- Email: [email protected]
Effects of two newly synthetized cholecystokinin tetrapeptides on the activity of single hippocampal and thalamic neurons
Neuropeptides (1995) 29, 83 87 © Pearson Professional Ltd 1995
Effects of Two Newly Synthetized Cholecystokinin Tetrapeptides on the Activity of Single Hippocampal and Thalamic Neurons U. ZIPPEL* and P. HENKLEINt
*Institute of Physiology, Tucholskystr.2 and tlnstitute of Pharmacology, Clara-Zetkin-Str.94, CharitY, Humboldt-University, 10117 Berlin, Germany (Reprint requests to UZ)
Abstract--The influence of two iontophoretically administered newly developed cholecystokinin (CCK) tetrapeptides with high selectivity and affinity to CCK-B receptors on the impulse activity of single hippocampal and thalamic neurons were tested in in-vivo experiments, in comparison to the effect of the sulfated octapeptide (CCK8$). A very similar responsiveness to the compared drugs was found. Most neurons responded with an increase of their discharge frequency. Only a few suppressive effects were elicited by each drug and in each of the structures. There was a good correspondence between the compared drugs concerning the direction and relative response amplitude, resulting in a highly significant correlation of the effects of both CCK4s with the CCK8S effects. On a subsample of neurons, the blocking effect of the selective CCK-B receptor blocker PD135 was tested and found to be effective in 16 out of 20 CCK4 responses, including also one inhibition. The results show that the new compounds act as effective CCK agonists binding to the B-type CCK receptor. The few inhibitory effects obtained could be explained by possible indirect effects mediated via inhibitory interneurons which are known to exist in both investigated structures.
Introduction
of anxiety and panic disorders, ~8 in the control of pain,9 ~0 and the interactions of CCK with dopaminergic functions. H For many years, various attempts have been made to use CCK or its agonists or antagonists for the treatment of some diseases in these fields 1~14 Further progress in understanding the role of CCK in brain functions depends among others on the development of substances which bind highly selectively and with high affinity to only one of the two known CCK receptors, A or B. Both types were identified in the brain, ~5-~6 but the A receptors have been found to be restricted to only
Cholecystokinin (CCK) is known to be the most common neuropeptide. It was found in nearly all brain regions. Its concentration exceeds in most cases by far that of other neuropeptides, 14 and its functional role has been widely discussed. The main fields of interest seem to be the involvement of CCK in the regulation of feeding, 5-6 in the mechanisms
Date received 3 January 1995 Date accepted 4 March 1995
83
84 a few regions 17 which are especially related to the alimentary behaviour. The B receptor which is much more common seems to be involved in diverse functions. The brain itself synthetizes diverse fragments of CCK. The sulfated octapeptide CCK2(~33 (CCK8S) represents 90% of the CCK contained in the brain. 18 It binds with equal affinity to both A and B receptors. The CCK4 fragment (CCK30~33) also present in the brain binds with 1000 times higher affinity to B than to A receptors. The recently developed artificial CCK4s (Suc-Trp-Met-AspPhe-NH2 and Suc-Trp-Nme-NLe-Asp-Phe-NH2, here designated as compound 1 and 2, respectively) show a further increase of the affinity and selectivity to the B receptor (K~27 nM, A/B ratio > 3000019 and Ki4 nM A/B ratio > 10000.2o The aim of the present work was to compare the effects of these new compounds with those of the natural CCK8S on the neuronal level and to describe their agonistic or antagonistic effectiveness.
Materials and methods
Animals Male Wistar rats (300-400 g) housed six per cage on diurnal light-dark cycle with food and water ad libitum were anesthetized with urethane (1.2 g/kg i.p., further injections as needed). A hole was drilled into the exposed skull at the position 5.0 mm anterior to the lambdoid suture and 3.5 mm lateral to the midline suture. The animals were positioned in a stereotaxic frame. The rectal temperature was kept constant at 37-38°C by a heating pad.
NEUROPEPTIDES barreled micropipette was glued to the recording electrode with tip distance of about 30-50 pm. The barrels were filled with CCK8S, one of the above mentioned CCK4s (both synthetized by P. Henklein), the selective CCK-B receptor blocker PD135 (RBI), 0.25 raM, pH 7.8 each, with sodium chloride (165 raM) for current balance and with phosphate buffer solution as control channel. Retaining currents of 3-5 nA were used.
Data analysis The mean discharge frequency 1 min ahead and during every iontophoresis was computed and a change of more than 50% regarded as a relevant influence of the applied drug. The influence on the whole investigated neuronal population was tested using the Wilcoxon rank sum test. The blocking effect of PD135 was estimated by comparing the impulses/rain during iontophoresis of the applied substance with the impulse frequency during combined iontophoresis of PD 135 and CCK4. Usually PD135 was given 2 rain before the onset of the concomitant CCK iontophoresis. The effects of CCK8S and CCK4 were compared with each other using the Spearman's rank correlation test.
Verification of recordin9 sites At the end of the experiment the rat was sacrificed by an overdose of urethane, decapitated and the brain fixed in 10% formaldehyde solution. Frontal slices of the frozen brain were prepared and the localization of the dye points were identified by light microscope using the stereotaxic atlas of Koenig and Klippel. 21
Recordin9 of unit activity The spontaneous discharge activity of single hippocampal and thalamic neurones was extracellularly recorded by means of micropipettes (tip resistance 10-30 MOhm) filled with saturated trypan blue solution for iontophoretic marking of the recorded site. The electric signals were conventionally amplified, the spikes transferred in standardized pulses and analysed with the spike 2 software program.
Application of substances All drugs were iontophoretically applied by negative currents up to 90 nA, usually 30-50 nA. The 5-
Results
The sample sizes when testing compound 1 (series 1) and 2 (series 2) consisted of 24 hippocampal and 11 thalamic and 25 hippocampal and 43 thalamic neurones, respectively. The results are summarized in Table 1. All CCK fragments used predominantly evoked excitatory effects, but each fragment elicited suppression of the activity of individual neurones, too, in all investigated regions (in the first series: CCK8S 2 and CCK4 4 neurons, in the second series: CCK8S 3 and CCK4 4 neurons). However, statistically the activity of the whole neuron population
EFFECTS OF TWO NEWLY SYNTHETIZED CHOLECYSTOKININ TETRAPEPTIDES
85
Proportion of neurons reliably (see text) effected by iontophoretically applicated CCK (CCK8S and two artificial CCK4s termed compound 1 and 2), significance of drug effects on the whole population of investigated neurons (Wilcoxon rank sum test) and correlation between the effects of two drugs tested on the same neurons (Spearman's rank correlation)
Table
Structure
n
CCK8S
CCK4
Correlation
influenced
Wilcoxon
influenced
Wilcoxon
rho/p
Compound 1 Hipp Thal
24 11
14 5
p = 0.024 p = 0.013
12 7
p = 0.012 p = 0.021
0.72/0.0000 0.92/0.0000
Compound 2 Hipp Thai
25 43
15 15
p = 0.002 p = 0.006
16 16
p = 0.006 p = 0.008
0.63/0.0003 0.74/0.0000
tested was significantly increased in each sample as shown by the Wilcoxon test which also includes all seemingly accidental changes of discharge fi'equencies, To compare the effects of CCK8S and the CCK4s the relative changes of the discharge rate of each neuron during the iontophoresis of one drug was plotted against those of the other (Figs. 1 & 2). The dots are well grouped around a straight line showing the good coincidence of both influences on most neurons. The correlation coefficient (Spear-
o'
~" ~ 60 ~" o o ~0 -o -~ ~> (1)
40
(/~
30
~= o
20
r-
i
% • •
%
%
._ 40
_N 10
--
e.
¢i
10
(.3 0 "0
50
i 60
20
~9 r,(t5 tO
0 F
i 40
Fig. 2 Comparison of effects of C C K 4 (compound 2) and CCK8S, plotted as explained in Fig. 1. n = 68 hippocampal and thalamic neurons.
J~
0 >
I 30
relative changes evoked by CCK8s [ranks]
O0
30
i
20
1o
> 05 OO
10
2o
30'
40
relative changes evoked by CCK8s [ranks] Fig. 1 Scatter plot of the relative change of the impulse rate during C C K 4 (compound 1) iontophoresis [ordinate:(impulse frequency during iontophoresis minus impulse frequency before iontophoresis)/impulse frequency before iontophoresis] against that during CCK8S iontophoresis (abscissa), both transformed in ranks, n = 35 hippocampal and thalamic neurons.
man's rank correlation) was found between 0.6 and 0.9 (see Table) which is highly significant (p <0.001). The blocking capacity of PD 135 was investigated in 21 neurons which were significantly influenced by CCK4. The concomitant iontophoresis of both drugs resulted in a strong reduction or disappearance of the CCK-induced changes of the discharge frequency in 17 cells including one inhibitory effect of CCK. An example is shown in Figure 3. Discussion
In the majority of neurones the observed effect of the compared drugs was qualitatively equal. There-
86
NEUROPEPTIDES
CCK8S 50nA imp.Is 15
CCK4 20hA
PD 135 50nA
CCK4 30hA
[-I__[-]__]T[--]
0,,,_..&.
I-"] OCK4
...... &
.... , .................
500s
Fig. 3 Example of the influence of CCK8S and CCK4 on the neuronal discharge rate and the blocking effect of PD 135. Abscissa: time [s], ordinate: impulse frequency, bin width 2 s.
c o u l d be excited b y the i o n t o p h o r e t i c a l l y a p p l i e d d r u g a n d in t u r n e v o k e G A B A e r g i c i n h i b i t i o n o f the n e u r o n u n d e r study. I n this case b l o c k i n g o f the G A B A r e c e p t o r s s h o u l d a b o l i s h the inhibition. In a n o t h e r s t u d y we were able to b l o c k C C K 8 S - i n d u c e d i n h i b i t i o n s o f L G N d n e u r o n e s with bicuculline. 28 F u r t h e r investigations are still necessary to solve this problem.
Acknowledgement fore the new C C K t e t r a p e p t i d e s can be r e g a r d e d as agonists acting on the C C K receptors. This conclusion is s u p p o r t e d b y the b l o c k i n g efficacy o f PD135, too. P D 1 3 5 b i n d s exclusively to C C K - B receptors, 22 w h i c h m e a n s t h a t the drugs m u s t also use these b i n d i n g sites as it has been f o u n d in f o r m e r investigations.a9-2° This r e c e p t o r t y p e p r e d o m i n a t e s in the b r a i n as a w h o l e a n d its existence has also been d e m o n s t r a t e d in the same structures we investigated, the h i p p o c a m p u s 23 a n d the t h a l a m u s . 24 The C C K - B r e c e p t o r m e d i a t e s e x c i t a t i o n Y I n agreem e n t w i t h this result, the o v e r w h e l m i n g m a j o r i t y o f o u r n e u r o n e s were excited b y the diverse C C K fragments. H o w e v e r , a few suppressive effects were f o u n d in b o t h structures a n d w i t h each o f the drugs, too. This result c o u l d be e x p l a i n e d b y two h y p o t h eses: (1) T h e r e c o u l d exist s o m e A receptors, too, which seem to m e d i a t e inhibition. T h e m a j o r i t y o f the t h a l a m i c n e u r o n e s (43 o u t o f 54) were l o c a t e d in the d o r s a l lateral geniculate nucleus ( L G N d ) . W e f o u n d some evidence for the existence o f b o t h types o f r e c e p t o r s in this structure in o u r f o r m e r i n v e s t i g a t i o n s ) 6 C o n c e r n i n g the occurrence o f C C K - A r e c e p t o r s in the h i p p o c a m p a l r e g i o n L a v o i e et al,27 j u s t gave a first hint. H o w ever, k e e p i n g in m i n d the high b i n d i n g selectivity o f the new C C K 4 c o m p o u n d s to B-type r e c e p t o r s this e x p l a n a t i o n seems to be unlikely. R e m a r k a b l y we were able to b l o c k one inhib i t i o n with the C C K - B r e c e p t o r blocker. A t least this one was e v o k e d via B-receptors. (2) T h e i n h i b i t o r y effects c o u l d have been indirectly m e d i a t e d via i n h i b i t o r y interneurones. I f B r e c e p t o r s are p r e s e n t at i n t e r n e u r o n s l o c a t e d in the vicinity o f the i n v e s t i g a t e d n e u r o n they
The authors wish to thank Mrs U. Seider for her excellent technical assistence, K. Leiterer for help in English language and U. Heinemann for critical reading. The experimental work was supported by grant 01229101 of the Bundesministerium ffir Forschung und Technologie, Germany.
References 1. Rehfeld, J. F. Neuronal cholecystokinin: One or multiple transmitters. J. Neurochem. 1985; 44: 1-10. 2. Albus, M. Cholecystokinin. Prog. Neuropsychopharmacol. Biol. Psychiatry 1988; 12: $5-$21. 3. Fallon, J. H. and Seroogy, K. B. The distribution and some connections of cholecystokinin neurons in the rat brain. In: Vanderhaegen, J. J. and Crawley, J. N. (Eds.). Ann. N.Y. Acad. Sci. 1985; 121 132. 4. Beinfeld, M. C., Meyer, D. K., Eskay, R. L., Jensen, R. T. and Brownstein, M. J. The distribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay. Brain Res. 1981; 212: 51-57. 5. Corwin, R. L., Gibbs, J. and Smith, G. P. Increased food intake after type A but not type B cholecystokinin receptor blockade. Physiol. Behav. 1991; 50: 255--258. 6. Dourish, C. T. Behavioural analysis of the role of CCKA and CCKB receptors in the control of feeding in rodents. In: Dourish, C. T., Cooper, S. J., Iversen, S. D. and Iversen, L. L., eds. Multiple cholecystokinin receptors in the CNS, Oxford, Oxford Science Publications, 1992: 234-253. 7. Harro, J., Vasar, E. and Bradwejn, J. CCK in animal and human research on anxiety. Trends Pharmacol. Sci. 1993; 14: 244-249. 8. Hendrie, C. A. and Neill, J. C. Ethological analysis of the role of CCK in anxiety. In: Dourish, C. T., Cooper, S. J., Iversen, S. D. and Iversen, L L., eds. Multiple chosecystokinin receptors in the CNS. Oxford, Oxford Science Publishers, 5992: 132-142. 9. Baber, N. S., Dourish, C. T. and Hill, D. R. The role of CCK, caerulein and CCK antagonists in nociception. Pain 1989; 39: 307-328. 10. Kellstein, D. E. and Mayer, D. J. CCK and opioid analgesia. In: Dourish, C. T., Cooper, S. J., Iversen, S. D. and Iversen, L. L., eds. Multiple cholecystokinin receptors in the CNS. Oxford, Oxford Science Publications, 1992: 439-454. 11, Kuiper, M. A., van Kamp, G. J. and Wolters, E. Ch. CCK: its role in dopamine-related disorders. In: Dourish, C. T.,
EFFECTS OF TWO NEWLY SYNTHETIZED CHOLECYSTOKININ TETRAPEPT1DES
12.
13.
14. 15.
16. 17.
18. 19.
20.
Cooper, S. J., Iversen, S. D. and Iversen, L. L., eds. Multiple cholecystokinin receptors in the CNS. Oxford, Oxford Science Publications, 1992: 395~400. Boyce, S., Rupniak, N. M., Tye, S., Steventon, M. J. and Iversen, S. D. Modulatory role for CCK-B antagonists in Parkinson's disease. Clin. Neuropharmacol. 1990; 13: 339347. Nishikawa, T., Tanaka, M., Tsuda, A., Kuwahara, H., Koga, I. and Uchida, Y. Effect of caeruletide on tardive dyskinesia: a pilot study of quantitative computer analysis on electromyogram and microvibration. Psychopharmacology 1986; 90: 5-8. Stoessl, A. J., Mak, E. and Calne, D. B. (+)-4-propyl9-hydroxynaphthoxazine (PHNO), a new dopamimetic in treatment of Parkinsonism. Lancet 1985; 2: 1330-1331. Moran, T. H., Robinson, P. H., Goldrich, M. S. and McHugh, P. R. Two brain cholecystokinin receptors: implications for behavioural actions. Brain Res. 1986; 362: 175179. Dourish, C. T. and Hill, D. R. Classification and function of CCK receptors. Trends Pharmacol. Sci. 1987; 8: 207-208. Hill, D. R., Campbell, N. J., Shaw, T. M. and Woodruff, G. N. Auto-radiographic localization and biochemical characterization of peripheral type CCK receptors in rat CNS using highly selective non-peptide CCK antagonists. J. Neurosci. 1987; 7: 2967-2976. Dockray, G. J. Cholecystokinin in rat cerebral cortex: identification, purification and characterization by immunochemical methods. Brain Res. 1980; 188: 155-165. Harhammer, R., ScMfer, U., Henklein, P., Ott, T. and Repke, H. CCK-8-related C-terminal tetrapeptides: affinities for central CCKB and peripheral CCKA receptors. Eur. J. Pharmacol. 1991; 209:263 266. Kaufmann, R., Lindschau, C., Henklein, P., Boomgaarden, M., Haller, H. and Sch6neberg, T. Studies with succinylated CCK-4 derivatives: characterization of CCKB receptor bind-
21.
22.
23.
24.
25.
26.
27.
28.
87
ing and measurement of [Ca2+]i mobilization. Mol. Neuropharmacol. 1993; 3: 147--151. Koenig, J. F. R. and Klippel, R. A. In: The rat brain: A stereotaxic atlas of the forebrain and lower parts of the brain stem. Baltimore, The Williams and Wilkins Company, 1963. Hughes, J., Boden, P., Costall, B. et al Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity. Proc. Natl. Acad. Sci. USA 1990; 87: 6728-6732. Boden, P. and Hill, R. G. Evidence for the cholecystokinin (CCK) fragments CCK-8S, CCK4 and pentagastrin sharing a common site of action on rat hippocampal neurons. J. Physiol. 1986; 381: 48. Pelaprat, D., Broer, Y., Studler, J. M. et al. Autoradiography of CCK receptors in the rat brain using [3H]Boc[Nle2831]CCKzT_33 and [125I]Bolton-HunterCCK4: functional significance of subregional distributions. Neurochem. Int. 1987; 10: 495-508. Branchereau, P., BOhme, G. A., Champagnat, J. et al. CholecystokininA and cholecystokininB receptors in neurons of the brainstem solitary complex of the rat: Pharmacological identification. J. Pharmacol. Exp. Ther. 1992; 260: 14331440. Albrecht, D., Mfiller, R., Zippel, U., Gabriel, H.-J., Henklein, P. and Davidowa, H. Cholecystokinin-induced activity changes of dorsal lateral geniculate neurons in rats. Neuroscience 1994; 59: 953-956. Lavoie, N., Gronier, B. and Debonnel, G. Implication of CCK in the potentiation of the NMDA response induced by sigma ligands in the rat dorsal hippocampus. 24th Meeting Soc. Neurosci., Miami Beach 1994, (abstr. 708.1). Albrecht, D., Zippel, U. and Davidowa, H. Effects of cholecystokinin on pairs of geniculate neurons recorded simultaneously - - Are interneurons involved? Brain Res. 1994; (in press).
Effects of Two Newly Synthetized Cholecystokinin Tetrapeptides on the Activity of Single Hippocampal and Thalamic Neurons U. ZIPPEL* and P. HENKLEINt
*Institute of Physiology, Tucholskystr.2 and tlnstitute of Pharmacology, Clara-Zetkin-Str.94, CharitY, Humboldt-University, 10117 Berlin, Germany (Reprint requests to UZ)
Abstract--The influence of two iontophoretically administered newly developed cholecystokinin (CCK) tetrapeptides with high selectivity and affinity to CCK-B receptors on the impulse activity of single hippocampal and thalamic neurons were tested in in-vivo experiments, in comparison to the effect of the sulfated octapeptide (CCK8$). A very similar responsiveness to the compared drugs was found. Most neurons responded with an increase of their discharge frequency. Only a few suppressive effects were elicited by each drug and in each of the structures. There was a good correspondence between the compared drugs concerning the direction and relative response amplitude, resulting in a highly significant correlation of the effects of both CCK4s with the CCK8S effects. On a subsample of neurons, the blocking effect of the selective CCK-B receptor blocker PD135 was tested and found to be effective in 16 out of 20 CCK4 responses, including also one inhibition. The results show that the new compounds act as effective CCK agonists binding to the B-type CCK receptor. The few inhibitory effects obtained could be explained by possible indirect effects mediated via inhibitory interneurons which are known to exist in both investigated structures.
Introduction
of anxiety and panic disorders, ~8 in the control of pain,9 ~0 and the interactions of CCK with dopaminergic functions. H For many years, various attempts have been made to use CCK or its agonists or antagonists for the treatment of some diseases in these fields 1~14 Further progress in understanding the role of CCK in brain functions depends among others on the development of substances which bind highly selectively and with high affinity to only one of the two known CCK receptors, A or B. Both types were identified in the brain, ~5-~6 but the A receptors have been found to be restricted to only
Cholecystokinin (CCK) is known to be the most common neuropeptide. It was found in nearly all brain regions. Its concentration exceeds in most cases by far that of other neuropeptides, 14 and its functional role has been widely discussed. The main fields of interest seem to be the involvement of CCK in the regulation of feeding, 5-6 in the mechanisms
Date received 3 January 1995 Date accepted 4 March 1995
83
84 a few regions 17 which are especially related to the alimentary behaviour. The B receptor which is much more common seems to be involved in diverse functions. The brain itself synthetizes diverse fragments of CCK. The sulfated octapeptide CCK2(~33 (CCK8S) represents 90% of the CCK contained in the brain. 18 It binds with equal affinity to both A and B receptors. The CCK4 fragment (CCK30~33) also present in the brain binds with 1000 times higher affinity to B than to A receptors. The recently developed artificial CCK4s (Suc-Trp-Met-AspPhe-NH2 and Suc-Trp-Nme-NLe-Asp-Phe-NH2, here designated as compound 1 and 2, respectively) show a further increase of the affinity and selectivity to the B receptor (K~27 nM, A/B ratio > 3000019 and Ki4 nM A/B ratio > 10000.2o The aim of the present work was to compare the effects of these new compounds with those of the natural CCK8S on the neuronal level and to describe their agonistic or antagonistic effectiveness.
Materials and methods
Animals Male Wistar rats (300-400 g) housed six per cage on diurnal light-dark cycle with food and water ad libitum were anesthetized with urethane (1.2 g/kg i.p., further injections as needed). A hole was drilled into the exposed skull at the position 5.0 mm anterior to the lambdoid suture and 3.5 mm lateral to the midline suture. The animals were positioned in a stereotaxic frame. The rectal temperature was kept constant at 37-38°C by a heating pad.
NEUROPEPTIDES barreled micropipette was glued to the recording electrode with tip distance of about 30-50 pm. The barrels were filled with CCK8S, one of the above mentioned CCK4s (both synthetized by P. Henklein), the selective CCK-B receptor blocker PD135 (RBI), 0.25 raM, pH 7.8 each, with sodium chloride (165 raM) for current balance and with phosphate buffer solution as control channel. Retaining currents of 3-5 nA were used.
Data analysis The mean discharge frequency 1 min ahead and during every iontophoresis was computed and a change of more than 50% regarded as a relevant influence of the applied drug. The influence on the whole investigated neuronal population was tested using the Wilcoxon rank sum test. The blocking effect of PD135 was estimated by comparing the impulses/rain during iontophoresis of the applied substance with the impulse frequency during combined iontophoresis of PD 135 and CCK4. Usually PD135 was given 2 rain before the onset of the concomitant CCK iontophoresis. The effects of CCK8S and CCK4 were compared with each other using the Spearman's rank correlation test.
Verification of recordin9 sites At the end of the experiment the rat was sacrificed by an overdose of urethane, decapitated and the brain fixed in 10% formaldehyde solution. Frontal slices of the frozen brain were prepared and the localization of the dye points were identified by light microscope using the stereotaxic atlas of Koenig and Klippel. 21
Recordin9 of unit activity The spontaneous discharge activity of single hippocampal and thalamic neurones was extracellularly recorded by means of micropipettes (tip resistance 10-30 MOhm) filled with saturated trypan blue solution for iontophoretic marking of the recorded site. The electric signals were conventionally amplified, the spikes transferred in standardized pulses and analysed with the spike 2 software program.
Application of substances All drugs were iontophoretically applied by negative currents up to 90 nA, usually 30-50 nA. The 5-
Results
The sample sizes when testing compound 1 (series 1) and 2 (series 2) consisted of 24 hippocampal and 11 thalamic and 25 hippocampal and 43 thalamic neurones, respectively. The results are summarized in Table 1. All CCK fragments used predominantly evoked excitatory effects, but each fragment elicited suppression of the activity of individual neurones, too, in all investigated regions (in the first series: CCK8S 2 and CCK4 4 neurons, in the second series: CCK8S 3 and CCK4 4 neurons). However, statistically the activity of the whole neuron population
EFFECTS OF TWO NEWLY SYNTHETIZED CHOLECYSTOKININ TETRAPEPTIDES
85
Proportion of neurons reliably (see text) effected by iontophoretically applicated CCK (CCK8S and two artificial CCK4s termed compound 1 and 2), significance of drug effects on the whole population of investigated neurons (Wilcoxon rank sum test) and correlation between the effects of two drugs tested on the same neurons (Spearman's rank correlation)
Table
Structure
n
CCK8S
CCK4
Correlation
influenced
Wilcoxon
influenced
Wilcoxon
rho/p
Compound 1 Hipp Thal
24 11
14 5
p = 0.024 p = 0.013
12 7
p = 0.012 p = 0.021
0.72/0.0000 0.92/0.0000
Compound 2 Hipp Thai
25 43
15 15
p = 0.002 p = 0.006
16 16
p = 0.006 p = 0.008
0.63/0.0003 0.74/0.0000
tested was significantly increased in each sample as shown by the Wilcoxon test which also includes all seemingly accidental changes of discharge fi'equencies, To compare the effects of CCK8S and the CCK4s the relative changes of the discharge rate of each neuron during the iontophoresis of one drug was plotted against those of the other (Figs. 1 & 2). The dots are well grouped around a straight line showing the good coincidence of both influences on most neurons. The correlation coefficient (Spear-
o'
~" ~ 60 ~" o o ~0 -o -~ ~> (1)
40
(/~
30
~= o
20
r-
i
% • •
%
%
._ 40
_N 10
--
e.
¢i
10
(.3 0 "0
50
i 60
20
~9 r,(t5 tO
0 F
i 40
Fig. 2 Comparison of effects of C C K 4 (compound 2) and CCK8S, plotted as explained in Fig. 1. n = 68 hippocampal and thalamic neurons.
J~
0 >
I 30
relative changes evoked by CCK8s [ranks]
O0
30
i
20
1o
> 05 OO
10
2o
30'
40
relative changes evoked by CCK8s [ranks] Fig. 1 Scatter plot of the relative change of the impulse rate during C C K 4 (compound 1) iontophoresis [ordinate:(impulse frequency during iontophoresis minus impulse frequency before iontophoresis)/impulse frequency before iontophoresis] against that during CCK8S iontophoresis (abscissa), both transformed in ranks, n = 35 hippocampal and thalamic neurons.
man's rank correlation) was found between 0.6 and 0.9 (see Table) which is highly significant (p <0.001). The blocking capacity of PD 135 was investigated in 21 neurons which were significantly influenced by CCK4. The concomitant iontophoresis of both drugs resulted in a strong reduction or disappearance of the CCK-induced changes of the discharge frequency in 17 cells including one inhibitory effect of CCK. An example is shown in Figure 3. Discussion
In the majority of neurones the observed effect of the compared drugs was qualitatively equal. There-
86
NEUROPEPTIDES
CCK8S 50nA imp.Is 15
CCK4 20hA
PD 135 50nA
CCK4 30hA
[-I__[-]__]T[--]
0,,,_..&.
I-"] OCK4
...... &
.... , .................
500s
Fig. 3 Example of the influence of CCK8S and CCK4 on the neuronal discharge rate and the blocking effect of PD 135. Abscissa: time [s], ordinate: impulse frequency, bin width 2 s.
c o u l d be excited b y the i o n t o p h o r e t i c a l l y a p p l i e d d r u g a n d in t u r n e v o k e G A B A e r g i c i n h i b i t i o n o f the n e u r o n u n d e r study. I n this case b l o c k i n g o f the G A B A r e c e p t o r s s h o u l d a b o l i s h the inhibition. In a n o t h e r s t u d y we were able to b l o c k C C K 8 S - i n d u c e d i n h i b i t i o n s o f L G N d n e u r o n e s with bicuculline. 28 F u r t h e r investigations are still necessary to solve this problem.
Acknowledgement fore the new C C K t e t r a p e p t i d e s can be r e g a r d e d as agonists acting on the C C K receptors. This conclusion is s u p p o r t e d b y the b l o c k i n g efficacy o f PD135, too. P D 1 3 5 b i n d s exclusively to C C K - B receptors, 22 w h i c h m e a n s t h a t the drugs m u s t also use these b i n d i n g sites as it has been f o u n d in f o r m e r investigations.a9-2° This r e c e p t o r t y p e p r e d o m i n a t e s in the b r a i n as a w h o l e a n d its existence has also been d e m o n s t r a t e d in the same structures we investigated, the h i p p o c a m p u s 23 a n d the t h a l a m u s . 24 The C C K - B r e c e p t o r m e d i a t e s e x c i t a t i o n Y I n agreem e n t w i t h this result, the o v e r w h e l m i n g m a j o r i t y o f o u r n e u r o n e s were excited b y the diverse C C K fragments. H o w e v e r , a few suppressive effects were f o u n d in b o t h structures a n d w i t h each o f the drugs, too. This result c o u l d be e x p l a i n e d b y two h y p o t h eses: (1) T h e r e c o u l d exist s o m e A receptors, too, which seem to m e d i a t e inhibition. T h e m a j o r i t y o f the t h a l a m i c n e u r o n e s (43 o u t o f 54) were l o c a t e d in the d o r s a l lateral geniculate nucleus ( L G N d ) . W e f o u n d some evidence for the existence o f b o t h types o f r e c e p t o r s in this structure in o u r f o r m e r i n v e s t i g a t i o n s ) 6 C o n c e r n i n g the occurrence o f C C K - A r e c e p t o r s in the h i p p o c a m p a l r e g i o n L a v o i e et al,27 j u s t gave a first hint. H o w ever, k e e p i n g in m i n d the high b i n d i n g selectivity o f the new C C K 4 c o m p o u n d s to B-type r e c e p t o r s this e x p l a n a t i o n seems to be unlikely. R e m a r k a b l y we were able to b l o c k one inhib i t i o n with the C C K - B r e c e p t o r blocker. A t least this one was e v o k e d via B-receptors. (2) T h e i n h i b i t o r y effects c o u l d have been indirectly m e d i a t e d via i n h i b i t o r y interneurones. I f B r e c e p t o r s are p r e s e n t at i n t e r n e u r o n s l o c a t e d in the vicinity o f the i n v e s t i g a t e d n e u r o n they
The authors wish to thank Mrs U. Seider for her excellent technical assistence, K. Leiterer for help in English language and U. Heinemann for critical reading. The experimental work was supported by grant 01229101 of the Bundesministerium ffir Forschung und Technologie, Germany.
References 1. Rehfeld, J. F. Neuronal cholecystokinin: One or multiple transmitters. J. Neurochem. 1985; 44: 1-10. 2. Albus, M. Cholecystokinin. Prog. Neuropsychopharmacol. Biol. Psychiatry 1988; 12: $5-$21. 3. Fallon, J. H. and Seroogy, K. B. The distribution and some connections of cholecystokinin neurons in the rat brain. In: Vanderhaegen, J. J. and Crawley, J. N. (Eds.). Ann. N.Y. Acad. Sci. 1985; 121 132. 4. Beinfeld, M. C., Meyer, D. K., Eskay, R. L., Jensen, R. T. and Brownstein, M. J. The distribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay. Brain Res. 1981; 212: 51-57. 5. Corwin, R. L., Gibbs, J. and Smith, G. P. Increased food intake after type A but not type B cholecystokinin receptor blockade. Physiol. Behav. 1991; 50: 255--258. 6. Dourish, C. T. Behavioural analysis of the role of CCKA and CCKB receptors in the control of feeding in rodents. In: Dourish, C. T., Cooper, S. J., Iversen, S. D. and Iversen, L. L., eds. Multiple cholecystokinin receptors in the CNS, Oxford, Oxford Science Publications, 1992: 234-253. 7. Harro, J., Vasar, E. and Bradwejn, J. CCK in animal and human research on anxiety. Trends Pharmacol. Sci. 1993; 14: 244-249. 8. Hendrie, C. A. and Neill, J. C. Ethological analysis of the role of CCK in anxiety. In: Dourish, C. T., Cooper, S. J., Iversen, S. D. and Iversen, L L., eds. Multiple chosecystokinin receptors in the CNS. Oxford, Oxford Science Publishers, 5992: 132-142. 9. Baber, N. S., Dourish, C. T. and Hill, D. R. The role of CCK, caerulein and CCK antagonists in nociception. Pain 1989; 39: 307-328. 10. Kellstein, D. E. and Mayer, D. J. CCK and opioid analgesia. In: Dourish, C. T., Cooper, S. J., Iversen, S. D. and Iversen, L. L., eds. Multiple cholecystokinin receptors in the CNS. Oxford, Oxford Science Publications, 1992: 439-454. 11, Kuiper, M. A., van Kamp, G. J. and Wolters, E. Ch. CCK: its role in dopamine-related disorders. In: Dourish, C. T.,
EFFECTS OF TWO NEWLY SYNTHETIZED CHOLECYSTOKININ TETRAPEPT1DES
12.
13.
14. 15.
16. 17.
18. 19.
20.
Cooper, S. J., Iversen, S. D. and Iversen, L. L., eds. Multiple cholecystokinin receptors in the CNS. Oxford, Oxford Science Publications, 1992: 395~400. Boyce, S., Rupniak, N. M., Tye, S., Steventon, M. J. and Iversen, S. D. Modulatory role for CCK-B antagonists in Parkinson's disease. Clin. Neuropharmacol. 1990; 13: 339347. Nishikawa, T., Tanaka, M., Tsuda, A., Kuwahara, H., Koga, I. and Uchida, Y. Effect of caeruletide on tardive dyskinesia: a pilot study of quantitative computer analysis on electromyogram and microvibration. Psychopharmacology 1986; 90: 5-8. Stoessl, A. J., Mak, E. and Calne, D. B. (+)-4-propyl9-hydroxynaphthoxazine (PHNO), a new dopamimetic in treatment of Parkinsonism. Lancet 1985; 2: 1330-1331. Moran, T. H., Robinson, P. H., Goldrich, M. S. and McHugh, P. R. Two brain cholecystokinin receptors: implications for behavioural actions. Brain Res. 1986; 362: 175179. Dourish, C. T. and Hill, D. R. Classification and function of CCK receptors. Trends Pharmacol. Sci. 1987; 8: 207-208. Hill, D. R., Campbell, N. J., Shaw, T. M. and Woodruff, G. N. Auto-radiographic localization and biochemical characterization of peripheral type CCK receptors in rat CNS using highly selective non-peptide CCK antagonists. J. Neurosci. 1987; 7: 2967-2976. Dockray, G. J. Cholecystokinin in rat cerebral cortex: identification, purification and characterization by immunochemical methods. Brain Res. 1980; 188: 155-165. Harhammer, R., ScMfer, U., Henklein, P., Ott, T. and Repke, H. CCK-8-related C-terminal tetrapeptides: affinities for central CCKB and peripheral CCKA receptors. Eur. J. Pharmacol. 1991; 209:263 266. Kaufmann, R., Lindschau, C., Henklein, P., Boomgaarden, M., Haller, H. and Sch6neberg, T. Studies with succinylated CCK-4 derivatives: characterization of CCKB receptor bind-
21.
22.
23.
24.
25.
26.
27.
28.
87
ing and measurement of [Ca2+]i mobilization. Mol. Neuropharmacol. 1993; 3: 147--151. Koenig, J. F. R. and Klippel, R. A. In: The rat brain: A stereotaxic atlas of the forebrain and lower parts of the brain stem. Baltimore, The Williams and Wilkins Company, 1963. Hughes, J., Boden, P., Costall, B. et al Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity. Proc. Natl. Acad. Sci. USA 1990; 87: 6728-6732. Boden, P. and Hill, R. G. Evidence for the cholecystokinin (CCK) fragments CCK-8S, CCK4 and pentagastrin sharing a common site of action on rat hippocampal neurons. J. Physiol. 1986; 381: 48. Pelaprat, D., Broer, Y., Studler, J. M. et al. Autoradiography of CCK receptors in the rat brain using [3H]Boc[Nle2831]CCKzT_33 and [125I]Bolton-HunterCCK4: functional significance of subregional distributions. Neurochem. Int. 1987; 10: 495-508. Branchereau, P., BOhme, G. A., Champagnat, J. et al. CholecystokininA and cholecystokininB receptors in neurons of the brainstem solitary complex of the rat: Pharmacological identification. J. Pharmacol. Exp. Ther. 1992; 260: 14331440. Albrecht, D., Mfiller, R., Zippel, U., Gabriel, H.-J., Henklein, P. and Davidowa, H. Cholecystokinin-induced activity changes of dorsal lateral geniculate neurons in rats. Neuroscience 1994; 59: 953-956. Lavoie, N., Gronier, B. and Debonnel, G. Implication of CCK in the potentiation of the NMDA response induced by sigma ligands in the rat dorsal hippocampus. 24th Meeting Soc. Neurosci., Miami Beach 1994, (abstr. 708.1). Albrecht, D., Zippel, U. and Davidowa, H. Effects of cholecystokinin on pairs of geniculate neurons recorded simultaneously - - Are interneurons involved? Brain Res. 1994; (in press).