Distinct cellular properties of identified dopaminergic and GABAergic neurons in the mouse ventral tegmental area.

The midbrain ventral tegmental area (VTA) contains neurons largely with either a dopaminergic (DAergic) or GABAergic phenotype. Physiological and pharmacological properties of DAergic neurons have been determined using tyrosine hydroxylase (TH) immunohistochemistry but many properties overlap with non-DAergic neurons presumed to be GABAergic. This study examined properties of GABAergic neurons, non-GABAergic neurons and TH-immunopositive neurons in VTA of GAD67-GFP knock-in mice. Ninety-eight per cent of VTA neurons were either GAD-GFP or TH positive,with the latter being five times more abundant. During cell-attached patch-clamp recordings, GAD-GFP neurons fired brief action potentials that could be completely distinguished from those of non-GFP neurons. Pharmacologically, the µ-opioid agonist DAMGO inhibited firing of action potentials in 92% of GAD-GFP neurons but had no effect in non-GFP neurons. By contrast, dopamine invariably inhibited action potentials in non-GFP neurons but only did so in 8% of GAD-GFP neurons. During whole-cell recordings, the narrower width of action potential in GAD-GFP neurons was also evident but there was considerable overlap with non-GFP neurons. GAD-GFP neurons invariably failed to exhibit the potassium-mediated slow depolarizing potential during injection of positive current that was present in all non-GFP neurons. Under voltage-clamp the cationic current, I(h), was found in both types of neurons with considerable overlap in both amplitude and kinetics. These distinct cellular properties may thus be used to confidently discriminate GABAergic and DAergic neurons in VTA during in vitro electrophysiological recordings.

Cellular morphine tolerance produced by ßarrestin-2-dependent impairment of µ-opioid receptor resensitization.

Chronic morphine treatment produces behavioral and cellular opioid tolerance that has been proposed to be caused by attenuated µ-opioid receptor (MOR) recovery from desensitization (resensitization). The process of MOR resensitization is thought to require ßarrestin-2 (ßarr-2)-dependent trafficking of desensitized receptors to endosomal compartments, followed by recycling of resensitized receptors back to the plasma membrane. However, there is little direct evidence for this, particularly in native neurons. This study used whole-cell patch-clamp recording in locus ceruleus (LC) neurons from wild-type (w.t.) and ßarr-2 knock-out (k.o.) mice to examine whether ßarr-2/dynamin-dependent trafficking is required for MOR resensitization in neurons from opioid-naive and morphine-treated mice. Surprisingly, recovery of MOR from acute desensitization in LC neurons does not require ßarr-2- or dynamin-dependent trafficking. To the contrary, MOR resensitization was accelerated by disruption of either ßarr-2 or dynamin function. Chronic morphine treatment caused cellular MOR tolerance and concurrently impaired MOR resensitization in neurons from w.t. mice, as expected from previous studies, but neither occurred in neurons from ßarr-2 k.o. mice. Moreover, the impairment of MOR resensitization caused by chronic morphine was reversed in w.t. neurons when G-protein-coupled receptor kinase-2 (GRK2) or dynamin function was disrupted. Together, these results establish that ßarr-2/dynamin-dependent receptor regulation is not required for MOR resensitization in LC neurons. Furthermore, chronic morphine treatment modifies GRK2-ßarr-2-dynamin-dependent MOR trafficking to impair receptor resensitization, thereby contributing to opioid tolerance in LC neurons by reducing the number of functional receptors on the surface membrane.

Complex actions of neurotensin in ascending and sigmoid colonic muscle: Involvement of enteric mediators.

The brain-gut peptide neurotensin has complex effects on gastrointestinal smooth muscle. Our objective was to elucidate the mechanisms underlying neurotensin contractions in human colon. Discrete concentration response curves to neurotensin were obtained in strips of circular muscle and taenia coli from "normal" ascending and sigmoid colon segments, in the presence and absence of various pharmacological inhibitors. Potency of neurotensin in all regions was similar (pD(2) ~7). Atropine and the selective muscarinic receptor antagonists, methoctramine and darifenacin, had no effect on neurotensin contractions. In ascending colon circular muscle, responses were enhanced by indomethacin (indicating inhibitory prostaglandin mechanisms) and by tetrodotoxin (TTX), hexamethonium and L-NAME, suggesting nicotinic and enteric inhibitory neurotransmission, with involvement of nitric oxide. In sigmoid circular muscle, neurotensin responses were also enhanced by TTX and hexamethonium, but were attenuated in the presence of mepyramine, MEN10627 and CP99994, suggesting inhibitory neuronal mechanisms and involvement of histamine and tachykinins, respectively; L-NAME and the GABA(B) receptor antagonist, CGP36742, were without effect. The transcripts of NTS1 and NTS3 receptors, but not NTS2 receptors, were detected in sigmoid colon circular muscle and taenia coli. No age and gender differences in NTS1 mRNA expression were found. In conclusion, neurotensin contracts circular muscle strips from ascending and sigmoid regions of the human colon via direct (muscle) and indirect (neuronal/non-neuronal mechanisms). The enteric mediators influenced by neurotensin are regionally specific. In taenia coli strips from both ascending and sigmoid colon, neurotensin contractions were unchanged in the presence of inhibitors, suggesting direct actions only.