Cell-type-specific excitatory and inhibitory circuits involving primary afferents in the substantia gelatinosa of the rat spinal dorsal horn in vitro

The substantia gelatinosa (SG) of the spinal dorsal horn shows significant morphological heterogeneity and receives primary afferent input predominantly from Aδ- and C-fibres. Despite numerous anatomical and physiological studies, correlation between morphology and functional connectivity, particularly in terms of inhibitory inputs, remains elusive. To compare excitatory and inhibitory synaptic inputs on individual SG neurones with morphology, we performed whole-cell recordings with Neurobiotin-filled-pipettes in horizontal slices from adult rat spinal cord with attached dorsal roots. Based on dendritic arborization patterns, four major cell types were confirmed: islet, central, radial and vertical cells. Dorsal root stimulation revealed that each class was associated with characteristic synaptic inputs. Islet and central cells had monosynaptic excitatory inputs exclusively from C-afferents. Islet cells received primary-afferent-evoked inhibitory inputs only from Aδ-fibres, while those of central cells were mediated by both Aδ- and C-fibres. In contrast, radial and vertical cells had monosynaptic excitatory inputs from both Aδ- and C-fibres and inhibitory inputs mediated by both fibre types. We further characterized the neurochemical nature of these inhibitory synaptic inputs. The majority of islet, central and vertical cells exhibited GABAergic inhibitory inputs, while almost all radial cells also possessed glycinergic inputs. The present study demonstrates that SG neurones have distinct patterns of excitatory and inhibitory inputs that are related to their morphology. The neurotransmitters responsible for inhibitory inputs to individual SG neurones are also characteristic for different morphological classes. These results make it possible to identify primary afferent circuits associated with particular types of SG neurone.     

Comparison of chemosensitivity tests: clonogenic assay versus MTT assay

When the development of chemotherapeutic agents reaches the clinical trial stage, it is necessary to perform drug sensitivity tests quickly in order to select the most promising agents for the treatment of cancer. In order to assess the possibility of using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a substitute for the human tumor clonogenic assay (HTCA), we evaluated the correlation between the results obtained by these 2 assays in 5 human lung cancer cell lines. The correlation coefficient between the results of the HTCA and the MTT assay was 0.673, indicating a relatively good correlation. The correlation was most prominent in platinum analogues (r = 0.939) and good in anthracyclines/anthracenedione (r = 0.611). However, no significant correlation was observed in vinca alkaloids, etoposide, irinotecan, SN-38 (an active metabolite of irinotecan), and rhizoxin. The results of the MTT assay showed a high degree of correlation with those of the HTCA in predicting the sensitivity of cancer cell lines to platinum analogues, and anthracyclines/anthracenedione. These results suggest that the MTT assay may be more convenient and quickly performed than the HTCA and can replace HTCA in evaluating the effects of anticancer agents, especially the platinum analogues and anthracyclines/anthracenedione.     

Adult-onset type II citrullinemia and idiopathic neonatal hepatitis caused by citrin deficiency: involvement of the aspartate glutamate carrier for urea synthesis and maintenance of the urea cycle

Citrin is a mitochondrial aspartate glutamate carrier primarily expressed in the liver, heart, and kidney. We found that adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin. In this report, we describe the frequency of SLC25A13 mutations, the roles of citrin as a member of the urea cycle and as a member of the malate-aspartate shuttle, the relationship between its functions and symptoms of citrin deficiency, and therapeutic issues.     

Use of collagen sponge incorporating transforming growth factor-beta1 to promote bone repair in skull defects in rabbits.

The objective of this study was to evaluate the potential of collagen sponge incorporating transforming growth factor-beta1 (TGF-beta1) to enhance bone repair. The collagen sponge was prepared by freeze-drying aqueous foamed collagen solution. Thermal cross-linking was performed in a vacuum at 140 degrees C for periods ranging from 1 to 48 h to prepare a number of fine collagen sponges. When collagen sponges incorporating 125I-labeled TGF-beta1 were placed in phosphate-buffered saline (PBS) solution at 37 degrees C, a small amount of TGF-beta1 was released for the first hour, but no further release was observed thereafter, irrespective of the amount of cross-linking time the sponges had received. Collagen sponges incorporating 125I-labeled TGF-beta1 or simply labeled with 125I were implanted into the skin on the backs of mice. The radioactivity of the 125I-labeled TGF-beta1 in the collagen sponges decreased with time; the amount of TGF-beta1 remaining dependent on the cross-linking time. The in vivo retention of TGF-beta1 was longer in those sponges that had been subjected to longer cross-linking times. The in vivo release profile of the TGF-beta1 was matched with the degradation profile of the sponges. Scanning electron microscopic observation revealed no difference in structure among sponges subjected to different cross-linking times. The TGF-beta1 immobilized in the sponges was probably released in vivo as a result of sponge biodegradation because TGF-beta1 release did not occur in in vitro conditions in which sponges did not degrade. We applied collagen sponges incorporating 0.1 microg of TGF-beta1 to skull defects in rabbits in stress-unloaded bone situations. Six weeks later, the skull defects were covered by newly formed bone, in marked contrast to the results obtained with a TGF-beta1 free empty collagen sponge and 0.1 microg of free TGF-beta1. We concluded that the collagen sponges were able to release biologically active TGF-beta1 and were a promising material for bone repair.     

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis

In the prespermatogenesis period, male germ cells (gonocytes) begin to reproliferate and move to the basement membrane of the seminiferous tubule. Although these two events-reproliferation and relocation-are important for establishment of spermatogenesis, they have not been greatly analyzed both in a mechanical and in an endocrine or paracrine aspect. In this study, the relationship between reproliferation and relocation of gonocytes was examined, using the thymidine analog bromodeoxyuridine (BrdU) labeling method and transmission electron microscopy (TEM). BrdU was injected into the fetuses [day 13.5 post coitus (dpc) to 18.5 dpc] and pups [day 0. 5 post partum (dpp) to 6.5 dpp] of C57BL/6J mice. Two hours later, BrdU positive gonocytes were examined immunohistochemically and these data were analyzed. TEM and LM observation was carried out as well. Gonocytes began to relocate on the basement membrane from 18.5 dpc (1.4%) while BrdU-labeled gonocytes were first detected on 1.5 dpp (13.6%). Relocated BrdU-negative gonocytes were recognized from 18.5 dpc (1.4%), and relocated BrdU-labeled gonocytes were recognized from 1.5 dpp (8.4%). On the other hand, non-relocated BrdU-labeled gonocytes were detected from 1.5 dpp (5.2%). Gonocyte relocation began 2 days earlier than reproliferation during the late fetal period. After birth, the two events occurred at random. These results indicate that the reproliferation of the gonocyte does not correlate with relocation. The two events may be regulated by different mechanisms.     

ADRENAL PHEOCHROMOCYTOMA WITH BOTH BENIGN AND MALIGNANT COMPONENTS

A case of adrenal pheochromocytoma Is reported characterized by polygonal basophilic granular cells of benign type, plump eosinophilic granular cells of transitional type, and spindle-shaped cells of malignant type. In the primary tumor the neoplastic cells of each type revealed a distinctive topographical distribution. All gradations between the latter two varieties of cells were found, especially in the vicinity of the transitional cell area. Multiple metastases were present in the liver and lungs, where only anaplastic spindle-shaped cells could be found. Ultrastructurally, the benign cells contained predominantly large, rather light, secretory granules with a coarsely granulated core. In the malignant cells, the number, size, and intensity of granules varied considerably from cell to cell or even within a single cell. In general, the malignant cells had a higher frequency of smaller granules with electron-dense homogeneous cores. Moderate amounts of noradrenalin but not significant dopamine or adrenalin could be demonstrated from a metastatic nodule In liver. We postulate that this adrenal medullary pheochromocytoma was benign originally and underwent malignant transformation shortly before the patient's death. This is based upon the patient's clinical features and the peculiar structures of the primary tumor.     

Physiological properties of sensory trigeminal neurons projecting to mesencephalic parabrachial area in the cat

1. One hundred forty-one trigeminomesencephalic neurons in the sensory trigeminal nucleus of cats anesthetized with alpha-chloralose were identified by antidromic stimulation of the mesencephalic parabrachial area (PBA) which includes the nucleus cuneiformis, lateral periaqueductal gray matter, and the region between the inferior colliculus and brachium conjunctivum. 2. Neurons were categorized based on their responses to non-noxious and noxious mechanical and heat stimuli delivered to their peripheral receptive fields (RFs) including skin, mucosa, guard hairs, vibrissae, cornea, and tooth pulps. They were classified into three types: 48 nociceptive-specific (NS) neurons which responded to heavy pressure and/or noxious mechanical stimuli, and/or noxious radiant heat; 19 wide dynamic range (WDR) neurons which had a graded response to light tactile stimuli, noxious pinch, and/or noxious radiant heat; and 36 low-threshold mechanoreceptive (LTM) neurons which responded maximally to innocuous tactile stimuli. In subnucleus caudalis (Vc), NS and WDR neurons were the majority (75%) among the three types, while in the rostral subnuclei they were about one-half (54%) of the population. 3. The RFs were distributed over the orofacial and head region but mainly in the facial region. Twelve neurons (33% of the LTM neurons) responded to deflection of vibrissae and only 1 NS neuron out of 52 NS and WDR neurons tested responded to electrical stimulation of a tooth pulp. 4. A contralateral projection was dominant (57%), 30% projected ipsilaterally and 13% projected bilaterally.(ABSTRACT TRUNCATED AT 250 WORDS)