Origins of endomorphin-immunoreactive fibers and terminals in different columns of the periaqueductal gray in the rat.

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are endogenous ligands for mu-opioid receptors (MOR). In the central nervous system, EM-immunoreactive (IR) neuronal cell bodies are located mainly in the hypothalamus and the nucleus tractus solitarius (NTS). EM-IR fibers and terminals are found widely distributed in many brain areas, including the different columns of the periaqueductal gray (PAG). The hypothalamus, NTS, and PAG are closely involved in modulation of vocalization, autonomic and neuroendocrine functions, pain, and defensive behavior through endogenous opioid peptides that bind to the MOR in these regions. Projections exist from both the hypothalamus and the NTS to the PAG. In order to examine whether there are EM1- and/or EM2-ergic projections from the hypothalamus and NTS to the PAG, immunofluorescence histochemistry for EM1 and/or EM2 was combined with fluorescent retrograde tracing. In rats that had Fluoro-Gold (FG) injected into different columns of the PAG, some of the EM1- or EM2-IR neurons in the hypothalamus, but none in the NTS, were labeled retrogradely with FG. The majority of the EM1/FG and EM2/FG double-labeled neurons in the hypothalamus were distributed in the dorsomedial nucleus, areas between the dorsomedial and ventromedial nucleus, and arcuate nucleus; a few were also seen in the ventromedial, periventricular, and posterior nucleus. The present results indicate that the EM-IR fibers and terminals in the PAG originate principally from the hypothalamus. They also suggest that EMs released from hypothalamus-PAG projecting neurons might mediate or modulate various functions of the PAG through binding to the MOR.

Endomorphin 1- and endomorphin 2-like immunoreactive neurons in the hypothalamus send axons to the parabrachial nucleus in the rat.

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are the endogenous peptides with high affinity and selectivity for the mu-opioid receptor (MOR). We examined whether or not EM1- and EM2-expressing hypothalamic neurons might send their axons to the parabrachial nucleus (PBN), where many MOR-expressing neurons have been observed. Immunofluorescence histochemistry was combined with fluorescent retrograde tract-tracing method. In the rats injected with Fluoro-Gold (FG) into the PBN, some of EM1- and EM2-immunoreactive hypothalamic neurons were labeled retrogradely with FG. The majority of the EM1/FG and EM2/FG double-labeled neurons were distributed in the dorsomedial hypothalamus nucleus, centromedial hypothalamic region, and arcuate nucleus; a few of them were also seen in the periventricular hypothalamic nucleus and posterior hypothalamic nucleus. Endomorphins released from PBN-projecting hypothalamic neurons may modulate the gustatory, autonomic and nociceptive functions through MOR-expressing PBN neurons.

Fos expression in serotonergic neurons in the rat brainstem following noxious stimuli: an immunohistochemical double-labelling study.

In order to detect whether there were different expression patterns of Fos protein induced by somatic or visceral noxious stimulation in the serotonergic neurons in the rat brainstem, an immunohistochemical double-labelling technique for serotonin (5-HT) and Fos was employed after subcutaneous or stomach injection of formalin. The two stimuli were matched in pilot experiments to produce maximum Fos expression. The expression of Fos protein in 5-HT-containing neurons (5-HT/Fos co-localized neurons) could be observed in the ventrolateral subdivision of the midbrain periaqueductal grey, interpeduncular nucleus, paramedian raphe nucleus, all of the brainstem raphe nuclei, the alpha part of the gigantocellular reticular nucleus and the lateral paragigantocellular reticular nucleus. The locations of the 5-HT/Fos co-localized neurons in the brainstem of animals subjected to somatic noxious stimulation were similar to those subjected to visceral noxious stimulation. However, the number and proportion of the 5-HT/Fos co-localized neurons in the median raphe nucleus and nucleus raphe obscurus of the rat subjected to visceral noxious stimulation were statistically greater than those in rats subjected to somatic noxious stimulation. These results suggest that serotonergic neurons in median raphe nucleus and nucleus raphe obscurus have a tendency to higher neuronal activity after visceral noxious stimulation.

Pre- and postsynaptic inhibition mediated by GABA(B) receptors in rat ventrolateral periaqueductal gray neurons.

The present study examined the actions of a GABA(B)-receptor agonist, baclofen, on synaptic transmission in rat ventrolateral periaqueductal gray (PAG) neurons of brainstem slices by using whole-cell voltage-clamp recordings. Baclofen (10 microM) induced a slow outward current (peak amplitude: 30.1+/-3.1pA, n=13) at -70mV, which persisted in the presence of tetrodotoxin (0.5 microM) and was diminished in the presence of postsynaptic intracellular K(+)-channel blockers (Cs(+) and TEA) and GDP-beta-S, indicating a direct postsynaptic depression mediated by K(+) channels and G proteins. Baclofen (10 microM) also decreased the frequency of both glutamatergic spontaneous EPSC (by 36+/-7%, n=11) and GABAergic spontaneous IPSC (by 37+/-12%, n=6) without changes in their amplitudes, indicating its presynaptic inhibitions. Taken together, the activation of postsynaptic GABA(B) receptors inhibits ventrolateral PAG neurons directly. At the same time, activating presynaptic GABA(B) receptors on glutamatergic and GABAergic nerve terminals inhibits glutamate and GABA release, respectively. The overall effects might influence an output of ventrolateral PAG neurons that build up the descending pain control system to the spinal dorsal horn.

Origins of GABA(B) receptor-like immunoreactive terminals in the rat spinal dorsal horn.

By means of immunohistochemistry for gamma-aminobutyric acid receptor B subtype (GABA(B)R), the origins of GABA(B)R-like immunoreactive (GABA(B)R-LI) terminals in the rat spinal dorsal horn were investigated. After dorsal root rhizotomy and/or spinal cord hemisection, the densities of GABA(B)R-LI terminals were remarkably depleted in the ipsilateral superficial dorsal horn of relevant segments, whereas GABA(B)R-LI neurons and sparsely distributed GABA(B)R-LI terminals remained. After injection of Fluoro-Gold (FG) into the left side of superficial lumbar dorsal horn, FG retrograde-labeled neurons were mainly observed in the ipsilateral rostral ventromedial medulla (RVM) and brainstem raphe nuclei. Some of the FG-labeled neurons, especially in the RVM, exhibited GABA(B)R-like immunoreactivity. Additionally, immunofluorescence histochemical double-staining revealed that the majority of GABA(B)R-LI neurons in the periaqueductal gray (PAG), RVM and brainstem raphe nuclei showed 5-hydroxytryptamine (5-HT)-like immunoreactivity. The present study morphologically proves that GABA(B)R-LI terminals in the spinal dorsal horn originate from peripheral afferents, intrinsic neurons and supraspinal structures; GABA(B)R and 5-HT co-exist in many neurons in the PAG, RVM and brainstem raphe nuclei. Considering that PAG, RVM, brainstem raphe nuclei and spinal dorsal horn are important structures involved in the pain modulation, we suggest that the descending pain modulation system might be mediated, at least in part, by GABA(B)R.