Discussion (Stress-induced reinstatement of Fix-C and Esc-C CPP)

Stress is known to precipitate relapse to drug use (Sinha, 2008) but the exact
signaling molecules that govern these behavioral responses are unclear. The FST is one
stressor that can be used to model stress-induced reinstatement outside the conditioning
context in animal models (Kreibich & Blendy, 2004). The present study shows that
exposure to FST successfully reinstates previously extinguished Fix-C and Esc-C CPP
(Fig. 4.1, 4.2 and 4.4). The reinstatement of CPP was not a spontaneous event since mice
that were re-exposed to the testing context without being subjected to the FST did not
show recovery of place preference (Fig. 4.5). Thus, there appear to be a requirement for
stress hormones and other signaling molecules working in concert to remind the animal
of the associations learned during conditioning and thus promote reinstatement of CPP.
Studies from our laboratory (Itzhak & Anderson, 2012) and others (Conrad et al.,
2013) have shown that mice trained by daily increases in the dose of cocaine (Esc-C)
produced higher magnitudes and more persistent CPP than mice trained by daily fixed
dose of cocaine (Fix-C). These findings suggest that different neural mechanisms may
contribute to the development of cocaine-associated memory. Consequently, this study
was undertaken to elucidate the contribution of signaling molecules to stress-induced
reinstatement of extinguished Fix-C and Esc-C CPP.
Because the NMDAR has a role in synaptic plasticity and the effects of cocaine
(Collingridge, 1987; Kelley et al., 2007; Alaghband & Marshall, 2013), we investigated
the effects of the non-competitive NMDAR antagonist MK-801 on stress-induced
reinstatement of Fix-C and Esc-C CPP. We found that NMDAR antagonism attenuated
stress-induced reinstatement of Fix-C but not Esc-C CPP (Fig 4.1A, B). This suggests a
more central role for an interaction between stress and the NMDAR in Fix-C CPP but not
in Esc-C CPP. With respect to Fix-C CPP, MK-801 may have interfered with the ability
of stress to remind the animal of the associations learned during conditioning and as such
prevented reinstatement (Ma et al., 2007). Studies have shown that CRH, the primary
regulator of the neuroendocrine response to stress, facilitates pre-synaptic glutamate
release (Hollrigel et al., 1998). Thus MK-801 may have attenuated stress-induced
reinstatement of Fix-C CPP by blocking the stimulating effect of increased synaptic
glutamate. However, because Esc-C memory is more stable and more resistant to
pharmacological manipulation (Itzhak & Anderson, 2012; Liddie et al., 2012; Liddie &
Itzhak, 2014, Chapter 2) it is likely that a dose of 0.3mg/kg MK-801 was ineffective at
preventing stress-induced NMDAR activation and as such could not attenuate
reinstatement of Esc-C CPP. Higher doses of MK-801 were not tested because of marked
effects on motor behavior. Previous work from our laboratory has shown that higher
doses of MK-801 were required to disrupt Esc-C memory reconsolidation when
administered post-memory retrieval (Liddie & Itzhak, 2014, Chapter 2). Thus, the
conditioning schedule determines the degree to which NMDAR channel blockade
influences cocaine-associated contextual memory.
We have previously shown that a) mice conditioned by Esc-C showed marked
increases in hippocampal expression levels of the NR2B subunit of the NMDAR
compared to mice conditioned by Fix-C and b) NR2B-containing NMDARs contribute
significantly to the acquisition and reconsolidation of Fix-C and Esc-C memory (Liddie
& Itzhak, 2014, Chapter 2). However, the present study found that there is no specific
requirement for NR2B-containing NMDARs in stress-induced reinstatement of Fix-C and
Esc-C memory (Fig. 4.1C, D). These results suggest that NR2B-mediated mechanisms
underlying the acquisition and reconsolidation of cocaine-associated memory are
dissociable from those required for stress-induced reinstatement of CPP. Our results are
in accordance with others who found that while ifenprodil attenuated the reinstating
effect of morphine priming, it had no effect on stress-induced reinstatement of
extinguished morphine CPP (Ma et al., 2007). Taken together, it appears that while the
NMDAR is involved in stress-induced reinstatement, albeit with differential effects on
Fix-C and Esc-C CPP, the NR2B subunit is not critical for this response.
Given the disparities in the effect of MK-801 on stress-induced reinstatement of
Fix-C and Esc-C CPP (Fig. 4.1), we aimed to elucidate the contribution of signaling
molecules downstream of the NMDAR. nNOS is functionally coupled to NMDAR
activity (Christopherson et al., 1999; Sattler et al., 1999) and we have previously found
differential effects of nNOS inhibition on Fix-C and Esc-C memory acquisition and
reconsolidation (Liddie & Itzhak, 2014, Chapter 2). Therefore, we investigated whether
nNOS inhibition would have differential effects on stress-induced reinstatement of Fix-C
and Esc-C CPP. We found that the nNOS inhibitor 7-NI prevented stress-induced
reinstatement for Fix-C but not Esc-C CPP (Fig. 4.2). An additional FST trial carried out
3 weeks later showed that the acute administration of 7-NI prior to FST-1 provided long
lasting protection since stress-induced reinstatement was significantly attenuated (data
not shown). This suggests that along with acquisition and reconsolidation, stress-induced
reinstatement of Fix-C CPP is NO-dependent while Esc-C CPP bypasses the dependence
on NO activity and is mediated by alternative signaling molecules. Previous studies
suggest that nNOS inhibitors possess anxiolytic and antidepressant properties (Harkin et
al., 1999; Yildiz et al., 2000). In the current study, there were no overt behavioral
anxiolytic or antidepressant effects of 7-NI with respect to immobility during FST and
locomotor activity; a result possibly due to the use of a relatively low dose of 7-NI.
However, there is still the possibility that 7-NI’s effect on reinstatement of Fix-C CPP
was due to a) antidepressant activity that was not manifested in motor behavior or b) a
reduction in the influence of NO at modulating the neuroendocrine response to stress
(Gulati et al., 2006). Indeed it has been suggested that NO may play a physiological role
in regulating the secretion of hypothalamic and pituitary hormones (Kim & Rivier, 1998;
Rivier, 1995). Because Esc-C CPP is potentially NO-independent (Liddie & Itzhak, 2014,
Chapter 2), it is not surprising that inhibition of nNOS was ineffective at disrupting
stress-induced reinstatement of Esc-C CPP. Thus, future experiments should be geared
toward identifying molecular factors which contribute to stress-induced reinstatement of
Esc-C CPP.
Although the FST is often used to measure depression-like behavior in
experimental animals, an alternative interpretation of behavior is that the immobility
observed reflects effects of learning and memory, a reflection of learned habituation,
rather than despair (West, 1990). This idea is supported by the observation that
administration of an amnesic agent (anisomycin) during the first phase of FST increased
mobility in the second FST (De Pablo et al., 1989). Thus anisomycin may have interfered
with the memory consolidation process rather than attention or activity. Based on the
latter interpretation, results demonstrate that 7-NI does not interfere/disrupt memory
associated with the FST since mice treated with 7-NI prior to FST-1 show similar
immobility as vehicle-treated mice (Fig. 4.3).
We next investigated the role of CRH-R1 in swim-induced reinstatement of Fix-C
and Esc-C CPP. The CRH-R1 plays a role in mediating stress responsivity (Smith et al.,
1998); thus highlighting an anxiogenic role for this receptor. Additionally, oral
administration of the CRH-R1 antagonist antalarmin has been shown to attenuate
behavioral, neuroendocrine, and autonomic responses to stress in primates (Habib et al.,
2000). We found that pretreatment with antalarmin attenuated stress-induced
reinstatement in Fix-C but not Esc-C CPP (Fig. 4.4). The effect of antalarmin on Fix-C
CPP may be due to its anxiolytic nature which may have attenuated the influence of
stress on reinstatement of extinguished Fix-C CPP. Our finding that CRH-R1 is involved
in stress-induced reinstatement of Fix-C CPP is in accordance with others who showed
that systemic administration of antalarmin blocked swim-induced reinstatement of CPP
acquired by a 4 day 15mg/kg fixed cocaine administration schedule (McReynolds et al.,
2014). The observation that antalarmin had no effect on Esc-C CPP is unclear but it
suggests that while stress-induced reinstatement of Fix-C CPP is mediated by CRH-R1,
Esc-C CPP engages additional or distinct molecular factors that contribute to the reestablishment
of the extinguished cocaine-associated memory. As such further research is
necessary to elucidate the disparate contribution of CRH-R1 in stress-induced
reinstatement of Fix-C and Esc-C CPP.


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