Stephen A. Schnell

Reduction of Lipofuscin-like Autofluorescence in Fluorescently Labeled Tissue

The 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 CuSO4 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 CuSO4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.

Spinal Synthesis of Estrogen and Concomitant Signaling by Membrane Estrogen Receptors Regulate Spinal κ- and μ-Opioid Receptor Heterodimerization and Female-Specific Spinal Morphine Antinociception

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.

Coexpression of α2A-adrenergic and δ-opioid receptors in substance P-containing terminals in rat dorsal horn

Agonists acting at α2‐adrenergic and opioid receptors (α2ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, 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 heterooligomers has been proposed as a molecular basis for interactions between neuronal G‐protein‐coupled receptors. The relevance of heterooligomer 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 whether coexpression could be demonstrated between these receptors. We observed extensive colocalization between α2A‐adrenergic and δ‐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 colocalized in subcellular structures of 0.5 μm or less in diameter in isolated nerve terminals. Furthermore, coincubation 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 coexpression of the synergistic receptor pair α2AAR‐DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps as a result of protein–protein interactions such as heterooligomerization. J. Comp. Neurol. 513:385–398, 2009.

Coexpression of alpha 2A-adrenergic and delta-opioid receptors in substance P-containing terminals in rat dorsal horn.

Agonists acting at α2‐adrenergic and opioid receptors (α2ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, 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 heterooligomers has been proposed as a molecular basis for interactions between neuronal G‐protein‐coupled receptors. The relevance of heterooligomer 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 whether coexpression could be demonstrated between these receptors. We observed extensive colocalization between α2A‐adrenergic and δ‐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 colocalized in subcellular structures of 0.5 μm or less in diameter in isolated nerve terminals. Furthermore, coincubation 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 coexpression of the synergistic receptor pair α2AAR‐DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps as a result of protein–protein interactions such as heterooligomerization. J. Comp. Neurol. 513:385–398, 2009.

Molecular cloning and tissue distribution of an avian D2 dopamine receptor mRNA from the domestic turkey (Maleagris gallopavo)

The reverse transcriptase‐polymerase chain reaction (RT‐PCR), in combination with 5′ and 3′ rapid amplification of cDNA ends (RACE), was used to clone a G protein‐coupled receptor from turkey brain mRNA. This cDNA clone has an open reading frame of 1,311 base pairs encoding a 436‐residue protein with seven transmembrane‐spanning domains and exhibits high homology with previously cloned mammalian D2 dopamine receptors. Northern blot analysis of turkey brain mRNA detected an approximate 2.4‐kb transcript. RT‐PCR and subsequent nucleotide sequence analysis of turkey brain and peripheral tissue mRNA also demonstrated the presence of an alternatively spliced mRNA corresponding to the predicted D2 short isoform. RT‐PCR experiments demonstrated a widespread distribution of alternatively spliced D2 dopamine receptor transcripts throughout the turkey brain and in select peripheral tissues as well. In situ hybridization experiments detected strong autoradiographic signals over much of the turkey telencephalon, diencephalon, mesencephalon, cerebellum, pituitary, and pineal gland. Dopamine has several important functions as a neurotransmitter and hormone in mammals and may have similar actions in avian species. The cloning and tissue distribution of the D2 receptor subtype should enable the investigation of any functional role dopamine and dopamine receptors exert on the physiology and behavior of birds. J. Comp. Neurol. 407:543–554, 1999.

Relationship of Spinal Dynorphin Neurons to δ-Opioid Receptors and Estrogen Receptor α: Anatomical Basis for Ovarian Sex Steroid Opioid Antinociception

Pharmacological and behavioral studies suggest that spinal δ- and κ-opioid antinociceptive systems are functionally associated with ovarian sex steroids. These interactions can be demonstrated specifically during pregnancy or hormone-simulated pregnancy (HSP). The analgesia associated with both conditions can be abolished by blockade of either spinal κ-opioid receptors or δ-opioid receptors (DOR). Furthermore, both dynorphin (DYN) release (J Pharmacol Exp Ther 298:1213–1220, 2001) and the processing of the DYN precursor (J Neurochem 65:1374–1380, 1995) are significantly increased in the spinal cord during HSP. We undertook the current study to determine whether DYN, DOR, and estrogen receptor α (ERα) share anatomical relationships that permit their direct interaction. Coexpression of DOR or ERα by DYN neurons was assessed using fluorescence immunohistochemistry and a synaptosomal release assay. Findings show that ERα and DYN are coexpressed. Moreover, in the spinal cord of HSP animals, there were significant increases in the number of DYN-immunoreactive (DYN-ir) cells, ERα-ir cells, cells double-labeled for DYN-ir and ERα-ir and the proportion of DYN-ir cells coexpressing ERα. Some varicose fibers in the spinal cord dorsal horn and intermediate gray matter that expressed DYN-ir also expressed DOR-ir. Activation of DORs located on DYN terminals was sufficient to inhibit K+-evoked DYN release. These data define, at least in part, the anatomical substrates that may be relevant to the antinociception of gestation and its hormonal simulation. Furthermore, they provide a framework for understanding sex-based nociception and antinociception and suggest novel strategies for treating pain.

Bisbenzimide: a fluorescent counterstain for tissue autoradiography.

Interpretation of the data from experiments using autoradiography (e.g. using in situ hybridization histochemistry, receptor binding, neuronal tract-tracing etc.) is aided when the autoradiographic grains can be seen in the context of cellular boundaries. Studies making use of autoradiography in the central nervous system have sometimes used tinctorial stains, such as cresyl violet, as counterstains to visualize the labeling. Tinctorial stains are excellent Nissl stains however, under bright-field illumination such dyes tend to obscure autoradiographic grains. In addition, dark-field illumination provides a common means of visualizing autoradiographic grains but tictorial stains are not optimally visible under these conditions. In an effort to find a counterstain that would be compatible with dark-field illumination, we have investigated the use of fluorescent dyes. Of the fluorescent dyes tested, bisbenzimide (Hoechst 33258) in pH 2.0 buffer was found to be optimal. Bisbenzimide counterstaining gave good resolution of cellular boundaries and appeared not to interfere with the ability to visualize autoradiographic grains. Furthermore, the illumination of bisbenzimide and of the autoradiographic grains could be controlled independently, making it easy to visualize or photograph the bisbenzimide Nissl staining and the autoradiographic grains simultaneously. Thus bisbenzimide is well suited for use as a fluorescent counterstain in autoradiographic studies.

Sex, Pain, and Opioids: Interdependent Influences of Sex and Pain Modality on Dynorphin-Mediated Antinociception in Rats

The role of dynorphin A (1-17; Dyn) and its associated kappa opioid receptor (KOR) in nociception represents a longstanding scientific conundrum: Dyn and KOR (Dyn/KOR) have variously been reported to inhibit, facilitate, or have no effect on pain. We investigated whether interactions between sex and pain type (which are usually ignored) influenced Dyn/KOR-mediated antinociception. Blockade of the spinal α2-noradrenergic receptor (α2-NAR) using yohimbine elicited comparable spinal Dyn release in females and males. Nevertheless, the yohimbine-induced antinociception exhibited sexual dimorphism that depended on the pain test used: in the intraperitoneal acetic acid–induced writhing test, yohimbine produced antinociception only in females, whereas in the intraplantar formalin-induced paw flinch test, antinociception was observed only in males. In females and males, both intrathecal Dyn antibodies and spinal KOR blockade eliminated the yohimbine-induced antinociception, indicating that Dyn/KOR mediated it. However, despite the conditional nature of spinal Dyn/KOR-mediated yohimbine antinociception, both intraplantar formalin and intraperitoneal acetic acid activated spinal Dyn neurons that expressed α2-NARs. Moreover, Dyn terminals apposed KOR-expressing spinal nociceptive neurons in both sexes. This similar organization suggests that the sexually dimorphic interdependent effects of sex and pain type may result from the presence of nonfunctional (silent) KORs on nociceptive spinal neurons that are responsive to intraplantar formalin (in females) versus intraperitoneal acetic acid (in males). Our findings that spinal Dyn/KOR-mediated antinociception depends on interactions between sex and pain type underscore the importance of using both sexes and multiple pain models when investigating Dyn/KOR antinociception.

Regulation of Spinal Dynorphin 1-17 Release by Endogenous Pituitary Adenylyl Cyclase-Activating Polypeptide in the Male Rat: Relevance of Excitation via Disinhibition

Opioids inhibit release of primary afferent transmitters but it is unclear whether the converse occurs. To test the hypothesis that primary afferent transmitters influence opioid-ergic tone, we studied the functional and anatomical relationships between pituitary adenylyl cyclase-activating polypeptide (PACAP) and dynorphin 1-17 (Dyn) in spinal cord. We found that activation of the PACAP-specific receptor PAC1 (PAC1R) inhibited, whereas PAC1R blockade augmented, spinal release of Dyn. It is noteworthy that in the formalin-induced pain model PAC1R blockade (via PACAP6-38) also resulted in antinociception that was abolished by spinal κ-opioid receptor blockade. These findings indicate that Dyn release is tonically inhibited by PACAP and that blocking this inhibition, which increases the spinal release of Dyn, results in antinociception. Consistent with this conclusion, we found in the spinal dorsal horn that Dyn-immunoreactive neurons 1) expressed PAC1R and 2) were apposed by PACAP terminals. Present results, in combination with the previous demonstration that the release of spinal Dyn is tonically inhibited by opioid- and nociceptin/orphanin FQ-coupled pathways (J Pharmacol Exp Ther 298:1213–1220, 2001), indicate that spinal Dyn-ergic neurons integrate multiple inhibitory inputs, the interruption of any one of which (i.e., disinhibition) is sufficient to enhance spinal Dyn release and generate antinociception. Gaining a better understanding of the role of primary afferent neurotransmitters in negatively modulating the spinal release of Dyn and the physiological use of disinhibition to increase spinal Dyn activity could suggest novel clinically useful approaches for harnessing endogenous Dyn for pain control.

Coexpression of the mu-opioid receptor splice variant MOR1C and the vesicular glutamate transporter 2 (VGLUT2) in rat central nervous system.

It has been reported that mu‐opioid agonists depress glutamate release in some neurons but the specific receptor subtype mediating this effect is unclear. The purpose of the present study was to examine whether a particular mu‐opioid receptor (MOR) splice‐variant, MOR1C, is expressed in rat central nervous system (CNS) by terminals expressing the vesicular glutamate transporter2 (VGLUT2), a marker of glutamatergic neurons. Several MOR splice variants have been identified in mice and MOR1C appears mainly to be localized to fibers and terminals, from which most neurotransmitter release would be expected. In addition, VGLUT2 has been found in the CNS and antibodies to it are reliable markers for glutamatergic terminals. Using fluorescence immunohistochemistry and confocal microscopy to examine spatial relationships between MOR1C and VGLUT2, we found that MOR1C and VGLUT2 puncta were widely distributed throughout the rat CNS; moreover, many regions contained terminals that expressed both. Thus, it appears that coexpression of MOR1C and VGLUT2 is common in the rat CNS. We hypothesize that activation of MOR1C by mu‐opioid agonists at some glutamatergic terminals may be a mechanism by which glutamate release is inhibited. J. Comp. Neurol. 508:542–564, 2008.