Interstitial cells of Cajal contain signalling molecules for transduction of nitrergic stimulation in guinea pig caecum.

Nitric oxide (NO) is an inhibitory signalling molecule in the gastrointestinal (GI) tract that is released from neurons and from leucocytes during inflammation. NO stimulates soluble guanylate cyclase (sGC), elevates cyclic guanosine 3',5'-monophospate (cGMP), and subsequently activates cGMP-dependent protein kinase (PKG). Targets for NO in the guinea pig caecum were investigated by characterizing the cellular distribution of sGC, cGMP and PKG. Immunoreactivity for both isoforms of sGC, sGCalpha1 and sGCbeta1, was observed in the interstitial cells of Cajal (ICC) and enteric neurons in the tunica muscularis. Double labelling with anti-Kit and anti-sGC antibodies showed sGCalpha1 and sGCbeta1-like immunoreactivity (LI) in almost all intramuscular (IM) and myenteric ICC. Neuronal processes with neuronal NO synthase were closely apposed to ICC expressing sGC-LI. Cells with sGC-LI possessed ultrastructural features of ICC-IM: caveolae, close association with nerve bundles and contacts with smooth muscle cells (SMC). Sodium nitroprusside, added with the phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine and zaprinast), enhanced cGMP-LI in almost all ICC and in some enteric neurons. Nerve stimulation also increased cGMP-LI in ICC and enteric neurons. In contrast, no resolvable increase in cGMP-LI was observed in any cells when the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was present. ICC and SMC also expressed PKG type I-LI. These data show that ICC express the downstream signalling molecules necessary to transduce nitrergic signals and activate inhibitory pathways and thus are primary targets for NO released from neurons and other cells in the GI tract.

Interstitial cells of Cajal are innervated by nitrergic nerves and express nitric oxide-sensitive guanylate cyclase in the guinea-pig gastrointestinal tract.

Nitric oxide (NO) is a major signaling molecule in the gastrointestinal tract, and released NO inhibits muscular contraction. The actions of NO are mediated by stimulation of soluble guanylate cyclase (sGC, NO-sensitive GC) and a subsequent increase in cGMP concentration. To elucidate NO targets in the gastrointestinal musculature, we investigated the immunohistochemical localization of the beta1 and alpha1 subunits of sGC and the distribution of neuronal NO synthase (nNOS) -containing nerves in the guinea-pig gastrointestinal tract. Distinct immunoreactivity for sGCbeta1 and sGCalpha1 was observed in the interstitial cells of Cajal (ICC), fibroblast-like cells (FLC) and enteric neurons in the musculature. Double immunohistochemistry using anti-c-Kit antibody and anti-sGCbeta1 antibody revealed sGCbeta1 immunoreactivity in almost all intramuscular ICC throughout the entire gastrointestinal tract. Immunoelectron microscopy revealed that sGCbeta1-immunopositive cells possessed some of the criteria for intramuscular ICC: presence of caveolae; frequently associated with nerve bundles; and close contact with smooth muscle cells. sGCbeta1-immunopositive ICC were closely apposed to nNOS-containing nerve fibers in the muscle layers. Immunohistochemical and immunoelectron microscopical observations revealed that FLC in the musculature also showed sGCbeta1 immunoreactivity. FLC were often associated with nNOS-immunopositive nerve fibers. In the myenteric layer, almost all myenteric ganglia contained nNOS-immunopositive nerve cells and were surrounded by myenteric ICC and FLC. Myenteric ICC in the large intestine and FLC in the entire gastrointestinal tract showed sGCbeta1 immunoreactivity in the myenteric layer. Smooth muscle cells in the stomach and colon showed weak sGCbeta1 immunoreactivity, and those in the muscularis mucosae and vasculature also showed evident immunoreactivity. These data suggest that ICC are primary targets for NO released from nNOS-containing enteric neurons, and that some NO signals are received by FLC and smooth muscle cells in the gastrointestinal tract.

Muscarinic M(2) acetylcholine receptor distribution in the guinea-pig gastrointestinal tract.

In the enteric nervous system, acetylcholine is the most common neurotransmitter to induce gastrointestinal smooth muscle contractions. Cholinergic signaling is mediated by muscarinic acetylcholine receptors on the surface of smooth muscle cells. Five different muscarinic receptor subtypes (M(1)-M(5)) have been identified and characterized, all of which belong to the superfamily of the G-protein-coupled receptor. The muscarinic M(2) acetylcholine receptor is the major muscarinic receptor subtype expressed by smooth muscle tissues in the gastrointestinal tract, where it is coexpressed with a smaller population of M(3) receptor. In this study, we examined the immunohistochemical distribution of the M(2) receptor using a specific antibody in the guinea-pig gastrointestinal tract. M(2) receptor-like immunoreactivity was mainly observed as associated with smooth muscle cells in the gastrointestinal tract. M(2) receptor-like immunoreactivity in smooth muscle cells was distributed throughout the cell membrane associated with caveolae. In the proximal colon, M(2) receptor-like immunoreactivity in the smooth muscle cells was weak. In the small intestine, interstitial cells of Cajal that possessed neurokinin 1 receptor-like immunoreactivity had intense M(2) receptor-like immunoreactivity. In the proximal colon, intramuscular and myenteric interstitial cells of Cajal exhibited M(2) receptor-like immunoreactivity. These findings indicate that, in the gastrointestinal musculature, M(2) receptors are distributed both in the smooth muscle cells and interstitial cells of Cajal, suggesting that the M(2) receptor elicits smooth muscle cell contraction and the interstitial cells of Cajal are the sites of innervation by enteric cholinergic neurons.

Alterations in enzyme histochemical characteristics of the masseter muscle caused by long-term soft diet in growing rabbits.

OBJECTIVE: Recently young people have an increasing tendency to intake an easily chewable diet and spend less time on mastication. The aim of the present study was to investigate the histochemical effects of long-term soft diet on the masseter muscle in growing rabbits. MATERIALS AND METHODS: Twelve young male Japanese white rabbits were divided into two groups (n = 6 each) at weaning (1 month after birth) and fed a solid diet (control group) or a powder diet (soft-diet group). The duration of the experimental period was 6 months. Masseter fibers from the superficial and the deep portions were histochemically defined as type 1, 2A, 2B, or 2C fibers. RESULTS: As compared with that of the control, the deep masseter of the soft-diet group showed a significantly lower ratio of type 1 fiber cross-sectional area to total area (6.3 and 10.1% for the soft-diet and control group, respectively), significantly more type 2A fibers (74.0%vs 50.3%) and significantly fewer type 2B fibers (4.3%vs 12.5%). However, fiber size did not differ between the two groups. NADH-tetrazolium-reductase (NADH-TR) of the masseter was less reactive in the soft-diet group, reflecting a lower oxidative capacity. CONCLUSIONS: These findings indicate that the alteration of the functional activities contributed to selective disuse influences on the type 1 and type 2B fibers, and a resultant increase in type 2A fibers. This study suggests that long-term alteration of jaw function induced by a soft diet can lead to adaptations of the masseter muscle.

Heterogeneous distribution of a gap junction protein, connexin43, in the gastroduodenal junction of the guinea pig.

The gastroduodenal junction differs in morphology and function from the stomach and the duodenum. We studied the immunohistochemical distribution of the gap junction protein, connexin43, and the nerve terminal proteins, SNAP-25 and synaptotagmin, in the musculature of the guinea pig gastroduodenal junction. Connexin43-immunopositive structures were distributed throughout the circular layer of the gastroduodenal junction, most densely in the duodenal circular layer. The difference in the distribution patterns of these structures between the stomach and the duodenum was readily observed in the gastroduodenal junction. In the inner part of the circular muscle layer of the gastroduodenal junction, the connexin43-immunopositive structures were relatively few or non-existent, whereas the SNAP-25-containing nerve fibers and synaptotagmin-containing nerve terminals, clearly observed, were numerous. These findings show a heterogeneous distribution of the gap junctions and nerves in the gastroduodenal junction. The results suggest that the gastroduodenal junction has heterogeneous electrical connections among smooth muscle cells via gap junctions, and specific nerve innervation, which regulates gastroduodenal motility.

Guidance of glial precursor cell migration by secreted cues in the developing optic nerve.

Oligodendrocyte precursors are produced in restricted foci of the germinative neuroepithelium in embryo brains and migrate to their sites of function, while astrocytes are produced in a wider area in the neuroepithelium. We investigated the guidance mechanisms of glial precursor (GP) cell migration in the optic nerve. GP cell migration in newborn rat optic nerve was monitored by the UV-thymine-dimer (TD) method. A double labeling study using NG2 and TD revealed that many of these in vivo migrating cells were NG2 positive, while some of them with large TD-positive nuclei were NG2 negative. An in vitro cell migration study using optic nerve with chiasma and/or eyeball tissue revealed that the GP cells migrated under the guidance of repulsive cues secreted from the optic chiasma. We detected the expression of netrin 1 and Sema3a in the optic chiasma, and that of Unc5h1 and neuropilin 1 in the optic nerve. Co-culture experiments of the optic nerve with cell clusters expressing guidance cues revealed that the migrating GP cells in the optic nerve were heterogeneous. Netrin 1 repelled a subtype of NG2-positive and PLP-positive GP cells with small nuclei. Sema3a repelled a subtype of GP cells with large nuclei.

Distribution of preganglionic terminals in the cervical sympathetic ganglia detected by the expression of c-Fos like protein after electric stimulation of the ventral root.

To determine the segmental relationship between the upper thoracic spinal cord and cervical sympathetic ganglia, we observed the distribution pattern of postganglionic cells which expressed c-Fos like protein, one of the products of immediate early genes, after electrical stimulation of ventral roots at the T1-T3 spinal segments. We recognized a clear segmental arrangement of postganglionic cells in the stellate ganglion along its rostrocaudal direction corresponding to the segmental arrangement of preganglionic neurons in the spinal cord. That is, postganglionic neurons which expressed c-Fos like protein after stimulation of the T1 ventral root were distributed in the middle region of the stellate ganglion in the rostrocaudal direction. The c-Fos like protein-positive neurons after stimulation of the T2 ventral root were distributed in a more caudal region of the stellate ganglion than after T1 ventral root stimulation. C-Fos like protein-positive neurons after stimulation of the T3 ventral root were mainly situated in a more caudal region of the stellate ganglion than after T2 ventral root stimulation. There was, however, no segmental relationship between the upper thoracic levels of the spinal cord and superior cervical ganglion in the rostrocaudal direction. These results indicate that the segmental innervation of the upper thoracic spinal cord exists in the stellate ganglion, but not in the superior cervical ganglion.

Ultrastructure of NADPH diaphorase-positive nerve fibers and their terminals in the rat cerebral arterial system.

To investigate how perivascular NO synthase (NOS)-containing nerves in the cerebral arterial system are involved in controlling the cerebral circulation, we observed the ultrastructure of NOS-containing nerve fibers and their terminals by means of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) histochemistry. We also observed the correlation between NADPH-d stained perivascular nerves and the perivascular sympathetic nerves, by means of double staining with NADPH-d histochemistry and tyrosine hydroxylase (TH) immunohistochemistry at the light microscopic level. NADPH-d-positive nerve fibers showed dense distribution mainly in the rostral portion of the circle of Willis and proximal portions of its main branches, where some of the NADPH-d-positive fibers coexisted with TH-positive fibers in a single nerve bundle. NADPH-d-positive nerve fibers were unmyelinated and had close contact with NADPH-d-negative myelinated and unmyelinated nerve fibers in a single nerve bundle, and NADPH-d-positive nerve terminals also existed closely with NADPH-d-negative nerve terminals. The number of NADPH-d-positive nerve terminals and their ratio to all other terminals were significantly higher in the rostral portion of the circle of Willis and the proximal portion of its branches, than the caudal portion of the circle of Willis and the distal portion of its branches. Nerve terminals were observed to locate within 250 nm from the basal lamina of arterial smooth muscle cells in the rostral portion of the circle of Willis and proximal portion of its branching arteries. The present observation confirmed that NOS-containing nerve fibers truly innervate the smooth muscle cells of the arterial wall in the circle of Willis and its main branches. Close contact between NADPH-d-positive and -negative nerve fibers and terminals in these arterial portions may indicate that NOS-containing perivascular nerves may work to modulate the rest of the other perivascular nervous system, such as the sympathetic nerves, to regulate the homeostasis of the arterial tonus.

Expression of L1 and TAG-1 in the corticospinal, callosal, and hippocampal commissural neurons in the developing rat telencephalon as revealed by retrograde and in situ hybridization double labeling.

In the telencephalon, the corticospinal (CS), callosal, and hippocampal commissural neurons are the major types of neurons that have axons crossing the midline of the brain. To understand the mechanisms involved in crossing the midline structure and to examine whether the expression patterns of L1 and TAG-1 in the commissural neurons are similar to those in the spinal cord, we investigated L1 and TAG-1 expression in these neurons in rats by using a double-labeling technique involving retrograde labeling and in situ hybridization. Expression of L1 messenger RNA was detected in the retrogradely labeled CS projection neurons by 1,1;-dioctadecyl-3,3, 3;,3;-tetramethylindocarbocyanine perchlorate (DiI) injection into the pons at embryonic day (E) 19, but expression of TAG-1 messenger RNA was not detected in these neurons. Also, after their axons crossed the pyramidal decussation, continued expression of L1 but no expression of TAG-1 in the CS projection neurons was shown by an additional double-labeling experiment involving DiI injection into the spinal cord at postnatal day (P) 1. An immunohistochemical study showed that L1 was continuously present in each level of the CS tract at E21 and P3, but TAG-1 immunoreactivity was not found in any level at any stage. Finally, we examined the expression of L1 and TAG-1 messenger RNAs in the callosal and hippocampal commissure neurons after their axons had crossed the midline by using the double-labeling technique. In both cases, hybridization signals of the L1 and TAG-1 messenger RNAs were observed in the retrogradely labeled neurons at P3. These results suggest that the roles of L1 and TAG-1 in the formation of the commissures in the forebrain are different from their roles in the spinal cord.

Femoral intercondylar notch measurements in osteoarthritic knees.

METHODS: We measured the dimensions of the intercondylar notch of the femur in 32 patients with primary severe osteoarthrosis (OA) of the knee and 54 embalmed cadaveric knees. RESULTS: There were 56 knees with morphologically normal anterior cruciate ligament (ACL), 11 knees with lax or partially ruptured ACL and 19 knees with missing ACL. The average width of the intercondylar notch in knees with lax and missing ACL was significantly narrower than that of knees with normal ACL. In addition, knees with missing ACL had a significantly smaller notch depth than knees with normal ACL. In medial compartment OA (56 knees), the notch width and depth in knees with severe OA (37 knees) were significantly smaller than those in normal (19 knees) and mild to moderate OA groups (19 knees). CONCLUSION: Our results indicate that osteophyte growth in the femoral intercondylar notch seems to correlate with the progression of medial compartment OA of the knee.

Expression of Hex mRNA in early murine postimplantation embryo development.

The onset of Hex expression and its role in early murine development was analyzed using in situ hybridization. Hex mRNA was first detected in the chorion of the ectoplacental cavity and weakly at the visceral endoderm of the future yolk sac at embryonic age (E) 7.5. Expression in embryonic tissues was detected exclusively in the hepatic anlage and thyroid primordium at E 9.5. At E 12.5 and E 15.5, Hex expression persisted in the fetal liver and thyroid, and was also detected in the fetal lung. These results suggest that Hex has its role in differentiation and/or organogenesis of several embryonic tissues.

Arborization pattern of sympathetic preganglionic axons in the rat superior cervical and stellate ganglia.

Anterograde labeling technique with Phaseolus Vulgaris leucoagglutinin (PHA-L) was employed to observe how a single preganglionic axon arborizes in the superior cervical ganglion (SCG) and stellate ganglion (STG) of rats. PHA-L was injected into the intermediolateral nucleus of the spinal cord at the middle point between segments T1 and T2, and labeled axons were detected immunohistochemically in serial sections. We traced and drew three preganglionic axons over their full length in the SCG and STG. In SCG, the labeled axons bifurcated repeatedly and extended to a length of 600-700 microns in the rostrocaudal direction, and about 200 microns in the transverse direction. These three preganglionic axons made 11, 14 and 11 dense terminal plexus regions along their trajectory. The pattern of the most dense terminal plexus corresponded to the pericellular type dendritic plexus, one of the plexus patterns of dendritic collaterals of SCG neurons. In the STG, the extent of axonal arborization was more variable than that in the SCG, ranging from 400 to 800 microns in the rostrocaudal direction and about 400 microns in the transverse direction. The three analyzed axons made 21, 19 and 20 dense terminal plexus regions along their trajectory, with a similar pattern to those in SCG. These results indicated that there might be a columnar or ellipsoidal organization of postganglionic neurons which are innervated by single preganglionic axons.

Distribution of vagal preganglionic neurons in the rat brain innervating thoracic and abdominal organs revealed by retrograde DiI tracing.

To investigate the distribution and number of preganglionic neurons which regulate motility and secretion in thoracic and abdominal organs in the vagal parasympathetic nervous system, the neuronal tracer DiI was injected into the organs and the distribution of retrogradely labeled neurons was examined in the rat brainstem. The stomach received the vast majority of efferent projections from the dorsal motor nucleus of the vagus nerve (DMV). The cecum and the duodenum also received projections from the DMV, but they originated from a smaller number of preganglionic neurons. Preganglionic neurons projecting to the stomach occupied the middle part of the DMV, those projecting to the cecum occupied the lateral part of the DMV, and those projecting to the duodenum were found in the medial edge of the DMV. The ventral and dorsal sides of the stomach wall were innervated by the left and right vagus nerves, respectively. However, immediately after passing the boundary between the stomach and duodenum, the left and right vagal nerve fibers mixed in the ventral and dorsal walls of the distal gastrointestinal tract. The nucleus ambiguous is a mixture of parasympathetic preganglionic neurons and motoneurons. In this study, we revealed that the major targets of these preganglionic neurons were the lungs and other thoracic organs.

NOS-positive preganglionic neurons innervate a subpopulation of postganglionic neurons in superior cervical ganglion in rats.

To determine the postganglionic targets of NOS-containing preganglionic neurons, we studied the association of NADPH-diaphorase positive preganglionic fibers and retrogradely labeled postganglionic neurons in the superior cervical ganglion (SCG) in rats. Wheat germ agglutinin-horseradish peroxidase solution was applied to the anterior chamber of the eye, middle cerebral artery, subcutaneous layer of the facial skin, or submucosal layer of the inside of the lip. Two days after tracer application, the rats were perfused with fixative solution. Serial sections of the SCG were stained histochemically for NADPH-diaphorase followed by diaminobenzidine reaction. More than 80% of the labeled postganglionic neurons innervating the structures in the subcutaneous or submucosal layer showed close association with NADPH-diaphorase positive preganglionic nerve terminals; approximately one-third of these labeled neurons were encircled by dense baskets of pericellular terminals. On the other hand, most of the postganglionic neurons innervating the iris (69%) or the cerebral artery (90%) did not show a distinct association with NADPH-diaphorase positive terminals. These results suggest that one of the principal roles of the NOS-containing preganglionic neurons may be in controlling the postganglionic neurons which innervate the structures in the subcutaneous or submucosal layer.

Restriction of the fate of early migrating trunk neural crest in gangliogenesis of avian embryos.

Trunk neural crest is the source of peripheral nervous tissue, the adrenal medulla and pigment cells. To quantitatively assay the potency of neural crest to form each derivative tissue, we isolated fragments of neural crest from quail embryos and transplanted them into the migration pathways of chicken embryos. In the resultant chimeras, we counted the quail cells derived from grafts distributed in the dorsal root ganglia, the sympathetic tissues around the aorta and the spinal nerves. Descendant cells of quail neural crest derived from the brachial or lumbosacral and lower levels were more abundant in the dorsal root ganglia than in the sympathetic tissue, while those derived from adreno-medullary levels were more abundant in the sympathetic tissue than in the dorsal root ganglia. No correlation was seen between the distribution pattern of quail cells and the rostrocaudal levels of graft site in chick embryos. These findings suggest that the developmental potency of truncal neural crest in gangliogenesis is restricted in the early phase of their migration and differs along the rostrocaudal axis, although it is not clear whether this restriction reflects determination of each crest cell. The size of the rudiments of the dorsal root ganglia in the normal embryo differed along the rostrocaudal axis, these differences being consistent with those in the fate of the neural crest at a given somite level.

Heterogeneity of neurogenic responses in intra- and extrameningeal arteries of dogs.

1. Neurogenic responses to transmural electrical stimulation were examined in endothelium-denuded extrameningeal (vertebral and carotid) and intrameningeal (spinal, basilar and middle cerebral) arteries isolated from dogs. 2. In the extrameningeal arteries, transmural electrical stimulation produced a phasic contraction. This contraction was abolished by tetrodotoxin, prazosin and guanethidine. However, alpha,beta-methylene ATP and NG-nitro-L-arginine (L-NOARG) had no significant effect on the contractile responses. 3. In the intrameningeal arteries, the neurogenic responses to electrical stimulation were composed of a transient contraction and relaxation. The transient contraction was selectively inhibited by guanethidine L-NOARG abolished the relaxation but not the contraction induced by electrical stimulation. Prazosin had no effect on either neurogenic response. 4. Noradrenaline produced a large contraction in the extrameningeal arteries which was selectively inhibited by prazosin. alpha,beta-Methylene ATP produced neither contraction nor inhibition of the response to noradrenaline in the extrameningeal arteries. 5. In the intrameningeal arteries, alpha,beta-methylene ATP produced a greater contraction than noradrenaline. The response to alpha,beta-methylene ATP was selectively abolished by desensitization of P2x-purinoceptors with alpha,beta-methylene ATP itself. The contractile response to noradrenaline was inhibited by rauwolscine but not by prazosin. 6. ATP produced endothelium-dependent relaxations in the extrameningeal and intrameningeal arteries, which were attenuated by endothelium removal. 7. NADPH diaphorase-positive fibres were dense in the middle cerebral and basilar arteries but rare or absent in the spinal artery. In the extrameningeal arteries diaphorase-positive traces were observed in the vasa vasorum. 8. The present findings indicate that the neurogenic responses of intrameningeal arteries of dogs are composed of NO-ergic and sympathetic purinergic components, while the extrameningeal arteries tested produced only sympathetic adrenergic responses, suggesting that regional heterogeneity may be associated with a sudden transition in innervation and receptor expression at the meninx.

[Study on the distribution of muscle spindles in the rat extensor digitorum longus muscle].

The distribution of muscle spindles (MS) in the extensor digitorum longus (EDL) muscle of the rat hind limb was morphologically studied on a series of longitudinal cryostat sections. The intrafusal muscle fibers were brownishly stained with the acetylcholinesterase reaction, and the mucopolysaccharide contained in the equatorial periaxial space was stained with alcian blue. This double-staining method made it easy for us to find the MS and to decide the equatorial portion and the whole extension of MS. From a series of camera lucida drawings the distribution of all MS was reconstructed on a sheet of paper and three-dimensionally imaged on a personal computer using image reconstructing software. The MS were distributed mainly in the superficial and lateral part of EDL muscle. Additionally, an ATPase reaction was employed to detect the red muscle fibers, and it was confirmed that their distribution of them is similar to that of MS.

Preservation of topography in the connections between the subiculum, field CA1, and the entorhinal cortex in rats.

In order to examine whether the entorhinal-hippocampal-entorhinal circuit is reciprocal and topographic, the connections between the subiculum, the CA1 field, and the entorhinal cortex were studied with the carbocyanine dye (Dil), which moves in both retrograde and anterograde directions. We investigated the organization of reciprocal connections revealed by injections of Dil in the entorhinal cortex along the rhinal sulcus. Anterograde fluorescent labeling showed the same pattern reported in previous studies of the dorsal hippocampus. When the injection site of DiI extended into the deep layers (IV-VI) of the same cortical column, the anterograde labeling of the perforant path was accompanied by retrograde labeling of the subicular neurons and the CA1 neurons. The distribution of labeled cells overlapped the distribution of labeled fibers, and the distribution of labeled cells paralleled that of the labeled fibers in the CA1 field. DiI injection into the medial entorhinal cortex revealed fewer retrogradely labeled subicular neurons than injection into the lateral entorhinal cortex, whereas the number of labeled CA1 neurons was not dependent on the injection site. The number of labeled CA1 neurons was always several times greater than the number of subicular neurons. Thus, the amount of information conveyed by the CA1 projection might be higher than that conveyed by the subicular projection. These results indicate that the entorhinal cortex, CA1, and the subiculum are connected reciprocally and topographically. We believe that the framework of the major hippocampal circuit proposed in previous studies should be reconsidered. We propose that the CA1 projection, rather than the subicular projection, is the main projection that feeds back information from the hippocampus to the entorhinal cortex.

Quantitative description of morphologic changes effected by antileukemic agents in L1210 leukemia cells.

The effect of antileukemic agents is explained mainly by the inhibition of DNA synthesis, which may reflect the structural alteration of the target cells. To identify such cellular alterations, in particular morphologic changes associated with the exposure to antileukemic agents were investigated in the murine leukemic L1210 cell line using flow cytometry and image analysis. Cells were cultured with eight kinds of antileukemic agents in cytostatic concentrations which are clinically achievable. Living cells were observed in culture without fixation or staining. Two parameters related to cell size and shape were measured simultaneously by the image analysing computer. These parameters provided us with different criteria for characterizing the antimetabolites, DNA-strand-damage inducers and other agents tested. Such morphologic studies may make it possible to estimate the characteristics of various antileukemic agents.

Electron microscopic study of endogenous peroxidase activity in human liver macrophages.

We evaluated the conditions of fixation for ultrastructurally demonstrating the endogenous peroxidase (PO) activity of macrophages in biopsied human liver. The application of microwaving and immersion fixation with tannic acid and aldehydes allowed excellent visualization of PO activity in the nuclear envelope (NE), rough endoplasmic reticulum (rER), and cytoplasmic granules (CG), with good preservation of cellular ultrastructures. The macrophages with PO activity showed one of the following five patterns of PO localization: positive in both the NE and rER but negative in the CG (type 1); negative in both the NE and rER but positive in the CG (type 2); negative in the NE but positive in both the rER and CG (type 3); positive in all three (type 4); PO negative (type 5). The type 1 cells resembled typical Kupffer cells, type 2 cells monocytes, and type 3 and 4 cells the exudate-resident macrophages considered to be a transitional form between exudate and resident macrophages. Type 5 cells may also be a transitional form between the exudate and resident macrophage, or an end-stage macrophage derived from exudate macrophages which have lost their PO activity. Tannic-acid-aldehyde immersion fixation with microwaving may be a useful method in the study of the PO activities of macrophages in biopsied human liver specimens.