GABAA Receptors of Cerebellar Granule Cells in Culture: Interaction with Benzodiazepines

GABA_A receptor mediated inhibition plays an important role in modulating the input/output dynamics of cerebellum. A characteristic of cerebellar GABA_A receptors is the presence in cerebellar granule cells of subunits such as α6 and δ which give insensitivity to classical benzodiazepines. In fact, cerebellar GABA_A receptors have generally been considered a poor model for testing drugs which potentially are active at the benzodiazepine site. In this overview we show how rat cerebellar granule cells in culture may be a useful model for studying new benzodiazepine site agonists. This is based on the pharmacological separation of diazepam-sensitive α1 β2/3 γ2 receptors from those which are diazepam-insensitive and contain the α6 subunit. This is achieved by utilizing furosemide/Zn²⁺ which block α6 containing and incomplete receptors.     

Unorthodox view of the functioning of a GABAA synapse

1. Studies about the permeation of labelled chloride and GABA across single plasma membranes microdissected from vestibular Deiters' neurons have yielded two unexpected results: (a) intracellular GABA stimulates chloride permeation in an asymmetric fashion (efflux being favoured); (b) under certain conditions GABA permeates by a diffusion mechanism in the out in direction across these plasma membranes.2. These two main results have been obtained over many years together with a host of other indications about the fine mechanism of these events. Thus, a picture has emerged of their physiological meaning within the context of the functioning of the GABA_A synapses between the Purkinje cells and the Deiters' neurons.3. In short, it is proposed that at these synapses GABA accumulates into the postsynaptic neuron after its release and activation of the postsynaptic receptors. GABA accumulated in the Deiters' neurons is involved in the process of chloride extrusion to build an inward directed electrochemical gradient for chloride.     

Modulation of the expression of GABA(A) receptors in rat cerebellar granule cells by protein tyrosine kinases and protein kinase C

The expression of GABA(A) receptors in rat cerebellar granules in culture has been studied by beta(2/3) subunit immunocytochemistry and fluorescence confocal microscopy. These cells show labeling all over the cell bodies' plasma membrane and dendrites. Treatment with the protein tyrosine kinase (PTK) inhibitor genistein results in a decrease of the labeling associated with the beta(2/3) subunit in both cell bodies and dendrites. No effect was found with an inactive genistein analogue, daidzein. A similar effect was found with a protein kinase C (PKC) activator, phorbol myristate acetate (PMA). The effects of genistein and PMA are additive.The interpretation of the results is that PTK inhibition blocks exocytotic deposit of newly synthesized GABA(A) receptors onto the neuronal plasma membrane. On the other hand, PKC activation speeds up endocytotic removal of GABA(A) receptors.     

Assessment of fertility protection and ovarian reserve with GnRH antagonist in rats undergoing chemotherapy with cyclophosphamide

Background  

Reproductive function following chemotherapy is of increasing importance given that survival rates are improving. We assessed whether a gonadotropin-releasing hormone antagonist (GnRHant; cetrorelix) could promote ovarian protection against damage due to chemotherapy.     

Entacapone Reduces Cortical Activation in Parkinson's Disease with Wearing-Off: A f-MRI Study

Background and Purpose

Wearing-off is one of the most frequent problems encountered by levodopa-treated patients. Entacapone, a peripheral inhibitor of catechol-O-methyltransferase (COMT), reduces this motor complication by prolonging the effect of levodopa. We sought to understand the impact of COMT-inhibition on movement execution in PD patients with wearing-off by comparing functional magnetic resonance imaging (f-MRI) activation patterns prior to and during entacapone treatment. Our hypothesis was to determine whether changes in cortical activation are associated to COMT-inhibitor treatment.

Methods

Nine levodopa-treated non-demented PD patients with wearing-off were prospectively studied in two f-MRI session, prior to and during entacapone treatment. A group of control subjects were also studied for comparison.

Results

The patients significantly improved under COMT-inhibitor treatment based on home diaries. F-MRI results showed that at baseline the patients presented a bilateral activation of the primary motor, controlateral premotor cortex and supplementary motor area, as well as ipsilateral cerebellum. During treatment with entacapone, PD patients showed reductions in the activations of these cortical areas and a decreased activation in the ipsilateral cerebellum.

Conclusions

Our preliminary findings indicate that f-MRI is able to detect cortical activation changes during long-term modulation of dopaminergic treatment in PD patients with wearing-off, and thus, this technique could be further investigated in advanced PD patients.     

Decrease of Serotonin Transporters in Blood Platelets after Epileptic Seizures

Serotonin transporter (SERT) was studied by [³H]-paroxetine binding in blood platelets from controls and epileptic patients with generalized convulsive seizures. The average K_D and B_Max were not different in the two cases. However, a significant decrease was found in the serotonin transporter density in the platelet membranes from patients having undergone an epileptic seizure less than 4 days before. This circumstance may indicate a homeostatic reaction to the epileptic attack.     

A Quantum-Dot Nanocrystal Study of GABA<Subscript>A</Subscript> Receptor Subunits in Living Cerebellar Granule Cells in Culture

Quantum dots (QDs) are semiconductor nanocrystals emerging as a new class of fluorescent labels with large brightness, multi color fluorescence emission and resistance against photobleaching. Here we have used QDs as biological markers in an immunofluorescence approach.In this work GABA(A )receptors of rat cerebellar granule cells have been studied and in particular we have visualized the beta(2/3) and delta subunits in live cells. The results obtained were compared to those gathered with conventional probes.The images of the delta subunit in living cells appear to correspond to those expected for a subunit part of GABA(A )receptors mediating tonic inhibition in the granules cell bodies.