Linking the periphery with the CNS: Do pupil dilation and skin conductance responses correlate with locus coeruleus and amygdala activity?

THE LC-NE SYSTEM FUNCTION AND MALFUNCTION
The neuromodulator norepinephrine (NE) is involved in multiple cognitive
processes including attention, learning, and emotions, and has been shown to be
disturbed in psychiatric disorders such as anxiety disorder, post-traumatic
stress disorder (PTSD), schizophrenia and others. Most of the NE released in
the brain originates from the locus coeruleus (LC), a brainstem nucleus with
noradrenergic projections to almost all brain regions, including amygdala. This
neuroanatomical formation of the noradrenergic system makes it well suited to
rapidly and globally modulate brain function in response to changes in the
environment, when cognitive flexibility and increased attention are required
(such as when a stressor is presented). Arousal, vigilance and cognitive
flexibility in these situations increase the chances of survival and prepare
the organism for immediate action. Nonetheless, if this state of arousal and
vigilance is prolonged, as it happens in cases of chronic stress, the same
mechanism becomes problematic and leads to disorders such as PTSD, anxiety
disorders etc instead of being beneficial for survival. Therefore the LC-NE
system can be functional and promote survival or contribute and be the main
mechanism behind a disorder.

THE LC-NE SYSTEM: CONNECTING THE FORBRAIN, MIDBRAIN AND THE PERIPHERY
The LC is activated in parallel with the autonomic system. NE released in the
forebrain facilitates attention, processing and cognitive flexibility. Central
activation of neuromodulatory neurons in concert with peripheral arousal
(related to LC via the vagus nerve, the PGi and from the PVN of the
hypothalamus) prepare the organism for a reorientation or reset of cortical
networks and an adaptive response (Nieuwenhuis, De Geus, & Aston-Jones, 2011;
Sara & Bouret, 2012).
In case of stress and fear this is translated as involvement of NE both at a
higher cognitive level by modulating forebrain regions involved in attention
and vigilance, and at a more peripheral level, by preparing the body for the
fight-and-flight response and controlling autonomic responses.

THE LC & AMYGDALA IN STRESS
Stressful and fearful events including psychological stress and conditioned
fear cause significant increases in NE release selectively in the amygdala,
hypothalamus, and LC. This increase is significantly attenuated by anxiolytic
drugs (e.g benzodiazepines) (Tanaka, Yoshida, Emoto, & Ishii, 2000).
Finally, the LC is connected with the basal lateral amygdala (Loughlin, Foote,
& Grzanna, 1986) and the central nucleus of the amygdala (Cedarbaum &
Aghajanian, 1978). The amygdala, like the LC, is also involved in triggering
autonomic reactions to conditioned stressors (LeDoux, Iwata, Cicchetti, & Reis,
1988). Electrical stimulation of the central amygdala elicits an excitatory
response of the LC (Bouret, Duvel, Onat, & Sara, 2003) and lesions in amygdala
block stress associated with increased release of monoamines in the prefrontal
cortex (Goldstein, Rasmusson, Bunney, & Roth, 1996).

THE PERIPHERY: PUPIL & SKIN CONDUCTANCE RESPONSES
Stimulus-evoked dilation of the pupil has been suggested to correlate with
phasic activity of the LC (Aston-Jones & Cohen, 2005). Increased phasic LC-NE
system activity is associated with improved task performance and stimulus
processing efficiency as shown by in vivo stimulation or recording of LC
activity in monkeys and rodents performing sensory and memory tasks
(Aston-Jones & Cohen, 2005; Berridge & Waterhouse, 2003; Samuels & Szabadi,
2008; Usher, Cohen, Servan-Schreiber, Rajkowski, & Aston-Jones, 1999). Based on
these findings, many studies have used stimulus-evoked pupil dilation as an
indirect measure of phasic activity of the human LC-NE system in the absence of
more direct measures in humans (e.g., (Einhäuser, Stout, Koch, & Carter, 2008);
(Gilzenrat, Nieuwenhuis, Jepma, & Cohen, 2010); (Jepma & Nieuwenhuis, 2010).
Although these studies assume a correlation of LC activity and pupil dilation
in humans, a direct relation has not been shown yet.
Another measure of autonomic activity that has been related to the LC-NE system
is the skin-conductance response (SCR), a broadly accepted measure of arousal.
As such, it can be related also to amygdala activation in fear paradigms.
Indeed, it has been shown that patients with unilateral or bilateral lesions of
the amygdala have deficits in classical fear conditioning and this is reflected
in their SCRs (Davis, 2001). Additionally, amygdala activity has been shown to
be correlated with SCR in fear conditioning in humans (Phelps, Delgado,
Nearing, & LeDoux, 2004). Apart from being a measure of arousal, the SCR is
also used to assess orientation of attention. It has been shown that perceived
mental effort correlates with changes in autonomic responses including heart
rate and SCR and it has been suggested that mental effort may reflect tonic
activity of the LC-NE system in healthy individuals (Howells, Stein, & Russell,
2010).
All in all, these results present tantalizing evidence that SCR and pupil
diameter responses can be used as physiological correlates of LC-NE system
activity. Nonetheless, up to date there has not been a direct link between
these measures and LC neuronal activity in humans. Therefore, we propose a more
direct approach using fMRI methods in order to a) reliably measure LC neural
activity, b) validate the relationship of LC activity with pupil dilation in
humans, and c) investigate the relationship between the SCR and LC and amygdala
activity.

GENOMIC AND INDIVIDUAL DIFFERENCES: INFLUENCE OF NORADRENALINE, CORTISOL AND
STRESS
When studying the role of LC-NE system in cognition, one must consider the role
of individual differences. The LC-NE system varies from person to person, and
these variations have to be linked to individual characteristic traits. A
correlation has been found between variations in a norepinephrine transporter
gene (which regulates presynaptic levels of NE) and novelty seeking personality
(Lee et al., 2008; Suzuki et al., 2008).
Central NE activity is genetically determined by polymorphisms of the NET, DBH
and COMT genes, and the alpha-1, alpha-2, beta-1 and beta-2 adrenergic receptor
genes. Central NE activity, in turn, is an important predictor of trait anxiety
(Hughes, Watkins, Blumenthal, Kuhn, & Sherwood, 2004; Ressler & Nemeroff, 2000;
White & Depue, 1999). Therefore, we will measure genotype and trait anxiety to
examine whether these measures are predictive of task performance, which would
provide additional evidence for a noradrenergic modulation of the cognitive
processes examined.
Adrenaline, noradrenaline and cortisol are the three major hormones secreted in
situations of elevated stress (Bremner, 2006). The experimental paradigm
applied in this protocol is a stress induction paradigm (fear conditioning).
Cortisol is a valid biomarker of stress activation (Bozovic, Racic, & Ivkovic,
2013), therefore assessing cortisol will allow us to verify that the
experimental paradigm is indeed effective.
Additionally, the adrenergic and the corticosteroid systems are influencing
each other as they are part of same axis, namely the
hypothalamic-pituitary-adrenergic (HPA) axis. This study targets to investigate
activity in two brain regions that play a key role in this stress network: the
locus coeruleus (main producer of noradernaline) and the amygdala (brain region
shown to drive stressful responses). Therefore we will measure salivary
cortisol to examine whether activity in these brain regions are correlated with
cortisol levels and whether these measures are predictive of task performance.


CONCLUSION
Animal research has indicated an important role for the neuromodulatory LC-NE
system in fear responses and in the optimization of performance. In absence of
more direct measures, SCR and pupil diameters have been used as measurements to
assess the LC-NE system activity at a peripheral physiological level. More
direct empirical studies of LC-NE function in humans have been scarce, in part
because of the methodological challenges involved. Recent methodological
developments, however, suggest that these challenges can be overcome and open
the way for functional imaging studies of the LC.

AIMS
Aim of this study is to: a) reliably measure LC neural activity; b) validate
the relationship of LC activity with pupil dilation in humans; c) investigate
the relationship between the SCR and LC and amygdala activity; and d) compare
how these measures vary between individuals with different levels of trait
anxiety.

SUBAIMS
Genotype, salivary cortisol and trait anxiety will be measured to examine
whether these measures are predictive of task performance, which would provide
additional evidence for LC-NE involvement in the cognitive processes examined.

DESIGN
The proposed study will use a four-session within-subjects experimental design.
Participants* brain activation patterns (especially in the LC) will be measured
using event-related fMRI.

GENERAL PROCEDURE
The proposed study will consist of 4 sessions.
In the first session structural scans will be acquired and the participants
will be required to just stay still in the MRI scanner. They will have to do
nothing else (duration of 1st session aprox 41 min).

In the second session the remaining part of the experimental protocol will take
place: the session will consist of three task procedures that have been shown
to elicit LC activity in animal studies. The study will start in the behavioral
lab (*interview room* located few rooms far away from the fMRI scanning room)
on the ground floor of the LUMC, and move to the fMRI room about 39 minutes
after the subject first arrives. Total scanning time will constitute
approximately 41 minutes (with additional time of 30 min for preparing the
psychophysiological measurements, removing the participant from the scanner
etc), and the subject will return to the behavioral lab after being scanned.
Administration of questionnaires will last approx. 42 minutes and will be
administered outside of the scanner before and after the fMRI scanning. With
breaks, time for explanations and questionnaires the second session will last
approximately 112 min. The total duration of the experiment (summing up the 2
sessions) will be two hours and a half.

Session 3 will be similar to session 1. Structural scans will be acquired and
the participants will be required to just stay still in the MRI scanner. They
will have to do nothing else. Similar scan sequences will be used as session 1
but the scans will be oriented towards a different direction for better
visualization of the regions of interest. The total duration of this session
will be less than 1 hour (approx. 45 min).

Session 4 will be similar to session 1 and 3 but this session will take place
at the 7 Tesla scanner. Structural scans will be acquired and the participants
will be required to just stay still in the MRI scanner. Total duration 1 hour.
The same principles apply for this protocol as the ones mentioned in the
protocol P07.096, Versie 3.0 * 16/04/2012 (Research at the 7 Tesla scanner).

The total duration of the experiment (summing up the 4 sessions) will be five
hours and a half.


At the end of the fourth session, the subject will be debriefed about the
purpose of the experiment.


TASK 1
Fear Conditioning
In the fMRI scanner, participants will undergo a classical Pavlovian
conditioning procedure. During this procedure, participants will be presented
with two different stimuli. One stimulus will be electric current reinforced in
30% of the cases (CS+) and the other will be never followed by a shock (CS-).
To reduce spontaneous SCRs, the conditioning task will be preceded by a
habituation phase during which each stimulus will be presented twice (2CS+ and
2CS-) without shock. After the habituation phase participants will be presented
12 CS+, 12 CS- and 6 CS+US (contaminated by shock). Heart rate and pupil size
will be measured throughout the procedure. In total, the conditioning procedure
will last no more than 15 minutes.

TASK 2
Fear Extinction
The fear extinction task will be similar to the fear conditioning task (same
visual stimuli) with the difference that no shock will be administered.
Heart rate and pupil size will be measured throughout the procedure. In total,
the fear extinction procedure will last no more than 15 minutes.

TASK 3
Oddball task during safe and during threat-of-shock periods
After the fear extinction task, participants will perform a visual oddball
task; they will be required to detect and respond to unpredictable, infrequent
target stimuli in a stream of frequent distracting stimuli. The oddball task
will be divided into *safe* blocks (in which the task will be performed and no
shock will be administered) and *threat-of-shock* blocks (in which the task
will be performed under the threat of shock). To this end, participants will be
told that shocks can be administered unexpectedly at any point during the
threat-of-shock blocks and that no shock will be administered during the safe
blocks. During the threat-of-shock blocks, shocks will be delivered on the
participant*s right index and middle finger (see below for details regarding
current shock administration). A maximum of 4 shocks will be delivered during
this task. In total the task will last no more than 20 min.
After the end of all tasks, the participants' awareness of the reinforcement
contingencies and evaluation of the shocker intensity will be assessed outside
the MRI scanning room by means of questionnaires and interview. Additionally,
participants will report on a Likert scale questionnaire how much overall
anxiety (0-10, no anxious * highly anxious) they felt during each task.

fMRI
There are no known risks associated with participating in an fMRI study. This
is a noninvasive technique involving no catheterizations or introduction of
exogenous tracers. Numerous human subjects have undergone magnetic resonance
studies without apparent harmful consequences. Radiofrequency power levels and
gradient switching times used in these studies are within the FDA approved
ranges. Some people become claustrophobic while inside the magnet and in these
cases the study will be terminated immediately at the subject's request.

7 Tesla MRI
It is estimated that at the moment more than 5000 subjects have been scanned on
7 or 8 Tesla MRI scanners without reports of clinical significant adverse
events. At the 7 Tesla scanner of the LUMC subjects are being scanned at a
daily basis and no severe adverse effects have been reported. On the same
system type as it has been installed in the LUMC (Philips Achieva 7.0 Tesla MRI
scanner) already more than 1000 subjects have been scanned without severe
adverse effects. At the moment the scientific community agrees that 7.0 or 8.0
Tesla MRI is safe for human use. For additional information please refer to the
protocol with Protocol-nummer: P07.096, Versie 3.0- 16/04/2012 which has been
already approved by the CME. For the 7 Tesla session of this study a similar
protocol will be applied. In case of questions please contact:

Dr.Ir. Thijs van Osch
Leids Universitair Medisch Centrum
Gebouw 1, Kamer J-00-040

Postzone C2S
Postbus 9600
2300 RC Leiden

Bezoekadres:
Albinusdreef 2
2333 ZA Leiden

Tel: +31 71 526 3678
M.J.P.van_Osch@lumc.nl


Mild electrical stimulations
Mild electrical stimulations will be delivered transcutaneously over
participant*s right index and middle finger or over the wrist. The discomfort
caused to the participants is kept to the lowest levels possible. More
specifically, the exact level of the shock intensity will be determined by the
participant himself by undergoing a stair case procedure until reaching the
level that the shock feels *uncomfortable but not painful*. The *common
knowledge* sensation most closely mimicking the sensation of the electrical
shocks is hitting one*s funny bone (medial epicondyle of the humerus). These
levels are typically used in diagnostic neurophysiologic protocols and the
procedure is similar to protocols approved and used in behavioral studies at
Leiden University and fMRI studies at other Dutch and international
Universities.

Eye tracker, SCR and heart rate
The eye tracker and the MR-compatible device for recording SCR and heart rate
have been used in previous 3-Tesla MRI studies and pose no significant risk of
injury or discomfort.