A novel rat robot controlled by electrical stimulation of the nigrostriatal pathway.

OBJECTIVE: Artificial manipulation of animal movement could offer interesting advantages and potential applications using the animal's inherited superior sensation and mobility. Although several behavior control models have been introduced, they generally epitomize virtual reward-based training models. In this model, rats are trained multiple times so they can recall the relationship between cues and rewards. It is well known that activation of one side of the nigrostriatal pathway (NSP) in the rat induces immediate turning toward the contralateral side. However, this NSP stimulation-induced directional movement has not been used for the purpose of animal-robot navigation. In this study, the authors aimed to electrically stimulate the NSP of conscious rats to build a command-prompt rat robot. METHODS: Repetitive NSP stimulation at 1-second intervals was applied via implanted electrodes to induce immediate contraversive turning movements in 7 rats in open field tests in the absence of any sensory cues or rewards. The rats were manipulated to navigate from the start arm to a target zone in either the left or right arm of a T-maze. A leftward trial was followed by a rightward trial, and each rat completed a total of 10 trials. In the control group, 7 rats were tested in the same way without NSP stimulation. The time taken to navigate the maze was compared between experimental and control groups. RESULTS: All rats in the experimental group successfully reached the target area for all 70 trials in a short period of time with a short interstimulus interval (< 0.7 seconds), but only 41% of rats in the control group reached the target area and required a longer period of time to do so. The experimental group made correct directional turning movements at the intersection zone of the T-maze, taking significantly less time than the control group. No significant difference in navigation duration for the forward movements on the start and goal arms was observed between the two groups. However, the experimental group showed quick and accurate movement at the intersection zone, which made the difference in the success rate and elapsed time of tasks. CONCLUSIONS: The results of this study clearly indicate that a rat-robot model based on NSP stimulation can be a practical alternative to previously reported models controlled by virtual sensory cues and rewards.

Rostral Agranular Insular Cortex Lesion with Motor Cortex Stimulation Enhances Pain Modulation Effect on Neuropathic Pain Model.

It is well known that the insular cortex is involved in the processing of painful input. The aim of this study was to evaluate the pain modulation role of the insular cortex during motor cortex stimulation (MCS). After inducing neuropathic pain (NP) rat models by the spared nerve injury method, we made a lesion on the rostral agranular insular cortex (RAIC) unilaterally and compared behaviorally determined pain threshold and latency in 2 groups: Group A (NP + MCS; n = 7) and Group B (NP + RAIC lesion + MCS; n = 7). Also, we simultaneously recorded neuronal activity (NP; n = 9) in the thalamus of the ventral posterolateral nucleus and RAIC to evaluate electrophysiological changes from MCS. The pain threshold and tolerance latency increased in Group A with "MCS on" and in Group B with or without "MCS on." Moreover, its increase in Group B with "MCS on" was more than that of Group B without MCS or of Group A, suggesting that MCS and RAIC lesioning are involved in pain modulation. Compared with the "MCS off" condition, the "MCS on" induced significant threshold changes in an electrophysiological study. Our data suggest that the RAIC has its own pain modulation effect, which is influenced by MCS.

Neuroprotection of posttreatment with risperidone, an atypical antipsychotic drug, in rat and gerbil models of ischemic stroke and the maintenance of antioxidants in a gerbil model of ischemic stroke.

Risperidone, an atypical antipsychotic drug, has been discovered to have some beneficial effects beyond its original effectiveness. The present study examines the neuroprotective effects of risperidone against ischemic damage in the rat and gerbil induced by transient focal and global cerebral ischemia, respectively. The results showed that pre- and posttreatment with 4 mg/kg risperidone significantly protected against neuronal death from ischemic injury. Many NeuN-immunoreactive neurons and a few F-J B-positive cells were found in the rat cerebral cortex and gerbil hippocampal CA1 region (CA1) in the risperidone-treated ischemia groups compared with those in the vehicle-treated ischemia group. In addition, treatment with risperidone markedly attenuated the activation of microglia in the gerbil CA1. On the other hand, we found that treatment with risperidone significantly maintained the antioxidants levels in the ischemic gerbil CA1. Immunoreactivities of superoxide dismutases 1 and 2, catalase, and glutathione peroxidase were maintained in the stratum pyramidale of the CA1; the antioxidants were very different from those in the vehicle-treated ischemia groups. In brief, our present findings indicate that posttreatment as well as pretreatment with risperidone can protect neurons in the rat cerebral cortex and gerbils CA1 from transient cerebral ischemic injury and that the neuroprotective effect of risperidone may be related to attenuation of microglial activation as well as maintenance of antioxidants.

Comparison of α-synuclein immunoreactivity in the hippocampus between the adult and aged beagle dogs.

Alpha-synuclein (α-syn), as a neuroprotein, is expressed in neural tissue, and it is related to a synaptic transmission and neuronal plasticity. In this study, we compared the distribution and immunoreactivity of α-syn and related gliosis in hippocampus between young adult (2-3 years) and aged (10-12 years) beagle dogs. In both groups, α-syn immunoreactivity was detected in neuropil of all the hippocampal sub-regions, but not in neuronal somata. In the aged hippocampus, α-syn immunoreactivity was apparently increased in mossy fibers compared to that in the adult dog. In addition, α-syn protein level was markedly increased in the aged hippocampus. On the other hand, GFAP and Iba-1 immunoreactivity in astrocytes and microglia, respectively, were increased in all the hippocampal sub-regions of the aged group compared to that in the adult group: especially, their immunoreactivity was apparently increased around mossy fibers. In addition, in this study, we could not find any expression of α-syn in astrocytes and microglia. These results indicate that α-syn immunoreactivity apparently increases in the aged hippocampus and that GFAP and Iba-1 immunoreactivity are also apparently increased at the regions with increased α-syn immunoreactivity. This increase in α-syn expression might be a feature of normal aging.

Comparison of expression of inflammatory cytokines in the spinal cord between young adult and aged beagle dogs.

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2-3 years old) and aged (10-12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.

Effects of transient cerebral ischemia on the expression of DNA methyltransferase 1 in the gerbil hippocampal CA1 region.

DNA methylation is a key epigenetic modification of DNA that is catalyzed by DNA methyltransferases (Dnmt). Increasing evidences suggest that DNA methylation in neurons regulates synaptic plasticity as well as neuronal network activity. In the present study, we investigated the changes in DNA methyltransferases 1 (Dnmt1) immunoreactivity and its protein levels in the gerbil hippocampal CA1 region after 5 min of transient global cerebral ischemia. CA1 pyramidal neurons were well stained with NeuN (a neuron-specific soluble nuclear antigen) antibody in the sham-group, Four days after ischemia-reperfusion (I-R), NeuN-positive ((+)) cells were significantly decreased in the stratum pyramidale (SP) of the CA1 region, and many Fluro-Jade B (a marker for neuronal degeneration)(+) cells were observed in the SP. Dnmt1 immunoreactivity was well detected in all the layers of the sham-group. Dnmt1 immunoreactivity was hardly detected only in the stratum pyramidale of the CA1 region from 4 days post-ischemia; however, at these times, Dnmt1 immunoreactivity was newly expressed in GABAergic interneurons or astrocytes in the ischemic CA1 region. In addition, the level of Dnmt1 was lowest at 4 days post-ischemia. In brief, both the Dnmt1 immunoreactivity and protein levels were distinctively decreased in the ischemic CA1 region 4 days after transient cerebral ischemia. These results indicate that the decrease of Dnmt1 expression at 4 days post-ischemia may be related to ischemia-induced delayed neuronal death.

Neuronal Responses in the Globus Pallidus during Subthalamic Nucleus Electrical Stimulation in Normal and Parkinson's Disease Model Rats.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) has been widely used as a treatment for the movement disturbances caused by Parkinson's disease (PD). Despite successful application of DBS, its mechanism of therapeutic effect is not clearly understood. Because PD results from the degeneration of dopamine neurons that affect the basal ganglia (BG) network, investigation of neuronal responses of BG neurons during STN DBS can provide informative insights for the understanding of the mechanism of therapeutic effect. However, it is difficult to observe neuronal activity during DBS because of large stimulation artifacts. Here, we report the observation of neuronal activities of the globus pallidus (GP) in normal and PD model rats during electrical stimulation of the STN. A custom artifact removal technique was devised to enable monitoring of neural activity during stimulation. We investigated how GP neurons responded to STN stimulation at various stimulation frequencies (10, 50, 90 and 130 Hz). It was observed that activities of GP neurons were modulated by stimulation frequency of the STN and significantly inhibited by high frequency stimulation above 50 Hz. These findings suggest that GP neuronal activity is effectively modulated by STN stimulation and strongly dependent on the frequency of stimulation.

Comparison of glial activation in the hippocampal CA1 region between the young and adult gerbils after transient cerebral ischemia.

It has been reported that young animals are less vulnerable to brain ischemia. In the present study, we compared gliosis in the hippocampal CA1 region of the young gerbil with those in the adult gerbil induced by 5 min of transient cerebral ischemia by immunohistochemistry and western blot for glial cells. We used male gerbils of postnatal month 1 (PM 1) as the young and PM 6 as the adult. Neuronal death in CA1 pyramidal neurons in the adult gerbil occurred at 4 days post-ischemia; the neuronal death in the young gerbil occurred at 7 days post-ischemia. The findings of glial changes in the young gerbil after ischemic damage were distinctively different from those in the adult gerbil. Glial fibrillary acidic protein-immunoreactive astrocytes, ionized calcium-binding adapter molecule (Iba-1), and isolectin B4-immunoreactive microglia in the ischemic CA1 region were activated much later in the young gerbil than in the adult gerbil. In brief, very less gliosis occurred in the hippocampal CA1 region of the young gerbil than in the adult gerbil after transient cerebral ischemia.

Increased immunoreactivities of cleaved αII-spectrin and cleaved caspase-3 in the aged dog spinal cord.

The activation of caspase-3 is considered to be a reliable marker for apoptotic cell death, and a 120-kDa fragment of αII-spectrin is generated by caspase-3 mediated cleavage of this structural protein. In the present study, we compared cleaved αII-spectrin (120-kDa) and cleaved caspase-3-immunoreactive cells and their protein levels in the cervical (C5-C6) and lumbar (L3-L4) levels of the spinal cord in adult (1-2 year-old) and aged (10-12 year-old) dogs (German shepherds). Weak cleaved αII-spectrin and cleaved caspase-3 immunoreactivity was found in neurons of the adult group; however, their immunoreactivity was distinctively increased in the neuronal cytoplasm in the aged group compared to those in the adult group, although the distribution pattern of their neurons was similar between the adult and age group. In addition, cleaved αII-spectrin and cleaved caspase-3 levels in the aged spinal cord were markedly increased compared to those in the adult group. These findings suggest that the increases of cleaved αII-spectrin and cleaved caspase-3 immunoreactivity may be related to aging of the spinal cord in dogs.

Comparison of GAD65 and 67 immunoreactivity in the lumbar spinal cord between young adult and aged dogs.

We investigated distribution and age-related changes in two isoforms of GABA synthesizing enzymes, glutamic acid decarboxylase (GAD) 65 and 67, in the lumbar levels (L(5)-L(6)) of the dog spinal cord. Male German shepherds were used at 1-2 years (young adult dogs) and 10-12 years (aged dogs) of age. GAD65 immunoreaction was observed in neuropil, not in cell bodies, in all laminae of the adult lumbar spinal cord: Many punctate GAD65-immunoreactive structures were shown in all laminae. The density of GAD65 immunoreactive structures was highest in laminae I-III, and lowest in lamina VII. In the aged dog, the distribution pattern of GAD65 immunoreactivity was similar to that in the adult dog; however the density of GAD65-immunoreactive structures and its protein levels were significantly increased in the aged lumbar spinal cord. GAD67 immunoreaction in the adult dog was also distributed in all laminae of the lumbar spinal cord like GAD65; however, we found that small GAD67-immunoreactive cell bodies were observed in laminae II, III and VIII. In the aged dogs, GAD67 immunoreactivity and its protein levels were also increased compared to those in the adult group. In conclusion, our results indicate that the distribution of GAD65-immunoreactive structures is different from GAD67-immunoreactive structures and that their immunoreactivity in the aged dogs is much higher than the adult dogs.

Relation among neuronal death, cell proliferation and neuronal differentiation in the gerbil main olfactory bulb after transient cerebral ischemia.

Neurogenesis occurs during the embryonic stage and throughout life. Brain injuries such as ischemic insults enhance cell proliferation in some areas of the brain. We examined proliferation of newly generated cells in each layer of the gerbil main olfactory bulb (MOB) after 5 min of transient cerebral ischemia using bromodeoxyuridine (BrdU) immunohistochemistry. Ischemia-related neuronal death in the MOB was not detected using Fluoro-Jade B histofluorescence and TUNEL staining. Many BrdU-positive ((+)) cells were found in the rostral migratory stream in control and ischemic MOBs. Significant increase of BrdU(+) cells was observed in the granule cell layer (GCL) and glomerular layer (GL) from 15 days post-ischemia, and BrdU(+) cells were very much higher than those of the control group 30 days post-ischemia. At this time point after ischemia/reperfusion, a few BrdU(+) cells in the GL and GCL were co-localized with calretinin(+) cells, and many BrdU(+) cells expressed doublecortin, a marker of immature neurons. These results indicate that cell proliferation is increased in the GCL and GL without apparent neuronal loss from 15 days after transient cerebral ischemia in gerbils.

Age-related changes in calbindin-D28k, parvalbumin, and calretinin immunoreactivity in the dog main olfactory bulb.

Expression and age-related changes of calbindin-D28k (CB), parvalbumin (PV), and calretinin (CR) in the main olfactory bulb of the dog were investigated by immunohistochemistry and western blot analysis. Neurons that expressed these calcium-binding proteins showed a characteristic laminar distribution. Most of CB-immunoreactive neurons were observed in the glomerular layer (GL) and the inner sublayer of the external plexiform layer (EPL). Most of PV-immunoreactive neurons were observed in the outer sublayer of the EPL. CR-immunoreactive neurons were mainly distributed in the GL and the granule cell layer. With regard to age-related changes, CB-immunoreactive neurons in the GL were stable among all age groups; however, in the EPL they decreased with age. PV-immunoreactive neurons decreased in middle-aged and aged groups. However, CR-immunoreactive neurons were not decreased in middle-aged and aged groups. These results suggest that CB-immunoreactive neurons in the EPL were most sensitive to aging, and that their reduction may be related to aging in the dog.

Transient cerebral ischemia induces active astrocytosis without distinct neuronal death in the gerbil main olfactory bulb: a long-term analysis.

In the present study, we examined ischemia-induced neuronal and glial changes in the gerbil MOB at various time points during 60 days after 5 min of transient cerebral ischemia. The number of neuronal neuclei-immunoreactive neurons was not changed after ischemia/reperfusion (I/R). Myelin basic protein immunoreaction was well preserved after I/R. Five days after I/R, reactive form of GFAP-immunoreactive astrocytes began to increase in the external plexiform layer and granule cell layer: These reactive astrocytes peaked 10 days after I/R, thereafter, they decreased with time after I/R. Iba-1-immunoreactive microglia were ubiquitously distributed in all layers of the MOB. After I/R, significant changes in their morphology and immunoreactivity were not detected. The results of western blot analyses for GFAP, Iba-1 and MBP were similar to the immunohistochemical data. In addition, 8-hydroxy-2'-deoxyguanosine (a marker for DNA damage) immunoreactivity and SOD1, an antioxidant, protein levels were not changed in the ischemic MOB. These results indicate that neurons in the MOB are resistant to ischemic insult, showing that astrocytes are activated late in the ischemic MOB.

Age-dependent changes in calretinin immunoreactivity and its protein level in the gerbil hippocampus.

Calretinin (CR)-immunoreactive interneurons are well known as the interneuron specific interneurons in the hippocampus. CR-immunoreactive neurons form cellular network and regulate the activity of other GABAergic inhibitory interneurons in the hippocampus. In the present study, we investigated age-related changes in CR-immunoreactive neurons and protein levels in the gerbil hippocampus during normal aging. In all subregions of the gerbil hippocampus, the number of CR-immunoreactive neurons was significantly decreased in the postnatal month 6 (PM 6) group compared to that in the PM 1 group. Thereafter, CR-immunoreactive neurons were decreased with age. In addition, the number of CR-immunoreactive cells in the subgranular zone were significantly decreased in the PM 6 group. We also observed that CR protein levels were decreased gradually with age. These results indicate that both CR immunoreactivity and its protein level were decreased with age in the gerbil hippocampus during normal aging.

(-)-Epigallocatechin-3-gallate increases cell proliferation and neuroblasts in the subgranular zone of the dentate gyrus in adult mice.

Neurogenesis is regulated by several factors such as age, stress and pharmacological agents. We observed the effects of (-)-epigallocatechin-3-gallate (EGCG), a major catechin of green tea, on neurogenesis in mice. The animals were orally administered EGCG for 4 weeks. Brain sections were stained using a marker for cell proliferation (Ki67 and BrdU) and neuroblasts (doublecortin, DCX). In all groups, Ki67, BrdU and DCX immunoreaction were observed in the subgranular zone of the dentate gyrus. Oral administration of EGCG significantly increased the number of Ki67-, BrdU- and DCX-immunoreactive cells as well as BrdU/DCX-colabled cells in the subgranular zone when compared to those in the vehicle-treated group. These results indicate that oral administration of EGCG can enhance cell proliferation and increase the number of neuroblasts in mice hippocampal dentate gyrus.

Species-difference of cyclooxygenase-2 in the hippocampus of rodents.

Cyclooxygenase (COX) generates free radicals and it is important in inflammatory response. In this study, we observed the immunoreactivity in mice (ICR and C57BL/6 strain), rats and gerbils. In these animals, COX-2 immunoreactivity was mainly detected in pyramidal cells of the hippocampal CA2/3 region and in granule cells of the dentate gyrus. COX-2 immunoreactivity in the CA2/3 region was the highest in ICR mice, while in gerbils COX-2 immunoreactivity was the lowest; COX-2 immunoreactivity in the dentate gyrus was the highest in rats and the lowest in gerbils. The protein levels of COX-2 were similar to the immunohistochemical data. COX-2 mRNA transcript was the highest in the gerbil and the lowest in the rat. In brief, COX-2 protein, not mRNA, in the hippocampus is generally higher in mice (ICR and C57BL/6 strain) than rats and gerbils.

Age-related changes in the insulin receptor ß in the gerbil hippocampus.

The insulin receptor has been reported to be associated with memory formation via the hippocampus. In this study, we observed age-related changes in the insulin receptor ß immunoreactivity and its protein levels in the hippocampus of gerbils of various ages in order to identify the correlation between the insulin receptor ß and aging processes in the hippocampus. Insulin receptor ß immunoreactivity was mainly detected in the molecular and polymorphic layers of the dentate gyrus, and in mossy fibers, Schaffer collaterals, alveus and stratum lacunosum-moleculare of the hippocampus proper (CA1-3) of gerbils at postnatal month 1 (PM 1). Insulin receptor ß immunoreactivity decreased with age in all of these structures, except for the alveus. Reduction of the insulin receptor ß immunoreactivity was prominent in the molecular layer of the dentate gyrus at PM 6 and in the stratum lacunosum-moleculare of the CA1 region at PM 12, while insulin receptor ß immunoreactivity was decreased in other regions in the PM 18 groups. In addition, insulin receptor ß protein level in the whole hippocampus was slightly increased at PM 3, and it decreased in an age-dependent manner from PM 6 to PM 24. These reductions of the insulin receptor ß in the hippocampus may be associated with age-related memory deficits in gerbils.

Classification of BMI control commands from rat's neural signals using extreme learning machine.

A recently developed machine learning algorithm referred to as Extreme Learning Machine (ELM) was used to classify machine control commands out of time series of spike trains of ensembles of CA1 hippocampus neurons (n = 34) of a rat, which was performing a target-to-goal task on a two-dimensional space through a brain-machine interface system. Performance of ELM was analyzed in terms of training time and classification accuracy. The results showed that some processes such as class code prefix, redundancy code suffix and smoothing effect of the classifiers' outputs could improve the accuracy of classification of robot control commands for a brain-machine interface system.

Immunohistochemical changes in orexigenic and anorexigenic neuropeptides in the rat hypothalamus after capsaicin administration.

Capsaicin has effects on the adiposity by increasing energy and lipid metabolism, and decreases appetite and fat intake. In the present study, we investigated changes in food intake and body weight after capsaicin treatment. We also observed changes in orexigenic and anorexigenic neuropeptides-agouti-related peptide (AgRP), alpha-melanocyte-stimulating hormone (alpha-MSH), adrenocorticotropic hormone (ACTH) and orexin-immunoreactivities in the rat hypothalamus after capsaicin administration. Only one day after capsaicin treatment, the mean food intake was significantly decreased. There was no significant difference in the mean body weight between vehicle- and capsaicin-treated groups. In addition, after capsaicin treatment, numbers of AgRP- and orexin-immunoreactive ((+)) cells were significantly decreased in the arcuate nucleus (ARC) and lateral hypothalamic area, respectively. In contrast, the number of alpha-MSH(+) and ACTH(+) cells in the ARC of the capsaicin-treated rats was higher than in the vehicle-treated rats. These results indicate that capsaicin reduces food intake, not body weight, transiently, and decreases AgRP and orexin immunoreactivities, whereas it increases alpha-MSH and ACTH immunoreactivities in rat hypothalamic nuclei.

Neuronal degeneration and microglial activation in the ischemic dentate gyrus of the gerbil.

In the present study, we investigated the time-course changes of neuronal degeneration and microglial activation in the gerbil dentate gyrus after transient cerebral ischemia using Fluoro-Jade B histofluorescence staining and immunohistochemistry for Iba-1. Fluoro-Jade B positive cells were observed from 6 hr and markedly increased 1 day after ischemia/reperfusion. Iba-1-immunoreactive microglia were increased and hypertrophied at early time, and Iba-1 immunoreactivity was highest at 2 days after ischemia/reperfusion. These results may be direct evidence on neuronal degeneration and microglial activation in the gerbil dentate gyrus after ischemia/reperfusion.