Voltage-dependent calcium channels and currents in native neurons and other cells have been divided into high voltage activated (HVA) and low voltage activated (LVA) (Carbone & Lux, 1984;Nowycky et al. 1985). LVA currents can be distinguished by their activation at smaller depolarizations, near to the resting potential, and by their rapid inactivation (Huguenard, 1996). At the single channel level, channels with a small unitary conductance activate over the same voltage range (Carbone & Lux, 1984). Native T-type channels are heterogeneous (Kobrinsky et al. 1994;Huguenard, 1996), suggesting that they comprise more than one subtype of channel. A new subfamily of voltage-dependent calcium channel á1 subunit genes (comprising á1G, á1H and á1I) has recently been cloned, whose structure is superficially similar to the previously cloned HVA á1 subunits A, B, C, D, E and S (Perez-Reyes et al. 1998;Lee et al. 1999), having four domains, each with a voltage sensor and a pore-forming P loop. However, there are a number of regions where the homology is very low, particularly in the intracellular linkers and the N and C termini. These novel channels, when expressed, form rapidly inactivating LVA currents that also have a small single channel conductance and slowly deactivating tail currents like native T-type currents (Carbone & Lux, 1984;Armstrong & Matteson, 1985). Recently, using an antisense approach, evidence has been obtained that T-type currents in primary sensory neurons are generated by the á1G, H and I family (Lambert et al. 1998). The HVA channels are all thought to form heteromeric channels with the accessory subunits á2-ä, â and possibly ã. The accessory subunits, particularly â subunits, have marked effects on the assembly of functional channels at the plasma membrane. In expression systems, the â subunits increase the number of plasma membrane channels (Chien et al. 1995;Shistik et al. 1995;Brice et al. 1997) and also affect the voltage dependence and kinetics of activation and inactivation (Jones et al. 1998). Inactivation kinetics are

BackgroundThe choice and appropriate use of animal models in drug discovery for Alzheimer’s disease (AD) is pivotal to successful clinical translation of novel therapeutics, yet true alignment of research is challenging. Current models do not fully recapitulate the human disease, and even exhibit various degrees of regional pathological burden and diverse functional alterations. Given this, relevant pathological and functional endpoints must be determined on a model-by-model basis. The present work explores the rTg4510 mouse model of tauopathy as a case study to define best practices for the selection and validation of cognitive and functional endpoints for the purposes of pre-clinical AD drug discovery.MethodsMale rTg4510 mice were first tested at an advanced age, 12 months, in multiple behavioural assays (step 1). Severe tau pathology and neurodegeneration was associated with profound locomotor hyperactivity and spatial memory deficits. Four of these assays were then selected for longitudinal assessment, from 4 to 12 months, to investigate whether behavioural performance changes as a function of accumulation of tau pathology (step 2). Experimental suppression of tau pathology—via doxycycline administration—was also investigated for its effect on functional performance.ResultsProgressive behavioural changes were detected where locomotor activity and rewarded alternation were found to most closely correlate with tau burden and neurodegeneration. Doxycycline initiated at 4 months led to a 50% suppression of transgene expression, which was sufficient to prevent subsequent increases in tau pathology and arrest related functional decline.ConclusionsThis two-step approach demonstrates the importance of selecting assays most sensitive to the phenotype of the model. A robust relationship was observed between pathological progression, development of phenotype, and their experimental manipulation—three crucial factors for assessing the translational relevance of future pre-clinical findings.

No abstract

The molecular identity of a gene which encodes the pore-forming subunit (alpha1G) of a member of the family of low-voltage-activated, T-type, voltage-dependent calcium channels has been described recently. Although northern mRNA analyses have shown alpha1G to be expressed predominantly in the brain, the detailed cellular distribution of this protein in the central nervous system (CNS) has not yet been reported. The current study describes the preparation of a subunit specific alpha1G riboprobe and antiserum which have been used in parallel in situ mRNA hybridization and immunohistochemical studies to localize alpha1G in the mature rat brain. Both alpha1G mRNA and protein were widely distributed throughout the brain, but variations were observed in the relative level of expression in discrete nuclei. Immunoreactivity for alpha1G was typically localized in both the soma and dendrites of many neurons. Whilst alpha1G protein and mRNA expression were often observed in cells known to exhibit T-type current activity, some was also noted in regions, e.g. cerebellar granule cells, in which T-type activity has not been described. These observations may reflect differences between the subcellular distribution of channels that can be identified by immunohistochemical methods compared with electrophysiological techniques.

The role of Ca2+ entry in determining the electrical properties of cerebellar Purkinje cell (PC) dendrites and somata was investigated in cerebellar slice cultures. Immunohistofluorescence demonstrated the presence of at least three distinct types of Ca2+ channel proteins in PCs: the α1A subunit (P/Q type Ca2+ channel), the α1G subunit (T type) and the α1E subunit (R type). In PC dendrites, the response started in 66 % of cases with a slow depolarization (50 ± 15 ms) triggering one or two fast (∼1 ms) action potentials (APs). The slow depolarization was identified as a low‐threshold non‐P/Q Ca2+ AP initiated, most probably, in the dendrites. In 16 % of cases, this response propagated to the soma to elicit an initial burst of fast APs. Somatic recordings revealed three modes of discharge. In mode 1, PCs display a single or a short burst of fast APs. In contrast, PCs fire repetitively in mode 2 and 3, with a sustained discharge of APs in mode 2, and bursts of APs in mode 3. Removal of external Ca2+ or bath applications of a membrane‐permeable Ca2+ chelator abolished repetitive firing. Tetraethylammonium (TEA) prolonged dendritic and somatic fast APs by a depolarizing plateau sensitive to Cd2+ and to ω‐conotoxin MVII C or ω‐agatoxin TK. Therefore, the role of Ca2+ channels in determining somatic PC firing has been investigated. Cd2+ or P/Q type Ca2+ channel‐specific toxins reduced the duration of the discharge and occasionallyinduced the appearance of oscillations in the membrane potential associated with bursts of APs. In summary, we demonstrate that Ca2+ entry through low‐voltage gated Ca2+ channels, not yet identified, underlies a dendritic AP rarelyeliciting a somatic burst of APs whereas Ca2+ entry through P/Q type Ca2+ channels allowed a repetitive firing mainly by inducing a Ca2+‐dependent hyperpolarization.