Characterization of the expression of gastrin-releasing peptide and its receptor in the trigeminal and spinal somatosensory systems of Japanese macaque monkeys: Insight into humans.

Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.

Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at -25 °C for Several Years.

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 °C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

Variation of pro-vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin-related glycopeptide copeptin.

Arginine vasopressin (AVP) is synthesized in parvocellular- and magnocellular neuroendocrine neurons in the paraventricular nucleus (PVN) of the hypothalamus. Whereas magnocellular AVP neurons project primarily to the posterior pituitary, parvocellular AVP neurons project to the median eminence (ME) and to extrahypothalamic areas. The AVP gene encodes pre-pro-AVP that comprises the signal peptide, AVP, neurophysin (NPII), and a copeptin glycopeptide. In the present study, we used an N-terminal copeptin antiserum to examine copeptin expression in magnocellular and parvocellular neurons in the hypothalamus in the mouse, rat, and macaque monkey. Although magnocellular NPII-expressing neurons exhibited strong N-terminal copeptin immunoreactivity in all three species, a great majority (~90%) of parvocellular neurons that expressed NPII was devoid of copeptin immunoreactivity in the mouse, and in approximately half (~53%) of them in the rat, whereas in monkey hypothalamus, virtually all NPII-immunoreactive parvocellular neurons contained strong copeptin immunoreactivity. Immunoelectron microscopy in the mouse clearly showed copeptin-immunoreactivity co-localized with NPII-immunoreactivity in neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa-d-arginine amide resulted in a marked reduction of copeptin-immunoreactivity in the NPII-immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro-AVP could explain the disproportionally low levels of N-terminal copeptin expression in rodent AVP (NPII)-expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration.

Vasopressin gene products are colocalised with corticotrophin-releasing factor within neurosecretory vesicles in the external zone of the median eminence of the Japanese macaque monkey (Macaca fuscata).

Arginine vasopressin (AVP), when released into portal capillaries with corticotrophin-releasing factor (CRF) from terminals of parvocellular neurones of the hypothalamic paraventricular nucleus (PVH), facilitates the secretion of adrenocorticotrophic hormone (ACTH) in stressed rodents. The AVP gene encodes a propeptide precursor containing AVP, AVP-associated neurophysin II (NPII), and a glycopeptide copeptin, although it is currently unclear whether copeptin is always cleaved from the neurophysin and whether the NPII and/or copeptin have any functional role in the pituitary. Furthermore, for primates, it is unknown whether CRF, AVP, NPII and copeptin are all colocalised in neurosecretory vesicles in the terminal region of the paraventricular CRF neurone axons. Therefore, we investigated, by fluorescence and immunogold immunocytochemistry, the cellular and subcellular relationships of these peptides in the CRF- and AVP-producing cells in unstressed Japanese macaque monkeys (Macaca fuscata). Reverse transcription-polymerase chain reaction analysis showed the expression of both CRF and AVP mRNAs in the monkey PVH. As expected, in the magnocellular neurones of the PVH and supraoptic nucleus, essentially no CRF immunoreactivity could be detected in NPII-immunoreactive (AVP-producing) neurones. Immunofluorescence showed that, in the parvocellular part of the PVH, NPII was detectable in a subpopulation (approximately 39%) of the numerous CRF-immunoreactive neuronal perikarya, whereas, in the outer median eminence, NPII was more prominent (approximately 52%) in the CRF varicosities. Triple immunoelectron microscopy in the median eminence demonstrated the presence of both NPII and copeptin immunoreactivity in dense-cored vesicles of CRF-containing axons. The results are consistent with an idea that the AVP propeptide is processed and NPII and copeptin are colocalised in hypothalamic-pituitary CRF axons in the median eminence of a primate. The CRF, AVP and copeptin are all co-packaged in neurosecretory vesicles in monkeys and are thus likely to be co-released into the portal capillary blood to amplify ACTH release from the primate anterior pituitary.

Identification of neuron-type specific promoters in monkey genome and their functional validation in mice.

Viral gene delivery is one of the most versatile techniques for elucidating the mechanisms underlying brain dysfunction, such as neuropsychiatric disorders. Due to the complexity of the brain, expression of genetic tools, such as channelrhodopsin and calcium sensors, often has to be restricted to a specified cell type within a circuit implicated in these disorders. Only a handful of promoters targeting neuronal subtypes are currently used for viral gene delivery. Here, we isolated conserved promoter regions of several subtype-specific genes from the macaque genome and investigated their functionality in the mouse brain when used within lentiviral vectors (LVVs). Immunohistochemical analysis revealed that transgene expression induced by the promoter sequences for somatostatin (SST), cholecystokinin (CCK), parvalbumin (PV), serotonin transporter (SERT), vesicular acetylcholine transporter (vAChT), substance P (SP) and proenkephalin (PENK) was largely colocalized with specific markers for the targeted neuronal populations. Moreover, by combining these results with in silico predictions of transcription factor binding to the isolated sequences, we identified transcription factors possibly underlying cell-type specificity. These findings lay a foundation for the expansion of the current toolbox of promoters suitable for elucidating these neuronal phenotypes.

Distinct Functions of the Primate Putamen Direct and Indirect Pathways in Adaptive Outcome-Based Action Selection.

Cortico-basal ganglia circuits are critical regulators of reward-based decision making. Reinforcement learning models posit that action reward value is encoded by the firing activity of striatal medium spiny neurons (MSNs) and updated upon changing reinforcement contingencies by dopamine (DA) signaling to these neurons. However, it remains unclear how the anatomically distinct direct and indirect pathways through the basal ganglia are involved in updating action reward value under changing contingencies. MSNs of the direct pathway predominantly express DA D1 receptors and those of the indirect pathway predominantly D2 receptors, so we tested for distinct functions in behavioral adaptation by injecting D1 and D2 receptor antagonists into the putamen of two macaque monkeys performing a free choice task for probabilistic reward. In this task, monkeys turned a handle toward either a left or right target depending on an asymmetrically assigned probability of large reward. Reward probabilities of left and right targets changed after 30-150 trials, so the monkeys were required to learn the higher-value target choice based on action-outcome history. In the control condition, the monkeys showed stable selection of the higher-value target (that more likely to yield large reward) and kept choosing the higher-value target regardless of less frequent small reward outcomes. The monkeys also made flexible changes of selection away from the high-value target when two or three small reward outcomes occurred randomly in succession. DA D1 antagonist injection significantly increased the probability of the monkey switching to the alternate target in response to successive small reward outcomes. Conversely, D2 antagonist injection significantly decreased the switching probability. These results suggest distinct functions of D1 and D2 receptor-mediated signaling processes in action selection based on action-outcome history, with D1 receptor-mediated signaling promoting the stable choice of higher-value targets and D2 receptor-mediated signaling promoting a switch in action away from small reward outcomes. Therefore, direct and indirect pathways appear to have complementary functions in maintaining optimal goal-directed action selection and updating action value, which are dependent on D1 and D2 DA receptor signaling.

Coding of the long-term value of multiple future rewards in the primate striatum.

Decisions maximizing benefits involve a tradeoff between the quantity of a reward and the cost of elapsed time until an animal receives it. The estimation of long-term reward values is critical to attain the most desirable outcomes over a certain period of time. Reinforcement learning theories have established algorithms to estimate the long-term reward values of multiple future rewards in which the values of future rewards are discounted as a function of how many steps of choices are necessary to achieve them. Here, we report that presumed striatal projection neurons represent the long-term values of multiple future rewards estimated by a standard reinforcement learning model while monkeys are engaged in a series of trial-and-error choices and adaptive decisions for multiple rewards. We found that the magnitude of activity of a subset of neurons was positively correlated with the long-term reward values, and that of another subset of neurons was negatively correlated throughout the entire decision-making process in individual trials: from the start of the task trial, estimation of the values and their comparison among alternatives, choice execution, and evaluation of the received rewards. An idiosyncratic finding was that neurons showing negative correlations represented reward values in the near future (high discounting), while neurons showing positive correlations represented reward values not only in the near future, but also in the far future (low discounting). These findings provide a new insight that long-term value signals are embedded in two subsets of striatal neurons as high and low discounting of multiple future rewards.

Dopamine neurons learn to encode the long-term value of multiple future rewards.

Midbrain dopamine neurons signal reward value, their prediction error, and the salience of events. If they play a critical role in achieving specific distant goals, long-term future rewards should also be encoded as suggested in reinforcement learning theories. Here, we address this experimentally untested issue. We recorded 185 dopamine neurons in three monkeys that performed a multistep choice task in which they explored a reward target among alternatives and then exploited that knowledge to receive one or two additional rewards by choosing the same target in a set of subsequent trials. An analysis of anticipatory licking for reward water indicated that the monkeys did not anticipate an immediately expected reward in individual trials; rather, they anticipated the sum of immediate and multiple future rewards. In accordance with this behavioral observation, the dopamine responses to the start cues and reinforcer beeps reflected the expected values of the multiple future rewards and their errors, respectively. More specifically, when monkeys learned the multistep choice task over the course of several weeks, the responses of dopamine neurons encoded the sum of the immediate and expected multiple future rewards. The dopamine responses were quantitatively predicted by theoretical descriptions of the value function with time discounting in reinforcement learning. These findings demonstrate that dopamine neurons learn to encode the long-term value of multiple future rewards with distant rewards discounted.

Neuronal basis for evaluating selected action in the primate striatum.

Humans and animals optimize their behavior by evaluating outcomes of individual actions and predicting how much reward the actions will yield. While the estimated values of actions guide choice behavior, the choices are also governed by other behavioral norms, such as rules and strategies. Values, rules and strategies are represented in neuronal activity, and the striatum is one of the best qualified brain loci where these signals meet. To understand the role of the striatum in value- and strategy-based decision-making, we recorded striatal neurons in macaque monkeys performing a behavioral task in which they searched for a reward target by trial-and-error among three alternatives, earned a reward for a target choice, and then earned additional rewards for choosing the same target. This task allowed us to examine whether and how values of targets and strategy, which were defined as negative-then-search and positive-then-repeat (or win-stay-lose-switch), are represented in the striatum. Large subsets of striatal neurons encoded positive and negative outcome feedbacks of individual decisions and actions. Once monkeys made a choice, signals related to chosen actions, their values and search- or repeat-type actions increased and persisted until the outcome feedback appeared. Subsets of neurons exhibited a tonic increase in activity after the search- and repeat-choices following negative and positive feedback in the last trials as the task strategy monkeys adapted. These activity profiles as a heterogeneous representation of decision variables may underlie a part of the process for reinforcement- and strategy-based evaluation of selected actions in the striatum.

Inactivation of the putamen selectively impairs reward history-based action selection.

Behavioral decisions and actions are directed to achieve specific goals and to obtain rewards and escape punishments. Previous studies involving the recording of neuronal activity suggest the involvement of the cerebral cortex, basal ganglia, and midbrain dopamine system in these processes. The value signal of the action options is represented in the striatum, updated by reward prediction errors, and used for selecting higher-value actions. However, it remains unclear whether dysfunction of the striatum leads to impairment of value-based action selection. The present study examined the effect of inactivation of the putamen via local injection of the GABA(A) receptor agonist muscimol in monkeys engaged in a manual reward-based multi-step choice task. The monkeys first searched a reward target from three alternatives, based on the previous one or two choices and their outcomes, and obtained a large reward; they then earned an additional reward by choosing the last rewarded target. Inactivation of the putamen impaired the ability of monkeys to make optimal choices during third trial in which they were required to choose a target different from those selected in the two previous trials by updating the values of the three options. The monkeys normally changed options if the last choice resulted in small reward (lose-shift) and stayed with the last choice if it resulted in large reward (win-stay). Task start time and movement time during individual trials became longer after putamen inactivation. But monkeys could control the motivation level depending on the reward value of individual trial types before and after putamen inactivation. These results support a view that the putamen is involved selectively and critically in neuronal circuits for reward history-based action selection.

Atomic-scale distribution of water molecules at the mica-water interface visualized by three-dimensional scanning force microscopy.

We have developed a method referred to as three-dimensional scanning force microscopy (3D-SFM) which enables us to visualize water distribution at a solid-liquid interface with an atomic-scale resolution in less than 1 min. The 3D-SFM image obtained at a mica-water interface visualizes 3D distributions of adsorbed water molecules above the center of hexagonal cavities and the laterally distributed hydration layer. The atomically resolved 3D-SFM image showing mirror symmetry suggests the existence of surface relaxation of the cleaved mica surface next to the aqueous environment.

Representation of action-specific reward values in the striatum.

The estimation of the reward an action will yield is critical in decision-making. To elucidate the role of the basal ganglia in this process, we recorded striatal neurons of monkeys who chose between left and right handle turns, based on the estimated reward probabilities of the actions. During a delay period before the choices, the activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. Fewer neurons were tuned to relative values or action choice. These results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit.

Encoding of direction and combination of movements by primate putamen neurons.

To perform multiple movements in a preprogrammed order, the brain needs to compute both the visuospatial and temporal organization of such multiple movements. Previous studies revealed participation of the parietal and premotor areas in visuospatial processing, and of the supplementary motor and presupplementary motor areas in temporal structuring of multiple movements. In the basal ganglia, on the other hand, relatively little has been known about how the neuronal processing of the visuospatial and temporal structuring of multiple movements occurs. In the present study, monkeys performed combinations of hand movements, either a lever turn-lever turn or lever turn-button press. Combinations of the two movements were performed under visually instructed condition first, then under remembered condition. We found that activity of 43% (30/69) and 60% (42/69) of putamen neurons was selective to the preprogrammed combination of movements and to the direction of the first movement. The neurons preferring remembered condition were mainly observed in the dorsomedial part of the putamen, where most of neurons were also selective to both direction and combination of movements, while those in the ventrolateral part of the putamen were not selective to the instructed and remembered conditions. The results supported a hypothesis that the movement direction-selective and the movement combination-selective neuron activities in the striatum may play an indispensable role in the visuospatial and temporal organization of movements through the cortico-basal ganglia loop system.

Goal-directed, serial and synchronous activation of neurons in the primate striatum.

To study roles of cortico-basal ganglia loops in action planning, we examined interactions between the activities of simultaneously recorded neurons in the striatum of monkeys performing sequence motor tasks by cross-correlation analysis. Serial activation occurred between projection neurons in a motor sequence-dependent manner, and was in the direction of a neuron encoding an early event in the sequence to a neuron encoding the same event or later, but closer event to the reward. Synchronous activation occurred between pairs of interneurons. The serial activation seems to originate through the cortico-basal ganglia loops, because projection neurons are inhibitory. We propose that the task-dependent serial and synchronous activation of striate neurons may be a neural substrate for goal-directed planning through the basal ganglia.