Immunocytochemical methods, both light and electron microscopic, were used to identify the cellular and subcellular locations of octadecaneuropeptide-like immunoreactivity (ODN-LI) in rat brains serially sectioned in total. ODN-LI... more
Immunocytochemical methods, both light and electron microscopic, were used to identify the cellular and subcellular locations of octadecaneuropeptide-like immunoreactivity (ODN-LI) in rat brains serially sectioned in total. ODN-LI includes a newly discovered family of rat brain neuropeptides that are processing products of a common endogenous neuropeptide precursor, diazepam binding inhibitor (DBI). The members of this neuropeptide family have been shown to displace benzodiazepines and beta carbolines from their specific recognition sites located on the allosteric modulatory centers of GABAA receptors. We have previously examined the distribution of DBI-LI in rat brain. The anti-ODN antiserum used in this study does not cross-react with rat DBI, and thus allows a distinct analysis of ODN-LI as opposed to DBI-LI, in rat brain. Neuronal perikarya with ODN-LI were located in many brain nuclei, such as the pontine n., reticular thalamic n., subgeniculate n., supraoptic n. and suprachias...
Research Interests: Neuroscience, Chemistry, Immunohistochemistry, Transmission Electron Microscopy, Medicine, and 14 moreHippocampus, Brain, Neuropeptides, Animals, Spinal Cord, Male, Colchicine, Rats, Olfactory Bulb, Subcellular Fractions, Neurosciences, Neuropeptide, Circumventricular Organs, and Chemical Neuroanatomy
Exposure of primary cultures of rat cerebellar granule cells to specific antagonists of the N-methyl-D-aspartate (NMDA)-selective glutamate receptor reduces the steady state levels of mRNAs encoding various gamma-aminobutyric acidA... more
Exposure of primary cultures of rat cerebellar granule cells to specific antagonists of the N-methyl-D-aspartate (NMDA)-selective glutamate receptor reduces the steady state levels of mRNAs encoding various gamma-aminobutyric acidA (GABAA) receptor subunits. These neurons are glutamatergic and require a depolarizing concentration of K+ (25 mM) for optimal development and survival. When the neuronal differentiation rate is retarded by lowering of the extracellular [K+] (to 12.5 mM), a persistent stimulation of the same glutamate receptors with nonneurotoxic doses of NMDA increases the expression of these GABAA receptor subunits. This suggests that the lowered K+ concentration reduces neuronal depolarization and the consequent release of glutamate from the cells. These results show that the neuronal content of selected GABAA receptor subunit mRNAs is optimized by certain levels of glutamate in the culture medium, suggesting a neurotrophic action of this neurotransmitter at certain dev...
Research Interests: Biology, Molecular Pharmacology, Medicine, Glutamate receptors, Animals, and 15 morePolymerase Chain Reaction, Rats, Receptor, Base Sequence, Neurosciences, Glutamic Acid, Gene Expression Regulation, Muscimol, Glutamate Receptor, Down-Regulation, Depolarization, Molecular Sequence Data, NMDA receptor, GABAA receptor, and Pharmacology and pharmaceutical sciences
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Previously, we have shown that erbB-3 expression is restricted to the ensheathing cells of the olfactory nerve layer, while erbB-4 is found in the periglomerular and mitral/tufted cells of the olfactory bulb and in cells coming out from... more
Previously, we have shown that erbB-3 expression is restricted to the ensheathing cells of the olfactory nerve layer, while erbB-4 is found in the periglomerular and mitral/tufted cells of the olfactory bulb and in cells coming out from the rostral migratory stream of the subependymal layer. In the present work, we have treated adult mice with zinc sulfate intranasal irrigation and analyzed erbB-3 and erbB-4 expression in the deafferented olfactory bulb. Following treatment, olfactory axons undergo degeneration, as indicated by the loss of OMP expression in the deafferented olfactory bulb. The thickness of the olfactory nerve layer is reduced, but the specific intensity of erbB-3 labeling in the remaining olfactory nerve layer is increased with respect to control. Interestingly, following deafferentation, erbB-4 immunoreactivity decreases specifically in cell types that normally make synaptic contacts with primary olfactory neurons in the glomeruli, i.e. periglomerular and mitral/tu...
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Research Interests: Psychology, Cognitive Science, Stem Cells, Neurogenesis, Biology, and 15 moreStem Cell, Medicine, Transgenic Mice, Cell Differentiation, Mice, Animals, Green Fluorescent Protein, Phenotype, Isoenzymes, Olfactory Bulb, Transgenic Mouse Technology, Subventricular Zone, Neurosciences, Glutamate decarboxylase, and Gamma-Aminobutyric Acid
Background A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium... more
Background A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium fluctuations play central roles in neuronal motility. Stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation. In this work we analyzed the potential interactions between the NRG1/ErbB4 system and NMDARs in the ST14A migratory process as well as its calcium dependence. Results RT-PCR studies have shown that both native ST14A cells (non-expressing ErbB4), as well as ErbB4-transfected cells express low levels of a restricted number of NMDAR subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form Ca2+ channels characterized by low Mg2+-sensitivity and low Ca2+-permeability, generating small, long-lasting currents. Ca2+-imaging experiments showed slow [Ca2+]i incr...
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The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering... more
The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron’s axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search i...
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In the past thirty years, cytochemical methods have allowed neuroscientists to identify and localize neuroactive molecules (neurotransmitters and neuropeptides), their receptors and their synthetic enzymes, and have advanced the... more
In the past thirty years, cytochemical methods have allowed neuroscientists to identify and localize neuroactive molecules (neurotransmitters and neuropeptides), their receptors and their synthetic enzymes, and have advanced the understanding of many neuronal functions. Classic methods (histochemical and immunohistochemical techniques) have been used extensively to draw neurochemical maps in adult and developing nervous systems. As a consequence, many neuroactive molecules have become specific biochemical markers for neuronal systems. Double labelling techniques have greatly contributed to the discovery of the coexistence of two or more chemical compounds in the same cell. The in situ hybridization technique has recently become a productive addition to the tools available to the neuroscientist, especially when combined with immunocytochemistry to correlate mRNAs and protein expression. Even today, innovative roles for neurocytochemistry continue to be found. The newest approaches ba...
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The endocannabinoid system (ECS) has a well-documented pivotal role in the control of mammalian reproductive functions, by acting at multiple levels, that is, central (CNS) and local (gonads) levels. Since studies performed in animal... more
The endocannabinoid system (ECS) has a well-documented pivotal role in the control of mammalian reproductive functions, by acting at multiple levels, that is, central (CNS) and local (gonads) levels. Since studies performed in animal models other than mammals might provide further insight into the biology of these signalling molecules, in the present paper we review the comparative data pointing toward the endocannabinoid involvement in the reproductive control of non-mammalian vertebrates, focussing in particular on the central regulation of teleost and amphibian reproduction. The morphofunctional distribution of brain cannabinoid receptors will be discussed in relation to other crucial signalling molecules involved in the control of reproductive functions, such as GnRH, dopamine, aromatase, and pituitary gonadotropins.
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Research Interests: Neuroscience, Psychology, Electron Microscopy, Localization, Immunohistochemistry, and 15 moreHeterogeneity, Glutamate, Glutamate receptors, Animals, Polymerase Chain Reaction, Rat, NMDA Receptors, Olfactory Bulb, Neurosciences, Postsynaptic Density, AMPA receptors, Glutamate Receptor, Functional Properties, NMDA receptor, and In Vitro Techniques
Olfactory neuroepithelial (OE) cells were dissociated from late stage embryonic mice and analysed for carnosine expression. The yield of carnosine neurones was twice as high when the OE cells were seeded along with the olfactory bulb... more
Olfactory neuroepithelial (OE) cells were dissociated from late stage embryonic mice and analysed for carnosine expression. The yield of carnosine neurones was twice as high when the OE cells were seeded along with the olfactory bulb cells. Carnosine neurones resulted from both in vitro survival and neurogenesis, and were associated with clusters of underlying flat cells immunopositive for keratin. Our results demonstrate that olfactory neurones expressing their neurotransmitter carnosine can be studied in culture, and the close association with keratin-immunopositive basal cells suggests that they are dependent on these cells for survival and/or differentiation.
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We studied the expression and distribution of the polypeptide diazepam binding inhibitor (DBI) in rat peripheral organs by immunocytochemistry, radioimmunoassay, Northern blot analysis and binding assay. Variable amounts of the DBI... more
We studied the expression and distribution of the polypeptide diazepam binding inhibitor (DBI) in rat peripheral organs by immunocytochemistry, radioimmunoassay, Northern blot analysis and binding assay. Variable amounts of the DBI peptide and DBI mRNA were found in all the tissues examined (liver, duodenum, testis, kidney, adrenal gland, heart, ovary, lung, skeletal muscle and spleen), with the highest level of expression in liver (220 pmol of DBI/mg protein) and the lowest in spleen (11 pmol of DBI/mg protein). A good correlation between DBI-like immunoreactivity (DBI-LI) and mRNA content was found in all tissues except the heart. The immunohistochemical analysis revealed discrete localization of DBI-LI in cell types with specialized functions: for example, the highest DBI-LI content was found in steroid-producing cells (glomerulosa and fasciculata cells of adrenal cortex, Leydig cells of testis); lower DBI-LI immunostaining was found in epithelial cells specialized for water and electrolyte transport (intestinal mucosa, distal convoluted tubules of kidney). Hepatic cells contained moderate immunoreactivity however the total content of DBI in liver is relatively high and is due to the diffuse presence of DBI in every hepatocyte. Cells with high expression of DBI have been shown to contain a high density of mitochondrial benzodiazepine (BZ) binding sites. This observation led us to perform a competitive binding assay between DBI and [3H]PK11195 (a ligand for the mitochondrial BZ binding sites) on mitochondrial membranes of adrenal cortical cells. In this experiment, DBI yielded an apparent competitive inhibition of the binding of PK11195 to the BZ binding sites. Our data support a possible role for DBI as endogenous regulator of intracellular metabolic functions, such as steroidogenesis, via the mitochondrial BZ receptors.
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Research Interests: Gene regulation, Gene expression, Multidisciplinary, Neuropeptides, Beta decay, and 15 moreAnimals, Nucleic acid hybridization, Central Nervous System, Arcuate Nucleus, Human Brain, Enzyme, High Density Concrete, Nervous System, Membrane Protein, Brain Chemistry, Binding Site, Nucleic Acid, Germinal Center, Gene Expression Regulation, and High density
Methamphetamine produces locomotor activation and typical stereotyped motor patterns, which are commonly related with increased catecholamine activity within the basal ganglia, including the dorsal and ventral striatum. Since the... more
Methamphetamine produces locomotor activation and typical stereotyped motor patterns, which are commonly related with increased catecholamine activity within the basal ganglia, including the dorsal and ventral striatum. Since the cerebellum is critical for movement control, and for learning of motor patterns, we hypothesized that cerebellar catecholamines might be a target of methamphetamine. To test this experimental hypothesis we injected methamphetamine into C57 Black mice at the doses of 5 mg/kg two or three times, 2 h apart. This dosing regimen is known to be toxic for striatal dopamine terminals. However, we found that in the cerebellum, methamphetamine increased the expression of the primary transcript of the tyrosine hydroxylase (TH) gene, followed by an increased expression of the TH protein. Increased TH was localized within Purkinje cells, where methamphetamine increased the number of TH-immunogold particles, and produced a change in the distribution of the enzyme by increasing the cytoplasmic percentage. Increased TH expression was accompanied by a slight increase in noradrenaline content. This effect was highly site-specific for the cortex of posterior vermal lobules, while only slight effects were detectable in the hemispheres. The present data indicate that the cerebellum does represent a target of methamphetamine, which produces specific and fine alterations of the catecholamine system involving synthesis, amount, and compartmentalization of TH as well as increased noradrenaline levels. This may be relevant for motor alterations induced by methamphetamine. In line with this, inherited cerebellar movement disorders in various animal species including humans are associated with increased TH immunoreactivity within intrinsic neurons of the same lobules of the cerebellar cortex.
Research Interests: Neuroscience, Psychology, Gene expression, Dopamine, Mice, and 15 moreAnimals, Methamphetamine, Male, Polymerase Chain Reaction, Endoplasmic Reticulum, Neurons, Enzyme, Norepinephrine, Analysis of Variance, Basal ganglia, Locomotor Activity, Cerebellar Cortex, Neurosciences, Movement Disorder, and Purkinje cell
The distribution of diazepam binding inhibitor (DBI), a multi-function peptide which has recently been discovered, was studied in the rat and human central nervous system and in peripheral organs of the rat by light and electron... more
The distribution of diazepam binding inhibitor (DBI), a multi-function peptide which has recently been discovered, was studied in the rat and human central nervous system and in peripheral organs of the rat by light and electron microscopical immunohistochemistry. In the central nervous system, DBI-LI was localized in many glial cells and glial tumors, and in some neurons. In the periphery, DBI-LI was found in many tissues but it was expressed selectively in specialized cell types. Intense DBI-LI was observed in some endocrine, steroid-producing cells such as glomerular cells of the adrenal gland and Leydig cells of the of the testis. Different types of epithelial cells, for instance distal convoluted tabular cells of the kidney and mucosal cells of the small intestine, displayed moderate DBI-LI. Some supporting cells, such as Schwann cells and Sertoli cells, were also immunopositive. The frequent localization of DBI in cells, also known to contain large amounts of mitochondrial benzodiazepine receptors, indicates that DBI may play an important role as an endogenous regulator of intracellular metabolic functions via the mitochondrial benzodiazepine receptor.
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The present study has analyzed the effect of progesterone and its derivatives (dihydroprogesterone and tetrahydroprogesterone) on the gene expression of the peripheral myelin protein 22 utilizing in vivo and in vitro models. The data... more
The present study has analyzed the effect of progesterone and its derivatives (dihydroprogesterone and tetrahydroprogesterone) on the gene expression of the peripheral myelin protein 22 utilizing in vivo and in vitro models. The data obtained indicate that tetrahydroprogesterone is able to stimulate the gene expression of peripheral myelin protein 22 both in vivo (in adult but not in old animals) and in Schwann cell cultures. An effect of this steroid, which is known to interact with the GABA(A) receptor, would not be surprising, since in the present study we show the presence in Schwann cells and in the sciatic nerve of the messengers for several subunits (alpha2, alpha3, beta1, beta2, and beta3) of the GABA(A) receptor. An effect of tetrahydroprogesterone is also evident on the gene expression of another myelin protein, the peripheral myelin protein zero. However, in this case also dihydroprogesterone, which is able to bind the progesterone receptor, is involved, both in old and adult animals, in the stimulation of messengers levels of this myelin protein. In conclusion, the present data show that the gene expression of two important peripheral myelin proteins can be influenced by progesterone derivatives. The hypothesis has been put forward that part of their effects might occur not through the classical progesterone receptor, but rather via an interaction with the GABA(A) receptor.
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In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small... more
In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes increasing amounts of acetylcholine (ACh) from the third day in vitro (DIV), which reaches a plateau around the 10 day of culture. Muscarinic cholinergic receptors (mAChR), measured by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB), are detectable from the fifth DIV and increase linearly during the time of culture. At the twelfth DIV, the density of mAChRs (approximately 600 fmol/mg protein) is comparable to the density of mAChR in adult rat cortex. These receptors are coupled to second messenger systems, since muscarinic agonists inhibit adenylate cyclase activity and stimulate phosphoinositide breakdown with efficacies and potencies similar to those found in adult rat cortex. Moreover, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, we were able to demonstrate the presence of the m1, m3, and m4 mAChR subtype mRNAs in this neuronal culture at 12 DIV. Our data suggest that corticostriatal neuronal cultures develop in vitro ACh-synthesizing neurons and functionally active cholinergic receptors. This therefore makes them ideally suited to study the development and properties of brain mAChR subtypes.
Research Interests: Cognitive Science, Kinetics, Molecular Neuroscience, Molecular, Cerebral Cortex, and 14 moreAnimals, Polymerase Chain Reaction, Acetylcholine, Rat Brain, Neurons, Rats, Radioligand Assay, Primary Culture, Muscarinic Receptor, Second Messengers, Neurosciences, Choline Acetyltransferase, Corpus striatum, and reverse transcriptase polymerase chain reaction
Glutamate (Glu) released by olfactory nerve axons acts on postsynaptic ionotropic and metabotropic glutamate receptors expressed by principal neurones and interneurones of the olfactory bulb (OB). Using ZnSO4 lesioning of the rat... more
Glutamate (Glu) released by olfactory nerve axons acts on postsynaptic ionotropic and metabotropic glutamate receptors expressed by principal neurones and interneurones of the olfactory bulb (OB). Using ZnSO4 lesioning of the rat olfactory mucosa and semiquantitative RT-PCR, we examined the effect of removal of the glutamatergic input to the OB on the expression of mGluR1a, mGluR1b and GluR1 mRNAs. Two days after lesioning, mGluR1a mRNA levels in OB increased by 45%. At this time, the expression of tyrosine hydroxylase (TH) mRNA, which is strictly dependent on olfactory nerve input, was still unchanged. In contrast, 16 days after lesioning, deafferented OB exhibited a decrease in both mGluR1a (-30%) and TH (-40%) mRNAs. GluR1 and mGluR1b mRNA levels were not affected at either time point. These results suggest that alterations in glutamatergic input to OB selectively modulate the expression of the mGluR1 splicing form possessing a longer C-terminal domain.