Effect of potassium on hippocampal ribonucleic acid concentration

EXPERIMENTAL

Effect

NEUROLOGY

25,

626-631 (1969)

of Potassium

on Hippocampal

Ribonucleic

Acid

Concentration IVAN IZQUIERDO, ERNA S. A~ARICHICH, Departamento

de Farmacologia,

Institute Estafeta

Naciolzal de Cdrdoha, Received

June

11, lP69;

Revision

AND ANTONIA

de Ciencias 32, Ctirdoba. Received

G. NL’ASELLO 1

Q&micas, Universidad drgentina August

14,1969

A cannula with normal (3.2 mEq) or higher than normal (7, 11, 25 mEq) K+ concentrations was placed on the hippocampus; this was considered artificially to cause corresponding modifications of (K+),. Up to 11 mEq, K+ produced an increase of total hippocampal RNA concentration, and 25 mEq caused a decrease. The increase wa6 attributed to an enhancement of spike-dependent transmitter release, as it was antagonized by 15 mEq Mg++ and by 30 mEq procaine. The possibility was considered that the increase of RNA was dependent on pyramidal cell firing, which would be enhanced by increased synaptic efficiency. Procaine, in the presence of normal K+, lowered the hippocampal RNA concentration. The decrease of hippocampal RN.4 found with 2.5 mEq K+ was related to the induction of seizures and to stimulation of the Na+-K+ pump. Introduction

Hippocampal extracellular space is small (11)) and neurons firing within it are prone to cause K+ accumulation (10, 19). Afferent stimulation is often followed, in the hippocampus, by heterosynaptic facilitation (31)) and if it is repetitive, by heterosynaptic post-tetanic potentiation ( 15, 16). This is enhanced by veratrine and depressed by tetraethylammonium (26), which are known to respectively increase and decrease the K+ outflow per spike (17). Perfusion of the hippocampus with high K+ enhances evoked responses (34) and may lead to seizures (33)) as does veratrine (26). Potentiation and facilitation of hippocampal postsynaptic responses are attributed (1.5, 16, 26, 31) to the known enhancing effect of high (K+)O on spike-induced transmitter release (27). Heterosynaptic potentiation (1.5, 16) and facilitation (31) constitute, by definition, paradigms of learning (18, 20). The hippocampus typically fea1 This work was supported by fellowships (E. S. Marichich and A. G. Nasello) and a grant (I. Izquierdo, No. 2389a) from the Consejo National de Investigaciones Cientificas y T&nicm, Argentina. The first author is an Established Investigator of that institution. We acknowledge the collaboration of R. Rodriguez during some of the experiments. 626

HIPPOCAMPAL

RNA

627

tures “theta” or faster rhythms during acquisition (1, 9)) which, if interfered with, result in impaired learning (3). These rhythms are caused by activity of a pacemaker in the medial septum, which sends repetitive impulses to the hippocampus by the dorsal fornix (30). Faradization of that pathway causes heterosynaptic potentiation in the hippocampus (15), and systemic or intraseptal administration of amphetamine, nicotine, or eserine, which activate the pacemaker (30) and generally favor learning (7, 23, 32), induce potentiation of hippocampal responses to commissural and subicular stimulation ( 16). We further reported that conditioning, or single injections of amphetamine and nicotine, increase hippocampal ribonucleic acid (RNA) concentratration, whereas other drugs or pseudoconditioning do not (25). We attributed this effect to a (K+),O increase, which learning-if it involves heterosynaptic potentiation or facilitation (15, 31)-amphetamine and nicotine were considered to produce. The present paper deals with the effect of artificially increased (K+)* on hippocampal RNA concentration. Material

and

Methods

One-hundred-and-seven adult female albino rats (155-250 g) were used. They were anesthetized with urethane (l-l.5 g/kg, ip) and placed in a stereotaxic machine. The dorsal hippocampus was exposed by suction and a polyethylene cannula of 3 mm internal diameter was (14-16,26,30) gently placed on the alveus of one side with a micromanipulator. The cannula contained electrolyte solutions whose hydrostatic pressure was kept at zero by way of a syringe on the other end. The solutions had varying K+ concentrations (3.2, 7, 11, 25 mEq/l), with or without Mg++ (15 mEq/l) or procaine (30 mEq/l) (Table l), and their ionic strength was completed to 151.6 mEq/l with Na+, Cl- being the only anion present. This ionic strength and the K+ concentration of 3.2 mEq/l were considered normal values, according to figures (29) for rat cerebrospinal fluid. It was assumed that the extracellular space was an aqueous medium and that diffusion between it and the cannula would be free; therefore, electrolyte concentrations between both would equilibrate very rapidly (33). An interval of 15-20 min was allowed to pass between the end of the surgical procedure and placement of the cannula, as we did in our electrophysiological experiments (14-16, 26, 31), before starting to record. During this interval, the exposed wound was kept covered with cellophane paper. The cannula was left in place 30 min, after which the underlying hippocampus was quickly dissected, freed from the dentate gyrus, fimbria, and fornix, weighed, and homogenized in ice-cold 0.2 N perchloric acid. Next, the Schmidt-Thannhauser procedure for total RNA assay was carried out, according to Munro and Fleck (24) ; RNA was measured by ab-

628

IZQUIERDO,

MARICHICH

AND

TABLE RNA

COKCENTRATION

OF HIPPOCAMPUS

AFTER PLACEMENT CONCENTRATIONS ON

NASELLO

I (mg.‘lOO g fresh tissue f. SE) DIFFERENT K+

OF A CANNULA CONTAINING IT (NUMBER OF EXPERIMENTS

IN

PARENTHESES)

Plus: Kf concentration (mEq/l) 3.2

7 11

___--

-

15 mEq/l

236 f 5 !14) 253 f 5"

“Significant

---___ procaine

204 f

5"

(10)

-

(14) 272 l @*c 216 f

7

115) 230 & 8

(16)

(16) 25

Mg++

247 f

30 mEq/l

-

.5bJ

difference from group treated with preceding K+

204 f 6c (10) -

concentration;

p <

0.02.

%ignificant difference from group treated with preceding K+ concentration; 0.0005. “Significant difference from group treated with 3.2 mEq/l K+; p < 0.0005. %ignificant difference from group treated with 3.2 mEq/l K+; p < 0.005.

p <

sorption at 260 mp in a Beckman DU spectrophotometer. The absorption spectrum between 210 and 320 rnp of several randomly chosen samples was measured in a Cary automatic apparatus by Dr. F. Cumar, of the Biochemistry Department of this Institute. No other peak but the expected one at 260 mp was ever observed. Results

Results are summarized in Table 1. Increasing K+ concentration, up to 11 mEq/l, was associated with increased hippocampal RNA, but a further increase to 25 mEq/l actually lowered it. The effect of 11 mEq/l K+ did not occur in the presence of either Mg++ or procaine; Mg++ had no significant effect of its own, but procaine alone (i.e., in the presence of normal K+) caused a decrease of hippocampal RNA concentration. The control value for hippocampal RNA in the present paper (236 f 5 mg/lOO g fresh tissue; Table 1) was considerably higher than any of the control values of a previous research (25). Animals in the present series were subjected to various experimental procedures (anesthesia, placement in the stereotaxic apparatus, surgical exposure of the hippocampus) to which those of the previous study were not; furthermore, removal of the hippocampus from the rest of the brain was done under anesthesia and in the whole animal in this series, whereas in the previous paper it was carried out in heads separated from the body.

HIPPOCAMPAL

RNA

629

Discussion

Isolated rat cerebral cortex responded to 15 rnM K+ in a bath with enhanced incorporation of uridine into RN,4, whereas 50 mM resulted in a fall of such incorporation (28). In view of this, we entertained the possibility that the effect of K+ on RNA concentration reflects variation in the rate of synthesis. The depressant effect of 25 mEq/l K* on hippocampal RNA may be related to the tendency to induce seizures of K+ concentrations of that order (33). Seizures, by an unknown mechanism, are known to lower total brain RNA (5). The mechanism might be intense stimulation of the Na+-K+pump by high ( K+)0 ( 17), which would reduce cellular ATP levels and, in consequence, depress RNA synthesis, which is dependent on ATP concentration (13). There is an increased Na+-K+-ATPase activity in rat brain during seizures (E. de Robertis, personal communication). The increase of hippocampal RNA by K+ up to 11 mEq/l might, in principle, be ascribed to any of the known effects of this ion on neurons. Some of these effects, however (depolarization, pump stimulation, direct activation of the genome) may be ruled out, in this connection, on various grounds. To begin with, none of these would be expected to be blocked by Mg++ or procaine, as the effect on RNA was. Besides, either depolarization or pump stimulation would, be proportional to (K+)” up to probably well over 25 mEq/l (17)) w h ereas the effect on RNA clearly was not. Furthermore, stimulation of the pump would rather tend to decrease RNA (see above). As to the possibility of a direct effect of K+ on the genome (6, 21), and in spite of us having originally proposed it in this connection (25), it is unlikely for two reasons : (a) it should perhaps not have been antagonized by Mg++, whose activating effect on isolated nuclei is similar to that of K+ (6, 21) ; (b) it is highly improbable that the range of (K+), we studied carried (K’) i to below 40 or above 100 mEq/l, which are the extreme limits between which rat brain cell isolated nuclei respond to K+ with maximal RNA synthesis (6). The antagonism by Mg++ on the effect of 11 mEq/l Ii+ on RNA points out to a mediation of the latter increased transmitter release, on which both ions have opposite effects (8, 27). Since procaine also blocked the effect of K+ on RNA, and since spontaneous miniature postsynaptic potentials of pyramidal cells have not been described (lo), it seems more likely that the K+ effect was mediated by an enhancement of spike-induced (27) and not of spontaneous transmitter release (8). The depressant effect of procaine alone on RNA suggests that the synthesis of the latter may be dependent on firing even in steady-state conditions. Indeed, probably all the situations which we found to increase hippo-

630

IZQUIERDO,

MARICHICH

AND

NASELLO

campal RNA involve increased pyramidal cell firing: conditioning amphetamine, and nicotine, through theta rhythm (1, 9, 10, 30) and heterosynaptic facilitation or potentiation (15, 16, 31), and high K+, through enhanced synaptic efficiency (27). Pyramidal firing originates apparently in the apical dendrites (2, 31), and since it is conducted into the axons (31) , it must traverse the region of the cells where the nuclei are. It is conceivable that when spikes pass by, charged molecules, such as the acidic proteins described by Berendes (4), enter the nuclei and activate the DNA to produce RNA. In this connection, one notes that both hippocampal pyramids and Purkinje cells, whose spikes originate in dendrites antipodal to the axons (22), have tetraploid amounts of nuclear DNA (12). References 1. ADE~, W. R. 1961. Studies of hippocampal electrical activity during approach learning, pp. 577-588. In “Brain mechanisms and learning.” A. Fessard, R. W. Gerard, J. Konorski, J. F. Delafresnaye teds.]. Blackwell, Oxford. 2. ANDERSEN, P., T. W. BLACKSTAD, and T. LBMO. 1966. Location and identification of excitatory synapses on hippocampal pyramidal cells. Exptl. Brabt Res. 1: 236-248. 3. AVIS, H. H., and P. L. CARLTOK. 1968. Retrograde amnesia produced by hippocampal spreading depression. Sciolce 161: 73-75. 4. BERENDES, H. D. 1968. Factors involved in the expreslsion of gene activity in polytene chromosomes. CI~ro~rloso~rn 24 : 418-437. 5. CHITRE, V. S., S. P. CHOPRA, and G. P. TALWAR. 1964. Changes in the ribonucleic acid content of the brain during experimentally induced convulsions. J. A’EIITOchcrtr. 11: 439-448. 6. DUTTON, G. R., and H. R. MAMLER. 1968. “In vitro” RSA synethesis by intact rat brain nuclei. J. j!relLl-oclzcrlr 15 : 765-780. 7. EVANGELISTA, A. M., and I. IZQUIERDO. 1968. Efecto de anfetamina, nicotina y atropina sobre adquisicibn y consolidation de memoria, I Rewio’rt, Sot. Arg. Farnzacol. Exptl., A4bstracts, p. 11. 8. GAGE, P. W., and D. M. J. QUASTEL. 1965. Dual effect of potassium on transmitter release. Nutztre 206: 625-626. 9. GRASTYAN, E., K. LISSAK, I. M;ZDARP;SZ, and H. DONHIXWER. 19.59. Hippocampal e!ectrical activity dur’ng the development of conditioned reflexes. Elecryocncephalog. Cliti. Ncrlvo~l~~siol. 11 : 109430. 10. GREEN, J. D. 1964. The hippocampus. Physiol. Rcz. 44 : 561-608. 11. GREEN. J. D., and 1). S. M.?S\VELL. 1961. Hippocampal electrical activity. I. Morphological aspects. Elcctrorncephalog. Cl&. Neurophysiol. 13 : 837-846. 12. HERMAN, C. J., and L. \v. LAPHAM. 1968. DNA contents of neurons in the cat hippocampus. Scicncc 160 : 537. 13. ITOH, T., and J. H. QUASTEL. 1969. Ribonucleic acid biosynthesis in adult and infant rat brain in vitro. Scirnce 164: 79-80. 14. IZQUIERDO, I. 1967. Effect of drugs on the spike complication of hippocampal field potentiabs. Exptl. Newel. 19 : I-10. 15. IZQUIERDO, I., and B. J. VASQUEZ. 1968. Field potentials in rat hippocampus : man-

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