Optocapacitive Generation of Action Potentials by Microsecond Laser Pulses of Nanojoule Energy.

Millisecond pulses of laser light delivered to gold nanoparticles residing in close proximity to the surface membrane of neurons can induce membrane depolarization and initiate an action potential. An optocapacitance mechanism proposed as the basis of this effect posits that the membrane-interfaced particle photothermally induces a cell-depolarizing capacitive current, and predicts that delivering a given laser pulse energy within a shorter period should increase the pulse's action-potential-generating effectiveness by increasing the magnitude of this capacitive current. Experiments on dorsal root ganglion cells show that, for each of a group of interfaced gold nanoparticles and microscale carbon particles, reducing pulse duration from milliseconds to microseconds markedly decreases the minimal pulse energy required for AP generation, providing strong support for the optocapacitance mechanism hypothesis.

Re-engineering a neuroprotective, clinical drug as a procognitive agent with high in vivo potency and with GABAA potentiating activity for use in dementia.

BACKGROUND: Synaptic dysfunction is a key event in pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) where synapse loss pathologically correlates with cognitive decline and dementia. Although evidence suggests that aberrant protein production and aggregation are the causative factors in familial subsets of such diseases, drugs singularly targeting these hallmark proteins, such as amyloid-ß, have failed in late stage clinical trials. Therefore, to provide a successful disease-modifying compound and address synaptic dysfunction and memory loss in AD and mixed pathology dementia, we repurposed a clinically proven drug, CMZ, with neuroprotective and anti-inflammatory properties via addition of nitric oxide (NO) and cGMP signaling property. RESULTS: The novel compound, NMZ, was shown to retain the GABAA potentiating actions of CMZ in vitro and sedative activity in vivo. Importantly, NMZ restored LTP in hippocampal slices from AD transgenic mice, whereas CMZ was without effect. NMZ reversed amnestic blockade of acetylcholine receptors by scopolamine as well as NMDA receptor blockade by a benzodiazepine and a NO synthase inhibitor in the step-through passive avoidance (STPA) test of learning and working memory. A PK/PD relationship was developed based on STPA analysis coupled with pharmacokinetic measures of drug levels in the brain: at 1 nM concentration in brain and plasma, NMZ was able to restore memory consolidation in mice. CONCLUSION: Our findings show that NMZ embodies a promising pharmacological approach targeting synaptic dysfunction and opens new avenues for neuroprotective intervention strategies in mixed pathology AD, neurodegeneration, and dementia.

Reduction of amyloid-beta levels in mouse eye tissues by intra-vitreally delivered neprilysin.

Amyloid-beta (Aß) is a group of aggregation-prone, 38- to 43-amino acid peptides generated in the eye and other organs. Numerous studies suggest that the excessive build-up of low-molecular-weight soluble oligomers of Aß plays a role in the progression of Alzheimer's disease and other brain degenerative diseases. Recent studies raise the hypothesis that excessive Aß levels may contribute also to certain retinal degenerative diseases. These findings, together with evidence that a major portion of Aß is released as monomer into the extracellular space, raise the possibility that a technology enabling the enzymatic break-down of monomeric Aß in the living eye under physiological conditions could prove useful for research on ocular Aß physiology and, perhaps ultimately, for therapeutic applications. Neprilysin (NEP), an endopeptidase known to cleave Aß monomer into inactive products, is a membrane-associated protein. However, sNEP, a recombinant form of the NEP catalytic domain, is soluble in aqueous medium. With the aim of determining the Aß-cleaving activity of exogenous sNEP in the microenvironment of the intact eye, we analyzed the effect of intra-vitreally delivered sNEP on ocular Aß levels in mice that exhibit readily measurable, aqueous buffer-extractable Aß40 and Aß42, two principal forms of Aß. Anesthetized 10-month wild-type (C57BL/6J) and 2-3-month 5XFAD transgenic mice received intra-vitreal injections of sNEP (0.004-10 µg) in one eye and were sacrificed at defined post-treatment times (30 min - 12 weeks). Eye tissues (combined lens, vitreous, retina, RPE and choroid) were homogenized in phosphate-buffered saline, and analyzed for Aß40 and Aß42 (ELISA) and for total protein (Bradford assay). The fellow, untreated eye of each mouse served as control, and concentrations of Aß (pmol/g protein) in the treated eye were normalized to that of the untreated control eye. In C57BL/6J mice, as measured at 2 h after sNEP treatment, increasing amounts of injected sNEP yielded progressively greater reductions of Aß40, ranging from 12% ± 3% (mean ± SEM; n = 3) with 4 ng sNEP to 85% ± 13% (n = 5) with 10 µg sNEP. At 4 ng sNEP the average Aß40 reduction reached >70% by 24 h following treatment and remained near this level for about 8 weeks. In 5XFAD mice, 10 µg sNEP produced an Aß40 decrease of 99% ± 1% (n = 4) and a substantial although smaller decrease in Aß42 (42% ± 36%; n = 4) within 24 h. Electroretinograms (ERGs) were recorded from eyes of C57BL/6J and 5XFAD mice at 9 days following treatment with 4 ng or 10 µg sNEP, conditions that on average led, respectively, to an 82% and 91% Aß40 reduction in C57BL/6J eyes, an 87% and 92% Aß40 reduction in 5XFAD eyes, and a 23% and 52% Aß42 reduction in 5XFAD eyes. In all cases, sNEP-treated eyes exhibited robust ERG responses, consistent with a general tolerance of the posterior eye tissues to the investigated conditions of sNEP treatment. The sNEP-mediated decrease of ocular Aß levels reported here represents a possible approach for determining effects of Aß reduction in normally functioning eyes and in models of retinal degenerative disease.

In vivo electroretinographic studies of the role of GABAC receptors in retinal signal processing.

All three classes of receptors for the inhibitory neurotransmitter GABA (GABAR) are expressed in the retina. This study investigated roles of GABAR, especially GABACR (GABA(A)-ρ), in retinal signaling in vivo by studying effects on the mouse electroretinogram (ERG) of genetic deletion of GABACR versus pharmacological blockade using receptor antagonists. Brief full-field flash ERGs were recorded from anesthetized GABACR(-/-) mice, and WT C57BL/6 (B6) mice, before and after intravitreal injection of GABACR antagonists, TPMPA, 3-APMPA, or the more recently developed 2-AEMP; GABAAR antagonist, SR95531; GABABR antagonist, CGP, and agonist, baclofen. Intravitreal injections of TPMPA and SR95531 were also made in Brown Norway rats. The effect of 2-AEMP on GABA-induced current was tested directly in isolated rat rod bipolar cells, and 2-AEMP was found to preferentially block GABACR in those cells. Maximum amplitudes of dark (DA) and light-adapted (LA) ERG b-waves were reduced in GABACR(-/-) mice, compared to B6 mice, by 30-60%; a-waves were unaltered and oscillatory potential amplitudes were increased. In B6 mice, after injection of TPMPA (also in rats), 3-APMPA or 2-AEMP, ERGs became similar to ERGs of GABACR(-/-) mice. Blockade of GABAARs and GABABRs, or agonism of GABABRs did not alter B6 DA b-wave amplitude. The negative scotopic threshold response (nSTR) was slightly less sensitive in GABACR(-/-) than in B6 mice, and unaltered by 2-AEMP. However, amplitudes of nSTR and photopic negative response (PhNR), both of which originate from inner retina, were enhanced by TPMPA and 3-APMPA, each of which has GABAB agonist properties, and further increased by baclofen. The finding that genetic deletion of GABACR, the GABACR antagonist 2-AEMP, and other antagonists all reduced ERG b-wave amplitude, supports a role for GABACR in determining the maximum response amplitude of bipolar cells contributing to the b-wave. GABACR antagonists differed in their effects on nSTR and PhNR; antagonists with GABAB agonist properties enhanced light-driven responses whereas 2-AEMP did not.

Cysteine-terminated B-domain of Staphylococcus aureus protein A as a scaffold for targeting GABA(A) receptors.

This study reports the preparation and characterization of cysteine-terminated B-domain (Bd-cys) of Staphylococcus aureus protein A, in combination with immunoglobulin G (IgG) antibodies directed against the ρ1 and α1 subunits of GABA(A) receptors, for localizing reagents of interest to the target receptor. A cysteine residue was inserted at the C terminus of the cysteine-lacking B-domain (Bd) and used for conjugating maleimide-containing compounds. As determined by enzyme-linked immunosorbent assay (ELISA), binding of a Bd-cys-S-fluorescein conjugate to polyclonal guinea pig anti-GABA(A)-ρ1 and rabbit anti-GABA(A)-α1 IgG was similar to that exhibited by full-length protein A. Surface plasmon resonance analysis of the interaction of Bd-cys-S-PEG3400-biotin conjugate (where PEG is polyethylene glycol) with anti-GABA(A)-ρ1 and anti-GABA(A)-α1 yielded K(D) values of 6.4 ± 1.9 and 0.4 ± 0.1 nM, respectively. Fluorescence anisotropy analysis of the binding of Bd-cys-S-fluorescein to the two antibodies yielded EC50 values of 65 and 18 nM, respectively. As determined with biotin-reactive fluorescent reagents, Bd-cys-S-PEG3400-biotin specifically bound to the plasma membrane of Xenopus laevis oocytes that expressed α1ß2γ2 or homomeric ρ1 GABA(A) receptors and were pretreated with the corresponding anti-GABA(A) IgG. The IgG-binding specificity and high affinity of Bd-cys conjugates illustrate the potential of these conjugates, in combination with a selected IgG, to localize compounds of interest at specific cell surface proteins.

2-Aminoethyl methylphosphonate, a potent and rapidly acting antagonist of GABA(A)-ρ1 receptors.

2-Aminoethyl methylphosphonate (2-AEMP), an analog of GABA, has been found to exhibit antagonist activity at GABA(A)-ρ1 (also known as ρ1 GABA(C)) receptors. The present study was undertaken to elucidate 2-AEMP's action and to test the activities of 2-AEMP analogs. Whole-cell patch-clamp techniques were used to record membrane currents in neuroblastoma cells stably transfected with human GABA(A)-ρ1 receptors. The action of 2-AEMP was compared with that of 1,2,5,6-tetrahydropyridin-4-yl methylphosphinic acid (TPMPA), a commonly used GABA(A)-ρ1 antagonist. With 10 µM GABA, 2-AEMP's IC(50) (18 µM) differed by less than 2.5-fold from that of TPMPA (7 µM), and results obtained were consistent with a primarily competitive mode of inhibition by 2-AEMP. Terminating the presentation of 2-AEMP or TPMPA in the presence of GABA produced a release from inhibition. However, the rate of inhibition release upon the termination of 2-AEMP considerably exceeded that determined with termination of TPMPA. Moreover, when presented at concentrations near their respective IC(50) values, the preincubation period associated with 2-AEMP's onset of inhibition was much shorter than that for TPMPA. Analogs of 2-AEMP possessing a benzyl or n-butyl rather than a methyl substituent at the phosphorus atom, as well as analogs bearing a C-methyl substituent on the aminoethyl side chain, exhibited reduced potency relative to 2-AEMP. Of these analogs, only (R)-2-aminopropyl methylphosphonate significantly diminished the response to 10 µM GABA. Structure-activity relationships are discussed in the context of molecular modeling of ligand binding to the antagonist binding site of the GABA(A)-ρ1 receptor.

Subunit-specific polyclonal antibody targeting human ρ1 GABA(C) receptor.

The GABA(C) receptor, a postsynaptic membrane receptor expressed prominently in the retina, is a ligand-gated ion channel that consists of a combination of ρ subunits. We report characterization of a novel guinea pig polyclonal antibody, termed GABA(C) Ab N-14, directed against a 14-mer peptide (N-14) of the extracellular domain of the human ρ1 subunit. The antibody exhibits high sensitivity for N-14 by ELISA. In Western blots, GABA(C) Ab N-14 shows reactivity with the ρ1 subunit of preparations obtained from ρ1 GABA(C)-expressing neuroblastoma cells, Xenopus oocytes, and mammalian retina and brain. Flow cytometry reveals a rightward shift in mean fluorescence intensity of GABA(C)-expressing neuroblastoma cells probed with GABA(C) Ab N-14. Immunostaining of neuroblastoma cells and oocytes with GABA(C) Ab N-14 yields fluorescence only with GABA(C)-expressing cells. Antibody binding has no effect on GABA-elicited membrane current responses. Immunostaining of human retinal sections shows preferential staining within the inner plexiform layer. GABA(C) Ab N-14 appears well suited for investigative studies of GABA(C) ρ1 subunit in retina and other neural tissues.

GABAC receptor binding of quantum-dot conjugates of variable ligand valency.

Highly fluorescent CdSe quantum dots (qdots) can serve as a platform for tethering multiple copies of a receptor-targeted ligand, affording study of how the level of multivalency affects receptor binding. We previously showed that qdots conjugated with long PEG chains terminated by muscimol, a known GABA(C) agonist, exhibit specific binding to the surface membrane of GABA(C) receptor-expressing Xenopus oocytes. The present report addresses the effect of varying the number, i.e., valency, of muscimol- (M-) terminated PEG chains attached to the qdot on binding of the resulting conjugate to GABA(C) receptors. M-PEG-qdots of differing muscimol valency were prepared by conjugating AMP-CdSe/ZnS qdots with muscimol-terminated and methylamine-terminated PEG chains in proportions designed to yield varying percentages of muscimol-terminated chains among the total approximately 150-200 chains bound to the qdot. The investigated valencies represented 0%, approximately 25%, approximately 50%, and 100% loading with muscimol (preparations termed M-PEG-qdot0, M-PEG-qdot25, M-PEG-qdot50, and M-PEG-qdot100, respectively. Binding of a given conjugate to surface membranes of GABA(C) receptor-expressing oocytes was analyzed by quantitative fluorescence microscopy following defined incubation with approximately 30 nM of the conjugate. With 5-20 min incubation, the fluorescence signal resulting from incubation with M-PEG-qdot25 exceeded, by approximately 6-fold, the fluorescence level obtained with M-PEG-qdot preparations that lacked muscimol-terminated chains (M-PEG-qdot0). M-PEG-qdot50 yielded a net signal roughly similar to that of M-PEG-qdot25, and that produced by M-PEG-qdot100 exceeded, by approximately 30-50%, those for M-PEG-qdot25 and M-PEG-qdot50. The time course of changes in oocyte surface membrane fluorescence resulting from the introduction of and removal of M-PEG-qdots in the medium bathing the oocyte indicated only a modest dependence of both binding and wash-out kinetics on muscimol valency. The results demonstrate a dependence of the binding activity of the M-PEG-qdot conjugates on muscimol valency, presumably reflecting higher GABA(C) avidity and/or affinity of the muscimol at high valency, and provide insight on the interactions of membrane receptor proteins with qdot conjugates containing multiple copies of a receptor-targeting ligand.

Amyloid-ß-Dependent Inactivation of the Mitochondrial Electron Transport Chain at Low Transmembrane Potential: An Ameliorating Process in Hypoxia-Associated Neurodegenerative Disease?

Cerebral hypoperfusion-induced hypoxia, a condition that impairs oxygen utilization and thus ATP production by mitochondrial oxidative phosphorylation (oxphos), is thought to contribute to neural degeneration in Alzheimer's disease. However, hypoxia upregulates the generation of amyloid-ß (Aß), a group of peptides known to impair/inhibit the electron transport chain (ETC) of reactions that support oxphos in the inner mitochondrial membrane (IMM). This is a hypothesis paper that reconciles the hypoxia-induced upregulation of Aß with Aß's ETC-inhibiting action and, specifically, posits an oxphos-enhancing effect of this inhibition under conditions of newly developing or otherwise mild hypoxia. This effect is typically transient; that is, under conditions of prolonged or severe hypoxia, the oxphos-enhancing activity is overwhelmed by Aß's well-known toxic actions on mitochondria and other cellular components. The hypothesis is motivated by evidence that the IMM transmembrane potential Ψm, an important determinant of ETC activity, exhibits heterogeneity, i.e., a range of values, among a given local population of mitochondria. It specifically proposes that during oxygen limitation, Aß selectively inactivates ETC complexes in mitochondria that exhibit relatively low absolute values of Ψm, thereby suppressing oxygen binding and consumption by complex IV of the ETC in these mitochondria. This effect of Aß on low-Ψm mitochondria is hypothesized to spare hypoxia-limited oxygen for oxphos-enabling utilization by the ETC of the remaining active, higher-Ψm local mitochondria, and thereby to increase overall ATP generated collectively by the local mitochondrial population, i.e., to ameliorate hypoxia-induced oxphos reduction. The protective action of Aß hypothesized here may slow the early development of hypoxia-associated cellular deterioration/loss in Alzheimer's disease and perhaps other neurodegenerative diseases.

Cholesterol Functionalization of Gold Nanoparticles Enhances Photoactivation of Neural Activity.

Gold nanoparticles (AuNPs) attached to the extracellular leaflet of the plasma membrane of neurons can enable the generation of action potentials (APs) in response to brief pulses of light. Recently described techniques to stably bind AuNP bioconjugates directly to membrane proteins (ion channels) in neurons enable robust AP generation mediated by the photoexcited conjugate. However, a strategy that binds the AuNP to the plasma membrane in a non protein-specific manner could represent a simple, single-step means of establishing light-responsiveness in multiple types of excitable neurons contained in the same tissue. On the basis of the ability of cholesterol to insert into the plasma membrane, here we test whether AuNP functionalization with linear dihydrolipoic acid-poly(ethylene) glycol (DHLA-PEG) chains that are distally terminated with cholesterol (AuNP-PEG-Chol) can enable light-induced AP generation in neurons. Dorsal root ganglion (DRG) neurons of rat were labeled with 20 nm diameter spherical AuNP-PEG-Chol conjugates wherein ∼30% of the surface ligands (DHLA-PEG-COOH) were conjugated to PEG-Chol. Voltage recordings under current-clamp conditions showed that DRG neurons labeled in this manner exhibited a capacity for AP generation in response to microsecond and millisecond pulses of 532 nm light, a property attributable to the close tethering of AuNP-PEG-Chol conjugates to the plasma membrane facilitated by the cholesterol moiety. Light-induced AP and subthreshold depolarizing responses of the DRG neurons were similar to those previously described for AuNP conjugates targeted to channel proteins using large, multicomponent immunoconjugates. This likely reflected the AuNP-PEG-Chol's ability, upon plasmonic light absorption and resultant slight and rapid heating of the plasma membrane, to induce a concomitant transmembrane depolarizing capacitive current. Notably, AuNP-PEG-Chol delivered to DRG neurons by inclusion in the buffer contained in the recording pipet/electrode enabled similar light-responsiveness, consistent with the activity of AuNP-PEG-Chol bound to the inner (cytofacial) leaflet of the plasma membrane. Our results demonstrate the ability of AuNP-PEG-Chol conjugates to confer timely stable and direct responsiveness to light in neurons. Further, this strategy represents a general approach for establishing excitable cell photosensitivity that could be of substantial advantage for exploring a given tissue's suitability for AuNP-mediated photocontrol of neural activity.

Recovery of rod photoresponses in ABCR-deficient mice.

PURPOSE: The ABCR protein of the rod outer segment is thought to facilitate movement of the all-trans retinal photoproduct of rhodopsin bleaching out of the disc lumen. This study was undertaken to investigate the extent to which ABCR deficiency affects the post-bleach recovery of the rod photoresponse in ABCR-deficient (abcr-/-) mice. METHODS: Electroretinographic (ERG) a-wave responses were recorded from abcr-/- mice and two control strains. A bright probe flash was used to examine the course of rod recovery after fractional rhodopsin bleaches of approximately 10(-6), approximately 3 x 10(-5), approximately 0.03, and approximately 0.30 to approximately 0.40. RESULTS: Dark-adapted abcr-/- mice and control animals exhibited similar normalized near-peak amplitudes of the paired-flash-ERG-derived, weak-flash response. Response recovery after approximately 10(-6) bleaching exhibited an average exponential time constant of 319, 171, and 213 ms, respectively, in the abcr-/- and the two control strains. Recovery time constants determined for approximately 3 x 10(-5) bleaching did not differ significantly among strains. However, those determined for the approximately 0.03 bleach indicated significantly faster recovery in abcr-/- mice (2.34 +/- 0.74 minutes) than in the controls (5.36 +/- 2.20 and 5.92 +/- 2.44 minutes). After approximately 0.30 to approximately 0.40 bleaching, the initial recovery in the abcr-/- mice was, on average, faster than in control mice. CONCLUSIONS: By comparison with control animals, abcr-/- mice exhibit faster rod recovery after a bleach of approximately 0.03. The data suggest that ABCR in normal rods may directly or indirectly prolong all-trans retinal clearance from the disc lumen over a significant bleaching range, and that the essential function of ABCR may be to promote the clearance of residual amounts of all-trans retinal that remain in the discs long after bleaching.

Preservation of retinoid influx into eye tissues of ABCR-deficient mice.

Using an in vivo radiolabeling technique, we investigated the movement of retinoid into the retinal pigment epithelium (RPE) of the abcr-/- mouse, which lacks the photoreceptor ABCR protein and is a model for Stargardt disease. Eye tissues and serum obtained from dark-adapted, 5- to 8-month-old abcr-/- and control mice following the intraperitoneal injection of all-trans ((3)H)retinol were analyzed to determine the inferred influx of retinoid from the serum into the RPE. At 4.5 hr post-injection, the inferred all-trans retinol influx in abcr-/- mice, which possess the leucine 450 variant of RPE65 protein, was 0.011 +/- 0.004 nmol (n = 3). This value did not differ significantly from that determined in age-matched controls possessing the methionine 450 variant of RPE65 (0.015 +/- 0.003 nmol; n = 3) or from 3-month-old wildtype mice that possess the leucine 450 RPE65 variant (0.020 +/- 0.007; n = 4). Thus, the absence of ABCR does not significantly compromise the passage of retinoid from the serum into the RPE under dark-adapted conditions.

Stimulus-evoked outer segment changes occur before the hyperpolarization of retinal photoreceptors.

Transient retinal phototropism (TRP) has been predominantly observed in rod photoreceptors activated by oblique visible light stimulation. Dynamic confocal microscopy and optical coherence tomography (OCT) have revealed rod outer segment (ROS) movement as the physical source of TRP. However, the physiological source of ROS movement is still not well understood. In this study, concurrent near-infrared imaging of TRP and electroretinogram (ERG) measurement of retinal electrophysiology revealed that ROS movement occurs before the onset of the ERG a-wave, which is known to reflect the hyperpolarization of retinal photoreceptors. Moreover, substitution of normal superfusing medium with low-sodium medium reversibly blocked the photoreceptor ERG a-wave, but largely preserved the stimulus-evoked ROS movements. Our experimental results and theoretical analysis indicate that early, disc-based stages of the phototransduction cascade, which occur before the hyperpolarization of retinal photoreceptors, contribute to the TRP associated ROS movement.

Activation of membrane receptors by neurotransmitter released from temperature-sensitive hydrogels.

The present paper describes the design, construction and testing of a temperature-sensitive N-isopropylacrylamide hydrogel device for studying the controlled presentation of gamma-aminobutyric acid (GABA) to GABA(C) membrane receptors expressed in Xenopus laevis oocytes. Upon temperature lowering, the GABA-loaded hydrogel positioned near the surface of the GABA(C)-expressing oocyte elicits a membrane current response resembling that induced by superfusion of the oocyte with free GABA. The response to cooling is not observed when GABA is omitted from the hydrogel loading solution. In addition, picrotoxin, a known GABA(C) receptor antagonist, inhibits the oocyte membrane current response associated with temperature lowering of GABA-loaded hydrogels. The data indicate that the present system affords a temperature-regulated release of GABA from the hydrogel and a resulting activation of the expressed GABA(C) receptors.

Synthesis and characterization of an electroactive surface that releases gamma-aminobutyric acid (GABA).

We report the synthesis and characterization of a new electroactive surface capable of releasing the neurotransmitter gamma-aminobutyric acid (GABA) upon reduction. The GABA was anchored to an alkanethiol via electrochemically active quinone (abbreviation, TM-GABA). The quinone unit, upon reduction to the hydroquinone, cyclizes to release GABA into solution. The half-life is 99 s. The self-assembled monolayer (SAM) of TM-GABA on gold was prepared and characterized with several surface sensitive techniques. X-ray photoelectron spectroscopy (XPS) explored the SAM formation of TM-GABA on Au surfaces. Cyclic voltammograms showed the ability to electrochemically control the quinone unit at the distal end of the chain. GABA was selectively released upon electrochemical reduction at a potential of -700 mV. The functional GABA terminal group was detected by surface plasmon resonance measurements of anti-GABA antibody binding.

Activation of membrane receptors by a neurotransmitter conjugate designed for surface attachment.

The derivatization of surfaces with bioactive molecules is a research area of growing importance for cell and tissue engineering. Tetherable molecules used in such applications must contain an anchoring moiety as well as the biofunctional group, typically along with a spacer to prevent steric clashes between the target molecule and the tethering surface. Post-synaptic membrane receptors at chemical synapses in neural tissue mediate signaling to the post-synaptic neuron and are activated by the binding of diffusible neurotransmitter molecules released by the pre-synaptic neuron. However, little attention has been directed at developing neurotransmitter analogs that might retain functionality when tethered to a surface that could be interfaced with post-synaptic receptor proteins. Muscimol (5-aminomethyl-3-hydroxyisoxazole), an analog of GABA (gamma-aminobutryic acid), is a known potent agonist of GABA(A) and GABA(C) post-synaptic receptors found in retina and other central nervous system tissue. The present paper reports experiments testing the electrophysiological activity of "muscimol-biotin" on cloned GABA receptors expressed in Xenopus oocytes. This compound, which is potentially suitable for tethering at avidin-coated surfaces, consists of muscimol conjugated through an N-acyl linkage to a 6-aminohexanoyl chain that is distally terminated by biotin. We find that muscimol-biotin, as well as a structurally similar compound (muscimol-BODIPY) containing a bulky fluorophore at the distal end of the aminohexanoyl chain, exhibits substantial agonist activity at GABA(A) and GABA(C) receptors. Muscimol-biotin and other similarly biotinylated neurotransmitter analogs, in combination with surface functionalization using avidin-biotin technology, may be useful in applications involving the controlled activation of neuronal post-synaptic receptors by surface-attached molecules.

Photosensitivity of neurons enabled by cell-targeted gold nanoparticles.

Unmodified neurons can be directly stimulated with light to produce action potentials, but such techniques have lacked localization of the delivered light energy. Here we show that gold nanoparticles can be conjugated to high-avidity ligands for a variety of cellular targets. Once bound to a neuron, these particles transduce millisecond pulses of light into heat, which changes membrane capacitance, depolarizing the cell and eliciting action potentials. Compared to non-functionalized nanoparticles, ligand-conjugated nanoparticles highly resist convective washout and enable photothermal stimulation with lower delivered energy and resulting temperature increase. Ligands targeting three different membrane proteins were tested; all showed similar activity and washout resistance. This suggests that many types of ligands can be bound to nanoparticles, preserving ligand and nanoparticle function, and that many different cell phenotypes can be targeted by appropriate choice of ligand. The findings have applications as an alternative to optogenetics and potentially for therapies involving neuronal photostimulation.

Acute radiolabeling of retinoids in eye tissues of normal and rpe65-deficient mice.

PURPOSE: Mice with a targeted disruption of the gene encoding RPE65, a protein ordinarily highly expressed in the retinal pigment epithelium (RPE), accumulate abnormally high levels of all-trans retinyl ester in the RPE and exhibit very little 11-cis retinal in the retina. The present study was undertaken to determine whether the Rpe65-deficient mouse exhibits an abnormal flux of retinoid between the systemic circulation and the eye tissues. METHODS: Dark-adapted Rpe65-deficient mice (Rpe65(-/-)) and wild-type control mice (Rpe65(+/+)) of approximate ages 1 and 3 months received an intraperitoneal injection of all-trans ((3)H)retinol. The mice were maintained in darkness for a defined period ( approximately 1.5, 4.5, 24, or 48 hours) and then anesthetized, exsanguinated, and killed. Retinoids contained in the retina, RPE, serum, and liver were extracted and analyzed for ((3)H) radioactivity and molar level. RESULTS: The specific activity (SA, in counts per minute per nanomole) of serum all-trans ((3)H)retinol in all mice exhibited a peak at postinjection times of 1.5 or 4.5 hours, and by 48 hours declined to approximately 7% or less of the peak. In Rpe65(+/+) mice, the average SA of RPE ((3)H)retinyl ester similarly exhibited an early peak (4.5 hours) and by 48 hours declined to approximately 6% to 10% of the peak. By contrast, the average SA of RPE ((3)H)retinyl ester in Rpe65(-/-) mice exhibited a peak at 24 or 48 hours. Radioactivity and molar data for serum all-trans retinol and RPE retinyl ester obtained at 4.5 hours were analyzed to infer the molar influx of all-trans retinol from the circulation into the RPE. Levels of all-trans retinol influx derived from this analysis (mean +/- SD: 0.014 +/- 0.004 nmol in 1-month Rpe65(+/+) mice; 0.021 +/- 0.009 nmol in 1-month Rpe65(-/-) mice; 0.016 +/- 0.013 nmol in 3-month Rpe65(+/+) mice; 0.026 +/- 0.018 nmol in 3-month Rpe65(-/-) mice) did not differ significantly from one another (P > 0.169). However, the inferred fractional influx (molar amount of entering all-trans retinol divided by the molar amount of RPE retinyl ester) in Rpe65(+/+) animals (0.34 +/- 0.04 and 0.10 +/- 0.03, respectively, for 1- and 3-month mice) substantially exceeded that for Rpe65(-/-) animals (0.055 +/- 0.023 and 0.015 +/- 0.006, respectively, for 1- and 3-month mice). Significant levels of ((3)H)retinaldehydes were detected in the retinas of Rpe65(+/+) mice, but not in those of Rpe65(-/-) mice, after the longer postinjection periods. CONCLUSIONS: The results indicate preservation of a substantial inward flux of all-trans retinol from the circulation into the RPE of Rpe65(-/-) mice, despite the presence of abnormally high molar levels of RPE retinyl ester. They further imply the occurrence of a robust outward movement of all-trans retinol from the RPE into the circulation in Rpe65(+/+) mice, and substantial impairment of this efflux process in Rpe65(-/-) mice. These findings raise the hypothesis that in normal RPE, 11-cis retinal and/or 11-cis retinol stimulate the efflux of all-trans retinol at the RPE basolateral membrane. In 3-month Rpe65(+/+) mice, the observed relationship between the SAs of retinaldehydes in the retina and of RPE retinyl ester is consistent with a last-in/first-out processing of all-trans retinol to 11-cis retinal within normally functioning RPE.

Dark adaptation of rod photoreceptors in normal subjects, and in patients with Stargardt disease and an ABCA4 mutation.

PURPOSE: Psychophysical and electroretinographic (ERG) studies indicate that patients with Stargardt disease exhibit abnormally slow rod dark adaptation after illumination that bleaches a substantial fraction of rhodopsin. However, relatively little information is available concerning rod recovery in this disease after weaker adapting (i.e., conditioning) light. With the use of a paired-flash ERG method, properties of the derived rod response to a low-bleach (<1%) but rod-saturating conditioning flash were investigated in seven normal subjects and in five Stargardt patients with identified sequence variations in the ABCA4 gene. METHODS: In the first of two experiments, the interval between a fixed conditioning flash (67 or 670 scotopic cd s m(-2)) and a bright probe flash of fixed strength was varied to determine the falling-phase kinetics of the derived rod response to the conditioning flash. In the second, the instantaneous amplitude-intensity function for the rod response at an intermediate stage of recovery from the conditioning flash was determined by presenting a test flash of various strengths at a fixed time after the conditioning flash, and a probe flash at 200 ms after the test flash. RESULTS: The maximum peak amplitude of the dark-adapted, rod-mediated a-wave determined in Stargardt patients (211 +/- 87 microV) was on average lower than that determined in normal subjects (325 +/- 91 microV; P = 0.06). The derived rod response to the 670 scotopic cd s m(-2) conditioning flash determined in normal subjects and Stargardt patients exhibited a biphasic recovery, and the kinetics of the early stage of this recovery were similar in the two subject groups. For both normal subjects and patients, normalized amplitude-intensity functions describing the dark-adapted derived rod response exhibited half-saturation at approximately 1.5 log scotopic troland second. In both groups, the normalized amplitude-intensity function determined at approximately 2 seconds after the 67 scotopic cd s m(-2) conditioning flash and at approximately 9 seconds after the 670 scotopic cd s m(-2) conditioning flash exhibited an average desensitization (i.e., an increase of test flash strength at half-saturation) of approximately 0.5 to 0.6 log unit relative to that determined under dark-adapted conditions. CONCLUSIONS: The results indicate that, despite a reduction in the average dark-adapted maximum a-wave amplitude in the Stargardt/ABCA4 patients, the early-stage recovery kinetics of the derived rod response to a low-bleaching conditioning flash as well as the lingering rod desensitization produced by such a flash are similar to those determined in normal subjects.

Bleaching desensitization: background and current challenges.

"Bleaching desensitization" in rod photoreceptors refers to the prolonged depression of phototransduction sensitivity exhibited by rods after their exposure to bright light, i.e., after photolysis (bleaching) of a substantial fraction of rhodopsin in the outer segments. Rod recovery from bleaching desensitization depends critically on operation of the retinoid visual cycle: in particular, on the removal of all-trans retinal bleaching product from opsin and on the delivery of 11-cis retinal to opsin's chromophore binding site. The present paper summarizes representative findings that address the mechanism of bleaching desensitization.