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Activation of identified septo-hippocampal neurons by noxious peripheral stimulation
Brain Research, 328 (1985) 15-21
15
Elsevier BRE 10541
Activation of Identified Septo-Hippocampal Neurons by Noxious Peripheral Stimulation P. DUTAR, Y. LAMOUR and A. JOBERT Unit~ de Recherches de Neurophysiologie Pharmacologique de I'INSERM (U. 161), 2 rue d'Al~sia, 75014 Paris (France)
(Accepted May 22nd, 1984) Key words: rat - - septo-hippocampal neurons - - noxious mechanical stimulation - - noxious heat - - encoding
Septo-hippocampal neurons (SHNs) were recorded from the medial septum-diagonal band area of rats anaesthetized with either urethane or fluothane. They were identified by their antidromic response to the electrical stimulation of the fimbria. Their responses to peripheral somatic noxious and non-noxious stimulation were studied. Non-noxious natural stimulations were relatively ineffective. In contrast, 68% of the SHNs were driven by noxious stimulation. The SHNs could be driven either by mechanical or thermal stimulation. Intraperitoneal injection of bradykinin excited about half of the SHNs. Some neurons were able to encode stimulus intensity (strength of the mechanical stimulation and/or temperature of the thermal stimulation). The receptive fields of the SHNs were large, usually involving half of the body or the whole body surface. These results suggest that SHNs, which are at the origin of the cholinergic septo-hippocampal pathway, might be involved in cerebral mechanisms related to nociception. INTRODUCTION The cholinergic nature of the s e p t o - h i p p o c a m p a l pathway is s u p p o r t e d by n u m e r o u s e x p e r i m e n t a l data 14. W e showed previously that septo-hippocampal neurons (SHNs) can be identified by their antidromic response to the electrical stimulation of the fimbria t2. W e show in the present study, that a majority of such SHNs are responsive to p e r i p h e r a l somatic stimulation and specially to noxious stimulation. Previous studiesl,2,5,6,8,13,17,19,26,28,37,38 have shown that neurons at the origin of o t h e r diffusely projecting systems such as the n o r a d r e n e r g i c and serotoninergic systems do receive p e r i p h e r a l inputs and specially nociceptive information. O u r results on SHNs will be c o m p a r e d with those of other authors obtained in these systems. MATERIALS AND METHODS Male S p r a g u e - D a w l e y albino rats (220-320 g) were anaesthetized with either urethane (1.5 g/kg or 2 g/kg i.p.) or fluothane (halothane 0.75% in a mix-
ture of 1/3 02, 2/3 N20). Rats ventilated spontaneously under urethane anaesthesia whereas they were paralyzed with an i.v. infusion of gallamine triethiodide (Flaxedil) and artificially ventilated under fluothane. In the fluothane p r o c e d u r e the animals were maintained under d e e p anaesthesia during the surgical procedure ( 2 - 2 . 5 % halothane). The percentage of halothane was then r e d u c e d to 0.75% and maintained at this level during the recording period. The level of anaesthesia was systematically checked using an electrocorticogram. U n d e r these conditions, the E C o G recorded longitudinally by two silver ball electrodes on the dura m a t e r consisted of theta waves associated with a few spindles. Higher proportions of halothane induced slower high voltage activities with periods of depressed activities. H e a r t rate was continuously monitored. W e could thus d e t e r m i n e if the responses o~ SHNs to peripheral stimulation were d e p e n d e n t on the type of anaesthesia and if it was possible to observe responses to noxious stimulation without any arousal reaction on the electrocorticographic recording. Furt h e r m o r e , we also wanted to be able to c o m p a r e our
Correspondence: Y. Lamour, Unit6 de Recherches de Neurophysiologie Pharmacologique de I'INSERM (U.161), 2 rue d'Al6sia, 75014 Paris, France.
0006-8993/85/$03.30 (~ 1985 Elsevier Science Publishers B .V.
16 data with other studies of nociceptive responses in supraspinal structures, especially in the thalamus 16, the cerebral cortex 23 and raphe nuclei 17 obtained in the rat under the same experimental conditions. Central temperature was kept at 37.5 °C by a thermostatically regulated heating pad. Animals were placed in a stereotaxic frame. Two monopolar stimulating electrodes were positioned in the fimbria on each side of the midline at the coordinates A 6060, H + 1.5 of the atlas of KOnig and Klippe118. The basal forebrain was then exposed using a ventral approach. Extracellular single unit recordings were obtained from medial septal-nucleus of the diagonal band of Broca area (MS-nDBB) neurons. Conventional amplification and displaying methods were used. Electrodes were glass micropipettes filled with a mixture of 1 M NaC1 and 2% pontamine sky blue (impedance: 8-12 Mr2). Units were identified as antidromically driven by fimbria stimulation according to the following criteria: collision of the antidromic with orthodromic spikes, fixed latency and high frequency following. When a unit was isolated, its peripheral receptive field was characterized. The mechanical stimulations consisted of a systematic stimulation of various areas of the body, by brushing, rubbing or pinching. Once located, the peripheral receptive field (RF) was examined to determine what kind of stimulation drove the neuron, by using a brush, by stroking or tapping, pressing with a blunt wooden probe, moving joints and finally pinching with surgical forceps (as strong as necessary to obtain a noxious sensation when applied to the skin of the investigator). When a unit was excited by a noxious stimulus, its response to thermal
stimuli was investigated using a hot water bath following a procedure described elsewhere in detail 29. Incrementally increasing temperatures were tested (45, 48, 50, 53, 55 °C). Stimulations were separated by at least 3 min intervals. The standard stimulus was the immersion of the tail in a water bath for 15 s. The vascularization of the peripheral cutaneous receptive fields was checked by observing the colour of the extremities and their ability to return quickly to the previous state after application of pressure or heat. The complete sequence of noxious heat applications was used only once in a given animal, in order to avoid damage to the tissues and/or sensitization of the receptors. The final recording site of each penetration was marked with a dye deposit (20 # A for 20 rain). At the end of the experiment, the animal was perfused through the heart with saline followed by a solution of 10% formaldehyde in saline. Frozen 100/~m thick sections of the whole brain were cut and stained with cresyl violet. Electrode penetrations were reconstructed on camera lucida drawings of these sections.
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/ TABLE I Proportion o f SHNs driven by noxious or non-noxious stimulations, under different conditions o f anaesthesia Number o f neurons tested
Urethane (1.5 g/kg) Urethane (2 g/kg) Fluothane Total
Responses to intense mechanical stimulation
Responses to light cutaneous stimulation
58
37 (64%)
7 (12%)
23 50 131
20 (87%) 32 (54%) 89 (68%)
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Fig. 1. Distribution in the medial septum-diagonal band area of septo-hippocampal neurons (identified by their antidromic response to fimbrial stimulation) which were driven by peripheral noxious stimulation (filled circles). All response types have been included (i.e. excitations, inhibition and triggering of the bursting activity). Data from experiments under urethane and fluothane have been pooled. Two antero-posterior levels of the septal area are shown (ant, anterior; post, posterior). CC, corpus callosum; V, ventricle; CP, caudate-putamen; LS, lateral septum; IC, Island of Calleja; Ac, nucleus accumbens; CA, commissura anterior.
17 LH
RH
T
LF 50
A CH
T
IF
IF
IH 25
El lmin
Fig. 2. Response of two septo-hippocampal neurons (SHNs), recorded under urethane anaesthesia (1.5 g/kg i.p.), to peripheral noxious stimulation. Vertical bar: number of action potentials/s. A: this SHN was located on the midline of the septum. It was excited by a noxious pinch applied to the left hindpaw (LH), right hindpaw (RH) and tail (T), but not to the left forepaw (LF). B: SHN located in the left medial septum excited by a noxious pinch applied on contralateral hindpaw (CH), tail (T), ipsilateral forepaw (IF) and hindpaw (IH). Notice that the second response to IF pinch was much larger than the first one. This was due to the fact that the second stimulation was stronger than the first, indicating some ability of this neuron to encode stimulus intensity.
In some experiments (n = 16) an intraperitoneal catheter was used for bradykinin injection (10/~g in 1 ml saline). RESULTS A total of 155 SHNs were r e c o r d e d from 38 animals: under fluothane (n = 50) or urethane (1.5 g/kg: n = 58, 2 g/kg: n = 47) anaesthesia.
Characteristics of the SHNs SHNs were antidromically driven from the fimbria with a mean latency of 1.46 + 0.1 ( S . E . M . ) ms (n = 155, range 0.4-11 ms). Their mean spontaneous firing frequency was 14 + 1 impulses/s (urethane anaesthesia) and 18.3 + 3 impulses/s (fluothane anaesthesia).
Nature of responses A m a j o r i t y of SHNs (68%), regardless of the conditions of anaesthesia, were driven by natural peripheral stimulation. In most of the cases the units were not driven by light cutaneous stimulation such as hair movements or light taps but only by strong, noxious mechanical or thermal stimulations (Table I). No segregation of the SHNs driven by noxious stimulation was observed in the M S - n D B B area (Fig. 1). The neurons were evenly distributed in the MS and vertical limb of the n D B B .
The nature of the stimulus driving the SHNs was difficult to define, i.e. it could not be clearly identified as superficial or deep (with the exception of a few SHNs driven by non-noxious cutaneous stimulation, see below). The most efficient stimulus was usually a pinch applied on the paws and/or the tail (Figs. 2, 3). Nine neurons were driven by non-noxious stimulation. In 5 cases these neurons were also driven by noxious stimulation (Fig. 3), whereas in 4 cases they were not. Unidentified neurons in the M S - n D B B area were less often responsive to peripheral stimulation: only 50% of them could be driven from the periphery. F u r t h e r m o r e the responses were often w e a k and/or poorly reproducible.
Topography of the receptive fields The most frequent receptive field o b s e r v e d comprised the two hindlimbs and the tail (45%). The next frequent type of R F comprised the whole body (38%). RFs located on the ipsi- or contralateral hindlimb were much less frequent (14%). R F s restricted to the tail were rare (1 case). Interestingly whole body R F s were more frequent under fluothane anaesthesia, whereas RFs restricted to the caudal part of the body were more frequent u n d e r urethane anaesthesia. The t o p o g r a p h y of the few R F s of neurons which were driven by non-noxious stimulation a p p e a r e d to be similar to those of neurons driven by noxious stimulation.
18 IH
IF
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CF
25
I 46 °
50*
54 °
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min
Fig. 3. Responses of a SHN (urethane 2 g/kg i.p.) to noxious stimulation (pinch) applied to ipsilateral hindpaw (IH), forepaw (IF), tail (T) and contralateral forepaw (CF). Notice that this neuron was not driven by forepaw stimulation. Non-noxious stimulation (LT, light touch) applied on the caudal part of the body was also effective. This neuron was also excited by a hot water bath applied on the tail: there was a slight excitation at 46 °C, and stronger responses at 50 and 54 °C. Notice the progressive increase in the amplitude and the duration of the response with increasing temperatures. The regression curve calculated for these responses to noxious thermal stimulation was linear (r = 1, P < 0.05, see Fig. 5).
Types of responses to peripheral stimulation Mechanical stimulation. In most of the cases the n e u r o n a l r e s p o n s e to p e r i p h e r a l s t i m u l a t i o n was an excitation. In a few cases (8% of the S H N s r e s p o n sive to p e r i p h e r a l s t i m u l a t i o n ) , an i n h i b i t i o n was observed (Fig. 4). In s o m e o t h e r cases an initial excitation was followed by inhibition. In a b o u t o n e - t h i r d of the cases (27 out of 81 S H N s u n d e r u r e t h a n e anaesthesia), the r e s p o n s e was associated with the accent u a t i o n or the a p p e a r a n c e of a r h y t h m i c b u r s t i n g activity at 4/s. In most of the cases the responses to in-
CF
Brady i.p.
tense m e c h a n i c a l s t i m u l a t i o n were of the tonic type a n d often outlasted the p e r i o d of s t i m u l a t i o n (Fig. 2). R e s p o n s e s lasting for 1 or 2 m i n were n o t rare, following a 15 s stimulation. P r o l o n g e d post-effects could s o m e t i m e s be observed. I n c r e a s i n g the strength of the m e c h a n i c a l s t i m u l a t i o n resulted in a n increase in the a m p l i t u d e a n d d u r a t i o n of the response (Fig. 2). These responses could be observed in the absence of a n y E C o G change.
Thermal stimulation. F o r t y S H N s were tested for their r e s p o n s e to t h e r m a l s t i m u l a t i o n (hot water b a t h 51°
54 °
T
48 °
25
I 1 min
Fig. 4. SHN inhibited by noxious stimulation (fluothane anaesthesia). This SHN was inhibited by noxious pinch applied to the contralateral forelimb (CF), tail (T), as well as bradykinin i.p. injection and application of hot water on the tail. Notice the very long inhibition following the application of 51 °C hot water on the tail.
19 applied on the tail). The SHNs studied were not responsive to non-noxious thermal stimulation. The threshold for response was between 46 °C and 48 °C. Responses were excitatory in 29 cases (72%) (Fig. 3), sometimes followed by an inhibition and inhibitory in 3 cases (Fig. 4). No responses were observed in 8 cases. Thus the majority (80%) of the SHNs tested were driven by noxious thermal stimulation. In 20 cases in which several values of noxious temperatures were tested (18 excitations, 2 inhibitions), a parallel increase in the number of spikes with increasing water temperature was observed in 7 cases and a regular decrease with increasing temperatures was observed in 1 case. Among these 8 neurons (Fig. 5), a linear relationship (r = 1, P < 0.05, linear regression analysis) could be calculated in 4 cases. The relationship was non-linear in the other 4 cases (r < 1). In a few cases a second series of stimulations with increasing temperatures were applied. In these cases some evidence of sensitization was obtained.
Noxious visceral stimulation by intraperitoneal bradykinin injections Bradykinin (10/~g in 1 ml saline) was injected intraperitoneally in 16 cases. Neuronal excitation was observed in 7 cases, inhibition in 2 cases and no effect in 7 cases. The responses to bradykinin were relatively long (1 to several minutes).
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Fig. 5. Relationships between temperature of the hot water bath applied on the tail and the number of spikes in the neuronal responses. Spikes were counted over a 2 min period following the beginning of the stimulation (15 s). A: 4 SHNs whose relationship was linear (r = 1, P < 0.05). Curves were traced by hand and do not correspond to the regression lines equations. B: 4 SHNs whose relationship was not linear, though these neurons displayed a regular relation between responses intensity and stimulus intensity.
DISCUSSION
Our results provide evidence that septo-hippocampal, presumably cholinergic, neurons are responsive to peripheral, especially noxious, stimulation. Responses of neurons to stimulation in the noxious range have already been reported in locus coeruleUS 8"19"28"37, raphe nuclei 1,2,17,26 and substantia nigra 5.38. The responses of SHNs to noxious stimulation seem to be quite different from those reported in the locus coeruleus (LC): Cedarbaum and Aghajanian s observed that all LC units studied responded to noxious stimuli applied anywhere on the body and that non-noxious stimuli were ineffective. Furthermore, they were able to drive LC neurons easily by sciatic nerve stimulation. In contrast, we observed 68% of responses to noxious stimulation among SHNs with some topographic organization of the RFs, and sciatic nerve stimulation was quite ineffective (Lamour et al., unpublished observations), Results obtained in the raphe nuclei and substantia nigra by several authors are somewhat contradictory: some authors reported a majority of excitatory responses 2.5.6,13,17.26, whereas others described only inhibitory responses 38, especially in presumed serotonergic dorsal raphe neurons I. We observed a prevalence of excitatory responses to peripheral noxious stimulation in SHNs. Most of the RFs were large, including the caudal part of the body or the whole body. Such large RFs were also observed in the raphe nuclei z.17. That septal neurons are sensitive to somatic stimulation was known for a long time 3,4.30. These authors observed that somatosensory stimulation could elicit bursting activity in the septum. Similarly the hippocampal theta rhythm can also be triggered by peripheral stimulation 15. However, no study dealing specifically with the effect of peripheral stimulation on identified septal neurons was available. An evoked release of acetylcholine (ACh) from the cerebral cortex following peripheral nerves stimulation has been reported by Mitchell z5 and Mullins and Phillis zT. Dudar et al. II observed that the release of ACh from the hippocampus was increased during sensory stimulation and running. The type of sensory stimulation used was in the non-noxious range. Therefore the septo-hippocampal pathway might be more respon-
20 sive to light mechanical stimulation in awake animals. They also report that the increased release of ACh elicited by sensory stimulation was blocked by systemic administration of atropine in animals anaesthetized with urethane but not in freely moving animals. These observations suggest that the cholinergic septo-hippocampal system is responsive to peripheral stimulation and that this response is, under certain conditions, atropine-sensitive. They are consistent with our results showing that SHNs are driven by peripheral stimulation. Our results show that, under our experimental conditions, non-noxious stimulation were relatively ineffective in evoking responses in SHNs. One of the main peripheral input to SHNs might therefore be a noxious input. These neuronal responses do not seem to be related to unspecific effects such as a general arousal reaction, since they could be recorded in the absence of any cortical arousal reaction. However, we cannot exclude that SHNs would be responsive to non-noxious peripheral stimulation in awake animals. A wider range of stimulation could indeed be effective in the absence of anaesthesia. An important piece of data shown by our experiments is that some SHNs were able to code the intensity of the noxious stimulus (see Figs. 2 and 5). This suggests that the response of SHNs to noxious stimulation is functionally important and that the output of SHNs in response to such stimulation is graded. Such an ability to encode the intensity of the noxious stimulation has been observed in other supraspinal structures, e.g. thalamus 29 and cerebral cortex 23. However, the responses of SHNs to peripheral stimulation were not similar to those observed in the ventrobasal nucleus of the thalamus 16 or cerebral cortex23: very few SHNs were driven by light tactile stimulation; no small, contralateral, somatotopically organized RFs were observed. These obvious differences suggest that SHNs and thalamic or cortical responses to peripheral stimulation subserve different functions. Responses to noxious stimulation were of two main types: (i) increase in discharge frequency, and (ii) appearance or accentuation of the rhythmic bursting activity. These two types of responses could
be associated (e.g. increase in discharge frequency and appearance of the bursting activity). The origin of the bursting activity in the septum is unknown. We observed that this type of activity is resistant to various pharmacological substances 22. This activity is, however, dependent on the anaesthesia used 20,33. The present data indicate that peripheral stimulation is able to trigger or modify the bursting activity, suggesting that it is at least in part under control of extraneous influences. The afferent projections to the MS-nDBB are only partially known. This area apparently receives projections from the lateral preoptic-lateral hypothalamic area 34.36. It also receives inputs from brainstem aminergic neurons 4,32 (see also references in Costa et al., 1983) 9. Direct and/or indirect influences are.also exerted by other structures, especially hippocampus 24.31,35. One could hypothesize that the responses of SHNs to noxious peripheral stimulation are due to the input coming from locus coeruleus, raphe nuclei, and/or other brainstem structures. However, it does not seem to be so simple since the properties of locus coeruleus or dorsal raphe neurons responses to noxious stimulation are rather different from those we observed in SHNs 1,s. The predominance among SHNs of excitatory responses to peripheral noxious stimulation suggests that the activity of SHNs is increased during such stimulation. Since the septo-hippocampal pathway is known to be a cholinergic pathway (though probably not all SHNs are cholinergic), it follows that this cholinergic pathway is stimulated during noxious stimulation. This would probably result in an increased release of ACh in the hippocampus. Since the prominent effect of ACh in the hippocampus is to facilitate pyramidal cells firing by various mechanisms7.10, 21, it follows that noxious stimulation should result in an increased activity of hippocampal neurons.
ACKNOWLEDGEMENTS We thank J. Carrou6, M. Cayla and E. Dehausse for technical assistance.
21 REFERENCES 1 Aghajanian, G. K., Wang, R. Y. and Baraban, J., Serotonergic and non-serotonergic neurons of the dorsal raphe: reciprocal changes in firing induced by peripheral nerve stimulation, Brain Research, 153 (1978) 169-175. 2 Anderson, S. D., Basbaum, A. I. and Fields, H. L., Response of medullary raphe neurons to peripheral stimulation and to systemic opiates, Brain Research, 123 (1977) 363-368. 3 Apostol, G. and Creutzfeldt, O. D., Crosscorrelation between the activity of septal units and hippocampal EEG during arousal, Brain Research, 67 (1974) 65-75. 4 Assaf, S. Y. and Miller, J. J., The role of a raphe serotonin system in the control of septal unit activity and hippocampal desynchronization, Neuroscience, 3 (1978) 539-550. 5 Barasi, S., Responses of substantia nigra neurones to noxious stimulation, Brain Research, 171 (1979) 121-130. 6 Behbehani, M. M. and Pomeroy, S. L., Effect of morphine injected in periaqueductal gray on the activity of single units in nucleus raphe magnus of the rat, Brain Research, 149 (1978) 266-269. 7 Ben-Aft, Y., Krnjevi6, K., Reinhardt, W. and Ropert, N., Intracellular observations on the disinhibitory action of acetylcholine in the hippocampus, Neuroscience, 6 (1981) 2475-2484. 8 Cedarbaum, J. M. and Aghajanian, G. K., Activation of locus coeruleus neurons by peripheral stimuli: modulation by a collateral inhibitory mechanism, Life Sci., 23 (1978) 1383-1392. 9 Costa, E., Panula, P., Thompson, H. K. and Cheney, D. L., The transsynaptic regulation of the septal-hippocampal cholinergic neurons, Life Sci., 32 (1983) 165-179. 10 Dodd, J., Dingledine, R. and Kelly, J. S., The excitatory action of acetylcholine on hippocampal neurones of the guinea pig and rat maintained in vitro, Brain Research, 207 (1981) 109-127. 11 Dudar, J. D., Whishaw, I. Q. and Szerb, J. C., Release of acetylcholine from the hippocampus of freely moving rats during sensory stimulation and running, Neuropharmacology, 18 (1979) 673-678. 12 Dutar, P., Lamour, Y. and Jobert, A., Acetylcholine excites identified septo-hippocampal neurons in the rat, Neurosci. Lett., 43 (1983) 43-47. 13 Feger, J., Jacquemin, J. and Ohye, C., Peripheral excitatory input to substantia nigra, Exp. Neurol., 59 (1978) 351-360. 14 Fibiger, H. C., The organization and some projections of cholinergic neurons of the mammalian forebrain, Brain Res. Rev., 4 (1982) 327-388. 15 Green, J. D. and Arduini, A. A., Hippocampal electrical activity in arousal, J. Neurophysiol., 17 (1954) 533-557. 16 Guilbaud, G., Peschanski, M., Gautron, M. and Binder, D., Neurones responding to noxious stimulation in VB complex and caudal adjacent regions in the thalamus of the rat, Pain, 8 (1980) 303-318. 17 Guilbaud, G., Peschanski, M., Gautron, M. and Binder, D., Responses of neurons of the nucleus raphe magnus to noxious stimuli, Neurosci. Lett., 17 (1980) 149-154. 18 K6nig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 19 Korf, J., Bunney, B. S. and Aghajanian, G. K., Noradrenergic neurons: morphine inhibition of spontaneous activity, Europ. J. Pharmacol., 25 (1974) 165-169. 20 Kramis, R., Vanderwolf, C. H. and Bland, B. H., Two types of hippocampal rhythmical slow activity in both the
rabbit and the rat: relations to behavior and effects of atropine, diethyl ether, urethane and pentobarbital, Exp. Neurol., 49 (1975) 58-85. 21 Krnjevi~, K., Reiffenstein, R. J. and Ropert, N., Disinhibitory action of acetylcholine in the rat's hippocampus: extracellular observations, Neuroscience, 6 (1981) 2465-2474. 22 Lamour, Y., Dutar, P. and Jobert, A., Septo-hippocampal and other medial septum-diagonal band neurons: electrophysiological and pharmacological properties, Brain Research, 309 (1984) 227-239. 23 Lamour, Y., Wilier, J. C. and Guilbaud, G., Rat somatosensory (SmI) cortex: I. Characteristics of neuronal responses to noxious stimulation and comparison with responses to non-noxious stimulation, Exp. Brain Res., 49 (1983) 35-45. 24 McLennan, H. and Miller, J. J., The hippocampal control of neuronal discharges in the septum of the rat, J. Physiol. (Lond.), 237 (1974) 607-624. 25 Mitchell, J. F., The spontaneous and evoked release of acetylcholine from the cerebral cortex, J. Physiol. (Lond.), 165 (1963) 98-116. 26 Moolenaar, G. M., Holloway, J. A. and Trouth, C. O., Responses of caudal raphe neurons to peripheral somatic stimulation, Exp. Neurol., 53 (1976) 304-313. 27 Mullins, W. J. and Phillis, J. W., The effect of graded forelimb afferent volleys on acetylcholine release from cat sensorimotor cortex, J. Physiol. (Lond.), 244 (1975) 741-756. 28 Nakamura, S., Some electrophysiological properties of neurones of rat locus coeruleus. J. Physiol. (Lond.), 267 (1977) 641-658. 29 Peschanski, M., Guilbaud, G., Gautron, M. and Besson, J. M., Encoding of noxious heat messages in neurons of the ventrobasal thalamic complex of the rat, Brain Research, 197 (1980) 401-413. 30 Petsche, H., Stumpf, C. and Gogolak, G., The significance of the rabbit's septum as a relay station between the midbrain and the hippocampus. 1. The control of hippocampus arousal activity by the septum cells, Electroenceph. clin. Neurophysiol., 14 (1962) 202-211. 31 Raisman, G., The connexions of the septum, Brain, 89 (1966) 317-348. 32 Segal, M., Brain stem afferents to the rat medial septum, J. Physiol. (Lond.), 261 (1976) 617-631. 33 Stumpf, C., Petsche, H. and Gogolak, G., The significance of the rabbit's septum as a relay station between the midbrain and the hippocampus. II. The differential influence of drugs upon both the septal cell firing pattern and the hippocampus theta activity, Electroenceph. clin. Neurophysiol., 14 (1962) 212-219. 34 Swanson, L. W., An autoradiographic study of the efferent connections of the preoptic region in the rat, J. comp. Neurol., 167 (1976) 227-256. 35 Swanson, L. W., The anatomy of the septo-hippocampal pathway. In S. Corkin, K. L. Davis, J. H. Growdon, E. Usdin and R. J. Wurtman (Eds.), Alzheimer's Disease: A Report of Progress in Research. Raven Press, New York, 1982, pp. 207-212. 36 Swanson, L. W. and Cowan, W. M., The connections of the septal region in the rat. J. comp. Neurol., 186 (1979) 621-656. 37 Takigawa. M. and Mogenson, G. J., A study of inputs to antidromically identified neurons of the locus coeruleus. Brain Research, 135 (1977) 217-230. 38 Tsai. C. T., Nakamura, S. and Iwama, K., Inhibition of neuronal activity of the substantia nigra by noxious stimuli and its modification by the caudate nucleus, Brain Research, 195 (1980) 299- 311.
15
Elsevier BRE 10541
Activation of Identified Septo-Hippocampal Neurons by Noxious Peripheral Stimulation P. DUTAR, Y. LAMOUR and A. JOBERT Unit~ de Recherches de Neurophysiologie Pharmacologique de I'INSERM (U. 161), 2 rue d'Al~sia, 75014 Paris (France)
(Accepted May 22nd, 1984) Key words: rat - - septo-hippocampal neurons - - noxious mechanical stimulation - - noxious heat - - encoding
Septo-hippocampal neurons (SHNs) were recorded from the medial septum-diagonal band area of rats anaesthetized with either urethane or fluothane. They were identified by their antidromic response to the electrical stimulation of the fimbria. Their responses to peripheral somatic noxious and non-noxious stimulation were studied. Non-noxious natural stimulations were relatively ineffective. In contrast, 68% of the SHNs were driven by noxious stimulation. The SHNs could be driven either by mechanical or thermal stimulation. Intraperitoneal injection of bradykinin excited about half of the SHNs. Some neurons were able to encode stimulus intensity (strength of the mechanical stimulation and/or temperature of the thermal stimulation). The receptive fields of the SHNs were large, usually involving half of the body or the whole body surface. These results suggest that SHNs, which are at the origin of the cholinergic septo-hippocampal pathway, might be involved in cerebral mechanisms related to nociception. INTRODUCTION The cholinergic nature of the s e p t o - h i p p o c a m p a l pathway is s u p p o r t e d by n u m e r o u s e x p e r i m e n t a l data 14. W e showed previously that septo-hippocampal neurons (SHNs) can be identified by their antidromic response to the electrical stimulation of the fimbria t2. W e show in the present study, that a majority of such SHNs are responsive to p e r i p h e r a l somatic stimulation and specially to noxious stimulation. Previous studiesl,2,5,6,8,13,17,19,26,28,37,38 have shown that neurons at the origin of o t h e r diffusely projecting systems such as the n o r a d r e n e r g i c and serotoninergic systems do receive p e r i p h e r a l inputs and specially nociceptive information. O u r results on SHNs will be c o m p a r e d with those of other authors obtained in these systems. MATERIALS AND METHODS Male S p r a g u e - D a w l e y albino rats (220-320 g) were anaesthetized with either urethane (1.5 g/kg or 2 g/kg i.p.) or fluothane (halothane 0.75% in a mix-
ture of 1/3 02, 2/3 N20). Rats ventilated spontaneously under urethane anaesthesia whereas they were paralyzed with an i.v. infusion of gallamine triethiodide (Flaxedil) and artificially ventilated under fluothane. In the fluothane p r o c e d u r e the animals were maintained under d e e p anaesthesia during the surgical procedure ( 2 - 2 . 5 % halothane). The percentage of halothane was then r e d u c e d to 0.75% and maintained at this level during the recording period. The level of anaesthesia was systematically checked using an electrocorticogram. U n d e r these conditions, the E C o G recorded longitudinally by two silver ball electrodes on the dura m a t e r consisted of theta waves associated with a few spindles. Higher proportions of halothane induced slower high voltage activities with periods of depressed activities. H e a r t rate was continuously monitored. W e could thus d e t e r m i n e if the responses o~ SHNs to peripheral stimulation were d e p e n d e n t on the type of anaesthesia and if it was possible to observe responses to noxious stimulation without any arousal reaction on the electrocorticographic recording. Furt h e r m o r e , we also wanted to be able to c o m p a r e our
Correspondence: Y. Lamour, Unit6 de Recherches de Neurophysiologie Pharmacologique de I'INSERM (U.161), 2 rue d'Al6sia, 75014 Paris, France.
0006-8993/85/$03.30 (~ 1985 Elsevier Science Publishers B .V.
16 data with other studies of nociceptive responses in supraspinal structures, especially in the thalamus 16, the cerebral cortex 23 and raphe nuclei 17 obtained in the rat under the same experimental conditions. Central temperature was kept at 37.5 °C by a thermostatically regulated heating pad. Animals were placed in a stereotaxic frame. Two monopolar stimulating electrodes were positioned in the fimbria on each side of the midline at the coordinates A 6060, H + 1.5 of the atlas of KOnig and Klippe118. The basal forebrain was then exposed using a ventral approach. Extracellular single unit recordings were obtained from medial septal-nucleus of the diagonal band of Broca area (MS-nDBB) neurons. Conventional amplification and displaying methods were used. Electrodes were glass micropipettes filled with a mixture of 1 M NaC1 and 2% pontamine sky blue (impedance: 8-12 Mr2). Units were identified as antidromically driven by fimbria stimulation according to the following criteria: collision of the antidromic with orthodromic spikes, fixed latency and high frequency following. When a unit was isolated, its peripheral receptive field was characterized. The mechanical stimulations consisted of a systematic stimulation of various areas of the body, by brushing, rubbing or pinching. Once located, the peripheral receptive field (RF) was examined to determine what kind of stimulation drove the neuron, by using a brush, by stroking or tapping, pressing with a blunt wooden probe, moving joints and finally pinching with surgical forceps (as strong as necessary to obtain a noxious sensation when applied to the skin of the investigator). When a unit was excited by a noxious stimulus, its response to thermal
stimuli was investigated using a hot water bath following a procedure described elsewhere in detail 29. Incrementally increasing temperatures were tested (45, 48, 50, 53, 55 °C). Stimulations were separated by at least 3 min intervals. The standard stimulus was the immersion of the tail in a water bath for 15 s. The vascularization of the peripheral cutaneous receptive fields was checked by observing the colour of the extremities and their ability to return quickly to the previous state after application of pressure or heat. The complete sequence of noxious heat applications was used only once in a given animal, in order to avoid damage to the tissues and/or sensitization of the receptors. The final recording site of each penetration was marked with a dye deposit (20 # A for 20 rain). At the end of the experiment, the animal was perfused through the heart with saline followed by a solution of 10% formaldehyde in saline. Frozen 100/~m thick sections of the whole brain were cut and stained with cresyl violet. Electrode penetrations were reconstructed on camera lucida drawings of these sections.
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Urethane (1.5 g/kg) Urethane (2 g/kg) Fluothane Total
Responses to intense mechanical stimulation
Responses to light cutaneous stimulation
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Fig. 1. Distribution in the medial septum-diagonal band area of septo-hippocampal neurons (identified by their antidromic response to fimbrial stimulation) which were driven by peripheral noxious stimulation (filled circles). All response types have been included (i.e. excitations, inhibition and triggering of the bursting activity). Data from experiments under urethane and fluothane have been pooled. Two antero-posterior levels of the septal area are shown (ant, anterior; post, posterior). CC, corpus callosum; V, ventricle; CP, caudate-putamen; LS, lateral septum; IC, Island of Calleja; Ac, nucleus accumbens; CA, commissura anterior.
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Fig. 2. Response of two septo-hippocampal neurons (SHNs), recorded under urethane anaesthesia (1.5 g/kg i.p.), to peripheral noxious stimulation. Vertical bar: number of action potentials/s. A: this SHN was located on the midline of the septum. It was excited by a noxious pinch applied to the left hindpaw (LH), right hindpaw (RH) and tail (T), but not to the left forepaw (LF). B: SHN located in the left medial septum excited by a noxious pinch applied on contralateral hindpaw (CH), tail (T), ipsilateral forepaw (IF) and hindpaw (IH). Notice that the second response to IF pinch was much larger than the first one. This was due to the fact that the second stimulation was stronger than the first, indicating some ability of this neuron to encode stimulus intensity.
In some experiments (n = 16) an intraperitoneal catheter was used for bradykinin injection (10/~g in 1 ml saline). RESULTS A total of 155 SHNs were r e c o r d e d from 38 animals: under fluothane (n = 50) or urethane (1.5 g/kg: n = 58, 2 g/kg: n = 47) anaesthesia.
Characteristics of the SHNs SHNs were antidromically driven from the fimbria with a mean latency of 1.46 + 0.1 ( S . E . M . ) ms (n = 155, range 0.4-11 ms). Their mean spontaneous firing frequency was 14 + 1 impulses/s (urethane anaesthesia) and 18.3 + 3 impulses/s (fluothane anaesthesia).
Nature of responses A m a j o r i t y of SHNs (68%), regardless of the conditions of anaesthesia, were driven by natural peripheral stimulation. In most of the cases the units were not driven by light cutaneous stimulation such as hair movements or light taps but only by strong, noxious mechanical or thermal stimulations (Table I). No segregation of the SHNs driven by noxious stimulation was observed in the M S - n D B B area (Fig. 1). The neurons were evenly distributed in the MS and vertical limb of the n D B B .
The nature of the stimulus driving the SHNs was difficult to define, i.e. it could not be clearly identified as superficial or deep (with the exception of a few SHNs driven by non-noxious cutaneous stimulation, see below). The most efficient stimulus was usually a pinch applied on the paws and/or the tail (Figs. 2, 3). Nine neurons were driven by non-noxious stimulation. In 5 cases these neurons were also driven by noxious stimulation (Fig. 3), whereas in 4 cases they were not. Unidentified neurons in the M S - n D B B area were less often responsive to peripheral stimulation: only 50% of them could be driven from the periphery. F u r t h e r m o r e the responses were often w e a k and/or poorly reproducible.
Topography of the receptive fields The most frequent receptive field o b s e r v e d comprised the two hindlimbs and the tail (45%). The next frequent type of R F comprised the whole body (38%). RFs located on the ipsi- or contralateral hindlimb were much less frequent (14%). R F s restricted to the tail were rare (1 case). Interestingly whole body R F s were more frequent under fluothane anaesthesia, whereas RFs restricted to the caudal part of the body were more frequent u n d e r urethane anaesthesia. The t o p o g r a p h y of the few R F s of neurons which were driven by non-noxious stimulation a p p e a r e d to be similar to those of neurons driven by noxious stimulation.
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Fig. 3. Responses of a SHN (urethane 2 g/kg i.p.) to noxious stimulation (pinch) applied to ipsilateral hindpaw (IH), forepaw (IF), tail (T) and contralateral forepaw (CF). Notice that this neuron was not driven by forepaw stimulation. Non-noxious stimulation (LT, light touch) applied on the caudal part of the body was also effective. This neuron was also excited by a hot water bath applied on the tail: there was a slight excitation at 46 °C, and stronger responses at 50 and 54 °C. Notice the progressive increase in the amplitude and the duration of the response with increasing temperatures. The regression curve calculated for these responses to noxious thermal stimulation was linear (r = 1, P < 0.05, see Fig. 5).
Types of responses to peripheral stimulation Mechanical stimulation. In most of the cases the n e u r o n a l r e s p o n s e to p e r i p h e r a l s t i m u l a t i o n was an excitation. In a few cases (8% of the S H N s r e s p o n sive to p e r i p h e r a l s t i m u l a t i o n ) , an i n h i b i t i o n was observed (Fig. 4). In s o m e o t h e r cases an initial excitation was followed by inhibition. In a b o u t o n e - t h i r d of the cases (27 out of 81 S H N s u n d e r u r e t h a n e anaesthesia), the r e s p o n s e was associated with the accent u a t i o n or the a p p e a r a n c e of a r h y t h m i c b u r s t i n g activity at 4/s. In most of the cases the responses to in-
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Brady i.p.
tense m e c h a n i c a l s t i m u l a t i o n were of the tonic type a n d often outlasted the p e r i o d of s t i m u l a t i o n (Fig. 2). R e s p o n s e s lasting for 1 or 2 m i n were n o t rare, following a 15 s stimulation. P r o l o n g e d post-effects could s o m e t i m e s be observed. I n c r e a s i n g the strength of the m e c h a n i c a l s t i m u l a t i o n resulted in a n increase in the a m p l i t u d e a n d d u r a t i o n of the response (Fig. 2). These responses could be observed in the absence of a n y E C o G change.
Thermal stimulation. F o r t y S H N s were tested for their r e s p o n s e to t h e r m a l s t i m u l a t i o n (hot water b a t h 51°
54 °
T
48 °
25
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Fig. 4. SHN inhibited by noxious stimulation (fluothane anaesthesia). This SHN was inhibited by noxious pinch applied to the contralateral forelimb (CF), tail (T), as well as bradykinin i.p. injection and application of hot water on the tail. Notice the very long inhibition following the application of 51 °C hot water on the tail.
19 applied on the tail). The SHNs studied were not responsive to non-noxious thermal stimulation. The threshold for response was between 46 °C and 48 °C. Responses were excitatory in 29 cases (72%) (Fig. 3), sometimes followed by an inhibition and inhibitory in 3 cases (Fig. 4). No responses were observed in 8 cases. Thus the majority (80%) of the SHNs tested were driven by noxious thermal stimulation. In 20 cases in which several values of noxious temperatures were tested (18 excitations, 2 inhibitions), a parallel increase in the number of spikes with increasing water temperature was observed in 7 cases and a regular decrease with increasing temperatures was observed in 1 case. Among these 8 neurons (Fig. 5), a linear relationship (r = 1, P < 0.05, linear regression analysis) could be calculated in 4 cases. The relationship was non-linear in the other 4 cases (r < 1). In a few cases a second series of stimulations with increasing temperatures were applied. In these cases some evidence of sensitization was obtained.
Noxious visceral stimulation by intraperitoneal bradykinin injections Bradykinin (10/~g in 1 ml saline) was injected intraperitoneally in 16 cases. Neuronal excitation was observed in 7 cases, inhibition in 2 cases and no effect in 7 cases. The responses to bradykinin were relatively long (1 to several minutes).
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Fig. 5. Relationships between temperature of the hot water bath applied on the tail and the number of spikes in the neuronal responses. Spikes were counted over a 2 min period following the beginning of the stimulation (15 s). A: 4 SHNs whose relationship was linear (r = 1, P < 0.05). Curves were traced by hand and do not correspond to the regression lines equations. B: 4 SHNs whose relationship was not linear, though these neurons displayed a regular relation between responses intensity and stimulus intensity.
DISCUSSION
Our results provide evidence that septo-hippocampal, presumably cholinergic, neurons are responsive to peripheral, especially noxious, stimulation. Responses of neurons to stimulation in the noxious range have already been reported in locus coeruleUS 8"19"28"37, raphe nuclei 1,2,17,26 and substantia nigra 5.38. The responses of SHNs to noxious stimulation seem to be quite different from those reported in the locus coeruleus (LC): Cedarbaum and Aghajanian s observed that all LC units studied responded to noxious stimuli applied anywhere on the body and that non-noxious stimuli were ineffective. Furthermore, they were able to drive LC neurons easily by sciatic nerve stimulation. In contrast, we observed 68% of responses to noxious stimulation among SHNs with some topographic organization of the RFs, and sciatic nerve stimulation was quite ineffective (Lamour et al., unpublished observations), Results obtained in the raphe nuclei and substantia nigra by several authors are somewhat contradictory: some authors reported a majority of excitatory responses 2.5.6,13,17.26, whereas others described only inhibitory responses 38, especially in presumed serotonergic dorsal raphe neurons I. We observed a prevalence of excitatory responses to peripheral noxious stimulation in SHNs. Most of the RFs were large, including the caudal part of the body or the whole body. Such large RFs were also observed in the raphe nuclei z.17. That septal neurons are sensitive to somatic stimulation was known for a long time 3,4.30. These authors observed that somatosensory stimulation could elicit bursting activity in the septum. Similarly the hippocampal theta rhythm can also be triggered by peripheral stimulation 15. However, no study dealing specifically with the effect of peripheral stimulation on identified septal neurons was available. An evoked release of acetylcholine (ACh) from the cerebral cortex following peripheral nerves stimulation has been reported by Mitchell z5 and Mullins and Phillis zT. Dudar et al. II observed that the release of ACh from the hippocampus was increased during sensory stimulation and running. The type of sensory stimulation used was in the non-noxious range. Therefore the septo-hippocampal pathway might be more respon-
20 sive to light mechanical stimulation in awake animals. They also report that the increased release of ACh elicited by sensory stimulation was blocked by systemic administration of atropine in animals anaesthetized with urethane but not in freely moving animals. These observations suggest that the cholinergic septo-hippocampal system is responsive to peripheral stimulation and that this response is, under certain conditions, atropine-sensitive. They are consistent with our results showing that SHNs are driven by peripheral stimulation. Our results show that, under our experimental conditions, non-noxious stimulation were relatively ineffective in evoking responses in SHNs. One of the main peripheral input to SHNs might therefore be a noxious input. These neuronal responses do not seem to be related to unspecific effects such as a general arousal reaction, since they could be recorded in the absence of any cortical arousal reaction. However, we cannot exclude that SHNs would be responsive to non-noxious peripheral stimulation in awake animals. A wider range of stimulation could indeed be effective in the absence of anaesthesia. An important piece of data shown by our experiments is that some SHNs were able to code the intensity of the noxious stimulus (see Figs. 2 and 5). This suggests that the response of SHNs to noxious stimulation is functionally important and that the output of SHNs in response to such stimulation is graded. Such an ability to encode the intensity of the noxious stimulation has been observed in other supraspinal structures, e.g. thalamus 29 and cerebral cortex 23. However, the responses of SHNs to peripheral stimulation were not similar to those observed in the ventrobasal nucleus of the thalamus 16 or cerebral cortex23: very few SHNs were driven by light tactile stimulation; no small, contralateral, somatotopically organized RFs were observed. These obvious differences suggest that SHNs and thalamic or cortical responses to peripheral stimulation subserve different functions. Responses to noxious stimulation were of two main types: (i) increase in discharge frequency, and (ii) appearance or accentuation of the rhythmic bursting activity. These two types of responses could
be associated (e.g. increase in discharge frequency and appearance of the bursting activity). The origin of the bursting activity in the septum is unknown. We observed that this type of activity is resistant to various pharmacological substances 22. This activity is, however, dependent on the anaesthesia used 20,33. The present data indicate that peripheral stimulation is able to trigger or modify the bursting activity, suggesting that it is at least in part under control of extraneous influences. The afferent projections to the MS-nDBB are only partially known. This area apparently receives projections from the lateral preoptic-lateral hypothalamic area 34.36. It also receives inputs from brainstem aminergic neurons 4,32 (see also references in Costa et al., 1983) 9. Direct and/or indirect influences are.also exerted by other structures, especially hippocampus 24.31,35. One could hypothesize that the responses of SHNs to noxious peripheral stimulation are due to the input coming from locus coeruleus, raphe nuclei, and/or other brainstem structures. However, it does not seem to be so simple since the properties of locus coeruleus or dorsal raphe neurons responses to noxious stimulation are rather different from those we observed in SHNs 1,s. The predominance among SHNs of excitatory responses to peripheral noxious stimulation suggests that the activity of SHNs is increased during such stimulation. Since the septo-hippocampal pathway is known to be a cholinergic pathway (though probably not all SHNs are cholinergic), it follows that this cholinergic pathway is stimulated during noxious stimulation. This would probably result in an increased release of ACh in the hippocampus. Since the prominent effect of ACh in the hippocampus is to facilitate pyramidal cells firing by various mechanisms7.10, 21, it follows that noxious stimulation should result in an increased activity of hippocampal neurons.
ACKNOWLEDGEMENTS We thank J. Carrou6, M. Cayla and E. Dehausse for technical assistance.
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