0306-4522/91$3.00+ 0.00 Pergamon Press plc 0 1991IBRG

Neuroscience Vol. 44, No. 2, pp. 449455, 1991 Printed in Great Britain

PHYS~ULO~ICAL INTE~CT~~NS BETWEEN ENKEPHAL~N AND EXCITATORY AMINO ACIDS IN THE CEREBELLAR CORTEX OF THE OPOSSUM ~~~~L~~~~ ~A~SU~IAL~~ ~r~GI~~A~A G. A. BISHOP Department of Cell Biology, Neurobiology and Anatomy, The Ohio State University, 333 W. 10th Avenue, Columbus, OH 43210, U.S.A.


opiate peptide enkephahn has been immunohistochemi~lly localized within specific populations of climbing fibers and mossy fibers in the opossum’s cerebellum. The intention of the present study was to determine the physiology effects of this peptide on Purkinje cell activity as well as to examine interactions between this peptide and the excitatory amino acids glutamate and nspartate. Iontophoretic appli~tion of enkephalin onto Furkinje cells in the posterior lobe vermis and adjacent hemisphere suppressed activity in nine of 16 (56%) spontaneously active units. Enkephalin increased the spontaneous activity of one unit and had no effect on six (38%) of the units. In addition, this peptide blocked the excitatory effects elicited by iontophoretic application of glutamate in 34 of 35 (97%) units tested and of aspartate in all cases. Enkephafin had no effect on one cell activated by ~~tamate. Simultaneous application of naloxone, a nonspecific opiate receptor antagonist, did not block the suppressive effects of enkephalin. Rather, there was a potentiation of suppressive responses as compared to that seen when each is applied alone. The results suggest that classically defined excitatory afferent projections to the cerebellum may be capable of both exciting and suppressing the activity of their target neurons The excitatory action is likely mediated by an amino acid, wbereas the release of the peptide enkephalln results in a decrease in unit activity. Further, it appears that enkephalin mediates its suppressive effect through mechanisms that are not mediated by opioid receptors.

The major

extrinsic projections to the cerebellar cortex have been identified as climbing fibers, derived

exclusively from neurons located within the inferior olivary complex, mossy fibers, arising from cells located in specific brainstem and spinal cord nuclei, and a beaded plexus of serotoninergic fibers that originate from specific reticular nuclei within the brainstem.‘~10*‘t~31 Climbing fibers and mossy fibers are excitatory to their respective target neurons,1° whereas serotonin appears to have a variable effect on Purkinje cell act~vity~~~“~*‘~** To mediate these excitatory effects, climbing fibers are thought to use the amino acid aspartate, whereas the mossy fiberparallel fiber system appears to release ghnamate.‘o In addition, i~~o~st~herni~ studies have identified several peptides within climbing fibers including enkepbalin (ENK), i2,31corticotropin releasing factor (CRF),4,6 and cholecystokinin.i3 Cummings and King5 have further shown that ENK and CRF are co-localized in discrete populations ofclimbing fibers. Mossy fibers have also been shown to contain peptides including ENK,*2~23*s’CRF,4*6 calcitonin generelated peptide= and ~hol~ystoki~i~.i3 Ahhough these peptides have been anatomically localized in cerebellar projections little is known about their physiolo~ca1 effect(s) in regulating cerebellar activity. -_ &hrevia&rns: CRF, corticotropin releasing factor; ENR,

The focus of the present study is to determine the physiolo~cal effects of ENK on Purkinje cell activity as well as to examine interactions between this pep tide and the excitatory amino acids aspartate and glutamate.


Adult opossums (n = 18) of both sexes were anesthetized with a combination of sodium thiaiymal (40 mg/kg) and aipha~h~oraIose (70 mg/kg) and secured in a stereotaxic frame. The occipital bone over the posterior lobe vermis and adjacent hemisphere was removed for plant of recording electrodes. The animal’s temperature was monitored and maintained at 36°C with a heating pad. The animal’s heart rate was also monitored #roughout the experiment. In addition, reiiexes were routinely tested and supplemental doses of anesthesia were given as needed. Recording electrodes One barrel of a multibarrel micropipette was filled with 4 M NaCi for purposes of recording neural activity. A second barrel was also filled with 4 M NaCl and functioned in an automatic balancing circnit that eliminated the possibility that the observed responses were caused by the appiied current rather than the substance being tasted. The remaining barrels were filled with combination of the fohowing substances: 0.01 M ENK, pH 4.2; 0.2 M glutamate, pH 7.5; 0.2 M aspartate, pH 7.5; and 0.05 M naloxone, pH 4.0. During advancement of the electrode, retaining currents of at least 10 nA af appropriate polarity were applied to prevent

enkephalin. 449




leakage of the various chemicals. Followmg extraceliular

isolation of a unit the currents were reversed to eject the substances. Gfutamate and aspartate were ejected with negative current whereas ENK and na~oxune were iontophoresed with positive current with respect to preparation ground. The extracellular responses were amplified on a Dagan pre-arn~lj~er, monitored on an oscilloscope and converted to a uniform voltage pulse by passage through a window discriminator. These +ses were counted by a ratemeter over 1-s intervals and dispfayed on a strip chart


RESULTS In &is study, 50 units were isolated extrace~~ularly in the posterior vermis or adjacent hemisphere. Histoiogicahy there are five different neurons within the cerebeliar cortex including granule cells, Golgi cells, basket cefis, stellate ceils, and Purkinje cells. The identity of isolated units was determined, in part, on the basis of their response properties. Units were identified as Purkinje cells if they gave rise to a high frequency burst of action potentials” (Fig. IA, B, arrow) superimposed on a firing pattern consisting of unitary spikes. This high frequency response is elicited by activation of the climbing fiber system which is known to give rise to a complex action potential that is restricted to the Purkinje cell. However, chmbing fibers have a tow firing rate (5--10/s) and thus the complex spike is infrequentfy seen. Further, only 32% of the units in this study showed spontaneous activity. Thus, identi~~at~on of the remaining units as Purkinje cells was not conclusive. However, for the following reasons the neurons from which the data for this paper were obtained were tentatively deignated as Purkinje cells. Firstly, given that granule cells are only 5-8 pm in diameter, it is unlikely that they would be isolated by a muftibarrel pipette with a tip diameter of 1-4 pm. Secondly, basket cells and stellate cells are also relatively small (10-15 pm) and are scattered in the molecular layer; thus, they are also difFicuh to isolate. Thirdly, whereas Golgi ceils have a large diameter, they are relatively few in number and scattered throu~haut the granule cell payer. Finally, the consistent response of isolated neurons, both those with and without complex spike activity, suggests that they belong to a single population of neurons. However, it is possible that all isolated neurons were not Purkinje cells. Thus, the genereiized term “unit” will be used in this text. elf the isolated units, 32% exhibited spontaneous activity. The absence of spontaneous activity in the remaining units is presumably due to effects of anesthesia in this in v&o preparat.ion. Firing of nonspontaneously active units or those with a low ievef of spontaneous activity was achieved by iontophoresis of either glutamate (Fig. 1C) or aspartate (Fig. IF, G). Data are illustrated in two ways in this paper. Firstiy, OsciIlographi~ tracings are shown in which each trace represents a single sweep of the oscilloscope beam. In addition. chart recordings are used to document

the long-term effects of the various neuroactive compounds. fn these recordings each upward deflection represents the number of action potentiafs generated

by a unit in a I-s intervai as counted by a window discriminator. Iontop~oretic application of ENK reduced the firing rate of the fast majority of s~n~neo~s~~ active units (Figs 13, E, 3E, open arrows). In addition, this peptide interacts with the excitatory amino acids glutamate and aspartate. Iontophoresis of either amino acid alone results in an enhancement of spontaneous activity (Fig. 1C. E-G). Simultaneous application of ENM blocked the augmenting action of either glutamate or aspartate (Fig. ID-G) in a dose-dependent manner. However, it should be noted that currents that completely suppressed spontaneous activity (e.g. Fig. 1E, +20, ~30 nA) only partially biockrrd the amino ~~~d-~ndu~~ activity (Fig. IE). fn some units the suppressive effects of ENK appeared almost ~nstantal~~Q~sIy (Fig. 1E); as soon as the current was applied to the barrel the neuron’s activity decreased. However, for the majority of cells (Fig. IF, G) there was a more gradual time-course characterized by an initial slowing of activity foIlowed by a more intense suppression of unit activity. These differences in response properties may represent di~eren~al numbers ofreceptorsor sensitivity ofreceptors present. Further, differences in response may reflect physical constraints of the experiment such as the position of the electrode relative to the ceif or amount of peptide rekdsed during initial versus Later stages of current apghcatian. Recovery of the cell to the control level of activity was also gradual for most cells (Fig. IF, G) and is likely related to mechanisms for uptake and clearance of the peptide from the area of the isolated neuron. In one isolated unit, iontophores~s of ENK resulted in an increase in activity {Fig. IH). The response properties of this ceil suggest that it is likely not a Purkinje cell. The histograms in Fig. 2 summarize the effects elicited following iontophoresis of ENK on spontaneously active units. In addition. the histograms sum~rj~e interactive effects between the peptide and two exc~tat~~~ amino acids, glutamate and aspartate. Briefly, ENK suppressed spike activity in nine out of 16 spontaneously active units (56%) and had no effect on six of these cells (38%). In contrast, ENK effectively blocked glutamate-induced activity in 34 of 35 units (97%) and all five units activated by aspartate. Naloxone. a nonspecific opiate receptor antagonist was s~mu~taneo~ly applied to eight isolated units to determine if it would block the ENK-induced suppression af unit activity. In seven of the eight units, naloxonc itself decreased both spontaneous and g~utam~te~~~duced activity. Further, when naloxone and ENK were simultaneously applied, the suppressive effects of either applied alone was increased. The results of an experiment which shows the effects of


Physiological effects of enkephalin SPON +ENK


; T


L lOmaw




ASP -22

Fig. 1. Effects on ENK on unit activity. (A-D) Oscillographic tracings that illustrate the suppressive effect of ENK on spontaneous (A, B) as well as glutamate-induced activity (C, D). (E-H) Chart recordings that illustrate ENK’s effect on spontaneous as well as glutamate- and aspartate-induced unit activity. Each upward deflection represents the number of action potentials fired by the unit in a l-s interval. In some cells application of glutamate results in an immediate suppression of activity (E). However, in most cells

onset of the effectas well as recovery to control levels is gradual (F, G). The effectis dose dependent (E, F). In one cell ENK increased spontaneous activity (H). naloxone on glutamate-induced firing are shown in Fig. 3. The effects of iontophoresis of naloxone or ENK are shown in Fig. 3E. In the first part of the chart recording, glutamate was continuously applied to the

unit. Both naloxone and ENK applied alone decreased activity in a dose-dependent manner (Fig. 3E, open arrow). Application of naloxone during the time ENK is applied or vice versa further reduced the firing frequency of the unit. The second half of the chart


G.A. BlSHOP n=5

n--35 100 90 80


70 n=




I decreased activity

16 no effect increased activity

50 O-

40 30


20 10 0





Fig. 2. Histograms that summarize the effects of iontophoretic application of ENK to isolated units in the cerebellar cortex. The effects of ENK on spontaneous- (Spon), glutamate- (Glut) and aspartate (Asp)-induced activity are shown. (See text for details.)


5mV l lOmsec Glut + ENK

Glut + ENK + Nal

E | m

Nal +50


Nal +10



Nal +10

Nal+5 ENK +32

ENK +20 Glut - 1 7


~lal+5 ~ ENK +15


Nal+5 ENK +15


Nal+15 Nal÷5 "--" ENK +70

ENK +21

Nal+5 ~ ENK +7

5 s

~ ENK ÷7


Fig. 3. Oscillographic tracings and chart recordings that illustrate interactions between ENK and naloxone. (A-D) Series of oscillographic traces that illustrate the firing rate of a unit following application of different combinations of chemicals. Glutamate activates the unit (A). Simultaneous application of glutamate and either ENK (B) or naloxone (Nal, C) depresses the glutamate-induced activity. Simultaneous application of both naloxone and ENK have a summative effect in completely blocking glutamate-induced activity (D). (E) Chart recording that illustrates the effects of naloxone (Nal) and ENK on the firing rate of the unit. Naloxone blocks glutamate activity in a dose-dependent manner. (Compare amount of suppression induced by application of naloxone at 50 versus 10 nA.) Application of ENK at 20 nA suppresses glutamate-induced activity. Simultaneous application of naloxone potentiates the suppressive effect of ENK. Reversal of the order of application of naloxone and ENK have similar results. Similar interactions occur when the unit is spontaneously active (open arrows).


Physiological effects of enkephalin


(see Ref. 10 for review} have shown that this excitation is likely to be mediated by the amino acid transmitters aspartate, in climbing fibers, and glutamate, for the mossy fiber-parallel fiber system. The presence of ENK in discrete populations of climbing fibers and mossy fibers is thus somewhat paradoxical on the basis of data derived from the present physiolo~~al study, as this peptide powerfully suppressed spontaneous Purkinje cell activity, as well as glutamate- and aspartate-induced activation of the neuron. ENK has been shown to exert a suppressive effect in other areas DISCUSSION of the CNS including the ~p~ampus,*~4 hypothalamus,3 striatum,‘4 inferior olive, 2z locus coeruleus t32 ENK is a member of the opioid peptide family that and spinal cord.33 has been ascribed roles in pain perception, stress It is not known at present, when or under what mechanisms, respiratory regulation, temperature concircumstances ENK is released from axon terminals. trol, and behavioral patterns such as feeding, drinking, In the sympathetic nervous system peptides have been grooming, and sexual activity (see Ref. 25 for review). co-localized with excitatory amino acids.‘G’8 In this Recent i~~o~st~he~~~ studies’2*u*31 have shown that this peptide is present in climbing fibers and mossy peripheral system the release of peptides from the terminal is dependent on firing frequency. If the axon fibers, classically defined aRerent systems within the is firing at low frequency the amino acid is released. cerebellar cortex of the opossum. King et ~1.‘~have described the distribution of this It is not until the neuron achieves a higher firing peptide based on an analysis of serial sections of the frequency that the peptide is released. Studies on release mechanisms in the CNS have not been carried opossum’s cerebellum. Not all climbing fibers or mossy fibers are immunor~ctive for this peptide; rather there out; however, these data suggest that the level of are discrete ~p~ations of both afferents which have activity in a particular afferent system may determine which neuroactive substance(s) is (are) released and a precise topographic distribution.‘2 Enkephaliner~c climbing fibers are restricted almost exclusively to consequently whether the target cell is activated or three parasagittal bands within the anterior lobe suppressed. In the cerebellum, ENK appears to exert its effect(s) vermis, to iobules VIII and X of the posterior vermis through mechanisms that are different from those used and to the lateral aspect of the flocculus. Immunoreactive mossy fibers have a similar lobular distribuby opiate peptides in the pain pathways. In the present tion; however, within the folia they are not confined study the suppressive effects of ENK were not blocked to specific parasagittal zones. In addition to these by simultaneous application or pretreatment with the opioid receptor antagonist naloxone. Although someclassicalIy defined alTerent terminals, ENK immunoreactivity is also present in a beaded system of fibers’* what paradoxical, the results of the present study within the Purkinje cell layer. The dist~bution of this are consistent with other investigations3,7-9,24~30 which beaded system is homogeneous throughout all cere- have shown that naloxone does not always block bellar lobules, although there are differences in the opiate effects. intensity of staining in disparate regions of the cortex. In the hippocampus, Fry et al.” presented similar This heterogeneity in distribution may account for results to those described in the present study. Nalsome of the variability of peptide effects seen in the oxone not only failed to antagonize the inhibitory present study. For example, ENK had no effect on effects of applied opioid peptides, but the chemical the firing rate of six spontaneously active units or on frequently potentiated an excitatory response to these one unit activated by appli~atjon of glutamate, It is peptides. They defined these effects as nonspecific possible that these neurons were not located in areas opioid actions. Finally, naloxone was also ineffective in which enkephalinergic afferents are present. Other in blocking the suppressive effect of morphine on possibilities for the variation may be related to medial thalamic neurons7 as well as tu~rojnfundibular receptor distribution or sensitivity. At present no neurons3 These findings suggest that the receptors in detailed data are available on the ~st~bution of the cerebellum res~nsible for the suppressive effects opioid receptors in the opossum’s o~&llum. Finally, of ENK must be different from classically defined, the population of neurons that showed the greatest naloxone-sensitive opioid receptors.3s32 To date, no variability was those that were spontaneously active. data are available with respect to the distribution ENK effectively blocked glutamate (96% of units or type of opiate receptors in the cerebellum of the opossum. Although opiate receptors have been tested) and aspartate (100% of tested units) activated cells. These data suggest that ENK may specifically identified in the cerebelli of several species, it is di&ult interact with receptors for these amino acids. to extrapolate these data to the opossum as species Functionally, climbing fibers and mossy fibers are variations have been reported. In the rabbit, 80% of defined as being excitatory to Purkinje cells and the opiate receptors in the cerebellum are of the mu granule cells: their respective targets. Several studies type. In contrast, the kappa type was the predominant recording illustrates comparable effects on spontaneous activity. It should be noted, however, that the individual as well as the combined effects of naloxone and ENK are more potent in the absence of glutamate (Fig. 3, cf. naloxone +5 with and without glutamate present). Currents of comparable or greater intensity applied to the control barrel of the electrode had no effect on the firing rate of the isolated unit (not illustrated).


G. A

receptor (> 80%) in the guinea-pig cerebellum.2’ Several different opiate receptors have been localized in the rat’s cerebellum including mu, kappa, and sigma. I9 Future studies are needed to determine the type and density of distribution of opioid receptors in the opossum’s cerebellum. Finally, it is possible that the effects may be related to the presence of different types of naloxone-~nsitive receptors in the cerebellum. Two distinct naloxone binding sites have been identified in the rat’s brain, labeled Type I and Type II, which have different characteristics and regional distributions.26 The Type II receptor, which is predominant in the cerebellum has a lower affinity for naloxone (& of 16 nM versus a I& of 2 nM for the Type I receptor). Further, binding to Type II sites decreases rapidly with increasing incubation temperature; in contrast, binding at Type I sites is unaffected by changes in temperature. These data suggest that naloxone and several other opiate agonists and antagonists bind to two distinct receptor sites. It is also possible that some of the observed effects in the opossum’s cerebellum are nonspecific8 or not mediated by opiate receptors. In the striatum, it has been postulated that ENK may non-competitively block glutamate from binding to its postsynaptic receptors.14 These investigators also found that the opioid antagonist naloxone was ineffective in blocking this effect. Naloxone failed to block the suppressive effects of morphine, an ENK agonist, in the cat’s cerebellum.30 However, naloxone did antagonize excitatory responses. The results of this study suggested that the excitatory effects were mediated by the opiate system, whereas the inhibition may have been related to the GABAergic system of the cat’s cerebellum. This would not be inconsistent with the findings of the present study. One cell was found that responded with an increase in activity. It is likely that this unit was not a Purkinje cell and may have been one of the interneurons in the cerebellar cortex. If this is confirmed in future studies, it suggests that the suppressive effects of ENK on Purkinje cells may be mediated via these inhibitory interneurons. These observations


could also be used to explain the observation that the effects of ENK were not always instantaneous in suppressing Purkinje cell activity. Since more than one neuron is involved in the circuit there may be a delay until the effect is relayed to the isolated Purkinje cell. Future intracellular-iontophoretic studies are needed to confirm this su~estion. Several hypotheses as to how ENK mediates its effects have been postulated. In a study carried out in the rat’s locus coeruleus3* it was suggested that ENK functions by increasing K + conductances which result in hyperpolarization of the neurons with subsequent suppression of activity. LlinPs and Sugimori15 have described several K+ conductances in Purkinje cells which when activated are thought to play a role in decreasing neuronal activity. If ENK does act by increasing K + conductance it may be involved in generating the rhythmic firing pattern characteristic of Purkinje cells. It has also been suggested” that ENK blocks adenylate cyclase activity in neurons which leads to suppression of activity. This latter mechanism of action has been described in the cerebellum of the rabbit and guinea-pig. Future studies in the opossum cerebellum are needed to determine if different receptor types are present and how ENK exerts its suppressive effect on Purkinje cell activity.


The data obtained in this study suggest that the classically defined afferent systems to the cerebellum are capable of both exciting and suppressing the activity of their target neurons. The precise conditions under which either or both effects are elicited and the mechanisms involved remain to be determined. Acknowledgements-This work was supported by NIH grant NS08798 awarded to Dr James S. King. I wish to

thank Drs King and Richard Rogers for reading the manuscript and offering editorial suggestions. I gratefully acknowledge the technical expertise of Mrs Katharine Dillingham and the photographic assistance of Mr Karl Rubin.

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Physiological effects of enkepbalin


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17 March 1991)

Physiological interactions between enkephalin and excitatory amino acids in the cerebellar cortex of the opossum Didelphis marsupialis virginiana.

The opiate peptide enkephalin has been immunohistochemically localized within specific populations of climbing fibers and mossy fibers in the opossum'...
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