1990 ACNP Past-President's Lecture. Where do we go from here.
Explore the source record for details and available documents.
Psychopharmacology↗
Biomedical subjects
F E Bloom
Publications and source records attributed to F E Bloom.
Molecular approaches to genes of the CNS.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Oxytocin neurons in the rat hypothalamus exhibit c-fos immunoreactivity upon osmotic stress.
In order to evaluate the responses to osmotic stress of oxytocinergic neurons in vivo, we have studied oxytocin (OXY) and c-fos protein expression in the brain by means of double-immunostaining. C-fos immunoreactivity was detected in a subset of OXY neurons, as well as in other neurons non-immunoreactive for OXY, as early as 90 min after intraperitoneal injection of a hypertonic saline solution. C-fos expression was found in approx. 70% of OXY-immunoreactive neurons in the supraoptic (SON), lateral subcommisural (LSN) and paraventricular (PVN) nuclei, and not in OXY neurons in other hypothalamic areas. The expression of c-fos may be used as a means to map the circuitry by which osmotic stimulation activates OXY-containing neurons, and thus provide further insights into the functions with which OXY may be associated.
Animals↗
Parvalbumin immunoreactivity in the rat retina.
The distribution of the Ca2+ binding protein parvalbumin was studied in the rat retina with immunocytochemistry using a mouse monoclonal antibody. Specific parvalbumin immunoreactivity was identified within a subpopulation of ganglion cells and a subpopulation of amacrine cells. The topographical data provided by the present study may serve as a basis for a functional characterization of parvalbumin's role in the nervous system.
Animals↗
Subtractive cDNA cloning of RC3, a rodent cortex-enriched mRNA encoding a novel 78 residue protein.
A rodent cortex-enriched mRNA, RC3, was identified by screening a rat brain cDNA library with a cortex-minus-cerebellum subtracted cDNA probe. Conceptual translation of RC3 cDNA sequences indicates that the rat and mouse mRNAs encode identical, novel 78 amino acid proteins. The RC3 protein amino terminus contains a cysteine-rich domain similar to those found in snake venom neurotoxins; the carboxyl terminus contains a collagen-like motif that may function in the assembly of RC3 subunits into a multimeric protein. Western blot experiments with an antiserum to a synthetic peptide corresponding to 27 residues of the 78 residue sequence identify an immunoreactive polypeptide with 18 kDa gel mobility that is likely to correspond to the RC3 protein. Northern blot analysis and in situ hybridization experiments show that RC3 mRNA is highly enriched in rat brain, with restricted expression in neuronal subsets primarily in the cortex, striatum, and hippocampus as well as certain nuclei within the thalamus, hypothalamus, the olfactory bulb.
Amino Acid Sequence↗
Free-choice responding for ethanol versus water in alcohol preferring (P) and unselected Wistar rats is differentially modified by naloxone, bromocriptine, and methysergide.
The role of opioids, dopamine and serotonin in ethanol (EtOH) reward and preference was investigated in non-deprived, Alcohol-Preferring (P), and genetically heterogenous Wistar rats. Operant responding for ethanol was initiated using sweet-solution substitution procedures. The rats were then trained in 30-min daily sessions to respond for ethanol (10% v/v) versus water under a two-lever, free-choice contingency. All testing was conducted in the absence of water and food deprivation or addition of sweeteners to the ethanol drinking solution. Rats of both strains developed stable preferences in responding for ethanol over water and consumed ethanol at quantities sufficient to produce pharmacologically relevant mean blood alcohol concentrations (P-Rats: 98 +/- 19.6 mg%; unselected Wistars: 41.7 +/- 8.5 mg%). In P-rats, systemic naloxone (NAL; 0.125, 0.25 and 0.5 mg/kg) pretreatments resulted in a dose-dependent suppression in responding for both ethanol and water, but did not alter ethanol preference (expressed as percent ethanol of total intake). In contrast, bromocriptine (BRO; 1.0, 2.0 and 4.0 mg/kg) produced a significant, dose-dependent shift in preference from ethanol toward water by inhibiting responding for ethanol while enhancing water consumption. In unselected Wistar rats, NAL and BRO treatments produced changes in ethanol preference patterns similar to those observed in P-rats. However, compared to P-rats, these changes were smaller and not consistently dose dependent. No changes in ethanol preference and water or ethanol intake were observed with methysergide (MET; 2.5, 5.0, 10.0 mg/kg) in either strain of rat. Together, the results suggest a possible involvement of dopaminergic mechanisms in the reinforcing properties of ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcohol Drinking↗
In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes.
We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding reserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electron-microscopical level.
Animals↗
Identification of two nerve growth factor-induced polypeptides in PC12 cells.
We have identified two nerve growth factor (NGF)-induced polypeptides (Mr 80,000 and 90,000) in PC12 cells that are heat stable and not sulfated. Indirect immunofluorescence localization of these polypeptides in NGF-treated PC12 cells reveals a punctate pattern concentrated in neurites. Immunoperoxidase staining of rat tissue shows that these polypeptides are found throughout the brain in selected subsets of neurons and are absent in the pituitary or adrenal medulla. Several of these characteristics are similar to a recently described NGF-induced secretory protein (VGF8a), which has sequence similarities to secretogranins. The property of NGF inducibility and the distribution of these polypeptides within rat tissues are both novel features for secretogranin proteins.
Adrenal Gland Neoplasms↗
mRNA coding for oxytocin is present in axons of the hypothalamo-neurohypophysial tract.
Neuronal mRNA is thought to be restricted to perikaryal and dendritic compartments containing rough endoplasmic reticulum. We have used both in situ hybridization and DNA polymerase chain reaction methods to determine the precise intracellular distribution of oxytocin mRNA. Using light- and electron-microscopic detection of in situ hybridization with 5'-bromo-2'-deoxyuridine-labeled oligonucleotide probes, we found oxytocin mRNA in axons and Herring bodies in the lateral and ventral hypothalamus, the median eminence, and the posterior lobe of the pituitary in postpartum lactating rats. Southern blot analysis of the amplification products confirmed the presence of oxytocin mRNA in all three tissue samples. The present findings indicate that oxytocin mRNA can be transported axonally. Such transport could reflect an adventitious compartmentalization or a functional storage in Herring bodies for subsequent secretion.
Animals↗
Funding health sciences research: a strategy to restore balance.
Explore the source record for details and available documents.
Financing, Government↗
1B1075: a brain- and pituitary-specific mRNA that encodes a novel chromogranin/secretogranin-like component of intracellular vesicles.
The rat 1B1075 mRNA encodes a 533-residue novel chromogranin/secretogranin-like acidic protein that contains an apparent secretion signal, several pairs of tandem basic residues, and internally repeated sequence elements. 1B1075 transcripts are detected, by blotting and in situ hybridization, at the highest levels in the neocortex, hippocampus, cerebellar cortex, selected pontine and diencephalic nuclei, and presumptive pituitary corticotrophs, at lower levels in specific nuclei in most other brain regions, but in none of several other tissues. Utilizing antisera to several nonoverlapping synthetic peptide fragments of the predicted protein sequence, we detect a brain- and pituitary-specific 57-kDa protein in cellular processes and fiber tracts, generally consistent with axonal transport from the cell bodies identified by in situ hybridization. Ultrastructural studies demonstrate that this protein is a component of intraneuronal vesicles in axons and vesicle-like structures in dendrites. Based on these data, we suggest the name Secretogranin III for the 1B1075 gene product. In related collaborative studies, a mouse deleted for the 1B1075-homologous gene has been produced that should allow assessment of its physiological role.
Aging↗
Nucleus accumbens as a substrate for the aversive stimulus effects of opiate withdrawal.
Previous studies from our laboratory using methylnaloxonium, a hydrophilic antagonist, showed that opiate receptors in the region of the nucleus accumbens are important for the acute reinforcing effects of heroin in non-dependent rats. A similar increased sensitivity to the response disruptive effects of intracerebrally injected methylnaloxonium in opiate dependent rats was observed in a fixed-ratio (FR) baseline of operant behaviors. These results suggest that the same opiate receptors in the region of the nucleus accumbens important for the positive reinforcing stimulus properties of opiates may also be responsible for the response disruptive, aversive stimulus properties of opiate withdrawal. These results also suggest that the neural substrates of some aspects of dependence may be partly related to those of the reinforcing effects of opiates. In particular, it is hypothesized that "euphoria" and "dysphoria" induced by opiates may reflect opponent motivational processes operating at a cellular level within the nucleus accumbens.
Animals↗
Cellular localization of 1B236/myelin-associated glycoprotein mRNA during rat brain development.
The protein encoded by the rat brain cDNA 1B236 has been shown to be identical to myelin-associated glycoprotein (MAG). In this report we describe the cellular distribution of 1B236/MAG mRNA transcripts in rat brain by using in situ hybridization. At postnatal day 20, large numbers of 1B236/MAG mRNA-containing oligodendrocytes are concentrated in myelinated fiber tracts and throughout gray matter regions. The presence of high levels of 1B236/MAG mRNA within oligodendrocytes at postnatal day 20 is consistent with the proposed role of MAG in formation of the myelin sheath during development. In the adult brain, our results suggest that not only is 1B236/MAG mRNA expressed at reduced levels within oligodendrocytes but also 1B236/MAG or a 1B236/MAG-like mRNA is present within neurons. This localization is consistent with the results of previous immunocytochemical studies using antibodies against the 1B236/MAG mRNA with different cell-type-specific patterns of expression suggests that oligodendrocytes and neurons employ different mechanisms for regulating the same gene. Thus, different cell types may use a similar cell adhesion molecule both during myelinogenesis and in the mature nervous system.
Animals↗
Regulation of neuronal oxytocin mRNA by ovarian steroids in the mature and developing hypothalamus.
We have examined the changes in neuronal expression of oxytocin mRNA in the perinatal and mature female rat as a function of endogenous gonadal steroids. Northern blot analysis demonstrated a significant developmental increase in the abundance of oxytocin mRNA in the female brain concomitant with puberty. Ovariectomy of adult females decreased total brain oxytocin mRNA to significantly lower levels. In contrast, lactating mothers had increased levels of neuronal oxytocin mRNA. In situ hybridization analysis of neuronal oxytocin mRNA in adolescent, mature virgin, and ovariectomized virgin female brains demonstrated that the location and number of neurons expressing oxytocin mRNA was unchanged and that total brain oxytocin mRNA differences were attributable to amounts expressed per neuron. Differences in mRNA abundance were noted in oxytocin neurons throughout the hypothalamus, including those known to project as magnocellular neurons to the neurohypophysis and those of parvocellular origin thought to make wholly intracerebral connections. This developmental and dynamic regulation of oxytocin mRNA levels during gonadal maturation may coordinate the peripheral and central effects of this peptide on the reproductive biology of the female rat.
Aging↗
The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.
cDNA clones of a neuronal-specific mRNA encoding a novel 25-kD synaptosomal protein, SNAP-25, that is widely, but differentially expressed by diverse neuronal subpopulations of the mammalian nervous system have been isolated and characterized. The sequence of the SNAP-25 cDNA revealed a single open reading frame that encodes a primary translation product of 206 amino acids. Antisera elicited against a 12-amino acid peptide, corresponding to the carboxy-terminal residues of the predicted polypeptide sequence, recognized a single 25-kD protein that is associated with synaptosomal fractions of hippocampal preparations. The SNAP-25 polypeptide remains associated with synaptosomal membrane components after hypoosmotic lysis and is released by nonionic detergent but not high salt extraction. Although the SNAP-25 polypeptide lacks a hydrophobic stretch of residues compatible with a transmembrane region, the amino terminus may form an amphiphilic helix that may facilitate alignment with membranes. The predicted amino acid sequence also includes a cluster of four closely spaced cysteine residues, similar to the metal binding domains of some metalloproteins, suggesting that the SNAP-25 polypeptide may have the potential to coordinately bind metal ions. Consistent with the protein fractionation, light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus. The mRNA was found to be enriched within neurons of the neocortex, hippocampus, piriform cortex, anterior thalamic nuclei, pontine nuclei, and granule cells of the cerebellum. The distribution of the SNAP-25 mRNA and the association of the protein with presynaptic elements suggest that SNAP-25 may play an important role in the synaptic function of specific neuronal systems.
Amino Acid Sequence↗
Cellular and molecular mechanisms of drug dependence.
The molecular and cellular actions of three classes of abused drugs--opiates, psychostimulants, and ethanol--are reviewed in the context of behavioral studies of drug dependence. The immediate effects of drugs are compared to those observed after long-term exposure. A neurobiological basis for drug dependence is proposed from the linkage between the cellular and behavioral effects of these drugs.
Animals↗
Immunohistochemical analysis of the development of somatostatin in the reeler neocortex.
The development of somatostatin (SS) neurons and fibers has been examined in the dorsolateral cortex of the mouse mutant reeler. Immunohistochemistry was performed using antisera directed primarily against SS28 or SS28(1-12). In the normal mouse at postnatal day 5 (P5), somatostatin (SS) neurons are concentrated in the ventral half of the cortex, in the developing layers V and VI. In the reeler mutant, SS neurons are scattered throughout the radial extent of the cortex, being concentrated in the dorsal half of the cortex in the polymorphic and large pyramidal cell layers. By P20, when the adult pattern of SS neuron distribution is evident in the normal mouse cortex, the distribution of similar neurons in the reeler appears inverted: immunoreactive neurons are concentrated in the dorsal half of the cortex. Immunoreactive fiber distribution follows a developmental pattern similar to that observed for SS neurons. At P5, SS fibers are most dense in layer I and V-VI of the normal cortex, while in the reeler, fibers are predominant in the polymorphic and large pyramidal cell layers. By P10, many fewer immunoreactive fibers can be detected in either normal or reeler mice than at P5. Nevertheless, while SS fibers in the normal cortex are most dense in layers I and V-VI, the reeler cortex exhibits little laminar heterogeneity in the distribution of these fibers. Thus, the SS fiber distribution appears less organized in the reeler cortex. These results suggest that whatever the nature of the genetic alteration resulting in cortical cellular developmental malposition in the reeler, SS cells and fibers, representing a completely intrinsic neocortical cellular system, behave as do all other cortical elements.
Aging↗
Synaptic organization of serotonin-immunoreactive fibers in primary visual cortex of the macaque monkey.
The macaque neocortex is very densely innervated by serotonin-containing fibers. The highest density of these fibers is in primary sensory regions such as the primary visual cortex. By using an antibody against serotonin, we analyzed the distribution and morphology of serotonin-immunoreactive fibers and synapses in the primary visual cortex of the adult cynomolgus monkey. In addition, we quantified the laminar distribution of labeled varicosities and the distances between varicosities in single fibers. While serotonin-immunoreactive fibers are found in all cortical layers, at least three bands of heightened density of innervation were readily recognized that were coincident with 1) layer IIIB to IVC alpha, 2) layer VA, and 3) layer VIB. Layer IVC alpha of area 17 contained more varicosities per unit area than any other sublayer. There was a high degree of variability in the intervaricosity distances along single fibers; more than half were longer than 10 microns. At the electron microscopic level, synaptic contacts were also observed throughout the entire thickness of area 17, with the highest frequency in layer IV. The labeled varicosities were packed with electron-lucent synaptic vesicles and formed synaptic complexes with small, but conspicuous, post-synaptic densities. Dendritic shafts were the most common postsynaptic target of the labeled synapses. Among these characteristically slender post-synaptic shafts, profiles with structural features of both spiny and smooth dendrites were observed. The small diameter of most of the postsynaptic dendrites indicated that distal dendrites were preferentially contacted by serotonin-immunoreactive varicosities. Although direct identification of the postsynaptic neurons will be required for complete characterization of this circuitry, the distribution of serotonin-immunoreactive varicosities suggests that serotoninergic interactions in the primary visual cortex of the cynomolgus monkey are directed predominantly at the distal dendrites of granular and infragranular neurons rather than at targets in the supragranular layers.
Animals↗