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Neuroblastoma cell lines are heterogeneous, comprised of at least three distinct cell phenotypes; neuroblastic N-type cells, non-neuronal substrate-adherent S-type cells and intermediate I-type cells. N- and S-type cell populations were enriched from the parental SH-SY5Y neuroblastoma cell line and induced to differentiate by the addition of retinoic acid (RA), a drug used in the treatment of neuroblastoma. N- and S-type cells were identified based on their differential expression of β-tubulin III, vimentin and Bcl-2. Store-operated Ca(2+) entry (SOCE) was then measured in proliferating and differentiated N- and S-type cell populations and the expression of STIM1, Orai1 and TRPC1, three proteins reported to play a key role in SOCE, was determined. In N-type cells the RA-induced switch from proliferation to differentiation was accompanied by a down-regulation in SOCE. STIM1 and Orai1 expression became down-regulated in differentiated cells, consistent with their respective roles as ER Ca(2+) sensor and store-operated Ca(2+) channel (SOC). TRPC1 became up-regulated suggesting that TRPC1 is not involved in SOCE, at least in differentiated N-type cells. In S-type cells SOCE remained active following the RA-induced switch from proliferation to differentiation and the expression of STIM1 and Orai1 remained unchanged. TRPC1 was not expressed in S-type cells. Our results indicate that differentiation of neuronal cells is associated with a remodelling of SOCE. Therapeutic targeting of SOCE proteins could potentially be a means of promoting neuronal differentiation in the treatment of neuroblastoma., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Activation of histamine H3 receptors (H3Rs) reduces inflammation and nociception, but the existence of H3Rs on peripheral innervation has never been demonstrated. Here we use antibodies to locate H3Rs in whisker pads, hairy and glabrous hind paw skin, dorsal root ganglia (DRGs), and spinal cords of rats, wild type mice, and H3R knockout (H3KO) mice. Although H3Rs have been hypothesized to be on C and sympathetic fibers, H3R-like immunoreactivity (H3R-LI) was only detected on presumptive periarterial A delta fibers and on A beta fibers that terminated in Meissner's corpuscles and as lanceolate endings around hair follicles. The H3R-positive periarterial fibers were thin-caliber and coexpressed immunoreactivity for calcitonin gene-related peptide (CGRP), substance P, acid sensing ion channel 3, and 200 kDa neurofilament protein (NF). H3R-LI was also detected on epidermal keratinocytes and Merkel cells, but not on Merkel endings, C fibers, any other A delta fibers, or sympathetic fibers. In DRGs, H3R-LI was preponderantly on medium to large neurons coexpressing NF-LI and mostly CGRP-LI. In dorsal horn, CGRP-positive fibers with and without H3R-LI ramified extensively in lamina II; many of the former formed a plexus in lamina V. Low levels of H3R-LI were also present on A beta fibers penetrating superficial and into deeper laminae. The distribution of H3R-LI was similar in rats and wild type mice, but was eliminated or strongly reduced in A delta fibers and A beta fibers, respectively, in H3KO mice. Taken with recently published behavioral results, the present findings suggest that periarterial, peptidergic, H3R-containing A delta fibers may be sources of high threshold mechanical nociception.

Stargazer (stg) mutant mice fail to express stargazin [transmembrane AMPA receptor regulatory protein gamma2 (TARPgamma2)] and consequently experience absence seizure-like thalamocortical spike-wave discharges that pervade the hippocampal formation via the dentate gyrus (DG). As in other seizure models, the dentate granule cells of stg develop elaborate reentrant axon collaterals and transiently overexpress brain-derived neurotrophic factor. We investigated whether GABAergic parameters were affected by the stg mutation in this brain region. GABA(A) receptor (GABAR) alpha4 and beta3 subunits were consistently upregulated, GABAR delta expression appeared to be variably reduced, whereas GABAR alpha1, beta2, and gamma2 subunits and the GABAR synaptic anchoring protein gephyrin were essentially unaffected. We established that the alpha4 betagamma2 subunit-containing, flunitrazepam-insensitive subtype of GABARs, not normally a significant GABAR in DG neurons, was strongly upregulated in stg DG, apparently arising at the expense of extrasynaptic alpha4 betadelta-containing receptors. This change was associated with a reduction in neurosteroid-sensitive GABAR-mediated tonic current. This switch in GABAR subtypes was not reciprocated in the tottering mouse model of absence epilepsy implicating a unique, intrinsic adaptation of GABAergic networks in stg. Contrary to previous reports that suggested that TARPgamma2 is expressed in the dentate, we find that TARPgamma2 was neither detected in stg nor control DG. We report that TARPgamma8 is the principal TARP isoform found in the DG and that its expression is compromised by the stargazer mutation. These effects on GABAergic parameters and TARPgamma8 expression are likely to arise as a consequence of failed expression of TARPgamma2 elsewhere in the brain, resulting in hyperexcitable inputs to the dentate.

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.