We have used cell-attached patch-clamp electrophysiology to characterize the activation and distribution of an 85 pS K+ channel on freshly dissociated rat striatal (caudate-putamen) neurons. In recordings from 643 cells, openings of this channel showed an absolute dependence on the presence of dopamine or the D2-like dopamine receptor agonist quinpirole in the cell-attached patch pipette, but were never seen when the D2 antagonist domperidone was applied along with quinpirole, or in the absence of drug. This channel displayed inward rectification at depolarized membrane potentials, but its activation was otherwise voltage insensitive. It was largely restricted to a subset of dissociated cells with diameters > or = 10 microns, with channel openings seen in about 25% of patches. When present, there were typically multiple channels per patch. Cells of this size were immunocytochemically stained for neuron-specific enolase but not glial fibrillary acidic protein; about 40% were also labeled for gamma-amino butyric acid (GABA) and about 60% for NADPH diaphorase, with GABAergic cells displaying a shape most similar to that of cells expressing the channel. A large number of distinct types of other channels were also present, comprising inwardly rectifying channels of 5-35 pS conductance and voltage-activated channels of 100-250 pS, but the frequencies of occurrence and fractional open times of these channels were independent of the presence or absence of dopaminergic agonists. Thus, the 85 pS K+ channel uniquely requires activation by a D2-like dopamine receptor on rat striatal neurons, and is selectively expressed by a subset of these cells, which are most likely to be GABAergic neurons.
Corpus striatum neurons acutely dissociated from the brains of young adult rats had membrane surfaces suitable for Gi-seal recording. Whole-cell current-clamp and voltage-clamp recordings indicated that the cells remained electrically excitable after dissociation. Cell-attached recordings frequently revealed single-channel openings in the presence of dopamine or of the D2 dopamine agonist quinpirole. Channel openings were rarely or never observed in the absence of drugs or in the presence of quinpirole plus the dopamine antagonist haloperidol. The D2 antagonist spiperone was more potent at blocking the appearance of the channel than was the D1 antagonist SCH-23390. The channel reversal potential varied with the extracellular K+ concentration as predicted by the Nernst equation. The channel currentvoltage relationship was linear, with a conductance of m85 pS in the presence of 140 mM KCL. These results are consistent with the opening of single K+ channels following D2 dopamine receptor activation.The corpus striatum (caudate and putamen) is a major postsynaptic target of dopaminergic projections in mammalian brain (1, 2). Because of the role of dopamine in the actions of antipsychotic drugs (1, 2) and because of the potential usefulness of studying these actions with single-molecule resolution, we have studied the effects of dopamine at the singlechannel level.Patch-clamp recording in adult mammalian brain has been hampered by the need for dissociated cells. One way to obtain dissociated cells is to work with cultured embryonic tissue (3). However, rat striatal dopamine receptor radioligand binding sites are largely absent in the neonate and do not reach adult levels until =28 days after birth (4). A method has been described for enzymatically splitting the hippocampus along the pyramidal cell layer (5) but may not be useful for brain regions such as the striatum, which are not organized into cellular layers.Kay and Wong (6) have described a method for acutely dissociating adult guinea pig brain, based upon trypsin treatment in a slightly acidic Pipes buffer. Kay et al. (7) and Huguenard and Alger (8) have used this method to record from guinea pig hippocampal pyramidal neurons. We have made minor modifications in their procedure to dissociate striatal neurons of young adult rats. We report here that we have used these dissociated cells to obtain an initial description at the single-channel level of the actions of dopamine in mammalian brain. post partum, were the tissue source. The corpus striatum was rapidly and carefully dissected in a beeswax-coated Petri dish under ice-cold Pipes saline [120 mM NaCI, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 25 mM D-glucose, 20 mM Pipes-NaOH (pH 7.0 at 25°C, preequilibrated with 100% 02)1 and hand-minced with a scalpel blade into blocks of =2 mm per edge. The tissue blocks were transferred by using a wide-bore pipette to a 25-ml jacketed double-side-armr Wheaton Celstir stirring chamber, containing 130,000 benzoylarginine ethyl ester units of trypsin (Sigma type XI) in 12 m...
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 ...
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