Cholecystokinin-8S-Induced Intracellular Calcium Signaling in Acutely Isolated Periaqueductal Gray Neurons of the Rat

Abstract

The midbrain periaqueductal gray (PAG) plays a crucial role in the integration of an animal's behavioral, somatic, and autonomic responses to threat, stress, and pain. 1,2) In particular, the PAG is a major site for modulation of nociception and a part of descending antinociceptive systems that relay via the rostral ventromedial medulla (RVM) to the spinal cord. 3,4) In addition, stimulation of dorsal PAG in several species produced defense reaction, anxiety, and fear, and lesions of the PAG produced anxiolytic effect. 5,6) Cholecystokinin (CCK) is a bioactive peptide that functions as a gastrointestinal hormone and a neuropeptide in both the peripheral and central nervous systems. As one of the most abundant neuropeptides in the brain, CCK has also been known to influence a variety of behaviors such as satiety, nociception, and anxiety/panic action in the brain. [7][8][9] Immunocytochemical studies have demonstrated that CCK is a major neurotransmitter or modulator in the PAG, and CCK receptors are heterogeneously distributed within the PAG.10,11) Microinjection of CCK or CCK receptor antagonists into the PAG antagonized or potentiated the acute antinociceptive effect of microinjected morphine, respectively. [12][13][14][15] In addition, Netto et al. 16) reported that microinjection of CCK into the PAG produced an anxiogenic effect through CCK 2 receptors using an animal model of anxiety. Based on these behavioral studies, the PAG could be a key site of anti-analgesic and anxiogenic actions of CCK, and it is possible that these effects of CCK are mediated through an alteration of neuronal activities in the PAG.CCK exerts its physiological effects via two subtypes of receptors, CCK 1 and CCK 2 receptors. 8,9) Both CCK receptors belong to a superfamily of G protein-coupled receptors (GPCR). 7,17,18) Direct coupling of CCK 1 and CCK 2 receptors onto Ga q to activate phospholipase C (PLC) and cause intracellular Ca 2ϩ signaling has been well demonstrated in pancreatic acinar cells [19][20][21] and several cell lines. [22][23][24] However, this effect is not clearly demonstrated in the central nervous system (CNS). We recently reported that CCK excited PAG neurons at both pre-and postsynaptic loci via the activation of CCK 2 receptors using slice preparations and whole-cell patch-clamp recordings.25) Therefore, in order to examine whether CCK could modulate intracellular Ca 2ϩ mobilizations in PAG neurons, we examined the effects of a sulfated form of CCK octapeptide (CCK-8S) using fura-2-based intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and whole-cell patch-clamp recordings in acutely isolated PAG neurons of the rat. MATERIALS AND METHODSAcute Isolation of PAG Neurons PAG neurons with synaptic boutons were acutely isolated using the technique modified from Hahm et al. 26) Briefly, 12 to 21-d-old SpragueDawley rats were anesthetized and decapitated in accordance with the NIH Guide for the Care of Use of Laboratory Animals (revised 1996). The brains were quickly removed and immersed in an ice-cold artifici...

Cholecystokinin Exerts an Effect via the Endocannabinoid System to Inhibit GABAergic Transmission in Midbrain Periaqueductal Gray

“…Together, these results suggest that the cholecystokinin-induced inward current is largely mediated by CCK1 receptors; however, a role for CCK2 receptors cannot be excluded. The present results differ to previous studies in the PAG where the postsynaptic effects of CCK-S were largely abolished by the CCK2 receptor antagonist L-365, 260 (10 mM), but not by 10 mM devazepide (Yang et al, 2006(Yang et al, , 2007. It might also be noted that CCK2 antagonists have been reported to block the increase in firing rate of PAG neurons, also the anxietyrelated activity, defensive rage, and the pro-nociceptive/ anti-opioid effects produced by CCK-S and pentagastrin within the PAG (Bertoglio and Zangrossi, 2005;Brack and Lovick, 2007;Chen et al, 1998;Liu et al, 1994;Luo et al, 1998;Netto and Guimaraes, 2004;Tortorici et al, 2003).…”

“…The CCK-S-induced inward current was likely to be mediated by activation of a non-selective cation conductance and inhibition of an inwardly rectifying potassium conductance, because it was associated with a near parallel shift in the current-voltage relationship. Although this differs to a previous study, in which it was found that CCK-S exerted an effect solely by inhibiting an inwardly rectifying potassium conductance in dorsolateral PAG neurons (Yang et al, 2006(Yang et al, , 2007, it is similar to our previous findings for neurotensin and substance P (Drew et al, 2005;Mitchell et al, 2009).…”

“…All agents were diluted to working concentrations in ACSF (solvent p0.03% v/v) immediately before use and applied by superfusion. Cholecystokinin, cannabinoid, and mGluR agents were used at concentrations that have previously been shown to be maximally effective in PAG slices (Drew et al, 2009;Drew et al, 2008;Yang et al, 2007). The entire system was washed in 30% ethanol after AM251 experiments.…”

“…Cholecystokinin, however, has complex effects on PAG neurons, including inhibition of voltagedependent calcium currents, a potassium current and the apamin-sensitive after-hyperpolarization (Yang et al, 2006(Yang et al, , 2007. In addition, cholecystokinin increases spontaneous miniature glutamatergic synaptic transmission within the PAG via a presynaptic mechanism (Yang et al, 2006).…”

Cholecystokinin receptors regulate sperm protein tyrosine phosphorylation via uptake of HCO3−

“…More and more evidence indicated that CCK and native or recombinant CCK1 and CCK2 receptors were coupled to the mobilization of intracellular Ca 2C (Matozaki et al 1988, Lee et al 1993, Dunlop et al 1996, Shimazoe et al 2008. The antagonist of CCK1 or CCK2 receptor could significantly affect physiological functions of different cell types by modulating intracellular Ca 2C concentration (Muller et al 1997, Simasko et al 2002, Yang et al 2007). Ca 2C is essential for the activation of the sAC/cAMP/PKA pathway in sperm.…”