SUMMARYThe fluorescent pigment lipofuscin accumulates with age in the cytoplasm of cells of the CNS. Because of its broad excitation and emission spectra, the presence of lipofuscin-like autofluorescence complicates the use of fluorescence microscopy (e.g., fluorescent retrograde tract tracing and fluorescence immunocytochemistry). In this study we examined several chemical treatments of tissue sections for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 1-10 mM CuSO 4 in 50 mM ammonium acetate buffer (pH 5) or 1% Sudan Black B (SB) in 70% ethanol reduced or eliminated lipofuscin autofluorescence in sections of monkey, human, or rat neural tissue. These treatments also slightly reduced the intensity of immunofluorescent labeling and fluorescent retrograde tract tracers. However, the reduction of these fluorophores was far less dramatic than that for the lipofuscin-like compound. We conclude that treatment of tissue with CuSO 4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.

We previously demonstrated that the spinal cord κ-opioid receptor (KOR) and μ-opioid receptor (MOR) form heterodimers (KOR/MOR). KOR/MOR formation and the associated KOR dependency of spinal morphine antinociception are most robust during proestrus. Using Sprague Dawley rats, we now demonstrate that (1) spinal synthesis of estrogen is critical to these processes, and (2) blockade of either estrogen receptor (ER) α-, β-, or G-protein-coupled ER1 or progesterone receptor (PR) substantially reduces KOR/MOR and eliminates mediation by KOR of spinal morphine antinociception. Effects of blocking ERs were manifest within 15 min, whereas those of PR blockade were manifest after 18 h, indicating the requirement for rapid signaling by estrogen and transcriptional effects of progesterone. Individual or combined blockade of ERs produced the same magnitude of effect, suggesting that they work in tandem as part of a macromolecular complex to regulate KOR/MOR formation. Consistent with this inference, we found that KOR and MOR were coexpressed with ERα and G-protein-coupled ER1 in the spinal dorsal horn. Reduction of KOR/MOR by ER or PR blockade or spinal aromatase inhibition shifts spinal morphine antinociception from KOR dependent to KOR independent. This indicates a sex steroid-dependent plasticity of spinal KOR functionality, which could explain the greater analgesic potency of KOR agonists in women versus men. We suggest that KOR/MOR is a molecular switch that shifts the function of KOR and thereby endogenous dynorphin from pronociceptive to antinociceptive. KOR/MOR could thus serve as a novel molecular target for pain management in women.

The rostral ventromedial medulla (RVM) forms part of a descending pathway that modulates nociceptive neurotransmission at the level of the spinal cord dorsal horn. However, the involvement of descending RVM systems in opioid analgesia are a matter of some debate. In the present study, patch-clamp recordings of RVM neurons were made from rats that had received retrograde tracer injections into the spinal cord. More than 90% of identified spinally projecting RVM neurons responded to opioid agonists. Of these neurons, 53% responded only to the mu-opioid agonist D-Ala2, N-Me-Phe4, Gly-ol5 enkephalin, 14% responded only to the kappa-opioid agonist U-69593, and another group responded to both mu and kappa opioids (23%). In unidentified RVM neurons, a larger proportion of neurons responded only to mu opioids (75%), with smaller proportions of kappa- (4%) and mu/kappa-opioid (13%) responders. These RVM slices were then immunostained for tryptophan hydroxylase (TPH), a marker of serotonergic neurons. Forty-percent of spinally projecting neurons and 11% of unidentified neurons were TPH positive. Of the TPH-positive spinally projecting neurons, there were similar proportions of mu- (33%), kappa- (25%), and mu/kappa-opioid (33%) responders. Most of the TPH-negative spinally projecting neurons were mu-opioid responders (67%). These findings indicate that functional opioid receptor subtypes exist on spinally projecting serotonergic and nonserotonergic RVM neurons. The proportions of mu- and kappa-opioid receptors expressed differ between serotonergic and nonserotonergic neurons and between retrogradely labeled and unlabeled RVM neurons. We conclude that important roles exist for both serotonergic and nonserotonergic RVM neurons in the mediation of opioid effects.

Agonists acting at α2-adrenergic and opioid receptors (α2ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When co-administered, agonists activating these receptors interact in a synergistic manner. Although the existence of α2AR/OR synergy has been well characterized, its mechanism remains poorly understood. The formation of hetero-oligomers has been proposed as a molecular basis for interactions between neuronal G-protein-coupled receptors. The relevance of hetero-oligomer formation to spinal analgesic synergy requires demonstration of the expression of both receptors within the same neuron as well as the localization of both receptors in the same neuronal compartment. We used immunohistochemistry to investigate the spatial relationship between α2ARs and ORs in the rat spinal cord to determine if co-expression could be demonstrated between these receptors. We observed extensive co-localization between α2A-adrenergic and delta-opioid receptors (DOP) on substance P (SP)-immunoreactive (ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. α2AAR- and DOP-ir elements were co-localized in subcellular structures of 0.5 μm or less in diameter in isolated nerve terminals. Furthermore, co-incubation of isolated synaptosomes with α2AR and DOP agonists resulted in a greater-than-additive increase in the inhibition of K+-stimulated neuropeptide release. These findings suggest that co-expression of the synergistic receptor pair α2AAR-DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps due to protein-protein interactions such as hetero-oligomerization.

The dorsal raphe nucleus (DRN) projects serotonergic axons throughout the brain and is involved in a variety of physiological functions. However, it also includes a large population of cells that contain other neurotransmitters. To clarify the physiological and pharmacological differences between the serotonergic and nonserotonergic neurons of the DRN, their postsynaptic responses to 5-hydroxytryptamine (5-HT, serotonin) and to selective activation of 5-HT1A or 5-HT2A/C receptors and their action potential characteristics were determined using in vitro patch-clamp recordings. The slices containing these neurons were then immunostained for tryptophan hydroxylase (TPH), a marker of serotonergic neurons. It was found that subpopulations of both serotonergic and nonserotonergic neurons responded to 5-HT with outward (i.e., inhibitory) and inward (i.e., excitatory) currents, responded to both 5-HT1A and 5-HT2A/C receptor activation with outward and inward currents, respectively, and displayed overlapping action potential characteristics. These findings suggest that serotonergic and nonserotonergic neurons in the DRN are both heterogeneous with respect to their individual pharmacological and electrophysiological characteristics. The findings also suggest that the activity of the different populations of DRN neurons will display heterogeneous changes when the serotonergic tone in the DRN is altered by neurological disorders or by drug treatment.