Synthetic ShK-like Peptide from the Jellyfish Nemopilema nomurai Has Human Voltage-Gated Potassium-Channel-Blocking Activity.

We identified a new human voltage-gated potassium channel blocker, NnK-1, in the jellyfish Nemopilema nomurai based on its genomic information. The gene sequence encoding NnK-1 contains 5408 base pairs, with five introns and six exons. The coding sequence of the NnK-1 precursor is 894 nucleotides long and encodes 297 amino acids containing five presumptive ShK-like peptides. An electrophysiological assay demonstrated that the fifth peptide, NnK-1, which was chemically synthesized, is an effective blocker of hKv1.3, hKv1.4, and hKv1.5. Multiple-sequence alignment with cnidarian Shk-like peptides, which have Kv1.3-blocking activity, revealed that three residues (3Asp, 25Lys, and 34Thr) of NnK-1, together with six cysteine residues, were conserved. Therefore, we hypothesized that these three residues are crucial for the binding of the toxin to voltage-gated potassium channels. This notion was confirmed by an electrophysiological assay with a synthetic peptide (NnK-1 mu) where these three peptides were substituted with 3Glu, 25Arg, and 34Met. In conclusion, we successfully identified and characterized a new voltage-gated potassium channel blocker in jellyfish that interacts with three different voltage-gated potassium channels. A peptide that interacts with multiple voltage-gated potassium channels has many therapeutic applications in various physiological and pathophysiological contexts.

4-EA-NBOMe, an amphetamine derivative, alters glutamatergic synaptic transmission through 5-HT1A receptors on cortical neurons from SpragueDawley rat and pyramidal neurons from C57BL/6 mouse.

New psychoactive substances (NPSs) are compounds designed to mimic illegal recreational drugs. In particular, there are difficulties in legal restrictions because there is no fast NPS detection method to suppress the initial spread of NPS with criminal records; thus, they expose the public to serious health threats, including toxicity and dependence. However, the effects of NPSs on the brain and the related cellular mechanisms are well unknown. One of the recently emerging drugs is 4-ethylamphetamine-NBOMe (4-EA-NBOMe), a member of the 2 C phenylalanine family with a similar structure to methamphetamine (methA). In this study, we tested the effect of methA analogs on the glutamatergic synaptic transmission on primary cultured cortical neurons of SpragueDawley (SD) rats and C57BL/6 mice, and also layer 2/3 pyramidal neurons of the medial prefrontal cortex (mPFC) of C57BL/6 mice. We found that acute treatment with 4-EA-NBOMe inhibits spontaneous excitatory postsynaptic currents (EPSCs) and that withdrawal after chronic inhibition by 4-EA-NBOMe augments glutamatergic synaptic transmission. These modifications of synaptic responses are mediated by 5-HT1A receptors. These findings suggest that 4-EA-NBOMe directly affects the central nervous system by changing the efficacy of glutamatergic synaptic transmission.

SELENBP1 overexpression in the prefrontal cortex underlies negative symptoms of schizophrenia.

The selenium-binding protein 1 (SELENBP1) has been reported to be up-regulated in the prefrontal cortex (PFC) of schizophrenia patients in postmortem reports. However, no causative link between SELENBP1 and schizophrenia has yet been established. Here, we provide evidence linking the upregulation of SELENBP1 in the PFC of mice with the negative symptoms of schizophrenia. We verified the levels of SELENBP1 transcripts in postmortem PFC brain tissues from patients with schizophrenia and matched healthy controls. We also generated transgenic mice expressing human SELENBP1 (hSELENBP1 Tg) and examined their neuropathological features, intrinsic firing properties of PFC 2/3-layer pyramidal neurons, and frontal cortex (FC) electroencephalographic (EEG) responses to auditory stimuli. Schizophrenia-like behaviors in hSELENBP1 Tg mice and mice expressing Selenbp1 in the FC were assessed. SELENBP1 transcript levels were higher in the brains of patients with schizophrenia than in those of matched healthy controls. The hSELENBP1 Tg mice displayed negative endophenotype behaviors, including heterotopias- and ectopias-like anatomical deformities in upper-layer cortical neurons and social withdrawal, deficits in nesting, and anhedonia-like behavior. Additionally, hSELENBP1 Tg mice exhibited reduced excitabilities of PFC 2/3-layer pyramidal neurons and abnormalities in EEG biomarkers observed in schizophrenia. Furthermore, mice overexpressing Selenbp1 in FC showed deficits in sociability. These results suggest that upregulation of SELENBP1 in the PFC causes asociality, a negative symptom of schizophrenia.

Generation of human iPSCs derived heart organoids structurally and functionally similar to heart.

Currently, due to the increasing demand for 3D culture, various organoids that mimic organs are being actively studied. Despite active reports, information on heart organoids (HOs), which are the first functional organs, is still insufficient. Parameters for reproducing hearts are: chamber formation, organization with cardiac cells, vascularization, and simulation of electrophysiological signals. In particular, since the heart reflects complex factors, it is necessary to develop HOs that can be simulated in depth. In this study, we have created self-organized HOs using human iPSCs, and validated mimicry of cardiac structures such as chamber and epicardium/myocardium and atrium/ventricle-similar areas. Furthermore, mechanical/electrophysiological features were verified through multiple analyzes after inhibition of ion channels. More importantly, the HOs function, due to the cardiovascular characteristics of HOs, was maintained through vascularization after in vivo transplantation. In conclusion, this study has the advantage of being able to easily and closely recapitulate morphological/functional aspects of the heart.

Anti-stress Effect of Octopus Cephalotocin in Rats.

Cephalotocin is a bioactivity-regulating peptide expressed in octopus (Octopus vulgaris). The peptide sequence of cephalotocin is very similar to the peptide sequence of mammalian vasopressin, and cephalotocin has been proposed to mainly activate arginine vasopressin 1b receptor (Avpr1b) in the brain. However, the effects of cephalotocin on mammalian behavior have not been studied. In the current study, cephalotocin significantly reduced both the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from not only cultured neuronal cells from postnatal Sprague-Dawley (SD) rats but also hippocampal slices from 4-week-old male C57BL/6 mice. Intraperitoneal (IP) injection did not affect the open field behaviors of C57BL/6 mice. Cephalotocin was directly infused into the hippocampus because the normalized Avpr1b staining intensity divided by the DAPI staining intensity indicated that Avpr1b expression tended to be high in the hippocampus. A hippocampal infusion of 1 mg/kg cephalotocin via an implanted cannula exerted an anti-stress effect, significantly reducing the immobility time in the tail suspension test (TST). The present results provide evidence that the effects of cephalotocin on the activity of hippocampal neurons are related to ameliorating stress, suggesting that cephalotocin may be developed as an anti-stress biomodulator that functions by affecting the brain.

Establishment of Neurotoxicity Assessment Using Microelectrode Array (MEA) with hiPSC-Derived Neurons and Evaluation of New Psychoactive Substances (NPS).

Background and Objectives: Currently, safety pharmacological tests for the central nervous system depend on animal behavioral analysis. However, due to the subjectivity of behavioral analysis and differences between species, there is a limit to appropriate nervous system toxicity assessment, therefore a new neurotoxicity assessment that can simulate the human central nervous system is required. Methods and Results: In our study, we developed an in vitro neurotoxicity assessment focusing on neuronal function. To minimize the differences between species and fast screening, hiPSC-derived neurons and a microelectrode array (MEA) that could simultaneously measure the action potentials of the neuronal networks were used. After analyzing the molecular and electrophysiological characters of our neuronal network, we conducted a neurotoxicity assessment on neurotransmitters, neurotoxicants, illicit drugs, and new psychoactive substances (NPS). We found that most substances used in our experiments responded more sensitively to our MEA-based neurotoxicity assessment than to the conventional neurotoxicity assessment. Also, this is the first paper that evaluates various illicit drugs and NPS using MEA-based neurotoxicity assessment using hiPSC-derived neurons. Conclusions: Our study expanded the scope of application of neurotoxicity assessment using hiPSC-derived neurons to NPS, and accumulated evaluation data of various toxic substances for hiPSC-derived neurons.

An Octopus-Derived Peptide with Antidiuretic Activity in Rats.

Discovering new drug candidates with high efficacy and few side effects is a major challenge in new drug development. The two evolutionarily related peptides oxytocin (OXT) and arginine vasopressin (AVP) are known to be associated with a variety of physiological and psychological processes via the association of OXT with three types of AVP receptors. Over decades, many synthetic analogs of these peptides have been designed and tested for therapeutic applications; however, only a few studies of their natural analogs have been performed. In this study, we investigated the bioactivity and usefulness of two natural OXT/AVP analogs that originate from the marine invertebrate Octopus vulgaris, named octopressin (OTP) and cephalotocin (CPT). By measuring the intracellular Ca2+ or cyclic AMP increase in each OXT/AVP receptor subtype-overexpressing cell, we found that CPT, but not OTP, acts as a selective agonist of human AVP type 1b and 2 receptors. This behavior is reminiscent of desmopressin, the most widely prescribed antidiuretic drug in the world. Similar to the case for desmopressin, a single intravenous tail injection of CPT into Sprague-Dawley rats reduced urine output and increased urinary osmolality. In conclusion, we suggest that CPT has a significant antidiuretic effect and that CPT might be beneficial for treating urological conditions such as nocturia, enuresis, and diabetes insipidus.

Identification of integrative hepatotoxicity induced by lysosomal phospholipase A2 inhibition of cationic amphiphilic drugs via metabolomics.

Drug-induced liver injury (DILI) is a condition caused by drugs that leads to abnormal hepatic function. Hepatotoxicity caused by DILI has been shown to be due to cellular stress, mitochondrial dysfunction, cell necrosis and apoptosis and many types of hepatotoxicity, such as phospholipidosis, steatosis and hepatitis, commonly share intracellular molecular mechanisms. Metabolomics can be useful for mechanism-based toxicity evaluations and has been recently utilized as a scientific technique that can effectively predict the risk factors for chemical substances. To evaluate the key events in hepatotoxicity associated with lysosomal phospholipase A2 (LPLA2) inhibition by cationic amphiphilic drugs (CADs), LPLA2 inhibition assays and phospholipid accumulation assays were performed in HepG2 cells. Additionally, to suggest the integrative molecular mechanisms of hepatotoxicity by CADs, we profiled intracellular metabolites. Cell-based metabolomics was performed using an UPLC-Orbitrap-MS instrument equipped with heated electrospray ionization in positive and negative ion modes. As a result, CADs such as amiodarone, fluoxetine, chlorpromazine and tamoxifen significantly inhibited LPLA2 and accumulated phospholipids. In metabolomics, a total of 17 significant metabolites were identified, and the changed metabolite types were as follows: nucleotide sugars, conjugated bile acids, branched-chain amino acids, polyamine biosynthesis, and long-chain fatty acid and glycerophospholipid metabolism. From these data, it was suggested that the integrative mechanism of DILI could be verified and that a toxicological approach is possible using metabolomics.

Surgical removal of a telemetry system in a cynomolgus monkey (Macaca fascicularis): a 12-month observation study.

BACKGROUND: Telemetry is a wireless implanted device that measures biological signals in conscious animals and usually requires surgery for its removal when the study is finished. After removing the device, the animals are either used for other studies or euthanatized. CASE PRESENTATION: Herein, we report the case of a living cynomolgus monkey (Macaca fascicularis) that was used for the entire experimental period, instead of euthanasia, after surgical removal of an implanted telemetry system. Radiography was used to determine the status of the implanted telemetry, following which, a repair surgery was performed for removing the system; clinical signs were used to preserve the life of the cynomolgus monkey. Postoperative clinical signs, food consumption, hematology, and serum biochemistry were examined during the 12-month observational period. No abnormal readings or conditions were observed in the subject after implant removal. CONCLUSIONS: This study may be a useful case report for living cynomolgus monkeys in telemetry implantations used throughout the study period. We suggest minimizing the suffering and improving the welfare of these animals.

Quinacrine directly dissociates amyloid plaques in the brain of 5XFAD transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common type of dementia characterized by the abnormal accumulation of amyloid-ß (Aß) in the brain. Aß misfolding is associated with neuroinflammation and synaptic dysfunction, leading to learning and memory deficits. Therefore, Aß production and aggregation have been one of the most popular drug targets for AD. Failures of drug candidates regulating the aforementioned Aß cascade stimulated development of immunotherapy agents for clearance of accumulated Aß in the brain. Here, we report that quinacrine, a blood-brain barrier penetrating antimalarial chemical drug, dissociates Aß plaques in the brain of AD transgenic mice. When co-incubated with pre-formed Aß fibrils, quinacrine decreased thioflavin T-positive ß-sheets in vitro, on top of its inhibitory function on the fibril formation. We confirmed that quinacrine induced dissociation of high-molecular-weight Aß aggregates into low-molecular-weight species by dot blots in association with size cut-off filtrations. Quinacrine was then administered to adult 5XFAD transgenic mice via weekly intravenous injections for 6 weeks, and we found a significant reduction of Aß plaques and astrocytosis in their cortex and hippocampus. In western blots of quinacrine-administered mouse brains, amelioration of AD-related biomarkers, glial fibrillary acidic protein, postsynaptic protein 95, phosphorylated cAMP response element-binding protein, phosphorylated c-Jun N-terminal kinase were observed. Lastly, quinacrine-stimulated dissociation of misfolded aggregates induced recovery of synaptic function associated with Aß in excitatory post-synaptic current recordings of primary rat cortical neurons treated with Aß aggregates and quinacrine. Collectively, quinacrine can directly dissociate Aß fibrils and alleviate decreased synaptic functions.

A nonsense TMEM43 variant leads to disruption of connexin-linked function and autosomal dominant auditory neuropathy spectrum disorder.

Genes that are primarily expressed in cochlear glia-like supporting cells (GLSs) have not been clearly associated with progressive deafness. Herein, we present a deafness locus mapped to chromosome 3p25.1 and an auditory neuropathy spectrum disorder (ANSD) gene, TMEM43, mainly expressed in GLSs. We identify p.(Arg372Ter) of TMEM43 by linkage analysis and exome sequencing in two large Asian families segregating ANSD, which is characterized by inability to discriminate speech despite preserved sensitivity to sound. The knock-in mouse with the p.(Arg372Ter) variant recapitulates a progressive hearing loss with histological abnormalities in GLSs. Mechanistically, TMEM43 interacts with the Connexin26 and Connexin30 gap junction channels, disrupting the passive conductance current in GLSs in a dominant-negative fashion when the p.(Arg372Ter) variant is introduced. Based on these mechanistic insights, cochlear implant was performed on three subjects, and speech discrimination was successfully restored. Our study highlights a pathological role of cochlear GLSs by identifying a deafness gene and its causal relationship with ANSD.

Thirteen-week oral toxicity study of fermented ginseng, GBCK25, in Sprague-Dawley rats.

Ginseng (Panax ginseng) is commonly used in Asia as a medicinal herb. In particular, fermented ginseng, GBCK25, has been recently developed to increase ginsenoside absorption. It also has other beneficial biological effects such as hemodynamic and anti-inflammation functions. Here, we investigated the potential toxicity of GBCK25 in Sprague-Dawley rats following 13 weeks of GBCK25 treatment by oral gavage at doses of 250, 500, or 1000 mg/kg/day and reversible toxic effects over a 4-week recovery phase. Ten male and female rats per group were randomly allocated to the main toxicology groups and five male and female rats per group were allocated to the 0 and 1000 mg/kg/day recovery groups, respectively. There was no mortality; significant clinical toxicity or microscopic findings; and changes in body weight, food consumption, hematological parameters, serum biochemistry, or absolute and relative organ weights in any of the groups. In conclusion, there were no toxicological changes upon repeated oral gavage of GBCK25 at doses of 250, 500, or 1000 mg/kg/day in Sprague-Dawley rats over 13 weeks. The no-observed-adverse-effect level of GBCK25 was 1000 mg/kg/day in both sexes of Sprague-Dawley rat.

Aberrant Tonic Inhibition of Dopaminergic Neuronal Activity Causes Motor Symptoms in Animal Models of Parkinson's Disease.

Current pharmacological treatments for Parkinson's disease (PD) are focused on symptomatic relief, but not on disease modification, based on the strong belief that PD is caused by irreversible dopaminergic neuronal death. Thus, the concept of the presence of dormant dopaminergic neurons and its possibility as the disease-modifying therapeutic target against PD have not been explored. Here we show that optogenetic activation of substantia nigra pars compacta (SNpc) neurons alleviates parkinsonism in acute PD animal models by recovering tyrosine hydroxylase (TH) from the TH-negative dormant dopaminergic neurons, some of which still express DOPA decarboxylase (DDC). The TH loss depends on reduced dopaminergic neuronal firing under aberrant tonic inhibition, which is attributed to excessive astrocytic GABA. Blocking the astrocytic GABA synthesis recapitulates the therapeutic effect of optogenetic activation. Consistently, SNpc of postmortem PD patients shows a significant population of TH-negative/DDC-positive dormant neurons surrounded by numerous GABA-positive astrocytes. We propose that disinhibiting dormant dopaminergic neurons by blocking excessive astrocytic GABA could be an effective therapeutic strategy against PD.

Inhibitors of synaptic vesicle exocytosis reduce surface expression of postsynaptic glutamate receptors.

Bafilomycin A1, a vacuolar H+-ATPase inhibitor, and botulinum toxin B and tetanus toxin, both vesicle fusion inhibitors, are widely known exocytosis blockers that have been used to inhibit the presynaptic release of neurotransmitters. However, protein trafficking mechanisms, such as the insertion of postsynaptic receptors and astrocytic glutamate-releasing channels into the plasma membrane, also require exocytosis. In our previous study, exocytosis inhibitors reduced the surface expression of astrocytic glutamate-releasing channels. Here, we further investigated whether exocytosis inhibitors influence the surface expression of postsynaptic receptors. Using pH-sensitive superecliptic pHluorin (SEP)-tagged postsynaptic glutamate receptors, including GluA1, GluA2, GluN1, and GluN2A, we found that bafilomycin A1, botulinum toxin B, and/or tetanus toxin reduce the SEP fluorescence of SEP-GluA1, SEP-GluA2, SEP-GluN1, and SEP-GluN2A. These findings indicate that presynaptic vesicle exocytosis inhibitors also affect the postsynaptic trafficking machinery for surface expression. Finally, this study provides profound insights assembling presynaptic, postsynaptic and astrocytic viewpoints into the interpretation of the data obtained using these synaptic vesicle exocytosis inhibitors.

The potent psychomotor, rewarding and reinforcing properties of 3-fluoromethamphetamine in rodents.

3-fluoromethamphetamine (3-FMA), a derivative of methamphetamine (METH), produces behavioral impairment and deficits in dopaminergic transmission in the striatum of mice. The abuse potential of 3-FMA has not been fully characterized. The aim of this study was to evaluate the effects of 3-FMA on locomotor activity as well as its rewarding and reinforcing properties in the conditioned place preference (CPP) and self-administration procedures. Intravenous (i.v.) administration of 3-FMA (0.5 and 1.0 mg/kg) significantly increased locomotor activity in a dose-dependent manner in rats. In the CPP procedure, intraperitoneal administration of 3-FMA (10 and 30 mg/kg) produced a significant alteration in place preference in mice. In the self-administration paradigms, 3-FMA showed drug-taking behavior at the dose of 0.1 mg/kg/infusion (i.v.) during 2 hr sessions under fixed ratio schedules and high breakpoints at the dose of 0.3 and 1.0 mg/kg/infusion (i.v.) during 6 hr sessions under progressive ratio schedule of reinforcement in rats. A priming injection of 3-FMA (0.4 mg/kg, i.v.), METH (0.2 mg/kg, i.v.), or cocaine (2.0 mg/kg, i.v.) reinstated 3-FMA-seeking behavior after an extinction period in 3-FMA-trained rats during 2 hr session. Taken together, these findings demonstrate robust psychomotor, rewarding and reinforcing properties of 3-FMA, which may underlie its potential for compulsive use in humans.

Ultrasonic Neuromodulation via Astrocytic TRPA1.

Low-intensity, low-frequency ultrasound (LILFU) is the next-generation, non-invasive brain stimulation technology for treating various neurological and psychiatric disorders. However, the underlying cellular and molecular mechanism of LILFU-induced neuromodulation has remained unknown. Here, we report that LILFU-induced neuromodulation is initiated by opening of TRPA1 channels in astrocytes. The Ca2+ entry through TRPA1 causes a release of gliotransmitters including glutamate through Best1 channels in astrocytes. The released glutamate activates NMDA receptors in neighboring neurons to elicit action potential firing. Our results reveal an unprecedented mechanism of LILFU-induced neuromodulation, involving TRPA1 as a unique sensor for LILFU and glutamate-releasing Best1 as a mediator of glia-neuron interaction. These discoveries should prove to be useful for optimization of human brain stimulation and ultrasonogenetic manipulations of TRPA1.

Activation of Astrocytic µ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes.

Recently, µ-opioid receptor (MOR), one of the well-known Gi-protein coupled receptors (Gi-GPCR), was reported to be highly expressed in the hippocampal astrocytes. However, the role of astrocytic MOR has not been investigated. Here we report that activation of astrocytic MOR by [D-Ala2,N-MePhe4,Gly-ol]-enkephalin (DAMGO), a selective MOR agonist, causes a fast glutamate release using sniffer patch technique. We also found that the DAMGO-induced glutamate release was not observed in the astrocytes from MOR-deficient mice and MOR-short hairpin RNA (shRNA)-expressed astrocytes. In addition, the glutamate release was significantly reduced by gene silencing of the TREK-1-containing two-pore potassium (K2P) channel, which mediates passive conductance in astrocytes. Our findings were consistent with the previous study demonstrating that activation of Gi-GPCR such as cannabinoid receptor CB1 and adenosine receptor A1 causes a glutamate release through TREK-1-containing K2P channel from hippocampal astrocytes. We also demonstrated that MOR and TREK-1 are significantly co-localized in the hippocampal astrocytes. Furthermore, we found that both MOR and TREK-1-containing K2P channels are localized in the same subcellular compartments, soma and processes, of astrocytes. Our study raises a novel possibility that astrocytic MOR may participate in several physiological and pathological actions of opioids, including analgesia and addiction, through astrocytically released glutamate and its signaling pathway.

Regulation of myelin structure and conduction velocity by perinodal astrocytes.

The speed of impulse transmission is critical for optimal neural circuit function, but it is unclear how the appropriate conduction velocity is established in individual axons. The velocity of impulse transmission is influenced by the thickness of the myelin sheath and the morphology of electrogenic nodes of Ranvier along axons. Here we show that myelin thickness and nodal gap length are reversibly altered by astrocytes, glial cells that contact nodes of Ranvier. Thrombin-dependent proteolysis of a cell adhesion molecule that attaches myelin to the axon (neurofascin 155) is inhibited by vesicular release of thrombin protease inhibitors from perinodal astrocytes. Transgenic mice expressing a dominant-negative fragment of VAMP2 in astrocytes, to reduce exocytosis by 50%, exhibited detachment of adjacent paranodal loops of myelin from the axon, increased nodal gap length, and thinning of the myelin sheath in the optic nerve. These morphological changes alter the passive cable properties of axons to reduce conduction velocity and spike-time arrival in the CNS in parallel with a decrease in visual acuity. All effects were reversed by the thrombin inhibitor Fondaparinux. Similar results were obtained by viral transfection of tetanus toxin into astrocytes of rat corpus callosum. Previously, it was unknown how the myelin sheath could be thinned and the functions of perinodal astrocytes were not well understood. These findings describe a form of nervous system plasticity in which myelin structure and conduction velocity are adjusted by astrocytes. The thrombin-dependent cleavage of neurofascin 155 may also have relevance to myelin disruption and repair.

Visual stimulation-induced mild stress enhances cognitive behavior in cynomolgus monkey.

Cortisol is a well-known endogenous glucocorticoid that serves as a stress indicator. It is normally released under stressful condition to warn about imminent danger and thus is critical for survival of the species. However, it is unclear how cortisol relates to cognitive process under physiological condition in high-order primates such as non-human primates (NHP). Here, we report that a slight but significant increase in blood cortisol level by mild stress is positively correlated with the cognitive function in cynomolgus monkey. We stimulated 3 groups of monkeys by viewing consecutive series of pictures of monkeys, pictures of humans, or animation still pictures. We first found that the blood cortisol level was significantly higher during the stimulation session and returned to normal after stimulation session. Among the three types of pictures, the monkeys which were stimulated with monkey pictures showed the most significant increase in cortisol level during stimulation. Furthermore, the monkeys showed significantly enhanced manipulation, suggesting that cortisol affected cognitive processes. Overall, our study demonstrates that visual stimulation both increases blood cortisol and enhances manipulating behavior. Therefore, unlike the common notion that cortisol is a stress indicator, our data supports that a mild increase of cortisol enhances cognition in NHP.