Christopher L. Thompson

Immunohistochemical localization of N-methyl-d-aspartate receptor NR1, NR2A, NR2B and NR2C/D subunits in the adult mammalian cerebellum

The distributions of the N-methyl-D-aspartate (NMDA) receptor NR1, NR2A, NR2B and NR2C/D subunits were mapped in adult mouse cerebellum using subunit-specific antibodies. Immunostaining with anti-NR1 antibodies was prominent in cell bodies and dendritic arbors of Purkinje cells, was light to moderate in cerebellar granule cells, Golgi interneurons and interneurons in the molecular layer. Anti-NR2A subunit-specific antibody staining of mouse cerebellum was moderate in the granule cells, and moderate to dense in Purkinje neurons and Bergmann glia. However, Purkinje neurons were not immunolabelled in adult rat brain. Anti-NR2B subunit-specific immunostaining was prominent in Purkinje cell bodies and dendrites but absent from the granule cell layer. Anti-NR2C/D subunit-specific immunostaining was largely restricted to cerebellar granule cells. These studies reveal that NMDA receptor subunits display distinct but overlapping expression patterns in the adult mammalian cerebellum. Furthermore, we have observed some differences between rats and mice in terms of the NMDA receptor subunits expressed in specific cerebellar cell types.

Mapping of GABAA receptor α5 and α6 subunit-like immunoreactivity in rat brain

The distribution of the α5 and α6 subunits of the GABAA receptor has been mapped in rat brain using affinity-purified antibodies generated against peptide sequences unique to the respective polypeptides. α5 Subunit-like immunoreactivity was of low density but was distributed across several cell groups including cortical interneurones, hippocampal CA3 pyramidal neurones, the anterior thalamic reticular nucleus and cerebellar Purkinje neurones. α6 Subunit-like immunoreactivity was observed in high density in cerebellar granule cells. These patterns are compatible with in situ hybridisation studies and provide a further anatomical substrate for GABAA receptor heterogeneity in the CNS.

Immunohistochemical localization of N-methyl-D-aspartate receptor subunits in the adult murine hippocampal formation: evidence for a unique role of the NR2D subunit.

NMDA receptors were immunopurified from adult mouse forebrain and screened by immunoblotting. NR1 was co-associated with NR2A, NR2B and NR2D but not NR2C, nor was NR2C detected in adult mouse hippocampal membranes. The anatomical distribution of NR1, 2A, 2B and 2D was mapped in the adult murine hippocampal formation. NR1-like immunoreactivity was localised to cell bodies of pyramidal neurons, granule cells and hilar cells of the dentate gyrus. Apical dendrites of the CA subfields and hilar cells were also immunopositive. NR2A- and NR2B-like immunoreactivity essentially co-localised with that of NR1 implying co-assembly of all three subunits in this brain structure. NR2D-like immunoreactivity was distinct, being totally excluded from pyramidal, granule and hilar cell bodies. Strong, punctate staining was restricted to the oriens layer of CA1 and the stratum lucidum of CA3 consistent with labelling of presynaptic receptors. Less intense staining was also observed in the internal third of the molecular layer of the dentate gyrus.

Studies on the subtype selectivity of CP-101,606 : evidence for two classes of NR2B-selective NMDA receptor antagonists.

The subtype-selectivity of racemic [(3)H]CP-101,606, a novel high-affinity NMDA receptor radioligand was determined using defined recombinant NMDA receptor subunits expressed in HEK 293 cells. [(3)H]CP-101,606 binds to adult rodent forebrain and NR1/NR2B receptors expressed in HEK 293 cells with K(D)=4.2 nM and 6.0 nM, respectively. In contrast, no high affinity specific binding was detected to NR1, NR2A, NR2B subunits expressed alone or NR1/NR2A receptors. HEK 293 cells were transfected with NR1, NR2A and NR2B receptor subunits and complexes comprising all three subunits were isolated by anti-NR2A immunoaffinity chromatography. Based on immunoblotting with subunit-selective antibodies, the immunopurified material contained all three NMDA receptor subunit polypeptides. However, in contrast to parallel studies in which high affinity [(3)H]Ro-25,6981 binding activity was observed, no high affinity [(3)H]CP-101,606 binding sites were detected to the immunopurified material. This study provides further evidence for two distinct classes of NR2B-directed NMDA receptor antagonists, one which binds with high affinity irrespective whether another NR2 subunit type is present (Ro-25,6981) and a second class which is affected significantly by the presence of another NR2 subunit type within the receptor complex, exemplified by CP-101,606.

Immunological identification of the mammalian H3 histamine receptor in the mouse brain

Affinity-purified antibodies raised against the peptide sequence H3 (349–358) receptor specifically recognized two protein species with Mr 62 000 and 93 000 in adult mouse forebrain membranes. Both immunoreactive species were suppressed greatly by preincubation of the antibody with the respective peptide. Immunohistochemical analysis using affinity-purified anti-H3 (349–358) antibodies yielded a high degree of coincidence with ligand-autoradiographical information, with high levels detected in the CA3 and dentate gyrus of the hippocampus, laminae V of the cerebral cortex, the olfactory tubercle, Purkinje cell layer of the cerebellum, substantia nigra, globus pallidus, thalamus and striatum. This study suggests further biochemical evidence for multiple H3 receptor subtypes and the widespread distribution of the H3 receptor in the mammalian brain.

GABAA α6-Containing Receptors Are Selectively Compromised in Cerebellar Granule Cells of the Ataxic Mouse, Stargazer

Stargazer mice fail to express the γ2 isoform of transmembrane α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor regulatory proteins that has been shown to be absolutely required for the trafficking and synaptic targeting of excitatory AMPA receptors in adult murine cerebellar granule cells. Here we show that 30 ± 6% fewer inhibitory γ-aminobutyric acid, type A (GABAA), receptors were expressed in adult stargazer cerebellum compared with controls because of a specific loss of GABAA receptor expression in the cerebellar granule cell layer. Radioligand binding assays allied to in situ immunogold-EM analysis and furosemide-sensitive tonic current estimates revealed that expression of the extrasynaptic (α6βxδ) α6-containing GABAA receptor were markedly and selectively reduced in stargazer. These observations were compatible with a marked reduction in expression of GABAA receptor α6, δ (mature cerebellar granule cell-specific proteins), and β3 subunit expression in stargazer. The subunit composition of the residual α6-containing GABAA receptors was unaffected by the stargazer mutation. However, we did find evidence of an ∼4-fold up-regulation of α1βδ receptors that may compensate for the loss of α6-containing GABAA receptors. PCR analysis identified a dramatic reduction in the steady-state level of α6 mRNA, compatible with α6 being the primary target of the stargazer mutation-mediated GABAA receptor abnormalities. We propose that some aspects of assembly, trafficking, targeting, and/or expression of extrasynaptic α6-containing GABAA receptors in cerebellar granule cells are selectively regulated by AMPA receptor-mediated signaling.

Differential cell surface expression of GABAA receptor α1, α6, β2 and β3 subunits in cultured mouse cerebellar granule cells – influence of cAMP‐activated signalling

In this study we have used mature, primary cultured mouse cerebellar granule cells (CGCs) to initiate our studies on the mechanisms governing neuronal trafficking of GABAA receptors (GABARs). Initially the steady‐state distribution of GABAR α1, α6, β2 and β3 subunits between the cell surface and cell interior was quantified. Cell surface proteins were modified with a membrane‐impermeable cross‐linking agent, bis(sulfosuccinimidyl)suberate (BS3) or the proteolytic enzyme, chymotrypsin. The proportion of unmodified (intracellular) and modified (cell surface) subunits was quantified by immunoblotting. We found that 51% of α6, 74% of α1, and 83% of β2/3 were expressed at the cell surface, thus identifying a sizeable intracellular pool of α6 in contrast to the low levels of intracellular α1 and β2/3. Chronic activation of protein kinase A (PKA) in CGCs in vitro, post‐transcriptionally up‐regulated expression of α1, β2 and β3 but not α6. This was paralleled by an increase in the BZ‐S subtype of [3H]Ro15–4513 binding sites. GABAR α1 was increased at the cell surface and in the cell interior, β2 was increased almost exclusively at the cell surface whilst β3 was increased almost exclusively in the cell interior. The intracellular pool of α6 was not affected. Thus, GABAR subunits are subject to differentially regulated trafficking, affording yet greater scope for GABAR diversity and plasticity.

Aberrant cerebellar granule cell-specific GABAA receptor expression in the epileptic and ataxic mouse mutant, Tottering

The Tottering (cacna1a(tg)) mouse arose as a consequence of a spontaneous mutation in cacna1a, the gene encoding the pore-forming subunit of the pre-synaptic P/Q-type voltage-gated calcium channel (VGCC, Ca(V)2.1). The mouse phenotype includes ataxia and intermittent myoclonic seizures which have been attributed to impaired excitatory neurotransmission at cerebellar granule cell (CGC) parallel fiber-Purkinje cell (PF-PC) synapses [Zhou YD, Turner TJ, Dunlap K (2003) Enhanced G-protein-dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca(2+)-channel mutant mouse, tottering. J Physiol 547:497-507]. We hypothesized that the expression of cerebellar GABA(A) receptors may be affected by the mutation. Indeed, abnormal GABA(A) receptor function and expression in the cacna1a(tg) forebrain has been reported previously [Tehrani MH, Barnes EM Jr (1995) Reduced function of gamma-aminobutyric acid A receptors in tottering mouse brain: role of cAMP-dependent protein kinase. Epilepsy Res 22:13-21; Tehrani MH, Baumgartner BJ, Liu SC, Barnes EM Jr (1997) Aberrant expression of GABA(A) receptor subunits in the tottering mouse: an animal model for absence seizures. Epilepsy Res 28:213-223]. Here we show a deficit of 40.2+/-3.6% in the total number of cerebellar GABA(A) receptors expressed (gamma2+delta subtypes) in adult cacna1a(tg) relative to controls. [(3)H]Muscimol autoradiography identified that this was partly due to a significant loss of CGC-specific alpha6 subunit-containing GABA(A) receptor subtypes. A large proportion of this loss of alpha6 receptors was attributable to a significantly reduced expression of the CGC-specific benzodiazepine-insensitive Ro15-4513 (BZ-IS) binding subtype, alpha6betagamma2 subunit-containing receptors. BZ-IS binding was reduced by 36.6+/-2.6% relative to controls in cerebellar membrane homogenates and by 37.2+/-3.7% in cerebellar sections. Quantitative immunoblotting revealed that the steady-state expression level of alpha6 and gamma2 subunits was selectively reduced relative to controls by 30.2+/-8.2% and 38.8+/-13.1%, respectively, alpha1, beta3 and delta were unaffected. Immunohistochemically probed control and cacna1a(tg) cerebellar sections verified that alpha6 and gamma2 subunit expression was reduced and that this deficit was restricted to the CGC layer. Thus, we have shown that abnormal cerebellar P/Q-type VGCC activity results in a deficit of CGC-specific subtype(s) of GABA(A) receptors which may contribute to, or may be a consequence of the impaired cerebellar network signaling that occurs in cacna1a(tg) mice.

AMPA and kainate receptors mediate mutually exclusive effects on GABAA receptor expression in cultured mouse cerebellar granule neurones

Studies on animal models of epilepsy and cerebellar ataxia, e.g., stargazer mice (stg) have identified changes in the GABAergic properties of neurones associated with the affected brain loci. Whether these changes contribute to or constitute homeostatic adaptations to a state of altered neuronal excitability is as yet unknown. Using cultured cerebellar granule neurones from control [+/+; α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazolepropionate receptor (AMPAR)‐competent, Kainate receptor (KAR)‐competent] and stg (AMPAR‐incompetent, KAR‐competent), we investigated whether non‐NMDA receptor (NMDAR) activity regulates GABAA receptor (GABAR) expression. Neurones were maintained in 5 mmol/L KCl‐containing basal media or depolarizing media containing either 25 mmol/L KCl or the non‐NMDAR agonist kainic acid (KA) (100 μmol/L). KCl‐ and KA‐mediated depolarization down‐regulated GABAR α1, α6 and β2, but up‐regulated α4, β3 and δ subunits in +/+ neurones. The KCl‐evoked but not KA‐evoked effects were reciprocated in stg neurones compatible with AMPAR‐regulation of GABAR expression. Conversely, GABAR γ2 expression was insensitive to KCl‐mediated depolarization, but was down‐regulated by KA‐treatment in a 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX)‐reversible manner in +/+ and stg neurones compatible with a KAR–mediated response. KA‐mediated up‐regulation of GABAR α4, β3 and δ was inhibited by L‐type voltage‐gated calcium channel (L‐VGCC) blockers and the Ca2+/calmodulin‐dependent protein kinase inhibitor, 4‐[(2S)‐2‐[(5‐isoquinolinylsulfonyl)methylamino]‐3‐oxo‐3‐(4‐phenyl‐1‐piperazinyl)propyl] phenyl isoquinoline sulfonic acid ester (KN‐62). Up‐regulation of GABAR α4 and β3 was also prevented by calcineurin (CaN) inhibitors, FK506 and cyclosporin A. Down‐regulation of GABAR α1, α6 and β2 was independent of L‐VGCC activity, but was prevented by inhibitors of CaN. Thus, we provide evidence that a KAR‐mediated and at least three mutually exclusive AMPAR‐mediated signalling mechanisms regulate neuronal GABAR expression.

Differential Cerebellar GABAA Receptor Expression in Mice with Mutations in CaV2.1 (P/Q-type) Calcium Channels

Ataxia is the predominant clinical manifestation of cerebellar dysfunction. Mutations in the human CACNA1A gene, encoding the pore-forming α1 subunit of CaV2.1 (P/Q-type) calcium channels, underlie several neurological disorders, including Episodic Ataxia type 2 and Familial Hemiplegic Migraine type 1 (FHM1). Several mouse mutants exist that harbor mutations in the orthologous Cacna1a gene. The spontaneous Cacna1a mutants Rolling Nagoya (tg(rol)), Tottering (tg) and Leaner (tg(ln)) mice exhibit behavioral motor phenotypes, including ataxia. Transgenic knock-in (KI) mouse strains with the human FHM1 R192Q and S218L missense mutations have been generated. R192Q KI mice are non-ataxic, whereas S218L KI mice display a complex behavioral phenotype that includes cerebellar ataxia. Given the dependence of γ-aminobutyric acid type A (GABAA) receptor subunit functioning on localized calcium currents, and the functional link between GABAergic inhibition and ataxia, we hypothesized that cerebellar GABAA receptor expression is differentially affected in Cacna1a mutants and contributes to the ataxic phenotype. Herein we quantified functional GABAA receptors and pharmacologically dissociated cerebellar GABAA receptors in several Cacna1a mutants. We did not identify differences in the expression of GABAA receptor subunits or in the number of functional GABAA receptors in the non-ataxic R192Q KI strain. In contrast, tg(rol) mice had a ∼15% decrease in the number of functional GABAA receptors, whereas S218L KI mice showed a ∼29% increase. Our data suggest that differential changes in cerebellar GABAA receptor expression profile may contribute to the neurological phenotype of cerebellar ataxia and that targeting GABAA receptors might represent a feasible complementary strategy to treat cerebellar ataxia.