Background History

The Parkinson's-Reversing Breakthrough

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Cell theory profoundly changed biology and medicine in the mid 19th century. Cells and the germ theory of disease gradually displaced the old humoral theories of health and disease. However, many authorities, including ES Russell and AN Whitehead in the 1920s, feared that a parliament of separate cells meant chaos. Nobody imagined the thousands of macromolecules and signaling molecules that we know today.

Early microscopists examined unstained brain tissue and reported "globules", often uniform in size. Cell theory had to wait for better optics. Achromatic microscopes became available in about 1830 and brought major changes. Ehrenberg reported the existence of cerebral ganglion cells in 1833 and Purkyne visualized neurons and dendrites (Figure 1). He presented drawings of neurons to scientific meetings [1].

Theodor Schwann proclaimed that the animal body was an organized collection of cells [2]. This implied mechanisms of intercellular communication and coordination, which were unknown at the time. Neural and endocrine mechanisms of communication and coordination began to emerge in the late 19th century. The neuromuscular junction was identified and studied before Sherrington developed the concept of synapses. Vulpian theorized in 1866 that curare acted on an intermediate zone between the nerve and the muscle [3]. DuBois-Reymond noted in 1877 that nerves might use chemical or electrical messages to communicate with effector elements [4]. The first systematic neuron theory and the word neuron came from von Waldeyer-Hartz in 1891 [5]. Waldeyer organized the observations of others into a coherent scheme without any new data of his own. His contemporaries Santiago Ramon y Cajal and Charles Sherrington played major roles in promoting and expanding the neuron concept. Cajal stressed the idea of neuronal polarization: axons carried outgoing messages, while dendrites processed incoming messages. Messages crossed the synapse in one direction only. His drawings often included arrows to stress this polarization. However, Cajal knew that retinal amacrine cells lacked axons and that conduction might go in the "wrong direction" in pathological states [6].

Figure 1. These drawings of nerve cells were used by Purkyne in his 1837 lecture to the Prague Congress. Purkyne outlined a cell theory (Kornchen) in advance of Schwann's famous book. This drawing includes cells from the brain stem (#16), thalamus (#17) and cerebellum containing what we now call Purkinje cells (#18). It is reproduced from reference [1] with the permission and assistance of author Larry Swanson who reproduced it from an original.

Figure 1. These drawings of nerve cells were used by Purkyne in his 1837 lecture to the Prague Congress. Purkyne outlined a cell theory (Kornchen) in advance of Schwann's famous book. This drawing includes cells from the brain stem (#16), thalamus (#17) and cerebellum containing what we now call Purkinje cells (#18). It is reproduced from reference [1] with the permission and assistance of author Larry Swanson who reproduced it from an original.

Neurophysiologist Charles Sherrington first wrote of synapses in 1897. His influential book, The Integrative Action of the Nervous System appeared in 1906 [7]. Sherrington's functional and physiological emphasis offset the limitations of neuroanatomy; light microscopy could not visualize the synaptic cleft. Sherrington and his students thought primarily in terms of electrical communication between cells.

Adrenaline1 was identified in 1899; it reproduced the features of some forms of endocrine and neural communication. The autonomic nervous system became a critical focus of monoamine pharmacology and theoretical development. Immunologist Paul Ehrlich and

1 I use adrenaline, noradrenaline, A, NA in this paper instead of the U.S. nomenclature (epinephrine) because of the historical emphasis of the paper and the importance of European pharmacologists in identifying these exceptions.

Cambridge physiologist John Langley were the first receptor theorists. They knew each other's work and were influenced by the Emil Fischer's well known studies of enzymes. Fischer suggested that enzyme-substrate specificity depended on something like a lock-and-key mechanism. Langley first mentioned "receptive substances" in 1878 and "the autonomic nervous system" in 1898. He showed that nicotine and curare acted on the same site or mechanism at the neuromuscular junction, with opposite actions [8]. Langley said in his 1906 Croonian lecture to the Royal Society:

"...stimuli passing the nerve can only affect the contractile molecules by the radical which combines with nicotine and curare. And this seems in its turn to require that the nervous impulse should not pass from nerve to muscle by an electrical discharge, but by the secretion of a special substance at the end of the nerve " [9].

Figure 2. This drawing of a hypothetical reticular network is reproduced from reference [1] with the permission of author Larry Swanson. There are no synapses; nerve cells are shown in direct continuity with muscle cells as well as other nerve cells. He reproduced it from an original in Landois and Sterling's Textbook of Human Physiology (1891).

Figure 2. This drawing of a hypothetical reticular network is reproduced from reference [1] with the permission of author Larry Swanson. There are no synapses; nerve cells are shown in direct continuity with muscle cells as well as other nerve cells. He reproduced it from an original in Landois and Sterling's Textbook of Human Physiology (1891).

Figure 3. This 1894 drawing of a spinal reflex arc shows Cajal's functional emphasis and his use of arrows to illustrate the flow of information. Compare it to Figure 2, from the same era. It was figure 9 in reference [1] and is reproduced with permission and assistance of Larry Swanson, who reproduced it from an original.

Figure 3. This 1894 drawing of a spinal reflex arc shows Cajal's functional emphasis and his use of arrows to illustrate the flow of information. Compare it to Figure 2, from the same era. It was figure 9 in reference [1] and is reproduced with permission and assistance of Larry Swanson, who reproduced it from an original.

Langley had a broader theory of chemical neurotransmission than did his student, Thomas Elliott, who suggested in an abstract that sympathetic nerve impulses might liberate adrenaline [10]. Walter Dixon proposed in 1907 that muscarine might be the vagus nerve messenger. Henry Dale reported in 1914 that adrenaline and acetylcholine duplicated the effects of sympathetic and parasympathetic nerve stimulation [11]. Dale and Otto Loewi constructed an outline of autonomic pharmacology between 1915-1945, which assumed that each cell used the same message at all its branches. Loewi showed that vagus nerve stimulation caused either an inhibitory substance or an excitatory one, depending upon the experimental details, to appear in the Ringer's solution perfusing a frog heart, as judged by the effect on a second heart. Loewi repeated this experiment at the 1926 International Congress of Physiology in Stockholm, but this was not enough to establish acetylcholine as "vagustoff", the vagus nerve neurotransmitter. Many authorities thought that transmission was too fast at ganglia and the neuromuscular junction to be chemically mediated. Acetylcholine wasn't demonstrated in animal tissues until 1929. Feldberg and Krayer used the leech bioassay to demonstrate that vagal stimulation produced acetylcholine-like material in the coronary venous blood of dogs and cats in 1933 [12]. Acetylcholine effects on muscle were nicotine-sensitive, its cardiovascular effects were not [13]. Dale proposed a division of autonomic pharmacology into cholinergic and adrenergic domains (he invented these words). John Eccles elevated Dale's principle to a formal theory of neurotransmission later, in the 1950s, after intracellular recording demonstrated a chemical basis for CNS neurotransmission. Dale himself never ruled out multiple transmitters. After observing atropine-resistant effects of vagus nerve stimulation, he said:

"We may suppose that vagus effects not paralysed by atropine are not humorally transmitted at all, or that the transmitter is not a choline ester, but in the latter case, we shall have to postulate not one, but several other transmitters with different degrees of liability to the antagonism of atropine." [14].

After World War II, intracellular recording demonstrated quantal transmitter release [15] and several different electron microscopic laboratories discovered synaptic vesicles. Now the skeptics were convinced. The standard model was summarized in classical publications of Bernard Katz and John Eccles. Excitatory transmission produced currents that depolarized the postsynaptic membrane, while inhibitory transmission hyperpolarized it, changing the membrane potential in the opposite direction [16,17]. These studies and theories considered only rapid responses, on a millisecond time scale. They didn't study stability of transmitter properties over periods longer than a day or the effects of disease and development.

The first paper in the new 1949 Pharmacological Reviews was Bacq's synthesis of adrenergic transmitter metabolism, hinting at the coming era of transmitter uptake and storage [18]. Reserpine was used by psychiatrists (it was approved by the US Food and Drug administration for clinical use in 1953) before its mode of action was understood. Bernard Brodie and his colleagues reported that reserpine released monoamines [19] and ascribed reserpine effects to serotonin depletion, which was only partly correct. Arvid Carlsson, who had studied with Brodie, showed that DOPA antagonized reserpine effects in rodents, whereas the serotonin precursor 5-hydroxytryptophan did not. [20]. This suggested the presence of a previously unknown brain amine and led to Carlsson's discovery that dopamine was a CNS neurotransmitter [21].

Reserpine could deplete all stored monoamine transmitters [22]. The first specific study of the storage granule transporter in 1962 [23] used adrenal chromaffin cells. Transporter specificity was limited; uptake was coupled to ATP hydrolysis. Carlsson found that reserpine was the most potent inhibitor of vesicular amine transport [22]. Tetrabenazine and ketanserin are also potent inhibitors. Tetrabenazine has little effect on the peripheral vesicular monoamine transporter; its depletion of CNS vesicular amine content lasts hours rather than days, unlike reserpine effects [24]. Once used extensively for treatment of psychosis and hypertension, reserpine is little used today because of many side effects, including depression and even suicide. Tetrabenazine is used today for chorea and other hyperkinetic movement disorders. It was never used for hypertension. The ketanserin derivative, (Cndihydrotetrabenazine [nC-CIDTBZ) has been used as a ligand for positron emission tomography (PET scanning) for in vivo studies of the type 2 vesicular monoamine transporter (VMAT2) in the human brain. Studies with DTBZ indicated fewer VMAT2 binding sites in the striatum of Parkinsonian patients than in controls [25]. Binding decreased during normal aging, less so than in Parkinson's disease. However, DTBZ binding is altered by dopamine depletion; it provides only an approximate measure of vesicle number [26].

Parkinson's disease [PD] is generally sporadic, but some patients have documented single gene mutations. PET studies of two kindreds with LRRK2 gene mutations revealed heterogeneous forms of presynaptic DA dysfunction, similar to the findings in sporadic Parkinson's disease. A small number of asymptomatic relatives with the mutation showed presynaptic abnormalities, often related to the plasma membrane DA transporter [27]. Only fifteen family members were studied, enough to suggest that multiple different presynaptic factors may contribute to PD. Follow-up studies showed progression of these presynaptic abnormalities as symptoms progressed [28]. No patients had evidence of postsynaptic D2 receptor dysfunction. There is a mouse model of PD produced by mutations of the vesicular monoamine transporter [29], but no analogous human mutations have been reported.

Two different genes encode vesicular monoamine transporters, VMAT1 and VMAT2 [30]. Very little VMAT1 is found in the adult CNS, and VMAT1 is inhibited only very weakly by tetrabenazine. Vesicular transporter expression is one factor regulating the neurotransmitter content of secretory vesicles and influences the amount of transmitter released [31]. Pothos et al showed that more DA molecules were released per quantum in cultured neurons treated with glial-derived neurotrophic factor (GDNF) and with DOPA [32]. This was the first demonstration that presynaptic factors could modulate quantal transmitter size of CNS neurons. Quantal size (the number of neurotransmitter molecules released by a single synaptic vesicle during exocytosis) was assumed to be invariant in the classical model. Quoting from Del Castillo and Katz,

"transmission at a nerve-muscle junction takes place in all-or-none quanta whose sizes are indicated by the spontaneously occurring miniature discharges...The average'quantum content' of the e.p.p. depends on the probability of response of the individual units, and this varies with the external Ca andMg concentrations " [33]

Figure 4. These are amperometric recordings of quantal DA release from cultured rat neurons [34]. Control (A) and experimental neurons overexpressing the VMAT2 gene (E) were stimulated with 40 mM potassium to increase quantal release. The experimental neurons have increased amplitude, mean of 7800 molecules © and 11800 molecules (E), and frequency (increased about 10 fold) both of which were statistically significant at the P<.01 level. Permission for reproduction obtained from Elsevier Limited, publisher of Behavioral Brain Research.

Figure 4. These are amperometric recordings of quantal DA release from cultured rat neurons [34]. Control (A) and experimental neurons overexpressing the VMAT2 gene (E) were stimulated with 40 mM potassium to increase quantal release. The experimental neurons have increased amplitude, mean of 7800 molecules © and 11800 molecules (E), and frequency (increased about 10 fold) both of which were statistically significant at the P<.01 level. Permission for reproduction obtained from Elsevier Limited, publisher of Behavioral Brain Research.

However, GDNF and DOPA have complex actions and the role of VMAT2 was not clear in the Pothos experiment. Pathos then used VAMT2 transfections into cultured midbrain and hippocampal neurons to show that more transporter protein caused increased quantal size [34]. Transporter protein reduction had the opposite effect, as shown in Figure 4.

Hippocampal nondopaminergic neurons became able to store and release DA when VMAT2 was expressed. Vesicular monoamine storage is also regulated by G proteins [35,36].

Mice with deletions of the G protein Go2a have a decreased motor response to cocaine and less stored cerebral DA [37]. G proteins appear to interact with calcium dependent activator proteins CAPS I and II, which regulate monoamine uptake and storage, perhaps by determining when filling is complete [36]. Cocaine is a well known inhibitor of the plasma membrane DA transporter [38]; many drugs act at both plasma and vesicular monoamine transporters.

DMPP Tyramine Noradrenillne

DMPP Tyramine Noradrenillne

Figure 5. This figure, modified from Haefely [42] and reproduced with permission, shows blood pressure responses to three different treatments in cats treated with two false transmitter precursors (amethyl dopa and a-methyl meta tyrosine) and reserpine. DMPP (dimethyl phenylpiperazinium), a nicotinic ganglionic stimulant; tyramine, an indirect acting "sympathomimetic drug"; and NA, the natural transmitter, were studied. Reserpinized cats had the greatest BP response to DMPP & NA; responses to the false transmitters were similar with NA and divergent with tyramine- false transmitters explain only part of these results.

Figure 5. This figure, modified from Haefely [42] and reproduced with permission, shows blood pressure responses to three different treatments in cats treated with two false transmitter precursors (amethyl dopa and a-methyl meta tyrosine) and reserpine. DMPP (dimethyl phenylpiperazinium), a nicotinic ganglionic stimulant; tyramine, an indirect acting "sympathomimetic drug"; and NA, the natural transmitter, were studied. Reserpinized cats had the greatest BP response to DMPP & NA; responses to the false transmitters were similar with NA and divergent with tyramine- false transmitters explain only part of these results.

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