The green fluorescence protein (GFP) gene recombinant virus labeling is a new method for the neuroanatomical studies. It is effective to investigate the morphological features of neurons, the chemical architectures (neural active substances and their receptors) of the neurons, the relationships between the neuronal morphological features and their termination areas or their functions, and local circuits related to specific functions. This method remedies the defects of previous morphological methods and could provide direct morphological evidence for the functional investigations.

Objective To investigate the connections between ?\|aminobutyric acid(GABA)\|or glycine (Gly)\|containing terminals and Fos\|positive projection neurons from the medullary dorsal horn (MDH) to the thalamus or parabrachial nuclei(PBN). Methods Tetramethyl rhodamine(TMR) retrograde tracing,combined with immunofluorescence histochemical triple\|staining for TMR/Fos/GABA or TMR/Fos/Gly was used. Results GABA\|or Gly\|immunoreactive terminals were chiefly located in the superficial laminae(laminae Ⅰ and Ⅱ) of the MDH.After orofacial noxious stimulation.Fos\|positive neurons were also mainly observed in the superficial laminae.After injecting TMR into the unilateral thalamus or PBN,TMR retrogradely labeled neurons were mainly distributed in the superficial laminae of the controlateral or ipsilateral MDH,respectively.Some of these TMR\|labeled neurons also exhibited Fos\|immunoreactivities.GABA\| or Gly\|containing terminals made close contacts with Fos positive retrogradely labeled neurons.Conclusion\ In the superficial laminae of the MDH,some of the orofacial nociceptive neurons send project fibers directly to the thalamus or PBN,GABA and Gly might exert their inhibitory effects on these nociceptive projection neurons.\;[

Objective To observe the distribution of GABAB receptor subtype 1 (GABABR1 ) in the rat nervous system. Method Immunocytochemical staining technique by using specific antibody against GABABR1 was used. Results Intensely and densely stained GABABR1-like immunoreactive neurons were observed in the V layer of cerebral cortex. islands of Calleja, caudate putamen, septohippocampal nucleus, hippocampus, basolateral amygdaloid nucleus, medial habenular nucleus, anterodorsal thalamic nucleus, mediodorsal thalamic nucleus, dorsal lateral geniculate nucleus. preoptic nuclei, supraoptic nucleus, lateral magnocellular part of paraven- tricular nucleus, anterior commisural nucleus, median eminence, arcuate nucleus, pars compacta of substantia nigra, ventral tegmental area, interpeduncular nucleus, Edinger-Westphal nucleus, mesencephalic trigeminal nucleus, locus coeruleus, nucleus of the trapezoid they, superficial layers of the caudalis subnucleus of the spinal trigeminal nucleus, dorsal motor nucleus of vagus, Purkinje cell layer of cerebellar cortex, laminae Ⅰ- Ⅲ, Ⅸ and X of spinal gray matter, lateral spinal nucleus, Onuf's nucleus and spinal dorsal root ganglion. Specially, in the cholinergic and monoaminergic nuclei of the brain. Conclusion These results indicate that GABABR1-like im- munoreactive structures are widely located in the rat brain. GABA might exert its principal inhibitory effects through these GABABR.

Calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV) are the most common calcium-binding proteins (CaBPs). In the present study, FOS immunoreactivity was first induced in neurons of the medullary dorsal horn (MDH) of the rat by noxious orofacial stimulation; CaBPs (CB, CR and PV) in these neurons were then identified by imumunofluorescence histochemistry, and then, in addition, afferents to CaBPs/FOS double-labeled neurons were showed by immunofluorescence histochemical staining for the γ-aminobutyric acid (GABA) , glycine transporter 2 (GlyT2) , enkephalin (ENK) , serotonin (5-HT) or substance P (SP). Under the light microscope,we observed that: (1) neuronal cell bodies exhibiting FOS-immunoreactivity were present throughout all laminae of the MOH, with the highest concentration in lamina Ⅱ; (2) most CB-, CR- and PV-immunoreactive (IR) neurons were located in lamina Ⅱ , but some were also encountered in laminae Ⅰ and Ⅲ; (3) 5-HT-, GABA-, GlyT2-, ENK- and SP-IR fibers and terminals were also chiefly located in laminae Ⅰ and Ⅱ of the MDH; (4) some FOS-IR neurons showed CB-, CR-, or PV-immunoreactivity; (5) 5-HT-, GABA-, GlyT2- and ENK-IR terminals made close contacts with FOS/CB, FOS/CR or FOS/PV double-labeled neurons; (6) SP-IR terminals, as well as 5-HT-, GABA-, GlyT2- or ENK-IR terminals, closely contacted CB-, CR- or PV-containing neurons. Under the electron microscope, 5-HT-, GABA-, GlyT2- and ENK-IR terminals principally made symmetric (inhibitory) synaptic connections with CB-, CR- and PV-containing neurons were observed. These results suggest that 5-HT, GABA, glycine (Gly) and ENK may modulate transmission of orofacial noxious information by inhibiting nociceptive neurons that contain CaBPs in the rat MDH.

Objective To observe the morphologic features of neurons labeled with green fluorescent protein (GFP) gene recombinant Sindibis virus in the deep part of lamina Ⅲ in the medullary dorsal horn (MDH) of the rat. Methods Neurons were infected and labeled with GFP gene recombinant Sindibis virus injected into lamina Ⅲ.Immunohistchemical staining showed the labeling results.The morphologic features of the GFP labeled neurons were revealed after reconstruction. Results A few GFP-labeled single neurons were found in the distal portions away from the virus injection site within the deep part of lamina Ⅲ of the MDH.According to the morphological features,especially their axons and axonal collaterals,GFP labeled neurons were classified into projection neurons and intrinsic neurons.The axons and their collaterals of the projection neurons entered to the superficial part of lamina Ⅲ,lamina Ⅱ and/or the medullary reticular formation.Most of the axons and their collaterals of the intrinsic neurons extended mainly within lamina Ⅲ.Conclusions According to the morphological features of axons and their axonal collaterals,GFP labeled neurons were classified into projection neurons and intrinsic neurons in the deep part of lamina Ⅲ of the MDH.GFP gene recombinant virus labeling technique is an effective method to investigate the morphological features of neurons.

After injecting retrograde tracer fiuoro-gold (FG) into the parabrachial region(PB), caudal ventrolateral medulla(CVLM) and the fourth segment of cervical spinal cord (C4), respectively, neurons in laminae I ～ Ⅱ of the medullary dorsalhorn projecting to the above mentioned brain areas were observed. PB received projections from bilateral laminae I and Ⅱ withan ipsilateral dominance; CVLM and C4 received projections from ipsilateral laminae I and Ⅱ. Neurons projecting to C4 werevery sparsely distributed in laminae I and Ⅱ of the medullary dorsal horn. The projecting neurons in outer part of lamina Ⅱwere more than those in inner part of lamina Ⅱ . Combined with immunofluorescence histochemistry for calbindin-D28k(CB) andparvalbumin(PV), it was demonstrated that a part of neurons projecting to PB or CVLM showed CB-like immunoreactivity, butnone of them exhibited PV-like immunoreactivity. There were only a few neurons in lamina Ⅱ projecting to C4 and they exhibitedneither CB- nor PV-like immunoreactivity. The present study provides further evidence for the existence of projecting neurons inlamina Ⅱ and suggests that immunostaining against CB and PV may distinguish two neuronal subpopulations in lamina Ⅱ .

A cerebellar afferent connection from the periaqueductal grey (PAG) has been demonstrated in the rat by means of retrograde transport of horseradish peroxidase (HRP) in the present study. The projection is bilateral, but the projection from the ipsilateral side is predominant (3:1). Its main origin is the ventromedial and ventrolateral regions of middle and caudal parts of PAG (98.8％), and the fibers reach different cerebellar cortical regions: culmen, declive, folium vermis, tuber vermis, pyramis vermis, uvula vermis, lobulus quadrangularis, crus Ⅰ, crus Ⅱ, and paraflocculus. Most labelled neurons are medium sized, but some small neurons also appear to project to cerebellum. Only a few large neurons are retrogradely labelled at the most caudal end of the caudal part. Functionally, both cerebellum and PAG are related to visceral activities. Consulting the present experiment, we discussed the significant role of the PAG-cerebellar projection.

In order to study the bifurcate projections of midbrain periaqueductal gray and nucleus raphe dorsalis to nucleus accumbens and nucleus raphe magnus, the fluorescence double-labeled method was used in the present study. Bisbenzimide (Bb) and propidium iodide (PI) were injected into nucleus raphe magnus and unilateral nucleus accumbens stereotaxically according to the time period necessary for their axonal transport. The percentages of double-labeled neurons were 21％; PI single labeled neurons were 32％; Bb single labeled neurons were 47％. Most of the labeled neurons were located in the middle and caudal parts of periaqueductal gray and the nucleus raphe dorsalis, and most were medium sized and fusiform and triangular in shape.

The distributions of 5-HT-like and Met-ENK-like immunoreactive (5-HT-LI and Met-ENK-LI) structures in the nucleus accumbens (Acb) of rats were studied by immunohistochemical technique in the present study. Under light microscope, 5-HT-LI fibers and terminals could be seen in each subnucleus at different planes of Acb, but the 5-HT-LI fibers and terminals in the medial and ventral subnuclei were more than the dorsal and lateral subnuclei, the amount of 5-HT-LI fibers and terminals in the caudal segment were more than the rostral segment. According to the diameter, pathway, and number of varicosity, 5-HT-LI fibers could be divided into 3 types: (A) thick fiber (0.35—0.40?m); (B) medium fiber(0.20—0.30?m); (C) thin fiber (about 0.10?m). These 3 types of 5-HT-LI fibers were remarkable in the medial and ventral subnuclei of Acb. 5-HT-LI neuronal bodies did not observed in the Acb. A few scattered Met-ENK-LI neuronal bodies were seen in the ventral subncleus and ventral part of the medial subnucleus. Met-ENK-LI fibers and terminals distributed in all subnuclei and predominant in the medial and ventral subnuclei. The distributions of Met-ENK-LI structures were no differences between the rostral and caudal segments. All of the Met-ENK-LI fibers were thin and irregular and villi-like in shape. There were only a few varicosities on the MetENK-LI fibers. Part of Met-ENK-LI fibers looked like discontinued varicosities. Under electron microscope, 5-HT-LI axonal boutons formed symmetric and asymmetric synapses with non-5-HT-LI dendrites. Met-ENK-LI dendrites formed symmetric and asymmetric axo-dendritc synapses with non-Met-ENK-LI axonal boutons. These synapses were mainly observed in the medial and ventral subnuclei of Acb. The identity of 5-HT-LI and Met-ENK-LI structures, especially in the medial and ventral subnuclei, supported the physiological studies that 5-HT-LI ascending efferent fibers activated the Met-ENK-LI neurons and then the latter sent descending efferent fibers to lower brainstem structures to take part in antinociceptive functions.