Orexin and Fear Memory Luke

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)

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

What about… Memory? Specifically: Fear related memory

Importance Role in memory processes PTSD Stress and anxiety disorders

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

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

Also looked at colocalization of: TH 5-HT GABA

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

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

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

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

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

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

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

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

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

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

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

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

Results – Regulation of Threat Learning Test the hypothesis that  orexin important for aversive memory formation Block OrxR1 using SB 334867 (ICV) OrxR2 via TCS-OX2-29 Different times during conditioning

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

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

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

Results – Direct LC injection

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

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

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 334867 (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 334867. (Right) Bar plot indicating a decrease in area under the synaptic response after application of CNQX and CNQX + SB 334867. 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.

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

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)

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

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

Results – circuit disconnection To test whether the orexin-LC circuit influences aversive learning downstream (amygdala) SB 334867 Propranolol (βAR antagonist)

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

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

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

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

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

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

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.)

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

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

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

Results - Contextual

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

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

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

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

Results – AAV restoration

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

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

Results – LA activation?

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

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)

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

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)

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

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

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)