Schematic representation of the main sources of volume transmission involving dopamine and nitric oxide signals in the central nervous system (Inspired by Zoli et al., ; Syková, ). (A) Synaptic transmission: Synaptic vesicular release followed by the diffusion of the transmitter outside the synaptic cleft at an effective concentration. Neurotransmitters can escape from the synaptic cleft (especially during repetitive stimulation and/or the massive release of a transmitter) and affect extrasynaptic receptors on glia or neurons or reach a neighboring synapse. These phenomena have been called “transmitter spillover” and “synaptic crosstalk.” This loss of synaptic independence means that transmitter release at one synapse can lead to the activation of receptors in nearby synapses. (B) Volume transmission: vesicular release from non-junctional varicosities, i.e. varicosities lacking pre-synaptic specializations and post-synaptic densities. Axons both in the central nervous system and in the autonomic nervous system form varicose (boutons-en-passant) branches that may release and uptake neurotransmitters. The transmitter release from varicosity without synaptic contact diffuses far away from the release site and activates remote receptors of high affinity. (C,D) Release of gaseous transmitters, e.g. nitric oxide release from neurons. The inside cell squares are representative of a local synthesis of the neurotransmitter, frequently by enzymatic regulated activity. Nitric oxide presumably operates within a 3-dimensional volume of tissue rather than, more conventionally, between immediately adjacent cellular partners (Kiss and Vizi, ). Glu, glutamate vesicles; NOS, nitric oxide synthase; NO, nitric oxide; NMDA, N-Methyl-d-Aspartate.

A schematic illustrating the method used to visualize processes present in the cell periphery. The cell body (red) was thresholded (A) to generate a binary mask and then enlarged [blue line in (B,C)] to occupy an additional 2 μm around its periphery. Areas occupied within the blue periphery by the green processes are shown in [(C,D); yellow areas]. In (D), only the pixels where processes are within the 2 μm proximity of the cell body are shown. A 2D scatter plot (E) was generated using Imaris 7.5 (Bitplane) revealing a pixel population (outlined with yellow line) with correlating intensities (Pearson ∼0.7), out of which a new channel was generated.

Confocal laser scanning microscopy of TH(+) and nNOS(+) double-immunostained sections from the rat cingulate cortex. The TH(+) immunoreactivity (A,C) is shown in green, whereas the nNOS(+) immunoreactivity (A,B) is red, in the same double-stained section. In (B,C), the binary masks of the nNOS(+) and TH(+) signals are shown, respectively, while (D) additionally shows the 2 μm-wide periphery (yellow) of the nNOS(+) cell body and processes (red). The yellow areas in (E) indicate the location of TH(+) processes within the 2 μm periphery of the nNOS(+) cell. There is absence of complete colocalization between nNOS(+) and TH(+) immunoreactivity in the rat cortex but a large number of fibers are present within the 2 μm nNOS(+) cell periphery. Scale bar is 50 μm.

Photomicrographs of nNOS(+) immunoreactive neurons (A) and TH(+) nerve fibers (B) in sections from the caudate–putamen/globus pallidus [merge in (C)]. nNOS(+) immunoreactive neurons appear polygonal or fusiform in shape with long smooth dendrites and a plexus of nerve fibers in the neuropil. The low-power photomicrograph shows the intense staining of the TH(+) immunoreactivity and the large number of nNOS(+) cell body in the caudal striatum. Scale bar = 200 μm.

Confocal laser scanning microscopy of TH(+) and nNOS(+) immunoreactive double-immunostained sections from the rat striatum. Fibers immunoreactive for TH(+; green) are presented in (B,C,C′). A neuron immunoreactive for nNOS(+)(red) is presented in (A,C,C′). Using CLSM optical sections acquired at the resolution limit, colocalized regions (Pearson correlation coefficient of ≥0.7) were identified (yellow). The yellow areas in (C,D,C′) also indicate the location of TH(+) processes within the 2 μm periphery of the nNOS(+) cell. Detail of the cell body is presented in (C′). In the rat striatum a large number of TH(+) fibers are present within the 2 μm nNOS(+) cell body. Scale bar in (D,C′) = 5 μm.

Confocal laser scanning microscopy of TH(+) and nNOS(+) immunoreactive double-immunostained sections from the rat striatum: a proximity analysis study. Distribution pattern of immunoreactivity for TH(+) and a nNOS(+) in double-stained sections of the striatum. Both, nNOS (red) and TH (green) fluorescent signals are shown in (A), while (B,C) show the corresponding binary masks. The 2 μm periphery (yellow) is shown in (D) and the area occupied by TH(+) immunoreactive fibers around the nNOS(+) immunoreactive cell body is shown in (E). The yellow areas in E indicate the location of TH(+) and nNOS(+) possible interaction. Scale bar in (A) is 10 μm.

The fluorescence pattern in a double-stained section of the nucleus accumbens, showing nNOS(+) [(A,C) – cell body) and TH(+) [(B,C) axons] immunoreactivities. Immunoreactivity for TH(+) and a nNOS(+) in double-stained sections of the nucleus accumbens. Both, nNOS (red) and TH (green) fluorescent signals are shown in (C). Whereas the TH(+) fiber immunoreactivity appears homogeneously distributed throughout the whole nuclei, the nNOS(+) immunoreactive neurons and fibers are stained much more sparsely. Scale bar in (A) = 10 μm, it applies to (B,C) also.

Internal globus pallidus [entopeduncular nucleus-(A,B), merge in (C)] and external globus pallidus (D) photomicrographs of nNOS(+) immunoreactive neurons and TH(+) nerve fibers. (A–C) TH(+) and nNOS(+) immunoreactive fiber staining representative of the typical honeycomb-like pattern in the entopeduncular nucleus [merge in (C)]. TH(+) immunoreactivity corresponds to the medial forebrain bundle. (D) Photomicrography of nNOS(+) immunoreactive neurons and TH(+) nerve fibers (merge) from a section of the external globus pallidus/caudal striatum. Note the nNOS(+) immunoreactive cells (red), the dark staining surrounds areas of lighter immunoreactivity (external globus pallidus) and the dense TH(+) fibers in the striatum. nNOS(+) immunoreactive neurons appear polygonal in shape with long smooth dendrites and a plexus of nerve fibers in the neuropil. Scale bars, 100 μm.

Confocal laser scanning microscopy of the external globus pallidus in TH(+) and nNOS(+) immunoreactive double-immunostained sections. The axons are immunoreactive for TH(+) [green, (B,C)] and the neurons/axons immunoreactive for NOS(+) [(A,C), red] in double-stained sections of the external globus pallidus. Both, TH(+) and nNOS(+) immunoreactivities are presented in (C). Observe the lower fluorescence of nNOS(+) immunoreactive cells surrounded by areas of lighter immunoreactivity of the TH(+)fibers. The nNOS(+) immunoreactivity delineating a neuronal cell body structure is covered by dots of TH(+) immunolabeling in yellow (C,D). Using CLSM, zones of cellular colocalization were ascertained [yellow, (C,D)]. Scale bar is 25 μm.

The substantia nigra compacta and ventral tegmental area nNOS(+) and TH(+) immunoreactive neurons and fibers. Distribution pattern of axons/cells immunoreactive for nNOS(+) (A) and TH(+) (B) in double-stained sections of the substantia nigra [(C), merge]. Cell nuclei are labeled in blue with DAPI. Using CLSM (A–C), zones of cellular colocalization were demonstrated in a single optical section of the substantia nigra compacta [(C), scale bar is = 5 μm]. Observe the dense staining of the TH(+) immunoreactive cells and fibers homogeneously distributed throughout the substantia nigra compacta and the ventral tegmental area (A). In the substantia nigra, dopaminergic cells are arranged in two bands (Fallon and Loughlin, ). The substantia nigra reticulate caudoventral emits cell bridges that make contact with the substantia nigra compacta rostrodorsal [González-Hernández and Rodríguez, ; (A,B)]. The substantia nigra compacta is characterized by a high density of dopaminergic somata as well as a dense network of overlapping dopaminergic dendrites. Scattered neurons in both substantia nigra compacta and reticulata display immunoreactivity to nNOS [Rodrigo et al., ; (A,B)]. Cell colocalization between nNOS(+) and TH (+) immunoreactivity is almost absent but a double-labeled cell is visualized in (C). Scale bars, 50 μm (A–C).

Substantia nigra compacta nNOS(+) immunoreactive neuron and TH(+) fibers. CLSM analysis of a double-immunostained section. (A) Show in detail a nNOS(+)immunoreactive neuron (red) surrounded by TH(+) immunoreactive fibers (green). (B,C) Show the binary masks of the TH(+) and nNOS(+) stains, respectively. In (D), the 2 μm proximity of the nNOS(+) cell is shown, while in (E) the area occupied by the TH(+) processes within the 2 μm proximity is visualized. Zones of colocalization were demonstrated in a single optical section of the substantia nigra compacta acquired at the resolution limit. Scale bar 10 μm.

Subthalamic nuclei of a control rat brain: distribution pattern of axons/cells immunoreactive for TH(+; green) and NOS(+; red) in double-stained sections. The cell nuclei are labeled with DAPI. Observe the dense staining of the TH(+) immunoreactive fibers of the medial forebrain bundle (green fibers). nNOS(+) immunoreactive neurons appear massively at the subthalamic nuclei with long smooth dendrites. The area occupied by TH(+) immunoreactive fibers around the nNOS(+) immunoreactive cell body is shown in the inset. Cell colocalization between nNOS(+) and TH (+) immunoreactivity is absent. Scale bar is 100 μm.

Confocal laser scanning microscopy of the pedunculopontine nuclei in a TH(+) and nNOS(+) double-immunostained section. Distribution pattern of axons/cells immunoreactive for TH(+) [green in (B,C)] and NOS(+) [red in (A,C)] is presented in this double-stained section. Observe the nNOS(+) immunoreactive neurons surrounded by sparse TH(+) fibers. TH(+) immunoreactive fibers appear scattered all around the cell soma. Scale bar 50 μm. Scale bar in (D) is 5 μm.