Martin W. Wessendorf

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.

Characterization of an immunofluorescence technique for the demonstration of coexisting neurotransmitters within nerve fibers and terminals.

Neurotransmitters have been shown to coexist in cell bodies, but demonstrating their coexistence within nerve fibers and terminals has been more difficult. However, two recent reports outlined a simple light-microscopic method by which two neurotransmitters can be shown to coexist in fibers and terminals. The method was identical to that used for immunohistochemical localization of one antigen, except that two primary--secondary antibody systems labeled with two different fluorochromes were used simultaneously. In the present study, a method for the simultaneous visualization of serotonin and substance P was characterized. This method employed an antiserum to serotonin generated in goat in combination with a rabbit-generated antiserum to substance P. These antisera were visualized with secondary antisera raised in swine and conjugated with rhodamine and fluorescein respectively. Spinal cord sections stained by this protocol showed large numbers of fibers fluorescing both red and green. Many of them were in the ventral horn, fewer were around the central canal, and virtually none were in the dorsal horn. The apparent double labeling could be shown not to be the result of cross-reactivity among the antisera, of any inappropriate affinity among the antisera, of green fluorescence by rhodamine, or of red fluorescence by fluorescein. It is concluded that the method provides a simple technique for visualizing fibers and terminals in which serotonin and substance P coexist.

Transneuronal labeling of CNS neuropeptide and monoamine neurons after pseudorabies virus injections into the stellate ganglion

The viral transneuronal labeling method was used in combination with immunohistochemical procedures to identify CNS neuropeptide and monoamine neurons that innervate the sympathetic preganglionic neurons (SPNs) which project to the stellate ganglion--the principal source of the sympathetic supply to the heart. Transneuronal labeling was found at three CNS levels: spinal cord, brainstem, and hypothalamus. In the thoracic spinal cord, apart from the pseudorabies virus (PRV)-labeled stellate SPNs, PRV-labeled neurons were localized in laminae I/II, IV, V, VII, and X as well as in the lateral spinal nucleus and lateral funiculus. In the C1-C4 spinal segments, labeled neurons were found in the lateral funiculus as well as laminae V and VII of the spinal gray matter. PRV-labeled cells were identified in lamina V and the dorsolateral funiculus of the lumbar spinal cord. Three medullary areas were consistently labeled: rostral ventromedial medulla (RVMM), rostral ventrolateral medulla (RVLM), and caudal raphe nuclei. The greatest concentration of labeling was found in the RVMM. This projection arose from adrenergic, serotonergic (5-HT), thyrotropin releasing hormone (TRH), substance P, somatostatin, enkephalin, and vasoactive intestinal peptide (VIP) immunoreactive neurons. The RVLM projection originated mainly from C1 adrenergic neurons, some of which contained immunoreactive neuropeptide Y (NPY). C3 adrenergic-NPY neurons lying near the floor of the 4th ventricle were also labeled. Enkephalin-, somatostatin- and VIP-immunoreactive RVLM neurons also contributed to this projection. 5-HT neurons of the caudal raphe nuclei (raphe pallidus, raphe obscurus, and raphe magnus) were labeled; some of these contained substance P or TRH-immunoreactivity with an occasional neuron staining for all three putative neurotransmitters. In the pons, catecholamine neurons in the A5 cell group, subcoeruleus and Kolliker-Fuse nuclei were labeled. The midbrain contained relatively few infected cells, but some were present in the Edinger-Westphal and precommissural nuclei. Forebrain labeling was concentrated in the paraventricular hypothalamic nucleus (PVN) with lesser amounts in the lateral hypothalamic area (LHA) and the perifornical region. In the PVN, oxytocin-immunoreactive neurons accounted for the greatest chemically-defined projection while corticotrophin releasing factor (CRF), vasopressin-, and angiotensin II-immunoreactive neurons provided successively lesser inputs. In the LHA, angiotensin II-immunoreactive neurons were labeled. In summary, this study provides the first detailed map of the chemically-coded CNS neurons involved in the control of the cardiosympathetic outflow.

Spinal analgesic actions of the new endogenous opioid peptides endomorphin-1 and -2

TWO highly-selective μ-opioid receptor agonists, endomorphin-1 and -2, were recently purified from bovine brain and are postulated to be endogenous μ-opioid receptor ligands. We sought to determine the effects of these ligands at the spinal level in mice. Endomorphin1 and -2 produced short acting, naloxone-sensitive antinociception in the tail flick test and inhibited the behavior elicited by intrathecally injected substance P. Both endomorphin-1 and -2 were anti-allodynic in the dynorphin-induced allodynia model. Although acute tolerance against both endomorphins developed rapidly, endomorphin-1 required a longer pretreatment time before tolerance was observed. We conclude that the endomorphins are potent spinal antinociceptive and anti-allodynic agents and that they or related compounds may prove therapeutically useful as spinal analgesics.

Evidence for co-existence of thyrotropin-releasing hormone, substance P and serotonin in ventral medullary neurons that project to the intermediolateral cell column in the rat

The present study was conducted to determine if substance P-, thyrotropin-releasing hormone- and/or serotonin-immunoreactivities coexist in ventral medullary neurons that project to the intermediolateral cell column in the rat. Neurons that projected to the intermediolateral cell column were identified by the presence of retrogradely transported rhodamine bead-labeled microspheres in the cell body after an injection of the microspheres into the intermediolateral cell column of the third thoracic spinal cord segment. Co-existence was determined by using a combination of dual color immunohistochemistry and serial 4-microns sections that were immunostained with different antibodies. Antibodies to substance P, serotonin, and pre-pro-thyrotropin releasing hormone160-169 were used to identify substance P, serotonin and thyrotropin-releasing hormone, respectively. Neurons that contained substance P-, thyrotropin-releasing hormone- and/or serotonin-immunoreactivities and that projected to the intermediolateral cell column were present in the nucleus raphe magnus, the nucleus raphe pallidus, the nucleus reticularis magnocellularis pars alpha, the paragigantocellular reticular nucleus and the parapyramidal region. Neurons that projected to the intermediolateral cell column, in each of these regions, were found to contain each of the following combinations of immunoreactive neurochemicals: substance P and thyrotropin-releasing hormone: substance P and serotonin; thyrotropin-releasing hormone and serotonin; or substance P, thyrotropin-releasing hormone and serotonin. In addition, most of the regions also contained neurons that appeared to contain only one of the neurochemicals and that also projected to the intermediolateral cell column. The greatest number of neurons that projected to the intermediolateral cell column and that also contained two or more co-existing neurochemicals was present in the midline regions. This study demonstrates the presence of neurons in the ventral medulla that project to the intermediolateral cell column and contain three co-existing neurochemicals. This study also demonstrates the use of a new method for the localization of three neurochemicals in single projection-specific neurons.

Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations.

Publisher Summary Advances in fluorescent probe chemistry, economical laser availability, and confocal microscope instrumentation are making the enormous potential of multicolor laser scanning confocal microscopy (LSCM) available to a wider range of biologists. This chapter outlines the requirements for performing multicolor immunofluorescence studies with LSCM. The principles of immunofluorescence histochemistry necessary for the preparation of multilabeled specimens are summarized. The chapter examines the technical aspects of confocal microscope instrumentation that affect its application to multicolor studies. The practical application and limitations of this technology are demonstrated for multicolor LSCM, using the inexpensive, air-cooled argon ion and krypton-argon ion lasers as light sources are presented and various aspects of confocal microscopy that affect the comparison of images acquired with different excitation and/or emission wavelengths are examined. The chapter provides a better understanding of the compromises needed to effect the accurate imaging of specimens stained with multiple fluorophores. The chapter also discusses the applications and limitations of multicolor laser scanning confocal immunofluorescence microscopy.

Relationship of μ- and δ-opioid receptors to GABAergic neurons in the central nervous system, including antinociceptive brainstem circuits

Inhibition of neurons containing γ‐aminobutyric acid (GABA) may underlie some of the excitatory effects of opioids in the central nervous system (CNS). In the present study, we examined the relationship of the cloned μ‐ and δ‐opioid receptors (MOR1 and DOR1, respectively) to GABAergic neurons in brain and spinal cord. This was done by combining immunofluorescent staining for MOR1 or DOR1 with that for GABA or glutamic acid decarboxylase (GAD); fluorescent retrograde tract‐tracing was used in some cases to identify neurons with particular projections. In rats, cells double labeled for GABA and MOR1 were observed in layers II–VI of the parietal cortex and in layers II–IV of the piriform cortex. In the hippocampus, double labeling was observed in the dentate gyrus and in regions CA1 and CA3. Double labeling was very prominent in the striatum and in the reticular nucleus of the thalamus; it was also observed in other portions of the diencephalon. However, double labeling for GABA and MOR1 was never observed in the cerebellar cortex.

Immunoreactivity for endomorphin-2 occurs in primary afferents in rats and monkey

ANTISERA were raised against endomorphin-2, a recently isolated endogenous opioid peptide that binds potently and selectively to the μ-opioid receptor. When sections of spinal cord were stained immunocytochemically, a dense plexus of fibres and varicosities was visualized in the superficial dorsal horn of rats and one monkey. Following unilateral multiple dorsal rhizotomy, labeling for endomorphin-2 was markedly reduced ipsilateral to the lesion. In sections stained for both endomorphin-2 and CGRP, double-labeling was observed. Taken together, these data suggest that endomorphin-2 occurs in small diameter primary afferent fibres in rodents and primates. It appears possible that the release of neurotransmitters from nociceptive primary afferents might be regulated by release of endomorphin-2 from primary afferent terminals.

Thyrotropin-releasing hormone in spinal cord: coexistence with serotonin and with substance P in fibers and terminals apposing identified preganglionic sympathetic neurons.

In this study we utilized the technique of simultaneous immunofluorescent double-labeling to investigate possible coexistence of the putative neurotransmitter thyrotropin-releasing hormone (TRH) with serotonin (5-HT) and with substance P (SP) in the intermediolateral cell column (IML) of rat spinal cord. We observed fibers and terminals immunoreactive for both TRH and 5-HT or TRH and SP in IML. In addition, this technique was used in animals in which we retrogradely labeled, with fluorescent tracer dyes, preganglionic sympathetic neurons within IML from either the adrenal medulla or the proximal cut end of the cervical sympathetic trunk. In these animals, fibers and terminals containing these combinations of neurotransmitters appeared to oppose identified preganglionic sympathetic neurons in IML. These data represent the first direct immunohistochemical demonstration of fibers and terminals in spinal cord which display coexistence of TRH- with either 5-HT- or SP-immunoreactivity. In addition, the proximity of TRH-immunoreactive fibers and terminals to sympathetic preganglionic neurons in IML support a role for TRH in the regulation of central sympathetic outflow.

Immunocytochemical mapping of endomorphin-2-immunoreactivity in rat brain

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) is a novel endogenous opioid with high affinity and selectivity for the mu-opioid receptor. Immunocytochemical studies have located this peptide in spinal cord, brainstem and selected brain regions. However, there are disagreements regarding its distribution between published reports. Furthermore, the distributions reported for the endomorphins resemble that of neuropeptide FF, suggesting that some of the previous findings might be due to cross-reactivity with the latter substance. In the present study, the distribution of endomorphin-2-immunoreactivity (ir) was examined throughout the entire rat brain using an affinity-purified antiserum that appeared not to cross-react with neuropeptide FF. Endomorphin-2-ir cell somata were most prominent in the hypothalamus and the nucleus of the solitary tract (NTS). Endomorphin-2-ir varicose fibers were observed in such areas as the bed nucleus of the stria terminalis, the septal nuclei, the periaqueductal gray, the locus coeruleus, the lateral parabrachial nucleus, the NTS, and the substantia gelatinosa of the medulla. More modest immunoreactivity was seen in substantia nigra, nucleus raphe magnus, the ventral tegmental area, the pontine nuclei and the amygdala. Fibers were also observed in the ventral cerebellum. Of note was the negligible immunoreactivity in the striatum, a region known to express high levels of mu-opioid receptors. Thus, endomorphin-2-ir was widely, but not uniformly, distributed throughout the central nervous system and was associated largely, but not exclusively, with regions expressing mu-opioid receptors. Based on its distribution, it may have a role in the control of neuroendocrine, cardiovascular and respiratory functions, and mood, feeding, sexual behavior and pain.