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Effects of morphine and naloxone on hippocampal CA3 field potentials following systemic administration in the freely-moving rat
Brain Reseurch Bulletin,
Vol.
13,pp. 241-245,1984.0
0361-9230/84 $3.00+ .OO
Ankho Internationai inc. Printed in the U.S.A.
Effects of Morphine and Naloxone on Hippo~ampal CA3 Field Potentials Following Systemic Administration in the Freely-Moving Rat M. A. LINSEMAN’
Neurobiology
Section, Addiction
Research Foundation,
Received LINSEMAN,
M. A. AND W. A. CORRIGALL.
AND W. A. CORRIGALL
l#ecrs
23 January
qf’morphine rat. BRAIN
Toronto, Canada M5S 2SI
1984
and naloxcme
on hippocampal
CA3,field
potentials
RES BULL 13(2) 241-245, 1984.-The effect of IV morphine, 2, 6 and hilar-evoked CA3 field potentials was studied to determine if this area would be more sensitive to mu-type opiate agonists than the CA1 or dentate regions. In addition, the effect of IV naloxone, 2 and 25 mgikg, on the same responses was studied to determine if endogenous opiates reported to be present in the mossy fibers are released by electrical stimulation of this pathway. Neither morphine nor naloxone had an effect on CA3 field potentials at any dose used. The CA1 region of the hippocampus is the area most sensitive to morphine, and this effect of morphine correlates best, anatomically, with the localization of mu-receptors identified by the binding of dihydromorphine. Physiological release of endogenous opiates from the hip~ampus remains to be shown.
fb//o\~Gzg
systemic
Hippocampus
udministration 15 m&kg, on
CA3
in the ,frwly-moving
Mossy fibers
Opiate
Morphine
Field potentials
in CAL and CA2 and less so in CA3 and dentate ([18], based upon di-hydromorphine binding). Kappa sites have been located in the dentate [4,19]. There is however a question of ligand specificity, both with respect to ligands used in these binding studies and in the electrophysiological studies, and the occurrence of generally similar electrophysiological data from all subregions of the hippocampus may reflect overiap of the ligands used with other receptor subtypes. Clearly, though, the distribution of binding sites is widespread. In contrast, recent studies of ligand distributions have indicated that within the hippocampus both enkephalin-like and dyno~hin-like immunoreactivity is localized predominantly to the mossy fiber system which projects to CA3 [3,7, 11, 17, 21, 22, 241. This observation raises two issues of relevance to the study reported here. First, because of this localization of endogenous ligands predominantly to the mossy fiber system, one might expect the most substantial interaction of exogenous opiates within the hippocampus to occur in the CA3 area. Secondly, leu-enkephalin immunoreactivity in the mossy fibers could derive either from pro-enkephalin or pro-dynorphin precursors, and therefore either or both of these precursors could be present in the mossy fibers. While opioid peptides derived from prodynorphin appear to act as kappa or delta ligands, those from pro-enkephalin mossy fibers may be mu, delta or kappa selective (251.
EARLY electrophysiological studies of opiate effects in the hippocampus were undertaken in the absence of substantial information regarding distribution of receptor sites or of endogenous ligands. These studies, using prima~ly tissue slice methodology, focussed on the CA1 region of the hippocampus, and demonstrated that opiates produce a marked augmentation of pyramidal cell activity [l]. A few studies have also shown opiate effects in the CA3 and dentate fields, and these generally parallel those in CA1 [5,9, 161but see [S, 10, 201. (See Fig. l(a) for illustration of hippocampal subfields.) We have confirmed these opiate effects in vim, in a study examining the effects of intravenously ~ministered morphine on evoked field potentials in CA1 and dentate in unanaesthetized freely moving rats [14]. Based upon electrophysiological observations, therefore, one might conclude that the more or less uniform effects of exogenously administered opiates across the hippocampal fields reflect a more or less homogeneous opioid receptor distribution. However, this is not the case. Binding studies have indicated that sites for D-ala’-, D-leu5-enkephalin (a putative delta-preferring tigand) predominate in the CA2 field, with fewer sites in CA3 and none in dentate [2]. Presumptive mu-type sites have been reported to be distributed either primarily throughout the pyramidal cell layer and less densely in stratum radiatum and the dentate region ([2], based upon etorphine binding), or primarily ‘Requests for reprints should be addressed Street, Toronto, Ontario, Canada M5S 251.
Naloxone
to Dr. M. A. Linseman, Neurobiology
241
Section, Addiction Research Foundation,
33 Russell
I.INSEMhN
AND C:ORRIG;AI.I
To address these issues, we have examined the effects of morphine (a presumed mu-agonist) and naloxone on evoked field potentials in the mossy fiber-to-CA3 pathway of the hippocampus in chronically-implanted rats to determine ( I ) whether, in comparison with our previous study of CA1 and dentate, more prominent effects would occur in the CA3 subfield. and, (2) whether endogenous upiuids are released by the mossy fibers.
M& Sprague-Dawley rats, 300-400 g, were prepared with a chronic intravenous jugular catheter and with chronic stimulating and recording electrodes implanted bilaterally in the mossy fibres and CA3 areas respectively. in a mannet similar to that previously described 1141, After a one-week recovery period, animals were examined for a criterion field potential response (a negative-going population spike superimposed on a positive slow wave): if this was present at either recording site, the animal was included in the experiment (e.g_, see Fig. t(b)). Rats were pIaced in a f2” square Plexiglas box and connected to the recording equipment via a cable and commutator mounted on a counterbalanced arm to permit frcedom of movement. The intravenous catheter was attached to a line leading to a remote infusion pump. The rat was allowed to adapt to the box for approx~ately 30 minutes, during which time three stimulus currents (hereafter referred to as A, B, and C) were chosen to generate a 3-point input/output (I/O) curve consisting of a response just above threshold for eliciting a population spike (current A), a second at midrange (current B) and a third at asymptote (current C) for the primary population spike (see Fig. I(b)). Stimulation consisted of siogIe monophasic negative square pulses, 0.1 msec duration, 0.33 Hz, at intensities up to 4000 pa providing no overt behavioral response was elicited in the animal. Baseline I/O curves were generated from lowest to highest current strength, with a sample of n=8 at each. As a test of the amount of recurrent inhibition. responses to paired pulses at current level C at separations of 15,30.60, 100, I50 and 200 msec immediately followed veneration of 110 data (sample n=4 at each paired pulse separation). The 15 msec interval was added in this experiment (as compared to ou1 earlier study of CAI and dentate) to take into account the fact that there was often a shorter duration of inhibition following a single pulse in CA3 than had been observed in CA1 and dentate responses. Following the coIIection ofbasefine data. a saiine infusion was administered to the animaf. In one-half of the animills this was followed at half-hour intervals by a series of incremental doses of morphine, 2, 6 and I5 mg/kg (IV, 1 ml/kg, over 1 min). Five minutes after the end each infusion, Ii0 curves and paired pulse tests were run as for baseline. Immediately following the data collection after the largest dose of morphine, the rat was infused with 2 mg/kg naloxone, and the stimulation tests were run again starting 5 min after the infusion to assess reversibility. lo the second half of the animals, the saline infusion was followed at half-hour intervals by infusions of 2 and 25 m&g naioxone. (The largest dose of naloxone was included to antagonize effects of any kappa Iigands refeased by mossy fiber stimutaGon [23].) Three minutes and twenty minutes foilowing the end of each infusion, plus 40 minutes following
of
f.3
Current B (700
j@
I 2 mv
5 msec. FIG. I _taf Schematicrepresentation of’ the various subfield+--fA I . the hippocampus and the m&n iibrr pathway which Connects them. Abbreviations: dent=dentate; f.42, CA3 and dentate-within
&mb=fimhria. fb) Example showing baseline fiild pcrtential responses from a CA3 site f&owing mossy Gbre stimulation at three current intensities which make up a 3-point inpul!output curve. Stimulus occurs at the break in the fine. Positive is up.
the largest dose, I/O curves and paired pulse tests were run. The twenty and forty-minute tests were included to assess possible recovery. After a period of at least three days following the first experiment, if responses were still present an electrodes, those animals that had previously received morphine. were given naloxone and vice versa, and the same procedures were followed. AII morphine and aII naloxone data were eventually combined. Bipolar hipp~am~~ EEG’s from CA3
OPIATES
243
AND CA3 FIELD POTENTIALS
(b) fa~~~rn --
(c) pa&rant
CAI
8
o
bl
l
sol
path - - dentote
;
6-
_-.-A
-__--y--l
6
c
stimulus current
200
100
a 0
IO0
200
FIG. 2, (a) Above: Mean X/Ocurves showing the effect of several incremental doses of morphine on mean ~o~ul~ion spike amplitude in field potentials recorded
in CA3 (n=6) foliowing stimulation of the ipsilateral mossy fiber pathway. Below: Mean curves showing the effect of morphine on the amplitude of CA3 population spikes in the test response of a paired-pulse paradigm following various. conditioning-test (C-T) pulse intervals. (b/c) Similar data obtained in a previous study when field potentials were recorded in CAYfollowing strmulatron of the stratum radiatum, or in dentate following stimulation of the perforaat path. Key: bl, baseline; Sal, saline; m2, 2 m&kg morphine: m6, 6 n&kg morphine; ml5, 15 mgLkg morphine; nal, 2 n&kg naloxone. Solid lines represent amplitudes of the primary population spike; dashed lines represent a secondary popufation spike. *indicates effects that were significantly diEerent from both basehne and saiine conditions fp
and from frontal cortex were recorded ~~ougho~& the periods of data collection. All data were monitored on line, but for analysis, were recorded on magnetic tape for subsequent signal averaging. Locations of stimulating and recording electrodes were later verified histologically. Data was included only if the tip of the stimulating electrode was within the area of distribution of the mossy fibers (see, for example, [7]f, and the corresponding recording electrode was located within the pyramidal ceil layer of CA3.
The amplitude of field potentials was measured as the average of peak population spike negativity to preceding and following maximum positivities in signal-averaged responses. I/O and paired pulse data obtained after each drug were evaluated using a Z-way analysis of variance, repeated measures design. Since there were no significant main effects or interactions, post-hoc tests were unnecessary.
244
L~NS~~AN
(4
(b)
n s h
A _
A
B
AND CURRIGALL
c
0
bl
sa nal nai
2mq/kg 25 *.
.“_ ---- r..---.., - ..--i 100 200
interpulse interval (msec)
stimulus current
FIG. 3. (a) Mean I:0 curves showing the lack of a sign&cant effect of both low and high doses of naloxone on mean ~pulation spike amplitudes in mossy fiber-to-CA3 field potentials (n=?). (b) Mean curves showing the effect of naloxone on the amplitude of CA3 population spikes in the test response of a paired pulse paradigm following conditioning-t& intervals ranging from 15-200 msec
RESULTS
The effects of morphine on mossy fiber-to-CA3 responses are shown in Fig. 2(a). For purposes of comparison, the effects of identical doses of morphine on CA1 and dentate responses are shown in Fig. 2(b) and (c). In contrast to its effects in CA1 and dentate, morphine at doses up to IS mg/kg IV had no significant effects on either the I/O or paired pulse responses recorded in CA3. This was true in spite of the fact that there were other indications of a drug effect, i.e., the animal was behaviorally very rigid and high amplitude activity dominated in the cortical EEG following the higher doses of drug. In addition, neither dose of naloxone had an effect on CA3 as shown in the f/U and paired pulse data in Fig. 3. What is remarkable about the CA3 data is its homogeneity. particularly that obtained in the paired pulse tests. It should be noted that there appeared to be less paired pulse inhibition than had been obtained in the other areas. DiSCUSSiON In contrast to results obtained previously in CA I, and in dentate, systemically administered morphine had no significant effect on CA3 field potentials evoked by mossy fiber stimulation. Although other investigators have reported excitatory effects of opiates, including morphine, in CA3 [5,9, f6f, in all of these cases administration of drug was by local application to the hippocampus (stice petision, pressure ejection or microiontophoresis) so the comp~biIity of drug concentrations to that obtained following systemic administration cannot be ascertained. It is also possible, however. that since the drug was administered systemically, the lack of effect could have resulted from the summation of opposite effects on different elements contributing to the CA3 response, e.g., d~erenti~ drug effects on different cell types in CA3 or on the excitability of mossy fibers and CA3 cells etc., possibilities that cannot be resolved in the present experiment. In any case, of all anatomical data cited above regarding localization of receptors and ligands within the hippocam-
pus.
the localization of mu-receptors as identified by dihydromorphine binding [ISj (but not etorphine binding [2li appears to correlate best with the effects of systemic morphine on the neural activity of the hippocampus. That is, mu-receptors identified by dihydromorphine binding were most densely localized in CA2 and CA1 and less so in CA3 and dentate. Similarly there were more profound effects of morphine in CA1 than in other areas, and morphine had ;I significant effect in CAI at a lower dose (6 mg&g in the paired pulse test) than in dentate. Although endogenous opiate ligands within the hippocampus have been localized especially to the mossy fiber system, naioxone had no effect on field potentials elicited by stimulation in the hilus. This stimulation should have activated the mossy fibers, although it is possibte it may additionally have stimulated others, e.g., commissural fibers. insofar as the doses of naloxone used are effective as antagonists, release of the endogenous opiates could not be demonstrated by single or paired pulse stimulation. Similarly, in other experiments, no evidence of opiate release was obtained using other neurophy~olo~ca1 measures of hippocampai activity, e.g., naloxone had no effect on potentiation of CA1 field potentials by high frequency stimulation of the stratum radiatum [ 131, or on duration of afterdischarge produced by high frequency stimulation of dentate or CAl, or on post-ictal depression caused by such stimulation (unpublished observations). In the event that opiate-containing cells might be innervated by ext~hi~camp~ fibers, the effects of naloxone on the potentiation of hippocampal responses by priming stimuli delivered to septum, median raphe and the reticular formation were also studied but were found to be insignticant [15]. The only indication of opiate release by the hippocampus we have observed to date is a behaviorat one-that wet dog shakes produced by high frequency stimulation were reduced in animals pretreated with naloxone (unpublished observations). Similarly wet-dog shakes produced by intrahippocarnpai kainic acid, and therefore presumably by stimulation of CA3 neurons, have been reported to be reduced by naloxone [121. And, indeed.
OPIATES
AND CA3 FIELD POTENTIALS
245
intrahippoc~palIy-applied morphine does produce wet-dog shakes ~unpublished observat~ons~. In addition to opiates, a number of other ~euro~ptides including somatostatin, choiecystokinin, vasoactive intestinal polypeptide, angiotensin and vasopressin have also been shown to enhance the electrical activity of the hippocampus although not necessarily by the same mechanisms [6]. Like opiates, they are present in small quantities within the hip pocampus, but ~onditjons for their release and physiological correlates of their activity are as yet unspecified. A solution
to
this enigma could be of importance to our understanding not only of the function of neuro~ptides but also of the hippo~~pus. ACKNOWLEDGEMENTS
We acknowledge the valuable technical assistance provided by Ms. Myrnalee ERiott and Ms. RoseMarie D’Gnofrio, and we are grateful to E. I. Du Pont De Nemours & Company for suppIyin8 the naioxone used in this study.
REFERENCES 1. Carrigall, W. A. Opiates and the hip~campus: A review of the functionaf and morphotogical evidence. ~~~~~~ffc~~~ 3~~~~~~~~? Bchur 18: 255-262, 1983, 2. Duka, T., M. Wuster, P. Schubert, R. Stoiber and A. Herz. Selective localization of different types of opiate receptors, in hippocampus as revealed by in rirro autoradiography. Brain !?e.s 20% 18I-186, 1981. 3, Fitzpatrick, D. and R. P. Johnson. ~nkepha~in-like immunoreactivity in the mossy fiber pathway of the hip~campal formation of the tree shrew. Neurosrience 6: 2485-2494, 1981. 4. Foote, R. W. and R. Maurer. Autoradiographic localization of opiate K-receptors in the guinea-pig brain. l&r J Pfz~rrrnff~(~t 1115: 99103, 1982. 5. French, E. D. and G. R. Siggins. An iontophoretic survey of opioid peptide actions in the rat limbie system: In sear& of opioid eu~~eptogenic mechanisms. Rvg~rf P@ 1: 127-146, 1980. 6. G&hwiler. B. H. The action of neuropeptides on the bioelectric activity of hippocampal neurons. In: The ~ellrob~~lu~~ qf’ the Nippocumprrs, edited by E. Seifert. Toronto: Academic Press, 1983, pp. 157-173. 7. Gall, C., N. Brecha, H. J. Karten and K. .I. Chang. Localization of enkeph~lin-hike immunoreact~vity to identified axonal and neoronal ~pu~ati~ns of the rat hippocampus. J Corn9 A%ur& 198: 335-350, 1981. 8. Gruel, 0, L., C. Chavkin, R. J. Valentine and G. R. S&gins. Dynorphin-A alters the excitability of pyramidal neurons of the rat hip~campus in vitro. .L$> Sci 33: 533-536, 1983. 9. Haas, H. L. and R. W. Ryall. Is excitation by enkephalins of hippocampal neurones in the rat due to presynaptic facilitation or to dis~nbibit~on?~ Pfwkof 308: 315-330. 1980. JO. Henriksen. S. J., G. Chouvet and F. E. Bloom. In r&o cehuiar responses to el~t~phoretically applied dynorphin in the rat trippocampus. tif> Sci 31: 1785-1788, 1982. Il. Khachaturian, H., M. E. Lewis, V. Hollt and S. J. Watson. Telencephalic enkephalinergic systems in the rat brain J Nertmsci 3: 844-855, 1983. 12. Lanthorn, T. and R. L. isaacson. Studies of k~i~ate-induced wet-dog shakes in the rat. t$& Sci 22: 173-178, 1978. 13. Linseman, M. A. and W. A. Corrigafl. Are endogenous opiates involved in potentiatian of field potentials in the hippocampus of the rat? Neurosci L,erf 27: 319-324, 1981.
14. Linseman, M. A. and W. A. CorrigaII. Effects of morphine on CA1 versus dentate ~~p~~~l fiifd potentials fotlowing systemic administration in freely-moving rats. ~ef~r~~harrnffc~~u~~ 21: 361-366, 1982. 15. Linseman, M. A. and W. A. Conigall. Release of enkephalin within the hippocampus-inabihty to demonstrate using naloxone blockade. Sot Netrrosci Abstr 8: 230, 1982. 16. Masukawa, L. M, and D. A. Prince. Enkephahn inhibition of inhibitory input to CA2 and CA3 pyramidal neurons in the hip pocampus. Brain Res 249: 271-280, 1982. 17. McGinty, J. F., S. J. Henriksen, A. Goldstein, L. Terenius and F. E. Bloom. Dyno~hin is contained within hip~~p~ mossy fibers: lmmunoch~micai alterations after kainic acid administration and colchicine-induced neurotoxicity. Pruc Nat1 Acud Sci USA so: 589-593, 1983. IS. Meibach, R. C. and S. Ma~yani. Localization of naloxonesensitive f”H] d~hydromorphjne binding sites within the hippocampus of the rat. Eur J Pburrnff&[~f 68: 175-179, 198% 19. Guirion. R., A. S. Weiss and C. B. Pert. Comparative pharmacologicai properties and autoradiographic distribution of [SH]ethylketocyclazocine binding sites in rat and guinea pig brain. Life Sci 33: 183-186, 1983. 20. Tieien, A. EA., F. H. Lopes da Silva, W, 3. Moffevanger and F. H. de Jonge. Di~erentia~ effects of enkephahn within hippocampal areas. Exp Brflin Res 44: 343-346, 1981. 21. Tielen, A. M., F. W. van Leeuwen and F. H. Lopes da Silva. The localization of leucine~~eph~in ~muno~~tivity within the guinea pig hippocampus. i%p Bruin RPS 48: 288-295, 1982. 22. Vincent, S. R., T. Hokfelt, I. Christensson and L. Terenius. Dyno~h~n-~mmunoreactjve neurons in the central nervous system of the rat. Neurosci Lett 33: 185-190, 1982. 23. Walker, 3. M., H. C. Moises, D. H. Coy, G. Bafdrighi and H. Akil. Non-opiate effects of dynorphin and des-tyr~yno~bin. Science 218: 1136-i-1138,1982. 24. Watson, S. J., H. Khachaturian, H. Akil, D. H. Coy and A. Goldstein. Comparison of the distribution ofdynorphin systems and enkephaiin systems in brain. Science 218: 1134-I f36, 1982. 25. Weber, E., C. 3. Evansand J. D. Barchas. MuItipleendo~~us ligands for opioid receptors. Trends hipttrosci 6: 333-336, 1983.
Vol.
13,pp. 241-245,1984.0
0361-9230/84 $3.00+ .OO
Ankho Internationai inc. Printed in the U.S.A.
Effects of Morphine and Naloxone on Hippo~ampal CA3 Field Potentials Following Systemic Administration in the Freely-Moving Rat M. A. LINSEMAN’
Neurobiology
Section, Addiction
Research Foundation,
Received LINSEMAN,
M. A. AND W. A. CORRIGALL.
AND W. A. CORRIGALL
l#ecrs
23 January
qf’morphine rat. BRAIN
Toronto, Canada M5S 2SI
1984
and naloxcme
on hippocampal
CA3,field
potentials
RES BULL 13(2) 241-245, 1984.-The effect of IV morphine, 2, 6 and hilar-evoked CA3 field potentials was studied to determine if this area would be more sensitive to mu-type opiate agonists than the CA1 or dentate regions. In addition, the effect of IV naloxone, 2 and 25 mgikg, on the same responses was studied to determine if endogenous opiates reported to be present in the mossy fibers are released by electrical stimulation of this pathway. Neither morphine nor naloxone had an effect on CA3 field potentials at any dose used. The CA1 region of the hippocampus is the area most sensitive to morphine, and this effect of morphine correlates best, anatomically, with the localization of mu-receptors identified by the binding of dihydromorphine. Physiological release of endogenous opiates from the hip~ampus remains to be shown.
fb//o\~Gzg
systemic
Hippocampus
udministration 15 m&kg, on
CA3
in the ,frwly-moving
Mossy fibers
Opiate
Morphine
Field potentials
in CAL and CA2 and less so in CA3 and dentate ([18], based upon di-hydromorphine binding). Kappa sites have been located in the dentate [4,19]. There is however a question of ligand specificity, both with respect to ligands used in these binding studies and in the electrophysiological studies, and the occurrence of generally similar electrophysiological data from all subregions of the hippocampus may reflect overiap of the ligands used with other receptor subtypes. Clearly, though, the distribution of binding sites is widespread. In contrast, recent studies of ligand distributions have indicated that within the hippocampus both enkephalin-like and dyno~hin-like immunoreactivity is localized predominantly to the mossy fiber system which projects to CA3 [3,7, 11, 17, 21, 22, 241. This observation raises two issues of relevance to the study reported here. First, because of this localization of endogenous ligands predominantly to the mossy fiber system, one might expect the most substantial interaction of exogenous opiates within the hippocampus to occur in the CA3 area. Secondly, leu-enkephalin immunoreactivity in the mossy fibers could derive either from pro-enkephalin or pro-dynorphin precursors, and therefore either or both of these precursors could be present in the mossy fibers. While opioid peptides derived from prodynorphin appear to act as kappa or delta ligands, those from pro-enkephalin mossy fibers may be mu, delta or kappa selective (251.
EARLY electrophysiological studies of opiate effects in the hippocampus were undertaken in the absence of substantial information regarding distribution of receptor sites or of endogenous ligands. These studies, using prima~ly tissue slice methodology, focussed on the CA1 region of the hippocampus, and demonstrated that opiates produce a marked augmentation of pyramidal cell activity [l]. A few studies have also shown opiate effects in the CA3 and dentate fields, and these generally parallel those in CA1 [5,9, 161but see [S, 10, 201. (See Fig. l(a) for illustration of hippocampal subfields.) We have confirmed these opiate effects in vim, in a study examining the effects of intravenously ~ministered morphine on evoked field potentials in CA1 and dentate in unanaesthetized freely moving rats [14]. Based upon electrophysiological observations, therefore, one might conclude that the more or less uniform effects of exogenously administered opiates across the hippocampal fields reflect a more or less homogeneous opioid receptor distribution. However, this is not the case. Binding studies have indicated that sites for D-ala’-, D-leu5-enkephalin (a putative delta-preferring tigand) predominate in the CA2 field, with fewer sites in CA3 and none in dentate [2]. Presumptive mu-type sites have been reported to be distributed either primarily throughout the pyramidal cell layer and less densely in stratum radiatum and the dentate region ([2], based upon etorphine binding), or primarily ‘Requests for reprints should be addressed Street, Toronto, Ontario, Canada M5S 251.
Naloxone
to Dr. M. A. Linseman, Neurobiology
241
Section, Addiction Research Foundation,
33 Russell
I.INSEMhN
AND C:ORRIG;AI.I
To address these issues, we have examined the effects of morphine (a presumed mu-agonist) and naloxone on evoked field potentials in the mossy fiber-to-CA3 pathway of the hippocampus in chronically-implanted rats to determine ( I ) whether, in comparison with our previous study of CA1 and dentate, more prominent effects would occur in the CA3 subfield. and, (2) whether endogenous upiuids are released by the mossy fibers.
M& Sprague-Dawley rats, 300-400 g, were prepared with a chronic intravenous jugular catheter and with chronic stimulating and recording electrodes implanted bilaterally in the mossy fibres and CA3 areas respectively. in a mannet similar to that previously described 1141, After a one-week recovery period, animals were examined for a criterion field potential response (a negative-going population spike superimposed on a positive slow wave): if this was present at either recording site, the animal was included in the experiment (e.g_, see Fig. t(b)). Rats were pIaced in a f2” square Plexiglas box and connected to the recording equipment via a cable and commutator mounted on a counterbalanced arm to permit frcedom of movement. The intravenous catheter was attached to a line leading to a remote infusion pump. The rat was allowed to adapt to the box for approx~ately 30 minutes, during which time three stimulus currents (hereafter referred to as A, B, and C) were chosen to generate a 3-point input/output (I/O) curve consisting of a response just above threshold for eliciting a population spike (current A), a second at midrange (current B) and a third at asymptote (current C) for the primary population spike (see Fig. I(b)). Stimulation consisted of siogIe monophasic negative square pulses, 0.1 msec duration, 0.33 Hz, at intensities up to 4000 pa providing no overt behavioral response was elicited in the animal. Baseline I/O curves were generated from lowest to highest current strength, with a sample of n=8 at each. As a test of the amount of recurrent inhibition. responses to paired pulses at current level C at separations of 15,30.60, 100, I50 and 200 msec immediately followed veneration of 110 data (sample n=4 at each paired pulse separation). The 15 msec interval was added in this experiment (as compared to ou1 earlier study of CAI and dentate) to take into account the fact that there was often a shorter duration of inhibition following a single pulse in CA3 than had been observed in CA1 and dentate responses. Following the coIIection ofbasefine data. a saiine infusion was administered to the animaf. In one-half of the animills this was followed at half-hour intervals by a series of incremental doses of morphine, 2, 6 and I5 mg/kg (IV, 1 ml/kg, over 1 min). Five minutes after the end each infusion, Ii0 curves and paired pulse tests were run as for baseline. Immediately following the data collection after the largest dose of morphine, the rat was infused with 2 mg/kg naloxone, and the stimulation tests were run again starting 5 min after the infusion to assess reversibility. lo the second half of the animals, the saline infusion was followed at half-hour intervals by infusions of 2 and 25 m&g naioxone. (The largest dose of naloxone was included to antagonize effects of any kappa Iigands refeased by mossy fiber stimutaGon [23].) Three minutes and twenty minutes foilowing the end of each infusion, plus 40 minutes following
of
f.3
Current B (700
j@
I 2 mv
5 msec. FIG. I _taf Schematicrepresentation of’ the various subfield+--fA I . the hippocampus and the m&n iibrr pathway which Connects them. Abbreviations: dent=dentate; f.42, CA3 and dentate-within
&mb=fimhria. fb) Example showing baseline fiild pcrtential responses from a CA3 site f&owing mossy Gbre stimulation at three current intensities which make up a 3-point inpul!output curve. Stimulus occurs at the break in the fine. Positive is up.
the largest dose, I/O curves and paired pulse tests were run. The twenty and forty-minute tests were included to assess possible recovery. After a period of at least three days following the first experiment, if responses were still present an electrodes, those animals that had previously received morphine. were given naloxone and vice versa, and the same procedures were followed. AII morphine and aII naloxone data were eventually combined. Bipolar hipp~am~~ EEG’s from CA3
OPIATES
243
AND CA3 FIELD POTENTIALS
(b) fa~~~rn --
(c) pa&rant
CAI
8
o
bl
l
sol
path - - dentote
;
6-
_-.-A
-__--y--l
6
c
stimulus current
200
100
a 0
IO0
200
FIG. 2, (a) Above: Mean X/Ocurves showing the effect of several incremental doses of morphine on mean ~o~ul~ion spike amplitude in field potentials recorded
in CA3 (n=6) foliowing stimulation of the ipsilateral mossy fiber pathway. Below: Mean curves showing the effect of morphine on the amplitude of CA3 population spikes in the test response of a paired-pulse paradigm following various. conditioning-test (C-T) pulse intervals. (b/c) Similar data obtained in a previous study when field potentials were recorded in CAYfollowing strmulatron of the stratum radiatum, or in dentate following stimulation of the perforaat path. Key: bl, baseline; Sal, saline; m2, 2 m&kg morphine: m6, 6 n&kg morphine; ml5, 15 mgLkg morphine; nal, 2 n&kg naloxone. Solid lines represent amplitudes of the primary population spike; dashed lines represent a secondary popufation spike. *indicates effects that were significantly diEerent from both basehne and saiine conditions fp
and from frontal cortex were recorded ~~ougho~& the periods of data collection. All data were monitored on line, but for analysis, were recorded on magnetic tape for subsequent signal averaging. Locations of stimulating and recording electrodes were later verified histologically. Data was included only if the tip of the stimulating electrode was within the area of distribution of the mossy fibers (see, for example, [7]f, and the corresponding recording electrode was located within the pyramidal ceil layer of CA3.
The amplitude of field potentials was measured as the average of peak population spike negativity to preceding and following maximum positivities in signal-averaged responses. I/O and paired pulse data obtained after each drug were evaluated using a Z-way analysis of variance, repeated measures design. Since there were no significant main effects or interactions, post-hoc tests were unnecessary.
244
L~NS~~AN
(4
(b)
n s h
A _
A
B
AND CURRIGALL
c
0
bl
sa nal nai
2mq/kg 25 *.
.“_ ---- r..---.., - ..--i 100 200
interpulse interval (msec)
stimulus current
FIG. 3. (a) Mean I:0 curves showing the lack of a sign&cant effect of both low and high doses of naloxone on mean ~pulation spike amplitudes in mossy fiber-to-CA3 field potentials (n=?). (b) Mean curves showing the effect of naloxone on the amplitude of CA3 population spikes in the test response of a paired pulse paradigm following conditioning-t& intervals ranging from 15-200 msec
RESULTS
The effects of morphine on mossy fiber-to-CA3 responses are shown in Fig. 2(a). For purposes of comparison, the effects of identical doses of morphine on CA1 and dentate responses are shown in Fig. 2(b) and (c). In contrast to its effects in CA1 and dentate, morphine at doses up to IS mg/kg IV had no significant effects on either the I/O or paired pulse responses recorded in CA3. This was true in spite of the fact that there were other indications of a drug effect, i.e., the animal was behaviorally very rigid and high amplitude activity dominated in the cortical EEG following the higher doses of drug. In addition, neither dose of naloxone had an effect on CA3 as shown in the f/U and paired pulse data in Fig. 3. What is remarkable about the CA3 data is its homogeneity. particularly that obtained in the paired pulse tests. It should be noted that there appeared to be less paired pulse inhibition than had been obtained in the other areas. DiSCUSSiON In contrast to results obtained previously in CA I, and in dentate, systemically administered morphine had no significant effect on CA3 field potentials evoked by mossy fiber stimulation. Although other investigators have reported excitatory effects of opiates, including morphine, in CA3 [5,9, f6f, in all of these cases administration of drug was by local application to the hippocampus (stice petision, pressure ejection or microiontophoresis) so the comp~biIity of drug concentrations to that obtained following systemic administration cannot be ascertained. It is also possible, however. that since the drug was administered systemically, the lack of effect could have resulted from the summation of opposite effects on different elements contributing to the CA3 response, e.g., d~erenti~ drug effects on different cell types in CA3 or on the excitability of mossy fibers and CA3 cells etc., possibilities that cannot be resolved in the present experiment. In any case, of all anatomical data cited above regarding localization of receptors and ligands within the hippocam-
pus.
the localization of mu-receptors as identified by dihydromorphine binding [ISj (but not etorphine binding [2li appears to correlate best with the effects of systemic morphine on the neural activity of the hippocampus. That is, mu-receptors identified by dihydromorphine binding were most densely localized in CA2 and CA1 and less so in CA3 and dentate. Similarly there were more profound effects of morphine in CA1 than in other areas, and morphine had ;I significant effect in CAI at a lower dose (6 mg&g in the paired pulse test) than in dentate. Although endogenous opiate ligands within the hippocampus have been localized especially to the mossy fiber system, naioxone had no effect on field potentials elicited by stimulation in the hilus. This stimulation should have activated the mossy fibers, although it is possibte it may additionally have stimulated others, e.g., commissural fibers. insofar as the doses of naloxone used are effective as antagonists, release of the endogenous opiates could not be demonstrated by single or paired pulse stimulation. Similarly, in other experiments, no evidence of opiate release was obtained using other neurophy~olo~ca1 measures of hippocampai activity, e.g., naloxone had no effect on potentiation of CA1 field potentials by high frequency stimulation of the stratum radiatum [ 131, or on duration of afterdischarge produced by high frequency stimulation of dentate or CAl, or on post-ictal depression caused by such stimulation (unpublished observations). In the event that opiate-containing cells might be innervated by ext~hi~camp~ fibers, the effects of naloxone on the potentiation of hippocampal responses by priming stimuli delivered to septum, median raphe and the reticular formation were also studied but were found to be insignticant [15]. The only indication of opiate release by the hippocampus we have observed to date is a behaviorat one-that wet dog shakes produced by high frequency stimulation were reduced in animals pretreated with naloxone (unpublished observations). Similarly wet-dog shakes produced by intrahippocarnpai kainic acid, and therefore presumably by stimulation of CA3 neurons, have been reported to be reduced by naloxone [121. And, indeed.
OPIATES
AND CA3 FIELD POTENTIALS
245
intrahippoc~palIy-applied morphine does produce wet-dog shakes ~unpublished observat~ons~. In addition to opiates, a number of other ~euro~ptides including somatostatin, choiecystokinin, vasoactive intestinal polypeptide, angiotensin and vasopressin have also been shown to enhance the electrical activity of the hippocampus although not necessarily by the same mechanisms [6]. Like opiates, they are present in small quantities within the hip pocampus, but ~onditjons for their release and physiological correlates of their activity are as yet unspecified. A solution
to
this enigma could be of importance to our understanding not only of the function of neuro~ptides but also of the hippo~~pus. ACKNOWLEDGEMENTS
We acknowledge the valuable technical assistance provided by Ms. Myrnalee ERiott and Ms. RoseMarie D’Gnofrio, and we are grateful to E. I. Du Pont De Nemours & Company for suppIyin8 the naioxone used in this study.
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