Neurons in the rostral ventromedial medulla (RVM) are important in the opioid modulation of dorsal horn nociceptive transmission. Systemically administered morphine inhibits one class of RVM cells, the on-cells; excites a second class of RVM cells, the off-cells; and has no effect on a third class, neutral cells. In contrast, iontophoretic application of morphine inhibits on-cells but does not alter the activity of either off- or neutral cells. The present study addresses whether the differential sensitivity to exogenous opioids is correlated with a differential termination pattern onto the three classes of RVM neurons by afferents containing endogenous opioids. Intracellular recordings were made from RVM neurons in rats under light halothane anesthesia. Physiologically characterized neurons were injected with Neurobiotin and then subsequently visualized with a Texas red fluorophore. Thick (50 microns) sections containing labeled RVM cells were processed for enkephalin immunoreactivity (ENK-IR) using an FITC fluorophore and then optically sectioned at 1.5 micron intervals using a dual-channel confocal laser scanning microscope. ENK-IR appositions were found on the somata and dendrites of all on-cells. Although ENK-IR varicosities were also apparently apposed to off- and neutral cells, the density of such appositions was significantly less than the density of ENK-IR appositions onto on-cells. The greater overall density of ENK-IR appositions onto on-cells was apparently due to a concentration of appositions on the soma and proximal dendrites of these neurons. These results support a model of RVM function in which endogenous opioid peptides produce an antinociceptive action by a direct inhibitory action on on-cells that facilitate nociceptive transmission. This on-cell inhibition may produce an additional antinociceptive effect by removing a possible on-cell inhibition of off-cells, which are thought to inhibit nociceptive transmission.
We have developed a novel fluorescent histochemical method to localize the enzyme neutral endopeptidase-24.11 (NEP, E.C. 3.4.24.11, enkephalinase) in the rat brain in order to directly compare the relative distributions of the enzyme and its putative peptide substrate, the enkephalins. The method is based on the sequential cleavage of the synthetic peptide substrate, glutaryl-alanyl-alanyl-phenylanyl-4-methoxy-2-naphthylamide, by NEP and exogenous aminopeptidase M to yield free 4-methoxy-2-naphthylamine (MNA). In the presence of nitrosalicylaldehyde, free MNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of NEP activity. The specificity of the method was demonstrated using the selective NEP inhibitors thiorphan, phosphoramidon, and JHF26. All NEP staining throughout the brain was abolished using a 50-nM concentration of these inhibitors. The enzyme was richly localized to many regions, including the cerebral cortex, caudate putamen, globus pallidus, hippocampus, substantia nigra, periaqueductal gray, several cranial nerve nuclei, nuclei of the reticular formation of the medulla. In most regions, reaction product was associated with cell bodies of varying size and morphology. In a number of regions, colchicine increased the amount of NEP staining, particularly in cell processes. The regional distribution pattern of the enzyme, however, did not change in response to colchicine and was similar to that of untreated animals. The histochemical localization of NEP was combined with fluorescent immunocytochemical visualization of the enkephalins in order to localize both in the same tissue section. In the globus pallidus, this combined fluorescent technique revealed numerous NEP-positive cell bodies surrounded by fiber pathways displaying intense enkephalin-like immunoreactivity. The source of the NEP in the globus pallidus was studied using the neurotoxic agent, N-methyl-D-aspartate (NMDA). A pronounced decrease in NEP cellular staining was observed within 7 d in response to NMDA, persisted for at least 16 weeks, and correlated with injury of pallidal neurons. There was no apparent change in enkephalin-like immunoreactivity in the globus pallidus in response to NMDA. These data provide evidence that NEP and enkephalin in the globus pallidus derive from different sources. This study supports the hypothesis that NEP localizes to enkephalin-rich regions of the rat brain, and that the enzyme may be involved in the inactivation of synaptically released enkephalins.
Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): National Institute of Health Objectives Patients with Alzheimer’s Disease (AD) display cerebral white matter (WM) hyperintensities in association with microvascular brain injury (mVBI) and cerebral microinfarcts. We determined the extent to which vasomotor dysfunction of WM penetrating arterioles is associated with mVBI in autopsy brains from patients with low or high AD neuropathology, and determined if these changes correlate with quantitative indices of MRI-defined WM integrity. Methods We analyzed 28 consecutive cases of prefrontal rapid autopsies in a population-based cohort with low or high AD neuropathological changes, and with or without cerebral microinfarcts (mVBI). WM penetrating and pial surface arteriolar responses to the endothelium-dependent agonist, bradykinin were assessed ex vivo with videomicroscopy. Expression of vascular endothelial nitric oxide synthase (eNOS) and NAD(P)H-oxidase (Nox1,2 and 4 isoforms) was measured with quantitative PCR. Diffusion tensor imaging was used to measure mean apparent diffusion coefficient (ADC) and fractional anisotropy (FA) in post-mortem prefrontal WM. Results Patients with high AD neuropathology and mVBI exhibited a significantly reduced dilation in response to bradykinin selectively in WM arterioles, when compared to low or high AD cases without mVBI or to pial surface arterioles. Expression of eNOS was reduced, whereas Nox-1 expression was increased in WM arterioles in AD and mVBI cases. Moreover, we found that in cases with low AD pathology the magnitude of bradykinin-induced WM arteriole dilation was correlated with higher FA and lower ADC. In contrast, dilation to bradykinin was associated with lower FA and higher ADC in cases with high AD neuropathology. Interpretation: Selectively impaired vasodilation to the endothelium-dependent agonist, bradykinin occurs in WM penetrating arterioles in AD patients with WM microinfarcts, which is likely due to reduced nitric oxide and an increase in Nox1-derived reactive oxygen species production. Notably, the bradykinin response of WM arterioles strongly correlated with MRI-defined WM integrity suggesting that impaired bradykinin-mediated vasodilation in WM penetrating arterioles contributes to disrupted white matter microstructural integrity as defined by MRI.
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