Kindling induces a long-term enhancement in the density of N-type calcium channels in the rat hippocampus

How seizures arise and recur in epilepsy is unknown. Recent genetic, pharmacological and electrophysiological data indicate a significant but undisclosed role for voltage-dependent calcium channels. Since the contribution such channels make to nerve function reflects the targeting of discrete subtypes to distinct cellular regions, we hypothesized that epilepsy reflects alterations in their spatiotemporal patterns of expression at the cell surface. To test this possibility, we examined the expression and distribution of hippocampal N-type calcium channels in an animal seizure model: kindling. Confocal microscopy of N-type calcium channels labeled with a new fluorescent ligand, coupled with a novel technique for analysing multiple images, revealed a 20-40% increase in their expression in CA1 and CA3 within 24 h post-seizure. These increases persisted in the dendritic fields of CA1, but had dissipated in CA3 by 28 days post-seizure. Such changes correlate poorly with cell number or synaptogenesis, but are consistent with increased N-type calcium channel expression on presynaptic terminals or, more likely, dendrites. These data rationalize recent electrophysiology and in situ hybridization data, and suggest that kindling alters N-type calcium channel trafficking mechanisms to cause a persistent, local, remodeling of their distributions in CA1 dendrites. The persistent induction of N-type calcium channels may be part of a mechanism for, and a hallmark of, synaptic plasticity, in which kindling represents a reinforcement of synapses en masse.     

Activity-dependent changes in synaptophysin immunoreactivity in hippocampus,piriform cortex,and entorhinal cortex of the rat

Synaptophysin, an integral membrane glycoprotein of synaptic vesicles, has been widely used to investigate synaptogenesis in both animal models and human patients. Kindling is an experimental model of complex partial seizures with secondary generalization, and a useful model for studying activation-induced neural growth in adult systems. Many studies using Timm staining have shown that kindling promotes sprouting in the mossy fiber pathway of the dentate gyrus. In the present study, we used synaptophysin immunohistochemistry to demonstrate activation-induced neural sprouting in non-mossy fiber cortical pathways in the adult rat. We found a significant kindling-induced increase in synaptophysin immunoreactivity in the stratum radiatum of CA1 and stratum lucidum/radiatum of CA3, the hilus, the inner molecular layer of the dentate gyrus, and layer II/III of the piriform cortex, but no significant change in layer II/III of the entorhinal cortex, 4 weeks after the last kindling stimulation. We also found that synaptophysin immunoreactivity was lowest in CA3 near the hilus and increased with increasing distance from the hilus, a reverse pattern to that seen with Timm stains in stratum oriens following kindling. Furthermore, synaptophysin immunoreactivity was lowest in dorsal and greatest in ventral sections of both CA3 and dentate gyrus in both kindled and non-kindled animals. This demonstrates that different populations of sprouting axons are labeled by these two techniques, and suggests that activation-induced sprouting extends well beyond the hippocampal mossy fiber system.     

Low-threshold L-type calcium channels in rat dopamine neurons

Ca(2+) channel subtypes expressed by dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) were studied using whole cell patch-clamp recordings and blockers selective for different channel types (L, N, and P/Q). Nimodipine (Nim, 2 microM), omega-conotoxin GVIA (Ctx, 1 microM), or omega-agatoxin IVA (Atx, 50 nM) blocked 27, 36, and 37% of peak whole cell Ca(2+) channel current, respectively, indicating the presence of L-, N-, and P-type channels. Nim blocked approximately twice as much Ca(2+) channel current near activation threshold compared with Ctx or Atx, suggesting that small depolarizations preferentially opened L-type versus N- or P-type Ca(2+) channels. N- and L-channels in DA neurons opened over a significantly more negative voltage range than those in rat dorsal root ganglion cells, recorded from using identical conditions. These data provide an explanation as to why Ca(2+)-dependent spontaneous oscillatory potentials and rhythmic firing in DA neurons are blocked by L-channel but not N-channel antagonists and suggest that pharmacologically similar Ca(2+) channels may exhibit different thresholds for activation in different types of neurons.     

Neurotensin enhances GABAergic activity in rat hippocampus CA1 region by modulating L-type calcium channels

Neurotensin (NT) is a tridecapeptide that interacts with three NT receptors; NTS1, NTS2, and NTS3. Although NT has been reported to modulate GABAergic activity in the brain, the underlying cellular and molecular mechanisms of NT are elusive. Here, we examined the effects of NT on GABAergic transmission and the involved cellular and signaling mechanisms of NT in the hippocampus. Application of NT dose-dependently increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from CA1 pyramidal neurons with no effects on the amplitude of sIPSCs. NT did not change either the frequency or the amplitude of miniature (m)IPSCs recorded in the presence of tetrodotoxin. Triple immunofluorescent staining of recorded interneurons demonstrated the expression of NTS1 on GABAergic interneurons. NT increased the action potential firing rate but decreased the afterhyperpolarization (AHP) amplitude in identified CA1 interneurons. Application of L-type calcium channel blockers (nimodipine and nifedipine) abolished NT-induced increases in action potential firing rate and sIPSC frequency and reduction in AHP amplitude, suggesting that the effects of NT are mediated by interaction with L-type Ca(2+) channels. NT-induced increase in sIPSC frequency was blocked by application of the specific NTS1 antagonist SR48692, the phospholipase C (PLC) inhibitor U73122, the IP(3) receptor antagonist 2-APB, and the protein kinase C inhibitor GF109203X, suggesting that NT increases gamma-aminobutyric acid release via a PLC pathway. Our results provide a cellular mechanism by which NT controls GABAergic neuronal activity in hippocampus.     

L-type calcium channels in the hippocampus and cerebellum of Alzheimer's disease brain tissue

There is growing evidence that the selective neuronal cell death observed in Alzheimer's Disease (AD) is the result of dysregulation of intracellular calcium (Ca2+) homeostasis. In the present study, L-type voltage sensitive calcium channels (L-VSCCs) were examined in the cerebellum and hippocampus of AD (n = 6; postmortem interval less than 5 h) and age-matched control (n = 6) tissue by homogenate binding techniques and quantitative in vitro receptor autoradiography using [3H]isradipine (PN200-110). Saturation analyses of the cerebellum revealed unaltered [3H]isradipine binding parameters (Kd and Bmax) between AD and control subjects. Analysis of AD and control hippocampus demonstrated significant differences as [3H]isradipine binding increased (62%) in AD, whereas hippocampal cell density decreased (29%) in AD, relative to control subjects. Moreover, AD differentially affected L-VSCC in area CA1 and dentate gyrus. The dentate gyrus had greatly increased binding (77%) with little cell loss (16%) in AD brains, whereas area CA1 had increased binding (40%) with significant cell loss (42%) in AD brains, relative to controls. The results of the present study suggest that hippocampal area CA1 may experience greater cell loss in response to increased L-VSCCs in AD relative to other brain regions.     

The involvement of L-type calcium channels in heterosynaptic long-term depression in the hippocampus.

The involvement of L-type calcium channels in heterosynaptic long-term depression (LTD) of the stratum radiatum input to area CA1 was studied in rat hippocampal slices. LTD of the radiatum field excitatory postsynaptic potential (EPSP) and population spike, produced by tetanization of the alveus in the presence of picrotoxin, was blocked by the calcium antagonist nimodipine and by a monoclonal antibody to the L-type calcium channel. LTD was produced in the absence of picrotoxin when the L-type calcium channel agonist, BAY-K8644, was applied. This effect was also blocked by nimodipine. These results indicate that L-type calcium channels are involved in heterosynaptic long-term depression.     

Cholinergic modulation of synaptic transmission and plasticity in entorhinal cortex and hippocampus of the rat

Effects of cholinergic agents on synaptic transmission and plasticity were examined in entorhinal cortex and hippocampus. Bath application of carbachol (0.25-0.75 microM) induced transient depression of field potential responses in all cases tested (24/24 in layer III of medial entorhinal cortex slices and 24/24 in CA1 of hippocampal slices; 11.0+/-1.9% and 7.8+/-2.5%, respectively) and long-lasting potentiation in some cases (4/24 in entorhinal cortex and 12/24 in hippocampus; 33.7+/-3.7% and 32.1+/-9.9%, respectively, in successful cases). Carbachol (0.5 microM) induced transient depression, but not long-lasting potentiation, of N-methyl-D-aspartate receptor-mediated responses in entorhinal cortex. At 5 microM, carbachol induced transient depression only (55. 9+/-4.7% in entorhinal cortex and 41.4+/-2.9% in hippocampus), which was blocked by atropine. Paired-pulse facilitation was not altered during carbachol-induced potentiation but enhanced during carbachol-induced depression. These results suggest that the underlying mechanisms of carbachol-induced depression and potentiation are decreased transmitter release and selective enhancement of non-N-methyl-D-aspartate receptor-mediated responses, respectively. Long-term potentiation could be induced in the presence of 10 microM atropine by theta burst stimulation. The magnitude was significantly lower (15.2+/-5.2%, n=9) compared with control (37.2+/-6.1%, n=8) in entorhinal cortex, however.These results demonstrate similar, but not identical, cholinergic modulation of synaptic transmission and plasticity in entorhinal cortex and hippocampus.     

P/Q-type, but not N-type, calcium channels mediate GABA release from fast-spiking interneurons to pyramidal cells in rat prefrontal cortex

The Cav2.1 (P/Q-) and Cav2.2 (N-type) voltage-gated calcium channels (VGCCs) play a predominant role in neurotransmitter release at central synapses, but their distribution is not uniform across different types of synapses. Although the functional significance of the differential distribution of N- and P/Q-type VGCCs is poorly understood, distinct types of VGCCs appear to differentially affect synaptic properties. For example, P/Q-type VGCCs are located closer to release sites and are less affected by G-protein-mediated inhibition than are N-type VGCCs. Thus P/Q-type VGCCs might be beneficial at synapses with high probability of release and precise timing of neurotransmission, such as the inhibitory inputs from parvalbumin-containing fast-spiking (FS) interneurons to pyramidal cells (PCs) in the neocortex. To determine whether VGCCs types predominate at synapses from FS interneurons to PCs in rat prefrontal cortex, whole cell paired recordings (n = 14) combined with intracellular labeling and fluorescence immunohistochemistry for parvalbumin were performed in acute slices. Bath application of the specific N-type VGCC blocker omega-conotoxin-GVIa (1 microM) did not alter inhibitory postsynaptic potential amplitude, failure rate, or synaptic dynamics; in contrast, application of P/Q-type VGCC blocker omega-agatoxin-IVa (0.5 microM) completely and irreversibly blocked neurotransmission. These results indicate that P/Q-type VGCCs mediate the GABA release from parvalbumin-positive FS interneurons to PCs in the rat neocortex.     

Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre-and postsynaptic calcium fluxes in rat hippocampal slices

The contribution of T-, L-, N-, P-, and Q-type Ca²⁺ channels to pre-and postsynaptic Ca²⁺ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca²⁺ concentration ([Ca²⁺]₀). [Ca²⁺]₀ was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca²⁺ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca²⁺]₀ decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca²⁺ entry (Ca_pre), while [Ca²⁺]⁰ decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca²⁺ entry (Ca_post). Ethosuximide and trimethadione were ineffective m concentrations up to 1 mM. At 10 mM, they reduced Ca_post and, much less, also Ca_pre Nimodipine (25 M) reduced Ca_post and, to a minor extent, Ca_pre. -Agatoxin IVA (0.4–1 M) and -conotoxin MVIIC (1 M) also reduced both Ca_pre and Ca_post, but with a stronger action on Ca_pre. -Conotoxin GVIA (3–8 M) reduced Ca_post without effect on Ca_pre. We conclude that during stimulus-induced, high-frequency neuronal activity Ca_post is carried by P/Q-, N-, and L-type channels and probably a further channel type different from these channels. Ca_pre includes at least P/Q-and possibly L-type channels. N-type channels did not contribute to Ca_pre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca²⁺ entry in this situation.     

ORL1 receptor-mediated internalization of N-type calcium channels

The inhibition of N-type calcium channels by opioid receptor like receptor 1 (ORL1) is a key mechanism for controlling the transmission of nociceptive signals. We recently reported that signaling complexes consisting of ORL1 receptors and N-type channels mediate a tonic inhibition of calcium entry. Here we show that prolonged ( approximately 30 min) exposure of ORL1 receptors to their agonist nociceptin triggers an internalization of these signaling complexes into vesicular compartments. This effect is dependent on protein kinase C activation, occurs selectively for N-type channels and cannot be observed with mu-opioid or angiotensin receptors. In expression systems and in rat dorsal root ganglion neurons, the nociceptin-mediated internalization of the channels is accompanied by a significant downregulation of calcium entry, which parallels the selective removal of N-type calcium channels from the plasma membrane. This may provide a new means for long-term regulation of calcium entry in the pain pathway.     

Age effects on atrophy rates of entorhinal cortex and hippocampus

The effects of age, subcortical vascular disease, apolipoprotein E (APOE) epsilon4 allele and hypertension on entorhinal cortex (ERC) and hippocampal atrophy rates were explored in a longitudinal MRI study with 42 cognitively normal (CN) elderly subjects from 58 to 87 years old. The volumes of the ERC, hippocampus, and white matter hyperintensities (WMH) and the presence of lacunes were assessed on MR images. Age was significantly associated with increased atrophy rates of 0.04+/-0.02% per year for ERC and 0.05+/-0.02% per year for hippocampus. Atrophy rates of hippocampus, but not that of ERC increased with presence of lacunes, in addition to age. WMH, APOE epsilon4 and hypertension had no significant effect on atrophy rates. In conclusion, age and presence of lacunes should be taken into consideration in imaging studies of CN subjects and AD patients to predict AD progression and assess the response to treatment trials.     

Glutamatergic receptor expression changes in the Alzheimer's disease hippocampus and entorhinal cortex

Alzheimer''s Disease (AD) is the leading form of dementia worldwide. Currently, the pathological mechanisms underlying AD are not well understood. Although the glutamatergic system is extensively implicated in its pathophysiology, there is a gap in knowledge regarding the expression of glutamate receptors in the AD brain. This study aimed to characterize the expression of specific glutamate receptor subunits in post‐mortem human brain tissue using immunohistochemistry and confocal microscopy. Free‐floating immunohistochemistry and confocal laser scanning microscopy were used to quantify the density of glutamate receptor subunits GluA2, GluN1, and GluN2A in specific cell layers of the hippocampal sub‐regions, subiculum, entorhinal cortex, and superior temporal gyrus. Quantification of GluA2 expression in human post‐mortem hippocampus revealed a significant increase in the stratum (str.) moleculare of the dentate gyrus (DG) in AD compared with control. Increased GluN1 receptor expression was found in the str. moleculare and hilus of the DG, str. oriens of the CA2 and CA3, str. pyramidale of the CA2, and str. radiatum of the CA1, CA2, and CA3 subregions and the entorhinal cortex. GluN2A expression was significantly increased in AD compared with control in the str. oriens, str. pyramidale, and str. radiatum of the CA1 subregion. These findings indicate that the expression of glutamatergic receptor subunits shows brain region‐specific changes in AD, suggesting possible pathological receptor functioning. These results provide evidence of specific glutamatergic receptor subunit changes in the AD hippocampus and entorhinal cortex, indicating the requirement for further research to elucidate the pathophysiological mechanisms it entails, and further highlight the potential of glutamatergic receptor subunits as therapeutic targets.     

人骨髓间充质干细胞L型钙离子通道的研究

目的:研究分离培养的人骨髓间充质干细胞(hMSCs)膜表面L型钙离子通道的表达。方法:采用密度梯度离心、贴壁筛选及单克隆培养法获得hMSCs,利用流式细胞分析、免疫荧光法鉴定,应用全细胞膜片钳技术记录hMSCs膜表面L钙离子通道的变化。结果:体外分离、培养出高度同源性的具有独特细胞免疫学表型hMSCs;40%的hMSCs表达钙离子电流,峰值电流(ICaPeak)在+20～+30mV为(102.67±19.06)pA,此电流可被Cd2+(20μmolL)阻断。结论:在未分化的hMSCs上L型钙离子通道表达较少,说明L型钙离子通道表达的增多可能是hMSCs定向分化的条件之一,调节hMSCs膜表面L型钙离子通道的表达将有助于促进其定向分化。     

Agonist-independent modulation of N-type calcium channels by ORL1 receptors

We have investigated modulation of voltage-gated calcium channels by nociceptin (ORL1) receptors. In rat DRG neurons and in tsA-201 cells, nociceptin mediated a pronounced inhibition of N-type calcium channels, whereas other calcium channel subtypes were unaffected. In tsA-201 cells, expression of N-type channels with human ORL1 resulted in a voltage-dependent G-protein inhibition of the channel that occurred in the absence of nociceptin, the ORL1 receptor agonist. Consistent with this observation, native N-type channels of small nociceptive dorsal root ganglion (DRG) neurons also had tonic inhibition by G proteins. Biochemical characterization showed the existence of an N-type calcium channel-ORL1 receptor signaling complex, which efficiently exposes N-type channels to constitutive ORL1 receptor activity. Calcium channel activity is thus regulated by changes in ORL1 receptor expression, which provides a possible molecular mechanism for the development of tolerance to opioid receptor agonists.     

Adrenergic facilitation of GABAergic transmission in rat entorhinal cortex

Whereas the entorhinal cortex (EC) receives noradrenergic innervations from the locus coeruleus of the pons and expresses adrenergic receptors, the function of norepinephrine (NE) in the EC is still elusive. We examined the effects of NE on GABA(A) receptor-mediated synaptic transmission in the superficial layers of the EC. Application of NE dose-dependently increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from the principal neurons in layer II/III through activation of alpha(1) adrenergic receptors. NE increased the frequency and not the amplitude of miniature IPSCs (mIPSCs) recorded in the presence of TTX, suggesting that NE increases presynaptic GABA release with no effects on postsynaptic GABA(A) receptors. Application of Ca(2+) channel blockers (Cd(2+) and Ni(2+)), omission of Ca(2+) in the extracellular solution, or replacement of extracellular Na(+) with N-methyl-D-glucamine (NMDG) failed to alter NE-induced increase in mIPSC frequency, suggesting that Ca(2+) influx through voltage-gated Ca(2+) or other cationic channels is not required. Application of BAPTA-AM, thapsigargin, and ryanodine did not change NE-induced increase in mIPSC frequency, suggesting that Ca(2+) release from intracellular stores is not necessary for NE-induced increase in GABA release. Whereas alpha(1) receptors are coupled to G(q/11) resulting in activation of the phospholipase C (PLC) pathway, NE-mediated facilitation of GABAergic transmission was independent of PLC, protein kinase C, and tyrosine kinase activities. Our results suggest that NE-mediated facilitation of GABAergic function contributes to its antiepileptic effects in the EC.     

Lymphocyte calcium signaling involves dihydropyridine-sensitive L-type calcium channels: facts and controversies

Calcium influx into lymphocytes is essential for activation, differentiation, and effector functions. While several channel- and receptor-types contribute to calcium influx, voltage-gated calcium channels (VGCC) mediate a well-characterized calcium influx pathway that is most exclusively identified in excitable cells. The role of L-type VGCCs, which belong to high-voltage activated calcium channels and are defined as dihydropyridine (DHP) receptors in excitable cells, is well documented. Interestingly, while lymphocytes do not range in the excitable cell category, the modulatory role of DHP agonists and antagonists and the identification of L-type VGCC-related molecules in B and T lymphocytes, mainly in Th2 cells, suggest these proteins are involved in the calcium response of these cells. Because the identity and the regulation of DHP receptors/channels in lymphocytes is far from being solved, we will discuss the challenging issues of demonstrating a role of L-type VGCCs in nonexcitable cells and the arguments supporting their role in lymphocytes. We will comment on the limitation of the use of DHP agonists and antagonists to ascertain a specific involvement of L-type VGCCs in lymphocyte calcium signaling. Finally, we will provide new clues on the interest of a potential use of DHP antagonists in Th2-cell-mediated pathology.