presented by justin achua. supporting paper orexin (orx) also known as hypocretin plays a role in...

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

Positive Feedback

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