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(a) Schematic illustrating the topography of the high voltage-activated calcium channel complex showing the main pore-forming α₁ subunit and ancillary subunits. The α₁ and δ subunits are integral membrane proteins, the β subunit is intracellular and binds directly to the α_1, while the α₂ subunit is thought to be largely extracellular. (b) Schematic diagram showing structure of the calcium channel α₁ subunit with its four domain structure. (c) The left panel shows the phylogenic relationship between the ten known calcium channel α₁ subunits. Ca_V1 subunits form the L-type subfamily, Ca_V2 channels the P/Q-type, N-type and R-type, and the Ca_V3 subunits form the low voltage-activated T-type calcium channels. The right panel shows representative traces of calcium currents recorded from reticular thalamic neurons in response to depolarization of the membrane potential. The upper trace shows a slow inactivating high voltage-activated current and the lower trace shows the fast inactivating low voltage-activated current.

Approximate positions of spontaneous loss of function mutations in tottering (tg) and it’s alleles (rolling, rocker, leaner, and tg 4J) are shown by black circles in pore forming alpha subunit (Cacna1a). Positions of two loss of function human mutations producing absence epilepsy, ataxia +/− episodic dyskinesia are shown with stars. Lethargic mutation interrupts interaction of cytoplasmic regulatory beta subunit (CacnB4) with the alpha subunit, and ducky results in defective membrane spanning a2delta subunit (Cacna2d2). Stargazer mutation shown in transmembrane gamma subunit (Cacng2) originally considered to be a member of the voltage-gated calcium channel as found in heteromeric muscle channels.

(A) Snapshot of calcium dye loaded slice (B) Change in fluorescence was measured for multiple cells and shown as a raster plot. Insert shows zoomed image of onset of synchronous network burst. The images were captured at 100 Hz by fast confocal microscopy equipped with EM-CCD camera (128×128 pixels). Note the stereotypic activation order for individual cells in multiple bursts.

A. Epistatic masking of spike wave seizures in double mutants bearing P/Q^tg and Kv1.1 −/− mutations. There is a striking semi-dominant effect showing an allelic dose-dependent decrease in seizure frequency with the removal of a single copy of the Kcna1a gene (middle), and complete suppression of seizure activity upon full ablation of the Kcna1a gene. In B, phenocopy experiments demonstrated suppression of cortical spike-wave absence seizures in adult tottering mutants following intraperitoneal injections of low doses of 4-aminopyridine, a non-specific potassium channel blocker. From Glasscock et al, 2007. (88).

(a-b) In the GAERS rodent model of absence epilepsy an arginine to proline missense mutation at position 1584 (R1584P) correlates with the expression of seizure activity. Mating GAERS with a non-epileptic strain (NEC) through two generations produces offspring that have no copies (wt/wt), one copy (wt/m) or two copies (m/m) of the R1584P mutation in an otherwise similar genetic background. Animals with two copies of the mutation spend increased time in seizure activity and experience more seizures than animals with no copies of the R1584P mutation. The missense GAERS mutation does not affect either the duration or morphology of individual seizures. (c) The WAG/Rij model of absence epilepsy displays increased expression of the Ca_V3.1 T-type calcium channel in thalamic centrolateral (CL) and lateral geniculate (LGN) neurons and of the Ca_V3.3 T-type in CL and RTN neurons. (d) In mice with a genetically enhanced expression of Ca_V3.1 channels (Tg1 and Tg2) larger T-type currents are observed in lateral dorsal (LDN) and ventrobasal (VB) thalamic neurons and the mice display spontaneous bilateral spike-wave discharges in EEG recordings.

a, interaction between mutations in a sodium channel and a potassium channel; b, interaction between mutations in two sodium channels; c, interaction between mutations in a calcium channel and a potassium channel. adapted from ref.¹⁶

Photolysis of caged glutamate directed at the terminal dendrite evokes a spatially restricted plateau potential. Photolysis directed at the main apical trunk near the base of the tuft evokes higher amplitude depolarizations that are dominated by calcium spikes.

(A) Progressive increase in the strength of glutamate photolysis stimulus directed at a thin terminal dendrite leads to a non-linear response. (B) Calcium imaging with Fluo-3 during such a plateau potential reveals that the electrogenesis is largely confined to the terminal dendritic compartment.

Following hypoxia, excitotoxicity and/or seizures, calcium entry into the mitochondria and/or energy failure contribute to mitochondrial swelling, outer membrane rupture, release of mitochondrial death factors and activation of the intrinsic pathway. Alternatively, an inflammatory process may induce the activation of the extrinsic pathway. Adapted from Niquet et al, 2006.

The brain lesion arising from the genetic loss of a cytosolic calcium ion channel regulatory Beta4 subunit, one of a 4 member family, is more complex that arising from the mutation of a single pore-forming alpha subunit. In A, due to subunit promiscuity with alpha subunits, the absence of a single regulatory subunit (β4) allows compensatory reshuffling and binding of alternate family members. In B, the distinct brain expression patterns of β1–4 family members in situ means that P/Q-type alpha/beta heteromeric channel complexes may differ in subunit composition in different brain regions. In C, co-immunoprecipitation patterns in lethargic brain show that α1a subunits, deprived of β4 interactions in lethargic mice, bind β1 and β3 partners instead, allowing functional rescue of the P/Q type current in cerebellar Purkinje cells. D. Patch clamp recordings confirm normal P/Q type currents in lethargic Purkinje cells. Different patterns of partial P/Q channel rescue throughout brain networks may contribute to the spike-wave and ataxic phenotype in these mice. Adapted from Burgess et al. 1999. (54).

Image examples, typical image size and microscopy objective lens to be used to study the dynamic function of these components. From left to right: (1) 3D reconstruction of neuronal processes expressing GFP molecule (2) CA1 pyramidal cell in a slice filled with a dye (3) CA3 pyramidal cells in a slice loaded with a calcium indicator dye (4) Hippocampus slice loaded with a voltage sensitive dye.

(a) Diagram of the thalamocortical network showing connections between the somatosensory cortex (SCX), the sensory relay neurons of the ventrobasal posterior thalamic groups (VB) and the reticular thalamic nucleus (RTN). (b) Under normal physiological conditions as sensory signals from the periphery are relayed to the cortex the VB and RTN neurons fire tonically in response to depolarization. In this state there is minimal T-type calcium channel activity. During epileptiform activity burst-firing becomes predominant (c) and the thalamocortical network becomes locked in a self-propagating oscillatory loop (Vm=membrane potential). (c: Inset) In the absence of sodium channel (600nM tetrodotoxin applied) activity the low threshold spike that underlies burst-firing is evident. (d) Burst-firing in the RTN of the GAERS epileptic rat model of absence epilepsy correlates with burst-firing in the RTN. Upper panels in show EEG recordings and lower panels show corresponding intracellular recordings in an RTN neuron with expanding timescales from on right side. Note that tonic neuronal firing does not correspond to spikes in the EEG trace (1), however burst-firing correlates closely with spikes observed on the EEG trace during spike-wave discharges (2).

Top left: NKCC1 accumulates intracellular Cl⁻ in immature neurons. Neurons with high intracellular Cl⁻ levels will lose negatively charged chloride ions when GABA binds to GABA_A receptors, opening a channel that is selectively permeable to anions. This loss of negative charge depolarizes neurons and may initiate excitatory processes such as action potentials and calcium waves. Top right: Allosteric modulators of the GABA_A receptor such as anticonvulsant barbiturates and benzodiazepines increase the open probability of the anion channel, resulting in increased Cl⁻ efflux, increased depolarization, and increased probability of initiating frankly excitatory processes. Bottom left: Blocking NKCC1 is a better anticonvulsant strategy, because it blocks Cl⁻ accumulation and the net loss of Cl⁻ when the GABA_A receptor channel opens. Although the neuron is not hyperpolarized by GABA_A receptor activations, the membrane conductance is increased, reducing the probability of initiating action potentials or calcium waves. Bottom right: the best anticonvulsant strategy may be to block NKCC1, thereby removing chloride accumulation, and increasing the open probability of the GABA_A receptor. This maximizes the increase in membrane conductance initiated by GABA binding, and minimizes Cl⁻ loss that might otherwise initiate action potentials.

The section was 25 μm thick and was prepared and α₂δ-1 was visualized as previously described. ⁵⁹ Note the lack of α₂δ-1 staining in the hippocampal granule and pyramidal cell body layers. The scale bar is 1 mm.

Region A is encoded by a separate exon, here called 18*. Region B is part of exon 19, generated by an alternative splice site. Main tissues showing expression of these splice variants involving regions A, B and C are given: SM (smooth muscle), A (aorta), L (lung), B (brain), H (heart).

A, A schematic of the hippocampus illustrates the major afferent pathways to the hippocampus and the position of the stimulation electrode used to activate the perforant path (PP). A. VSD image of a hippocampal slice from a control animal, containing most of each dentate blade and CA3. A snap shot at 25 ms after a burst stimulus applied to the perforant path generates a strong voltage response (red color) in the dentate gyrus that is minimally transmitted as excitation in CA3. B. Calcium imaging of dentate gyrus from control animal and animal 7 days after injection with pilocarpine. PP pathway was stimulated while imaging response from calcium indicator loaded tissues. Images on the left show baseline fluorescence and second images show normalized fluorescence change after electrical stimulation. Traces show normalized fluorescence changes on a randomly selected population of 10 cells from the images. Asterisks (*) indicate cells that responded to the electrical stimulation. Despite strong electrical stimulation majority of dentate granule cells did not have a response in control animal. Middle panels. In the presence of 100 μM picrotoxin in imaging solution, the same electrical stimulation recruited more responsive cells, demonstrating disruption of inhibitory mechanisms. Lower panel. In a slice prepared from an animal 1 week after pilocarpine injection (during the latent period), there was a significant increase in dentate granule cell activation following perforant path stimulation (Image Courtesy of Dr. Cho).

Split composite image of in situ hybridization patterns of three genes, Cacna1g (red), Cacna1h (green) and Cacna1i (blue) in adult mouse brain. Left side of brain depicts levels of expression and representative baseline cortical EEG activity in wild type mouse. Right side of brain shows elevated transcript levels in bac-transgenic Cacna1g overexpressing mouse strain, with typical cortical spike-wave EEG discharge above. The bac transgene drives Cacna1g under the control of its endogenous promoter, selectively elevating T-type current to conform with its native pattern in brain circuitry. At right, current traces from patch clamp recordings of neurons in the lateral dorsal nucleus (A) and ventrobasal nucleus (B) in wild type and transgenic mice show representative elevations in T-type currents in mutant neurons overexpressing Cacna1g. From Ernst et al. (74)(Figure 54.3).