Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2. I. The developing rat central nervous system

We used in situ hybridization histochemistry to study the expression of the mRNA of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic and postnatal development of the central nervous system (CNS) in the rat. In the adult rat, VMAT2 mRNA is present exclusively in monoaminergic cell groups of the CNS and VMAT1 mRNA was reported to be present in the adrenal medulla and certain intestinal epithelial cells. In contrast to the above, the expression of VMAT1 mRNA has previously never been detected in the central nervous system. This study shows the first evidence that both transporter molecules are expressed in CNS during ontogenesis. We here demonstrate four main expression patterns detected during development: 1. VMAT2 mRNA expression in monoaminergic neurons of the brainstem beginning as early as embryonic day E13. 2. Expression of VMAT2 mRNA in all major sensory relay nuclei of central nervous system. 3. Co-expression of VMAT1 and VMAT2 mRNA in most limbic structures, basal ganglia, as well as in some hypothalamic nuclei. 4. Exclusive expression of VMAT1 mRNA in the neocortical subventricular zone, in the amygdala at early (E15-18) and late (P1-P28) timepoints, the granular cell layer of cerebellum, and in several brainstem motor nuclei. Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect wiring of sensory and also motor circuits. VMAT1 mRNA expression may reflect a specific effect of monoamines in glial differentiation and cerebellar granule cell migration and/or differentiation.     

Ketamine inhibits monoamine transporters expressed in human embryonic kidney 293 cells

BACKGROUND: Ketamine has been characterized as having psychotomimetic and sympathomimetic effects. These symptoms have raised the possibility that ketamine affects monoaminergic neurotransmission. To elucidate the relation between ketamine and monoamine transporters, the authors constructed three cell lines that stably express the norepinephrine, dopamine, and serotonin transporters and investigated the effects of ketamine on these transporters. METHODS: Human embryonic kidney cells were transfected using the Chen-Okayama method with the human norepinephrine, rat dopamine, and rat serotonin transporter cDNA subcloned into the eukaryotic expression vector. Using cells stably expressing these transporters, the authors investigated the effects of ketamine on the uptake of these compounds and compared them with those of pentobarbital. RESULTS: Inhibition analysis showed that ketamine significantly inhibited the uptake of all three monoamine transporters in a dose-dependent manner. The Ki (inhibition constant) values of ketamine on the norepinephrine, dopamine, and serotonin transporters were 66.8 microM, 62.9 microM, and 162 microM, respectively. Pentobarbital, a typical general anesthetic agent with no psychotic symptoms, did not affect the uptake of monoamines, however. Further, neither the glycine transporter 1 nor the glutamate/aspartate transporter was affected by ketamine, indicating that ketamine preferentially inhibits monoamine transporters. CONCLUSIONS: Ketamine inhibited monoamine transporters expressed in human embryonic kidney cells in a dose-dependent manner. This result suggests that the ketamine-induced inhibition of monoamine transporters might contribute to its psychotomimetic and sympathomimetic effects through potentiating monoaminergic neurotransmission.     

Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine

Vesicular monoamine transporters are known to transport monoamines from the cytoplasm into secretory vesicles. We have used homologous recombination to generate mutant mice lacking the vesicular monoamine transporter 2 (VMAT2), the predominant form expressed in the brain. Newborn homozygotes die within a few days after birth, manifesting severely impaired monoamine storage and vesicular release. In heterozygous adult mice, extracellular striatal dopamine levels, as well as K+- and amphetamine-evoked dopamine release, are diminished. The observed changes in presynaptic homeostasis are accompanied by a pronounced supersensitivity of the mice to the locomotor effects of the dopamine agonist apomorphine, the psychostimulants cocaine and amphetamine, and ethanol. Importantly, VMAT2 heterozygous mice do not develop further sensitization to repeated cocaine administration. These observations stress the importance of VMAT2 in the maintenance of presynaptic function and suggest that these mice may provide an animal model for delineating the mechanisms of vesicular release, monoamine function, and postsynaptic sensitization associated with drug abuse.     

Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2. II. Expression in neural crest derivatives and their target sites in the rat

We used in situ hybridization histochemistry to study the expression of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic development in the rat. In the adult rat VMAT2 is present exclusively in neuronal tissues and VMAT1 is present in the adrenal medulla and in certain intestinal endocrine cells. We found that both transporter molecules are more widely expressed during development. We demonstrate a complete overlap of the two VMAT mRNAs in the sympathetic nervous system between E13 and E21 days. In addition, VMAT2 (and to some extent VMAT1) mRNA is expressed in ganglionic cells of the parasympathetic nervous system and in cranial ganglia (trigeminal, vestibular and spiral ganglia) between E12 and E21. The sensory neurons of the dorsal root ganglia, which are also neural crest derivatives, express VMAT2 mRNA (E11-E21), exclusively. Both VMAT mRNAs are found in the developing GI system, but in different cells. VMAT1 mRNA was detected in organs of the endocrine system (pituitary gland, adrenal gland, testis, seminal vesicle), some connective tissue cells, and the thymus. We observed expression of both VMAT mRNAs in two separate cell groups in the placenta (E8-E10). Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect migration and differentiation of neural crest derivatives.     

VMAT2 knockout mice: heterozygotes display reduced amphetamine-conditioned reward, enhanced amphetamine locomotion, and enhanced MPTP toxicity

The brain vesicular monoamine transporter (VMAT2) pumps monoamine neurotransmitters and Parkinsonism-inducing dopamine neurotoxins such as 1-methyl-4-phenyl-phenypyridinium (MPP+) from neuronal cytoplasm into synaptic vesicles, from which amphetamines cause their release. Amphetamines and MPP+ each also act at nonvesicular sites, providing current uncertainties about the contributions of vesicular actions to their in vivo effects. To assess vesicular contributions to amphetamine-induced locomotion, amphetamine-induced reward, and sequestration and resistance to dopaminergic neurotoxins, we have constructed transgenic VMAT2 knockout mice. Heterozygous VMAT2 knockouts are viable into adult life and display VMAT2 levels one-half that of wild-type values, accompanied by smaller changes in monoaminergic markers, heart rate, and blood pressure. Weight gain, fertility, habituation, passive avoidance, and locomotor activities are similar to wild-type littermates. In these heterozygotes, amphetamine produces enhanced locomotion but diminished behavioral reward, as measured by conditioned place preference. Administration of the MPP+ precursor N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to heterozygotes produces more than twice the dopamine cell losses found in wild-type mice. These mice provide novel information about the contributions of synaptic vesicular actions of monoaminergic drugs and neurotoxins and suggest that intact synaptic vesicle function may contribute more to amphetamine-conditioned reward than to amphetamine-induced locomotion.     

Transport of histamine by vesicular monoamine transporter-2

Histamine mediates signalling by a wide range of neural and non-neural cells including mast cells. Like other biogenic amines, histamine is released from specialized secretory vesicles and requires transport from the cytoplasm into these vesicles. Of the two vesicular monoamine transporters, histamine potently inhibits 3H-serotonin transport by one (VMAT2) but not the other (VMAT1). In addition, histamine-containing cells in both neural and non-neural cells express VMAT2. However, histamine lacks the hydroxyl groups generally considered necessary for recognition as a substrate by the vesicular monoamine transporters. Using a heterologous expression system, we now report that VMAT2 not only shows inhibition by histamine but also transports 3H-histamine. Interestingly, histamine differs from other monoamine transmitters and does not inhibit 3H-reserpine binding to VMAT2, indicating interaction at a distinct site. Surprisingly, reserpine inhibits histamine transport with much less potency than serotonin transport, suggesting a different transport mechanism. However, replacement of serines in the third transmembrane domain of VMAT2 that have been shown to be essential for recognition of other monoamines also eliminate 3H-histamine transport, suggesting that these serine residues may do more than simply recognize the hydroxyl groups on a monoamine substrate.     

The vesicular monoamine transporter: from chromaffin granule to brain

All characterized monoaminergic cells utilize the same transport system for the vesicular accumulation of monoamines prior to their release. This system operates in neuronal (catecholaminergic, serotoninergic or histaminergic) as well as in endocrine or neuroendocrine cells. For several decades, chromaffin granules from bovine adrenal medulla have been used as a model system, allowing progress in the understanding of the biophysics, the biochemistry and the pharmacology of the monoamine vesicular transporter. The transporters from rat, bovine and man have been cloned. Surprisingly, two genes encode different isoforms of the protein which are differentially expressed in monoaminergic systems. The conjunction of recombinant DNA techniques and expression in secretory or non-secretory cells with the large body of data obtained on the chromaffin granule transporter has allowed rapid progress in the study of the protein. But interestingly enough, this progress has open new possibilities in the study of biological problems, especially in the brain. The transporter is useful for the determination of the relationship between small and large dense core vesicles, for the understanding of the mechanism of the drugs such as 1-methyl-4-phenylpyridinium (MPP+), tetrabenazine or amphetamines, and as a marker in brain development. The possibility of regulations at the vesicular transporter level and of their effect on the quantum size has to be investigated. The vesicular monoamine transporter is also an important target for brain imaging.     

Vesicular neurotransmitter transporters. Potential sites for the regulation of synaptic function

Neurotransmission depends on the regulated release of chemical transmitter molecules. This requires the packaging of these substances into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific vesicular transporters. The family of genes encoding the vesicular transporters for biogenic amines and acetylcholine have recently been cloned. Direct comparison of their transport characteristics and pharmacology provides information about vesicular transport bioenergetics, substrate feature recognition by each transporter, and the role of vesicular amine storage in the mechanism of action of psychopharmacologic and neurotoxic agents. Regulation of vesicular transport activity may affect levels of neurotransmitter available for neurosecretion and be an important site for the regulation of synaptic function. Gene knockout studies have determined vesicular transport function is critical for survival and have enabled further evaluation of the role of vesicular neurotransmitter transporters in behavior and neurotoxicity. Molecular analysis is beginning to reveal the sites involved in vesicular transporter function and the sites that determine substrate specificity. In addition, the molecular basis for the selective targeting of these transporters to specific vesicle populations and the biogenesis of monoaminergic and cholinergic synaptic vesicles are areas of research that are currently being explored. This information provides new insights into the pharmacology and physiology of biogenic amine and acetylcholine vesicular storage in cardiovascular, endocrine, and central nervous system function and has important implications for neurodegenerative disease.     

Individual residues contribute to multiple differences in ligand recognition between vesicular monoamine transporters 1 and 2

Molecular cloning has identified two vesicular monoamine transporters (VMATs), one expressed in non-neural cells of the periphery (VMAT1) and the other by multiple monoamine cell populations in the brain (VMAT2). Functional analysis has previously shown that VMAT2 has a higher affinity than VMAT1 for monoamine neurotransmitters as well as the inhibitor tetrabenazine. The analysis of chimeric transporters has also identified two major regions required for the high affinity interactions of VMAT2 with these ligands. We have now used site-directed mutagenesis to identify the individual residues responsible for these differences. Focusing on the region that spans transmembrane domains 9 through 12, we have replaced VMAT2 residues with the corresponding residues from VMAT1. Many residues in this region had no effect on the recognition of these ligands, but substitution of Tyr-434 with Phe and Asp-461 with Asn reduced the affinity for tetrabenazine, histamine, and serotonin. Although the ability to affect recognition of multiple ligands suggests a general structural role for these residues, the mutations did not affect dopamine recognition, indicating a more specific role, possibly in recognition of the ring nitrogen that occurs in tetrabenazine, histamine, and serotonin but not dopamine. The mutation K446Q reduced the affinity of VMAT2 for tetrabenazine and serotonin but not histamine, whereas F464Y reduced serotonin affinity and perhaps histamine recognition but not tetrabenazine sensitivity, providing more evidence for specificity. Interestingly, the Vmax of both VMATs for dopamine exceeded that for serotonin by 3-5-fold, indicating a difference in the speed of packaging of these two neurotransmitters. We also found that VMAT1 has a higher affinity for tryptamine than VMAT2. This mutually exclusive interaction with serotonin and tryptamine also suggests a physiological rationale for the existence of two VMATs. Surprisingly, the residue responsible for this difference, Tyr-434, also accounts for the higher affinity interaction of VMAT2 with tetrabenazine, histamine, and serotonin. Interestingly, replacement of Tyr-434 with alanine increases the affinity of VMAT2 for both serotonin and dopamine and reduces the rate of dopamine transport.     

Localization and function of five glutamate transporters cloned from the salamander retina

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. Native glutamate transporters have been well characterized in several retinal neurons, particularly from the salamander retina. We have cloned five distinct glutamate transporters from the salamander retina and examined their localization and functional properties: sEAAT1, sEEAAT2A, sEAAT2B, sEAAT5A and sEAAT5B. sEAAT1 is a homologue of the glutamate transporter EAAT1 (GLAST), sEAAT2A and sEAAT2B are homologues of EAAT2 (GLT-1) and sEAAT5A and sEAAT5B are homologues of the recently cloned human retinal glutamate transporter EAAT5. Localization was determined by immunocytochemical techniques using antibodies directed at portions of the highly divergent carboxy terminal. Glutamate transporters were found in glial, photoreceptor, bipolar, amacrine and ganglion cells. The pharmacology and ionic dependence were determined by two-electrode voltage clamp recordings from Xenopus laevis oocytes which had previously been injected with one of the glutamate transporter mRNAs. Each of the transporters behaved in a manner consistent with a glutamate transporter and there were some distinguishing characteristics which make it possible to link the function in native cells with the behavior of the cloned transporters in this study.     

Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells

To clarify the mechanisms of the antiepileptic activity of phenytoin (PHI), the effects of PHT on extracellular and total levels of monoamines (dopamine and serotonin), in rat striatum and hippocampus were studied. The plasma concentrations of PHT associated with therapeutic activity did not affect striatal and hippocampal extracellular levels of monoamines, whereas supratherapeutic concentrations of PHT decreased striatal and hippocampal extracellular levels of monoamines, in a concentration dependent manner. Toxic concentrations of PHT produced generalized seizures 'paradoxical intoxication' and an initial drastic decrease in striatal and hippocampal extracellular levels of monoamines before seizure onset, whereas the extracellular monoamines levels increased after seizures. In addition, the therapeutic concentrations of PHT did not affect monoamine turnover, whereas supratherapeutic concentrations of PHT inhibited monoamine turnover. These results suggest that monoaminergic transmission may not be involved in the antiepileptic mechanism of action of PHT, and that dysfunction of monoaminergic transmission can produce generalized tonic-clonic convulsions. Thus, the present study suggests that 'Paradoxical Intoxication' induced by toxic concentrations of PHT, at least partially, can be mediated by hypo-monoaminergic function in the brain.     

Co-expression of dopamine transporter mRNA and tyrosine hydroxylase mRNA in ventral mesencephalic neurones

Radioactive in situ hybridization was used to map the cellular localization of dopamine (DA) transporter mRNA-containing cells in the adult rat central nervous system. The distribution of DA transporter mRNA-containing cells was compared to adjacent sections processed to visualize tyrosine hydroxylase (TH) mRNA, a marker of catecholamine containing neurones. TH mRNA-containing cells, visualized using an alkaline phosphatase labelled probe, were detected in the hypothalamus, midbrain and pons; the strongest hybridization signals being detected in the substantia nigra, ventral tegmental area and locus coeruleus. The distribution of DA transporter mRNA-containing cells was more restricted; a strong signal being detected in the substantia nigra pars compacta and ventral tegmental area only. No hybridization signal was detected in the locus coeruleus. By simultaneously hybridizing mesencephalic tissue with both the alkaline phosphatase-labelled TH probe and the 35S-labelled DA transporter probe we were able to demonstrate that both DA transporter and TH mRNAs are expressed by the same cells in the substantia nigra and ventral tegmental area. The restricted anatomical localization of DA transporter mRNA-containing cells and the lack of expression in the locus coeruleus and other adrenergic and noradrenergic cell groups confirms the DA transporter as a presynaptic marker of DA containing nerve cells in the rat brain.     

Differential localization of vesicular acetylcholine and monoamine transporters in PC12 cells but not CHO cells

Previous studies have indicated that neuro-endocrine cells store monoamines and acetylcholine (ACh) in different secretory vesicles, suggesting that the transport proteins responsible for packaging these neurotransmitters sort to distinct vesicular compartments. Molecular cloning has recently demonstrated that the vesicular transporters for monoamines and ACh show strong sequence similarity, and studies of the vesicular monoamine transporters (VMATs) indicate preferential localization to large dense core vesicles (LDCVs) rather than synaptic-like microvesicles (SLMVs) in rat pheochromocytoma PC12 cells. We now report the localization of the closely related vesicular ACh transporter (VAChT). In PC12 cells, VAChT differs from the VMATs by immunofluorescence and fractionates almost exclusively to SLMVs and endosomes by equilibrium sedimentation. Immunoisolation further demonstrates colocalization with synaptophysin on SLMVs as well as other compartments. However, small amounts of VAChT also occur on LDCVs. Thus, VAChT differs in localization from the VMATs, which sort predominantly to LDCVs. In addition, we demonstrate ACh transport activity in stable PC12 transformants overexpressing VAChT. Since previous work has suggested that VAChT expression confers little if any transport activity in non-neural cells, we also determined its localization in transfected CHO fibroblasts. In CHO cells, VAChT localizes to the same endosomal compartment as the VMATs by immunofluorescence, density gradient fractionation, and immunoisolation with an antibody to the transferrin receptor. We have also detected ACh transport activity in the transfected CHO cells, indicating that localization to SLMVs is not required for function. In summary, VAChT differs in localization from the VMATs in PC12 cells but not CHO cells.     

Patch-clamp and amperometric recordings from norepinephrine transporters: channel activity and voltage-dependent uptake

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.     

Computational methods in molecular diversity and combinatorial chemistry

(+)-MK-801 is known to be a specific non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors. However, besides having an anticonvulsant effect, this compound possesses a central sympathomimetic effect and an anxiolytic-like action, raising the possibility that (+)-MK-801 might affect monoamine uptake systems. To elucidate this possibility, we investigated the effects of (+)-MK-801 on monoamine transporters expressed in HEK cells. (+)-MK-801 significantly inhibited the uptake of all three monoamine transporters in a dose-dependent manner and the inhibitions were competitive with respect to monoamines. The Ki values of (+)-MK-801 on the norepinephrine, dopamine and serotonin transporters were 3.2 microM, 40 microM and 43 microM, respectively. In addition, (-)-MK-801, a less potent antagonist of NMDA receptors, also inhibited monoamine transporters with a similar potency as that of (+)-MK-801. These results clearly indicate that MK-801, a non-competitive antagonist of NMDA receptors, competitively inhibits monoamine transporters without stereoselectivity.     

The vesicular monoamine transporter, in contrast to the dopamine transporter, is not altered by chronic cocaine self-administration in the rat

Although much evidence suggests that the brain dopamine transporter (DAT) is susceptible to dopaminergic regulation, only limited information is available for the vesicular monoamine transporter (VMAT2). In the present investigation, we used a chronic, unlimited-access, cocaine self-administration paradigm to determine whether brain levels of VMAT2, as estimated using [3H]dihydrotetrabenazine (DTBZ) binding, are altered by chronic exposure to a dopamine uptake blocker. Previously, we showed that striatal and nucleus accumbens DAT levels, as estimated by [3H]WIN 35,428 and [3H]GBR 12,935 binding, are altered markedly using this animal model (Wilson et al., 1994). However, in sequential sections from the same animals, [3H]DTBZ binding was normal throughout the entire rostrocaudal extent of the basal ganglia (including striatum and nucleus accumbens), cerebral cortex, and diencephalon, as well as in midbrain and brainstem monoamine cell body regions, both on the last day of cocaine access and after 3 weeks of drug withdrawal. These data provide additional evidence that VMAT2, unlike DAT, is resistant to dopaminergic regulation.     

Oocytes are a source of catecholamines in the primate ovary: evidence for a cell-cell regulatory loop

Catecholamines, thought to derive from the extrinsic innervation of the ovary, participate in the regulation of ovarian development and mature gonadal function. Recently, intraovarian neurons containing tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, were described in the ovary of nonhuman primates. We now show that the primate ovary expresses both the genes encoding TH and dopamine beta-hydroxylase (DBH), the key enzymes in norepinephrine (NE) biosynthesis. Ovarian neurons were identified as a site of TH and DBH gene expression, and surprisingly, oocytes were identified as an exclusive site of DBH synthesis. Oocytes contain neither TH mRNA nor protein, indicating that they are unable to synthesize dopamine (DA). They did, however, express a DA transporter gene identical to that found in human brain. The physiological relevance of this transporter system and DBH in oocytes was indicated by the ability of isolated oocytes to metabolize exogenous DA into NE. Isolated follicles containing oocytes-but not those from which the oocytes had been removed-responded to DA with an elevation in cAMP levels; this elevation was prevented by propranolol, a beta-adrenoreceptor antagonist. The results suggest that oocytes and somatic cells are linked by a neuroendocrine loop consisting of NE synthesized in oocytes from actively transported DA and cAMP produced by somatic follicular cells in response to NE-induced beta-adrenoreceptor activation.     

EEG-based communication: improved accuracy by response verification

We used (+)[11C]dihydrotetrabenazine, a new ligand for the type 2 vesicular monoamine transporter, with positron emission tomography to study striatal monoaminergic presynaptic terminals in 7 male severe chronic alcoholic subjects without Wernicke-Korsakoff disease compared with 7 male normal controls of similar ages. We found reduced specific binding in the caudate nucleus and putamen in the alcoholic group, and the difference reached significance in the putamen. Specific binding was not decreased in the thalamus, which was examined as a reference structure. We also detected deficits in blood-to-brain transfer rate, K1, in the same regions of the alcoholic group, with a significant difference in the putamen. K1 was unchanged in the thalamus. The finding of reduced striatal VMAT2 in severe chronic alcoholic patients suggests that nigrostriatal monoaminergic terminals are reduced, with or without loss of neurons from the substantia nigra. The findings suggest that the damaging effects of severe chronic alcoholism on the central nervous system are more extensive than previously considered.     

Cyclic HIV-1 protease inhibitors derived from mannitol: synthesis, inhibitory potencies, and computational predictions of binding affinities

The molecular characteristics of midbrain dopamine (DA) neurons have been extensively studied in Parkinson's disease (PD). No such studies of the characteristics of midbrain DA neurons in Alzheimer's disease (AD) or Alzheimer's disease with parkinsonism (AD/Park) have been published. We examined the levels of tyrosine hydroxylase (TH) protein, and the expression of TH and dopamine transporter (DAT) mRNAs, in midbrain neurons of PD, AD, and AD/Park cases. In PD, the loss of TH protein in the ventral tier of the substantia nigra pars compacta (SNpc) of the PD group in accompanied by severe losses in the number of neurons that express TH mRNA and DAT mRNA (74% loss). Remaining neurons show a shift to higher concentrations of TH mRNA but a shift to lower concentrations of DAT mRNA per cell. Hence, there is evidence that compensation in the remaining neurons can elevate concentrations of TH mRNA and lower DAT mRNA. Alternatively, there may be a predilection for a loss of neurons with high levels of DAT mRNA and low TH mRNA levels within the SNpc of PD cases. There was no change in TH protein but an elevation of TH mRNA concentrations per neuron without any change in concentrations of DAT mRNA in the AD group. The AD/Park group did not exhibit changes in the level of TH protein, but showed a small loss (26%) of neurons in the SNpc and a greater loss in other regions of the midbrain (43-53%). Remaining DA neurons showed a marked shift to lower concentrations of DAT mRNA per neuron and a nonsignificant shift in cellular concentration of TH mRNA to higher levels. This is consistent with our previous work showing that with AD/Park there is a significant reduction in the number of DAT sites located on DA terminals in the striatum, but the midbrain neurons have not died. Our results indicate that the differential regulation of mRNAs encoding TH and DAT is similar in the parkinsonian disorders (PD and AD/Park) even though the degree of cell death is very different. This might suggest that compensatory events occur in these DA neurons in AD/Park that are similar to those in PD and that result in differential effects on mRNAs encoding TH and DAT proteins.     

Cocaine reward models: conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice

Cocaine and methylphenidate block uptake by neuronal plasma membrane transporters for dopamine, serotonin, and norepinephrine. Cocaine also blocks voltage-gated sodium channels, a property not shared by methylphenidate. Several lines of evidence have suggested that cocaine blockade of the dopamine transporter (DAT), perhaps with additional contributions from serotonin transporter (5-HTT) recognition, was key to its rewarding actions. We now report that knockout mice without DAT and mice without 5-HTT establish cocaine-conditioned place preferences. Each strain displays cocaine-conditioned place preference in this major mouse model for assessing drug reward, while methylphenidate-conditioned place preference is also maintained in DAT knockout mice. These results have substantial implications for understanding cocaine actions and for strategies to produce anticocaine medications.