Two-photon imaging of neuronal activity in motor cortex of marmosets during upper-limb movement tasks.

Two-photon imaging in behaving animals has revealed neuronal activities related to behavioral and cognitive function at single-cell resolution. However, marmosets have posed a challenge due to limited success in training on motor tasks. Here we report the development of protocols to train head-fixed common marmosets to perform upper-limb movement tasks and simultaneously perform two-photon imaging. After 2-5 months of training sessions, head-fixed marmosets can control a manipulandum to move a cursor to a target on a screen. We conduct two-photon calcium imaging of layer 2/3 neurons in the motor cortex during this motor task performance, and detect task-relevant activity from multiple neurons at cellular and subcellular resolutions. In a two-target reaching task, some neurons show direction-selective activity over the training days. In a short-term force-field adaptation task, some neurons change their activity when the force field is on. Two-photon calcium imaging in behaving marmosets may become a fundamental technique for determining the spatial organization of the cortical dynamics underlying action and cognition.

Dopamine and glutamate release in the anterior default system during rest: A monkey microdialysis study.

Human neuroimaging studies have demonstrated the presence of a default system in the brain, which shows a default mode of brain activity, i.e., greater activity during rest than during an attention-demanding cognitive task. Our previous study on monkeys has revealed a default mode of brain activity in medial cortical areas. We have observed an increase in dopamine (DA) release during a working memory (WM) task compared with that during rest in the monkey lateral prefrontal cortex (LPFC). However, no previous study has examined DA release related to the default mode of brain activity. We used a microdialysis technique to investigate changes in DA release in the medial prefrontal cortex (MPFC), which constitutes the anterior default system, during the WM task and rest. Because DA and glutamate (Glu) release in the LPFC is interrelated, we also examined Glu release in the MPFC. We observed a significant increase in DA release, but no significant change in Glu release during rest compared with that during the WM task. We also observed an inhibitory relationship between the two transmitters in the MPFC. Considering that human default brain activity is related to internal thought processes and increased DA release in the LPFC plays an important role in executive control, increase in DA release during rest in the monkey anterior default system may be related to some form of internal thought process.

Stereotaxic atlas of the goat brain for an accurate approach to the hypothalamic nuclei.

While the external acoustic meatus of mice, rats and cats are at right angles to the side of the head, they are angled forward in ruminants. Because of this, the coordinates of stereotaxic atlases created using the same methods as for small animals were not suitable for practical use and made it very difficult to place electrodes in hypothalamic nuclei. The aim of this study was to produce a highly accurate stereotaxic atlas for goats that enables precise placement of electrodes in the hypothalamic nuclei. A method of fixing the head in place so as to maintain the basis cranii interna in a horizontal position was established allowing right angled three dimensional axes to be superimposed in the brains of Japanese Saanen goats. This research attempted to establish a new landmark alternative to the external acoustic meatus. The new landmark is the extremitas posterior clivus presphenoidale (EPCP) at the base of cranial bone. This landmark can easily be visualized through lateral radiography. This enabled the AP (anterior-posterior) and H (height) coordinates of the position to be fixed at 30 and 5, respectively. The A30 and H5 coordinates of the serial frontal, sagittal, and horizontal stereotaxic atlases in the present experiment were set to coincide with the position of the EPCP. In order to clarify whether the deviation of the stereotaxic brain coordinates due to inconsistent immobilization the goat's head was eliminated, the AP and H coordinates of the extremitas anterior fossa hypophysialis and the extremitas posterior fossa hypophysialis were measured using the corrected axes. In the three dimensional stereotaxic atlas created using the new landmark, it became possible to approach the hypothalamic nuclei of goats accurately. This method significantly differentiates the stereotaxic atlas from its predecessors.

Higher dopamine release induced by less rather than more preferred reward during a working memory task in the primate prefrontal cortex.

An optimal level of dopamine (DA) in the mammalian prefrontal cortex (PFC) is critical for higher cognitive control of behavior. Too much or too little DA in the PFC induces impairment in working memory (WM) task performance. PFC DA is also concerned with motivation. When reward is anticipated and/or delivered, an increase in PFC DA release is observed. In the primate, more preferred reward induces enhanced WM-related neuronal activity in the dorsolateral PFC (DLPFC). We hypothesized that there would be more DA release in the primate DLPFC when more preferred, as compared with less preferred, reward is delivered during a WM task. Contrary to our hypothesis, we found higher DA release in the DLPFC when less rather than more preferred reward was used during a WM task, while unpredictable free reward delivery induced an increase in DLPFC DA release irrespective of the difference in the incentive value of the reward. Behaviorally, the monkey was more motivated with preferred than with less preferred reward, although it performed the task almost without error irrespective of the difference in the reward. Considering that mild stress induces an increase in DA release in the mammalian PFC, performing a WM task for less preferred reward could have been mildly stressful, and this mild stress may have induced more DLPFC DA release in the present study. The higher DA release in the DLPFC with less preferred reward may be beneficial for monkeys to cope with mildly stressful and unfavorable situations to achieve proficient WM task performance.

Default mode of brain activity demonstrated by positron emission tomography imaging in awake monkeys: higher rest-related than working memory-related activity in medial cortical areas.

Human neuroimaging studies have demonstrated the presence of a "default system" in the brain, which shows a "default mode of brain activity," i.e., greater activity during the resting state than during an attention-demanding cognitive task. The default system mainly involves the medial prefrontal and medial parietal areas, including the anterior and posterior cingulate cortex. It has been proposed that this default activity is concerned with internal thought processes. Recently, it has been indicated that chimpanzees show high metabolic levels in these medial brain areas during rest. Correlated low-frequency spontaneous activity as measured by functional magnetic resonance imaging was observed between the medial parietal and medial prefrontal areas in the anesthetized monkey. However, there have been few attempts to demonstrate a default system that shows task-induced deactivation in nonhuman primates. We conducted a positron emission tomography study with [(15)O]H(2)O to demonstrate a default mode of brain activity in the awake monkey sitting on a primate chair. Macaque monkeys showed higher level of regional blood flow in these medial brain areas as well as lateral and orbital prefrontal areas during rest compared with that under a working memory task, suggesting the existence of internal thought processes in the monkey. However, during rest in the monkey, the highest level of blood flow relative to that in other brain regions was observed not in the default system but in the dorsal striatum, suggesting that regions with the highest cerebral blood flow during rest may differ depending on the resting condition and/or species.

Reward expectancy-related prefrontal neuronal activities: are they neural substrates of "affective" working memory?

Primate prefrontal delay neurons are involved in retaining task-relevant cognitive information in working memory (WM). Recent studies have also revealed primate prefrontal delay neurons that are related to reward/omission-of-reward expectancy. Such reward-related delay activities might constitute "affective WM" (Davidson, 2002). "Affective" and "cognitive" WM are both concerned with representing not what is currently being presented, but rather what was presented previously or might be presented in the future. However, according to the original and widely accepted definition, WM is the "temporary storage and manipulation of information for complex cognitive tasks". Reward/omission-of-reward expectancy-related neuronal activity is neither prerequisite nor essential for accurate task performance; thus, such activity is not considered to comprise the neural substrates of WM. Also, "affective WM" might not be an appropriate usage of the term "WM". We propose that WM- and reward/omission-of-reward expectancy-related neuronal activity are concerned with representing which response should be performed in order to attain a goal (reward) and the goal of the response, respectively. We further suggest that the prefrontal cortex (PFC) plays a crucial role in the integration of cognitive (for example, WM-related) and motivational (for example, reward expectancy-related) operations for goal-directed behaviour. The PFC could then send this integrated information to other brain areas to control the behaviour.

Functional significance of delay-period activity of primate prefrontal neurons in relation to spatial working memory and reward/omission-of-reward expectancy.

The lateral prefrontal cortex (LPFC) is important in cognitive control. During the delay period of a working memory (WM) task, primate LPFC neurons show sustained activity that is related to retaining task-relevant cognitive information in WM. However, it has not yet been determined whether LPFC delay neurons are concerned exclusively with the cognitive control of WM task performance. Recent studies have indicated that LPFC neurons also show reward and/or omission-of-reward expectancy-related delay activity, while the functional relationship between WM-related and reward/omission-of-reward expectancy-related delay activity remains unclear. To clarify the functional significance of LPFC delay-period activity for WM task performance, and particularly the functional relationship between these two types of activity, we examined individual delay neurons in the primate LPFC during spatial WM (delayed response) and non-WM (reward-no-reward delayed reaction) tasks. We found significant interactions between these two types of delay activity. The majority of the reward expectancy-related neurons and the minority of the omission-of-reward expectancy-related neurons were involved in spatial WM processes. Spatial WM-related neurons were more likely to be involved in reward expectancy than in omission-of-reward expectancy. In addition, LPFC delay neurons observed during the delayed response task were not concerned exclusively with the cognitive control of task performance; some were related to reward/omission-of-reward expectancy but not to WM, and many showed more memory-related activity for preferred rewards than for less-desirable rewards. Since employing a more preferred reward induced better task performance in the monkeys, as well as enhanced WM-related neuronal activity in the LPFC, the principal function of the LPFC appears to be the integration of cognitive and motivational operations in guiding the organism to obtain a reward more effectively.

Long- and short-range reward expectancy in the primate orbitofrontal cortex.

The orbitofrontal cortex (OFC) is important in motivation and emotion. We previously reported reward expectancy-related delay activities during a delayed reaction time task in primate OFC neurons. To further investigate the significance of the OFC in motivational operations, we examined pre-instruction, baseline activities of OFC neurons in relation to reward expectancy during the delayed reaction time task. In this task, an instruction cue indicated whether reward would be present or absent in the trial. Each set of four consecutive trials constituted one block within which three different kinds of rewards and one trial with no reward were given in a fixed order that differed from the monkey's reward preference. We identified two types of OFC neurons with reward expectancy-related pre-instruction activities: Step-type neurons showed stepwise changes (increase or decrease) in pre-instruction activity toward the trial with a particular outcome, which usually was the most or least attractive within a block; Pref-type neurons showed pre-instruction activity changes according to the monkey's preference for each trial's outcome. We propose that Step-type and Pref-type neurons are related to long-range and short-range reward expectancies of a particular outcome, respectively. The OFC is considered to play important roles in goal-directed behaviour by adjusting the motivational level toward a certain (current or future) outcome of a particular motivational significance based on the two kinds of reward expectancy processes. Impairments in goal-directed behaviour by OFC patients may be caused by a lack of long-range expectancy or by a deficit in compromising between short-range and long-range expectancies.

Release of neurotransmitters in the monkey frontal cortex is related to level of attention.

Attention is reported to be maintained by monoamines, acetylcholine and amino acids systems. Changes in the releases of these neurotransmitters during the three stages comprising quiet wake (QW) and two arousal states (AW), which are activated from different sources, were investigated. Norepinephrine releases during AW were significantly higher than that during QW. Conversely, the levels of acetylcholine and serotonin that were released did not change significantly among these three stages. The interesting observation was the dissociation of the increase between glutamate and dopamine releases in the two AW states. The results indicate that attention level is related to the amount of norepinephrine release, and that attention quality is related to the interaction between dopamine and glutamate releases.

Differential changes in glutamate concentration in the primate prefrontal cortex during spatial delayed alternation and sensory-guided tasks.

Glutamate is a major neurotransmitter in the mammalian brain and glutamatergic neurotransmission in the frontal cortex is indicated to play important roles in cognitive operations. We previously examined changes in extracellular dopamine in the primate frontal cortex in cognitive tasks, and in this paper we extend this to glutamate. We employed, as cognitive tasks, a delayed alternation task where the animal must retain information in working memory, and a sensory-guided task in which there is no working memory requirement but there may be more sensory processing requirements. Using the in vivo microdialysis method, we examined changes in extracellular glutamate concentration in the dorsolateral, arcuate, orbitofrontal, and premotor areas of the primate frontal cortex. Compared to basal rest levels, we observed significant increases in glutamate concentration in dorsolateral and arcuate areas of the prefrontal cortex during the sensory-guided task, but did not find significant changes in any of the frontal areas examined during the delayed alternation task. When glutamate concentration was compared between the delayed alternation and sensory-guided tasks, difference was observed only in the dorsolateral prefrontal cortex, especially in the ventral lip area of the principal sulcus. The results indicate the importance of glutamate in processing sensory information but not in retaining information in working memory in the primate dorsolateral and arcuate prefrontal cortex. We also compared the concentration of glutamate and dopamine in the tasks. We found a double dissociation in the concentration of glutamate and dopamine in the dorsolateral area: there was an increase in glutamate but no change in dopamine during the sensory-guided task, whereas there was an increase in dopamine but no change in glutamate during the delayed alternation task. It is thus suggested that in the primate dorsolateral prefrontal cortex, increased glutamate tone without dopamine increase facilitates sensory-guided task performance, while increased dopamine tone without glutamate increase is beneficial for working memory task performance.

Coding and monitoring of motivational context in the primate prefrontal cortex.

The prefrontal cortex is involved in acquiring and maintaining information about context, including the set of task instructions and/or the outcome of previous stimulus-response sequences. Most studies on context-dependent processing in the prefrontal cortex have been concerned with such executive functions, but the prefrontal cortex is also involved in motivational operations. We thus wished to determine whether primate prefrontal neurons show evidence of representing the motivational context learned by the monkey. We trained monkeys in a delayed reaction task in which an instruction cue indicated the presence or absence of reward. In random alternation with no reward, the same one of several different kinds of food and liquid rewards was delivered repeatedly in a block of approximately 50 trials, so that reward information would define the motivational context. In response to an instruction cue indicating absence of reward, we found that neurons in the lateral prefrontal cortex not only predicted the absence of reward but also represented more specifically which kind of reward would be omitted in a given trial. These neurons seem to code contextual information concerning which kind of reward may be delivered in following trials. We also found prefrontal neurons that showed tonic baseline activity that may be related to monitoring such motivational context. The different types of neurons were distributed differently along the dorsoventral extent of the lateral prefrontal cortex. Such operations in the prefrontal cortex may be important for the monkey to maximize reward or to modify behavioral strategies and thus may contribute to executive control.