Biomedical subjects

P Bovolin

Publications and source records attributed to P Bovolin.

At least 19 recordsLinked to original sources

PubMed2004

GABAergic phenotypic differentiation of a subpopulation of subventricular derived migrating progenitors.

Olfactory bulb interneurons are continuously generated throughout development and in adulthood. These neurons are born in the subventricular zone (SVZ) and migrate along the rostral migratory stream into the olfactory bulb where they differentiate into local interneurons. To investigate the differentiation of GABAergic interneurons of the olfactory bulb we used a transgenic mouse which expresses green fluorescent protein (GFP) under the control of the glutamic acid decarboxylase 65 kDa (GAD65) promoter. During development and in adulthood GFP was expressed by cells in the SVZ and along the entire length of its rostral extension including the distal portion within the olfactory bulb. The occurrence of GAD65 mRNA in these zones was confirmed by PCR analysis on microdissected regions along the pathway. Polysialic acid neural cell adhesion molecule, a marker of migrating neuroblasts in adults, was coexpressed by the majority of the GFP-positive SVZ-derived progenitor cells. Cell tracer injections into the SVZ indicated that approximately 26% of migrating progenitor cells expressed GFP. These data show the early differentiation of migrating SVZ-derived progenitors into a GAD65-GFP-positive phenotype. These cells could represent a restricted lineage giving rise to GAD65-positive GABAergic olfactory bulb interneurons.

Animals↗

PubMed2001

ErbB-4 and neuregulin expression in the adult mouse olfactory bulb after peripheral denervation.

ErbB-4 is expressed by the periglomerular and the mitral/tufted cells of the adult mouse olfactory bulb (OB) and in the present work we tested whether this expression is regulated by the olfactory nerve input to the OB. Reversible zinc sulphate lesions of the olfactory mucosa were made in adult mice and the deafferented OB analysed by immunohistochemistry, Western blotting and semiquantitative RT-PCR. Following deafferentation, the expression of erbB-4, erbB-2 and neuregulin-1 (NRG-1) mRNAs in the OB was altered. At early stages (7-14 days) after lesion the levels of expression of olfactory marker protein (OMP), tyrosine hydroxylase (TH), erbB-4 and NRG-1 mRNAs were decreased, whilst expression of erbB-2 increased and that of NRG-2 was not significantly altered. We observed at least two distinct time courses for these expression changes. The lowest amounts of mRNA for erbB-4 and NRG-1 were observed at day 7 after lesion, whilst mRNAs for TH and OMP were lowest at day 14. At day 28 after the lesion, when olfactory receptor neuron axons had reinnervated the olfactory bulb, the expression levels of OMP, TH, erbB-2, erbB-4 and NRG-1 were identical to control values. These results indicate that the expression of erbB-4 mRNA and protein in periglomerular and mitral cells is controlled by peripheral olfactory innervation. The tight correlation in NRG-1 and erbB-4 expression levels also suggests a possible functional link that deserves further exploration.

Animals↗

PubMed1999

Progesterone derivatives are able to influence peripheral myelin protein 22 and P0 gene expression: possible mechanisms of action.

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.

20-alpha-Dihydroprogesterone↗

PubMed1999

Transregulation of erbB expression in the mouse olfactory bulb.

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/tufted cells. Partial lesion of the olfactory epithelium allows regenerative axon growth of olfactory neurons to the olfactory bulb. Following olfactory axon regeneration, erbB-3 and erbB-4 immunoreactivity in the olfactory bulb is similar to control. Thus, like tyrosine hydroxylase, the down regulation of erbB-4 expression in the periglomerular cells is reversible.

Administration, Intranasal↗

PubMed1998

ErbB-3 and ErbB-4 expression in the mouse olfactory system.

The interplay between neuregulins and the ErbB receptor family has a pivotal role in the development of several tissues, including the nervous system, and is maintained in the adult olfactory system where extensive plasticity and neurogenesis are retained. In the present work we show the immunohistochemical localization of ErbB-3 and ErbB-4 in the olfactory system of adult normal and lesioned mice. The expression of ErbB-3 is demonstrated to be restricted to the ensheathing cells of the olfactory nerve and to a few substentacular cells of the olfactory epithelium (OE). ErbB-3 staining circumvents the glomeruli but is never observed inside them or elsewhere in the adult olfactory bulb. Conversely, ErbB-4 immunoreactivity is found in all the periglomerular and mitral/tufted cells of the olfactory bulb (OB) and to a minor extent in the olfactory neurons and basal cells of the OE. Interestingly enough, cells coming out from the rostral migratory stream of the subependymal layer (SEL), as well as isolated cells in the granule cell layer, possibly migrating cells, strongly stained for ErbB-4 expression. Lesions of the olfactory epithelium have been performed by unilateral intranasal irrigation with ZnSO4 and 3 weeks after the irrigation, the olfactory bulbs were analyzed for olfactory marker protein (OMP), tyrosine hydroxylase (TH), ErbB-3 and ErbB-4 expression. In the deafferented OB, the drastic loss of immunoreactivity for OMP was accompanied by a strong reduction of ErbB-3 expression. Most of the deafferented dopaminergic interneurons switched off TH expression. In the deafferented periglomerular and mitral/tufted cells ErbB-4 expression was turned off and down, respectively. No differences were noted at the granular cell layer level in the deafferented OB with respect to control. Taken together our results suggest that, in normal conditions, neuregulins are involved in the survival of the ensheathing cells of the olfactory nerve through ErbB-3 activation and in the functional activity of postsynaptic neurons through ErbB-4 activation.

Animals↗

PubMed1998

Deafferentation up-regulates the expression of the mGlu1a metabotropic glutamate receptor protein in the olfactory bulb.

Chemical lesion of olfactory neuroepitheium induced an up-regulation of the mGlu1a metabotropic glutamate receptor protein in the olfactory bulb, as shown by Western blot analysis. At 2 days after the lesion, the increase in the receptor protein was associated with an increase in mGlu1a mRNA levels; in contrast, at longer times after the lesion (16 days), mRNA levels were reduced in spite of the high expression of the receptor protein, perhaps as a result of product-inhibition of mGlu1 gene expression. Immunohistochemical analysis showed that the increase in mGlu1a induced by olfactory denervation was confined to the glomeruli, which occupy the external portion of the olfactory bulb. Within these structures, mGlu1a receptors are mainly localized on the distal dendrites of mitral cells, which are innervated by the glutamatergic axons of the olfactory nerve. These results demonstrate that the expression of mGlu1a receptors is up-regulated in response to glutamatergic deafferentation, supporting a role for this particular receptor subtype in the physiology of synaptic transmission.

Amino Acid Sequence↗

PubMed1998

Cytochemistry of neurotransmitters: from statical analysis to the understanding of dynamic processes.

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 based on RT-PCR (reverse transcriptase-polymerase chain reaction) promise levels of sensitivity never reached before in in situ studies, and can provide simultaneous expression/functional data at the single-cell level.

Animals↗

PubMed1997

Glutamatergic deafferentation of olfactory bulb modulates the expression of mGluR1a mRNA.

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.

Animals↗

PubMed1997

Glutamate receptors in the olfactory bulb synaptic circuitry: heterogeneity and synaptic localization of N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1.

In this study, we analysed the molecular heterogeneity and synaptic localization of the N-methyl-D-aspartate receptor subunit 1 and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit 1 in the olfactory bulb glomerular synaptic circuitry. Semiquantitative reverse transcriptase polymerase chain reaction showed that approximately 40% of the N-methyl-D-aspartate receptor subunit 1 messenger RNA splice variants contain the N1 exon, which conveys specific functional properties on the channel. In other forebrain and hindbrain regions that we examined, the ratio of the N1-containing (receptor subunit 1(1XX)) to N1-lacking (receptor subunit 1(0XX)) N-methyl-D-aspartate receptor subunit 1 messenger RNAs varied considerably. The cellular and subcellular distribution of N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 was investigated with antibodies generated against the C-terminal domain of the individual subunits [Petralia R. S. et al. (1994) J. Neurosci. 14, 667 696; Wenthold R. J. et al. (1992) J. biol Chem. 267, 501 507]. Both N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 were localized to the postsynaptic density of asymmetric synapses established by olfactory receptor neuron terminals with the dendrites of mitral and tufted cells. Not all of these synapses, however, were labelled. These results are consistent with the notion that glutamate is the neurotransmitter at the olfactory nerve to mitral and tufted cell synapses, and suggest a high heterogeneity in the expression of the postsynaptic glutamate receptors.

Animals↗

PubMed1994

In vitro study of olfactory receptor neurones expressing the dipeptide carnosine.

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.

Animals↗

PubMed1993

Developmental expression of the alpha 6 GABAA receptor subunit mRNA occurs only after cerebellar granule cell migration.

Using a competitive polymerase chain reaction (PCR) and appropriate internal standards, we have analyzed absolute amounts of the alpha 6 GABAA receptor subunit mRNA in the postnatally developing cerebellum and neocortex. The PCR data have shown that absolute amounts of the alpha 6 receptor subunit mRNA in the cerebellum increase dramatically (nearly 100-fold) during the second postnatal week, reaching maximal levels by postnatal day 21 (1 fmol/microgram total RNA). The absolute amount of the alpha 6 GABAA receptor subunit mRNA in the cortex at postnatal day 1 was 2 amol/microgram total RNA and increased to 7 amol/micrograms total RNA by postnatal day 14. No further increase in alpha 6 mRNA expression in the adult cortex was observed. Microscopic analysis of emulsion coated and counterstained sections indicated that alpha 6 GABAA receptor subunit mRNA labeling was only detected in the internal granule cell layer and not in either the external granule cell layer or in migrating granule cells. The alpha 1 GABAA receptor subunit mRNA increased in the cerebellar cortex with a similar temporal profile, although its distribution extended to additional cell types (Purkinje cells, stellate/basket cells and possibly cerebellar astrocytes). The temporal expression of these two GABAA receptor subunit mRNAs is coincident with the formation of synaptic contacts in the granule cell dendrites suggesting that afferent pathways innervating these neurons following cell migration may play a critical role in increasing the expression of mRNAs encoding the alpha 1 and alpha 6 GABAA receptor subunits.

Aging↗

PubMed1992

Expression patterns of gamma-aminobutyric acid type A receptor subunit mRNAs in primary cultures of granule neurons and astrocytes from neonatal rat cerebella.

Using a competitive polymerase chain reaction assay, we have quantitated the absolute amounts of mRNA encoding 14 distinct subunits of the gamma-aminobutyric acid type A (GABAA) receptor in primary cultures of rat cerebellar granule neurons and cerebellar astrocytes. We found that the total amount of GABAA receptor subunit mRNA in astrocytes was 2 orders of magnitude lower than in neuronal cells. Furthermore, granule cell cultures expressed all 14 different GABAA subunit mRNAs, while the astroglial cultures contained detectable amounts of all the subunits expressed by granule cells except the alpha 6 and the gamma 2L subunits. Of the alpha subunit family members, the alpha 1, alpha 5, and alpha 6 mRNAs were prominent in granule cells, while the alpha 1 and alpha 2 mRNAs were abundant in astrocytes. Of the beta receptor subunit mRNAs, the beta 1 and beta 3 mRNAs were abundantly expressed in both cultures. The gamma 2S and gamma 2L mRNAs constituted the great majority of gamma subunit mRNAs in neurons, while the gamma 1 subunit mRNA was the most abundant gamma subunit mRNA in astrocytes. When various allosteric modulators of GABAA receptors were tested electrophysiologically, methyl 6,7-dimethoxy-4-ethyl-beta-carboline- 3-carboxylate (DMCM) was the only one to modulate chloride currents elicited by GABA in a significantly different manner in granule cells (negative modulation) compared with astrocytes (positive modulation). The latter effect was previously observed in transiently expressed recombinant GABAA receptors containing a gamma 1 instead of a gamma 2 subunit. Our quantitative mRNA results suggest that an important molecular determinant responsible for the DMCM-positive modulatory effect on astroglial native GABAA receptors is the presence of the gamma 1 subunit in the receptor assembly.

Animals↗

PubMed1992

Distinct developmental patterns of expression of rat alpha 1, alpha 5, gamma 2S, and gamma 2L gamma-aminobutyric acidA receptor subunit mRNAs in vivo and in vitro.

We have quantitated the alpha 1, alpha 5, gamma 2S, and gamma 2L gamma-aminobutyric acidA (GABAA) receptor subunit mRNAs in the maturing cerebellum in vivo and in cerebellar granule neurons differentiating in vitro. Absolute amounts of mRNA were measured by reverse transcription and competitive polymerase chain reaction (PCR) analysis with appropriate internal standards. The alpha 1 and gamma 2L mRNA content increased continuously during postnatal cerebellar maturation and their changes with time matched very closely those of the cerebellar granule cells differentiating in vitro. The gamma 2S subunit mRNA showed a relatively constant pattern of expression both in vivo and in vitro, with comparable absolute concentrations in both developmental paradigms. The alpha 5 mRNA was initially high in vivo and decreased (eight-fold) to adult levels as postnatal cerebellar development progressed. In vitro the amount of alpha 5 GABAA receptor subunit mRNA was higher than in vivo at 3 days, increased by more than twofold by 8 days, and declined to approximately the initial values at 23 and 28 days in vitro. Collectively, the results indicate that the alpha 1, alpha 5, gamma 2S, and gamma 2L GABAA receptor subunit mRNAs are regulated differentially in a temporal manner during in vivo and in vitro maturation. Moreover, a comparison of the ontogenetic profiles of the gamma 2S and gamma 2L mRNAs indicates that alternative splicing of the gamma 2 primary RNA transcript is regulated developmentally during postnatal maturation of the rat cerebellum.

Animals↗

PubMed1991

Immunohistochemistry of diazepam binding inhibitor (DBI) in the central nervous system and peripheral organs: its possible role as an endogenous regulator of different types of benzodiazepine receptors.

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.

Adrenal Glands↗

PubMed1991

Regulation of gamma-aminobutyric acidA receptor subunit expression by activation of N-methyl-D-aspartate-selective glutamate receptors.

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 developmental stages of granule cells in culture.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1990

Distribution and characterization of diazepam binding inhibitor (DBI) in peripheral tissues of rat.

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.

Animals↗

PubMed1990

Diazepam binding inhibitor (DBI) processing: immunohistochemical studies in the rat brain.

Diazepam Binding Inhibitor (DBI) is an endogenous 11-kDa peptide originally isolated from rat brain. In rat brain DBI coexists with at least three different processing products and the members of this peptide family have been shown to displace benzodiazepines and beta carbolines from recognition sites located on the allosteric modulatory centers of GABAA receptors. Immunocytochemical methods were used to study the location of DBI and two of the processing products, octadecaneuropeptide (ODN) DBI 33-50 and triakontatetraneuropeptide (TTN) DBI 17-50, in rat brain. DBI-LI was found in selected neuronal perikarya and in many glia and glial-like cells. All circumventricular organs displayed a strong DBI like immunoreactivity (LI). The distribution and cellular location of the ODN-LI and TTN-LI differed from that of DBI because they were preferentially associated with DBI in neurons, but not in glia or glial-like cells. The presence of DBI, but not of its processing products, in glial cells, circumventricular organs, and cells of peripheral tissues suggests that the function of this peptide may extend to other yet unknown function in addition to an action on the allosteric modulatory center of GABAA receptors located in neurons.

Animals↗

PubMed1990

Primary cultures of corticostriatal cells from newborn rats: a model to study muscarinic receptor subtypes regulation and function.

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.

Acetylcholine↗