Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory‐related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High‐affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate‐limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well‐documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide‐based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK‐N‐SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease. Curr. Protoc. Neurosci . 60:7.23.1‐7.23.16. © 2012 by John Wiley & Sons, Inc.

Cholinergic neurons are a major constituent of the mammalian central nervous system. Acetylcholine, the neurotransmitter used by cholinergic neurons, is synthesized from choline and acetyl CoA by the enzymatic action of choline acetyltransferase (ChAT). The transport of choline into the cholinergic neurons, which results in synthesis of ACh, is hemicholinium-sensitive and is referred to as high-affinity choline uptake (HACU). Thus, the formation of acetylcholine in cholinergic neurons largely depends on both the levels of choline being transported into the cells from the extracellular space and the activity of ChAT. Several methods were described previously to measure HACU and ChAT simultaneously in synaptosomes, but the same for cultured cells is lacking. We describe a procedure to measure HACU and ChAT at the same time in cultured cells by simple techniques employing radionuclides. In this procedure, we determined quantitatively hemicholinium-sensitive choline uptake and ChAT enzyme activity in a small number of differentiated human neuroblastoma (SK-N-SH) cells. We also determined the kinetics of choline uptake in the SK-N-SH cells. We believe that these simple methods can be used for neurochemical and drug discovery studies in several models of neurodegenerative disorders including Alzheimer's disease.

Previous microarray data indicates that EtOH administered by self-infusion into the VTA alters gene expression in the nucleus accumbens (Acb), genes associated with synaptic plasticity, neurite outgrowth, and dendritic spine formation. The objective of this study was to examine the effect of intracranial self-administration (ICSA) of EtOH in the VTA on protein expression in the Acb. Adult Wistar rats were surgically implanted with guide cannulae aimed at the posterior VTA and self-administered 150 mg% EtOH for 7 consecutive sessions after which the Acb was dissected and homogenized. Synaptosomes were prepared from pooled samples via discontinuous sucrose gradient centrifugation, and proteins in the enriched samples were subjected to trypsin digestion. Tryptic peptides from each sample were analyzed serially via linear ion-trap LC-MS/MS. Proteins were identified via SEQUEST and X!Tandem. Protein quantification was based on total ion chromatograms whereby the integral volume under each selected peptide peak was measured, normalized, and compared for relative abundance. In this study, 1120 proteins were identified and quantified. The identities of significantly altered proteins will be presented in terms of the functional significance of their EtOH-mediated changes. Supported by NIH Grant R21AA015698-01.

The phenotypic effect of theweaver mutation in the ventral midbrain of homozygous mutants is associated with the progressive loss of dopaminergic neurons. To discover whether the number of mesencephalic dopaminergic cells is altered in weaver heterozygotes (wv/+), we studied mice between 20 and 365 days of age. We counted tyrosine hydroxylase (TH)-immunopositive cells in the substantia nigra (SN), retrorubral nucleus (RRN), and ventral tegmental area (VTA), and measured cross-sectional areas of neuronal somata in the SN ofwv/+ and age-matched wild-type controls (+/+). The number of TH-positive cells in thewv+ ventral midbrain was on average 13% lower than normal. Cell loss was detected selectively in the SN (12%) and VTA (23%). The areas of somatic profiles in thewv/+ nigral neurons were on average reduced by 9.8%. The neuronal losses in the SN and VTA correlated with a 13.8% reduction in dopamine level in the ventral striatum inwv/+ mice at 14–16 months of age. Our findings imply that a single dose of theweaver gene in the mouse is associated with cellular damage leading to a chronic deficiency in the mesostriatal dopaminergic system.

Past research has indicated that chronic ethanol exposure enhances dopamine (DA) neurotransmission in several brain regions. The present study examined the effects of chronic ethanol drinking on dopamine transporter (DAT) function in the nucleus accumbens (Acb) of High-Alcohol-Drinking replicate line 1 (HAD-1) rats. HAD rats were given concurrent 24-h access to 15% ethanol and water or water alone for 8 weeks. Subsequently, DA uptake and the Vmax of the DAT were compared between the two groups using homogenates of the nucleus accumbens. DA uptake was measured following a 2min incubation at 37°C in the presence of 8nM [3H]DA. For kinetic analyses, DA uptake was assessed in the presence of 5 concentrations of [3H]DA ranging from 8nM to 500nM. Analyses of the data revealed a significant increase in DA uptake in the ethanol group compared to water controls. Kinetic analyses revealed the change in DA uptake to be a consequence of an increase in the Vmax of transport. These findings demonstrate that chronic free-choice oral ethanol consumption in HAD-1 female rats increases DA uptake in the Acb by increasing the Vmax of the transporter. However, it is not known whether the ethanol-induced change in Vmax is caused by differences in the actual number of available transporter sites or from a difference in the velocity of operation of a similar number of transporters. Overall, the data indicate that chronic ethanol consumption by HAD-1 rats produces prolonged neuroadaptations within the mesolimbic DA system, which may be important for the understanding of the neurobiological basis of alcoholism.

Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia. are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly > 900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LCMS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin 1, 11, and V, VAMP-2, syntaxin 1A and 1 B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.

Renal blood flow falls in both kidneys following delivery of a clinical dose of shockwaves (SW) (2000 SW, 24 kV, Dornier HM3) to only one kidney. The role of renal nerves in this response was examined in a porcine model of renal denervation. Six-week-old pigs underwent unilateral renal denervation. Nerves along the renal artery of one kidney were identified, sectioned and painted with 10% phenol. Two weeks later the pigs were anesthetized and baseline renal function was determined using inulin and PAH clearances. Animals then had either sham-shockwave lithotripsy (SWL) (group 1), SWL to the innervated kidney (group 2) or SWL to the denervated kidney (group 3). Bilateral renal function was again measured 1 and 4 h after SWL. Both kidneys were then removed for analysis of norepinephrine content to validate the denervation. Renal plasma (RPF) flow was significantly reduced in shocked innervated kidneys (group 2) and shocked denervated kidneys (group 3). RPF was not reduced in the unshocked denervated kidneys of group 2. These observations suggest that renal nerves play a pivotal role in modulating the vascular response of the contralateral unshocked kidney to SWL, but only a partial role, if any, in modulating that response in the shocked kidney.

Treating one kidney with high-energy acoustic shock waves reduces blood flow 65% in that kidney and 33% in the contralateral unshocked kidney. We tested the hypothesis that renal nerves were involved in this response. Six-week old pigs underwent unilateral renal denervation. Nerves along the renal artery of one kidney were cut and the artery was painted with 10% phenol. After 2 weeks, the pigs were anesthetized and bilateral renal function was determined using inulin and PAH (para-aminohippurate) clearance. Glomerular filtration rate (GFR, inulin clearance) and renal blood flow (PAH clearance) were measured both before and after lithotripsy to the lower pole of the innervated kidney (2000 shocks, 24 kV, unmodified Dornier HM-3) or sham lithotripsy. Both kidneys were then removed to measure norepinephrine content in the tissue. Norepinephrine levels were significantly reduced (more than 90%) in denervated kidneys indicating complete denervation. As expected, blood flow and GFR fell (approximately 50%) after lithotripsy in the innervated shocked kidneys, but did not fall significantly in denervated contralateral kidneys or in sham lithotripsy animals. Results suggest that renal nerves mediate the fall in contralateral renal function after lithotripsy. [Work supported by NIH P01 DK43881.]

The most prominent structural feature of the G protein-coupled receptor superfamily is their seven hydrophobic domains, which are postulated to form membrane-spanning alpha helices, Some members of the G protein-coupled receptor family, specifically several serotonin (5-HT) receptors, possess eight hydrophobic domains. The importance of this extra hydrophobic domain, located at the N terminus of the receptor, is unknown. This question was addressed by deleting the extra hydrophobic region from the 5-HT2C receptor and comparing its function and topology with those of the wild-type receptor. Immunofluorescence microscopy was used to determine the location of the N terminus of the epitope-tagged wild-type and mutant receptors, The N terminus of both receptors was extracellular, suggesting that the extra hydrophobic domain does not change the topology of this receptor and is unlikely to be a membrane-spanning alpha helix. Radioligand-binding studies in transfected cells and expression studies in Xenopus oocytes demonstrated that seven hydrophobic domains were sufficient for normal function in these assays. Interestingly, the mutant receptor, now containing seven hydrophobic domains, is expressed at higher levels in transfected cells than the wild-type receptor containing eight hydrophobic domains, suggesting that the extra hydrophobic domain does impact the activity of this receptor by regulating its expression.

The present investigation was undertaken to test the hypothesis that a reduction in the activity of protein tyrosine kinases would result in an alteration in dopamine transport. Genistein, a broad-spectrum inhibitor of protein tyrosine kinases, inhibited dopamine uptake into mouse striatal homogenates with an IC50 of 18 μM. The inhibition by genistein was rapid, reversible and somewhat selective, in that genistein did not inhibit the uptake of choline or GABA under similar conditions. Kinetic analyses indicated that genistein was a non-competitive inhibitor. Another protein tyrosine kinase inhibitor, tyrphostin 23, also inhibited transport but was significantly less potent than genistein. Tyrphostin 25 and lavendustin A were without major effect on dopamine uptake. In addition, the inactive structural analog of genistein, genistin, had no significant effect on dopamine uptake. The inhibition of dopamine transport by 50 μM genistein was accompanied by a reduction in the level of a 110-kDa band of tyrosine phosphoprotein. It is suggested that protein tyrosine kinases play a role in the cascade of events which ultimately lead to regulation of neuronal dopamine transport.

The cholinergic septohippocampal system plays an important role in spatial learning and memory functions. Transections of the septohippocampal pathway have been shown to result in a near complete loss of cholinergic innervation in the hippocampus and induce severe spatial memory impairments. In this article, we have reviewed the studies which demonstrate the ability of intrahippocampal septal grafts to reinnervate the hippocampal formation and ameliorate spatial learning and memory deficits. Neuroanatomical studies suggest that grafts of cholinergic tissue can innervate the host hippocampal formation in a pattern that mimics that of the normal septohippocampal pathway. This innervation, in turn, is associated with the formation of graft-to-host synaptic connections. Neurochemical studies reveal that intrahippocampal grafts of septal cells can restore choline acetyltransferase activity, acetylcholine synthesis, and high affinity choline uptake in presynaptic terminals of grafted neurons. In addition, these grafts can normalize the upregulation of cholinergic muscarinic receptors seen postsynaptically in the hippocampus following lesions of the septohippocampal pathway. The functional nature of these grafts is also substantiated by electrophysiological recordings which demonstrate stimulus-evoked graft-to-host synaptic transmission as well as the reinstatement of EEG activity typical of septohippocampal connectivity. In addition to graft-to-host connections, behavioral and neurochemical studies also provide evidence for host-to-graft connections that can regulate the activity of grafted cholinergic neurons during the performance of specific behavioral tasks requiring spatial memory function. Together, these studies suggest that grafts of cholinergic neurons from the medial septal nucleus can become anatomically and functionally incorporated into the circuitry of the host hippocampal formation.

The ability of the 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide to bind to the dopamine transporter and inhibit [3H]dopamine uptake was investigated in rat brain synaptosomes from the nucleus accumbens and caudate putamen. Competitive displacement experiments showed that 1-(m-chlorophenyl)-biguanide inhibited the binding of [3H]GBR-12935 in a biphasic manner (IC50 values of 0.4 and 2.0 μM [high affinity] and 34.8 and 52.7 μM [low affinity] for caudate putamen and nucleus accumbens, respectively), and the high affinity binding site differed between brain regions. Serotonin was ineffective at competing for [3H]GBR-12935 binding, while the selective 5-HT3 receptor antagonist ICS 205–930 exhibited an IC50 > 100 μM. The maximum density of [3H]GBR-12935 binding sites was more than two-fold greater in the caudate putamen than in the nucleus accumbens (6.9 vs. 2.7 pmol/mg protein), and KD values were similar (4.7 and 4.2 nM). 1-(m-chlorophenyl)-biguanide was able to inhibit [3H]dopamine uptake into synaptosomes of both brain regions, however it was significantly more potent in the caudate putamen (IC50: 5.1 vs. 6.5 μM). The results demonstrate that some of the reported dopamine releasing effects of 1-(m-chlorophenyl)-biguanide may be due in part to activity at the dopamine transporter, and further suggest a possible difference in dopamine uptake parameters between the caudate putamen and nucleus accumbens.

In addition to an altered dopaminergic input, the striatum of the weaver mutant mouse (wv/wv) has increased serotonin tissue content and uptake compared to the wild-type mouse (+/+). To gain information regarding the functional status of serotonergic inputs to the wv/wv striatum, endogenous serotonin release from wv/wv and +/+ striatum was measured under basal conditions as well as in the presence of fenfluramine or elevated concentrations of potassium (K+). Fractional basal release of serotonin from the +/+ striatum was significantly greater than that from the wv/wv striatum. In the presence of K+, evoked release (stimulated release minus basal release) was greater from the +/+ striatum than from the wv/wv striatum. In the presence of fenfluramine, evoked serotonin release was greater from the wv/wv striatum compared to the +/+ striatum. These data are consistent with the involvement of an additional transmitter(s) in modulating serotonin release to a greater extent in the wv/wv than the +/+ striatum. The data on fenfluramine-stimulated serotonin release suggest that the additional serotonin content found in the wv/wv striatum is in a releasable pool but that striatal serotonin release might be attenuated more in wv/wv than in +/+ mice.

Abstract: The weaver mutant mouse (wv/wv) has an ∼70% loss of nigrostriatal dopamine (DA) neurons, but the fractional DA release evoked by amphetamine (but not a high potassium level) has been shown to be greater from striatal slices of the weaver compared with +/+ mice. In the present work we tested the hypothesis that fractional DA release from weaver striatum would be greater when release was mediated by the DA transporter. Serotonin (5‐HT)‐stimulated fractional DA release was greater from weaver than from +/+ striatum. The release evoked by 5‐HT in the presence of 10 µM nomifensine (an antagonist of the DA transporter) was less than in its absence, but the difference between weaver and +/+ striatum remained. In the presence of nomifensine, 1‐(m‐chlorophenyl)biguanide, classified as a 5‐HT3 agonist, also induced a greater fractional release from weaver compared with +/+ striatum. When veratridine was used at a low concentration (1 µM), the fractional evoked release of DA was higher from the weaver in the presence and absence of nomifensine. These findings suggest that the reason for the difference in the responsiveness of the two genotypes to these release‐inducing agents is not related to DA transporter function.

The cholinergic projections from basal forebrain nuclei to the retrosplenial cortex (RSC) have previously been studied using a variety of histological approaches. Studies using acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) immunocytochemistry have demonstrated that this projection travels via the cingulum on route to the RSC. Preliminary studies from our laboratory, however, have shown that the fornix may also be involved in this projection. The present study uses the combination of pathway lesions, and the analysis of cholinergic neurochemical markers in the RSC to determine the role of the fornix in the cholinergic projection to the RSC. High affinity choline uptake (HACU) and ChAT activity were measured in the RSC of control rats, animals with cingulate lesions, and animals with fornix plus cingulate lesions. Fornix plus cingulate lesions resulted in significant deceases in HACU and ChAT activity in comparison to cingulate lesions alone. Muscarinic receptor binding was also evaluated in combination with the various lesions, and a significant increase in retrosplenial receptor binding was noted following fornix lesions. Together, these results support the concept of a fornix-mediated cholinergic pathway to the RSC.

The dopamine (DA) uptake system was investigated in the mesostriatal system of normal and weaver mutant mice, which lose mesencephalic DA neurons, as well as in weaver mutants with ventral mesencephalic grafts to the striatum. Assays of [3H]DA uptake in striatal synaptosomal fractions in vitro and autoradiography of [3H]mazindol binding in brain sections were carried out in wild-type mice (+/+) and in the two hemispheres of homozygous weaver mutants (wv/wv) that had received unilateral grafts of mesencephalic cell suspensions to the right side. Net [3H]DA uptake, expressed as pmol/mg-protein/2-min, was on the average 50.6 in the striatum of wild-type mice, 7.9 in the non-grafted, and 10.1 in the transplanted striatum of weaver mutants. [3]DA uptake in wild-type mice differed significantly from both the grafted and non-grafted weaver striata (P<0.001). Paired comparisons for [3H]DA uptake between right and left sides of recipient weaver mice showed a significant side effect (P<0.02), the right side being 28–38% higher than the left side [mean of all individual (R-L)/L values]. The results of amphetamine-induced turning behavior tests were compared with the biochemical findings. Mice with grafts to the right side rotated an average of 22 turns to the left and 7 turns to the right during the five one-minute sessions; the mean value L/(L+R) was 64%. A plot of (L-R) rotations against (R-L) [3H]DA uptake gave a correlation coefficient of 0.552 (P<0.05), indicating that animals with a strong rotational bias to the left tended to have higher [3H]DA on the right. Similarly, the animals that were used for [3H]mazindol binding autoradiographic studies displayed on the average 72% rotations to the left side. In the [3H]mazindol binding data, non-grafted weaver mutants showed the severest depletion relative to wild-type in the dorsomedial and dorsolateral caudate-putamen (86% and 87%, respectively). Mice with unilateral grafts to the right side showed an increase in [3H]mazindol binding signal in the transplanted side of 40–64% (depending on dorsoventral topography) over the contralateral, non-grafted side. These findings attest to the functional effects of the grafts at the anatomical, biochemical, and behavioral levels. The parallel measurements of motor performance and DA uptake in the same animals offers an index of behavioral recovery as a function of transmitter-related activity. Furthermore, by conducting measurements of the synaptosomal DA uptake in vitro and of the binding characteristics of mazindol in brain slices by autoradiography, one has the advantage of combining the anatomical resolution of uptake site visualization with a dynamic indicator of function for DA uptake in the nerve terminal.

Mice of different ages and homozygous or heterozygous for the weaver gene ( wv ) were used to study the time course for the effect of the weaver gene on several striatal dopaminergic parameters. Dopamine uptake was decreased in the homozygous weaver at all ages examined. The deficit in uptake at the earliest age studied, postnatal day 3, was approximately 50% and increased to greater than 70% at older ages. In control mice, dopamine uptake reached a maximum by postnatal day 22, but in homozygous weaver mice, development of uptake activity was curtailed by postnatal day 7. Dopamine content and tyrosine hydroxylase activity were significantly decreased in the homozygous weaver at all ages studied except postnatal days 7 and 10. The magnitude of the deficit in dopamine content ranged from approximately 40% at postnatal days 3 and 5 to about 70% in adults (6 months to 1 year of age). The magnitude of the deficit in tyrosine hydroxylase activity ranged from 40 to 70%. In general, no major differences between heterozygotes and controls were observed for any of the dopaminergic parameters investigated. The results of the present investigation indicate that neurochemical alterations can be observed in the striata of weaver mice as early as postnatal day 3 and raise the possibility that the striatal dopamine transporter may be an early target of the weaver mutation.

The norepinephrine (NE), dopamine (DA), and serotonin (5-HT) systems are thought to be important in the development of anxiety and stress. The dorsomedial hypothalamus (DMH) of rats has been implicated in the regulation of physiological and behavioral responses associated with fear and anxiety. In order to elucidate the interactions between the monoamine systems and the DMH, we studied the effects of subjecting rats to the fear-potentiated startle test, a commonly used test of anxiety in rats, on the NE, DA, and 5-HT levels in the DMH. Rats in the potentiated startle test, but not those exposed to just foot shocks or acoustic startle, showed significantly higher levels of NE and DA in the DMH compared to cage controls. In contrast, foot shocks significantly elevated the 5-HT levels in the DMH.