Central nervous system myelin is a specialized structure produced by oligodendrocytes that ensheaths axons, allowing rapid and efficient saltatory conduction of action potentials. Many disorders promote damage to and eventual loss of the myelin sheath, which often results in significant neurological morbidity. However, little is known about the fundamental mechanisms that initiate myelin damage, with the assumption being that its fate follows that of the parent oligodendrocyte. Here we show that NMDA (N-methyl-d-aspartate) glutamate receptors mediate Ca2+ accumulation in central myelin in response to chemical ischaemia in vitro. Using two-photon microscopy, we imaged fluorescence of the Ca2+ indicator X-rhod-1 loaded into oligodendrocytes and the cytoplasmic compartment of the myelin sheath in adult rat optic nerves. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor antagonist NBQX completely blocked the ischaemic Ca2+ increase in oligodendroglial cell bodies, but only modestly reduced the Ca2+ increase in myelin. In contrast, the Ca2+ increase in myelin was abolished by broad-spectrum NMDA receptor antagonists (MK-801, 7-chlorokynurenic acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing receptors (NVP-AAM077 and ifenprodil). In vitro ischaemia causes ultrastructural damage to both axon cylinders and myelin. NMDA receptor antagonism greatly reduced the damage to myelin. NR1, NR2 and NR3 subunits were detected in myelin by immunohistochemistry and immunoprecipitation, indicating that all necessary subunits are present for the formation of functional NMDA receptors. Our data show that the mature myelin sheath can respond independently to injurious stimuli. Given that axons are known to release glutamate, our finding that the Ca2+ increase was mediated in large part by activation of myelinic NMDA receptors suggests a new mechanism of axo-myelinic signalling. Such a mechanism may represent a potentially important therapeutic target in disorders in which demyelination is a prominent feature, such as multiple sclerosis, neurotrauma, infections (for example, HIV encephalomyelopathy) and aspects of ischaemic brain injury.

Analgesic therapies are still limited and sometimes poorly effective, therefore finding new targets for the development of innovative drugs is urgently needed. In order to validate the potential utility of blocking T-type calcium channels to reduce nociception, we explored the effects of intrathecally administered oligodeoxynucleotide antisenses, specific to the recently identified T-type calcium channel family (Ca V 3.1, Ca V 3.2, and Ca V 3.3), on reactions to noxious stimuli in healthy and mononeuropathic rats. Our results demonstrate that the antisense targeting Ca V 3.2 induced a knockdown of the Ca V 3.2 mRNA and protein expression as well as a large reduction of 'Ca V 3.2-like' T-type currents in nociceptive dorsal root ganglion neurons. Concomitantly, the antisense treatment resulted in major antinociceptive, anti-hyperalgesic, and anti-allodynic effects, suggesting that Ca V 3.2 plays a major pronociceptive role in acute and chronic pain states. Taken together, the results provide direct evidence linking Ca V 3.2 T-type channels to pain perception and suggest that Ca V 3.2 may offer a specific molecular target for the treatment of pain.

The pituitary adenylate cyclase-activating polypeptide (PACAP)/ glucagon superfamily includes nine hormones in humans that are related by structure, distribution (especially the brain and gut), function (often by activation of cAMP), and receptors (a subset of seven-transmembrane receptors). The nine hormones include glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, glucose-dependent insulinotropic polypeptide (GIP), GH-releasing hormone (GRF), peptide histidine-methionine (PHM), PACAP, secretin, and vasoactive intestinal polypeptide (VIP). The origin of the ancestral superfamily members is at least as old as the invertebrates; the most ancient and tightly conserved members are PACAP and glucagon. Evidence to date suggests the superfamily began with a gene or exon duplication and then continued to diverge with some gene duplications in vertebrates. The function of PACAP is considered in detail because it is newly (1989) discovered; it is tightly conserved (96% over 700 million years); and it is probably the ancestral molecule. The diverse functions of PACAP include regulation of proliferation, differentiation, and apoptosis in some cell populations. In addition, PACAP regulates metabolism and the cardiovascular, endocrine, and immune systems, although the physiological event(s) that coordinates PACAP responses remains to be identified.

T-type calcium channels are thought to transform neuronal output to a burst mode by generating low voltage-activated (LVA) calcium currents and rebound burst discharge. In this study we assess the expression pattern of the three different T-type channel isoforms (Cav3.1, Cav3.2, and Cav3.3) in cerebellar neurons and focus on their potential role in generating LVA spikes and rebound discharge in deep cerebellar nuclear (DCN) neurons. We detected expression of one or more Cav3 channel isoforms in a wide range of cerebellar neurons and selective expression of different isoforms in DCN cells. We further identify two classes of large-diameter DCN neurons that exhibit either a strong or weak capability for rebound discharge, despite the ability to generate LVA spikes when calcium currents are pharmacologically isolated. By correlating the Cav3 channel expression pattern with the electrophysiological profile of identified DCN cells, we show that Cav3.1 channels are expressed in isolation in DCN-burst cells, whereas Cav3.3 is expressed in DCN-weak burst cells. Cav3.1-expressing DCN cells correspond to excitatory or GABAergic neurons, whereas Cav3.3-expressing cells are non-GABAergic. The Cav3 class of LVA calcium channels is thus expressed in specific combinations in a wide range of cerebellar neurons but contributes to rebound burst discharge in only a select number of cell classes.Purkinje cells ͉ cerebellum ͉ rebound discharge

Voltage-gated calcium channels represent a heterogenous family of calcium-selective channels that can be distinguished by their molecular, electrophysiological, and pharmacological characteristics. We report here the molecular cloning and functional expression of three members of the low voltage-activated calcium channel family from rat brain (␣ 1G , ␣ 1H , and ␣ 1I ). Northern blot and reverse transcriptase-polymerase chain reaction analyses show ␣ 1G , ␣ 1H , and ␣ 1I to be expressed throughout the newborn and juvenile rat brain. In contrast, while ␣ 1G and ␣ 1H mRNA are expressed in all regions in adult rat brain, ␣ 1I mRNA expression is restricted to the striatum. Expression of ␣ 1G , ␣ 1H , and ␣ 1I subunits in HEK293 cells resulted in calcium currents with typical T-type channel characteristics: low voltage activation, negative steady-state inactivation, strongly voltage-dependent activation and inactivation, and slow deactivation. In addition, the direct electrophysiological comparison of ␣ 1G , ␣ 1H , and ␣ 1I under identical recording conditions also identified unique characteristics including activation and inactivation kinetics and permeability to divalent cations. Simulation of ␣ 1G , ␣ 1H , and ␣ 1I T-type channels in a thalamic neuron model cell produced unique firing patterns (burst versus tonic) typical of different brain nuclei and suggests that the three channel types make distinct contributions to neuronal physiology.

The mechanisms of Ca(2+) release from intracellular stores in CNS white matter remain undefined. In rat dorsal columns, electrophysiological recordings showed that in vitro ischemia caused severe injury, which persisted after removal of extracellular Ca(2+); Ca(2+) imaging confirmed that an axoplasmic Ca(2+) rise persisted in Ca(2+)-free perfusate. However, depletion of Ca(2+) stores or reduction of ischemic depolarization (low Na(+), TTX) were protective, but only in Ca(2+)-free bath. Ryanodine or blockers of L-type Ca(2+) channel voltage sensors (nimodipine, diltiazem, but not Cd(2+)) were also protective in zero Ca(2+), but their effects were not additive with ryanodine. Immunoprecipitation revealed an association between L-type Ca(2+) channels and RyRs, and immunohistochemistry confirmed colocalization of Ca(2+) channels and RyR clusters on axons. Similar to "excitation-contraction coupling" in skeletal muscle, these results indicate a functional coupling whereby depolarization sensed by L-type Ca(2+) channels activates RyRs, thus releasing damaging amounts of Ca(2+) under pathological conditions in white matter.

T-type calcium channels play critical roles in cellular excitability and have been implicated in the pathogenesis of a variety of neurological disorders including epilepsy. Although there have been reports that certain neuroleptics that primarily target D2 dopamine receptors and are used to treat psychoses may also interact with T-type Ca channels, there has been no systematic examination of this phenomenon. In the present paper we provide a detailed analysis of the effects of several widely used neuroleptic agents on a family of exogenously expressed neuronal T-type Ca channels (alpha1G, alpha1H, and alpha1I subtypes). Among the neuroleptics tested, the diphenylbutylpiperidines pimozide and penfluridol were the most potent T-type channel blockers with Kd values (approximately 30-50 nm and approximately 70-100 nm, respectively), in the range of their antagonism of the D2 dopamine receptor. In contrast, the butyrophenone haloperidol was approximately 12- to 20-fold less potent at blocking the various T-type Ca channels. The diphenyldiperazine flunarizine was also less potent compared with the diphenylbutylpiperadines and preferentially blocked alpha1G and alpha1I T-type channels compared with alpha1H. The various neuroleptics did not significantly affect T-type channel activation or kinetic properties, although they shifted steady-state inactivation profiles to more negative values, indicating that these agents preferentially bind to channel inactivated states. Overall, our findings indicate that T-type Ca channels are potently blocked by a subset of neuroleptic agents and suggest that the action of these drugs on T-type Ca channels may significantly contribute to their therapeutic efficacy.

Glutamate release from rod photoreceptors is dependent on a sustained calcium influx through L-type calcium channels. Missense mutations in the CACNA1F gene in patients with incomplete X-linked congenital stationary night blindness implicate the Ca v 1.4 calcium channel subtype. Here, we describe the functional and pharmacological properties of transiently expressed human Ca v 1.4 calcium channels. Ca v 1.4 is shown to encode a dihydropyridine-sensitive calcium channel with unusually slow inactivation kinetics that are not affected by either calcium ions or by coexpression of ancillary calcium channel ␤ subunits. Additionally, the channel supports a large window current and activates near Ϫ40 mV in 2 mM external calcium, making Ca v 1.4 ideally suited for tonic calcium influx at typical photoreceptor resting potentials. Introduction of base pair changes associated with four incomplete X-linked congenital night blindness mutations showed that only the G369D alteration affected channel activation properties. Immunohistochemical analyses show that, in contrast with previous reports, Ca v 1.4 is widely distributed outside the retina, including in the immune system, thus suggesting a broader role in human physiology.