Journal of Neuroscience Cellular

Input-Specific GABAergic Signaling to Newborn Neurons in Adult Dentate Gyrus

Markwardt, S. J., Wadiche, J. I., Overstreet-Wadiche, L. S. — Wed, 02 Dec 2009 10:02:09 PST

Adult neurogenesis is the multistage process of generating neurons from adult neural stem cells. Accumulating evidence indicates that GABAergic depolarization is an important regulator of this process. Here, we examined GABAergic signaling to newly generated granule cells (GCs) of the adult mouse dentate gyrus. We show that the first synaptic currents in newborn GCs are generated by activation of GABA_A receptors by GABA with a spatiotemporal profile suggestive of transmitter spillover. However, the GABAergic response is not attributable to spillover from surrounding perisomatic synapses. Rather, our results suggest that slow synaptic responses in newborn GCs are generated by dedicated inputs that produce a relatively low concentration of GABA at postsynaptic receptors, similar to slow IPSCs in mature GCs. This form of synaptic signaling drives robust phasic depolarization of newborn GCs when the interneuron network is synchronously active, revealing a potential mechanism that translates hippocampal activity into regulation of adult neurogenesis via synaptic release of GABA.

Photoreceptor Coupling Is Controlled by Connexin 35 Phosphorylation in Zebrafish Retina

Li, H., Chuang, A. Z., O'Brien, J. — Wed, 02 Dec 2009 10:02:09 PST

Electrical coupling of neurons is widespread throughout the CNS and is observed among retinal photoreceptors from essentially all vertebrates. Coupling dampens voltage noise in photoreceptors and rod–cone coupling provides a means for rod signals to enter the cone pathway, extending the dynamic range of rod-mediated vision. This coupling is dynamically regulated by a circadian rhythm and light adaptation. We examined the molecular mechanism that controls photoreceptor coupling in zebrafish retina. Connexin 35 (homologous to Cx36 of mammals) was found at both cone–cone and rod–cone gap junctions. Photoreceptors showed strong Neurobiotin tracer coupling at night, extensively labeling the network of cones. Tracer coupling was significantly reduced in the daytime, showing a 20-fold lower diffusion coefficient for Neurobiotin transfer. The phosphorylation state of Cx35 at two regulatory phosphorylation sites, Ser110 and Ser276, was directly related to tracer coupling. Phosphorylation was high at night and low during the day. Protein kinase A (PKA) activity directly controlled both phosphorylation state and tracer coupling. Both were significantly increased in the day by pharmacological activation of PKA and significantly reduced at night by inhibition of PKA. The data are consistent with direct phosphorylation of Cx35 by PKA. We conclude that the magnitude of photoreceptor coupling is controlled by the dynamic phosphorylation and dephosphorylation of Cx35. Furthermore, the nighttime state is characterized by extensive coupling that results in a well connected cone network.

A Selective Glial Barrier at Motor Axon Exit Points Prevents Oligodendrocyte Migration from the Spinal Cord

Kucenas, S., Wang, W.-D., Knapik, E. W., Appel, B. — Wed, 02 Dec 2009 10:02:09 PST

Nerve roots have specialized transition zones that permit axon extension but limit cell movement between the CNS and PNS. Boundary cap cells prevent motor neuron soma from following their axons into the periphery, thereby contributing to a selective barrier. Transition zones also restrict movement of glial cells. Consequently, axons that cross the CNS–PNS interface are insulated by central and peripheral myelin. The mechanisms that prevent the migratory progenitors of oligodendrocytes and Schwann cells, the myelinating cells of the CNS and PNS, respectively, from crossing transition zones are not known. Here, we show that interactions between myelinating glial cells prevent their movements across the interface. Using in vivo time-lapse imaging in zebrafish we found that, in the absence of Schwann cells, oligodendrocyte progenitors cross ventral root transition zones and myelinate motor axons. These studies reveal that distinct mechanisms regulate the movement of axons, neurons, and glial cells across the CNS–PNS interface.

Interneurons Produced in Adulthood Are Required for the Normal Functioning of the Olfactory Bulb Network and for the Execution of Selected Olfactory Behaviors

Breton-Provencher, V., Lemasson, M., Peralta, M. R., Saghatelyan, A. — Wed, 02 Dec 2009 10:02:09 PST

Olfactory bulb (OB) interneurons are continuously renewed throughout an animal's lifespan. Despite extensive investigation of this phenomenon, little is known about bulbar circuitry functioning and olfactory performances under conditions of ablated arrival of new neurons into the adult OB. To address this issue we performed morphological, electrophysiological, and behavioral analysis in mice with suppressed bulbar neurogenesis. Infusion of the antimitotic drug AraC to the lateral ventricle via 28 d osmotic minipumps abolished the arrival of newly born neurons into the adult OB without affecting the total number of granule cells. The number, dendritic arborization, and spine density of interneurons generated in adulthood, before pump installation, were also not affected by AraC treatment. As a result of ablated neurogenesis, mitral cells—the principal output neurons in the OB—receive fewer inhibitory synapses, display reduced frequency of spontaneous IPSCs, experience smaller dendrodendritic inhibition, and exhibit decreased synchronized activity. Consequently, short-term olfactory memory was drastically reduced in AraC-treated mice. In contrast, olfactory performances of AraC-treated animals were undistinguishable from those of control mice in other odor-associated tests, such as spontaneous odor discrimination and long-term odor-associative memory tasks. Altogether, our data highlight the importance of adult neurogenesis for the proper functioning of the OB network and imply that new bulbar interneurons are involved in some, but not all, odor-associated tasks.

Preferred Sites of Exocytosis and Endocytosis Colocalize during High- But Not Lower-Frequency Stimulation in Mouse Motor Nerve Terminals

Gaffield, M. A., Tabares, L., Betz, W. J. — Wed, 02 Dec 2009 10:02:09 PST

The spatial relationship of exocytosis and endocytosis in motor nerve terminals has been explored, with varied results, mostly in fixed preparations and without direct information on the utilization of each exocytic site. We sought to determine these spatial properties in real time using synaptopHluorin (spH) and FM4-64. Earlier we showed that nerve stimulation elicits the appearance of spH fluorescence hot spots, which mark preferred sites of exocytosis. Here we show that nerve stimulation in the presence of the styryl dye FM4-64 evokes hot spots of FM4-64 fluorescence. Their size, density, and rate of appearance are similar to the spH hot spots, but their rate of disappearance after stimulation was much slower (t_1/2 ~9 min vs ~10 s for spH hot spots), consistent with FM4-64 spots identifying bulk endocytosis and subsequent slow intracellular dispersion of nascent vesicles. Simultaneous imaging of both fluorophores revealed a strong colocalization of spH and FM4-64 spots, but only during high (100 Hz) stimulation. At 40 Hz stimulation, exocytic and endocytic spots did not colocalize. Our results are consistent with the hypothesis that hot spots of endocytosis, possibly in the form of bulk uptake, occur at or very near highly active exocytic sites during high-frequency stimulation.

Estradiol-Induced Estrogen Receptor-{alpha} Trafficking

Bondar, G., Kuo, J., Hamid, N., Micevych, P. — Wed, 02 Dec 2009 10:02:09 PST

Estradiol has rapid actions in the CNS that are mediated by membrane estrogen receptors (ERs) and activate cell signaling pathways through interaction with metabotropic glutamate receptors (mGluRs). Membrane-initiated estradiol signaling increases the free cytoplasmic calcium concentration ([Ca²⁺]_i) that stimulates the synthesis of neuroprogesterone in astrocytes. We used surface biotinylation to demonstrate that ER has an extracellular portion. In addition to the full-length ER [apparent molecular weight (MW), 66 kDa], surface biotinylation labeled an ER-immunoreactive protein (MW, ~52 kDa) identified by both COOH- and NH₂-directed antibodies. Estradiol treatment regulated membrane levels of both proteins in parallel: within 5 min, estradiol significantly increased membrane levels of the 66 and 52 kDa ER. Internalization, a measure of membrane receptor activation, was also increased by estradiol with a similar time course. Continuous treatment with estradiol for 24–48 h reduced ER levels, suggesting receptor downregulation. Estradiol also increased mGluR1a trafficking and internalization, consistent with the proposed ER–mGluR1a interaction. Blocking ER with ICI 182,780 or mGluR1a with LY 367385 prevented ER trafficking to and from the membrane. Estradiol-induced [Ca²⁺]_i flux was also significantly increased at the time of peak ER activation/internalization. These results demonstrate that ER is present in the membrane and has an extracellular portion. Furthermore, membrane levels and internalization of ER are regulated by estradiol and mGluR1a ligands. The pattern of trafficking into and out of the membrane suggests that the changing concentration of estradiol during the estrous cycle regulates ER to augment and then terminate membrane-initiated signaling.

Outwardly Rectifying Tonically Active GABAA Receptors in Pyramidal Cells Modulate Neuronal Offset, Not Gain

Pavlov, I., Savtchenko, L. P., Kullmann, D. M., Semyanov, A., Walker, M. C. — Wed, 02 Dec 2009 10:02:09 PST

Hippocampal pyramidal cell excitability is regulated both by fast synaptic inhibition and by tonically active high-affinity extrasynaptic GABA_A receptors. The impact of tonic inhibition on neuronal gain and offset, and thus on information processing, is unclear. Offset is altered by shunting inhibition, and the gain of a neuronal response to an excitatory input can be modified by changing the level of "background" synaptic noise. Therefore, tonic activation of GABA_A receptors would be expected to modulate offset and, in addition, to alter gain through a shunting effect on synaptic noise. Here we show that tonically active GABA_A receptors in CA1 pyramidal cells show marked outward rectification, while the peaks of IPSCs exhibit a linear current–voltage relationship. As a result, tonic GABA_A receptor-mediated currents have a minimal effect upon subthreshold membrane potential variation due to synaptic noise, but predominantly affect neurons at spiking threshold. Consistent with this, tonic GABA_A receptor-mediated currents in pyramidal cells exclusively affect offset and not gain. Modulation of tonically active GABA_A receptors by fluctuations in extracellular GABA concentrations or neuromodulators acting on high-affinity receptors potentially provides a powerful mechanism to alter neuronal offset independently of neuronal gain.

Proneurotrophin-3 Is a Neuronal Apoptotic Ligand: Evidence for Retrograde-Directed Cell Killing

Yano, H., Torkin, R., Martin, L. A., Chao, M. V., Teng, K. K. — Wed, 25 Nov 2009 10:03:10 PST

Although mature neurotrophins are well described trophic factors that elicit retrograde survival signaling, the precursor forms of neurotrophins (i.e., proneurotrophins) can function as high-affinity apoptotic ligands for selected neural populations. An outstanding question is whether target-derived proneurotrophins might affect neuronal survival/death decisions through a retrograde transport mechanism. Since neurotrophin-3 (NT-3) is highly expressed in non-neural tissues that receive peripheral innervation, we investigated the localized actions of its precursor (proNT-3) on sympathetic neurons in the present study. Pharmacological inhibition of intracellular furin proteinase activity in 293T cells resulted in proNT-3 release instead of mature NT-3, whereas membrane depolarization in cerebellar granule neurons stimulated endogenous proNT-3 secretion, suggesting that proNT-3 is an inducible bona fide ligand in the nervous system. Our data also indicate that recombinant proNT-3 induced sympathetic neuron death that is p75^NTR- and sortilin-dependent, with hallmark features of apoptosis including JNK (c-Jun N-terminal kinase) activation and nuclear fragmentation. Using compartmentalized culture systems that segregate neuronal cell bodies from axons, proNT-3, acting within the distal axon compartment, elicited sympathetic neuron death and overrode the survival-promoting actions of NGF. Together, these results raise the intriguing possibility that dysregulation of proneurotrophin processing/release by innervated targets can be deleterious to the neurons projecting to these sites.

Dopamine-Stimulated Dephosphorylation of Connexin 36 Mediates AII Amacrine Cell Uncoupling

Kothmann, W. W., Massey, S. C., O'Brien, J. — Wed, 25 Nov 2009 10:03:10 PST

Gap junction proteins form the substrate for electrical coupling between neurons. These electrical synapses are widespread in the CNS and serve a variety of important functions. In the retina, connexin 36 (Cx36) gap junctions couple AII amacrine cells and are a requisite component of the high-sensitivity rod photoreceptor pathway. AII amacrine cell coupling strength is dynamically regulated by background light intensity, and uncoupling is thought to be mediated by dopamine signaling via D₁-like receptors. One proposed mechanism for this uncoupling involves dopamine-stimulated phosphorylation of Cx36 at regulatory sites, mediated by protein kinase A. Here we provide evidence against this hypothesis and demonstrate a direct relationship between Cx36 phosphorylation and AII amacrine cell coupling strength. Dopamine receptor-driven uncoupling of the AII network results from protein kinase A activation of protein phosphatase 2A and subsequent dephosphorylation of Cx36. Protein phosphatase 1 activity negatively regulates this pathway. We also find that Cx36 gap junctions can exist in widely different phosphorylation states within a single neuron, implying that coupling is controlled at the level of individual gap junctions by locally assembled signaling complexes. This kind of synapse-by-synapse plasticity allows for precise control of neuronal coupling, as well as cell-type-specific responses dependent on the identity of the signaling complexes assembled.

Cleavage of proBDNF to BDNF by a Tolloid-Like Metalloproteinase Is Required for Acquisition of In Vitro Eyeblink Classical Conditioning

Keifer, J., Sabirzhanov, B. E., Zheng, Z., Li, W., Clark, T. G. — Wed, 25 Nov 2009 10:03:10 PST

The tolloid/bone morphogenetic protein-1 family of metalloproteinases have an important role in the regulation of embryonic pattern formation and tissue morphogenesis. Studies suggest that they participate in mechanisms of synaptic plasticity in adults, but very little is known about their function. Recently, we isolated a reptilian ortholog of the tolloid gene family designated turtle tolloid-like gene (tTll). Here, we examined the role of tTLL in an in vitro model of eyeblink classical conditioning using an isolated brainstem preparation to assess its role in synaptic plasticity during conditioning. Analysis by real-time reverse transcription-PCR shows that an extracellularly secreted form of tTLL, tTLLs, is transiently expressed in the early stages of conditioning during conditioned response acquisition, whereas a cytosolic form, tTLLc, is not. Short interfering RNA (siRNA)-directed gene knockdown and rescue of tTLL expression demonstrate that it is required for conditioning. Significantly, we show that tTLLs cleaves the precursor proBDNF into mature BDNF in cleavage assay studies, and application of recombinant tTLLs protein alone to preparations results in induction of mature BDNF expression. The mature form of BDNF is minimally expressed in preparations treated with anti-tTLL siRNA, and the synaptic incorporation of both GluR1- and GluR4-containing AMPA receptors is significantly reduced, resulting in suppression of conditioning. This is the first study to demonstrate that expression of an extracellularly secreted tolloid-like metalloproteinase is regulated in the early stages of classical conditioning and functions in the conversion of proBDNF to mature BDNF. The mature form of BDNF is required for synaptic delivery of AMPA receptors and acquisition of conditioned responses.

Schwann Cells Inhibit Ectopic Clustering of Axonal Sodium Channels

Voas, M. G., Glenn, T. D., Raphael, A. R., Talbot, W. S. — Wed, 18 Nov 2009 10:02:12 PST

The clustering of voltage-gated sodium channels at the axon initial segment (AIS) and nodes of Ranvier is essential for the initiation and propagation of action potentials in myelinated axons. Sodium channels localize to the AIS through an axon-intrinsic mechanism driven by ankyrin G, while clustering at the nodes requires cues from myelinating glia that interact with axonal neurofascin186 (Sherman et al., 2005; Dzhashiashvili et al., 2007; Yang et al., 2007). Here, we report that in zebrafish mutants lacking Schwann cells in peripheral nerves (erbb2, erbb3, and sox10/colorless), axons form numerous aberrant sodium channel clusters throughout their length. Morpholino knockdown of ankyrin G, but not neurofascin, reduces the number of sodium channel clusters in Schwann cell-deficient mutants, suggesting that these aberrant clusters form by an axon-intrinsic mechanism. We also find that gpr126 mutants, in which Schwann cells are arrested at the promyelinating stage (Monk et al., 2009), are deficient in the clustering of neurofascin at the nodes of Ranvier. When Schwann cell migration in gpr126 mutants is blocked, there is an increase in the number of neurofascin clusters in peripheral axons. Our results suggest that Schwann cells inhibit the ability of ankyrin G to cluster sodium channels at ectopic locations, restricting its activity to the AIS and nodes of Ranvier.

Complementary Theta Resonance Filtering by Two Spatially Segregated Mechanisms in CA1 Hippocampal Pyramidal Neurons

Hu, H., Vervaeke, K., Graham, L. J., Storm, J. F. — Wed, 18 Nov 2009 10:02:12 PST

Synaptic input to a neuron may undergo various filtering steps, both locally and during transmission to the soma. Using simultaneous whole-cell recordings from soma and apical dendrites from rat CA1 hippocampal pyramidal cells, and biophysically detailed modeling, we found two complementary resonance (bandpass) filters of subthreshold voltage signals. Both filters favor signals in the theta (3–12 Hz) frequency range, but have opposite location, direction, and voltage dependencies: (1) dendritic H-resonance, caused by h/HCN-channels, filters signals propagating from soma to dendrite when the membrane potential is close to rest; and (2) somatic M-resonance, caused by M/Kv7/KCNQ and persistent Na⁺ (NaP) channels, filters signals propagating from dendrite to soma when the membrane potential approaches spike threshold. Hippocampal pyramidal cells participate in theta network oscillations during behavior, and we suggest that that these dual, polarized theta resonance mechanisms may convey voltage-dependent tuning of theta-mediated neural coding in the entorhinal/hippocampal system during locomotion, spatial navigation, memory, and sleep.

SR1, a Mouse Odorant Receptor with an Unusually Broad Response Profile

Wed, 18 Nov 2009 10:02:12 PST

The current consensus model in mammalian olfaction is that the detection of millions of odorants requires a large number of odorant receptors (ORs) and that each OR interacts selectively with a small subset of odorants, which are typically related in structure. Here, we report the odorant response properties of an OR that deviates from this model: SR1, a mouse OR that is abundantly expressed in sensory neurons of the septal organ and also of the main olfactory epithelium. Patch-clamp recordings reveal that olfactory sensory neurons (OSNs) that express SR1 respond to many, structurally unrelated odorants, and over a wide concentration range. Most OSNs expressing a gene-targeted SR1 locus that lacks the SR1 coding sequence do not show this broad responsiveness. Gene transfer in the heterologous expression system Hana3A confirms the broad response profile of SR1. There may be other mouse ORs with such broad response profiles.

Neuronal Transporters Regulate Glutamate Clearance, NMDA Receptor Activation, and Synaptic Plasticity in the Hippocampus

Scimemi, A., Tian, H., Diamond, J. S. — Wed, 18 Nov 2009 10:02:12 PST

In the mammalian brain, the specificity of excitatory synaptic transmission depends on rapid diffusion of glutamate away from active synapses and the powerful uptake capacity of glutamate transporters in astrocytes. The extent to which neuronal glutamate transporters influence the lifetime of glutamate in the extracellular space remains unclear. Here we show that EAAC1, the predominant neuronal glutamate transporter at excitatory synapses in hippocampal area CA1, buffers glutamate released during synaptic events and prolongs the time course of its clearance by astrocytes. EAAC1 does not significantly alter activation of receptors in the synaptic cleft. Instead, it reduces recruitment of perisynaptic/extrasynaptic NR2B-containing NMDARs, thereby facilitating induction of long-term potentiation by short bursts of high-frequency stimulation. We describe novel roles of EAAC1 in regulating glutamate diffusion and propose that NMDARs at different subsynaptic locations can make distinct contributions to the regulation of synaptic strength.

Novel Repression of Kcc2 Transcription by REST-RE-1 Controls Developmental Switch in Neuronal Chloride

Yeo, M., Berglund, K., Augustine, G., Liedtke, W. — Wed, 18 Nov 2009 10:02:13 PST

Transcriptional upregulation of Kcc2b, the gene variant encoding the major isoform of the KCC2 chloride transporter, underlies a rapid perinatal decrease in intraneuronal chloride concentration (chloride shift), which is necessary for GABA to act inhibitory. Here we identify a novel repressor element-1 (RE-1) site in the 5' regulatory region of Kcc2b. In primary cortical neurons, which recapitulate the chloride shift in culture, the novel upstream RE-1 together with a known intronic RE-1 site function in concerted interaction to suppress Kcc2b transcription. With critical relevance for the chloride shift, only in the presence of the dual RE-1 site could inhibition of REST upregulate Kcc2b transcription. For this, we confirmed increased KCC2 protein expression and decreased intraneuronal chloride. Kcc2b developmental upregulation was potentiated by BDNF application, which was fully dependent on the presence of dual RE-1. In addition, the developmental chloride shift and GABA switch, from excitatory to inhibitory action, was accelerated by REST inhibition and slowed by REST overexpression. These results identify the REST–dual RE-1 interaction as a novel mechanism of transcriptional Kcc2b upregulation that significantly contributes to the ontogenetic shift in chloride concentration and GABA action in cortical neurons, which is fundamental for brain function in health and disease. Thus, we present here a new logic for the perinatal chloride shift, which is critical for establishment of GABAergic cortical inhibitory neurotransmission.

In Vivo Quantification of Myelin Changes in the Vertebrate Nervous System

Wed, 18 Nov 2009 10:02:13 PST

Destruction or changes associated with myelin membranes in the CNS play a key role in the pathogenesis of multiple sclerosis and other related neurodegenerative disorders. A long-standing goal has been to detect and quantify myelin content in vivo. For this reason, we have developed a myelin-imaging technique based on positron emission tomography (PET). PET is a quantitative imaging modality that has been widely used in clinical settings for direct assessment of biological processes at the molecular level. However, lack of myelin-imaging probes has hampered the use of PET for imaging of myelination in the CNS. Here, we report a myelin-imaging agent, termed Case Imaging Compound (CIC) that readily penetrates the blood–brain barrier and preferentially localizes to myelinated regions of the brain. After radiolabeling with positron-emitting carbon-11, [¹¹C]CIC–PET was conducted in longitudinal studies using a lysolethicin-induced rat model of focal demyelination and subsequent remyelination. Quantitative analysis showed that the retention of [¹¹C]CIC correlates with the level of demyelination/remyelination. These studies indicate that, for the first time, [¹¹C]CIC–PET can be used as an imaging marker of myelination, which has the potential to be translated into clinical studies in multiple sclerosis and other myelin-related diseases for early diagnosis, subtyping, and efficacy evaluation of therapeutic treatments aimed at myelin repair.

Functional Heterogeneity at Dopamine Release Sites

Daniel, J. A., Galbraith, S., Iacovitti, L., Abdipranoto, A., Vissel, B. — Wed, 18 Nov 2009 10:02:13 PST

Although drugs used to treat several neurological diseases are presumed to target synapses that secrete dopamine (DA), relatively little is known about synaptic vesicle (SV) release mechanisms at single DA synapses. We found that the relative probability of release (Pr) varied between individual DA synapses. Furthermore, DA terminals generally exhibited lower Pr than glutamatergic hippocampal (Hpc) terminals, suggesting that DA release is less reliable than the release of glutamate. Our mathematical model of fluorescence loss shows that Pr is regulated by two independent and heterogeneous elements. First, the size of the recycling SV pool regulates Pr. Second, Pr is also independently regulated by additional factors, which are reflected in the time constant of FM 1-43 destaining, . We found that the observed difference in Pr between Hpc and DA neurons results because the recycling SV pool is smaller in DA neurons than in Hpc neurons. However, does not vary between these two neuron populations. We also identified a population of functional nonsynaptic boutons in DA axons, which are not associated with a postsynaptic element and which are not functionally different from boutons that formed conventional synapses. Our work provides a new approach to the study of SV exocytosis in DA neurons and shows that synaptic terminals of DA neurons are functionally heterogeneous and differ from excitatory terminals in terms of Pr.

Individual Calcium Syntillas Do Not Trigger Spontaneous Exocytosis from Nerve Terminals of the Neurohypophysis

McNally, J. M., De Crescenzo, V., Fogarty, K. E., Walsh, J. V., Lemos, J. R. — Wed, 11 Nov 2009 10:02:14 PST

Recently, highly localized Ca²⁺ release events, similar to Ca²⁺ sparks in muscle, have been observed in neuronal preparations. Specifically, in murine neurohypophysial terminals (NHT), these events, termed Ca²⁺ syntillas, emanate from a ryanodine-sensitive intracellular Ca²⁺ pool and increase in frequency with depolarization in the absence of Ca²⁺ influx. Despite such knowledge of the nature of these Ca²⁺ release events, their physiological role in this system has yet to be defined. Such localized Ca²⁺ release events, if they occur in the precise location of the final exocytotic event(s), may directly trigger exocytosis. However, directly addressing this hypothesis has not been possible, since no method capable of visualizing individual release events in these CNS terminals has been available. Here, we have adapted an amperometric method for studying vesicle fusion to this system which relies on loading the secretory granules with the false transmitter dopamine, thus allowing, for the first time, the recording of individual exocytotic events from peptidergic NHT. Simultaneous use of this technique along with high-speed Ca²⁺ imaging has enabled us to establish that spontaneous neuropeptide release and Ca²⁺ syntillas do not display any observable temporal or spatial correlation, confirming similar findings in chromaffin cells. Although these results indicate that syntillas do not play a direct role in eliciting spontaneous release, they do not rule out indirect modulatory effects of syntillas on secretion.

Differential Activity-Dependent Secretion of Brain-Derived Neurotrophic Factor from Axon and Dendrite

Wed, 11 Nov 2009 10:02:14 PST

Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival and differentiation during development and for synaptic function and plasticity in the mature brain. BDNF-containing vesicles are widely distributed and bidirectionally transported in neurons, and secreted BDNF can act on both presynaptic and postsynaptic cells. Activity-dependent BDNF secretion from neuronal cultures has been reported, but it remains unknown where the primary site of BDNF secretion is and whether neuronal activity can trigger BDNF secretion from axons and dendrites with equal efficacy. Using BDNF fused with pH-sensitive green fluorescent protein to visualize BDNF secretion, we found that BDNF-containing vesicles exhibited markedly different properties of activity-dependent exocytic fusion at the axon and dendrite of cultured hippocampal neurons. Brief spiking activity triggered a transient fusion pore opening, followed by immediate retrieval of vesicles without dilation of the fusion pore, resulting in very little BDNF secretion at the axon. On the contrary, the same brief spiking activity induced "full-collapse" vesicle fusion and substantial BDNF secretion at the dendrite. However, full vesicular fusion with BDNF secretion could occur at the axon when the neuron was stimulated by prolonged high-frequency activity, a condition neurons may encounter during epileptic discharge. Thus, activity-dependent axonal secretion of BDNF is highly restricted as a result of incomplete fusion of BDNF-containing vesicles, and normal neural activity induces BDNF secretion from dendrites, consistent with the BDNF function as a retrograde factor. Our study also revealed a novel mechanism by which differential exocytosis of BDNF-containing vesicles may regulate BDNF–TrkB signaling between connected neurons.

Overexpressing Temperature-Sensitive Dynamin Decelerates Phototransduction and Bundles Microtubules in Drosophila Photoreceptors

Wed, 11 Nov 2009 10:02:14 PST

shibire^ts1, a temperature-sensitive mutation of the Drosophila gene encoding a Dynamin orthologue, blocks vesicle endocytosis and thus synaptic transmission, at elevated, or restrictive temperatures. By targeted Gal4 expression, UAS-shibire^ts1 has been used to dissect neuronal circuits. We investigated the effects of UAS-shibire^ts1 overexpression in Drosophila photoreceptors at permissive (19°C) and restrictive (31°C) temperatures. At 19°C, overexpression of UAS-shi^ts1 causes decelerated phototransduction and reduced neurotransmitter release. This phenotype is exacerbated with dark adaptation, age and in white mutants. Photoreceptors overexpressing UAS-shibire^ts1 contain terminals with widespread vacuolated mitochondria, reduced numbers of vesicles and bundled microtubules. Immuno-electron microscopy reveals that the latter are dynamin coated. Further, the microtubule phenotype is not restricted to photoreceptors, as UAS-shibire^ts1 overexpression in lamina cells also bundles microtubules. We conclude that dynamin has multiple functions that are interrupted by UAS-shibire^ts1 overexpression in Drosophila photoreceptors, destabilizing their neural communication irreversibly at previously reported permissive temperatures.

Surviving Hilar Somatostatin Interneurons Enlarge, Sprout Axons, and Form New Synapses with Granule Cells in a Mouse Model of Temporal Lobe Epilepsy

Wed, 11 Nov 2009 10:02:14 PST

In temporal lobe epilepsy, seizures initiate in or near the hippocampus, which frequently displays loss of neurons, including inhibitory interneurons. It is unclear whether surviving interneurons function normally, are impaired, or develop compensatory mechanisms. We evaluated GABAergic interneurons in the hilus of the dentate gyrus of epileptic pilocarpine-treated GIN mice, specifically a subpopulation of somatostatin interneurons that expresses enhanced green fluorescence protein (GFP). GFP-immunocytochemistry and stereological analyses revealed substantial loss of GFP-positive hilar neurons (GPHNs) but increased GFP-positive axon length per dentate gyrus in epileptic mice. Individual biocytin-labeled GPHNs in hippocampal slices from epileptic mice also had larger somata, more axon in the molecular layer, and longer dendrites than controls. Dual whole-cell patch recording was used to test for monosynaptic connections from hilar GPHNs to granule cells. Unitary IPSCs (uIPSCs) recorded in control and epileptic mice had similar average rise times, amplitudes, charge transfers, and decay times. However, the probability of finding monosynaptically connected pairs and evoking uIPSCs was 2.6 times higher in epileptic mice compared to controls. Together, these findings suggest that surviving hilar somatostatin interneurons enlarge, extend dendrites, sprout axon collaterals in the molecular layer, and form new synapses with granule cells. These epilepsy-related changes in cellular morphology and connectivity may be mechanisms for surviving hilar interneurons to inhibit more granule cells and compensate for the loss of vulnerable interneurons.

Polar Residues in the Second Transmembrane Domain of the Rat P2X2 Receptor That Affect Spontaneous Gating, Unitary Conductance, and Rectification

Cao, L., Broomhead, H. E., Young, M. T., North, R. A. — Wed, 11 Nov 2009 10:02:14 PST

Membrane ion channels activated by extracellular ATP (P2X receptors) are widely distributed in the nervous system. Their molecular architecture is fundamentally distinct from that of the nicotinic or glutamate receptor families. We have measured single-channel currents, spontaneous gating, and rectification of rat P2X2 receptor in which polar and charged residues of the second transmembrane domain (TM2) were systematically probed by mutagenesis. The results suggest that Asn³³³ and Asp³⁴⁹ lie respectively in external and internal vestibules. Substitutions at Asn³³³, Thr³³⁶, and Ser³⁴⁰ were particularly likely to cause spontaneously active channels. At Thr³³⁶, Thr³³⁹, and Ser³⁴⁰, the introduction of positive charge (Arg, Lys, or His, or Cys followed by treatment with 2-aminoethyl methanethiosulphonate) greatly enhanced outward currents, suggesting that side-chains of these three residues are exposed in the permeation pathway of the open channel. These functional findings are interpreted in the context of the recently reported 3.1 Å crystal structure of the zebrafish P2X4.1 receptor in the closed state. They imply that the gate is formed by residues Asn³³³ to Thr³³⁹ and that channel opening involves a counter-clockwise rotation and separation of the TM2 helices.

Dynorphin Opioid Peptides Enhance Acid-Sensing Ion Channel 1a Activity and Acidosis-Induced Neuronal Death

Sherwood, T. W., Askwith, C. C. — Wed, 11 Nov 2009 10:02:15 PST

Acid-sensing ion channel 1a (ASIC1a) promotes neuronal damage during pathological acidosis. ASIC1a undergoes a process called steady-state desensitization in which incremental pH reductions desensitize the channel and prevent activation when the threshold for acid-dependent activation is reached. We find that dynorphin A and big dynorphin limit steady-state desensitization of ASIC1a and acid-activated currents in cortical neurons. Dynorphin potentiation of ASIC1a activity is independent of opioid or bradykinin receptor activation but is prevented in the presence of PcTx1, a peptide which is known to bind the extracellular domain of ASIC1a. This suggests that dynorphins interact directly with ASIC1a to enhance channel activity. Inducing steady-state desensitization prevents ASIC1a-mediated cell death during prolonged acidosis. This neuroprotection is abolished in the presence of dynorphins. Together, these results define ASIC1a as a new nonopioid target for dynorphin action and suggest that dynorphins enhance neuronal damage following ischemia by preventing steady-state desensitization of ASIC1a.