Evidence to suggest that extracellular acetate is accumulated by rat hippocampal cholinergic nerve terminals for acetylcholine formation and release.

It is well established that extracellular choline is transported into central cholinergic nerve terminals by 'high' and 'low' affinity processes to form the neurotransmitter acetylcholine (ACh). The intent of the present investigation was to ascertain whether extracellular acetate might also be transported into central cholinergic nerve terminals to form ACh. To test this possibility, rat hippocampal tissue was incubated with varying concentrations of extracellular [1-(14)C]acetate (0.1-100 microM) and the uptake of [1-(14)C]acetate and the amount of [14C]ACh formed by the tissue determined. The results indicated that the uptake of extracellular [1-(14)C]acetate was temperature-dependent and saturable having an apparent Michaelis constant (Km) of 22 microM. The formation of [14C]ACh in the tissue as a function of extracellular [1-(14)C]acetate appeared to occur by both 'high' and 'low' affinity processes with apparent Km values of 0.5 and 19.6 microM, respectively. In other experiments, three inhibitors (lithium, allicin and sodium) of acetyl CoA synthetase (EC 6.2.1.1 acetate: CoA ligase), the enzyme which converts acetate to acetyl CoA when ATP and CoA are present, inhibited [1-(14)C]acetate uptake and the amount of [14C]ACh formed from that [1-(14)C]acetate. Additionally, vesamicol, an inhibitor of ACh transport into synaptic vesicles, blocked the filling of a synaptic vesicle-enriched fraction of hippocampal tissue with newly synthesized [14C]ACh formed from extracellular [1-(14)C]acetate. High K+ depolarization of hippocampal tissue loaded with extracellular [1-(14)C]acetate not only increased the synthesis but also the release of [14C]ACh. These results suggest that extracellular acetate is recycled by rat hippocampal cholinergic nerve terminals for the formation and release of ACh. They also suggest that the enzyme acetyl CoA synthetase mediates extracellular acetate uptake into hippocampal cholinergic nerve terminals by metabolizing it to acetyl CoA and thereby creating a diffusion gradient for it to follow.

Evidence to suggest that cytosolic acetylcholine in rat hippocampal nerve terminals is not directly transferred into synaptic vesicles for release.

Rat hippocampal minces were loaded with [acetyl 1-14C]acetylcholine ([14C]ACh) in the presence of the "poorly penetrating" acetylcholinesterase (EC 3.1.1.7; AChE) inhibitor echothiophate and the effect of high K+ depolarization determined on the subcellular storage and release of [14C]ACh and its metabolites. Results indicated that high K+ did not augment the release of [14C]ACh. Rather, it increased the release of [14C]acetate while simultaneously reducing the level of [14C]ACh in the cytosolic (S3) fraction. When the identical experiment was performed with paraoxon, a "penetrating" AChE inhibitor, high K+ still did not increase the release of [14C]ACh. However, paraoxon prevented the K(+)-induced loss of [14C]ACh from the cytosolic fraction as well as the K(+)-induced gain of [14C]acetate in the release medium. When minces were loaded with [14C]ACh in the presence of echothiophate and subsequently subjected to high K+ depolarization in the absence or presence of vesamicol (AH5183; (-)-trans-2-[4-phenylpiperidino] cyclohexanol), a drug which blocks the refilling of synaptic vesicles with ACh, the amount of endogenous ACh released was reduced approximately 50%. Conversely, the amount of [14C]ACh released was not reduced at all. These results suggest that cytosolic ACh is not directly transported into synaptic vesicles for release when hippocampal nerve terminals are depolarized. Rather, its hydrolysis is accelerated in response to depolarization. A working hypothesis explaining the importance of the depolarization-induced breakdown of cytosolic ACh to central ACh metabolism is presented.

Membrane-bound choline-O-acetyltransferase in rat hippocampal tissue is associated with synaptic vesicles.

Some of the choline-O-acetyltransferase (EC 2.3.1.6; ChAT) in rat hippocampal nerve terminals is non-ionically associated with membranes. The intent of the present report was to ascertain whether any of this membrane-bound ChAT might be associated with synaptic vesicles. To test this possibility, synaptosomal (P2) fractions were hypo-osmotically shocked in water, salt washed to remove ionically-bound ChAT, subjected to sucrose density gradient centrifugation, and the activity of ChAT compared with the amount of occluded ACh in the various subcellular fractions. A peak of ChAT and occluded ACh occurred in that fraction of the gradient (0.4 M sucrose) acknowledged to be enriched in synaptic vesicles. In other experiments, Immunobeads coated with an antibody directed against the synaptic vesicle specific SV2 protein immunoprecipitated both ChAT and occluded ACh from the 0.4 M sucrose fraction, but no other fraction. Immunobeads coated with an anti-ChAT antiserum immunoprecipitated synaptophysin from the 0.4 M sucrose fraction, an effect which was blocked by pretreatment of the anti-ChAT Immunobeads with purified ChAT. These results suggest that some of the membrane-bound ChAT in rat hippocampal nerve terminals is associated with cholinergic synaptic vesicles.

Membrane-bound choline-O-acetyltransferase in rat hippocampal tissue is anchored by glycosyl-phosphatidylinositol.

In an earlier study, we presented evidence to suggest that some of the particulate choline-O-acetyltransferase (ChAT) in rat hippocampal tissue might be linked to membranes by a glycosyl-phosphatidylinositol (GPI) anchor. In the present report, we attempted to determine if any of this GPI-anchored ChAT might be intracellular. Internalization of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis into rat hippocampal synaptosomes by the DMSO (dimethyl sulfoxide) freeze/thawing procedure caused an increase in cytosolic and a decrease in membrane-bound ChAT. Incubation of a plasma membrane enriched subcellular fraction at 16 degrees C relative to 4 degrees C led to a conversion of the membrane-bound, amphiphilic ChAT into hydrophilic ChAT. This conversion was blocked by zinc, an inhibitor of GPI-PLC. The cytosolic fraction of ChAT immunoreacted on western blots with an antibody directed against the cross-reacting determinant (CRD) of the GPI anchor. We suggest that some of the membrane-bound ChAT in rat hippocampal tissue is GPI-anchored intracellularly; also, that an endogenous GPI-PLC-like enzyme acts to release it into the cytosol.

Effect of phospholipase C from Bacillus cereus on the release of membrane-bound choline-O-acetyltransferase from rat hippocampal tissue.

Some of the enzyme choline-O-acetyltransferase (ChAT) associated with central cholinergic nerve terminals appears to be non-ionically associated with membranes. In the present study, we tested the possibility that some membrane-bound ChAT might be anchored to membranes by a phosphatidylinositol linkage by incubating rat hippocampal tissue with phospholipase C (PLC) from Bacillus cereus. The PLC selectively augmented the release of ChAT; also, the glycosylphosphatidylinositol-PLC inhibitor, zinc, blocked this increase in release. When control and PLC-treated hippocampal tissues were subjected to Triton X-114 phase separation, a procedure that separates amphiphilic from hydrophilic proteins, the detergent-soluble, membrane-bound fraction of tissue ChAT appeared to be the source of the ChAT released by PLC into the incubation medium. Zinc also blocked the temperature-dependent release of ChAT, but not lactic dehydrogenase, from hippocampal tissue. Extracellular membrane-bound ChAT appeared to be the source of the ChAT released by a low exogenous concentration of PLC, as well as that released by a temperature-dependent process during tissue incubation. Phosphatidylinositol-specific PLC from Bacillus thuringiensis released ChAT, but not lactic dehydrogenase, from a crude synaptosomal fraction prepared from rat hippocampal tissue. These results suggest that some of the membrane-bound ChAT in rat hippocampal tissue may be extracellular and anchored to the membrane by phosphatidylinositol, and also that an endogenous factor in hippocampal tissue may function to remove this extracellular ChAT from the membrane.

Effect of 2-(4-phenylpiperidino)cyclohexanol (AH 5183) on the veratridine-induced increase in acetylcholine synthesis by rat hippocampal tissue.

The intent of this study was to determine whether the drug 2-(4-phenylpiperidino)cyclohexanol (AH 5183 or vesamicol) might inhibit the veratridine-induced increase in acetylcholine (ACh) synthesis by reducing the veratridine-induced activation of a detergent-soluble choline-O-acetyltransferase (EC 2.3.1.6; ChAT) fraction associated with a vesicle-bound store of ACh. When minces of rat hippocampal tissue were loaded with [14C]choline and subsequently depolarized with veratridine, an increase in the synthesis of [14C]ACh occurred that could be abolished by L-AH 5183 (75 nM). When minces were depolarized with veratridine in the presence of L-AH 5183 (75 nM), the depolarization-induced activation of a detergent-soluble ChAT fraction associated with a vesicle-bound store of ACh was blocked. Conversely, the veratridine-induced activation of a water-soluble ChAT fraction believed to be cytosolic was not. AH 5183 also blocked the repletion of the vesicle-bound store with newly synthesized ACh following veratridine-induced depletion of ACh, a result that appeared to be mediated by an effect on the synthesis of ACh at the vesicular surface. These results suggest that veratridine depolarization of rat hippocampal nerve terminals stimulates the synthesis of ACh by activating a detergent-soluble fraction of ChAT closely associated with synaptic vesicle release sites. ACh synthesis and transport at the vesicular surface may be influenced by a common AH 5183-sensitive regulatory protein.

Evidence to suggest that the spontaneous release of acetylcholine from rat hippocampal tissue is carrier-mediated.

The effect of L- and D-stereoisomers of 2-(4-phenylpiperidino) cyclohexanol (AH 5183) on the spontaneous release of acetylcholine (ACh) from rat hippocampal tissue was studied. L-AH 5183 was approximately 100 times more potent than was D-AH 5183 in reducing spontaneous ACh release. Spontaneous ACh release was also temperature dependent. These results may suggest that the spontaneous release of ACh from brain tissue is carrier-mediated.

Veratridine-induced activation of choline-O-acetyltransferase activity in rat hippocampal tissue: relationship to the veratridine-induced release of acetylcholine.

The effect of veratridine depolarization on the activity of 3 choline-O-acetyltransferase (ChAT) fractions in rat hippocampal tissue was investigated. Those concentrations of veratridine which augmented acetylcholine (ACh) release also increased the activity of water and detergent soluble ChAT fractions. These results may suggest that the depolarization induced release of ACh is linked to an activation of ChAT activity.

Veratridine-induced breakdown of cytosolic acetylcholine in rat hippocampal minces: an intraterminal form of acetylcholinesterase or choline O-acetyltransferase?

Rat hippocampal minces were loaded with N-methyl-[3H]acetylcholine ([3H]ACh) in the presence of the 'poorly penetrating' acetylcholinesterase (EC 3.1.1.7, AChE) inhibitor echothiophate and the effect of the depolarizing agent veratridine determined on the subcellular storage and release of [3H]ACh and [3H]choline. Results indicated that veratridine stimulated the release of [3H]ACh from a crude vesicular fraction (P3) by a Ca2+-dependent process, while simultaneously accelerating the breakdown of cytosolic (S3) [3H]ACh. A portion of the [3H]choline derived from the hydrolyzed S3 [3H]ACh was donated to the P3 fraction for [3H]ACh formation and release. When the identical experiment was done using hippocampal minces from septal lesioned rats, veratridine did not stimulate either the Ca2+-dependent release of [3H]ACh or the hydrolysis of cytosolic [3H]ACh. Incubation of control hippocampal minces with paraoxon, an AChE inhibitor which can penetrate cholinergic nerve terminals more rapidly than echothiophate, prevented veratridine from stimulating the Ca2+-dependent release of [3H]ACh from the P3 fraction. Instead, it then stimulated the Ca2+-independent release of [3H]ACh from the S3 fraction. When minces were incubated with the choline O-acetyltransferase (EC 2.3.1.6, ChAT) inhibitor 4-(1-naphthyl)vinyl pyridine (NVP), veratridine was no longer able to stimulate the Ca2+-dependent release of labelled ACh either. Instead, veratridine stimulated the Ca2+-independent release of labelled ACh from the S3 fraction. NVP also abolished the veratridine-induced, Ca2+-dependent release of total ACh. Both paraoxon and NVP inhibited the reversible reaction of ionically bound ChAT prepared from rat brain when tested in vitro, yet paraoxon was much less potent than NVP, and was unable to inhibit this reaction at the low concentration which prevented the veratridine induced breakdown of S3 [3H]ACh during mince incubation. Veratridine depolarization of hippocampal minces stimulated the activity of a membrane-bound fraction of ChAT associated with the P3 fraction, but this fraction of ChAT did not become more sensitive to inhibition by paraoxon during tissue incubation. Veratridine depolarization of minces also increased the activity of membrane-bound AChE, but this enzyme was not inhibited by the low NVP concentration which prevented the veratridine-induced breakdown of S3 [3H]ACh. The veratridine-induced increase in membrane-bound ChAT activity was dependent on the presence of extracellular Ca2+ in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunological, isoelectric, hydrophobic and molecular weight differences between soluble and ionically membrane-bound fractions of choline-o-acetyltransferase prepared from mouse and rat brain.

Choline-O-acetyltransferase (EC 2.3.1.6; ChAT) was prepared from synaptosomal fractions (P(2)) of mouse and rat brain in the presence of proteolytic inhibitors by the method of Gray and Whittaker (1962) as modified by (Salehmoghaddam and Collier, 1976). The P(2) fraction was hypo-osmotically shocked with glass distilled water and centrifuged to separate the cytoplasmic (S(3)) and vesicle-bound (P(3)) fractions. Fraction S(3) was saved for ChAT assay and compared with the ChAT fraction eluted from the P(3) by salt at a pH 7.4 or by detergent (Benishin and Carroll, 1983). These three fractions of ChAT were then compared by molecular weights, isoelectric points, immunoblotting with monoclonal or polyclonal antibodies and hydrophobicity. The results show that the S(3) fraction of ChAT has a molecular weight of 66 K(d), whereas the ionically-bound fraction of ChAT has a molecular weight of 73-78 K(d). SDS-PAGE of these two ChAT fractions followed by immunoblotting revealed the presence of two immunoreactive bands at 28-29 K(d) and 50-51 K(d) for the ionically bound ChAT fraction. Conversely, none of these antibodies immunostained any protein bands for the S(3) ChAT fraction even though one monoclonal antibody had been prepared against this ChAT fraction and the S(3) ChAT fraction had a similar specific activity prior to SDS-PAGE as did the salt solubilized ChAT fraction. However, anti-ChAT monoclonal antibody MB16 binds the native S(3) ChAT fraction in the co-precipitation assay. The S(3) fraction of ChAT had only one isoelectric point at pH 7.8, whereas the ionically bound and detergent soluble ChAT fractions had two isoelectric points at pH 8.1-8.15 and 7.45-7.5. The S(3) ChAT fraction also differed in hydrophobicity from the other two ChAT fractions. These differences between the S(3) and salt soluble ChAT fractions were not obviated by addition of Triton X-100 and thus could not be attributed to the association of lipids with either of the fractions. We conclude that the water soluble fraction of ChAT in central nerve terminals differs in its physical properties and its subcellular location from that which ionically binds to membranes.

The effect of the acetylcholine transport blocker 2-(4-phenylpiperidino) cyclohexanol (AH5183) on the subcellular storage and release of acetylcholine in mouse brain.

The effect of the acetylcholine (ACh) transport blocker 2-(4-phenylpiperidino) cyclohexanol (AH5183) on the subcellular storage and release of acetylcholine was studied in mouse forebrain. Results indicated that AH5183 reduced the amount of ACh released from mouse forebrain minces by high K+ and veratridine over the identical concentration range as it inhibits the active transport of ACh into synaptic vesicles isolated from the electric organ of Torpedo. However, AH5183 did not block the K+- or veratridine-induced reduction of cytoplasmic (S3) ACh. Also, it did not block the loss of vesicular (P3) ACh caused by these depolarizing agents. It did, however, cause a disappearance of nerve ending ACh which was partially matched by a selective gain in the choline content of the P3 fraction. When minces of mouse forebrain were pretreated in high K+ to deplete the S3 and P3 fractions of their ACh content and then subsequently incubated in normal Krebs with [14C]choline, AH5183, at a concentration which reduces ACh release by 50%, did not affect the repletion of P3 stores with newly synthesized [14C]ACh. At somewhat higher concentrations, however, AH5183 reduced the amount of [14C]ACh in the P3 fraction without affecting the amount of [14C]ACh in the S3 fraction. At these concentrations it did not inhibit extracellular choline transport or ChAT activity. These results suggest that AH5183 may reduce the amount of ACh released from central cholinergic nerve terminals in response to depolarization through a combination of effects: (1) it may facilitate the breakdown or loss of ACh stored in the vesicular fraction; (2) it may also block the transport of newly synthesized ACh into the vesicular fraction.

Molecular weight determinations of soluble and membrane-bound fractions of choline O-acetyltransferase in rat brain.

Three fractions of choline O-acetyltransferase (ChAT) (EC 2.3.1.6) were solubilized from a nerve ending fraction of rat forebrain using three sequential washes of an increasingly chaotrophic nature (100 mM sodium phosphate, pH 7.4; 500 mM NaCl; 2% Triton DN-65) as previously described (Benishin, C.G., and P.T. Carroll (1983) J. Neurochem. 41: 1030-1039). The molecular weights of the soluble (NaP) and membrane-bound fractions (NaCl and 2% Triton DN-65) of ChAT, following partial purification, were determined using either gel filtration on Sephadex G-200, G-100 Superfine, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by "Western blotting" and immunochemical visualization of ChAT with four different anti-ChAT monoclonal antibodies (Ab8, Ab9, 4D7, and 1E6). Results obtained with gel filtration indicated that the NaP- and Triton DN-65-solubilized fractions of ChAT had molecular weights in the range of 73,000 to 78,000, whereas the NaCl-solubilized fraction of ChAT had a molecular weight in the range of 230,000 to 240,000. Results obtained with SDS-PAGE and Western blotting indicated that all three fractions of ChAT were composed of the same nonidentical subunits.

Veratridine-induced release of acetylcholine from mouse forebrain minces: dependence on the hydrolysis of cytoplasmic acetylcholine for a source of choline.

The importance of depolarization induced hydrolysis of cytoplasmic acetylcholine (ACh) in providing choline for the veratridine-and high K+-induced release of acetylcholine was studied in mouse forebrain minces. Results indicated that a loss of hydrolyzable cytoplasmic ACh prior to depolarization reduced the amount of ACh released by veratridine but not the amount released by high K+. The reduction in the veratridine-induced release of ACh did not occur during the first 5 min of incubation. Loss of vesicular ACh prior to depolarization reduced both the veratridine- and K+-induced release of ACh during the first 5 min of incubation. Blockade of extra-cellular choline transport by hemicholinium (HC-3) did not affect the veratridine-induced release of ACh during a 10 min incubation period unless the cytoplasmic pool of ACh had first been depleted and was unavailable as a source of choline. In contrast, HC-3 reduced the K+-induced release of ACh from brain tissue with normal stores of cytoplasmic ACh. These results indicate that both depolarizing agents primarily stimulate the release of preformed ACh from a vesicular fraction during the first 5 min of mince incubation. Thereafter, they both stimulate the release of newly synthesized ACh, however, they differ in one important respect. The principal source of choline for the veratridine-induced release of newly synthesized ACh appears to be the cytoplasmic pool of ACh, whereas the major source of choline for the K+-induced release of newly synthesized ACh appears to be extracellular choline.

Developmental differences between soluble and membrane-bound fractions of choline-O-acetyltransferase in neonatal mouse brain.

Three fractions (one soluble and two membrane-bound) of choline acetyltransferase (ChAT) isolated from a nerve ending fraction of mouse forebrain, which have previously been reported to differ in several biochemical and physical aspects, were also found to differ in their rates of postnatal development. At 2 days of age, the activity in all three fractions was very low. Sodium phosphate buffer-soluble (cytoplasmic) ChAT activity increased significantly by 8 days of age, whereas the ChAT activity of the two membrane-bound fractions (NaCl- and Triton-soluble) did not increase until 13 days of age. These results suggested that the differences observed between the three fractions of ChAT prepared from mouse brain are not solely artifacts of the isolation procedure.

Depolarization of mouse forebrain minces with veratridine and high K+: failure to stimulate the Ca2+ independent, spontaneous release of acetylcholine from the cytoplasm due to hydrolysis of the acetylcholine stored there.

Both high K+ and veratridine, depolarizing agents with different mechanisms of action, lowered the ACh content of the cytoplasmic (S3) fraction of mouse forebrain minces incubated in a Ca2+-free Krebs solution, without stimulating ACh release or altering the level of ACh in the vesicle-bound (P3) fraction. Veratridine increased the level of choline in the P3 fraction by the same amount as it reduced the level of ACh in the S3 fraction, and these changes did not occur in the presence of tetrodotoxin (TTX). Pretreatment of minces in normal Krebs increased the ACh but not the choline content of the S3 fraction. Following this expansion of the S3 ACh content, veratridine caused an even greater loss of S3 ACh, and increased the Ca2+-independent release of ACh slightly. Under these conditions, veratridine also stimulated the Ca2+ independent release of choline, and this increase exceeded that obtained for the Ca2+-independent release of ACh. Preincubation in normal Krebs with paraoxon did not alter the S3 ACh content after 5 min, but raised it by 78% after 30 min. Under the latter conditions of pretreatment, veratridine then stimulated the Ca2+-independent release of ACh even more, but did not stimulate the release of choline. These results suggest that depolarization of brain tissue does not facilitate the Ca2+-independent release of ACh from the cytoplasm because a portion of ACh stored there is hydrolyzed. When the cytoplasmic level of ACh is sufficiently elevated prior to depolarization, then some ACh escapes hydrolysis and is released independently of Ca2+. It is suggested that the depolarization-induced hydrolysis of cytoplasmic ACh may be mediated by an intraterminal form of AChE and may, in addition to the hydrolysis of extracellular ACh, provide substrate for the formation and release of ACh by the vesicle-bound fraction.

Multiple forms of choline-O-acetyltransferase in mouse and rat brain: solubilization and characterization.

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.

Spontaneous release of acetylcholine and acetylhomocholine from mouse forebrain minces: cytoplasmic or vesicular origin.

The objective of this study was to determine the subcellular origin of cholinergic transmitter released spontaneously from mouse forebrain minces. To accomplish this objective, minces were pretreated in ionic media and then loaded with [14C]homocholine, an analog of choline, to form the false transmitter [14C]acetylhomocholine [( 14C]AHCh). The ratio of the false transmitter [14C]AHCh to the true transmitter ACh was then used as an index of cholinergic transmitter contents for both the cytoplasmic (S3) and vesicle-bound (P3) fractions. Three different pretreatment procedures were used to cause the following changes in S3 and P3 false to true transmitter ratios prior to spontaneous release: 1) a small increase in the S3 ratio of [14C]AHCh to acetylcholine (ACh) and a large increase in the P3 ratio of [14C] AHCh to ACh; 2) a decrease in the S3 ratio of [14C]AHCh to ACh and an increase in the P3 ratio of [14C]AHCh to ACh; 3) an increase in the P3 ratio of [14C]AHCh to ACh without affecting the S3 ratio of [14C]AHCh to ACh. The influence of each pretreatment on these subcellular ratios was then compared with its influence on the spontaneous release ratio of [14C]AHCh to ACh. In all 3 instances, the influence of pretreatment on the ratio of spontaneously released false and true cholinergic transmitters from minces coincided with the effect of pretreatment on the pre-release ratio of false to true transmitter in the S3 fraction. These results suggest that much of the cholinergic transmitter which is spontaneously released from mouse forebrain occurs from the cytroplasmic fraction.

Acetylcholine levels and choline acetyltransferase activity in turtle cortex.

Histochemical localization of acetylcholine esterase (AChE) shows that the reaction product is in the outer half of the molecular layer of dorsal cortex of the turtle Pseudemys. Thalamic and noradrenergic locus coeruleus fibers are found in the same location. Two hypotheses could account for this apparent overlap of inputs. First, a cholinergic fiber system could exist in turtle cortex that occupies the same portion of the molecular layer. On the other hand, the AChE enzyme could be associated with a non-cholinergic fiber system, for example the adrenergic fibers. In the latter alternative nlo cholinergic fiber input would need to be present in turtle cortex at all. Our experiments analyzed the levels of acetylcholine (ACh) and the activity of its synthetic enzyme, choline acetyltransferase (ChAT) in adult turtle thalamic input to cortex as a first step toward distinguishing between these alternatives. The results show that turtle cortex contains ACh and exhibits ChAT activity. These biochemical results support the idea that the AChE staining pattern in the outer half of the molecular layer may reflect the laminar distribution of cholinergic fiber activity in this simple cortex.