Corticosterone impairs flexible adjustment of spatial navigation in an associative place-reward learning task.

Cognitive challenges are often accompanied by a discharge of stress hormones, which in turn modulate multiple brain areas. Among these, the medial temporal lobe and the prefrontal cortex are critically involved in high-order cognitive functions such as learning, memory, and decision-making. Previous studies assessing the effects of corticosterone on spatial memory found an increase or a decrease in performance depending on the timing of stress hormone discharge relative to the behavioral task. Most of these studies, however, made use of aversively motivated behaviors, whereas less is known about corticosteroid effects on flexible learning during reward-driven spatial navigation. To study how corticosterone modulates flexible spatial learning, we tested rats on a place-reward association task where hormone treatment was administered immediately after a session presenting a change in reward locations. The corticosterone-treated group showed delayed learning during the initial sessions and suboptimal memory consolidation throughout testing. Repeated training on the novel reward positions improved performance and eliminated differences from the control group. We conclude that a marked increase in plasma corticosterone levels immediately after training impairs the flexible formation of new place-reward associations.

Reward Expectancy Strengthens CA1 Theta and Beta Band Synchronization and Hippocampal-Ventral Striatal Coupling.

The use of information from the hippocampal memory system in motivated behavior depends on its communication with the ventral striatum. When an animal encounters cues that signal subsequent reward, its reward expectancy is raised. It is unknown, however, how this process affects hippocampal dynamics and their influence on target structures, such as ventral striatum. We show that, in rats, reward-predictive cues result in enhanced hippocampal theta and beta band rhythmic activity during subsequent action, compared with uncued goal-directed navigation. The beta band component, also labeled theta's harmonic, involves selective hippocampal CA1 cell groups showing frequency doubling of firing periodicity relative to theta rhythmicity and it partitions the theta cycle into segments showing clear versus poor spike timing organization. We found that theta phase precession occurred over a wider range than previously reported. This was apparent from spikes emitted near the peak of the theta cycle exhibiting large "phase precessing jumps" relative to spikes in foregoing cycles. Neither this phenomenon nor the regular manifestation of theta phase precession was affected by reward expectancy. Ventral striatal neuronal firing phase-locked not only to hippocampal theta, but also to beta band activity. Both hippocampus and ventral striatum showed increased synchronization between neuronal firing and local field potential activity during cued compared with uncued goal approaches. These results suggest that cue-triggered reward expectancy intensifies hippocampal output to target structures, such as the ventral striatum, by which the hippocampus may gain prioritized access to systems modulating motivated behaviors. SIGNIFICANCE STATEMENT: Here we show that temporally discrete cues raising reward expectancy enhance both theta and beta band activity in the hippocampus once goal-directed navigation has been initiated. These rhythmic activities are associated with increased synchronization of neuronal firing patterns in the hippocampus and the connected ventral striatum. When transmitted to downstream target structures, this expectancy-related state of intensified processing in the hippocampus may modulate goal-directed action.

Spike-Based Functional Connectivity in Cerebral Cortex and Hippocampus: Loss of Global Connectivity Is Coupled to Preservation of Local Connectivity During Non-REM Sleep.

UNLABELLED: Behavioral states are commonly considered global phenomena with homogeneous neural determinants. However, recent studies indicate that behavioral states modulate spiking activity with neuron-level specificity as a function of brain area, neuronal subtype, and preceding history. Although functional connectivity also strongly depends on behavioral state at a mesoscopic level and is globally weaker in non-REM (NREM) sleep and anesthesia than wakefulness, it is unknown how neuronal communication is modulated at the cellular level. We hypothesize that, as for neuronal activity, the influence of behavioral states on neuronal coupling strongly depends on type, location, and preceding history of involved neurons. Here, we applied nonlinear, information-theoretical measures of functional connectivity to ensemble recordings with single-cell resolution to quantify neuronal communication in the neocortex and hippocampus of rats during wakefulness and sleep. Although functional connectivity (measured in terms of coordination between firing rate fluctuations) was globally stronger in wakefulness than in NREM sleep (with distinct traits for cortical and hippocampal areas), the drop observed during NREM sleep was mainly determined by a loss of inter-areal connectivity between excitatory neurons. Conversely, local (intra-area) connectivity and long-range (inter-areal) coupling between interneurons were preserved during NREM sleep. Furthermore, neuronal networks that were either modulated or not by a behavioral task remained segregated during quiet wakefulness and NREM sleep. These results show that the drop in functional connectivity during wake-sleep transitions globally holds true at the cellular level, but confine this change mainly to long-range coupling between excitatory neurons. SIGNIFICANCE STATEMENT: Studies performed at a mesoscopic level of analysis have shown that communication between cortical areas is disrupted in non-REM sleep and anesthesia. However, the neuronal determinants of this phenomenon are not known. Here, we applied nonlinear, information-theoretical measures of functional coupling to multi-area tetrode recordings from freely moving rats to investigate whether and how brain state modulates coordination between individual neurons. We found that the previously observed drop in functional connectivity during non-REM (NREM) sleep can be explained by a decrease in coupling between excitatory neurons located in distinct brain areas. Conversely, intra-area communication and coupling between interneurons are preserved. Our results provide significant new insights into the neuron-level mechanisms responsible for the loss of consciousness occurring in NREM sleep.

Effects of Arc/Arg3.1 gene deletion on rhythmic synchronization of hippocampal CA1 neurons during locomotor activity and sleep.

The activity-regulated cytoskeletal-associated protein/activity regulated gene (Arc/Arg3.1) is crucial for long-term synaptic plasticity and memory formation. However, the neurophysiological substrates of memory deficits occurring in the absence of Arc/Arg3.1 are unknown. We compared hippocampal CA1 single-unit and local field potential (LFP) activity in Arc/Arg3.1 knockout and wild-type mice during track running and flanking sleep periods. Locomotor activity, basic firing and spatial coding properties of CA1 cells in knockout mice were not different from wild-type mice. During active behavior, however, knockout animals showed a significantly shifted balance in LFP power, with a relative loss in high-frequency (beta-2 and gamma) bands compared to low-frequency bands. Moreover, during track-running, knockout mice showed a decrease in phase locking of spiking activity to LFP oscillations in theta, beta and gamma bands. Sleep architecture in knockout mice was not grossly abnormal. Sharp-wave ripples, which have been associated with memory consolidation and replay, showed only minor differences in dynamics and amplitude. Altogether, these findings suggest that Arc/Arg3.1 effects on memory formation are not only manifested at the level of molecular pathways regulating synaptic plasticity, but also at the systems level. The disrupted power balance in theta, beta and gamma rhythmicity and concomitant loss of spike-field phase locking may affect memory encoding during initial storage and memory consolidation stages.

Offline orbitofrontal cortex reactivation depends on recency of place-reward changes and coheres with hippocampal replay.

The orbitofrontal cortex, one of the key neocortical areas in valuation and emotion, is critical for cognitive flexibility but its role in the consolidation of recently acquired information remains unclear. Here, we demonstrate orbitofrontal offline replay in the context of a place-reward association task on a maze with varying goal locations. When switches in place-reward coupling were applied, replay was enhanced relative to sessions with stable contingencies. Moreover, replay strength was positively correlated with the subsequent overnight change in behavioral performance. Interrogating relationships between orbitofrontal and hippocampal activity, we found that orbitofrontal and hippocampal replay could occur independently but became coordinated during a type of cortical state with strong spiking activity. These findings reveal a structured form of offline orbitofrontal ensemble activity that is correlated with cognitive flexibility required to adapt to changing task contingencies, and becomes associated with hippocampal replay only during a specific state of high cortical excitability.

Reward cues in space: commonalities and differences in neural coding by hippocampal and ventral striatal ensembles.

Forming place-reward associations critically depends on the integrity of the hippocampal-ventral striatal system. The ventral striatum (VS) receives a strong hippocampal input conveying spatial-contextual information, but it is unclear how this structure integrates this information to invigorate reward-directed behavior. Neuronal ensembles in rat hippocampus (HC) and VS were simultaneously recorded during a conditioning task in which navigation depended on path integration. In contrast to HC, ventral striatal neurons showed low spatial selectivity, but rather coded behavioral task phases toward reaching goal sites. Outcome-predicting cues induced a remapping of firing patterns in the HC, consistent with its role in episodic memory. VS remapped in conjunction with the HC, indicating that remapping can take place in multiple brain regions engaged in the same task. Subsets of ventral striatal neurons showed a "flip" from high activity when cue lights were illuminated to low activity in intertrial intervals, or vice versa. The cues induced an increase in spatial information transmission and sparsity in both structures. These effects were paralleled by an enhanced temporal specificity of ensemble coding and a more accurate reconstruction of the animal's position from population firing patterns. Altogether, the results reveal strong differences in spatial processing between hippocampal area CA1 and VS, but indicate similarities in how discrete cues impact on this processing.

A new mathematical pharmacodynamic model of clonogenic cancer cell death by doxorubicin.

Previous models for predicting tumor cell growth are mostly based on measurements of total cell numbers. The purpose of this paper is to provide a new simple mathematical model for calculating tumor cell growth focusing on the fraction of cells that is clonogenic. The non-clonogenic cells are considered to be relatively harmless. We performed a number of different types of experiments: a long-term drug "treatment", several concentrations/fixed time experiments and time-series experiments, in which human MCF-7 breast cancer cells were exposed to doxorubicin and the total number of cells were counted. In the latter two types, at every measurement point a plating efficiency experiment was started. The final number of colonies formed is equal to the number of clonogenic cells at the onset of the experiment. Based on the intracellular drug concentration, our model predicts cell culture effects taking clonogenic ability and growth inhibition by neighboring cells into account. The model fitted well to the experimental data. The estimated damage parameter which represents the chance of an MCF-7 cell to become non-clonogenic per unit time and per unit intracellular doxorubicin concentration was found to be 0.0025 ± 0.0008 (mean ± SD) nM(-1) h(-1). The model could be used to calculate the effect of every doxorubicin concentration versus time (C-t) profile. Although in vivo parameters may well be different from those found in vitro, the model can be used to predict trends, e.g. by comparing effects of different in vivo C-t profiles.

Paper-based analytical device for electrochemical flow-injection analysis of glucose in urine.

This article describes a new design for a paper-based electrochemical system for flow-injection analysis. Capillary wicking facilitates a gravity-driven flow of buffer solution continuously through paper and nitrocellulose, from a buffer reservoir at one end of the device to a sink at the other. A difference in height between the reservoir and the sink leads to a continuous and constant flow. The nitrocellulose lies horizontally on a working electrode, which consists of a thin platinum layer deposited on a solid support. The counter and reference electrodes are strategically positioned upstream in the buffer reservoir. A simple pipetting device was developed for reliable application of (sub)microliter volumes of sample without the need of commercial micropipets; this device did not damage the nitrocellulose membrane. Demonstration of the system for the determination of the concentration of glucose in urine resulted in a noninvasive, quantitative assay that could be used for diagnosis and monitoring of diabetes. This method does not require disposable test strips, with enzyme and electrodes, that are thrown away after each measurement. Because of its low cost, this system could be used in medical environments that are resource-limited.

Hippocampus leads ventral striatum in replay of place-reward information.

Associating spatial locations with rewards is fundamental to survival in natural environments and requires the integrity of the hippocampus and ventral striatum. In joint multineuron recordings from these areas, hippocampal-striatal ensembles reactivated together during sleep. This process was especially strong in pairs in which the hippocampal cell processed spatial information and ventral striatal firing correlated to reward. Replay was dominated by cell pairs in which the hippocampal "place" cell fired preferentially before the striatal reward-related neuron. Our results suggest a plausible mechanism for consolidating place-reward associations and are consistent with a central tenet of consolidation theory, showing that the hippocampus leads reactivation in a projection area.

Theta-band phase locking of orbitofrontal neurons during reward expectancy.

The expectancy of a rewarding outcome following actions and cues is coded by a network of brain structures including the orbitofrontal cortex. Thus far, predicted reward was considered to be coded by time-averaged spike rates of neurons. However, besides firing rate, the precise timing of action potentials in relation to ongoing oscillations in local field potentials is thought to be of importance for effective communication between brain areas. We performed multineuron and field potential recordings in orbitofrontal cortex of rats performing olfactory discrimination learning to study the temporal structure of coding predictive of outcome. After associative learning, field potentials were marked by theta oscillations, both in advance and during delivery of reward. Orbitofrontal neurons, especially those coding information about upcoming reward with their firing rate, phase locked to these oscillations in anticipation of reward. When established associations were reversed, phase locking collapsed in the anticipatory task phase, but returned when reward became predictable again after relearning. Behaviorally, the outcome anticipation phase was marked by licking responses, but the frequency of lick responses was dissociated from the strength of theta-band phase locking. The strength of theta-band phase locking by orbitofrontal neurons robustly follows the dynamics of associative learning as measured by behavior and correlates with the rat's current outcome expectancy. Theta-band phase locking may facilitate communication of outcome-related information between reward-related brain areas and offers a novel mechanism for coding value signals during reinforcement learning.

Learning-associated gamma-band phase-locking of action-outcome selective neurons in orbitofrontal cortex.

Gamma oscillations (30-100 Hz) correlate to a variety of neural functions, including sensory processing, attention, and action selection. However, they have barely been studied in relation to emotional processing and valuation of sensory signals and actions. We conducted multineuron and local field potential recordings in the orbitofrontal cortex (OFC) of rats performing a task in which they made go or no-go decisions based on two olfactory stimuli predicting appetitive or aversive outcomes. Gamma power was strongest during the late phase of odor sampling, just before go/no-go movement, and increased with behavioral learning. Learning speed was correlated to the slope of the gamma power increment. Spikes of OFC neurons were consistently timed to the gamma rhythm during odor sampling, regardless of the associated outcome. However, only a specific subgroup of cells showed consistent phase timing. These cells showed action-outcome selective activity, not during stimulus sampling but during subsequent movement responses. During sampling, this subgroup displayed a suppression in firing rate but a concurrent increment in the consistency of spike timing relative to gamma oscillations. In addition to gamma rhythm, OFC field potentials were characterized by theta oscillations during odor sampling. Neurons phase-locked to either theta or gamma rhythms but not to both, suggesting that they become associated with separate rhythmic networks involving OFC. Altogether, these results suggest that OFC gamma-band synchronization reflects inhibitory control over a subpopulation of neurons that express information about the emotional valence of actions after a motor decision, which suggests a novel mechanism for response inhibition.

Towards using high-performance liquid chromatography at home.

In order to make high-performance liquid chromatography (HPLC) more widely available at home and in small-scale settings, we have simplified two of its most costly modules, namely the pump and the detector. This should make the setup affordable for home or small laboratory use. A manual HPLC pump was constructed so as to fit into a caulk gun from a local hardware store enabling the generation of 100-150 bar of pressure. In order to limit the pressure drop during the running of a chromatogram, a pulse dampener was developed. We further modified the electrochemical detection (ECD) system so as to use a cheap boron-doped diamond electrode with an overlay of thin filter paper, causing an eluent flow over the electrode by wicking and gravity. Both the pump and the detector are at least ten times cheaper than conventional HPLC modules. Using a home-packed JupiterⓇ Proteo reversed phase capillary column we show how this low-cost HPLC system generates well resolving chromatograms after direct injection of fresh urine. The ECD did not lose its sensitivity during regular use over more than half a year. For homovanillic acid (HVA), which is of medical interest, we measured a linear dynamic range of two orders of magnitude, a detection limit of HVA in the injected sample of 3 µM and a coefficient of variation <10%. The contribution to peak broadening by the detector was much smaller than the contributions by the injector and by the column. After consumption of table olives containing hydroxytyrosol (HT), its metabolite HVA in the corresponding urine could be measured quantitatively. An approach to quantify HT in table olives is presented, as well. This method provides a new tool for investigating physiology of oneself or of dear ones at home.

Single-cell and population coding of expected reward probability in the orbitofrontal cortex of the rat.

The orbitofrontal cortex (OFC) has been implicated in decision-making under uncertainty, but it is unknown how information about the probability or uncertainty of future reward is coded by single orbitofrontal neurons and ensembles. We recorded neuronal ensembles in rat OFC during an olfactory discrimination task in which different odor stimuli predicted different reward probabilities. Single-unit firing patterns correlated to the expected reward probability primarily within an immobile waiting period before reward delivery but also when the rat executed movements toward the reward site. During these pre-reward periods, a subset of OFC neurons was sensitive to differences in probability but only very rarely discriminated on the basis of reward uncertainty. In the reward period, neurons responded during presentation or omission of reward or during both types of outcome. At the population level, neurons were characterized by a wide divergence in firing-rate variability attributable to expected probability. A population analysis using template matching as reconstruction method indicated that OFC generates a distributed representation of reward probability with a weak dependence on neuronal group size. The analysis furthermore confirmed that predictive information coded by OFC populations was quantitatively related to reward probability, but not to uncertainty.

Reward-associated gamma oscillations in ventral striatum are regionally differentiated and modulate local firing activity.

Oscillations of local field potentials (LFPs) in the gamma range are found in many brain regions and are supposed to support the temporal organization of cognitive, perceptual, and motor functions. Even though gamma oscillations have also been observed in ventral striatum, one of the brain's most important structures for motivated behavior and reward processing, their specific function during ongoing behavior is unknown. Using a movable tetrode array, we recorded LFPs and activity of neural ensembles in the ventral striatum of rats performing a reward-collection task. Rats were running along a triangle track and in each round collected one of three different types of rewards. The gamma power of LFPs on subsets of tetrodes was modulated by reward-site visits, discriminated between reward types, between baitedness of reward locations and was different before versus after arrival at a reward site. Many single units in ventral striatum phase-locked their discharge pattern to the gamma oscillations of the LFPs. Phase-locking occurred more often in reward-related than in reward-unrelated neurons and LFPs. A substantial number of simultaneously recorded LFPs correlated poorly with each other in terms of gamma rhythmicity, indicating that the expression of gamma activity was heterogeneous and regionally differentiated. The orchestration of LFPs and single-unit activity by way of gamma rhythmicity sheds light on the functional architecture of the ventral striatum and the temporal coordination of ventral striatal activity for modulating downstream areas and regulating synaptic plasticity.

Fast-spiking interneurons of the rat ventral striatum: temporal coordination of activity with principal cells and responsiveness to reward.

Although previous in vitro studies revealed inhibitory synaptic connections of fast-spiking interneurons to principal cells in the striatum, uncertainty remains about the nature of the behavioural events that correlate with changes in interneuron activity and about the temporal coordination of interneuron firing with spiking of principal cells under natural conditions. Using in vivo tetrode recordings from the ventral striatum in freely moving rats, fast-spiking neurons were distinguished from putative medium-sized spiny neurons on the basis of their spike waveforms and rates. Cross-correlograms of fast-spiking and putative medium-sized spiny neuron firing patterns revealed a variety of temporal relationships, including peaks of concurrent firing and transient decrements in medium-sized spiny neuron spiking around fast-spiking unit activity. Notably, the onset of these decrements was mostly in advance of the fast-spiking unit firing. Many of these temporal relationships were dependent on the sleep-wake state. Coordinated activity was also found amongst pairs of the same phenotype, both fast-spiking units and putative medium-sized spiny neurons, which was often marked by a broad peak of concurrent firing. When studying fast-spiking neurons in a reward-searching task, they generally showed a pre-reward ramping increment in firing rate but a decrement specifically when the rat received reward. In conclusion, our data indicate that various forms of temporally coordinated activity exist amongst ventral striatal interneurons and principal cells, which cannot be explained by feed-forward inhibitory circuits alone. Furthermore, firing patterns of ventral striatal fast-spiking interneurons do not merely correlate with the general arousal state of the animal but display distinct reward-related changes in firing rate.

Population coding of reward magnitude in the orbitofrontal cortex of the rat.

Although single-cell coding of reward-related information in the orbitofrontal cortex (OFC) has been characterized to some extent, much less is known about the coding properties of orbitofrontal ensembles. We examined population coding of reward magnitude by performing ensemble recordings in rat OFC while animals learned an olfactory discrimination task in which various reinforcers were associated with predictive odor stimuli. Ensemble activity was found to represent information about reward magnitude during several trial phases, namely when animals moved to the reward site, anticipated reward during an immobile period, and received it. During the anticipation phase, Bayesian and template-matching reconstruction algorithms decoded reward size correctly from the population activity significantly above chance level (highest value of 43 and 48%, respectively; chance level, 33.3%), whereas decoding performance for the reward delivery phase was 76 and 79%, respectively. In the anticipation phase, the decoding score was only weakly dependent on the size of the neuronal group participating in reconstruction, consistent with a redundant, distributed representation of reward information. In contrast, decoding was specific for temporal segments within the structure of a trial. Decoding performance steeply increased across the first few trials for every rewarded odor, an effect that could not be explained by a nonspecific drift in response strength across trials. Finally, when population responses to a negative reinforcer (quinine) were compared with sucrose reinforcement, coding in the delivery phase appeared to be related to reward quality, and thus was not based on ingested liquid volume.

Preferential reactivation of motivationally relevant information in the ventral striatum.

Spontaneous "off-line" reactivation of neuronal activity patterns may contribute to the consolidation of memory traces. The ventral striatum exhibits reactivation and has been implicated in the processing of motivational information. It is unknown, however, whether reactivating neuronal ensembles specifically recapitulate information relating to rewards that were encountered during wakefulness. We demonstrate a prolonged reactivation in rat ventral striatum during quiet wakefulness and slow-wave but not rapid eye movement sleep. Reactivation of reward-related information processed in this structure was particularly prominent, and this was primarily attributable to spike trains temporally linked to reward sites. It was accounted for by small, strongly correlated subgroups in recorded cell assemblies and can thus be characterized as a sparse phenomenon. Our results indicate that reactivated memory traces may not only comprise feature- and context-specific information but also contain a value component.

A split microdrive for simultaneous multi-electrode recordings from two brain areas in awake small animals.

Complex cognitive operations such as memory formation and decision-making are thought to be mediated not by single, isolated brain structures but by multiple, connected brain areas. To facilitate studies on the neural communication between connected brain structures, we developed a multi-electrode microdrive for chronically recording ensembles of neurons in two different brain areas simultaneously. The "split drive" contains 14 independently movable microdrivers that were designed to hold tetrodes and to permit day-to-day adjustment of dorsoventral position in the brain. The limited weight of the drive allowed rats to adjust well to the headstage after recovering from surgery and permitted stable recording sessions across at least several weeks. In addition to describing the design and assembly of the split drive, we also discuss some important individual parts of microdrives used for tetrode recordings in general. Furthermore, the split drive was applied to two widely separated and connected brain structures, the hippocampus and ventral striatum. From these two areas, stable ensemble recordings were conducted in rats performing a reward-searching task on a triangular track, yielding group sizes of about 15 and 25 units in the dorsal hippocampus and ventral striatum, respectively.

Simple, rapid, and sensitive liquid chromatography-fluorescence method for the quantification of tranexamic acid in blood.

Tranexamic acid (TA) is a synthetic antifibrinolytic agent that is being considered as a candidate adjuvant drug for site-specific pharmaco-laser therapy of port wine stains. For drug utility studies, a high-performance liquid chromatography (HPLC)-fluorescence method was developed for the quantification of TA in blood. Platelet-poor plasma was prepared, size-separated using 3kDa cut-off centrifuge filters, and derivatized with naphthalene-2-3-dicarboxaldehyde (NDA) and cyanide. The excess of NDA was quenched after 2 min by adding tryptophan. The derivatives were separated on a 2.1mm C18 column using an acetate buffer/acetonitrile gradient. Excellent separation from plasma background was obtained at pH 5.5. Quantification was carried out at 440/520 nm. The limit of detection was 0.5 microM and the mean+/-SD recovery from whole blood was 81.7+/-10.9%. Derivatized TA samples were stable for at least 36 h at 4 degrees C. The method was successfully applied to a heat-induced TA release study from thermosensitive liposomes.

Epidermal growth factor receptor-induced activator protein 1 activity controls density-dependent growth inhibition in normal rat kidney fibroblasts.

Density-dependent growth inhibition secures tissue homeostasis. Dysfunction of the mechanisms, which regulate this type of growth control is a major cause of neoplasia. In confluent normal rat kidney (NRK) fibroblasts, epidermal growth factor (EGF) receptor levels decline, ultimately rendering these cells irresponsive to EGF. Using an activator protein (AP)-1 sensitive reporter construct, we show that AP-1 activity is strongly decreased in density-arrested NRK cells, but is restored after relaxation of densitydependent growth inhibition by removing neighboring cells. EGF could not induce AP-1 activity or S-phase entry in density-arrested cells, but could do so after pretreatment with retinoic acid, which enhances EGF receptor expression. Our results support a model in which the EGF receptor regulates density-dependent growth control in NRK fibroblasts, which is reflected by EGF-induced mitogenic signaling and consequent AP-1 activity.