The neurobiological research community greeted with great excitement the discovery of endogenous opioid-like substances in the brain that bind to the same receptors as morphine. Before the discovery of endorphins, dynorphins, and enkephalins, physiological studies showed that animals that received electrical stimulation directly in the central gray exhibited analgesia that was reversed by the opioid antagonist naloxone (Figure 5.7). Methionine and leucine enkephalin were first shown to bind to the opioid receptor and exert opioid-like activity in vivo. β-endorphin and dynorphin were then isolated, and the precursor molecules for enkephalins and endorphins were identified. Three distinct families of peptides have so far been identified: enkephalins, endorphins, and dynorphins. Each of these peptides has a distinct polypeptide precursor and a distinct but overlapping neuroanatomical distribution (Table 5.7, Figure 5.8). These endogenous opioid peptides produce self-administration, analgesia, locomotor activation, and place preference when administered directly into the brain.
FIGURE 5.7 The effects of naloxone (1 mg/kg) on stimulation-produced analgesia in (A) rats showing an initial degree of analgesia of 100% and (B) animals showing a mean initial degree of analgesia of 58%. These data show that the opioid antagonist naloxone can reverse stimulation-induced analgesia in rodents. The stimulation was in the periaqueductal gray, producing analgesia. Naloxone reversed analgesia, suggesting that there must be an endogenous opioid-like compound in the brain, which of course we now know there is. [Taken with permission from Akil H, Mayer DJ, Liebeskind JC. Antagonism of stimulation-produced analgesia by naloxone, a narcotic antagonist. Science, 1976, (191), 961–962.]
Opioid Receptors are G-Protein-Coupled Receptors with Seven Transmembrane-Spanning Regions
Opioid peptides produce analgesia when injected directly into the raphe, periaqueductal gray, nucleus reticularis gigantocellularis of the medulla, and spinal cord, among other sites. Microinjection of an enkephalinase inhibitor into the brain, which blocked the degradation of enkephalin, had analgesic effects when injected into the central nucleus of the amygdala, periaqueductal gray, and ventral medulla.
Endogenous opioid peptides and non-peptide opioids, such as morphine, injected directly into the nucleus accumbens and ventral tegmental area produce locomotor activation, and animals will also self-administer them both intracerebroventricularly (which is a nonspecific injection into the ventricles of the brain that does not target any particular brain site) and directly into several brain sites, notably the lateral hypothalamus and nucleus accumbens. β-endorphin injected intracerebroventricularly produced conditioned place preference in rats, and enkephalin injected into the ventral tegmental area produced conditioned place preference. Another opioid peptide, endomorphin-1 (this peptide is hypothesized to be an endogenous compound, but the gene and synthetic pathway have yet to be discovered) has high selectivity for μ opioid receptors and produces locomotor activity when injected into the ventral tegmental area but not the nucleus accumbens.
FIGURE 5.8 Synthesis of endogenous opioid peptides.
One question that remains to be answered is the way that opioids act at the cellular level to engage reward circuits. One hypothesis is that opioids excite neurons by disinhibiting inhibitory interneurons. For example, μ opioid agonists inhibit the firing of a subgroup of neurons (likely γ-aminobutyric acid [GABA] neurons) in the central nucleus of the amygdala.
Pharmacologists initially identified three putative opioid receptors – μ, δ, and κ – based on differential pharmacological actions, differential antagonism, and ultimately radioligand binding. Differential opioid receptor density was localized to different brain areas using autoradiography. High concentrations were found in regions that overlap with the endogenous opioid and are associated with pain, such as the periaqueductal gray, medial thalamus, and amygdala (Figure 5.9).
The subsequent cloning and characterization of the genes that encode the μ, δ, and κ receptors confirmed the pharmacological categories. The amino acid sequences of these three receptors are very similar and share approximately 70% sequence identity. However, each receptor is derived from different chromosomes, suggesting high conservation and a lack of divergence in recent evolutionary history. A nociceptin receptor was discovered later, culminating in a four-member gene subfamily of the G-protein-coupled receptor family. The endogenous peptide nociceptin binds to the nociceptin receptor, but rather than having opioid effects it appears to have more anti-stress like effects.
Mouse strains that lack the genes of the opioid system have been generated by utilizing homologous recombination technology. Molecular studies have provided important confirmation and extension of pharmacological studies of the molecular basis of the effects of opioids. The most important receptor for the effects of opioids in relation to addiction is the μ receptor. Morphine reinforcement, measured by conditioned place preference and self-administration, is absent in μ receptor knockout mice. These mice also fail to exhibit the development of somatic signs of morphine dependence. In fact, all of the effects of morphine tested so far, including analgesia, hyperlocomotion, respiratory depression, and inhibition of gastrointestinal motility, are abolished in μ knockout mice (Figure 5.10).
Studies of δ opioid receptor knockout mice found increased levels of anxiety-like behavior in numerous behavioral tests. δ Opioid receptor knockout mice also show less tolerance to the analgesic effects of morphine, suggesting that these receptors may contribute to this adaptive aspect of chronic opioid exposure. κ Opioid receptor knockout mice, in contrast, show effects that support the hypothesis that activation of the dynorphin-κ receptor systems in the brain can produce dysphoric-like actions