The discovery of the central dopamine (DA), noradrenaline (NA), and 5-hydroxytryptamine (5-HT) pathways was made in 1963–1965 with the Falck-Hillarp technique (Falck et al., 1962) and represented my thesis work (see Fuxe, 1965; Fuxe et al., 2007a). These monoamine brainstem systems were unique in having ascending monosynaptic connections with the tel- and diencephalon including the cerebral cortex giving rise to global networks all over the tel- and diencephalon with collaterals forming terminal networks in the lower brainstem. They also produced descending connections to the spinal cord innervating its entire gray matter at all rostro-caudal levels. The locus coeruleus built up of NA cell bodies could even send out both ascending and descending NA projections as well as NA projections to the cerebellum resulting in a global modulation of the entire central nervous system (CNS) from a single nerve cell nucleus through the vast NA nerve terminal plexa formed. This unique architecture of the central monoamine neurons gave thoughts on if in fact these neurons communicated via synaptic transmission.
In 1969–1970 we observed the appearance of extraneuronal diffuse (catecholamine) fluorescence around midbrain DA nerve cell bodies after amphetamine, a catecholamine releasing drug, in reserpine-nialamide-L-dopa treated rats (Fuxe and Ungerstedt, 1970a). In parallel, a similar diffuse extraneuronal 5-HT fluorescence was found among the 5-HT cell bodies of the dorsal raphe nucleus after 5-HT uptake blockade with chlorimipramine in reserpine and nialamide pretreated rats (Fuxe et al., 1970b). We did not, in the beginning, understand these observations but we asked the question if they reflected a communication different from the synaptic one. We also observed that microinjected DA in the striatum could diffuse (Ungerstedt et al., 1969). This observation was confirmed in 1992 with DA immunohistochemistry (Agnati et al., 1992).
Such observations and the existence of global monoamine terminal networks together with several other observations in the literature, especially the demonstration of non-junctional monoamine varicosities by Descarries et al. (1975) led Agnati et al. (1986) to propose the concept of volume transmission (VT). The major observation in this paper was the failure to see a correlation between the regional distribution of central encephalin and beta-endorphin immunoreactive terminal networks and of central opioid receptors. The existence of two main modes of intercellular communication in the CNS was proposed and called wiring transmission (WT; prototype: synaptic transmission) and VT (Agnati et al., 1986). The major criterion for this classification was the different characteristics of the communication channel with physical boundaries well delimited in the case of WT (axons and their synapses; gap junctions) (Froes et al., 1999; Ozog et al., 2002; Bennett et al., 2003; Duan et al., 2004; Schools et al., 2006) but not in the case of VT (the extracellular fluid (ECF) filled tortuous channels of the extracellular space (ECS) and the cerebrospinal fluid (CSF) filled ventricular space and sub-arachnoidal space). Of special importance for us was the demonstration of relative transmitter-receptor mismatches in the opioid peptide systems (Agnati et al., 1986; Fuxe et al., 1988c). In parallel, Descarries et al. (1991) developed his concept of diffuse transmission based on the existence of non-junctional monoamine varicosities which is in line with our concept of VT.
Volume transmission
Volume transmission is defined as “A widespread mode of intercellular communication that occurs in the ECF and in the CSF of the CNS with VT signals moving from source to target cells via energy gradients leading to diffusion and convection.” The communication channels are diffuse; there exists a reserved or private communication mainly mediated via high affinity G protein-coupled receptors (GPCRs); the safety of communication is low as a result of the diffusion process and the connectivity of the diffusion channels is dynamic from open to closed (see Fuxe and Agnati, 1991b; Agnati et al., 2000, 2006; Chen et al., 2002a; Fuxe et al., 2005, 2007a). There may exist three subtypes of VT: extrasynaptic fast VT (100 msec-sec), the long-distance slow VT (sec-hour), and the roamer type of VT-mediated via microvesicles (Agnati et al., 2010a; Fuxe et al., 2010a). Two modes of microvesicle release exist namely the exocytosis of internal luminal vesicles formed in the multivesicular bodies (exosomes) and the direct budding of small vesicles from the plasma membrane (shedding vesicles). Exosomes represent a subclass of such membrane vesicles which are released by cells upon fusion of the multivesicular bodies with the plasma membrane (Guescini et al., 2012).
Migration (diffusion and flow) of VT signals (transmitters, modulators, trophic factors, ions, etc.) takes place via concentration gradients, temperature gradients, and pressure waves.
Wiring transmission, the prototype being synaptic transmission is characterized by private channels (axons) and a private or reserved communication mainly mediated by ion-channel receptors and operates with high speed (msecs); the safety is high and the connectivity static and/or dynamic. One subtype of WT is the gap junction the communication of which has no privacy but can be described as having a broadcasting character.
The overall evidence indicates that vast majority of neurons possess the fundamental property of operating via both WT and VT but the balance between them varies from one nerve cell population to the other and with the dynamic state of the neuron. In contrast, glial cells and endothelial cells operate exclusively via VT. The source of the neuronal VT signal is mainly from nerve terminal networks but also from soma and dendrites.
The ECS is characterized by three parameters (see Nicholson and Sykova, 1998). Volume fraction is the relative size of the ECS compared to total brain tissue volume. Tortuosity is the increase in path length that is imposed on a migrating molecule by cellular structures and extracellular matrix components. These two parameters are unit-less. Then, there is the clearance which is a constant that reflects the removal or disappearance of a compound from the ECS, and has the unit per second. Also a mathematical theory of diffusion in the brain with dual-probe microdialysis has been developed and the theory was able to fit the experimental data (Chen et al., 2002b; Hoistad et al., 2002). The diffusion of 3H-mannitol using dual-probe microdialysis enabled a quantification of the classical diffusion parameters discussed above in the rat striatum.
Extrasynaptic transmission (a subtype of VT)
Extrasynaptic VT including perisynaptic transmission is linked to synaptic transmission and likely often takes place due to incomplete diffusion barriers (transmitter spillover or extrasynaptic release) with the synaptic transmitter reaching extrasynaptic domains of the pre and postsynaptic membrane of the synapse, the astroglia, and even adjacent synapses (Vizi, 1980; Vizi et al., 2004; Harvey and Svoboda, 2007). Other terminal varicosities fail to form synapses and are asynaptic and release the transmitter directly into the ECS. This subtype of VT operates at the local circuit level mainly through binding and activation of extrasynaptic high affinity GPCRs on neuronal and glial cells (Carmignoto, 2000) but also receptor tyrosine kinase, ion-channel receptors, and cytokine receptors are involved in linking together regulation of gene expression, firing and trophism, blood flow, and immune responses in the neuroglial networks of the CNS. In this integrative process, receptor–receptor interactions in different types of receptor heteromers play a major role (see Fuxe et al., 2007a; Borroto-Escuela et al., 2010, 2011; Fuxe et al., 2010a,b; Kenakin et al., 2010).
Extrasynaptic glutamate and GABA transmission
The classical synaptic transmitters glutamate and GABA can be measured extracellularly with microdialysis (see Ferraro et al., 1998) and reach receptors at surrounding astroglia, and at neighboring synapses, modulating the synaptic transmission process and leading to astroglial release of glutamate being dependent on the efficacy of the glial and neuronal transporters of glutamate (see Del Arco et al., 2003; Fuxe et al., 2007b). Thus, the extrasynaptic transmission can be blocked by glial sheaths and extracellular matrix through their formation of diffusion barriers e.g., perineuronal nets and the astrocytic isolation may change over a short or long time-scale. The fate of escaping transmitters is determined not only by the transporters but also by inactivating enzymes. It seems clear that one and the same transmitter, like glutamate and GABA, can be released both as a synaptic signal and as an extrasynaptic signal producing a rapid form of VT.
As an extrasynaptic signal, glutamate can increase calcium mediated astroglial glutamate release via activation of astroglial metabotropic glutamate receptors which may involve both exocytosis and transporters (Del Arco et al., 2003). The increase of extracellular glutamate levels can be detected by microdialysis (Del Arco et al., 2003). In this way, metabolic and trophic adjustments can develop in the neuron-astrocytic unit upon activation of its glutamate synapses including increases in blood flow.
Recently, imaging of extrasynaptic glutamate VT has been accomplished by Okubo et al. (2010) during synaptic activity. The glutamate optical sensor (EOS) is a hybrid-type fluorescent indicator consisting of the glutamate-binding domain of the AMPAR subunit GluR2 and a fluorescent small molecule conjugated near the glutamate-binding pocket. EOS changes its fluorescence intensity upon binding of glutamate, for which it has both high affinity and high selectivity. The electron microscopy analysis indicated that labeled K716A-EOS was distributed throughout the extracellular surface of cells without any accumulation in the synaptic cleft, and that > 97% of the indicator molecules were present extrasynaptically. They imaged the K716A-glutamate optical signal (EOS) fluorescence at various depths of the brain slice in response to parallel fiber stimulation. The magnitude and duration of the extrasynaptic dynamics were adequate for the activation of the extrasynaptic glutamate receptors and crosstalk between synapses was established (Okubo et al., 2010; Okubo and Iino, 2011).
Extrasynaptic 5-HT transmission
Double immunolabeling of 5-HT immunoreactivity (IR) in nerve terminals and 5-HT2A IR in apical dendritic shafts of pyramidal nerve cells of the cerebral cortex have demonstrated their relationships (Jansson et al., 2001). In the horizontal sections, the mean distance from a 5-HT2A-IR apical dendrite to the closest 5-HT-IR terminal-like varicosity was calculated to be 7.7 μm. Thus, these observations indicate the existence of an extrasynaptic 5-HT2A mediated VT in the cerebral cortex. The 5-HT terminal-extrasynaptic 5-HT receptor subtype relationships have been reviewed by Jansson et al. (2002). These observations are in line with the early work of Fuxe et al. (1970b) on 5-HT diffusion in the raphe dorsalis and of Descarries et al. (1975) on the existence of large numbers of non-junctional 5-HT terminal varicosities which vary from one region to the other (Descarries and Mechawar, 2000). De-Miguel and Trueta (2005) have also demonstrated extrasynaptic exocytosis of 5-HT involving multiple mechanisms including both clear vesicles and dense core vesicles. Somatic exocytosis of 5-HT in cultured leech neurons takes place exclusively from dense core vesicles (Trueta et al., 2003). It is of substantial interest that 5-HT released from cultured leech neurons produces inhibition of the firing of the 5-HT neurons through activation of 5-HT autoreceptors (Cercos et al., 2009).
Extrasynaptic DA transmission
Dopamine transmitter/D1 receptor mismatches have been observed by receptor autoradiography and dual immunolabeling of the pre and postjunctional structures of the ascending DA neurons (Fuxe et al., 1988a,b). Thus, release of DA is often observed not to be in strict contiguity with postsynaptic membranes, and a high incidence of non-junctional DA terminal varicosities is observed in the neostriatum (Descarries et al., 1996; Descarries and Mechawar, 2000). The ultrastructural analysis, based on transmitter and receptor immunocytochemistry, has repeatedly demonstrated short distance transmitter/receptor mismatches for the DA transmitter with the major part of the D1 and D2 receptor labeling being outside the DA synapses in the local striatal circuits, in which the DA synapses and/or non-junctional DA terminals are found (Dana et al., 1989; Levey et al., 1993; Sesack et al., 1994; Smiley et al., 1994; Hersch et al., 1995, 1997; Yung et al., 1995; Caille et al., 1996; for review see Jansson et al., 2002). The evidence therefore indicates that DA mainly acts as a VT transmitter being directly released into the ECF or reaching it via leaking DA synapses (Fuxe and Agnati, 1991a,b; Zoli and Agnati, 1996; Fuxe et al., 2007a). Rice and Cragg (2008) have modeled DA spillover after quantal release based on a large number of experimental data. In the updated DA synapse the DA release is unconstrained by the extrasynapic dopamine transporter (DAT), the diffusion process being too fast for the DAT which mainly increases clearance of DA and thus its half-life. A cloud of DA is formed and can reach the extrasynaptic DA receptors which are in majority. Thus, the primary mode of DA communication is VT. The effective radius for high affinity DA receptors is 7–8 μm which is compatible with extrasynaptic VT. This is the extrasynaptic short distance subtype of VT. The spheres obtained would encompass tens to thousands of synapses and the action of DA involves the activation of extrasynaptic receptors on terminals, dendrites, soma, and axons.
The D2 receptors are of special interest since the KiH (the dissociation constant of the high affinity state of the receptor) value for DA at [3H] raclopride-labeled D2-like receptors in dorsal striatum was 12 nM (Marcellino et al., 2011), and this can help explain PET findings that amphetamine-induced increases in DA release can produce an up to 50% decrease of [11C] raclopride binding in the dorsal striatum in vivo (Dewey et al., 1993; Laruelle, 2000; Seneca et al., 2006). These combined results give indications for the existence of striatal D2-like receptor-mediated extrasynaptic form of DA VT at the local circuit level in vivo in the human striatum (Marcellino et al., 2011). However, in Parkinson disease also a long-distance form of DA VT likely develops due to the development of DA receptor supersensitivity (Fuxe et al., 2003, 2010c). Thus, larger distances of diffusion of released DA can still maintain DA transmission since the supersensitive DA receptors are sensitive to very low concentrations of DA. Parkinson disease develops when too few DA terminal networks remain and distance from the DA release site to the vast majority of DA becomes too distant for DA receptor activation (Fuxe, 1979). At this stage l-DOPA treatment and/or D2 receptor agonist treatment are introduced producing antiparkinson actions through activation of the supersensitive striatal DA (Fuxe et al., 2003, 2010c). Thus, this treatment substitutes for the loss of DA VT in Parkinson disease.
Extrasynaptic noradrenaline transmission
The low synaptic incidence of cortical NA nerve terminal plexa has been demonstrated in the rat and monkey being in the order of 7–18% (Seguela et al., 1990; Aoki et al., 1998), which is clearly lower than the synaptic incidence in cortical (56–92%) and neostriatal (30–40%) DA nerve terminal plexa in the rat (Descarries and Mechawar, 2000).
The relationships of NA nerve terminal to extrasynaptic adrenergic receptors have been established (Aoki et al., 1998). Dual immunolabeling of β-adrenergic receptors and CA nerve terminal networks in the cerebral cortex using electron microscopic immunocytochemistry have demonstrated membrane contacts between CA nerve terminals rich in vesicles and β-adrenergic IR astrocytes, giving evidence for neuroglia communication via VT (Aoki, 1992; Aoki and Pickel, 1992). It was of interest that these β-adrenergic receptor IR astrocytes also surrounded asymmetric axo-spinous synapses, where the astroglia β-adrenergic receptors may modulate e.g., glutamate spillover by modulating the activity of the glial glutamate transporters and/or the permeability of the astroglial gap junctions, and thus the sphere of astroglia activation (Aoki, 1992). In another study with similar techniques, Aoki et al. (1998) also demonstrated that prefrontal NA terminal networks can interact via VT with astroglia, dendritic shafts, and axon terminals through their α2-adrenergic receptors as well as via synaptic transmission through α2-adrenergic receptors located in postsynaptic membranes at spines of pyramidal cells.
A dysfunction of the locus coeruleus NA system together with meso-cortical DA system may contribute to attention deficit hyperactivity disorders (ADHD). High levels of D4 IR have been found in many cortical regions like motor, somatosensory, temporal association, and cingulate cortices with synaptic and extrasynaptic locations (Rivera et al., 2008). In addition, these receptors have a high affinity for NA (Newman-Tancredi et al., 1997), and the D4 IR is in fact more closely related to the widespread NA terminal plexa than the restricted DA terminal plexa (mainly limbic cortex).
It should therefore be considered that CAs may be released from the cortical NA nerve terminals and may, via extrasynaptic VT, reach and activate dopamine D4 receptors located on pyramidal and non-pyramidal nerve cells in many regions of the cerebral cortex (Rivera et al., 2008). Thus, methylphenidate drugs used in the treatment of ADHD are known to release DA and NA and may therefore act in part by restoring synaptic and extrasynaptic DA and NA transmission in the cerebral cortex (see Smiley et al., 1992; Smiley et al., 1997) involving not only classical D1, D2, and adrenergic receptors but also D4 receptors which may be activated also by extrasynaptic NA transmission (Rivera et al., 2008).
Extrasynaptic acetylcholine transmission
Widespread plexa of cholinergic nerve terminals exist in the CNS with high densities especially in the striatum and have, like the NA and 5-HT nerve terminal networks, been found to have a low synaptic incidence in the cerebral cortex and neostriatum and thus mainly operate via volume (diffuse) transmission (Descarries and Mechawar, 2000). This was in the beginning found to be surprising since acetylcholinesterase was believed to quickly degrade acetylcholine in the synaptic cleft. The explanation is that this effective degradation is performed by the A12 isoform of acetylcholinesterase while brain acetylcholinesterase mainly consists of the G4 isoform (Gisiger and Stephens, 1988) as discussed by Descarries et al. (1997). Thus, the extrasynaptic form of VT can develop but long distance VT is prevented by the brain acetylcholinesterase.
Extracellular monoamine and acetylcholine transmission and vesicular glutamate transporters
Large numbers of monoamine and acetylcholine neurons have been shown to contain a vesicular glutamate transporter (VGLUT; see El Mestikawy et al., 2011) which may have relevance for extrasynaptic monoamine and acetylcholine transmission. Thus, the VGLUT is mainly driven by the vesicular transmembrane potential component of the proton gradient which leads to more acidified vesicles. This allows the vesicular monoamine transporter (VMAT2) and vesicular acetylcholine transporters (VAChT) to accumulate higher amounts of monoamines and acetylcholine, respectively since these transporters are mainly dependent on the pH gradient over the vesicular membrane (El Mestikawy et al., 2011). Three types of VGLUT exist namely VGLUT1, VGLUT2, and VGLUT3 and are present in central monoamine and acetylcholine neurons besides GABA and glutamate neurons. This has led to discussions on whether glutamate can be co-transmitter in these neurons enabling glutamate co-release. It has been especially discussed if glutamate in DA neurons is linked to the formation of junctional DA terminals (synapses; Descarries et al., 2008). However, it appears to be largely a developmental feature since double labeling of tyrosine hydroxylase (TH) and VGLUT2 in DA terminals is hardly observed in adult rats. It is of substantial interest that a heterogeneity in DA, 5-HT and acetylcholine terminals may exist with regard to co-expression of VGLUTs and VMAT2 or VAChT (see El Mestikawy et al., 2011). In some of them it seems possible that there may exist synaptic vesicles containing both VMAT2 and VGLUT2 (DA varicosity), VMAT2 and VGLUT3 (5-HT varicosity), and VAChT and VGLUT3 (acetylcholine varicosity). In fact, evidence exists that the presence of VGLUT can lead to so-called vesicular synergy meaning increased accumulation of the primary transmitter (monoamine or acetylcholine; Gras et al., 2008; Amilhon et al., 2010; Hnasko et al., 2010). The molecular mechanism for vesicular synergy is likely that the VGLUT is driven by the vesicular transmembrane potential with the accumulation of glutamate (acid) leading to an acidification of the vesicle. In view of the fact that the VMAT2 and VAChT is dependent on a pH gradient over the vesicular membrane this will allow increased amounts of monoamines and acetylcholine to accumulate in the monoamine and acetylcholine vesicles (El Mestikawy et al., 2011). This is likely of relevance for the extrasynaptic monoamine and acetylcholine transmission since higher amounts of transmitter can be released into the ECF directly or via synaptic spillover from these varicosities and a larger volume can be reached with sufficient concentrations of monoamines and acetylcholine to activate their extrasynaptic high affinity receptors. Increased quantal contents of vesicles of the primary transmitter with increased release has in fact been shown for VGLUT3 in 5-HT and acetylcholine neurons (Gras et al., 2008; Amilhon et al., 2010). Thus, an increase in extrasynaptic transmission is obtained. The VGLUT research will represent an exciting field for understanding VT in the CNS.
Long distance VT
Long distance DA VT up to 30–50 μm may also exist in the striatum of the intact rat. Thus, we have observed substantial D1-TH terminal mismatches (in the maximal range of 30–50 μm) in the nucleus accumbens shell with D1 rich areas surrounded by highly dense DA nerve terminal plexa (Jansson et al., 1999). It may be that here the existence of energy gradients, especially temperature gradients created by the uncoupling protein 2 (UCP2) in the DA terminal regions, increase the migration of DA into the D1 rich and TH poor region (Rivera et al., 2006). UCP2 produces a disappearance of the H+ gradient in the mitochondria with generation of heat leading to a temperature gradient vs. the D1 receptor rich cellular network. This may lead to mass movement of the ECF (flow) carrying DA into the mismatch region which is more rapid than DA diffusion alone. It should be mentioned that DA can also be released into the portal blood vessels from the tuberoinfundibular DA neurons (Fuxe, 1963, 1964; Fuxe et al., 1967; MacLeod and Lehmeyer, 1974; Andersson et al., 1981) to activate the D2 receptors on the prolactin cells of the anterior pituitary. Thus, DA can act as a synaptic transmitter, VT transmitter, and as a hypothalamic hormone.
Long distance peptide VT and CSF VT
Peptide neurons likely operate via long distance VT with distances over 1 mm involving also flow in the CSF (Fuxe et al., 2010a). One of the best examples is CSF delivered beta-endorphin (2500 pmol in 5 μl) which could accumulate in nerve cell body subpopulations and their dendrites all over the paraventricular hypothalamus as seen 15 min after the CSF injection (Agnati et al., 1992; Bjelke and Fuxe, 1993). The likely mechanism is that beta-endorphin is internalized into discrete subependymal and periventricular nerve terminal plexa and then undergoes retrograde transport to the nerve cell bodies developing beta-endorphin IR. These results indicate that beta-endorphin CSF VT may exist which – via opioid receptors located on periventricular nerve terminal plexa – may become internalized and retrogradely transported to potentially modulate the gene expression and firing of such neurons (Agnati et al., 1992). Striatally microinjected beta-endorphin can also reach the CSF as an intact peptide as shown with mass-spectrometry (Hoistad et al., 2005). So, beta-endorphin may not only employ long distance diffusion in the ECF but also CSF mediated VT as a complementary communication pathway, to also reach and activate distant opioid receptors. These results are in line with the work of Duggan’s group on beta-endorphin based on antibody microprobes (MacMillan et al., 1998). They observed that beta-endorphin IR could be detected in CSF and remote brain areas lacking beta-endorphin IR terminals, like the cerebral cortex, 60–90 min after the initial stimulation of the arcuate nucleus where the beta-endorphin cell bodies are located.
These results taken together give strong indications that beta-endorphin could migrate for long distances in the ECF and CSF after its release from beta-endorphin IR nerve terminal networks.
These results are also of relevance for understanding cotransmission in monoamine neurons often containing neuropeptides (see book by Hökfelt et al., 1986). The release of neuropeptides may allow the monoamine neurons to send VT signals to cellular networks further away from the monoamine terminals. Peptides and proteins may have a high stability and/or act via active peptide fragments which make long distance VT possible involving also CSF VT (Fuxe et al., 2010a and see above). The temporal code of VT related to dynamic changes in release of transmitters is likely to exist especially at short distances, while with long distances of diffusion/convection observed with peptides/proteins the dynamic changes in VT modulating the wired networks may be less pronounced. However, it seems likely that peptide release may be exceptionally enhanced with burst firing based on the work of Lundberg (1991) indicating that long-distance peptides/protein VT can be markedly increased under specific physiological conditions.
In the case of monoamine/peptide co-storing neurons it thus seems likely that the co-released monoamines and peptides from the same nerve terminal networks only interact via receptor-receptor interactions in the range of short diffusion distances. At long distances the migrating peptides/proteins probably along preferential VT channels (paravascular ECF channels or ECF channels along myelinated fiber bundles) instead activate receptors which interact with receptors stimulated by transmitters belonging to other types of neurons.
Understanding the role of extrasynaptic and long distance VT in the functional modules (neuronal networks) of the CNS giving rise to the neuronal circuits
The neuronal–astroglial networks form functional modules which give rise to several outputs/efferents that is several types of axon bundles forming fiber tracts that influence in various ways other functional modules linked to it. The resulting activity changes in these functional modules will, by forming dynamic brain circuits, have an impact on brain function e.g., behavioral changes. A major role of the VT signals is to modulate the WT, especially the synaptic transmission, in the neuronal networks forming the functional modules. In this way, via changes in VT, it becomes possible for the same neuronal network to change the balance of activity in its different projection neurons to other functional modules (neuronal networks) and thus in their brain circuits and in brain function. The mechanism involved is likely the ability of the diffusing VT signals in ECF to upregulate and/or downregulate synaptic transmission in different parts of the neuronal network via activation of e.g., monoamine, acetylcholine, and/or peptide receptors mainly located in the local circuits of the neuronal–astroglial network. Both neuronal extrasynaptic and long distance VT signaling can be involved and the architecture of the diverse high affinity receptor distributions on the neurons and glia will determine the outcome on the balance of activity in the different types of projection neurons from the functional module. These neuronal VT signals can be released from terminal networks within the functional module with nerve cell bodies located outside the functional module or from interneurons within the module. Also the projection neurons of the module can mainly via recurrent collaterals give rise to VT signals. There also exists soma-dendritic extrasynaptic exocytosis of VT signals within the neurons of the module contributing inter alia to the activation and regulation of the high affinity soma-dendritic autoreceptors at this level (see De-Miguel and Trueta, 2005). Finally, VT signals arise also from the glial cells of the module like adenosine (Ferre and Fuxe, 2000) and kynurenic acid (Schwarcz and Pellicciari, 2002) from the astroglia. The major molecular mechanism appears to be the modulation of synaptic receptors through activation of extrasynaptic receptors by VT signals which modulate the synaptic receptors via allosteric receptor–receptor interactions in receptor heteromers built up of synaptic and extrasynaptic receptors (Agnati et al., 2010a; Fuxe et al., 2010a). The formation of receptor heteromers by synaptic ion-channel receptors like NMDA and GABA receptors and GPCRs have been demonstrated by Fang Liu and colleagues (see Liu et al., 2000; Lee et al., 2002; Liu et al., 2006). In addition, also VT signals can interact and integrate their signaling via GPCR–GPCR interactions in heteromers e.g., A2A-D2 heteromers at the extrasynaptic level (Fuxe et al., 1998, 2010b).