Federico F. Trigo

Axonal GABAA receptors

Type A GABA receptors (GABAARs) are well established as the main inhibitory receptors in the mature mammalian forebrain. In recent years, evidence has accumulated showing that GABAARs are prevalent not only in the somatodendritic compartment of CNS neurons, but also in their axonal compartment. Evidence for axonal GABAARs includes new immunohistochemical and immunogold data: direct recording from single axonal terminals; and effects of local applications of GABAAR modulators on action potential generation, on axonal calcium signalling, and on neurotransmitter release. Strikingly, whereas presynaptic GABAARs have long been considered inhibitory, the new studies in the mammalian brain mostly indicate an excitatory action. Depending on the neuron that is under study, axonal GABAARs can be activated by ambient GABA, by GABA spillover, or by an autocrine action, to increase either action potential firing and/or transmitter release. In certain neurons, the excitatory effects of axonal GABAARs persist into adulthood. Altogether, axonal GABAARs appear as potent neuronal modulators of the mammalian CNS.

Laser photolysis of caged compounds at 405 nm: Photochemical advantages, localisation, phototoxicity and methods for calibration

Rapid, localised photolytic release of neurotransmitters from caged precursors at synaptic regions in the extracellular space is greatly hampered at irradiation wavelengths in the near-UV, close to the wavelength of maximum absorption of the caged precursor, because of inner-filtering by strong absorption of light in the cage solution between the objective and cell. For this reason two-photon excitation is commonly used for photolysis, particularly at multiple points distributed over large fields; or, with near-UV, if combined with local perfusion of the cage. These methods each have problems: the small cross-sections of common cages with two-photon excitation require high cage concentrations and light intensities near the phototoxic limit, while local perfusion gives non-uniform cage concentrations over the field of view. Single-photon photolysis at 405 nm, although less efficient than at 330-350 nm, with present cages is more efficient than two-photon photolysis. The reduced light absorption in the bulk cage solution permits efficient wide-field uncaging at non-toxic intensities with uniform cage concentration. Full photolysis of MNI-glutamate with 100 micros pulses required intensities of 2 mW microm(-2) at the preparation, shown to be non-toxic with repeated exposures. Light scattering at 405 nm was estimated as 50% at 18 microm depth in 21-day rat cerebellum. Methods are described for: (1) varying the laser spot size; (2) photolysis calibration in the microscope with the caged fluorophore NPE-HPTS over the wavelength range 347-405 nm; and (3) determining the point-spread function of excitation. Furthermore, DM-Nitrophen photolysis at 405 nm was efficient for intracellular investigations of Ca2+-dependent processes.

Laser photolysis of DPNI-GABA, a tool for investigating the properties and distribution of GABA receptors and for silencing neurons in situ

Laser photolysis to release GABA at precisely defined times and locations permits investigation of the distribution of functional GABA(A) receptors in neuronal compartments, the activation kinetics and pharmacology of GABA(A) receptors in situ, and the role of individual neurons in neural circuits by selective silencing with low GABA concentrations. We describe the experimental evaluation and applications of a new nitroindoline-caged GABA, DPNI-GABA, modified to minimize the pharmacological interference commonly found with caged GABA reagents, but retaining the advantages of nitroindoline cages. Unlike the 5-methoxycarbonylmethyl-7-nitroindolinyl-GABA tested previously, DPNI-GABA inhibited GABA(A) receptors with much lower affinity, reducing peak GABA-evoked responses with an IC(50) of approximately 0.5 mM. Most importantly, the kinetics of receptor activation, determined as 10-90% rise-times, were comparable to synaptic events and were little affected by DPNI-GABA present at 1mM concentration, permitting photolysis of DPNI-GABA to mimic synaptic activation of GABA(A) receptors. With a laser spot of 1 microm applied to cerebellar molecular layer interneurons, the spatial resolution of uncaging DPNI-GABA in dendrites was estimated as 2 microm laterally and 7.5 microm focally. Finally, at low DPNI-GABA concentration, photorelease restricted to the area of the soma suppressed spiking in single Purkinje neurons or molecular layer interneurons for periods controlled by the flash intensity and duration. DPNI-GABA has properties better adapted for fast kinetic studies with laser photolysis at GABA(A) receptors than previously reported caged GABA reagents, and can be used in experiments where spatial resolution is determined by the dimensions of the laser light spot.

Enhancement of GABA Release through Endogenous Activation of Axonal GABAA Receptors in Juvenile Cerebellum

Recent evidence indicates the presence of presynaptic GABAA receptors (GABAARs) in the axon domain of several classes of central neurons, including cerebellar basket and stellate cells. Here, we investigate the possibility that these receptors could be activated in the absence of electrical or chemical stimulation. We find that low concentrations of GABA increase the frequency of miniature GABAergic synaptic currents. Submaximal concentrations of a GABAAR blocker, gabazine, decrease both the miniature current frequency and the probability of evoked GABA release. Zolpidem, an agonist of the benzodiazepine binding site, and NO-711 (1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), a blocker of GABA uptake, both increase the frequency of miniature currents. These effects occur up to postnatal day 14, but not later. Immunohistochemistry indicates the presence of α1-containing GABAARs in interneuron presynaptic terminals with a similar age dependence. We conclude that, under resting conditions, axonal GABAARs are significantly activated, that this activation results in enhanced GABA release, and that it can be augmented by increasing the affinity of GABAARs or reducing GABA uptake. Our findings suggest the existence of a positive-feedback mechanism involving presynaptic GABAARs that maintains a high release rate and a high local GABA concentration in the immature cerebellar network.

Somatic Depolarization Enhances GABA Release in Cerebellar Interneurons via a Calcium/Protein Kinase C Pathway

In cortical and hippocampal neurons, tonic somatic depolarization is partially transmitted to synaptic terminals, where it enhances transmitter release. It is not known to what extent such “analog signaling” applies to other mammalian neurons, and available evidence concerning underlying mechanisms is fragmentary and partially controversial. In this work, we investigate the presence of analog signaling in molecular layer interneurons of the rat cerebellum. GABA release was estimated by measuring autoreceptor currents in single recordings, or postsynaptic currents in paired recordings of synaptically connected neurons. We find with both assays that moderate subthreshold somatic depolarization results in enhanced GABA release. In addition, changes in the calcium concentration were investigated in the axon compartment using the calcium-sensitive dye OGB-1 (Oregon Green BAPTA-1). After a step somatic depolarization, the axonal calcium concentration and the GABA release probability rise with a common slow time course. However, the amount of calcium entry that is associated to one action potential is not affected. The slow increase in calcium concentration is inhibited by the P/Q calcium channel blocker ω-agatoxin-IVA. The protein kinase C inhibitor Ro 31-8220 (3-[3-[2,5-dihydro-4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-1H-pyrrol-3-yl]-1H-indol-1-yl]propyl carbamimidothioic acid ester mesylate) did not affect the calcium concentration changes but it blocked the increase in GABA release. EGTA was a weak blocker of analog signaling, implicating a close association of protein kinase C to the site of calcium entry. We conclude that analog signaling is prominent in cerebellar interneurons and that it is triggered by a pathway involving activation of axonal P/Q channels, followed by calcium entry and local activation of protein kinase C.

Readily releasable pool of synaptic vesicles measured at single synaptic contacts

To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7.

Vesicular release statistics and unitary postsynaptic current at single GABAergic synapses.

The existence of vesicular docking sites in central synapses is supported by morphological and biochemical evidence, but their functional role remains elusive. To investigate this role we have studied single depressing GABAergic synapses where multivesicular release and postsynaptic receptor saturation have been documented. We used failure/success patterns to estimate the number of vesicular docking sites, which varied from one to six among synapses. Variations of docking site numbers account for differences in release probability, as well as in the amplitude and decay kinetics of unitary postsynaptic currents. Upon repetitive stimulation, decreasing docking site occupancy likewise accounts for changes both in presynaptic and postsynaptic parameters. Finally steady-state docking site occupancy during train stimulations can be modulated by applying subthreshold presynaptic conditioning potential steps. The results suggest that differences in docking site numbers determine intersynaptic variability and that docking site occupancy is a key parameter controlling single synapse signaling.

Synthesis and biological evaluation of bis-CNB-GABA, a photoactivatable neurotransmitter with low receptor interference and chemical two-photon uncaging properties.

Photoactivatable “caged” neurotransmitters allow optical control of neural tissue with high spatial and temporal precision. However, the development of caged versions of the chief vertebrate inhibitory neurotransmitter, γ-amino butyric acid (GABA), has been limited by the propensity of caged GABAs to interact with GABA receptors. We describe herein the synthesis and application of a practically useful doubly caged GABA analog, termed bis-α-carboxy-2-nitrobenzyl-GABA (bis-CNB-GABA). Uncaging of bis-CNB-GABA evokes inward GABAergic currents in cerebellar molecular layer interneurons with rise times of 2 ms, comparable to flash duration. Response amplitudes depend on the square of flash intensity, as expected for a chemical two-photon uncaging effect. Importantly, prior to uncaging, bis-CNB-GABA is inactive at the GABAA receptor, evoking no changes in holding current in voltage-clamped neurons and showing an IC50 of at least 2.5 mM as measured using spontaneous GABAergic synaptic currents. Bis-CNB-GABA is stable in solution, with an estimated half-life of 98 days in the light. We expect that bis-CNB-GABA will prove to be an effective tool for high-resolution chemical control of brain circuits.

Axonal GABAA receptors depolarize presynaptic terminals and facilitate transmitter release in cerebellar Purkinje cells

GABAA receptors have been described in the axonal compartment of neurons; contrary to dendritic GABAA receptors, axonal GABAA receptors usually induce depolarizing responses. In this study we describe the presence of functional axonal GABAA receptors in cerebellar Purkinje cells by using a combination of direct patch‐clamp recordings from the axon terminals and laser GABA photolysis. In Purkinje cells, axonal GABAA receptors are depolarizing and induce an increase in neurotransmitter release that results in a change of short‐term synaptic plasticity. These results contribute to our understanding of the cellular mechanisms of action of axonal GABAA receptors and highlight the importance of the presynaptic compartment in neuronal computation.