Search

Cajal recognized that the elaborate shape of neurons is fundamental to their function in the brain. However, there are no simple and generalizable genetic methods to study neuronal or glial cell morphology in the mammalian brain. Here, we describe four mouse lines conferring Cre-dependent sparse cell labeling based on mononucleotide repeat frameshift (MORF) as a stochastic translational switch. Notably, the optimized MORF3 mice, with a membrane-bound multivalent immunoreporter, confer Cre-dependent sparse and bright labeling of thousands of neurons, astrocytes, or microglia in each brain, revealing their intricate morphologies. MORF3 mice are compatible with imaging in tissue-cleared thick brain sections and with immuno-EM. An analysis of 151 MORF3-labeled developing retinal horizontal cells reveals novel morphological cell clusters and axonal maturation patterns. Our study demonstrates a conceptually novel, simple, generalizable, and scalable mouse genetic solution to sparsely label and illuminate the morphology of genetically defined neurons and glia in the mammalian brain.

Feedback inhibition by horizontal cells regulates rod and cone photoreceptor calcium channels that control their release of the neurotransmitter glutamate. This inhibition contributes to synaptic gain control and the formation of the center-surround antagonistic receptive fields passed on to all downstream neurons, which is important for contrast sensitivity and color opponency in vision. In contrast to the plasmalemmal GABA transporter found in non-mammalian horizontal cells, there is evidence that the mechanism by which mammalian horizontal cells inhibit photoreceptors involves the vesicular release of the inhibitory neurotransmitter GABA. Historically, inconsistent findings of GABA and its biosynthetic enzyme, L-glutamate decarboxylase (GAD) in horizontal cells, and the apparent lack of surround response block by GABAergic agents diminished support for GABA's role in feedback inhibition. However, the immunolocalization of the vesicular GABA transporter (VGAT) in the dendritic and axonal endings of horizontal cells that innervate photoreceptor terminals suggested GABA was released via vesicular exocytosis. To test the idea that GABA is released from vesicles, we localized GABA and GAD, multiple SNARE complex proteins, synaptic vesicle proteins, and Cav channels that mediate exocytosis to horizontal cell dendritic tips and axonal terminals. To address the perceived relative paucity of synaptic vesicles in horizontal cell endings, we used conical electron tomography on mouse and guinea pig retinas that revealed small, clear-core vesicles, along with a few clathrin-coated vesicles and endosomes in horizontal cell processes within photoreceptor terminals. Some small-diameter vesicles were adjacent to the plasma membrane and plasma membrane specializations. To assess vesicular release, a functional assay involving incubation of retinal slices in luminal VGAT-C antibodies demonstrated vesicles fused with the membrane in a depolarization- and calcium-dependent manner, and these labeled vesicles can fuse multiple times. Finally, targeted elimination of VGAT in horizontal cells resulted in a loss of tonic, autaptic GABA currents, and of inhibitory feedback modulation of the cone photoreceptor Cai, consistent with the elimination of GABA release from horizontal cell endings. These results in mammalian retina identify the central role of vesicular release of GABA from horizontal cells in the feedback inhibition of photoreceptors.

How neurons in the eye feed signals back to photoreceptors to optimize sensitivity to patterns of light appears to be mediated by one or more unconventional mechanisms. Via these mechanisms, horizontal cells control photoreceptor synaptic gain and enhance key aspects of temporal and spatial center-surround receptive field antagonism. After the transduction of light energy into an electrical signal in photoreceptors, the next key task in visual processing is the transmission of an optimized signal to the follower neurons in the retina. For this to happen, the release of the excitatory neurotransmitter glutamate from photoreceptors is carefully regulated via horizontal cell feedback, which acts as a thermostat to keep the synaptic transmission in an optimal range during changes to light patterns and intensities. Novel findings of a recently described model that casts a classical neurotransmitter system together with ion transport mechanisms to adjust the alkaline milieu outside the synapse are reviewed. This novel inter-neuronal messaging system carries feedback signals using two separate, but interwoven regulated systems. The complex interplay between these two signaling modalities, creating synaptic modulation-at-a-distance, has obscured it's being defined. The foundations of our understanding of the feedback mechanism from horizontal cells to photoreceptors have been long established: Horizontal cells have broad receptive fields, suitable for providing surround inhibition, their membrane potential, a function of stimulus intensity and size, regulates inhibition of photoreceptor voltage-gated Ca2+ channels, and strong artificial pH buffering eliminates this action. This review compares and contrasts models of how these foundations are linked, focusing on a recent report in mammals that shows tonic horizontal cell release of GABA activating Cl- and HCO3 - permeable GABA autoreceptors. The membrane potential of horizontal cells provides the driving force for GABAR-mediated HCO3 - efflux, alkalinizing the cleft when horizontal cells are hyperpolarized by light or adding to their depolarization in darkness and contributing to cleft acidification via NHE-mediated H+ efflux. This model challenges interpretations of earlier studies that were considered to rule out a role for GABA in feedback to cones.

The stream of visual information sent from photoreceptors to second-order bipolar cells is intercepted by laterally interacting horizontal cells that generate feedback to optimize and improve the efficiency of signal transmission. The mechanisms underlying the regulation of graded photoreceptor synaptic output in this nonspiking network have remained elusive. Here, we analyze with patch clamp recording the novel mechanisms by which horizontal cells control pH in the synaptic cleft to modulate photoreceptor neurotransmitter release. First, we show that mammalian horizontal cells respond to their own GABA release and that the results of this autaptic action affect cone voltage-gated Ca2+ channel (CaV channel) gating through changes in pH. As a proof-of-principle, we demonstrate that chemogenetic manipulation of horizontal cells with exogenous anion channel expression mimics GABA-mediated cone CaV channel inhibition. Activation of these GABA receptor anion channels can depolarize horizontal cells and increase cleft acidity via Na+/H+ exchanger (NHE) proton extrusion, which results in inhibition of cone CaV channels. This action is effectively counteracted when horizontal cells are sufficiently hyperpolarized by increased GABA receptor (GABAR)-mediated HCO3- efflux, alkalinizing the cleft and disinhibiting cone CaV channels. This demonstrates how hybrid actions of GABA operate in parallel to effect voltage-dependent pH changes, a novel mechanism for regulating synaptic output.

The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGAT(flox/flox) mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT(-/-) mice were the same as Cx57-VGAT(+/+) controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABAA receptor agonist muscimol in Cx57-VGAT(-/-) mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT(-/-) mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors.

The ability of light to cause pain is paradoxical. The retina detects light but is devoid of nociceptors while the trigeminal sensory ganglia (TG) contain nociceptors but not photoreceptors. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are thought to mediate light-induced pain but recent evidence raises the possibility of an alternative light responsive pathway independent of the retina and optic nerve. Here, we show that melanopsin is expressed in both human and mouse TG neurons. In mice, they represent 3% of small TG neurons that are preferentially localized in the ophthalmic branch of the trigeminal nerve and are likely nociceptive C fibers and high-threshold mechanoreceptor Aδ fibers based on a strong size-function association. These isolated neurons respond to blue light stimuli with a delayed onset and sustained firing, similar to the melanopsin-dependent intrinsic photosensitivity observed in ipRGCs. Mice with severe bilateral optic nerve crush exhibit no light-induced responses including behavioral light aversion until treated with nitroglycerin, an inducer of migraine in people and migraine-like symptoms in mice. With nitroglycerin, these same mice with optic nerve crush exhibit significant light aversion. Furthermore, this retained light aversion remains dependent on melanopsin-expressing neurons. Our results demonstrate a novel light-responsive neural function independent of the optic nerve that may originate in the peripheral nervous system to provide the first direct mechanism for an alternative light detection pathway that influences motivated behavior.

An inner retinal microcircuit composed of dopamine (DA)-containing amacrine cells and melanopsin-containing, intrinsically photosensitive retinal ganglion cells (M1 ipRGCs) process information about the duration and intensity of light exposures, mediating light adaptation, circadian entrainment, pupillary reflexes, and other aspects of non-image-forming vision. The neural interaction is reciprocal: M1 ipRGCs excite DA amacrine cells, and these, in turn, feed inhibition back onto M1 ipRGCs. We found that the neuropeptide somatostatin [somatotropin release inhibiting factor (SRIF)] also inhibits the intrinsic light response of M1 ipRGCs and postulated that, to tune the bidirectional interaction of M1 ipRGCs and DA amacrine cells, SRIF amacrine cells would provide inhibitory modulation to both cell types. SRIF amacrine cells, DA amacrine cells, and M1 ipRGCs form numerous contacts. DA amacrine cells and M1 ipRGCs express the SRIF receptor subtypes sst(2A) and sst4 respectively. SRIF modulation of the microcircuit was investigated with targeted patch-clamp recordings of DA amacrine cells in TH-RFP mice and M1 ipRGCs in OPN4-EGFP mice. SRIF increases K(+) currents, decreases Ca(2+) currents, and inhibits spike activity in both cell types, actions reproduced by the selective sst(2A) agonist L-054,264 (N-[(1R)-2-[[[(1S*,3R*)-3-(aminomethyl)cyclohexyl]methyl]amino]-1-(1H-indol-3-ylmethyl)-2-oxoethyl]spiro[1H-indene-1,4'-piperidine]-1'-carboxamide) in DA amacrine cells and the selective sst4 agonist L-803,087 (N(2)-[4-(5,7-difluoro-2-phenyl-1H-indol-3-yl)-1-oxobutyl]-L-arginine methyl ester trifluoroacetate) in M1 ipRGCs. These parallel actions of SRIF may serve to counteract the disinhibition of M1 ipRGCs caused by SRIF inhibition of DA amacrine cells. This allows the actions of SRIF on DA amacrine cells to proceed with adjusting retinal DA levels without destabilizing light responses by M1 ipRGCs, which project to non-image-forming targets in the brain.

Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury.

To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained ∼470 vesicles that were hemi-fused and ∼187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3-15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.

The aim of the present study was to evaluate the expression of two high affinity GABA transporters (GAT-1 and GAT-3) in the rat cerebellum using immunocytochemistry and affinity purified antibodies. GAT-1 immunoreactivity was prominent in punctate structures and axons in all layers of the cerebellar cortex, and was especially prominent around the somata of Purkinje cells. In contrast, the deep cerebellar nuclei showed few if any GAT-1 immunoreactive puncta. Weak GAT-3 immunoreactive processes were present in the cerebellar cortex, whereas GAT-3 immunostaining was prominent around the somata of neurons in the deep cerebellar nuclei. Electron microscopic preparations of the cerebellar cortex demonstrated that GAT-1 immunoreactive axon terminals formed symmetric synapses with somata, axon initial segments and dendrites of Purkinje cells and the dendrites of granule cells. Astrocytic processes in the cerebellar cortex were also immunolabeled for GAT-1. However, Purkinje cell axon terminals that formed symmetric synapses with neurons in the deep cerebellar nuclei lacked GAT-1 immunoreactivity. Instead, weak GAT-1 and strong GAT-3 immunoreactivities were expressed by astrocytic processes that enveloped the Purkinje cell axon terminals. In addition, GAT-3-immunoreactivity appeared in astrocytic processes in the cerebellar cortex. These observations demonstrate that GAT-1 is localized to axon terminals of three of the four neuronal types that were previously established as being GABAergic, i.e. basket, stellate and Golgi cells. GAT-1 and GAT-3 are expressed by astrocytes. The failure to identify a GABA transporter in Purkinje cells is consistent with previous data that indicated that Purkinje cells lacked terminal uptake mechanisms for GABA. The individual glial envelopment of Purkinje cell axon terminals in the deep cerebellar nuclei and the dense immunostaining of GAT-3, and to a lesser extent GAT-1, expressed by astrocytic processes provide a compensatory mechanism for the removal of GABA from the synaptic cleft of synapses formed by Purkinje cell axon terminals.

Increasing spike rates drive greater neuronal energy demand. In turn, mitochondrial ATP production leads to the generation of reactive oxygen species (ROS) that can modulate ion channel gating. Does ROS production autoregulate the excitability of a neuron? We investigated the links between retinal ganglion cell (RGC) excitability and spike activity-driven ROS production in male and female mice. Changes to the light-evoked and current-evoked spike patterns of functionally identified aRGC subtypes, along with their NaV channel-gating properties, were recorded during experimentally induced decreases and increases of intracellular ROS. During periods of highest spike rates (e.g., following light onset in ON sustained RGCs and light offset in OFF sustained RGCs), these aRGC subtypes responded to reductions of ROS (induced by catalase or glutathione monoethyl ester) with higher spike rates. Increases in ROS (induced by mercaptosuccinate, antimycin-A, or H2O2) lowered spike rates. In ON and OFF transient RGCs, there were no changes in spike rate during ROS decreases but increased ROS increased spiking. This suggests that endogenous ROS are intrinsic neuromodulators in RGCs having high metabolic demands but not in RGCs with lower energy needs. We identified ROS-induced shifts in the voltage- dependent gating of specific isoforms of NaV channels that account for the modulation of ON and OFF sustained RGC spike frequency by ROS-mediated feedback. ROS-induced changes to NaV channel gating, affecting activation and inactivation kinetics, are consistent with the differing spike pattern alterations observed in RGC subtypes. Cell-autonomous generation of ROS during spiking contributes to tuning the spike patterns of RGCs. [ABSTRACT FROM AUTHOR]

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the [Mu] opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the [Delta] or [Kappa] receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesiclelike structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.