Institute of Experimental Medicine, Budapest

1. Institute of Experimental Medicine, Budapest Brain mechanisms of PTSD Glutamatergic plasticity and PTSD Long-term effects of trauma exposure Long-term effects of trauma exposure Novel approches to PTSD Cannabinoids in PTSD József Haller 1/22

2. "Incubation" phase (1-6 month) The development of persistent symptoms Trauma Early response Acute Stress Disorder ~1 month Silent period What is PTSD? PTSD = Post-Traumatic Stress Disorder An anxiety disorder that develops in response to strong stressors 2/22

3. re-experiencing avoiding cue-associated cues hyperarousal Trauma (life-threatening stress) + psychosis depression violence drug addiction anxiety, etc. How does it look like? Is it simply anxiety? DSM-IV criteria for PTSD Triggering factor (Cluster A):traumatic event(1) life threatening(2) helpless Reexperiencing the trauma (ClusterB)(1) distressing recollections(2) distressing dreams (3) reliving the experience by illusions, hallucinations, and dissociative flashbacks (4) psychological distress when faced with trauma-related cues(5)psychological distress when faced withtrauma-relatedcues Avoidance and numbing (ClusterC)(1) avoidingtrauma-related thoughts, feelings, or conversations(2) avoidingtrauma-related activities, places, or people(3) inability to recall an important aspect of the trauma (4) diminished interest in significant activities (5) detachment from others (6) restricted range of affect (7) sense of a foreshortened future Hyperarousal (ClusterD) (1) sleep problems (2) irritability (3) difficulty concentrating (4) hypervigilance (5) exaggerated startle response Duration is more than 1 month (ClusterE) Number of symptoms "required" within a cluster 2 2 3 2 1 A single exposure to a traumatic stress induces a dramatic worsening of in psychological functioning A remarkable example of neuronal plasticity It usully lasts years or decades 3/22

4. Prevalence of PTSD (%) Risk factor Combat Prevalence in the general population Terrorist attack 90 100 60 70 80 10 20 30 40 50 0 Natural disaster (earthquake) Forced migration 10 Assaultive violence Family violence 8 6 and in their mothers Prevalence of PTSD in the general population (%) Trafic accidents, children Diabetes, children 4 and in their mothers 2 AIDS Certain profesions (ambulance) 0 1 2 3 4 Study Nr. 5 6 7 8 Average likelyhood Unemployment Trauma in mothers, PTSD in child How important it is? Risk factors and risks similar to the prevalence of other anxiety disorders General perception: People in general are not at risk. I'm safe. Factors prenatal stress early life stress traumatic stressors in adulthood Factors leading to PTSD are parts of daily life Certain stressors predictably lead to PTSD Breslau et al., 1991; Breslau et al., 1999; Clohessy and Ehlers, 1999; Creamer et al., 2001; Goowin and Davidson, 2004; Kessler et al., 1995; Landolt et al., 2005; Marshall et al., 2005; Olley et al., 2005; Perkonnig et al., 2000; Resnik et al., 1993; Rosenman, 2002; Seedat et al., 2005; Shalev and Freedman, 2005 4/22

5. plasticity of glutamatergic neurotransmission 300 15 * 200 a 10 a open arm exploration (%time) opponent time (sec) b 100 5 b 0 0 Kontroll 0 0.05 0.1mg/kg MK-801 Kontroll 0 0.05 0.1mg/kg MK-801 electric shock 24 hours earlier cat exposure 24 h earlier Adamec et al., 1999 How does it develop? Glutamatergic plasticity Trauma Behavioral deficits The blockade of NMDA receptors by MK-801 blocks the development of trauma-related deficits 5/22 Haller et al., 2006

6. plasticity of glutamatergic neurotransmission 300 a a serotonin serotonin 200 opponent time (sec) b b - - 100 c plasticity of glutamatergic neurotransmission 0 Sham 5-HTx NAx xx Behavioral deficits + noradrenalin Treatment NMDA blockers are highly debilitating and addictive ("angel dust") SSRIs Problems with these treatments less well tolerated than placebo efficacy: significant but often poor certain symptoms respond less well effects are often gender-dependent Trauma Behavioral deficits There are better ways to control glutamatergic plasticity alpha 1 or beta blockers Serotonin and noradrenaline modulate but not mediate trauma-induced deficits 6/22

7. Detailed functional maping of cortical networks Department of functional and cellular neuroanatomy (T.F. Freund) Theory on the role of particular neurons Theory-based approach Developing a behavioral model Unbiased approach Detailed maping of neuronal activation patterns Detailed analysis of activated neurons (e.g. chemotype identification, network analysis, etc.) Testing the theory by behavioral pharmacology Department of Behavioral Neurobiology (J. Haller) Describing the neural control of the behavior in question - new treatment approaches If the theory is right: new treatment approaches Theory driven approach Unbiased approach (applied earlier in identifying the neural background of violent forms of aggression and proposing NK1 blockers for treatment) 7/22

8. The theory: a neuron made for anxiety Specifically involved in anxiety Calretinin cell CCK - B receptors CCK basket cell 5-HT3 receptors Parvalbumin basket cell CB1 cannabinoid receptors serotonergic innervation M2 muscarinic receptors CB1 cannabinoid receptorok Nicotinic receptors with α4 and α7 subunits GABAA receptorok α2 alegységgel GABAA receptors mainly with α1 subunits GABAA receptors mainly with α2 subunits 8/22

9. Endocannabinoids CB1 receptors Cannabinoid signaling and anxiety Specifically involved in anxiety Calretinin cell CCK - B receptors GABA GABA CCK basket cell 5-HT3 receptors Parvalbumin basket cell CB1 cannabinoid receptors Axon terminal Postsynaptic membrane serotonergic innervation M2 muscarinic receptors CB1 cannabinoid receptorok Nicotinic receptors with α4 and α7 subunits GABAA receptorok α2 alegységgel GABAA receptors mainly with α1 subunits GABAA receptors mainly with α2 subunits 9/22

10. 40 30 20 Open arm exploration (% time) 10 * 0 * * * * * WT CB1-KO WT CB1-KO WT CB1-KO WT CB1-KO 40 CD1 mice Saline WIN-55,212 AM-251 AM+WIN * 30 20 Open arm exploration (% time) 10 0 Sal Ana 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Sal Ana minutes Saline minutes Andamide minutes Saline minutes Andamide Testing the hypothesis and ...strange findings with endogenous cannabinoids Coward No changes in locomotion Brave mice called by phone Subtle changes in endogenous cannabinoid signalling change the response of mice to environmental stimuli 10/22

11. CB1/x receptors Glutamate receptors CB1 cannabinoid receptors PTSD and the response to environmental stimuli re-experiencing(e.g. exaggerated responses to cues) avoiding cue-associated cues hyperarousal(e.g. hypervigilence) Trauma (life-threatening stress) Trauma-related deficits are glutamate-dependent Cannabinoids (i) control the response to stimuli (neurobehavioral evidence) (ii) control glutamatergic discharges (neurophysiological evidence) Cannabinoids must have a role in PTSD 11/22

12. 100 100 80 80 60 60 * Exploration (%time) Resting (%time) * 40 40 * * 20 20 0 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Time (min) Time (min) * * 100 * * 80 60 * Freezing (%time) * 40 o 20 24 hours 0 1 2 3 4 5 6 7 8 9 10 Time (min) The conditioned fear test as model of PTSD Model of post-traumatic stress disorder Symptom modelled Cluster "B" re-experiencing the trauma by symptom 4 intense psychological distress in response to trauma-associated cues 12/22

13. 40 * 30 20 freezing (%time) 40 * * * CB1 gene disruptionabolishes conditioned freezing 10 # 30 * 0 20 freezing (%time) 1 2 3 4 5 time (min) 10 Open-field (mice shocked 24h earlier) 400 control shock AM251 1mg/kg 0 1 2 3 4 5 shock shock AM251 3mg/kg 300 time (min) counts WT-control KO-control 200 KO-shock WT-shock 100 # # 100 # # 80 # 0 # 60 WIN0 WIN03 WIN1 WIN3 freezing (%time) 40 * * 20 * * Cannabinoids promote shock-induced freezing (the trauma-related behavioral dysfunction) 0 CB1 blockade slightly decreases conditioned freezing CB1 activation increases conditioned freezing The antagonist blocks the effects of the agonist 1 2 3 4 5 time (min) shock WIN55,212 1mg/kg control shock WIN55,212 3mg/kg shock # # 100 # # 80 # 60 freezing (%time) 40 20 0 5 1 2 3 4 time (min) shock shock WIN03 shockWIN03AM1 shockWIN3 shockWIN3AM1 The interaction between cannabinoid signaling and conditioned fear AM-251 had no effect on locomotion up to 55 mg/kg (literature) 13/22

14. Contextual conditioned fear (hippocampus-specific) Cue-induced conditioned fear (amygdala-specific) Cannabinoids and contextual and cue-induced conditioned fear Cannabinoids promote behavioral dysfunctions induced by traumatic experience this appears to be valid for behaviors largely controlled by the hippocampus Cannabinoids promote the extinction of aversive memories this is apparent in behaviors largely controlled by the amygdala Different distribution ofCB1 receptors in,and/or different rolesof cannabinoids in controlling the,amygdala and hippocampus? Involvement of other corticalstructures (e.g prefrontal cortex)in the differential effects? Specific involvement in trauma? Unpecific effect (consequence of memory-releted effects)? Haller et al., 2006 Marsicano et al., 2002 The involvement of cannabinoids in trauma-related behavioral dysfunctions suggests that cannabinoid signaling is involved in post-traumatic stress disorder Opposed to effects on memory Consistent with effects on memory Cannabinoids promote the expression of fear Cannabinoids promote the extinction of fear (no effect on expression) 14/22

15. Interactions between 5-HT3 and CB1 receptors in conditioned fear 5-HT3 receptors CB1 cannabinoid receptors 15/22

16. Elevated plus-maze 40 30 Sal 1 mg/kg 20 3 mg/kg 10 mg/kg 10 Conditioned fear vehicle, no shock 0 shock, vehicle Closed entries % time open arms % open entries 35 shock, 3 mg/kg mCPBG # # 30 # # shock, 10 mg/kg mCPBG 25 # 20 freezing (% time) 15 10 5 0 1 2 3 4 5 6 7 8 9 10 min The effects of the 5-HT3 agonist mCPBG on anxiety The 5-HT3 agonist does not affect plus-maze anxiety (natural fear) The same agonist prolongs the conditioned fear response without affecting amplitude 16/22

17. 40 * 30 20 freezing (%time) * 10 # 0 1 2 3 4 5 35 time (min) 30 control shock AM251 1mg/kg 25 shock shock AM251 3mg/kg 20 Freezing (%time) 15 10 5 0 1 2 3 4 5 6 7 8 9 10 min For comparison: the effects of AM-251 anxiolytic CCK interneuron GABA CCK anxiogenic Concurrent 5-HT3 agonism and CB1 blockade abolishes conditioned fear Conditioned fear vehicle, no shock Cannabinoids -alone or in combination- are potentially important in the treatment of trauma-induced behavioral deficits shock, vehicle shock, mCPBG3mg/kg shock, mCPBG 3 mg/kg + AM-251 0.3 mg/kg shock, mCPBG 3 mg/kg + AM-251 1 mg/kg One problem: was the model good enough to draw this conclusion? Conclusion There is an unexpectedly strong interaction between mechanisms mediated by 5-HT3 and CB1 receptors, that may be involved in ther control of behavioral dysfunctons induced by trauma exposure 17/22

18. Glucocorticoid excess Helplessness Behavioral suppression Mania Anhedonia Despair Etc. The usual way of modeling psychiatric disorders (1) Tearing symptoms apart (depressionis just an example) (3) Bringing the bits together - theoretical perfection (3) What often happens in practice A better solution: modeling the disorder. Is this possible? This may be feasible in PTSD where all symptoms have one single cause: the trauma (2) Building models for each symptom Learned helplessness Forced swimming Chronic mild stress Bulbectomy /open field Chronic restraint Forced submission Etc. 18/22

19. A symptom by symtom analysis of a PTSD model "New pharmacological approaches need new behavioral methods" Traumas employed in the laboratory Electric shocks of various strength, durations, aggravated or not with cue reminders Cat exposure Suffocation stress Restraint + forced swim + halothane or ether Behavioreal deficits investigated Limited number (usually one), often after a limited time interval Can a complex disorder be modelled simply? Question asked: can we create a rat in which PTSD can be DIAGNOSED? 19/22

20. 1 day after shock 28 days after shock 100 unshocked 0.8 mA shocks 3 mA shocks unshocked 0.8 mA shocks 3 mA shocks 80 * * DSM-IV criteria for PTSD Triggering factor (Cluster A):traumatic event(1) life threatening(2) helpless Reexperiencing the trauma (ClusterB)(1) distressing recollections(2) distressing dreams (3) reliving the experience by illusions, hallucinations, and dissociative flashbacks (4) psychological distress when faced with trauma-related cues(5) psychological distress when faced withtrauma-relatedcues Avoidance and numbing (ClusterC)(1) avoidingtrauma-related thoughts, feelings, or conversations(2) avoidingtrauma-related activities, places, or people(3) inability to recall an important aspect of the trauma (4) diminished interest in significant activities (5) detachment from others (6) restricted range of affect (7) sense of a foreshortened future Hyperarousal (ClusterD) (1) sleep problems (2) irritability (3) difficulty concentrating (4) hypervigilance (5) exaggerated startle response Duration is more than 1 month(ClusterE) Number of symptoms "required" within a cluster 2 2 3 2 1 * 60 Duration (%time) * 25 days after shocks 1 day before shocks 40 460 * eD= early dark phase mD = mid dark phase lD= late dark phase eL= early light phase mL = mid light phase lL= late light phase Unshocked control Rats shocked with 3 mA currents 20 * 100 420 0 Freezing Escape jumps Freezing Escape jumps 80 *# 100 Heart rate 380 * 60 unshocked 0.8 mA shocks 3 mA shocks % time in opponent's chamber * 40 80 340 control *# 20 12 Elevated plus-maze 10 8 6 4 2 0 400 Open field 3mA 300 unshocked, object 1 unshocked object 2 shocked (3 mA) object 1 shocked (3 mA) object 2 * 60 0 Heart rate changes compared to baseline lLAverage eD 1 day after shock mD lD eL mL 28 days after shock eD mD lD eL mL lL Average 300 % time * * * * 120 200 * Closeed arm entries 40 Line crossings (exploration) * * * * 100 80 * * 20 0 40 12 Days after shock 6 24 12 Days after shock 6 24 0 0 Average Object Object -5 T 5 10 20 30 minutes manipulation burying 25 Rez. intr. + * 20 + 120 + 15 + 80 + % time * 10 40 5 0 0 SOC AGO Average -5 T 5 10 20 30 Deficits seen in rats receiving 10, 1 sec long, 0.8 or 3 mA shocks over 5 min The duration of the deficits is at least 4 weeks Also consider the life-span of rats 12 days after shock 20/22

21. DSM-IV criteria for PTSD Triggering factor (Cluster A):traumatic event(1) life threatening(2) helpless Reexperiencing the trauma (ClusterB)(1) distressing recollections(2) distressing dreams (3) reliving the experience by illusions, hallucinations, and dissociative flashbacks (4) psychological distress when faced with trauma-related cues(5) psychological distress when faced withtrauma-relatedcues Avoidance and numbing (ClusterC)(1) avoidingtrauma-related thoughts, feelings, or conversations(2) avoidingtrauma-related activities, places, or people(3) inability to recall an important aspect of the trauma (4) diminished interest in significant activities (5) detachment from others (6) restricted range of affect (7) sense of a foreshortened future Hyperarousal (ClusterD) (1) sleep problems (2) irritability (3) difficulty concentrating (4) hypervigilance (5) exaggerated startle response Duration is more than 1 month(ClusterE) Number of symptoms "required" within a cluster 2 2 3 2 1 Can the disorder be modelled as a whole?           Rats shocked with 3 mA currents reach a state that can be considered an animal variant of PTSD 21/22

22. The next step Sound theory supported by experimental evidence GO! Good model 22/22