A Noncholinergic Action of Acetylcholinesterase (ACHE) in the Brain: from Neuronal Secretion to the Generation of Movement

Cellular and Molecular Neurobiology, Vol. 11, No. 1, 1991

Review

A Noncholinergic Action of Acetylcholinesterase (ACHE) in the Brain: From Neuronal Secretion to the Generation of Movement

Susan A. Greenfield ~

Received November 20, 1989; accepted March 21, 1990

KEY WORDS: acetylcholinesterase; substantia nigra; dendrites; calcium conductances; release; neuromodulation; movement.

SUMMARY

1. In various brain regions, there is a puzzling disparity between large amounts of acetylcholinesterase and low levels of acetylcholine. One such area is the substantia nigra. Furthermore, within the substantia nigra, a soluble form of acetylcholinesterase is released from the dendrites of dopamine-containing nigrostriatal neurons, independent of cholinergic transmission. These two issues have prompted the hypothesis that acetylcholinesterase released in the substantia nigra has an unexpected noncholinergic function. 2. Electrophysiological studies demonstrate that this dendritic release is a function, not of the excitability of the cell from which the acetylcholinesterase is released, but of the inputs to it. In order to explore this phenomenon at the behavioral level, a novel system has been developed for detecting release of acetylcholinesterase "on-line." It can be seen that release of this protein within the substantia nigra can reflect, but is not causal to, movement. 3. Once released, the possible actions of acetylcholinesterase can be studied at both the cellular and the behavioral level. Independent of its catalytic site, acetylcholinesterase has a "modulatory" action on nigrostriatal neurons. The functional consequences of this modulation would be to enhance the sensitivity of the cells to synaptic inputs. 4. Many basic questions remain regarding the release and action of acetylcholinesterase within the substantia nigra and, indeed, within other areas of

1 University Department of Pharmacology, South Parks Road, Oxford OX1 3QT, U.K. 55 0272-4340/91/0200-0055506.50/0~ 1991 Plenum Publishing Corporation 56 Greenfield the brain. Nonetheless, tentative conclusions can be formulated that begin, in a new way, to provide a link between cellular mechanisms and the control of movement.

NONCHOLINERGIC FUNCTIONS OF ACETYLCHOLINESTERASE

"The specificity of an enzyme," Davenport once remarked, "is in inverse proportion to its familiarity." Acetylcholinesterase (ACHE) must surely be the most apposite illustration of this idea. The concept of AChE performing a function or functions other than the hydrolysis of acetylcholine has already been pivotal to five reviews (Karczmar, 1969; Greenfield, 1984a, 1985; Chatonnet and Lockridge, 1989; Small, 1989) and four book chapters (Silver, 1974; Greenfield, 1984b,c, 1986). Whereas it is a relatively straightforward task to document diverse tissues where the presence and/or properties of AChE deviate from classical expectations of cholinergic transmission, there is no obvious strategy for identifying what these novel, noncholinergic actions might be. Nonetheless, we review here studies that have been attempting this very task, within one particular brain area. The two basic reasons for attributing AChE with a function beyond hydrolysis of acetylcholine are distribution and solubility. The distribution of AChE in tissues with little or no corresponding acetylcholine and choline acetyltransferase has long proved a puzzle to the biochemist (Karczmar, 1969; Silver, 1974). More recently the disparity has been thrown into sharper and indeed more aesthetic focus with the advent of immunocytochemistry, where choline acetyltransferase can now be visualized in neurons and compared directly with the presence of AChE (Levey et al., 1983; Cuello and Sofroniew, 1985). Considering the brain alone, large quantities of AChE but relatively little of the more faithful cholinergic marker choline acetyltransferase are found in diverse areas, which include the hypothalamus, globus pallidus, raphe nucleus, red nucleus, cerebellum (Greenfield, 1984a), and the subject of this review, substantia nigra (Fig. 1). The substantia nigra is probably best known, unfortunately, due to the close association between Parkinson's disease and degeneration of the dopamine- containing cells in this nucleus. These dopaminergic nitrostriatal cells were essentially the "prototype" neurons for the demonstration that AChE could be colocalized with a catecholamine (Lehmann and Fibiger, 1978; Butcher et al. 1975). It is perhaps not surprising that the presence of AChE in this region is still the focus of intense investigation. The result of this recent research has been to identify a cholinergic projection to the pars compacta of the substantia nigra from the pedunculopontine nucleus (Henderson and Greenfield, 1987; Beninato and Spencer, 1987, 1988; Gould et al. 1989; Martinez-Murillo et al. 1989a, b). However, the discovery of this pathway does not preclude noncholinergic functions for nigral AChE since the projection is relatively sparse (Martinez- MuriUo et al. 1989) and to a part of the substantia nigra (pars compacta) A NoncholinergicAction of AChE in the Brain 57

Fig. 1. Disparity between the distribution of AChE and that of CHAT. Photomicrographs of sections of ferret substantia nigra stained histochemicallyfor AChE or immunocytochemicallyfor CHAT. Adjacent sections of ferret brain stem stained for (A) AChE or (B) CHAT; (C) nonspecific staining involved in ChAT immunocytochemicalprocedure. The substantia nigra, arrowed in A, shows intense staining for ACHE. In B, ChAT-positive staining is visible in the interpeduncular necleus, in a cranial nerve nucleus, and in the superior coUiculus, but not in the substantia nigra, all of which nonetheless stain intensely for AChE (A). Bar = I mm. From Henderson and Greenfield (1987).

(Henderson and Greenfield, 1987) that in any event receives relatively little of the total nigral synaptic input (Rinvik and Grofova, 1970; Henderson and Greenfield, 1984). The fact remains that the presence of AChE in large amounts throughout the substantia nigra does not correspond to the low, albeit now localized, levels of choline acetyltransferase (Fig. 1). It would thus seem that a prime function of AChE in the substantia nigra is a noncholinergic one: this hypothesis is corroborated further by the second surprising property of ACHE, solubility. Almost 15 years ago Chubb and Smith (1975a) provided elegant evidence that soluble AChE was not an experimental artefact. Indeed, the soluble form of AChE was secreted in a physiological manner (Chubb and Smith, 1975b). Although this initial work was performed in the adrenal medulla, it was soon shown that a similar phenomenon occurred in the CNS: AChE derived from the brain is detectable in cerebrospinal fluid (Chubb et al., 1976). However, levels of AChE secreted from the brain into cerebrospinal fluid are dramatically reduced following an electrolytic lesion of the substantia nigra: this region, it seemed, must therefore be one of the main origins within the brain, of secreted AChE (Greenfield and Smith, 1979). At that time, however, it was not known from which cell type or types the protein was derived. Within the substantia nigra, dopamine-containing nigrostriatal neurons contain AChE in various subcellular compartments, i.e., the RER and the Golgi apparatus (Henderson and Greenfield, 1984). What would be the signficance of AChE being stored in these parts of the neuron? Unlike nondopaminergic neurons in the substantia nigra, which can also contain ACHE, nigrostriatal neurons are characterized by an abundance of RER (Henderson and 58 Greenfield

Greenfield, 1984), purportedly to manufacture protein such as AChE at a very high rate (Domesick et al., 1983). In muscle Rotundo (1984) has shown that soluble, secreted AChE has a much higher synthesis rate than the membrane- bound form and that, prior to release, the protein is processed via the Golgi apparatus. The large amounts of AChE-reactive RER and Golgi apparatus might thus betoken the cellular machinery for the secretion of AChE from dopamine- containing nigrostriatal neurons specifically (Henderson and Greenfield, 1984; Greenfield, 1985). Fortunately, it has been possible to text this hypothesis directly, by means of localized perfusion of the substantia nigra in vivo in control animals compared to those where the nigrostriatal pathway has been selectively destroyed with the neurotoxin 6-hydroxydopamine (Ungerstedt, 1971). It can be seen that AChE is released locally and directly within the substantia nigra (Greenfield et al., 1980), is independent of stimulation of the ACh receptor (Burgun et al., 1985), and occurs more or less exclusively from nigrostriatal neurons (Greenfield et al., 1983a). It would seem, then, that in the substantia nigra, release of AChE is indeed a noncholinergic phenomenon, associated rather with local dopamine systems. But this finding immediately entails a puzzle: in the substantia nigra there are very few dopamine-containing axon terminals or collaterals (Wassef et al., 1981). If AChE is indeed derived from nigrostriatal neurons, we are therefore forced to conclude that the protein is secreted from the somata/dendrites of these neurons. Yet this hypothesis is not quite as heretical as it might first seem, in the light of two exciting and singularly pioneering studies: Kreutzberg et al. (1975) suggested from cytochemical evidence that AChE was secreted from the dendrites of the guinea pig facial nucleus, while Glowinski and his colleagues showed that release of dopamine occurred from the dendrites of nigrostriatal neurons (Nieoullon et al., 1977). The dendritic release of AChE from nigrostriatal cells seems therefore quite feasible. Within the substantia nigra dendritic release of dopamine and AChE has many similar features. The reasons for this idea (see Table I), and the

Table I. Similarities Between Dopamine and Acetylcholinesterase in the Substantia Nigra

(i) Colocalized in nigrostriatal neurons ~ (ii) Not stored in vesicles, b but SER of dendrites c'd'e (iii) Accumulate in parallel during development a (iv) Spontaneous and evoked release from somata/dendrites of nigrostriatal cells r (v) Release not related to firing frequency e (vi) Release not blocked by TI'X or gamma-butyrylactone g (vii) Release potassium-evoked, calcitlm- dependent h (viii) Release evoked by amphetamine' (ix) Slow diffusion rates through the extracellular space r

a Source: Butcher et al. (1975). b Source: Wassef et al. (1981). c Source: Mercer et al. (1978). d Source: Liesli et al. (1980). e Source: Henderson and Greenfield (1984). Y Source: Greenfield (1985). g Source: Weston and Greenfield (1986). h Source: Greenfield et al. (1983b). i Source: Greenfield and Shaw (1982). A Noneholinergic Action of AChE in the Brain 59 implications arising from it, have already been reviewed (Greenfield, 1985). For our immediate purposes it is necessary only to summarize some basic conclusions: first, the characteristics of dendritic release of dopamine and AChE are strikingly similar; and second, the phenomena do not appear to correspond to classical descriptions of release from the axon terminal. The picture that emerges is of a sustained nonquantal release of transmitter and protein from the SER, independent of somatic excitability. Since there are very few dendrodendritic synapses in the pars reticulata of the substantia nigra (Wassef et al., 1981), it would seem that ACHE, and to a lesser extent the more fragile molecule dopamine, would diffuse in an indiscriminate and slow fashion through the extracellular space. This description of a possible form of nonclassical "neuromodulation" brings us to the central question: Why is AChE secreted from the dendrites of nigrostriatal neurons? We have tackled this question by addressing it in two stages, release of AChE and subsequent action. Each of these two stages has been examined in turn at two levels, the cellular and behavioral. We explore what can thus be regarded as four distinct approaches and then attempt to synthesize the data into a unified theory for a noncholinergic function for ACHE.

RELEASE OF AChE

Cellular Level Within the substantia nigra, dendritic release of AChE appears sensitive to transient events, i.e., secretion can be modified by diverse treatments (Table II). Yet the final conditions triggering this release have long proved a puzzle, in that dendritic release of AChE appears unrelated to the excitability of the cell (Llinas et al., 1984; Greenfield, 1985) and is indeed resistant to blockade of impulse flow by TFX (Weston and Greenfield, 1986): The actual ionic basis for release of AChE thus seemed nonclassical and elusive. However, a clue as to the possible "final common path" for dendritic release of AChE first came when we recorded intracellularly from nigrostriatal neurons. Intracellular recordings revealed that within nigrostriatal cells, there is a conductance carried by calcium ions and resistant to blockade of sodium channels

Table II. Evoked Release of AChE in the Substantia Nigra Treatment Species Reference

Veratridine Rat (in vitro) Cuello et al. (1981) Potassium ions Rat (in vitro) Cuello et al. (1981) Rat Greenfield et al. (1983a) Cat Greenfield et al. (1980) Rabbit Greenfield et al. (1983) Guinea pig Taylor et al. (1988) Haloperidol Rat Weston and Greenfield (1986) Amphetamine Rabbit Greenfield and Shaw (1982) Guinea pig Taylor et al. (1988) Gamma-hydroxyburyrate Rat Weston and Greenfield (1986) 5-HT Guinea pig Taylor et al. (1988) Peripheral ES Guinea pig Taylor and Greenfield (1989b) Central ES Guinea pig Taylor and Greenfield (1989a) 611 Greenfield

a: Control b: TTX

c: TTX,TEA d: TTX,TEA,Cd ++

._12o mV

__j 0.2 nA

50 msec

e: ® PRE 5-HT POST ®

2--I ~ L, o,o, ® t 50 msec 300"

w, 200 M,I IT-

100

20 40 60 80 100 TIME/MINS

Fig. 2. Visualization of the "HTSgCa." Intercellular recordings from a single nigrostriatal neuron: depolarization from resting membrane potential (-57 mV) following blockade of various ion channels. (a) Control: family of current pulses from a control period showing the voltage threshold for sodium-dependent action potential. (b) TI'X: in the presence of 10 -6 tetrodotoxin, strong currents evoke "spikes" of short (5- to 10-msec) duration (HTS gCa), indicated by the pointer. (c) TI'X and TEA: in the A Noncholinergic Action of AChE in the Brain 61 by T'FX (Llinas et al., 1984) (Figs. 2a-d). This conductance had a high threshold for generation, which was lowered by a TEA-induced increase in the length constant (Llinas et al., 1984) (Figs. 2a-d). It thus seemed that this "high- threshold calcium spike' (HTSgCa) was generated remote from the soma, i.e., in the dendrite. Given that the HTSgCa has similar ionic and spatial properties to dendritic release itself, it was clearly tempting to suggest that the former was the ultimate cause of the latter (Llinas et al., 1984; Greenfield, 1985). More recently we have been able to test this idea by employing the following strategy: identify a transmitter system directly afferent on to nigrostriatal cell dendrites, then test whether this transmitter modifies dendritic release of ACHE, and finally, observe whether the transmitter also modifies the HTSgCa in the same way. One of the major inputs to the substantia nigra is a 5-HT containing projection from the Raphe nucleus (Steinbusch, 1981): by using a double immunocytochemical procedure, it has been demonstrated that many of the 5-HT-containing axon terminals form direct contact with the perikarya and dendrites of nigral dopaminergic cells (Nedergaard et al., 1988) (Fig. 2e). Furthermore, local application of 5-HT to the substantia nigra leads to an increase in release of AChE (Taylor et al., 1988) (Fig. 2e). The next step was to see whether 5-HT had any effect on the HTSgCa: during intracellular recordings, the transmitter was applied by iontophoresis to either the proximal or the distal portions of the nigrostriatal cell dendrite. There was a 5-HT-induced enhancement of the HTSgCa, following selective application to the distal dendrites (Fig. 2e), which is the part of the dendrite thought to recieve most afferent synaptic input (Rinvik and Grofova, 1970). This effect was a true receptor-mediated phenomenon leading to a net calcium influx into the dendrite, independent of sodium- or potassium-channel activation (Nedergaard et al., 1988a). It seemed then that the HTSgCa was indeed the ionic basis for dendritic

Figure 2 legend continued. presence of TFX and TEA (10 -2 M), the membrane input resistance has increased. Smaller current pulses are now needed to evoke the HTSgCa: the amplitude, duration, and indeed number of these transients are now enhanced. (d) TTX, TEA, and Cd: when 10-3M Cd is added with both Trx and TEA present, the HTSgCa is markedly attenuated. From Nedergaard et al. (1988a). (e) steps involved in evoked release of AChE by 5-HT. (1) Dopaminergic dendrites of nigrostriatal neurons receive direct synaptic input from a 5-HT-containing nerve terminal. 5-HT-immunoreactivc boutons form asymmetrical synaptic contact (arrows) with a dendrite containing benzidine dihydrochioride reaction product granules (single arrow), thus identifying this dendrite as tyrosine hydroxylase immunoreactive. Bar = 0.5/tm. From Nedergaard et al., 1988a. (2) 5-HT enhances HTSgCa. Averaged membrane potential responses to intracellular current pulses showing the HTSgCa, before, during, and after application of 5-HT to the distal dendrites of a nigrostriatat neuron. When 5-HT is applied, the amplitude of the HTSgCa is increased by 61%, i.e., an increased amount of Ca ions is entering the dendrite. From Nedergaard et al. (1988a). (3) 5-HT enhances release of ACHE. The effects of 5-HT applied directly to the substantia nigra on local release of ACHE. The arrow indicates the time when 5-HT (10 -7 M) was applied to the substantia nigra (allowing for dead space in the perfusion circuit). The treatment caused a significant increase in release of AChE six animals (mean values of group shown by filled squares), compared to four animals not receiving the treatment (mean values of group shown by open squares). From Taylor et al. (1988). 62 Greenfield release: the short duration of the conductance, and its modification in the distal dendrite, electrically remote from the soma, would explain the disparity between dendritic release of AChE and overall cell excitability. Furthermore, it would appear that the HTSgCa really is a "final common path" since subsequent studies have employed diverse secretogues to show a clear correlation between enhancement of the HTSgCa and increases in release of AChE (Nedergaard et al., 1989). However, perhaps the most intriguing interim conclusion that can be drawn is that the phenomenon does not reflect the activity of the cell which secretes the protein but, rather, the excitability of the inputs to it. Since AChE is a protein, it will be presumably more robust than a transmitter in the extracellular space and, as we have seen, will diffuse slowly throughout a population of nigral neurons. An incoming synaptic signal would thus be triggering an event that, in both space and time, vastly exceeded its own duration and the mere one-to-one communica- tion of classic transmission. The next step is to identify when such signals would normally occur, i.e., In what context would afferent fibres themselves normally evoke the dendritic release of ACHE?

Behavioral Level

Within the substantia nigra, the dendritic release of AChE is apparently sensitive to diverse experimental treatments (Table II). But as yet we have no idea as to the physiological context in which the phenomenon would occur. A principal reason for this lacuna is a technical one: there is no established assay for AChE that is sufficiently sensitive to measure release of the protein on the same time scale as behavioral events, i.e., "on-line." However, in 1981, Israel and Lesbats described a new light-emitting reaction for acetylcholine. This chemiluminescent assay exploited a cascade reaction such that choline produced from the hydrolysis of the ester could be oxidized to yield hydrogen peroxide which in turn could catalyze the oxidation of luminol-peroxidase to emit light (Israel and Lesbats, 1981). Since this assay is essentially measuring the hydrolysis of acetylcholine, it follows that the principle could be used just as easily to monitor the activity of AChE (Birman, 1985). A further possibility is that the reaction might be sensitive enough to detect release of AChE from the substantia nigra, not in the usual, discrete 10-rain aliquots, but continuously in "real time." It was this rationale that prompted Llinas and myself to develop a system for actually visualizing the release of AChE from the substantia nigra in vitro (Llinas and Greenfield 1987) (Fig. 3). By using this new approach, we can observe the phenomenon of the release of AChE more convincingly than ever before and perhaps eventually visualize events over a much higher spatial resolution. But two big drawbacks remain: first, because the reagents are in direct contact with the tissue, they are not necessarily under optimal chemical conditions, which would entail potentially hostile pH values and temperatures; and second, it goes without saying that a brain slice has a very poor behavioral repertoire! If we wish to use this luminescent assay to study the release of AChE in a physiological context, then the system has to be A Noncholinergic Action of AChE in the Brain 63

Fig. 3. Visualization of spontaneous and evoked release of AChE within the substantia nigra in vitro. Successive photographs taken of a quadrant of brain slice at 1-min intervals. In the upper photograph, the slice was perfused with solution containing choline oxidase, microperoxidase, and luminol: no signal is apparent. However, following the addition of acetylcholine (subsequent four photographs), AChE activity toward this substrate is detectable, in the region of the substantia nigra, as a light-emitted signal caused by the eventual oxidation of luminol. After approximately 4 min, i.e. in the fifth photograph, a steady state is reached. The sixth picture shows increased light emission by the addition of 30 mM KC1 to the bathing solution. Low intensities of light appear here as paler than the inner darker area of higher light emission. From Llinas and Greenfield (1987). 64 Greenfield developed yet again to monitor as sensitively as possible release of the protein in the substantia nigra of the freely moving animal. Recently, we have developed a flow circuit which incorporates a continuous supply of perfusate from the substantia nigra in vivo: an instantaneous readout of AChE activity can thus be obtained without any potentially toxic reagents having direct contact with the brain tissue (Taylor et al., 1989). It could be argued that this signal corresponds to fluctuations in AChE release that occurs some time previously, due to the "dead space" of the circuit. However, this preliminary evidence suggests that temporal lag can be readily overcome by resynchronizing behavioral events on video tape with the light-emitted signal on a second recorder (Taylor et al., 1989). Using this novel system, we are just starting to gain insights into the physiological release of AChE in the substantia nigra; e.g., release appears to be pulsatile (Taylor and Greenfield, 1989a). In the anaesthetized state, there is less AChE released than when an animal is awake and freely moving; furthermore, bouts of transient movement can be seen to be associated with very distinct periods of transient increases in AChE release. However, the reverse is not true, i.e., release of AChE from the substantia nigra can be evoked locally and directly, without causing any ensuing movement. It therefore appears that increases in dendritic release of AChE may, at the most, reflect, but not cause, movement (Fig. 4). We have seen already that changes in AChE release can reflect the activity of incoming synaptic inputs. It is tempting to complete this apparent syllogism with the extrapolation that at least some incoming afferent signals to the substantia nigra reflect, i.e., are corollaries of, movement. The phenomenon of AChE release is thus providing us with a powerful means of studying the role of the substantia nigra in the control of movement and, more immediately, of bridging the gap between cellular and behavioral events.

ACTION OF AChE

Cellular Level

Although release of AChE is proving a valuable tool for providing a physiological index of events in the substantia nigra, the phenomenon has most probably evolved for a particular and immediate purpose within the extracellular space. So what could be the action, if any, of released ACHE? This question has been approached by introducing exogenous AChE into the extracellular space in an attempt to simulate the presence of the secreted substance. The hypothesis that AChE could have noncholinergic actions at the cellular level has been tested by applying three different cholinesterase preparations to nigrostriatal neurons during intercellular recordings. These preparations have included not only AChE itself (originating from electric eel), but also AChE rendered catalytically inactive by pretreatment with Soman, as well as butyrylcholinesterase, which of course will also hydrolyze acetylcholine. These preparations have been applied either in a single bulk aliquot to the perfusate of brain A Noncholinergic Action of AChE in the Brain 65

A 2.5' B 2.5'~

2.0' 2, 0 ANAESTHETIZED SPONTA NEODS START OF HYDROLYSIS I>ERFUSION ~ 1,5' OF Ach E 1.5

~ 1,0' 1.0

• 0.5' ~ 0.5' g O. ,, , • ,,, ' ~ ~b ~ 20 25 ~'o ~; ,o .o ,; 5?""5; 6; a; ; ~-~"o TIM~ rmln5]

C 2.5, * D 25'

~.0" (,3 FREELYMOVING 2.0" E 5E 1.5' 1.5" E Y' I > 1.0. g ~.o. 1 zs> 0,5" i w 0,5. L 6 O" 0- 4 . ORINK~NG-~ • , ,,, , . , ,,.,, 80 8"5 9"0 9; 1;0 1;5 1;0 115 120 120 125 130 135 140 145 TtME (mins)

E 2.5' F 2.025t 2.0' ts,

.~ 1.0'

Lu 0.5' 8 6 O" O' 41'-"RAISE D K~'EONS *'P , , ,. 1,50 155 1(~0 165 170 175 175 180 185 190 195 200 205 210 215 t45

Fig. 4. Relation between release of AChE from the substantia nigra and generation of movement. Six consecutive traces (A-F) showing on-line chemiluminescent measurement of AChE release within the substantia nigra of a guinea pig in vivo. Perfusion of the substantia nigra started at 17 rain (A) and was continuous for almost 200rain (F). Values for the AChE activity detected were calibrated using commercial enzyme (Sigma Type VI-S). The six traces each show the effect of a particular experimental paradigm on AChE release. (a) Perfusion of the substantia nigra of the anesthetized animal leads to an initial large signal that decays of the basal level clearly above that attributable to the spontaneous hydrolysis of acetylcholine. (B) In the anesthetized state, a stable and spontaneous release of AChE is observed of approximately 0.2 mU/ml. (C) As the animal awakens and begins to move freely, an increase in basal release of AChE is seen, and in addition, transient periods of large amounts of phasic release become apparent. The tonic release of AChE increased to approximately 0.3-0.4 mU/ml, whereas phasic release levels reached values as high as 2.5 mU/ml. It was noted that clear bouts of movement ( * ) were correlated with a transient increase in AChE release, although the reverse was not necessarily true (note unmarked peaks). (D) A water bottle is presented for 10 rain (125-135 rain). As the animal drinks, a large increase in AChE release is seen throughout this period. (E) A depolarizing concentration of potassium ions (30 raM) is applied locally to the stubstantia nigra, evoking a large increase in AChE release but with no discernible effect on behavior. (F) The animal is reanesthetized, and a marked decrease in phasic, though not tonic, release of AChE is seen. From Taylor and Greenfield (unpublished records). 66 Greenfield slices (Greenfield et al., 1988) or via pressure ejection in much smaller quantities to the dendritic region of nigrostriatal cells (Greenfield et al., 1989). In both cases, the qualitative effect has been the same: AChE and Soman-AChE have both induced a hyperpolarization of the nigrostriatal cell membrane (Fig. 5), which was due ultimately to an opening of potassium channels (Greenfield et al., 1988, 1989). On the other hand, butyrylcholinesterase has proved inefficacious (Fig. 5), suggesting that the effect observed is not due to the nonspecific effects of exogenous protein or, indeed, to the hydrolysis of acetylcholine.

a: AChE

b: Soman AChE v -54 mV

c: BuChE

-65 mV ..... • .... -- ---~'~ 110 mV

2 min

d: e: ;!lq '~,1

i ,,, mll

]I 20mY

_I t

J 0.1 nA

100 ms Fig. 5. Noncholinergic action of AChE in the substantia nigra and the consequences. (a-c) Typical effects of various cholinesterase preparations, pressure ejected in the vicinity of the distal dendrites of three nigrostriatal cells. (a) Application of AChE (Sigma electric eel Type V-S) for period denoted by line leads to a reversible hyperpolarization of approximately 4 mV. (b) AChE (Sigma electric eel Type VI-S) pretreated with Soman leads to a hyperpolarization comparable with that seen in a. (c) BuChE, at a concentration in the electrode 10 times that in a and b and applied for a comparable period of time, is inefficacious. Scale bars refer to a-c. From Greenfield et al. (1989). (d, e) Intracellular records on a faster time scale than above, showing the effects of hyperpolarization on a nigrostriatal cell. (d) Increasing depolarization from a normal resting membrane potential leads to repetitive firing action potentials. (e) When the neuron is hyperpolarized by approximately 15 mV, via sustained negative current injection, depolarizing pulses now lead to a marked and long-lasting depolarization (LTS gCa) from which action potentials are generated in bursts. From Greenfield et al. (1988). A Noncholinergic Action of AChE in the Brain 67

What would be the functional effects of this AChE-induced hyperpolarization? Again, we have a clue from studying intracellular recordings of nigrostriatal cells. If the neuron is hyperpolarized, most easily by a direct injection of negative current, then depolarization leads to generation of a long lasting conductance, from which action potentials occur in bursts (Llinas et al., 1984) (Fig. 5). This long-lasting conductance is resistant to TTX but is mediated by calcium ions; during hyperpolarization, it has a relatively low threshold for generation and hence is referred to as "low-threshold calcium conductance" (LTSgCa). We now know that this LTSgCa is located primarily in the distal dendrites (Harris et al., 1989). Functionally, it would be a valuable part of the repertoire of the neuron, since it causes burst firing, which in turn leads to a disproportionate amount of dopamine release from nigrostriatal terminals in the striatum (Gonon, 1988). In the light of the above, it is therefore tempting to ascribe a noncholinergic "modulatory" role to released ACHE. By inducing an initial hyperpolarization to a group of cells, AChE could be viewed as the agent responsible for the potential generation (deinactivation) of the LTSgCa. This potential would be realized, i.e., the LTSgCa activated, only if there was depolarizing input at some point during the hyperpolarization. Normally, this depolarization trigger could come from synaptic inputs to a particular cell within the hyperpolarized group, which would initiate an LTSgCa and, hence, burst firing and an increase in striatal dopamine release. Hence, the action of AChE alone would not lead to burst firing; similarly, incoming synaptic signals to a nonhyperpolarized cell would be relatively ineffective. Rather, only synaptic signals occurring while AChE was present would instigate the LTSgCa; put another way, AChE could be viewed as enhancing the sensivitity of the nigrostriatal cell to synaptic input. The idea that AChE could be pivotal to a modulatory mechanism of this sort was first proposed as the "low-pass filter" hypothesis (Greenfield, 1985). At that time, several of the basic premises of this theory had yet to be established, i.e., that incoming synaptic signals would modify the HTSgCa and that AChE could act as the hyperpolarizing agent for deinactivation of the LTSgCa. As we have seen, however, recent findings have provided much stronger grounds for accepting these premises, and a modified version of the original theory is shown in Fig. 6. Perhaps the most urgent issue, however, is to test this idea. If AChE really is acting in the fashion outlined in Fig. 6, then we should see it enhance the pattern of response to synaptic activation following, say, stimulation of a major afferent pathway such as the striatum. This finding is exactly what is obtained if AChE is applied to the substantia nigra in vivo, during striatal stimulation (Last and Greenfield, 1987) (Fig. 6). It now remains to be seen whether this noncho!in- ergic action of AChE is mere phenomenology or would have any significance at the behavioral level.

Behavioral Level

The strategy in these experiments has been a very simple one. Exogenous AChE has been introduced either unilaterally or bilaterally into the substantia nigra, and the effect observed in the freely moving rat. Since AChE could 68 Greenfield

10 impul~es

Ill 0 ~Ii <,. i,~ r-i ~> ! ~'~ ~, |

i | i i 0

illD. t I! I

I:I I ell

•!~II 4-

! O

¢II

I I,--

lzl 0 9

"~ e'-

O A Noncholinergic Action of AChE in the Brain 69 purportedly be enhancing the activity of nigrostriatal neurons, we have looked in particular at behaviors reflecting activity of the dopaminergic nigrostriatal pathway. Stereotypy is an indication of enhanced dopamine release in the striatum (Robbins, 1977; Joyce and Iversen, 1984). Following bilateral administration of AChE to the substantia nigra, stereotyped behavior is seen that is not apparent following treatment with butyrylcholinesterase (Weston and Greenfield, 1985). In 1971, Ungerstedt demonstrated that an imbalance of dopamine in the striatum led to circling movements toward the dopamine-improverished side, i.e., that a rat would circle away from the side where the nigrostriatal pathway was most active. Following injection of AChE into one substantia nigra, we have found that animals display circling behavior away from the treated side. Again, this effect was not seen following administration of butyrylcholinesterase and was maximal following treatment with a purified preparation (Chubb et al., 1980) of AChE derived from fetal calf serum and consisting only of the secreted form (Greenfield et al., 1984). In summary, then, it would appear that secretion of AChE from nigrostriatal cell dendrites is not a mere disposal process for a soluble product: rather it seems that the secretion of AChE has a purpose, namely, the enhancement of response of the cell to incoming information, which will in turn lead to enhanced dopamine availability in the striatum.

QUESTIONS AND CONCLUSIONS

The issue of a noncholinergic function of AChE in the substantia nigra appears to resemble the mythical hydra, where severing of one head led to the generation of seven more. Even if we accepted the above as a superficial answer to the question "Why do nigrostriatal neurons secrete ACHE?" then many more questions immediately arise. Some of the more obvious of these should now be considered.

The "Docking Mechanism" of Dendritic AChE Release We have seen that local release of AChE within the substantia nigra is not analogous to classical release of transmitter from the axonal terminal. Perhaps one of the biggest discrepancies lies in the size of the membrane surface area from which release can take place: in the case of the nigrostriatal cell dendrite, the length could be up to a thousand microns (Juraska et al., 1977). There must therefore be some docking mechanism, i.e., an intracellular signal that pre- disposes an AChE-containing SER to fuse with a particular segment of plasmamembrane. One hypothesis would dispense with the complex issue of intracellular signaling altogether~ by subscribing to the idea that "released" AChE is in fact derived from the membrane. Grounds for this idea: are provided from a study on erythrocytes and Torpedo, where AChE could be dissociated from the detergent- treated membrane by breaking the inositol phosphate anchor in the membrane 70 Greenfield with a selective form of phospholipase C (Taguchi and Ikezawa, 1987; Toutant et al., 1989). In the brain, however, this phenomenon is unlikely to occur since treatment with phospholipase C does not "release" AChE (Inestrosa et al., 1987), and indeed, within the striatum, AChE appears to be covalently attached to the lipid by a link that is insensitive to phospholipase C (Inestrosa et al., 1987). In addition, this hypothesis does not embrace facts, some of which have been discussed earlier, that are nonetheless consistent with the concept of the secretion of AChE from within the cell: abundant RER for rapid (i.e., soluble) protein synthesis (Domesick et al., 1983); continuity of AChE along the entire surface of the membrane wall, not just opposite sites of stimulation, i.e., synaptic boutons (Henderson and Greenfield, 1984); Ca-dependent release (Greenfield et al., 1983b; Weston and Greenfield 1986); concomitant release of other proteins (Greenfield et al., 1983b; Greenfield and Shaw, 1982); evoked decreases in release (Greenfield et al., 1980; Burgun et al., 1985; Weston and Greenfield, 1986). It is conceivable, however, that while evoked release of AChE is dependent on intraceUular events, spontaneous release is not. That these two forms of release may be very different is evidenced by the observation that only release of AChE can be evoked, whereas spontaneous release of AChE occurs in parallel with nonspecific cholinesterase and is even decreased in a similar way following 6-OHDA lesions (Greenfield et al., 1983a). Perhaps membrane-bound cholinesterases can be spontaneously derived from the membrane, but if so the insensitivity of this phenomenon to acute signals would render it of little or obscure physiological relevance. In any event, we should consider what type of intracellular signal would evoke release of ACHE. It is conceivable that the heterogeneous distribution of synaptic inputs along the dendrite in some way determine from where the AChE should ultimately be released. However, we have seen that 5-HT, for example, modifies the HTSgCa only when applied to the distal dendrites, even though 5-HT-reactive boutons can also make contact with proximal dendrites and soma (Nedergaard et al., 1988). Conversely, activation of acetylcholine receptors leads to an enhanced HTSgCa (Nedergaard et al., 1989) but no concomitant increase in AChE release (Burgun et al., 1985). Clearly, more factors must be involved in the docking mechanism than chance proximity of AChE-containing SER to a receptor-activated calcium channel.

Mechanism of Action of AChE Protein bioactivity of the type described here for AChE is such a novel phenomenon that precise mechanistic descriptions of its action can only be negative (i.e., what is not the case), general, or speculative. In the first, negative category, it appears that we really can rule out hydrolysis of acetylcholine, despite the minor cholinergic input to the substantia nigra: butyrylcholinesterase has proved inefficacious, AChE rendered catalytically inactive still has an effect, and a purified preparation of the secreted form is far more potent even when the net hydrolyzing ability of the preparation is far lower than commercial enzyme (Greenfield et al., 1984). A Noncholinergic Action of AChE in the Brain 71

Furthermore, it s~s tha~'~w~~:c~i~~i~fiat~:":ahefiz~,mafie a~tion of AChE toward substance P (Chubb et al., 1980). The hydrolysis rates reported by Chub and colleagues are too slow to be of plausible value physiologically; nigrostriatal cells already secrete an aminopeptidase electrophoretically distinct from AChE (Greenfield and Shaw, 1982); exogenous AChE does not inhibit (Greenfield et al., 1981), and in fact enhances (Last and Greenfield, 1987), substance P- mediated synaptic activity; substance P (James and Starr, 1977; Olpe and Koella, 1977; Pycock, 1980) and AChE (Greenfield et al., 1984) cause similar, not opposite, behavioral effects; and finally, Substance P does not evoke release of AChE within the substantia nigra (Cuello et al., 1981). Another possible mechanism of AChE ~action that can be discounted is interaction with dopamine receptors (D2) located on the nigrostriatal somata and dendrites (Nedergaard et al., 1988b). In view of the close parallels between dendritic release of dopamine and that of AChE (Table I), it is tempting to suggest that AChE either acts directly on an allosteric part of the D2 receptor to enhance the action of dopamine or at least, via some form of protective action, increases the availability of endogenous dopamine (see Greenfield, 1985). However, we now know that, unlike the action of dopamine (Lacey et al., 1987), the action of AChE persists in the presence of the dopamine (D2) antagonist sulpiride (Greenfield et al., 1989). Nonetheless, an interaction of the transmitter and protein cannot be excluded: however, it seems more probable that such an interaction might be at the prerelease stage, where the release of one substance acts as a trigger for release of the other. Yet this reaction need not necessarily be mandatory: it should not be forgotten that it is possible to demonstrate a clear differential release of AChE and dopamine from the same substantia nigra at the same time (Greenfield et al., 1980). In terms of "general" theories of noncholinergic action of ACHE, most of the more realistic possibilities have already been listed (Greenfield, 1985), namely, enzymatic toward an unknown substrate; direct and noncholinergic interaction with acethylcholine receptors (see Fossier et al., 1983); modification of the turnover or affinity of receptors other than those for acetylcholine or dopamine, direct interaction with, or insertion in the membrane as, an ion channel (Kaufman and Silman, 1980); direct but less specific interaction with the membrane; and indirect action as a carrier protein for a neuroactive yet vulnerable substance. Of the more speculative hypotheses as to the noncholinergic action of ACHE, two of the most attractive are woefully incomplete. One possibility is that a breakdown product, i.e., a peptide fragment, is in fact the active substance. Were this to be the case, however, we would still be faced with the problems of explaining why a small molecule should be encased in such a familiar glycoprotein with a well-established catalytic site, and indeed how the small molecule actually exerts its effects. Another incomplete and speculative conjecture that is nonetheless worthy of mention concerns the potassium channel that finally mediates the AChE-induced hyperpolarization (Greenfield et al., 1988, 1989). A recent study (Mourre et al., 1989) reports that there is an extraordinarily high number of 72 Greenfield sulfonylurea (glibenclamide) binding sites in the substantia nigra: glibenclamide is a specific blocker (Schmid-Antomarchi et al., 1987) of an ATP-sensitive potassium channel, the activity of which is related very closely to cell metabolism (Ashcroft, 1988). Perhaps AChE therefore exerts its action via this channel: exactly how this would be done, of course, is still unknown.

Termination of Action of AChE If released AChE does indeed have a noncholinergic "modulatory" action, then there should also be some means of terminating this action, i.e., of breaking down the active part of the AChE molecule. Presumably, the protein could either be taken up into neurons or be destroyed in the extracellular space. Endocytosis cannot be ruled out as yet: indeed, it has long proved puzzling that nondopaminergic cells in the pars reticulata of the substantia nigra contain ACHE, despite the fact that RER is relatively sparse and, more significantly, the Golgi apparatus is not AChE positive (Henderson and Greenfield, 1984; and see Noncholinergic Functions of Acetylcholinesterase, first section above). Perhaps the AChE seen within these neurons has been taken up from the extracellular space (Jessen et al., 1978). Nonspecific destruction of AChE by diverse proteases is not a very satisfying hypothesis, as it invokes a large random element in the proposed mechanism. An alternative explanation might be, however, that proteolytic activity is so slow that it does not feature as a rate-limiting step in the action of ACHE. There are three separate pieces of indirect evidence that might support this hypothesis. First, released ACHE, derived from the substantia nigra, is nonetheless detectable in cerebrospinal fluid up to 100 min after the stimulation that evoked it (Greenfield and Smith, 1979; Appleyard et al., 1987). Second, soluble AChE constitutes only about 10% of the total nigral AChE (Greenfield and Smith, 1979), and it is possible that the fate of the secreted protein, which does not initially reside on the membrane (see Rotundo, 1984), is eventually to become attached to it. Third, it is clear that AChE can have a noncholinergic action lasting for days (Greenfield et al., 1986) or more (Greenfield et al., 1984): however, adaptive secondary mechanisms cannot, of course, be excluded.

Ubiquity of "Noncholinergic" AChE It would seem against the biological principle of economy for AChE to evolve as a modulatory neurosecretory protein in only one area of the brain. Within the brain alone areas where AChE is released and/or where AChE may have noncholinergic actions include cerebellum (Appleyard et al., 1988; Apple- yard and Jahnsen, 1989), striatum (Greenfield, 1984; De Sarno et al., 1987), hippocampus (Appleyard and Smith, 1987), hypothalamus (Greenfield and Smith, 1979; Romero and Smith, 1980), and locus coeruleus (Greenfield, 1984; Green- field et al., 1986). Clearly, a discussion of all these different brain regions is beyond the scope of this review. Nonetheless, it should be noted that there is no obvious single neurochemical, physiological, anatomical, or pathological factor linking all of these regions. Either AChE has multiple and completely distinct A Noncholinergic Action of AChE in the Brain 73 noncholinergic actions or, perhaps, one' cellular action (e.g., opening of potassium channels) which has diverse effects at the behavioral or "organizational" level, depending on the neuronal context of the particular brain area in which it takes place.

Conclusions

Many different types of evidence suggest that AChE could have noncholinergic functions in diverse tissues. But as yet, identification of such function has been most intensively investigated specifically in relation to the dopamine-containing neurons of the Substantia nigra. In this region at least, release of AChE could be viewed as a powerful, yet variable, amplifier. A single signal, transmitted briefly across an axodendritic junction, can be magnified in both space and time by the secreted protein, which in turn will possibly facilitate burst firing and amounts of dopamine release in the striatum: this cascade of events could never have been achieved by one-to-one signaling along a relay of neuronal contacts arranged in series and employing classical neurotransmission. The relevance of this phenomenon might be that AChE is released as different components of a movement are occurring: more movement causes more AChE to be released (see Fig. 6), so that it would diffuse further and a larger population of cells would be involved. It follows, then, that the role of the substantia nigra might therefore not be strictly as a "low-pass filter" (Greenfield, 1985) but, rather, might be linked to the continuous monitoring of the duration of a movement once initiated. We would now need to know, however, exactly what the "components of a movement" are which are registered with afferent nigral signals; indeed the anatomical and chemical specificity of these pathways surely imply a potential diversity in the quality and quantity of reception of movement-related signals. We can pursue the idea of pathway selectivity still further. As we have seen, it is probable that only certain signals will release ACHE, possibly with varying potency; however, an exciting aspect of the revised model is that, once released, i.e., during or following a movement, AChE will enhance the sensitivity to a group of cells to any subsequent, even non-movement-related, signal impinging on that particular population. In this way the proposed AChE-mediated mechanism could be one not just of motor-motor sequencing but of sensorimotor or even a cognitive-motor integration, not otherwise achievable via conventional synaptic transmission. At the very least this type of reasoning illustrates the problem of tracing from a cellular right through to a systems level why a cell should secrete a protein. Perhaps one of the most vital first steps is to be careful to distinguish between the action of a substance and its eventual function. For example, the action of the transmitter acetylcholine may be to increase membrane conductance to potassium, sodium, or calcium ions, whereas the function of these events could have a significance only in the full context of the system in which the acetylcholine was operating to lower heart rate, contract skeletal muscle, induce salivation, etc. Nonetheless, both the action and the function of acetylcholine are understood in 74 Greenfield such cases. When we turn to the brain, the situation is always more problematic. We may well be able to describe an action of acetylcholine, e.g., facilitation of cell firing by blockade of the "m" current in hippocampal pyramidal cells (Cole and Nicoll, 1983); yet despite the precise narrative we might give of this sequence of physicochemical events underpinning this phenomenon, from receptor attach- ment through to channel closure, the actual function of even acetylcholine- facilitated excitation is still elusive, since it depends on an as yet incompletely understood context. If we now return to the situation in the brain where even the physicochemical basis of the noncholinergic action of AChE is unknown, we can see the problem of trying to understand its "function." We will really know the noncholinergic function of AChE in the substantia nigra only when we truly understand the function of the substantia nigra in the generation of movement, and vice versa. Perhaps we should be content with aiming to explain the noncholinergic action of AChE with particular target membranes, in this case nigrostriatal cells. Yet at least this approach of studying action within the context of neuronal populations will enable us to see that the possibility for diverse noncholinergic functions of AChE with the brain would be enough to make all the Hydra's heads spin.

REFERENCES

Appleyard, M., and Jahnsen, H. (1989). Non-cholinergic effects of acetylcholinesterase upon the membrane properties of mammalian cerebeller Purkinje cells. Eur. J. Neurosci. Suppl. 129. Appleyard, M., and Smith, A. D. (1987). Spontaneous and carbachol-evoked in vivo secretion of acetylcholinesterase from the hippocampus of the rat. Neurochem. Int. 11:397-406. Appleyard, M., Green, A. R., and Greenfield, S. A. (1987). Acetylcholinesterase activity rises in rat cerebrospinal fluid postictally; Effect of a substantia nigra lesion on this rise and on seizure threshold. Br. J. Pharmacol. 91:149-154. Appleyard, M., Vercher, J.-L., and Greenfield, S. A. (1988). Release of acetylcholinesterase from the guinea pig cerebellum in vivo. Neuroscience 25:133-138. Ashcroft, F. M. (1988). Adenosine 5'-triphosphate-sensitive potassium channels. Annu. Rev. Neurosci. 11:97-118. Beninato, M., and Spencer, R. F. (1987). A cholinergic projection to the rat substantia nigra from the pedunculopontine tegmental nucleus. Brain Res. 412:169-174. Beninato, M., and Spencer, R. F. (1988). The cholinergic innervation of the rat substantia nigra: A light and electron microscopic immunohistochemical study. Exp. Brain Res. 72:178-184. Birman, S. (1985). Determination of acetylcholinesterase activity by a new chemiluminescent assay with the natural substrate. Biochem. J. 225:825-828. Burgun, C., Greenfield, S. A., Waksman, A., and Weston, J. (1985). Differential effects of cholinergic agonists on acetylcholinesterase release from the rat substantia nigra in vivo. J. Physiol. 369:66P. Butcher, L. L., Talbot, K., and Bilezikjian, L. (1975). Acetylcholinesterase neurons in dopamine- containing regions of the brain. J. Neural Trans. 37:127-153. Chatonnet, A., and Lockridge, O. (1989). Comparison of acetylcholinesterase and butyrylcholinesterase. Biochem. J. 260-625-634. Chubb, I. W., and Smith, A. D. (1975a). Isoenzymes of soluble and membrane-bound acetylcholinesterase in bovine splanchnic nerve and adrenal medulla. Proc. R. Soc. Lond. 191B:245-261. Chubb, I. W., and Smith, A. D. (1975b), Release of acetylcholinesterase into the perfusate from the ox adrenal gland. Proc. R. Soc. Lond. 191B:263-269. Chubb, I. W., Goodman, S., and Smith, A. D. (1976). Is acetylcholinesterase secreted from central neurons into cerebrospinal fluid? Neuroscience 1:57-62. Chubb, I. W., Hodgson, A. J., and White, G. H. (1980). Acetylcholinesterase hydrolyses substance P. Neuroscience 5:2065-2072. A Noncholinergic Action of AChE in the Brain 75

Cole, A. E., and Nichol!;_R; A. (1983)~;~:Ac~|d10fi~ .meA~tes,.a.SMv¢i~Synaptic potential in hippocampal pyramidal ce~ils. Science 22ii1299Li30L ~c ...... Cuello, A. C., and Sofroniew, M. V. (1985). The anatomy of the CNS cholinergic neurons. In Neurotransmitters in Action (D. Bousfield, Ed.), Elsevier, Amsterdam, pp. 309-318. Ceullo, A. C., Romero, E., and Smith, A. D. (1981). In vitro release of acetylcholinesterase from the rat substantia nigra. J. Physiol. 312:14-15. De Sarno, P., Giacobini, E., and Downen, M. (1987). Release of acetylcholinesterase from the caudate nucleus of the rat. J. Neurosci. Res. 18:578-590. Domesick, V. B., Stinus, L., and Paskevich, P. A. (1983). The cytology of dopaminergic and nondopaminergic neurons in the substanfia nigra and ventral tegmental area of the rat--a light-microscopic and electron-microscopic study. Neuroscience 8:743P. Fossier, P., Baux, G., and Tauc, L. (1983). Possible role of acetylcholinesterase in regulation of post-synaptic receptor efficacy at a central inhibitory synapse of Aplysia. Nature 301:710-712. Gonon, G. G. (1988). Nonlinear relationship b~tween impulse flow and dopamine released by rat midbrain dopaminergic neurons as studied ~y ~fi~Viv~6"eleetrochemistry. Neuroscience 24:19-28. Gould, E., Woolf, N. J., and Butcher, L. L. (1989). Cholinergic projections to the substantia nigra from the pedunculopontine and laterdorsal tegmental nuclei. Neuroscience 28-611-623. Greenfield, S. A. (1984a). Acetylcholinesterase may have novel functions in the brain. Trends Neurosci. 7:364-368. Greenfield, S. A. (1984b). A novel function for acetylcholinesterase in nigro-striatal neurons. In Cholinesterases, Walter de Gruyter, Berlin, New York, pp. 289-303. Greenfield, S. A. (1984c). Can enzymes released from the nigro-striatal pathway act as neuromodula- tors? In The Basal Ganglia Advances in Behavioural Biology 27 (J. S. McKenzie, R. E. Kemm, and L. N. Wilcock, Eds.), Plenum Press, New York, London, pp. 319-332. Greenfield, S. A. (1985). The significance of dendritic release of transmitter and protein in the substantia nigra. Neurochem. Int. 7:887-901. Greenfield, S. A. (1986). Release of acetylcholinesterase from nigrostriatal neurons. In Cellular Biology of Ectoenzymes (G. W. Kreutzberg, M. Roddington, and H. Zimmerman, Eds.), Springer-Verlag, New York. Greenfield, S. A., and Shaw, S. G. (1982). Release of acetylcholinesterase and aminopeptidase in vivo following infusion of amphetamine into the substantia nigra. Neuroscience 7:2883-2893. Greenfield, S. A., and Smith, A. D. (1979). The influence of electrical stimulation of certain brain regions on the concentration of acetylcholinesterase in rabbit cerebrospinal fluid. Brain Res. 177:445-459. Greenfield, S. A., Cheramy, A., Leviel, V., and Glowinski, J. (1980). In vivo release of acetylcholinesterase in the cat substantiae nigrae and caudate nuclei. Nature 284"355-357. Greenfield, S. A., Stein, J. F., Hodgson, A. J., and Chubb, I. W. (1981). Depression of nigral pars compacta cell discharge by exogenous acetylcholinesterase. Neuroscience 6:2287-2295. Greenfield, S. A., Grunewald, R. A., Foley, P., and Shaw, S. G. (1983a). Origin of various enzymes released from the substantia nigra and caudate nucleus: Effects of 6-hydroxydopamine lesions of the nigro-striatal pathway. J. Comp. Neurol. 214:87-92. Greenfield, S. A., Cheramy, A., and Glowinski, J. (1983b). Evoked release of proteins from central neurons in vivo. J. Neurochem. 40:1048-1057. Greenfield, S. A. Chubb, I. W., Grunewald, R. A., Henderson, Z., May, J., Portnoy, S., Weston, J., and Wright, M. C. (1984). A non-cholinergic function for acetylcholinesterase in the substantia nigra: Behavioural evidence. Exp. Brain Res. 54:513-520. Greenfield, S. A., Appleyard, M. E., and Bloomfield, M. R. (1986). 6-Hydroxydopamine-induced turning behaviour in the rat: The significance of acetylcholinesterase in cerebrospinal fluid. Behav. Brain Res. 21:47-54. Greenfield, S. A., Jack, J. J. B., Last, A. T. J., and French M. (1988). An electrophysiological action of acetylcholinesterase independent of its catalytic site. Exp. Brain Res. 70:441-444. Greenfield S. A., Nedergaard, S., Webb, C., and French, M. (1989). Pressure ejection of aeetylcholinesterase within the guinea pig substantia nigra has non-classical actions on the pars compacta cells independent of selective receptor and ion channel blockade. Neuroscience 29:21-25. Harris, N. C., Ramsay, S., Kelion, A., and Greenfield, S. A., (1989). Electrophysiologocai evidence for a dendritic localization of a calcium conductance in guinea pig substantia nigra neurons in vitro. Exp. Brain Res. 74"411-416. Henderson, Z., and Greenfield, S. A. (1984). Ultrastructural localization of acetylcholinesterase in substantia nigra: A comparison between rat and guinea pig. J. Comp. Neurol. 230:278-286. Henderson, Z., and Greenfield S. A. (1987). Does the substantia nigra have a cholinergic innervation? Neurosci. Lett. 73:109-113. 76 Greenfield

Inestrosa, N. C., Roberts, W. L., Marshall, T. L., and Rosenberry, T. L. (1987). Acetylcholines- terase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylocholinesterase in other tissues. J. Biol. Chem. 262:4441-4444. Israel, M., and Lesbats, B. (1981). Chemiluminescent determination of acetylcholine, and continuous detection of its release from Torpedo electric organ synapses and synaptosomes. Neurochem. Int. 3:81-90. James, T. A., and Starr, M. S. (1979). Effects of substance P injected into the substantia nigra. Br. J. Pharmacol. 65:423-429. Jessen, K. R., Chubb, I. W., and Smith, A. D. (1978). Intracellular localization of acetylcholinesterase in nerve terminals and capillaries of the rat superior cervical ganglion. J. Neurocytol. 7:145-154. Joyce, E. M., and Iversen, S. D. (1984). Dissociable effects of 6-OHDA-induced lesions of neostriatum on anorexia, locomotor activity and stereotyping. The role of behavioural competi- tion. Physchopharmcolog 83:363-366. Juraska, J. M., Wilson, C. J., and Groves, P. M. (1977). The substantia nigra of the rat: A Golgi study. J. Comp. Neurol. 172:585-600. Karczmar, A. G. (1969). Is the central cholinergic nervous system overexploited? Fed. Proc. 28:147-157. Kaufman, K., and Silman, I. (1980). The induction of ion channels through excitable membranes by acetylcholinesterase. Naturwissenschaften 67:608-610. Kreutzberg, G. W., and Kaija, H. (1974). Exogenous acetylcholinesterase as tracer for extracellular pathways in the brain. Histochemistry 42:233-237. Kreutzberg, G. Q., Troth, L., and Kaija, H. (1975). Acetylcholinesterase as a marker of dendritic transport and dendritic secretion. Adv. Neurol. 12:269-281. Lacey, M. G., Mercuri, N. B., and North, R. A. (1987). Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta. J. Physiol. 392:397-416. Last, A. T. J., and Greenfield, S. A. (1987). Acetylcholinesterase has a non-cholinergic neuromodu- latory action on the guinea-pig substantia nigra. Exp. Brain Res. 67:445-448. Lehmann, J., and Fibiger, H. C. (1978). Acetylcholinesterase in the substantia nigra and caudate- putamen of the rat: Properties and localization in dopaminergic neurons. J. Neurochem. 30:615-624. Levey, A. I., Wainer, B. H., Mufson, E. J., and Mesulam, M.-M. (1983). Colocalization of acetylcholinesterase and choline acetyltransferase in the rat cerebrum. Neuroscience 9:9-22. Liesli~ P., Panuala, P., and Rechardt, L. (1980). Ultrastructural localization of acetylcholinesterase activity in primary cultures of rat substantia nigra. Histochemistry 70:7-18. Llinas, R. R., and Greenfield, S. A. (1987). On-line visualization of dendritic release of acetylcholinesterase from mammalian substantia nigra neurons. Proc. Natl. Acad. Sci. USA 84:3047-3050. Llinas, R., Greenfield, S. A., and Jahnsen, H. J. (1984). Electrophysiology of pars compacta cells in the in vitro substantia nigra--a possible mechanism for dendritic release. Brain Res. 294:127-132. Martinez-MuriUo, R., Villalba, R., Montero-Caballero, M. I., and Rodrigo, J. (1989a). Cholinergic somata and terminals in the rat substantia nigra: An immunocytochemical study with optical and electron microscopic techniques. J. Comp. Neurol. 281:397-415. Martinez-Murillo, R., Villalba, R. M., and Rodrigo, J. (1989b). Electron microscopic localization of cholinergic terminals in the rat substantia nigra: An immunocytochemical study. Neurosci. Lett. 96:121-126. Mercer, L., del Fiacco, M., and Cuello, A. C. (1978). The smooth endoplasmic reticulum as a possible storage site for dendritic dopamine in substantial nigra neurones. Experientia 35:101- 103. Mercer, L., Del Fiacco, M., and Cuello, A. C. (1979). The smooth endoplasmic recticulum as a possible storage site for dendritic dopamine in substantia nigra neurones. Experientia 35:101-103. Mourre, C., Yehezkel, B. A., Bernardi, H., Fosset, M., and Lazdunski, M. (1989). Antidiabetic sulfonylureas: Localization of binding sites in the brain and effects on the hyperpolarization induced by anoxia in hippocampai slices. Brain Res. 486:159-164. Nedergaard, S., Bolam, J. P., and Greenfield, S. A. (1988a). Facilitation of a dendritic calcium conductance by 5-HT in the substantia nigra. Nature 333:174-177. Nedergaard, S., Hopkins, C., and Greenfield, S. A. (1988b). Do nigrostriata neurons possess a discrete dendritic modulatory mechanism? Electrophysiological evidence from guinea-pig brain slices. Exp. Brain Res. 69:444-448. Nedergaard, S., Webb, C., and Greenfield, S. A. (1989). A possible ionic basis for dendritic release of dopamine in the guinea pig substantia nigra. Acta Physiol. Scand. 135:67-68. A Noncholinergic Action of AChE in the Brain 77

Nieoullon, A., Cheramy, A., and Glowinski, J. (1977). Release of dopamine in vivo from cat substantia nigra. Nature 2,66:375-377. Olpe, H. R., and Koella, W. P. (1977). Rotatory behaviour in rats by intranigral application of substance P and an eledoisin fragment. Brain Res. 126:576-579. Pycock, C. J. (1980). Turning behavior in animals. Neuroscience 5:461-514. Rinvik, E., and Grofova, I. (1970). Observations on the fine structure of the substantia nigra in the cat. Exp. Brain Res. 11:229-248. Robbins, T~ W. (1977). A critique of the methods available for the measurement of spontaneous locomotor activity. In Handbook of Psychopharmacology (L. L. Iversen, S. D. Iversen, and S. H. Snyder, Eds.), Plenum Press, New York, Vol. 7, pp. 37-82. Romero, E., and Smith, A. D. (1980). Release of acetylcholinesterase from superfused slices of rat hypothalamus. J. physiol. 301:52P. Rotundo, R. (1984). Synthesis, assembly and processing of the AChE in tissue cultured muscle. In Cholinesterases: Fundamental and Applied Aspects (M. Brzin, T. Kiauta, and E. A. Barnard, Eds.), Walter de Gruyter, The Hague, pp. 203-218. Schmid-Antomarchi, H., De WeiUe, J. R., Fosset, M., and Lazdunski, M. (1987). The receptor for antidiabetic sulfonylureas controls the activity of the ATP-modulated K channel in insulin- secreting cells. J. Biol. Chem. 262:15840-15844. Silver, A. (1974). The Biology of the Cholinesterases, Elsevier, Amsterdam, pp. 117-303,428-431. Small, D. H. (1989). Acetylcholinesterase: Zymogens or neuropeptide processing enzymes? Neuroscience 29:241-249. Steinbusch, H. W. M., Nieuwenhuys, R., Verhofstad, A. A. K., and Van der Kooy, D. (1981). The nucleus raphe dorsalis of the rat and its projections upon the caudate putamen. A combined cytotectonic immunohistochemical and retrograde transport study. J. Physiol. (Paris) 77:157- 174. Taguchi, R., and Ikezwa, H. (1987). Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholiphase. J. Biochem. 102:803-811. Taylor, S. J., and Greenfield, S. A. (1989a). Pulsatile release of acetylcholinesterase following electrical stimulation of the striatum. Brain Res. 505:153-156. Taylor, S. J., and Greenfield, S. A. (1989b). Release of acetylcholinesterase from the guinea-pig substantia nigra during peripheral nerve stimulation. Brain Res. 482:356-358. Taylor, S. J., Haggblad, J., and Greenfield, S. A. (1989). Measurement of cholinesterase activity released from the brain "on-line" and in vivo. Neurochem. Int. 15:199-205. Taylor, S. J., Bartlett, M. J., and Greenfield, S. A. (1988). Release of acetylcholinesterase within the guinea-pig substantia nigra: Effects of 5-hydroxytryptamine and amphetamine. Neuropharmacology 27:507-514. Toutant, J. P., Roberts, W. L., Murray, N. R., and Rosenberry, T. L. (1989). Conversion of human erythrocyte acetylcholinesterase from an amphiphilic to a hydrophilic form by phosphatidylinositol-specific phospholiphase-C and serum phospholipase-D. Eur. J. Biochem. 180:503-508. Ungerdstedt, U. (1971). Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system. Acta Physiol. Scand. Suppl. 367:69-93. Wassef, M., Berod, A., and Sotelo, C. (1981). Dopaminergic dendrites in the pars reticulata of the rat substantia nigra and their striatal input. Combined immunocytochemical localization of tyrosine hydroxylase and anterograde degeneration. Neuroscience 6:2125-2139. Weston, J., and Greenfield, S. A. (1985). Application of acetylcholinesterase to the substantia nigra induces stereotypy in rats. Behav. Brain Res. 18:71-74. Weston, J., and Greenfield, S. A. (1986). Release of acetylcholinesterase in the rat nigrostriatal pathway: relation to receptor activation and firing rate. Neuroscience 17:1079-1088.