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Glucose d erived A strocytal Lactate mediates Orexin neurons during wakefulness. Presented by Justin Achua. Orexin. Supporting Paper. Orexin and its functions. Orexin ( Orx ) also known as Hypocretin Plays a role in circadian rhythms Sleep/wake cycles Feeding cycles Promotes Arousal - PowerPoint PPT Presentation
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Presented by Justin Achua
Supporting Paper
• Orexin (Orx) also known as Hypocretin
• Plays a role in circadian rhythms• Sleep/wake cycles• Feeding cycles• Promotes Arousal• Reward systems
• Diminished levels of Orx leads to symptoms of narcolepsy• Failure to wake in response to lack of food
• Orx wards off sleep• Injections of Orx shown to interrupt slept and promote
wakeful periods
Narcolepsy - The body’s inability to regulate sleep/wake cycles• Abnormal daytime sleep…
• Disturbed nocturnal sleep
• Fire during active waking
• Decrease firing during quiet waking• Almost cease firing during sleep
aW – active wakefulnessqW – quiet wakefulnesstSWS – transition to non-REM sleepSWS – non-REM sleeptPS – transition to REM sleepPS – REM sleep
• Several seconds before the end of rapid eye movement (REM) sleep firing begins again
• Definite change in skeletal muscle firing during aW• Observed at end of REM sleep (PS)
• EEG recording reading?
• Stained hypothalamic neurons• Response recorded cells stained red• Orx cells stained green
• Measured Orx cells colored yellow (red + green)• All Orx cells located in LH and PFA
Supporting Paper
• Two monocarboxylate transporters• MCT1 transporters distributed in astrocytes• MCT2 transporters distributed in axons
• Lactate levels increase in a cell• MCTs transport excess lactate
across cell membrane• MCT1 best for lactate export• MCT2 best for lactate uptake
Metabolized lactate• In vitro brain
preparations• Survive on lactate • Inherent capacity to take
up and metabolize lactate
Glutamate bursts• Astrocytes generate
lactate• Could be used by
neighboring neurons following bursts of glutamate
• Could other astrocytes also use the lactate?
Main Paper
Active neurons need vast amounts of energy substrates•Brain is ~2% of body weight, 25% of total body glucose utilization•Neurons can use glucose
• Glucose excited neurons• Glucose inhibited neurons
•Lactose is main energy substrate
• Astrocytes are main cells that metabolize glucose• Orx neurons detect rapid change in glucose levels
• Glucose inhibited• Adapt to new glucose level within several minutes
• Glucose fluctuations in later hypothalamus are slow and long
• How do these fit together?
Circadian Rhy thms
Glucose
Orx
• Orx neurons are excited by lactate…
• Astrocytes release lactate…
• Astrocytes are EVERYWHERE in the brain…
• Orx neurons are in the brain!
Circadian Rhy thms
Glucose
Orx
lactate
Astro cytes
• Electrode placed around receptor/channel• Stimulation• Neurotransmitter
administration• Membrane sampling
• Cell-attached measures a single receptor/channel
• Whole-cell ruptures membrane, allows for change/measurement of intracellular fluid
• α-cyano-4-hydroxycinnamate (4-CIN)• Specific monocarboxylate transporter (MCTs) inhibitor
• Prevents lactate from crossing plasma membrane
• When applied to Orx neurons, firing significantly inhibited
• Orx neurons DO use lactate!
• Extracellular glucose was removed for 20 min• Inability of Orx neurons to fire• Very similar effects observed in 4-CIN firing experiment
• Extracellular glucose is NECESSARY for spontaneous firing
• Tetrodotoxin (TTX) used in 0mM glucose environment
• TTX blocks neurochemical factor reuptake into presynaptic terminal
• Hyperpolarization observed in postsynaptic terminal• Suggesting direct postsynaptic effect
• Astrocytes are excited by both lactate and acetate• Neurons do not use acetate
• If direct activation both lactate and acetate should excite astrocyte• Astrocyte should in turn activate Orx neuron
• Synapse remained silenced in presence of acetate
Lactate Acetate
Unsilenced Silenced
• Astrocytes can metabolize glucose into lactate• Can be released into extracellular space
• Hypothalamic brain slices treated with fluoroacetate (FAC)• FAC is a glial toxin
• Extracellular glucose removed during final 20 min• Glucose or lactate applied in presence of FAC
• Recordings found FAC prevented glucose from reversing firing inhibition
• Lactate restored firing in presence of FAC
• Lactate is NEEDED to maintain spontaneous firing in Orx neurons
• Lactate IS synthesized from glucose and released endogenously by astrocytes
• ATP-sensitive potassium channels (KATP)• Blocking of channel (gibenclamide) blocked
hyperpolarization due to deprivation of glucose• Also lead to irreversible cell damage (over-firing)
• KATP mediates lactate effect on firing frequency• May play a neuroprotective role in Orx neurons
• KATP channel blocker reduced the effects of 4-CIN• Effect not seen in various other tissues
• Lactate availability is MONOITORED by KATP channels
• Kir6.1 and SUR1 subunits comprised Orx KATP channels
• Cytosolic solution diluted with ATP-free pipette solution• Induced glibenclamide-sensitive outward curent
• Mitochondrial uncoupler m-chloropheny-lhydrazone (CCCP) was bath applied• Inhibits metabolism • Effects also blocked by glibenclamide
• KATP channels in Orx neurons are SENSITIVE to metabolic state
• Hypothalamic slices of Orx neurons bathed in various concentrations of lactate• In absence of glucose• ~20 minutes to incubate
• Effect of firing rate found to be concentration dependent• Orx neurons CAN act as lactate sensors!
• Glucose conc. changed from 2.5mM – 1mM• Hypothalamic slices allowed to adjust 30 – 60 min
• Firing activity independent of glucose concentration
• Thought that 1mM glucose past lactate conc. Plateau
• Administration of 4-CIN to measure glucose levels• 4-CIN competitive blocker, competes against lactate on
membrane transporter
• 4-CIN shown to significantly decrease firing at 1mM glucose• Orx cells can be depolarized using tolbutamide• Suggests activation of KATP channels
• Saturated at >1mM glucose
• Brain glucose levels drop to 0.2 and 0.7 mM• Insulin-induced hypoglycemia• Overnight fasting
• Firing frequency and inhibition of neurons conc. dependent• Firing frequency plateaus at 2.5mM
• Latency to inhibit independent of glucose conc.• Neurons have individual energy stores
• KATP channels can be ACTIVATED by low conc. of endogenous glucose
• Orx neuron response to positive current injections• Recorded in presence/absence of lactate
• Presence of lactate• Baseline activity increased• Firing frequency increased
• Low levels of available energy substrates• Decreases basal firing rate• Blunts pulsatile firing
• Orx neurons co-express glutamate• Primary excitatory neurotransmitter
• Activated Orx neuron can trigger positive feedback system• Recruit additional lactate releasing Orx neurons• Lactate release accompanies decreased pH
• Orx neurons are excited at low pH• Additive excitatory effects
• Orx neurons activated more efficiently with lactate• Lactate signals adequate energy substrates• Orx promotes wakefulness, food intake, and glucose
production
• Lactate is an important regulator of the Orexin system• Depends on astrocyte derived lactate• Concentration dependent
• KATP channels reduced excitability• Plays a neuroprotective role
• Orx neurons can temporarily maintain activity in absence of energy substrates• Neurons may have energy stores
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