1. Orexin and Fear Memory
Luke

2. We have seen that:
Orx modifies processes in thermogenesis
Sleep deprivation modifies Orx sensitivity to GABAergic inhibition
Exercise restores Orx response in the male PFA after early life stress (but not females)

3. Orexin/Hypocretin Involved in: Reward Emotion
Affects heart rate/blood pressure/metabolic rate
Promotes spontaneous physical activity
Influences neuroprotection against ischemic damage
Arousal states

4. What about…
Memory?
Specifically:
Fear related memory

5. Importance
Role in memory processes
PTSD
Stress and anxiety disorders

6. Conditioned-Fear Stress Increases Fos Expression in Monoaminergic and GABAergic Neurons of the Locus Coeruleus and Dorsal Raphe Nuclei
Ishida et al. 2002

7. Introduction LC Fos ↑ in brain tissue from stressors
Restraint
Footshock
Anxiety
Will conditioned fear (CF) cause Fos expression in brainstem monoaminergic nuclei?
VTA DR LC

8. Also looked at colocalization of:TH
5-HT
GABA

9. Methods Male Wistar rats 3 groups
Conditioned-reexposed
Conditioned-not reexposed
Not conditioned-reexposed
Conditioning was contextual cues from testing apparatus

10. Immunohistochemical examination
For Fos
Double-labeling
Fos and: TH
5-HT
GABA

12. Results
Just saying: Yes, they were conditioned.
Mann-Whitney U-test

13. Fos+ cells significantly increased in DR & LC, but not VTA
Newman-Keuls test

14. Double-labeled brain sections of conditioned reexposed
Double-labeled brain sections of conditioned reexposed. A: DR Fos+/5-HT B: LC Fos+/TH C: DR Fos+/GABA D: LC Fos+/GABA
Black = fos white = other arrow = coexpression

15. LC: Fos+/TH 63% Fos+/GABA 60% DR: Fos+/5-HT 52% Fos+/GABA 60%

16. Summary
CF may induce intense Fos expression in serotonergic DR and noradrenergic LC neurons
Not dopaminergic VTA neurons (possibly because nonspecific contextual stimuli, mild stressor, only two sessions)
To show activity in LC from conditioned/learned fear

17. Orexin/Hypocretin System Modulates Amygdala-dependent Threat Learning Through the Locus Coeruleus
Sears et al. 2013

18. Introduction Activation of OrxR1/2 commonly increases excitability by:
Reducing potassium channel conductance
Enhancing presynaptic glutamate release
Increasing postsynaptic NMDA conductance
OrxR2  maintenance of arousal or wakefulness
OrxR1  responses to environmental stimuli

19. Overactivity 
Exacerbate panic-like episodes
Anxiety-like phenotype in rats
Conversely, almorexant can blunt autonomic and behavioral responses to stress
Evidence suggests orexin neurons modulate LC responses to salient sensory events
Almorexant = OrxR1/2 antagonist

20. Orexin could contribute to aversive learning
Through LC
Norephinephrine’s importance to aversive memory processes in the amygdala
Pavlovian threat (fear) conditioning
Well established paradigm to assess the formation, storage, and expression of aversive memories

21. Methods Sprague-Dawley rats Weak training protocol ICV….
CS = series of auditory pips
US = footshock/blue light laser
ICV….
Direct injections
Optogenetics
Immunohistochemistry
Whole cell clamp recordings

22. Results – Regulation of Threat Learning
Test the hypothesis that  orexin important for aversive memory formation
Block OrxR1 using SB (ICV)
OrxR2 via TCS-OX2-29
Different times during conditioning

23. A = timeline. B = Mean freezing data for OrxR1antagonist
A = timeline B = Mean freezing data for OrxR1antagonist C= to rule out nonselective effect of OrxR1antagonist, use OrxR2antagonist

24. SB 334867 SB pre-LTM training  impaired freezing
SB post-LTM training  no effect
SB pre-LTM testing  no effect
OrxR1 activation required for normal formation of threat memories
Not due to absence of activation during consolidation

25. TCS-OX2-29 TCS pre-LTM training  No effect
Suggests no nonselective effect of SB on OrxR2

26. Results – Direct LC injection

27. SB 300 ng pre-LTM training no significance
SB 1 ug pre-LTM training reduced freezing
SB 1 ug post-LTM training  no significance
SB 1 ug pre-STM training  reduced freezing
Suggests OrxR1 in LC required for proper aversive memory formation
STM test  LC OrxR1 important for acquisition
Supplemental materials: not due to shock sensitivity between ICV vs direct
Also did in lateral nucleus of the amygdala (LA)
no significance

28. Results – Optogenetic orexin fibers
Channelrhodopsin-2 (ChR2) expressed in medial and perifornical hypothalamus orexin fields  LC
Infection efficiency ≈ 45%
mCherry = control  no optogenetic activation
Application of AMPA & OrxR1 blockers
Fast and slow depolarization components measured

29. In vitro stimulation of ChR2-expressing fibers from orexin-immunopositive neurons activates LC neurons. (A) Schematic depicting strategy for targeting orexin neurons in medial PFH and projections to LC. (B) Neurons in the hypothalamus transduced with LV-Hcrt::ChR2-mCherry colabel with mCherry (red) and orexin (green). Image depicts infected (arrow) and uninfected (arrowhead only) orexin-A immunopositive neurons. All infected cells were orexin-A immunoreactive. (Scale bar, 50 μM.) (C) mCherry-immunopositive fibers project to and innervate DBH-immunopositive neurons in LC. (Scale bar, 50 μM.) (D) (Upper) Schematic of recording from LC cells in a horizontal brainstem slice. (Lower) Spontaneous firing characteristic of LC neurons used to identify cells after patching. Calibration bars: 200 ms and 40 mV. (E) Averaged traces (five sweeps each) from a representative LC cell receiving orexin inputs; stimulation is a 10-pulse train of illumination at 20 Hz (pulse width = 10 ms). The blue line indicates the mean baseline response (in 0.1% DMSO), the orange line the response from the same cell after 15 min in CNQX (10 μM), and the black line the response after 10 additional min bathed in CNQX and SB (10 μM). Calibration bars: 100 ms and 5 mV. (F) (Left) Bar plot indicating the mean decrease in initial EPSP amplitude after application of CNQX and CNQX + SB (Right) Bar plot indicating a decrease in area under the synaptic response after application of CNQX and CNQX + SB The right bars in each plot represent values after 28 min of washout in control ACSF (0.1% DMSO). All values are normalized as a percent of the baseline response and are presented as mean ± SEM. One-way ANOVA, *P < 0.01 CNQX vs. baseline, #P < 0.05 CNQX vs. SB + CNQX.

30. CNQX addition CNQX + SB addition
Blocked ≈ 79% of fast EPSP
Blocked ≈ 77% of slow EPSP
CNQX + SB addition
Blocked ≈ 93% of fast EPSP
Blocked ≈ 99% of slow EPSP
Demonstrate that orexin expressing cells of the PFH corelease both orexin and glutamate to mediate direct, rapid, and robust depolarization of LC cells

31. Supplemental materials:
SB alone
Fast -No significant difference but was a negative trend
Slow – no significant difference
SNR shows synaptic signature of orexin is small relative to AMPA in co-expressing neurons
Taken together, may be small, yet can still make the difference (enhance learning)

32. Results – optogenetic activation in vivo
Cannulae above LC. B: cfos expression after light C: cfos increase only on light stimulation side of brain D: increased freezing from baseline levels E: to ensure that this mechanism occurred through OrxR1

33. Optogenetic stimulation in vivo
Increased freezing behavior
OrxR1 antagonism with optogenetic stimulation
Reduced freezing behavior

34. Results – circuit disconnection
To test whether the orexin-LC circuit influences aversive learning downstream (amygdala) SB
Propranolol (βAR antagonist)

36. Ipsilateral drug infusions in LC & LA  no effect
Contralateral  reduced freezing
Hypothalamus – LC – LA circuit necessary for normal threat memory formation
Enhances threat memory via NE release to LA

37. Summary
OrxR1 activation required for normal formation of threat memories
Not due to absence of activation during consolidation
Suggests OrxR1 in LC required for proper aversive memory formation
STM test  LC OrxR1 important for acquisition

38. Summary
Demonstrate that orexin expressing cells of the PFH co-release both orexin and glutamate to mediate direct, rapid, and robust depolarization of LC cells
Optogenetic stimulation of PFH Orexin neurons enhances threat memory
Hypothalamus – LC – LA circuit necessary for normal threat memory formation
Enhances threat memory via NE release to LA

39. Orexin Receptor-1 in the Locus Coeruleus Plays an Important Role in Cue-Dependent Fear Memory Consolidation
Soya et al. 2013

40. Introduction Orexin potently excites LC-NA neurons
Conditioned fear stress causes a robust increase in NA LC neuron firing
NA input from LC to LA is a key factor in fear memory formation

41. Methods 12-14 week old male mice OrxR1 KO mice OrxR2 KO mice
Experiments performed during light phase

42. Cued fear-conditioning test
Contextual fear-conditioning test
CS  80 dB tone
US  mild foot shock
Tested 24 hr after training (LTM in Sears et al.)

43. Adeno-associated virus (AAV)
Rat OrxR1 for restoration
ChR2 for control
Only used if OrxR1 found precisely in LC-NA neurons
Zif268 to assess amygdala activity
AAV used to reintroduce OrxR1

44. Results - Cued
A: timeline for cued test B: freezing every 30 sec during conditioning C: freezing every 30 sec during testing D: total freezing time during testing

45. OrxR1 KO  decreased freezing during conditioning
OrxR2 KO  negative trend during conditioning
OrxR1 KO  decreased freezing during testing w/ and w/out CS
OrxR2 KO  no significance
Also tested sensitivity to shock due to KOs
Excluded possibility that decreased response due to decreased sensitivity

46. Results - Contextual

47. OrxR1 KO  decreased freezing during conditioning
OrxR1 KO  decreased freezing during testing
OrxR2 KO  decreased freezing during testing

48. Results suggest:
OrxR1 involved in cued and contextual fear conditioning
Further suggests: abnormalities in both evoking of fear related behavior and formation of fear memory
OrxR2 involved only in contextual

49. Results – double-labeling
A: timeline TH/Fos Saying: fewer NA LC neurons activated with OrxR1 KO

50. OrxR1 KO  fewer double labeled cells after all but homecage
Meaning:
Fewer LC-NA neurons activated after cued and contextual fear in OrxR1 KO mice
OrxR1 mediated pathway activates LC-NA neurons in emotionally relevant situations

51. Results – AAV restoration

52. Cued Conditioning: Cued Testing:
KO restoration had no significance compared to KO control
Both lower freezing time compared to WT
Cued Testing:
KO control significantly lower freezing time than KO restoration
Suggests: OrxR1 importance in consolidation, retrieval, and presentation of cue-dependent fear memory rather than emergence of fear-related behavior

53. Contextual conditioning:
KO restoration group had lower freezing in conditioning and testing than WT
Suggests: OrxR1 restoration not sufficient to rescue formation of fear memory in contextual situations
Emergence of a behavioral response to US do not depend on OrxR1 in LC-NA neurons
Depend on OrxR1 in other brain regions

54. Results – LA activation?

55. Homecage  no difference in Zif268
After Cued  fewer Zif268 labeled cells
After Contextual  no significance but trend of fewer Zif268 labeled cells
Meaning:
Reduced activation of LA in OrxR1 KO after cued testing

57. Cued
OrxR1 KO restoration:
Restored Fos expression in LC
Restored Zif268 expression in LA
OrxR1 KO control
No effect
Contextual
OrxR1 KO restoration
Did not restore Zif268
Suggests: OrxR1 important for amygdala activation through LC-NA neurons in cued fear memory (not contextual)

58. Summary
Results suggest OrxR1 involved in both evoking of fear related behavior and formation of fear memory
Fewer LC-NA neurons activated after cued and contextual fear in OrxR1 KO mice
OrxR1 mediated pathway activates LC-NA neurons in emotionally relevant situations
OrxR1 importance in consolidation, retrieval, and presentation of cue-dependent fear memory rather than emergence of fear-related behavior

59. Summary
Suggests: OrxR1 restoration not sufficient to rescue formation of fear memory in contextual situations
Emergence of a behavioral response to US do not depend on OrxR1 in LC-NA neurons
Depend on OrxR1 in other brain regions
Reduced activation of LA in OrxR1 KO after cued testing
OrxR1 important for amygdala activation through LC- NA neurons in cued fear memory (not contextual)

60. Overall Discussion Sears LC OrxR1 Soya  LC OrxR1
OrxR1 not important for consolidation
Important for acquisition
Soya  LC OrxR1
OrxR1 important for consolidation
Behavioral responses to US not due to LC OrxR1

61. Take Home Messages
CF may induce intense Fos expression in serotonergic DR and noradrenergic LC neurons
Demonstrate that orexin expressing cells of the PFH co- release both orexin and glutamate to mediate direct, rapid, and robust depolarization of LC cells
Suggests OrxR1 in LC required for proper aversive memory formation
Optogenetic stimulation of PFH Orexin neurons enhances threat memory

62. Take Home Messages
Hypothalamus – LC – LA circuit necessary for normal threat memory formation
Results suggest OrxR1 involved in both evoking of fear related behavior and formation of fear memory
OrxR1 importance in consolidation, retrieval, and presentation of cue-dependent fear memory rather than emergence of fear-related behavior
OrxR1 important for amygdala activation through LC-NA neurons in cued fear memory (not contextual)