From synaptically localized to volume transmission by nitric oxide

Abstract

Nitric oxide (NO) functions widely as a transmitter/diffusible second messenger in the central nervous system, exerting physiological effects in target cells by binding to specialized guanylyl cyclase-coupled receptors, resulting in cGMP generation. Despite having many context-dependent physiological roles and being implicated in numerous disease states, there has been a lack of clarity about the ways that NO operates at the cellular and subcellular levels. Recently, several approaches have been used to try to gain a more concrete, quantitative understanding of this unique signalling pathway. These approaches have included analysing the kinetics of NO receptor function, real-time imaging of cellular NO signal transduction in target cells, and the use of ultrasensitive detector cells to record NO as it is being generated from native sources in brain tissue. The current picture is that, when formed in a synapse, NO is likely to act only very locally, probably mostly within the confines of that synapse, and to exist only in picomolar concentrations. Nevertheless, closely neighbouring synapses may also be within reach, raising the possibility of synaptic crosstalk. By engaging its enzyme-coupled receptors, the low NO concentrations are able to stimulate physiological (submicromolar) increases in cGMP concentration in an activity-dependent manner. When many NO-emitting neurones or synapses are active simultaneously in a tissue region, NO can act more like a volume transmitter to influence, and perhaps coordinate, the behaviour of cells within that region, irrespective of their identity and anatomical connectivity.

Figure 1. Synaptic spread of NO

The zone of NO formation was modelled as a disc (0.4 μm diameter) that constantly emits 40 NO molecules per second, using the equation for an instantaneous disc source (Carslaw & Jaeger, 1986, eqn (10.3.9), p. 260) modified to include inactivation of NO as a first‐order decay term, and differentiated with respect to time. The profiles are taken 40 ms after the start, when the concentrations are at steady state. The plume of NO above a 1.5 μm × 1.5 μm grid (A) is shown compressed into 2‐dimensions in B and superimposed on an electron micrograph of an excitatory synapse with the plane of the disc centred on the synaptic cleft. In C, the profile in the plane of the disc surface is shown with rate constants for NO decay of 0, 50 and 150 s⁻¹, corresponding to half‐lives of approximately 0, 14 and 5 ms, respectively.

Figure 2. Compartmental analysis of synaptic NO signal transduction

In the model A, which is an extension of one described previously (Wood et al. 2011), the synaptic space is divided into multiple concentric hemispheres, a group of which (outlined in red; radius = 0.6 μm) are designated the NO target structure. NO is generated in a 0.4 μm diameter zone (thick black line) at the base of the target structure and the colour‐coded NO profile from Fig. 1 B is shown centred on this zone. Distances correspond to the x‐axis in B. The target contains NO‐activated guanylyl cyclase (usually 3.3 μm) and cGMP‐stimulated phosphodiesterase‐5 having a maximal activity of 106 μm s⁻¹ and a basal activity of 0.2% of this value. The kinetic schemes describing both these components were as published (Batchelor et al. 2010; Wood et al. 2011). NO is produced as a pulse having the shape depicted in the inset in B and peaking at a rate of 40 molecules s⁻¹, with half the NO flowing each side of the emission zone through all available surfaces. The fluxes of NO in each hemisphere and of cGMP within the target hemispheres are calculated similarly to the method adopted for Ca²⁺ (Nowycky & Pinter, 1993; McHugh & Kenyon, 2004). The diffusion coefficients for NO and cGMP were those used previously (Wood et al. 2011) and NO was subject to first‐order decay (rate constant = 150 s⁻¹) in each compartment. In B, the peak NO concentration in each hemisphere is plotted as a function of the concentration of NO‐activated guanylyl cyclase (GC) together with the concentrations obtained by solving analytically the equation for diffusion from a disc surface (Carslaw & Jaeger, 1986, eqn (10.3.10), p. 260) modified to include first‐order decay. The inset shows sample NO concentration profiles within the target structure, 0.2 μm from the emitting zone (orange line: profile from disc; dashed black line, green line and magenta line: profiles from model assuming NO‐activated guanylyl cyclase concentrations of 3.3 nm, 3.3 μm and 33 μm, respectively. The cGMP responses (blue lines; right‐hand ordinates) to either a single NO pulse (C) or to repeated pulses at 10 Hz (D) illustrate the likely activity dependence of NO‐mediated synaptic transmission effected largely by temporal summation at the level of guanylyl cyclase/cGMP. The sample NO traces (red lines; left‐hand ordinates) are from the central hemisphere. E shows the time‐courses of cGMP accumulation in response to 10 NO pulses delivered at different frequencies.

Figure 3. Conditions for volume transmission with multiple small NO emitters

Spheres of radius = 0.2 μm and numbering 25 (A), 49 (B) and 81 (C) are arranged in 2‐dimensional arrays within a fixed area (16 μm × 16 μm). The spheres generate NO at their surfaces at the rate of 40 molecules s⁻¹ and the resultant NO concentrations throughout the array at steady state are calculated as described (Bellefontaine et al. 2014). The upper panels illustrate the distributions of NO within and outside the area of emitters and the traces below (black lines) are sample concentrations taken through the centre of each array (marked by arrows in A, upper panel). The red trace (C) extends the array into 3 dimensions.