Electrophysiological evidence for a non-opioid interaction between dynorphin and GABA in the substantia nigra of the rat

Vol. 23, No. 2, pp. 483490, Great Britain

Neuroscience

Printed in

0306-4522/87 $3.00 + 0.00 Pergamon Journals Ltd

1987

ELECTROPHYSIOLOGICAL EVIDENCE FOR A NON-OPIOID INTERACTION BETWEEN DYNORPHIN AND GABA IN THE SUBSTANTIA NIGRA OF THE RAT B. C. ROBERTSON,D. W. HOMMER and L. R. SKIRBOLL Electrophysiology

Unit, Clinical Neuroscience Branch, NIMH, Bethesda, MD 20892, U.S.A.

Abstract-Interactions between neuronal responses mediated by dynorphin Al-8 and GABA were investigated in the substantia nigra zona reticulata. Extracellular recordings and microiontophoresis were performed using five-barrel microelectrodes in chloral hydrate-anesthetized male rats. When iontophoresed alone, dynorphin Al-8 significantly inhibited the firing of 22% of the neurons tested. The inhibition was rapid in onset and recovery and was dose-dependent. In another 22% of the cells, iontophoretic dynorphin produced an increase in the baseline firing rate which was slow in both onset and offset; the remaining 56% were unaffected by dynorphin. When GABA and dynorphin Al-8 were applied in conjunction, the inhibitory action of GABA was attenuated in 61% of the cells; whereas, when dynorphin and GABA were ejected simultaneously onto the cells that were inhibited by dynorphin Al-8, the respective inhibitory effects of dynorphin and GABA appeared to be additive. The kappa antagonist, MR-2266, failed to block the ability of dynorphin Al-8 to attenuate the action of GABA. In addition, the non-opiate peptide des-tyr-dynorphin A2-17, produced effects similar to that of dynorphin Al-8. The role of dynorphin in the basal ganglia and its interaction with the other major transmitter in the substantia nigra zona reticulata, GABA, is discussed.

Dynorphin is a 17-amino acid opioid peptide originally extracted and purified from porcine pituitary.r2 It contains a [Leulenkephalin (Leu-ENK) sequence at the amino acid terminus and is distinguished by its extraordinary potency as an opiate agonist in the in vitro guinea-pig myenteric plexus muscle bioassay.5.6,‘2 In addition, there is also good evidence that dynorphin is the specific endogenous ligand for the kappa opiate receptor.5~8~24 In the brain, the amino acid terminal fragment, dynorphin l-8, is present in up to IO-fold higher concentrations than dynorphin 1-17.4o The highest concentration of dynorphin in the brain is found in the substantia nigra,42,43specifically in the substantia nigra zona reticulata (SNZr) were dynorphin Al-8 is present in a 16-fold higher concentration than dynorphin A1-17.9 The major neuronal input into the SNZr arises from cells in the striatum and pallidum, the axons of which descend in the striatonigral pathway. Striatal-nigral fibers contain GABA,“, substance P3 and dynorphin.‘0~2’*25~‘4~35 Studies have also shown co-localizations of GABA and enkephalin in the caudate.‘s23 The striatum is important in regulation of a variety of motor behaviors. Since the pars reticulata, along Address for correspondence: Dr L. R. Skirboll, Electrophysiology Unit, Clinical Neuroscience Branch, NIMH, Bldg 10, Rm 4N-214, Bethesda, MD 20892, U.S.A. Abbreuiutions: Leu-ENK, [Leulenkephalin; Met-ENK, [Metlenkephalin; MR-2266, (-)2-(3_furylmethyl)-5,9,diethyl-2’-hydroxy-6,7benzomorphan; SN, substantia nigra; SNZc, substantia nigra zona compacta; SNZr, substantia nigra zona reticulata.

with the globus pallidus, receives virtually all of the efferents from the striatum, it is not surprising that the SNZr is an important relay station for information relating to posture and motility. Several lines of evidence indicate that the muscular rigidity observed after the administration of morphine in rats is mediated through opiate receptors in the striatumr5 and/or the SNZr.32x33These data, taken together with the evidence of dense dynorphin projections in the SNZr, led us to undertake an electrophysiological study of the interactions between dynorphin and GABA in the SNZr. EXPERIMENTAL PROCEDURES Recordings were performed in male Sprague-Dawley rats weighing 2o(r250 g anesthetized with chloral hydrate (400 mg/kg i.p.). A 3 mm burr hole was drilled at coordinates overlying the substantia nigra, i.e. - 5.0 mm from bregma and 2.2 mm lateral to the midline according to the atlas of Paxinos and Watson.26 Supplementary chloral hydrate was given via a lateral tail vein. Temperature was continuously monitored with a rectal probe and maintained at between 3637°C. Standard single-unit extracellular recording techniques were employed and described in detail elsewhere.3’ Fivebarrel glass micropipettes were used for recording and for ejection of drugs. Micropipettes were preloaded with glass filaments. After being pulled, pipette tips were broken back to a diameter of approximately 5-10pm. The central recording barrel was filled with 0.2% Pontamine Sky Blue in 2 M NaCl. The balance channel was filled with 2 M NaCl. The three remaining barrels were each filled with a combination of the following drug solutions: 1 mM dynorphin Al-8 (Peninsula Labs) in 0.16 M NaCl, nH 6.0: 1 mM des-tyr-dynorphin A2-17 (Peninsula Labs) in 0.16 M NaCl pH 6.0: I mM [Metlenkephalin (Met-ENK; Peninsula Labs) in 0.16M NaCl, pH4.5; 1 mM GABA (Sigma) in 0.16M

484

B. C.

ROBERTSONet ul

NaCI, pH 4.0; 0.16 M NaCI, pH 6.0. The in oitro impedance of the recording barrel ranged from 2.0 to 4.0 MB; for the drug-containing and balance channels it was 2&60 MO. Between periods of iontophoresis, negative retaining cmrents of - 10 to - 20 nA were passed through the drug barrels. All drugs were ejected with positive currents of 2-90 nA. Mu and kappa opiate antagonists, naloxone and MR-2266 I(-)2-(3-furylmethyl)-5,9-die~yl-~-hydroxy-6,7benzomorphan], a gift from Boeringher Ingelheim, were respectively delivered via the lateral tail vein in doses of 0.1-S mg/kg. Effects of antagonists were examined from 5 to I5 min post-injection. Electrodes were lowered through the burr hole into the brain using a hydraulic microdrive (Kopf). Electrode potentials were passed through a high-impedance amplifier and monitored on an oscilloscope and audiomonitor. The tiring rate of single neurons was counted by a rate averaging instrument over 5 s intervals and recorded as a histogram on a strip chart recorder. Recordings were obtained from either SNZr or zona compacta (SNZc) neurons identified on the basis of firing rate, pattern and spike duration and morphology,4.‘3 Responses to repeated iontophoretic pulses of GABA (15 s duration at 30 s intervals) were examined before, during and after simultaneous iontophoresis of either dynorphin Al -8, des-tvr-dvnornhin A2-17. Met-ENK or NaCl. In other cases: responses to iontophoretic pulses of dynorphin A l-8 (15s duration of 30 s intervals) were examined. At the conclusion of the experiment, the location of the electrode tip was marked by the iontophoretic ejection of Pontamine Sky Blue. The brain was removed and placed in 4% paraformaldehyde. Frozen serial sections were cut at 20 pm intervals on a cryostat and examined for the location of the recording spot. RESULTS

Effects of GABA and dyrzorphin A 1-8 on baseline activity GABA, iontophoretically applied consistently and effectively, depressed the firing rates of every SNZr

DYNORPHIN 20 -0-m

1

20

30

3OnA

-z

3

3c

0

Fig. I. Response of SNZr cell to the iontophoretic administration of dynorphin Al-8. The peptide produced a dosedependent decrease in cell firing during iontophoresis. Horizontal bars indicate duration of ejection and numbers indicate ejection current.

neuron tested. Uniform current pulses of’ I5 s duration (2-15 nA) repeatedly applied at 30 s intervab produced a 5@IOO% inhibition of SNZr firing rates. The responses

to iontophoretic

dynorphin

Al -8

were variable. Of the 88 SNZr cells tested 19 (22%) showed a 50-100% inhibitIon in response to dynorphin Al-8 (IS-60 nA; Fig. 1) while another 19 (22%) showed a significant increase (18 + 2.2%; r-test, t = 9.82, P < 0.001; Fig. 2B) in baselme firing rate. The baseline activities of the remaining 56?b were not significantly altered at currents of up to 80 nA. The inhibitory and excitatory profiles of dynorphin Al -8 response were quite different from each other. In the majority of cases, the inhibitory response was rapid in onset and offset (within 5 s) and was dosedependent, i.e. increasing nA current produced successively greater inhibition. In contrast. the excitatory response to dynorphin Al -8 was slow in onset and offset and was not always dose-dependent. In most cases, the peak excitatory effect was observable only after as much as I min of continuous iontophoretic application. Interactions between CABA and dynorphin To investigate the possible interactions between dynorphin AI-8 and GABA, two subpopulations of SNZr neurons were examined. The first group was composed of cells which showed a rapid inhibition in response to dynorphin Al-K Ejection currents were applied to the GABA barrel which, when administered alone, were sufficient to depress neuronal Bring by approximately 50%. After at least six pulses of GABA, peptide was delivered simultaneously with GABA. Under these condttions, the respective inhibitory effects of dyno~hin Al-8 and GABA appeared to be additive. In the second group, 62 cells which did not show a rapid inhibitory response to dynorphin received six warm-up pulses of GABA; the GABA was subsequently turned off and dynorphin Al -8 was applied alone for 30-90 s. After this the GABA current was turned on again while dynorphin continued to be administered. GABA was delivered for three more cycles and dynorphin Al-8 was turned off. GABA pulses were continued to monitor full recovery. In 38 (61%) cells, the inhibitory effect of GABA on SNZr neurons was significantly attenuated (paired t-test, I = 9.96, P < 0.001; Fig. 2). GABA-induced inhibition was blocked b> 14100% (mean = 38 + 3.4%). In addition, the ability of dynorphin to attenuate the actions of GABA appeared to be dose (current)-dependent, i.e. increasing currents of dynorphin A 1-8 (20-90 nA) produced increasing degrees of GABA block. Full recovery of the GABA inhibition was obtained in most cells 30-60 s after dynorphin was turned off. The ability of dynorphin Al-8 to block the effects of GABA appeared to be independent of its actions on baseline activity. There was no stgnificant correlation (Pearson product moment) between baseline firing rate and GABA

GABA and dynorphin interactions in the substantia nigra

485

unaffected by dynorphin A1-8. Finally, in at least 15 cells dynorphin A1-8 failed to have any effect on G A B A inhibition. ] B

J

GIABA ÷ OYN A(I-e)

EffEcts of NaCl and [Met]enkephalin on firing rate and GABA response

DYNORPHIN 40nA 10

10

10

lOnA

'~s

Fig. 2. Interaction between GABA and dynorphin (DYN). (A) Simultaneous administration of GABA and dynorphin AI-8 produced a significant attenuation of the inhibitory effects of GABA. (*, paired t = 9.96, P < 0.001, n = 38). (B) Representative response of an SNZr cell to the administration of both GABA and dynorphin A1 8. GABA alone significantly inhibited the firing of the cell. Simultaneous administration of dynorphin during the GABA pulses attenuated GABA-induced inhibition. Note slow increase in baseline firing during dynorphin ejection. Horizontal bars indicate duration of ejection and numbers indicate ejection current. inhibition in either the absence (r = 0.17) or presence (r = 0.02) of iontophoretically applied dynorphin A I - 8 . This blocking action was observed both in neurons that showed the slow excitation in response to the peptide (n = 19) as well as in cells that showed no change (n = 21) in baseline activity. G A B A inhibition was potentiated (50 + 2.3%) in only 9 cells (19%). Two of these cells demonstrated the slow excitation in baseline activity while seven were

To gain insight into the specificity of the effects of dynorphin on S N Z r activity and its ability to modulate G A B A responsivity, the ability of M e t - E N K and NaC1 to interact with G A B A was examined. To test the effect of the vehicle (1.6 M NaCI), an equimolar solution of NaCI was iontophoretically administered during pulses of G A B A . In six cells in which dynorphin A1-8 attenuated the G A B A response, NaCI had no significant effect on G A B A induced inhibition (Fig. 3). In eight additional cells in which NaC1 was tested without prior administration of dynorphin A1-8, only one cell was attenuated while the others were not significantly affected~ In 10 cells, the effects of the iontophoretic administration of M e t - E N K was examined. M e t - E N K administered in currents up to 50 nA had no significant effect on baseline firing rate. In experiments in which the interactive actions of G A B A and M e t - E N K were examined, paradigms identical to those described for d y n o r p h i n - G A B A interaction were employed. U n d e r these conditions, the peptide had no effect on the ability of G A B A to inhibit neuronal activity (paired t-test, t = - 1.09).

Evidence for a non-opiate effect of dynorphin To examine whether the inhibitory effect of dynorphin A I - 8 was related to opioid function, either the mu antagonist, naloxone or the kappa antagonist, MR-2266, were given intravenously (cumulative

DYN 50nA I

10

10

NaCI 60nA

l

10

10

I

10

10

I

10

10

10 nA GABA

20-

°~

0

I

i 60s

Fig. 3. Response of a single SNZr unit to the simultaneous administration of dynorphin (DYN) A1-8 or NaCI and GABA. Record shows the ability of dynorphin to reduce the GABA response while NaC1 has no effect. Horizontal bars indicate duration of ejection and numbers indicate ejection current.

486

B. C . ROBERTSON e t al.

NALOXONE 0.2 0.4 0.8

1.6 mg/kg

! 1 1 1--20

15

I,~,5 --20 --20 --20

20 nADYNA

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0 I

I

60s Fig. 4. The inhibitory effects of iontophoretic dynorphin (DYN) are not blocked by the i.v. administration of naloxone. Repeated administration of dynorphin for up to 30 min post-naloxone administration yielded similar results. Horizontal bars indicate ejection time and numbers indicate ejection current. Arrows denote i.v. administration of naloxone in doses indicated.

doses of 0.1 5mg/kg). Dynorphin responses were tested up to 30 min after administration of the antagonist. Neither naloxone (n = 5; Fig. 4) nor MR-2266 (n = 5) blocked the inhibitory actions of dynorphin A 1-8. In a separate set of experiments, MR-2266 also failed to block the ability of dynorphin A 1 - 8 to attenuate the actions of G A B A (n = 5; Fig. 5). In contrast, des-tyr-dynorphin A2-17 (20-60nA), a dynorphin analogue which has been shown to act at a non-opioid site, produced a significant attenuation of the G A B A response (paired t-test, t = 2.447,

P < 0.01) similar to that seen with dynorphin A1 8 (n = 7; Fig. 6).

Effects o f dynorphin A 1-8 on dopamine neurons In order to determine the specificity of the effects of dynorphin with regard to neuroanatomical loci, the effects of this peptide on activity in the SNZc were examined. These neurons are effectively inhibited by G A B A but this area of the brain is devoid of dynorphin immunoreaetivity. In 10 cells, dynorphin A1-8 in currents of 2 0 - 8 0 n A was ineffective in

MR-2266 (2 mg/kg) d,yn4OnA ~ dyn,4OnA 10

10

I

60s

10

10

10

10 nA GABA

I

Fig. 5. Ability of dynorphin (DYN) to attenuate GABA-induced inhibition is not blocked by the administration of the kappa antagonist, MR-2266. Repeated administration of dynorphin up to 30 min after antagonist administration yielded similar results. Horizontal bars indicate ejection time and numbers ejection currents. Arrow denotes i.v. administration of MR-2266 (2 mg/kg).

GABA and dyno~hin 40nA DYN A(1 -8)

I GABA

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487

interactions in the substantia nigra

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40nA des-tyr-DYN (2-17) &

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Fig. 6. Interaction of des-tyr-dioxin 2-17 with GABA. Representative responses of a single SNZr cell to the administration of either dynorphin Al-8 or the non-opiate des-tyr-dynorphin 2-17 during GABA iontophoresis. The record shows that both peptides are effective in attenuating the inhibitory response to GABA. Horizontal bars indicate ejection time and number indicate current. DYN, dynorphin. altering the baseline firing rate. in three cells, GABA pulses sufficient to produce inhibition similar to that described for the SNZr system were applied. In no case did dynorphin have an effect on GABA-induced inhibition. DISCUSSION

A rich plexus of both GABA and dynorphincontaining fibers and terminals is present in the SNZr.10*‘4~2’~U~3S There are also several lines of evidence that dynorphin acts as a neurotransmitter. Chavkin and co-workers’ report that dynorphin is released from hippocampal slices in vitro by a Ca2+-dependent mechanism. Several groups have reported the inhibitory effects of dynorphin in the hippocampus and substantia nigra.z*20~37~38~39 In this study, we report that dynorphin appears to act both as a neurotransmitter and/or neuromodulator in the SNZr and that these effects appear to be non-opioid in origin. The baseline activity of less than half of the spontaneously active neurons in the SNZr were effected by the iontophoretic administration of dynorphin; 44% of the cells were either significantly inhibited (22%) or excited (22%) by the peptide. The majority of spontaneously active cells were not, however, directly inhibited by application of dynorphin. Rather, dynorphin produced a significant and dose-dependent attenuation of the GABAinduced inhibition. The specificity of this response was conthmed by the inability of either saline or Met-ENK to produce a similar attenuation of the GABA response. Thus, dynorphin appears to have two contradictory actions: (1) direct inhibition; and (2) attenuation of the inhibition produced by GABA. Certainly, one would predict that a putative inhibitory transmitter such as dynorphin might potentiate

rather than attenuate the actions of GABA. In fact, when GABA and dyno~hin were applied simultaneously to neurons which responded to dynorphin with a clear inhibitory response, the effects of the two inhibitions were additive. In addition, dynorphininduced attenuation was observed both in neurons which showed the slow excitation in response to dynorphin as well as in cells which demonstrate no significant response to the peptide. In fact, the ability of dynorphin to attenuate the GABAergic inhibition might well explain why a population of neurons was excited by continuous dynorphin application. Given the density of GABA in this subnucleus, it is likely that spontaneously active neurons are exposed to some endogenous GABA release during the recording period. If dynorphin is serving to attenuate the actions of endogenous GABA, then the baseline tiring rate would be expected to increase. This idea is supported by the slow onset and offset of the effect. Given the immediate and potent inhibitory effects of dynorphin, it is unlikely that this slow excitatory action reflects a delayed ejection of the peptide from the pipette. Although there is evidence that dynorphin binds to an opiate receptor,5~6*8~24~4L we were unable to block the inhibitory effects of dynorphin with naloxone (up to 4mg/kg). The inability of naloxone to block the pharmacologic effects of dynorphin have been reported elsewhere. 16~‘9*37 Walker and co-workers3* have shown that dynorphin produces marked motor and electroencephalogram changes but fails to produce reliable analgesia in the tail-flick assay, a test that tends to select for opiaie activity. They have also shown that dynorphin l-17 is also capable of depressing the firing rate of hippocampal neurons in contrast to the effects observed with other opioid agents such as enkephalin. None of these effects of dynorphin were blocked by naloxone.37+38In contrast,

488

B. C. ROBERTSON CI uf.

Lavin and Gar~a-~unoz20 report that naloxone reversed the dynorphin in~bition of SNZr activity in doses of 0.2 mg/kg, i.v. In their study, pressure injection of dynorphin produced a long-lasting inhibition (mean = 6 min) with a latency of up to 4 min. Given the susceptibility of dynorphin Al-9 to peptidases,* it is possible that these investigators were not looking at the direct effects of the peptide. The inhibitory responses reported in our study were both rapid in onset and offset (within 5 s). It may be argued, however, that since dynorphin is thought to bind p~ferentiaIly to the kappa receptor, naloxone’s inability to block dynorphin reflects its weaker antagonism at the kappa site.S”,8*24 To address this question, we examined the effect of the kappa opiate antagonist, MR-2266, in our system. This agent has been shown to be a more specific kappareceptor antagonist than naloxone.24.30.36MR-2266 has been shown to block the antinociception effects of dynorphin A in doses of 0.4 mg/kg intrathecally” and 5 mg/kg, s.c.~’ In our study, this compound failed to reverse either the d~o~hin-indu~d inhibition or GABA attenuation in doses up to 5 mg/kg, i.v. These data suggest that the actions of dynorphin in the SNZr may reflect a non-opiate action of this peptide. Further evidence for a non-opioid action of dynorphin is provided by evidence that des-tyr-dynorphin A2-17 proved equipotent in attenuating GABAinduced inhibition. This peptide is a fragment of dynorphin which fails to bind to opiate receptors in c&o but produces pha~acologic effects similar to the parent compound. 37.38These authors report that dyno~hin and des-tyr-dyno~hin produce similar effects on electroencephalogram, motor function and hippocampal unit firing which could not be blocked with naloxone. Similarly, Herrera-Marschitz and coworkers” report that both dynorphin Al-8 and the non-opioid dynorphin (dynorphin 6-17) both induced a marked contralateral rotation after injection into SN. Thus, it seems unlikely that the attenuation of the GABA response by dynorphin reflects an action at an opioid receptor. The physiolo~cal significance of a non-opiate site for dynorphin is unknown. Thus, several laboratories have reported this unique feature of dynorphin pharmacology, i.e. an ability to act at two distinct biological sites, an opiate and a non-opiate site. Many of these studies have been performed using dynorphin l-17. It has been suggested that this larger dynorphin

is rapidly hydrolysed into some non-opiate but highly potent substance.* In this regard, it is intesting that dynorphin AI-8 is the predominant form of dynorphin in the SN.42 In addition: Dores et

molecule

a1.9 report non-opioid equimolar

that a bridge peptide which is a major end product of prodynorphin is present in concentration with dynorphin A in the

SN. The mechanism underlying the observed nonopioid interaction between dynorphin and GABA are not yet understood. However, it seems unlikely that the attenuation of the GABA responses involves a presynaptic action such as the ability of morphine to decrease GABA release in the SN.18 Such an action would be expected to uniformly cause fixed increases in firing with every dynorphin application rather than the attenuation observed during recording from cells

with no apparent change in baseline activity. Also, it is unlikely that any presynaptic action on GABAergic neurons could override the effects of iontophoretic GABA administration. A postsynaptic mechanism unrelated to any opiate receptor seems likely. For example, this apparent modulation may reflect a dynorphin-me~ated change in the GABA receptor binding kinetics or in the ion currents elicited by the interactions of the two transmitters at the membrane level. However, at present. the site of the observed interaction between GABA and dynorphin remains to be elucidated.

CONCLUSIONS

These results suggest that dynorphin agonists and/or antagonists may be of value in the treatment of neuropsychiatric disorders which are characterized by dysfunction in the basal ganglia. In this regard, chronic haloperidol has been shown to increase dynorphin-related peptides in the caudate and SN while having no effect on mu, delta and kappa opiate binding site densities.28*” These data suggest that chronic neuroleptic treatment may increase prodyno~hin-~tide levels in a manner unrelated to the opiate receptor. Under these conditions, the development of substances which can block the nonopiate actions of dynorphin may be of value in the treatment of neuroleptic-induced tardive dyskinesia. Acknowledgements-We wish to thank Robert Long for his excellent technical assistance.

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of substance P

4. Bunney B. S., Walters J. R., Roth R. H. and Aghajanian G. K. (1973) Dopaminergic neurons: effects of antipsychotic drugs and amphetamine on single unit activity. J. Pharmac. exp. 7’her. 185, 560-571.

GABA and dyno~h~n interactions in the su~~ntia

nigra

5. Chavkin C., and Goldstein A. (1981) Specific receptor for the opioid peptide DYN: structure-activity Proc. natn. Acad. Sci. U.S.A. 76, 65436541.

489 relationships.

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DYN A l-8 in discrete nuclei

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