Glutamatergic neurotransmission from melanopsin retinal ganglion cells is required for neonatal photoaversion but not adult pupillary light reflex.

Melanopsin-expressing retinal ganglion cells (mRGCs) in the eye play an important role in many light-activated non-image-forming functions including neonatal photoaversion and the adult pupillary light reflex (PLR). MRGCs rely on glutamate and possibly PACAP (pituitary adenylate cyclase-activating polypeptide) to relay visual signals to the brain. However, the role of these neurotransmitters for individual non-image-forming responses remains poorly understood. To clarify the role of glutamatergic signaling from mRGCs in neonatal aversion to light and in adult PLR, we conditionally deleted vesicular glutamate transporter (VGLUT2) selectively from mRGCs in mice. We found that deletion of VGLUT2 in mRGCs abolished negative phototaxis and light-induced distress vocalizations in neonatal mice, underscoring a necessary role for glutamatergic signaling. In adult mice, loss of VGLUT2 in mRGCs resulted in a slow and an incomplete PLR. We conclude that glutamatergic neurotransmission from mRGCs is required for neonatal photoaversion but is complemented by another non-glutamatergic signaling mechanism for the pupillary light reflex in adult mice. We speculate that this complementary signaling might be due to PACAP neurotransmission from mRGCs.

Melanopsin-dependent light avoidance in neonatal mice.

Melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) form a light-sensitive system separate from rods and cones. Direct light stimulation of ipRGCs can regulate many nonimage-forming visual functions such as photoentrainment of circadian rhythms and pupil responses, and can intensify migraine headache in adults. In mice, ipRGCs are light responsive as early as the day of birth. In contrast, their eyelids do not open until 12-13 d after birth (P12-13), and light signaling from rods and cones does not begin until approximately P10. No physiological or behavioral function is established for ipRGCs in neonates before the onset of rod and cone signaling. Here we report that mouse pups as young as P6 will completely turn away from a light. Light-induced responses of ipRGCs could be readily recorded in retinas of pups younger than P9, and we found no evidence for rod- and cone-mediated visual signaling to the RGCs of these younger mice. These results confirm that negative phototaxis is evident before the onset of rod- and cone-mediated visual signaling, and well before the onset of image-forming vision. Negative phototaxis was absent in mice lacking melanopsin. We conclude that light activation of melanopsin ipRGCs is necessary and sufficient for negative phototaxis. These results strongly suggest that light activation of ipRGCs may regulate physiological functions such as sleep/wake cycles in preterm and neonatal infants.

Glycinergic transmission in the Mammalian retina.

Glycine and gamma-aminobutyric acid (GABA) are the major inhibitory neurotransmitters in the retina. Approximately half of the amacrine cells release glycine at their synapses with bipolar, other amacrine, and ganglion cells. Glycinergic amacrine cells are small-field amacrine cells with vertically oriented dendrites and comprise more than 10 different morphological types. The retinal distributions of glycine receptor (GlyR) alpha1, alpha2, alpha3 and alpha4 subtypes have been mapped with subunit-specific antibodies. GlyRs were clustered at postsynaptic hot spots which showed selective distributions for the different subunits. As a rule, only one alpha subunit was expressed at a given postsynaptic site. The kinetic properties of GlyRs were measured by recording spontaneous inhibitory postsynaptic currents (sIPSCs) from identified retinal neurons in wild-type, Glra1(spd-ot), Glra2 and Glra3 knockout mice. From observed differences of sIPSCs in wild-type and mutant mice, the cell-type specific subunit composition of GlyRs could be defined. OFF-cone bipolar cells and A-type ganglion cells receive prominent glycinergic input with fast kinetics that is mainly mediated by alpha1beta GlyRs (decay time constant tau approximately 5 ms). By contrast, AII amacrine cells express alpha3beta GlyRs with medium fast kinetics (tau approximately 11 ms). Narrow-field (NF) and wide-field amacrine cells contain predominantly alpha2beta GlyRs with slow kinetics (tau approximately 27 ms). Lastly, ON-starburst, narrow-field and wide-field amacrine cells in Glra2 knockout mice express alpha4beta GlyRs with very slow kinetics (tau approximately 70 ms).

Glycinergic input of widefield, displaced amacrine cells of the mouse retina.

Glycine receptors (GlyRs) of displaced amacrine cells of the mouse retina were analysed using whole cell recordings and immunocytochemical staining with subunit-specific antibodies. During the recordings the cells were filled with a fluorescent tracer and 11 different morphological types could be identified. The studies were performed in wild-type mice and in mutant mice deficient in the GlyRalpha1 (Glra1(spd-ot), 'oscillator' mouse), the GlyRalpha2 (Glra2(-/-)) and the GlyRalpha3 subunit (Glra3(-/-)). Based on their responses to the application of exogenous glycine in the retinas of wild-type and mutant mice, the cells were grouped into three major classes: group I cells (comprising the morphological types MA-S5, MA-S1, MA-S1/S5, A17, PA-S1, PA-S5 and WA-S1), group II cells (comprising the morphological types PA-S4, WA-S3 and WA-multi) and ON-starburst cells. For further analysis, spontaneous inhibitory postsynaptic currents (sIPSCs) were measured both in wild-type and mutant mouse retinas. Glycinergic sIPSCs and glycine induced currents of group I cells remained unaltered across wild-type and the three mutant mice (mean decay time constant of sIPSCs, tau approximately 25 ms). Group II cells showed glycinergic sIPSCs and glycine induced currents in wild-type, Glra1(spd-ot) and Glra3(-/-) mice (tau approximately 25 ms); however, glycinergic currents were absent in group II cells of Glra2(-/-) mice. Glycine induced currents and sIPSCs recorded from ON-starburst amacrine cells did not differ significantly between wild-type and the mutant mouse retinas (tau approximately 50-70 ms). We propose that GlyRs of group II cells are dominated by the alpha2 subunit; GlyRs of ON-starburst amacrine cells appear to be dominated by the alpha4 subunit.

Mirror-symmetrical populations of wide-field amacrine cells of the macaque monkey retina.

Retinas of macaque monkeys were immunostained for glycogen phosphorylase (glypho). Glypho was localized to regular and displaced amacrine cells. Their processes occupied two narrow strata within the inner plexiform layer (IPL). The labeling pattern is reminiscent of cholinergic amacrine cells; however, double immunostaining of the retinas for choline acetyltransferase and glypho revealed two different cell populations. Intracellular injection of DiI showed that glypho-immunoreactive amacrine cells are wide-field amacrine cells with straight, radially oriented, and sparsely branched dendrites. The density of the cells increased from approximately 70/mm(2) in the peripheral retina to approximately 700/mm(2) in the central retina. The regular glypho-immunoreactive amacrine cells branch in sublamina 2 of the IPL, where they receive input from OFF-cone bipolar cells. The displaced cells branch in sublamina 3/4 and receive input from ON-cone bipolar cells. This suggests that the regular cells are OFF-cells and the displaced cells are ON-cells. The cells express gamma-aminobutyric acid (GABA)-like immunoreactivity and receive glycinergic input through synapses expressing preferentially the glycine receptor alpha2 subunit. The close proximity of the dendritic strata of glypho-immunoreactive amacrine cells, cholinergic amacrine cells, and direction-selective ganglion cells suggests a possible role of the cells in the generation of direction-selective light responses of the monkey retina.

Type 4 OFF cone bipolar cells of the mouse retina express calsenilin and contact cones as well as rods.

Immunocytochemical discrimination of distinct bipolar cell types in the mouse retina is a prerequisite for analyzing retinal circuitry in wild-type and transgenic mice. Here we demonstrate that among the more than 10 anatomically defined mouse bipolar cell types, type 4 bipolar cells are specifically recognized by anti-calsenilin antibodies. Axon terminals in the inner plexiform layer are not readily identifiable because calsenilin is also expressed in a subset of amacrine and ganglion cells. In contrast, in the outer plexiform layer calsenilin immunoreactivity allows the analysis of photoreceptor to type 4 bipolar cell contacts. A dense plexus of calsenilin-positive dendrites makes several basal contacts at cone pedicles. An individual calsenilin-positive bipolar cell contacts five to seven cones. In addition, some calsenilin-positive dendrites contact rod photoreceptors. On average we counted 10 rod spherule contacts per type 4 bipolar cell, and approximately 10% of rods contacted type 4 bipolar cells. We suggest that type 4 bipolar cells, together with the recently described type 3a and b cells, provide an alternative and direct route from rods to OFF cone bipolar cells. In the Bassoon DeltaEx4/5 mouse, a mouse mutant that shows extensive remodeling of the rod system including sprouting of horizontal and rod bipolar cells into the outer nuclear layer due to impaired synaptic transmission, we found that in addition mixed-input (type 3 and 4) OFF bipolar cells sprout to ectopic sites. In contrast, true cone-selective type 1 and 2 OFF cone bipolar cells did not show sprouting in the Bassoon mouse mutant.

Periodicity in Na(+) channel properties alters excitability of a model neuron.

The voltage gated Na channels play vital role in action potential waveform shaping and propagation. We have shown earlier that the duration and amplitude of a prolonged depolarization alter all the steady state and kinetic parameters of rNa(v)1.2a voltage gated Na channel in a pseudo-oscillatory fashion. In the present study, we show that the Hodgkin-Huxley voltage and time dependent rate constants of activation (alpha(m) and beta(m)) and fast inactivation (alpha(h) and beta(h)), obtained from the analyses of Na currents and steady state activation and inactivation plots, following application of prepulses in both slow (1-100s) and fast (100-1000ms) ranges, vary with the duration of a prepulse in a pseudo-oscillatory manner. Using these Hodgkin-Huxley kinetic parameters in simulation, the excitability and firing pattern of a model neuron are shown to vary in a history dependent periodic fashion.

Glycine receptors of A-type ganglion cells of the mouse retina.

A-type ganglion cells of the mouse retina represent the visual channel that transfers temporal changes of the outside world very fast and with high fidelity. In this study we combined anatomical and physiological methods in order to study the glycinergic, inhibitory input of A-type ganglion cells. Immunocytochemical studies were performed in a transgenic mouse line whose ganglion cells express green fluorescent protein (GFP). The cells were double labeled for GFP and the four alpha subunits of the glycine receptor (GlyR). It was found that most of the glycinergic input of A-type cells is through fast, alpha1-expressing synapses. Whole-cell currents were recorded from A-type ganglion cells in retinal whole mounts. The response to exogenous application of glycine and spontaneous inhibitory postsynaptic currents (sIPSCs) were measured. By comparing glycinergic currents recorded in wildtype mice and in mice with specific deletions of GlyRalpha subunits (Glra1spd-ot, Glra2-/-, Glra3-/-), the subunit composition of GlyRs of A-type ganglion cells could be further defined. Glycinergic sIPSCs of A-type ganglion cells have fast kinetics (decay time constant tau = 3.9 +/- 2.5 ms, mean +/- SD). Glycinergic sIPSCs recorded in Glra2-/- and Glra3-/- mice did not differ from those of wildtype mice. However, the number of glycinergic sIPSCs was significantly reduced in Glra1spd-ot mice and the remaining sIPSCs had slower kinetics than in wildtype mice. The results show that A-type ganglion cells receive preferentially kinetically fast glycinergic inputs, mediated by GlyRs composed of alpha1 and beta subunits.

G-protein activation modulates pseudo-periodic oscillation of Na channel.

We have shown before that the duration and amplitude of both prolonged (1-160 s) and short (100-1000 ms) depolarizing prepulse altered all the steady-state and kinetic parameters of rNav1.2a voltage-gated sodium channel in a pseudo-oscillatory fashion with variable time period and amplitude, often superimposed on a linear trend. In this study, we have examined the effect of G-protein activation on pseudo-oscillatory properties of the rNav1.2a sodium channel alpha subunit, heterologously expressed in Chinese hamster ovary cells. G-protein modification caused insignificant changes in the slow pseudo-periodic oscillation of the activation properties of sodium channel; only the time period of the oscillation was altered from approximately 30 to 21s. In contrast, G-protein activation abolished the faster component of pseudo-periodic oscillation in steady-state inactivation properties of sodium channel; the conditioning duration dependence of steady-state inactivation becomes monotonic in nature.

Ionotropic glutamate receptors of amacrine cells of the mouse retina.

The mammalian retina contains approximately 30 different morphological types of amacrine cells, receiving glutamatergic input from bipolar cells. In this study, we combined electrophysiological and pharmacological techniques in order to study the glutamate receptors expressed by different types of amacrine cells. Whole-cell currents were recorded from amacrine cells in vertical slices of the mouse retina. During the recordings the cells were filled with Lucifer Yellow/Neurobiotin allowing classification as wide-field or narrow-field amacrine cells. Amacrine cell recordings were also carried out in a transgenic mouse line whose glycinergic amacrine cells express enhanced green fluorescent protein (EGFP). Agonist-induced currents were elicited by exogenous application of NMDA, AMPA, and kainate (KA) while holding cells at -75 mV. Using a variety of specific agonists and antagonists (NBQX, AP5, cyclothiazide, GYKI 52466, GYKI 53655, SYM 2081) responses mediated by AMPA, KA, and NMDA receptors could be dissected. All cells (n = 300) showed prominent responses to non-NMDA agonists. Some cells expressed AMPA receptors exclusively and some cells expressed KA receptors exclusively. In the majority of cells both receptor types could be identified. NMDA receptors were observed in about 75% of the wide-field amacrine cells and in less than half of the narrow-field amacrine cells. Our results confirm that different amacrine cell types express distinct sets of ionotropic glutamate receptors, which may be critical in conferring their unique temporal responses to this diverse neuronal class.

Induction of pseudo-periodic oscillation in voltage-gated sodium channel properties is dependent on the duration of prolonged depolarization.

The neuronal voltage-gated sodium channels play a vital role in the action potential waveform shaping and propagation. Here, we report the effects of prolonged depolarization (1-160 s) on the detailed kinetics of activation, fast inactivation and recovery from slow inactivation in the rNa(v)1.2a voltage-gated sodium channel alpha-subunit expressed in Chinese hamster ovary (CHO) cells. Wavelet analysis revealed that the duration and amplitude of a prolonged sustained depolarization altered all the steady state and kinetic parameters of the channel in a pseudo-oscillatory fashion with time-variable period and amplitude, often superimposed on a linear trend. The half steady state activation potential showed a reversible depolarizing shift of 5-10 mV with duration of prolonged depolarization, while half steady state inactivation potential showed a hyperpolarizing shift of 43-55 mV. The time periods for most of the parameters relating to activation and fast and slow inactivation, lie close to 28-30 s, suggesting coupling of these kinetic processes through an oscillatory mechanism. Co-expression of the beta1-subunit affected the time periods of oscillation (close to 22 s for alpha + beta1) in steady state activation parameters. Application of a pulse protocol that mimicked paroxysmal depolarizing shift (PDS), a kind of depolarization seen in epileptic discharges, instead of a sustained depolarization, also caused oscillatory behaviour in the rNav1.2a alpha-subunit. This inherent pseudo-oscillatory mechanism may regulate excitability of the neurons, account for the epileptic discharges and subthreshold membrane potential oscillation and offer a molecular memory mechanism intrinsic to the neurons, independent of synaptic plasticity.

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail.

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.