Although Dale offered a very good lead in the peripheral nervous system to search for putative transmitters released from dendritic processes, it was the central nervous system which offered us the first unequivocal example of such a phenomenon. A great deal of this is due to the cytoarchitecture of the substantia nigra (SN) dopaminergic cell neurons. The majority of their cell bodies are neatly packed in the SN pars compacta, and their axonal processes project dorsally and rostrally toward the neostriatum (caudate and putamen, Ungerstedt, 1971), while their long, branched dendrites radiate profusely through the pars reticulata. This description of the SN dopaminergic neurons corresponds well with Ramon y Cajal's (1904) description: "The Golgi method shows these neurons to have different shapes, predominantly triangular and provided with very long, shaggy and discretely divided dendrites, which expand throughout almost all the nucleus (meaning the area)". The identification of dopamine as the transmitter of the nigro-striatal pathway occupies an interesting and most influencial chapter in the history of modern neuroscience. Arvid Carlsson in the 1950s forcefully promoted dopamine as a transmitter (see Carlsson 1958; and Carlsson's autobiographic account, 1998). The fact that dopamine was found highly concentrated in the basal ganglia (Bertler and Rosengreen, 1964) led Sourkes in Canada and Hornykiewicz in Canada and Austria to demonstrate the dopamine deficits in Parkinson's disease and the consequent proposal for L-DOPA replacement therapy (Sourkes and Poirier, 1965; Hornykiewicz, 1972; and for personal accounts see Hornykiewicz, 1992, and Sourkes, 2000).
The actual anatomical demonstration of the nigral dopaminergic neurons was provided by the revolutionary technique of catecholamine-induced fluorescence (Dahlstrom and Fuxe, 1964). The possibility of some local dendritic synthesis of dopamine was implied in the elegant reports of the occurrence of immunoreactivity to catecholamine synthesizing enzymes in SN dendrites (Pickel et al., 1976; Hokfelt et al., 1973). However, attention to the fate of dopamine in dendritic compartments was provided by the provocative short communication from Bjorklund and Lindvall (1975) which demonstrated glyoxylic acid-induced fluorescence in SN cell bodies and in the long dendrites extending into the rat SN pars reticulata. This report implied a functional role for dendritic dopamine, as the reserpine depletion of the dendritic fluorescence could be re-established by incubating substantia nigra slices in vitro with dopamine, and its uptake prevented with desimipramine and benztropine. These observations provoked Laurie Geffen, Thomas Jessell (at the time a Cambridge graduate student), Leslie Iversen
Fig. 2. Composite schematic, illustrating the sites of dopamine incorporation, storage and release from dendritic processes of the rat substantia nigra, A) scheme illustrating the segregation of axonal and dendritic processes in the nigrostriatal pathway. SN, substantia nigra; c, pars compacta; IP, nucleus interpeduncularis; MGB, medial geniculate body; sc, superior colliculus; CS, corpus striatum; Sept, septum; CX, cortex; Hyp, hypothalamus B) illustration of the microdissection of the pars reticulata of the rat substantia nigra from live, unfixed, unstained brain stem tissue slices. The open (dissected out pars reticulata) is observed by transillumination between the crus cerebri (below) and the SN pars compacta (above). On the right the exit of the 3rd cranial nerve can be observed. The darker (myelinated) region in the upper, right corner is part of the medial forebrain bundle C) Illustrates the rate constant outflow of previously incorporated 3H-dopamine, from superfused slices of the rat substantia nigra (top panels, a and c) and corpus striatum (b and d). Panels a and b illustrate repeated K+ stimulated release of dopamine from (microdissected) substantia nigra dendrites, and the marked inhibition of dendritic and axonal release in the absence of Ca2+ (open squares in c and d) or presence of high magnesium molarity (reproduced with permission from Geffen et al, 1976). D) Radioautography illustrating the incorporation of 3H-dopamine in a number of substantia nigra neurons (labeled I to V) and long dendritic processes, radiating towards the pars reticulata, dark field micrograph. E) Phase contrast micrograph showing the localization of neurons labeled I to V, as well as the dendritic profiles incorporating radioactive dopamine as displayed with arrows. F) Electron microscopic radioautography illustrating silver grains (circles), depicting subcellular sites of tritiated dopamine in the perisomatic region and the dendritic process of a substantia nigra neuron. Scale bar = 5nm, D-F from (Cuello and Kelly, 1977). G) Electron micrograph, electron dense products (arrow heads) illustrating the subcellular storage sites of incorporated 5-OH-dopamine within tubular profiles resembling the smooth endoplasmic reticulum cisterns (ser) of a substantia nigra dendritic profile ( Reproduced with permission from Mercer et al, 1979).
and myself to investigate whether dendrites are also able to release neurotransmitters. The neat organization of the SN dopaminergic cell bodies in the pars compacta and dopaminergic dendrites in the pars reticulata permitted simple microdissection, taking advantage of the fact that fresh, unstained, live tissue slices could be obtained by a procedure developed at the time (for review see Cuello and Carson, 1983) and also of the refinement in the micro superperfusion system developed by Jessell and Iversen. We embarked on the project by comparing the uptake and release of 3H-dopamine in the area of the nerve terminals (caudate putamen) and the dendrites (substantia nigra) in microdissected rat CNS tissue section. In these experiments (Geffen et al., 1976), we provided the first direct evidence for a dopamine uptake and release mechanism in dendrites of the SN neurons. We were able to show that short pulses of high molarity KC1 provoked a Ca2+-dependent release of newly incorporated [3H] dopamine from substantia nigra slices (dopaminergic dendrites), analogous to that seen in dopaminergic nerve terminals of the neostriatum. Simultaneously, and to our great surprise, in the same Nature issue Korf et al. (1976) indicated that the antidromic stimulation of the substantia nigra projection to the neostriatum resulted in an elevated amount of dopamine metabolites providing indirect evidence that a release of the amine could occur in the SN somatodendritic area following neuronal stimulation. Soon after these reports the Glowinsky group in Paris provided additional in vivo evidence of this mechanism using the push-pull cannula approach (Nieoullon et al., 1977) and later, in 1981, a well quoted review article emphasizing the TTX-insensitive nature of this dendritic monoamine release and its incipient pharmacology (Cheramy et al., 1981).
The rat and human brain contain approximately the same concentrations of dopamine in the substantia nigra, in the order of 0.40 (ig/g of wet tissue (Sourkes and Poirier, 1965; Hornykiewicz, 1972; Cuello and Iversen, 1978). At the time we developed a highly sensitive radioenzymatic technique (Cuello et al., 1973) which permitted us to determine monoamine concentrations in minute, separate, microdissected regions of the rat substantia nigra. We observed relatively high concentrations (1.52 Hg/gi of dopamine in the SN pars compacta where the dopaminergic elements are densely concentrated in cell bodies and short dendrites, while smaller amounts (0.28 Hg/g,) were found in the pars reticulata where the long-branched dopaminergic dendrites are present (Cuello and Iversen, 1978). As the weight of the pars reticulata is almost 3 times that of the compacta, it can be concluded that the pars reticulata contributes significantly to the total dopamine content of the substantia nigra.
We were interested in defining the dendritic storage sites of dopamine. We were able to demonstrate by high resolution radioautography that radiolabeled dopamine is incorporated into substantia nigra dendrites (Cuello and Kelly, 1977), however, the electron microscopical analysis of this material did not reveal the expected "synaptic vesicle" localization as the potential site of monoamine storage. The question then was where is dopamine stored in the SN dendritic processes if not in synaptic vesicles? Synaptic vesicles had been described in dendrites of substantia nigra neurons (Hajdu et al., 1973) but were not seen either by Sotelo (1971) using radiolabeled noradrenaline or by us stereotaxically applying 3H-dopamine in the SN (Cuello and Kelly, 1977; Cuello and Iversen, 1978; Cuello, 1982).
We resorted to the use of false transmitters which render an electron dense signal upon incorporation into subcellular compartments. Typically, these were small and large core synaptic vesicles of sympathetic neurons. We stereotaxically injected small amounts of the false transmitter 5-OHD into the rat substantia nigra. This again did not demonstrate amine storage in synaptic vesicles but generated evidence of amine uptake and storage in short cisterns of the smooth endoplasmic reticulum (SER) within dendritic profiles of the substantia nigra (Mercer et al.,1979). Such subcellular compartamentalization would be compatible with what had been previously proposed by Tranzer (1972) as an "immature" catecholaminergic compartment in the axonal shaft of sympathetic nerves. The involvement of these structures was elegantly confirmed by Pickel and collaborators (Nirenberg et al., 1996) using the immunogold technique for the ultrastructural localization of the vesicular monoamine transporter VMAT2. These authors found clear evidence of VMAT2-immunoreactive sites in tubulo-vesicular profiles related to the SER within dendritic processes of ventrotegmental dopaminergic neurons. Such non-synaptic vesicle localization of dendritic dopamine could explain the ionic and electrophysiological characteristics observed for the dopamine release in these neuronal processes (Cheramy et al 1981; Rice et al., 1997) and most importantly, it provided a structural basis for the recent and well supported proposition, that the dendritic dopamine release is the consequence of a functional dopamine transport reversal (Falkenburger et al., 2001). These authors have proposed the interesting possibility of the therapeutic inhibition of the dendritic VMAT2 transporter in early stages of Parkinson disease, preventing dopamine-induced autoinhibition at the nigra level.
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