Elsevier

Neuropeptides

Volume 41, Issue 6, December 2007, Pages 453-463
Neuropeptides

Cholecystokinin enhances GABAergic inhibitory transmission in basolateral amygdala

https://doi.org/10.1016/j.npep.2007.08.001Get rights and content

Abstract

The neuropeptide cholecystokinin (CCK) is anxiogenic in studies of human and animal behavior. As the amygdala formation has been implicated in generation of emotional states such as anxiety, we tested the effect of CCK on spontaneous synaptic events in the basolateral amygdala (BLA) using whole cell patch recordings in rat brain slice preparation. We found that CCK increased the frequency of spontaneous inhibitory postsynaptic potentials (sIPSPs) and currents (sIPSCs). This effect was blocked by the fast sodium channel blocker tetrodotoxin (TTX), indicating that the CCK effect is likely mediated by direct excitation of GABAergic interneurons. The CCKB receptor subtype antagonist, CR2945, blocked the CCK effect, while CCK4, a specific CCKB agonist, increased sIPSC frequency. We hypothesize that these actions may underlie the anxiogenic effects of CCK observed in behavioral studies.

Introduction

The neuropeptide cholecystokinin (CCK) was first discovered in pig small intestine and later in peripheral and central nervous system. CCK and its receptors are widely distributed in the central nervous system (Noble et al., 1999, Dunlop, 1998). Centrally, CCK is implicated in various functions including emotional states, pain, learning, memory, and satiety (Crawley and Corwin, 1994). CCK is anxiogenic in humans and animals when administered peripherally or centrally (Rotzinger and Vaccarino, 2003). Intravenous injection of the CCK agonist CCK4 caused panic in humans both with and without panic disorder (de Montigny, 1989, Bradwejn et al., 1990). In animal models CCK-related peptides are anxiogenic whereas CCK antagonists are anxiolytic (Harro, 2006). The acute anxiogenic effects of CCK appear to be the result of activation of CCKB receptor subtypes (Matto et al., 1997, Hernandez-Gomez et al., 2002, Rotzinger and Vaccarino, 2003). In addition, systemic administration of a CCKB receptor antagonist blocked acquisition and expression of conditioned fear (Tsutsumi et al., 1999). In transgenic mice, overexpression of CCKB receptors in forebrain increased anxiety-like behaviors which were attenuated with diazepam (Chen et al., 2006).

The amygdala is a brain region involved in generation of fear and anxiety (LeDoux, 2000) and may be a site of action mediating the anxiogenic effect of CCK. In the acoustic startle paradigm, injection of the CCK agonist pentagastrin into BLA increased the startle response, and this effect was blocked by a CCKB antagonist (Frankland et al., 1997).

In contrast to the behavioral studies, little data is available regarding CCK effects on synaptic transmission in the amygdala. We therefore tested CCK effects on synaptic transmission and membrane properties using whole cell recording in a brain slice preparation.

Section snippets

Brain slice preparation

All procedures were approved by the Duke University and Durham VAMC Animal Care and Use Committees. Male Sprague–Dawley rats (14–21 days old) were decapitated under isoflurane anesthesia. Brains were quickly removed and placed into chilled (4 °C) solution containing (in mM) 2.5 KCl, 1.25 NaH2PO4, 10.0 MgSO4, 0.5 CaCl2, 26.0 NaHCO3, 11.0 glucose, and 234.0 sucrose bubbled with 95% O2/5% CO2. Coronal slices (350 μM) containing the amygdala region were cut using a vibrating tissue slicer. The slices

Results

Using current clamp recording techniques in regular spiking BLA pyramidal neurons (Fig. 1A), we found that CCK8S (1 μM, 30 s application) increased the frequency of sIPSPs (Fig. 1B). This was generally accompanied by a slight hyperpolarization of membrane potential (−1.2 ± 0.3 mV, n = 9; Fig. 1B). The IPSP frequency increase lasted 4–14 min (8.1 ± 1.5, n = 9). In 5 of 9 cells, rhythmic “compound” IPSPs with long duration and large amplitude also occurred at the beginning of the effect (Fig. 1B(b)). The

Discussion

In this study we found that superfusion of CCK8S increased the frequency of spontaneous inhibitory synaptic responses (sIPSPs and sIPSCs) in the BLA. The sIPSCs were completely blocked by GABAA antagonist. The sIPSCs were action potential dependent because CCK did not change frequency of spontaneous synaptic events (mIPSCs) in the presence of TTX. The CCKB receptor antagonist, CR2945, blocked the CCK effect; the CCKB receptor agonist CCK4 increased sIPSC frequency, and this effect was blocked

CCK increases IPSC frequency by activating interneurons

Because TTX blocked the increase in frequency of spontaneous synaptic events by CCK8S, the source of the increased sIPSC frequency would appear to be a direct excitatory action of CCK on local interneurons in BLA. A similar mechanism has been previously reported in hippocampus and thalamus. In the hippocampus, CCK8S increased sIPSC frequency in CA1 pyramidal neurons by activating local interneurons in an action potential dependent way (Miller et al., 1997). In the thalamus, CCK8S increased IPSC

Role of CCK receptor subtypes

At least two subtypes of CCK receptors have been previously described, CCKA and CCKB (Wank, 1995). In rodents, CCKA receptors are found both in the peripheral and central nervous system whereas CCKB receptors are located primarily in the central nervous system (Noble et al., 1999). CCKB receptors and CCKB receptor mRNA are found in significant amounts in BLA (Mercer et al., 2000), although CCKA receptors appear to be lacking (Mercer and Beart, 1997). The majority of the available behavioral

Functional significance

In general, decreasing GABAergic inhibition in the BLA increases anxious behaviors (Sanders and Shekhar, 1995). Decreasing GABA transmission in the BLA enhances aversive conditioning, while increasing GABA transmission retards aversive conditioning and induces anxiolytic responses (Davis et al., 1994). CCK appears to not fit this general scheme in that CCK is anxiogenic in behavioral tests yet increases inhibitory transmission in the BLA. Thus, it is as yet difficult to fully reconcile our data

Acknowledgements

This study is supported by a VA Merit Review (SDM) and the VISN 6 MIRECC. We thank Dr. Maeng-Hee Kang-Park for additional technical assistance.

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