Kei Oyama

Reward Prediction Error Coding in Dorsal Striatal Neurons

In the current theory of learning, the reward prediction error (RPE), the difference between expected and received reward, is thought to be a key factor in reward-based learning, working as a teaching signal. The activity of dopamine neurons is known to code RPE, and the release of dopamine is known to modify the strength of synaptic connectivity in the target neurons. A fundamental interest in current neuroscience concerns the origin of RPE signals in the brain. Here, we show that a group of rat striatal neurons show a clear parametric RPE coding similar to that of dopamine neurons when tested under probabilistic pavlovian conditioning. Together with the fact that striatum and dopamine neurons have strong direct and indirect fiber connections, the result suggests that the striatum plays an important role in coding RPE signal by cooperating with dopamine neurons.

Long-lasting single-neuron labeling by in vivo electroporation without microscopic guidance.

In order to make a direct link between the morphological and functional study of the nervous system, we established an experimental protocol for labeling individual neurons persistently without microscopic guidance by injecting a plasmid encoding fluorescent protein electroporatively after recording their activity extracellularly. Using a glass pipette filled with electrolyte solution containing a plasmid encoding green fluorescent protein (GFP), single-neuron recording and electroporation were performed on anesthetized rats. When performing the electroporation at the completion of recording, the degree of contact between the target neuron and the electrode tip was adjusted by monitoring the change of the trace of recorded action potentials and the increase of electrode resistance. The expression of GFP and its immunostaining with a polyclonal antibody enabled us to clearly see the basic structural components such as cell bodies, axons, dendrites, and even smaller components such as spines. Identification of the morphological subtypes of neurons was possible with every labeled neuron. The optimum condition for labeling was a 30% increase of the electrode resistance, and the labeling success rate evaluated 3 days after labeling was 40%. The rate evaluated one month after labeling was only slightly lower (33%). We also confirmed experimentally that this recording and labeling procedure can be similarly successful in head-fixed behaving rats. This new experimental protocol will be a breakthrough in systems neuroscience because it makes a direct link between the morphology and behavior-related activity of single neurons.

Discrete coding of stimulus value, reward expectation, and reward prediction error in the dorsal striatum

To investigate how the striatum integrates sensory information with reward information for behavioral guidance, we recorded single-unit activity in the dorsal striatum of head-fixed rats participating in a probabilistic Pavlovian conditioning task with auditory conditioned stimuli (CSs) in which reward probability was fixed for each CS but parametrically varied across CSs. We found that the activity of many neurons was linearly correlated with the reward probability indicated by the CSs. The recorded neurons could be classified according to their firing patterns into functional subtypes coding reward probability in different forms such as stimulus value, reward expectation, and reward prediction error. These results suggest that several functional subgroups of dorsal striatal neurons represent different kinds of information formed through extensive prior exposure to CS-reward contingencies.

Rabies Virus Vector Transgene Expression Level and Cytotoxicity Improvement Induced by Deletion of Glycoprotein Gene

The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level.

Comparative Overview of Visuospatial Working Memory in Monkeys and Rats

Neural mechanisms of working memory, particularly its visuospatial aspect, have long been studied in non-human primates. On the other hand, rodents are becoming more important in systems neuroscience, as many of the innovative research methods have become available for them. There has been a question on whether primates and rodents have similar neural backgrounds for working memory. In this article, we carried out a comparative overview of the neural mechanisms of visuospatial working memory in monkeys and rats. In monkeys, a number of lesion studies indicate that the brain region most responsible for visuospatial working memory is the ventral dorsolateral prefrontal cortex (vDLPFC), as the performance in the standard tests for visuospatial working memory, such as delayed response and delayed alternation tasks, are impaired by lesions in this region. Single-unit studies revealed a characteristic firing pattern in neurons in this area, a sustained delay activity. Further studies indicated that the information maintained in the working memory, such as cue location and response direction in a delayed response, is coded in the sustained delay activity. In rats, an area comparable to the monkey vDLPFC was found to be the dorsal part of the medial prefrontal cortex (mPFC), as the delayed alternation in a T-maze is impaired by its lesion. Recently, the sustained delay activity similar to that found in monkeys has been found in the dorsal mPFC of rats performing the delayed response task. Furthermore, anatomical studies indicate that the vDLPFC in monkeys and the dorsal mPFC in rats have much in common, such as that they are both the major targets of parieto-frontal projections. Thus lines of evidence indicate that in both monkeys and rodents, the PFC plays a critical role in working memory.

Robust, highly customizable, and economical multi-channel electrode for chronic multi-unit recording in behaving animals

Multi-unit recording has been one of the most widely used techniques to investigate the correlation between multiple neuronal activities and behavior. However, a common problem of currently used multi-channel electrodes is their physical weakness. In this study, we developed a novel multi-channel electrode with sufficient physical strength to penetrate a thickened dura mater. This electrode consists of low-cost materials and is easily fabricated, and it enables recording without removing dura mater, thereby reducing the risk of inflammation, infection, or brain herniation. The low-cost multi-channel electrode developed in this study would be a useful tool for chronic recording in behaving animals.

Reward- and conditioned stimulus-related activity of rat striatal neurons are affected by time discounting

The prefrontal cortex is credited with contributing to relational reasoning, or the ability to integrate multiple acquired associations to generate new relationships. We have recorded single-unit activity from the lateral prefrontal cortex (LPFC) and the striatum while the monkeys performed a sequential paired-association task with asymmetric reward schedule. In the task, the monkeys learned two sequences of associated stimuli: A1-B1-C1 and A2-B2C2. The asymmetric reward rule was instructed by pairing C1 (or C2) with large (or small) reward block by block. The monkey also learned associations between new stimuli (e.g. N1, N2) and B1 and B2. The new stimuli were presented as the first cue in sequential paired-association trials instead of the old stimuli (A1 and A2). The findings from single-unit activity suggest that the LPFC can use an internal model of category to transfer reward information associated with one stimulus to other stimuli, even to new stimuli that had never been paired with different amount of reward. The striatum only uses direct experience between conditioned stimuli and reward to predict reward. One prediction from this hypothesis is that if the LPFC is inactivated, the monkey still correctly predicts reward for old stimuli through the striatal pathway, but has deficits in predicting reward for new stimuli. We injected muscimol to locally inactivate the LPFC, and also saline into the LPFC as control. In saline sessions, the monkey had significantly higher choice accuracy for new stimuli in large than in small reward trials, but this difference disappeared in muscimol session, consistent with the prediction. Together with single-unit activity data, our results suggest that the LPFC play a critical role in category-based reward inference.

Reward prediction error coding in striatal neurons

Retinoic acid (RA), Vitamin A metabolite, functions as a specific ligand for retinoic acid receptors (RARs) and regulates various biological phenomena through transcriptional regulation. RARs highly and ubiquitously express in brain. Previous studies showed that RA-deficient mice and RARs-knockout mice exhibit impairments of hippocampal LTP and spatial memory, suggesting that RARs may play an important role in hippocampal synaptic plasticity and memory. However, molecular mechanisms in which RARs regulate synaptic plasticity and memory remain unclear. To ask this question, we have examined effects of loss-of-functions of RARs on memory and LTP by generating and analyzing conditional mutant mice that enable to regulate forebrain-specific overexpression of dominant negative RARalpha mutant (alpha-402) lacking C-terminal region of RARalpha (aa 403–462). Our recent studies have shown that these mutant mice displayed impairments of hippocampal AMPA receptor-mediated excitatory postsynaptic potentials, LTP and memory formation. To understand mechanisms of these impairments at the molecular level, we performed biochemical analyses. Our expression analyses revealed that inducible overexpression of alpha-402 led to decreases in expression of GluR1 and PSD-95 in hippocampus. These results suggest that RARs regulate memory formation and LTP via changing in expression of synaptic plasticity-related genes. We are also analyzing expression levels of RAR target genes in these mutant mice. To further understand mechanisms in which RARalpha regulates learning and memory and LTP, we generated mutant mice expressing another dominant negative RARalpha mutant (alpha-391) that displays an additional truncation containing a nuclear export signal compared to alpha-402 mutant. We are now performing biochemical, electrophysiological and behavioral analyses using these mutant mice.

The effects of dopamine receptor antagonists on the activity of striatal neurons in rats during a probabilistic Pavlovian conditioning task

Retrieval of contextual fear memory by re-exposure to conditioned stimulus (CS) initiates two processes; reconsolidation and extinction. Previous our studies showed that short re-exposure to the CS inducing memory reconsolidation activated CREBdependent gene expression in hippocampus, while prolonged re-exposure inducing memory extinction inhibited this gene expression, suggesting that hippocampus plays distinct roles in reconsolidation and extinction of contextual fear memory. As a first step to understand the mechanism by which hippocampal gene expression are oppositely regulated in response to short and long re-exposure, we here performed biochemical analyses of glutamate receptors in hippocampus following short and long re-exposure. We found that long re-exposure to the CS inducing extinction decreased in expression of NMDA receptors including NR1, NR2A and NR2B and phosphorylation of GluR1 subunit of AMPA receptors at S831 and S845. These down-regulations of glutamate receptors might play critical roles in extinction of contextual fear memory by inhibiting hippocampal gene expression.