Proc. Natl. Acad. Sci. USAVol. 77, No. 9, pp. 5527-5531, September 1980Neurobiology

Morphine and opioid peptides reduce paraventricular neuronalactivity: Studies on the rat hypothalamic slice preparation

(neurohypophysial neurons/opiates/naloxone)

QUENTIN J. PITTMAN, JAMES D. HATTON, AND FLOYD E. BLOOMArthur V. Davis Center for Behavioral Neurobiology, The Salk Institute, P.O. Box 85800, San Diego, California 92138

Contributed by Floyd E. Bloom, June 3,1980

ABSTRACT Extracellular discharges of neurons in theparaventricular nucleus (PVN) were recorded from slices of rathypothalamus in vitro. PVN neurons (n = 14) were identifiedby the criteria of (i) phasic activity patterns and (ii) antidromicinvasion from the neurohypophysial tract. Neurons notdisplaying either of these features were considered unidentifiedwith respect to physiological function (n = 85) The majority ofunidentified neurons responded to bath application of morphine(1 pM), [D-Ala2, Met5]enkephalin (1 pM), or,-endorphin (0.01-1,AM) with a prompt, reversible, dose-related reduction in spikedischarge frequency. Naloxone (1 pM) antagonized the op-ioid-induced depressions in some, but not all, cases. At theconcentrations tested, no tachyphylaxis to the effects of theopioids was observed. The opioid effects on putative neurohy-pophysial neurons were less pronounced; while 2 were de-pressed, the remaining 12 displayed no change in frequency orpattern of discharge to micromolar concentrations of morphine,[D-Aa2, Met5Jenkephalin, or ,-endorphin. Our results indicatethat opioids depress neuronal activity in the rat PVN via an in-teraction with a specific opiate receptor but that this effect ismore pronounce on unidentified neurons than on putativeneurohypophysial neurons in the slice.

Modification of neurohypophysial secretion by opiates wasinferred many years ago from the work of DeBodo (1), whoobserved antidiuresis after morphine administration. Despitenumerous studies since that time, effects of opiate adminis-tration on vasopressin secretion are still controversial. A numberof investigations in rats (2-5) have demonstrated an opioid-induced antidiuresis attributed to stimulation of vasopressinrelease from the neurohypophysis. These studies are contrasted,however, by recent reports indicating reduced immunoreactivevasopressin in rat plasma after administration of morphine oropioid peptides (6, 7) and by reduced release of vasopressinfrom the neural lobe in vitro (8).

These contradictory findings prompted us to examine theeffects of morphine and the opioid peptides [D-Ala2, Me*]-enkephalin (an enkephalin analog more resistant to degrada-tion) and ,B-endorphin on neuronal activity within the hypo-thalamic paraventricular nucleus (PVN). This nucleus containscell bodies of vasopressin- and oxytocin-synthesizing neurons(among others) whose discharge frequency has been shown tocorrelate with hormone release from their terminals in theneurohypophysis (9, 10). Therefore, one might expect opiate-induced alterations in neurohypophysial secretion to be re-flected in changes in the electrical activity of these neurons. Wehave looked for such effects, using the in vitro hypothalamicslice preparation, which demonstrates neuronal activity similarto that seen in vivo (11-13).

The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

5527

METHODSSlice Preparation. Male Sprague-Dawley rats weighing

125-200 g were decapitated and their brains were quickly re-moved. The hypothalamus was blocked out and 500-,gm sliceswere cut in the coronal plane on a McIlwain tissue chopper.Slices containing the PVN were placed in a slice chamber(adapted from ref. 14), where their lower surfaces were bathedin an oxygenated, heated (350C) solution (composition in mM:NaCl, 138; NaHCO3, 4.2; Na2HPO4, 0.3; KCI, 5.35 or 8.03;KH2PO4, 0.4; CaCl2, 0.75; MgCl2, 0.5; MgSO4, 0.4; glucose, 11;phenol red, 0.03; Hepes, 10) and their upper surfaces wereexposed to warmed, humidified oxygen. After a period of 1 hr,the level of the medium was raised above the surface of theslices, and oxygenated, warmed (35-370C) solution was per-fused through the chamber at 1.5 ml/min. A stopcock manifoldplaced on the inflow line permitted introduction of drug-con-taining solutions without disrupting the flow of the perfusate.Stable, extracellular recordings could be obtained from thesingle neurons in the slices for periods of 8-10 hr.

Recording. Extracellular recording in the PVN was carriedout with glass micropipettes (5-10 MU) filled with 4% pon-tamine sky blue in 0.5 M sodium acetate. Action potentials wereamplified and filtered, and, on occasion, photographed fromthe face of the oscilloscope. A variable voltage gate with a visible"window" selected suitable action potentials for integration ofactivity (displayed as firing rate on a chart recorder) or for spiketrain analysis on a MINC LAB II minicomputer (DigitalEquipment, Maynard, MA; software by K. Liebold).

Stimulation. A bipolar stimulation electrode of nichromewire (outside diameter 100 ,um, insulated to the tips; tip sepa-ration 0.3-0.5 mm) was positioned with a micromanipulatoronto the surface of the slice ventral-lateral to the PVN in thearea where neurohypophysial axons pass on their way to theposterior pituitary. Single current pulses, 0.05 msec in durationand at 50-150 ,A intensity, were applied at 1 Hz to this elec-trode from isolated stimulation units controlled by a pro-grammable clock. Evoked potentials recorded in PVN neuronsafter stimulation were classified as antidromic if they fulfilledthe following criteria: (i) ability to follow two suprathresholdstimuli at constant latency and at frequencies in excess of 150Hz; (ii) evidence of cancellation of antidromic potentials acti-vated at appropriate collision intervals after a spontaneousaction potential.

Localization. Recording electrodes were visually directedto the area of the PVN after its localization in the transillumi-nated slice through a dissecting microscope. At the end of anexperiment, and on occasion when cells of particular interestwere encountered, pontamine sky blue was iontophoresed from

Abbreviation: PVN, paraventricular nucleus.

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the electrode. After fixation of the slice in 5% formalin, frozensections were cut on a cryostat at 40 ,m, stained with neutralred, and examined for discrete pontamine sky blue spots.Drugs and Chemicals. Perfusion medium prepared by the

Salk media laboratory was sterile at the start of each experiment.Drugs were dissolved in it immediately before use. Morphinesulfate was purchased from Merck and naloxone hydrochloridewas a gift from Endo Laboratories. All peptides were a gift ofN. Ling, Neuroendocrinology Laboratory, Salk Institute.

RESULTSExtracellular recordings were obtained from slice preparationsfrom 35 different animals, and all data pertain to cells localizedin the PVN after histology. The majority of the cells encoun-tered in these preparations displayed spontaneous action po-tentials at frequencies of 1-7 Hz, although individual cellsdisplaying continuous activity in excess of 10 Hz were infre-quently encountered. Those cells displaying continuous activityand not demonstrating evoked potentials after electrical stim-ulation of the neurohypophysial tract were considered un-identified with respect to physiological function. Upon histo-logical examination of recording loci, the majority (but rnot all)of these cells were localized in parvicellular parts of PVN. Asmaller group of cells was tentatively identified as putativeneurohypophysial neurons; inclusion of a cell in this group re-quired that it demonstrate at least one of the following criteria:(i) antidromic activation from the stimulating electrode in thearea of the neurohypophysial tract (n = 3) or (ii) evidence ofphasic, bursting activity (n = 12) such as has been attributedto vasopressinergic neurons in vivo (10). Only 1 of the 14 put-ative neurohypophysial neurons encountered displayed bothphasic activity and evidence of antidromic invasion from theneurohypophysial tract. Histological localization of these put-ative neurohypophysial neurons placed them in, or close to,groups of magnocellular neurons.

Table 1. Effects of morphine, [D-Ala2, Met5]enkephalin, andf-endorphin on activity of PVN neurons

Type of cellPutative

neurohypo-Unidentified physial

Naloxone NaloxoneCompound t reversal t- reversal

Morphine (1 AtM) 28 0 16 . 21/27 1 0 7 0/1[D-Ala2, Met5]-Enkephalin (1 ptM) 15 0 10 5/9 1 0 3

3-Endorphin (0.01-1 pM) 8 0 8 3/5 0 0 2

Changes of action potential frequency of PVN neurons to bathapplication of morphine, [D-Ala2, Met5]enkephalin, or i3-endorphin.Description of unidentified and putative neurohypophysial neuronsis in the text. Arrows indicate response: 1, depression; t, excitation;-, no change. Numbers under the arrows indicate the number ofneurons that exhibited that response. The naloxone reversal columnsrefer to number of drug-induced depressions that were antagonizedby 1 IAM naloxone (numerator) out of total number of cells tested(denominator).

Pharmacological Studies: Unidentified Neurons. Morphine(1 AM) was applied by bath perfusion to 44 PVN neurons (Table1). On 28 of these cells, exposure to morphine was associatedwith a reduction in activity that was rapid in onset and wasreversed after wash-out of the opiate from the bath. At thismorphine concentration, desensitization or tachyphylaxis wasnot encountered during drug applications up to 15 min in du-ration (n = 4). The reduction in activity was not accompaniedby obvious changes in the amplitude or of the shape of the ac-tion potential (although the latter feature was not criticallyevaluated during the majority of the drug applications). In cellsdisplaying sensitivity to 1 MM morphine, threshold (defined asa visually detectable reduction in activity on the rate meterrecord) when tested was greater than 0.1 MM.

Naloxone, 1,M

Morphine, 1 gM

101 ~ ~~~ 1 Spikes/secIL~~m.~~ fi"6"~

Naloxone, 1 gM[D-Ala2, Mets] Enk, 1 M

B

Spikes/secn

Naloxone, 1 pM

p-Endorphin, 0.01 IiM10

21,i i~i~i11111,1 ~ Spikes/sec

5 min

FIG. 1. Rate meter records of three spontaneously active PVN neurons in slice preparations from three different rats. (A) Activity of a neuronlocated in the central part of the nucleus, just medial to the lateral mass of magnocellular neurons. Addition of 1 uM morphine to the perfusateis associated with a reduction in activity that is reversed with concurrent perfusion of the opiate antagonist naloxone. (B) Record from a cellin the parvicellular anterior-dorsal PVN that demonstrates a naloxone-reversible depression of activity to 1 MAM [D-Ala2, Met5]enkephalin. (C)Neuron in the posterior parvicellular PVN that is depressed by 0.01 MuM 3-endorphin; the depression is antagonized by 1 MAM naloxone. Thehorizontal bar above the traces represents the duration of drug application; actual admittance of the perfused substance to the slice chamberis delayed approximately 2 min from the onset indicated by the bar. Lower time scale applies to all three records.

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[D-Ala2, Met5]Enk, 0.1 pM

mlmalmI IIIm iJ1111mm1ImIm II J il 1111

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FIG. 2. Discontinuous rate meter records of a PVN neuron located in the medial parvicellular part of PVN, which demonstrates a variablethreshold response to [D-Ala2, Met5]enkephalin at 0.1 ,uM but a potent depressant response to this peptide at 1 ,uM.

The majority of the morphine-induced depressions were

quickly reversed by addition of 1 MM naloxone to the perfusate(Fig. 1A). Naloxone application in the absence of morphine didnot affect cell firing rates at this concentration. It was also in-effective in reversing 7y-aminobutyric acid-induced depressions(10-100 MM; data not shown).

In 16 of 44 PVN cells, morphine application for a minimumof 5 min was without apparent effect on either the frequencyor the pattern of cellular activity. The lack of response to 1 AMmorphine did not appear to be associated with the previousexposure of the cell either to naloxone or to the opioids. Theseunresponsive cells were not tested with concentrations ofmorphine greater than 1 ,M.

Perfusion of [D-Ala2, Met5]enkephalin onto unidentified PVNcells reduced neuronal activity in 15 of 25 cells (Table 1). Thepeptide-induced depressions resembled those observed aftermorphine administration in that they were fast in onset, re-

versible, and antagonized by naloxone (Fig. 1B). However, a

proportion of these depressions were not reversed by 1 ,Mnaloxone (Table 1), with opioid responses of cells encounteredwithin the same penetration of the microelectrode sometimesdemonstrating different sensitivities to naloxone. Thresholdsensitivities of cells to [D4Ala2, Met5]enkephalin were similarto those observed for morphine; Fig. 2 demonstrates both thelack of consistent effect with 0.1 MuM [D-Ala2, Met5]enkephalinand the complete depression of activity of this same cell with1 MM.

f3-endorphin was tested on 16 unidentified cells, and it re-

duced spontaneous activity in 8. Action potential size remainedunaltered (Fig. 3) in the presence of up to 1 MM f3-endorphin.In f3-endorphin-sensitive cells, effects could be detected byusing concentrations of f3-endorphin as low as 0.01 MM, and thesubsequent depressions were reversed by naloxone (1 MM) in3 of 5 cells tested (Fig. 1C). Of the 8 cells that were unresponsiveto f3-endorphin (Table 1), 5 were tested at 0.01 MM, while theother 3 were exposed to 1 MM.

Putative Neurohypophysial Neurons. In contrast to thepotent depressant effects of morphine and the opioid peptideson unidentified PVN cells, these substances were ineffectivein modifying the frequency or pattern of discharge of themajority of putative neurohypophysial neurons (Table 1). Onone occasion, however, morphine at 1 AM caused a decreasein activity of a phasically firing PVN neuron and in anotherexperiment, [D-Ala2, Met5]enkephalin at 1 MM abolished phasicdischarges of another putative neurohypophysial neuron (Fig.

4). Nevertheless, 7 of 8 putative neurohypophysial neuronsshowed no obvious response to morphine at 1 tiM, 3 of 4 werenot affected by [D-Ala2, Met5]enkephalin at 1 AM, and dis-charges of 2 others were not changed by f3-endorphin at 1 M(n = 1) or 0.01AM (n = 1).

DISCUSSIONThe predominant effect of both morphine and the opioidpeptides when applied by iontophoresis or pressure to centralneurons in vivo has been to reduce neuronal discharge fre-quency (reviewed in refs. 15 and 16). In agreement with thesein vivo findings the present data obtained in vitro indicate thatmorphine, [D-Ala2, Met5]enkephalin, and 3-endorphin all de-press neuronal activity in the PVN, also. Furthermore, the useof bath perfusion for drug application reveals that these sub-

Control (CSF)

,-Endorphin, 1 MM

B-Endorphin, 1 MM + naloxone, 1 MM

Recovery (CSF)

10 secFIG. 3. Four discontinuous oscillograph records illustrate the

results of perfusion of (3-endorphin and naloxone on the spontaneouselectrical activity of an unidentified PVN neuron in the slice. Theupper trace shows the activity of this cell during perfusion of artificialcerebrospinal fluid (CSF). The following traces illustrate the abolitionof spontaneous activity after perfusion of f3-endorphin and the an-tagonism of this depression by naloxone. The fourth trace demon-strates the return to control activity after wash-out of the ,B-endorphinand naloxone. Note lack of effect of these agents on spike size.

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5530 Neurobiology: Pittman et al.

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B

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3 minFIG. 4. Rate meter records of two putative neurohypophysial neurons, both located in the lateral, magnocellular parts of the PVN. (A)

Phasic activity of this cell was reversibly depressed by [D-Ala2, Met5]enkephalin at 1 uM. Naloxone was not tested on this cell. (B) Phasic activityof this cell is abolished by morphine at 1 pM; the morphine effect is slightly antagonized by naloxone at 1 ,M.M

stances are effective in reducing spontaneous activity at con-centrations in the micromolar range or lower. These concen-trations are similar to those used in other in vitro electrophys-iological studies on myenteric plexus neurons (17), neurons inculture (18, 19), and hippocampal (20, 21) and locus coeruleus(22) slices.

Naloxone was effective in antagonizing the majority of theopiate-mediated depressions. Although some reports indicatenonspecificity of naloxone action, in particular when testedagainst 'y-aminobutyric acid actions at relatively high con-centrations (23, 24), the micromolar doses of naloxone used inour slice preparation were ineffective in reversing y-amino-butyric acid depression. Therefore, the ready reversibility bynaloxone of most of our morphine-induced depressions and aproportion of peptide-induced depressions suggests an inter-action with a specific opiate receptor. Nevertheless, in agree-ment with previous studies using iontophoretic techniques invivo (25-27), a small number of the morphine-induced de-pressions and nearly half of the tested peptide-associated de-pressions were resistant to naloxone blockade. Although suchresponses have been termed "nonspecific" in some iontopho-retic studies (27), our use of bath perfusion rather than ionto-phoresis to apply drugs circumvents at least two possible tech-nical problems of the iontophoretic techniques: (i) current ar-tifacts and (ii) presence of extremely high concentrations ofdrugs adjacent to receptor sites and causing "nonspecific," (i.e.,naloxone-resistant) responses. Thus, it is less likely that theseresponses, which were relatively resistant to naloxone blockade,represent nonspecific opioid actions. It is possible that theyrepresent interactions with a different type of opiate receptor(28-31) or with a type of receptor requiring higher doses ofnaloxone for opioid blockade. The existence of multiple re-ceptor populations within the PVN is supported by our obser-vations that morphine and the enkephalin analog appeared tobe of approximately equal potency, whereas /-endorphin wasoften effective in reducing excitability at concentrations 1/10thto 1/100th of this. Further studies with selective agonists andantagonists are required to clarify the nature of these recep-tors.

There is evidence from iontophoretic studies (32, 33) thatcentral neurons show tachyphylaxis to repeated applications

of enkephalin and morphine. With these observations in mind,we examined our data to determine if cells failing to respondto opiates were those encountered during the latter parts of anexperiment, when they had already been exposed several timesto opiates. This was not the case. Even cells that had been ex-posed up to six times during the course of a particular experi-ment continued to respond to opiate application with reduceddischarge activity. Furthermore, on individual cells, continuousapplication of morphine for up to 15 min at a time did not causedesensitization. Thus, we did not observe acute tolerance to thedepressant effect of opiates on electrical activity in the PVN.

There is considerable evidence suggesting that morphine andopioid peptides can act presynaptically to alter transmitterrelease (34, 35) or that they can have indirect actions vianeighboring interneurons (36). The consistent pattern of de-pressant responses in our data suggests that these do not resultfrom indirect or secondary effects of opiate interaction withadjacent neurons. Nevertheless, further investigations utilizingintracellular recording will be helpful to differentiate betweenpossible pre- and postsynaptic opiate actions. Such intracellularstudies may also prove valuable in resolving the issue of whetheropiates depress cellular activity by hyperpolarizing the mem-brane and increasing conductance, as suggested for myentericplexus (37) and locus coeruleus (22) neurons, or whether theyact (also postsynaptically) to block a chemically excitable Nachannel without altering membrane resting potential or resis-tance (38, 39). In the present extracellular studies, opiate de-pressions were not associated with increases in action potentialamplitude.Our studies demonstrate an interesting contrast between the

depressant responses to morphine and the opioid peptides ob-served in unidentified cells and the less-pronounced effect ofthese compounds on putative neurohypophysial neurons. Thus,even though the PVN contains immunoreactive enkephalin (40)and f3-endorphin (41) fibers, the reported interactions of opiateswith vasopressin secretion may take place via -a mechanismother than a direct effect on cellular activity. A presynapticinteraction within the posterior pituitary has been suggestedon the basis of binding (42, 43) studies. Our immunohisto-chemical and physiological studies have prompted us to hy-pothesize an opiate-neurohypophysial interaction at the pitu-

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itary level (44, 45), and recent studies in vivo (46) and in vitro(8) support this possibility.The present findings do not exclude an opiate action within

the PVN on neurohypophysial secretion, because 2 of 14 cellsdid show depression after opioid exposure. Some property ofphasic cells may also require a higher dose of opiates for ef-fective depression. Our inability to definitely identify neuro-hypophysial neurons in the slice makes it possible that some ofthe unidentified neurons that demonstrated a response to op-iates were, in fact, neurohypophysial neurons. Furthermore,in the slice preparation, most of the afferent pathways to thePVN are probably severed and a possible action of opiates viainteraction with these pathways would not be detectable invitro. In vivo electrophysiological studies on neurohypophysialneurons with their afferent inputs intact may provide more

information on this matter (cf. refs. 46-48). Such investigationswill also be particularly relevant for the identification of thosepresently unidentified cells which, in the slice, were highlyresponsive to morphine, [D-Ala2, Met'lenkephalin, or 1-en-

dorphin.

We thank Dr. Glenn Hatton for demonstrating his hypothalamicslice preparation to us. We acknowledge Drs. G. Siggins, C. Pepper,S. Henriksen, and D. Gruol for helpful discussions, Carol Shoemakerfor histological assistance, Dr. N. Ling for providing us with opioidpeptides, and Ms. Nancy Callahan for typing the manuscript. This workwas supported by National Institute on Drug Abuse Grant DA-01785.Q.J.P. is supported by the Medical Research Council of Canada.

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