Optogenetic Manipulation of Cerebellar Purkinje Cell Activity In Vivo

Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex. Although their anatomical connections and physiological response properties have been extensively studied, the causal role of their activity in behavioral, cognitive and autonomic functions is still unclear because PC activity cannot be selectively controlled. Here we developed a novel technique using optogenetics for selective and rapidly reversible manipulation of PC activity in vivo. We injected into rat cerebellar cortex lentiviruses expressing either the light-activated cationic channel channelrhodopsin-2 (ChR2) or light-driven chloride pump halorhodopsin (eNpHR) under the control of the PC-specific L7 promoter. Transgene expression was observed in most PCs (ChR2, 92.6%; eNpHR, 95.3%), as determined by immunohistochemical analysis. In vivo electrophysiological recordings showed that all light-responsive PCs in ChR2-transduced rats increased frequency of simple spike in response to blue laser illumination. Similarly, most light-responsive PCs (93.8%) in eNpHR-transduced rats decreased frequency of simple spike in response to orange laser illumination. We then applied these techniques to characterize the roles of rat cerebellar uvula, one of the cardiovascular regulatory regions in the cerebellum, in resting blood pressure (BP) regulation in anesthetized rats. ChR2-mediated photostimulation and eNpHR-mediated photoinhibition of the uvula had opposite effects on resting BP, inducing depressor and pressor responses, respectively. In contrast, manipulation of PC activity within the neighboring lobule VIII had no effect on BP. Blue and orange laser illumination onto PBS-injected lobule IX didn't affect BP, indicating the observed effects on BP were actually due to PC activation and inhibition. These results clearly demonstrate that the optogenetic method we developed here will provide a powerful way to elucidate a causal relationship between local PC activity and functions of the cerebellum

Regional genome transcriptional response of adult mouse brain to hypoxia

<p>Abstract</p> <p>Background</p> <p>Since normal brain function depends upon continuous oxygen delivery and short periods of hypoxia can precondition the brain against subsequent ischemia, this study examined the effects of brief hypoxia on the whole genome transcriptional response in adult mouse brain.</p> <p>Result</p> <p>Pronounced changes of gene expression occurred after 3 hours of hypoxia (8% O₂) and after 1 hour of re-oxygenation in all brain regions. The hypoxia-responsive genes were predominantly up-regulated in hindbrain and predominantly down-regulated in forebrain - possibly to support hindbrain survival functions at the expense of forebrain cognitive functions. The up-regulated genes had a significant role in cell survival and involved both shared and unshared signaling pathways among different brain regions. Up-regulation of transcriptional signaling including hypoxia inducible factor, insulin growth factor (IGF), the vitamin D3 receptor/retinoid X nuclear receptor, and glucocorticoid signaling was common to many brain regions. However, many of the hypoxia-regulated target genes were specific for one or a few brain regions. Cerebellum, for example, had 1241 transcripts regulated by hypoxia only in cerebellum but not in hippocampus; and, 642 (54%) had at least one hepatic nuclear receptor 4A (HNF4A) binding site and 381 had at least two HNF4A binding sites in their promoters. The data point to HNF4A as a major hypoxia-responsive transcription factor in cerebellum in addition to its known role in regulating erythropoietin transcription. The genes unique to hindbrain may play critical roles in survival during hypoxia.</p> <p>Conclusion</p> <p>Differences of forebrain and hindbrain hypoxia-responsive genes may relate to suppression of forebrain cognitive functions and activation of hindbrain survival functions, which may coordinately mediate the neuroprotection afforded by hypoxia preconditioning.</p

Study on the bile pigment originating from hemogrobin

I made some experiments on the bile pigment formed at the destruction of the hemogrobin. Method. 1-2% O-Hb Ringer's solution or 1-2% CO-Hb Ringer's solution were perfused through the liver of toads (Bufo japonicos), and then the bile. pigment in the bile duct was measured by the absorption band of the ultraviolet ray, by the absorption band of the visible ray and by the variation of colour. Summary. 1) When O-Hb Ringer's solution was perfused through the liver the bile pigment was elimineted in the bile duct. It is just as the normal bile pigment in colour, in the absorption band of the ultraviolet ray and in the absorption band of the visible ray. 2) The elimination of the bile pigment from the liver is influenced by the poisoning with cyanic acid, i.e. it ceases entirely. 3) If CO-Hb Ringer's solution is perfused through the liver bile pigment is not eliminated

Study on the elimination of dyes from the liver

I made some experiment on the liver as I had done on the kidneys and summarize them as follows. Method. For this purpose I made use of the following dyes; phenolsulphone phthalein, rohodamin patent blue sup. patent blue V, chrysoidin, safranin, methylviolet, pironin, methylen blue, lithione carmin kongored, indulin, alkali blue. They were injected into the arterial circulation the blood being supplied by 0.56 Ringer' ssolution through the liver of toads (Bufo japonicus), and them the amount eliminated in the bile duct was measured. Summary. 1) 1 Phenol-sulphone phthalein, 2 rhodamin, 3 patent blue sup., 4 chrysoidin, 5 safranin, 6 methylviolet, 7 pironin, are eliminated from the liver and the amount eliminated is in the order indicated. 2) Hethylen blue, lithion carmin, kongored and indulin are eliminated from the liver only in small quantities or not at all. 3 Patent blue V. and alkali blue are not at all eriminated from the liver. 4) Elimination of the dyes from the liver is influenced by the poisoning with cyanic acid and stop. Accordingly, I have summalized as follows. The elimination of dyes I used, is not always in proportion with the degree of diffusion measured by gelation and agar-method, and it is always accompanied with oxydation

On the vaso-motor nerves supplying the stomach, the intestines, the colon, the spleen, and the kidneys

I made some experiments on toads (Bufo japonicus) for the purpose of the studying vaso-motor nerves supplying the stomach, the nitestines, the colon, the spleen, and the kidneys. These experiments were the followong; Method. I introduced Ringer's solution under constant pressure into one of the above mentioned organs through the vessels which supply it. The vessls going to other organs were completely ligatured. By this precaution the amount of Ringer's solution flowing out from the vein is made to imdicate the width of the perfused vessels. Then their nerves were stimulated by pinching or induction current. The results wer marked by recording the number of drops. 1) All vaso-motor nerve fibers leave the spinal cord through the anterior roots. 2) The vaso-motor nerve fibers going the stomach are found in the anterior roots of the 3rd, 4th, 5th, and 6th spinal nerve. These fibers passing through the 4th, 5th and 6th sympathetic ganglion concentrate in the soral ganglion passing through it and innervate to the stomach. As a rule, in the 4th spinal nerve the most vaso-motor nerve fibers are found, but are sometimes distributed over the 3rd and 5th. 3) The vaso-motor nerve fibers going to the intestine and the colon are found in the anterior roots of the 3rd, 4th, 5th, and 6th spinal nerve. These fibers passing through the 4th, 5th, and 6th sympathetic ganglion concentrate in the soral ganglion passing through it and innervate the intestine and the colon. As a rule, in the 3rd and 4th spinal nerve the most vaso-motor nerve fibers are found. 4) The vaso-motor nerve fibers going to the spleen are found in the anterior roots of the 3rd and 4th spinal nerve. These fibers passing through the 4th, 5th and 6th sympathetic ganglion concentrate in the soral ganglion passing through it and innervate the spleen. 5) The vaso-motor nerve fibers going to the kidney are found in the anterior roots of the 3rd, 4th, 5th, 6th, and 7th spinal nerve. These fibers passing through the 6th, 7th, 8th, and 9th, sympathetic ganglion, and innervate the kidney. As a rule, in the 6th spinal nerve the most vaso-motor nerve fibers are found

Study on the elimination of dyes from the kidneys

In spite of the many investigations regarding the elimination of the several dyes from the blood from the kidneys, since Heidenhain, there is as far as I know still a lack of systhematic investigation in relation to the diffusibity of dyes. Hoping to fill up this gap I made the following experiments on toads (Bufo japonicus).Method.For my purpose I used the following dyes; Phenol-sulphone phthalein, picric acid, chrysoidin, bismarckbrown, auranin, pyronin, eosin, methylen blue, safranin, patent blue V., rhodamin, extra B., methylviolet, fuchsin, neutralred, alkali blue, indulin, kongored, indigocarmin, carmin extra B,. As a preminary experiment I measured the rate of diffusion of these dyes on the gelatin and agar. The following list is arranged in the descending order of the diffusion rate of the dyes.1. phenol-sulphone phtalein, 2. picric acid, 3. rhodamin, 4. patent blue V., 5. chrysoidin, 6. pyronin, 7. safranin, 8. eosin, 9. fuchsin, 10. bismarckbrown, 11. methylen blue, 12. methylviolet B., 13. carmin, 14. neutralred, 15. kongored, 16. alkaliblue, 17. indulin. Summary. 1) 1. picric acid, 2. phenol-sulphone phthalein, 3. rhodamin, 4. patent blue V., 5. chrysoidin, 6. pyronin, 7. safranin, 8. eosin, 9. fuchsin, 10. bismarckblown, 11. methylen blue, 12. methylviolet, 13. carmin,. The above mentioned dyes are very easily eliminaten from the glomerular capsules but from the tubles the elimination is less in amount and the order is the one indicated above. 2) Neutralred, indulin, alkali blue and kongored are eliminated from the glomerular capsules, but not from the tubles. 3) The more elimination of dyes from the tubles abounds the greater is the diffusion. 4) When perfused from the renal portal vein only, the dyes were eliminated in the lumen, but there was either no elimination of water at all or it had been absorbed by the epithelium of the convoluted tubles. 5) The elimination of the dyes from the glomerular capsules is influenced neither by the supply of oxygen in the fluid-flow nor by poisoning with cyanic acid. The elimination of dyes is not always accompanied by oxydation. 6) The elimination of the dyes from the tubles is influenced both by th supply of oxygen in the fluid-flow, and by poisoning with cyanic acid, and elimination of the dyes is accompanied by oxydation. Accordingly, I summarize as follows.The glomerural capsules allow the diffusion into them of all the dyes, which were made use of in the experiments; and regarding the elimination of dyes from the tubles there can hardly be found an explanation in terms of physical chemistory

Cerebellar Control of Defense Reactions under Orexin-mediated Neuromodulation as a Model of Cerebellohypothalamic Interaction

Recent evidence has indicated that, when an animal is exposed to harmful stimuli, hypothalamic orexinergic neurons are activated via the amygdala and in turn tune the neuronal circuits in the spinal cord, brainstem, and an area of the cerebellum (folium-p of the flocculus) by neuromodulation. The animal would then initiate “defense reactions” composed of complex movements and associated cardiovascular responses. To investigate neuronal mechanisms of the defense reactions, Nisimaru et al. (2013) analyzed cardiovascular responses induced by an electric foot shock stimulus to a rabbit and found two major effects. One is redistribution of arterial blood flow from visceral organs to active muscles, and the other is a modest increase in blood pressure. Kainate-induced lesions of folium-p impaired these two effects. Moreover, folium-p Purkinje cells were shown to project to the parabrachial nucleus, one of the major cardiovascular centers in the brainstem. These data indicate that folium-p Purkinje cells regulate cardiovascular defense reactions via parabrachial nucleus under orexin-mediated neuromodulation. In this article, we review these data from the viewpoint that the defense reactions are expressions of “anger and anxiety”, which respectively lead to “fight and flight” behaviors. The present orexin case may provide a model of cerebellohypothalamic interactions via neuropeptides or amines of hypothalamic origin, which may underlie various types of emotion and behavior