Alterations in Intracellular Calcium Ion Concentrations in Cerebellar Granule Cells of the CACNA1A Mutant Mouse, Leaner, During Postnatal Development

Maintaining calcium ion (Ca²⁺) homeostasis is crucial for normal neuronal function. Altered Ca²⁺ homeostasis interferes with Ca²⁺ signaling processes and affects neuronal survival. In this study, we used homozygous leaner and tottering mutant mice, which carry autosomal recessive mutations in the gene coding for the α_1A pore forming subunit of Ca_V2.1 (P/Q-type) voltage-gated calcium channels (VGCC). Leaner mice show severe ataxia and epilepsy, while tottering mice are less severely affected. Leaner cerebellar granule cells (CGC) show extensive apoptotic cell death that peaks at postnatal (P) day 20 and continues into adulthood. Intracellular Ca²⁺ ([Ca²⁺]_i) concentrations in leaner and tottering mouse Purkinje cells have been described, but [Ca²⁺]_i concentrations have not been reported for granule cells, the largest neuronal population of the cerebellum. Using the ratiometric dye, Fura-2 AM, we investigated the role of Ca²⁺ homeostasis in CGC death during postnatal development by demonstrating basal [Ca²⁺]_i, depolarization induced Ca²⁺ transients, and Ca²⁺ transients after completely blocking Ca_V2.1 VGCC. From P20 onward, basal [Ca²⁺]_i levels in leaner CGC were significantly lower compared to age-matched wild-type CGC. We also compared basal [Ca²⁺]_i levels in leaner and wild-type CGC to basal [Ca²⁺]_i in tottering CGC. Potassium chloride induced depolarization revealed no significant difference in Ca²⁺ transients between leaner and wild-type CGC, indicating that even though leaner CGC have dysfunctional P/Q-type VGCC, Ca²⁺ transients after depolarization are the same. This suggests that other VGCC are compensating for the dysfunctional P/Q channels. This finding was further confirmed by completely blocking Ca_V2.1 VGCC using ω-Agatoxin IV-A.     

Prolonged IL‐1β exposure alters neurotransmitter and electrically induced Ca2+ responses in the myenteric plexus

Background Infection and inflammatory diseases of the gut results in profound changes of intestinal motor function. Acute administration of the pro‐inflammatory cytokine interleukin‐1β (IL‐1β) was shown to have excitatory and neuromodulatory roles in the myenteric plexus. Here we aimed to study the effect of prolonged IL‐1β incubation on the response of myenteric neurones to different stimuli. Methods Longitudinal muscle myenteric plexus preparations (LMMP’s) of the guinea pig jejunum were incubated for 24 h in medium with or without IL‐1β. After loading with Fluo‐4, calcium imaging was used to visualize activation of neurones. The response to application of serotonin (5‐HT), substance P (SP) and ATP or to electrical fibre tract stimulation (eFTS) was tested. Expression of nNOS, HuD, calbindin and calretinin was compared by immunohistochemistry. Key Results IL‐1β concentration‐dependently influenced the neuronal responsiveness and duration of the [Ca²⁺]_i rises to 5‐HT and ATP, while it also affected the Ca²⁺‐transient amplitudes induced by 5‐HT, ATP and SP. Ca²⁺‐transients in response to eFTS were observed in significantly more neurones per ganglion after IL‐1β (10^?10 and 10^?11 mol L^?1). Peak [Ca²⁺]_i rise after eFTS was concentration‐dependently decreased by IL‐1β. The duration of the [Ca²⁺]_i rise after eFTS was prolonged after IL‐1β 10^?12 mol L^?1. IL‐1β (10^?9 mol L^?1) incubation did not affect the number of nNOS, calretinin and calbindin expressing neurones, nor did it induce neuronal loss (HuD). Conclusions & Inferences In this study, IL‐1β differentially modulates the neuronal response to eFTS and neurotransmitter application in the myenteric plexus of guinea pigs. This cytokine could be implicated in the motility disturbances observed during gastrointestinal inflammation.     

Interactions with PDZ proteins diversify voltage‐gated calcium channel signaling

Voltage‐gated Ca²⁺ (Ca_V) channels are crucial for neuronal excitability and synaptic transmission upon depolarization. Their properties in vivo are modulated by their interaction with a variety of scaffolding proteins. Such interactions can influence the function and localization of Ca_V channels, as well as their coupling to intracellular second messengers and regulatory pathways, thus amplifying their signaling potential. Among these scaffolding proteins, a subset of PDZ (postsynaptic density‐95, Drosophila discs‐large, and zona occludens)‐domain containing proteins play diverse roles in modulating Ca_V channel properties. At the presynaptic terminal, PDZ proteins enrich Ca_V channels in the active zone, enabling neurotransmitter release by maintaining a tight and vital link between channels and vesicles. In the postsynaptic density, these interactions are essential in regulating dendritic spine morphology and postsynaptic signaling cascades. In this review, we highlight the studies that demonstrate dynamic regulations of neuronal Ca_V channels by PDZ proteins. We discuss the role of PDZ proteins in controlling channel activity, regulating channel cell surface density, and influencing channel‐mediated downstream signaling events. We highlight the importance of PDZ protein regulations of Ca_V channels and evaluate the link between this regulatory effect and human disease.     

Ca(2+) transients in submucous neurons during the colonic migrating motor complex in the isolated murine large intestine

Background The colonic migrating motor complex (CMMC) is a spontaneous, rhythmic, and neurally mediated motor pattern generated by myenteric neurons, which can propel fecal pellets in mice. Our aim was to determine whether submucous neurons were also activated during the CMMC. Methods The isolated murine colon was opened and sections of mucosa were removed to expose the submucous ganglia, which were then loaded with Fluo‐4. Key Results Colonic migrating motor complexes, which occurred spontaneously or by mechanically stimulating the mucosa, were identified by displacement of the tissue (duration = 23.3 s). Between CMMCs, spontaneous Ca²⁺ transients (frequency = 0.9 Hz) were observed in 55% (n = 8) of submucous neurons. During the CMMC, 98% (seven ganglia, n = 7) of submucous neurons within the same ganglion exhibited rapid Ca²⁺ transients (1.6 Hz) superimposed on a sustained rise in Ca²⁺ (duration ～23 s) that occurred 1.7 s following the mucosal stimulus; whereas other neurons exhibited a similar, but delayed response that occurred either at 7 or 13 s following the stimulus. The activity in submucous neurons was correlated with activity in adjacent nerve varicosities. Ondansetron (1 mm ; 5‐HT₃ antagonist) significantly reduced the frequency and duration of the Ca²⁺ transient responses. Conclusions & Inferences Activity in the submucous neurons appears to be secondary to that in the myenteric plexus and appears to be generated largely by activity in myenteric descending (serotonergic) interneurons. During the CMMC, there is likely to be an increase in secretion to lubricate and facilitate fecal pellet propulsion.     

l-DOPA induces concentration-dependent facilitation and inhibition of presynaptic acetylcholine release in the guinea-pig submucous plexus

Neurotransmitter- or neuromodulator-like actions ofl-DOPA were investigated with intracellular recordings from submucous plexus neurons of the guinea-pig caecum.l-DOPA at 30 nM augmented the amplitude of fast EPSPs, but did not affect depolarizations elicited by puff application of acetylcholine (ACh). The augmenting effect ofl-DOPA on the fast EPSPs was counteracted byl-DOPA methyl ester. The fast EPSPs were depressed by 10 μMl-DOPA, but transiently augmented after rinsing the drug.l-DOPA methyl ester did not affect the inhibitory action ofl-DOPA on the fast EPSPs, but antagonized the potentiation following the inhibition. The depolarization elicited by exogenously applied. ACh was inhibited by 10 μMl-DOPA. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) of the neuronal soma were measured with fura-2 microfluorophotometry. The transient increase in the [Ca²⁺]_i evoked by the somatic action potential (Δ[Ca²⁺]_AP) was facilitated by 30 nMl-DOPA, but decreased by the drug at 10 μM. It is concluded thatl-DOPA at low concentrations enhances the Δ[Ca²⁺]_AP, increasing the neurotransmitter release, but at high dose diminishes the Δ[Ca²⁺]_AP, inhibiting the neurotransmission.     

Paradoxical increase in survival of newborn neurons in the dentate gyrus of mice with constitutive depletion of serotonin

Increased adult neurogenesis is a major neurobiological correlate of the beneficial effects of antidepressants. Indeed, selective serotonin (5‐HT) re‐uptake inhibitors, which increase 5‐HT transmission, enhance adult neurogenesis in the dentate gyrus (DG) of the hippocampus. However, the consequences of 5‐HT depletion are still unclear as studies using neurotoxins that target serotonergic neurons reached contradictory conclusions on the role of 5‐HT on DG cell proliferation. Here, we analysed two genetic models of 5‐HT depletion, the Pet1^?/? and the VMAT2^f/f; SERT^cre/+ mice, which have, respectively, 80 and 95% reductions in hippocampal 5‐HT. In both models, we found unchanged cell proliferation of the neural precursors in the DG subgranular zone, whereas a significant increase in the survival of newborn neurons was noted 1 and 4 weeks after BrdU injections. This pro‐survival trait was phenocopied pharmacologically with 5‐HT synthesis inhibitor PCPA treatment in adults, indicating that this effect was not developmental. Furthermore, a 1‐week administration of the 5‐HT_1A receptor agonist 8‐OH‐DPAT in Pet1^?/? and PCPA‐treated mice normalised hippocampal cell survival. Overall, our results indicate that constitutive 5‐HT depletion does not alter the proliferation of neural precursors in the DG but promotes the survival of newborn cells, an effect which involves activation of postsynaptic 5‐HT_1A receptors. The role of 5‐HT in selective neuronal elimination points to a new facet in its multiple effects in controlling neural circuit maturation.     

Submucous rather than myenteric neurons are activated by mucosal biopsy supernatants from irritable bowel syndrome patients

Background We previously showed that colonic mucosal biopsy supernatants from patients with irritable bowel syndrome (IBS) activate neurons of the human submucous plexus, an area with densely packed immune cells. Based on the concept that mucosa‐nerve signaling is altered in IBS, we tested in this study whether the nerve sensitizing effect of IBS mucosal biopsy supernatants is more prominent in the submucous than myenteric plexus. Methods Fast neuroimaging with the voltage‐sensitive dye Di‐8‐ANEPPS was used to record activity of guinea‐pig submucous and myenteric neurons after application of constipation (C)‐ and diarrhea (D)‐IBS supernatants (three each) and four supernatants from healthy control subjects. Results are based on recordings from 4731 neurons. Key Results Control supernatants did not evoke significant responses in submucous or myenteric neurons. In contrast, all IBS supernatants evoked a significant spike discharge (median 3.6 Hz) in 46% of submucous neurons. This activation was significantly stronger than in the myenteric plexus where even twice the amount of supernatants evoked a lower spike frequency (median 2.1 Hz) in only 8.5% of neurons. Pharmacological studies revealed serotonin, histamine, and proteases as components mediating neuronal activation. Individual application of these components revealed that only serotonin evoked a significantly stronger activation of submucous compared with myenteric neurons. Conclusions & Inferences Direct neuronal activation by IBS mucosal biopsy supernatants is primarily a feature of submucous rather than myenteric neurons. This is associated with a stronger excitation of submucous neurons by serotonin. The plexus‐specific effects support the concept that altered mucosa‐nerve signaling underlies disturbances in IBS.     

Local anesthetics inhibit glutamate release from rat cerebral cortex synaptosomes

Local anesthetics have been widely used for regional anesthesia and the treatment of cardiac arrhythmias. Recent studies have also demonstrated that low‐dose systemic local anesthetic infusion has neuroprotective properties. Considering the fact that excessive glutamate release can cause neuronal excitotoxicity, we investigated whether local anesthetics might influence glutamate release from rat cerebral cortex nerve terminals (synaptosomes). Results showed that two commonly used local anesthetics, lidocaine and bupivacaine, exhibited a dose‐dependent inhibition of 4‐AP‐evoked release of glutamate. The effects of lidocaine or bupivacaine on the evoked glutamate release were prevented by the chelation of extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl ‐threo‐beta‐benzyl‐oxyaspartate did not have any effect on the action of lidocaine or bupivacaine. Both lidocaine and bupivacaine reduced the depolarization‐induced increase in [Ca²⁺]_C but did not alter 4‐AP‐mediated depolarization. Furthermore, the inhibitory effect of lidocaine or bupivacaine on evoked glutamate release was prevented by blocking the Ca_v2.2 (N‐type) and Ca_v2.1 (P/Q‐type) channels, but it was not affected by blocking of the ryanodine receptors or the mitochondrial Na⁺/Ca²⁺ exchange. Inhibition of protein kinase C (PKC) and protein kinase A (PKA) also prevented the action of lidocaine or bupivacaine. These results show that local anesthetics inhibit glutamate release from rat cortical nerve terminals. This effect is linked to a decrease in [Ca²⁺]_C caused by Ca²⁺ entry through presynaptic voltage‐dependent Ca²⁺ channels and the suppression of the PKA and PKC signaling cascades. Synapse 67:568–579, 2013 . © 2013 Wiley Periodicals, Inc.     

Differential Kv1.3, KCa3.1, and Kir2.1 expression in “classically” and “alternatively” activated microglia

Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) promote differentiation into classically activated M1‐like microglia, which produce high levels of pro‐inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL‐4 in contrast induces a phenotype associated with anti‐inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K⁺ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL‐4) microglia and studying their K⁺ channel expression by whole‐cell patch‐clamp, quantitative PCR and immunohistochemistry. We identified three major types of K⁺ channels based on their biophysical and pharmacological fingerprints: a use‐dependent, outwardly rectifying current sensitive to the K_V1.3 blockers PAP‐1 and ShK‐186, an inwardly rectifying Ba²⁺‐sensitive K_ir2.1 current, and a Ca²⁺‐activated, TRAM‐34‐sensitive K_Ca3.1 current. Both K_V1.3 and K_Ca3.1 blockers inhibited pro‐inflammatory cytokine production and iNOS and COX2 expression demonstrating that K_V1.3 and K_Ca3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN‐γ microglia exhibited high K_V1.3 current densities (～50 pA/pF at 40 mV) and virtually no K_Ca3.1 and K_ir currents, while microglia differentiated with IL‐4 exhibited large K_ir2.1 currents (～ 10 pA/pF at ?120 mV). K_Ca3.1 currents were generally low but moderately increased following stimulation with IFN‐γ or ATP (～10 pS/pF). This differential K⁺ channel expression pattern suggests that K_V1.3 and K_Ca3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106–121     

The Expression of Voltage-Gated Ca2+ Channels in Pituicytes and the Up-Regulation of L-Type Ca2+ Channels During Water Deprivation

The primary components of the neurohypophysis are the neuroendocrine terminals that release vasopressin and oxytocin, and pituicytes, which are astrocytes that normally surround and envelop these terminals. Pituicytes regulate neurohormone release by secreting the inhibitory modulator taurine in an osmotically‐regulated fashion and undergo a marked structural reorganisation in response to dehydration as well as during lactation and parturition. Because of these unique functions, and the possibility that Ca²⁺ influx could regulate their activity, we tested for the expression of voltage‐gated Ca²⁺ channel α1 subunits in pituicytes both in situ and in primary culture. Colocalisation studies in neurohypophysial slices show that pituicytes (identified by their expression of the glial marker S100β), are immunoreactive for antibodies directed against Ca²⁺ channel α1 subunits Ca_V2.2 and Ca_V2.3, which mediate N‐ and R‐type Ca²⁺ currents, respectively. Pituicytes in primary culture express immunoreactivity for Ca_V1.2, Ca_V2.1, Ca_V2.2, Ca_V2.3 and Ca_V3.1 (which mediate L‐, P/Q‐, N‐, R‐ and T‐type currents, respectively) and immunoblotting studies confirmed the expression of these Ca²⁺ channel α1 subunits. This increase in Ca²⁺ channel expression may occur only in pituicytes in culture, or may reflect an inherent capability of pituicytes to initiate the expression of multiple types of Ca²⁺ channels when stimulated to do so. We therefore performed immunohistochemistry studies on pituitaries obtained from rats that had been deprived of water for 24 h. Pituicytes in these preparations showed a significantly increased immunoreactivity to Ca_V1.2, suggesting that expression of these channels is up‐regulated during the adaptation to long‐lasting dehydration. Our results suggest that Ca²⁺ channels may play important roles in pituicyte function, including a contribution to the adaptation that occurs in pituicytes when the need for hormone release is elevated.     

R‐type Ca2+ channels contribute to fast synaptic excitation and action potentials in subsets of myenteric neurons in the guinea pig intestine

Background R‐type Ca²⁺ channels are expressed by myenteric neurons in the guinea pig ileum but the specific function of these channels is unknown. Methods In the present study, we used intracellular electrophysiological techniques to determine the function of R‐type Ca²⁺ channels in myenteric neurons in the acutely isolated longitudinal muscle‐myenteric plexus. We used immunohistochemical methods to localize the Ca_V2.3 subunit of the R‐type Ca²⁺ channel in myenteric neurons. We also studied the effects of the non‐selective Ca²⁺ channel antagonist, CdCl₂ (100 μmol L^?1), the R‐type Ca²⁺ channel blockers NiCl₂ (50 μmol L^?1) and SNX‐482 (0.1 μmol L^?1), and the N‐type Ca²⁺ channel blocker ω‐conotoxin GVIA (CTX 0.1 μmol L^?1) on action potentials and fast and slow excitatory postsynaptic potentials (fEPSPs and sEPSPs) in S and AH neurons in vitro. Key Results Ca_V2.3 co‐localized with calretinin and calbindin in myenteric neurons. NiCl₂ and SNX‐482 reduced the duration and amplitude of action potentials in AH but not S neurons. NiCl₂ inhibited the afterhyperpolarization in AH neurons. ω‐conotoxin GVIA, but not NiCl₂, blocked sEPSPs in AH neurons. NiCl₂ and SNX‐482 inhibited cholinergic, but not cholinergic/purinergic, fEPSPs in S neurons. Conclusions and Inferences These data show that R‐type Ca²⁺ channels contribute to action potentials, but not slow synaptic transmission, in AH neurons. R‐type Ca²⁺ channels contribute to release of acetylcholine as the mediator of fEPSPs in some S neurons. These data indicate that R‐type Ca²⁺ channels may be a target for drugs that selectively modulate activity of AH neurons or could alter fast synaptic excitation in specific pathways in the myenteric plexus.     

Serotonin 5‐HT1B receptor‐mediated calcium influx‐independent presynaptic inhibition of GABA release onto rat basal forebrain cholinergic neurons

Modulatory roles of serotonin (5‐HT) in GABAergic transmission onto basal forebrain cholinergic neurons were investigated, using whole‐cell patch‐clamp technique in the rat brain slices. GABA_A receptor‐mediated inhibitory postsynaptic currents (IPSCs) were evoked by focal stimulation. Bath application of 5‐HT (0.1–300 μm ) reversibly suppressed the amplitude of evoked IPSCs in a concentration‐dependent manner. Application of a 5‐HT_1B receptor agonist, CP93129, also suppressed the evoked IPSCs, whereas a 5‐HT_1A receptor agonist, 8‐OH‐DPAT had little effect on the evoked IPSCs amplitude. In the presence of NAS‐181, a 5‐HT_1B receptor antagonist, 5‐HT‐induced suppression of evoked IPSCs was antagonised, whereas NAN‐190, a 5‐HT_1A receptor antagonist did not antagonise the 5‐HT‐induced suppression of evoked IPSCs. Bath application of 5‐HT reduced the frequency of spontaneous miniature IPSCs without changing their amplitude distribution. The effect of 5‐HT on miniature IPSCs remained unchanged when extracellular Ca²⁺ was replaced by Mg²⁺. The paired‐pulse ratio was increased by CP93129. In the presence of ω‐CgTX, the N‐type Ca²⁺ channel blocker, ω‐Aga‐TK, the P/Q‐type Ca²⁺ channel blocker, or SNX‐482, the R‐type Ca²⁺ channel blocker, 5‐HT could still inhibit the evoked IPSCs. 4‐AP, a K⁺ channel blocker, enhanced the evoked IPSCs, and CP93129 had no longer inhibitory effect in the presence of 4‐AP. CP93129 increased the number of action potentials elicited by depolarising current pulses. These results suggest that activation of presynaptic 5‐HT_1B receptors on the terminals of GABAergic afferents to basal forebrain cholinergic neurons inhibits GABA release in Ca²⁺ influx‐independent manner by modulation of K⁺ channels, leading to enhancement of neuronal activities.     

KCa2 channels transiently downregulated during spatial learning and memory in rats

Small‐conductance calcium‐activated potassium channels (K_Ca2) are essential components involved in the modulation of neuronal excitability, underlying learning and memory. Recent evidence suggests that K_Ca2 channel activity reduces synaptic transmission in a postsynaptic NMDA receptor‐dependent manner and is modulated by long‐term potentiation. We used radioactive in situ hybridization and apamin binding to investigate the amount of K_Ca2 subunit mRNA and K_Ca2 proteins in brain structures involved in learning and memory at different stages of a radial‐arm maze task in naive, pseudoconditioned, and conditioned rats. We observed significant differences in K_Ca2.2 and K_Ca2.3, but not K_Ca2.1 mRNA levels, between conditioned and pseudoconditioned rats. K_Ca2.2 levels were transiently reduced in the dorsal CA fields of the hippocampus, whereas K_Ca2.3 mRNA levels were reduced in the dorsal and ventral CA fields of the hippocampus, entorhinal cortex, and basolateral amygdaloid nucleus in conditioned rats, during early stages of learning. Levels of apamin‐binding sites displayed a similar pattern to K_Ca2 mRNA levels during learning. Spatial learning performance was positively correlated with levels of apamin‐binding sites and K_Ca2.3 mRNA in the dorsal CA1 field and negatively correlated in the dorsal CA3 field. These findings suggest that K_Ca2 channels are transiently downregulated in the early stages of learning and that regulation of K_Ca2 channel levels is involved in the modification of neuronal substrates underlying new information acquisition. © 2009 Wiley‐Liss, Inc.     

Heat But Not Mechanical Hypersensitivity Depends on Voltage-Gated CaV2.2 Calcium Channel Activity in Peripheral Axon Terminals Innervating Skin

Voltage-gated Ca_V2.2 calcium channels are expressed in nociceptors at presynaptic terminals, soma, and axons. Ca_V2.2 channel inhibitors applied to the spinal cord relieve pain in humans and rodents, especially during pathologic pain, but a biological function of nociceptor Ca_V2.2 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is underappreciated. Here, we demonstrate that functional Ca_V2.2 channels in peripheral axons innervating skin are required for capsaicin-induced heat hypersensitivity in male and female mice. We show that Ca_V2.2 channels in TRPV1-nociceptor endings are activated by capsaicin-induced depolarization and contribute to increased intracellular calcium. Capsaicin induces hypersensitivity of both thermal nociceptors and mechanoreceptors, but only heat hypersensitivity depends on peripheral Ca_V2.2 channel activity, and especially a cell-type-specific Ca_V2.2 splice isoform. Ca_V2.2 channels at peripheral nerve endings might be important therapeutic targets to mitigate certain forms of chronic pain.SIGNIFICANCE STATEMENT It is generally assumed that nociceptor termini in the spinal cord dorsal horn are the functionally significant sites of Ca_V2.2 channel in control of transmitter release and the transmission of sensory information from the periphery to central sites. We show that peripheral Ca_V2.2 channels are essential for the classic heat hypersensitivity response to develop in skin following capsaicin exposure. This function of Ca_V2.2 is highly selective for heat, but not mechanical hypersensitivity induced by capsaicin exposure, and is not a property of closely related Ca_V2.1 channels. Our findings suggest that interrupting Ca_V2.2-dependent calcium entry in skin might reduce heat hypersensitivity that develops after noxious heat exposure and may limit the degree of heat hypersensitivity associated with certain other forms of pain.     

Deletion of Cav2.1(α1A) subunit of Ca2+‐channels impairs synaptic GABA and glutamate release in the mouse cerebellar cortex in cultured slices

Deletion of both alleles of the P/Q‐type Ca²⁺‐channel Ca_v2.1(α_1A) subunit gene in mouse leads to severe ataxia and early death. Using cerebellar slices obtained from 10 to 15 postnatal days mice and cultured for at least 3 weeks in vitro, we have analysed the synaptic alterations produced by genetically ablating the P/Q‐type Ca²⁺‐channels, and compared them with the effect of pharmacological inhibition of the P/Q‐ or N‐type channels on wild‐type littermate mice. Analysis of spontaneous synaptic currents recorded in Purkinje cells (PCs) indicated that the P/Q‐type channels play a prominent role at the inhibitory synapses afferent onto the PCs, with the effect of deleting Ca_v2.1(α_1A) partially compensated. At the granule cell (GC) to PC synapses, both N‐ and P/Q‐type Ca²⁺‐channels were found playing a role in glutamate exocytosis, but with no significant phenotypic compensation of the Ca_v2.1(α_1A) deletion. We also found that the P/Q‐ but not N‐type Ca²⁺‐channel is indispensable at the autaptic contacts between PCs. Tuning of the GC activity implicates both synaptic and sustained extrasynaptic γ‐aminobutyric acid (GABA) release, only the former was greatly impaired in the absence of P/Q‐type Ca²⁺‐channels. Overall, our data demonstrate that both P/Q‐ and N‐type Ca²⁺‐channels play a role in glutamate release, while the P/Q‐type is essential in GABA exocytosis in the cerebellum. Contrary to the other regions of the CNS, the effect of deleting the Ca_v2.1(α_1A) subunit is partially or not compensated at the inhibitory synapses. This may explain why cerebellar ataxia is observed at the mice lacking functional P/Q‐type channels.     

Serotonin (5‐HT) regulates neurite outgrowth through 5‐HT1A and 5‐HT7 receptors in cultured hippocampal neurons

Serotonin (5‐HT) production and expression of 5‐HT receptors (5‐HTRs) occur early during prenatal development. Recent evidence suggests that, in addition to its classical role as a neurotransmitter, 5‐HT regulates neuronal connectivity during mammalian development by modulating cell migration and neuronal cytoarchitecture. Given the variety of 5‐HTRs, researchers have had difficulty clarifying the specific role of each receptor subtype in brain development. Signalling mediated by the G‐protein‐coupled 5‐HT_1AR and 5‐HT₇R, however, has been associated with neuronal plasticity. Thus, we hypothesized that 5‐HT promotes neurite outgrowth through 5‐HT_1AR and 5‐HT₇R. The involvement of 5‐HT_1AR and 5‐HT₇R in the morphology of rat hippocampal neurons was evaluated by treating primary cultures at 2 days in vitro with 5‐HT and specific antagonists for 5‐HT_1AR and 5‐HT₇R (WAY‐100635 and SB269970, respectively). The stimulation of hippocampal neurons with 100 nM 5‐HT for 24 hr produced no effect on either the number or the length of primary neurites. Nonetheless, after 5HT₇R was blocked, the addition of 5‐HT increased the number of primary neurites, suggesting that 5HT₇R could inhibit neuritogenesis. In contrast, 5‐HT induced secondary neurite outgrowth, an effect inhibited by 1 μM WAY‐100635 or SB269970. These results suggest that both serotonergic receptors participate in secondary neurite outgrowth. We conclude that 5‐HT_1AR and 5‐HT₇R regulate neuronal morphology in primary hippocampal cultures by promoting secondary neurite outgrowth. © 2014 Wiley Periodicals, Inc.     

5‐HT1B autoreceptor regulation of serotonin transporter activity in synaptosomes

Serotonin‐1B (5‐HT_1B) autoreceptors are located in serotonin (5‐HT) terminals, along with serotonin transporters (SERT), and play a critical role in autoregulation of serotonergic neurotransmission and are implicated in disorders of serotonergic function, particularly emotional regulation. SERT modulates serotonergic neurotransmission by high‐affinity reuptake of 5‐HT. Alterations in SERT activity are associated with increased risk for depression and anxiety. Several neurotransmitter receptors are known to regulate SERT K_m and V_max, and previous work suggests that 5‐HT_1B autoreceptors may regulate 5‐HT reuptake, in addition to modulating 5‐HT release and synthesis. We used rotating disk electrode voltammetry to investigate 5‐HT_1B autoreceptor regulation of SERT‐mediated 5‐HT uptake into synaptosomes. The selective 5‐HT_1B antagonist SB224289 decreased SERT activity in synaptosomes prepared from wild‐type but not 5‐HT_1B knockout mice, whereas SERT uptake was enhanced after pretreatment with the selective 5‐HT_1B agonist CP94253. Furthermore, SERT activity varies as a function of 5‐HT_1B receptor expression—specifically, genetic deletion of 5‐HT_1B decreased SERT function, while viral‐mediated overexpression of 5‐HT_1B autoreceptors in rat raphe neurons increased SERT activity in rat hippocampal synaptosomes. Considered collectively, these results provide evidence that 5‐HT_1B autoreceptors regulate SERT activity. Because SERT clearance rate varies as a function of 5‐HT_1B autoreceptor expression levels and is modulated by both activation and inhibition of 5‐HT_1B autoreceptors, this dynamic interaction may be an important mechanism of serotonin autoregulation with therapeutic implications. Synapse, 2012. © 2012 Wiley Periodicals, Inc.     

Galanin inhibition of voltage‐dependent Ca2+ influx in rat cultured myenteric neurons is mediated by galanin receptor 1

Galanin activates three receptors, the galanin receptor 1 (GalR1), GalR2, and GalR3. In the gastrointestinal tract, GalR1 mediates the galanin inhibition of cholinergic transmission to the longitudinal muscle and reduction of peristalsis efficiency in the small intestine. Galanin has also been shown to inhibit depolarization‐evoked Ca²⁺ increases in cultured myenteric neurons. Because GalR1 immunoreactivity is localized to cholinergic myenteric neurons, we hypothesized that this inhibitory action of galanin on myenteric neurons is mediated by GalR1. We investigated the effect of galanin 1‐16, which has high affinity for GalR1 and GalR2, in the presence or absence of the selective GalR1 antagonist, RWJ‐57408, and of galanin 2‐11, which has high affinity for GalR2 and GalR3, on Ca²⁺ influx through voltage‐dependent Ca²⁺ channels in cultured myenteric neurons. Myenteric neurons were loaded with fluo‐4 and depolarized by high K⁺ concentration to activate voltage‐dependent Ca²⁺ channels. Intracellular Ca²⁺ levels were quantified with confocal microscopy. Galanin 1‐16 (0.01–1 μM) inhibited the depolarization‐evoked Ca²⁺ increase in a dose‐dependent manner with an EC₅₀ of 0.172 μM. The selective GalR1 antagonist, RWJ‐57408 (10 μM), blocked the galanin 1‐16 (1 μM)‐mediated inhibition of voltage‐dependent Ca²⁺ channel. By contrast, the GalR2/GalR3 agonist, galanin 2‐11 did not affect the K⁺‐evoked Ca²⁺ influx in myenteric neurons. GalR1 immunoreactivity was localized solely to myenteric neurons in culture, as previously observed in intact tissue. These findings indicate that the inhibition of depolarization‐evoked Ca²⁺ influx in myenteric neurons in culture is mediated by GalR1 and confirm the presence of functional GalR1 in the myenteric plexus. This is consonant with the hypothesis that GalR1 mediates galanin inhibition of transmitter release from myenteric neurons. © 2008 Wiley‐Liss, Inc.