Intracellular recording of cerebral cortical actions of prostaglandin F2α (PGF2α)

PROSTAGLANDINS INTRACELLULAR RECORDING OF CERHHRAL CORTICAL ACTIONS OF PROSTAGLANDIN F2u (PGF 1 2a Nary Ann Narrassi and Chuong C. Huang Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201 and University of Missouri Institute of Psychiatry, St. Louis, Missouri 63139. ABSTRACT Uur reported data on the cortical inhibitory actions of prosta) and the diversity of data in the literature on glandin F2 (PGF cerebral P8 act&s are examined here in the light of intracellular recording which provides the requisite membrane data for the first time. Thus, 1) intracellular recording from the cat cerebral cortex is obtained for the actions of PGF and for norepinephrine (NE) and serotonin (5HT). 2) The parallel Ganges in firing and polarization and the simultaneous transmembrane conductance changes are qualitatively identical for PGF NE and 5HT. 3) The reduction in firing accompanied by 22' hyperpolarisa ion indicates that PGF , NE and 5HT all inhibit these cells. 4) The ionic species responsi g le for this inhibition is such that it increased the tranmaembrane resistance, and this was true for all three. 5) The changes in membrane parameters, identical in direction for PGFx, and NH, but stronger for the latter, constitute conditions that can lead to competitive inhibition and therefore connote, presumably, actions at the same or related receptors. Such competition with evoked cortical field potentials is shown in the preceding paper. INTRODUCTION In the preceding paper (11, we have re-examined the multiplicity of reported prostaglandin (PG) effects on central nervous system (CNS) field and extracellular unit potentials in view of the consistent reduction of cerebral evoked potentials that we obtained on intracarotid injection of PGFza in the cat. Dur interpretation of our data as a cerebral synaptic inhibition similar to that reported by Marraszi (2,3) for norepinephrine(NE) and serotonin (5IiT).with a capability for competitive inhibition with NE- requires confirmation by direct membrane data from intracellular recording. This was not previously available, and we, therefore, supply it for the cerebral action of PG in the intact cortex of the cat. Preliminary reports of this data have appeared (4.5). MATERIAL.9 AND METHODS The solutions were prepared and administered as in the preceding paper (1). The intracarotid route was used for comparison with our evoked potential results. It provides a sharp blood to tissue concentration gradient to promote passage through the blood brain barrier. The required local concentration thus achieved becomes so diluted in the general circulation that it is subthreshold for peripheral effects, which would complicate the CNS data. Address reprint requests to Dr. Mary Ann Marrassi, Wayne State Univ. We wish to thank the Upjohn Company for the generous supply of PGFZa.

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PROSTAGLANDINS The.recording site was the ipsilateral pericruciate coxtex, which contains sufficiently large cells to be impaled readily by glass microelectrodes of less than 1 micron tip size. (In initial experiments, these large cells were located and identified by antidranic stimulation.) The micropipets were filled with 3.0 M KC1 and inserted to a depth of up to one millimeter. The electrode was advanced through a restraining foot resting gently on the cortex, until either a negative shift of the resting (SO-SCnW) potential indicated penetration and/ or firing was recorded with a sharp increase in spike heights and reduction in biphasicity. Male cats under 3 kg were prepared by initial minimal doses of thiopental intravenously (IV) and then flaxedilized (IV) and artifically respired, with IV flaxedil supplements and dextran solution as needed to maintain BP. All wound edges and pressure points were kept locally anesthetized with xylocaine. The skull was trephined, and the frontal cortex including the pericruciate area was exposed and coated with agar to reduce pulsation of vascular and respiratory origin. Respiratory excursions were further reduced by pneumothorax. Blood pressure was monitored continuously frcnn the femoral artery. A battery operated heating pad helped maintain body temperature monitored by a rectal thermister. The neuronal signal was passed through a negative capacity preamplifier affording suitable neutralization and was displayed after pushpull amplification in parallel AC and DC channels on a dual beam oscilloscope. The image was fed through a reflexor to a Grass Camera, and the AC channel was also monitored with a Grass filtered speaker. Manentary constant current injection of 6-10 x 10m4 uamps allowed determination of membrane resistance by the size of the resulting non-stimulating pulses in the DC potential. Both polarities were non-stimulating. RRSDLTS In this manner, 67 units in 23 cats were examined. Thirty-one of these units (14 cats) in which the most ccsaplete data.:was obtained are tabulated in Fig. 1. Representative examples in Figs. 2-4 show the membrane polarization, firing and transmembrane resistance monitored simultaneously and continuously pre- and post-injection. The rapidly reversible inhibitory action of 5liTwas used as a measure of sensitivity and of successful maintenance of the baseline or control condition preceding and following the PG testing. This allowed comparison of data from field and unit potentials and between individual cats. Of the units in the table, 26 (marked with an asterisk) were held long enough to record both PG and 5HT actions in the same unit. In every case, these actions ware qualitatively identical. Since the question of competitive inhibition between NR and PGF2c, as described in the previous companion paper (l), was to be considered, the membrane data were obtained for NE. As seen in the typical ezperiment in Fig. 2, NE reduces spontaneous firing (upper AC channel) as it hyperpolarizes (lower DC channel) and increases membrane resistance (increased amplitude of non-stimulating DC pulses). A rebound depolarisation, increased firing and decreased transmembrane resistance intervenes before recovery of all parameters to control level. Thus, the parallel recording of the three membrane parameters indicates inhibition

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Fig. 1 : ProstaglandinF2q (9 2a) Cortical Inhfbition- Concurrent Unit Changes. 14 of the 23 cats used are picked because of the completeness _. _ of the data. This represents31 of the 67 units recorded.As indicated in 26 of these, SElTinhibitionwas shown in the same unit. Primby PI* ary (1 ), i.e. direct effects on the recordedunits, are shown by the solid arrows, and secondary (2O), or indirectones, by dashed arrows, and no change by a dash.

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Cat

FIG, 2

common

& TRANSMEMBRANE Injection-ipsilateral

carotid

artery

RESISTANCE

Fiq. 2 : Norepinephrine (NE) Inhibition of Spontaneous Unit Discharge Cortical Intracellular Continuous Monitoring. Spontaneous firing is intracellularly recorded on the upper AC channel and polarization changes in the lower DC channel (at lower gain). The transmembrane resistance is proportional to the CC shift induced by Intracarotid injection is at zero non-stimulating injected -rent. time. Recovery is associated with an intervening rebound depolarization to beyond the control level with return of firing to a rate faster than the preinjection 20 sec. control period compared with the period frcrm 30 to 110 sec. post-injection. Beyond 130 sec. post-injection the rate slowed to preinjection control level accompanied by a corresponding return of the polarization level and transmembrane resistance.

Flaxedilized

HYPERPDLARIZATION

IN PERICRUCIATE AREA=CONTINUOUS MONITORING

NOREPINEPHRINE INHIBITION OF SPONTANEOUS UNIT DISCHARGE

DC

AC

PROSTAGLANDINS followedby post-inhibitoryrebound, and return to control level, i.e, ccanplete recovery. The qualitativelyidenticalsequenceof concommittentchanges in firing,polarization,and tranmaembraneresistanceis presented in Fig. 3 for PGF2c in the upper half of the figure and for SEIT. in the lower half. A similar experiment (withoutthe transmembraneresistancemeasurements) in a somewhatless sensitiveunit* is shown in Fig. 4 as an example where post-inhibitoryrebound is not conspicuous.The 5HT dose (lowerhalf of the figure) is about 60% of that in Fig. 3, but the latency to initial or primary inhibitionis about seven fold greater, while with the PGP , in approximatelythe same dose, thke is about twice the latency.2a In these typical units, which for PG are representativeof the 45 positive findingsin 67 trials, the initial reductionof firing is identifiedby the acccmpanyinghyperpolarisation as true inhibition. , usually seen with inhibition,we The post-inhibitoryfacilitation identifiedas of secondaryorigin, i.e. a disinhibitionor release from inhibition,to be consideredin the discussion. The summary table (Fig. 1) shows that the effect of PGF did not vary qualitativelyover a dose range of 11-65 us/kg, i.e. t&t of the 31 units tabulated,all exhibitedthe primary inhibitionand all but 7 showed the secondarypost-inhibitoryrebound. One cat (C 100) even shows the possiblepreliminarysecondaryeffect that is predictablefrom the explanationpresented in the discussion.The expectedprimary hyperpolarizationwas present in all but one unit. The secondarydepolarization occurred a little less regularly,i.e. 13 out of 31 units. In no case were either primary or secondarychanges in a directiondifferent from expectation.Similarly,the primary tranmuembraneresistance,when measured, increasedin all but one instancewhere no change was detected,and the secondarydecreasedor showed no change. The BP changes,on the other hand, when they did occur, were small to moderate and variable.A vehicle control,as defined in the precedingpaper (l), again had no effect. DISCTJSSION The intracellularrecordingof the action of PGF2c on the cat cerebral cortex given here makes some requiredmembraneparameters availablefor the first time. This allows us, by using the associated changes in membrane polarization(cf. Fig. 5), to clearly characterize the decrease in unit firing as a primary inhibition.Consistencycan then be brought to the apparentlydiverse effects reported for extracellularlyrecordedunit firing, since they can be accountedfor by the same process of inhibition. Such a plausible explanationcan be seen to result from the operationof the two factors brought out in Fig. 6. These are 11 the systematicallyvarying concentrationsof drugs associatedwith onset, maintenance,and offset of action and 2) their action first on the lower * The higher gain required in the DC channel of this experimentcauses the firing to appear on the DC channel, meant only for the membrane potential,as well as on the AC channel.

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Flaxedilized

AREA

FIG, 3

*PGF2d(THAM

Salt)Inj.

- ipsilateral

common

OF SPONTANEOUS UNIT CONTINUOUS MONITORING - LOWER HALF

(PGF ) Inhibition of Spontaneous Unit Fiq. 3 : Prostsqldndin F Discharge - Cor gq xcal -g In acellular Continuous Monitoriq. Recordings as in Fig. 2. The upper 2 traces show the course of PGF~~ actions,and the lower,the course of 5HT actions in the same unit.

Cat PERICRUCIATE

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DISCHARGE

sec.

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PROSTAGLANDINS

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CORTICAL

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Cat

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CONTINUOUS

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Disc-e - Cc&al Intracellularcontinous~~,,itori,,g. RecordingsaB in Figs. 2 C 3 except that gain is the s-e for both channels.Consequentlythe firing appears on both channels,causing overlap of AC and DC recorded spikes,which preventedrecordingof DC pulses to measure tranm&ane resistance.

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PROSTAGLANDINS thresholdfor inhibitionof inhibitorycells, i,e, disinhibition leading to secondaryapparentexcitation (increasedfiringI,andthen on the higher thresholdfor inhibitionof excitatorycells as well (decreasedfiring), i.e. primary inhibition.'Aninhibitoryagent can thus manifest its action in diametricallyoppositeways dependingon what part of the concentration- thresholdinteractioncurve is sampledin the recording. By matching the changes in unit firing with the attendentpolarization, it is now possible to identifythe true nature of the action (Fig. 5). A decrease in firing associatedwith hyperpolarization defines inhibition(primaryinhibition,Fig. 5 #l), whereas primary excitation would be associatedwith depolarization( Fig. 5 #2). Apparent excitation as.a result of disinhibitionis also associatedwith depolarization, but this secondaryeffect is recognizablebecause it ordinarilyfollows primary inhibition (Fig. 5, #l). Post-excitatorydepression,an apparent inhibition,on the other hand, exhibitsno change from the depolarization during the primary excitationthat it follows (Fig.5#2). Fig. 6 also predicts a short-livedpreliminarydisinhibitionor secondary excitation.This is infrequentbut one instanceoccurred,cat #lOO, Fig. 1. All of these changesmight be expected,if the secondaryeffects are meant to be protective.Thus, the initial corticaldisinhibition (diagonallyhatched left area, Fig. 61, which is short-livedand often not seen because of the fast rising concentration,would tend to offset any spuriousor premature tendencyto inhibition.The longer post-inhibitory rebound (rightdiagonallyhatched area, Fig. 6) would tend to terminatethe primary inhibitionfaster, even though the concentration is falling slowly at the tail end of the curve. These secondaryeffects would thereforetend to accentuateor sharpen the onset and offset of the primary action (7).

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Resting

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LEVEL

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observed rate changes indicated by solid arrdws and expected polarization changes indicated by dashed arrows. Interpretation # 1 presents sequence of inhibitory effects. Interpretation # 2 presents sequence of excitation, secondary depression, and continued depression.

FIG. 5 Sequences of observed firing rates and corresponding expected polarization changes identifying inhibition and disinhibition (Interpretation #l) and excitation and depression(Interp. #2)

UNIT

CELL

INTRA

UNIT

CELL

INTRA

ANALYSIS

FIG.

6.

thresholci

(2O effect)

concentration

2O excitation

= disinhibition

1" inhibition

2' action

= action during onset and offset of drug concentration curve (i.e., on modifying of negative feedback circuits or other inhibitory circuits)

= action during effective concentration plateau and imediately adjacent region of drug concentration curve (i.e., on final pathway)

Theoretical curves for inhibition by developing drug concentration (ordinate) intersecting over time (abscissa) with low threshold for negative feedback and higher threshold for primary effects.

Definition:

lo action

INTERSECTIONOF DRUG CONCENTRATIONAND THRESHOLD CURVES

low threshold

higher

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PROSTAGLANDINS

1.

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4. 5.

6.

7. 8.

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REFERENCES Marrassi, M.A. and J.H. Daugherty. Effects of Prost,aglandin F2a (PGFza) on Cerebral Cortical Evoked Potentials Correlated with Local Dxygen Tension. Prostaglandins, preceeding paper. Marrassi, A.S. The Effect of Drugs on Neurons and Synapses. In Brain Mechanism and Drug Action (W.C. Fields, ed.1, C.C. Thomas, Springfield, Ill., 1957, p. 45. Marrazsi, A.S. The Way of a NeuroExploring With Drugs: pharmacologist. In Legacies in the Study of Behavior: The Wisdom and Experience of Many (J.W. Cullen, ea.), C.C. Thomas, Springfield, Ill., 1975, p. 96. Prostaglandin Cerebral InhibMarrazzi, M.A. and C.C. Huang. ition, Membrane Potentials. Fed. Proc. 34: 764, 1975. Marrazsi, M.A. Cerebral PGFZo Action on Cortical Field Potentials and Intracellular Potentials. Presented at the Fourth Annual Prostaglandin Conference, Vail, Colorado, March, 1975. Prostaglandin - NorepineSiggins, G., B. Hoffer and F. Bloom. Microelectrophoretic and Histophrine Interactions in Brain: chemical Correlates. Ann. N.Y. Acad. Sci. 180: 302, 1971. Inhibition as a Determinant ofsynaptic and Marrazzi, A.S. Ann. N.Y. Acad. Sci. 92: 998, 1961. Behavioral Patterns. Membrane Effzts - Analysis of Huang, C.C. and A.S. Marrazzi. Cerebral 5HT, LSD and CPZ Interactions, Neurosciences Abstracts, Qualitative Identify of 1972 and Huang, C.C. and A.S. Marrazzi. Cerebral Neuronal Membrane Actions of 5HT, LSD, and CPZ, in preparation. Biological Psychiatry 5: Exploring With Drugs. Marrazzi, A.S. 171, 1973. . . Cyclic Siggins, G.R., A.P. Oliver, B.J. Hoffer and F.E. Bloom. Effects on Transmembrane Properties of AMP and Norepinephrine. Cerebellar Purkinje Cells. Science 171: 192, 1971. AnalyG of Drug Block of LSD/S Huang, C.C. and A.S. Marraszi. Fed. Proc. HT/GABA by Monitoring the Role of Membrane Changes. 32: 303, 1973. -

Received

3/11/77

--Approved

7/l/77

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