment 1) even when the context was manipulated to favor the expression of extinction (Experiment 2). Next, we assessed whether extinction influenced the excitatory effect of a stimulus when it was trained as a discriminative stimulus. Extinction of this stimulus alone had no effect on its ability to control instrumental performance; however, when extinguished with its associated lever press response, discriminative control was lost (Experiments 3 and 4). Finally, after instrumental and Pavlovian training, we extinguished a Pavlovian stimulus predicting one food outcome with a lever press response that delivered a different outcome. In a general PIT test, we found this extinction abolished the ability of the Pavlovian stimulus to elevate responding on a lever trained with a different outcome, revealing for the first time that extinction can abolish the general PIT effect. We conclude that extinction can produce an inhibitory association between the stimulus and the general response type, whether Pavlovian or instrumental, performed during the extinction training.

Reversal learning is thought to involve an extinction-like process that inhibits the expression of the initial learning. However, behavioral evidence for this inhibition remains difficult to interpret as various procedures have been employed to study reversal learning. Here, we used a discrimination task in rats to examine whether the inhibition produced by reversal learning is as sensitive to the passage of time as the inhibition produced by extinction. Experiment 1 showed that when tested immediately after reversal training, rats were able to use the reversed contingencies to solve the discrimination task in an outcome-specific manner. This ability to use outcome-specific information was lost when a delay was inserted between reversal training and test. However, interpretation of these data was made difficult by a potential floor effect. This concern was addressed in Experiment 2 in which it was confirmed that the passage of time impaired the ability of the rats to use the reversed contingencies in an outcome-specific manner to solve the task. Further, it revealed that the delay between initial learning and test was not responsible for this impairment. Additional work demonstrated that solving the discrimination task was unaffected by Pavlovian extinction but that the discriminative stimuli were able to block conditioning to a novel stimulus, suggesting that Pavlovian processes were likely to contribute to solving the discrimination. We therefore concluded that the expression of reversal and extinction learning do share the same sensitivity to the effect of time. However, this sensitivity was most obvious when we assessed outcome-specific information following reversal learning. This suggests that the processes involved in reversal learning are somehow distinct from those underlying extinction learning, as the latter has usually been found to leave outcome-specific information relatively intact. Thus, the present study reveals that a better understanding of the mechanisms supporting reversal training requires assessing the impact that this training exerts on the content of learning rather than performance per se.

Precise identification of neuronal populations is a major challenge in neuroscience. In the striatum, more than 95% of neurons are GABAergic medium-sized spiny neurons (MSNs), which form two intermingled populations distinguished by their projections and protein content. Those expressing dopamine D(1)-receptors (D1Rs) project preferentially to the substantia nigra pars reticulata (SNr), whereas those expressing dopamine D(2)- receptors (D2Rs) project preferentially to the lateral part of the globus pallidus (LGP). The degree of segregation of these populations has been a continuous subject of debate, and the recent introduction of bacterial artificial chromosome (BAC) transgenic mice expressing fluorescent proteins driven by specific promoters was a major progress to facilitate striatal neuron identification. However, the fraction of MSNs labeled in these mice has been recently called into question, casting doubt on the generality of results obtained with such approaches. Here, we performed an in-depth quantitative analysis of striatal neurons in drd1a-EGFP and drd2-EGFP mice. We first quantified neuronal and non-neuronal populations in the striatum, based on nuclear staining with TO-PRO-3, and immunolabeling for NeuN, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein Mr approximately 32,000), and various markers for interneurons. TO-PRO-3 staining was sufficient to identify MSNs by their typical nuclear morphology and, with a good probability, interneuron populations. In drd1a-EGFP/drd2-EGFP double transgenic mice all MSNs expressed EGFP, which was driven in about half of them by drd1a promoter. Retrograde labeling showed that all MSNs projecting to the SNr expressed D1R and very few D2R (<1%). In contrast, our results were compatible with the existence of some D1R-EGFP-expressing fibers giving off terminals in the LGP. Thus, our study shows that nuclear staining is a simple method for identifying MSNs and other striatal neurons. It also unambiguously confirms the degree of segregation of MSNs in the mouse striatum and allows the full exploitation of results obtained with BAC-transgenic mice.

The basolateral amygdala (BLA) complex receives dense cholinergic projections from the nucleus basalis of Meynert (NBM) and the horizontal limb of the diagonal band of Broca (HDB). The present experiments examined whether these projections regulate the formation, extinction, and renewal of fear memories. This was achieved by employing a Pavlovian fear conditioning protocol and optogenetics in transgenic rats. Silencing NBM projections during fear conditioning weakened the fear memory produced by that conditioning and abolished its renewal after extinction. By contrast, silencing HDB projections during fear conditioning had no effect. Silencing NBM or HDB projections during extinction enhanced the loss of fear produced by extinction, but only HDB silencing prevented renewal. Next, we found that systemic blockade of nicotinic acetylcholine receptors during fear conditioning mimicked the effects produced by silencing NBM projections during fear conditioning. However, this blockade had no effect when given during extinction. These findings indicate that basal forebrain cholinergic signaling in the BLA plays a critical role in fear regulation by promoting strength and durability of fear memories. We concluded that cholinergic compounds may improve treatments for post-traumatic stress disorder by durably stripping fear memories from their fear-eliciting capacity.

Psychostimulants such as amphetamine (AMPH) target dopamine (DA) neuron synapses to engender drug-induced plasticity. While DA neurons modulate the activity of striatal (Str) cholinergic interneurons (ChIs) with regional heterogeneity, how AMPH affects ChI activity has not been elucidated. Here, we applied quantitative fluorescence imaging approaches to map the dose-dependent effects of a single dose of AMPH on ChI activity at 2.5 and 24 h after injection across the mouse Str using the activity-dependent marker phosphorylated ribosomal protein S6 (p-rpS6240/244). AMPH did not affect the distribution or morphology of ChIs in any Str subregion. While AMPH at either dose had no effect on ChI activity after 2.5 h, ChI activity was dose dependently reduced after 24 h specifically in the ventral Str/nucleus accumbens (NAc), a critical site of psychostimulant action. AMPH at either dose did not affect the spontaneous firing of ChIs. Altogether this work demonstrates that a single dose of AMPH has delayed regionally heterogeneous effects on ChI activity, which most likely involves extra-Str synaptic input.

Over 30% of ERα breast cancer patients develop relapses and progress to metastatic disease despite treatment with endocrine therapies. The pioneer factor PBX1 translates epigenetic cues and mediates estrogen induced ERα binding. Here we demonstrate that PBX1 plays a central role in regulating the ERα transcriptional response to epidermal growth factor (EGF) signaling. PBX1 regulates a subset of EGF-ERα genes highly expressed in aggressive breast tumours. Retrospective stratification of luminal patients using PBX1 protein levels in primary cancer further demonstrates that elevated PBX1 protein levels correlate with earlier metastatic progression. In agreement, PBX1 protein levels are significantly upregulated during metastatic progression in ERα-positive breast cancer patients. Finally we reveal that PBX1 upregulation in aggressive tumours is partly mediated by genomic amplification of the PBX1 locus. Correspondingly, ERα-positive breast cancer patients carrying PBX1 amplification are characterized by poor survival. Notably, we demonstrate that PBX1 amplification can be identified in tumor derived-circulating free DNA of ERα-positive metastatic patients. Metastatic patients with PBX1 amplification are also characterized by shorter relapse-free survival. Our data identifies PBX1 amplification as a functional hallmark of aggressive ERα-positive breast cancers. Mechanistically, PBX1 amplification impinges on several critical pathways associated with aggressive ERα-positive breast cancer.

Information processing in the striatum requires the postsynaptic integration of glutamatergic and dopaminergic signals, which are then relayed to the output nuclei of the basal ganglia to influence behavior. Although cellularly homogeneous in appearance, the striatum contains several rare interneuron populations which tightly modulate striatal function. Of these, cholinergic interneurons (CINs) have been recently shown to play a critical role in the control of reward-related learning; however how the striatal cholinergic network is functionally organized at the mesoscopic level and the way this organization influences striatal function remains poorly understood. Here, we systematically mapped and digitally reconstructed the entire ensemble of CINs in the mouse striatum and quantitatively assessed differences in densities, spatial arrangement and neuropil content across striatal functional territories. This approach demonstrated that the rostral portion of the striatum contained a higher concentration of CINs than the caudal striatum and that the cholinergic content in the core of the ventral striatum was significantly lower than in the rest of the regions. Additionally, statistical comparison of spatial point patterns in the striatal cholinergic ensemble revealed that only a minor portion of CINs (17%) aggregated into cluster and that they were predominantly organized in a random fashion. Furthermore, we used a fluorescence reporter to estimate the activity of over two thousand CINs in naïve mice and found that there was a decreasing gradient of CIN overall function along the dorsomedial-to-ventrolateral axis, which appeared to be independent of their propensity to aggregate within the striatum. Altogether this work suggests that the regulation of striatal function by acetylcholine across the striatum is highly heterogeneous, and that signals originating in external afferent systems may be principally determining the function of CINs in the striatum.

The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain's lipid landscape remain largely unexplored. The levels of saturated FFAs, particularly of myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the PLA1 isoform DDHD2 in mice dramatically reduces saturated FFA responses to memory acquisition across the brain. Furthermore, DDHD2 loss also decreases memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits that mirror human spastic paraplegia. Via pulldown-mass spectrometry analyses, we find that DDHD2 binds to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1+/- mouse model of human early infantile encephalopathy associated with intellectual disability and motor dysfunction, we show that STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated FFAs in the brain. These findings suggest key roles for DDHD2 and STXBP1 in lipid metabolism and in the processes of synaptic plasticity, learning, and memory.

Streamlined action sequences must remain flexible should stable contingencies in the environment change. By combining analyses of behavioral structure with a circuit-specific manipulation in mice, we report on a relationship between action timing variability and successful adaptation that relates to post-synaptic targets of primary motor cortical (M1) projections to dorsolateral striatum (DLS). In a two-lever instrumental task, mice formed successful action sequences by, first, establishing action scaffolds and, second, smoothly extending action duration to adapt to increased task requirements. Interruption of DLS neurons in M1 projection territories altered this process, evoking higher-rate actions that were more stereotyped in their timing, reducing opportunities for success. Based on evidence from neuronal tracing experiments, we propose that DLS neurons in M1 projection territories supply action timing variability to facilitate adaptation, a function that may involve additional downstream subcortical processing relating to collateralization of descending motor pathways to multiple basal ganglia centers.

The acquisition of motor skills involves implementing action sequences that increase task efficiency while reducing cognitive loads. This learning capacity depends on specific cortico-basal ganglia circuits that are affected by normal ageing. Here, combining a series of novel behavioural tasks with extensive neuronal mapping and targeted cell manipulations in mice, we explored how ageing of cortico-basal ganglia networks alters the microstructure of action throughout sequence learning. We found that, after extended training, aged mice produced shorter actions and displayed squeezed automatic behaviours characterised by ultrafast oligomeric action chunks that correlated with deficient reorganisation of corticostriatal activity. Chemogenetic disruption of a striatal subcircuit in young mice reproduced age-related within-sequence features, and the introduction of an action-related feedback cue temporarily restored normal sequence structure in aged mice. Our results reveal static properties of aged cortico-basal ganglia networks that introduce temporal limits to action automaticity, something that can compromise procedural learning in ageing.

Contemporary theories emphasize the involvement of the endogenous opioid system in assigning hedonic values to rewards. Although earlier research supports this view, recent findings suggest that opioids play a larger and more complex role in reward processes than these theories suggest. For example, opioid activity in the basolateral amygdala is required for encoding incentive learning, a process by which the value of goal-directed actions is updated. Outside the amygdala, opioid receptors in the ventral striatum have been found to promote choice between different courses of action. Specifically, μ opioid receptors in the nucleus accumbens core and δ opioid receptors in the nucleus accumbens shell have been reported to mediate distinct aspects of incentive motivation; the core regulating the effect of experienced reward and the shell of predicted reward on choice. In both cases, the involvement of opioid receptors was restricted to the time of choice, although changes in their expression pattern could be observed prior to that point. This time-restricted involvement of opioid receptor-related processes is consistent with the view that opioids in the nucleus accumbens are central components of the limbic-motor interface, integrating reward-related information with instrumental learning to guide decision-making, particularly the selection and execution of goal-directed actions.

Evidence indicates that the infralimbic cortex (IL) encodes and retrieves the inhibitory memory produced by fear extinction. Recently, we have shown that the IL is also involved in the inhibitory memory generated by stimulus pre-exposure that causes latent inhibition. These results are surprising because a stimulus undergoing fear extinction carries aversive motivational value, whereas a pre-exposed stimulus is neutral. The present experiments tested the hypothesis that the IL encodes inhibition irrespective of the motivational information about the stimulus. Using rats, we first confirmed that IL activity during stimulus pre-exposure is required for latent inhibition. Then, we found that pharmacological stimulation of the IL facilitated aversive extinction to a stimulus that had been trained and extinguished as an appetitive stimulus. This facilitation was stimulus specific and required appetitive extinction. The same facilitation was found when appetitive extinction was replaced with random presentations of the stimulus and an appetitive outcome. Together, these findings indicate that non-reinforced stimulus presentations establish an inhibitory memory that is reactivated and strengthened in the IL during subsequent aversive extinction. This is consistent with the view that the IL encodes inhibition irrespective of motivational value, suggesting that this brain region plays a general role in inhibitory learning.

Predictive learning exerts a powerful influence over choice between instrumental actions. Nevertheless, how this learning is encoded in a sufficiently stable manner to influence choices that can occur much later in time is unclear. Here, we report that the basolateral amygdala (BLA) encodes predictive learning and establishes the memory necessary for future choices by driving the accumulation of delta-opioid receptors (DOPRs) on the somatic membrane of cholinergic interneurons in the nucleus accumbens shell (NAc-S). We found that the BLA controls DOPR accumulation via its influence on substance P release in the NAc-S, and that although DOPR accumulation is not necessary for predictive learning per se, it is necessary for the influence of this learning on later choice between actions. This study uncovers, therefore, a novel GPCR-based form of memory that is established by predictive learning and is necessary for such learning to guide the selection and execution of specific actions.