Stimulation of chemokine CXC receptor 4 induces synaptic depression of evoked parallel fibers inputs onto Purkinje neurons in mouse cerebellum

In the present work, we studied the effects of the stimulation of the chemokine CXC receptor 4 (CXCR4) by the stromal-derived cell growth factor-1alpha (SDF-1alpha) on the evoked excitatory postsynaptic current. This was generated in Purkinje neurons (PN) from mouse cerebellar slices by the stimulation of parallel fibers. It was found that the amplitude of EPSC was reversibly reduced by SDF-1alpha application. This effect was dose-dependent (IC(50)=0.34 nM) and was abolished by the anti-CXCR4 monoclonal antibody (mAb) 12G5. This SDF-1alpha-induced synaptic depression was caused by a decrease of evoked glutamate release, rather than a decrease in the postsynaptic glutamate receptor (GluR) sensitivity, as the mean amplitude of the spontaneous EPSCs was not influenced by chemokine application. Moreover, NMDA receptors (NMDARs) are involved in EPSC depression being inhibited by the NMDAR blocker 2-amino-5-phosphonopentanoic acid (AP-5). The mechanisms by which SDF-1alpha modulates neurotransmission in the cerebellar cortex are discussed.     

Short-term plasticity of Bergmann glial cell extrasynaptic currents during parallel fiber stimulation in rat cerebellum

Bergmann glial cells (BGC) enclose the synapses of Purkinje neurons (PN) and interneurons in the molecular layer of the cerebellar cortex. During synaptic transmission, glutamate evokes inward currents in the glia by activation of Ca2+-permeable aminohydroxymethylisoxazole propionic acid receptors (AMPAR) and electrogenic transporters. We describe the plasticity of BGC currents during paired-pulse and repetitive stimulation of parallel fibers in cerebellar slices. Paired-pulse facilitation (PPF) of BGC AMPAR currents was 4-fold, twice that of PN PPF. Experiments with a low-affinity AMPAR antagonist showed an increase in extrasynaptic glutamate concentration during the second pulse of the pair. PPF of glial transporter currents was 1.8-fold, similar to synaptic PPF. Tetanic stimulation revealed that facilitation of BGC AMPAR currents is not sustained during high-frequency stimulation, and substantial depression is observed after a few pulses. Consequently, Ca2+ influx through glial AMPARs would initially be facilitated but subsequently depressed, generating a transient Ca2+ influx in response to a sustained tetanus. This pattern of plasticity may be important in enabling Bergmann glial cell processes to detect and support synapses with high-frequency input. Finally, a new current was observed in BGC during repetitive stimulation. It was blocked by NBQX and intracellular GDP-beta-S, increased by glutamate uptake inhibition, had PPF similar to synaptic PPF, and was unaffected by an inhibitor of fast glial AMPAR currents. The evidence suggests that activation of neuronal AMPARs causes the release of a paracrine messenger to activate a G-protein coupled receptor in the BGC.     

Chemokine receptor CXCR2 regulates the functional properties of AMPA-type glutamate receptor GluR1 in HEK cells

Experiments were conducted in both HEK cells and cerebellar neurons to investigate whether CXC chemokine receptor 2 (CXCR2) is functionally coupled to GluR1. The co-expression of CXCR2 with GluR1 in HEK cells increased (i) the GluR1 "apparent" affinity for the transmitter; (ii) the GluR1 channel open probability; and (iii) GluR1 binding site cooperativity upon CXCR2 stimulation with CXC chemokine ligand 2 (CXCL2). The affinity of C-terminal-deleted GluR1 for glutamate (Glu) remained stable instead. Furthermore, CXCL2 increased the binding site cooperativity of AMPA receptors in rat cerebellar granule cells; and the amplitude of spontaneous excitatory postsynaptic current (sEPSCs) in Purkinje neurons (PNs). Our findings indicate that the coupling of CXCR2 with GluR1 may modulate glutamatergic synaptic transmission.     

Elevation of intradendritic sodium concentration mediated by synaptic activation of metabotropic glutamate receptors in cerebellar Purkinje cells

Cerebellar Purkinje cells express both ionotropic glutamate receptors and metabotropic glutamate receptors. Brief tetanic stimulation of parallel fibers in rat and mouse cerebellar slices evokes a slow excitatory postsynaptic current in Purkinje cells that is mediated by the mGluR1 subtype of metabotropic glutamate receptors. The effector system underlying this mGluR1 EPSC has not yet been identified. In the present study, we recorded the mGluR1 EPSC using the whole-cell patch-clamp technique in combination with microfluorometric recordings of the intracellular sodium concentration ([Na+]i) by means of the fluorescent sodium indicator SBFI. The mGluR1 EPSC was induced by local parallel fibre stimulation in the presence of the ionotropic glutamate receptor antagonists NBQX and D-APV and the GABAA receptor antagonists bicuculline or picrotoxin. The mGluR1 EPSC was associated with an increase in [Na+]i that was restricted to a specific portion of the dendritic tree. The mGluR1 EPSC as well as the increase in [Na+]i were inhibited by the mGluR antagonist S-MCPG. In the presence of NBQX, D-APV, pictrotoxin and TTX, bath application of the selective mGluR agonist 3,5-DHPG induced an elevation in [Na+]i which extended over the whole dendritic field of the Purkinje cell. This finding demonstrates that the mGluR1-mediated postsynaptic current leads to a significant influx of sodium into the dendritic cytoplasm of Purkinje cells and thereby provides a novel intracellular signalling mechanism that might be involved in mGluR1-dependent synaptic plasticity at this synapse.     

Trans-ACPD-induced Ca2+ signals in cerebellar Purkinje cells.

A combination of intracellular recording and fluorometric measurements of cytosolic calcium [( Ca2+]i) was used to locate changes in [Ca2+]i induced by the specific metabotropic glutamate receptor (mGluR) agonist trans-D,L-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), in Purkinje cells of rat cerebellar slices. Under voltage-clamp conditions, application of t-ACPD (100 microM) induced an inward current accompanied by a large increase in [Ca2+]i located primarily in the soma but also, to a lesser degree, in restricted parts of the dendrites. In contrast, elevations of [Ca2+]i associated with calcium spikes were confined to the dendrites and inward currents of a similar amplitude induced by (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), an agonist of ionotropic glutamate receptors, did not raise [Ca2+]i.     

Afferent-target cell interactions in the cerebellum: negative effect of granule cells on Purkinje cell development in lurcher mice.

Lurcher (Lc) is a gain-of-function mutation in the delta2 glutamate receptor gene that results in a large, constitutive inward current in the cerebellar Purkinje cells of +/Lc mice. +/Lc Purkinje cells fail to differentiate fully and die during postnatal development. In normal mice, interactions with granule cells promote Purkinje cell dendritic differentiation. Partial destruction of the granule cell population in young +/Lc mice by x irradiation resulted in a significant increase in Purkinje cell dendritic growth and improved cytoplasmic structure but did not prevent Purkinje cell death. These results indicate two components to Purkinje cell abnormalities in +/Lc mice: a retardation/blockade of dendritic development that is mediated by interactions with granule cells and the death of the cell. Thus, the normal trophic effects of granule cell interaction on Purkinje cell development are absent in the +/Lc cerebellum, suggesting that granule cells are powerful regulators of Purkinje cell differentiation.     

Close homologue of adhesion molecule L1 promotes survival of Purkinje and granule cells and granule cell migration during murine cerebellar development

Several L1‐related adhesion molecules, expressed in a well‐coordinated temporospatial pattern during development, are important for fine tuning of specific cerebellar circuitries. We tested the hypothesis that CHL1, the close homologue of L1, abundantly expressed in the developing and adult cerebellum, is also required for normal cerebellar histogenesis. We found that constitutive ablation of CHL1 in mice caused significant loss (20–23%) of Purkinje and granule cells in the mature 2‐month‐old cerebellum. The ratio of stellate/basket interneurons to Purkinje cells was abnormally high (+38%) in CHL1‐deficient (CHL1?/?) mice compared with wild‐type (CHL1+/+) littermates, but the γ‐aminobutyric acid (GABA)ergic synaptic inputs to Purkinje cell bodies and dendrites were normal, as were numbers of Golgi interneurons, microglia, astrocytes, and Bergmann glia. Purkinje cell loss occurred before the first postnatal week and was associated with enhanced apoptosis, presumably as a consequence of CHL1 deficiency in afferent axons. In contrast, generation of granule cells, as indicated by in vivo analyses of cell proliferation and death, was unaffected in 1‐week‐old CHL1?/? mice, but numbers of migrating granule cells in the molecular layer were increased. This increase was likely related to retarded cell migration because CHL1?/? granule cells migrated more slowly than CHL1+/+ cells in vitro, and Bergmann glial processes guiding migration in vivo expressed CHL1 in wild‐type mice. Granule cell deficiency in adult CHL1?/? mice appeared to result from decreased precursor cell proliferation after the first postnatal week. Our results indicate that CHL1 promotes Purkinje and granule cell survival and granule cell migration during cerebellar development. J. Comp. Neurol. 513:496–510, 2009. © 2009 Wiley‐Liss, Inc.     

Localization of endoplasmic reticulum and plasma membrane Ca2+-ATPases in subcellular fractions and sections of pig cerebellum

Subcellular fractions and sections of the cerebellum were analysed to evaluate the relative activity and distribution of organellar and plasma membrane Ca2+-ATPases (SERCA and PMCA). Western blot analysis of the fractions with IID8 or Y/1F4 SERCA-specific antibodies or else with 5F10 or pbPMCA antibodies, specific to PMCA pump, revealed a major content of SERCA protein in microsomes and of PMCA protein in plasma membrane vesicles. The Ca2+-ATPase activity of microsomes was more sensitive to thapsigargin, a SERCA-specific inhibitor, whereas the activity of the plasma membrane vesicle fraction was inhibited more by vanadate, a blocker of PMCA activity. The SERCA and PMCA distribution analysed in cerebellar sections revealed IID8 antibody reactions in Purkinje cell cytoplasm, granule cells and cerebellar glomeruli. Y/1F4 gave immunostaining in Purkinje cells, molecular layer interneurons (basket and stellate cells) and glomeruli, but granule cells were not labelled. The 5F10 antibody reacted with Purkinje cells, including their dendritic spines, as well as cerebellar glomeruli, whereas the pbPMCA antibody labelled several processes in all three layers and some synaptic interaction sites. The differential content and localization of the two types of Ca2+ pumps in specific neuronal areas of pig cerebellum indicate precise Ca2+ requirements of specific cellular regions.     

Roles of glutamate transporters in shaping excitatory synaptic currents in cerebellar Purkinje cells

Several subtypes of glutamate transporters are abundantly expressed near the excitatory synapses on cerebellar Purkinje cells. We investigated the roles of the glutamate transporters in shaping the excitatory postsynaptic currents (EPSCs) and regulating the levels of extracellular glutamate in the mouse cerebellum using a potent blocker of glutamate transporters, dl-threo-beta-benzyloxyaspartate (dl-TBOA). This drug markedly prolonged AMPA receptor-mediated EPSCs in Purkinje cells evoked by stimulating both parallel fibres and climbing fibres. The decay phase of the prolonged EPSCs was fitted by double exponentials, of which the slower component was preferentially inhibited by a low-affinity competitive antagonist of AMPA receptors, gamma-d-glutamyl-glycine, indicating that the slow component induced by dl-TBOA was the AMPA receptor-mediated current activated by lower concentrations of glutamate than those contributing to the peak of the EPSC. This result suggests that dl-TBOA prolongs the stay of synaptically released glutamate in the synaptic cleft and also induces glutamate spillover to extrasynaptic targets as well as neighbouring synapses. Furthermore, high concentrations of dl-TBOA in the presence of cyclothiazide generated a continuous inward current in Purkinje cells, of which the amplitude reached the peak level of the climbing-fibre EPSC. This continuous inward current was abolished by the blocker of AMPA receptors, indicating that the strong inhibition of glutamate uptake causes the rapid accumulation of glutamate in the extracellular space. These results highlight the importance of glutamate transporters in maintaining the proper glutamatergic transmission in Purkinje cell synapses.     

Depression of parallel and climbing fiber transmission to Bergmann glia is input specific and correlates with increased precision of synaptic transmission

In the cerebellar cortex, Bergmann glia enclose the synapses of both parallel and climbing fiber inputs to the Purkinje neuron. The glia express Ca(2+)-permeable AMPA receptors, and the GLAST and GLT-1 classes of glutamate transporter, which are activated by glutamate released during synaptic transmission. We have previously reported that parallel fiber to Bergmann glial transmission in rat cerebellar slices exhibits a form of frequency-dependent plasticity, namely long-term depression, following repetitive stimulation at 0.1-1 Hz. Here, we report that this form of plasticity is also present at the climbing fiber input, that climbing and parallel fibers can be depressed independently, that discrete parallel fiber inputs can also be depressed independently, and that depression is maintained when a distributed array of parallel fibers are stimulated (in contrast to several forms of synaptic plasticity at the Purkinje neuron). Depression of glutamate transporter currents does not correlate with a decrease in the stringency with which Purkinje neuron synapses are isolated. Rather, postsynaptic currents in Purkinje neurons decay more rapidly and perisynaptic metabotropic glutamate receptors are activated less effectively after stimulation at 0.2 and 1 Hz, suggesting that depression arises from a decrease in extrasynaptic glutamate concentration and not from impairment of glutamate clearance in and around the synapse. These results indicate that neuron-glial plasticity is activity dependent, input specific and does not require spillover between adjacent synapses to manifest. They also argue against a withdrawal of the glial sheath from synaptic regions as the putative mechanism of plasticity.     

Integrin subunit gene expression is regionally differentiated in adult brain.

Integrins are a diverse family of heterodimeric (alphabeta) adhesion receptors recently shown to be concentrated within synapses and involved in the consolidation of long-term potentiation. Whether neuronal types or anatomical systems in the adult rat brain are coded by integrin type was studied in the present experiments by mapping the relative densities of mRNAs for nine alpha and four beta subunits. Expression patterns were markedly different and in some regions complementary. General results and areas of notable labeling were as follows: alpha1-limited neuronal expression, neocortical layer V, hippocampal CA3; alpha3 and alpha5-diffuse neuronal and glial labeling, Purkinje cells, hippocampal stratum pyramidale, locus coeruleus (alpha3); alpha4- discrete limbic regions, olfactory cortical layer II, hippocampal CA2; alpha6-most prominently neuronal, neocortical subplate, endopiriform, subiculum; alpha7-discrete, all neocortical layers, hippocampal granule cells and CA3, cerebellar granule and Purkinje cells, all efferent cranial nerve nuclei; alpha8-discrete neuronal, deep cortex, hippocampal CA1, basolateral amygdala, striatum; alphaV-all cortical layers, striatum, Purkinje cells; beta4-dentate gyrus granule cells; beta5-broadly distributed, neocortex, medial amygdala, cerebellar granule and Purkinje cells, efferent cranial nerve nuclei; alpha2, beta2, and beta3-mRNAs not detected. These results establish that brain subfields express different balances of integrin subunits and thus different integrin receptors. Such variations will determine which matrix proteins are recognized by neurons and the types of intraneuronal signaling generated by matrix binding. They also could generate important differences in synaptic plasticity across brain systems.     

Voltage clamp analysis of the effect of excitatory amino acids and derivatives on Purkinje cell dendrites in rat cerebellar slices maintained in vitro

A voltage clamp analysis of the effects ofl-aspartate,l-glutamate and related derivatives on Purkinje cell dendrites was performed in rat cerebellar slices maintained in vitro. Short iontophoretic pulse applications ofl-aspartate andl-glutamate in the dendritic field of Purkinje cells induced dose-dependent inward currents with fast onset and recovery. Quisqualate application also gave rise to well developed inward currents with fast onset and slow recovery, whereas N-methyl-d,l-aspartate had no or little effect on Purkinje cell membranes unless prolonged (several seconds) applications were used. Steady applications of low doses of N-methyl-d,l-aspartate much more severely depressedl-aspartate thanl-glutamate mediated responses, whereas inward currents due to quisqualate were unaffected. Inward currents due to quisqualate were often more reduced than those due tol-aspartate by steady applications of 2-amino-5-phosphonovalerate, and the antagonistic action of this drug on responses due tol-glutamate was very weak. These results suggest that receptors of Purkinje cells for glutamate and aspartate are different, and are also different from N-methyl-d-aspartate and quisqualate receptors.     

Acute ethanol alters the firing pattern and glutamate response of cerebellar Purkinje neurons in culture

Modified explant cultures derived from the cortical region of fetal rat cerebellum, and extracellular recording techniques were used to examine the sensitivity and response or cerebellar neurons, isolated from extracellular afferent input, to acute ethanol (EtOH) exposure. Recordings were made from Purkinje neurons (PNs) and granule cells maintained in culture for several weeks, with the emphasis on the PN. Both the PNs and granule cells exhibited spontaneous activity inculture, but, unlike the PNs, not all of the granule cells were spontaneously active. The majority of PNs studied exhibited a high frequency, regular simple spike firing pattern, previously shown to be endogenously generated by voltage-sensitive mechanisms intrinsic to the PN. The granule cells exhibited slow, irregular patterns of activity. EtOH at doses as low as 22 mM (100 mg%), a concentration that reflects blood levels during EtOH intoxication, altered the spontaneous activity of both neuronal types, demonstrating that EtOH has direct actions on cerebellar neurons. In the PNs, acute EtOH (20–80 mM) produced an increase in the regularity of the spontaneous activity and either a transient increase or no change in firing rate. Acute EtOH also significantly altered the response of PNs to the excitatory transmitter glutamate. In the granule cells, acute EtOH altered firing pattern with small and variable effects on firing rate. These data demonstrate that there are multiple sites of EtOH action in the cerebellum and that changes in PN activity with acute EtOH exposure may occur via direct actions on the PN and indirect actions via synaptically connected cerebellar neurons. The demonstration of EtOH-sensitive sites intrinsic to the cerebellum suggests that EtOH actions at these sites contribute to alterations in PN activity that occur in vivo after acute EhOH exposure.     

Organization of cerebellar cortex secondary to deficit of granule cells in weaver mutant mice

This study deals with some consequences of the early postnatal abnormalities of cerebellar Bergmann glial fibers and granule cell neurons. (1) Cerebellar size is mildly reduced in heterozygous weaver (+/wv) mice and markedly reduced in homozygotes (wv/wv), but the pattern of fissures is essentially normal. Comparison with other mutants displaying small cerebella suggests that cell proliferation rate in the external granular layer is a key deteminant of cerebellar cortical folding. (2) Mossy fiber terminals differentiate on schedule despite the reduced number and abnormal positions of granule cells. However, many of them enter the modified molecular layer, and as noted especially in noninbred wv/wv mice one to two years old, form synapses with dendrites of aberrant granule cells. Where granule cells are absent, mossy fibers form more than the normal number of synapses with dendrites of Golgi type II neurons. (3) Purkinje cells are only mildly affected by the disorder of neighbouring cells. Their dendrites grow abnormally into the territory occupied by external granule cells, reach the external surface, and may turn inward. They form few tertiary branches. Dendritic spines are present in profusion and show membrane thickenings akin to normal postsynaptic elements. Although they receive no axonal contacts, the spines persist, enveloped by glial processes, for at least two years. Apart from the absence of parallel fiber contacts, afferent and intrinsic axons form the normal classes of synaptic connections with Purkinje cells. (4) Interneurons of the molecular layer are generated on schedule. At the time of their earliest recognition, they reside in the external granular layer, where they receive synaptic contacts from climbing fibers and other interneurons. In the absence of parallel fibers, interneurons differentiate in situ but their dendrites are abortive and randomly oriented. Growth of their dendrites, in contrast to that of Purkinje cell dendrites, appears to be markedly influenced by the organization of the local cellular milieu.     

Long-term depression of neuron to glial signalling in rat cerebellar cortex

Bergmann glial cells enclose synapses throughout the molecular layer of the cerebellum and express extrasynaptic AMPA receptors and glutamate transporters. Accordingly, stimulation of parallel fibres leads to the generation of inward currents in the glia due to AMPA receptor activation and electrogenic uptake of glutamate. Elimination of AMPA receptor Ca(2+) permeability leads to the withdrawal of glial processes and synaptic dysfunction, suggesting that AMPA receptor-mediated Ca(2+) signalling is essential for glial support of the neuronal network. Here we show that glial extrasynaptic currents (ESCs) exhibit activity-dependent plasticity, specifically, long-term depression during repetitive stimulation of parallel fibres at low frequencies (0.033-1 Hz) -- conditions in which Purkinje neuron excitatory postsynaptic currents (EPSCs) remain stable. Both the rate of onset and the magnitude of ESC depression increased with stimulation frequency. Depression was reversible following brief periods of stimulation, but became increasingly persistent as the duration of repetitive stimulation increased. All glial currents -- AMPA receptors, glutamate transporter and a recently discovered slow 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide (NBQX)-sensitive current -- were depressed. Increasing presynaptic release probability by doubling external Ca(2+) concentration did not affect the time course of depression, suggesting that neither decreased release probability nor fatigue of release sites contribute to depression. Inhibition of glutamate uptake caused a dramatic enhancement of the rate of depression, implicating glutamate in the underlying mechanism. The strength of neuron to glial signalling in the cerebellum is therefore dynamically regulated, independently of adjacent synapses, by the frequency of parallel fibre activity.     

Complex effects of stromal cell-derived factor-1 alpha on melanin-concentrating hormone neuron excitability

Stromal cell-derived factor 1alpha (SDF-1alpha), a chemoattractant for leucocytes and neurons, and its receptor, CXCR4 are expressed in subsets of neurons of specific brain areas. In rat lateral hypothalamic area (LHA) we show, using immunocytochemistry, that CXCR4 is localized within melanin-concentrating hormone (MCH)-expressing neurons, mainly involved in feeding behaviour regulation. We investigated whether SDF-1alpha may control MCH neuronal activity. Patch-clamp recordings in rat LHA slices revealed multiple effects of SDF-1alpha on the membrane potential of MCH neurons, indirect through glutamate/GABA release and direct through GIRK current activation. Moreover, SDF-1alpha at 0.1-1 nM decreased peak and discharge frequency of action potential evoked by current pulses. These effects were further confirmed in voltage-clamp experiments, SDF-1alpha depressing both potassium and sodium currents. At 10 nM, however, SDF-1alpha increased peak and discharge frequency of action potential evoked by current pulses. Using a specific CXCR4 antagonist, we demonstrated that only the depressing effect on AP discharge was mediated through CXCR4 while the opposite effect was indirect. Together, our studies reveal for the first time a direct effect of SDF-1alpha on voltage-dependent membrane currents of neurons in brain slices and suggest that this chemokine may regulate MCH neuron activity.     

PLCgamma signaling underlies BDNF potentiation of Purkinje cell responses to GABA

Brain-derived neurotrophic factor (BDNF) regulates neuronal survival, neurite outgrowth, and excitatory synaptic transmission. We reported recently that acute BDNF exposure decreased gamma-aminobutyric acid (GABA) responses in cultured mouse cerebellar granule cells through tyrosine receptor kinase B (TrkB) receptor-mediated signaling. In the present study, we extend this work to investigate BDNF-induced modulation of GABA responses and GABA(A) receptor-mediated synaptic events in cerebellar slices. Thin (200 microm) parasagittal slices of cerebellum were prepared from postnatal Day 7 and 14 mice. Purkinje cells and granule cells, both of which express TrkB-like immunoreactivity, were identified for whole-cell recording. BDNF promptly enhanced GABA responses in Purkinje cells but, consistent with our previous finding in culture, attenuated those recorded in granule cells. In Purkinje cells, BDNF exposure shifted rightward the cumulative peak amplitude distribution for miniature inhibitory postsynaptic currents (mIPSCs) without changing the mIPSC frequency. BDNF-induced potentiation of Purkinje cell responses to GABA was blocked by TrkB-Fc (receptor body that sequesters BDNF), K252a (inhibitor of TrkB receptor autophosphorylation), U73122 (inhibitor of phospholipase-Cgamma [PLCgamma]), KN62 (specific inhibitor of calcium/calmodulin-dependent kinase), KT5720 (specific cyclic AMP-dependent kinase inhibitor), and by intracellular dialysis of Rp-cyclic AMP or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid). Overall, our results indicate that BDNF acutely potentiates GABA(A) receptor function in cerebellar Purkinje cells via the TrkB receptor-PLCgamma signal transduction cascade. In addition, we propose that cyclic AMP-mediated intracellular signaling mechanisms may facilitate manifestation of the BDNF-induced modulatory outcome.     

Role of the alpha-chemokine stromal cell-derived factor (SDF-1) in the developing and mature central nervous system

alpha-chemokines, which control the activation and directed migration of leukocytes, participate in the inflammatory processes in host defense response. One of the alpha-chemokines, CXCL12 or stromal cell-derived factor 1 (SDF-1), not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development as we discuss here. SDF-1 indeed activates the CXCR4 receptor expressed in a variety of neural cells, and this signaling results in diverse biological effects. It enhances migration and proliferation of cerebellar granule cells, chemoattracts microglia, and stimulates cytokine production and glutamate release by astrocytes. Moreover, it elicits postsynaptic currents in Purkinje cells, triggers migration of cortical neuron progenitors, and produces pain by directly exciting nociceptive neurons. By modulating cell signaling and survival during neuroinflammation, SDF-1 may also play a role in the pathogenesis of brain tumors, experimental allergic encephalitis, and the nervous system dysfunction associated with acquired immunodeficiency syndrome.     

Activation of presynaptic cAMP-dependent protein kinase is required for induction of cerebellar long-term potentiation.

Cerebellar long-term potentiation (LTP) is a persistent increase in the strength of the granule cell-Purkinje neuron synapse that occurs after brief stimulation of granule cell axons at 2-8 Hz. Previous work has indicated that cerebellar LTP induction requires presynaptic Ca influx, stimulation of Ca-sensitive adenylyl cyclase, and activation of PKA. The evidence implicating PKA has come from bath application of drugs during LTP induction, an approach that does not distinguish between PKA activation in the presynaptic or postsynaptic cell. Although bath application of PKA inhibitor drugs (KT5720, Rp-8CPT-cAMP-S) blocked LTP induction in granule cell-Purkinje neuron pairs in culture, selective application to granule cell or Purkinje neuron somata via patch pipettes did not. We hypothesized that presynaptic PKA activation is required for LTP induction but that drugs applied to the granule cell soma cannot diffuse to the terminal within this timescale. To test this hypothesis, we transfected cerebellar cultures with an expression vector encoding a peptide inhibitor of PKA [Rous sarcoma virus (RSV)-protein kinase A inhibitor (PKI)]. Transfection of RSV-PKI into presynaptic granule cells, but not postsynaptic Purkinje neurons or glial cells, blocked LTP induction produced by either synaptic stimulation or an exogenous cAMP analog. An expression vector encoding a control peptide with no PKA inhibitory activity was ineffective. These results show that induction of cerebellar LTP requires a presynaptic signaling cascade, including Ca influx, stimulation of Ca-sensitive adenylyl cyclase, and activation of PKA, and argue against a requirement for postsynaptic Ca signals or their sequelae.