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Cyclic AMP-generating systems in rat hippocampal slices
Brain Research, 213 (1981) 351-364 © Elsevier/North-HollandBiomedicalPress
351
CYCLIC AMP-GENERATING SYSTEMS IN RAT HIPPOCAMPAL SLICES
M. SEGAL,V. GREENBERGERand R. HOFSTEIN Isotope Department, The Weizmann Institute of Science, Rehovot (Israel)
(Accepted October 16th, 1980) Key words: hippocampus-- cAMP-- kainate-- norepinephrine
SUMMARY Properties of the norepinephrine- (NE) stimulated, cAMP-generating system were studied in rat hippocampal slices. NE but not other putative neurotransmitters, caused a 3-4-fold rise in cAMP levels in the slices. All 3 main subdivisions of the hippocampus (HPC), the dentate gyrus, areas CA3 and CA 1, possessed the capacity to produce cAMP. The latency to the NE stimulation of cAMP formation was about 20 see but maximal stimulation was reached only after 5-10 rain of incubation. Intrahippocampal injection of kainic acid (KA) caused a nearly complete destruction of hippocampal neurons and a marked increase in number of glial cells. NE caused a 12-15-fold rise in cAMP levels in KA-treated HPC. Compared to normal HPC where potency order of noradrenergic agonists indicated activation of a beta-1 receptor type, the pattern for the KA-treated HPC indicated the dominance of beta-2 receptors. The beta-1 antagonist, practolol, and the beta-2 antagonist, H35/25, were about equipotent in blocking the NE-stimulated cAMP formation in normal HPC. In KA-treated HPC, on the other hand, H35/25 was much more potent than practolol in inhibiting NE-stimulated cAMP formation. It is suggested that in the HPC beta-1 adrenergic receptors are primarily neuronal and beta-2 receptors, glial, and that activation of both receptor species results in activation of a cAMP-generating system.
INTRODUCTION Norepinephrine (NE) can stimulate formation of cyclic adenosine monophosphate (cAMP) in a variety of tissues including the brain1,a,11,17,27. A decade of
352 extensive studies have characterized the cAMP-generating system in the brain yet the physiological relevance of this system remained unclear. It has been postulated that cAMP serves as a second messenger for the action of NE 1,2,1~,29 and that accumulation of cAMP promotes phosphorylation of certain membranous proteins, leading somehow to hyperpolarization of the affected neurons 1,12,17. Evidence in support of this contention has thus far been largely indirect; it is not clear whether the changes in cAMP produced by NE precede the physiological changes; it is not clear if the changes in cAMP are sufficient or even necessary for the formation of the physiological changes, and it was not clear, until recently, what exactly the physiological effects of NE in the forebrain are ~,~7,29. This current state of knowledge is partly due to the lack of a central model system, where physiological and biochemical events can be monitored under similar biological conditions. The rat hippocampal slice preparation can be used as a model system for such an analysis. The hippocampus (HPC) receives a dense noradrenergic innervation from the nucleus locus coeruleus2L Stimulation of the latter or iontophoresis of NE can supp~ ess spontaneous activity of HPC neurons 29. Such stimulation can also cause elevation of cAMP levels in the HPC of the freely moving rat (unpublished observations). We have started to use the slice preparation aiming at the correlation of the physiological and biochemical changes produced by NE. It was found that NE causes hyperpolarization of neurons in the rat HPC with a delay of 15-20 sec, and that this effect was mimicked by application of cAMP 2s. These observations encouraged the search for correlative cAMP changes produced by NE in these slices. Specifically, the stimulation parameters (time course, dose), the noradrenergic receptor type involved and the neuronal origin of the cAMP response to NE were explored. Preliminary experiments were done with guinea pig HPC slices which demonstrated considerable stinmlation of cAMP formation by NE. All experiments reported herein were done with rat HPC slices to allow comparisons with previous physiological experiments 2s, 29
METHODS
Adult (250-350 g) male Wistar rats of a local breeding colony were used. Rats were decapitated, their brains removed rapidly and 500/~m transverse hippocampal slices were cut with a Mcllwain tissue chopper. Using a fine brush, slices were transferred into the incubation vials which were placed in ice throughout the procedure. Care was taken to randomize placement of the slices in the vials such that slices of various dorsoventral locations would be evenly distributed. Incubation vials containing 5-7 slices in 2.0 ml cold Krebs solution were then placed in a 37 °C heated bath in an atmosphere saturated with 95 % 02-5 % COz. Half of the medium was replaced twice at 30 and 60 min after onset of the incubation. Antagonists were added to the incubation medium at 50 min. In control vials only an equal volume of Krebs was added. NE or other agonists were added at 60 min in a volume of 100/tl Krebs containing 12 mM ascorbate; control vials received only Krebs-ascorbate solution. Incubation with the agonist lasted 10 min and the vials were transferred thereafter into
353 a I00 °C water bath for 10 min. The boiled slices were separated from the supernatant fluid by centrifugation at 1000 × g for 10 min and the content of protein in the slices was determined by the Lowry method is. cAMP levels were determined by the saturation assay based on Brown et al.4,12. A binding protein prepared from calf adrenal cortex according to Brown et al. 4 was added, at concentrations which bind at least 30 % of total [3H]cAMP present in the tube, to reaction tubes containing 1 pmol [SH]cAMP and a sample of the supernatant fluid. Incubation lasted for 90 rain at 0 °C, and was terminated by addition of 0.1 ml 10 % charcoal/2 % bovine serum albumin. Reaction tubes were stirred and centrifuged at 1000 × g for 10 min and samples were taken from the supernatant for counting. Some rats were pretreated with kainic acid (KA) 9. This was injected bilaterally into hippocampus of chloral hydrate-anesthetized rats. Two 0.4 #1 injections containing 1 mg/ml KA were made into each HPC, aimed at the dorsal and ventral HPC (coordinates from bregma --3.5, 2.5, 4.0 mm and --6.0, 5.5 and 7.5 ram, respectively). Animals were sacrificed at various intervals thereafter. Some injected rats were perfused and their brains sectioned and stained for histological verification of the magnitude of the KA-induced lesions. Some rats were injected with 6-hydroxydopamine (6-OHDA) to destroy noradrenergic terminals. 6-OHDA (400 #g/rat) was injected intracisternally twice, 72 h apart, as previously described 29. The following materials were used: (--)arterenol hydrochloride (NE, Sigma), Lisoproterenol-HC1 (ISO, Sigma), L-epinephrine bitartarate (EPI, Sigma), 3-isobutyl-1methylxanthine (IBMX, Sigma) adenosine 3',5'-cyclic monophosphoric acid (cAMP, Sigma), [6-3H]cAMP (24.1 Ci/mmol, NEN), propranolol (abic), phentolamine (Regitine, Ciba), D,L-erytro-4'-methyl-a-(1-isopropyl amine ethyl) benzyl alcohol-HCl (H35/25, Astra) practolol (a gift from Dr. Y. Gutman, Hadassah Medical School, Jerusalem), kainic acid (KA, Sigma). The Krebs solution contained (in raM) NaCI 124, KCI 5, KH2PO4 1.25, NaHCO3 26, CaClz 2, MgSO4 2, glucose 10 and had a pH of 7.4. The solution was equilibrated with a gas mixture of 95 % 02-5 % CO2 before use.
Statistical comparisons of the grouped data were done using analysis of variance followed by Duncan's multiple comparisons tests. RESULTS
Technical considerations Initial experiments were performed to delineate some optimal conditions for detection of stimulated cAMP formation in hippocampal slices. The following variables were studied: (a) tissue thickness; the physiological experiments28 were performed with 350-#m thick transverse slices. In comparative experiments, NE caused a 378 ~ stimulation of cAMP formation in 500 #m slices whereas the results with 350-/~m thick slices were more variable and the best stimulation amounted to 344 % above basal level in one experiment. Since 500-#m thick slices were easier to handle and yielded more consistant results, they were preferred; (b) temperature effects; a comparison was made between slices maintained at 22 °C and at 37 °C. Both
354 the basal and the stimulated cAMP levels were higher at the lower temperature. The difference in basal activity contributed to the lower apparent stimulation ratio of c A M P formation at 22 °C - - 250 ~o compared to 378 ~ in slices maintained at 37 °C. Experiments were performed thereafter at 37 °C, (c) phosphodiesterase inhibition; the possible contribution of modulation of phosphodiesterase (PDE), the enzyme responsible for the breakdown of cAMP, to the observed accumulation of cAMP, was measured using IBMX, a P D E inhibitor. In two of 3 experiments, the addition of I B M X augmented the N E stimulation of c A M P formation by 30 ~ . With a few exceptions (see below) most further experiments were performed without IBMX.
Neurotransmitter comparisons A comparison was made among the main substances assumed to play a neurotransmitter role in the HPC. An agonist concentration of 100 ffM was used for all tests. Among the 7 substances tested, N E produced a 4-fold elevation of c A M P levels in the slices. Adenosine produced a 2.4-fold increase in cAMP level, histamine (HA) produced only a small (82 ~ ) increase in c A M P level whereas the other 4 agents had no effect. A tendency for a reduction in basal c A M P levels by glutamate and G A B A was detected (Fig. 1). Regional distribution of cAMP responses to NE To determine basal and NE-stimulated cAMP levels in the various regions of the HPC, slices were dissected as described, followed by placement of the slices in cooled petri dishes. Under a stereomicroscope, the slices were dissected into region CA1, dentate gyrus (DG) including CA4 and the hilus, and region CA3. Following the dissection, the slices were collected and incubated as before. It was preferred to dissect the slices before incubation rather than to incubate and then dissect, so as to minimize the traumatic effect of the dissection on c A M P accumulation. There were differences
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Fig. 1. Effects of putative neurotransmitters on cAMP levels in rat hippocampal slices. The substances were incubated for 10 min at a concentration of 100 ~M. Each bar represents the mean d: S.E.M. of the ratio of cAMP formation in the presence and absence of the agonist. Each mean consists of 4-6 cAMP determinations in two separate experiments. The basal cAMP levels were similar in all tests and amounted to 5-10 pmol/mg protein. Abbreviations: GABA, gamma amino-butyric acid; Glu, Lglutamic acid; ACH, acetylcholine; 5-HT, 5-hydroxy tryptamine; HA, histamine; AD, adenosine.
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Fig. 2. Regional distribution of NE-stimulated cAMP formation in hippocampal slices. The HPC was dissected into regions CA1, CA3 and the dentate gyrus (DG). The values are mean dz S.E.M. of cAMP formed in 5 separate experiments. Each experiment was done in the presence and absence of 100 # M NE and propranolol (100 #M).
in basal and NE-stimulated cAMP levels in the regions of the HPC. The basal level was about the same in CA1 and CA3 regions but was twice as large in the DG. NE caused a 3-fold rise in cAMP levels in CA1, a 4-fold rise in CA3 region and less than a 2-fold rise in DG (Fig. 2). The reduced NE stimulation of cAMP formation in D G can be explained by the high basal cAMP level there. In all 3 regions propranolol (100/zM) nearly abolishes the rise in cAMP above basal levels. These results indicate that the NE-stimulated cAMP-generating system is present in all regions of the HPC as was recently suggested for the beta adrenergic receptor distribution t°. The noradrenergic receptor type associated with the cAMP system The effects of 3 noradrenergic agonists, ISO, NE and EPI, on cAMP formation were studied with different agonist concentrations ranging from 10 nM to 0.1 mM in 7-14 different experiments. There were marked differences in the maximal cAMP stimulation attained by the different agonists. Whereas ISO caused only an averaged 33 ~ increase in cAMP formation, NE stimulated cAMP formation by a maximum of 2.88-fold above basal levels, and EP1 stimulated cAMP formation by 3.39 fold - above basal levels. Although the effects of ISO were fairly small, they were statistically significant at the highest concentrations used (10 -5 M and 10-4 M, 13 and 9 experiments, respectively). ECs0 values were calculated from dose-response curves (Fig. 3) and amounted to approximately 6 × 10-7 M for ISO, 3 × 10-6 M for NE and 2 × 10-5 M for EPI. This order of potency being ISO > NE > EPI indicates that the cAMP-coupled noradrenergic receptor in the rat hippocampus is probably of the beta1 type 16,a°. The time course of N E action The time course of NE action on the cAMP-generating system was determined by incubation of the slices with I00/~M NE for various intervals followed by the termination of the reaction by boiling. Due to equilibration times, the intervals are accurate only to within 2-3 sec. The first indication for an increase in NE stimulation of cAMP formation was seen 20 sec after application of NE (Fig. 4). The amount of
356 I
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-~ 6 5 4 Agonis! Concenlr0fion(-log M) Fig. 3. Dose-response curves of adrenergic effects on cAMP concentrations. Each data point is a mean of 5-12 separate cAMP determinations. Ordinate: percentage of maximal cAMP increase above basal level seen with the particular agonist. Statistical analysis of the data by means of analysis of variance has revealed a significant agonist effect (F2,167) = 24.7, P < 0.0001); a significant concentration difference (F(7,167) = 20.3, P < 0.001); and a significant interaction (F(10,167) = 5.6, P < 0.001).
c A M P f o r m e d g r a d u a l l y rose to reach an averaged 2-fold increase by 60 sec. A later c o m p o n e n t was evident some 5 min after a p p l i c a t i o n o f N E at which time there was a 3fold rise a b o v e basal c A M P levels.
cAMP responses in KA-treated hippocampi W i t h i n 3 - 4 days after the K A injection, the H P C was slightly h y p e r t r o p h i e d . This was n o t associated with a noticeable increase in p r o t e i n content a n d was p r o b a b l y caused b y edemic a c c u m u l a t i o n o f water in the tissue. It was followed by a g r a d u a l r e d u c t i o n in the size o f the H P C which fell to less t h a n 50 ~ o f its original size some 2-3 weeks after the injection. Histological e x a m i n a t i o n (Fig. 5) o f the K A - t r e a t e d H P C revealed t h a t the injection resulted in a nearly c o m p l e t e n e u r o n a l loss in regions C A 1 a n d CA3. Some parts o f the dentate gyrus ( D G ) a n d m o s t o f the n e u r o n s in C A 2 survived the d a m a g e b u t the surviving neurons c o m p r i s e d only roughly 5-15 ~ o f the I
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Fig. 4. Time course of the effect of NE on cAMP levels. Each point represents mean + S.E.M. of 5-12 separate determinations. An analysis of variance test revealed a significant difference among the various time points (F(7,50) = 9.93, P < 0.0001). Duncan's comparisons indicated no differences within the following groups: 0-10, 20-40, 60-120 and 300 see.
357 n e u r o n a l p o p u l a t i o n in the HPC. There was a marked proliferation of glial cells in the K A - t r e a t e d H P C (Fig. 5). This proliferation was p r o n o u n c e d especially in the regions completely devoid of n e u r o n s but was present also in the h i p p o c a m p a l areas - - where some n e u r o n s were still intact.
Fig. 5. Morphological changes induced by KA in rat HPC. A and B: region CA1 of normal and KAtreated HPC, respectively. Cresyl violet stain. Note the accumulation of small nuclei of glial cells evenly distributed throughout the tissue section, and the disappearance of the large, neuronal elements. Magnification 160 ×. C and D: sections through the dentate gyrus of normal and KA-treated HPC, respectively. 63 ×. The rat was sacrificed 3 weeks after unilateral injection of I ~g KA into the HPC.
358 100 80
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Fig. 6. Changes in N E stimulation of cAMP formation as a function of time after K A injection. Each point is a mean o f 4-12 determinations. Control experiments consist of pooled data of sham-operated rats which were treated similar to the K A rats with the exception of the injection of the drug. The right column consists of experiments performed 1-3 weeks after K A injection. There were no systematic differences among rats sacrificed 1-3 weeks after K A injection.
For the biochemical experiments, rats were decapitated at various intervals after the injection of KA. As can be seen in Fig. 6, there was no effect of KA on basal and NE-stimulated cAMP formation shortly after the injection. Starting at 4 days after KA injection and reaching a peak more than 6 days after the injection there was a marked, up to 15-fold, rise in NE-stimulated cAMP production with no effect on basal cAMP levels. This rise in cAMP production was high even 3-4 weeks after the KA injection, indicating that it is not a transient effect related to the fast proliferation phase of the glial cells. The marked elevation of NE-stimulated cAMP formation is not likely to have taken place solely on proliferating noradrenergic terminals in the KA-injected HPC. When KA was injected into 6-OHDA-pretreated rats which are devoid of noradrenergic terminals, NE was still capable of causing a large increase in cAMP formation in the HPC (Fig. 7). Note that 6-OHDA pretreatment did not induce an apparent
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Fig. 7. N E stimulation of cAMP formation in normal, 6 - O H D A and KA-treated rats. Each bar represents 3-8 c A M P determinations. A significant difference among treatments (F(3,51) = 16.1, P < 0.0001), concentrations (F(2,51) = 66.1,P < 0.0001)and their interactions (F(6,51) = 14.2,P < 0.0001 ) were found.
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Fig. 8. Effects of noradrenergic agonists on c A M P formation in KA-treated HPC. Each data point represents a mean of 4-10 c A M P determinations. A n analysis of variance test revealed a significant treatment effect (F(2,67) = 7.0, P < 0.002),'a significant concentration effect (F(3,67) = 41.6, P < 0.0001) and a significant interaction (F(6,67) = 15.5, P < 0.0001).
supersensitivity when the highest concentration (10 -~ M) NE was compared. However, with lower concentrations 0 0 -6 M) of NE, 6-OHDA-treated slices are clearly more efficient in producing cAMP than normal controls. In two other experiments (data not shown) 6-OHDA-treated slices produced 20-30% more cAMP in the presence of NE than normal controls. The NE-triggered rise in cAMP formation was not a general feature of the cAMP-generating system in the KA-treated HPC. In comparison, HA which caused an averaged 80 % rise in cAMP formation in the intact HPC, failed to do so in the KAtreated one and caused a non-significant averaged increase of only 8 % above basal levels (6 determinations). This observation confirms an earlier report on the HAstimulated cAMP formation in the HPCT,25. The noradrenergic receptor type involved in stimulation of cAMP formation in KA-treated HPC was assessed using the agonists ISO, NE and EPI. Marked differences between the KA and the normal HPC were
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Fig. 9. Effects of propranolol and phentolamine on NE- stimulated c A M P formation. N E and the antagonists were applied in a concentration of 100/2M. The results represent means of two separate experiments. A significant treatment effect (F(1,6) = 29.5, P < 0.001), antagonist effect (F(2,6) 19.1, P < 0.0025) and interaction (F(2,6) = 14.1, P < 0.005) were found. It appears that while phentolamine was ineffective in the normal, it became potent in antagonizing some N E effects in KA-treated HPC.
360 found. ISO at a concentration of 1 × 10 -7 M stimulated cAMP formation by 6.8-fold above basal level, with an ECs0 of about 10 -8 M. EPI stimulated cAMP formation by 13-fold with an EC50 of about 3 × 10 -5 M whereas NE stimulation amounted to 18fold, with an ECs0 value similar to that of the normal, 3 × 10-6 M (Fig. 8). It appears that the order of potency was different from that of the normal case and had a beta-2 pattern of ISO > EPI > NE 5,21,s°.
Effects of adrenergic antagonists The pharmacological profile of the NE-stimulated cAMP formation in normal and KA-treated HPC was further investigated using adrenergic antagonists. In the normal case, propranolol, a beta antagonist markedly reduced the NE-stimulated cAMP formation whereas phentolamine, an alpha antagonist, was nearly ineffective. The co-existence of alpha and beta components in the cAMP response, could be demonstrated by the usage of the combination of both antagonists; when phentolamine and propranolol were used together, their effects were additive (in two experiments NE stimulation was 3.7-fold above basal cAMP levels; with propranolol 1.85-fold above basal levels, with phentolamine 2.6-fold above basal cAMP levels and with both propranolol and phentolamine, 1.4 above basal levels). Phentolamine was more effective in blocking responses to NE in KA-treated HPC (Fig. 9). This observation suggests the possible enrichment of KA-treated HPC with an alpha adrenergic receptor type s but still indicates that the main receptor associated with the cAMP-generating system is of the beta type. An attempt to estimate the beta receptor species associated with the cAMP system was made using practolol, a beta-1 antagonistS,6, 22,80, and H35/25, a less specific beta-2 antagonist tg. In the normal HPC, practolol and H35/25 were about equipotent in blocking the NE stimulation of cAMP formation (Fig. 10). In KAtreated rats, H35/25 was far more potent in antagonizing cAMP formation than [ ] +NO A N T . ~÷PRACIOLOL ~ ~-H35/25 --
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Fig. 10. Effects of a beta-1 antagonist, practolol, and a beta-2 antagonist, H35/25, on NE-stimulated cAMP formation. All drugs were applied at a concentration of 100 # M . Results are means of 5-12 determinations. Analysis of variance indicates a significant treatment effect (F(1,47) = 206.8, P < 0.0001) a significant antagonist effect (F(2,47) = 33.0, P < 0.0001) and interaction (F(2,47) = 13.5, P < 0.0001). While practolol and H35/25 were about equipotent in the normal HPC, H35/25 was more potent in the KA treated HPC (right).
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Fig. 11. Dose-response curves for antagonist supressinn of NE-stimulated cAMP formation. Agonist (NE) concentration was 100 FM and antagonists were varied between 5 and 200/~M. Results are means of two separate experiments. practolol. On the basis of a dose-response curve (Fig. 11) using various concentrations of the antagonists, the IC50 values for practolol and H35/25 in the normal H P C were about the same (7 × 10-6 M) whereas they were markedly different in the K A - H P C ; 2 × 10-5 M and 1.5 times 10-4 M, for H35/25 and practolol respectively. These data indicate that the adrenergic receptor associated with c A M P stimulation in KA-treated H P C is of the beta-2 type. DISCUSSION
The present experiments were designed to analyze some properties of the cAMPgenerating system in the rat hippocampus. The slice preparation was selected to allow comparisons between the physiological and biochemical effects of NE. Such comparisons can not be made in an intact, in vivo situation. NE appears to be unique among the putative neurotransmitters known to innervate the HPC, in that it is the only one which could markedly stimulate cAMP formation. Most of the other excitatory (ACh, glutamate) or inhibitory (GABA, 5HT) neurotransmitters were not effective or even caused a reduction in cAMP levels. The only other agent found to stimulate cAMP formation was histamine (HA), as previously reported 25. Interestingly, the HA-stimulated cAMP-generating system was not active in KA-treated rats, indicating a different distribution from that stimulated by NE. The time course of NE-stimulated cAMP formation introduces an interesting difference from the physiological effect of N E on hippocampal pyramidal cells. It was recently found ~s that NE produces a hyperpolarization of CA1 pyramidal neurons, which reaches its maximum within 20-30 see of NE application and is maintained for
362 several minutes, cAMP formation, while dearly stimulated some 20 sec after NE application, does not reach an asymptotic level until about 5 min later. This indicates that while part of the cAMP formed might be associated with the generation of the hyperpolarization, certainly not all, especially not the cAMP formed 1-5 min after NE application, is associated with the physiological response. The question is then, what might be the functional role of the large accumulation of cAMP in response to NE. Several possible explanations can be proposed. It is possible that the excess cAMP formed simply represents an overshoot of an activated system and that this overshoot has no particular significance. Alternately, the large accumulation of cAMP might subserve another physiological action that has not been detected yet. Finally, it is possible that the extra cAMP formed is associated with some types of biochemical processes not necessarily related to the immediate physiological function, e.g. the triggering of glycogenolysis in both glial and neuronal cells 13,24,31. The latter assumption would imply that the early (20-60 sec) rise in cAMP levels involves activation of one type of receptors which is different from the receptor type activated later. The large increase in the efficacy of NE in stimulating the formation of cAMP in KA-treated HPC is somewhat surprising. It has been thought that most if not all of cAMP is generated in neurons 1,2,15,17 and yet when neurons are being destroyed by KA, the cAMP formation triggered by NE is augmented rather than depressed. The excess cAMP formation can be caused by one of 3 possible mechanisms. It is possible that cAMP formation is accelerated in remaining neurons and/or glia by suppression of action of PDE, the enzyme responsible for the breakdown of cAMP. This is not a likely possibility since the use of isobutyryl methyl xantine (IBMX), a PDE inhibitor, was nearly as effective in normal as in KA-treated HPC (unpublished observations). A second possibility is that the excess cAMP is formed in proliferating presynaptic fibers innervating the injured HPC. This is also not likely, since 6-OHDA treatment, which destroys noradrenergic terminals, did not abolish the enhanced cAMP response. The third possibility is that in KA-treated HPC there is a proliferation of non-neuronal elements which contain noradrenergic receptor linked to a cAMP-generating system. Two non-neuronal elements can account for this: blood vessels which have a beta-2 adrenergic receptor 14 and glial cells which may also have a beta adrenergic receptor coupled to adenylate cyclase13,20,81. Histological examination of the KA-treated HPC has revealed a marked increase in number of glial cells and it is likely that the rise in glial noradrenergic receptors coupled to adenylate-cyclase is responsible for the observed elevated cAMP response. This was also suggested for the KA-treated striatum 2a. The non-neuronal proliferating elements contain both alpha and beta adrenoreceptors; phentolamine which was relatively inefficient in antagonizing NE-stimulated cAMP accumulation in the normal HPC, became efficient in KA-treated HPC. The possibility that the new alpha and beta receptors are present on the same or on different sites remains to be determined. In this respect it is interesting to note that alpha adrenoreceptors might regulate presynaptically, the release of amino acid neurotransmitters (F. Moroni, personal communication). Since it is likely that some
363 excitatory afferents proliferate in the KA-treated HPC, it is possible that alpha adrenoreceptors proliferate on these presynaptic fibers. This possibility is subject to further experimentations. The beta adrenergic receptor species present in the brain have been the subject o f intensive recent investigations. It has been suggested that the main neuronal noradrenergic receptor is o f the beta-1 type, at least in cortical structures~,16,21,2~, a0. It has also been suggested that the beta-2 type is associated with non-neuronal elements21, 26. These suggestions are consistent with our observations. The beta adrenergic receptor in the normal H P C is predominantly of the beta-1 type, while in KA-treated HPC, the noradrenergic receptor is o f the beta-2 type. These data indicate that the beta-1 receptor is probably located mainly on neurons and the beta-2 receptors on both neurons and glia and that both are associated with a cAMP-generating system. It is tempting to speculate that the two receptor systems have different functions as well as time courses; the one associated with the physiological response, i.e. neuronal hyperpolarization would have a faster response time than the other one which is probably associated with a metabolic response. Further experiments should test this hypothesis. Altogether, the hippocampal slice preparation proves to be a useful model system for the investigation o f the correlation between biochemical and physiological events underlying neurot~ansmitter action in the brain.
REFERENCES 1 Bloom, F. E., The role of cyclic nucleotides in central synaptic function, Rev. Physiol. Biochem. Pharmacol., 74 0975) 1-103. 2 Bloom, F. E., Hoffer, B. J., Battenberg, E. F., Siggins, G. R., Steiner, A. L., Parker, C. W. and Wedner, H. J., Adenosine 3',5'-monophosphate is localized in cerebellar neurons: immunofluorescence evidence, Science, 177 (1972) 436-438. 3 Blumberg, J. B., Vetulani, J., Stawarz, R. J. and Sulser, F., The noradrenergic cyclic AMP generating system in the limbic forebrain: pharmacological characterization in vitro and possible role of limbic noradrenergic mechanisms in the mode of action of antipsychotics, Europ. J. Pharmacok, 37 (1976) 357-366. 4 Brown, B. L., Albano, J. D. M., Ekins, R. P., Sgherzi, A. M. and Tampion, W., A simple and sensitive saturation assay method for the measurement of adenosine 3':5'-cyclic monophosphate, Biochem. J., 121 (1971) 561-562. 5 Burges, R. A. and Blackburn, K. J., Adenyl cyclase and the differentiation of fl adrenoreceptors, Nature New Biol., 235 (1972) 249-250. 6 Bylund, D. B. and Snyder, S. H., Beta adrenergic receptor binding in membrane preparations from mammalian brain, Molec. Pharmacol., 12 (1976) 568-580. 7 Chang, R. S. L., Tran, V. T. and Snyder, S. H., Neurotransmitter receptor Iocalizations: brain lesion induced alterations in benzodiazepine, GABA, fl-adrenergic and histamine Hi-receptor binding, Brain Research, 190 (1980) 95-110. 8 Chasin, M., Rivkin, I., Mamrak, F., Samaniego, G. and Hess, S. M., a- and fl-adrenergic receptors as mediators of accumulation of cyclic adenosine 3',5'-monophosphate in specific areas of guinea pig brain, J. biol. Chem., 246 (1971) 3037-3041. 9 Coyle, J. T. and Schwarcz, R., Lesion of striatal neurones with Kainic acid provides a model for Huntington's chorea, Nature (Lond.), 263 (1976) 244-246. 10 Crutcher, K. A. and Davis, J. N., Hippocampal a and fl adrenergic receptors: comparison of [aH]dihydroalprenolol and [aHIWB 4101 binding with noradrenergic innervafion in the rat, Brain Research, 182 (1980) 107-117. 11 Dolphin, A., Adrien, J., Hamon, M. and Bockaert, J., Identity of [aH]dihydroalprenolol binding
364 sites and fl-adrenergic receptors coupled with adenylate cyclase in the central nervous system: pharmacological properties, distribution and adaptive responsiveness, Molec. Pharmacol., 15 (1979) 1-15. 12 Forn, J., Krueger, B. K. and Greengard, P., Adenosine 3',5'-monophosphate content in rat caudate nucleus: demonstration of dopaminergic and adrenergic receptors, Science, 186 (1974) 1118-1120. 13 Gilman, A. G. and Nirenberg, M., Effect of catecholamines on the adenosine 3'5 cyclic monophosphate concentrations of clonal satellite cells of neurons, Proc. nat. Acad. Sci. (U.S.A.), 68 (1971) 2165-2168. 14 Herbst, T. J., Raichle, M. E. and Ferrendelli, J. A., fl adrenergic regulation of adenosine 3'5'monophosphate concentration in brain microvessels, Science, 204 (1979) 330-332. 15 Hoffer, B. J., Siggins, G. R., Oliver, A. P. and Bloom, F. E., Activation of the pathway from locus coeruleus to rat cerebellar purkinje neurons: Pharmacological evidence of noradrenergic central inhibition, J. Pharmacol. exp. Ther., 184 (1973) 553-569. 16 Horn, A. S. and Phillipson, O. T., A noradrenaline sensitive adenylate cyclase in the rat limbic forebrain: preparation, properties and the effects of agonists, adrenolytics and neuroleptic drugs, Europ. J. Pharmacol., 37 (1976) 1-11. 17 Iversen, L. L., Catecholamine-sensitive adenylate cyclases in nervous tissue, J. Neurochem., 29 (1977) 5-12. 18 Lowry, O. H., Rosebrough, N. J., Farr, A. and Randall, J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 19 Levy, B. and Wilkenfeld, B. E,, An analysis of selective beta receptor blockade, Europ. J. Pharmacol., 5 (1969) 227-234. 20 Maguire, M. E., Wiklund, R. A., Anderson, H. J. and Gilman, A. G., Binding of 125I-iodohydroxybenzylpindolol to putative fl adrenergic receptors of rat glioma cells and other cell clones, J. biol. Chem., 251 (1976) 1221-1231. 21 Minneman, K. P., Dibner, M. D., Wolfe, B. B. and Molinoff, P. B., fll and f12adrenergic receptors in rat cerebral cortex are independently regulated, Science, 204 (1979) 866-868. 22 Minneman, K. P., Hegstrand, L. R. and Molinoff, P. B., Simultaneous determination of beta-1 and beta-2 adrenergic receptors in tissues containing both receptor subtypes, Molec. Pharmacol., 16 (1979) 34-46. 23 Minneman, K. P., Quik, M. and Emson, P. C., Receptor-linked cyclic AMP systems in rat neostriatum: differential localization revealed by kainic acid injection, Brain Research, 151 (1978) 507-521. 24 Nahorski, S. R., Rogers, K. J. and Edwards, C., Cerebral glycogenolysis and stimulation of fladrenoreceptors and histamine H2 receptors, Brain Research, 92 (1975) 529-533. 25 Palacios, J. M., Garbard, M., Barbin, G. and Schwartz, J. C., Histamine receptors mediating cyclic AMP accumulation in slices from guinea pig hippocampus: characterization and localization, Agents and Actions, 9 (1979) 82-83. 26 Pittman, R. N., Minneman, K. P. and Molinoff, P. B., Ontogeny of fll and f12adrenergic receptors in rat cerebellum and cerebral cortex, Brain Research, 188 (1980) 357-367. 27 Robison, G. R., Butcher, R. W. and Sutherland, E. W., Cyclic AMP, Academic Press, New York, 1971. 28 Segal, M., The action of norepinephrine in the rat hippocampus; intracellular studies in the slice preparation, Brain Research, in press. 29 Segal, M. and Bloom, F. E., The action of norepinephrine in the rat hippocampus I, Iontophoretic studies, Brain Research, 72 (1974) 79-97. 30 U'Prichard, D. C., Bylund, D. B. and Snyder, S. H.,t(zL)-[SH]Epinephrine and (--)-[3H]dihydroalprenolol binding to fll and fl~- - noradrenergic receptors in brain, heart and lung membranes, J. biol. Chem., 253 (1978) 5090-5102. 31 Wilkening, D. and Makman, M. H., Activation of glycogen, phosphorylase in rat caudate nucleus slices by L-isopropylnorepinephrineand dibutyrylcyclic AMP, J. Neurochem., 28 (1977) 1001-1007.
351
CYCLIC AMP-GENERATING SYSTEMS IN RAT HIPPOCAMPAL SLICES
M. SEGAL,V. GREENBERGERand R. HOFSTEIN Isotope Department, The Weizmann Institute of Science, Rehovot (Israel)
(Accepted October 16th, 1980) Key words: hippocampus-- cAMP-- kainate-- norepinephrine
SUMMARY Properties of the norepinephrine- (NE) stimulated, cAMP-generating system were studied in rat hippocampal slices. NE but not other putative neurotransmitters, caused a 3-4-fold rise in cAMP levels in the slices. All 3 main subdivisions of the hippocampus (HPC), the dentate gyrus, areas CA3 and CA 1, possessed the capacity to produce cAMP. The latency to the NE stimulation of cAMP formation was about 20 see but maximal stimulation was reached only after 5-10 rain of incubation. Intrahippocampal injection of kainic acid (KA) caused a nearly complete destruction of hippocampal neurons and a marked increase in number of glial cells. NE caused a 12-15-fold rise in cAMP levels in KA-treated HPC. Compared to normal HPC where potency order of noradrenergic agonists indicated activation of a beta-1 receptor type, the pattern for the KA-treated HPC indicated the dominance of beta-2 receptors. The beta-1 antagonist, practolol, and the beta-2 antagonist, H35/25, were about equipotent in blocking the NE-stimulated cAMP formation in normal HPC. In KA-treated HPC, on the other hand, H35/25 was much more potent than practolol in inhibiting NE-stimulated cAMP formation. It is suggested that in the HPC beta-1 adrenergic receptors are primarily neuronal and beta-2 receptors, glial, and that activation of both receptor species results in activation of a cAMP-generating system.
INTRODUCTION Norepinephrine (NE) can stimulate formation of cyclic adenosine monophosphate (cAMP) in a variety of tissues including the brain1,a,11,17,27. A decade of
352 extensive studies have characterized the cAMP-generating system in the brain yet the physiological relevance of this system remained unclear. It has been postulated that cAMP serves as a second messenger for the action of NE 1,2,1~,29 and that accumulation of cAMP promotes phosphorylation of certain membranous proteins, leading somehow to hyperpolarization of the affected neurons 1,12,17. Evidence in support of this contention has thus far been largely indirect; it is not clear whether the changes in cAMP produced by NE precede the physiological changes; it is not clear if the changes in cAMP are sufficient or even necessary for the formation of the physiological changes, and it was not clear, until recently, what exactly the physiological effects of NE in the forebrain are ~,~7,29. This current state of knowledge is partly due to the lack of a central model system, where physiological and biochemical events can be monitored under similar biological conditions. The rat hippocampal slice preparation can be used as a model system for such an analysis. The hippocampus (HPC) receives a dense noradrenergic innervation from the nucleus locus coeruleus2L Stimulation of the latter or iontophoresis of NE can supp~ ess spontaneous activity of HPC neurons 29. Such stimulation can also cause elevation of cAMP levels in the HPC of the freely moving rat (unpublished observations). We have started to use the slice preparation aiming at the correlation of the physiological and biochemical changes produced by NE. It was found that NE causes hyperpolarization of neurons in the rat HPC with a delay of 15-20 sec, and that this effect was mimicked by application of cAMP 2s. These observations encouraged the search for correlative cAMP changes produced by NE in these slices. Specifically, the stimulation parameters (time course, dose), the noradrenergic receptor type involved and the neuronal origin of the cAMP response to NE were explored. Preliminary experiments were done with guinea pig HPC slices which demonstrated considerable stinmlation of cAMP formation by NE. All experiments reported herein were done with rat HPC slices to allow comparisons with previous physiological experiments 2s, 29
METHODS
Adult (250-350 g) male Wistar rats of a local breeding colony were used. Rats were decapitated, their brains removed rapidly and 500/~m transverse hippocampal slices were cut with a Mcllwain tissue chopper. Using a fine brush, slices were transferred into the incubation vials which were placed in ice throughout the procedure. Care was taken to randomize placement of the slices in the vials such that slices of various dorsoventral locations would be evenly distributed. Incubation vials containing 5-7 slices in 2.0 ml cold Krebs solution were then placed in a 37 °C heated bath in an atmosphere saturated with 95 % 02-5 % COz. Half of the medium was replaced twice at 30 and 60 min after onset of the incubation. Antagonists were added to the incubation medium at 50 min. In control vials only an equal volume of Krebs was added. NE or other agonists were added at 60 min in a volume of 100/tl Krebs containing 12 mM ascorbate; control vials received only Krebs-ascorbate solution. Incubation with the agonist lasted 10 min and the vials were transferred thereafter into
353 a I00 °C water bath for 10 min. The boiled slices were separated from the supernatant fluid by centrifugation at 1000 × g for 10 min and the content of protein in the slices was determined by the Lowry method is. cAMP levels were determined by the saturation assay based on Brown et al.4,12. A binding protein prepared from calf adrenal cortex according to Brown et al. 4 was added, at concentrations which bind at least 30 % of total [3H]cAMP present in the tube, to reaction tubes containing 1 pmol [SH]cAMP and a sample of the supernatant fluid. Incubation lasted for 90 rain at 0 °C, and was terminated by addition of 0.1 ml 10 % charcoal/2 % bovine serum albumin. Reaction tubes were stirred and centrifuged at 1000 × g for 10 min and samples were taken from the supernatant for counting. Some rats were pretreated with kainic acid (KA) 9. This was injected bilaterally into hippocampus of chloral hydrate-anesthetized rats. Two 0.4 #1 injections containing 1 mg/ml KA were made into each HPC, aimed at the dorsal and ventral HPC (coordinates from bregma --3.5, 2.5, 4.0 mm and --6.0, 5.5 and 7.5 ram, respectively). Animals were sacrificed at various intervals thereafter. Some injected rats were perfused and their brains sectioned and stained for histological verification of the magnitude of the KA-induced lesions. Some rats were injected with 6-hydroxydopamine (6-OHDA) to destroy noradrenergic terminals. 6-OHDA (400 #g/rat) was injected intracisternally twice, 72 h apart, as previously described 29. The following materials were used: (--)arterenol hydrochloride (NE, Sigma), Lisoproterenol-HC1 (ISO, Sigma), L-epinephrine bitartarate (EPI, Sigma), 3-isobutyl-1methylxanthine (IBMX, Sigma) adenosine 3',5'-cyclic monophosphoric acid (cAMP, Sigma), [6-3H]cAMP (24.1 Ci/mmol, NEN), propranolol (abic), phentolamine (Regitine, Ciba), D,L-erytro-4'-methyl-a-(1-isopropyl amine ethyl) benzyl alcohol-HCl (H35/25, Astra) practolol (a gift from Dr. Y. Gutman, Hadassah Medical School, Jerusalem), kainic acid (KA, Sigma). The Krebs solution contained (in raM) NaCI 124, KCI 5, KH2PO4 1.25, NaHCO3 26, CaClz 2, MgSO4 2, glucose 10 and had a pH of 7.4. The solution was equilibrated with a gas mixture of 95 % 02-5 % CO2 before use.
Statistical comparisons of the grouped data were done using analysis of variance followed by Duncan's multiple comparisons tests. RESULTS
Technical considerations Initial experiments were performed to delineate some optimal conditions for detection of stimulated cAMP formation in hippocampal slices. The following variables were studied: (a) tissue thickness; the physiological experiments28 were performed with 350-#m thick transverse slices. In comparative experiments, NE caused a 378 ~ stimulation of cAMP formation in 500 #m slices whereas the results with 350-/~m thick slices were more variable and the best stimulation amounted to 344 % above basal level in one experiment. Since 500-#m thick slices were easier to handle and yielded more consistant results, they were preferred; (b) temperature effects; a comparison was made between slices maintained at 22 °C and at 37 °C. Both
354 the basal and the stimulated cAMP levels were higher at the lower temperature. The difference in basal activity contributed to the lower apparent stimulation ratio of c A M P formation at 22 °C - - 250 ~o compared to 378 ~ in slices maintained at 37 °C. Experiments were performed thereafter at 37 °C, (c) phosphodiesterase inhibition; the possible contribution of modulation of phosphodiesterase (PDE), the enzyme responsible for the breakdown of cAMP, to the observed accumulation of cAMP, was measured using IBMX, a P D E inhibitor. In two of 3 experiments, the addition of I B M X augmented the N E stimulation of c A M P formation by 30 ~ . With a few exceptions (see below) most further experiments were performed without IBMX.
Neurotransmitter comparisons A comparison was made among the main substances assumed to play a neurotransmitter role in the HPC. An agonist concentration of 100 ffM was used for all tests. Among the 7 substances tested, N E produced a 4-fold elevation of c A M P levels in the slices. Adenosine produced a 2.4-fold increase in cAMP level, histamine (HA) produced only a small (82 ~ ) increase in c A M P level whereas the other 4 agents had no effect. A tendency for a reduction in basal c A M P levels by glutamate and G A B A was detected (Fig. 1). Regional distribution of cAMP responses to NE To determine basal and NE-stimulated cAMP levels in the various regions of the HPC, slices were dissected as described, followed by placement of the slices in cooled petri dishes. Under a stereomicroscope, the slices were dissected into region CA1, dentate gyrus (DG) including CA4 and the hilus, and region CA3. Following the dissection, the slices were collected and incubated as before. It was preferred to dissect the slices before incubation rather than to incubate and then dissect, so as to minimize the traumatic effect of the dissection on c A M P accumulation. There were differences
4.0 2.5
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Fig. 1. Effects of putative neurotransmitters on cAMP levels in rat hippocampal slices. The substances were incubated for 10 min at a concentration of 100 ~M. Each bar represents the mean d: S.E.M. of the ratio of cAMP formation in the presence and absence of the agonist. Each mean consists of 4-6 cAMP determinations in two separate experiments. The basal cAMP levels were similar in all tests and amounted to 5-10 pmol/mg protein. Abbreviations: GABA, gamma amino-butyric acid; Glu, Lglutamic acid; ACH, acetylcholine; 5-HT, 5-hydroxy tryptamine; HA, histamine; AD, adenosine.
355 c:
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Fig. 2. Regional distribution of NE-stimulated cAMP formation in hippocampal slices. The HPC was dissected into regions CA1, CA3 and the dentate gyrus (DG). The values are mean dz S.E.M. of cAMP formed in 5 separate experiments. Each experiment was done in the presence and absence of 100 # M NE and propranolol (100 #M).
in basal and NE-stimulated cAMP levels in the regions of the HPC. The basal level was about the same in CA1 and CA3 regions but was twice as large in the DG. NE caused a 3-fold rise in cAMP levels in CA1, a 4-fold rise in CA3 region and less than a 2-fold rise in DG (Fig. 2). The reduced NE stimulation of cAMP formation in D G can be explained by the high basal cAMP level there. In all 3 regions propranolol (100/zM) nearly abolishes the rise in cAMP above basal levels. These results indicate that the NE-stimulated cAMP-generating system is present in all regions of the HPC as was recently suggested for the beta adrenergic receptor distribution t°. The noradrenergic receptor type associated with the cAMP system The effects of 3 noradrenergic agonists, ISO, NE and EPI, on cAMP formation were studied with different agonist concentrations ranging from 10 nM to 0.1 mM in 7-14 different experiments. There were marked differences in the maximal cAMP stimulation attained by the different agonists. Whereas ISO caused only an averaged 33 ~ increase in cAMP formation, NE stimulated cAMP formation by a maximum of 2.88-fold above basal levels, and EP1 stimulated cAMP formation by 3.39 fold - above basal levels. Although the effects of ISO were fairly small, they were statistically significant at the highest concentrations used (10 -5 M and 10-4 M, 13 and 9 experiments, respectively). ECs0 values were calculated from dose-response curves (Fig. 3) and amounted to approximately 6 × 10-7 M for ISO, 3 × 10-6 M for NE and 2 × 10-5 M for EPI. This order of potency being ISO > NE > EPI indicates that the cAMP-coupled noradrenergic receptor in the rat hippocampus is probably of the beta1 type 16,a°. The time course of N E action The time course of NE action on the cAMP-generating system was determined by incubation of the slices with I00/~M NE for various intervals followed by the termination of the reaction by boiling. Due to equilibration times, the intervals are accurate only to within 2-3 sec. The first indication for an increase in NE stimulation of cAMP formation was seen 20 sec after application of NE (Fig. 4). The amount of
356 I
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c A M P f o r m e d g r a d u a l l y rose to reach an averaged 2-fold increase by 60 sec. A later c o m p o n e n t was evident some 5 min after a p p l i c a t i o n o f N E at which time there was a 3fold rise a b o v e basal c A M P levels.
cAMP responses in KA-treated hippocampi W i t h i n 3 - 4 days after the K A injection, the H P C was slightly h y p e r t r o p h i e d . This was n o t associated with a noticeable increase in p r o t e i n content a n d was p r o b a b l y caused b y edemic a c c u m u l a t i o n o f water in the tissue. It was followed by a g r a d u a l r e d u c t i o n in the size o f the H P C which fell to less t h a n 50 ~ o f its original size some 2-3 weeks after the injection. Histological e x a m i n a t i o n (Fig. 5) o f the K A - t r e a t e d H P C revealed t h a t the injection resulted in a nearly c o m p l e t e n e u r o n a l loss in regions C A 1 a n d CA3. Some parts o f the dentate gyrus ( D G ) a n d m o s t o f the n e u r o n s in C A 2 survived the d a m a g e b u t the surviving neurons c o m p r i s e d only roughly 5-15 ~ o f the I
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357 n e u r o n a l p o p u l a t i o n in the HPC. There was a marked proliferation of glial cells in the K A - t r e a t e d H P C (Fig. 5). This proliferation was p r o n o u n c e d especially in the regions completely devoid of n e u r o n s but was present also in the h i p p o c a m p a l areas - - where some n e u r o n s were still intact.
Fig. 5. Morphological changes induced by KA in rat HPC. A and B: region CA1 of normal and KAtreated HPC, respectively. Cresyl violet stain. Note the accumulation of small nuclei of glial cells evenly distributed throughout the tissue section, and the disappearance of the large, neuronal elements. Magnification 160 ×. C and D: sections through the dentate gyrus of normal and KA-treated HPC, respectively. 63 ×. The rat was sacrificed 3 weeks after unilateral injection of I ~g KA into the HPC.
358 100 80
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Fig. 6. Changes in N E stimulation of cAMP formation as a function of time after K A injection. Each point is a mean o f 4-12 determinations. Control experiments consist of pooled data of sham-operated rats which were treated similar to the K A rats with the exception of the injection of the drug. The right column consists of experiments performed 1-3 weeks after K A injection. There were no systematic differences among rats sacrificed 1-3 weeks after K A injection.
For the biochemical experiments, rats were decapitated at various intervals after the injection of KA. As can be seen in Fig. 6, there was no effect of KA on basal and NE-stimulated cAMP formation shortly after the injection. Starting at 4 days after KA injection and reaching a peak more than 6 days after the injection there was a marked, up to 15-fold, rise in NE-stimulated cAMP production with no effect on basal cAMP levels. This rise in cAMP production was high even 3-4 weeks after the KA injection, indicating that it is not a transient effect related to the fast proliferation phase of the glial cells. The marked elevation of NE-stimulated cAMP formation is not likely to have taken place solely on proliferating noradrenergic terminals in the KA-injected HPC. When KA was injected into 6-OHDA-pretreated rats which are devoid of noradrenergic terminals, NE was still capable of causing a large increase in cAMP formation in the HPC (Fig. 7). Note that 6-OHDA pretreatment did not induce an apparent
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359
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supersensitivity when the highest concentration (10 -~ M) NE was compared. However, with lower concentrations 0 0 -6 M) of NE, 6-OHDA-treated slices are clearly more efficient in producing cAMP than normal controls. In two other experiments (data not shown) 6-OHDA-treated slices produced 20-30% more cAMP in the presence of NE than normal controls. The NE-triggered rise in cAMP formation was not a general feature of the cAMP-generating system in the KA-treated HPC. In comparison, HA which caused an averaged 80 % rise in cAMP formation in the intact HPC, failed to do so in the KAtreated one and caused a non-significant averaged increase of only 8 % above basal levels (6 determinations). This observation confirms an earlier report on the HAstimulated cAMP formation in the HPCT,25. The noradrenergic receptor type involved in stimulation of cAMP formation in KA-treated HPC was assessed using the agonists ISO, NE and EPI. Marked differences between the KA and the normal HPC were
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Fig. 9. Effects of propranolol and phentolamine on NE- stimulated c A M P formation. N E and the antagonists were applied in a concentration of 100/2M. The results represent means of two separate experiments. A significant treatment effect (F(1,6) = 29.5, P < 0.001), antagonist effect (F(2,6) 19.1, P < 0.0025) and interaction (F(2,6) = 14.1, P < 0.005) were found. It appears that while phentolamine was ineffective in the normal, it became potent in antagonizing some N E effects in KA-treated HPC.
360 found. ISO at a concentration of 1 × 10 -7 M stimulated cAMP formation by 6.8-fold above basal level, with an ECs0 of about 10 -8 M. EPI stimulated cAMP formation by 13-fold with an EC50 of about 3 × 10 -5 M whereas NE stimulation amounted to 18fold, with an ECs0 value similar to that of the normal, 3 × 10-6 M (Fig. 8). It appears that the order of potency was different from that of the normal case and had a beta-2 pattern of ISO > EPI > NE 5,21,s°.
Effects of adrenergic antagonists The pharmacological profile of the NE-stimulated cAMP formation in normal and KA-treated HPC was further investigated using adrenergic antagonists. In the normal case, propranolol, a beta antagonist markedly reduced the NE-stimulated cAMP formation whereas phentolamine, an alpha antagonist, was nearly ineffective. The co-existence of alpha and beta components in the cAMP response, could be demonstrated by the usage of the combination of both antagonists; when phentolamine and propranolol were used together, their effects were additive (in two experiments NE stimulation was 3.7-fold above basal cAMP levels; with propranolol 1.85-fold above basal levels, with phentolamine 2.6-fold above basal cAMP levels and with both propranolol and phentolamine, 1.4 above basal levels). Phentolamine was more effective in blocking responses to NE in KA-treated HPC (Fig. 9). This observation suggests the possible enrichment of KA-treated HPC with an alpha adrenergic receptor type s but still indicates that the main receptor associated with the cAMP-generating system is of the beta type. An attempt to estimate the beta receptor species associated with the cAMP system was made using practolol, a beta-1 antagonistS,6, 22,80, and H35/25, a less specific beta-2 antagonist tg. In the normal HPC, practolol and H35/25 were about equipotent in blocking the NE stimulation of cAMP formation (Fig. 10). In KAtreated rats, H35/25 was far more potent in antagonizing cAMP formation than [ ] +NO A N T . ~÷PRACIOLOL ~ ~-H35/25 --
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Fig. 10. Effects of a beta-1 antagonist, practolol, and a beta-2 antagonist, H35/25, on NE-stimulated cAMP formation. All drugs were applied at a concentration of 100 # M . Results are means of 5-12 determinations. Analysis of variance indicates a significant treatment effect (F(1,47) = 206.8, P < 0.0001) a significant antagonist effect (F(2,47) = 33.0, P < 0.0001) and interaction (F(2,47) = 13.5, P < 0.0001). While practolol and H35/25 were about equipotent in the normal HPC, H35/25 was more potent in the KA treated HPC (right).
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Fig. 11. Dose-response curves for antagonist supressinn of NE-stimulated cAMP formation. Agonist (NE) concentration was 100 FM and antagonists were varied between 5 and 200/~M. Results are means of two separate experiments. practolol. On the basis of a dose-response curve (Fig. 11) using various concentrations of the antagonists, the IC50 values for practolol and H35/25 in the normal H P C were about the same (7 × 10-6 M) whereas they were markedly different in the K A - H P C ; 2 × 10-5 M and 1.5 times 10-4 M, for H35/25 and practolol respectively. These data indicate that the adrenergic receptor associated with c A M P stimulation in KA-treated H P C is of the beta-2 type. DISCUSSION
The present experiments were designed to analyze some properties of the cAMPgenerating system in the rat hippocampus. The slice preparation was selected to allow comparisons between the physiological and biochemical effects of NE. Such comparisons can not be made in an intact, in vivo situation. NE appears to be unique among the putative neurotransmitters known to innervate the HPC, in that it is the only one which could markedly stimulate cAMP formation. Most of the other excitatory (ACh, glutamate) or inhibitory (GABA, 5HT) neurotransmitters were not effective or even caused a reduction in cAMP levels. The only other agent found to stimulate cAMP formation was histamine (HA), as previously reported 25. Interestingly, the HA-stimulated cAMP-generating system was not active in KA-treated rats, indicating a different distribution from that stimulated by NE. The time course of NE-stimulated cAMP formation introduces an interesting difference from the physiological effect of N E on hippocampal pyramidal cells. It was recently found ~s that NE produces a hyperpolarization of CA1 pyramidal neurons, which reaches its maximum within 20-30 see of NE application and is maintained for
362 several minutes, cAMP formation, while dearly stimulated some 20 sec after NE application, does not reach an asymptotic level until about 5 min later. This indicates that while part of the cAMP formed might be associated with the generation of the hyperpolarization, certainly not all, especially not the cAMP formed 1-5 min after NE application, is associated with the physiological response. The question is then, what might be the functional role of the large accumulation of cAMP in response to NE. Several possible explanations can be proposed. It is possible that the excess cAMP formed simply represents an overshoot of an activated system and that this overshoot has no particular significance. Alternately, the large accumulation of cAMP might subserve another physiological action that has not been detected yet. Finally, it is possible that the extra cAMP formed is associated with some types of biochemical processes not necessarily related to the immediate physiological function, e.g. the triggering of glycogenolysis in both glial and neuronal cells 13,24,31. The latter assumption would imply that the early (20-60 sec) rise in cAMP levels involves activation of one type of receptors which is different from the receptor type activated later. The large increase in the efficacy of NE in stimulating the formation of cAMP in KA-treated HPC is somewhat surprising. It has been thought that most if not all of cAMP is generated in neurons 1,2,15,17 and yet when neurons are being destroyed by KA, the cAMP formation triggered by NE is augmented rather than depressed. The excess cAMP formation can be caused by one of 3 possible mechanisms. It is possible that cAMP formation is accelerated in remaining neurons and/or glia by suppression of action of PDE, the enzyme responsible for the breakdown of cAMP. This is not a likely possibility since the use of isobutyryl methyl xantine (IBMX), a PDE inhibitor, was nearly as effective in normal as in KA-treated HPC (unpublished observations). A second possibility is that the excess cAMP is formed in proliferating presynaptic fibers innervating the injured HPC. This is also not likely, since 6-OHDA treatment, which destroys noradrenergic terminals, did not abolish the enhanced cAMP response. The third possibility is that in KA-treated HPC there is a proliferation of non-neuronal elements which contain noradrenergic receptor linked to a cAMP-generating system. Two non-neuronal elements can account for this: blood vessels which have a beta-2 adrenergic receptor 14 and glial cells which may also have a beta adrenergic receptor coupled to adenylate cyclase13,20,81. Histological examination of the KA-treated HPC has revealed a marked increase in number of glial cells and it is likely that the rise in glial noradrenergic receptors coupled to adenylate-cyclase is responsible for the observed elevated cAMP response. This was also suggested for the KA-treated striatum 2a. The non-neuronal proliferating elements contain both alpha and beta adrenoreceptors; phentolamine which was relatively inefficient in antagonizing NE-stimulated cAMP accumulation in the normal HPC, became efficient in KA-treated HPC. The possibility that the new alpha and beta receptors are present on the same or on different sites remains to be determined. In this respect it is interesting to note that alpha adrenoreceptors might regulate presynaptically, the release of amino acid neurotransmitters (F. Moroni, personal communication). Since it is likely that some
363 excitatory afferents proliferate in the KA-treated HPC, it is possible that alpha adrenoreceptors proliferate on these presynaptic fibers. This possibility is subject to further experimentations. The beta adrenergic receptor species present in the brain have been the subject o f intensive recent investigations. It has been suggested that the main neuronal noradrenergic receptor is o f the beta-1 type, at least in cortical structures~,16,21,2~, a0. It has also been suggested that the beta-2 type is associated with non-neuronal elements21, 26. These suggestions are consistent with our observations. The beta adrenergic receptor in the normal H P C is predominantly of the beta-1 type, while in KA-treated HPC, the noradrenergic receptor is o f the beta-2 type. These data indicate that the beta-1 receptor is probably located mainly on neurons and the beta-2 receptors on both neurons and glia and that both are associated with a cAMP-generating system. It is tempting to speculate that the two receptor systems have different functions as well as time courses; the one associated with the physiological response, i.e. neuronal hyperpolarization would have a faster response time than the other one which is probably associated with a metabolic response. Further experiments should test this hypothesis. Altogether, the hippocampal slice preparation proves to be a useful model system for the investigation o f the correlation between biochemical and physiological events underlying neurot~ansmitter action in the brain.
REFERENCES 1 Bloom, F. E., The role of cyclic nucleotides in central synaptic function, Rev. Physiol. Biochem. Pharmacol., 74 0975) 1-103. 2 Bloom, F. E., Hoffer, B. J., Battenberg, E. F., Siggins, G. R., Steiner, A. L., Parker, C. W. and Wedner, H. J., Adenosine 3',5'-monophosphate is localized in cerebellar neurons: immunofluorescence evidence, Science, 177 (1972) 436-438. 3 Blumberg, J. B., Vetulani, J., Stawarz, R. J. and Sulser, F., The noradrenergic cyclic AMP generating system in the limbic forebrain: pharmacological characterization in vitro and possible role of limbic noradrenergic mechanisms in the mode of action of antipsychotics, Europ. J. Pharmacok, 37 (1976) 357-366. 4 Brown, B. L., Albano, J. D. M., Ekins, R. P., Sgherzi, A. M. and Tampion, W., A simple and sensitive saturation assay method for the measurement of adenosine 3':5'-cyclic monophosphate, Biochem. J., 121 (1971) 561-562. 5 Burges, R. A. and Blackburn, K. J., Adenyl cyclase and the differentiation of fl adrenoreceptors, Nature New Biol., 235 (1972) 249-250. 6 Bylund, D. B. and Snyder, S. H., Beta adrenergic receptor binding in membrane preparations from mammalian brain, Molec. Pharmacol., 12 (1976) 568-580. 7 Chang, R. S. L., Tran, V. T. and Snyder, S. H., Neurotransmitter receptor Iocalizations: brain lesion induced alterations in benzodiazepine, GABA, fl-adrenergic and histamine Hi-receptor binding, Brain Research, 190 (1980) 95-110. 8 Chasin, M., Rivkin, I., Mamrak, F., Samaniego, G. and Hess, S. M., a- and fl-adrenergic receptors as mediators of accumulation of cyclic adenosine 3',5'-monophosphate in specific areas of guinea pig brain, J. biol. Chem., 246 (1971) 3037-3041. 9 Coyle, J. T. and Schwarcz, R., Lesion of striatal neurones with Kainic acid provides a model for Huntington's chorea, Nature (Lond.), 263 (1976) 244-246. 10 Crutcher, K. A. and Davis, J. N., Hippocampal a and fl adrenergic receptors: comparison of [aH]dihydroalprenolol and [aHIWB 4101 binding with noradrenergic innervafion in the rat, Brain Research, 182 (1980) 107-117. 11 Dolphin, A., Adrien, J., Hamon, M. and Bockaert, J., Identity of [aH]dihydroalprenolol binding
364 sites and fl-adrenergic receptors coupled with adenylate cyclase in the central nervous system: pharmacological properties, distribution and adaptive responsiveness, Molec. Pharmacol., 15 (1979) 1-15. 12 Forn, J., Krueger, B. K. and Greengard, P., Adenosine 3',5'-monophosphate content in rat caudate nucleus: demonstration of dopaminergic and adrenergic receptors, Science, 186 (1974) 1118-1120. 13 Gilman, A. G. and Nirenberg, M., Effect of catecholamines on the adenosine 3'5 cyclic monophosphate concentrations of clonal satellite cells of neurons, Proc. nat. Acad. Sci. (U.S.A.), 68 (1971) 2165-2168. 14 Herbst, T. J., Raichle, M. E. and Ferrendelli, J. A., fl adrenergic regulation of adenosine 3'5'monophosphate concentration in brain microvessels, Science, 204 (1979) 330-332. 15 Hoffer, B. J., Siggins, G. R., Oliver, A. P. and Bloom, F. E., Activation of the pathway from locus coeruleus to rat cerebellar purkinje neurons: Pharmacological evidence of noradrenergic central inhibition, J. Pharmacol. exp. Ther., 184 (1973) 553-569. 16 Horn, A. S. and Phillipson, O. T., A noradrenaline sensitive adenylate cyclase in the rat limbic forebrain: preparation, properties and the effects of agonists, adrenolytics and neuroleptic drugs, Europ. J. Pharmacol., 37 (1976) 1-11. 17 Iversen, L. L., Catecholamine-sensitive adenylate cyclases in nervous tissue, J. Neurochem., 29 (1977) 5-12. 18 Lowry, O. H., Rosebrough, N. J., Farr, A. and Randall, J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 19 Levy, B. and Wilkenfeld, B. E,, An analysis of selective beta receptor blockade, Europ. J. Pharmacol., 5 (1969) 227-234. 20 Maguire, M. E., Wiklund, R. A., Anderson, H. J. and Gilman, A. G., Binding of 125I-iodohydroxybenzylpindolol to putative fl adrenergic receptors of rat glioma cells and other cell clones, J. biol. Chem., 251 (1976) 1221-1231. 21 Minneman, K. P., Dibner, M. D., Wolfe, B. B. and Molinoff, P. B., fll and f12adrenergic receptors in rat cerebral cortex are independently regulated, Science, 204 (1979) 866-868. 22 Minneman, K. P., Hegstrand, L. R. and Molinoff, P. B., Simultaneous determination of beta-1 and beta-2 adrenergic receptors in tissues containing both receptor subtypes, Molec. Pharmacol., 16 (1979) 34-46. 23 Minneman, K. P., Quik, M. and Emson, P. C., Receptor-linked cyclic AMP systems in rat neostriatum: differential localization revealed by kainic acid injection, Brain Research, 151 (1978) 507-521. 24 Nahorski, S. R., Rogers, K. J. and Edwards, C., Cerebral glycogenolysis and stimulation of fladrenoreceptors and histamine H2 receptors, Brain Research, 92 (1975) 529-533. 25 Palacios, J. M., Garbard, M., Barbin, G. and Schwartz, J. C., Histamine receptors mediating cyclic AMP accumulation in slices from guinea pig hippocampus: characterization and localization, Agents and Actions, 9 (1979) 82-83. 26 Pittman, R. N., Minneman, K. P. and Molinoff, P. B., Ontogeny of fll and f12adrenergic receptors in rat cerebellum and cerebral cortex, Brain Research, 188 (1980) 357-367. 27 Robison, G. R., Butcher, R. W. and Sutherland, E. W., Cyclic AMP, Academic Press, New York, 1971. 28 Segal, M., The action of norepinephrine in the rat hippocampus; intracellular studies in the slice preparation, Brain Research, in press. 29 Segal, M. and Bloom, F. E., The action of norepinephrine in the rat hippocampus I, Iontophoretic studies, Brain Research, 72 (1974) 79-97. 30 U'Prichard, D. C., Bylund, D. B. and Snyder, S. H.,t(zL)-[SH]Epinephrine and (--)-[3H]dihydroalprenolol binding to fll and fl~- - noradrenergic receptors in brain, heart and lung membranes, J. biol. Chem., 253 (1978) 5090-5102. 31 Wilkening, D. and Makman, M. H., Activation of glycogen, phosphorylase in rat caudate nucleus slices by L-isopropylnorepinephrineand dibutyrylcyclic AMP, J. Neurochem., 28 (1977) 1001-1007.