Altered gating of opiate receptor-modulated K ⁺ channels on amygdala neurons of morphine-dependent rats

Abstract

The molecular mechanism of tolerance to opiate drugs is poorly understood. We have used single-channel patch-clamp recordings to study opiate receptor effects on dissociated neurons from rat amygdala, a limbic region implicated in addiction processes. A 130-pS inwardly rectifying K ؉ -preferring cation channel was activated by opioid receptors in a membrane-delimited manner. After chronic treatment of the rats with morphine, channel gating changed markedly, with an approximately 100-fold decrease in open probability at a given morphine concentration. The change in channel gating correlated both in time course and in dose of morphine treatment with the development of functional opiate dependence and appeared to arise at a step after G-protein activation and before channel permeation by K ؉ . This decreased receptor-channel coupling appears to be large enough to account quantitatively for opiate tolerance and may represent one of the mechanisms through which tolerance occurs. D rug addiction is a complex process that includes tolerance (the need for increasing drug dosage to achieve the same effect) and dependence (the appearance of adverse effects on drug withdrawal). Human heroin addicts experience tolerance of more than 100-fold if drug availability is unlimited (1). Changes in the number, affinity, or membrane trafficking of opioid receptors (2-6), in the coupling of receptors to Gproteins (7, 8), or in associated second messenger systems (9) have been implicated in opiate tolerance mechanisms; however, these effects are usually on the order of 10 -20% and therefore appear unable to account quantitatively for tolerance. Consequently, the molecular basis of opiate tolerance remains poorly understood.We have used patch-clamp electrophysiology to resolve single ion channels that are activated by opioid receptors and to study changes in channel properties after chronic opiate treatment. We performed these studies by using freshly dissociated neurons from the amygdalohippocampal area (AHA) of 30-to 45-day-old rats. The AHA is an output nucleus of the amygdala (10, 11) and as such is part of a limbic region implicated in the motivational effects of opiates and other drugs of abuse (10-15). AHA neurons are known to display inhibitory responses to opiates in vivo (14). Furthermore, these cells exhibit a decreased inhibition by morphine after chronic treatment, consistent with tolerance, as well as an activation of firing after precipitated withdrawal, consistent with dependence (14). We now describe a K ϩ -permeable channel that is activated by opioid receptors and show that its gating undergoes changes after chronic morphine treatment in a manner that may account for opiate tolerance in limbic neurons. MethodsCell Preparation. Coronal 300-m sections of the posteriodorsal amygdala were cut on a vibrating tissue slicer under an ice-cold Pipes-buffered solution previously described (16) and were blocked to include the AHA (10) and some underlying amygdaloid nuclei, but excluded the hippocampus. After slowly warming the ...

Opioid Receptor Modulation of a Metabolically Sensitive Ion Channel in Rat Amygdala Neurons

“…As previously reported (Chen et al, 2000), a 130 pS channel was observed when met-enkephalin, endomorphin-1, or morphine were applied from within the patch pipette. Figure 4 A shows examples of channel recordings, with 130 pS channel activity in the presence of agonists, but no activity in the absence of drug, or when the relatively nonselective opiate antagonist naloxone or the -selective antagonist D-Phe-C ys-Tyr-D-Trp-OrnThr-Pen-Thr-NH 2 (CTOP) (Gulya et al, 1986) were present with the agonists.…”

“…The maximal amplitude of the hyperpolarization was 3.4 Ϯ 2.1 mV (n ϭ 36 trials on 15 cells) and rarely went all the way to the K ϩ reversal potential. Previous studies (Chen et al, 2000) showed that this effect was associated with a greater latency to the onset of action potentials, but did not cause an obvious change in action potential properties. At low concentrations (5-25 nM), the highly potent and selective -opioid peptide endomorphin-1 (Zadina et al, 1997) elicited responses similar to those for met-enkephalin (Fig.…”

“…Amygdala neurons were freshly dissociated by methods similar to those previously described (Greif et al, 1995;Chen et al, 2000). Male rats (Sprague Dawley VAF; Charles River Laboratories, Wilmington, M A), 30 -45 d old, were killed by rapid decapitation, and the brains were rapidly and gently removed into an ice-cold solution of (in mM): NaC l, 124; KC l, 4; C aC l 2 , 1; MgCl 2 , 1; MnCl 2 , 0.02; D-glucose, 25; and PI PES-Na, 20, pH 7.0, equilibrated with O 2 .…”

“…However, there is little direct evidence for this idea in the brain; for instance, the ability of a K ATP channel blocker to inhibit an opiate effect does not demonstrate direct coupling of the receptor to the channel. We have recently described an inwardly rectifying 130 pS K ϩ -preferring cation channel in dissociated AHA neurons that is activated by opioid receptors in a membrane-delimited manner (Chen et al, 2000;Murphy and Freedman 2001). The gating of this channel changes after chronic morphine treatment in a manner that suggests that it may play a role in opiate tolerance (Chen et al, 2000).…”

“…We have recently described an inwardly rectifying 130 pS K ϩ -preferring cation channel in dissociated AHA neurons that is activated by opioid receptors in a membrane-delimited manner (Chen et al, 2000;Murphy and Freedman 2001). The gating of this channel changes after chronic morphine treatment in a manner that suggests that it may play a role in opiate tolerance (Chen et al, 2000). We now show that this channel is specifically expressed by a subset of cells that appear to be AHA projection neurons and that it is unique among the K ϩ -permeable channels of these cells in its receptor sensitivity.…”