Comparison of cell expression formats for the characterization of GABA(A) channels using a microfluidic patch clamp system.

Ensemble recording and microfluidic perfusion are recently introduced techniques aimed at removing the laborious nature and low recording success rates of manual patch clamp. Here, we present assay characteristics for these features integrated into one automated electrophysiology platform as applied to the study of GABA(A) channels. A variety of cell types and methods of GABA(A) channel expression were successfully studied (defined as I(GABA)>500 pA), including stably transfected human embryonic kidney (HEK) cells expressing α(1)ß(3)γ(2) GABA(A) channels, frozen ready-to-assay (RTA) HEK cells expressing α(1)ß(3)γ(2) or α(3)ß(3)γ(2) GABA(A) channels, transiently transfected HEK293T cells expressing α(1)ß(3)γ(2) GABA(A) channels, and immortalized cultures of human airway smooth muscle cells endogenously expressing GABA(A) channels. Current measurements were successfully studied in multiple cell types with multiple modes of channel expression in response to several classic GABA(A) channel agonists, antagonists, and allosteric modulators. We obtained success rates above 95% for transiently or stably transfected HEK cells and frozen RTA HEK cells expressing GABA(A) channels. Tissue-derived immortalized cultures of airway smooth muscle cells exhibited a slightly lower recording success rate of 75% using automated patch, which was much higher than the 5% success rate using manual patch clamp technique by the same research group. Responses to agonists, antagonists, and allosteric modulators compared well to previously reported manual patch results. The data demonstrate that both the biophysics and pharmacologic characterization of GABA(A) channels in a wide variety of cell formats can be performed using this automated patch clamp system.

Ion channel pharmacology under flow: automation via well-plate microfluidics.

Automated patch clamping addresses the need for high-throughput screening of chemical entities that alter ion channel function. As a result, there is considerable utility in the pharmaceutical screening arena for novel platforms that can produce relevant data both rapidly and consistently. Here we present results that were obtained with an innovative microfluidic automated patch clamp system utilizing a well-plate that eliminates the necessity of internal robotic liquid handling. Continuous recording from cell ensembles, rapid solution switching, and a bench-top footprint enable a number of assay formats previously inaccessible to automated systems. An electro-pneumatic interface was employed to drive the laminar flow of solutions in a microfluidic network that delivered cells in suspension to ensemble recording sites. Whole-cell voltage clamp was applied to linear arrays of 20 cells in parallel utilizing a 64-channel voltage clamp amplifier. A number of unique assays requiring sequential compound applications separated by a second or less, such as rapid determination of the agonist EC(50) for a ligand-gated ion channel or the kinetics of desensitization recovery, are enabled by the system. In addition, the system was validated via electrophysiological characterizations of both voltage-gated and ligand-gated ion channel targets: hK(V)2.1 and human Ether-à-go-go-related gene potassium channels, hNa(V)1.7 and 1.8 sodium channels, and (α1) hGABA(A) and (α1) human nicotinic acetylcholine receptor receptors. Our results show that the voltage dependence, kinetics, and interactions of these channels with pharmacological agents were matched to reference data. The results from these IonFlux™ experiments demonstrate that the system provides high-throughput automated electrophysiology with enhanced reliability and consistency, in a user-friendly format.

IonFlux: a microfluidic patch clamp system evaluated with human Ether-à-go-go related gene channel physiology and pharmacology.

Ion channel assays are essential in drug discovery, not only for identifying promising new clinical compounds, but also for minimizing the likelihood of potential side effects. Both applications demand optimized throughput, cost, and predictive accuracy of measured membrane current changes evoked or modulated by drug candidates. Several competing electrophysiological technologies are available to address this demand, but important gaps remain. We describe the industrial application of a novel microfluidic-based technology that combines compounds, cells, and buffers on a single, standard well plate. Cell trapping, whole cell, and compound perfusion are accomplished in interconnecting microfluidic channels that are coupled to pneumatic valves, which emancipate the system from robotics, fluidic tubing, and associated maintenance. IonFlux™ is a state-of-the-art, compact system with temperature control and continuous voltage clamp for potential application in screening for voltage- and ligand-gated ion channel modulators. Here, ensemble recordings of the IonFlux system were validated with the human Ether-à-go-go related gene (hERG) channel (stably expressed in a Chinese hamster ovary cell line), which has established biophysical and pharmacological characteristics in other automated planar patch systems. We characterized the temperature dependence of channel activation and its reversal potential. Concentration response characteristics of known hERG blockers and control compounds obtained with the IonFlux system correlated with literature and internal data obtained on this cell line with the QPatch HT system. Based on the biophysical and pharmacological data, we conclude that the IonFlux system offers a novel, versatile, automated profiling, and screening system for ion channel targets with the benefit of temperature control.

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.

Synaptic and extrasynaptic factors governing glutamatergic retinal waves.

In the few days prior to eye-opening in mice, the excitatory drive underlying waves switches from cholinergic to glutamatergic. Here, we describe the unique synaptic and spatiotemporal properties of waves generated by the retina's glutamatergic circuits. First, knockout mice lacking vesicular glutamate transporter type 1 do not have glutamatergic waves, but continue to exhibit cholinergic waves, demonstrating that the two wave-generating circuits are linked. Second, simultaneous outside-out patch and whole-cell recordings reveal that retinal waves are accompanied by transient increases in extrasynaptic glutamate, directly demonstrating the existence of glutamate spillover during waves. Third, the initiation rate and propagation speed of retinal waves, as assayed by calcium imaging, are sensitive to pharmacological manipulations of spillover and inhibition, demonstrating a role for both signaling pathways in shaping the spatiotemporal properties of glutamatergic retinal waves.

Vesicular glutamate transporter 1 is required for photoreceptor synaptic signaling but not for intrinsic visual functions.

Glutamatergic neurotransmission requires vesicular glutamate transporters (VGLUTs) to sequester glutamate into synaptic vesicles. Generally, VGLUT1 and VGLUT2 isoforms show complementary expression in the CNS and retina. However, little is known about whether isoform-specific expression serves distinct pathways and physiological functions. Here, by examining visual functions in VGLUT1-null mice, we demonstrate that visual signaling from photoreceptors to retinal output neurons requires VGLUT1. However, photoentrainment and pupillary light responses are preserved. We provide evidence that melanopsin-containing, intrinsically photosensitive retinal ganglion cells (RGCs), signaling via VGLUT2 pathways, support these non-image-forming functions. We conclude that VGLUT1 is essential for transmitting visual signals from photoreceptors to second- and third-order neurons, but VGLUT1 is not necessary for intrinsic visual functions. Furthermore, melanopsin and VGLUT2 expression in a subset of RGCs immediately after birth strongly supports the idea that intrinsic vision can function well before rod- and cone-mediated signaling has matured.

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 gamma-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.

Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites.

Vesicular glutamate transporters (VGLUTs) 1 and 2 show a mutually exclusive distribution in the adult brain that suggests specialization for synapses with different properties of release. Consistent with this distribution, inactivation of the VGLUT1 gene silenced a subset of excitatory neurons in the adult. However, the same cell populations exhibited VGLUT1-independent transmission early in life. Developing hippocampal neurons transiently coexpressed VGLUT2 and VGLUT1 at distinct synaptic sites with different short-term plasticity. The loss of VGLUT1 also reduced the reserve pool of synaptic vesicles. Thus, VGLUT1 plays an unanticipated role in membrane trafficking at the nerve terminal.

Need rods? Get glycine receptors and taurine.

Taurine, a multifunctional amino acid prevalent in developing nervous tissues, regulates the number of rod photoreceptors in developing postnatal rodent retina. In this issue of Neuron, Young and Cepko show that taurine acts via GlyRalpha2 subunit-containing glycine receptors expressed by retinal progenitor cells at birth.

The H+-coupled electrogenic lysosomal amino acid transporter LYAAT1 localizes to the axon and plasma membrane of hippocampal neurons.

Recent work has identified a lysosomal protein that transports neutral amino acids (LYAAT1). We now show that LYAAT1 mediates H+ cotransport with a stoichiometry of 1 H+/1 amino acid, consistent with a role in the active efflux of amino acids from lysosomes. In neurons, however, LYAAT1 localizes to axonal processes as well as lysosomes. In axons LYAAT1 fails to colocalize with synaptic markers. Rather, axonal LYAAT1 colocalizes with the exocyst, suggesting a role for membranes expressing LYAAT1 in specifying sites for exocytosis. A protease protection assay and measurements of intracellular pH further indicate abundant expression at the plasma membrane, raising the possibility of physiological roles for LYAAT1 on the cell surface as well as in lysosomes.

Vesicular neurotransmitter transporter expression in developing postnatal rodent retina: GABA and glycine precede glutamate.

Vesicular transporters regulate the amount and type of neurotransmitter sequestered into synaptic vesicles and, hence, the kind of signal transmitted to postsynaptic neurons. Glutamate is the prominent excitatory neurotransmitter in retina; GABA and glycine are the main inhibitory neurotransmitters. Little is known about the ontogeny of vesicular neurotransmission in retina. We investigated expression of glutamatergic [vesicular glutamate transporter 1 (VGLUT1)] and GABA/glycinergic [vesicular GABA/glycine transporter (VGAT)] vesicular transporters in postnatal retina. VGLUT1 labels glutamatergic synapses. VGLUT1 and synaptic vesicle 2 colocalized to photoreceptor terminals. VGLUT1 colocalized with PKC to rod bipolar terminals and to ON bipolar terminals in metabotropic glutamate receptor 6+/- mice. Developmentally, VGAT expression precedes VGLUT1. In rat and mouse retina, VGAT occurred in the inner retina by postnatal day 1 (P1). In rat retina, VGLUT1 was in the outer retina by P5-P7 and the inner retina by P7. In the mouse retina, VGLUT1 expression was in the outer retina by P3 and the inner retina by P5. Both rat and mouse retina had an adult pattern of VGLUT1 expression by P14. VGLUT1 expression precedes ribbon synapses, which are first observed in the inner retina at P11 (Fisher, 1979) in mouse and P13 (Horsburgh and Sefton, 1987) in rat. The ribbon synapse marker RIBEYE was not detected in inner retina of P5 or P7 rat. Spontaneous EPSCs in mouse ganglion cells were recorded as early as P7. Together, these findings indicate that vesicular GABA and glycine transmission precedes vesicular glutamate transmission in developing rodent retina. Furthermore, vesicular glutamate transmission likely occurs before ribbon synapse formation in the inner retina.

The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate.

Quantal release of the principal excitatory neurotransmitter glutamate requires a mechanism for its transport into secretory vesicles. Within the brain, the complementary expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the release of glutamate by all known excitatory neurons. We now report the identification of VGLUT3 and its expression by many cells generally considered to release a classical transmitter with properties very different from glutamate. Remarkably, subpopulations of inhibitory neurons as well as cholinergic interneurons, monoamine neurons, and glia express VGLUT3. The dendritic expression of VGLUT3 by particular neurons also indicates the potential for retrograde synaptic signaling. The distribution and subcellular location of VGLUT3 thus suggest novel modes of signaling by glutamate.

Cell-specific expression of plasma membrane calcium ATPase isoforms in retinal neurons.

Ca(2+) extrusion by high-affinity plasma membrane calcium ATPases (PMCAs) is a principal mechanism for the clearance of Ca(2+) from the cytosol. The PMCA family consists of four isoforms (PMCA1-4). Little is known about the selective expression of these isoforms in brain tissues or about the physiological function conferred upon neurons by any given isoform. We investigated the cellular and subcellular distribution of PMCA isoforms in a mammalian retina. Mouse photoreceptors, cone bipolar cells and horizontal cells, which respond to light with a graded polarization, express isoform 1 (PMCA1) of the PMCA family. PMCA2 is localized to rod bipolar cells, horizontal cells, amacrine cells, and ganglion cells, and PMCA3 is predominantly expressed in spiking neurons, including both amacrine and ganglion cells but is also found in horizontal cells. PMCA4 was found to be selectively expressed in both synaptic layers. Optical measurements of Ca(2+) clearance showed that PMCAs mediate Ca(2+) extrusion in both rod and cone bipolar cells. In addition, we found that rod bipolar cells, but not cone bipolar cells possess a prominent Na(+)/Ca(2+) exchange mechanism. We conclude that PMCA isoforms are selectively expressed in retinal neurons and that processes of Ca(2+) clearance are different in rod and cone bipolar cells.