Differential expression of VGLUT1 or VGLUT2 in the trigeminothalamic or trigeminocerebellar projection neurons in the rat.

The vesicular glutamate transporters, VGLUT1 and VGLUT2, reportedly display complementary distribution in the rat brain. However, co-expression of them in single neurons has been reported in some brain areas. We previously found co-expression of VGLUT1 and VGLUT2 mRNAs in a number of single neurons in the principal sensory trigeminal nucleus (Vp) of the adult rat; the majority of these neurons sent their axons to the thalamic regions around the posteromedial ventral nucleus (VPM) and the posterior nuclei (Po). It is well known that trigeminothalamic (T-T) projection fibers arise not only from the Vp but also from the spinal trigeminal nucleus (Vsp), and that trigeminocerebellar (T-C) projection fibers take their origins from both of the Vp and Vsp. Thus, in the present study, we examined the expression of VGLUT1 and VGLUT2 in Vp and Vsp neurons that sent their axons to the VPM/Po regions or the cortical regions of the cerebellum. For this purpose, we combined fluorescence in situ hybridization (FISH) histochemistry with retrograde tract-tracing; immunofluorescence histochemistry was also combined with anterograde tract-tracing. The results indicate that glutamatergic Vsp neurons sending their axons to the cerebellar cortical regions mainly express VGLUT1, whereas glutamatergic Vsp neurons sending their axons to the thalamic regions express VGLUT2. The present data, in combination with those of our previous study, indicate that glutamatergic Vp neurons projecting to the cerebellar cortical regions express mainly VGLUT1, whereas the majority of glutamatergic Vp neurons projecting to the thalamus co-express VGLUT1 and VGLUT2.

Coexpression of VGLUT1 and VGLUT2 in trigeminothalamic projection neurons in the principal sensory trigeminal nucleus of the rat.

VGLUT1 and VGLUT2 have been reported to show complementary distributions in most brain regions and have been assumed to define distinct functional elements. In the present study, we first investigated the expression of VGLUT1 and VGLUT2 in the trigeminal sensory nuclear complex of the rat by dual-fluorescence in situ hybridization. Although VGLUT1 and/or VGLUT2 mRNA signals were detected in all the nuclei, colocalization was found only in the principal sensory trigeminal nucleus (Vp). About 64% of glutamatergic Vp neurons coexpressed VGLUT1 and VGLUT2, and the others expressed either VGLUT1 or VGLUT2, indicating that Vp neurons might be divided into three groups. We then injected retrograde tracer into the thalamic regions, including the posteromedial ventral nucleus (VPM) and posterior nuclei (Po), and observed that the majority of both VGLUT1- and VGLUT2-expressing Vp neurons were retrogradely labeled with the tracer. We further performed anterograde labeling of Vp neurons and observed immunoreactivies for anterograde tracer, VGLUT1, and VGLUT2 in the VPM and Po. Most anterogradely labeled axon terminals showed immunoreactivities for both VGLUT1 and VGLUT2 in the VPM and made asymmetric synapses with dendritic profiles of VPM neurons. On the other hand, in the Po, only a few axon terminals were labeled with anterograde tracer, and they were positive only for VGLUT2. The results indicated that Vp neurons expressing VGLUT1 and VGLUT2 project to the VPM, but not to the Po, although the functional differences of three distinct populations of Vp neurons, VGLUT1-, VGLUT2-, and VGLUT1/VGLUT2-expressing ones, remain unsettled.

Medullary dorsal horn neurons providing axons to both the parabrachial nucleus and thalamus.

It has often been suggested that the trigemino- and spino-thalamic pathways are highly implicated in sensory-discriminative aspects of pain, whereas the trigemino- and spino-parabrachial pathways are strongly implicated in affective/emotional aspects of pain. On the other hand, the superficial laminae of the spinal dorsal horn, where many nociceptive neurons are distributed, have been reported to contain projection neurons innervating both the parabrachial nucleus (PBN) and thalamus by way of axon collaterals (Hylden et al., 1989). For the medullary dorsal horn (caudal subnucleus of spinal trigeminal nucleus: Vc), however, the existence of such neurons has not been reported. Thus, in the present study, we examined whether the Vc might contain projection neurons sending their axons to both the thalamus and PBN. Dual retrograde labeling with fluorescence dyes was attempted. In each rat, tetramethylrhodamine-dextran amine and Fluoro-gold were stereotaxically injected into the PBN and thalamic regions, respectively. The proportion of the dually labeled Vc cells in the total population of all labeled Vc cells was about 20%. More than 90% of the dually labeled neurons were distributed in lamina I (marginal zone), less than 10% of them were located in lamina II (substantia gelatinosa), and only a few (about 1%) were found in lamina III (magnocellular zone). The results indicate that some Vc neurons in the superficial laminae mediate nociceptive information directly to the PBN and thalamus by way of axon collaterals and that the vast majority of them project to the ipsilateral PBN and contralateral thalamus.

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.