Glucose d erived A strocytal Lactate mediates Orexin neurons during wakefulness

1. Glucose derived Astrocytal Lactate mediates Orexin neurons during wakefulness Presented by Justin Achua

2. Orexin Supporting Paper

3. Orexin and its functions • Orexin (Orx) also known as Hypocretin • Plays a role in circadian rhythms • Sleep/wake cycles • Feeding cycles • Promotes Arousal • Reward systems

4. Narcolepsy… narcolepsy? • 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 …

5. ALSO! • Disturbed nocturnal sleep

6. Orexinergic neuron firing • 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

7. Orexinergic neuron firing • 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?

8. Location, location, location

9. Location, location, location • 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

10. Allen brain atlas

11. Orexinergic projections

12. Lactate Secreting Astrocytes Supporting Paper

13. Lactate production

14. Monocarboxylate transporters (MCT) • 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

15. Additional info Metabolized lactate Glutamate bursts Astrocytes generate lactate Could be used by neighboring neurons following bursts of glutamate Could other astrocytes also use the lactate? • In vitro brain preparations • Survive on lactate • Inherent capacity to take up and metabolize lactate

16. K+ channel mediated Lactate Effect on Orexinergic neurons Main Paper

17. Overview 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

18. How do we regulate all this? • 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? Orx Glucose Circadian Rhy thms

19. The missing piece • Orx neurons are excited by lactate… • Astrocytes release lactate… • Astrocytes are EVERYWHERE in the brain… • Orx neurons are in the brain! Orx Glucose Astro cytes lactate Circadian Rhy thms

20. Patch clamp recordings method • 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

21. Do Orx neurons use lactate? • 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!

22. Do we even need glucose? • 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

23. Which side is this happening on? • Tetrodotoxin (TTX) used in 0mM glucose environment • TTX blocks neurochemical factor reuptake into presynaptic terminal • Hyperpolarization observed in postsynaptic terminal • Suggesting direct postsynaptic effect

24. Direct astrocytal activation? • 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

25. Lactate Acetate Silenced Unsilenced

26. SO!? What is happening here? • 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

27. 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

28. What about non-spontaneous firing? • 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

30. KATP channels • Kir6.1 and SUR1 subunits comprised Orx KATP channels

32. KATP channels • Cytosolic solution diluted with ATP-free pipette solution • Induced glibenclamide-sensitive outward curent • Mitochondrial uncouplerm-chloropheny-lhydrazone (CCCP) was bath applied • Inhibits metabolism • Effects also blocked by glibenclamide • KATP channels in Orx neurons are SENSITIVE to metabolic state

33. KATP channels

34. Lactate sensors • 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!

35. Does concentration of glucose affect lactose? • Glucose conc. changed from 2.5mM – 1mM • Hypothalamic slices allowed to adjust 30 – 60 min • Firing activity independent of glucose concentration

36. How much glucose is too much? • 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

38. Natural glucose levels • Brain glucose levels drop to 0.2 and 0.7 mM • Insulin-induced hypoglycemia • Overnight fasting

39. Glucose conc. and firing rate • 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

41. Lactate potentiating firing • 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

43. Excitatory inputs • 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

44. Take home • 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

45. Big picture! Positive Feedback

46. Thank you! Have a great weekend!