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 wakefulness qW – quiet wakefulness tSWS – transition to non-REM sleep SWS – non-REM sleep tPS – transition to REM sleep PS – 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

LactateAcetate 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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