A Manca

Increase in expression of the GABA(A) receptor alpha(4) subunit gene induced by withdrawal of, but not by long-term treatment with, benzodiazepine full or partial agonists.

The effects of long-term exposure to, and subsequent withdrawal of, diazepam or imidazenil (full and partial agonists of the benzodiazepine receptor, respectively) on the abundance of GABA(A) receptor subunit mRNAs and peptides were investigated in rat cerebellar granule cells in culture. Exposure of cells to 10 microM diazepam for 5 days significantly reduced the amounts of alpha(1) and gamma(2) subunit mRNAs, and had no effect on the amount of alpha(4) mRNA. These effects were accompanied by a decrease in the levels of alpha(1) and gamma(2) protein and by a reduction in the efficacy of diazepam with regard to potentiation of GABA-evoked Cl- current. Similar long-term treatment with 10 microM imidazenil significantly reduced the abundance of only the gamma(2)S subunit mRNA and had no effect on GABA(A) receptor function. Withdrawal of diazepam or imidazenil induced a marked increase in the amount of alpha(4) mRNA; withdrawal of imidazenil also reduced the amounts of alpha(1) and gamma(2) mRNAs. In addition, withdrawal of diazepam or imidazenil was associated with a reduced ability of diazepam to potentiate GABA action. These data give new insights into the different molecular events related to GABA(A) receptor gene expression and function produced by chronic treatment and withdrawal of benzodiazepines with full or partial agonist properties.

Changes in GABAA Receptor Gene Expression Induced by Withdrawal of, but Not by Long-Term Exposure to, Ganaxolone in Cultured Rat Cerebellar Granule Cells

The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanolone, on the function and expression of GABAA receptors were determined. Electrophysiological recordings demonstrated that ganaxolone potentiated with a potency and efficacy similar to those of allopregnanolone the Cl−currents evoked by GABA at recombinant human GABAAreceptors (comprising α1β2γ2L or α2β2γ2L subunit assemblies) expressed in Xenopus oocytes. Exposure of cultured rat cerebellar granule cells to 1 μM ganaxolone for 5 days had no effect on the abundance of mRNAs encoding the α1, α2, α3, α4, α5, γ2L, or γ2S subunits of the GABAA receptor. Withdrawal of ganaxolone after such long-term treatment, however, induced an increase in the abundance of α2, α4, and α5 subunit mRNAs and a decrease in the amounts of α1, γ2L, and γ2S subunit mRNAs. These changes were maximal 3 to 6 h after drug withdrawal and were reversible, being no longer apparent after 24 h. These results suggest that long-term exposure of cerebellar granule cells to ganaxolone does not affect the sensitivity of the GABAAreceptor to several positive modulators. Nevertheless, the reduction in the amounts of the α1 and γ2 subunit mRNAs together with the increase in the abundance of the α4 subunit mRNA induced by abrupt discontinuation of long-term treatment with ganaxolone suggest that withdrawal of this drug might result in a reduced response to classic benzodiazepines.

Ret, GFRalpha-1, GFRalpha-2 and GFRalpha-3 receptors in the human hippocampus and fascia dentata.

The immunohistochemical occurrence and localization of the receptor components of the glial cell line‐derived neurotrophic factor (GDNF) family ligands, the Ret receptor tyrosine kinase and GDNF family receptor (GFR) alpha‐1 to ‐3, is described in the human post‐mortem hippocampal formation at pre‐ and full‐term newborn, and adult age. Two different antibodies for each of the four‐receptor molecules were used. Western blot analysis indicates that the availability of GFRalpha receptor proteins may vary with age and post‐mortem delay. The immunohistochemical detectability of GFRalpha‐1, GFRalpha‐2, GFRalpha‐3 and Ret receptor molecules is shown in the rat up to 72 h post‐mortem. In the human specimens, labelled neuronal perikarya were detectable for each receptor protein at all examined ages, with prevalent localization in the pyramidal layer of the Ammons horn and hilus and granular layer of the fascia dentata. In the adult subjects, abundant punctate‐like structures were also present. Labelled glial elements were identifiable. Comparison of the pattern of immunoreactive elements among young and adult subjects suggests that the intracellular distribution of the GDNF family ligands may vary between pre‐ and perinatal life and adult age. The results obtained suggest the involvement of the Ret and GFRalpha receptors signalling in processes subserving both the organization of this cortical region during development and the functional activity and maintenance of the mature hippocampal neurons.

High affinity neurotrophin receptors in the human pre-term newborn, infant and adult cerebellum

The immunohistochemical occurrence of the high affinity neurotrophin (NT) receptors trkA, trkB, and trkC is shown in the pre‐term newborn, infant, and adult human post‐mortem cerebellum. Immunoreactive neuronal perikarya and processes were observed in all specimens examined, where they appeared unevenly distributed in the cerebellar cortical layers and deep nuclei, and showed regional differences among cerebellar lobules and folia. The trk receptor‐antibodies, tested by Western blot on human cerebellum homogenates, revealed multiple immunoreactive bands for trkA and single bands for trkB and trkC. The results obtained show the tissue localization of the trk receptor‐like immunoreactivity in the human cerebellum from prenatal to adult age. The analysis for codistribution of the receptors with the relevant ligand and among the receptors in discrete cortical and deep nuclei tissue fields shows a wide variety of conditions, from a good similarity in terms of type and density of labeled structures, to a lack of correspondence, and suggests the possibility of colocalization of trk receptors with the relevant neurotrophin and among them in the cerebellar cortex. These results sustain the concept that the neurotrophin trophic system participates in the development, differentiation, and maintenance of the human cerebellar connectivity and support the possibility of a multifactorial trophic support for the neurotrophins through target‐derived and local mechanisms.

Neurturin, persephin, and artemin in the human pre- and full-term newborn and adult hippocampus and fascia dentata.

The immunochemical occurrence and localization of the Glial cell line-derived neurotrophic factor (GDNF) family ligands neurturin (NTN), persephin (PSP), and artemin (ART) is described in the human postmortem hippocampus and fascia dentata from subjects aged 21 weeks of gestation to 88 years. The detectability of NTN, PSP, and ART is shown in the rat by Western blot and immunohistochemistry up to 70 h postmortem. In the human tissue, labeled neuronal perikarya were detectable for each trophin at all examined ages, with prevalent localization in the pyramidal layer of the Ammons horn and hilus and granular layer of the fascia dentata. In the adult subjects, punctate elements were also present. Comparison of the pattern of immunoreactive structures among young and adult subjects suggests that intracellular distribution and/or trafficking of the GDNF family ligands may undergo age-related changes. Labeled glial elements were also identifiable. Western blot analysis indicates that the availability of the dimeric and monomeric forms of the trophins may vary with age and postmortem delay. The results obtained suggest the involvement of NTN, PSP, and ART in processes subserving both the organization of this cortical region during development and the functional activity and maintenance of the mature human hippocampal neurons.

Neurotrophin-like immunoreactivity in the human pre-term newborn, infant, and adult cerebellum

The immunohistochemical occurrence of the neurotrophin (NT) proteins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐4 (NT‐4), and neurotrophin‐3 (NT‐3) is shown in the pre‐term newborn, infant, and adult human post‐mortem cerebellum. The NT‐like immunoreactive structures were unevenly distributed and showed regional differences among cerebellar lobules and folia. NGF‐, NT‐4‐, and NT‐3‐positive neuronal perikarya were observed in all specimens examined. At variance with the other neurotrophins, the BDNF antiserum labelled neuronal cell bodies only in newborn life and infancy, as well as extensive nerve fibre systems, whose density increased with age. The NT‐antibodies, tested by Western blot on human cerebellum homogenates, revealed immunoreactive bands corresponding to proteins of heterogenous molecular weight. The results obtained provide a first demonstration of the tissue localization of the NTs in the human cerebellum from perinatal to adult age, thus suggesting their involvement in the development, differentiation and maintenance of the cerebellar connectivity. Codistribution of the four NTs or sets of them was observed in cortical and deep nuclei neurons. Multiple trophic roles for NTs, encompassing the classic target‐derived and local mechanisms of support, are envisaged as significant in development, differentiation, and maintenance of the human cerebellar connectivity.