Transcript Luke Fuhrman - USD Biology

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)
Involved in:
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Reward
Emotion
Affects heart rate/blood pressure/metabolic rate
Promotes spontaneous physical activity
Influences neuroprotection against ischemic
damage
• Arousal states
 Memory?
 Specifically:
 Fear related memory
 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
 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
 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
Mann-Whitney U-test
Newman-Keuls test
 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
 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
 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
 Sprague-Dawley rats
 Weak training protocol
 CS = series of auditory pips
 US = footshock/blue light laser
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ICV….
Direct injections
Optogenetics
Immunohistochemistry
Whole cell clamp recordings
 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
 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 pre-LTM training  No effect
 Suggests no nonselective effect of SB on OrxR2
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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
 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
 CNQX 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)
 Optogenetic stimulation in vivo
 Increased freezing behavior
 OrxR1 antagonism with optogenetic stimulation
 Reduced freezing behavior
 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
 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
 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
 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
 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
 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
 OrxR1 KO  decreased freezing during conditioning
 OrxR2 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
 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
 Cued Conditioning:
 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
 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
 Restored Fos expression in LC
 Did not restore Zif268
 Suggests: OrxR1 important for amygdala activation
through LC-NA neurons in cued fear memory (not
contextual)
 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
 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)
 Sears 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
 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
 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)