Alex H. Vielma

The elusive crypt olfactory receptor neuron: evidence for its stimulation by amino acids and cAMP pathway agonists.

SUMMARY Crypt olfactory receptor neurons (ORNs) are a third type of chemosensory neuron along with ciliated and microvillous ORNs in the olfactory epithelium of fishes, but their functional role is still unknown. To investigate their odorant response properties and possible transduction pathways, we recorded crypt ORN activity with calcium imaging and the patch clamp technique in its cell-attached mode in combination with odorant and agonist stimulation. Bile salts and putative fish pheromones did not elicit responses with either method, but the cells frequently responded to amino acids, with excitation and intracellular calcium signals. 8Br-cAMP and IBMX plus forskolin stimulated over 40% of crypt ORNs and triggered calcium signals in a similar percentage. Furthermore, crypt ORNs were immunoreactive to an antiserum against adenylate cyclase III. Together, these data suggest the presence of a cAMP transduction pathway, which might transduce odorants such as amino acids.

Nitric oxide signaling in the retina: What have we learned in two decades?

Two decades after its first detection in the retina, nitric oxide (NO) continues to puzzle visual neuroscientists. While its liberation by photoreceptors remains controversial, recent evidence supports three subtypes of amacrine cells as main sources of NO in the inner retina. NO synthesis was shown to depend on light stimulation, and mounting evidence suggests that NO is a regulator of visual adaptation at different signal processing levels. NO modulates light responses in all retinal neuron classes, and specific ion conductances are activated by NO in rods, cones, bipolar and ganglion cells. Light-dependent gap junction coupling in the inner and outer plexiform layers is also affected by NO. The vast majority of these effects were shown to be mediated by activation of the NO receptor soluble guanylate cyclase and resultant cGMP elevation. This review analyzes the current state of knowledge on physiological NO signaling in the retina.

Spectral sensitivities of photoreceptors and their role in colour discrimination in the green-backed firecrown hummingbird (Sephanoides sephaniodes)

We studied the photopic spectral sensitivity in the green-backed firecrown, Sephanoides sephaniodes, a South American hummingbird, and its possible ecological relationship with preferred flowers and body colouration. Avian colour vision is in general tetrachromatic with at least four types of cones, which vary in sensitivity from the near ultraviolet (UV) to the red wavelength range. Hummingbirds represent an important family of birds, yet little is known about their eye sensitivity, especially about the role of photoreceptors and their oil droplet complements. The photopic electroretinogram shows a main sensitivity peak at 560 nm and a secondary peak in the UV, and may be explained by the presence of four single cones (λmax at ~370, 440, 508 and 560 nm), and a double cone (λmax at 560 nm) screened by oil droplets. The flowers preferred by the firecrown are those in which the red–green wavelength region predominates and have higher contrast than other flowers. The crown plumage of males is highly iridescent in the red wavelength range (peak at 650 nm) and UV; when plotted in a high-dimensional tetrachromatic space, it falls in a “red + UV” purple hue line, suggesting a potential significant communication signal for sexual differentiation.

Retinal photoreceptors of two subterranean tuco-tuco species (Rodentia, Ctenomys): morphology, topography, and spectral sensitivity.

Traditionally, vision was thought to be useless for animals living in dark underground habitats, but recent studies in a range of subterranean rodent species have shown a large diversity of eye features, from small subcutaneous eyes to normal‐sized functional eyes. We analyzed the retinal photoreceptors in the subterranean hystricomorph rodents Ctenomys talarum and Ctenomys magellanicus to elucidate whether adaptation was to their near‐lightless burrows or rather to their occasional diurnal surface activity. Both species had normally developed eyes. Overall photoreceptor densities were comparatively low (95,000–150,000/mm2 in C. magellanicus, 110,000–200,000/mm2 in C. talarum), and cone proportions were rather high (10–31% and 14–31%, respectively). The majority of cones expressed the middle‐to‐longwave‐sensitive (L) opsin, and a 6–16% minority expressed the shortwave‐sensitive (S) opsin. In both species the densities of L and S cones were higher in ventral than in dorsal retina. In both species the tuning‐relevant amino acids of the S opsin indicate sensitivity in the near UV rather than the blue/violet range. Photopic spectral electroretinograms were recorded. Unexpectedly, their sensitivity profiles were best fitted by the linear summation of three visual pigment templates with λmax at 370 nm (S pigment, UV), at 510 nm (L pigment), and at 450 nm (an as‐yet unexplained mechanism). Avoiding predators and selecting food during the brief aboveground excursions may have exerted pressure to retain robust cone‐based vision in Ctenomys. UV tuning of the S cone pigment is shared with a number of other hystricomorphs. J. Comp. Neurol. 518:4001–4015, 2010.

Retinal photoreceptor arrangement, SWS1 and lws opsin sequence, and electroretinography in the South American marsupial Thylamys elegans (waterhouse, 1839)

We studied the retinal photoreceptors in the mouse opossum Thylamys elegans, a nocturnal South American marsupial. A variety of photoreceptor properties and color vision capabilities have been documented in Australian marsupials, and we were interested to establish what similarities and differences this American marsupial showed. Thylamys opsin gene sequencing revealed two cone opsins, a longwave‐sensitive (LWS) opsin and a shortwave‐sensitive (SWS1) opsin with deduced peak sensitivities at 560 nm and 360 nm (ultraviolet), respectively. Immunocytochemistry located these opsins to separate cone populations, a majority of LWS cones (density range 1,600–5,600/mm2) and a minority of SWS1 cones (density range 100–690/mm2). With rod densities of 440,000–590,000/mm2, the cones constituted 0.4–1.2% of the photoreceptors. This is a suitable adaptation to nocturnal vision. Cone densities peaked in a horizontally elongated region ventral to the optic nerve head. In ventral—but not dorsal—retina, roughly 40% of the LWS opsin‐expressing cones occurred as close pairs (double cones), and one member of each double cone contained a colorless oil droplet. The corneal electroretinogram (ERG) showed a high scotopic sensitivity with a rod peak sensitivity at 505 nm. At mesopic light levels, the spectral ERG revealed the contributions of a UV‐sensitive SWS1 cone mechanism and an LWS cone mechanism with peak sensitivities at 365 nm and 555 nm, respectively, confirming the tuning predictions from the cone opsin sequences. The two spectral cone types provide the basis for dichromatic color vision, or trichromacy if the rods contribute to color processing at mesopic light levels. J. Comp. Neurol. 518:1589–1602, 2010.

The GABAergic system in the retina of neonate and adult Octodon degus, studied by immunohistochemistry and electroretinography.

In the vertebrate retina, gamma‐aminobutyric acid (GABA) mediates inhibitory processes that shape the visual response and is also thought to have neurotrophic functions during retinal development. To investigate the role of GABAergic signaling at the beginning of visual experience, we used immunohistochemistry to compare the distribution of GABA, the two isoforms of glutamic acid decarboxylase GAD65/67, and the GABA receptor types A, B, and C, in neonate versus adult Octodon degus, a native South American rodent with diurnal‐crepuscular activity and a high cone‐to‐rod ratio. In parallel, we used electroretinography to evaluate retinal functionality and to test the contribution of fast GABAergic transmission to light responses at both developmental stages. Neonate O. degus opened their eyes on postnatal day (P)0 and displayed an adult‐like retinal morphology at this time. GABA, its biosynthetic sources, and receptors had a similar cellular distribution in neonates and adults, but labeling of the outer plexiform layer and of certain amacrine and ganglion cells was more conspicuous at P0. In neonates, retinal sensitivity was 10 times lower than in adults, responses to ultraviolet light could not be detected, and oscillatory potentials were reduced or absent. Blockade of GABAA/C receptors by bicuculline and TPMPA had no noticeable effect in neonates, while it significantly altered the electroretinogram response in adults. Conclusion: In spite of modest differences regarding retinal morphology and GABAergic expression, overall light response properties and GABAergic signaling are undeveloped in neonate O. degus compared to adults, suggesting that full retinal functionality requires a period of neural refinement under visual experience. J. Comp. Neurol. 514:459–472, 2009.

Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells

Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhβE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine >> RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the β4 subunit. Activation of nAChRs induced GABA release after Ca2+ accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca2+ stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing.

On Biophysical Properties and Sensitivity to Gap Junction Blockers of Connexin 39 Hemichannels Expressed in HeLa Cells

Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca2+. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.

Nitric oxide modulates the temporal properties of the glutamate response in type 4 OFF bipolar cells.

Nitric oxide (NO) is involved in retinal signal processing, but its cellular actions are only partly understood. An established source of retinal NO are NOACs, a group of nNOS-expressing amacrine cells which signal onto bipolar, other amacrine and ganglion cells in the inner plexiform layer. Here, we report that NO regulates glutamate responses in morphologically and electrophysiologically identified type 4 OFF cone bipolar cells through activation of the soluble guanylyl cyclase-cGMP-PKG pathway. The glutamate response of these cells consists of two components, a fast phasic current sensitive to kainate receptor agonists, and a secondary component with slow kinetics, inhibited by AMPA receptor antagonists. NO shortened the duration of the AMPA receptor-dependent component of the glutamate response, while the kainate receptor-dependent component remained unchanged. Application of 8-Br-cGMP mimicked this effect, while inhibition of soluble guanylate cyclase or protein kinase G prevented it, supporting a mechanism involving a cGMP signaling pathway. Notably, perfusion with a NOS-inhibitor prolonged the duration of the glutamate response, while the NO precursor L-arginine shortened it, in agreement with a modulation by endogenous NO. Furthermore, NO accelerated the response recovery during repeated stimulation of type 4 cone bipolar cells, suggesting that the temporal response properties of this OFF bipolar cell type are regulated by NO. These results reveal a novel cellular mechanism of NO signaling in the retina, and represent the first functional evidence of NO modulating OFF cone bipolar cells.

Role of connexin channels in the retinal light response of a diurnal rodent.

Several studies have shown that connexin channels play an important role in retinal neural coding in nocturnal rodents. However, the contribution of these channels to signal processing in the retina of diurnal rodents remains unclear. To gain insight into this problem, we studied connexin expression and the contribution of connexin channels to the retinal light response in the diurnal rodent Octodon degus (degu) compared to rat, using in vivo ERG recording under scotopic and photopic light adaptation. Analysis of the degu genome showed that the common retinal connexins present a high degree of homology to orthologs expressed in other mammals, and expression of Cx36 and Cx43 was confirmed in degu retina. Cx36 localized mainly to the outer and inner plexiform layers (IPLs), while Cx43 was expressed mostly in cells of the retinal pigment epithelium. Under scotopic conditions, the b-wave response amplitude was strongly reduced by 18-β-glycyrrhetinic acid (β-GA) (−45.1% in degu, compared to −52.2% in rat), suggesting that connexins are modulating this response. Remarkably, under photopic adaptation, β-GA increased the ERG b-wave amplitude in degu (+107.2%) while reducing it in rat (−62.3%). Moreover, β-GA diminished the spontaneous action potential firing rate in ganglion cells (GCs) and increased the response latency of ON and OFF GCs. Our results support the notion that connexins exert a fine-tuning control of the retinal light response and have an important role in retinal neural coding.