Giorgio Gorini

Vagus nerve stimulation increases norepinephrine concentration and the gene expression of BDNF and bFGF in the rat brain

Vagus nerve stimulation therapy, effective for treatment-resistant epilepsy, has recently been approved also for treatment-resistant depression; nevertheless, the molecular mechanism(s) underlying its therapeutic action remains unclear. Given that neurotrophic factors and monoamines could play a crucial role in the pathophysiology of depression, we tested whether vagus nerve stimulation increases the expression of brain-derived neurotrophic factor, fibroblast growth factor, and nerve growth factor as well as the concentration of norepinephrine in the rat brain. Rats were implanted with a vagus nerve stimulator device and the effects of acute stimulation were evaluated on the growth factors mRNA levels and norepinephrine concentration by ribonuclease protection assay and microdialysis, respectively. We found that acute vagus nerve stimulation increased the expression of brain-derived neurotrophic factor and fibroblast growth factor in the hippocampus and cerebral cortex, decreased the abundance of nerve growth factor mRNA in the hippocampus, and, similar to the antidepressant drug venlafaxine, increased the norepinephrine concentration in the prefrontal cortex. This study demonstrates that acute vagus nerve stimulation triggers neurochemical and molecular changes in the rat brain involving neurotransmitters and growth factors known to play a crucial role in neuronal trophism. These new findings contribute to the elucidation of the molecular mechanisms underlying the therapeutic actions of vagus nerve stimulation in both treatment-resistant depression and epilepsy.

Chronic vagus nerve stimulation induces neuronal plasticity in the rat hippocampus

Vagus nerve stimulation (VNS) is used to treat pharmacotherapy-resistant epilepsy and depression. However, the mechanisms underlying the therapeutic efficacy of VNS remain unclear. We examined the effects of VNS on hippocampal neuronal plasticity and behaviour in rats. Cell proliferation in the hippocampus of rats subjected to acute (3 h) or chronic (1 month) VNS was examined by injection of bromodeoxyuridine (BrdU) and immunohistochemistry. Expression of doublecortin (DCX) and brain-derived neurotrophic factor (BDNF) was evaluated by immunofluorescence staining. The dendritic morphology of DCX+ neurons was measured by Sholl analysis. Our results show that acute VNS induced an increase in the number of BrdU+ cells in the dentate gyrus that was apparent 24 h and 3 wk after treatment. It also induced long-lasting increases in the amount of DCX immunoreactivity and in the number of DCX+ neurons. Neither the number of BrdU+ cells nor the amount of DCX immunoreactivity was increased 3 wk after the cessation of chronic VNS. Chronic VNS induced long-lasting increases in the amount of BDNF immunoreactivity and the number of BDNF+ cells as well as in the dendritic complexity of DCX+ neurons in the hippocampus. In contrast to chronic imipramine treatment, chronic VNS had no effect on the behaviour of rats in the forced swim or elevated plus-maze tests. Both chronic and acute VNS induced persistent changes in hippocampal neurons that may play a key role in the therapeutic efficacy of VNS. However, these changes were not associated with evident behavioural alterations characteristic of an antidepressant or anxiolytic action.

Distinct patterns of expression and regulation of GABAA receptors containing the δ subunit in cerebellar granule and hippocampal neurons

Neuronal plasticity is achieved by regulation of the expression of genes for neurotransmitter receptors such as the type A receptor (GABAAR) for γ‐aminobutyric acid. We now show that two different rat neuronal populations in culture manifest distinct patterns of GABAAR plasticity in response to identical stimuli. Whereas prolonged exposure to ethanol had no effect on expression of the δ subunit of GABAARs at the mRNA or protein level in cerebellar granule neurons, it increased the abundance of δ subunit mRNA and protein in hippocampal neurons. Subsequent ethanol withdrawal transiently down‐regulated δ subunit expression in cerebellar granule neurons and gradually normalized that in hippocampal neurons. These effects of ethanol exposure and withdrawal were accompanied by corresponding functional changes in GABAARs. GABAARs containing the δ subunit were also distributed differentially in the cerebellar and hippocampal neurons. These findings reveal complex and distinct mechanisms of regulation of the expression of GABAARs that contain the δ subunit in different neuronal types.

Plastic neuronal changes in GABAA receptor gene expression induced by progesterone metabolites: In vitro molecular and functional studies

Expression of specific gamma-aminobutyric acid type A (GABA(A)) receptor subunit genes in neurons is affected by endogenous modulators of receptor function such as neuroactive steroids. Neuroactive steroids such as the progesterone metabolite allopregnanolone might thus exert differential effects on GABA(A) receptor plasticity in neurons, likely accounting for some of the physiological actions of these compounds. Here we summarise experimental data obtained in vitro that show how fluctuations in the concentration of progesterone regulate both the expression and function of GABA(A) receptors. The data described in this manuscript are in agreement with the notion that fluctuations in the concentrations of progesterone and its metabolite allopregnanolone play a major role in the temporal pattern of expression of various subunits of the GABA(A) receptor. Thus, rapid and long-lasting increases or decreases in the concentrations of these steroid derivatives observed in physiological and patho-physiological conditions, or induced by pharmacological treatments, might elicit selective changes in GABA(A) receptor gene expression and function in specific neuronal populations. Given both the importance of GABA(A) receptors in the regulation of neuronal excitability and the large fluctuations in the plasma and brain concentrations of neuroactive steroids associated with physiological conditions and the response to environmental stimuli, these compounds are likely among the most relevant endogenous modulators that could affect emotional and affective behaviors.

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.

Flumazenil selectively prevents the increase in α4‐subunit gene expression and an associated change in GABAA receptor function induced by ethanol withdrawal

The actions of ethanol on γ‐aminobutyric acid type A (GABAA) receptors are still highly controversial issues but it appears that some of its pharmacological effects may depend on receptor subunit composition. Prolonged ethanol exposure produces tolerance and dependence and its withdrawal alters GABAA receptor subunit gene expression and function. Whereas benzodiazepines are clinically effective in ameliorating ethanol withdrawal symptoms, work in our laboratory showed that benzodiazepines also prevent, in vitro, some of the ethanol withdrawal‐induced molecular and functional changes of the GABAA receptors. In the present work, we investigated the effects, on such changes, of the benzodiazepine receptor antagonist flumazenil that can positively modulate α4‐containing receptors. We here report that flumazenil prevented both the ethanol withdrawal‐induced up‐regulation of the α4‐subunit and the increase in its own modulatory action. In contrast, flumazenil did not inhibit ethanol withdrawal‐induced decrease in α1‐ and δ‐subunit expression as well as the corresponding decrease in the modulatory action on GABAA receptor function of both the α1‐selective ligand zaleplon and the δ‐containing receptor preferentially acting steroid allopregnanolone. These observations are the first molecular and functional evidence that show a selective inhibition by flumazenil of the up‐regulation of α4‐subunit expression elicited by ethanol withdrawal.

Chronic Intermittent Ethanol Regulates Hippocampal GABA(A) Receptor Delta Subunit Gene Expression

Chronic ethanol consumption causes structural and functional reorganization in the hippocampus and induces alterations in the gene expression of gamma-aminobutyric acid type A receptors (GABAARs). Distinct forced intermittent exposure models have been used previously to investigate changes in GABAAR expression, with contrasting results. Here, we used repeated cycles of a Chronic Intermittent Ethanol paradigm to examine the relationship between voluntary, dependence-associated ethanol consumption, and GABAAR gene expression in mouse hippocampus. Adult male C57BL/6J mice were exposed to four 16-h ethanol vapor (or air) cycles in inhalation chambers alternated with limited-access two-bottle choice between ethanol (15%) and water consumption. The mice exposed to ethanol vapor showed significant increases in ethanol consumption compared to their air-matched controls. GABAAR alpha4 and delta subunit gene expression were measured by qRT-PCR at different stages. There were significant changes in GABAAR delta subunit transcript levels at different time points in ethanol-vapor exposed mice, while the alpha4 subunit levels remained unchanged. Correlated concurrent blood ethanol concentrations suggested that GABAAR delta subunit mRNA levels fluctuate depending on ethanol intoxication, dependence, and withdrawal state. Using a vapor-based Chronic Intermittent Ethanol procedure with combined two-bottle choice consumption, we corroborated previous evidences showing that discontinuous ethanol exposure affects GABAAR delta subunit expression but we did not observe changes in alpha4 subunit. These findings indicate that hippocampal GABAAR delta subunit expression changes transiently over the course of a Chronic Intermittent Ethanol paradigm associated with voluntary intake, in response to ethanol-mediated disturbance of GABAergic neurotransmission.

Molecular mechanisms of tolerance to and withdrawal of GABA(A) receptor modulators

catenin, known to serve as a linker between the cadherin and actin cytoskeleton, results in destabilization of synaptic contacts. On the contrary, overexpression of this catenin causes excess spine formation and reduced spine turnover. Pharmacological suppression of neural activities in hippocampal cultures induces a release of a N-catenin from synapses, whereas elevation of neural activities have opposite effects, i.e., enrichment of this molecule in synapses, suggesting the existence of an activity-dependent mechanism to control the association of a Ncatenin with synapses. In addition, it is known that a number of different cadherin subtypes with distinct adhesive specificities, generated due to the sequence diversity of their extracellular domain, are expressed in the nervous system, and each neuron has a unique set of these cadherins. These observations suggest that the cadherin–catenin complex regulates synapse dynamics from the cytoplasmic side, and possibly synaptic specificity from the extracellular side, although the latter idea is awaiting experimental tests. PL1 CONTROL OF SYNAPTIC JUNCTION DYNAMICS: ROLES OF THE CADHERIN–CATENIN COMPLEX Takeichi, M. RIKEN Center for Developmental Biology, Kobe, Japan