Examining how arousal systems and stress exposure modulate fear and extinction memory processes
By Dayan Knox
Dayan Knox is an assistant professor in the behavioral neuroscience program in the Department of Psychology at the University of Delaware. Knox received his graduate training in the psychobiology program at The Ohio State University, where he examined the effects of physiological arousal on cortical activity and the role of basal forebrain corticopetal cholinergic neurons in modulating fear memory. His postdoctoral work, in the Department of Psychiatry at the University of Michigan Medical School, focused on the effects of traumatic stress on fear and extinction memory, and on neurobiological processes relevant to post-traumatic stress disorder. Knox continues to address these and related topics, using a combination of behavioral, molecular and electrophysiological methods.
Fear and extinction memory have important roles to play in our everyday lives. Forming fear memories based on prior threat exposure is adaptive and helpful. For example, seeing someone attacked by an unleashed dog in a park may generate fear when you see the park again. This fear memory may then drive avoidance of the park, which will lower your chances of being attacked by a dog. However, learning when fear behavior is inappropriate is also beneficial. To continue the example, if after walking by the same park for a month you witness no more dog attacks, the fear generated from your previous memory may subside. Believing you no longer have a reason to fear the park is indicative of the formation of a new extinction memory (i.e. the park is now safe), which may allow you to enjoy the pleasant features of this hypothetical park (e.g. swings, fish pond, flowering trees). Detrimental psychological functioning could also stem from aberrant fear and extinction memory. Enhanced fear memory and/or deficits in extinction memory have been implicated in anxiety disorders such as phobia and post-traumatic stress disorder (PTSD). Below, I describe some of my research investigating the neurobiology of fear and extinction memory.
The role of BFCCNs in facilitating the modulatory effect of physiological arousal on cortical activity
For my first year project as a graduate student I was assigned the task of investigating the role of basal forebrain corticopetal cholinergic neurons (BFCCNs) in facilitating the modulatory effect that physiological arousal has on cortical activity. BFCCNs are clusters of cholinergic neurons in the base of the forebrain that provide acetylcholine to the neocortex, and can serve as an extra-thalamic relay of sensory information (Jones, 2004). BFCCNs receive input from arousal systems such as noradrenergic neurons in the locus coeruleus (Zaborszky & Cullinan, 1996), have a powerful modulatory effect on cortical activity (Jones, 2004), and have been implicated in a number of psychological processes such as attention (Sarter & Bruno, 1997) and motor learning (Conner, Culberson, Packowski, Chiba, & Tuszynski, 2003).
Enhanced physiological arousal is a common characteristic of aversive emotions such as fear and anxiety, and previous labs have demonstrated that enhanced physiological arousal can enhance fear memory consolidation (Cahill & McGaugh, 1998; McGaugh, 2004). Because BFCCNs receive input from central arousal norepinephrine neurons in the locus coeruleus and project extensively to the neocortex, they are ideally positioned to facilitate the modulatory effects that physiological arousal has on cortical activity. This modulatory effect could then impact psychological processes (e.g. learning and memory) during fear and/or anxiety. We demonstrated that systemic epinephrine administration, which does not cross the blood brain barrier (Hardebo & Owman, 1980), enhanced cerebral auditory evoked potentials in the neocortex, and selective BFCCN lesions attenuated this effect (Berntson, Shafi, Knox, & Sarter, 2003). In our next study we used chronic indwelling cannulas to selectively block α1-noradrenergic receptors in the basal forebrain. Selective noradrenergic receptor blockade disrupted the enhancement systemic epinephrine administration had on cerebral auditory evoked potentials in the neocortex (Knox, Sarter, & Berntson, 2004). Together these studies provided support for a circuit proposed by McGaugh and colleagues (2004) and Berntson et al. (1998), by which enhanced physiological arousal can lead to changes in neural activity critical for altering emotional reactivity during fear and anxiety. The results of these studies led us to hypothesize that BFCCNs would be critical for fear memory. Addressing this question was the focus of my next set of studies.
How to conceptualize the role of BFCCNs in fear memory formation and expression?
If enhanced physiological arousal enhances fear memory consolidation and BFCCN lesion disrupts the modulatory effect that enhanced physiological arousal has on cortical activity, we hypothesized that BFCCN lesions should disrupt fear memory. Previous research in the lab suggested this might be a possibility (Stowell, Berntson, & Sarter, 2000), but in order to test this hypothesis, we examined the effects of BFCCN lesions on cued fear conditioned suppression and fear conditioned freezing. In both of these paradigms, an innocuous stimulus (e.g. light or tone) is paired with an electric shock, which renders the innocuous stimulus, "fearful." In the cued fear conditioned suppression paradigm, a cessation in bar pressing for reward, induced by presentation of the fear stimulus, is used to index fear memory. In the fear conditioned freezing paradigm, freezing behavior generated by presentation of the fear stimulus is used to index fear memory. We hypothesized that BFCCN lesions would disrupt cued fear conditioned suppression and freezing. In support of our hypothesis, BFCCN lesions abolished cued fear conditioned suppression, but to our surprise these lesions had no effect on cued fear conditioned freezing (Knox & Berntson, 2006). This was quite odd, since rats with the BFCCN lesions could not acquire fear memory in one behavioral test (i.e. conditioned suppression paradigm), but could in another (i.e. conditioned freezing paradigm). Since BFCCNs are not critical for bar pressing (Knox & Berntson, 2006, 2008; Stowell, et al., 2000), this dissociation would suggest neural processes critical for fear memory is somewhat dependent on the parameters of the fear learning task and the dependent variable used to measure fear memory. In other words, the neurobiological mechanism that encodes fear memory may not be universal for all fear events.
The toxin we used to induce BFCCN lesions targets cholinergic neurons that project to the neocortex, but left cholinergic neurons that project to the amygdala intact (Berntson, Hart, Ruland, & Sarter, 1996). This would suggest that cholinergic input to select cortical regions can be critical for fear memory in the fear conditioned suppression paradigm. To test this hypothesis, we recorded the electroencephalogram (EEG) in the prefrontal cortex during acquisition and expression of contextual fear conditioned suppression in lesion and control rats. In control rats, decreases in the slow-wave components of the prefrontal cortical EEG during acquisition of contextual fear conditioned suppression were associated with enhanced contextual fear conditioned suppression. BFCNN lesions attenuated this decrease in slow-wave EEG in the prefrontal cortex and contextual fear conditioned suppression (Knox & Berntson, 2008). These findings suggest that cholinergic input to select regions of the prefrontal cortex is critical for acquisition of contextual fear conditioned suppression. Given that human lesion studies also suggest that prefrontal cortical circuits can be critical for fear memory (Koenigs et al., 2008), exploring the role of cholinergic input to prefrontal cortical circuits in fear memory is actively being pursued in my laboratory.
Examining how traumatic stress exposure results in persistent fear expression
During my postdoctoral study, my research focus switched to examining the neurobiological processes critical for mediating post-traumatic stress disorder. PTSD is an anxiety disorder that is caused by exposure to trauma, and has three clusters of symptoms: avoidance, emotional numbing and arousal (APA, 1994). In order to model PTSD in rats, we used the single prolonged stress (SPS) paradigm, which was developed by Israel Liberzon. SPS involves serial exposure to restraint, forced swim and ether. It is believed that the combined effects of these stressors produce a unique stress response that is useful for modelling PTSD (Yamamoto et al., 2009). SPS exposure enhances fast negative feedback of the hypothalamic-pituitary-adrenal (HPA) axis and hippocampal glucocorticoid receptor mRNA expression (Liberzon, Lopez, Flagel, Vazquez, & Young, 1999). Because PTSD is associated with enhanced negative feedback of the HPA axis (Yehuda, Yang, Buchsbaum, & Golier, 2006) and enhanced glucocorticoid receptor expression (van Zuiden, Kavelaars, Geuze, Olff, & Heijnen, 2012), we used the SPS model to explore neurobiological and behavioral phenomena relevant to PTSD.
In our first set of studies we examined the effects of SPS on unconditioned fear expression, but exposure to SPS had no effects on unconditioned fear in the elevated plus maze, light enhanced startle and predator odor-induced freezing paradigms (laboratory observation). In our next set of studies we examined the effects of SPS on fear and extinction memory. To do this we first exposed rats to SPS or control stress, and then adopted a set of relatively simple behavioral procedures across three days. Rats were first presented with tones that were paired with an electrical shock (i.e. fear conditioning). As the number of tone-shock events increases, freezing behavior increases, and this increase in freezing behavior is indicative of acquisition of fear memory. In other words, rats learn that the tone predicts the shock. On the second day, rats were presented with tones in the absence of shocks (i.e. extinction learning). At first rats freeze when presented with the tone, which suggests rats recall the fear memory. However, as the fear stimulus is presented in the absence of shock, rats learn that the tone no longer predicts the shock, which is reflected by a decrease in freezing upon tone presentation (i.e. extinction memory). On the third day, rats were subjected to an extinction test where tones were presented again in the absence of shocks. If rats show little freezing, this is evidence of extinction memory recall. In other words, rats remember that the tone no longer predicts the shock. If rats freeze upon tone presentation during the extinction test, this is evidence of fear memory recall. In other words rats fail to recall the extinction memory or show an extinction retention deficit.
SPS had inconsistent effects on acquisition and recall of fear memory and had no effects on acquisition of extinction memory. However, SPS consistently enhanced conditioned freezing during the extinction test, which suggests SPS induced extinction retention deficits (Knox et al., 2012; Knox, Nault, Henderson, & Liberzon, 2012). We also showed that SPS-induced extinction retention deficits do not manifest unless there is a robust increase in glucocorticoid receptor expression in the hippocampus (Knox, Nault, et al., 2012). These findings suggest SPS-enhanced hippocampal glucocorticoid receptor expression leads to extinction retention deficits.
It should be noted that SPS also induces a number of other neurobiological effects that could contribute to extinction retention deficits. 1H-magnetic resonance spectroscopy (MRS) imaging technology can be used to measure the concentration of chemicals in solid tissue such as the brain. In 1H-MRS, a strong magnetic field is applied across a sample of tissue, and then the resonances at various frequencies that occur in the tissue can be used to quantify the concentration of certain chemicals in that tissue. Using 1H-MRS imaging we showed that SPS exposure decreased glutamate, glutamine and creatine levels in the medial prefrontal cortex (Knox, Perrine, George, Galloway, & Liberzon, 2010), which suggests a decrease in excitatory tone. This effect could contribute to extinction retention deficits in the SPS paradigm. In another study, we used single unit electrophysiology to measure the activity of locus coeruleus neurons. The locus coeruleus is the major source of norepinephrine in the central nervous system, and given that enhanced norepinephrine metabolism has been observed in PTSD patients (Southwick et al., 1993), we hypothesized that exposure to SPS would enhance the activity of locus coeruleus neurons. SPS decreased baseline single unit activity of locus coeruleus neurons, but enhanced the responsivity of these neurons to stimulation (George et al., 2013). These effects could also contribute to SPS-induced extinction retention deficits. The transfer of phosphate groups from one protein to another is critical for energy transfer in cells, cellular signaling in neurons, and has been implicated in fear memory consolidation (Schafe et al., 2000). We showed that SPS-enhanced glucocorticoid receptor expression is correlated with increased phosphorylation of protein kinase B (i.e. Akt) (Eagle et al., 2013). Because Akt phosphorylation has been implicated in fear memory (Ou & Gean, 2006), SPS-enhanced Akt phosphorylation could lead to SPS-induced extinction retention deficits. Thus, identifying the neurobiological mechanisms by which SPS induces extinction retention deficits require further investigation.
Also unclear are the psychological mechanisms by which SPS induces extinction retention deficits. Extinction retention deficits in the SPS model could emerge, because of the effects SPS has on fear memory (e.g. enhanced fear memory reconsolidation or resistance) or extinction memory (e.g. deficits in extinction memory consolidation and/or retrieval). Identification of the exact mechanisms by which SPS exposure results in extinction retention deficits is actively being pursued in my laboratory.
Finally it has been well established that the incidence of PTSD in women is higher than in men even though women have a lower probability of trauma exposure (Greenberg et al., 1999; Pratchett, Pelcovitz, & Yehuda, 2010). This observation suggests that females are more susceptible to the effects of traumatic stress. Especially because so little is known about the role of sex in traumatic stress susceptibility, this is a line of research we are currently pursuing.
Interesting findings along the way
Personal growth in the practice of science involves focus on answering a set of well-defined questions. However, in this pursuit, it is often the case that tangential interesting findings are observed. I would like to highlight two such findings that I was fortunate to observe during my postdoc study. Current models of the role of prefrontal circuits in fear and anxiety have traditionally been defined as inhibitory. A fairly large data set has demonstrated that the infralimbic region of the rat medial prefrontal cortex is critical for acquisition and maintenance of extinction memory (Quirk, Garcia, & Gonzalez-Lima, 2006; Quirk et al., 2010). However, previous studies have shown that the prelimbic region of the rat medial prefrontalcortex is critical for expression of conditioned (Corcoran & Quirk, 2007; Sierra-Mercado, Padilla-Coreano, & Quirk, 2011) and unconditioned (Stern, Do Monte, Gazarini, Carobrez, & Bertoglio, 2010) fear. In direct contrast to these experiments, we demonstrated that the prelimbic cortex, but not infralimbic cortex, is critical for inhibiting unconditioned fear induced by predator odor exposure (Fitzpatrick, Knox, & Liberzon, 2011). These findings raise the possibility that fear expression in different animal models of conditioned and unconditioned fear capture different aspects of fear reactivity, though very little is known about these differences. The results also suggest that a better understanding of how to conceptualize salient differences in neurobiological function in animal fear models is needed.
If an animal is conditioned in one context, then tested for fear conditioning in another context, robust levels of conditioned fear are usually observed. This well documented observation has been used to argue that expression of conditioned fear is context independent (i.e. is not very sensitive to contextual manipulations). However, for a long time it was unknown if expression of unconditioned fear is also context independent. In a series of experiments, we demonstrated that freezing induced by predator odor varies depending on contextual feature manipulation. While predator odor freezing is equivalent in novel and familiar contexts, this type of freezing is enhanced if the predator odor is presented in a context that was previously paired with food (i.e. appetitive context) (Knox, Fitzpatrick, George, Abelson, & Liberzon, 2012). Furthermore, this enhancement in predator odor-induced freezing was correlated with a failure to increase neural activity in the prelimbic cortex (Knox, Fitzpatrick, et al., 2012). To account for these findings, we proposed a contextual expectancy mismatch hypothesis. This hypothesis states that if rats detect a threat in a context where they expect a reward, this expectancy mismatch leads to enhanced expression of unconditioned fear. The possibility that such a nuanced cognitive process, with complementary changes in neurobiology, could be observed in rats is exciting and suggests that changes in fear expression are not solely determined by learning, memory and innate fear responsivity, but can be modulated by other cognitive processes as well.
I would like to thank my previous mentors Gary Berntson, The Ohio State University, and Israel Liberzon, University of Michigan — for helping me through my transformation from lay person to scientist. While I am grateful to the numerous undergraduates who helped me conduct experiments described in this brief, I would like to particularly thank Holly Brothers, Stephanie Stout and Christopher J. Fitzpatrick for their tireless help, encouragement and creativity. Lastly, I would like to thank the funding agencies whose financial support made all of this research possible. They are the Department of Defense, Department of Veteran Affairs and the Alumni for Graduate Research Foundation at The Ohio State University.
American Psychiatric Association. (1994). Diagnostic and Statistical Manual of Mental Disorders. (4th ed.). Washington D. C.: American Psychiatric Association.
Berntson, G. G., Hart, S., Ruland, S., & Sarter, M. (1996). A central cholinergic link in the cardiovascular effects of the benzodiazepine receptor partial inverse agonist FG 7142. Behav Brain Res, 74(1-2), 91-103.
Berntson, G. G., Sarter, M., & Cacioppo, J. T. (1998). Anxiety and cardiovascular reactivity: the basal forebrain cholinergic link. Behav Brain Res, 94(2), 225-248.
Berntson, G. G., Shafi, R., Knox, D., & Sarter, M. (2003). Blockade of epinephrine priming of the cerebral auditory evoked response by cortical cholinergic deafferentation. Neuroscience, 116(1), 179-186.
Cahill, L., & McGaugh, J. L. (1998). Mechanisms of emotional arousal and lasting declarative memory. Trends Neurosci, 21(7), 294-299.
Conner, J. M., Culberson, A., Packowski, C., Chiba, A. A., & Tuszynski, M. H. (2003). Lesions of the Basal forebrain cholinergic system impair task acquisition and abolish cortical plasticity associated with motor skill learning. Neuron, 38(5), 819-829.
Corcoran, K. A., & Quirk, G. J. (2007). Activity in prelimbic cortex is necessary for the expression of learned, but not innate, fears. J Neurosci, 27(4), 840-844.
Eagle, A. L., Knox, D., Roberts, M. M., Mulo, K., Liberzon, I., Galloway, M. P., & Perrine, S. A. (2013). Single prolonged stress enhances hippocampal glucocorticoid receptor and phosphorylated protein kinase B levels. Neurosci Res, 75(2), 130-137.
Fitzpatrick, C. J., Knox, D., & Liberzon, I. (2011). Inactivation of the prelimbic cortex enhances freezing induced by trimethylthiazoline, a component of fox feces. Behav Brain Res, 221(1), 320-323.
George, S. A., Knox, D., Curtis, A. L., Aldridge, J. W., Valentino, R. J., & Liberzon, I. (2013). Altered locus coeruleus-norepinephrine function following single prolonged stress. Eur J Neurosci, 37(6), 901-909.
Greenberg, P. E., Sisitsky, T., Kessler, R. C., Finkelstein, S. N., Berndt, E. R., Davidson, J. R., . . . Fyer, A. J. (1999). The economic burden of anxiety disorders in the 1990s. J Clin Psychiatry, 60(7), 427-435.
Hardebo, J. E., & Owman, C. (1980). Barrier mechanisms for neurotransmitter monoamines and their precursors at the blood-brain interface. Ann Neurol, 8(1), 1-31.
Jones, B. E. (2004). Activity, modulation and role of basal forebrain cholinergic neurons innervating the cerebral cortex. Prog Brain Res, 145, 157-169.
Knox, D., & Berntson, G. G. (2006). Effect of nucleus basalis magnocellularis cholinergic lesions on fear-like and anxiety-like behavior. Behav Neurosci, 120(2), 307-312.
Knox, D., & Berntson, G. G. (2008). Cortical modulation by nucleus basalis magnocellularis corticopetal cholinergic neurons during anxiety-like states is reflected by decreases in delta. Brain Res, 1227, 142-152.
Knox, D., Fitzpatrick, C. J., George, S. A., Abelson, J. L., & Liberzon, I. (2012). Unconditioned freezing is enhanced in an appetitive context: implications for the contextual dependency of unconditioned fear. Neurobiol Learn Mem, 97(4), 386-392.
Knox, D., George, S. A., Fitzpatrick, C. J., Rabinak, C. A., Maren, S., & Liberzon, I. (2012). Single prolonged stress disrupts retention of extinguished fear in rats. Learn Mem, 19(2), 43-49.
Knox, D., Nault, T., Henderson, C., & Liberzon, I. (2012). Glucocorticoid Receptors And Extinction Retention Deficits In The Single Prolonged Stress Model. Neuroscience, 223, 163-173.
Knox, D., Perrine, S. A., George, S. A., Galloway, M. P., & Liberzon, I. (2010). Single prolonged stress decreases glutamate, glutamine, and creatine concentrations in the rat medial prefrontal cortex. Neurosci Lett, 480(1), 16-20.
Knox, D., Sarter, M., & Berntson, G. G. (2004). Visceral afferent bias on cortical processing: role of adrenergic afferents to the basal forebrain cholinergic system. Behav Neurosci, 118(6), 1455-1459.
Koenigs, M., Huey, E. D., Raymont, V., Cheon, B., Solomon, J., Wassermann, E. M., & Grafman, J. (2008). Focal brain damage protects against post-traumatic stress disorder in combat veterans. Nat Neurosci, 11(2), 232-237.
Liberzon, I., Lopez, J. F., Flagel, S. B., Vazquez, D. M., & Young, E. A. (1999). Differential regulation of hippocampal glucocorticoid receptors mRNA and fast feedback: relevance to post-traumatic stress disorder. J Neuroendocrinol, 11(1), 11-17.
McGaugh, J. L. (2004). The amygdala modulates the consolidation of memories of emotionally arousing experiences. Annu Rev Neurosci, 27, 1-28.
Ou, L. C., & Gean, P. W. (2006). Regulation of amygdala-dependent learning by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol-3-kinase. Neuropsychopharmacology, 31(2), 287-296.
Pratchett, L. C., Pelcovitz, M. R., & Yehuda, R. (2010). Trauma and violence: are women the weaker sex? Psychiatr Clin North Am, 33(2), 465-474.
Quirk, G. J., Garcia, R., & Gonzalez-Lima, F. (2006). Prefrontal mechanisms in extinction of conditioned fear. Biol Psychiatry, 60(4), 337-343.
Quirk, G. J., Pare, D., Richardson, R., Herry, C., Monfils, M. H., Schiller, D., & Vicentic, A. (2010). Erasing fear memories with extinction training. J Neurosci, 30(45), 14993-14997.
Sarter, M., & Bruno, J. P. (1997). Cognitive functions of cortical acetylcholine: toward a unifying hypothesis. Brain Res Brain Res Rev, 23(1-2), 28-46.
Schafe, G. E., Atkins, C. M., Swank, M. W., Bauer, E. P., Sweatt, J. D., & LeDoux, J. E. (2000). Activation of ERK/MAP kinase in the amygdala is required for memory consolidation of pavlovian fear conditioning. J Neurosci, 20(21), 8177-8187.
Sierra-Mercado, D., Padilla-Coreano, N., & Quirk, G. J. (2011). Dissociable roles of prelimbic and infralimbic cortices, ventral hippocampus, and basolateral amygdala in the expression and extinction of conditioned fear. Neuropsychopharmacology, 36(2), 529-538.
Southwick, S. M., Krystal, J. H., Morgan, C. A., Johnson, D., Nagy, L. M., Nicolaou, A., . . . Charney, D. S. (1993). Abnormal noradrenergic function in posttraumatic stress disorder. Arch Gen Psychiatry, 50(4), 266-274.
Stern, C. A., Do Monte, F. H., Gazarini, L., Carobrez, A. P., & Bertoglio, L. J. (2010). Activity in prelimbic cortex is required for adjusting the anxiety response level during the elevated plus-maze retest. Neuroscience, 170(1), 214-222.
Stowell, J. R., Berntson, G. G., & Sarter, M. (2000). Attenuation of the bidirectional effects of chlordiazepoxide and FG 7142 on conditioned response suppression and associated cardiovascular reactivity by loss of cortical cholinergic inputs. Psychopharmacology (Berl), 150(2), 141-149.
van Zuiden, M., Kavelaars, A., Geuze, E., Olff, M., & Heijnen, C. J. (2012). Predicting PTSD: Pre-existing vulnerabilities in glucocorticoid-signaling and implications for preventive interventions. Brain Behav Immun, 30:12-21.
Yamamoto, S., Morinobu, S., Takei, S., Fuchikami, M., Matsuki, A., Yamawaki, S., & Liberzon, I. (2009). Single prolonged stress: toward an animal model of posttraumatic stress disorder. Depress Anxiety, 26(12), 1110-1117.
Yehuda, R., Yang, R. K., Buchsbaum, M. S., & Golier, J. A. (2006). Alterations in cortisol negative feedback inhibition as examined using the ACTH response to cortisol administration in PTSD. Psychoneuroendocrinology, 31(4), 447-451.
Zaborszky, L., & Cullinan, W. E. (1996). Direct catecholaminergic-cholinergic interactions in the basal forebrain. I. Dopamine-beta-hydroxylase- and tyrosine hydroxylase input to cholinergic neurons. J Comp Neurol, 374(4), 535-554.
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