A STUDY ON THE ABILITY OF PRECURSOR AND ACETYLATED PRODUCT TO REFILL CHOLINERGIC VESICLES INDEPENDENTLY OF THE CYTOPLASM

Mouse forebrain minces were incubated in a Krebs or a lithium high K+ (L.K.) Krebs medium, cytoplasmic (S 3 ) and crude vesicular (P 3 ) fractions prepared and the levels of choline and acetylcholine (ACh) in each, determined. ACh levels were depleted by 70% in the P3 fraction of L.K. Krebs as compared to Krebs, incubated minces; while, s3 ACh levels were not significantly altered by the lithium incubation. Lithium treatment lowered the s3 choline content by 29%, with no significant effect upon P3 choline levels, when compared to s3 and P3 fractions of contralateral minces incubated in Krebs media. Minces, depleted of P3 ACh, were subsequently incubated in Krebs containing paraoxon (0. luM) and 0. lmN concentrations of either 14c choline or 14c homocholine. Depletion of P3 ACh resulted in an enhanced transport of extracellular 14c choline (84%) and 14c homocholine (76%), into this fraction but not into the s3 fraction. Furthermore, the depleted P3 fraction was repleted with newly synthesized 14c ACh or 14c acetylhomocholine ( 14c AHc). The ratio of 14c ACh to the total ACh in the repleted P3 fraction (0.63), exceeded that attained in the s3 fraction, (0.35). Additionally, the ratio of 14c AHc to ACh in the repleted P3 fraction (7.26), was greater than that attained in the s3 fraction (0.44). Similar results were obtained when mouse brain hippocampal minces were depleted of P3 ACh and subsequently

incubated in Krebs containing 14 c homocholine (0. lmM). The ratio of 14 c AHc to ACh in the repleted P 3 fraction (1 .50), exceeded that ratio attained in the s 3 fraction. Incubation of depleted forebrain minces in Krebs containing the preformed products 14 c ACh or 14 c AHc, in 0. lmM concentrations, did not result in an increased transport, or the repletion of the P 3 fraction with labelled product. However, depletion of P 3 ACh in hippocampal minces, did result in a greater accumulation of preformed 14 c AHc into the repleted fraction, resulting in a higher ratio of 14 c AHc to ACh in the repleted P 3 fraction (0. 17), as compared to the P 3 fraction of nondepleted minces, (0. 11). The amount of preformed 14 c AHc transported into the repleted P 3 fraction was insufficient, however, to replace lost transmitter . These results indicate that depletion of P 3 transmitter stores, occurs independently of the cytoplasm with newly synthesized product formed from extracellular precursor. The majority of acetylcholine, in brain tissue, is located within the nerve terminals of cholinergic neurons (Hebb and Whittaker, 1958;Gray and Whittaker, 1962). Acetylcholine (ACh) is synthesized by the enzyme choline-0-acetyltransferase (ChAT; E.C. 2.3. 1.6.), from the precursor choline and acetyl-coenzyme A (Hebb, 1972) and, similar to ACh, the enzyme is primarily found within cholinergic nerve endings (Hebb and Smallman, 1956). The subcellular localization of nerve ending ACh and ChAT has been studied extensively, and there is general agreement that the former is bimodally distributed between the cytoplasm and synaptic vesicles (De Robertis ~ ~·· 1963;Whittaker~~ .• 1964;Whittaker and Sheridan, 1965). 1-.!! vitro, ChAT also appears bimodally distributed, existing in a soluble and a membrane associated form (Whittaker~~., 1964). The significance of the latter form, .:!...!!. vivo, has been the subject of much controversy.
The development of a unified model, mechanistically depicting ACh metabolism, requires the characterization of precursor uptake and neurotransmitter synthesis, storage and release. According to the current model, all ACh is synthesized by cytoplasmic ChAT and subsequently transported into synaptic vesicles (Fonnum, 1968(Fonnum, , 1970. Fonnum (1967Fonnum ( , 1968 has reported that in vitro, as much as 85% of the total nerve terminal ChAT can be solubilized by high ionic strength media, 2 the remainder being associated nonionically with membrane components of the terminal. Thus, given similar l.!!_ vivo conditions, the majority of ChAT may be cytoplasmic, suggesting that the bulk of ACh synthesis occurs here and that little physiologically significant synthesis occurs elsewhere. ACh, formed in the cytoplasm, is believed to be translocated into synaptic vesicles (Whittaker, 1959) where upon depolarization all release is said to originate in a ca 2 + dependent manner (del Castillo and Katz, 1954, 1955Hebb, 1972;Fonnum, 1973). The current hypothesis places little importance, in terms of maintaining releasable stores of ACh, upon the membrane associated enzyme. Essentially, according to this model, the vesicles are entirely dependent upon the cytoplasm as a source of releasable transmitter. In turn, the enzyme activity of cytoplasmic ChAT is dependent upon the existing vesicular transmitter levels. Thus the two compartments are envisioned as operating in unison and as being interdependent.
Recent investigations have provided evidence that certain aspects of the present model require some modification.
That all ACh is synthesized by cytoplasmic ChAT has been questioned on the basis of the observations of Hattori et al. (1976) and Feiganson and Barnett (1977), that ChAT also appears adsorbed to the outer vesicle membrane and possibly contained within the vesicles (Hattori~~., 1976). Others (Haubrich and Chippendale, 1977) have reported that ChAT may be associated with the neuronal membrane, possibly near the vesicular attachment sites. Smith and Carroll (1980) have demonstrated enzyme activity in a crude vesicular fraction capable of acetylating choline and certain choline analogs.
The authors have suggested that membrane associated ChAT is capable of maintaining vesicular transmitter levels.
Furthermore, investigations characterizing the release mechanism of ACh, have indicated that both compartments may function independently of each other. Release of ACh, both 3 centrally and peripherally, is believed to occur in two ways-spontaneously and by depolarization (Somogyi and Szerb, 1972;Grewaal and Quastel, 1973;Carroll and Goldberg, 1975).
Spontaneous release of ACh originates from the cytoplasm of nerve terminals (Carroll and Goldberg, 1976), occurs during and in the absence of depolarization and does not require extracellular choline or ca 2 +. In contrast the depolarized release of transmitter is dependent upon both extracellular 2+ choline and Ca , exceeds the amount released spontaneously, and appears to originate from the vesicles (Carroll and Goldberg, 1976 Marchbanks (1969), Marchbanks and Israel (1972) and Richter and Marchbanks (1971), using in vitro tissue preparations, have reported that most newly synthesized ACh formed from extracellular choline, appeared in the cytoplasm relative to the vesicles resulting in a greater sp. act. in the former compartment. Similar qualitative results were reported by Chakrin and Whittaker (1969) after injecting labelled choline into the cerebral cortex of anesthesized animals.
Systemic administration of labelled choline results in the synthesis of labelled ACh in brain tissue (Schuberth ~ ~., 1969(Schuberth ~ ~., , 1970Jenden ~ ~., 1974). However, different from the results reported above, the sp. act. found in the vesicular fraction was higher than that attained in the cytoplasm (Aquilonious ~ ~., Schuberth ~ ~., 1969Schuberth ~ ~., , 1970, indicating that newly synthesized and not preformed ACh can refill vesicular stores of transmitter. Katz~~· (1973), found that accumulation of pre- synthesis prior to exposure to labelled precursor or acetylated product. Depletion of vesicular ACh was attained by incubating brain minces in a lithium containing incubation medium essentially as described by Carroll and Nelson (1978).
The nature of vesicular refilling was further examined using the choline and ACh analogs -homocholine and acetylhomocholine, respectively. According to Barker and Mittag (1975) and Collier et al. (1977a, b), homocholine is not acetylated by cytoplasmic ChAT in vitro even though in the latter studies homocholine was taken up by rat brain 6 synaptosomes and the cat superior cervical ganglion, a percentage of which was acetylated. Release of the acetylated analog was also demonstrated from the ganglionic tissue by 2+ a Ca -dependent process . Thus if vesicles, previously depleted of ACh, could be refilled with newly synthesized acetylhomocholine, it would further suggest that a vesicular refilling process operates independently of the cytoplasm.
The significance of the present study is that if vesicular stores of transmitter can be refilled, as well as depleted, independently of the cytoplasm, the vesicles and the cytoplasm may represent two physiologically, as well as morphologically, separate compartments. 7 LITERATURE REVIEW Differential centrifugation of brain tissue which has been homogenized in iso-osmotic media, led to the observation that acetylcholine (ACh) is nonhomogeneously distributed within a cholinergic neuron (Hebb and Whittaker, 1958;Whittaker, 1959 The degree of interdependence is of pharmacological import. Several neural disorders, such as Huntington's chorea (Eckernas ~ ~., 1977) and tardive dyskinesia (Davis~~., 1975), have been associated with central cholinergic pathways. If the processes of ACh synthesis, storage and release occur separately within each compartment, then each transmitter pool may be differentially effected by disease processes.

Free ACh
Homogenization of brain tissue in 0.32M sucrose, liberates a soluble ACh containing fraction (S 1 ), not bound by membranes and referred to as 11 free 11 ACh. This fraction, derived primarily from that ACh originally contained within axons, represents 20-30% of the total tissue ACh store and requires a cholinesterase inhibitor present during homogenization to protect it from hydrolysis by acetylcholinesterase (AChE; E.C. 3. l. 1.7). Free ACh is thought to be formed by ChAT being transported from the cell body to the nerve terminals. At present no physiological function has been attached to this transmitter pool.

Bound ACh
Separation of the s 1 fraction at 17,000g gives rise to a pellet (P 2 in Fig. l), referred to as the crude mitochondrial fraction. Distribution studies by Hebb and Whittaker (1958), Whittaker (1959), and later by Gray and Whittaker (1962) ford, 1969). Purification of the P 2 fraction on discontinuous sucrose density gradients showed that essentially all of the P 2 ACh was recovered in the synaptosomal fraction (Marchbanks, 1966) and that little ACh was associated with mitochondria of this fraction.
When the synaptosomal pellet is resuspended in hypoosmotic media and homogenized, the synaptosomal cytoplasm is liberated along with other nerve ending organelles. High speed centrifugation of the synaptosomal homogenate produces a soluble fraction (S 3 ) corresponding to the nerve terminal cytoplasm. Whittaker (1959) and Whittaker~~-(1964) found that approximately 50-60% of the nerve ending ACh was liberated upon rupturing the synaptosomes. The ability to measure brain ACh in the s 3 fraction depends upon the use of a cholinesterase inhibitor during hypo-osmotic rupture of fraction P 2 and therefore s 3 ACh is referred to as "labi le-bound11 ACh. The remaining 40-50% of synaptosomal ACh is found associated with the high speed pellet (P 3 ) which consists of small mitochondria, partially disrupted synaptosomes and vesicles. Whittaker~~-(1964) and Whittaker and Sheridan (1965) have demonstrated that P 3 ACh is contained within the vesicles.
Much of what is presently understood regarding the sequence of events prior to, during and after release of ACh, has been derived from electrophysiological studies at peripheral synapses. Katz (1950, 1952), observed minute spontaneous fluctuations in membrane potentials (miniature endplate potentials; m.e.p.p. 15 ) that occurred with a mean frequency of approximately one per second, with a potential of less th an lmV. These m.e.p.p.'s are believed to be the result of ACh release in the form of discrete packets or quanta at the neuromuscular junction (Fatt and Katz, 1952) and probably at central synapses (Katz and Miledi, 1963). Each quantum, consisting of several thousand ACh molecules, has been equated with a m.e.p.p.; however, this relationship is not well established. Inorganic calcium, ca 2 +, is required for the quantal release of ACh and its role in neurotransmission has been examined by del Castillo and Katz (1954), Katz andMiledi (1967, 1971) and Miledi (1973). When tissue is depolarized, ca 2 + influx is increased and the quanta are released more frequently, producing an endplate potential (e.p.p.) postsynaptically.
At about the same time that the quantal nature of ACh release was demonstrated, electronmicroscopic analysis of brain tissue Bennett, 1954, 1955), and of the neuromuscular junction (Robertson, 1956), demonstrated 0 the existence of synaptic vesicles (400-500A in dia.), con-centr~ted at the presynaptic nerve terminal membrane. These l l vesicles became the structural counterpart of the release mechanism, providing an understanding as to how ACh is released as quanta. Each vesicle, depending upon the animal species used, may consist of as many as 1,500 to 2,000 molecules of ACh (Krnjevic and Phillis, 1963), although a much more conservative estimate of 306 molecules ACh/vesicle was derived by Whittaker and Sheridan (1965); however, the low distribution of cholinergic terminals in the tissue used was not considered by these investigators.  (Heuser, 1978). These investigators have f urther reported evidence, obtained through freeze fracturing techniques supporting an exocytotic-endocytotic release cycle.
Actual membrane alterations (indentation) have been observed where synaptic vesicles are believed to have fused with the neuronal membrane and released their contents. Excitation of such tissue in the absence of ca 2 +, which is necessary for quantal release (del Castillo and Katz, 1954), results in a lack of these membrane indentations.
Fractionation of brain (Barker~~., 1972) and electric organ (Zimmerman and Denston, 1977)  of ACh metabolism has been reported by several investigators (Schuberth, 1966;Schuberth ~ ~., 1970;Dross and Kewitz, 1972;Nordberg, 1977). Washing the brains in ice cold media minimizes this effect. The forebrains were then blotted dry, weighed, minced and placed on a chilled petri dish until the onset of incubation.

Incubation of Brain Tissue
Objectives Tissue incubations were routinely conducted at 37-38° under 95%0 2 -5% C0 2 in a Dubnoff Metabolic Shaker set at 90 cycles/min. The overall design of the tissue incubations, as described below, was to: 1) selectively reduce the ACh content of the vesicle-bound pool without altering cytoplasmic ACh levels; 2) determine whether the reduced vesiclebound pool could be refilled with newly synthesized product formed from extracellular precursors and/or with extra_ Several investigators have shown that lithium ions depolarize nerve terminals (Schou, 1957;Keynes and Swan, 1959) and by an unestablished mechanism blocks choline uptake presynaptically (Diamond and Kennedy, 1969;Simon and Kuhar, 1976;Jope, 1979). In an initial set of experiments (performed by Dr. P.J. Carroll), incubation of brain minces in a lithium Krebs medium, containing NaHC0 3 , resulted in a 70% reduction of vesicle-bound ACh relative to contralateral minces incubated in a Krebs medium. No significant alteration of cytoplasmic ACh levels occurred as a result of the incubations.
l 7 According to Schou (1957), lithium ions (Li+), appear to depress cellular respiration in several tissues (e.g. renal and cardiac tissues). Accordingly, a modified lithium Krebs medium was utilized in which KHC0 3 replaced NaHC0 3 (maintaining ios-osmolarity). Potassium, in concentrations up to 40 mM, is known to stimulate tissue respiration (Canzanelli, 1942) and when added to a lithium medium reverses the lithium-induced depression of respiration. However, it should be pointed out that in brain tissue, Li+ concentrations up to lOOmM also stimulate cellular respiration (Canzanelli, 1942).

Incubation of brain minces in lithium Krebs containing
NaHC0 3 or KHC0 3 has the same effect on lowering vesicle-bound ACh levels by 70% independently of the cytoplasm (Carroll and Nelson, 1978) and releases equivalent amounts of ACh into the incubation media (Nelson~~., 1980), thereby indicating that the inclusion of potassium is not disrupting central ACh metabolism. Therefore, KHC0 3 replaced NaHC0 3 in all lithium Krebs media used, unless otherwise stated. It should also be noted that Li+ are unique in that they can replace Na+ in several processes (Schou, 1957) and of particular importance Li+ can support ChAT activity whereas the absence of Na+ cannot (Potter~~., 1968).

Tissue Incubation
Minces were preincubated for a total of 30 minutes in 10 mls of normal Krebs (K.) or lithium K+ Krebs (L.K.) media.
The tissue was centrifuged (1000 g; 4°) and placed in fresh media after the initial 15 minutes to reduce the accumulation of extracellular choline in the media generated primarily from phospholipid turnover (Bhatnagar and Macintosh, 1967;Browning and Schulman, 1968;Browning, 1971;Collier~..!.!_., 1972). If the choline levels in the media become sufficiently elevated the lithium-induced inhibition of choline uptake may be partially reversed (although this effect was not examined in this investigation).
Tissue samples were placed on ice at the end of the preincubation, centrifuged (1000 g, 4°) and the pellets washed (2 x 5 mls) with ice cold K. media (the second wash contained 0. l uM paraoxon).
To ascertain whether the vesicle-bound fraction, pre- [ C]AHc and 0. l uM paraoxon.
Subsequent t-o: the 30 minute K. incubation in labelled precursor or preformed product, tissue samples were centrifuged (1000 g; 4°), the media discarded and the tissue washed (2 x 5 mls) with ice cold 0.32 M sucrose.

Hippocampus
Several investigators have determined that ACh uptake into brain tissue is not specific for cholinergic neurons (Katz!.!_~., 1973;Kuhar and Simon, 1974), suggesting that ACh uptake into central tissues is of little physiological import.
To determine whether an acetylated product could enter After surface washing the P 3 pellets (2 x 8 mls) with glass distilled water (pH 4.0), the pellets were transferred to ground glass homogenizers (Duall-20; Thomas Co.) and homogenized in 500 ul ice cold lN Formic Acid/Acetone (15/85; v/v) and allowed to set in ice, for 20 minutes. This procedure extracts nearly all tissue ACh and choline (Toru and Aprison, 1966). Subsequent to a 10,000 g 20 minute centrifugation in the cold, an aliquot (300 ul) of clear supernate was removed and stored at -20°.

Total Radiolabel Uptake
The total radiolabel uptake of labelled precursor or product into the s 3 or P 3 fractions was determined by Quenching did not appear to reduce the counting efficiency.

Determination of Tissue ACh and AHc
Total ACh and AHc levels were determined using the assay method of Goldberg and Mccaman (1973). In some cases, a modification of this method was employed where the extraction of tissue samples was omitted.

Extraction of Tissue
According to Goldberg and Mccaman (1973) and Mccaman and Stetzler (1977), consistent and reliable results derived from the assay described b~low are attainable only when tissue samples are initially extracted with a liquid cation exchange substance -sodium tetraphenylboron dissolved in 3-heptanone (TPB/3H). Using this procedure, Na-TPB exchanges Na+ for quaternary species present in the aqueous solution.
The resulting amine-TPB complex is insoluble in aqueous,but soluble in organic (e.g., 3-heptanone) solvents. were similarly assayed along with samples. Additionally, internal standards (100 pmoles AChBr) were added to some samples to determine if any tissue inhibition of the assay existed.

Conversion of Precursor to Product
When minces were incubated in K. containing the precursors [ 14 c]choline or [ 14 c]homocholine, the percent conversion (acetylation) of the total radiolabel uptake was determined by a method as described by Carroll and Goldberg (1975). and thus any activity observed in the P 3 sample represents cytoplasmic contamination.
Brain minces were incubated and subcellular fractions prepared as previously described. An aliquot of the hypoosmotically ruptured P 2 fraction was removed prior to ultracentrifugation and separation of the s 3 and P 3 fractions.
The P 3 fraction was subsequently homogenized in a total of 1.0 ml of water. Bovine serum albumen was added to the P 2 aliquot and the s 3 fraction to stabilize the enzyme. The  Table l indicate that although s 3 ACh content did not differ between treatments, lithium incubation did result in a 70% reduction of P 3 ACh levels, when compared to minces incubated in Krebs media. Conversely, s 3 choline levels were significantly reduced (29%) as a result of L. K. Krebs treatment, while the amount of choline in the P 3 fraction was only slightly (11%) lowered, (Table 3).
The transport of extracellular choline into the presynaptic nerve terminal for ACh synthesis has be-_en established as a sodium dependent process Simon, 1973: Kuhar et~., 1973;Simon~~., 1976). Thus the observed lowering of P 3 ACh levels of minces incubated in the lithium containing solutions with either low Na+ (NaHC0 3 , 28mM) or no added Na+ (KHC0 3 substituted for NaHC0 3 , 28mM), may reflect the lack of an adequate sodium ion concentration rather than the presence of lithium. To test this possibility, one set of forebrain minces were incubated in a normal Krebs medium (total Na+= 145mM), while contralateral minces were similarly incubated in a low Na+ Krebs medium (LiCl replaced by equiosmolar sucrose, total Na+ = ~BmM). Incubation of tissue in the low Na+ Krebs solution resulted in a 50% reduction of P 3 ACh levels relative to minces incubated in the normal Krebs medium. However, unlike that observed after L. K.
Krebs treatment, the levels of s 3 ACh were also reduced by nearly 30% relative to control tissue. The effect of lowered Na+ on s 3 and P 3 choline levels was not determined in this study.

Ability of Extracellular Choline to Refill A Vesicle-Bound
Transmitter Pool With Newly Synthesized ACh.
Incubation of forebrain minces in a Krebs solution containing lithium results in the depletion of P 3 , but not s 3 , ACh ( choline was found associated with the repleted P 3 fraction of minces previously exposed to the Krebs solution containing lithium (Table 5) Krebs preincubated minces.

Repletion of the P 3 Fraction with Newly Formed Acetylhomocholine Derived from the Extracellular Precursor-Homocholine
As previously stated, homocholine differs from the natural precursor choline in that it appears not to be a suitable substrate for solubilized (i.e., cytoplasmic) ChAT (Currier and Mautner, 1974;Barker and Mittag, 1975;Collier ~ ~., 1977). Thus if a depleted P 3 fraction was refilled with newly synthesized acetylhomocholine (AHc) formed from extracellular homocholine, an alternative site of synthesis would be proposed to exist which serves the purpose of vesicular refilling.
To examine this possibility, fo~~br a in mi nces were preincubated in Krebs or L. K. Krebs and incubated an additional 35 30 minutes in Krebs containing [ 14 c]homocholine (0. lmM). As seen in Table 4, the total amount of acetylated product (ACh and AHc) in the P 3 fraction of minces after Krebs or L. K.
Krebs pretreatment, was similar (9.3 nmoles/gm and 7.3 nmoles/ gm, respectively). However, the ratio of newly synthesized [ 14 c] AHc to ACh in the P 3 fraction of minces exposed to the lithium solution (7.26) exc~eded that attained in the same fraction of minces pretreated in Krebs media (0.46) (Fig. 3).
Similar results were obtained when mouse br.ain hippocampal minces were exposed to [ 14 c]homochol~ne (O.lmM). As seen in Table 8, the total amount of acetylated product in the P 3 fraction did not differ between Krebs (4.25 nmole/gm) and L. K. Krebs (4.0 nmoles/gm) pretreated minces. However, the amount of newly synthesized [ 14 c]AHc in the repleted P 3 fraction (2.03 nmoles/gm) was greater than that measured in the P 3 fraction of Krebs preincubated minces (0.61 nmoles/gm).
Thus the ratio of labelled AHc to ACh in the P 3 fraction of L. K. Krebs preincubated minces exceed that in the same frac-

~ccumulation of Extracellular [ 14 cJ ACh
The results shown in Table 6 indicate that after a 30 minute incubation in Krebs with [ 14 c] ACh, the total ACh content did not differ significantly between Krebs (15.8 nmoles/ gm) and L. K. Krebs (12.6 nmoles/gm), pretreatments in the P 3 fraction. However, unlike that observed with [ 14 c]choline, the .depleted P 3 fraction of L. K. Krebs exposed minces did not contain a greater amount of [ 14 c]ACh (1.8 nmoles/gm) when compared to the same fraction of contralateral minces (1.5 nmoles/gm) preincubated in Krebs media. The ratio of labelled to total ACh therefore, in the P 3 fraction of repleted tissues (0.20) did not differ significantly from the P 3 fraction of nondepleted minces (0. 14) (Fig. 4). The results in Figure 4 further indicate that the ratio of [ 14 c]ACh to total ACh in the P 3 fraction after either pretreatment did not exceed the ratios attained in the associated s 3 fractions.
Accumulation of Extracellular [ 14 c]AHc Table 6 indicates that similar amounts of acetylated product (ACh and AHc) existed in the P 3 fractions between pretreatments. Additionally, as was observed when forebrain tissues were incubated with preformed ACh, the repleted P 3 fraction did not accumulate significantly more [ 14 c] AHc when compared to the P 3 fraction of nondepleted minces. Thus, the ratio of [ 14 c]AHc to ACh, in the P 3 fraction did not differ significantly between pretreatments (0.09 and 0.09) (Fig. 5).
Similarly, the ratio of [ 14 c] AHc to ACh in the P 3 fraction  Table 8). Dowdall and Whittaker (1975) and Jenden ~ ~· (1976) have reported that the pre-existing nerve terminal levels of ACh regulate the transport of extracellular choline into the nerve ending. Table 7  The results presented in  (Salehmoghaddam and Collier, 1976). Lithium, however, in addition to depolarizing nerve terminals, also blocks choline transport presynaptically (Simon and Kuhar, 43 1976) thereby limiting the availability of precursor and thus preventing the repletion of P 3 stores of transmitter with newly synthesized ACh. It is important to note that lithium does support ChAT activity (Potter~~., 1968). The selective depletion of P 3 ACh levels may reflect a more immediate requirement by this compartment, relative to the s 3 fraction, for extracellular choline. Several investigators have reported that homocholine is not readily acetylated by cytoplasmic ChAT (Currier and Mautner, 1974;Barker and MiHog, 1975;Collier~..!~., 1977).

Depletion of P 3 ACh and Its Effect Upon the Transport of Extracellular Precursors and Products
Thus it was unexpected that nearly equivalent amounts of newly synthesized [ 14 c] AHc were measured in the P 3 (4.7 nmloles/ gm) and the s 3 (5.3 nmoles/gm) fractions of lithium pretreated minces. However, it is possible that the [ 14 c] AHc found associated with the s 3 fraction may have originated from that synthesized by a membrane-bound ChAT (located on or near the vesicular and/or neuronal membranes), which is released from the enzymes synthetic site into the cytoplasm. Further evidence that the depleted P 3 fraction is preferentially repleted with newly synthesized product formed from extracellular precursor rather than preformed s 3 ACh, is derived from the observation that the total amount of acetylated product in the repleted P 3 fraction of forebrain          Values represent the mean ± S,E , for rat~o of label to total ACh in subcellular fractions. An asterisk indicates a sis;nificant ~i~ference~ P < 0 .05 ( l..."lalysis of Variance : one way classification). l l= experimental animals used.
( 1 2 ) Values represent the mean ± S .E. for ratio of label to to al An in s bcellular fractions . ( ) = experimental animals used. .c