The cholinergic neurons and fibers of the hypothalamus could not be revealedsuccessfully in the past,therefore,there has been general agreement that the hypotha-lamus is very poorly innervated by cholinergic system.In this study,choline acetyl-trans ferase (ChAT)-like immunoreactive positive neurons and fibers of the hypo-thalamus were revealed successfully by using avidin-biotin immunocytochemical me-thod.This study demonstrated for the first time that the cat hypothalamus is richlyinnervated by cholinergic system.We found cholinergic neurons of varying numbersin the following areas:dorsomedial hypothalamic nucleus,paraventricular nucleus,dorsal hypothalamic area,the area of the tuber cinereum surrounding ventromedialhypothalamic nucleus,lateral hypothalamic area,anterior hypothalamic area,anteriorhypothalamic nucleus,parvocellular hypothalamic nucleus,tuber-mammillary nucl-eus,posterior hypothalamic area,anterior mammillary nucleus and supramammillarynucleus.There were a lot of ChAT-like positive fibers in the lateral hypothalamicarea,mammillary area,dorsal hypothalamic area,paraventricular nucleus,parvocel-lular hypothalamic nucleus,the area of the tuber cinereum.Three kinds of neuronperikarya related to cholinergic system were identified in the hypothalamus of thecat:1.cholinergic perikarya;2.noncholinergic-cholinoceptive perikarya;3.choli-nergic-cholinoceptive perikarya.There were also immunoreactive positive fiberswhich were non-varicose and varicose.Two kinds of varicose-fibers,one withstrong immunoreactivity and the other with weak immunoreactivity were distingui-shed.

WGA-HRP was injected into the inferior colliculus in the rat in order to study the non-auditorial afferents of the inferior colliculus. In the diencephalon and truncus encephali, retrograde labeling was observed in many non-auditorial regions: bilateral (with contralateral predominance) cuneate nucleus, gracile nucleus, nucleus of spinal tract of trigeminal nerve, nucleus medullae oblongatae centralis dorsalis; bilateral (with homolateral predominance) locus ceruleus, nucleus reticularis pontis oralis, laterodorsal tegmental nucleus, substantia nigra, sub-parafascicular nucleus, perifornical nucleus; bilateral medial parabrachial nucleus, deep tegmental nucleus of midbrain, lateral hypothalamic area; homolateral deep layer of superior colliculus, zona incerta; contralateral nucleus of solitary tract; and posteromedian septum, central tegmental nucleus, dorsal nucleus of the raphe. The afferent neurons from cuneate nucleus, gracile nucleus, nucleus of spinal tract of trigeminal nerve, nucleus of solitary tract mainly located below the level of obex. The results mentioned above suggested that the inferior colliculus is not only an auditorial relay nucleus, but also a complex body which is of different functions.

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 .

40 ?m in diameter) were accounted for about 15％, and the rest were medium-and small-sized cells.

In the present study the commissural projection between the two superior colliculi in the rat was examined with horseradish peroxidase method. The result shows that when HRP was injected into the superior colliculus of one side, HRP labeled cells could be found in every part of the contralateral superior colliculus. In each case the labeled cells were relatively concentrated in the region corresponding to the site of injection. It indicates that various parts of one superior colliculus may be connected chiefly with the corresponding part of the opposite side through the commissural projection.Most of the labeled cells were found in the middle layer of the superior colliculus, especially in its upper half, less in the deep layer, and the least in the superficial layer. Neurons in the superficial layer of one side project only to the superficial layer of the contralateral side, and so are the middle-deep layers. The connections between the superficial layers of both sides were independent from those of the middledeep layers.The commissural projection of the bilateral superior colliculi passes through the commissure of the superior colliculus which could be divided into a dorsal and a ventral fiberal fasciculns. The dorsal one was smaller, predominantly related to the superficial layer and the upper half of the middle layer; the ventral one was larger, part of its fibers related to the lower half of the middle layer and the deep layer, while the rest project to other nuclei of the contralateral region of the mesencephalon (e. g. nucleus cuneiforms, etc,).Most of the labeled cells were small in size, the rest were medium-sized, and no large ones were found.

It is demonstrated that the parabigeminal nucleus of the rat is subdivided intodorsal,middle and ventral groups.The parabigeminal nucleus sends fibers to bila-teral superior colliculus,the rostral half of it predominantly projects to the rostralhalf of the contralateral superior colliculus,but the rostral end projects only to thecontralateral one;the caudal half of this nucleus predominantly projects to thecaudal half of the ipsilateral superior colliculus,but the caudal end projects only tothe ipsilateral one.The superficial layer of the superior colliculus receives projec-tions from the ipsilateral dorsal and ventral groups and from the contralateralmiddle group of the parabigeminal nucleus.The middle and deep layers receive pro-jections from the ipsilateral middle group and contralateral dorsal and ventral groups,and probably from the other groups of both sides.The superior colliculus also sends fibers to both parabigeminal nuclei,predo-minantly the ipsilateral side.The lateral tegmental area sends fibers to the middle and deep layers of the su-perior colliculus.From the results described above,it could be concluded that the parabigeminalnucleus——tectum——parabigeminal nucleus connections are not only concerned withsuperficial layer,but also with the middle and deep layers of the superior colliculus.The tegmentum——tectum——tegmentum connections are only concerned with the mid-dle and deep layers of the superior colliculus.

Objective To express a mutated insulin gene in HepG-2 cell line to further research of insulin gene therapy. Methods Native human insulin cDNA was obtained from fetus pancreas with RT-PCR. Furin consensus cleavage sequence was introduced into proinsulin cDNA with site-directed mutagenesis (overlap extension PCR), and the new sequence was named as INS/furin. Subsequently, INS/furin was subcloned into the multiple clone sites of plasmid p(G1RE)3BP-1Luc. The new plasmid p(G1RE)3BP-11?furin was identified with the method of enzyme digestion by Hind Ⅲ and EcoR V. HepG-2 cells were transfected with the plasmid p(G1RE)3BP-11?furin by liposome-mediated method. The transfected HepG-2 cells were incubated for 48h in a glucose-containing medium (25mmol/L), and then the conditioned media were collected and HepG-2 cells were harvested respectively. The expression of INS/furin mRNA in transfected HepG-2 cells was examined by RT-PCR, the regained DNA was sequenced and insulin in conditioned media was investigated by radioimmunoassay. Results Two enzymes, Hind Ⅲ and EcoR V, digested p(G1RE)3BP-11?furin, and 2 fragments with length of 260 bp and 4 700bp, were obtained. The 260bp fragment was identified as insulin/furin, indicating that the target gene had been successfully inserted in specific sites. RT-PCR showed that insulin/furin mRNA was expressed in transfected HepG-2 cell, and the regained DNA was confirmed as insulin/furin by sequencing; while insulin was detected by radioimmunoassay in conditioned media. Conclusion The recombinant mammalian expression plasmid p(G1RE)3BP-11?furin has been successfully constructed, and transfected into HepG-2 cells, which therefore may efficiently secrete bioactive insulin.

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.