Form of the postsynaptic density. A serial section study.

Through the use of serial sectioning of dog cerebral cortex tissue, holes or perforations could be revealed in the larger postsynaptic densities (PSDs), in confirmation of the earlier work of Peters and Kaisermann-Abramof (1969. Z. Zellforsch. Mikrosk. Anat. 100:487-506). These holes appeared in serial sections which happened to be cut both parallel and normal to the plane of the synaptic junction. Cleft material was absent in that part of the synaptic cleft opposite this hole. Somestimes the presynaptic membrane opposite the hole was indented into the presynaptic cell. In addition, most of the synaptic vesicles in the presynaptic cell close to the membrane were clustered at that part of the membrane opposite the edge of the density disk. The meaning of the hole and of the other features mentioned above for the function of the density is not known at present.

The structure of postsynaptic densities isolated from dog cerebral cortex. II. Characterization and arrangement of some of the major proteins within the structure.

An attempt was made to identify some of the proteins of the postsynaptic density (PSD) fraction isolated from dog cerebral cortex. The major protein has been tentatively labeled "neurofilament" protein, on the basis of its 51,000 mol wt correspondence to a protein found in neurofilament preparations. Other proteins are akin to some dog myofibrillar proteins, on the basis if immunological crossreaction and equal sodium dodecyl sulfate (SDS)-gel electrophoretic mobilities. While a protein similar to dog muscle myosin is not present in the PSD fraction, a major protein present is actin, as evident from reactivity with antiactin serum, from SDS-gel mobility, and from amino acid composition. Only very little tubulin may be present in the PSD fraction, as determined by gel electrophoresis. Various treatments of the PSD fraction were attempted in order to extract some proteins, as revealed by gel electrophoresis, and to observe the structural changes of the PSD fraction residue after extraction of these proteins. The PSD is remarkably resistant to various extraction conditions, with only 4 M guanidine being found to extract most of the proteins, except the 51,000 mol wt protein. Disulfide reducing agents such as dithiothreitol (DTT), blocking agents such as p-chloromercuribenzoate (PCMB) (both in the presence of deoxycholate [DOC]), a Ca++ extractor, ethylene glycol-bis (beta- aminoethyl ether) N,N,N',N'-tetraacetate (EGTA), and guanidine caused an opening up of the native dense PSD structure, revealing approximately 10-nm filaments, presumably consisting of "neurofilament" protein. Both DTT-DOC and PCMB-DOC removed chiefly actin but also some other proteins. EGTA, in greatly opening up the structure, as observed in the electron microscope, revealed both 10-nm and 3- to 5-nm filaments; the later could be composed of actin, since actin was still in the residue after the treatment. EGTA removed a major 18,000 mol wt component and two minor proteins of 68,000 and 73,000 mol wt. Based on the morphological and biochemical evidence, a picture is presented of the PSD as a structure partly made up of 10-nm and 3- to 5-nm filaments, held together through Ca++ interaction and by bonds amendable to breakage by sulfhydrylblocking and disulfide-reducing reagents; either removal of Ca++ and/or rupture of these disulfide bonds opens up the structure. On the basis of the existence of filamentous proteins and the appearance of the PSD after certain treatments as a closed or open structure, a theory is presented with envisages the PSD to function as a modulator in the conduction of the nerve impulse, by movements of its protein relative.

The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition.

A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of methods of Davis and Bloom (21, 22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 or more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.

Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.

Postsynaptic densities (PSDs) have been isolated from cerebral cortex, midbrain, cerebellum, and brain stem by the Triton X-100 method previously used in the isolation of cerebral PSDs (Cohen et al., 1977, J. Cell Biol. 74:181). These PSDs have been compared in protein composition, protein phosphorylation, and morphology. Thin-section electron microscopy revealed that cerebral cortex and midbrain PSDs were identical, being approximately 57 nm thick and composed of apparent aggregates 20-30 nm in diameter. Isolated cerebellar PSDs appeared thinner (33 nm) than cerebral cortex PSDs and lacked the apparent 20- to 30-nm aggregates, but had a latticelike structure. In unidirectional and rotary-shadowed replicas, the cerebrum and midbrain PSDs were circular in shape with a large central perforation or hole in the center of them. Cerebellum PSDs did not have a large perforation, but did have numerous smaller perforations in a lattice like structure. Filaments (6-9 nm) were observed connecting possible 20- to 30-nm aggregates in cerebrum PSDs and were also observed radiating from one side of the PSD. Both cerebral cortex and midbrain PSDs exhibited identical protein patterns on SDS gel electrophoresis. In comparison, cerebellar PSDs (a) lacked the major 51,000 Mr protein, (b) contained two times less calmodulin, and (c) contained a unique protein at 73,000 Mr. Calcium plus calmodulin stimulated the phosphorylation of the 51,000 and 62,000 Mr bands in both cerebral cortex and midbrain PSDs. In cerebellar PSDs, only the 58,000 and 62,000 Mr bands were phosphorylated. In the PSDs from all brain regions, cAMP stimulated the phosphorylation of Protein Ia (73,000 Mr), Protein Ib (68.000 Mr), and a 60,000 Mr protein, although cerebrum and midbrain PSDs contained very much higher levels of phosphorylated protein than did the cerebellum. On the basis of the morphological criteria, it is possible that PSDs isolated from cerebrum and midbrain were derived from the Gray type I, or asymmetric, synapses, whereas cerebellum PSDs were derived from the Gray type II, or symmetric, synapses. Since there is some evidence that the type I synapses are involved in excitatory mechanisms while the type II are involved in inhibitory mechanisms, the role of the PSD and of some of its proteins in these synaptic responses is discussed.

Subcellular distribution in cerebral cortex of two proteins phosphorylated by a cAMP-dependent protein kinase.

The subcellular distribution of Proteins Ia and Ib, two proteins which serve as specific substrates for protein kinases present in mammalian brain, was studied in the dog cerebral cortex. Proteins Ia and Ib were found to be most highly enriched in synaptic vesicle fractions; they were also present in postsynaptic density and synaptic membrane fractions in significant amounts. Proteins Ia and Ib present in the synaptic vesicle fraction appear to be similar, if not identical, to those present in the postsynaptic density fraction as judged by several criteria: (a) the ability to serve as substrate for cAMP-dependent protein kinase, (b) electrophoretic mobility in the presence of sodium dodecyl sulfate, (c) extractability with NH4Cl or EGTA, and (d) fragmentation to electrophoretically similar peptides by a purified Staphylococcus aureus protease. In addition, the postsynaptic density fraction has been found to contain cAMP-dependent Protein Ia and Protein Ib kinase activity. The subcellular localization of Proteins Ia and Ib suggests a role for these proteins in the physiology of the synapse.

Isolation and properties of the mixed lipid micelles present in intestinal content during fat digestion in man.

To evaluate better the physicochemical characteristics of human fat digestion, a method was developed which allowed characterization of the bile acid-lipid mixed micelles of the aqueous phase of post-prandial duodenal fluid. Duodenal fluid was collected after a 36-g fat breakfast for two 90-min periods and for 60 min after i.v. cholecystokinin and was ultracentrifuged at 15,400,000 g-min. The aqueous phase was isolated, passed through a 200-nm filter, and the mixed micelles were concentrated by an ultrafiltration procedure using a 1.5-nm filter. The 1.5-nm retentate was eluted from Sepharose 6B columns with 1.5-nm filtrate for both preequilibration fluid and eluent. 1.5-nm filtrate approximated the monomer concentrations. Each sample was assayed for bile acid, fatty acid, lecithin, lysolecithin, protein, cholesterol, and counterions (pH, Na+, K+, Ca2+). Constituents were concentrated only on the 1.5-nm filter. On gel permeation chromatography, coincident peaks were observed for bile acid, fatty acid, lysolecithin, and cholesterol; and were eluted with a Kav range of 0.50-0.68 (corresponding to a Stokes radius of 2.3-3.5 nm). An average density of 1.25 and coincident peaks of bile acid and fatty acid were found for the mixed micelles on sucrose density gradients. The regression lines of micellar fatty acid, lysolecithin, and cholesterol vs. bile acid gave a stoichiometry of 1.4 mol fatty acid, 0.15 mol lysolecithin, and 0.06 mol cholesterol for each mole of bile acid. Mixed micelles were homogeneous in composition. These results provide direct evidence for the existence of the postprandial mixed micelle and describe several of its physicochemical properties.

The role of fs(1)K10 in the localization of the mRNA of the TGF alpha homolog gurken within the Drosophila oocyte.

A critical step in Drosophila dorsoventral patterning is the movement of gurken mRNA from the anterior cortex of the oocyte to the oocyte's anterodorsal corner at stage 8 of oogenesis. Such movement is dependent on fs(1)K10. It has been proposed that fs(1)K10 mediates gurken mRNA movement by down-regulating gurken mRNA levels, thus ensuring that gurken mRNA does not saturate its receptors located in the oocyte's anterodorsal corner. In contradiction to this model, we show here--both genetically and immunocytochemically--that GRK protein levels are lower in the anterodorsal region of fs(1)K10 mutant oocytes than in the anterodorsal region of fs(1)K10+ oocytes. From this and other data, we propose a more direct role for fs(1)K10 in the gurken mRNA localization process.

P element transformation vectors for studying Drosophila melanogaster oogenesis and early embryogenesis.

We have constructed six new P-element-based Drosophila melanogaster transformation vectors that specifically allow for the high-level accumulation of any RNA of interest in the developing egg and pre-blastoderm embryo. Such specificity results, in part, from the inclusion in the vectors of an enhancer active exclusively in nurse cells, the principal providers of RNA to the egg and early embryo. The nurse cell enhancer was derived from the hsp26 heat-shock (HS) gene, but its activity was neither dependent on nor sensitive to HS. In addition to the nurse cell enhancer, two of the vectors contain sequences from the K10 gene that promote the early transfer of RNAs from nurse cells into the oocyte; RNAs that contain the K10 sequence are transferred into the oocyte during the early to middle stages of oogenesis (i.e., during stages 2-9), while RNAs that lack such sequences are stored in nurse cells until stage 11. All of the vectors contain a tsp and a multiple cloning site (MCS) immediately downstream from the hsp26 nurse cell enhancer. In three of the vectors, the MCS is preceded by an ATG start codon. A wild-type copy of the white gene is included in all of the vectors as a selectable marker for transformation. The specificity of the vectors was demonstrated by the analysis of the expression patterns of lacZ derivatives.

Fine structure of squid (Loligo pealei) optic lobe synapses.

Cephalopod optic lobes are a well-known source of cholinergic nerve endings [Dowdall and Whittaker (1973) J. Neurochem, 20, 921-935]. In order to utilize this property for subsequent analyses of cholinergic mechanisms of transmission in the CNS, we describe the ultrastructure of the entire optic lobe of the squid (Loligo pealei) and relate the morphology of synaptosomes to the intact tissue. In the cortex, chemical junctions were found showing two basic forms. The first was an invaginated synapse, appearing only between presynaptic bags and spines which may originate from the trunks of amacrine cells of the outer granule layer. The second was that of a typical synapse, found in almost all layers except the upper portion of the first radial layer. Synapses in the medulla were predominantly of the second type, although a few photoreceptor endings extended to this region as well. The different types of terminals observed in the intact squid optic lobe corresponded to the different types of endings recognized in a synaptosome fraction derived from these lobes. Because of its high content of cholinergic endings and distinct synaptic types, the squid optic lobe may contribute to the elucidation of the mechanisms of cholinergic transmission in the central nervous system. In addition, electrotonic synapses were found between photoreceptor processes in the cortex, as well as other elements of the neuropil.

Transneuronal degeneration in the midbrain central gray following chemical lesions in the ventromedial nucleus: a qualitative and quantitative analysis.

In the preceding experiments with electrolytic lesions of the ventromedial nucleus of the hypothalamus, we showed pre- and postsynaptic degeneration in the midbrain central gray of the rat. The postsynaptic degeneration seen may indicate a transneuronal effect of the ventromedial nucleus on the midbrain central gray. Electrolytic lesions, however, destroy afferent endings and fibers in passage, so that the postsynaptic degeneration seen in the midbrain central gray may be due to retrograde degeneration of midbrain central gray afferents to the ventromedial nucleus or due to degeneration of fibers in passage. In order to distinguish among these possibilities, chemical, i.e. kainic acid and N-methyl aspartate, lesions were made in the ventromedial nucleus and the ultrastructure of the midbrain central gray and cerebral cortex was examined at various intervals following the lesions. Both of these excitotoxins have been shown to destroy neurons, sparing afferent terminals and fibers in passage. Animals receiving kainic acid lesions in the right ventromedial nucleus were allowed to survive for one week, and animals receiving N-methyl aspartate lesions in the right ventromedial nucleus were permitted to survive for four, eight, and 20 days. Midbrain central gray tissue of unlesioned animals served as a control for both kainic acid and N-methyl aspartate lesions. In addition, other control animals received injections of the same amount of N-methyl aspartate in the right parietal cortex and were permitted to survive for four and eight days. For each of the above injection and survival conditions, the left cortex and subdivisions of the midbrain central gray were removed and processed for electron microscopy. Animals receiving ventromedial hypothalamic lesions with both kainic acid and N-methyl aspartate showed signs of pre- and postsynaptic degeneration. A quantitative analysis (General Linear Model Procedure) of degeneration was performed on the cortex and midbrain central gray of animals receiving N-methyl aspartate lesions in the ventromedial nucleus and cortex, and several parameters were measured. Animals receiving ventromedial hypothalamic lesions and surviving for eight and 20 days show significantly higher ratios of degenerating presynaptic elements to total presynaptic elements, degenerating postsynaptic elements to total postsynaptic elements, and degenerating total elements to total elements, in the midbrain central gray than in the cortex. Furthermore, the ratio of degenerating postsynaptic elements to total postsynaptic elements is larger than the other ratios.(ABSTRACT TRUNCATED AT 400 WORDS)

Projections of ventromedial hypothalamic neurons to the midbrain central gray: an ultrastructural study.

Ventromedial hypothalamic projections to the midbrain central gray may be involved in the mediation of female reproductive behavior. In order to demonstrate and examine projections of the ventromedial nucleus in the midbrain central gray, in the rat, electrolytic lesions were placed in the ventromedial nucleus and the midbrain central gray was examined for ultrastructural signs of degeneration at various intervals, i.e. 27.5 h and two, four, six and eight days following the lesions. The fine structure of the midbrain central gray of unlesioned animals was also examined to characterize its normal morphology and to establish a baseline with which to compare the effects of the lesion. In unlesioned animals, the neuropil of midbrain central gray contained several synaptic types, with axodendritic synapses appearing to be the most predominant. Dendrites contained well-preserved microtubules. Synaptic endings contained many clear, round vesicles and some contained dense-cored vesicles as well. Neuropil synapses were both asymmetric and symmetric. Cell bodies were characterized by light cytoplasm and had asymmetric and symmetric synapses on their surface. Following electrolytic lesions in the ventromedial nucleus, various types of degenerative patterns were seen in the midbrain central gray, including electron-dense, flocculent, watery, and pinocytotic degeneration. Specific characteristics of degeneration included shrunken, dense axons and endings, clumped synaptic vesicles, abnormally large, dark mitochondria, membranous sacs of various sizes, swollen endings with reduced numbers of synaptic vesicles, and endings and processes containing large numbers of coated vesicles. Some of these signs were already evident at 27.5 h following the lesion. In addition, degenerating postsynaptic processes and cell bodies were seen in the midbrain central gray. At 27.5 h survival time, degenerating dendritic processes often appeared swollen, devoid of microtubules, and contained enlarged mitochondria. At longer survival times neuronal degeneration was observed in the midbrain central gray, characterized by electron-dense cell bodies and pycnotic nuclei. Both degenerating pre- and postsynaptic elements appeared to be engulfed by glial processes. Control lesions in non-hypothalamic regions which project to the midbrain central gray, i.e. nucleus gigantocellularis and pontine reticular formation and in a non-projecting region, i.e. parietal cortex, were performed.(ABSTRACT TRUNCATED AT 400 WORDS)

Once-daily gentamicin dosing in newborn infants.

OBJECTIVE: We developed a simplified gentamicin dosing protocol for all neonates using a loading dose and once-daily dosing that would have an equal or lower incidence of toxicity and an equal or improved effectiveness compared with a regimen with no loading dose that included use of divided daily dosing. METHODS: All neonatal intensive care unit patients with a postnatal age /=37 weeks; weight, >/=2500 g). One hundred percent of the initial and maintenance peak SDL in term protocol neonates were 5 to 12 micrograms/mL; compared with 84% of the initial and 61% of maintenance peak SDL in the term control group. One hundred percent of the initial and maintenance trough SDL were in the desired range of <2 micrograms/mL in term protocol neonates; compared with 70% of the initial and 94% of maintenance trough SDL in the term control group. No significant differences were found in any SDL in low birth weight neonates (gestational age <37 weeks or weight <2500 g and >1500 g) in the protocol compared with the control group. The very low birth weight (weight <1500 g) protocol neonates had a significantly higher mean initial trough SDL (2.3 +/- 0.7 micrograms/mL vs 1.5 +/- 0.6 micrograms/mL) and a lower incidence of initial trough SDL <2.0 micrograms/mL (30% vs 95%) than very low birth weight neonates in the control group. No differences were seen between groups in incidence of significant rise in serum creatinine or failure of hearing screen. CONCLUSION: A loading dose followed by once-daily dosing was shown to result in SDL in the safe and therapeutic range in all term neonates in this study. In low birth weight neonates, this regimen resulted in peak and trough SDL throughout therapy that were similar to those observed in the control group. Delaying the initiation of maintenance once-daily dosing until 36 to 48 hours after the loading dose would be expected to result in a higher incidence of initial trough SDL in target range for very low birth weight neonates.

Favorable results of neonatal intensive care for very low-birth-weight infants.

From 1961 to 1976, 229 infants with birth weights ranging from 751 to 1,000 gm were admitted to the Stanford University Hospital Intensive Care Nursery. The overall neonatal mortality for these infants was 63% (144/229), and there were ten late deaths. Before 1967, no infant in this group who required mechanical ventilation survived; thereafter, 30% (34/114) of the ventilated patients survived. Of the 75 long-term survivors 60 participated in a high-risk infant follow-up program; these included 23 infants who had received mechanical ventilation. The mean birth weight of these infants was 928 +/- 67 (SD) gm. Seventeen children (28%) had significant morbidity: seven (12%) with severe handicaps and ten (17%) with moderate handicaps. During this same period, seven infants weighing less than 750 gm at birth were also observed. The three infants who had not required ventilatory support thrived; the other four infants had required respirators and were significantly handicapped. More recently, neonatal mortality for infants with birth weights from 751 to 1,000 gm has improved: for 1977 to 1980, it was 28% (33/118). Furthermore, neonatal mortality for ventilated infants in this weight group was 27% (26/95). These data indicate an improved prognosis for very low-birth-weight infants, even with ventilatory support.

Trisomy 22 with congenital diaphragmatic hernia and absence of corpus callosum in a liveborn premature infant.

We report on a liveborn premature male with trisomy 22 who had multiple congenital anomalies, including congenital diaphragmatic hernia and absence of corpus callosum. He died of pulmonary hypoplasia associated with diaphragmatic hernia within 12 hours of age. Chromosome analysis by multiple banding techniques based on lymphocyte culture confirmed that he had trisomy 22. This may be the first report of congenital diaphragmatic hernia and isolated absence of corpus callosum associated with trisomy 22.

Regulation of neuronal nitric oxide synthase mRNA in lordosis-relevant neurons of the ventromedial hypothalamus following short-term estrogen treatment.

Short-term estrogenic regulation of neuronal nitric oxide synthase (nNOS) mRNA in the ventrolateral subdivision of the ventromedial nucleus (VLVMN), an area central to lordosis, was demonstrated using in situ hybridization. Estrogen-treated animals showed a significantly greater signal in the VLVMN, but not the arcuate or supraoptic nuclei, compared to ovariectomized controls. Neuronal NOS may be involved in early actions of estrogen in the VLVMN.

Estrogen affects the expression of Ca2+/calmodulin-dependent protein kinase IV in amygdala.

We examined the effects of long-term estradiol benzoate (E2) treatment on protein expression of Ca2+/calmodulin-dependent protein kinase IV (CaMK IV) in the amygdala of ovariectomized (OVX) rats. Western blot analysis revealed increased protein levels of CaMK IV in the nuclear but not in the membranal or cystolic fraction of total amygdala in E2-treated compared to OVX rats. Significant increases in levels of CaM kinase IV gold immunolabeling were seen in the medial and basomedial, but not in the central or basolateral, amygdala of E2 compared to OVX rats, indicating the neuroanatomical heterogeneity of the E2 effect. These results suggest that CaMK IV may act as a molecular target for actions of estrogen in the amygdala of rats.

Increased immunoreactive erythropoietin in cord plasma and neonatal bilirubin production in normal term infants after labor.

The purpose of this investigation was to compare immunoreactive erythropoietin levels in umbilical cord plasma and neonatal bilirubin production in infants born of normal women who delivered with or without labor. Two groups of term (38 to 42 weeks) singleton pregnancies were compared: 1) those delivered by repeat elective cesarean section without prior labor (N = 17), and 2) those delivered vaginally or by cesarean section after labor (N = 24). None of the infants was asphyxiated, and there was no difference in Apgar scores between the no-labor and labor groups. The cord plasma erythropoietin levels were lower in the infants of women who had repeat elective cesarean section without labor than in those whose mothers had labor before delivery (Wilcoxon rank sum test, P less than .025). The median erythropoietin for the no-labor group was 22.9 mU/mL compared with 38.8 mU/mL for the labor group. The pulmonary excretion rate of carbon monoxide (VeCO), an index of bilirubin production, for the no-labor group was 14.3 +/- 6.2 SD microL/kg per hour compared with 18.0 +/- 4.9 SD microL/kg per hour for the labor group (P less than .05). The hemoglobin concentration for the no-labor group was 16.0 +/- 1.5 SD g/dL compared with 17.7 +/- 2.2 SD g/dL for the labor group (P less than .05). The VeCO correlated with the hemoglobin concentration (N = 32, r = 0.37, P less than .05). The results of the present study suggest that labor is normally associated with increases in the cord plasma erythropoietin level.(ABSTRACT TRUNCATED AT 250 WORDS)

Inositol trisphosphate-induced calcium release in brain microsomes.

The effects of different ionic media on Ca2+ uptake and release in isolated brain microsomes were investigated. KCl (100 mM) provided the best medium for Ca2+ uptake in the presence of ATP. The effect of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ release was examined and was maximum at 0.2 microM. IP3-induced Ca2+ release was dependent on extramicrosomal free Ca2+ concentration with maximal release at 5.0 microM free Ca2+. Replacement of KCl by sucrose or NaCl did not show any response to IP3. Electron microscopy showed that the microsomal fraction consisted of characteristic endoplasmic reticulum-derived vesicular profiles and were free of mitochondria or plasma membrane contamination. Our results support the concept that the endoplasmic reticulum is the target for IP-3 induced mobilization of Ca2+ in the cell.

gamma-Aminobutyric acid and somatostatin immunoreactivity in the visual cortex of normal and dark-reared rats.

Our previous single unit and ultrastructural studies of visual cortex of dark-reared rats revealed an impairment of intracortical inhibitory mechanisms [2,3,5]. Neurochemical changes in inhibitory neurotransmitter and/or neuropeptides, such as gamma-aminobutyric acid (GABA) and somatostatin (SS), respectively, may contribute to the observed alterations. The present study was designed to measure GABA and SS alterations in the visual cortex of the same dark-reared preparation, as possible neurochemical correlates of the changes seen both physiologically and anatomically in previous companion studies. In the present investigation the mean densities of GABA- and SS-immunoreactive neurons in area 17 of dark-reared rats were determined and compared to the density of those of rats reared in normal lighting conditions. Dark-rearing resulted in a significant decrease in the density of GABA-immunoreactive neurons in all cell layers of area 17 of the rat visual cortex; not limited to the thalamorecipient layer(s). There was also a higher mean density of total cortical cells in dark-reared animals. No differences, however, were seen in the density of SS-immunoreactive neurons. The alterations of GABA-immunoreactive neurons in all cortical layers agree with the altered synaptic ultrastructure and physiological responses seen in all cortical layers as reported in our previous companion studies. Taken together, these studies further support the notion of a deficit in intracortical inhibitory mechanisms in the visual cortex of dark-reared adult rats.

NADPH diaphorase activity and nitric oxide synthase immunoreactivity in lordosis-relevant neurons of the ventromedial hypothalamus.

The distribution of the enzymes NADPH diaphorase and nitric oxide synthase in the ventromedial nucleus of the hypothalamus of cycling and ovariectomized/estrogen-treated and control female rats was demonstrated using histochemical and immunocytochemical methods. Serial section analysis of vibratome sections through the entire ventromedial nucleus showed that NADPH diaphorase cellular staining was localized primarily in the ventrolateral subdivision. NADPH diaphorase staining was visible in both neuronal perikarya and processes. Light microscopic immunocytochemistry using affinity-purified polyclonal antibodies to brain nitric oxide synthase revealed a similar pattern of labelling within the ventromedial nucleus and within neurons of the ventrolateral subdivision of the ventromedial nucleus. Control experiments involved omitting the primary antibodies; no labelling was visible under these conditions. Some, but not all, neurons in the ventrolateral subdivision of the ventromedial nucleus contained both NADPH diaphorase and brain nitric oxide synthase as demonstrated by co-localization of these two enzymes in individual cells of this area. That NADPH diaphorase and brain nitric oxide synthase were found in estrogen-binding cells was shown by co-localization of NADPH diaphorase and estrogen receptor and brain nitric oxide synthase and estrogen receptor at the light and ultrastructural levels, respectively. Our studies suggest that brain nitric oxide synthase is present and may be subject to estrogenic influences in lordosis-relevant neurons in the ventrolateral subdivision of the ventromedial nucleus. The hypothalamus is a primary subcortical regulatory center controlling sympathetic function. Therefore, not only is nitric oxide likely to be important for reproductive behavior, but also for the regulation of responses to emotional stress and other autonomic functions.