Mapping of endogenous morphine‐like compounds in the adult mouse brain: Evidence of their localization in astrocytes and GABAergic cells

Abstract

Endogenous morphine, morphine-6-glucuronide, and codeine, which are structurally identical to vegetal alkaloids, can be synthesized by mammalian cells from dopamine. However, the role of brain endogenous morphine and its derivative compounds is a matter of debate, and knowledge about its distribution is lacking. In this study, by using a validated antibody, we describe a precise mapping of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the mouse brain. First, a mass spectrometry approach confirmed the presence of morphine and codeine in mouse brain, but also, of morphine-6-glucuronide and morphine-3-glucuronide representing two metabolites of morphine. Second, light microscopy allowed us to observe immunopositive cell somas and cytoplasmic processes throughout the mouse brain. Morphine-like immunoreactivity was present in various structures including the hippocampus, olfactory bulb, band of Broca, basal ganglia, and cerebellum. Third, by using confocal microscopy and immunofluroscence co-localization, we characterized cell types containing endogenous opiates. Interestingly, we observed that morphine-like immunoreactivity throughout the encephalon is mainly present in γ-aminobutyric acid (GABA)ergic neurons. Astrocytes were also labeled throughout the entire brain, in the cell body, in the cytoplasmic processes, and in astrocytic feet surrounding blood vessels. Finally, ultrastructural localization of morphine-like immunoreactivity was determined by electron microscopy and showed the presence of morphine-like label in presynaptic terminals in the cerebellum and postsynaptic terminals in the rest of the mouse brain. In conclusion, the presence of endogenous morphine-like compounds in brain regions not usually involved in pain modulation opens the exciting opportunity to extend the role and function of endogenous alkaloids far beyond their analgesic functions.

Localization of endogenous morphine‐like compounds in the mouse spinal cord

“…This antibody has been used previously and extensively controlled (Charron et al, 2011;Glattard et al, 2010;Laux et al, 2011;Muller et al, 2008), demonstrating the specificity of the labeling. 1) In ELISA, the 6D6 antibody recognized morphine, codeine, M3G, and M6G, without any cross-reactivity with other morphine precursors or related compound (i.e., dopamine or THP).…”

“…This antiserum was used in our previous study on mouse brain and displayed a specific labeling of astrocytes cell bodies and processes (i.e., astrocytic foot around blood vessels; Laux et al, 2011).…”

“…In the mouse brain, morphine-like compounds were found in GABAergic cells and astrocytes (Charron et al, 2011;Laux et al, 2011). To characterize the cells and processes containing morphine-like compounds in the mouse spinal cord, we used confocal microscopy with different cell markers.…”

“…Endogenous alkaloids, which are structurally identical to vegetal alkaloids, are present in various mammalian tissues and cells (Donnerer et al, 1986(Donnerer et al, , 1987Gintzler et al, 1978;Glattard et al, 2010;Goldstein et al, 1985;Goumon et al, 2009;Hazum et al, 1981;Herbert et al, 2000;Laux et al, 2011;Muller et al, 2008;Neri et al, 2004;Stefano et al, 2000;Weitz et al, 1986;Zhu et al, 2001). Endogenous morphine biosynthesis pathways have been demonstrated in the SH-SY5Y human cell line and shown to be derived from dopamine (Boettcher et al, 2005;Muller et al, 2008;Neri et al, 2008;Poeaknapo, 2005;Poeaknapo et al, 2004).…”

“…Endogenous morphine biosynthesis pathways have been demonstrated in the SH-SY5Y human cell line and shown to be derived from dopamine (Boettcher et al, 2005;Muller et al, 2008;Neri et al, 2008;Poeaknapo, 2005;Poeaknapo et al, 2004). However, several reports have noted that endogenous alkaloids can be synthesized by nondopaminergic or noncatecholaminergic cells (i.e., pancreatic cells, hepatocytes, and placenta-derived cells; Molina et al, 1995;Poeaknapo et al, 2004;Weitz et al, 1987), suggesting that nondopaminergic cells may internalize dopamine or intermediate metabolites to finalize morphine biosynthesis (Charron et al, 2011;Goumon et al, 2009;Laux et al, 2011).…”