Counting Silver Grains Research Articles

A short history of the initial application of anti-5-BrdU to the detection and measurement of S phase.

Because this article is a bit of history rather than ascientific paper, we will depart from the formality of ajournalarticle.Thisarticlehastwopurposes.Thefirstistogive credit to two deceased individuals, Albert Castro,M.D., and Howard Gratzner, Ph.D. (1). Castro was the keyperson who was able to produce a polyclonal antibodythat was specific for bromodeoxyuridine (BrdU). For 10years, Gratzner took the lead in converting an idea into afunctional assay that affected DNA analysis and led to thedevelopment of many other assays with similar method-ologies. The second is to inform young scientists that (a)inventions or creations have utility in fields other than thepurpose for which they were created; (b) it is possible todo useful work in an institution that certainly would nothave been classified as one of the best; and (c) the pres-ence of talented people who are willing to collaborate isinvaluable.Scientifically, Gratzner is best known for the develop-ment of the first monoclonal antibody (mAb) to BrdU andiododeoxyuridine. This work was the culmination of pre-vious efforts with Robert Leif to develop polyclonal anti-bodies, which detected BrdU with variable specificity.Collaborations with scientists at the Lawrence LivermoreLaboratory encouraged the development of a widely usedflow cytometric test for the simultaneous measurementsof DNA and BrdU. The use of mAbs to halogenated pyri-midines was a significant scientific advance and currentlyis an integral tool for the analysis by flow and digitalmicroscopy of the cell cycle.On a personal level, Gratzner’s enthusiasm for scienceand his intelligence, curiosity, loyal interactions with fel-low collaborators, and friendship are sorely missed by allwhoknewhim.Hewasgenuinelyawarmandnicepersonwho was liked by almost everyone with whom he came incontact (1).Castro was a jovial man who interacted well with hiscollaborators. He was generous and very knowledgeable.He ran an immunodiagnostics laboratory at the universi-ties of Oregon and Miami and was an expert in the pro-duction of antibodies in animals. His warm smile, gener-ous giving of his knowledge, common sense, enthusiasmfor scientific research, and ability to get the job done wereessential to this project.Because this is a story of events that happened approx-imately 30 years ago and the existing records are limited,the accuracy of this article is limited by our memory andthose of our colleagues who have kindly helped with thisproject. Of course, this is our version of the events.Cell division and DNA replication are fundamental tobiology and specifically to the biology of cancer. Thisarticledescribeshowasimpleprocedureforthedetectionand quantitation of DNA synthesis was developed. Theprecise determination of when S phase occurs in the cellcycle is of use to maximize the selective killing of tumorcells with cycle-specific chemotherapeutic drugs. Ofgreater significance, studies of the control of the cellcycle, including its detour into apoptosis, can provideextremely useful insights into the creation of new thera-peutic regimens.The original process to detect S-phase cells included theuse of tritiated thymidine with autoradiography to mea-sure the labeling index (percentage of S-phase cells)and/or the fraction of labeled mitotic cells. Before and atthe infancy of flow cytometry, it was believed that thequantitative DNA analysis of normal and neoplastic cellsmight provide an objective marker for the diagnosis ofneoplasia. These measurements were performed with amicroscope on Feulgen-stained cells. In a normal popula-tion, it was established that there were two predominantpeaks in the DNA distribution, with a ratio of 1:2 in DNAcontent, and that cells that were synthesizing DNA (Sphase) were scattered in between, with a relative DNAcontent between 1 and 2. The detection of tritiated thy-

Metabolic mapping in mammalian retina: a biochemical and 3H-2-deoxyglucose autoradiographic study

It has long been known that mammalian retinas metabolize glucose aerobically to lactic acid and carbon dioxide. The classical view holds that glucose is the primary substrate for energy metabolism in all retinal cells, and that photoreceptor cells have the highest rates of glycolysis and respiration. A different and more recent view is that the Müller cells are the principal, if not sole aerobic producers of lactate, which then serves as the primary fuel for the mitochondria in photoreceptor cells and other retinal neurons. In this paper, we have examined these two competing hypotheses in rat and guinea pig retinas by identifying the cellular sites of glucose uptake and phosphorylation via hexokinase by means of autoradiographic studies with 3H-2-deoxyglucose ( 3H-2DG). The rat retina serves as a vascular model and the guinea pig retina serves as an avascular model. Rat and guinea pig eyecups were incubated in oxygenated, bicarbonate-buffered media containing glucose in the presence of labeled and unlabeled 2DG. Biochemical measurements of lactate production and ATP content were made on rat retinas incubated with different concentrations of glucose and 2DG in order to establish the optimal condition for conducting the autoradiographic studies with 3H-2DG. The optimal substrate concentrations were 1 m m glucose and 0·25 m m 2DG. Results showed that following incubation of dark-adapted rat eyecups for 1 hr in media containing 1 m m glucose/0·25 m m 2DG and supplemented with 3H-2DG, the label was distributed throughout all the layers of the retina, from the ganglion cell layer to the retinal pigment epithelium, with denser label associated with the outer retina (photoreceptors) relative to the density of label in the inner retina, as evaluated by counts of silver grains in individual retinal layers. Exposure of rat eyecups to light did not alter the relative distribution of label, but did increase total grain counts by 70%. However, uptake of labeled 2DG, as measured by scintillation counting of radioactivity in trichloroacetic acid extracts, was not significantly different between light- and dark-adapted rat retinas. In guinea pig eyecups, labeled 2DG was distributed throughout all the retinal layers. Addition of 10 m m lactate or pyruvate to the glucose/2DG media produced no measurable change in the density or distribution of label in the eyecups. Measurements of the activity of hexokinase in rat retinas revealed that this enzyme was present in both the mitochondrial and cytosolic fractions. The present results suggest that as long as the availability of ambient glucose is adequate, retinal neurons use glucose, rather than glial-derived lactate, as the major substrate for the production of high energy phosphates.

Granule cell proliferation and axon terminal degradation in the dentate gyrus of gerbils (Meriones unguiculatus) during maturation, adulthood and aging.

The objective of the present study was to examine both naturally occurring degrading events in axon terminals of the dentate gyrus and granule cell proliferation in the dentate gyrus of gerbils (Meriones unguiculatus) throughout postnatal life. For that purpose, (1) a selective silver staining technique was applied to analyze neuronal lysosome accumulation (LA), indicating synaptic degradation during development. LA was quantified by counting silver grains in the inner third and outer two thirds of the molecular layer, granular layer, subgranular layer and the hilus of the dentate gyrus. (2) Proliferation of granule cells was identified by in-vivo labeling with 5-bromo-2'-desoxyuridine (BrdU). BrdU-labeled granule cell nuclei were identified in consecutive horizontal slices along the mid-septotemporal axis of the hippocampus and light-microscopically quantified 4h after the BrdU-labeling. It was found (1) that in young animals LA significantly increased within all layers and reached adult levels after about 3 months. During subsequent development LA kept on this level throughout aging with highest values within the inner molecular layer. (2) There was a highly significant temporal gradient in granule cell proliferation with numbers of BrdU-labeled cells exponentially declining during juvenile life. Nevertheless, granule cell proliferation occurred throughout adult life and aging. The present results are discussed (1) with concepts of ongoing neuroplasticity and remodeling of neuronal networks in the developing and adult brain, and (2) with regard to pharmacologically induced neuromorphogenesis.

Expression of glycine receptor subunit mRNAs in the rat cochlear nucleus

The distribution of glycine receptor subunit mRNAs in the cochlear nucleus of the adult rat was examined using radioactive in situ hybridization. Expression was compared among six cell types by counting silver grains over somata. Expression of the immature α2 subunit was not above the threshold for detection in any neurons. Levels of expression of mature subunits varied among different cell types. Spherical bushy, small cell cap/shell neurons and fusiform cells had high expression of glycine receptor mRNA for α1, α3 and β subunits. Octopus cells and corn cells had high expression for α3 and β subunits, and only moderate expression for α1 subunit. Granule cells located between the dorsal and ventral cochlear nucleus had moderate expression of α3 and β subunits and no detectable α1 expression. These patterns of expression predict differences in glycinergic pharmacological properties between the cochlear nucleus neurons.

Differential distribution of N-methyl- d-aspartate receptor-2 subunit messenger RNA in the rat superior olivary complex

The distribution of N-methyl- D-aspartate receptor subunit messenger RNA was examined in the superior olivary complex of the rat using in situ hybridization methods. Non-radioactive methods showed N-methyl- D-aspartate receptor-1 and N-methyl- D-aspartate receptor-2A, -2B and -2C subunit expression to be abundant in principal cells of all major superior olivary complex regions, with N-methyl- D-aspartate receptor-1 expression higher than N-methyl- D-aspartate receptor-2. After autoradiographic in situ hybridization, counting silver grains over cells was used to quantitate and compare levels of expression of the N-methyl- D-aspartate receptor-2 subunits. N-methyl- D-aspartate receptor-2A, -2B and -2C expression was detected over cells in the lateral and medial superior olive, the medial, lateral, and ventral nucleus of the trapezoid body, and the superior paraolivary nucleus. N-methyl- D-aspartate receptor-2D expression was only observed in the lateral superior olive, lateral nucleus of the trapezoid body and ventral nucleus of the trapezoid body. Three distinct patterns of labeling were observed. The lateral superior olive and ventral nucleus of the trapezoid body showed higher expression of N-methyl- D-aspartate receptor-2A and -2C than other subunits; the medial nucleus of the trapezoid body showed higher N-methyl- D-aspartate receptor-2B expression than other subunits; and the medial superior olive, superior paraolivary nucleus and ventral nucleus of the trapezoid body showed relatively equivalent expression of N-methyl- D-aspartate receptor-2A, -2B and -2C subunits. N-methyl- D-aspartate receptor-2D had the lowest expression, with levels greater than background in only the lateral superior olive, ventral nucleus of the trapezoid body and lateral nucleus of the trapezoid body. Immunocytochemistry using antibodies to N-methyl- D-aspartate receptor-2A, -2B and -2C showed immunolabeling consistent with in situ hybridization results.

Calbindin D28K and parvalbumin gene expression in rat embryonic ventral forebrain grafts.

The present study characterizes expression of calbindin D28K (CB-D28K) and parvalbumin (PV) in ventral forebrain (VFB) grafts placed in the neocortex of adult rats bearing quisqualic acid lesions to the nucleus basalis magnocellularis. Three to nine months after transplantation surgery, rats were killed for in situ hybridization with probes to CB-D28K or PV and for immunohistochemistry with antibodies to CB-D28K or PV. In addition, an antibody to choline acetyltransferase (ChAT) was used to characterize the cholinergic component in the graft and an antibody to tyrosine hydroxylase (TH) to explore catecholaminergic innervation of the graft. Quantitative analysis of CB-D28K and PV messenger ribonucleic acid (mRNA) was based on counts of silver grains generated by emulsion autoradiography. Cells expressing CB-D28K mRNA were significantly larger than such cells in the adult VFB and the mean number of silver grains per cell was significantly greater than to such cells in the adult VFB. The level of CB-D28K mRNA expression as calculated by ratio of silver grains per unit area was also significantly increased. Quantification of PV mRNA showed no significant differences between the cells in the graft and in the adult VFB. In order to begin to interpret these findings, a comparison was made with such cells in the VFB of developing rats. Brain sections were sampled from embryonic day 17 and postnatal days 1, 5, 12, 19 and adult (6-12 months of age). Cells expressing CB-D28K mRNA were detected in ventral forebrain from postnatal day 5 and cells expressing PV mRNA were detected in ventral forebrain from postnatal day 19. In the course of normal development of the ventral forebrain, no CB-D28K cells were found that were as large or expressed such high levels of CB-D28K mRNA as observed in the grafts. We conclude that changes in grafted cells expressing CB-D28K do not reflect an arrest of developmental processes. TH immunohistochemistry revealed lack of catecholaminergic innervation of the graft, whereas adult mediolateral septal cells that express CB-D28K receive such innervation in addition to other neurotransmitter inputs. Imbalance in neurotransmitter inputs to grafted cells expressing CB-D28K is discussed as a possible factor in their increased size and gene expression.

Transient expression of acetylcholinesterase messenger RNA and enzyme activity in developing rat thalamus studied by quantitative histochemistry and in situ hybridization.

The molecular basis for transient expression of acetylcholinesterase in noncholinergic regions of the early postnatal rat brain was studied by in situ hybridization histochemistry. A 33P-labelled 63-mer DNA oligonucleotide was used to probe acetylcholinesterase messenger RNA in the brains of rat pups at one, two, six, nine, 12, 16 and 21 days of age (birth = day 0). Cryostat brain-sections were hybridized with probe and exposed to X-ray film or emulsion coatings. Acetylcholinesterase messenger RNA was quantitated by counting silver grains and by measuring X-ray film density with video imaging and computer-based densitometry. Adjacent sections were stained histochemically for acetylcholinesterase activity, also quantitated by video densitometry. Overall there was a significant correlation between apparent levels of acetylcholinesterase activity and acetylcholinesterase messenger RNA. Increases in message tended to accompany the surges of acetylcholinesterase activity that marked the maturation of thalamocortical sensory relay pathways. Acetylcholinesterase expression in the youngest rats was generally sparse but it increased markedly during the first postnatal week, especially in the sensory relay nuclei of the thalamus. Levels of message and enzyme activity in the medial and dorsolateral geniculate and the ventral posteromedial and ventral posterolateral nuclei rose to a peak, typically about day 9. Beyond this time there was a gradual decline. By day 21 the staining and in situ hybridization patterns resembled those in adult brains, whose thalamic relay nuclei are impoverished in acetylcholinesterase activity and messenger RNA. Thus, acetylcholinesterase expression is strongly modulated in certain thalamic systems as they undergo neural morphogenesis.

Parathyroid hormone restores bone mass and enhances osteoblast insulin-like growth factor I gene expression in ovariectomized rats

In the osteopenic rat model, estrogen deficiency results in increased bone turnover with net bone loss occurring during cancellous modeling. However, estrogen-deficient rats treated with parathyroid hormone (PTH) experience a net gain of bone tissue due to the anabolic effects of PTH. To evaluate the possibility that local insulinlike growth factor I (IGF-I) production modulates the in vivo balance of bone formation and resorption in ovariectomized (OVX) estrogen-deficient rats and in OVX rats treated with PTH, we have studied the expression of IGF-I mRNA in cancellous bone osteoblasts using in situ hybridization techniques. Three-month-old virgin rats were subjected to sham surgery or OVX. Two weeks later, half the OVX rats began treatment with hPTH(1–34), 5 gmg/100 g body weight, 5 days/week for 4 weeks. All animals were killed at the same time, providing three groups: sham surgery alone; OVX alone; and OVX + PTH. Bone histomorphometry performed in undecalcified sections of tibial metaphysis confirmed that OVX rats had significantly ( p < 0.05) increased bone surface formation rates (BFR/BS, μm 3/μm 2/year) with osteopenia while OVX + PTH rats had increased BFRBS with increased bone volumes compared to sham animals ( p < 0.05). Decalcified tissue from all three groups contained immunoreactive IGF-I. Similar tissue sections were hybridized with an 35S-labeled IGF-I antisense riboprobe. Evaluation of the specific signal over cancellous osteoblasts allowed a relative estimate of IGF-I mRNA transcript abundance in the three groups by counting silver grains per osteoblast, corrected for background activity. Despite evidence for increased BFR/BS, OVX animals had comparable frequency distributions of grain counts (after correction for background) to sham animals (88% of cells containing ⩽1 grain with a mean of 0.76 ± 0.28 grains/osteoblast vs. 81% containing ⩽1 grain with a mean of 0.61 ± 0.09, NS). However, OVX + PTH animals had over 40% of osteoblasts with ⩾2 grains per cell and twice the IGF-I mRNA abundance that either control group had (ANOVA, p < 0.001; OVX + PTH compared to either sham or OVX, p < 0.05). These data suggest tjat the anabolic effect pf PTH is associated with increased IGF-I mRNA steady state levels; while estrogen deficiency causes increase bone turnover, increased levels of IGF-I mRNA were not observed.

Unilateral Forelimb Amputation Affects Protein Synthesis in Ipsilateral and Contralateral Spinal Cord and Spinal Ganglia Neurons of the Newt in vivo

The aim of this investigation was to analyze the effect of urodele limb amputation and blastema growth on the protein synthesis in spinal cord and spinal ganglia neurons corresponding to the 3rd and 4th spinal segment of young, postmetamorphic Triturus vulgaris, in vivo. Protein synthesis was studied on both trunk sides as a function of right forelimb amputation at the distal stylopodium, and blastema development, during a critical period of 22 days post amputation (dpa). Protein synthesis was assessed by counting silver grains on autoradiographs obtained after pulse labeling with tritiated phenylalanine. An electronic image analyzer was used to evaluate the results. During the period of observation, several statistically significant, yet moderate changes were observed in the protein synthesis of motor neurons. In contrast, the studied metabolic parameter exhibited considerable augmentation in sensory neurons and spinal nerve. The main results may be summarized as follows: (a) Limb amputation triggers protein synthesis in both ipsilateral and contralateral neural elements; (b) protein synthesis is higher on the ipsilateral than on the contralateral side; (c) as the aftermath of forelimb amputation, the 4th ipsilateral ganglion responds with an approximately 20% higher protein synthesis in comparison to the 3rd one; (d) peaks in protein synthesis are encountered mainly at two time points: 3 hpa and 14 dpa. Protein synthesis returns to control levels after 22 days of limb regeneration.

Naturally occurring degrading events in axon terminals of the dentate gyrus and stratum lucidum in the spiny mouse (Acomys cahirinus) during maturation, adulthood and aging.

The naturally occurring dynamics of presynaptic axon terminals were investigated in the dentate gyrus and stratum lucidum of the spiny mouse (Acomys cahirinus) during maturation, adulthood and aging. A sensitive and selective silver-staining technique was applied to analyze neuronal lysosome accumulation (LA), indicating synaptic degradation during development. LA was quantified by counting silver grains in the inner third and outer two thirds of the molecular layer, granular layer, and the infragranular layer of the dentate gyrus, and in the strata oriens, pyramidale, lucidum and radiatum of the medial and distal regio inferior on postnatal days 21, 28, 95, 730, and 1,460. In young and adult animals, LA was most abundant within the inner molecular layer. When animals grew older, LA densities obviously decreased in the inner molecular layer but increased in the outer molecular layer. Within the stratum lucidum only the distal regio inferior showed an extremely high LA density on postnatal day 21, dramatically decreasing thereafter and reaching adult low values during the first postnatal month. By electron microscopy in the inner molecular layer we found LA in large synaptic boutons and small terminals both with distinct synaptic contact zones. Degrading presynaptic profiles may further accumulate dense bodies, zones with completely disorganized cytoplasm, and lamellarly organized whorled membrane debris. In the distal regio inferior comparable phenomena were observed in typical mossy fiber boutons. Despite these degrading events, no electron-dense degenerating terminals were found. These results on naturally occurring nondegenerative synaptic degradation are discussed with current concepts of synaptic turnover and remodelling in the developing, adult and aging brain.

The de novo and salvage pathways for the synthesis of pyrimidine residues of RNA predominate in different locations within the mouse duodenal epithelium.

RNA synthesis was examined by radioautography in mouse duodenal epithelium using 3H-uridine as a tracer of the salvage pathway and 3H-orotic acid as a tracer of the de novo pathway. The incorporation of the two precursors was estimated by counting silver grains in light-microscopic and electron-microscopic radioautographs at successive levels of crypt and villus. With both precursors, silver grains were found over all epithelial nuclei, but in numbers varying by location. Thus, after 3H-uridine injection, the number of grains was high over nucleolus and nucleoplasm in the base of the crypt, declined gradually in the middle and top of the crypt, and was low along the villus. After 3H-orotic acid, the number of grains was fairly low throughout, but peaked over the nucleoplasm in lower villus cells. The 3H-uridine reaction over nucleolus and nucleoplasm in crypt cells was interpreted as synthesis by the salvage pathway of ribosomal RNA and heterogeneous RNA, respectively, whereas the 3H-orotic acid reaction over the nucleoplasm of some villus cells indicated that these cells synthesized heterogeneous RNA by the de novo pathway.

Temporal profile of interneuron and pyramidal cell protein synthesis in rat hippocampus following cerebral ischemia

Cellular protein synthesis was investigated in the rat hippocampus 2-100 h following 20 min of cerebral ischemia induced by four-vessel occlusion. [3H]-Phenylalanine was retrogradely infused through the external carotid artery for 30 min. This method limited the distribution of the tracer to one hemisphere and required 1/50th of the tracer amount used for intravenous tracer infusion. Cellular [3H]phenylalanine incorporation was examined in hematoxyline and eosin-stained sections coated with nuclear emulsion. A score for relative protein synthesis was estimated from counts of silver grains across neuron somata with undamaged morphology. Shortly after ischemia a generalized complete arrest of protein synthesis was observed. In CA1 pyramidal cells, this was followed by a transient incomplete regeneration (9-20 h) and later (46-100 h) persistent cessation of protein synthesis. By contrast protein synthesis in interneurons, CA3c pyramidal cells and granule cells recovered to preischemic levels 9-100 h after ischemia, as did the CA3ab pyramidal cells 46-100 h postischemia. Moreover, eosinophilic cell changes were seen in hilar and CA3c neurons at all postischemic stages and in CA1 pyramidal cells 46-72 h after ischemia. [3H]Phenylalanine incorporation was absent in neurons demonstrating eosinophilic cell changes. From the rapid recovery of protein synthesis in hippocampal interneurons, we conclude that changes in interneuronal protein synthesis per se are not involved in the pathophysiology of the delayed ischemic CA1 pyramidal cell death.

An accurate method for automated counting of silver grains in autoradiographs

A grain counting algorithm has been developed for use with an image analysis system interfaced with a microscope. The algorithm consists of a sequence of tests involving thresholding, prospective grain center detection, contrast, and three kinds of artifact detection. The function of each of these parameters is described. This algorithm is accurate to within 4% for those fields most frequently encountered, and to within 9% for all fields examined. Furthermore, it operates very quickly and at relatively low magnification, making it possible to scan large areas efficiently.

Identification of human growth hormone messenger ribonucleic acid in pituitary adenoma cells by in situ hybridization.

The technique of in situ hybridization was used to examine human GH gene expression in human GH-secreting pituitary adenoma cells. Five somatotroph adenoma specimens obtained at surgery were dispersed into single cell suspensions. Hybridization experiments were performed on cells immediately after dispersal or on cells cultured for 48-72 h in a defined serum-free medium. Tritiated GH cDNA was used to probe fixed cells, and hybridization was determined by liquid autoradiography. Of the freshly dispersed adenoma cells probed with GH cDNA, more than 70% contained GH mRNA, as determined by counting silver grains per cell. Significant cellular grain counts were obtained for GH cDNA-probed cells from all five tumors, while negative controls showed negligible silver grain counts. In cultured cells derived from three of five tumors, an average of 40% contained detectable GH mRNA. Therefore, quantitative in situ hybridization is a useful technique to demonstrate the expression of GH mRNA in human pituitary adenoma cells.

Accuracy of an automated counting method by television-computer equipment in the quantitative autoradiography

Commercially available television-computer equipment was used in the counting of silver grains in autoradiograms. The counting was made very rapidly and easily. The counts were compared with the counts made visually. With some limitations both counts showed excellent agreement. The accuracy of the method used is discussed.

High resolution autoradiographic determination of the topographic distribution of radioactivity in the hippocampal formation after injection of [1- 14C]glucose or 2-deoxy[ 14C]glucose

Using high resolution autoradiography, the accumulation of radioactivity after intravenous injection of [1- 14C]glucose was measured in the corpus callosum, hippocampus, dorsal hippocampal commissure, somatosensory cortex, inferior colliculus and pontine periaqueductal grey. Autoradiograms were prepared by thaw-mounting 4μm frozen sections on nuclear emulsion-coated slides, and were evaluated quantitatively with a computer-assisted video system for automated counting of silver grains. In all brain regions examined, silver grain densities were greater in rats killed 30 min after injection of [1- 14C]glucose compared to rats killed 10 min after injection. After intravenous injection of [1- 14C]glucose or 2-deoxy[ 14C]glucose, the relative uptake and retention of radioactivity in different hippocampal subregions was compared. Striking differences were found in the hippocampus between 2-deoxy[ 14C]glucose and [1- 14]glucose autoradiograms. After injection of 2-deoxy[ 14C]glucose, there were large variations in the uptake and retention of radioactivity among different pyramidal cell fields. The CA 3 pyramidal cell field retained considerably more radioactivity than other pyramidal cell fields after injection of 2-deoxy[ 14C]glucose, while after injection of [1- 14C]glucose, the retention of radioactivity was similar in all pyramidal cell fields. After [1- 14C]glucose injection, the dentate gyrus contained relatively high levels of radioactivity and more 14C accumulated in the granular layer, compared to the molecular layer. In contrast, after 2-deoxy[ 14C]glucose injection, there was uniformly less radioactivity throughout the dentate gyrus when compared to rats injected with [1- 14C]glucose and there was no preferential accumulation of 2-deoxy[ 14C]glucose in the granular layer compared to the molecular layer. These results imply that, in the hippocampal formation, different cellular functions are assessed by the determination of the uptake and retention of radioactivity from [1- 14C]glucose and 2-deoxy[ 14C]glucose.

Regulation of glycine receptor binding in the mouse hypoglossal nucleus in response to axotomy

Recent studies have shown that muscarinic receptors in brain hypoglossal nuclei exhibit a loss of specific ligand binding in response to axotomy of the hypoglossal nerve. The mouse hypoglossal nucleus contains a high level of receptors for the inhibitory neurotransmitter, glycine; the ligand [ 3H]strychnine binds to the glycine receptor with high affinity. In the present study [ 3H]strychnine binding in mouse hypoglossal nuclei was examined at 1 to 150 days after unilateral lesions of the hypoglossal nerve. Brains were sectioned on a cryostat, thaw-mounted onto microscope slides, incubated with [ 3H]strychnine and processed for light microscopic autoradiography. Receptor density was assessed by counting silver grains in photomicrographs of operated and control nuclei. During the first 25 days after axotomy grain density fell to 50 percent of that of the control nucleus. After this time grain density slowly increased, returning to control levels by 150 days post lesion. These data indicate that glycine receptors on the axotomized cells of the hypoglossal nucleus are lost when connection with the target muscles of the tongue is interrupted, and that the receptors reappear when the hypoglossal nerve regenerates. It is suggested that excitatory and inhibitory neurotransmitter receptor systems may be regulated in a coordinated fashion by the functional state of the motoneuron.

Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine.

The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [3H]uridine injection or by the de novo pathway as shown after [3H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium--that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5-3H]uridine or [5-3H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloracetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [3H]uridine and 72% after [3H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A+ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [3H] uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant. After [3H]-orotic acid injection, the number of silver grains over the nucleolus was negligible at all levels, whereas over the nucleoplasm the number was low in crypt cells, but high in villus cells with a peak in mid villus. The interpretation is that, except for a small amount of label incorporated into DNA from either precursor by crypt cells, the bulk of the label is incorporated into RNA as follows. In the crypts, cells make almost exclusive use of uridine, that is, of the salvage pathway, for the synthesis of ribosomal RNA in the nucleolus and of messenger and transfer RNA in the nucleoplasm. However, when cells pass from crypt to villus, they mainly utilize orotic acid--i.e., the de novo pathway--for the synthesis of messenger and transfer RNA within the nucleoplasm.

Increases in protein-precursor incorporation in the denervated neuropil of the dentate gyrus during reinnervation

Cellular metabolic events accompanying postlesion synaptogenesis in the hippocampus were studied by analyzing incorporation of protein precursor ([ 3H]leucine) in the dentate gyrus. Adult rats were injected intravenously with [ 3H]leucine at periods from 2 to 60 days following unilateral destruction of the entorhinal cortex, and were killed 30 min later. Precursor incorporation was quantified autoradiographically by counting silver grains over the cell bodies and dendrites of dentate granule cells ipsiand contralateral to the lesion. The relative grain density was increased over the denervated portion of the neuropil at 6–12 days postlesion, corresponding to the early phase of terminal proliferation and reactive synaptogenesis. Whereas incorporation was increased over the denervated neuropil, the availability of [ 3H]leucine was decreased relative to the contralateral side in autoradiographic preparations designed to reveal the concentration of the unincorporated 3H-labeled precursor and its diffusible degradation products. Silver grains were not selectively associated with glial cell bodies or vascular elements, but rather were distributed diffusely throughout the neuropil. Increases in grain density over the denervated zone were observed when animals were killed 8 min after the leucine injection, suggesting that the increases were not due solely to rapid transport from granule cell bodies to dendrites. We propose that an increased incorporation of protein precursor occurs primarily within the denervated dendrites of granule cells during the early phase of reinnervation, and that protein synthetic activity in these cells might be involved in the process of reinnervation.

Effect of chromatolysis upon the incorporation of L-4,5-(3H)-leucine into axotomized neurons

Autoradiography was used to compare the rate of protein synthesis in neurons which showed different types of neuronal change after axotomy. Different types of axon reaction were obtained after crush lesion of the facial nerve in rats and young rabbits and after evulsion of the facial nerve in rats. The incorporation of (3H)-leucine into protein was determined after intraperitoneal injection by counting silver grains over facial neurons in autoradiograms prepared from paraffin sections. Crush lesion of the facial nerve in rats was followed by barely discernible changes in the neurons, no chromatolysis, and full functional recovery. The lesion caused a significant transitory increase in the protein synthesis. After nerve evulsion, the same neurons showed a severe central chromatolysis and ultimate nerve cell disintegration. The protein synthesis was unchanged during the early chromatolytic phase after evulsion and decreased at later stages. Crush lesion of the facial nerve in young rabbits was followed by severe central chromatolysis, full functional recovery, and no loss of neurons. There was a moderate increase in the protein synthesis during chromatolysis and a further increase during the phase of recovery. It is concluded that regeneration after axotomy is accompanied by an increase in protein synthesis, regardless of the type of morphologic change in the neurons. Chromatolysis is not a prerequisite for this increase, and the chromatolytic response per se seems to be of minor importance for the extent of incorporation of (3H)-leucine into neuronal proteins.