Calcium stores in hippocampal synaptic boutons mediate short-term plasticity, store-operated Ca2+ entry, and spontaneous transmitter release

Evoked transmitter release depends upon calcium influx into synaptic boutons, but mechanisms regulating bouton calcium levels and spontaneous transmitter release are obscure. To understand these processes better, we monitored calcium transients in axons and presynaptic terminals of pyramidal neurons in hippocampal slice cultures. Action potentials reliably evoke calcium transients in axons and boutons. Calcium-induced calcium release (CICR) from internal stores contributes to the transients in boutons and to paired-pulse facilitation of EPSPs. Store depletion activates store-operated calcium channels, influencing the frequency of spontaneous transmitter release. Boutons display spontaneous Ca2+ transients; blocking CICR reduces the frequency of these transients and of spontaneous miniature synaptic events. Thus, spontaneous transmitter release is largely calcium mediated, driven by Ca2+ release from internal stores. Bouton store release is important for short-term synaptic plasticity and may also contribute to long-term plasticity.     

Calcium transients in cerebellar granule cell presynaptic terminals.

Calcium ions act presynaptically to modulate synaptic strength and to trigger neurotransmitter release. Here we detect stimulus-evoked changes in residual free calcium ([Ca2+]i) in rat cerebellar granule cell presynaptic terminals. Granule cell axons, known as parallel fibers, and their associated boutons, were labeled with several calcium indicators. When parallel fibers were extracellularly activated with stimulus trains, calcium accumulated in the terminals, producing changes in the fluorescence of the indicators. During the stimulus train, the fluorescence change per pulse became progressively smaller with the high affinity indicators Fura-2 and calcium green-2 but remained constant with the low affinity dyes BTC and furaptra. In addition, fluorescence transients of high affinity dyes were slower than those of low affinity indicators, which appear to accurately report the time course of calcium transients. Simulations show that differences in the observed transients can be explained by the different affinities and off rates of the fluorophores. The return of [Ca2+]i to resting levels can be approximated by an exponential decay with a time constant of 150 ms. On the basis of the degree of saturation in the response of high affinity dyes observed during trains, we estimate that each action potential increases [Ca2+]i in the terminal by several hundred nanomolar. These findings indicate that in these terminals [Ca2+]i transients are much larger and faster than those observed in larger boutons, such as those at the neuromuscular junction. Such rapid [Ca2+]i dynamics may be found in many of the terminals in the mammalian brain that are similar in size to parallel fiber boutons.     

Minimal Change in the Cytoplasmic Calcium Dynamics in Striatal GABAergic Neurons of a DYT1 Dystonia Knock-In Mouse Model

DYT1 dystonia is the most common hereditary form of primary torsion dystonia. This autosomal-dominant disorder is characterized by involuntary muscle contractions that cause sustained twisting and repetitive movements. It is caused by an in-frame deletion in the TOR1A gene, leading to the deletion of a glutamic acid residue in the torsinA protein. Heterozygous knock-in mice, which reproduce the genetic mutation in human patients, have abnormalities in synaptic transmission at the principal GABAergic neurons in the striatum, a brain structure that is involved in the execution and modulation of motor activity. However, whether this mutation affects the excitability of striatal GABAergic neurons has not been investigated in this animal model. Here, we examined the excitability of cultured striatal neurons obtained from heterozygous knock-in mice, using calcium imaging as indirect readout. Immunofluorescence revealed that more than 97% of these neurons are positive for a marker of GABAergic neurons, and that more than 92% are also positive for a marker of medium spiny neurons, indicating that these are mixed cultures of mostly medium spiny neurons and a few (~5%) GABAergic interneurons. When these neurons were depolarized by field stimulation, the calcium concentration in the dendrites increased rapidly and then decayed slowly. The amplitudes of calcium transients were larger in heterozygous neurons than in wild-type neurons, resulting in ~15% increase in cumulative calcium transients during a train of stimuli. However, there was no change in other parameters of calcium dynamics. Given that calcium dynamics reflect neuronal excitability, these results suggest that the mutation only slightly increases the excitability of striatal GABAergic neurons in DYT1 dystonia.     

PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior

Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.     

ApoER2 and VLDLr Are Required for Mediating Reelin Signalling Pathway for Normal Migration and Positioning of Mesencephalic Dopaminergic Neurons

The migration of mesencephalic dopaminergic (mDA) neurons from the subventricular zone to their final positions in the substantia nigra compacta (SNc), ventral tegmental area (VTA), and retrorubral field (RRF) is controlled by signalling from neurotrophic factors, cell adhesion molecules (CAMs) and extracellular matrix molecules (ECM). Reelin and the cytoplasmic adaptor protein Disabled-1 (Dab1) have been shown to play important roles in the migration and positioning of mDA neurons. Mice lacking Reelin and Dab1 both display phenotypes characterised by the failure of nigral mDA neurons to migrate properly. ApoER2 and VLDLr are receptors for Reelin signalling and are therefore part of the same signal transduction pathway as Dab1. Here we describe the roles of ApoER2 and VLDLr in the proper migration and positioning of mDA neurons in mice. Our results demonstrate that VLDLr- and ApoER2-mutant mice have both a reduction in and abnormal positioning of mDA neurons. This phenotype was more pronounced in VLDLr-mutant mice. Moreover, we provide evidence that ApoER2/VLDLr double-knockout mice show a phenotype comparable with the phenotypes observed for Reelin- and Dab1- mutant mice. Taken together, our results demonstrate that the Reelin receptors ApoER2 and VLDLr play essential roles in Reelin-mediated migration and positioning of mDA neurons.     

Regulation of presynaptic neurotransmission by macroautophagy

mTOR is a regulator of cell growth and survival, protein synthesis-dependent synaptic plasticity, and autophagic degradation of cellular components. When triggered by mTOR inactivation, macroautophagy degrades long-lived proteins and organelles via?sequestration into autophagic vacuoles. mTOR further regulates synaptic plasticity, and neurodegeneration occurs when macroautophagy is deficient. It is nevertheless unknown whether macroautophagy modulates presynaptic function. We find that the mTOR inhibitor rapamycin induces formation of autophagic vacuoles in prejunctional dopaminergic axons with associated decreased axonal profile volumes, synaptic vesicle numbers, and evoked dopamine release. Evoked dopamine secretion was enhanced and recovery was accelerated in?transgenic mice in which macroautophagy deficiency was restricted to dopaminergic neurons; rapamycin failed to decrease evoked dopamine release in the striatum of these mice. Macroautophagy that follows mTOR inhibition in presynaptic terminals, therefore, rapidly alters presynaptic structure and neurotransmission.     

Specific Neurochemical Derangements of Brain Projecting Neurons in Apolipoprotein E-Deficient Mice

Abstract: Apolipoprotein E (apoE)-deficient mice provide a useful system for studying the role of apoE in neuronal maintenance and repair. Previous studies revealed specific memory impairments in these mice that are associated with presynaptic derangements in projecting forebrain cholinergic neurons. In the present study we examined whether dopaminergic, noradrenergic, and serotonergic projecting pathways of apoE-deficient mice are also affected and investigated the mechanisms that render them susceptible. The densities of nerve terminals of forebrain cholinergic projections were monitored histochemically by measurements of acetylcholinesterase activity, whereas those of the dopaminergic nigrostriatal pathway, the noradrenergic locus coeruleus cortical projection, and the raphe-cortical serotonergic tract were measured autoradiographically using radioligands that bind specifically to the respective presynaptic transporters of these neuronal tracts. The results obtained revealed that synaptic densities of cholinergic, noradrenergic, and serotonergic projections in specific brain regions of apoE-deficient mice are markedly lower than those of controls. Furthermore, the extent of presynaptic derangement within each of these tracts was found to be more pronounced the further away the nerve terminal is from its cell body. In contrast, the nerve terminal density of the dopaminergic neurons that project from the substantia nigra to the striatum was unaffected and was similar to that of the controls. The rank order of these presynaptic derangements at comparable distances from the respective cell bodies was found to be septohippocampal cholinergic > nucleus basalis cholinergic > locus coeruleus adrenergic > raphe serotonergic ? nigrostriatal dopaminergic, which interestingly is similar to that observed in Alzheimer's disease. These results suggest that two complementary factors determine the susceptibility of brain projecting neurons to apoE deficiency: pathway-specific differences and the distance of the nerve terminals from their cell body.     

Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals

Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.     

Fluorescence-Based Monitoring of In Vivo Neural Activity Using a Circuit-Tracing Pseudorabies Virus

The study of coordinated activity in neuronal circuits has been challenging without a method to simultaneously report activity and connectivity. Here we present the first use of pseudorabies virus (PRV), which spreads through synaptically connected neurons, to express a fluorescent calcium indicator protein and monitor neuronal activity in a living animal. Fluorescence signals were proportional to action potential number and could reliably detect single action potentials in vitro. With two-photon imaging in vivo, we observed both spontaneous and stimulated activity in neurons of infected murine peripheral autonomic submandibular ganglia (SMG). We optically recorded the SMG response in the salivary circuit to direct electrical stimulation of the presynaptic axons and to physiologically relevant sensory stimulation of the oral cavity. During a time window of 48 hours after inoculation, few spontaneous transients occurred. By 72 hours, we identified more frequent and prolonged spontaneous calcium transients, suggestive of neuronal or tissue responses to infection that influence calcium signaling. Our work establishes in vivo investigation of physiological neuronal circuit activity and subsequent effects of infection with single cell resolution.     

Effects of Gabapentin on Calcium Transients in Neurons of the Rat Dorsal Root Ganglia

In our experiments on rat dorsal root ganglia (DRG) neurons, we studied the effects of an antiepileptic agent, gabapentin, on calcium transients evoked by depolarization of the membrane using the fluorescence calciumsensitive dye Fura-2/AM. Application of gabapentin to neurons with large-diameter somata practically did not change the characteristics of calcium transients. In mid-sized neurons, the amplitude of transients decreased, on average, by 27% with respect to the control, while in small-sized neurons the transients changed insignificantly (on average, less than by 7%). The mid-sized neurons were additionally subjected to the capsaicin test, which allowed us to differentiate primary nociceptive neurons of this group where TRPV1-type channels are expressed. In capsaicin-sensitive neurons, application of gabapentin led to a decrease in the amplitude of calcium transients, on average, by 37%, while such a decrease was only 16% in capsaicininsensitive neurons. Based on our own data and findings of other researchers on the ability of gabapentin to demonstrate affine binding with the accessory α₂δ subunit of voltage-dependent calcium channels and also on the peculiarities of expression of these channels in somatosensory neurons of the corresponding types, we discuss the probable pattern of expression of subunits of the α₂δ-1 subtype in DRG cells of different sizes. We demonstrated that the effects of gabapentin on calcium transients in nociceptive and hypothetically nonnociceptive mid-sized DRG neurons are selective (the effects in neurons involved in the sensation of acute pain are probably more intense). Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 281–287, July–August, 2008.     

The Niemann-Pick C1 protein in recycling endosomes of presynaptic nerve terminals

Niemann-Pick type C (NPC) disease is a fatal, neurodegenerative disorder caused in 95% of cases by loss of function of NPC1, a ubiquitous endosomal transmembrane protein. A biochemical hallmark of NPC deficiency is cholesterol accumulation in the endocytic pathway. Although cholesterol trafficking defects are observed in all cell types, neurons are the most vulnerable to NPC1 deficiency, suggesting a specialized function for NPC1 in neurons. We investigated the subcellular localization of NPC1 in neurons to gain insight into the mechanism of action of NPC1 in neuronal metabolism. We show that NPC1 is abundant in axons of sympathetic neurons and is present in recycling endosomes in presynaptic nerve terminals. NPC1 deficiency causes morphological and biochemical changes in the presynaptic nerve terminal. Synaptic vesicles from Npc1(-/-) mice have normal cholesterol content but altered protein composition. We propose that NPC1 plays a previously unrecognized role in the presynaptic nerve terminal and that NPC1 deficiency at this site might contribute to the progressive neurological impairment in NPC disease.     

Neurotransmitter Modulation of Neuronal Calcium Channels

There are many different calcium channels expressed in the mammalian nervous system, but N-type and P/Q-type calcium channels appear to dominate the presynaptic terminals of central and peripheral neurons. The neurotransmitter-induced modulation of these channels can result in alteration of synaptic transmission. This review highlights the mechanisms by which neurotransmitters affect the activity of N-type and P/Q-type calcium channels. The inhibition of these channels by voltage-dependent and voltage-independent mechanisms is emphasized because of the wealth of information available on the intracellular mediators and on the effect of these pathways on the single-channel gating.     

Regulation of N-type Voltage-Gated Calcium Channels and Presynaptic Function by Cyclin-Dependent Kinase 5

N-type voltage-gated calcium channels localize to?presynaptic nerve terminals and mediate key events?including synaptogenesis and neurotransmission.?While several kinases have been implicated in the modulation of calcium channels, their impact on presynaptic functions remains unclear. Here we report that the N-type calcium channel is a substrate for cyclin-dependent kinase 5 (Cdk5). The pore-forming α(1) subunit of the N-type calcium channel is phosphorylated in the C-terminal domain, and phosphorylation results in enhanced calcium influx due to increased channel open probability. Phosphorylation of the N-type calcium channel by Cdk5 facilitates neurotransmitter release and alters presynaptic plasticity by increasing the number of docked vesicles at the synaptic cleft. These effects are mediated by an altered interaction between N-type calcium channels and RIM1, which tethers presynaptic calcium channels to the active zone. Collectively, our results highlight a molecular mechanism by which N-type calcium channels are regulated by Cdk5 to affect presynaptic function.     

Presynaptically localized cyclic GMP-dependent protein kinase 1 is a key determinant of spinal synaptic potentiation and pain hypersensitivity

Synaptic long-term potentiation (LTP) at spinal neurons directly communicating pain-specific inputs from the periphery to the brain has been proposed to serve as a trigger for pain hypersensitivity in pathological states. Previous studies have functionally implicated the NMDA receptor-NO pathway and the downstream second messenger, cGMP, in these processes. Because cGMP can broadly influence diverse ion-channels, kinases, and phosphodiesterases, pre- as well as post-synaptically, the precise identity of cGMP targets mediating spinal LTP, their mechanisms of action, and their locus in the spinal circuitry are still unclear. Here, we found that Protein Kinase G1 (PKG-I) localized presynaptically in nociceptor terminals plays an essential role in the expression of spinal LTP. Using the Cre-lox P system, we generated nociceptor-specific knockout mice lacking PKG-I specifically in presynaptic terminals of nociceptors in the spinal cord, but not in post-synaptic neurons or elsewhere (SNS-PKG-I(-/-) mice). Patch clamp recordings showed that activity-induced LTP at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) was completely abolished in SNS-PKG-I(-/-) mice, although basal synaptic transmission was not affected. Analyses of synaptic failure rates and paired-pulse ratios indicated a role for presynaptic PKG-I in regulating the probability of neurotransmitter release. Inositol 1,4,5-triphosphate receptor 1 and myosin light chain kinase were recruited as key phosphorylation targets of presynaptic PKG-I in nociceptive neurons. Finally, behavioural analyses in vivo showed marked defects in SNS-PKG-I(-/-) mice in several models of activity-induced nociceptive hypersensitivity, and pharmacological studies identified a clear contribution of PKG-I expressed in spinal terminals of nociceptors. Our results thus indicate that presynaptic mechanisms involving an increase in release probability from nociceptors are operational in the expression of synaptic LTP on spinal-PAG projection neurons and that PKG-I localized in presynaptic nociceptor terminals plays an essential role in this process to regulate pain sensitivity.     

Presynaptic action potential amplification by voltage-gated Na+ channels in hippocampal mossy fiber boutons

Action potentials in central neurons are initiated near the axon initial segment, propagate into the axon, and finally invade the presynaptic terminals, where they trigger transmitter release. Voltage-gated Na(+) channels are key determinants of excitability, but Na(+) channel density and properties in axons and presynaptic terminals of cortical neurons have not been examined yet. In hippocampal mossy fiber boutons, which emerge from parent axons en passant, Na(+) channels are very abundant, with an estimated number of approximately 2000 channels per bouton. Presynaptic Na(+) channels show faster inactivation kinetics than somatic channels, suggesting differences between subcellular compartments of the same cell. Computational analysis of action potential propagation in axon-multibouton structures reveals that Na(+) channels in boutons preferentially amplify the presynaptic action potential and enhance Ca(2+) inflow, whereas Na(+) channels in axons control the reliability and speed of propagation. Thus, presynaptic and axonal Na(+) channels contribute differentially to mossy fiber synaptic transmission.     

Progressive Axonal Degeneration of Nigrostriatal Dopaminergic Neurons in Calcium-Independent Phospholipase A2β Knockout Mice

Calcium-independent phospholipase A₂β (iPLA₂β, PLA2G6) is essential for the remodeling of membrane glycerophospholipids. Mutations in this gene are responsible for autosomal recessive, young onset, L-dopa-responsive parkinsonism (PARK14), suggesting a neurodegenerative condition in the nigrostriatal dopaminergic system in patients with PLA2G6 mutations. We previously observed slowly progressive motor deficits in iPLA₂β-knockout (KO) mice. To clarify whether a deficiency of iPLA₂β leads to the degeneration of nigrostriatal dopaminergic neurons, we analyzed the striatum of iPLA₂β-KO mice. At all clinical stages, nerve terminals in the striatum were immunopositive for tyrosine hydroxylase (TH) and dopamine transporter (DAT) in wild-type (WT) control mice. In iPLA₂β-KO mice, focal loss of nerve terminals positive for TH and DAT was found from 56 weeks (early clinical stage), although iPLA₂β-KO mice at 56 weeks showed no significant decrease in the number of dopaminergic neurons in the substantia nigra compared with age-matched WT mice, as reported previously. At 100 weeks (late clinical stage), greater decreases in DAT immunoreactivity were observed in the striatum of iPLA₂β-KO mice. Moreover, strongly TH-positive structures, presumed to be deformed axons, were observed in the neuropils of the striatum of iPLA₂β-KO mice starting at 15 weeks (preclinical stage) and increased with age. These results suggest that the degeneration of dopaminergic neurons occurs mainly in the distal region of axons in iPLA₂β-KO mice.     

Calcium transients in growth cones and axons of cultured Helisoma neurons in response to conditioning factors

Accumulating evidence indicates that cytosolic calcium levels regulate growth cone motility and neurite extension. The purpose of this study was to determine if intracellular calcium levels also influence the initiation of neurite extension induced by growth-promoting factors. An in vitro preparation of axotomized neurons that can be maintained in the absence of growth-promoting factors was utilized. The distal axons of cultured Helisoma neurons plated into defined medium do not extend neurites until they are exposed to Helisoma brain-conditioned medium. This provided the opportunity to study the intracellular changes associated with neurite extension. Cytosolic calcium levels were monitored with the calcium-sensitive dye fura 2 at the distal axon. In control medium calcium levels in the distal axon were constant. However, transient elevations in cytosolic calcium in the axonal growth cone occurred after addition of conditioned medium and coincident with the initiation of neurite extension. Application of calcium channel blockers showed that the transients resulted from calcium influx across the neuronal membrane. The transients, however, were not required for neurite extension, although they did influence the rate and extent of neurite outgrowth. Simultaneous extracellular patch recordings demonstrated that the calcium transients were correlated temporally with an increase in rhythmic spontaneous electrical activity of cells, suggesting that conditioned medium influences ionic membrane properties of these neurons. © 1995 John Wiley & Sons, Inc.     

Dendritic NMDA receptors activate axonal calcium channels

NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca(2+) and monovalent cations, they could alter release directly by increasing presynaptic Ca(2+) or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca(2+) channels (VSCCs). Using two-photon microscopy to measure Ca(2+) excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca(2+) transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca(2+) entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons, indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca(2+) -dependent forms of presynaptic plasticity and release.