David M. Sherry

Expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in development of rod vs. cone and ON vs. OFF circuits

Glutamatergic transmission is crucial to the segregation of ON and OFF pathways in the developing retina. The temporal sequence of maturation of vesicular glutamatergic transmission in rod and cone photoreceptor and ON and OFF bipolar cell terminals is currently unknown. Vesicular glutamate transporters (VGLUTs) that load glutamate into synaptic vesicles are necessary for vesicular glutamatergic transmission. To understand better the formation and maturation of glutamatergic transmission in the rod vs. cone and ON vs. OFF pathways of the retina, we examined the developmental expression of VGLUT1 and VGLUT2 immunocytochemically in the mouse retina. Photoreceptor and bipolar cell terminals showed only VGLUT1‐immunoreactivity (‐IR); no VGLUT2‐IR was present in any synapses of the developing or adult retina. VGLUT1‐IR was first detected in cone photoreceptor terminals at postnatal day 2 (P2), several days before initiation of ribbon synapse formation at P4–P5. Rod terminals showed VGLUT1‐IR by P8, when they invade the outer plexiform layer (OPL) and initiate synaptogenesis. Developing OFF bipolar cell terminals showed VGLUT1‐IR around P8, 2–3 days after bipolar terminals were first identified in the inner plexiform layer (IPL) by labeling for the photoreceptor and bipolar cell terminal marker, synaptic vesicle protein 2B. Although terminals of ON bipolar cells were present in the IPL by P6–P8, most did not show VGLUT1‐IR until P8–P10 and increased dramatically from P12. These data suggest a hierarchical development of glutamatergic transmission in which cone circuits form prior to rod circuits in both the OPL and IPL, and OFF circuits form prior to ON circuits in the IPL. J. Comp. Neurol. 465:480–498, 2003.

Vesicular glutamate transporter 3 expression identifies glutamatergic amacrine cells in the rodent retina.

Synaptic transmission from glutamatergic neurons requires vesicular glutamate transporters (VGLUTs) to concentrate cytosolic glutamate in synaptic vesicles. In retina, glutamatergic photoreceptors and bipolar cells exclusively express the VGLUT1 isoform, whereas ganglion cells express VGLUT2. Surprisingly, the recently identified VGLUT3 isoform was found in presumed amacrine cells, generally considered to be inhibitory interneurons. To investigate the synaptic machinery and conceivable secondary neurotransmitter composition of VGLUT3 cells, and to determine a potential functional role, we further investigated these putative glutamatergic amacrine cells in adult and developing rodent retina. Reverse transcriptase‐PCR substantiated VGLUT3 expression in mouse retina. VGLUT3 cells did not immunostain for ganglion or bipolar cell markers, providing evidence that they are amacrine cells. VGLUT3 colocalized with synaptic vesicle markers, and electron microscopy showed that VGLUT3 immunostained synaptic vesicles. VGLUT3 cells were not immunoreactive for amacrine cell markers γ‐aminobutyric acid, choline acetyltransferase, calretinin, or tyrosine hydroxylase, although they immunostain for glycine. VGLUT3 processes made synaptic contact with ganglion cell dendrites, suggesting input onto these cells. VGLUT3 immunostaining was closely associated with the metabotropic glutamate receptor 4, which is consistent with glutamatergic synaptic exocytosis by these cells. In the maturing mouse retina, Western blots showed VGLUT3 expression at postnatal day 7/8 (P7/8). VGLUT3 immunostaining in retinal sections was first observed at P8, achieving an adult pattern at P12. Thus, VGLUT3 function commences around the same time as VGLUT1‐mediated glutamatergic transmission from bipolar cells. Furthermore, a subset of VGLUT3 cells expressed the circadian clock gene period 1, implicating VGLUT3 cells as part of the light‐entrainable retina‐based circadian system. J. Comp. Neurol. 477:386–398, 2004.

Differential distribution and developmental expression of synaptic vesicle protein 2 isoforms in the mouse retina.

Synaptic vesicle protein 2 (SV2), a ubiquitous synaptic vesicle protein, is known to participate in the regulation of Ca2+‐mediated synaptic transmission, although its precise function has not been established. Three SV2 isoforms (SV2A, SV2B, SV2C) have been identified recently, each of which has a unique distribution in brain, suggesting synapse‐specific functions. To determine if SV2A, ‐B, and ‐C are differentially distributed among synapses in the retina and the sequence of their development, we examined their distribution and expression patterns immunocytochemically in adult and developing mouse retina. The three SV2 isoforms were differentially distributed in the synapses of the two plexiform layers in the adult retina. SV2A was present in cone, but not rod, terminals in the outer plexiform layer (OPL) and in many synaptic terminals in the inner plexiform layer (IPL). SV2B was present only in the ribbon synapse‐containing terminals of rod and cone photoreceptors and bipolar cells. SV2C was present in starburst amacrine cells, other conventional synapses in the IPL of unknown origin, and in presumptive interplexiform cell terminals in the INL and OPL. Each SV2 isoform was expressed in its distinct presynaptic terminals early and throughout postnatal development. In addition, SV2A was transiently expressed by developing horizontal cells. The unique distribution of each isoform suggests potentially distinct functions at different types of synapses, with SV2B having ribbon synapse‐specific functions, and SV2C being important for the functions of starburst amacrine cells. Rod and cone terminals contain different complements of SV2 isoforms, indicating that ribbon synapses are not all identical. The early expression of SV2 isoforms prior to initiation of synapse formation suggests that they may have important synapse‐specific roles during synaptogenesis. J. Comp. Neurol. 460:106–122, 2003.

SV2 Acts via Presynaptic Calcium to Regulate Neurotransmitter Release

Synaptic vesicle 2 (SV2) proteins, critical for proper nervous system function, are implicated in human epilepsy, yet little is known about their function. We demonstrate, using direct approaches, that loss of the major SV2 isoform in a central nervous system nerve terminal is associated with an elevation in both resting and evoked presynaptic Ca(2+) signals. This increase is essential for the expression of the SV2B(-/-) secretory phenotype, characterized by changes in synaptic vesicle dynamics, synaptic plasticity, and synaptic strength. Short-term reproduction of the Ca(2+) phenotype in wild-type nerve terminals reproduces almost all aspects of the SV2B(-/-) secretory phenotype, while rescue of the Ca(2+) phenotype in SV2B(-/-) neurons relieves every facet of the SV2B(-/-) secretory phenotype. Thus, SV2 controls key aspects of synaptic functionality via its ability to regulate presynaptic Ca(2+), suggesting a potential new target for therapeutic intervention in the treatment of epilepsy.

Distribution of plasma membrane-associated syntaxins 1 through 4 indicates distinct trafficking functions in the synaptic layers of the mouse retina

BackgroundSyntaxins 1 through 4 are SNAP receptor (SNARE) proteins that mediate vesicular trafficking to the plasma membrane. In retina, syntaxins 1 and 3 are expressed at conventional and ribbon synapses, respectively, suggesting that synaptic trafficking functions differ among syntaxin isoforms. To better understand syntaxins in synaptic signaling and trafficking, we further examined the cell- and synapse-specific expression of syntaxins 1 through 4 in the mouse retina by immunolabeling and confocal microscopy.ResultsEach isoform was expressed in the retina and showed a unique distribution in the synaptic layers of the retina, with little or no colocalization of isoforms. Syntaxin 1 was present in amacrine cell bodies and processes and conventional presynaptic terminals in the inner plexiform layer (IPL). Syntaxin 2 was present in amacrine cells and their processes in the IPL, but showed little colocalization with syntaxin 1 or other presynaptic markers. Syntaxin 3 was found in glutamatergic photoreceptor and bipolar cell ribbon synapses, but was absent from putative conventional glutamatergic amacrine cell synapses. Syntaxin 4 was localized to horizontal cell processes in the ribbon synaptic complexes of photoreceptor terminals and in puncta in the IPL that contacted dopaminergic and CD15-positive amacrine cells. Syntaxins 2 and 4 often were apposed to synaptic active zones labeled for bassoon.ConclusionThese results indicate that each syntaxin isoform has unique, non-redundant functions in synaptic signaling and trafficking. Syntaxins 1 and 3 mediate presynaptic transmitter release from conventional and ribbon synapses, respectively. Syntaxins 2 and 4 are not presynaptic and likely mediate post-synaptic trafficking.

Progression of Neuronal and Synaptic Remodeling in the rd10 Mouse Model of Retinitis Pigmentosa

The Pde6brd10 (rd10) mouse has a moderate rate of photoreceptor degeneration and serves as a valuable model for human autosomal recessive retinitis pigmentosa (RP). We evaluated the progression of neuronal remodeling of second‐ and third‐order retinal cells and their synaptic terminals in retinas from Pde6brd10 (rd10) mice at varying stages of degeneration ranging from postnatal day 30 (P30) to postnatal month 9.5 (PNM9.5) using immunolabeling for well‐known cell‐ and synapse‐specific markers. Following photoreceptor loss, changes occurred progressively from outer to inner retina. Horizontal cells and rod and cone bipolar cells underwent morphological remodeling that included loss of dendrites, cell body migration, and the sprouting of ectopic processes. Gliosis, characterized by translocation of Müller cell bodies to the outer retina and thickening of their processes, was evident by P30 and became more pronounced as degeneration progressed. Following rod degeneration, continued expression of VGluT1 in the outer retina was associated with survival and expression of synaptic proteins by nearby second‐order neurons. Rod bipolar cell terminals showed a progressive reduction in size and ectopic bipolar cell processes extended into the inner nuclear layer and ganglion cell layer by PNM3.5. Putative ectopic conventional synapses, likely arising from amacrine cells, were present in the inner nuclear layer by PNM9.5. Despite these changes, the laminar organization of bipolar and amacrine cells and the ON‐OFF organization in the inner plexiform layer was largely preserved. Surviving cone and bipolar cell terminals continued to express the appropriate cell‐specific presynaptic proteins needed for synaptic function up to PNM9.5. J. Comp. Neurol. 518:2071–2089, 2010.

Contribution of voltage‐gated sodium channels to the b‐wave of the mammalian flash electroretinogram

Voltage‐gated sodium channels (Nav channels) in retinal neurons are known to contribute to the mammalian flash electroretinogram (ERG) via activity of third‐order retinal neurons, i.e. amacrine and ganglion cells. This study investigated the effects of tetrodotoxin (TTX) blockade of Nav channels on the b‐wave, an ERG wave that originates mainly from activity of second‐order retinal neurons. ERGs were recorded from anaesthetized Brown Norway rats in response to brief full‐field flashes presented over a range of stimulus energies, under dark‐adapted conditions and in the presence of steady mesopic and photopic backgrounds. Recordings were made before and after intravitreal injection of TTX (∼3 μm) alone, 3–6 weeks after optic nerve transection (ONTx) to induce ganglion cell degeneration, or in combination with an ionotropic glutamate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 200 μm) to block light‐evoked activity of inner retinal, horizontal and OFF bipolar cells, or with the glutamate agonist N‐methyl‐d‐aspartate (NMDA, 100–200 μm) to reduce light‐evoked inner retinal activity. TTX reduced ERG amplitudes measured at fixed times corresponding to b‐wave time to peak. Effects of TTX were seen under all background conditions, but were greatest for mesopic backgrounds. In dark‐adapted retina, b‐wave amplitudes were reduced only when very low stimulus energies affecting the inner retina, or very high stimulus energies were used. Loss of ganglion cells following ONTx did not affect b‐wave amplitudes, and injection of TTX in eyes with ONTx reduced b‐wave amplitudes by the same amount for each background condition as occurred when ganglion cells were intact, thereby eliminating a ganglion cell role in the TTX effects. Isolation of cone‐driven responses by presenting test flashes after cessation of a rod‐saturating conditioning flash indicated that the TTX effects were primarily on cone circuits contributing to the mixed rod–cone ERG. NMDA significantly reduced only the additional effects of TTX on the mixed rod–cone ERG observed under mesopic conditions, implicating inner retinal involvement in those effects. After pharmacological blockade with CNQX, TTX still reduced b‐wave amplitudes in cone‐isolated ERGs indicating Nav channels in ON cone bipolar cells themselves augment b‐wave amplitude and sensitivity. This augmentation was largest under dark‐adapted conditions, and decreased with increasing background illumination, indicating effects of background illumination on Nav channel function. These findings indicate that activation of Nav channels in ON cone bipolar cells affects the b‐wave of the rat ERG and must be considered when analysing results of ERG studies of retinal function.

Endosomal Accumulation of the Activated Epidermal Growth Factor Receptor (EGFR) Induces Apoptosis

Background: EGF receptor (EGFR) signaling is regulated by endocytosis. Results: The intracellular localization of the EGFR affects its signaling. Conclusion: EGFRs on the limiting membrane of endosomes, but not from the intraluminal vesicles, can induce apoptosis. Significance: EGFR signaling is spatially regulated at multiple steps of the endocytic pathway. Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.

Localization of glutamate and glutamate transporters in the sensory neurons of Aplysia

The sensorimotor synapse of Aplysia has been used extensively to study the cellular and molecular basis for learning and memory. Recent physiologic studies suggest that glutamate may be the excitatory neurotransmitter used by the sensory neurons (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163–7167; Armitage and Siegelbaum [1998] J Neurosci. 18:8770–8779). We further investigated the hypothesis that glutamate is the excitatory neurotransmitter at this synapse. The somata of sensory neurons in the pleural ganglia showed strong glutamate immunoreactivity. Very intense glutamate immunoreactivity was present in fibers within the neuropil and pleural‐pedal connective. Localization of amino acids metabolically related to glutamate was also investigated. Moderate aspartate and glutamine immunoreactivity was present in somata of sensory neurons, but only weak labeling for aspartate and glutamine was present in the neuropil or pleural‐pedal connective. In cultured sensory neurons, glutamate immunoreactivity was strong in the somata and processes and was very intense in varicosities; consistent with localization of glutamate in sensory neurons in the intact pleural‐pedal ganglion. Cultured sensory neurons showed only weak labeling for aspartate and glutamine. Little or no γ‐aminobutyric acid or glycine immunoreactivity was observed in the pleural‐pedal ganglia or in cultured sensory neurons. To further test the hypothesis that the sensory neurons use glutamate as a transmitter, in situ hybridization was performed by using a partial cDNA clone of a putative Aplysia high‐affinity glutamate transporter. The sensory neurons, as well as a subset of glia, expressed this mRNA. Known glutamatergic motor neurons B3 and B6 of the buccal ganglion also appeared to express this mRNA. These results, in addition to previous physiological studies (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163–7167; Trudeau and Castellucci [1993] J Neurophysiol. 70:1221–1230; Armitage and Siegelbaum [1998] J Neurosci. 18:8770–8779)) establish glutamate as an excitatory neurotransmitter of the sensorimotor synapse. J. Comp. Neurol. 423:121–131, 2000.

Role of Melatonin and its Receptors in the Vertebrate Retina

Melatonin is a chemical signal of darkness that is produced by retinal photoreceptors and pinealocytes. In the retina, melatonin diffuses from the photoreceptors to bind to specific receptors on a variety of inner retinal neurons to modify their activity. Potential target cells for melatonin in the inner retina are amacrine cells, bipolar cells, horizontal cells, and ganglion cells. Melatonin inhibits the release of dopamine from amacrine cells and increases the light sensitivity of horizontal cells. Melatonin receptor subtypes show differential, cell-specific patterns of expression that are likely to underlie differential functional modulation of specific retinal pathways. Melatonin potentiates rod signals to ON-type bipolar cells, via activation of the melatonin MT2 (Mel1b) receptor, suggesting that melatonin modulates the function of specific retinal circuits based on the differential distribution of its receptors. The selective and differential expression of melatonin receptor subtypes in cone circuits suggest a conserved function for melatonin in enhancing transmission from rods to second-order neurons and thus promote dark adaptation.