A confocal laser microscopic study of enkephalin-immunoreactive appositions onto physiologically identified neurons in the rostral ventromedial medulla

Abstract

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.

Noradrenergic input to nociceptive modulatory neurons in the rat rostral ventromedial medulla

“…The major disadvantage of the CLSM method is the uncertainty about whether or not an apposition represents a synapse. This issue is discussed in our previous work (Mason et al, 1992). Currently, a study is underway in our laboratory to confirm at the EM level that some of the TH-ir appositions seen in this study are synapses.…”

“…This technique allows for the efficient and objective analysis of the density and pattern of cytochemically defined inputs onto physiologically identified and labeled neurons (Mason et al, 1992). In comparison, standard light microscopy would require multiple semi-thin sectioning to achieve the same level of z-axis resolution.…”

“…Appositions were judged by visual inspection from single optical sections. An apposition was judged to be present if the pixels between the THlabeled element and the TR-labeled neurons in a single optical section were greater than half maximal intensity (Mason et al, 1992).…”

“…To rule out the possibility that some profiles we counted as appositions were the result of chance, a modification of the frame-shifted merge method was used (Mason et al, 1992). Briefly, some frames of TR-labeled neuronal processes were randomly selected and merged with TH-ir frames from either the same or a distant optical section (displaced 6 µm in the Z-axis).…”

Enkephalinergic control of the secretory activity of neurons producing stereoisomers of crustacean hyperglycemic hormone in the eyestalk of the crayfish Orconectes limosus

“…In the analysis of confocal images, an immunoreactive profile was considered a fiber when it was observed on more than one optical section, and appositions between two neurons were defined by sites where the edges of a labeled immunoreactive cell and those of a fiber either occupied directly adjacent pixels or overlapped (Mason et al, 1992). As shown Figure 3G, one enk-IR profile could be followed over several optical sections, either in direct apposition (Fig.…”