Activation of gastrin-releasing peptide receptors at the infralimbic cortex elicits gastrin-releasing peptide release at the basolateral amygdala: Implications for conditioned fear
Graphical abstract
Introduction
The amygdala appears to be involved in all aspects of conditioned fear including the acquisition, expression and extinction of learned fear responses (Maren and Fanselow, 1996, Davis, 1997, Marek et al., 2013), whereas the infralimbic (IL) cortex is more specifically involved in the extinction of conditioned fear (Milad and Quirk, 2002, Milad et al., 2004, Marek et al., 2013). These brain regions share extensive reciprocal neural connections (McDonald et al., 1996, Sah et al., 2003), such that the IL cortex is thought to inhibit neuronal activity within the amygdala (Milad and Quirk, 2002, Milad et al., 2004, Pare et al., 2004). It seems that glutamatergic projections from the IL cortex to the lateral/basolateral amygdala (BLA) synapse on GABA interneurons located both within the BLA and intercalated cells located between the BLA and central nucleus of the amygdala (CeA) (Quirk et al., 2003, Pare et al., 2004), producing a feedward inhibitory mechanism to reduce conditioned fear responses when activated. What is less well understood, however, are the modulatory influences within this circuitry that might impact the excitatory/inhibitory balance and subsequent conditioned fear responses.
Gastrin-releasing peptide (GRP), the mammalian analog of the amphibian peptide bombesin, might have a modulatory influence within this IL-BLA circuitry. GRP (BB2) receptors are highly expressed at both the anterior cingulate cortex (which encompasses the IL cortex) and the lateral nucleus of the amygdala (LA), specifically on GABAergic interneurons, and the application of GRP to either of these sites causes increased inhibition through release of GABA (Shumyatsky et al., 2002, Cao et al., 2010). Increasing evidence suggests the involvement of GRP in both conditioned fear expression and extinction (Shumyatsky et al., 2002, Roesler et al., 2004, Roesler et al., 2012, Mountney et al., 2006, Mountney et al., 2008, Martel et al., 2012). For instance, mice lacking BB2 receptors showed enhanced expression of learned fear responses as well as impaired extinction of a conditioned emotional response (Shumyatsky et al., 2002, Martel et al., 2012), and GRP injected into either the IL cortex, the CeA or the BLA, reduced freezing in a conditioned emotional response paradigm (Mountney et al., 2006, Mountney et al., 2008). Unexpectedly, injections of the BB2 receptor antagonist(s) BW2258U89 or RC-3095 into the IL cortex or the BLA, respectively, also reduced freezing. Injection of BW2258U89 into the CeA had mixed results, as reduced freezing was elicited by a high dose (300 ng), whereas the opposite effect was provoked by a low dose (50 ng) (Mountney et al., 2006). It is possible that at higher doses, these BB2 receptor antagonists have intrinsic agonist properties, as previously demonstrated with BW2258U89 (Kirkham et al., 1995) as well as other BB2 receptor antagonists (Wang et al., 1990, Ryan et al., 1999, Gonzalez et al., 2009). Similarly Roesler’s group (Dantas et al., 2006) demonstrated opposite effects of low vs. high doses of RC-3095 injected into the dorsal hippocampus on memory retention, with the effects of high-dose administration (10 μg) being more in keeping with agonist activity.
Like GRP, corticotropin-releasing factor (CRF) also appears to be involved in conditioned fear as intra-LA injection of CRF enhanced the expression of learned fear (Isogawa et al., 2012), whereas systemic administration of a CRF antagonist impaired the expression of contextual fear memory (Hikichi et al., 2000). Substantial anatomical overlap exists between GRP and CRF including a high density of CRF1 receptors at the prefrontal cortex (including the IL cortex) and the BLA (De Souza et al., 1985). Moreover, several of the endocrine and behavioral effects of exogenous bombesin (the amphibian counterpart to GRP) administration, such as hypothalamic-pituitary hormone activation and anorectic actions, were blocked by pretreatment with a CRF receptor antagonist (Plamondon and Merali, 1997, Kent et al., 2001b). In addition, central administration of bombesin reduced endogenous CRF levels at several brain sites, including the CeA, while increasing CRF release from the median eminence and anterior pituitary (Kent et al., 2001a). Finally, a protracted and sustained elevation of GRP and CRF release was observed at the BLA 24 h after fear conditioning (Mountney et al., 2011), which may suggest that these two peptidergic systems act in a collaborative manner.
Given the robust anatomical connections between the IL cortex and the BLA, coupled with high BB2 receptor expression at these two sites, the primary objective of the present study was to determine whether a functional pathway utilizing GRP (and CRF) exists between the IL cortex and the BLA that could be relevant to conditioned fear. To this end, we assessed whether the activation of GRP receptors at the IL cortex (via GRP administration) provokes the downstream release of GRP and/or CRF at the BLA and whether this effect would be blocked by the co-administration of a BB2 receptor antagonist.
Section snippets
Subjects
Male Sprague–Dawley rats (Charles River Laboratories, St-Constant, Quebec, Canada) weighing approximately 275–300 g upon arrival, were maintained on a 12-h light/dark cycle (lights on at 07:00 h) in a climate-controlled environment (23 °C, relative humidity 60%). Animals were doubly housed in standard plastic cages (45 × 25 × 20 cm) until surgery and had free access to food (Purina Lab Chow; Charles River Laboratories) and water. All experimental procedures were performed in accordance with the
Results
Fig. 2A depicts the interstitial levels of ir-GRP (expressed as a percentage of baseline values) at the BLA under basal conditions and following bilateral IL microinjection of either GRP (n = 8), the BB2 receptor antagonist RC-3095 (n = 8), GRP and RC-3095 combined (n = 8), or vehicle (n = 6). Analysis of ir-GRP levels within the right side of the BLA revealed a significant GRP × RC-3095 × Samples interaction, F(8, 152) = 4.06, p < .001. The follow-up comparisons of the simple effects indicated that treatment
Discussion
In light of the facts that (1) BB2 receptors are highly expressed in the IL cortex and the BLA, two structures with robust anatomical connections, and (2) microinjection of GRP into either the IL cortex or the BLA reduced freezing in a conditioned fear paradigm (Mountney et al., 2006, Mountney et al., 2008), the present study sought to determine whether a functional pathway utilizing GRP exists between the IL cortex and the BLA. As expected, injection of GRP into the IL cortex elicited the
Conclusion
Although IL administration of GRP or RC-3095 appears to have no effect on CRF release at the BLA, endogenous GRP release at this site was enhanced by either the administration of GRP or RC-3095, an effect that was blocked when the agonist and antagonist were co-administered. The fact that the release profile was similar following administration of either GRP or its antagonist parallels our earlier behavioral findings (Mountney et al., 2006, Mountney et al., 2008) and supports the view that the
Role of funding source
Funding for this study was provided by the NSERC (Grant 119692); NSERC had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.
Conflict of interest
There are no conflicts of interest to report.
Acknowledgements
We kindly thank Jonathan James for his work on conducting the radioimmunoassay and Christine MacKay for the time she volunteered during the microdialysis experiments and her work on the histologies.
References (40)
- et al.
Role of NMDA receptors in the lateralized potentiation of amygdala afferent and efferent neural transmission produced by predator stress
Physiol Behav
(2005) - et al.
Opposite effects of low and high doses of the gastrin-releasing peptide receptor antagonist RC-3095 on memory consolidation in the hippocampus: possible involvement of the GABAergic system
Peptides
(2006) - et al.
Characterization of putative GRP- and NMB-receptor antagonist’s interaction with human receptors
Peptides
(2009) - et al.
Bombesin-induced HPA and sympathetic activation requires CRH receptors
Peptides
(2001) - et al.
Meal pattern analysis in rats reveals partial agonist activity of the bombesin receptor antagonist BW2258U89
Pharmacol Biochem Behav
(1995) - et al.
Interleukin-1 receptor antagonist exerts agonist activity in the hippocampus independent of the interleukin-1 type I receptor
J Neuroimmunol
(2003) - et al.
The amygdala and fear conditioning: has the nut been cracked?
Neuron
(1996) - et al.
Projections of the medial and lateral prefrontal cortices to the amygdala: a Phaseolus vulgaris leucoagglutinin study in the rat
Neuroscience
(1996) - et al.
Effects of intracerebral ventricular administration of gastrin-releasing peptide and its receptor antagonist RC-3095 on learned fear responses in the rat
Behav Brain Res
(2011) - et al.
In vivo levels of corticotropin-releasing hormone and gastrin-releasing peptide at the basolateral amygdala and medial prefrontal cortex in response to conditioned fear in the rat
Neuropharmacology
(2011)
Anorectic action of bombesin requires receptor for corticotropin-releasing factor but not for oxytocin
Eur J Pharmacol
Iodination of proteins, and peptides using solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3,6-diphenyl glycoluril (iodogen) radioiodine into proteins and peptides without damage
Anal Biochem
Identification of a signaling network in lateral nucleus of amygdala important for inhibiting memory specifically related to learned fear
Cell
Prefrontal cortical regulation of hypothalamic–pituitary–adrenal function in the rat and implications for psychopathology: side matters
Brain Res
Des-Met carboxyl-terminally modified analogues of bombesin function as potent bombesin receptor antagonists, partial agonists, or agonists
J Biol Chem
Mechanistic explanation for the unique pharmacologic properties of receptor partial agonists
Biomed Pharmacother
Facilitation of the inhibitory transmission by gastrin-releasing peptide in the anterior cingulate cortex
Mol Pain
Neurobiology of fear responses: the role of the amygdala
J Neuropsychiatry Clin Neurosci
Corticotropin-releasing factor receptors are widely distributed within the rat central nervous system: an autoradiographic study
J Neurosci
Suppression of conditioned fear by administration of CRF receptor antagonist CP-154,526
Pharmacopsychiatry
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