- Email: [email protected]
Environmental determination of neurotransmitter function
126
TINS- November 1978
Environmental determination of neurotransmitter function Paul H. Patterson in 'dec/Jing" which transmitter to produce, developing neurones are subject to a variety of influencesfrom their surroundings. Of particular importance 6re the findings that a group of neurones can alter their designation from, for example, adrenergic to ckalinergic, and back again, depending on the nature of their environment. Developing neurones in the vertebrate embryo are dependent on their environment for their survival and maintenance. in the most obvious sense,they rely on the Mood supply or cerebral spinal fluid for necessary nutrients and hormones. Gila have also been suggested to play a role in this support effort. On a more subtle level, a neurone's target tissues and its synaptic inputs can also exert life or death influences, it has been known for some time that developing neurongs can die if deprived of their normal synaptic targets. Mo;e recently, it has been shown that neurones which normally die during development can be "rescued' if additional target area is provided. Such target effects are often interpreted in terms of a growth factor which is necessary for survival, and permits further neuronal development. The best support for such a notion comes from studies of developing sympathetic neurones. if they are cut off from their targets by axotomy, they atrophy and die. However, if Nerve Growth Factor (NGF) is supplied to these cut neurones, they will survive and growa. Furthermore, sympathetic neurones that would die during normal development can be 'rescued' by systemic injections of NGF', in addition to such target influences, synaptic input also appears to be necessary for the normal development of some neurones. For instance, if one eye in a young kitten is covered, the cells that receive input from that eye in the lateral geniculate body will atrophy8. Such environmental factors can influence neuronal development in quite a different way as well. Recent studies have shown that developing autonomic neurones can become adrenerglc or cholinergic, [email protected]~oUuud~ t,m=19"re
ing on the siiptals they rec~ve from their surroundinlpt,'. In fact, it is possible to control experimentally this choice of differentiation pathways, either by transplanting neuronal precursor cells to different positions in the embryo, or by altering the environment of immature neurones under controlled conditions in cell culture. This transmitter choice is of course critical for the proper function of the autonomic system because acetylcholine (ACh) and noradrenaline (NA) usually have opposite effects on their target tissues. ACh slowi down the rate of the heart beat, for instance, while NA speeds it up. la ~ivo studies ll'he neural crest is a transient embryonic structure lying on the dorsal margin of the neural tube which gives rise to, among other derivatives, the neurones of the autonomic system. Using a biological cell marking technique to follow the fate of cells in avian embryos, LeDouarin and colleagues have studied the factors determiningthe choice of transmitter selected by autonomic neurones. This work has demonstrated strikingly the importance of the environment provided by tissues through which these cells migrate as well as the final sites of maturation in the target tissues'. Normally, some of the cells in the lumbar segments of the crest migrate past the ventral neural tube, mesenchyme, and notochord to form eventually sympathetic ganglia. These ganglia, of course, consist primarily of adrenergic neurones. On the other hand, some of the cells in the cranial segments of the crest migrate to the gut and become cholinergic neurones. These transmitter choices are not. com-
pletely preprogrammed in the crest cells because if cranial selpments of the crest (presumptive cholinergic) are transplanted to the lumbar region (presumptive adrenergh:), some of the cells migrate to the sympathetic ganglia and become adrenergic neurones. The transplanted celi~ were taken from quail embryos which have a distinctive nuclear morphology ano could thus be identified unambiguously in the host (chicken). The reverse transfortnation of a presumptive adrencrgic population into cholinerigic neurones was also p~sible. The influence of the gut itself was demonstrated in an experiment where presumptive adrenergic crest cells were transplanted directly into embryonic, aneural gut. In this isolated target tissue some of the crest cells formed a ganglion and differentiated into cholinerglc neurones. On the other hand, when crest plus neural tube and notochord (tissues which are part of the migratory pathway) were transplanted into the piece of gut, the crest cells became adrenergic. Thus both the target tissue and the migratory environment can influence the transmitter choice. Interestingly, the fate. of the neuronal precursor cells could also be changed even after they had halted their migration, formed a ganglion, and some bqpm differentiation. One example of this is the cholinergic ciliary gangl:.on, if it is "backtransplanted', just after it forms, to a presumptive adrenergic region of the crest, the ganglion cells do not stay together but undergo a second migration. Some of these cells then end up in sympathetic ganglia and become adrenergl~. Thus the transmitter produced by this neural crest population can be influenced even after the cells have migrated and localized themselves.
! 27
T I N S - November 1978
Had any of the cells that bec__ag~eadrenergic after the transplantation previously begun their differentiation as cholinergic neuroncs in the ciliary ganglion? To answer questions of this type, the development of single .,teurones, not populations of cells. must be followed. The population v. single ,:ell problem also means that the transplantation results can he explained by supposing ¢ither (a) that entironmentai factors induced a new or different transmitter in pluripotcnt or reversibly committed cells, or (b) that the environment selected among heterogeneous cells that were already irreversibly committed to one transmitter, in either case, the transplantation studies have clearly demonstrated the profound influence that the developmental environment can have on expression of transmitter functions in fifo. h t fitro stw#ks
A second line of inquiry involves cultures of dissociated rat sympathetic neurones; similar questions to those just considered are being investigated in single neurones and in populations of cellsTM. The type of transmitter (ACh or NA), and the type of synapses formed by these cells, depends on the fluid and cellular environment in which they develop. Because these neurones do not appear to divide in culture, such changgs are postmitotic. The neurones can be grown either in the near absence of other cell types, or in the presence of a variety of non.neuronal cells of known origin. When grown in the virtual absence of other cell types in an appropriate medium, these neurones can develop many of the properties expected of mature adrenersic neurones. They can synthesize, store, take up, and release NA, and form adrenersic synapses. In contrast, when the neurones are co-cultured with appropriate (see below) r,on.neuronal cells, they can produce as mue~ as 1000fold more ACh than cultures that contain only neurones, rhis ACh is used at functional cholinergic synapses between the neurones, or with skeletal or heart muscle cells. The neurones do not have to be in direct contact with the non-neuronal cells for those changes to occur; culture medium that has been conditioned by cultures of appropriate non-neuronal cells (CM) also causes dramatic increases i~.neuronal ACh production and the f r e q ~ of cholin. ersic ~Jnal~¢ formation. At the same time, however, growth in CM causes a marked
reduction in adrenersic prol~'~es. effects of CM are rather specilic in that CM does not affect appreciably either neuronal survival or growth. Thin, certain non-
neuronal cells can produce a diffusable l~lanted from ve~" young rats or in dissignal that has a profound and .,,elective sociated sympathetic neurones from effect on the type of transmitter chosen by animals of relatively )oung ages. if most of the cultured neurones are sympathetic peurones. adrenergic to begin with. and most can be made to become cholinergic, then it is A d r e m ~ drive in cultwe The constancy of survival suggests that possible that single neurone~ pass through CM is not acting by selectively allowing a a transition period, displaying both transclass of predetermined cholinergic neurones mitter properties simultaneously. Such to survive that would otherwise have died. neurones ha,,e been observed with electroExperiments with single cells have sup- physiological techniques: some young ported this conclusion. It is possible to single neurones appear to release both ACh grow single neurones in microcultures and NA at fun.,:!ionai synapses with under various conditions; as many as cardiac muscle cells-~. Such dual-f~nc!ion 80-90% of individual neurones grown on neurone~ ha~e not been obser~'ed reliably heart or skeletal muscle can be cholinergic, with biochemic.al t~hniques in mature while as few as 0% are cholinergic under cultured neurones. It v,iil clearly be of "control" conditions. These observations, interest to foll.:,g a .~ingle cell electroand the constancy of neuronal number and physiologically ~wer a period of becks to growth in the mass cultures, suggests that obse~e the timc- course of these changes. these sympathetic neurones have the capacity to become adrenergic or cholinergic, Why are in vitro t'hanges different from those depending on the signals they receive. As in rivo? One ob~.ious question raised by these with the case of the "back-transplanted" ciliary ganglion discussed earlier, the studies is ~hy is ~t possible in culture to get neurones have retained this plasticity even virtually all of the neurones to become after ganglion formation, in fact, it J~ likely cholinergic. ~hereas. in fifo. histochemical that they remain dcvelopmenta~.ly labile evidence suggeszs that onl) a small minoreven after they begin to expr~s one trans- ity ( --5 40) ofth~ s) mpathetic neurones are cholinergic. Among the ro.~sibIe explanamitter choice. Most, if not all, of the neuroncs express lions are the following: (a) the cholinergic adrenersic properties early in the culture signal m lifo i.~ selecti;el)r localized such period (I week postnatally), and many that onl) a few sympathetic neurone~ were adrenergic as much as a week before recei~,e it: (h) ,";GF exert~ an adren.-'r[ic birth, beginning to differentiate e~en while influence; or (,') ~napti¢ input to the still dividing. These observations are com- ganglion from the spinal cord plays a role patible with the evidence :hat the lumbar in this ,]¢¢i~ion. Considering ,:ach of the.~¢ explanation~ crest cells receive an early adrenergic signal while migrating past the notochord, neural in turn: if the cholinergic ~ignals were tube, and mesenchyme. This signal is selccti~..ly localized (at. for instance in a obviously reversible, however, since 80- targgt v,hich receives sympathetic cholinerg0% of the individual neurones can be- gic innervation, this could explain v,h~ come ~olinergic under appropriate condi- only" tl~,~¢ ,~ympathetlc neurones become tions. However, this plasticity probably cholinelgic. Thi~ possibility remains to be becomes much reduced as the neuroncs definitively tested. What has been done thus mature; cholinergic properties develop in far is to assess the ability of t arious organs culture only in sympathetic ganglia ex- to "condition cholinergically" the medium
Neuropacdia 4. Sympotkelic neurones
#a
128 in culture. The resultsare at once intriguing and puzzling. Targets normally receiving cholinergi~ innervation (skeletal and heart muscle) are far better at conditioning the medium t~an a target roc~'iving only adrenergic innervation Oiver). On the other hand, cell types not known to receive synapses at all (fibroblasts and gila) can also condition the medium. More" conelusive im'ormation may come from purification of the active factor in CM and producing an antiserum to it. Such :zntibudie~ could enable localization ofa sir~ilar factor in fifo. Preliminary evidence suggests that the factor is a heat stable, Imsic protein (M. Weber and P. Patterson, unpublished). NGF inheuee Wh~t is the role of. NGF in tire adrenergic-cholinergic decision (b)? NGF is known to be critical for the development of adrenergic :~ympathetic neurones. However. NGF ha.,, be~n found to .~timulate not only ad~nergic differentiation in the cultures, but also cholinergic differentmtion (in, the presence of CM). In quanlitative comparisons. NGF has been found to promote adrenergic and cholinergic development to the same extent. Thus NGF is a qualitatively different type of developmental signal th~,n CM. NGF is necessary for survival, and stimulates growth and differentiation, but does not tell neurones which transmitter to produce; all three of these properties are .different from those of CM. Symptie hqmts Another way to explain why there are so few cholinergic sympathetic cells in viro is to postulate another factor which prevents most of the neurones from responding to a cholinergic cue. The logical candidate for such a factor is the spinal cord input to the sympathetic ganglion (c), not only because it is normally present in viro, and not in ritro, but because it has been known that denervation of the ganglion in young mice greatly reduced adrenergic development. Therefore the effect of neuronal activity on the adrcnergic-cholinergic decision was studied in culture. The electrical effects of spinal cord innervation of the cultured sympathetic neurones were mimicked by direct electrical stimulation, growth in elevated K ~-, or in veratridine. AH three treatments would be expected to depolarize the cells, but by different mechanisms. Such depolarization resulted in up to 300row lower ratios of ACh production to N~ production than in controlss. Thus.
TINS- November 1978 evoking activity in the neurones made them more adrenergic and less susceptible to the cholinerglg signal, CM. Since the neurones were adrenergic to begin with, depolarization was interpreted to be stabilizing or fixing this initial transmitter choice. ~
- dmlgmslle a t e m e These results give rise to a model of how the neurones in a sympathetk ganglion decide wbeth~ to become adrencrgi¢ or cholinergic. The crest cells destined to form the ganglion are hypothesized to receive an early, but reversible, adrenersic signal, as discussed previously. Most of the ueurom~s are then innervated by their spinal cord inputs and this stabilizes the initial transmitter decision. Some of the neurones, however, are not innervated at this critical time (or t~eir innervation is relatively inactive electrically) and these cells will then be susceptible to a choliuergi¢ sigua~, which may or may not be selectively localized in particular target tissues. neurones will then become cholinergk. This scheme is tentative and oversimplified in many respects, but does make certain predictions which are testable in 1he developing embryo. Furthermore, it broadens our ideas of the possible role of activity in the development of the nervous system. Embodied in the theory is the idea that the specific synap~ formation between certain preganglionic axons and their designated postganglionic neuronal target cells (which is known to occur in these ganglia) would determine not. only the circuitry of the ganglion, but would d~termine what transmitter the postsynaptic cell will use. Thus the developmental information needed for specifu: synapse formation in construction of the appropriate wiring diagram could be used for anoiber developmental purpose as well. otlm mmmmmm~ ~ One question raised by the studies ofthe adrenergic-cholinerglc decision is how generalizable is the concept of environmental influences on the choice of trap.smitter? There are a number of intriguing places in the developing nervous system where the neurones within a particular group or layer look superficially like one another, but end up being heterogeneous as to transmitter content. For instance, the myenteric plexus in the gut contains, in addition to choUner~c neurones, neurones of at least five other transmitter classes. The retina is a highly stratified structure, and yet within several of the layers there is considerabl: heterogeneity of transmitters,
wnh at least ACh, aromatic amines, and pedmps several amino acid transmitters rel.l~-nted. Similarly, in the dmsal root ganglion, sever~ transmitters and/or modulators are represented. Substance P and somatostatin are highly concentrated in different neurones, even though both belong to the C-fibre class of afferents. The qumtion is (as it is with the retina and myenteric plexus) do these neurones make and receive different patterns of input and output connections, and, if so, do these differences influence the final choice of peptide or transmitter? Of interest in this context is the recent finding that the substance P to somatostatin ratio in dorsal root ganglion cultures can be altered as much as 30-fold by the presence of uon. neuronal cells (or CM), without changes in neuronal survival (A. Mudge, personal communication).
ne~U~ am 1. Btmge, R.. Johmon, M. and Rots, C. R. (1978) Science 199, 1409-1416. 2. Furshlmn, E. J., MacLemh, P. R., O'Lalgue, P. H. and Potter, D. D. (1976) Proc. Nat. Acad. StL U.S.A. 73, 422.5-4229. 3. Hendry. !. A. (1975J Bea/n Res. 94. 87-97. 4. Hcmdry, L A. and Campbell, J. (1976) J. Neurocytol. 5, 351-360. 5. LcDouarin, N. M., Teillgt, M. A., Zillm', C. and Smith, J. (1978) Proc. Nat. Acad. SoL U.S.A. 75, 20~)-2034. 6. Patterson, P. H. (1978) Annu. Rev. Net,.~¢i. I, 1-17. 7. Wslicke. P. A.. Cami~not. R. B. and Patterson, P. H. (1977) Prof. Nat. Acad. S¢I. U.S.A. 74. $767-5771.
P. H. Patterson b aa Anoclate Profetwr in tke Dei:artmeat of Nearobiology at Harvard Medical School, Boston, MA 02115, U.S.A.
To pmpmive m b o n of reviews M o s t o f the Neuroviews are written by invitation. Nevertheless unsolicited articles will also be considered f o r publication in T r e n d s in N e u r o S c i e n c e s . I f y o u wish to submit a review f o r publication, please first contact the S t a f f Editor in the Camb r i d g e editorial oJ~ce address on page !I1.
TINS- November 1978
Environmental determination of neurotransmitter function Paul H. Patterson in 'dec/Jing" which transmitter to produce, developing neurones are subject to a variety of influencesfrom their surroundings. Of particular importance 6re the findings that a group of neurones can alter their designation from, for example, adrenergic to ckalinergic, and back again, depending on the nature of their environment. Developing neurones in the vertebrate embryo are dependent on their environment for their survival and maintenance. in the most obvious sense,they rely on the Mood supply or cerebral spinal fluid for necessary nutrients and hormones. Gila have also been suggested to play a role in this support effort. On a more subtle level, a neurone's target tissues and its synaptic inputs can also exert life or death influences, it has been known for some time that developing neurongs can die if deprived of their normal synaptic targets. Mo;e recently, it has been shown that neurones which normally die during development can be "rescued' if additional target area is provided. Such target effects are often interpreted in terms of a growth factor which is necessary for survival, and permits further neuronal development. The best support for such a notion comes from studies of developing sympathetic neurones. if they are cut off from their targets by axotomy, they atrophy and die. However, if Nerve Growth Factor (NGF) is supplied to these cut neurones, they will survive and growa. Furthermore, sympathetic neurones that would die during normal development can be 'rescued' by systemic injections of NGF', in addition to such target influences, synaptic input also appears to be necessary for the normal development of some neurones. For instance, if one eye in a young kitten is covered, the cells that receive input from that eye in the lateral geniculate body will atrophy8. Such environmental factors can influence neuronal development in quite a different way as well. Recent studies have shown that developing autonomic neurones can become adrenerglc or cholinergic, [email protected]~oUuud~ t,m=19"re
ing on the siiptals they rec~ve from their surroundinlpt,'. In fact, it is possible to control experimentally this choice of differentiation pathways, either by transplanting neuronal precursor cells to different positions in the embryo, or by altering the environment of immature neurones under controlled conditions in cell culture. This transmitter choice is of course critical for the proper function of the autonomic system because acetylcholine (ACh) and noradrenaline (NA) usually have opposite effects on their target tissues. ACh slowi down the rate of the heart beat, for instance, while NA speeds it up. la ~ivo studies ll'he neural crest is a transient embryonic structure lying on the dorsal margin of the neural tube which gives rise to, among other derivatives, the neurones of the autonomic system. Using a biological cell marking technique to follow the fate of cells in avian embryos, LeDouarin and colleagues have studied the factors determiningthe choice of transmitter selected by autonomic neurones. This work has demonstrated strikingly the importance of the environment provided by tissues through which these cells migrate as well as the final sites of maturation in the target tissues'. Normally, some of the cells in the lumbar segments of the crest migrate past the ventral neural tube, mesenchyme, and notochord to form eventually sympathetic ganglia. These ganglia, of course, consist primarily of adrenergic neurones. On the other hand, some of the cells in the cranial segments of the crest migrate to the gut and become cholinergic neurones. These transmitter choices are not. com-
pletely preprogrammed in the crest cells because if cranial selpments of the crest (presumptive cholinergic) are transplanted to the lumbar region (presumptive adrenergh:), some of the cells migrate to the sympathetic ganglia and become adrenergic neurones. The transplanted celi~ were taken from quail embryos which have a distinctive nuclear morphology ano could thus be identified unambiguously in the host (chicken). The reverse transfortnation of a presumptive adrencrgic population into cholinerigic neurones was also p~sible. The influence of the gut itself was demonstrated in an experiment where presumptive adrenergic crest cells were transplanted directly into embryonic, aneural gut. In this isolated target tissue some of the crest cells formed a ganglion and differentiated into cholinerglc neurones. On the other hand, when crest plus neural tube and notochord (tissues which are part of the migratory pathway) were transplanted into the piece of gut, the crest cells became adrenergic. Thus both the target tissue and the migratory environment can influence the transmitter choice. Interestingly, the fate. of the neuronal precursor cells could also be changed even after they had halted their migration, formed a ganglion, and some bqpm differentiation. One example of this is the cholinergic ciliary gangl:.on, if it is "backtransplanted', just after it forms, to a presumptive adrenergic region of the crest, the ganglion cells do not stay together but undergo a second migration. Some of these cells then end up in sympathetic ganglia and become adrenergl~. Thus the transmitter produced by this neural crest population can be influenced even after the cells have migrated and localized themselves.
! 27
T I N S - November 1978
Had any of the cells that bec__ag~eadrenergic after the transplantation previously begun their differentiation as cholinergic neuroncs in the ciliary ganglion? To answer questions of this type, the development of single .,teurones, not populations of cells. must be followed. The population v. single ,:ell problem also means that the transplantation results can he explained by supposing ¢ither (a) that entironmentai factors induced a new or different transmitter in pluripotcnt or reversibly committed cells, or (b) that the environment selected among heterogeneous cells that were already irreversibly committed to one transmitter, in either case, the transplantation studies have clearly demonstrated the profound influence that the developmental environment can have on expression of transmitter functions in fifo. h t fitro stw#ks
A second line of inquiry involves cultures of dissociated rat sympathetic neurones; similar questions to those just considered are being investigated in single neurones and in populations of cellsTM. The type of transmitter (ACh or NA), and the type of synapses formed by these cells, depends on the fluid and cellular environment in which they develop. Because these neurones do not appear to divide in culture, such changgs are postmitotic. The neurones can be grown either in the near absence of other cell types, or in the presence of a variety of non.neuronal cells of known origin. When grown in the virtual absence of other cell types in an appropriate medium, these neurones can develop many of the properties expected of mature adrenersic neurones. They can synthesize, store, take up, and release NA, and form adrenersic synapses. In contrast, when the neurones are co-cultured with appropriate (see below) r,on.neuronal cells, they can produce as mue~ as 1000fold more ACh than cultures that contain only neurones, rhis ACh is used at functional cholinergic synapses between the neurones, or with skeletal or heart muscle cells. The neurones do not have to be in direct contact with the non-neuronal cells for those changes to occur; culture medium that has been conditioned by cultures of appropriate non-neuronal cells (CM) also causes dramatic increases i~.neuronal ACh production and the f r e q ~ of cholin. ersic ~Jnal~¢ formation. At the same time, however, growth in CM causes a marked
reduction in adrenersic prol~'~es. effects of CM are rather specilic in that CM does not affect appreciably either neuronal survival or growth. Thin, certain non-
neuronal cells can produce a diffusable l~lanted from ve~" young rats or in dissignal that has a profound and .,,elective sociated sympathetic neurones from effect on the type of transmitter chosen by animals of relatively )oung ages. if most of the cultured neurones are sympathetic peurones. adrenergic to begin with. and most can be made to become cholinergic, then it is A d r e m ~ drive in cultwe The constancy of survival suggests that possible that single neurone~ pass through CM is not acting by selectively allowing a a transition period, displaying both transclass of predetermined cholinergic neurones mitter properties simultaneously. Such to survive that would otherwise have died. neurones ha,,e been observed with electroExperiments with single cells have sup- physiological techniques: some young ported this conclusion. It is possible to single neurones appear to release both ACh grow single neurones in microcultures and NA at fun.,:!ionai synapses with under various conditions; as many as cardiac muscle cells-~. Such dual-f~nc!ion 80-90% of individual neurones grown on neurone~ ha~e not been obser~'ed reliably heart or skeletal muscle can be cholinergic, with biochemic.al t~hniques in mature while as few as 0% are cholinergic under cultured neurones. It v,iil clearly be of "control" conditions. These observations, interest to foll.:,g a .~ingle cell electroand the constancy of neuronal number and physiologically ~wer a period of becks to growth in the mass cultures, suggests that obse~e the timc- course of these changes. these sympathetic neurones have the capacity to become adrenergic or cholinergic, Why are in vitro t'hanges different from those depending on the signals they receive. As in rivo? One ob~.ious question raised by these with the case of the "back-transplanted" ciliary ganglion discussed earlier, the studies is ~hy is ~t possible in culture to get neurones have retained this plasticity even virtually all of the neurones to become after ganglion formation, in fact, it J~ likely cholinergic. ~hereas. in fifo. histochemical that they remain dcvelopmenta~.ly labile evidence suggeszs that onl) a small minoreven after they begin to expr~s one trans- ity ( --5 40) ofth~ s) mpathetic neurones are cholinergic. Among the ro.~sibIe explanamitter choice. Most, if not all, of the neuroncs express lions are the following: (a) the cholinergic adrenersic properties early in the culture signal m lifo i.~ selecti;el)r localized such period (I week postnatally), and many that onl) a few sympathetic neurone~ were adrenergic as much as a week before recei~,e it: (h) ,";GF exert~ an adren.-'r[ic birth, beginning to differentiate e~en while influence; or (,') ~napti¢ input to the still dividing. These observations are com- ganglion from the spinal cord plays a role patible with the evidence :hat the lumbar in this ,]¢¢i~ion. Considering ,:ach of the.~¢ explanation~ crest cells receive an early adrenergic signal while migrating past the notochord, neural in turn: if the cholinergic ~ignals were tube, and mesenchyme. This signal is selccti~..ly localized (at. for instance in a obviously reversible, however, since 80- targgt v,hich receives sympathetic cholinerg0% of the individual neurones can be- gic innervation, this could explain v,h~ come ~olinergic under appropriate condi- only" tl~,~¢ ,~ympathetlc neurones become tions. However, this plasticity probably cholinelgic. Thi~ possibility remains to be becomes much reduced as the neuroncs definitively tested. What has been done thus mature; cholinergic properties develop in far is to assess the ability of t arious organs culture only in sympathetic ganglia ex- to "condition cholinergically" the medium
Neuropacdia 4. Sympotkelic neurones
#a
128 in culture. The resultsare at once intriguing and puzzling. Targets normally receiving cholinergi~ innervation (skeletal and heart muscle) are far better at conditioning the medium t~an a target roc~'iving only adrenergic innervation Oiver). On the other hand, cell types not known to receive synapses at all (fibroblasts and gila) can also condition the medium. More" conelusive im'ormation may come from purification of the active factor in CM and producing an antiserum to it. Such :zntibudie~ could enable localization ofa sir~ilar factor in fifo. Preliminary evidence suggests that the factor is a heat stable, Imsic protein (M. Weber and P. Patterson, unpublished). NGF inheuee Wh~t is the role of. NGF in tire adrenergic-cholinergic decision (b)? NGF is known to be critical for the development of adrenergic :~ympathetic neurones. However. NGF ha.,, be~n found to .~timulate not only ad~nergic differentiation in the cultures, but also cholinergic differentmtion (in, the presence of CM). In quanlitative comparisons. NGF has been found to promote adrenergic and cholinergic development to the same extent. Thus NGF is a qualitatively different type of developmental signal th~,n CM. NGF is necessary for survival, and stimulates growth and differentiation, but does not tell neurones which transmitter to produce; all three of these properties are .different from those of CM. Symptie hqmts Another way to explain why there are so few cholinergic sympathetic cells in viro is to postulate another factor which prevents most of the neurones from responding to a cholinergic cue. The logical candidate for such a factor is the spinal cord input to the sympathetic ganglion (c), not only because it is normally present in viro, and not in ritro, but because it has been known that denervation of the ganglion in young mice greatly reduced adrenergic development. Therefore the effect of neuronal activity on the adrcnergic-cholinergic decision was studied in culture. The electrical effects of spinal cord innervation of the cultured sympathetic neurones were mimicked by direct electrical stimulation, growth in elevated K ~-, or in veratridine. AH three treatments would be expected to depolarize the cells, but by different mechanisms. Such depolarization resulted in up to 300row lower ratios of ACh production to N~ production than in controlss. Thus.
TINS- November 1978 evoking activity in the neurones made them more adrenergic and less susceptible to the cholinerglg signal, CM. Since the neurones were adrenergic to begin with, depolarization was interpreted to be stabilizing or fixing this initial transmitter choice. ~
- dmlgmslle a t e m e These results give rise to a model of how the neurones in a sympathetk ganglion decide wbeth~ to become adrencrgi¢ or cholinergic. The crest cells destined to form the ganglion are hypothesized to receive an early, but reversible, adrenersic signal, as discussed previously. Most of the ueurom~s are then innervated by their spinal cord inputs and this stabilizes the initial transmitter decision. Some of the neurones, however, are not innervated at this critical time (or t~eir innervation is relatively inactive electrically) and these cells will then be susceptible to a choliuergi¢ sigua~, which may or may not be selectively localized in particular target tissues. neurones will then become cholinergk. This scheme is tentative and oversimplified in many respects, but does make certain predictions which are testable in 1he developing embryo. Furthermore, it broadens our ideas of the possible role of activity in the development of the nervous system. Embodied in the theory is the idea that the specific synap~ formation between certain preganglionic axons and their designated postganglionic neuronal target cells (which is known to occur in these ganglia) would determine not. only the circuitry of the ganglion, but would d~termine what transmitter the postsynaptic cell will use. Thus the developmental information needed for specifu: synapse formation in construction of the appropriate wiring diagram could be used for anoiber developmental purpose as well. otlm mmmmmm~ ~ One question raised by the studies ofthe adrenergic-cholinerglc decision is how generalizable is the concept of environmental influences on the choice of trap.smitter? There are a number of intriguing places in the developing nervous system where the neurones within a particular group or layer look superficially like one another, but end up being heterogeneous as to transmitter content. For instance, the myenteric plexus in the gut contains, in addition to choUner~c neurones, neurones of at least five other transmitter classes. The retina is a highly stratified structure, and yet within several of the layers there is considerabl: heterogeneity of transmitters,
wnh at least ACh, aromatic amines, and pedmps several amino acid transmitters rel.l~-nted. Similarly, in the dmsal root ganglion, sever~ transmitters and/or modulators are represented. Substance P and somatostatin are highly concentrated in different neurones, even though both belong to the C-fibre class of afferents. The qumtion is (as it is with the retina and myenteric plexus) do these neurones make and receive different patterns of input and output connections, and, if so, do these differences influence the final choice of peptide or transmitter? Of interest in this context is the recent finding that the substance P to somatostatin ratio in dorsal root ganglion cultures can be altered as much as 30-fold by the presence of uon. neuronal cells (or CM), without changes in neuronal survival (A. Mudge, personal communication).
ne~U~ am 1. Btmge, R.. Johmon, M. and Rots, C. R. (1978) Science 199, 1409-1416. 2. Furshlmn, E. J., MacLemh, P. R., O'Lalgue, P. H. and Potter, D. D. (1976) Proc. Nat. Acad. StL U.S.A. 73, 422.5-4229. 3. Hendry. !. A. (1975J Bea/n Res. 94. 87-97. 4. Hcmdry, L A. and Campbell, J. (1976) J. Neurocytol. 5, 351-360. 5. LcDouarin, N. M., Teillgt, M. A., Zillm', C. and Smith, J. (1978) Proc. Nat. Acad. SoL U.S.A. 75, 20~)-2034. 6. Patterson, P. H. (1978) Annu. Rev. Net,.~¢i. I, 1-17. 7. Wslicke. P. A.. Cami~not. R. B. and Patterson, P. H. (1977) Prof. Nat. Acad. S¢I. U.S.A. 74. $767-5771.
P. H. Patterson b aa Anoclate Profetwr in tke Dei:artmeat of Nearobiology at Harvard Medical School, Boston, MA 02115, U.S.A.
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