Nociceptor-like rat dorsal root ganglion neurons express the angiotensin-II AT2 receptor throughout development

AT2 receptor (AT2R) plays a functional role in foetal development. Its expression declines in most tissues soon after birth but stays high in sensory areas of the adult nervous system. In the dorsal root ganglia (DRG) the expression pattern of AT2R during development and the identity of the subpopulation expressing it remain unknown. Using a combination of semi-quantitative PCR, western blotting and immunohistochemistry we examined the expression of AT2R at mRNA and protein levels in rat DRGs from embryonic day 15 (E15) until postnatal day 30 (PN30). We found that both AT2R mRNA and protein levels exhibited only minor (statistically non-significant) fluctuations from E15 to PN30. Detailed quantitative analysis of ABC/DAB AT2R staining showed a) that the receptor was present in most neurons at E15 and E18 and b) that postnatally it was predominantly expressed by small DRG neurons. Given that small neurons are putative C-nociceptors and the proposed role of AT2R in neuropathic pain, we next examined whether these AT2R-positive neurons co-localized with Ret and trkA embryonically and with IB4-binding postnatally. Most AT2R-positive neurons expressed trkA embryonically and bound IB4 postnatally. We found strong positive statistically highly significant correlations between AT2R cytoplasmic%intensities and trkA at E15/E18 and with Ret only at E18. Cytoplasmic AT2R also strongly and positively correlated with IB4-binding at PN3, 15 and 30. Our demonstration that a subpopulation of C-nociceptor-like neurons expresses AT2R during development supports a role for this receptor in neuropathic pain.     

Human and monkey cholinergic neurons visualized in paraffin-embedded tissues by immunoreactivity for VAChT,the vesicular acetylcholine transporter

The predicted C-terminal dodecapeptide of the human vesicular acetylcholine transporter (VAChT), deduced from the unique open reading frame of the recently cloned human VAChT cDNA, was conjugated through an N-terminal cysteine to keyhole limpet hemocyanin and used as an immunogen to generate polyclonal antihuman VAChT antibodies in rabbits. The distribution of the VAChT antigen in representative regions of the cholinergic nervous system was examined and compared to that of the acetylcholine biosynthetic enzyme choline acetyltransferase (ChAT), a specific marker for cholinergic neurons. VAChT immunoreactivity was localized in cell bodies of neurons in the basal forebrain and ventral horn of the spinal cord, regions in which major cholinergic projection systems to the cerebral cortex and to skeletal muscle, respectively, originate. The primate caudate nucleus contained numerous VAChT-positive interneurons. VAChT immunoreactivity was visualized in both cell bodies and extensive terminals in striatal interneurons, in contrast to formalin-fixed, deparaffinized sections stained for ChAT, in which cell bodies and fibers were stained but nerve terminals were less well visualized than with the VAChT antiserum. VAChT-positive nerve fibers were visualized in routinely immersion-fixed, paraffin-embedded human cerebral cortex, comparable to the density of fibers observed in perfusion-fixed Bouin’s-postfixed monkey cerebral cortex. Extensive investment of virtually all principal ganglion cells of thoracic sympathetic ganglia of monkey and human with VAChT-positive nerve terminals was observed. VAChT-positive cell bodies, presumably corresponding to cholinergic sympathetic sudomotor neurons, were a significant fraction of the total principal cell population in monkey and human thoracic sympathetic ganglia. VAChT is a specific marker for cholinergic neurons in human and rhesus monkey, visualizing especially nerve terminals more extensively than antibodies against the cholinergic biosynthetic enzyme ChAT, in routinely fixed tissue. VAChT immunoreactivity in cholinergic nerve terminals of the central and peripheral nervous systems ought to prove useful for visualizing cholinergic synapses and neuroeffector junctions, and their functional status during development and in neurodegenerative and autonomic disease.     

GABAergic neurons of the rat dorsal hippocampus express muscarinic acetylcholine receptors

The expression of muscarinic acetylcholine receptors (mAChRs) in glutamic acid decarboxylase (GAD)-positive cells in the different strata of CA1, CA3, and the dentate gyrus (DG) of the dorsal hippocampus is examined by way of quantitative immunofluorescent double labeling employing M35, the monoclonal antibody raised against purified mAChR protein. Of all GAD-positive neurons, 97.5% express mAChRs. Conversely, 92.9% of the muscarinic cholinoceptive nonpyramidal neurons express GAD. These results indicate that the vast majority of the γ-aminobutyric acid (GABA)ergic neurons express mAChRs. In addition to GAD, parvalbumin (PARV) and somatostatin (SOM) are two neurochemical substances notably expressed in GABAergic neurons. In order to examine whether the entire muscarinic cholinoceptive nonpyramidal cell group can be characterized by these three GABAergic markers, a cocktail of GAD, PARV, and SOM was used in a fluorescent double-labeling experiment with M35. These results show that 97.2% of all muscarinic cholinoceptive nonpyramidal neurons can be neurochemically characterized by the content of GAD, PARV, and SOM. In conclusion, nearly all GABAergic cells express mAChRs and, conversely, virtually the entire muscarinic cholinoceptive nonpyramidal cell group belongs to the GABAergic cell population. This study, therefore, provides anatomical evidence for an extensive neuronal connectivity of the hippocampal muscarinic cholinoceptive nonpyramidal system and the inhibitory GABAergic circuitry.     

Neurotransmitter phenotype plasticity in cultured dissociated adult rat dorsal root ganglia: an immunocytochemical study

Culturing sympathetic ganglion neurons in vitro may modify phenotypic expression of some neurotransmitters. For dorsal root ganglia (DRG), contradictory results have been reported; most studies have used immature material. We have therefore performed a detailed immunocytochemical analysis of the transmitter content of cultured adult rat DRG neurons. To demonstrate possible modifications of neurotransmitter phenotypes, we have compared the results obtained with the same techniques on neurons cultured for 3 days and on freshly dissociated DRG cells. Also, the transmitter profile of cultured neurons was compared with that known from in situ studies. Out of 22 antigens studied, 20 were detected in cultured DRG neurons. All of them were expressed in small and/or intermediate-sized cells. Large neurons only contained CGRP, VIP, NPY, beta-END, ENK, and GABA. The percentage of immunostained neurons varied for the various antisera: less than 10% of cultured neurons were positive for ENK, beta-LPH, beta-END, DYN, VASO, and OXY; 10-30% for SOM, CCK, CAT, and SP; and greater than 30% for NPY, CRF, GLU, NT, VIP, GABA, GRP, CGRP, 5-HT, and TRH. In the latter two groups of transmitters (except CGRP), the proportion of immunoreactive neurons was by far larger in cultured than in freshly dissociated DRG. The most pronounced (greater than 25%) increase in the proportion of positively stained neurons after culturing was observed for the GRP, CRF, TRH, and 5-HT antisera. Serotonin was the only transmitter identified in cultured but not in freshly dissociated cells. These data indicate, on one hand, that various antigens, for example, CAT, GABA, NT, TRH, NPY, beta-LPH, and beta-END, which up to now have not been described in DRG in situ, can be detected immunocytochemically a few hours after dissociation of adult rat DRG. On the other hand, several transmitters, for example, VIP, NPY, SP, GABA, GLU, NT, GRP, CRF, TRH, and 5-HT, are expressed in a significantly higher proportion of cells in cultured than in freshly dissociated preparations. This might reflect a change in the phenotypic expression of transmitters due to the new environment generated by the culture conditions, a hypothesis that can be tested by measuring specific mRNA levels. Moreover, considering the plasticity and multipotentiality of their transmitter phenotype, cultured adult DRG neurons might represent an interesting material for autografts into the injured central nervous system.     

Neuronal and non-neuronal cell populations of the avian dorsal root ganglia express muscarinic acetylcholine receptors

The distribution of muscarinic acetylcholine receptors was investigated by immuno-light and electron microscopy in the chick dorsal root ganglion during embryonic development (E12 and E18) and after hatching. The monoclonal antibody we used recognizes the acetylcholine binding site shared by all five muscarinic acetylcholine receptor subtypes. At E12, light microscopy reveals several immunopositive neurons with variable degrees of immunolabeling, heterogeneously distributed throughout the ganglion. Later in development and after hatching, the intensity of immunolabeling seems to decrease and the immunopositive neurons, of the small-medium-sized type, are located mostly in the medio-dorsal region of the ganglion. Under the electron microscope, the immunoreaction is associated with the Nissl bodies, budding Golgi cisterns and, especially at E12, with discrete loci along the neuronal plasma membrane. Unmyelinated nerve fibers, in both central and peripheral branches, are also immunopositive, suggesting that muscarinic acetylcholine receptors are transported towards the spinal cord and the periphery, respectively. A large number of perineuronal satellite cells and both myelinating and unmyelinating Schwann cells are intensely labeled. These observations, combined with previous data on the pharmacological and functional characterization of muscarinic acetylcholine receptors in the avian dorsal root ganglion, suggest that both sensory neurons and non-neuronal cells are able to respond to acetylcholine stimuli. Since muscarinic acetylcholine receptor-immunoreactivity is restricted to the small-medium-sized neurons and their unmyelinated fibers, of the nociceptive type, we suggest that these receptors are involved in modulating the transduction of noxious stimuli from the periphery.     

Purification and culture of adult rat dorsal root ganglia neurons

To study the trophic requirements of adult rat dorsal root ganglia neurons (DRG) in vitro, we developed a purification procedure that yields highly enriched neuronal cultures. Forty to fifty ganglia are dissected from the spinal column of an adult rat. After enzymatic and mechanical dissociation of the ganglia, myelin debris are eliminated by centrifugation on a Percoll gradient. The resulting cell suspension is layered onto a nylon mesh with a pore size of 10 microns. Most of the neurons, the diameter of which ranged from 17 microns to greater than 100 microns, are retained on the upper surface of the sieve; most of the non-neuronal cells with a caliber of less than 10 microns after trypsinization go through it. Recovery of neurons is achieved by reversing the mesh onto a Petri dish containing culture medium. Neurons to non-neurons ratio is 1 to 10 in the initial cell suspension and 1 to 1 after separation. When these purified neurons are seeded at a density of 3,000 neurons/cm2 in 6 mm polyornithine-laminin (PORN-LAM) coated wells, neuronal survival (assessed by the ability to extend neurites), measured after 48 hr of culture, is very low (from 0 to 16%). Addition of nerve growth factor (NGF) does not improve neuronal survival. However, when neurons are cultured in the presence of medium conditioned (CM) by astrocytes or Schwann cells, 60-80% of the seeded, dye-excluding neurons survive. So, purified adult DRG neurons require for their short-term survival and regeneration in culture, a trophic support that is present in conditioned medium from PNS or CNS glia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurogenesis of subpopulations of rat lumbar dorsal root ganglion neurons including neurons projecting to the dorsal column nuclei

The time of birth of subpopulations of dorsal root ganglion (DRG) neurons was studied with immunohistochemistry for 5-bromodeoxyuridine (BrdU). Pregnant rats were injected with BrdU i.p. to label the neurons on one of the embryonic days (E) E11-E16. When they were adults, the rats were given injections of Fluoro-Gold (FG) into the gracile nucleus to identify DRG neurons projecting to this structure. Following a 5 day survival period, the animals were perfused with aldehyde fixative. Sections from the L3-L5 DRGs were processed for BrdU immunohistochemistry followed by either immunostaining for the antineurofilament antibody RT97, as marker of the light neuronal subpopulation, or histochemical staining for the B4 isolectin from Griffonia simplicifolia I, as marker of the small dark subpopulation. The results indicated that the DRG neurons were generated between E12 and E16. The RT97⁺ neurons were generated on E12–E15, with a peak at E13. FG⁺ neurons, the majority of which were RT97⁺, were also generated on E12–E15. The B4⁺ neurons were generated on E13–E16, with a peak around E14. The overall pattern of neurogenesis of the DRG neurons showed that the RT97⁺ neurons were produced prior to the B4⁺ neurons. These findings are in agreement with earlier observations that the large DRG neurons are generated earlier than the small dark neurons. Our findings also suggest the existence of a third neuronal subpopulation that might be produced at the latest period of DRG neurogenesis at E15–E16. © 1996 Wiley-Liss, Inc.     

Estradiol attenuates the adenosine triphosphate-induced increase of intracellular calcium through group II metabotropic glutamate receptors in rat dorsal root ganglion neurons

Estradiol attenuates the ATP-induced increase of intracellular calcium concentration ([Ca(2+)](i)) in rat dorsal root ganglion (DRG) neurons by blocking the L-type voltage gated calcium channel (VGCC). Because ATP is a putative nociceptive signal, this action may indicate a site of estradiol regulation of pain. In other neurons, 17β-estradiol (E(2)) has been shown to modulate L-type VGCC through a membrane estrogen receptor-group II metabotropic glutamate receptor (mGluR(2/3)). The present study investigated whether the rapid estradiol attenuation of the ATP-induced increase in [Ca(2+)](i) requires mGluR(2/3). Previously we showed that DRG (L(1)-S(3)) express ERα, P2X(3), and mGluR(2/3) receptors. DRG were acutely dissociated by enzyme digestion and grown in short-term culture for imaging analysis. DRG neurons were stimulated twice, once with ATP (50 μM) for 5 sec and then again in the presence of E(2) (100 nM) or E(2) (100 nM) + LY341495 (100 nM), an mGluR(2/3) inhibitor. ATP induced a transient increase in [Ca(2+)](i) (216.3 ± 41.2 nM). This transient increase could be evoked several times in the same DRG neurons if separated by a 5-min washout. Treatment with estradiol significantly attenuated the ATP-induced [Ca(2+)](i) increase in 60% of the DRG neurons, to 163.3 ± 20.9 nM (P < 0.001). Coapplication of E(2) and the mGluR(2/3) inhibitor LY341495 blocked the 17β-estradiol attenuation of the ATP-induced [Ca(2+) ](i) transient (209.1 ± 32.2 nM, P > 0.05). These data indicate that the rapid action of E(2) in DRG neurons is dependent on mGluR(2/3) and demonstrate that membrane estrogen receptor-α-initiated signaling involves interaction with mGluRs.     

Colocalization of metabotropic glutamate receptors in rat dorsal root ganglion cells

Glutamate is the main excitatory transmitter in both central and peripheral nervous systems. Discovery of metabotropic glutamate receptors (mGluRs) made it clear that glutamate can have excitatory or inhibitory effects on neuronal function, with group I mGluRs enhancing cell excitability but group II and III mGluRs decreasing excitability. The present study investigated the colocalization of mGluR subtypes representing groups I, II, or III in rat L5 dorsal root ganglion (DRG) cells. The analyses show that group III has the highest expression, with 75.0% of DRG cells expressing mGluR8, followed by group II, with 51.6% expressing mGluR2/3, followed by group I, with only 6.8% expressing mGluR1alpha. mGluR8 is expressed by small, medium, and large diameter cells. In contrast, mGluR1alpha and mGluR2/3 are expressed by mainly small and medium cells. Approximately half of cells expressing group I mGluR1alpha also express either group II mGluR2/3 or group III mGluR8. These mGluR1alpha double-labeled populations are not likely to overlap since >1.0% of mGluR1alpha are triple-labeled. As expected from the high percentage of single-labeled mGluR2/3 and mGluR8 cells, there is a considerable population of double-labeled cells with approximately 30% of each population expressing both receptors. Due to the fact that the number of mGluR1alpha-expressing cells in the DRG is low, the percentage of triple-labeled cells is also low ( approximately 1-2%). The prevalence of groups II and III indicate that glutamate could have a substantial inhibitory effect of primary afferent function, reducing and/or fine-tuning sensory input before transmission to the spinal cord. These anatomical data highlight the potential inhibitory role glutamate may play in peripheral sensory transmission.     

Adult dorsal root ganglia sensory neurons express the early neuronal fate marker doublecortin

It has been widely accepted that doublecortin (DCX) may represent a neuronal fate marker transiently expressed by immature neurons during development of the central and peripheral nervous tissue and in neurogenic areas of the adult brain. Previous work described the presence of DCX in the developing dorsal root ganglia (DRG), structures of the peripheral nervous system originating from the neural crest, but no information is available on its expression in adulthood. To this purpose, we have performed an immunohistochemical and biochemical analysis for DCX expression in DRG from adult male mice and rats. To our surprise, we demonstrated that the majority of DRG neurons do express DCX, both in somata and in fibers. DCX(+) cells have been characterized morphologically and phenotypically with well-established markers of DRG neuronal subpopulations. A large number of DCX(+) cells belong to the small and medium-sized nociceptive neurons. Additionally, DCX immunoreactivity is present in the spinal cord dorsal horns, the projection area of DRG neurons. The novel and unexpected localization for DCX protein opens up new, interesting vistas on the functional role of this protein in mature neurons and in particular in sensory neurons.     

Serotoninergic dorsal raphe neurons possess functional postsynaptic nicotinic acetylcholine receptors

Very few neurons in the telencephalon have been shown to express functional postsynaptic nicotinic acetylcholine receptors (nAChRs), among them, the noradrenergic and dopaminergic neurons. However, there is no evidence for postsynaptic nAChRs on serotonergic neurons. In this study, we asked if functional nAChRs are present in serotonergic (5-HT) and nonserotonergic (non-5-HT) neurons of the dorsal raphe nucleus (DRN). In rat midbrain slices, field stimulation at the tegmental pedunculopontine (PPT) nucleus evoked postsynaptic currents (eEPSCs) with different components in DRN neurons. After blocking the glutamatergic and GABAergic components, the remaining eEPSCs were blocked by mecamylamine and reduced by either the selective alpha7 nAChR antagonist methyllycaconitine (MLA) or the selective alpha4beta2 nAChR antagonist dihydro-beta-eritroidine (DHbetaE). Simultaneous addition of MLA and DHbetaE blocked all eEPSCs. Integrity of the PPT-DRN pathway was assessed by both anterograde biocytin tracing and antidromic stimulation from the DRN. Inward currents evoked by the direct application of acetylcholine (ACh), in the presence of atropine and tetrodotoxin, consisted of two kinetically different currents: one was blocked by MLA and the other by DHbetaE; in both 5-HT and non-5-HT DR neurons. Analysis of spontaneous (sEPSCs) and evoked (eEPSCs) synaptic events led to the conclusion that nAChRs were located at the postsynaptic membrane. The possible implications of these newly described nAChRs in various physiological processes and behavioral events, such as the wake-sleep cycle, are discussed.     

Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: Effects of NGF or prior peripheral axotomy

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increase in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA. © 1994 Wiley-Liss, Inc.     

Aberrant trafficking of the high-affinity choline transporter in AP-3-deficient mice

The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha . In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.     

Epibatidine, a nicotinic acetylcholine receptor agonist, inhibits the capsaicin response in dorsal root ganglion neurons

In patch-clamp recordings from small-medium diameter dorsal root ganglion neurons in culture, (+/-)-epibatidine (1 microM) was able to inhibit the capsaicin response (IC(50)=0.32 microM) in neurons where there was no detectable direct nicotinic response. Thus, (+/-)-epibatidine may inhibit the vanilloid receptor in a manner that is not dependent upon nicotinic current activation, representing another mechanism by which such ligands could modulate vanilloid receptor signaling.     

Secretory pathways of neuropeptides in rat lumbar dorsal root ganglion neurons and effects of peripheral axotomy

Using immunocytochemistry combined with confocal and electron microscopy, the secretory pathways related to substance P (SP), calcitonin gene-related peptide (CGRP), galanin (GAL), and neuropeptide Y (NPY) were investigated in neurons in rat lumbar (L) 4 and L5 dorsal root ganglia (DRGs) before and after peripheral axotomy. All four peptides were processed through the regulated secretory pathway in many small neurons in normal DRGs, and CGRP through this pathway also in some large neurons. In many small neurons, two neuropeptides could be sorted into the same or separate large dense-core vesicles (LDCVs). The LDCVs had a significantly larger diameter in small as compared to large DRG neurons. Fourteen days after sciatic nerve cut, the levels of SP- and CGRP-like immunoreactivities (-LIs) and the number of LDCVs containing these peptides were markedly reduced, but SP- and CGRP-LIs were still seen in the regulated pathway. GAL-LI was markedly increased in many small neurons and some large neurons and NPY-LI mainly in large neurons. Both peptides were particularly abundant in the Golgi region. In small neurons, the number of LDCVs containing GAL- or NPY-LI was increased, but did not appear to reach the numbers containing SP- or CGRP-LI in normal DRG neurons. After axotomy, CGRP-LI and GAL-LI were often in separate LDCVs. One type of NPY-positive large neurons showed budding off of LDCVs after axotomy, but also some “scattered” labeling in the cytoplasm. In the second type, NPY-LI was mainly found in multivesicular bodies. In several myelinated nerve fibers a “diffuse” distribution of NPY was seen together with some LDCVs containing NPY-LI. In contrast, in unmyelinated nerve fibers, NPY-, GAL-, SP-, and CGRP-LIs were always observed in LDCVs. Thus, both in normal and axotomized DRG neurons, peptides are processed through the regulated pathway. However, in some large neurons, NPY is, in addition, secreted through the constitutive pathway, perhaps as a consequence of limited sorting mechanisms for NPY, i.e., the plasticity of the secretory mechanisms does not match the rate of peptide synthesis after axotomy. © 1995 Wiley-Liss, Inc.     

Brain vesicular acetylcholine transporter in human users of drugs of abuse

Limited animal data suggest that the dopaminergic neurotoxin methamphetamine is not toxic to brain (striatal) cholinergic neurons. However, we previously reported that activity of choline acetyltransferase (ChAT), the cholinergic marker synthetic enzyme, can be very low in brain of some human high-dose methamphetamine users. We measured, by quantitative immunoblotting, concentrations of a second cholinergic marker, the vesicular acetylcholine transporter (VAChT), considered to be a "stable" marker of cholinergic neurons, in autopsied brain (caudate, hippocampus) of chronic users of methamphetamine and, for comparison, in brain of users of cocaine, heroin, and matched controls. Western blot analyses showed normal levels of VAChT immunoreactivity in hippocampus of all drug user groups, whereas in the dopamine-rich caudate VAChT levels were selectively elevated (+48%) in the methamphetamine group, including the three high-dose methamphetamine users who had severely reduced ChAT activity. To the extent that cholinergic neuron integrity can be inferred from VAChT concentration, our data suggest that methamphetamine does not cause loss of striatal cholinergic neurons, but might damage/downregulate brain ChAT in some high-dose users. However, the finding of increased VAChT levels suggests that brain VAChT concentration might be subject to up- and downregulation as part of a compensatory process to maintain homeostasis of neuronal cholinergic activity. This possibility should be taken into account when utilizing VAChT as a neuroimaging outcome marker for cholinergic neuron number in human studies.