Postsynaptic Components Research Articles

GA-Binding Protein Is Dispensable for Neuromuscular Synapse Formation and Synapse-Specific Gene Expression

The mRNAs encoding postsynaptic components at the neuromuscular junction are concentrated in the synaptic region of muscle fibers. Accumulation of these RNAs in the synaptic region is mediated, at least in part, by selective transcription of the corresponding genes in synaptic myofiber nuclei. The transcriptional mechanisms that are responsible for synapse-specific gene expression are largely unknown, but an Ets site in the promoter regions of acetylcholine receptor (AChR) subunit genes and other "synaptic" genes is required for synapse-specific transcription. The Ets domain transcription factor GA-binding protein (GABP) has been implicated to mediate synapse-specific gene expression. Inactivation of GABPalpha, the DNA-binding subunit of GABP, leads to early embryonic lethality, preventing analysis of synapse formation in gabpalpha mutant mice. To study the role of GABP at neuromuscular synapses, we conditionally inactivated gabpalpha in skeletal muscle and studied synaptic differentiation and muscle gene expression. Although expression of rb, a target of GABP, is elevated in muscle tissue deficient in GABPalpha, clustering of synaptic AChRs at synapses and synapse-specific gene expression are normal in these mice. These data indicate that GABP is dispensable for synapse-specific transcription and maintenance of normal AChR expression at synapses.

Analysis of the factors influencing information transmission at the calyx of Held

The calyx of Held is a giant glutamatergic synapse located in the medial nucleus of the trapezoid body (MNTB) of the mammalian auditory brainstem. It is an important model system for studying short-term plasticity because each postsynaptic MNTB neuron receives only one giant synapse, and it is possible to record both pre- and postsynaptic events simultaneously. Physiological experiments show that the evoked excitatory postsynaptic current (EPSC) from the calyx displays a significant depression in response amplitude during a sustained stimulus train [1]. This observation is the result of interactions between various pre- and postsynaptic components occurring across multiple time-scales. Hundreds of readily releasable vesicles (RRVP) aggregate at the different active zones on the presynaptic terminal. The stochastic release of neurotransmitter from the RRVPs (triggered by influx of calcium ions during action potentials, APs), can exhaust the supply of vesicles. Simultaneously, the depleted RRVPs are constantly replenished by a large vesicle reserve pool, and this replenishment is also enhanced by a calcium-dependent process (mediated by the presynaptic APs). The amplitude of the presynaptic AP-evoked calcium ion concentration is affected by inactivation and facilitation of voltage-gated calcium channels as well as activation of presynaptic metabotropic glutamate receptors (mGluRs). In this study we extend our deterministic, multiple time-scale model of the calyx of Held [2,3] to a stochastic version. We use information theory to measure the amount of information transmitted between pre- and postsynaptic compartments [4]. A series of long, homogeneous Poisson spike trains with mean frequencies up to several hundred Hertz are used to stimulate the calyx model. This spike train is repeated many times, allowing the calculation of the conditional and unconditional entropy of the postsynaptic EPSC amplitude in response to presynaptic interspike intervals (ISI). The mutual information, a measure of the information content of the postsynaptic response (EPSC) about the afferent spike train (ISI), is then computed in terms of conditional and unconditional entropies. The results show that the information content in the postsynaptic response is influenced by the degree of variation of presynaptic calcium ion concentration. Rapid onset and recovery from facilitation maintains high information transfer rates across the frequency range. Suppression of calcium transients by slowly recovering inactivation and mGluR activation results in less information transmission, but prevents depletion of the RRVPs. Fast-acting postsynaptic receptor desensitisation also contributes to information transmission, but in a competitive way to facilitation.

Pre- and postsynaptic mechanisms in the clonidine- and oxymetazoline-induced inhibition of gastric motility in the rat

The inhibitory effect of clonidine (non-selective α 2-adrenoceptor agonist) and oxymetazoline (α 2A-adrenoceptor selective agonist) was compared on basal and stimulated gastric motor activity (gastric tone and contractions) using the balloon method in the rat. It was shown that oxymetazoline (0.2–1.7 μmol/kg, i.v.) decreased the basal motility, while clonidine (1.9–3.8 μmol/kg, i.v.) failed to affect it. When motility was stimulated centrally by insulin (5 IU/rat, i.v.), both clonidine (1.9–3.8 μmol/kg, i.v.) and oxymetazoline (0.1–3.4 μmol/kg, i.v.) inhibited the gastric motor activity. However, while the effect of clonidine was antagonized by the non-selective α 2-adrenoceptor antagonist yohimbine (5 μmol/kg, i.v.) and the α 2A-adrenoceptor selective antagonist BRL 44408 (3 μmol/kg, i.v.), the effect of oxymetazoline was only partially affected. Prazosin (α 1- and α 2B-adrenoceptor antagonist, 0.07–0.28 μmol/kg, i.v.) also failed to reverse the effect of oxymetazoline. Furthermore, when gastric motility was stimulated peripherally by activation of postsynaptic cholinergic muscarinic receptors by the combination of carbachol (0.14 μmol/kg, i.v.) and hexamethonium (37 μmol/kg, i.v.), clonidine (3.8 μmol/kg, i.v.) failed to affect the increased motor activity, however, oxymetazoline (0.8–3.4 μmol/kg, i.v.) exerted a pronounced inhibition. These results suggest that different mechanisms may be involved in the inhibitory effect of clonidine and oxymetazoline; while clonidine reduces the gastric motility by activation of presynaptic α 2-adrenoceptors, postsynaptic component in the effect of oxymetazoline has also been raised.

Neurofascin regulates the formation of gephyrin clusters and their subsequent translocation to the axon hillock of hippocampal neurons

Little is known about the role of cell adhesion molecules (CAMs) in inhibitory synapse development. In particular, a functional link between CAMs and the clustering of postsynaptic scaffold component gephyrin, which is a critical determinant of γ-aminobutyric acid A (GABA) receptor clustering, still needs to be elaborated. At early stages of inhibitory synapse formation, gephyrin and CAM neurofascin are diffusely expressed in the soma of hippocampal neurons. Subsequently, gephyrin clusters become localized to the axon hillock and neurofascin is observed all over the soma including the axon hillock suggesting a function for neurofascin in gephyrin clustering. Transfection of expression vectors for different isoforms and mutants of neurofascin revealed that neurofascin is required for the formation of gephyrin clusters presumably dependent on extracellular interactions. Furthermore, expression of neurofascin is necessary for the translocation of gephyrin clusters to the axon hillock of hippocampal neurons as shown by shRNA-mediated knockdown. In addition, overexpression of an embryonic neurofascin isoform is sufficient for functional rescue after knockdown of endogenous neurofascin.

Alterations in GABA-related transcriptome in the dorsolateral prefrontal cortex of subjects with schizophrenia.

In subjects with schizophrenia, impairments in working memory are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC). This dysfunction appears to be due, at least in part, to abnormalities in gamma-aminobutyric acid (GABA)-mediated inhibitory circuitry. To test the hypothesis that altered GABA-mediated circuitry in the DLPFC of subjects with schizophrenia reflects expression changes of genes that encode selective presynaptic and postsynaptic components of GABA neurotransmission, we conducted a systematic expression analysis of GABA-related transcripts in the DLPFC of 14 pairs of schizophrenia and age-, sex- and post-mortem interval-matched control subjects using a customized DNA microarray with enhanced sensitivity and specificity. Subjects with schizophrenia exhibited expression deficits in GABA-related transcripts encoding (1) presynaptic regulators of GABA neurotransmission (67 kDa isoform of glutamic acid decarboxylase (GAD(67)) and GABA transporter 1), (2) neuropeptides (somatostatin (SST), neuropeptide Y (NPY) and cholecystokinin (CCK)) and (3) GABA(A) receptor subunits (alpha1, alpha4, beta3, gamma2 and delta). Real-time qPCR and/or in situ hybridization confirmed the deficits for six representative transcripts tested in the same pairs and in an extended cohort, respectively. In contrast, GAD(67), SST and alpha1 subunit mRNA levels, as assessed by in situ hybridization, were not altered in the DLPFC of monkeys chronically exposed to antipsychotic medications. These findings suggest that schizophrenia is associated with alterations in inhibitory inputs from SST/NPY-containing and CCK-containing subpopulations of GABA neurons and in the signaling via certain GABA(A) receptors that mediate synaptic (phasic) or extrasynaptic (tonic) inhibition. In concert with previous findings, these data suggest that working memory dysfunction in schizophrenia is mediated by altered GABA neurotransmission in certain DLPFC microcircuits.

Plastic‐embedded semithin cross‐sections as a tool for high‐resolution immunofluorescence analysis of the neuromuscular junction molecules: Specific cellular location of protease‐activated receptor‐1

In the neuromuscular junction (NMJ), three cellular elements (nerve ending, postsynaptic muscle component, and teloglial Schwann cell) are closely juxtaposed and functionally interdependent. It is important to determine the precise location of the relevant molecules involved in structural stability and neurotransmission at the three cellular components of this synapse in order to understand the molecular mechanisms underlying NMJ formation, maintenance, and functionality. In this paper, we show that plastic-embedded 0.5-mum semithin cross-sections from whole-mount multiple-immunofluorescence-stained muscles provide a simple and sensitive high-resolution procedure for analyzing the cellular and subcellular distribution of molecules at the NMJ. We have used this procedure to resolve the location of protease-activated receptor 1 (PAR-1). Previously, by immunohistochemistry we had detected PAR-1 in muscle fibers concentrated in the synaptic area but could not determine whether PAR-1 is expressed only in the muscle fiber at the NMJ. Our present results demonstrate that PAR-1 is concentrated in the postsynaptic region but not in the presynaptic terminal and that the labelling pattern for PAR-1 overlapped with Schwann cell staining.

Retrograde modulation of presynaptic release probability through signaling mediated by PSD-95–neuroligin

The structure and function of presynaptic and postsynaptic components of the synapse are highly coordinated. How such coordination is achieved and the molecules involved in this process have not been clarified. Several lines of evidence suggest that presynaptic functionalities are regulated by retrograde mechanisms from the postsynaptic side. We therefore sought postsynaptic mechanisms responsible for trans-synaptic regulation of presynaptic function at excitatory synapses in rat hippocampal CA1 pyramidal neurons. We show here that the postsynaptic complex of scaffolding protein PSD-95 and neuroligin can modulate the release probability of transmitter vesicles at synapse in a retrograde way, resulting in altered presynaptic short-term plasticity. Presynaptic beta-neurexin serves as a likely presynaptic mediator of this effect. Our results indicate that trans-synaptic protein-protein interactions can link postsynaptic and presynaptic function.

The actin binding domain of ACF7 binds directly to the tetratricopeptide repeat domains of rapsyn

Formation of the neuromuscular junction requires the release of agrin from the presynaptic terminal of motor neurons. Clustering of acetylcholine receptors (AChRs) on the postsynaptic sarcolemma is initiated by agrin-dependent activation of the muscle-specific kinase. While the postsynaptic scaffolding protein rapsyn is vital for high density AChR aggregation, little is known about the mechanism through which AChRs are immobilized on the postsynaptic membrane. Ultrastructural and immunohistochemical studies of rat skeletal muscle have suggested that AChRs are anchored to a membrane-associated cytoskeleton that contains spectrin-like proteins and is thus similar to that of the human erythrocyte [Bloch RJ, Bezakova G, Ursitti JA, Zhou D, Pumplin DW (1997) A membrane skeleton that clusters nicotinic acetylcholine receptors in muscle. Soc Gen Physiol Ser 52:177–195]. We are studying a protein of the spectrin superfamily, ACF7 (also known as MACF), as a postsynaptic cytoskeletal component of the neuromuscular junction. ACF7 has multiple cytoskeleton-binding domains, including an N-terminal actin-binding domain that, we postulate, may interact with rapsyn, the scaffolding protein that binds directly to AChRs. To test this hypothesis, we co-expressed fragments of these molecules in cultured fibroblasts and assessed their co-distribution and interaction using confocal microscopy and co-immunoprecipitation. We demonstrate that the actin-binding domain of ACF7 specifically interacts with the tetratricopeptide repeat domains of rapsyn. Furthermore, we show using surface plasmon resonance and blot overlay that the actin-binding domain of ACF7 binds directly to rapsyn. These results suggest that, in mammalian skeletal muscle, AChRs are immobilized in the membrane through rapsyn-mediated anchoring to an ACF7-containing network that in turn is linked to the actin cytoskeleton.

Laminins containing the β2 chain modulate the precise organization of CNS synapses

Synapses are formed and stabilized by concerted interactions of pre-, intra-, and post-synaptic components; however, the precise nature of the intrasynaptic components in the CNS remains obscure. Potential intrasynaptic components include extracellular matrix molecules such as laminins; here, we isolate β2-containing laminins, including perhaps laminins 13 (α3β2γ3) and 14 (α4β2γ3), from CNS synaptosomes suggesting a role for these molecules in synaptic organization. Indeed, hippocampal synapses that form in vivo in the absence of these laminins are malformed at the ultrastructural level and this malformation is replicated in synapses formed in vitro, where laminins are provided largely by the post-synaptic neuron. This recapitulation of the in vivo function of laminins in vitro suggests that the malformations are a direct consequence of the removal of laminins from the synapse. Together, these results support a role for neuronal laminins in the structural integrity of central synapses.

Cbln1 Is Essential for Interaction-Dependent Secretion of Cbln3

Cbln1 and the orphan glutamate receptor GluRdelta2 are pre- and postsynaptic components, respectively, of a novel transneuronal signaling pathway regulating synapse structure and function. We show here that Cbln1 is secreted from cerebellar granule cells in complex with a related protein, Cbln3. However, cbln1- and cbln3-null mice have different phenotypes and cbln1 cbln3 double-null mice have deficits identical to those of cbln1 knockout mice. The basis for these discordant phenotypes is that Cbln1 and Cbln3 reciprocally regulate each other's degradation and secretion such that cbln1-null mice lack both Cbln1 and Cbln3, whereas cbln3-null mice lack Cbln3 but have an approximately sixfold increase in Cbln1. Unlike Cbln1, Cbln3 cannot form homomeric complexes and is secreted only when bound to Cbln1. Structural modeling and mutation analysis reveal that, by constituting a steric clash that is masked upon binding Cbln1 in a "hide-and-run" mechanism of endoplasmic reticulum retention, a single arginine confers the unique properties of Cbln3.

Synapse formation and elimination: Role of activity studied in different models of adult muscle reinnervation

Synapse competition and elimination are a general developmental process both in central and in peripheral nervous systems that is strongly activity dependent. Some common features regulate synapse competition, and one of these is an application to development of the Hebb's postulate of learning: repeated coincident spike activity in competing presynaptic inputs on the same target cell inhibits competition, whereas noncoincident activity promotes weakening of some of the inputs and ultimately their elimination. Here we report experiments that indicate that the development of muscle innervation (initial polyneuronal innervation and subsequent synapse elimination) follows the Hebb's paradigm. We utilized two different models of muscle reinnervation in the adult rat: 1) we crushed nerves going to soleus or extensor digitorum longus muscles, to activate regeneration of the presynaptic component of the neuromuscular junctions (NMJ), or 2) we injected the soleus muscle with Marcaine (a myotoxic agent) to activate regeneration of the postsynaptic component, the muscle fiber. A condition of transient polyneuronal innervation occurs during NMJ regeneration in both cases, although the two models differ insofar as the relative strength of the competing inputs is concerned. During the period of competition (a few days or weeks, in Marcaine or crush experiments, respectively), we imposed a synchronous firing pattern on the competing inputs by stimulating motor axons distal to a chronic conduction block and demonstrated that this procedure strongly inhibits synapse elimination, with respect to control muscles in which regeneration occurs under natural impulse activity of motoneurons.

Modeling the pre- and post-synaptic components involved in the synaptic modification between cones and horizontal cells in carp retina

In retinal synapses between cones and luminosity type horizontal cells (LHC), it was previously found in this laboratory that repetitive red flashes progressively strengthened the LHC's response to red flash, whereas weakened the LHC's response to green flash; repetitive green flash remarkably depressed the LHC's red response, but caused little changes in the cell's green response. However, the detailed mechanisms underlying these phenomena are not entirely clear. In the present study, based on an ion-channel model described mainly in the form of Hodgkin-Huxley equations, possible mechanisms of the short-term synaptic modification are investigated. The simulation results suggest that: (1) the auto-enhancement effect might be induced by the Ca2+-dependent process on the post-synaptic AMPA receptors, which could lead to changes of the ionic channel's properties; (2) the asymmetric response to red- and green-flashes and the mutual-chromatic suppression effects might be attributed to the regulatory effects on the presynaptic glutamate release.

The mGlu1 antagonist CPCCOEt enhances the climbing fibre response in Purkinje neurones independently of glutamate receptors

CPCCOEt (7-(hydroxyimino)cyclopropa[ b]chromen-1a-carboxylate ethyl ester) is frequently used to test for the involvement of mGlu1 receptors. Using whole-cell voltage recording from Purkinje cells in slices of rat cerebellum we find that CPCCOEt, at concentrations used to block mGlu1 receptors, causes an enhancement of the climbing fibre response. Application of alternative antagonists with activity at mGlu1 neither mimicked nor occluded the effects of CPCCOEt. Receptor antagonists demonstrated that this non-mGlu1 action of CPCCOEt was not mediated by other mGlu receptors or GABA B receptors. Voltage-clamped climbing fibre EPSCs are unaffected by CPCCOEt whilst application of a glutamate transport blocker did not occlude the CPCCOEt effect. This suggests that a postsynaptic voltage-dependent component of the complex climbing fibre response is the target. We have found no evidence for the involvement of the hyperpolarisation-activated current, I h, and calcium-activated conductances. Voltage-gated sodium, calcium and potassium channels are possible targets with inhibition of a potassium channel the most likely. Awareness of this non-mGlu-mediated effect of CPCCOEt is likely to be important for the correct interpretation of its actions.

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.

Autoimmune Channelopathies and Related Neurological Disorders

Ion channels are crucial elements in neuronal signaling and synaptic transmission, and defects in their function are known to underlie rare genetic disorders, including some forms of epilepsy. A second class of channelopathies, characterized by autoantibodies against ligand- and voltage-gated ion channels, cause a variety of defects in peripheral neuromuscular and ganglionic transmission. There is also emerging evidence for autoantibody-mediated mechanisms in subgroups of patients with central nervous system disorders, particularly those involving defects in cognition or sleep and often associated with epilepsy. In all autoimmune channelopathies, the relationship between autoantibody specificity and clinical phenotype is complex. But with this new information, autoimmune channelopathies are detected and treated with increasing success, and future research promises new insights into the mechanisms of dysfunction at neuronal synapses and the determinants of clinical phenotype.

Cholesterol and lipid microdomains stabilize the postsynapse at the neuromuscular junction

Stabilization and maturation of synapses are important for development and function of the nervous system. Previous studies have implicated cholesterol-rich lipid microdomains in synapse stabilization, but the underlying mechanisms remain unclear. We found that cholesterol stabilizes clusters of synaptic acetylcholine receptors (AChRs) in denervated muscle in vivo and in nerve-muscle explants. In paralyzed muscles, cholesterol triggered maturation of nerve sprout-induced AChR clusters into pretzel shape. Cholesterol treatment also rescued a specific defect in AChR cluster stability in cultured src(-/-);fyn(-/-) myotubes. Postsynaptic proteins including AChRs, rapsyn, MuSK and Src-family kinases were strongly enriched in lipid microdomains prepared from wild-type myotubes. Microdomain disruption by cholesterol-sequestering methyl-beta-cyclodextrin disassembled AChR clusters and decreased AChR-rapsyn interaction and AChR phosphorylation. Amounts of microdomains and enrichment of postsynaptic proteins into microdomains were decreased in src(-/-);fyn(-/-) myotubes but rescued by cholesterol treatment. These data provide evidence that cholesterol-rich lipid microdomains and SFKs act in a dual mechanism in stabilizing the postsynapse: SFKs enhance microdomain-association of postsynaptic components, whereas microdomains provide the environment for SFKs to maintain interactions and phosphorylation of these components.

Identification of developmentally regulated expression of MuSK in astrocytes of the rodent retina

One of the master regulators of postsynaptic neuromuscular synaptogenesis is the muscle-specific receptor tyrosine kinase (MuSK). In mammals prominent MuSK expression is believed to be restricted to skeletal muscle. Upon activation by nerve-derived agrin MuSK-dependent signalling participates in both the induction of genes encoding postsynaptic components and aggregation of nicotinic acetylcholine receptors (AChR) in the subsynaptic muscle membrane. Strikingly, expression of certain isoforms of nerve-derived agrin can also be detected in the CNS. In this study, we examined the expression of MuSK in the brain and eye of rodents. In the retina MuSK was expressed in astrocytes between postnatal days 7 and 14, i.e. at the time when the eyes open. We found that agrin was localized adjacent to MuSK-expressing astrocytes which in turn were detected close to the inner limiting membrane of the rodent retina. In summary, the presence of MuSK on retinal astrocytes suggests a novel role of MuSK signalling pathways in the CNS.

The Role of Nitric Oxide and GluR1 in Presynaptic and Postsynaptic Components of Neocortical Potentiation

In this study, we investigated the mechanisms underlying synaptic plasticity at the layer IV to II/III pathway in barrel cortex of mice aged 6-13 weeks. This pathway is one of the likely candidates for expression of experience-dependent plasticity in the barrel cortex and may serve as a model for other IV to II/III synapses in the neocortex. We found that postsynaptic autocamtide-2-inhibitory peptide is sufficient to block long-term potentiation (LTP) (IC50 of 500 nm), implicating postsynaptic calcium/calmodulin-dependent kinase II in LTP induction. AMPA receptor subunit 1 (GluR1) knock-out mice also showed LTP in this pathway, but potentiation was predominantly presynaptic in origin as determined by paired-pulse analysis, coefficient of variation analysis, and quantal analysis, whereas wild types showed a mixed presynaptic and postsynaptic locus. Quantal analysis at this synapse was validated by measuring uniquantal events in the presence of strontium. The predominantly presynaptic LTP in the GluR1 knock-outs was blocked by postsynaptic antagonism of nitric oxide synthase (NOS), either with intracellular N-omega-nitro-L-arginine methyl ester or N-nitro-L-arginine, providing the first evidence for a retrograde transmitter role for NO at this synapse. Antagonism of NOS in wild types significantly reduced but did not eliminate LTP (group average reduction of 50%). The residual LTP formed a variable proportion of the total LTP in each cell and was found to be postsynaptic in origin. We found no evidence for silent synapses in this pathway at this age. Finally, application of NO via a donor induced potentiation in layer II/III cells and caused an increase in frequency but not amplitude of miniature EPSPs, again implicating NO in presynaptic plasticity.

Inhibition of constitutive inward rectifier currents in cerebellar granule cells by pharmacological and synaptic activation of GABAB receptors

gamma-Aminobutyric acid (GABA)(B) receptors are known to enhance activation of Kir3 channels generating G-protein-dependent inward rectifier K(+)-currents (GIRK). In some neurons, GABA(B) receptors either cause a tonic GIRK activation or generate a late K(+)-dependent inhibitory postsynaptic current component. However, other neurons express Kir2 channels, which generate a constitutive inward rectifier K(+)-current (CIRK) without requiring G-protein activation. The functional coupling of CIRK with GABA(B) receptors remained unexplored so far. About 50% of rat cerebellar granule cells in the internal granular layer of P19-26 rats showed a sizeable CIRK current. Here, we have investigated CIRK current regulation by GABA(B) receptors in cerebellar granule cells, which undergo GABAergic inhibition through Golgi cells. By using patch-clamp recording techniques and single-cell reverse transcriptase-polymerase chain reaction in acute cerebellar slices, we show that granule cells co-express Kir2 channels and GABA(B) receptors. CIRK current biophysical properties were compatible with Kir2 but not Kir3 channels, and could be inhibited by the GABA(B) receptor agonist baclofen. The action of baclofen was prevented by the GABA(B) receptor blocker CGP35348, involved a pertussis toxin-insensitive G-protein-mediated pathway, and required protein phosphatases inhibited by okadaic acid. GABA(B) receptor-dependent CIRK current inhibition could also be induced by repetitive GABAergic transmission at frequencies higher than the basal autorhythmic discharge of Golgi cells. These results suggest therefore that GABA(B) receptors can exert an inhibitory control over CIRK currents mediated by Kir2 channels. CIRK inhibition was associated with an increased input resistance around rest and caused a approximately 5 mV membrane depolarization. The pro-excitatory action of these effects at an inhibitory synapse may have an homeostatic role re-establishing granule cell readiness under conditions of strong inhibition.

Morphological alterations of the synapses in the locus coeruleus in Parkinson's disease

Parkinson's disease is the most common movement disorder in the broad spectrum of neurodegenerative diseases, associated frequently with gradual decline of the higher mental faculties. From the morphological point of view it is characterized by the degeneration of a substantial number of dopaminergic neurons of the substantia nigra and a considerable degeneration of neuronal networks in locus coeruleus, putamen, globus pallidus, thalamus and some areas of the cortex of the brain hemispheres. Filamentous inclusions, in the form of Lewy bodies and Lewy neuritis, composed mainly of alpha synuclein, been the hallmark of diffuse Lewy body dementia, have been described in the neurons of the substantia nigra in many cases of Parkinson's disease associated with dementia. In previous studies we have described the morphological alterations in the synapses in the caudate nucleus and the globus pallidus in cases of Parkinson's disease. In the present study we attempted to describe the morphological and morphometric alterations of the locus coeruleus in patients who suffered from Parkinson's disease with normal cognitive function and in patients who suffered from Parkinson's disease associated with dementia, comparing them with normal controls. The morphological alterations of the neurons, the dendrites, the retrograde axonic collaterals and the synapses were more impressive in cases of Parkinson's disease associated with dementia than in Parkinson's disease with normal cognitive function. The majority of the synapses demonstrated changes in size and shape of the pre- and postsynaptic components, polymorphism of the synaptic vesicles and marked morphological alterations of the mitochondria. The morphological alterations of the synapses in cases of Parkinson's disease associated with dementia, plead in favor of the importance of the neuronal circuits of locus coeruleus in cognitive functions.