Interaction of cholecystokinin and glutamate agonists within the dLGN, the dentate gyrus, and the hippocampus

BrainResearchBulletin,Vol. 39, No. 6, pp. 381-389, 1996 Copyright © 1996ElsevierScienceInc. Printedin the USA.All rightsreserved 0361-9230/96 $15.00 + .00 ELSEVIER

PII S0361-9230(96) 00030-5

Interaction of Cholecystokinin and Glutamate Agonists within the dLGN, the Dentate Gyrus, and the Hippocampus SIEGRUN GABRIEL,.1 ROBERT GROTZMANN,* MATHIAS LEMKE,* HANS-JORGEN GABRIEL,* PETER HENKLEIN'I- AND HELGA DAVIDOWA*

*Department of Neurophysiology, Institute of Physiology, Tucholsky Str. 2 ?Institute of Biochemistry, Hessische Str. 3-4, Faculty of Medicine (CharitY), Humboldt University Berlin, 10117 Berlin, Germany [Received 1 August 1995; Accepted 11 January 1996] ABSTRACT: The interac'don of su~,at~d cholecystokinin (CCK-SS) with excitatory amino acids (EAA) was studied on single units of the dorsal lateral genicuiste nucleus (dLGN), the dentate gyrus, and the hippocampal CA3 region in rats anaesthe'dzedwith urethane. lontophoreUc co-adminislndion of small, individually ineffec'dve currents of CCK-8S and kainic acid or N-methyI-D-aspartate repeatedly elicited an increase of the discharge rate in needy all genicuista and half of the dentate neurons but not in those of the CA3 region. The effect could be reduced by the CCKB receptor antagonist PD 135,158 more often than by the CCKA antagonist KL 1001. The inreased firing due to co-administn~on of CCK and kainate could also be suppressed by the non-NMDA antagonist CNQX but not by the NMDA antagonists CPP or AP-5, which were otherwise able to prevent the neuron from responding to co-admini~u,~;on of CCK and NMDA. It is suggested that in distinct brain regions the effectivity of the"low level" EAA transmission may be enhanced by small amounts of CCK-8S. This is thought to be mediated by a coactivation of CCK and EAA receptors.

from a subpopulation of intrinsic GABAergic interneurons [28,47,49], whereas within the dLGN the CCK-like immunoreactivity disappears after lesions of the upper strata of the superior colliculus [41]. The sulfated octapeptide CCK-8S, the most common natural agonist of CCKA- and CCKB receptors, caused excitatory effects in the dorsal raphe nucleus [10], in the cerebral cortex [30], in the hippocampus [11,14,22], and in the dLGN [2]. However, inhibitory actions have also been observed in the solitary complex [13] and in geniculate units [3,24,25]. In the dLGN, the responsiveness to CCK-8S and the direction of effects on visual responses apparently vary with the kind of sensory stimulation (diffuse or restricted to the center of the receptive field) [ compare 2 and 25 ]. A number of patch clamp studies revealed that the inhibition mediated by the CCKA receptor is associated with the activation of an inhibitory potassium current [13], whereas the excitation mediated by CCKB receptors appears due to a decreased conductance of a transient potassium current [10,13,44]. Recently, it has been reported that in the thalamic reticular nucleus, the excitation by CCK is elicited by the reduction of a voltage-independent K ÷ leak conductance via CCKA receptors [15]. Furthermore, it should be noted that inhibitions by CCK could be an indirect consequence of exciting inhibitory interneurons by CCK-8S [3,24]. Since EAAs play an essential role in processing sensory information [ 45,46 ] and in the induction of synaptic plasticity [ 18 ], it is of interest whether CCK promotes or attenuates their effects. Although CCK causes an excitation of neurons in various brain regions, it has been shown that CCK is able to reduce EAA-induced depolarizations [29] and to counteract excitotoxic damage of cultured neurons [1,31 ] as well. Therefore, we studied possible interactions between CCK-8S and EAA agonists on single unit activity in vivo by iontophoretic administration at amounts subthreshold for individual excitation of the cells.

KEY WORDS: Dorsal lateral genicu|ata nucleus, Area dentata, Single unit activity, Excitatory amino acid, Cholecystokinin, Microiontophoresis, Rat.

INTRODUCTION Fast excitatory synaptic transmission within the dentate gyrus (DG), the hippocampal CA3 region (CA3), and the thalamic dorsal lateral geniculate nucleus (dLGN) is mediated by excitatory amino acids (EAAs) [16,17,45,46,50] activating ionophoric glutamate receptors (GIuR) [8,39 cited for review]. GluR are coupled to cationic multiconductance channels, mainly permeable for Na ÷ and K ÷ ions (AMPA/kainate) and for Na ÷ , Ca 2+ , and K ÷ ions (NMDA) [38]. The NMDA channel exhibits a voltage-dependent block by Mg 2÷ ions [4]. The neuronal activity can additionally be modulated, not only by other classical transmitters but also by several peptides, including Cholecystokinin (CCK). CCK is present in nerve terminals around principal ceils both in the hippocampus [32] and in the dLGN [ 23 ]. In the hippocampus, these terminals originate

METHOD All experimental procedures concerning surgery and animal care were approved by the Regional Berlin Animal Ethics Com-

Requests for reprints should be addressed to Dr. Siegrun Gabriel, Charit6, Department of Neurophysiology, Tucholsky Str. 2, D-10117 Berlin, Germany. 381

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mittee. The experiments were performed on male Wistar rats (250-350 g) anaesthetized with urethane ( 1.2 g/kg, IP). ECoG and ECG were continuously monitored, and the rectal temperature was kept constant with a heating pad. The skull was trepanated at a site 4 mm anterior to lambda and 3.5 mm lateral allowing us to record from the dentate gyrus, the CA3 region, and the dLGN along one track. Extracellularly recorded action potentials were conventionally amplified and discriminated, displayed on-line as a frequency time histogram, and stored on disk (CED interface, spike 2 software package). Recordings were made before, during, and after iontophoretic administration of EAA agonists, CCK-8S, and their respective antagonists, ejected individually and by co-administration. The following drugs were chosen and prepared as shown in parentheses: kainic acid (KA, 100 raM, dissolved in 100 mM NaOH, pH 8.00), N-methyl-D-aspartate (NMDA, 100 mM, in 100 mM NaOH, pH 8.00), the non-NMDA antagonists 6-cyano7-quinoxaline-2,3-dione (CNQX, 1 raM, in 50 mM NaC1, pH 8.00), the NMDA antagonists (±)-2-Amino-5-phospho nopentanoic acid (AP-5, 50 raM, in distilled water, pH 8.00) and (_+)3- ( 2-Carboxypiperazin-4-yl)-propyl- 1-phosphonic acid ( CPP. 25 raM, in 150 mM NaC1, pH 8.00), the CCKB antagonist PD 135,158 N-methyl-D-glucamine salt (PD, 0.25 raM, in 60 mM phosphate buffered saline (PBS), pH 7.8), the sulfated cholecystokinin octapeptide (CCK-8S, 0.25 raM, in 60 mM PBS, pH 7.8), and the CCK A antagonist Suc-CCK-27-31-PEA ( KL 1001, 0.25 mM, in 60 mM PBS, pH 7.8) [12]. Kainate, NMDA, and AP-5 were purchased from SIGMA and CNQX, CPP, and PD 135,158 from Research Biochemical International ( RBI ). The peptides CCK-8S and KL I001 were synthetized by P. Henklein (Institute of Biochemistry, Charit6). Iontophoretic ejection of the drugs was performed by means of a seven-barreled microcapillary (tip diameter about 7 /~m), glued to the recording micropipette (vertical tip separation about 30 #m). All substances were ejected with negative currents ( 1100 nA) and retained with positive currents (5-15 nA). Changes in overall retention current were usually balanced via a NaC1 (50-150 mM, pH 7.9 ) -filled barrel. The effects of switching the balance unit on and off were frequently controlled (a) during stepping the electrode through the overlying cortex, (b) before beginning the recording from a cell, (c) when the resistance of a barrel had apparently changed, or (d) when NaC1 was ejected to seek for possible artifacts caused by application of higher currents or by perineuronal changes in osmolarity and pH. Previous work in our laboratory [2,3,20,21,24,25] revealed that current balancing had no effect on responses to CCK-8S or on the blocking effects of KL1001 or PD135,158, provided that the control measurements yielded a stable discharge rate concomittant to ejections of NaCI with currents up to 150 nA in both directions, and appropriate values of barrel resistance (30-80 Mr/ for the iontophoresis barrels, 5 - 2 0 Mf~ for the recording capillary) which did not change, neither during ejection nor by switching the balance unit on and off. In some experiments, all barrels were filled with drugs in order to test almost all of them on one and the same cell. The effects described in the Results were observed in experiments with and without current balancing. Repeated iontophoretic administrations have only been performed when the control discharge rates were nearly recovered. The time to recovery varied with the drugs or with the combinations of drugs administered, particularly for CCK-8S and its antagonists, and with the cell investigated. Therefore, the interejection intervals could not be kept identical for all cells and drugs, though this may account for some degree of the variability of effects. The recording sites were marked by iontophoretic deposits of saturated Trypan Blue ( 10 ~zA, 10 min). Their positions were histologically verified from the dye marks in fixed and fro-

GABRIEL ET AL.

zen coronal sections of 80 #m thickness and then mapped on schematic drawings taken from the stereotaxic atlas [42]. Data were deft ved from frequency time histograms (bin width 1 s ). We calculated the mean value of the discharge frequency before (control value, analysis time 60-90 s) and during ejection of a drug (drug value, analysis time 15 s including the time of the maximum effect), as well as the difference between them (drug value minus control value). A difference which exceeded the control value by more than two standard deviations of the mean control was referred to as "response," a decrease of the response by 66% or more as "reduction" (block). The individual ejection of CCK-8S or of any antagonist always preceded a co-administration ( 20-120 s ), whereby the difference calculated for the preceding ejection period served as a reference value for the co-administration effect. We used the Wilcoxon signed rank test to compare control and drug values and the Mann-Whitney U-test for comparisons of effects between the different regions. lnterspike interval histograms were additionally computed for some cells. RESULTS We studied a total of 77 cells which responded to iontophoretic administration of CCK-8S as well as to one or both of the glutamate receptor agonists NMDA and kainate. The cells were located within the DG (N = 21, triangles), within the CA3 subfield iN = 17, circles), and within the dLGN (N = 39, squares), most of them in the latero-caudal shell of the nucleus ) (Fig. 7A ). Figure I shows eight trials (500 s in duration) of a frequency time histogram recorded from a geniculate unit. CCK-8S and kainate (KA) excited the neuron when administered with currents of 30 nA and 25 nA, respectively. Currents of 15 nA (CCK8S) and of 10 nA (KA) induced no visible changes of the discharge rate (A). In contrast, the co-administration of CCK-8S and KA with identical currents caused a strong rise of the discharge rate CA) which was not mimicked by co-administration of NaC1 (75 raM, 50 nA) and CCK ( 15-30 nA) nor by that of NaC1 and kainate (10 nA) (B). The effect could be repeatedly evoked (C), but it was not obtained after a preapplication of the CCKB antagonist PD 135,158 (D) which also abolished the response of the unit to CCK-8S alone (E,F). The CCKA antagonist KL 1001 (70 nA) failed to reduce the response to CCK (G) as well as that to co-administration of CCK and KA (H). In Figure 2, it is demonstrated that co-administration of CCK-8S (35 nA) with NMDA ( 18 nA) and of CCK-8S with kainate (20 nA) [all currents subthreshold for individual excitation of the cell, see ( B )] caused a comparable increase of the discharge frequency (C). In presence of the NMDA antagonist AP-5, the neuron did not respond to co-administration of CCK and NMDA (D), while the reaction to co-administration of CCK and kainate was stronger than before (E, second peak). It is possible, however, that this facilitation of the response may be due to an increased perineuronal concentration of KA, still enhanced after the preceding "suprathreshold" administration of KA (E, first peak). The response to co-administration of CCK-8S and KA was abolished by an additional application of the non-NMDA antagonist CNQX (F). Again, after a long recovery period of 24 min, the neuron increased its discharge frequency concomittant to co-administration of CCK and KA (G). Similarly as in Figure 1, the response was not obtained when the CCKB antagonist PD was additionally ejected (G), but could be evoked in presence of the CCKA antagonist KL 1001. As shown in Figure 3, a geniculate unit could increase its discharge frequency during co-administration of CCK and NMDA despite a preceding ejection of CNQX (G), whereas a similar ejection of CNQX led to a suppression of the response to co-administration of CCK and KA (K). Re-

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FIG. 1. Single unit activity displayed as discharge frequency (imp/s, y-axis) over time (t in s, x-axis). A ycalibration bar (50 imp/s) is given on the right side of (H). (A-H) eight trials (analysis time 500 s, bin width 1 s) recorded subsequently from a geniculate neuron. The compounds iontophoretically administered are indicated on the right in the same top to bottom sequence as the bars (stippled line for NaC1 75 mM) which mark the administration periods. Duration of the administration is given by the length of the bars, ejection current intensities in nA by the numbers left from the bars. Missing a number means that the current is the same as before. (A) Increases of the discharge frequency by administration of CCK-8S (30 nA) and kainate (KA, 25 nA), and by co-administration of 15 nA CCK-8S and 10 nA KA which were ineffective when individually ejected. (B) Coapplication of 50 nA NaCI (75 mM) with CCK-8S or KA did not cause a change of the discharge frequency in response to CCK-8S (15, 25, and 30 nA) and did not mimick the effect of CCK8S and KA co-administered. (C) Again, co-administration of CCK-8S and KA using the same currents as before caused an increase of the discharge rate. (D,E,F) The responses to co-administration (C) as well as to 30 nA CCK alone (E) disappeared when the CCKB antagonist PD 135,158 was additionally coapplied. (G) The response to CCK-8S recovered within 10 min and could still be obtained during an additional ejection of the CCKA antagonist KL1001. (It) Even the response to co-administration of CCK-8S and KA was not suppressed by KL ejected with 30, 50, and 70 nA (only shown for 70 nA). In the current experiment, NMDA was left absent from the iontophoresis barrels for using current balance via a barrel filled with NaC1. During recording, (A)-(D) current balance was off, in the other trials on.

cordings from two dentate units are depicted in Figure 4 ( A - H and I - K ) . In both, the relatively fast spiking neuron and the spontaneously silent one, the co-administration of CCK-8S and N M D A also induced a remarkable elevation of the firing frequency (C,J,K). The effect was completely blocked by the N M D A antagonist CPP (H) and clearly reduced by the CCKB antagonist PD135,158 (D,E), but not by the C C K A antagonist KL1001 (F). In the other cell ( I - K ) , the response to the preceding ejection of CCK-8S increased whereas that to the subsequent co-administration of CCK and N M D A decreased when the current of CCK was stepwise enhanced from 20 nA to 40 nA (J,K). We obtained similar effects in four cells which we tested with increasing currents for CCK while leaving the current constant for kainate (Fig. 5 ), Increasing the ejection current of CCK8S yielded increased discharge frequencies upon co-administration with kainate reaching a maximum and declining upon further enhancement of the current for CCK-8S. The response to CCK8S was unaltered in presence of CPP or C N Q X ejected with currents effective to block the effect to co-administration of CCK and N M D A or kainate (n = 10). The C C K A antagonist KL 1001 apparently facilitated the response to co-administration of CCK and N M D A or kainate in a small number of cells (4 out of 43). Furthermore, it should be mentioned that interval histograms of geniculate units computed for the period of co-administration mostly showed a lower frequency peak of short intervals ( < 5 ms) and an additional frequency peak of intervals in the range of 1 5 - 4 0 ms compared with the interval histogram of the preceding ejection period of CCK-8S alone. In interval histograms

of the hippocampal cells investigated, a frequency peak of intervals shorter than 5 ms has not been observed. In summary, co-administration of CCK with kainate and of CCK with N M D A yielded effects in the same direction and of roughly equal strength in 82% (28 out of 34) of the cells tested with both combination of drugs (Fig. 6). The difference between the effects of co-administered K A and N M D A found in the dLGN was statistically insignificant. This allowed us to pool data from cells tested with only one or both combinations. As shown in Figure 7B, 46 out of 77 cells increased their discharge rate by a value higher than the control value plus three standard deviations of the mean control (filled marks) when CCK-8S was coadministered with either K A or NMDA. The discharge frequency increased on average by a factor of 7 (p < 0.001, N = 77, Wilcoxon test). With reference to the three regions investigated, it is obvious that nearly all dLGN cells (36 out of the 39) and about one half of the DG cells ( 10 out of 21 ) responded in this way. The elevation of the discharge rate was significantly higher in the dLGN (squares) than in the DG (triangles) (32.1 ± 13.99 vs. 19.7+_7.89 imp/s; p < 0.01, M a n n - W h i t n e y U-test). In the CA3 region (circles) a similar augmentation of the discharge frequency was not seen. As analysed for 23 cells (dLGN and D G ) , the effect of co-administering CCK-8S with one of the EAAs [29.9 (16.06) i m p / s for mean value and (standard deviation) of the discharge frequency] was neither evoked by co-administration of NaC1 with CCK-8S [2.6 (1.95) imp/s vs. 2.5 (2.10) imp/ s for CCK-8S alone] nor with one of the two EAAs [4.1 (4.28) imp/s vs. 3.6 (4.25) imp/s for the E A A alone]. The currents

384

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FIG. 2 Recordings from a geniculate unit, denoted as in Fig. I. (A) lontophoretic administration of 55 nA NMDA, 60 nA CCK-8S, and 50 nA kainate (KA*) evoked strong responses. (B,C) Co-administrations with currents subthreshold to changes in firing (B) caused comparable increases of the discharge frequency for CCK-8S and NMDA and for CCK-8S and KA (second and third peak in C). (D,E) The response to CCK-8S and NMDA is absent in presence of the NMDA antagonist AP-5, but that to co-administration of CCK-8S and KA seems to be stronger than in (C). This response facilitation may be due to a higher perineuronal concentration of KA caused by the preceding ejection of KA. (F,G,tt) The response to CCK and KA was largely suppressed in presence of both the non-NMDA antagonist CNQX and AP-5 (F), the reaction recurred after 24 min but seems still to be reduced (G). The co-administration failed to evoke an increase of the discharge frequency when the CCKB antagonist was additionally applied, but revealed a strong effect in presence of the C C K A antagonist (H). (All trials without current balance.)

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FIG. 3 Recordings from a geniculate neuron denoted as in Fig. 1. (A,B,I) The currents used for co-administration (50 nA CCK-8S, 20 nA NMDA, and 20 nA kainate) were unable to cause firing when individually administered. (C,H,,J) Potentiating effects of CCK-8S and NMDA and of CCK-8S and KA are shown. (D,E,F,K) The effect of CCK-8S and NMDA co-administered was not observed in presence of the CCKB antagonist PD (D) and the NMDA antagonist CPP (F) but could be obtained in presence of the CCKA antagonist KL (E) and the non-NMDA antagonist CNQX (F), which was able to block the response to coadministration of CCK and KA (K).

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FIG. 4. Recordings from two dentate neurons (A-H, I-K) denoted as in Fig. 1. (A,B) The currents used to co-eject CCK and NMDA were even at the threshold (A,B) or subthreshold (I) for individual excitation of the cells. (CJ,K) Both the relatively fast spiking neuron and the more silent one showed an increase of the discharge rate during coadministration of CCK and NMDA (C, first peak in J and third peak in K, respectively). (D,E) A sequence of additional co-administrations of the CCKB antagonist was started after (C) using ejection currents of 20, 30, 40 and 50 nA, a reduction was noted with 40 and 50 nA, displayed in (D) and (E), respectively. (F) The response could again be evoked in presence of the CCKA antagonist KL. (G) The co-adminislration was performed using a smaller ejection current for CCK to avoid the prolongation of the response seen in (C). (H) The co-administration was repeated before and during an additional application of the NMDA antagonist CPP. CIJ,]K) The response of the other neuron to co-administration of CCK-8S and NMDA [ first peak in (J)] seems to be attenuated when the ejection current of CCK-8S has been stepwise enhanced, whereas the responses to CCK obtained within the preapplication time became increasingly stronger (J and K). After the ejection current for CCK was reduced below the value of the first co-administration, the response increment was comparably largest (last peak in K).

used to eject NaC1 and CCK-8S are comparable [47.7 (20.00) nA vs. 48.3 (27.10) nA]. An effect upon co-administration of CCK-8S and either kainate or N M D A could be reduced either by one of the CCK antagonists or by the specific E A A antagonist. The black bars in Figure 8 illustrate the percentage of cells in which the effect could be reduced by more than 66% ( " b l o c k e d " ) . The increase of the firing frequency caused by co-administration of CCK and N M D A or kainate was more often blocked by the CCKB antagonist than by the C C K A antagonist [Fig. 8, ( K L + P D ) : 63% vs. 12% (N = 41 ), p < 0.001, Pearson chi-square for cross tabulation]. The mean discharge frequency in response to co-administration [28.7 (11.14) imp/s] was lowered to 32% by PD and to 74% by KL [9.3 (10.79) imp/s and 21.8 (15.94) imp/s, respectively] when cells were considered in which the response was clearly blocked either by PD or by KL (N = 31 ). The effects of the two E A A - C C K combinations were reduced by the specific E A A antagonist (CPP or AP-5 for NMDA, C N Q X for K A ) in 73 % ( 16 / 22 ) and 79 % ( 11 / 14 ) of the cells tested for CCK with N M D A and CCK with KA, respectively. Thereby, the mean effects were completely abolished in these cells [23.8 (10.71) vs. - 0 . 9 (1.68) and 24.6 (10.44) vs. 1.9 (4.04) imp/s, respectively ]. However, the reaction to C C K - K A could not be reduced by the N M D A antagonists CPP or AP-5 [25.7 (9.75) vs. 23.2 (8.24) imp/s] in all 17 cells tested (Fig. 8, AP-5), though the

currents were usually appropriate to block the reaction to CCK and NMDA.

DISCUSSION We found a remarkable increase of the neuronal discharge frequency in response to simultaneous administration of CCK8S and either K A or N M D A ejected with currents subthreshold to individually exciting the cells. This effect was not mimicked by ejection of NaC1 with comparable currents, osmolarity, and pH. Both CCK and EAAs seem to be able to induce similar excitatory effects on units in the hippocampal slice preparation [14,22] and on units in the dLGN of anaesthetized animals [2], although the responses of geniculate neurons to CCK-8S mostly started slower and outlasted the time of administration. In previous experiments, we had observed an additive increase by CCK-8S and glutamate of "sluggish" responses to stimulation of the center of the receptive field in a small number of neurons tested (not published). In a subsample of neostriatal neurons, the responses to glutamate could be increased by CCK by a factor of two [21], which is smaller than the effect reported here. Now, we found a multiplication of the firing frequency indicating the induction of a nonlinear process by co-administration of CCK and E A A agonists in the "subthreshold" range. Taking into account that this result is most prominent in the dLGN, it

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FIG. 5 Relation between effects of co-administration of CCK-8S and KA (y-axis: drug value minus control value, imp/s) and the ejection current of CCK-8S (x-axis, nA) measured in four neurons. As marked below thc abscisa, the currents used to eject kainate were constant for the study of an individual neuron, but varied between the neurons. may be possible that the nonlinear increase in discharge frequency is due to peculiar intrinsic properties of thalamic neurons. i.e., the ability to generate burst discharges [19,36,50]. Burst firing modes add nonlinearity to visual responses [27,37]. If CCK-8S shifted the membrane potential towards values permitting the generation of low threshold (LT) Ca 2+ spikes, increased burst firing should be evoked by both a small ( individually subthreshold) fast kainate-EPSP or a slower NMDA-EPSP as well. Indeed, CCK-8S can induce burst firing via C C K A receptors [20]. However, we observed that the effect of the co-administration could rarely be blocked by C C K A receptor antagonists, indicating that bursting induced by CCK-8S via C C K A receptors is not a precondition for the effect. Moreover, our interval histograms did not provide any hint for increased burst firing during the period of co-administration. Thus, under these conditions, other features seem to be responsible for the high discharge rate. CCK is supposed to reduce a transient outward potassium current via CCKB receptors [13]. The additional inward current activated by individually subthreshold perineuronal concentrations of kainate or N M D A may then be strong enough to cause a depolarization reaching the threshold for a switch towards the relay mode. Alternatively, the discharge frequency might increase due to an additional enhancement of the basal release of aspartate [ 71 or of glutamate and aspartate [43] by CCK-8S or, conversely. due to an additional release of CCK by agonists of the EAA receptors [6,51]. However, increased firing by co-administering CCK-8S with both kainate and N M D A as well could be antagonized by blocking the receptor for the EAA ejected and not by the respective unspecific antagonist. This may be considered as an objection to the possibility that the release of aspartate or glutamate by CCK-8S is responsible for the effect. Otherwise, it should be noted that there is some similarity considering the "bell shape" of the concentration/release relationship found in the literature [7] and the current/effect relationship observed in our experiments (Fig. 5). The suggestion that an increased release of CCK by EAA agonists may contribute to the effect under discussion cannot be substantiated by decisive arguments. We demonstrated that the discharge frequency was enhanced by both N M D A and kainate co-administered with CCK-8S, whereas an increase of the basal release of CCK could be evoked by gluta-

mate and kainic acid [51] but not by N M D A [6]. Therefore, further experiments have to be performed using appropriate methods to test the implication of release mechanisms. Another process which could lead to an increase of the discharge frequency during co-administration is a sensitization of postsynaptic EAA receptors by CCK, possibly due to the induction of secondmessenger-dependent enzyme cascades [48 ]. We would expect that such a sensitization strengthens the effect of a repeated coadministration. This could not be detected in our experiments possibly due to masking by ejection of receptor antagonists or by other sources of decreased effectivity, such as the long interejection intervals sometimes used. The range of currents of CCK-8S over which the effect of the co-administration could be evoked was found to be limited to values ( sub)threshold for exciting the cell by CCK-8S alone. The attenuation of the effect by further increasing the ejection current of CCK-8S may be associated with reaching a higher perineuronal concentration of CCK-8S, which causes an additional depolarization of inhibitory intemeurons. This assumption is substantiated by the finding that inhibitory actions of CCK can partly be blocked by G A B A antagonists [3,24]. Though the mechanisms which mediate the effects of co-administering CCK-SS with NMDA and kainate remain to be elucidated, our results support the existence of an augmenting interaction of CCK and glutamate agonists apparently restricted to resting conditions, i.e., a " l o w level" transmission of excitatory amino acids and a small perineuroual concentration of CCK. The effect of this interaction was mainly antagonized by the CCKB antagonist PD 135,158, This is in accordance with results showing a preferential B-receptor-mediated action of CCK in hippocampal and geniculate fields [2,11,25]. The proportion of effects blocked by the C C K A antagonist is related to the distribution of C C K A receptors and to the chance of recording from cells which expressed C C K A receptors rather than to ineffective perineuronal concentrations for KL1001. This C C K A antagonist had blocked the effects of CCK-8S on visual responses of genic-

imp/s 40 W

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FIG. 6 Mean value (bars) plus standard error (symbols lined to the bars) of the co-administration effect (drug value minus control value) ['or CCK-8S with NMDA (3) and for CCK-8S with KA (4), The sum of the effects of CCK and NMDA (1) and of CCK and KA (2) ejected individually with the same below-threshold currents may serve as additional reference. All administrations were performed on each out of 18 cells in the dLGN, out of 8 cells in the dentate gyrus and out of 8 cells within the CA3 region. The two effects caused by co-administration of CCK with NMDA and by CCK with kainate did not differ significantly.

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administration could also be antagonized by blocking the receptor for the E A A ejected. The attempt to block the effect of coadministering kainate with C C K by AP-5 or CPP was a failure, though the antagonists were ejected with currents effective to block the responses to CCK-8S and N M D A . This is inconsistent with the idea that the N M D A - r e c e p t o r - g a t e d ion channel may be involved in the interaction of K A and CCK-8S. The results of the blocking experiments suggest the following conclusions: ( a ) a C C K receptor has to be activated; ( b ) the E A A receptor allowing binding the agonist administered has to be activated, i.e., the nonlinear process leading to the potentiation of the discharge frequency is not addressed to a specific E A A receptor; and ( c ) the mechanisms underlying the interaction should be mainly postsynaptic. Furthermore, we found regional differences represented by predominantly strong effects of the interaction in the d L G N and moderate effects in the DG, whereas the CA3 region was apparently devoid of them. Therefore, it should be presumed that the interaction occurred most frequently and strongest in a region where burst firing due to Ca 2+ spikes is common. Since evidence has been provided that low and high threshold Ca 2+ currents are also present in hippocampal neurons [ 9,40 ], an account of other intrinsic properties of thalamic cells [35] to the regional differences should be taken into consideration. A different density of CCK receptors (high density in the geniculate nuclei and moderate density within the hippocampus) [26] may additionally be responsible for the differences between the d L G N and the DG. The differences between the D G and the CA3 region might parrelative frequency

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FIG. 7. (A) Location of the cells investigated assigned according to two maps of coronal slices, 4.70 and 4.20 mm distant from the interaural level [27]. (B) Mean value and standard error for the co-administration effects (drug value minus control value in imp/s; y-axis) by CCK-8S and one of the glutamate agonists, separately demonstrated for neurons of the different regions (DdLGN, ADG, ©CA3) and for the strength of effects [ filled symbols (top): effect larger than the control value plus three standard deviations of the mean control, filled symbols (middle): effect within the range of the control value ± three standard deviations of the mean control; empty symbols (bottom): effect lesser than the control value minus three standard deviations of the mean control]. The number of cells included in the subsamples is indicated above the error bars. Data were pooled from experiments in which only one of the two combinations were tested with that from experiments in which each of the two could be investigated (using the larger value for the calculations). The ejection currents for CCK-8S and the glutamate agonists (mean + standard error) used in the different regions were 48 ± 4.12 and 33.4 ± 4.86 (dLGN), 46 ± 6.51 and 38.2 ___6.00 (DG), and 51.8 ± 6.81 and 38.7 ± 8.57 (CA3), respectively.

ulate units with higher frequency ( 17 out of 36 cells) [25 ] than reported here, though there were the same conditions of drug administration as in the present experiments. W h e t h e r the CCKB receptor is favored to mediate the interaction due to particular physiological properties has to be left open. The effect of the co-

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FIG. 8. Percentage of neurons in which the responses to co-administration were reduced (>66%, black bars)/not reduced (<66%, stippled bars) by antagonists. The groups of bars are mainly categorized by the antagonists used and by the number of cells investigated, given below the x-axis. In presence of the CCKA antagonist KL, the co-administration effect could be reduced in 8 out of 43 cells tested. In contrast, the CCKB antagonist caused a reduction in 31 out of 41 cells. Testing both antagonists in each of 41 cells, we obtained a reduction by PD 135,158 in 26 ceils (3), by KL in 5 cells (1), partially by both (>50%) in 4 cells (2), and "noreduction" in presence of each of the two in the remaining 6 cells (4). The effect of NMDA and CCK-8S was markedly reduced by AP-5 or CPP in 17 out of 22 cells. The response to kainate and CCK-8S could not be blocked by AP-5 or CPP in all cells tested, whereas it was abolished by CNQX in 11 out of 14 cells investigated. The mean values and (standard deviation) of currents in nA used to eject the antagonists were 60.8 (21.97) (KL1001), 38.9 (21.13) (PD 135,158), 37.8 (18.19) (CNQX), 27.6 (16.98) (AP-5 or CPP), and 57.9 (18.34) (AP-5 or CPP, tested against CCK-8S and kainate).

388

G A B R I E L ET AL.

tially be attributed to corresponding distinctions in the specific binding of [3H]CCK8, which is slightly higher in the DG than in the CA3 region, especially in the layer of the principal cells [33]. However, it seems unlikely that this is the reason for the absence of augmenting effects in CA3. Lavoie et al. [ 34 ] recently reported for the CA3 region a potentiation of neuronal responses to N M D A by CCK-8S mediated by C C K A receptors. Further experiments are required to find out the preconditions for such differences. In conclusion, small quantities of CCK and EAAs could result in a considerable, short-lasting augmentation of the neuronal activity. Thus, a small perineuronal concentration of CCK enables the projection neurons of distinct brain regions to respond to low presynaptic spike frequencies usually caused by stimuli of small size or low intensity ( d L G N ) or by low entorhinal activity ( D G ) . This promotion of glutamatergic transmission at a low activity level may be involved in the maintenance of functional plasticity as recently shown for the CA1 region [5]. The augmenting interaction is not necessarily in contradiction to suppression by CCK of responses to glutamate agonists [ 2 9 ] , of responses to optimal sensory stimulation [ 25 ], or of excitotoxic effects [ 1,31 I. Most of the suppressive interactions of CCK and E A A agonists were observed at the level of neuronal responses, whereas the augmenting interaction reported here was found under resting conditions. Therefore, the neuronal activity may be enhanced by CCK when the activation of ionic glutamate receptors t'GluR) is low, whereas the neuronal activity may be suppressed when the activation of GluRs is high. Thus, the spectrum of functions of CCK is extended towards an augmenting interaction with glutamate agonists via CCKB and ionic glutamate receptors, coactivated by small perineuronal concentrations of their ligands at a low level of neuronal activity, at least in the dLGN and the DG. ACKNOWLEDGEMENTS The authors thank Prof. U. Heinemann and Dr. R. M. Empson for critically reading the first draft of the manuscript. They gratefully acknowledge the excellent technical assistance of Ms. S. Latta and Ms. U. Seider, and the photographical assistence of Ms. W. Piepke and Ms. B. Formann. This research was supported by grant 01ZZ9101 of the Bundesministerium fiir Forschung und Technologic Germany.

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