Organization of endogenous opioids in the rostral agranular insular cortex of the rat

Abstract

The rostral agranular insular cortex (RAIC) of rats has opioid receptors and has been implicated in the analgesic and reinforcing effects of opiates. To help in understanding the function of endogenous opioids in this structure, we sought to identify and describe the opioid peptides intrinsic to the RAIC by using immunohistochemical methods. Immunolabeling for proopiomelanocortin (POMC), the precursor to beta-endorphin, and endomorphin 1 and 2 on sectioned rat forebrain revealed limited labeling consisting of individual varicose fibers. Immunolabeling for prodynorphin and enkephalin revealed numerous immunopositive cell bodies and fibers with distribution and morphology unique to each. Prodynorphin-immunopositive cell bodies consisted of two types: large, lightly labeled, pyramidal-shaped cell bodies in lamina V and more intensely labeled, small, ovoid cell bodies scattered in other lamina. Axonal fibers immunolabeled for prodynorphin varied in size and were found in all lamina. Immunolabeling for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) was rarely found in dynorphin-containing cell bodies (6%, 10/167) but was visible within a subpopulation of axons. Enkephalin immunolabeling was detected within a single morphological subpopulation of nonpyramidal neurons located predominantly in lamina II/III, 30% (33/109) of which were also GABA immunopositive. Axons immunolabeled for enkephalin were also abundant in lamina II/III. These results suggest that dynorphin and enkephalin peptides are the predominant endogenous opioids in the RAIC and their distinct distributions suggest divergent functional roles. The localization of prodynorphin immunoreactivity to pyramidal cells suggests the possibility that this neuropeptide may be used in RAIC projection neurons, whereas enkephalin distribution was more characteristic of a role in local networks.

Preproenkephalin mRNA is Expressed in a Subpopulation of GABAergic Neurons in the Spinal Dorsal Horn of the GAD67‐GFP Knock‐In Mouse

“…A greater degree of colocalization between ENK and GABA has also been revealed in different regions of the brain and in different species, such as in the avian retina (Watt et al, 1984), the rat cerebral cortex (Taki et al, 2000), the rat rostral agranular insular cortex (Evans et al, 2007), and the basal ganglia (McGinty et al, 2007). The widespread colocalization of ENK and GABA in cells of the central nervous system suggests the functional relevance, perhaps interdependence of these two neurotransmitters.…”

Search of the human proteome for endomorphin-1 and endomorphin-2 precursor proteins

“…Several groups have studied the immunocytochemical localization of endomorphin-like immunoreactivity in the spinal cord and brain (Martin-Schild et al, 1997, 1999Pierce et al, 1998;Schreff et al, 1998;Wessendorf, 2000, Wang et al, 2002;Sanderson Nydahl et al, 2004;Evans et al, 2007), however, the peptide/protein species that were recognized by the endomorphin antibodies were not characterized biochemically. Based on our bioinformatic search of the human proteome, it appeared likely that many proteins would be recognized by the endomorphin antibodies due to the presence of the endomorphin tetrapeptide sequence within them.…”

“…We therefore used western blotting to characterize the endomorphin-like immunoreactivity in mouse brain, and several other murine and human tissue samples, using a commercial polyclonal antibody generated against synthetic endomorphin-2 for detection. This is the same antibody that was used recently by Evans et al (2007) for immunohistochemical studies in rat brain. Mouse brain lysates contained four major immunoreactive protein bands with apparent molecular masses of 25, 28, 78, and 117 kilodaltons (kDa).…”

“…Membranes were blocked for 1 h in 3% dried milk, 50 mM Tris HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl 2 , 10% glycerol and 0.1% Tween 20, followed by overnight incubation at 4°C with a 1:2000 dilution of the antiendomorphin polyclonal antibody (AB 5106, Upstate/Chemicon, Temecula, CA) for detection of endomorphin-like immunoreactivity. The AB 5106 antibody was raised in rabbits against synthetic endomorphin-2 (Tyr-Pro-Phe-Phe-NH 2 ) conjugated to bovine serum albumin, and has been used by Evans et al (2007) to study endomorphin-2 immunoreactivity in the rat insular cortex using immunohistochemical methods. The vendor's specifications assert that the polyclonal antiserum cross-reacts 100% with endomorphin-2, 70% with endomorphin-1, <0.03% with Met-enkephalin, and <0.01% with Leu-enkephalin and β-endorphin.…”

Basic Mechanisms of Pain

“…Finally, the insula also has descending projections to the brainstem through which it exerts control over the autonomic nervous system (120) as well as apparently regulating the descending pain control systems. Recent studies have documented the presence of opioids such as dynorphin and enkephalin in the insular cortex and suggested a role for these opioid systems in the generation of cortically-mediated analgesia (121). One possible interpretation of this arrangement is that the insula helps to orchestrate the bodies physiological response to pain, including pain control or enhancement depending n the situation (118).…”