Plurichemical transmission and chemical coding of neurons in the digestive tract

GASTROENTEROLOGY 1995;108:554-563

SPECIAL REPORTS AND REVIEWS

Plurichemical Transmission and Chemical Coding of Neurons in the Digestive Tract

JOHN B. FURNESS, HEATHER M. YOUNG, SUELI POMPOLO, JOEL C. BORNSTEIN, WOLFGANG A. A. KUNZE, and KAREN MCCONALOGUE Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Australia

The enteric nervous system contains neurons with well- defined functions. However, when neurons of the same function are examined in different regions or species, they are found to show subtle differences in their phar- macologies of transmission and different chemical cod- ing. Individual enteric neurons use more than one trans- mitter, i.e., transmission is plurichemical. For example, enteric inhibitory neurons have three or more primary transmitters, including nitric oxide, vasoactive intesti- nal peptide, and possibly adenosine triphosphate and pituitary adenylyl cyclase activating peptide. Primary transmitters are highly conserved, although their rela- tive roles vary considerably between gut regions. Multi- ple substances, including transmitters and their syn- thesizing enzymes and nontransmitters (such as neurofilament proteins), provide neurons with a chemi- cal coding through which their functions and projec- tions can be identified. Although equivalent neurons in different regions have the same primary transmitters, other chemical markers differ substantially. Caution must be taken in extrapolating pharmacological and neurochemical observations between species or even between regions in the one species. On the other hand, careful interregion and interspecies comparisons lead to an understanding of the features of enteric neurons that are highly conserved and can be used in valid extrapolation.

S ometimes in the course of scientific exploration, ob- servations can accumulate so rapidly that we fumble

to assimilate them into a coherent view. We encounter such a period now; the number of possible neurotransmit- ters in peripheral neurons seems to exceed necessity by a large margin. Although it might be hypothesized that only two transmitters are necessary, one for excitation and one for inhibition, or perhaps that even one transmit- ter and two receptors is enough, about 30 transmitter candidates have been discovered in the enteric nervous system.* It has been difficult to make sense of the experi- mental data. Daniel et al. 2'~ and Fox-Threlkeld, 4 for ex- ample, have drawn attention to the variations in re-

sponses of intestinal preparations from differing regions and species to the same agonists and the substantial dif- ferences in responses to the same agonists delivered by different routes. Others have pointed out that the pres- ence of possible neurotransmitters in enteric neurons is inconsistent between species and regions) For example, neurons that are immunoreactive for substance P inner- vate the mucosa in some species but not in others. 6 An- other example is the directions that neurons project; so- matostatin immunoreactivity (IR) is in neurons with anally projecting axons in the guinea pig small intestine

and in orally projecting neurons in the large intestine of the same species/

A number of ideas that may assist in interpretation of the pharmacological and localization data have been developed. In summary, these are as follows. (1) The plurichemical transmission hypothesis. This proposition is that each neuron communicates by the release and action of combinations of messenger substances. Those familiar with blood clotting or the immune system (e.g., complement fixation) will be attuned to cell communica- tion involving multiple chemical interactions. Until re-

cently, neurotransmitters usually have been thought to act alone. (2) The chemical coding hypothesis. The hy- pothesis is that combinations of chemical attributes, principally related to the transmitters used by neurons, correlate with the functions and locations of neurons. The codes of functionally equivalent neurons have some chemical features in common that generally relate to their primary transmitters (see below). (3) Primary trans- mitters and neuromodulators. It is suggested that differ- ent substances released from the one neuron serve differ- ent purposes. Among transmitter roles are those of primary transmitters, a primary transmitter being the

Abbreviations used in this paper: ACh, acetylcholine; CHAT, choline acetyltransferase; IR, immunoreactivRy; PACAP, pituitary adenylyl cyclase activating peptide; TK, tachykinin(s).

© 3.995 by the American Gastroenterological Association 0016-5085/95/$3.00

February 1995 ENTERIC NEUROTRANSMITTERS 555

substance (or substances) that has the major role in

changing excitability of effector cells, and those of neuro- modulators, substances that modify transmission, for ex- ample, by adjusting transmitter release or changing the

efficacy of primary transmitters. The ideas that are expressed in this report have evolved

over a period of t ime 8-12 and are almost certain to change

as new discoveries are made. Meanwhile, they provide a

framework for considering the significance of existing data in relation to neural function in the enteric nervous

system and elsewhere.

In 1877, Du Bois Reymond reported that transmission from neurons was likely to be either electrical or chemi-

cal. 13 It took many years before adequate experiments

could be performed, but by the early 1950s, it became accepted that mammalian neurons communicate chemi-

cally. 14 At that time, only two peripheral transmitters,

norepinephrine (noradrenaline) and acetylcholine (ACh), were identified, and the consensus seemed to be that

transmission was either adrenergic or cholinergic. This view remained so strong that the defining acronym for an undetermined transmitter has become nonadrenergic,

noncholinergic. The realization that nonadrenergic, non- cholinergic transmission occurred in the periphery came

substantially from studies of transmission to gastrointes- tinal muscle. 15-17 In the period from the mid-1960s up to the present, substantial effort has been expended to identify nonadrenergic, noncholinergic transmitters in

the enteric nervous system, in most cases without consen- sus being reached. Most recently, it has been recognized that transmission from each neuron involves several chemicals. Such plurichemical transmission, explored for the inhibitory motor neurons, helps explain the difficulty in establishing transmitter identification.

Whereas pharmacological studies, to mimic or inter- fere with transmission, remain the most powerful means

to identify transmitters, histochemical methods have been instrumental in showing that the transmitters are produced by the relevant neurons and that the same neurons contain several potential transmitters. Moreover, the chemical coding shown by histochemical studies has been crucial for the analysis of the organization of nerve circuits.

To show the ideas that are presented in this review, we have concentrated on three groups of neurons: excitatory neurons to the muscle, inhibitory neurons to the muscle, and secretomotor neurons. Possible transmitters of other enteric neurons have been reviewed elsewhere. ~

Defining Transmitters and the Transmission Process

Chemical transmission is the process by which substances are released from nerve terminals and act on

effector cells to produce changes in the physiological or

biochemical state of the effector. Each substance that is

released from the nerve ending to affect cells that it innervates is a transmitter; the responses of the postjunc-

tional cells depend on the ways that transmitters or their

effects interact. This concept says nothing about the t ime

course of the effect; long-term effects, even lasting a day

or more, must be considered part of the transmission

process. However, only short-term effects, lasting milli- seconds to seconds, are considered in this review.

The transmitters with which we are most familiar

cause transient changes in the excitability of the inner-

vated cells. A good example is the primary transmitter,

ACh, released at the skeletal neuromuscular junction. As

is explained below, most enteric neurons use more than

one primary transmitter. In some cases, substances re-

leased from axons seem to have minor influences on

effector cells. For example, some blood vessels are

innervated by vasoconstrictor neurons that contain neuro-

peptide Y but use norepinephrine and adenosine triphos- phate (ATP) as primary transmitters, re-2° At some of

these neuroeffector junctions, neuropeptide Y has mini-

mal postjunctional effect and can be regarded as a second-

ary transmitter. Neurotransmitters may act to modify

the release or action of the primary transmitter; when

they act in this way, they are called neuromodulators.

Neuromodulators can act postsynaptically, as vasoactive

intestinal polypeptide (VIP) does on the salivary gland

cells of the cat, where it substantially increases the effec-

tiveness of ACh, the primary transmitter, in promoting secretion. 21'22 Presynaptic modulation is also common.

In this case, a neuromodulator acts on the nerve terminal

to modify transmitter release, usually reducing transmit-

ter output. These concepts of primary transmitters, sec-

ondary transmitters, and neuromodulators are still evolv-

ing. The distinction between the classes is not rigid, as

will be clear from the discussion below. Furthermore, a

transmitter can have more than one role at a neuroeffector

junction as norepinephrine does at many junctions where it is a primary transmitter and also has presynaptic, mod- ulatory effects. 12

A rather surprising outcome of the investigation of possible neurotransmitters is that, in some instances, no

function can be attributed to a biologically active sub- stance in a particular neuron. An example is substance P in preganglionic fibers supplying the amphibian heart. 23

Also surprising are cases in which a potential secondary transmitter or neuromodulator has an action opposite that of the primary transmitter, t° B o w e r s 24 has argued

that inactive substances should be expected due to imper- fect control of gene expression. He calls neurotransmit- ters that fall into this class superfluous transmitters.

556 FURNESS ET AL. GASTROENTEROLOGY Vol. 108, No. 2

The Use of Chemical Coding

The observation that neurons can be distinguished by their chemical code has been helpful in analysis of neural circuits. For example, the coding of vasomotor neurons allowed Gibbins and Morris 25 to show that suc-

cessive segments of the arterial tree are separately inner- vated. Chemical coding also showed that noradrenergic neurons that control intestinal secretion are separate from those that control motili ty or arterial resistance. 8 Immu-

noreactivity for calbindin in the guinea pig small intes- tine has allowed the projections of enteric sensory neurons to be deduced, even though the reason why calbindin is

specifically contained in these neurons is unknown.

Transmitters of Inhibitory Motor Neurons

The identity of the transmitter of these neurons has been hotly debated. Burnstock et al. 16 produced evi-

dence to suggest that ATP is the transmitter; a few years later, evidence for VIP was reported. 26'27 More recent

evidence for the involvement of nitric oxide in this trans- mission is compelling. 28 Contribution to transmission by

pituitary adenylyl cyclase activating peptide (PACAP) is also possible, 29'3° as is the participation of carbon monox- ide. 31

An explanation for the differences encountered by in- vestigators is that there are at least two, and in many regions three, primary transmitters of these neurons and that the relative roles of the primary transmitters differ between regions and species. Stimulation of the inhibi- tory neurons causes relaxation and hyperpolarization of the muscle as shown by mechanical recording, electrical recording, and pharmacological analysis. Mechanical re- cording, by virtue of its simplicity, has allowed a broad range of preparations to be surveyed, but it lacks the temporal resolution that can be achieved with intracellu- lar microelectrodes.

Electrophysiological evidence for more than one inhib- itory transmitter was obtained by Niel et al., 32 who dis-

tinguished two responses to inhibitory nerve stimulation in the guinea pig small intestine: a fast hyperpolarization that is blocked by apamin, which blocks a class of small conductance potassium channels, and a slower hyperpo- larization that is resistant to this drug. This observation was confirmed in the same tissue by Crist et al., 33 who reported the fast component to be partly reduced by desensitization with o~,~-methylene ATP and the slow component to be blocked by the antagonist of VIP recep- tors, VIP 10-28 . Crist et al. 33 concluded that ATP and VIP are cotransmitters and that other substances may be involved because pharmacological antagonism was in-

complete. It was subsequently reported, also in guinea pig small intestine, that the slow component is blocked by inhibition of N O synthase. 34'35 Because the effect of

VIP was also reduced by N O synthase inhibition, He and Goya134 concluded that VIP acts indirectly, i.e., VIP

causes N O to be produced in the tissue. Further evidence for an indirect action of VIP being mediated by NO release was provided by Grider et al. 36 However, in some

gut muscle, VIP seems to act directly in that its action is not inhibited by blocking NO synthase. 37 Grider et al. 36 also showed that NO amplifies VIP release.

The conclusion that N O contributes only to slow com- ponents of transmission cannot be extrapolated; the in- hibitory nerves of canine proximal colon cause fast hyper- polarizing potentials that are blocked by NO synthase inhibition. 38 N O could be acting directly on the muscle

of the canine colon or could be evoking the release of another substance. Although apamin seems to be selec- tive for the non-NO component of transmission in the

guinea pig intestine, this is not the case in the dog small intestine, where apamin antagonizes both the action of NO and the NO synthase-sensitive component of inhib- itory transmission. 39 Conversely, in the circular muscle

of the opossum esophagus, apamin does not affect the fast hyperpolarization caused by nerve stimulation. 4°

Pharmacological analysis of relaxations also provides evidence for plurichemical transmission and confirms that the relative roles of transmitters differ substantially between regions. 8'41-45 Variable contributions of different

transmitters are suggested by variations in the extent by which a component of transmission from the inhibitory

motor neurons is blocked by apamin between gut regions in the guinea pig. 4I'46 For example, inhibitory transmis-

sion to the taeniae coli is almost completely blocked by apamin, whereas transmission to the longitudinal muscle of the proximal colon of the same species is almost fully resistant. 41'46 Investigations of transmission in the rat

intestine indicated that ATP could contribute signifi- cantly to transmission in the duodenum but not in the ileum. 42 The degree of contribution of N O to transmis-

sion also varies; antagonism of NO-mediated transmis- sion is effective in reducing inhibitory transmission in many gut regions but is poorly effective in the taenia. 36'4v

Similarly, Suthamnatpong et al. 4~ found that inhibition of NO-mediated transmission is effective in the proximal colon but not in the distal colon of the rat. Conversely, VIP receptor antagonism is effective in the distal but not in the proximal colon. In another region, the circular muscle of the lower esophagus of the opossum, ATP does not seem to contribute to transmission, whereas VIP and N O probably do. 27'4°'48 Pharmacological studies in the gastric fundus of the rat also point to N O and VIP being

February 1995 ENTERIC NEUROTRANSMITTERS 557

cotransmitters. 4v'49 A third component of the inhibitory

response in the same muscle may be mediated by ATP because the response is reduced by desensitization with oq~-methylene ATP. 5° Three pharmacologically distin- guishable components of inhibitory transmission to the circular muscle of the guinea pig colon have also been

reported; one sensitive to NO synthase inhibition, one sensitive to apamin, and one insensitive to combined apamin and NO synthase inhibition. 45

Although VIP has been referred to as a possible trans- mitter, the closely related peptide, peptide histidine iso- leucine, is produced by the same gene; it is localized histochemically in the same neurons, and it also relaxes the muscle. Thus, peptide histidine isoleucine and VIP should be considered together as possible transmitters. 51

A further compound, PACAP, needs to be included as a possible inhibitory transmitter. This peptide is found in axons that innervate the muscle throughout the gastro- intestinal tract. 52 In the taeniae coli, these have been

shown to be the axons of inhibitory motor neurons to the muscle. 3° The direct action of PACAP on gastrointestinal muscle is to cause relaxation. However, electrophysiolog-

ical evidence for PACAP being responsible for the mem- brane hyperpolarization associated with inhibitory trans- mission is lacking. A neurotransmitter role also needs to be considered for CO that relaxes the internal anal sphincter in the opossum; moreover, when the inhibitory nerves are stimulated, the levels of the enzyme for CO synthesis, heine oxygenase, increases. 3. Inhibitory trans- mission was antagonized when heme oxygenase was in- hibited with zinc protoporphyrin IX. It is puzzling that

the inhibitory action of VIP was also antagonized; this may suggest that the action of zinc protoporphyrin is not specific for heme oxygenase.

In conclusion, the enteric inhibitory neurons do not have a single transmitter. It is probable that each of ATP, NO, peptide histidine isoleucine/VIP, and PACAP contributes to inhibitory transmission to gastrointestinal muscle but to differing extents depending on region and species. Moreover, significant interplay occurs between the transmitters in that they can influence each other's release and ac t ion . 34'36'53

Chemical Coding of Inhibitory Motor Neurons

In this context, aspects of the chemistry that can be revealed histochemically are considered. In a wide range of vertebrates, IR for VIP is in nerve fibers in- nervating the muscle (we are unaware of any situation in which it has been investigated and not been found), and N O synthase, revealed either by immunohistochem- istry or by histochemical localization of reduced nicotin-

amide adenine dinucleotide phosphate diaphorase, is also present.54 56 That these two inhibitory transmitters are

in the same fibers innervating the muscle can be deduced from double labeling experiments in guinea pig small intestine and t aen iae coli, 11'57'58 rat small intestine, 59 and

toad intestine) 5 Both pharmacological evidence and sin- gle labeling histochemistry suggests that this will prove

to be the case elsewhere. In contrast, other chemical attributes of the inhibitory neurons are highly variable (Table 1). In the small intestine of the guinea pig, inhibi- tory neurons with short projections along the intestine

have IR for enkephalin and neuropeptide Y, whereas neurons with long projections have gastrin-releasing pep- tide and dynorphin-IR. .1'6° However, in the taenia of the

guinea pig colon, the neurons contain both dynorphin and enkephalin-IR but do not show gastrin-releasing peptide or neuropeptide Y - I R . 5s In the large intestine

of the guinea pig and in the canine small intestine, neuro- peptide Y - I R nerve fibers project orally to supply the circular muscle; it is thus likely that the excitatory rather

than the inhibitory innervation is neuropeptide Y - I R in these regions. 7'61 In the rat and human intestine, neu-

ropeptide Y is colocalized with VIP in presumed inhibi- tory neurons that supply the muscle. 62'63 In the human

taeniae coli, galanin coexists with VIP in the inhibitory 64 neurons.

IR for PACAP is encountered in fibers innervating gastrointestinal muscle throughout the digestive tract of several species) 2'65 In the taenia of the guinea pig cecum,

PACAP-IR is in the N O synthase/VIP inhibitory nerve

fibers and in the small intestine and colon; the projections of the PACAP-IR neurons correspond to the projections of the inhibitory motor neurons. 3°'65 Thus, PACAP-IR

may be a marker of some inhibitory motor neurons. In conclusion, the chemical code of enteric inhibitory

motor neurons always seems to include N O synthase and peptide histidine isoleucine/VIP, whereas other markers that occur in these neurons do so in an apparently capri-

cious way and cannot be regarded as useful in predicting the function of any immunoreactive neurons without fur- ther data. PACAP is possibly a third marker of the inhib- itory motor neurons.

Transmitters of Excitatory Motor Neurons

There is incontrovertible evidence, extensively re- viewed in the past, that ACh is a primary transmitter of excitatory neurons that innervate gastrointestinal mus- cle. 2'41 However, these neurons also release tachykinins (TKs), which are direct excitants of the muscle and also contribute to transmission. The TKs are a group of closely related peptides: substance P, neurokinin A, neu-

558 FURNESS ET AL, GASTROENTEROLOGY Vol. 108 , No. 2

Table 1. Chemical Coding o f Inhibi tory Mo to r Neurons

VIP/peptide Gastrin- NO histidine releasing

Gut region synthase isoleucine Dynorphin Enkephalin Galanin peptide Neuropeptide Y PACAP References

Guinea pig ileum (short neurons) Yes Yes Yes Yes Yes No

Guinea pig ileum (long neurons) Yes Yes Yes No ? Yes

Guinea pig taeniae coil Yes Yes Yes Yes No No

Human intestine Yes Yes - - No Yes - - Rat small intestine Yes Yes - - No - - No Dog small intestine Yes Yes - - No (?) - - No (?) Toad intestine Yes Yes - - - - No - -

Yes Yes 11, 60, 65

No Yes 1 1 , 6 0 , 6 5

No Yes 11, 30 Yes Yes See t e~ Yes - - 59, 70 No - - 6 1 , 1 0 7 - - - - 55

Yes, IR or enzymatic activity present; no, not present; - - , undetermined; ?, uncertain; no (?), probably not present.

rokinin B, neuropeptide K, and neuropeptide 7. Each of these, except neurokinin B, seems to be produced by enteric neurons. 66 Consistent with this, receptors for the

neurokinins, except for neurokinin B, are found on intes- tinal muscle. 6v In histochemical studies made between 1976 and 1988 and in some more recent studies, antibod- ies to substance P were used to localize T K neurons. This work showed that the circular muscle of the mammalian gastrointestinal tract, independent of region or species,

and the longitudinal muscle, where that was examined, was innervated by T K immunoreactive nerve fibers. Mi- croscopical evidence that the TKs are actually in the cholinergic excitatory motor neurons has been difficult

to obtain directly because of the difficulty of localizing cholinergic neurons with the rather poor antibodies against choline acetyltransferase (CHAT) that have been available. Nevertheless, some success has been achieved in the guinea pig, where the excitatory motor neurons have been shown to contain TK-IR. 6°'68'69 Less direct

evidence comes from experiments that exclude the pres- ence of TK-IR in the inhibitory motor neurons by show- ing that TK-IR is in separate fibers from those that contain NO synthase and VIP. 11'63'v°'vl This coupled with

nerve fiber counts that indicate that only two substantial populations of axons innervate the muscle, the inhibitory fibers and those containing TK-IR, 11'vl makes it certain

that the majority of excitatory motor neurons contain both ACh and TKs. Nevertheless, a minority of motor neurons may contain only ACh or only TKs. 68

Because ACh and TKs are both released from the excit- atory neurons and because ACh seems to dominate trans- mission, studies of the TK component have examined residual excitation when neurons are stimulated in the presence of block of muscarinic receptors. In these cir- cumstances, nerve stimulation c a u s e s c o n t r a c t i o n 72'73 and evokes slow depolarization. 74'v5 The nonmuscarinic com-

ponent of excitatory transmission is blocked by antago-

nists of TK receptors, leaving little residual response. Blockade of muscarinic receptors also shows a contribu- tion of TKs to excitatory transmission in peristalsis, v3

Chemical Coding of Excitatory Motor Neurons

In addition to ACh and TKs, many excitatory muscle motor neurons contain opioid peptides (dynor- phin, enkephalin, and opioid gene-related peptides). This

has been shown by immunohistochemical double stain- ing or electron microscopic analysis in several gastroin- testinal regions. 5s'6°'63'71 As implied by the effectiveness

of muscarinic plus TK receptor blockade to inhibit excit- atory transmission, the opioid peptides do not seem to be primary transmitters. Moreover, opioid peptides gen- erally have no direct effect on gastrointestinal muscle) Nevertheless, opioids can modify transmission, in some cases enhancing but in most cases inhibiting transmission from the motor neurons; i.e., opioid peptides contained in enteric motor neurons are neuromodulators. 3'76'77

Some excitatory motor neurons, those innervating the longitudinal muscle of the guinea pig small intestine, have IR for the calcium-binding protein calretinin, but calretinin is not found in the corresponding neurons in- nervating the circular muscle. 6°

Chemistry of Secretomotor Neurons

The movement of water and electrolytes across the mucosal epithelium in the small and large intestines is controlled in part by secretomotor neurons, whose cell bodies are in submucous ganglia. These neurons are excit- atory, i.e., they enhance the secretion of electrolytes from the interstitium to the lumen. Transmission has choliner- gic and noncholinergic components, although the relative contribution from each varies with species and perhaps region. <v8 There has not been a sufficient number of studies in different species and regions to be confident

February 1995 ENTERIC NEUROTRANSMITTERS 559

Table 2. Chemical Coding of Submucous Neurons

Proportion Presumed Species Code (%) function

Guinea pig s±'84'8~'Sr Dynorphin/galanin/neuromedin U/peptide histidine isoleucine/VIP 45 Secretomotor ChAT/cholecystokinin/calcitonin gene-related peptide/dynorphin/_+galanin/

neuromedin U/neuropeptide Y/somatostatin 30 ChAT/(calbindin)/dynorphin/neuromedin U/substance P 10 ChAT/calretinin/dynorphin 15 Calbindin/neuropeptide Y/peptide histidine isoleucine/VIP 40 Calcitonin gene-related peptide/somatostatin/±substance P 18 Calcitonin gene-related peptide/substance P/neurofiiament protein 25 Neurofilament protein _+ substance P 15 Galanin 2

Rat88,89

Secretomotor Sensory Unknown Secretomotor Unknown Unknown Unknown Unknown

that all components of transmission are known or whether the cholinergic and noncholinergic neurons are separate. However, studies of transmitter presence, trans- mitter release, and pharmacological mimicry strongly implicate VIP as a primary noncholinergic transmitter, v9

The neurons of the submucosal plexus ganglia in guinea pig and rat small intestine provide an interesting study of differences of chemical coding between species. In these species, in contrast to larger animals such as pigs and humans, the submucosal plexus ganglia proba- bly only contain cell bodies of sensory neurons and neu- rons involved in secretomotor control. 8° In the guinea

pig, there are only four types of nerve cells in submucous ganglia (Table 2). 81'82 Neurons that contain ChAT and

substance P and react with some antibodies to calbindin are thought to be primary sensory neurons. 82-84 Choliner-

gic secretomotor neurons have the chemical code CHAT/

cholecystokinin/calcitonin gene-related peptide/dynor- phin/neuromedin U/neuropeptide Y/somatostatin (about 70% of these contain galanin), whereas the noncholiner- gic secretomotor neurons are VIP/dynorphin/galanin/ neuromedin U. 4t'81,sS-sv The fourth group has the coding

ChAT/calretinin/dynorphin. s1'85 In the rat small intes-

tine, neurons subserving the same functions are presumed to exist, but the chemical coding of the nerve cells is quite different 62'88'89 (Table 2).

Human Enter ic Neurons

The organization of the human and other mam- malian enteric nervous systems and the physiological control that enteric neurons exert over motility, secre- tion, and other digestive processes in different species are similar. 9°-92 In particular, drugs that modify neuro- transmission have similar effects in humans and other mammals. For example, muscarinic antagonists reduce acid secretion and suppress motili ty in humans and labo- ratory animals. Furthermore, the recent discovery that N O is an enteric inhibitory transmitter has been con- firmed pharmacologically in preparations of human gas-

trointestinal tract. 93 96 Thus, it can be anticipated that

the primary transmitters of human enteric neurons are the same as in other mammals. Based on comparative

studies (reviewed above), it might also be expected that substances that contribute to the chemical codes of hu- man enteric neurons will have different patterns of colo- calization from those of other species.

Muscle Motor Neurons

The two markers of motor neurons to gut muscle in other mammals, TKs and VIP, are also contained in neurons innervating the muscle throughout the human gastrointestinal tract. 54 As in other species, VIP-IR nerve

fibers project anally in human in tes t ines T K and VIP- IR are in separate fibers as is also the case in other mam- mals. 63 VIP relaxes human gastrointestinal muscle, whereas TKs cause it to contract.98-loo These observations

imply that TKs mark the excitatory neurons and VIP marks the inhibitory neurons. 63'97 The effectiveness of

muscarinic antagonists in inhibiting excitation and of

N O synthase antagonism in reducing inhibitory trans- mission indicates that the excitatory neurons are cholin- ergic and that the inhibitory neurons contain NO syn-

thase. The presence of N O synthase in nerve fibers innervating the muscle has been confirmed histochemi- cally. %'*°1-1°3 Double labeling has shown that the TK-

IR nerve fibers are immunoreactive for enkephalin and that many of the VIP-IR nerve fibers are immunoreactive for neuropeptide Y and galanin. 63'64 It has been recently

shown that PACAP-IR is also colocalized with VIP-IR in fibers innervating the muscle of the human intes- tine. t°4 Thus, the coding of excitatory muscle motor neu- rons in human intestine can be deduced to be CHAT/ TK/enkephalin and of inhibitory muscle motor neurons to be NO synthase/PACAP/VIP/+galanin/+neuropep - tide Y. As in other species, there may be regional differ- ences in chemical coding; such differences, if they occur in humans, are yet to be identified. It seems that the coding N O synthase/PACAP/VIP for inhibitory motor

560 FURNESS ET AL. GASTROENTEROLOGY Vol. 108, No. 2

neurons and ChAT/TK for excitatory motor neurons is

conserved among mammalian species.

Secretomotor Neurons

The only consistent marker of secretomotor neu-

rons that has been identified in mammals is VIP. VIP-

IR fibers, which are presumed to have a secretomotor

function, provide a rich innervation of the mucosa of the human small and large intestine. 1°5 By analogy with

other species, these would be expected to be noncholiner-

gic secretomotor neurons. However, there are also cholin-

ergic secretomotor neurons supplying the human mu- cosa, 1°6 but the chemical coding of these neurons is

unknown.

Conclusions

A small part of the extensive literature on the

enteric nervous system has been selected for review to

show that substantial variations in the chemistries of

neurons that have the same functions are encountered

when neurons of different regions and species are com-

pared, even though the primary transmitters are well

conserved between species. It is concluded that transmis-

sion from each enteric neuron involves the release of

several substances, although the relative roles of these

mult iple transmitters vary with region and species. Some

of the substances whose presence distinguishes popula-

tions of neurons are neuromodulators, not primary trans- mitters, or may even have no role in transmission. The

recognition and exploitation of chemical coding of neu-

rons has been instrumental in deciphering neural cir-

cuitry within the intestine and elsewhere in the auto- nomic nervous system. ~2'4I'6°'83

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Received May 20, 1994. Accepted August 30, 1994. Address requests for reprints to: John B. Furness, Ph.D., Depart-

ment of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3052, Australia. Fax: (61) 3-347-5219.

Supported by grants from the National Health and Medical Re- search Council of Australia and U.S. Public Health Service grant NS 23816.