Noxious stimuli produce prolonged changes in the CA1 region of the rat hippocampus

337

Pain, 39 (1989) 337-343 Elsevier

PAIN 01506

Noxious stimuli produce prolonged changes in the CA1 region of the rat hippocampus Sanjay Khanna

and John

G. Sinclair

Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B.C. V6T I W5 (Canada) (Received

25 April 1989, revision received 4 July 1989, accepted

24 July 1989)

Limbic structures including the hippocampus are thought to be involved in pain though Summary neuronal responses to noxious stimuli. In this report we show that a prolonged and substantial depression CA1 pyramidal cell population spike is produced by a brief but intense noxious stimulus applied to the rats. This depression is temperature-dependent and habituates to subsequent noxious stimuli applied more depression is absent when noxious heat is applied in the presence of hippocampal theta rhythm.

Key words:

Pain; Hippocampus;

Population

spike; Theta rhythm

Introduction Although the hippocampus has not been the focus of many studies on pain mechanisms, there are studies which suggest a hippocampus involvement in pain behaviour. For example, stimulation of the anterior hippocampus in cats and monkeys produces responses which mimick painful behaviour such as vocalization, offensive and defensive escape responses and autonomic manifestations [10,18]. Lesioning of the dorsal region or larger areas of the rat hippocampus has also been shown to increase threshold to vocalization [4] and decrease aggressive behaviour [12]. In humans, surgical section of the cingulum bundle, which connects the posterior frontal cortex to the hippocampus, results in a loss of ‘negative affect’

Correspondence to: Dr. John G. Sinclair, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, B.C. V6T lW5, Canada. 0304-3959/89/$03.50

not much is known of their of the dorsal hippocampal tail of lightly anaesthetized than 1 h later. Further, the

0 1989 Elsevier Science Publishers

associated with intractable pain [13]. Based on various lines of evidence, including some mentioned above, Melzack and Casey [19] proposed that the limbic forebrain structures, including the hippocampus, are involved in ‘aversive drive and affect that comprise the motivational dimension of pain.’ According to O’Keefe and Nadel [21], the hippocampus is involved in making of a cognitive map of the animal’s environment. This can indirectly affect the animal’s ‘motivational process.’ For example, the hippocampus can be involved in associating pain or fear with a particular place where the animal received a painful stimulus. This may explain deficits in avoidance behaviour to escape electric footshock in hippocampal lesioned animals [20,27] (also see Discussion). Evidence from hippocampal single cell and electroencephalogram (EEG) recording also showed that nociceptive stimuli can produce changes in hippocampal neuronal activity. For example, noxious stimulation of the tooth pulp [6] or heating of the tail [23] alters the background

B.V. (Biomedical

Division)

activity of hippocampal neurones. Soulairac et al. [24] reported that strong electrical stimulation ol the tail in conscious animals synchronizes the hippocampal EEG which lasts several seconds and is correlated with the animal vocalizing and biting the electrode. Similarly, hippocampal EEG synchronization, or theta rhythm of 5--6 Hz frequency, was reported in unanaesthetized rabbits after a strong noxious stimulus [17]. In the present study we have investigated the effect of a peripheral noxious stimulus on dorsal hippocampal CA1 pyramidal cell synaptic excitability. These cells are the major output neurones in the hippocampus. We found that a noxious stimulus produces profound and prolonged depression of the dorsal hippocampal CA1 population spike which habituated to repeated tests. Further, this depression of neuronal transmission was found to be dependent on the hippocampal EEG state of the animal. Materials and methods Male Sprague-Dawley rats (250-350 g) were lightly anaesthetized with urethane 1.0 g/kg, i.p. Deep anaesthesia during surgery was produced by supplementation with halothane. Most animals underwent very limited surgery. After placing the animal in a stereotaxic headholder, holes were drilled in the cranium to permit the positioning of stimulating and recording electrodes. In 4 animals, a carotid artery was also cannulated for blood pressure recording. Dorsal hippocampal (DH) CA1 population spikes were recorded with carbon fibre microelectrodes [3] and evoked by electrical stimulation (0.2 msec pulse duration, 0.2 Hz) in the DH region CA3 using a concentric bipolar electrode (David Kopf Inst., Model NE-100). The recording and stimulating electrodes were positioned relative to bregma, the midline and the surface of the cortex as follows: P 3.8, L 1.4, V 2.5 (CAl); P 2.3, L 1.4, V 3.5 (CA3). The recording electrode was adjusted to produce the maximal population spike upon CA3 stimulation. In addition, another concentric bipolar electrode was directed to the contralateral hippocampus to record the DH EEG. After determining the population spike amplitude vs. stimulus intensity relationship, the stimu-

lus Intensity was adjusted to produce a population spike which was about 80% of maximal. The output from the recording electrode was bandpasa filtered at 3 kHz. amplified and displayed on an oscilloscope. This output was also stored and later analyzed using the Waveman and Dataman programs [29] run on an Apple 1Ie computer. The latter program calculates the population spike amplitude by measuring the vertical from the peak of the spike to the tangent across the mouth of the spike. The Waveman program was also used to calculate the slope of the somatic field excitatory postsynaptic potential (fEPSP) at a fixed latency near the middle of the first positive wave. The contralateral DH EEG was continuously monitored on a polygraph (amplifier l/2 amplitude low and high filters set at 1 Hz and 35 Hz. respectively). To ensure recording stability the evoked CA1 population spike was monitored for about 1 h before a heat stimulus was applied. The noxious stimulus consisted of placing and maintaining 3.0 cm of the distal end of the tail in water at 50. 55 or 60°C for 15 sec. The animals generally reacted to noxious heat with reflex movements but did not vocalize. At 50” C/15 set, a tail-flick generally occurred with a latency of 5-10 set, whereas, the tail-flick latency in 55 “C water was always less than 5 sec. In the latter case the tail-flick was also more vigorous and the tail would have been withdrawn from the hot water if released by the experimenter. These reflexes were scored using the following scale: Score

Reactmn Mild tail-flick response (TFR), latency Mild TFR, latency < 5.0 set Vigorous TFR, latency < 5.0 set

> 5.0 set

-

1 7 3

A second application of noxious heat to the same receptive area was given 72 min after the first application. Subsequent noxious exposures were made at shorter time intervals. In 4 experiments the tail was exposed to 55°C water only until a tail-flick occurred. In these experiments a series of at least 5 tests, each at 4 min intervals, was performed while evaluating the effect on the CA1 population spike amplitude.

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At the end of each experiment the recording and stimulating sites were lesioned and histologically verified. The electrophysiological data were analyzed using l-way analysis of variance and the computed F ratio was used to determine significant difference among group means. Significant difference between different pairs of means was then determined using the Duncan multiple range test. The reflex-reaction score was compared within the same group of animals using the paired [ test and between different groups of animals with the unpaired r test. Statistically sig~ficant difference between compared values was accepted at P < 0.05.

The CA1 population spike, which was fairly stable over the control time period, occurred at a latency of 5-8 msec following the stimulation (Figs. 1 and 2). Exposure of the tail to noxious heat depressed the CA1 population spike amplitude in a temperature-dependent manner (Fig. 2). No consistent effect was seen at 50°C. However, 2-5 min following the application of noxious heat at 55 and 6O”C, a mean peak depression of 68 f 13% (range 27-100%) and 83 + 6% (range 63100%) occurred, respectively. The depression was long lasting in that the population spike amplitude remained significantly inhibited 8 min following heat exposure at 55°C and 18 min at 6O*C (Fig. 2B,C; F ratio of 3.23 and 7.29, respectively, P < 0.01; followed by Duncan test). The depression of population spike amplitude was not accompanied by any significant change in the slope of fEPSP (e.g., 5S°C, 15 set). An intense noxious stimulus was required to depress the CA1 population spike. If the tail remained in the water only long enough to evoke a tail-flick reflex no depression of the population spike occurred. The heat-evoked depression of the population spike was greatly attenuated or eliminated (i.e., habituated) on subsequent exposure(s) of noxious heat to the tail (Fig. 3B). This occurred whether the noxious stimulus was hot water at 55OC or 60°C.

Fig. 1. The effect of noxious heat (55°C. 15 set) applied to the tail on the dorsal hippocampal CA1 population spike amplitude. Each trace in the figure is an average of 4 consecutive sweeps. Positive is up in these traces. The control trace is an average of the population spikes within the minute prior to noxious heat application. The arrow under the control trace indicates the stimulus artifact. The other traces were collected after the noxious heat application around the times indicated above the trace. Note the marked and prolonged depression of spike amplitude following noxious heat exposure.

The DH EEG spontaneously alternated between irregular activity and a 4-6 Hz rhythmic sinusoidal wave-like activity which is commonly called theta rhythm. Interestingly, when noxious heat (SS’C, 15 set, tail) was applied with the hippocampal EEG in theta rhythm, no depression of the population spike was observed (Fig. 3C). If, however, the hippocampal EEG was in an irregular pattern at the time noxious heat was applied, a 4-6 Hz theta rhythm was produced (Fig. 3A,B) along with the depression of the population spike. However, it appears that there is a dissociation between the persistent depression of the population spike and the induction of theta rhythm in response to a noxious stimulus. For example, at 5O*C noxious heat exposure, a theta rhythm lasting an average of 6 + 1 set was produced with no significant change in population spike amplitude.

340 A

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8 4 I

o!

-60

I -40

-20

20 1

4iI

6II

B -.- _~..

2

A

0

-60

-. ..-- -_

-40

-20

20 I

bo

do

lo Time (min) Fig. 2. Temperature-dependent effect of noxious heat applied to the tail on the CA1 population spike amplitude. Files of population spikes were collected and their amplitude averaged pe~odically over the course of an experiment. These files were collected more frequently around the noxious heat stimulus. A, B and C show the effect of noxious heat applied for 15 XC (arrowhead) at 50, 55 and WC. respectively. Each point represents the meankS.E.M. population spike amplitude. N values for A, B and C are 6, 7 and 5, respectively. Significant differences from preheat controls are indicated by asterisks (P <: 0.05).

Furthermore, two successive exposures of 55°C evoked theta rhythms lasting 11 f 2 set and 12 rfi 2 set while only the first application resulted in a consistent depression of CA1 population spike (Fig. 3A,B). Table I shows the duration of the evoked theta rhythm and the reflex-reaction score in response to noxious stimuli for different test groups. As noted above the duration of the evoked theta rhythm was dependent on the intensity of the noxious stimulus and was not significantly changed with repeated exposures of the same stimulus. Similarly, the reflex reaction score was dependent

5

L

1

01

-40

-4 -50

A

1

i0 40 60 Time (min) Fig. 3. The influence of repeated noxious heat exposures and the hippocampal EEG state on the CA1 population spike. The upper traces in each section are contralateral hippocampal EEG records and each point in the graphs represents the mean It S.E.M. population spike amplitude. In these experiments noxious heat to the tail was applied at SS”C, 15 set indicated by solid bar below the trace and the arrowhead in each plot. A: the first application of noxious heat produces a long-lasting depression of the population spike when the EEG was in an irregular state at the time of application (n = 7). An example is shown in the upper trace. Also note that noxious stimulus induces theta rhythm. B: the time course in B is a continuation of A. Note that there is a habituation to the heat-induced depression of the population spike on repeated applications of the stimulus. However, theta rhythm is reproducibly evoked with each stimulus. The example used for illustration in the upper trace was obtained during the second application of noxious heat (72 min after the first application) in the same experiment shown in A. In this experiment there was a marked inhibition of the popuiation spike with the first application and none with the second application. C: in another set of animals the population spike failed to be inhibited by the first application of noxious heat to the tail (n = 5) if the hippocampal EEG was in theta rhythm at the time of heat a~pi~cation (upper trace). The large regular spikes in the EEG traces resulted from contralateral CA3 stimulation to evoke the CAI population spike. -60

341

TABLE

I

NOXIOUS

HEAT

INDUCED

AND REFLEX-REACTION

SCORE 55OC(n=5) with prior theta

55OC(n=7)

50°C(n=6)

Test group

Reflex-reaction

THETA

1st exposure

2nd exposure

1.3 * 0.2

1.5 * 0.2

6

7

1st exposure

2nd exposure

1st exposure

score

(* S.E.M.) Duration of evoked theta (set + S.E.M.)

k1

*1

on the stimulus intensity and did not habituate to a second exposure of noxious heat. It is perhaps noteworthy that the inhibition of the population spike was markedly attenuated in the 4 animals prepared for blood pressure recording. The mean inhibition upon placing the tail in 55’C water was 31+ 10% and was observed only during the application of the noxious stimulus. These experiments were not included in the data shown. Thus it appears that the additional surgery required for the cannulation of the carotid artery is responsible for the attenuation. Similarly, Clarke and Matthews [7] showed that trauma due to surgery is involved in increasing the jaw opening reflex threshold in cats. In our experiments two applications of noxious heat separated by more than 1 h produced similar increases in blood pressure lasting approximately 30 sec. Thus, the noxious stimulus-induced depression of the CA1 population spike is not due to blood pressure changes.

Discussion Numerous examples of synaptic plasticity in the form of long-term potentiation or depression have been reported in the hippocampus [for review see 15,281. Most of these result from activating a particular input to pyramidal or granule cells in the hippocampus using suitable stimulus parameters. There are, however, some reports of long-term potentiation occurring after certain behavioural tasks. We report here a rather prolonged depression of the CA1 population spike in response to noxious stimuli. This response was

2.7 + 0.2 11

*2

2.6 f 0.2 12 * 2

2.8 * 0.2 _

markedly attenuated or eliminated upon repeating the noxious stimulus to the same receptive field more than 1 h later. Others have shown that the CA1 population spike is depressed by non-noxious sensory stimuli such as stroking the fur in conscious rats [16]. We did not see comparable effects in lightly anaesthetized animals. The difference may have been due to the presence of the anaesthetic in our case or to the strength in afferent drive in evoking the population spike. Herreras et al. [16] reported the greatest effect of fur stroking when the CA1 population spike was near threshold. In our experiments, the stimulation intensity was adjusted to produce a population spike which was approximately 80% of maximal. There were also qualitative differences in the responses between these two studies. Noteably, fur stroking depressed the spike amplitude only for the period of stimulation, noxious heat produced a prolonged depression. Secondly, there was no habituation to repetitive applications of fur stroking. Thus, the hippocampus may differentially process non-noxious and noxious sensory information. The population spike reflects the synchronous discharge of pyramidal neurones following synaptic depolarization of the cell population [l]. A depression of population spike amplitude following a noxious stimulus might indicate a decrease in excitability of CA1 pyramidal neurones. This decrease in the population spike amplitude was unaccompanied by any change in somatic fEPSP. This raises the possibility that the observed PD is due to postsynaptic mechanisms. Similarly, during

34’

long-term potentiation the population spike amplitude increased when the size of the dendritic fEPSP was kept constant [2]. Nevertheless, a presynaptic decrease in synaptic efficacy following noxious heat stimulus cannot be ruled out. For example. a slight decrease in fEPSP amplitude may decrease the population spike which would be amplified due to reduced ephaptic effects [22,26]. The net effect would be a disproportionately greater decrease in population spike amplitude compared to the fEPSP. Here, it may be pointed out that, although not significant. the slope of fEPSP tended to be decreased following noxious heat exposure. Furthermore, in our experiments the electrical stimulation intensity of the afferents was sufficient to evoke a near maximal CA1 population spike. At this intensity of stimulation small changes in fEPSP might be obscured. Therefore, it may be necessary to evaluate changes in the somatic fEPSP at a lower population spike amplitude or record the dendritic fEPSP to evaluate changes in synaptic efficacy. The hippocampal response to noxious stimuli is markedly influenced by the state of the EEG. Noxious stimuli failed to produce an inhibition of the population spike if the EEG was in theta rhythm at the time of noxious stimulus application. Similarly, hippocampal tooth pulp stimulation evoked potentials were absent during theta activity in conscious animals [6]. It is known that during theta rhythm the hippocampal CA1 pyramidal cells undergo fluctuation in excitability related to different phases of the theta wave [14]. Possibly, this limits the extent or duration of influence of external input onto these cells. The hippocampal theta rhythm requires a medial septal input and involves a cholinergic link [5]. It is noteworthy that hippocampal theta rhythm is invariably elicited in response to noxious stimuli. A similar finding has been reported by others in response to an intense electrical stimulation [24] or pinching the tail [25]. Consistent with this notion is the finding that the septal-hippocampal neurones respond to intense peripheral noxious heat [Ill and receive a direct projection from the spinal cord [8]. There appears to be a relationship between the reflex-reaction score and the duration of theta

rhythm induced by different intensities of noxious heat stimuli and there is no habituation to these responses. This is in marked contrast to the population spike changes (persistent depression and then habituation) in response to the same noxious stimuli. These latter observations might be consistent with the view expressed by Vinogradova [30] that the hippocampal neurones are involved in registration of information leading to learning. She reported habituation of the hippocampal pyramidal cell depression accompanied by adaptive changes of the animal orienting response. Our findings may also be in line with O’Keefe and Nadel’s postulate that the hippocampus is involved in detection of a mismatch between the current milieu and the cognitive map [21]. Perhaps, in such an event. when a noxious stimulus is applied the prolonged depression of synaptic transmission initiates plasticity changes in the central nervous system which are involved in adaptive changes in animal behaviour. For example, both normal and hippocampal lesioned animals receiving an electric footshock escaped to a safe platform, but only normal animals showed a reluctance to step down from the safe area [27]. Of particular interest are the findings reported in a recent paper by Coderre and Wall [9]. They found that ankle joint urate arthritis in the rat was associated with a decreased response to noxious stimuli of the distal foot. Interestingly, they also noted that the forebrain was essential for the induction but not the maintenance of this reduced responsiveness. To conclude, a strong noxious stimulus to the tail produced a profound and persistent depression of dorsal hippocampal CA1 population spike which habituated upon subsequent exposures. Such neuronal changes might be involved in adaptive changes in animal behaviour following noxious stimuli. The excitability changes in the region CA1 of the hippocampus to noxious stimulus can be effectively modulated by prior input from the septal nuclei producing theta rhythm. In conscious animals theta rhythm is induced by a variety of stimuli and animal behaviour. Such conditions might be expected to modulate changes to noxious stimuli in the hippocampus and its participation in animal behaviour to such stimuli.

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