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The effect of septal nuclei stimulation on the release of acetylcholine from the rabbit hippocampus
Brain Research, 83 (1975) 123-133
123
© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
THE EFFECT OF SEPTAL NUCLEI STIMULATION ON THE RELEASE OF ACETYLCHOLINE FROM THE RABBIT HIPPOCAMPUS*
J. D. DUDAR Department of Physiology and Biophysics, Tupper Building, Dalhousie University, Halifax, N. S. B 3H 4H7 (Canada)
(Accepted September 2nd, 1974)
SUMMARY Acetylcholine (ACh) was collected from the alvear surface of the dorsal hippocampus in anesthetized rabbits by means of small cups (0.25 sq. cm) filled with artificial cerebrospinal fluid which contained eserine salicylate and atropine sulfate (1 /zg/ml of each). Stimulation of the medial septal nucleus (MS) at frequencies of 5, 10 and 50 Hz produced a similar increase in the release of ACh which was greater than the amount of ACh released with 1 Hz stimulation. However, the output per stimulus was highest with 1 Hz stimulation and it declined with higher frequencies. The increased release of ACh from the dorsal hippocampus could only be evoked by stimulating the MS, as stimulation of the lateral septal nucleus and of the contralateral hippocampus was without effect. The increase in ACh release with MS stimulation was abolished by sectioning the fimbria but was not altered by sectioning the alveus or dorsal fornix. These results support histochemical and anatomical data which indicates that there is a cholinergic pathway from the MS to the hippocampus and that the bulk of the fibers travel in the fimbria.
INTRODUCTION A variety of histochemical and biochemical studies indicate that the hippocampus receives a cholinergic input from the medial septal nucleus (MS) and nucleus of the diagonal band (NDB). Fibers staining for acetylcholinesterase (ACHE) originate from the MS and NDB and project to the hippocampal formation via the fimbria, the aiveus and the dorsal fornixlS, 19. The hippocampus itself has a characteristic * Some of this work was reported to the Canadian PhysiologicalSocietyWinter Meeting, January 17-29, 1974(Physiology Canada, 4 (1973) 175).
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staining pattern for ACh[~ 14,z7,29, and the choline acetylase (ChAc) distribution corresponds well to that of AChE la. Chronic lesions of the MS result in a decrease in ChAc activity and a reduction in the levels of acetylcholine (ACh) by at least 80 ~o (see ref. 17). Lesions of the fimbria also result in a large decrease in AChE and ChAc ls,19,27,2~. Furthermore, ACh has been collected from the dorsal surface of the hippocampus and the release can be increased by stimulating the surface of the septum 26. Since the cholinergic input to the hippocampus appears to originate in the MS and NDB and travels in definite pathways, the anatomical and histochemical findings can be tested by observing hippocampal ACh release while stimulating the various inputs or lesioning the pathways. MATERIALS AND METHODS
New Zealand white rabbits of either sex, weighing between 2.0 and 3.5 kg were used for these experiments. They were anesthetized with a mixture of urethane and pentobarbital (4 g urethane, 300 mg pentobarbital in 40 ml; 4 ml/kg, i.v.) or a mixture of chloralose and urethane (1 ~ chloralose, 25 ~ urethane; 3 ml/kg, i.v.) or a gaseous mixture of halothane and nitrous oxide (0.5-1 ~ halothane in 7 0 ~ N20 and 30°/ 02). After cannulating the trachea, the animals were placed in a stereotaxic frame and the skull was oriented according to the atlas of Sawyer et al. 24. The skull was opened and the overlying cerebral cortex was removed by suction to expose the septal and hippocampal regions. Oval perspex cylinders, the lower edges of which were shaped to fit the dorsal hippocampus, were positioned on the region overlying region CA1 and as far from the midline as possible. These cylinders covered an area of 0.25 sq. cm and held a volume of 0.30 ml. Once the cylinders were in place the remaining exposed brain was covered with warmed mineral oil. Initial AChE inhibition was produced with a 30-min exposure to the organophosphate echothiophate iodide (Ayerst) 0.5 mg/m110. A 0.25-ml aliquot of artificial CSF 12 which was warmed to 37 °C and which contained 1 /tg/ml of both atropine sulfate and eserine salicylate was used to collect ACh. The eserine was present to prevent ACh hydrolysis from AChE present in blood and other fluids which sometimes entered the cup and the atropine was used to ensure maximum ACh collection 1°,21. Samples were collected every 10 rain and assayed on the dorsal muscle o f the leech in a microbath 11. The values for ACh are expressed as AChC1 equivalents in ng/sq. cm.min. Some of the samples were tested on the curarized leech muscle (1 #g/ml) and they did not produce any contractions. Others were chromatographed (cellulose TLC plates; solvent system of n-butanol-formic acid-water, 60:15:35) along with ACh standards. Those sample areas which had the same RF value (0.8) as the standards assayed between 80 ~ and 90 ~ of their values before chromatography. These findings indicated that the contractions of the leech muscle produced by the samples were due to ACh. Small concentric stainless steel electrodes (0.5 mm diameter, 1.0 mm tip separation) were used for bipolar stimulation with pulses of 0.2 msec duration at 30 or 60 V (nominal voltage). Stimulation was continuous throughout the collection period.
HIPPOCAMPALAChRELEASE
125
For the stimulation of MS, the stimulating electrodes were placed under direct vision at a site 2.0 mm anterior to the junction of the septum and hippocampus. All septal stimulating sites were confirmed histologically and compared with the atlases of Andy and Stephan a and Urban and Richard al. The electrical activity of the hippocampus was monitored from the alvear surface by the square-cut ends of Tefloncoated silver wires referenced to a silver plate imbedded in the neck muscles. With the aid of a microscope, the alveus and dorsal fornix were sectioned by a shallow cut which was made with a small scalpel blade and which extended from the midline to the fimbria, anterior to the collecting cup. The fimbria was sectioned with a fine pair of scissors at the same level. RESULTS
The spontaneous release of ACh from the first 3 samples under chloraloseurethane anesthesia, the anesthetic most commonly used in these experiments, was 1.3 4- 0.1 ng/sq, cm.min (mean -4- S.E.M., 25 rabbits). This was identical to rate of release observed under halothane-N20 (7 rabbits) and slightly higher than that released under pentobarbital-urethane anesthesia, which was 1.1 --~ 0.1 ng/sq, cm. min (14 rabbits). The rates of release under the 3 types of anesthetic were not significantly different from each other. The release of ACh was relatively uniform in any one animal but it varied from animal to animal. Since the MS is thought to be the origin of the cholinergic input to the hippocampus, this nucleus was stimulated at frequencies of 1, 5, 10 and 50 Hz at 30 V (nominal voltage) to establish the effect of stimulus frequency on ACh release. For 50 Hz stimulation two procedures were used; one was continuous stimulation and other was stimulation in trains of 50 Hz for 2 sec repeated every 10 sec. This procedure allows one to compare the effect of continuous high frequency stimulation with a more physiological bursting pattern which delivers the same number of stimuli as 10 Hz. In each experiment stimulation was done sequentially from the lowest to the highest frequency.
--
]
6O STIM
1Hz
5Hz
l~) IOHz
180 MINUTES 5OHz
Fig. 1. An individual experiment showing the effect of stimulating the MS at various frequencies on A C h release from the dorsal hippocampus. Stimulation periods are marked by the filled bars. Stimulation at 50 Hz was in trains of 2 sec duration repeated every 10 sec. Chloralose-urethane anesthesia.
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TABLE I THE EFFECTOF MS STIMULATIONON A C h RELEASEFROMTHE DORSALHIPPOCAMPUS The effect of stimulation is the difference between the a m o u n t of A C h present in the resting sample immediately prior to stimulation and the a m o u n t present in the stimulated sample. Release per stimulus was calculated by dividing this difference with the total number of stimuli delivered. All values are means ± S.E.M. N -= 6. Stimulation at 30 V (nominal).
Frequency (Hz)
1
5
10
50 pooled
50 continuous
50 trains
A C h release ng/
2.5 ± 0.5*
5.8 ± 0.6
5.7 ~ 0.4
6.2 ~k 0.9
5.9 ± 1.7
6.5 -~ 1.1
sq. cm-min AChrelease/
418
i
78
193
± 22
95
-- 6
20
~ 6
107
~± 17
stimulus pg/sq, cm * Significantly different from the other values.
Fig. 1 shows one experiment and the effect o f the 4 frequencies on ACh release. The effect of stimulation on ACh was determined by subtracting the amount of ACh in ng/sq, cm.min in the sample immediately prior to stimulation from the amount of ACh present in the stimulated sample (Table I). It can be seen from this table that those frequencies higher than 1 Hz produced a similar release of ACh. However, when ACh release per stimulus was calculated, those stimuli delivered at 1 Hz were more effective in releasing ACh than were those stimuli delivered at higher frequencies. Continuous stimulation at 50 Hz released an amount of ACh similar to that produced by stimulation at 50 Hz in trains. Stimulation in trains, however, was a more effective form of stimulation since it released 5 times the amount of ACh per stimulus and was as effective as 10 Hz stimulation. Due to the consistent effectiveness of 5 Hz stimulation it was the frequency used in subsequent experiments. There was a possibility that the difference in output per stimulus would be reflected in the potentials evoked by stimulation. However, when the amplitude of the primary positive-negative wave was measured at a constant latency for each frequency in each experiment there was no significant difference in the amplitudes produced by l, 5 and 10 Hz. In order to determine whether the increased release of ACh was restricted to stimulation of the MS, two series of experiments were performed with low stimulus intensities of 30 V. The first series consisted o f moving the stimulating electrode in 1 mm steps from the surface of the septum down into the MS. The second set compared the effect of stimulating the adjacent lateral septal nucleus (LS) and the homotopic region of the contralateral dorsal hippocampus with MS stimulation. Since there was a variability in the resting release of ACh from animal to animal, the results were normalized by expressing the average resting release in the two samples prior to stimulation as 100K and converting all of the values to percentages. The mean resting value (_-k S.E.M.) is listed as the 100K values in Figs. 2 and 3.
HIPPOCAMPALACh RELEASE
3t
127
o
SURFACE I00'/,,-I.I _+0.20 nglcm21min
Imm I00~*,2.0+0.23 nglcm21min
2rnm I00~,-I.I +_0.05 nglcm21min
>2ram I00%=I.I +0.12 nglcm21min
n-8
n=3
n-4
n=3
Fig. 2. The effect of stimulating the MS at several depths on the release of ACh from the dorsal hippocampus. Stimulation at 5 Hz and 30 V (nominal) is marked by stippled bars. Each bar represents the per cent release (4- S.E.M.) in a 10 min collection period. In order to normalize the data, the average resting release value in the 2 samples prior to stimulation was taken as 100% and this average was used to convert all release values to percentages. Pentobarbital-urethane anesthesia.
The effect of moving the stimulating electrode down through MS is shown in Fig. 2. When the electrode was on the surface and stimulating the fibers of the dorsal fornix there was no increase in ACh release. However, when the electrode entered the MS and continued down through it, stimulation increased ACh release two-fold. The effect of stimulating sites other than the MS is shown in Fig. 3. Stimulation of both the LS and the contralateral dorsal hippocampus did not increase ACh release. As the MS had been shown to be the site of cholinergic input to the dorsal hippocampus, it was appropriate to investigate the distribution of the cholinergic fibers within the afferent input to the hippocampus. According to Lewis and Shute is the AChE-containing fibers which innervate the dorsal hippocampus travel in the dorsal fornix, the alveus and the fimbria. In order to investigate these pathways, A C h was collected bilaterally from both dorsal hippocampi while stimulating the MS of one side. Preliminary experiments had shown that high intensity stimulation at 60 V was adequate to produce a similar increase in ACh from both dorsal hippocampi with only one electrode. Thus, the contralateral hippocampus served as a control.
20o. t~ I00
0
I~EDIAL SITES 100~-1.5±0.14 n-ll
ng/cm2/min
LATERALSITES
nglcm2tmin
I00%,I.8~-0.24 n-4
CONTRALATHC
nglcm2/min
I00%-1.5_+0.2/ n-4
Fig. 3. The effect of stimulating the medial septal nucleus, lateral septal nucleus and the contralateral hippoeampus (contralat HC) on the release of ACh from the dorsal hippocampus. Stimulation at 5 Hz and 30 V (nominal) is marked by stippled bars. Each bar represents the per cent release (4S.E.M.) in a 10 min collection period. The data was normalized as in Fig. 2. Pentobarbital-urethane anesthesia.
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J.D. DUDAR CONTRALATERALTO 100T.-1.1 +_O.1ng/cmZ.min 800
700 600 500 400
300 200 100
120
60 TIME (rain) AL
FM
o
TIME (rain)
Fig. 4. Bilateral collection of ACh from the dorsal hippocampi showing the effects of unilateral sectioning of the alveus and dorsal fornix (AL) and the fimbria (FM) on the resting and evoked release of ACh. ACh release was evoked by stimulation of the MS of one side at 5 Hz (stippled bars) and the sectioning (marked by arrows) was carried out on the ipsilateral side following two control stimulation periods. The alveus and dorsal fornix were sectioned first and followed by section of the fimbria. Each bar represents a mean (4- S.E.M.) from 3 experiments. The data were normalized as in Fig12 except the average of the first 3 resting samples of each side were used for 100 ~o. Ch!oralose-urethane anesthesia. ~ = stimulation site.
HIPPOCAMPAL ACh RELEASE FM
IPSILATERALTO t 500-
100%• 0.6 _+0.(]6
129 AL
rlg/cm2, min
400-
300.
i!ii 200100-
TIME
rain )
120
180
Fig. 5. Similarexperiment to that of Fig. 4 showing only the results from the ipsilateral dorsal hippocampus and with the sectioning o f the fimbria (FM) preceding that of the alveus (AL), N = 3. Chloralose-urethane anesthesia. ~ = stimulation site.
The results from 3 experiments in which the alveus and dorsal fornix were sectioned before sectioning the fimbria are shown in Fig. 4, while Fig. 5 shows the effect of sectioning the fimbria first. In both cases, two control stimulation periods preceded the sectioning of the various pathways. As seen from Fig. 5 the increase in ACh release due to MS stimulation was not affected by the simultaneous sectioning of the alveus and dorsal fornix. However, after the fimbria was sectioned, stimulation failed to increase ACh release on that side. A similar failure of stimulation to increase ACh release following fimbrial section is shown in Fig. 5. Severing the alveus and dorsal fornix after sectioning the fimbria had no noticeable effect on the resting or evoked release of ACh. In some other experiments, a midline cut was made to try and section the hippocampal commissure between the two dorsal hippocampi in addition to the fimbrial and alvear lesions. This procedure was also without effect on resting or evoked ACh release. DISCUSSION
One of the tenents for a substance to be considered as a transmitter in a neural pathway is to demonstrate its release upon activation of the pathway. The data presented here show that the release of ACh from the hippocampus is increased by stimulation in the medial septum, that the increase varies with the stimulation frequency and that this release is abolished by transection of the fimbria. These data, considered with the anatomical and histochemical data, indicate that ACh is the transmitter from the medial septum to the dorsal hippocampus and that the major cholinergic pathway is through the fimbria. In the present experiments, the values obtained for spontaneous ACh release (1.1 and 1.3 ng/sq, cm.min)from the rabbit dorsal hippocampus are considerably greater than that reported by Smith ~6 who observed a mean resting release rate of 0.24 ng/sq, cm-min under urethane anesthesia and with atropine added to the collecting fluid. This difference might be due to different experimental conditions or to a
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less sensitive bioassay procedure. However, the present data for hippocampal resting ACh release are similar to the resting release of ACh from the cerebral cortex observed in rabbits under Dial anesthesia 9,15 but somewhat lower than the cortical release in unanesthetized rabbits 4. Furthermore, preliminary experiments from this laboratory have found the rate of spontaneous ACh release from the dorsal hippocampus and from the contralateral cerebral cortex of the same animal to be similar. Thus it appears that the spontaneous release of ACh from the dorsal hippocampus is of the same magnitude as the release from the cerebral cortex. The spontaneous release of ACh from the dorsal hippocampus was found to be quite uniform with the 3 types of anesthesia used. This is in contrast to the spontaneous ACh release from the cerebral cortex of cats where the rate of release appears to be dependent on the type of anesthetic and the depth of anesthesia obtained 1°. Phillis 23, however, found that there was little difference in the ACh release from cat cerebral cortex when he used halothane, Dial or chloralose anesthesia. It is possible that in the present experiments a similar depth of anesthesia was produced which resulted in the uniform rate of release. Another possibility may be that the cholinergic nerve elements of the hippocampus are less sensitive to different anesthetic agents than those of the cerebral cortex. Smith 26 has shown that the release of ACh from the dorsal hippocampus can be increased to 2.5 times the resting release by stimulating the surface of the septum. The results from the present experiments are different in that stimulating the surface of the septum did not increase ACh release. It was only when the medial nucleus of the septum was stimulated that there was an increase in ACh release. The difference may be due to the use of different intensities of stimulation current. With high intensity stimulation it is quite possible for the current to spread into the medial nucleus which lies close to the dorsal surface of the septum. The present results also show that the increased ACh release due to MS stimulation was frequency dependent. This is similar to what has been reported for ACh release from the rabbit visual cortex evoked by stimulation of the lateral geniculate body 9 and from the rabbit auditory cortex after stimulating the medial geniculate body 15. In both of these cases the total amount of ACh released by stimulation increased with frequency up to 10 or 20 Hz and did not increase further with higher stimulus frequencies. These findings are in contrast with the effect of peripheral nerve stimulation on ACh release from the somatosensory cortex where maximal release occurs at 1 Hz21, z3. When ACh release per impulse is considered, the evoked release o f ACh from the dorsal hippocampus resembles that released from the neuromuscular junction 8, ~6,80, the ileum 22 and from cerebral cortical slicesL As the stimulating frequency increases the amount of ACh released per impulse falls, particularly if the stimulation is prolonged. At the superior cervical ganglion, however, the output of ACh per stimulus is constant up to 16 Hz and then it decreases with higher frequencies 5. Presumably the quantum content is reduced as the stimulus frequency is increased, possibly due to a lag in mobilization of stored ACh rather than to a lag in synthesis of new ACh as suggested by Bourdois et aLL
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The data from the release of ACh per stimulus suggested that the evoked potentials recorded from the alvear surface overlying area CA1 might reflect the decrease in ACh per stimulus. However, this was not the case as there was no difference between the potentials produced by 1, 5 or 10 Hz. This finding is not too surprising since it is possible that the potentials are produced by deep cellular elements from which ACh does not get into the collecting fluid. Also there are probably many non-cholinergic afferent fibers passing through or originating in the septum which are activated and contribute to the evoked potentials. The ACh collected from the dorsal surface of the hippocampus probably originates from those terminals which contain AChE 1s,2°,27, and ChAc 1a,17,19. Both of these enzymes have their highest activity in the infrapyramidal layer of the hippocampus and the juxtagranular region of the area dentata 13,27. Since the infrapyramidal layer lies just below the alvear surface of the hippocampal formation there is less than 0.5 mm of tissue for ACh to diffuse to the surface. The upper blade of granule cells of the fascia dentata lies another 1-1.5 mm deeper which would make diffusion to the surface much slower. Acetylcholine is a possible excitatory transmitter at these two groups of cells as indicated by the response to the iontophoretic application of ACh to the cells in pyramidal and granule cell layers6. The medial nucleus and the vertical limb of the NDB stain heavily for AChE 19, 2o and lesions of the MS markedly reduce the levels of both AChE and ChAc in the hippocampus 17,20. In addition, the concentration of ACh falls after MS lesionslT, 25. However, lesions of the lateral septal nucleus or lateral hypothalamus do not change hippocampal AChE levels20. Furthermore, transection of the fimbria, the major afferent input to the hippocampus, results in a large loss of AChE 1s,27 and ChAc activities19. The other hippocampal afferents originating from the entorhinal cortex and contralateral hippocampus (commissural fibers) contain no ChAc or AChE 2s. These observations indicate that cholinergic fibers originate in the MS and travel principally in the fimbria to terminate on the pyramidal cells of the hippocampus and the granule cells of the dentate area. The present results support these findings since there is an increase in the release of ACh only when the MS is stimulated and this increase can be abolished by sectioning the fimbria. The supracallosal stria, dorsal fornix and alveus contribute AChE-containing fibers to the dorsal hippocampus 18. The lack of effect on ACh release after transecting these pathways shows that they are quantitatively unimportant sources for the areas sampled by the collecting cup. Electrophysiological studies in rabbits under chloralose-urethane anesthesia have shown that stimulation of the MS evoked potentials via possible monosynaptic pathways in both the dentate granule cells and the hippocampal CA1 pyramidal cells 1, 2. The granule cell potential is not abolished by sectioning the dorsal fornix, the alveus or the fimbria while the CA1 pyramidal cells potential is abolished by sectioning the alveus. These findings do not correlate with the findings of the present experiments in which the increase in ACh release due to MS stimulation was abolished by sectioning the fimbria. At the present time it is difficult to account for this difference unless one accepts that there are possibly two afferent pathways from the MS to the hippocampus; one cholinergic travelling principally through the fimbria and the other non-
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cholinergic travelling in pathways other t h a n the fimbria. U n f o r t u n a t e l y , the bulk of the available histochemical a n d the a n a t o m i c a l data are from the rat so that there m a y be some species difference c o n c e r n i n g the a n a t o m i c a l c o n n e c t i o n s between the MS a n d the h i p p o c a m p a l formation. ACKNOWLEDGEMENTS This work has been s u p p o r t e d by the C a n a d i a n Medical Research Council. I would like to t h a n k J. C. Szerb for his interest a n d criticism a n d Lee H o p k i n s for her technical assistance, especially in the A C h bioassay.
REFERENCES 1 ANDERSEN,P., BRULAND, H., AND KAADA, B. R., Activation of the dentate area by septal stimulation, Acta physiol, scand., 51 (1961) 17-28. 2 ANDERSEN,P., BRULAND,H., AND KAADA,B. R., Activation of the field CA1 of the hippocampus by septal stimulation, Actaphysiol. scand., 51 (1961) 29-40. 3 AND',',O. J., AND STEPHAN, H., The Septum of the Cat, Thomas, Springfield, Ill., 1964. 4 BEANI,L., BIANCHI,C., SANTINOCETO,L., AND MARCHETTI,P., The cerebral acetylcholine release in conscious rabbits with semi-permanently implanted epidural cups, Int. J. Neuropharmacol., 7 (1968) 469-481. 5 BIRKS,R. I., AND MACINTOSH,F. C., Acetylcholine metabolism of a sympathetic ganglion, Canad. J. Biochem., 39 (1961) 787-827. 6 BISCOE,T. J., AND STRAUGHAN,D. W., Micro-electrophoretic studies of neurons in the cat hippocampus, J. Physiol. (Lend.), 183 (1966) 341-359. 7 BOURDOIS,P. S., MITCHELL, J. F., SOMOGYI,G. T., AND SZERB, J. C., The output per stimulus of acetylcholine from cerebral cortical slices in the presence or absence of cholinesterase inhibition, Brit. J. Pharmacol., 52 (1974), in press. 8 BROOKS,V. B., AND THINS, R. E., Reduction of quantum content during neuromuscular transmission, J. Physiol. (Lend.), 162 (1962) 298-310. 9 COLLIER,B., AND MITCHELL,J. F., The central release of acetylcholine during stimulation of the visual pathway, J. Physiol. (Lend.), 184 (1966) 239-254. l0 DUDAR,J. D., AND SZERB, J. C., The effect of topically applied atropine on resting and evoked cortical acetylcholine release, J. Physiol. (Lend.), 203 (1969) 74t-762. 11 DUDAR,J. D., AND SZERB, J. C., A. The push-puU cannula. B. Bioassay of acetyicholine on leech muscle suspended in a microbath, Exp. Physiol. Biochem., 3 (1970) 341-350. 12 FELDBERG, W., AND MALCOLM, J. L., Experiments on the site of action of tubocurarine when applied via .the cerebral ventricles, J. Physiol. (Lend.), 149 (1959) 58-77. 13 FONNUM,F., Topographical and subcellular localization of choline acetyltransferase in rat hippocampal formation, J. Neurochem., 77 (1970) 1029-1037. 14 GENESER-JENSEN,F. A., Distribution of acetylcholinesterase in the hippocampal region of the guinea pig, Z. Zellforsch., 124 (1972) 546-560. 15 HEMSWORTH,B. A., AND MITCHELL,J. F., The characteristics of acetylcholine release mechanisms in the auditory cortex, Brit. J. Pharmacol., 36 (1969) 161-170. 16 KRNJEVI~,K., AND MITCHELL,J. F., The release of acetylcholine in the isolated rat diaphragm, J. Physiol. (Lend.), 155 (1961) 246-262. 17 KUI~AR,J. J., SETI'IY,V. H., ROTH, R. H., AND AOHAJANIAN,G. K., Choline: selective accumulation by central cholinergic neurons, J. Neurochem., 20 (1973) 581-593. 18 LEWIS, P. R., AND SHUTE, C. C. O., The cholinergic limbic system: projection to hippocampal formation, medial corteX, nuclei of the ascending cholinergic reticular system and the subfornical organ and supra-optic crest, Brain, 90 (1967) 521-540. 19 LEWm,P. R., SHUTS, C. C. D., AND SILVER,A., Confirmation from choline acetylase analyses of a massive cholinergic innervation to the rat hippocampus, J. Physiol. (Lend.), 191 (1967) 215-224.
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20 MELLGREN,S. I., AND SREBRO, B., Changes in acetylcholinesterase and distribution of degenerating fibres in the hippocampal region after septal lesions in the rat, Brain Research, 52 (1973)19-36. 21 MITCHELL,J. F., The spontaneous and evoked release of acetylcholine from the cerebral cortex, J. Physiol. (Lond.), 165 (1963) 98-116. 22 PATON, W. n . M., Cholinergic transmission and acetylcholine output, Canad. J. Biochem., 41 (1963) 2637-2653. 23 PHILLIS, J. W., Acetylcholine release from the cerebral cortex: its role in cortical arousal, Brain Research, 7 (1968) 378-389. 24 SAWYER, C. H., EVERETT, J. W., AND GREEN, J. D., The rabbit diencephalon in stereotaxic coordinates, J. comp. Neurol., 101 (1954) 801-824. 25 SETHY, V. H., KUHAR, i . J., ROTH, R. H., VANWOERT, i . H., AND AGHAJANIAN,G. K., Cholinergic neurons: effect of acute septal lesion on acetylcholine and choline content of rat hippocampus, Brain Research, 55 (1973) 481-484. 26 SMITH, C. M., The release of acetylcholine from rabbit hippocampus, Brit. J. Pharmacol., 45 (1972) 172P. 27 STORM-MATHISEN,J., Quantitative neurochemistry of acetylcholinesterase in rat hippocampal region correlated to histochemical staining, J. Nearochem., 17 (1970) 739-750. 28 STORM-MATHISEN,J., Glutamate decarboxylase in the rat hippocampal region after lesions of the afferent fiber systems. Evidence that the enzyme is located in intrinsic neurones, Brain Research, 40 (1972) 215-235. 29 STORM-MATHISEN,J., AND BLACKSTAD,T. W., Cholinesterase in the hippocampai region, ,4cta anat. (Basel), 56 (1964) 216-253. 30 STRAUGHAN,D. W., The release of acetylcholine from mammalian motor nerve endings, Brit. J. Pharmacol., 15 (1960) 417-424. 31 URBAN, I., AND RICHARD, P., ,4 Stereotaxic Atlas of the New Zealand Rabbit's Brain, Thomas, Springfield, Ill., 1972.
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© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
THE EFFECT OF SEPTAL NUCLEI STIMULATION ON THE RELEASE OF ACETYLCHOLINE FROM THE RABBIT HIPPOCAMPUS*
J. D. DUDAR Department of Physiology and Biophysics, Tupper Building, Dalhousie University, Halifax, N. S. B 3H 4H7 (Canada)
(Accepted September 2nd, 1974)
SUMMARY Acetylcholine (ACh) was collected from the alvear surface of the dorsal hippocampus in anesthetized rabbits by means of small cups (0.25 sq. cm) filled with artificial cerebrospinal fluid which contained eserine salicylate and atropine sulfate (1 /zg/ml of each). Stimulation of the medial septal nucleus (MS) at frequencies of 5, 10 and 50 Hz produced a similar increase in the release of ACh which was greater than the amount of ACh released with 1 Hz stimulation. However, the output per stimulus was highest with 1 Hz stimulation and it declined with higher frequencies. The increased release of ACh from the dorsal hippocampus could only be evoked by stimulating the MS, as stimulation of the lateral septal nucleus and of the contralateral hippocampus was without effect. The increase in ACh release with MS stimulation was abolished by sectioning the fimbria but was not altered by sectioning the alveus or dorsal fornix. These results support histochemical and anatomical data which indicates that there is a cholinergic pathway from the MS to the hippocampus and that the bulk of the fibers travel in the fimbria.
INTRODUCTION A variety of histochemical and biochemical studies indicate that the hippocampus receives a cholinergic input from the medial septal nucleus (MS) and nucleus of the diagonal band (NDB). Fibers staining for acetylcholinesterase (ACHE) originate from the MS and NDB and project to the hippocampal formation via the fimbria, the aiveus and the dorsal fornixlS, 19. The hippocampus itself has a characteristic * Some of this work was reported to the Canadian PhysiologicalSocietyWinter Meeting, January 17-29, 1974(Physiology Canada, 4 (1973) 175).
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J. I). I)UI)AR
staining pattern for ACh[~ 14,z7,29, and the choline acetylase (ChAc) distribution corresponds well to that of AChE la. Chronic lesions of the MS result in a decrease in ChAc activity and a reduction in the levels of acetylcholine (ACh) by at least 80 ~o (see ref. 17). Lesions of the fimbria also result in a large decrease in AChE and ChAc ls,19,27,2~. Furthermore, ACh has been collected from the dorsal surface of the hippocampus and the release can be increased by stimulating the surface of the septum 26. Since the cholinergic input to the hippocampus appears to originate in the MS and NDB and travels in definite pathways, the anatomical and histochemical findings can be tested by observing hippocampal ACh release while stimulating the various inputs or lesioning the pathways. MATERIALS AND METHODS
New Zealand white rabbits of either sex, weighing between 2.0 and 3.5 kg were used for these experiments. They were anesthetized with a mixture of urethane and pentobarbital (4 g urethane, 300 mg pentobarbital in 40 ml; 4 ml/kg, i.v.) or a mixture of chloralose and urethane (1 ~ chloralose, 25 ~ urethane; 3 ml/kg, i.v.) or a gaseous mixture of halothane and nitrous oxide (0.5-1 ~ halothane in 7 0 ~ N20 and 30°/ 02). After cannulating the trachea, the animals were placed in a stereotaxic frame and the skull was oriented according to the atlas of Sawyer et al. 24. The skull was opened and the overlying cerebral cortex was removed by suction to expose the septal and hippocampal regions. Oval perspex cylinders, the lower edges of which were shaped to fit the dorsal hippocampus, were positioned on the region overlying region CA1 and as far from the midline as possible. These cylinders covered an area of 0.25 sq. cm and held a volume of 0.30 ml. Once the cylinders were in place the remaining exposed brain was covered with warmed mineral oil. Initial AChE inhibition was produced with a 30-min exposure to the organophosphate echothiophate iodide (Ayerst) 0.5 mg/m110. A 0.25-ml aliquot of artificial CSF 12 which was warmed to 37 °C and which contained 1 /tg/ml of both atropine sulfate and eserine salicylate was used to collect ACh. The eserine was present to prevent ACh hydrolysis from AChE present in blood and other fluids which sometimes entered the cup and the atropine was used to ensure maximum ACh collection 1°,21. Samples were collected every 10 rain and assayed on the dorsal muscle o f the leech in a microbath 11. The values for ACh are expressed as AChC1 equivalents in ng/sq. cm.min. Some of the samples were tested on the curarized leech muscle (1 #g/ml) and they did not produce any contractions. Others were chromatographed (cellulose TLC plates; solvent system of n-butanol-formic acid-water, 60:15:35) along with ACh standards. Those sample areas which had the same RF value (0.8) as the standards assayed between 80 ~ and 90 ~ of their values before chromatography. These findings indicated that the contractions of the leech muscle produced by the samples were due to ACh. Small concentric stainless steel electrodes (0.5 mm diameter, 1.0 mm tip separation) were used for bipolar stimulation with pulses of 0.2 msec duration at 30 or 60 V (nominal voltage). Stimulation was continuous throughout the collection period.
HIPPOCAMPALAChRELEASE
125
For the stimulation of MS, the stimulating electrodes were placed under direct vision at a site 2.0 mm anterior to the junction of the septum and hippocampus. All septal stimulating sites were confirmed histologically and compared with the atlases of Andy and Stephan a and Urban and Richard al. The electrical activity of the hippocampus was monitored from the alvear surface by the square-cut ends of Tefloncoated silver wires referenced to a silver plate imbedded in the neck muscles. With the aid of a microscope, the alveus and dorsal fornix were sectioned by a shallow cut which was made with a small scalpel blade and which extended from the midline to the fimbria, anterior to the collecting cup. The fimbria was sectioned with a fine pair of scissors at the same level. RESULTS
The spontaneous release of ACh from the first 3 samples under chloraloseurethane anesthesia, the anesthetic most commonly used in these experiments, was 1.3 4- 0.1 ng/sq, cm.min (mean -4- S.E.M., 25 rabbits). This was identical to rate of release observed under halothane-N20 (7 rabbits) and slightly higher than that released under pentobarbital-urethane anesthesia, which was 1.1 --~ 0.1 ng/sq, cm. min (14 rabbits). The rates of release under the 3 types of anesthetic were not significantly different from each other. The release of ACh was relatively uniform in any one animal but it varied from animal to animal. Since the MS is thought to be the origin of the cholinergic input to the hippocampus, this nucleus was stimulated at frequencies of 1, 5, 10 and 50 Hz at 30 V (nominal voltage) to establish the effect of stimulus frequency on ACh release. For 50 Hz stimulation two procedures were used; one was continuous stimulation and other was stimulation in trains of 50 Hz for 2 sec repeated every 10 sec. This procedure allows one to compare the effect of continuous high frequency stimulation with a more physiological bursting pattern which delivers the same number of stimuli as 10 Hz. In each experiment stimulation was done sequentially from the lowest to the highest frequency.
--
]
6O STIM
1Hz
5Hz
l~) IOHz
180 MINUTES 5OHz
Fig. 1. An individual experiment showing the effect of stimulating the MS at various frequencies on A C h release from the dorsal hippocampus. Stimulation periods are marked by the filled bars. Stimulation at 50 Hz was in trains of 2 sec duration repeated every 10 sec. Chloralose-urethane anesthesia.
126
J.D. DUI)AR
TABLE I THE EFFECTOF MS STIMULATIONON A C h RELEASEFROMTHE DORSALHIPPOCAMPUS The effect of stimulation is the difference between the a m o u n t of A C h present in the resting sample immediately prior to stimulation and the a m o u n t present in the stimulated sample. Release per stimulus was calculated by dividing this difference with the total number of stimuli delivered. All values are means ± S.E.M. N -= 6. Stimulation at 30 V (nominal).
Frequency (Hz)
1
5
10
50 pooled
50 continuous
50 trains
A C h release ng/
2.5 ± 0.5*
5.8 ± 0.6
5.7 ~ 0.4
6.2 ~k 0.9
5.9 ± 1.7
6.5 -~ 1.1
sq. cm-min AChrelease/
418
i
78
193
± 22
95
-- 6
20
~ 6
107
~± 17
stimulus pg/sq, cm * Significantly different from the other values.
Fig. 1 shows one experiment and the effect o f the 4 frequencies on ACh release. The effect of stimulation on ACh was determined by subtracting the amount of ACh in ng/sq, cm.min in the sample immediately prior to stimulation from the amount of ACh present in the stimulated sample (Table I). It can be seen from this table that those frequencies higher than 1 Hz produced a similar release of ACh. However, when ACh release per stimulus was calculated, those stimuli delivered at 1 Hz were more effective in releasing ACh than were those stimuli delivered at higher frequencies. Continuous stimulation at 50 Hz released an amount of ACh similar to that produced by stimulation at 50 Hz in trains. Stimulation in trains, however, was a more effective form of stimulation since it released 5 times the amount of ACh per stimulus and was as effective as 10 Hz stimulation. Due to the consistent effectiveness of 5 Hz stimulation it was the frequency used in subsequent experiments. There was a possibility that the difference in output per stimulus would be reflected in the potentials evoked by stimulation. However, when the amplitude of the primary positive-negative wave was measured at a constant latency for each frequency in each experiment there was no significant difference in the amplitudes produced by l, 5 and 10 Hz. In order to determine whether the increased release of ACh was restricted to stimulation of the MS, two series of experiments were performed with low stimulus intensities of 30 V. The first series consisted o f moving the stimulating electrode in 1 mm steps from the surface of the septum down into the MS. The second set compared the effect of stimulating the adjacent lateral septal nucleus (LS) and the homotopic region of the contralateral dorsal hippocampus with MS stimulation. Since there was a variability in the resting release of ACh from animal to animal, the results were normalized by expressing the average resting release in the two samples prior to stimulation as 100K and converting all of the values to percentages. The mean resting value (_-k S.E.M.) is listed as the 100K values in Figs. 2 and 3.
HIPPOCAMPALACh RELEASE
3t
127
o
SURFACE I00'/,,-I.I _+0.20 nglcm21min
Imm I00~*,2.0+0.23 nglcm21min
2rnm I00~,-I.I +_0.05 nglcm21min
>2ram I00%=I.I +0.12 nglcm21min
n-8
n=3
n-4
n=3
Fig. 2. The effect of stimulating the MS at several depths on the release of ACh from the dorsal hippocampus. Stimulation at 5 Hz and 30 V (nominal) is marked by stippled bars. Each bar represents the per cent release (4- S.E.M.) in a 10 min collection period. In order to normalize the data, the average resting release value in the 2 samples prior to stimulation was taken as 100% and this average was used to convert all release values to percentages. Pentobarbital-urethane anesthesia.
The effect of moving the stimulating electrode down through MS is shown in Fig. 2. When the electrode was on the surface and stimulating the fibers of the dorsal fornix there was no increase in ACh release. However, when the electrode entered the MS and continued down through it, stimulation increased ACh release two-fold. The effect of stimulating sites other than the MS is shown in Fig. 3. Stimulation of both the LS and the contralateral dorsal hippocampus did not increase ACh release. As the MS had been shown to be the site of cholinergic input to the dorsal hippocampus, it was appropriate to investigate the distribution of the cholinergic fibers within the afferent input to the hippocampus. According to Lewis and Shute is the AChE-containing fibers which innervate the dorsal hippocampus travel in the dorsal fornix, the alveus and the fimbria. In order to investigate these pathways, A C h was collected bilaterally from both dorsal hippocampi while stimulating the MS of one side. Preliminary experiments had shown that high intensity stimulation at 60 V was adequate to produce a similar increase in ACh from both dorsal hippocampi with only one electrode. Thus, the contralateral hippocampus served as a control.
20o. t~ I00
0
I~EDIAL SITES 100~-1.5±0.14 n-ll
ng/cm2/min
LATERALSITES
nglcm2tmin
I00%,I.8~-0.24 n-4
CONTRALATHC
nglcm2/min
I00%-1.5_+0.2/ n-4
Fig. 3. The effect of stimulating the medial septal nucleus, lateral septal nucleus and the contralateral hippoeampus (contralat HC) on the release of ACh from the dorsal hippocampus. Stimulation at 5 Hz and 30 V (nominal) is marked by stippled bars. Each bar represents the per cent release (4S.E.M.) in a 10 min collection period. The data was normalized as in Fig. 2. Pentobarbital-urethane anesthesia.
128
J.D. DUDAR CONTRALATERALTO 100T.-1.1 +_O.1ng/cmZ.min 800
700 600 500 400
300 200 100
120
60 TIME (rain) AL
FM
o
TIME (rain)
Fig. 4. Bilateral collection of ACh from the dorsal hippocampi showing the effects of unilateral sectioning of the alveus and dorsal fornix (AL) and the fimbria (FM) on the resting and evoked release of ACh. ACh release was evoked by stimulation of the MS of one side at 5 Hz (stippled bars) and the sectioning (marked by arrows) was carried out on the ipsilateral side following two control stimulation periods. The alveus and dorsal fornix were sectioned first and followed by section of the fimbria. Each bar represents a mean (4- S.E.M.) from 3 experiments. The data were normalized as in Fig12 except the average of the first 3 resting samples of each side were used for 100 ~o. Ch!oralose-urethane anesthesia. ~ = stimulation site.
HIPPOCAMPAL ACh RELEASE FM
IPSILATERALTO t 500-
100%• 0.6 _+0.(]6
129 AL
rlg/cm2, min
400-
300.
i!ii 200100-
TIME
rain )
120
180
Fig. 5. Similarexperiment to that of Fig. 4 showing only the results from the ipsilateral dorsal hippocampus and with the sectioning o f the fimbria (FM) preceding that of the alveus (AL), N = 3. Chloralose-urethane anesthesia. ~ = stimulation site.
The results from 3 experiments in which the alveus and dorsal fornix were sectioned before sectioning the fimbria are shown in Fig. 4, while Fig. 5 shows the effect of sectioning the fimbria first. In both cases, two control stimulation periods preceded the sectioning of the various pathways. As seen from Fig. 5 the increase in ACh release due to MS stimulation was not affected by the simultaneous sectioning of the alveus and dorsal fornix. However, after the fimbria was sectioned, stimulation failed to increase ACh release on that side. A similar failure of stimulation to increase ACh release following fimbrial section is shown in Fig. 5. Severing the alveus and dorsal fornix after sectioning the fimbria had no noticeable effect on the resting or evoked release of ACh. In some other experiments, a midline cut was made to try and section the hippocampal commissure between the two dorsal hippocampi in addition to the fimbrial and alvear lesions. This procedure was also without effect on resting or evoked ACh release. DISCUSSION
One of the tenents for a substance to be considered as a transmitter in a neural pathway is to demonstrate its release upon activation of the pathway. The data presented here show that the release of ACh from the hippocampus is increased by stimulation in the medial septum, that the increase varies with the stimulation frequency and that this release is abolished by transection of the fimbria. These data, considered with the anatomical and histochemical data, indicate that ACh is the transmitter from the medial septum to the dorsal hippocampus and that the major cholinergic pathway is through the fimbria. In the present experiments, the values obtained for spontaneous ACh release (1.1 and 1.3 ng/sq, cm.min)from the rabbit dorsal hippocampus are considerably greater than that reported by Smith ~6 who observed a mean resting release rate of 0.24 ng/sq, cm-min under urethane anesthesia and with atropine added to the collecting fluid. This difference might be due to different experimental conditions or to a
130
J.D. DUDAR
less sensitive bioassay procedure. However, the present data for hippocampal resting ACh release are similar to the resting release of ACh from the cerebral cortex observed in rabbits under Dial anesthesia 9,15 but somewhat lower than the cortical release in unanesthetized rabbits 4. Furthermore, preliminary experiments from this laboratory have found the rate of spontaneous ACh release from the dorsal hippocampus and from the contralateral cerebral cortex of the same animal to be similar. Thus it appears that the spontaneous release of ACh from the dorsal hippocampus is of the same magnitude as the release from the cerebral cortex. The spontaneous release of ACh from the dorsal hippocampus was found to be quite uniform with the 3 types of anesthesia used. This is in contrast to the spontaneous ACh release from the cerebral cortex of cats where the rate of release appears to be dependent on the type of anesthetic and the depth of anesthesia obtained 1°. Phillis 23, however, found that there was little difference in the ACh release from cat cerebral cortex when he used halothane, Dial or chloralose anesthesia. It is possible that in the present experiments a similar depth of anesthesia was produced which resulted in the uniform rate of release. Another possibility may be that the cholinergic nerve elements of the hippocampus are less sensitive to different anesthetic agents than those of the cerebral cortex. Smith 26 has shown that the release of ACh from the dorsal hippocampus can be increased to 2.5 times the resting release by stimulating the surface of the septum. The results from the present experiments are different in that stimulating the surface of the septum did not increase ACh release. It was only when the medial nucleus of the septum was stimulated that there was an increase in ACh release. The difference may be due to the use of different intensities of stimulation current. With high intensity stimulation it is quite possible for the current to spread into the medial nucleus which lies close to the dorsal surface of the septum. The present results also show that the increased ACh release due to MS stimulation was frequency dependent. This is similar to what has been reported for ACh release from the rabbit visual cortex evoked by stimulation of the lateral geniculate body 9 and from the rabbit auditory cortex after stimulating the medial geniculate body 15. In both of these cases the total amount of ACh released by stimulation increased with frequency up to 10 or 20 Hz and did not increase further with higher stimulus frequencies. These findings are in contrast with the effect of peripheral nerve stimulation on ACh release from the somatosensory cortex where maximal release occurs at 1 Hz21, z3. When ACh release per impulse is considered, the evoked release o f ACh from the dorsal hippocampus resembles that released from the neuromuscular junction 8, ~6,80, the ileum 22 and from cerebral cortical slicesL As the stimulating frequency increases the amount of ACh released per impulse falls, particularly if the stimulation is prolonged. At the superior cervical ganglion, however, the output of ACh per stimulus is constant up to 16 Hz and then it decreases with higher frequencies 5. Presumably the quantum content is reduced as the stimulus frequency is increased, possibly due to a lag in mobilization of stored ACh rather than to a lag in synthesis of new ACh as suggested by Bourdois et aLL
HIPPOCAMPALACh RELEASE
131
The data from the release of ACh per stimulus suggested that the evoked potentials recorded from the alvear surface overlying area CA1 might reflect the decrease in ACh per stimulus. However, this was not the case as there was no difference between the potentials produced by 1, 5 or 10 Hz. This finding is not too surprising since it is possible that the potentials are produced by deep cellular elements from which ACh does not get into the collecting fluid. Also there are probably many non-cholinergic afferent fibers passing through or originating in the septum which are activated and contribute to the evoked potentials. The ACh collected from the dorsal surface of the hippocampus probably originates from those terminals which contain AChE 1s,2°,27, and ChAc 1a,17,19. Both of these enzymes have their highest activity in the infrapyramidal layer of the hippocampus and the juxtagranular region of the area dentata 13,27. Since the infrapyramidal layer lies just below the alvear surface of the hippocampal formation there is less than 0.5 mm of tissue for ACh to diffuse to the surface. The upper blade of granule cells of the fascia dentata lies another 1-1.5 mm deeper which would make diffusion to the surface much slower. Acetylcholine is a possible excitatory transmitter at these two groups of cells as indicated by the response to the iontophoretic application of ACh to the cells in pyramidal and granule cell layers6. The medial nucleus and the vertical limb of the NDB stain heavily for AChE 19, 2o and lesions of the MS markedly reduce the levels of both AChE and ChAc in the hippocampus 17,20. In addition, the concentration of ACh falls after MS lesionslT, 25. However, lesions of the lateral septal nucleus or lateral hypothalamus do not change hippocampal AChE levels20. Furthermore, transection of the fimbria, the major afferent input to the hippocampus, results in a large loss of AChE 1s,27 and ChAc activities19. The other hippocampal afferents originating from the entorhinal cortex and contralateral hippocampus (commissural fibers) contain no ChAc or AChE 2s. These observations indicate that cholinergic fibers originate in the MS and travel principally in the fimbria to terminate on the pyramidal cells of the hippocampus and the granule cells of the dentate area. The present results support these findings since there is an increase in the release of ACh only when the MS is stimulated and this increase can be abolished by sectioning the fimbria. The supracallosal stria, dorsal fornix and alveus contribute AChE-containing fibers to the dorsal hippocampus 18. The lack of effect on ACh release after transecting these pathways shows that they are quantitatively unimportant sources for the areas sampled by the collecting cup. Electrophysiological studies in rabbits under chloralose-urethane anesthesia have shown that stimulation of the MS evoked potentials via possible monosynaptic pathways in both the dentate granule cells and the hippocampal CA1 pyramidal cells 1, 2. The granule cell potential is not abolished by sectioning the dorsal fornix, the alveus or the fimbria while the CA1 pyramidal cells potential is abolished by sectioning the alveus. These findings do not correlate with the findings of the present experiments in which the increase in ACh release due to MS stimulation was abolished by sectioning the fimbria. At the present time it is difficult to account for this difference unless one accepts that there are possibly two afferent pathways from the MS to the hippocampus; one cholinergic travelling principally through the fimbria and the other non-
132
s.D. DUDAR
cholinergic travelling in pathways other t h a n the fimbria. U n f o r t u n a t e l y , the bulk of the available histochemical a n d the a n a t o m i c a l data are from the rat so that there m a y be some species difference c o n c e r n i n g the a n a t o m i c a l c o n n e c t i o n s between the MS a n d the h i p p o c a m p a l formation. ACKNOWLEDGEMENTS This work has been s u p p o r t e d by the C a n a d i a n Medical Research Council. I would like to t h a n k J. C. Szerb for his interest a n d criticism a n d Lee H o p k i n s for her technical assistance, especially in the A C h bioassay.
REFERENCES 1 ANDERSEN,P., BRULAND, H., AND KAADA, B. R., Activation of the dentate area by septal stimulation, Acta physiol, scand., 51 (1961) 17-28. 2 ANDERSEN,P., BRULAND,H., AND KAADA,B. R., Activation of the field CA1 of the hippocampus by septal stimulation, Actaphysiol. scand., 51 (1961) 29-40. 3 AND',',O. J., AND STEPHAN, H., The Septum of the Cat, Thomas, Springfield, Ill., 1964. 4 BEANI,L., BIANCHI,C., SANTINOCETO,L., AND MARCHETTI,P., The cerebral acetylcholine release in conscious rabbits with semi-permanently implanted epidural cups, Int. J. Neuropharmacol., 7 (1968) 469-481. 5 BIRKS,R. I., AND MACINTOSH,F. C., Acetylcholine metabolism of a sympathetic ganglion, Canad. J. Biochem., 39 (1961) 787-827. 6 BISCOE,T. J., AND STRAUGHAN,D. W., Micro-electrophoretic studies of neurons in the cat hippocampus, J. Physiol. (Lend.), 183 (1966) 341-359. 7 BOURDOIS,P. S., MITCHELL, J. F., SOMOGYI,G. T., AND SZERB, J. C., The output per stimulus of acetylcholine from cerebral cortical slices in the presence or absence of cholinesterase inhibition, Brit. J. Pharmacol., 52 (1974), in press. 8 BROOKS,V. B., AND THINS, R. E., Reduction of quantum content during neuromuscular transmission, J. Physiol. (Lend.), 162 (1962) 298-310. 9 COLLIER,B., AND MITCHELL,J. F., The central release of acetylcholine during stimulation of the visual pathway, J. Physiol. (Lend.), 184 (1966) 239-254. l0 DUDAR,J. D., AND SZERB, J. C., The effect of topically applied atropine on resting and evoked cortical acetylcholine release, J. Physiol. (Lend.), 203 (1969) 74t-762. 11 DUDAR,J. D., AND SZERB, J. C., A. The push-puU cannula. B. Bioassay of acetyicholine on leech muscle suspended in a microbath, Exp. Physiol. Biochem., 3 (1970) 341-350. 12 FELDBERG, W., AND MALCOLM, J. L., Experiments on the site of action of tubocurarine when applied via .the cerebral ventricles, J. Physiol. (Lend.), 149 (1959) 58-77. 13 FONNUM,F., Topographical and subcellular localization of choline acetyltransferase in rat hippocampal formation, J. Neurochem., 77 (1970) 1029-1037. 14 GENESER-JENSEN,F. A., Distribution of acetylcholinesterase in the hippocampal region of the guinea pig, Z. Zellforsch., 124 (1972) 546-560. 15 HEMSWORTH,B. A., AND MITCHELL,J. F., The characteristics of acetylcholine release mechanisms in the auditory cortex, Brit. J. Pharmacol., 36 (1969) 161-170. 16 KRNJEVI~,K., AND MITCHELL,J. F., The release of acetylcholine in the isolated rat diaphragm, J. Physiol. (Lend.), 155 (1961) 246-262. 17 KUI~AR,J. J., SETI'IY,V. H., ROTH, R. H., AND AOHAJANIAN,G. K., Choline: selective accumulation by central cholinergic neurons, J. Neurochem., 20 (1973) 581-593. 18 LEWIS, P. R., AND SHUTE, C. C. O., The cholinergic limbic system: projection to hippocampal formation, medial corteX, nuclei of the ascending cholinergic reticular system and the subfornical organ and supra-optic crest, Brain, 90 (1967) 521-540. 19 LEWm,P. R., SHUTS, C. C. D., AND SILVER,A., Confirmation from choline acetylase analyses of a massive cholinergic innervation to the rat hippocampus, J. Physiol. (Lend.), 191 (1967) 215-224.
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20 MELLGREN,S. I., AND SREBRO, B., Changes in acetylcholinesterase and distribution of degenerating fibres in the hippocampal region after septal lesions in the rat, Brain Research, 52 (1973)19-36. 21 MITCHELL,J. F., The spontaneous and evoked release of acetylcholine from the cerebral cortex, J. Physiol. (Lond.), 165 (1963) 98-116. 22 PATON, W. n . M., Cholinergic transmission and acetylcholine output, Canad. J. Biochem., 41 (1963) 2637-2653. 23 PHILLIS, J. W., Acetylcholine release from the cerebral cortex: its role in cortical arousal, Brain Research, 7 (1968) 378-389. 24 SAWYER, C. H., EVERETT, J. W., AND GREEN, J. D., The rabbit diencephalon in stereotaxic coordinates, J. comp. Neurol., 101 (1954) 801-824. 25 SETHY, V. H., KUHAR, i . J., ROTH, R. H., VANWOERT, i . H., AND AGHAJANIAN,G. K., Cholinergic neurons: effect of acute septal lesion on acetylcholine and choline content of rat hippocampus, Brain Research, 55 (1973) 481-484. 26 SMITH, C. M., The release of acetylcholine from rabbit hippocampus, Brit. J. Pharmacol., 45 (1972) 172P. 27 STORM-MATHISEN,J., Quantitative neurochemistry of acetylcholinesterase in rat hippocampal region correlated to histochemical staining, J. Nearochem., 17 (1970) 739-750. 28 STORM-MATHISEN,J., Glutamate decarboxylase in the rat hippocampal region after lesions of the afferent fiber systems. Evidence that the enzyme is located in intrinsic neurones, Brain Research, 40 (1972) 215-235. 29 STORM-MATHISEN,J., AND BLACKSTAD,T. W., Cholinesterase in the hippocampai region, ,4cta anat. (Basel), 56 (1964) 216-253. 30 STRAUGHAN,D. W., The release of acetylcholine from mammalian motor nerve endings, Brit. J. Pharmacol., 15 (1960) 417-424. 31 URBAN, I., AND RICHARD, P., ,4 Stereotaxic Atlas of the New Zealand Rabbit's Brain, Thomas, Springfield, Ill., 1972.