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Effect of olfactory bulb kindling on evoked potentials in the pyriform cortex
Brain Research, 361 (1985)61-69 Elsevier
61
BRE 11289
Effect of Olfactory Bulb Kindling on Evoked Potentials in the Pyriform Cortex R.D. RUSSELL and J.S. STRIPLING
Department of Psychology, Universityof Arkansas, Fayetteville, AR 72701 (U.S.A.) (Accepted April 5th, 1985)
Key words: evoked potential - - kindling - - olfactory bulb - - long-term potentiation - pyriform cortex - - rat - - post-seizure inhibition
The potential evoked in the pyriform cortex by single-pulse stimulation of the olfactory bulb was examined before and after single and repeated elieitation of an epileptiform afterdiseharge produced by stimulation of the olfactory bulb. A single afterdiseharge (AD) produced a rapid (i.e. within 5 min) increase in the amplitude of an early surface-negative wave and duration of a later surface-positive wave. These effects persisted at least 48-72 h. Repeated elicitation of ADs resulted in kindling. A large increase in the amplitude of a later surface-negative wave (approximately 25 ms latency) occurred during kindling. This wave remained significantly elevated for at least 72 h after the last AD. Long-term potentiation of the early surface-negative wave was produced by kindling or two focal ADs. A short-term effect which was consistently observed following a focal or generalized AD was a prolongation of a late surface-positive wave. These effects are discussed in relation to long-term potentiation, postseizure inhibition, and kindling development.
INTRODUCTION Sufficiently intense electrical stimulation of limbic system structures can readily trigger an epileptiform afterdischarge ( A D ) which does not propagate far from the site of stimulation. As A D s are elicited repeatedly, they gradually intensify. This p h e n o m e n o n has been termed 'kindling' and can be elicited by repeated stimulation of most, and possibly all, limbic structurest3,22, 26. Kindling development is evident electrophysiologically as an increase in the duration and amplitude of the A D , especially in secondary structures to which the seizure does not propagate strongly in the earliest stages of kindling 25. Kindling development is also evident behaviorally. The first few A D s produce no apparent behavioral manifestations whereas later ADs, although initially focal, propagate to distant sites and produce a behavioral convulsion characterized by forelimb clonus, rearing, and falling 25. This behavioral index of seizure generalization correlates with a large increase in A D amplitude recorded in sites distant from the area of stimulation 25. Racine et al. 28 were the first to demonstrate that kindling produced subsequent alterations in evoked
potentials (EPs) elicited via stimulation of the kindled site. Subsequent research has studied the effects of kindling on the E P both in viv05,10,2°, 21,30,31,37,43,44 and in vitro19,24,38 in the rat, and in vivo in other species such as the cat 42, rabbit is and monkey 12. The majority of these studies have examined changes in the EPs recorded in the hippocampal formation. However, Racine et al. 30 have demonstrated that kindling of numerous other limbic sites results in changes in non-hippocampal EPs elicited via stimulation of the kindled sites. One of the limbic circuits which Racine et al. 30 examined was the input to the pyriform cortex (PC) from the lateral olfactory tract (LOT), which consists of olfactory bulb (OB) mitral cell axons which synapse on pyramidal cells in the PC 36. Kindling stimulation applied to the L O T appeared to elicit a late spike-type component in the PC evoked potentiaP o. The present experiment further explores the effect of kindling on the PC evoked potential. However, the OB was chosen for electrical stimulation as opposed to the L O T because OB kindling has been of special interest in this laboratory for several years (e.g. refs. 39, 40). The projection from the OB to the PC appears to be well suited for studying the effect of kin-
Correspondence: J.S. Stripling, Department of Psychology, University of Arkansas, Fayetteville, AR 72701, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
62 dling on the EP for several reasons. First, the OB and PC are among the fastest kindling sites in the brain26, 27. Second, the highly laminar organization of the PC makes this paleocortical structure ideal for the recording and analysis of field potentials. In addition, the pyriform cortex EP is relatively well understood in terms of neuronal activity in the pC~,8, 9.14-16,34,35.
The present experiment assessed the effects of OB kindling on the PC potential evoked by OB stimulation. Changes in the EP were examined following both focal (i.e. localized) seizures triggered by OB stimulation at the start of kindling and generalized seizures triggered by OB stimulation at the end of kindling. The long-term effects of focal and generalized seizures were assessed by the comparison of animals which experienced only two focal seizures with others which were kindled to a criterion of 5 generalized seizures (as indicated by the presence of forelimb clonus). MATERIALS AND METHODS
Subjects The subjects were 9 male Long-Evans rats which weighed 333-367 g at the time of surgery. The rats were housed individually in clear plastic cages and given free access to Purina Rat Chow and tap water. The colony room was maintained on a 12/12 h lightdark cycle and all data were collected during the light phase.
Surgery Rats were injected with sodium pentobarbital (42.5 mg/kg, i.p.), chloral hydrate (100 mg/kg, i.p.), and atropine sulfate (4 mg/kg, i.p.) and placed in a Kopf stereotaxic instrument with the incisor bar positioned 5 mm above the plane of the interaural line. A single bipolar stimulation electrode was implanted in the left OB (8.9 mm anterior and 1.2 mm lateral to bregma, and 1.8 mm ventral to the dura) of each animal. The electrode was made by joining a 125¢tm and a 200 ~m diameter wire with Epoxylite varnish so that the 125/~m Wireextended 0.5 m m beyond the tip of the 200/~m wire. A single monopolar electrode (200 #m diameter) was lowered toward the left pyriform cortex (2.4 mm anterior and 2.8 mm lateral to bregma at the dura with a 14° lateral angle) and
placed at a depth dependent upon the amplitude and polarity of the pyriform cortex EP. Four rats received placements dorsal to the superficial pyramidal cell layer of the PC and the other 5 received ventral placements. These placements resulted in a positive or negative polarity, respectively, for the initial wave of the EP. The recording reference was a stainless steel screw inserted in the bone overlying the right anterior neocortex. The electrodes were cemented in place and the rats were allowed at least 3 weeks postoperative recovery before kindling began.
Electrical stimulation All electrical stimulation (for EPs and ADs) was delivered via the OB electrode. Constant-current stimulation was provided by a Grass $48 stimulator coupled to a Grass PSIU6 stimulus isolation unit. All stimulation consisted of monophasic square-wave pulses which were 0.2 ms in duration. The I25 #m lead of the OB electrode was the cathode and was positioned at surgery so that it was posterior to the 200/~m anode. EPs were elicited by single- or pairedpulse stimulation, while ADs were triggered by 2 s trains of 100 pulses delivered at a frequency of 50 pulses/s.
Electrophysiological recording All electrophysiological activity (EPs and ADs) was recorded via the PC electrode. The animal was placed in a Faraday cage during recording, and a field-effect transistor circuit was used to minimize cable movement artifacts 32. The EP signal was amplified by a Model 461D preamplifier and 411 power amplifier in a Beckman R 6 t l polygraph, using a 0.5-2500 Hz bandpass. Amplified EPs were digitized by a Tecmar Lab Master analog-to-digital converter sampling at 5 kHz. An IBM Personal Computer was used to average the digitized evoked potentials, either on-line or from evoked potentials stored previously on floppy diskette, and calculate the S.E.M. associated with each point of the averaged EP (AEP). A Hewlett Packard 7470A digital plotter was used to plot the AEPs and results of statistical analyses. ADs were recorded with the Beckman R611 polygraph.
Kindling procedure On the first day of kindling the AD threshold was
63 determined for all animals using an ascending staircase procedure. Stimulation intensity began at 100/~A and was delivered at 60 s intervals with 100/~A current increments until an AD was elicited. The animals were then divided into Kindled (n = 5) and Non-Kindled (n = 4) groups matched for AD threshold and EP polarity and amplitude. Two to three days after the first AD, a second AD was elicited in all animals by delivering a single train at a current intensity of 150% of the individual's AD threshold. Animals in the Kindled group were then kindled to criterion by daily stimulation at this current intensity. The kindling criterion was 5 consecutive ADs accompanied by bilateral forelimb clonus. Each animal in the Non-Kindled group experienced no further ADs but instead received a matched number of daily stimulations at a frequency (1 pulse/s) which did not elicit ADs. Thus, at the end of the experimental treatment, animals in the Kindled group had been kindled to the point of exhibiting generalized seizures, while animals in the Non-Kindled group had received the same total number of stimulation pulses, but administered in such a way that only two localized seizures were elicited at the beginning of the treatment.
Evoked potential tests Each AEP was calculated from 10 EPs elicited at 1 s intervals. During kindling, EPs were elicited by stimulating with 10 single pulses followed by 10 paired pulses (20 ms inter-pulse interval) at current intensities of 100,200,400 and 800/~A, in that order, with a 60 s interval separating each current intensity tested. For determining the effect of a focal OB seizure, EPs were collected from all animals immediately before and 5, 15, 30 min, 24 and 48-72 h after the first AD. For determining the effect of kindling, EPs were collected immediately before and 5, 15, 30 min, 24 and 72 h after the 5th AD accompanied by forelimb clonus (Kindled group) or matched control stimulation (Non-Kindled group). Immediately after the 72 h EP test, an AD was triggered in the Kindled group and the Non-Kindle d group received 1 pulse/s stimulation. Fo~y-eight h later, input/output (I/O) curves were measured for current intensities ranging from 25 to 1000/zA. These curves were compared with I/O curves measured 7 days before each animal
experienced its first AD.
Histology Electrode placement was verified histologically using the Prussian blue technique. All animals had good OB and PC placements. Examination of tissue slices with a light microscope revealed that one animal (No. 833) in the Non-Kindled group had a lesion along the OB electrode tract. This animal also required excessive current (originally 800/~A and subsequently greater than 2000 pA compared to 400 p A maximum for the other animals) to trigger an AD. Therefore, this animal's data were excluded from analysis. Data analysis Regions of statistically significant differences between two AEPs from the same animal were determined by conducting a t-test, with P < 0.01, between each of the corresponding points of the two AEPs (see Fig. 1A, B). A total of 100 ms (500 digitized points), which included 5 ms of baseline activity, was analyzed for each comparison. For determining short-term (5-30 min) and long-term (1-3 days) effects of a focal AD, each rat's AEP immediately preceding the first AD was compared to each of its AEPs collected at various times after that AD (5, 15, 30 min, 24 and 48-72 h). This AEP was also used as the basis for comparison in assessing long-term (1-3 days) effects of kindling (Kindled group) or two focal ADs followed by daily 1 pulse/s stimulation (NonKindled group). The AEP recorded immediately before the 5th AD with clonus (Kindled group) was used as the basis for comparison in assessing shortterm (i.e. 5-30 min) effects of a kindled AD. Comparable short-term comparisons were made in nonkindled animals for assessing the effects of 1 pulse/s stimulation. Paired-pulse EPs were not analyzed because kindled animals showed a large increase in a late component (C3, discussed below) of the EP elicited by the first pulse which obscured the response to the second pulse. RESULTS
The pyriform cortex evoked potential A typical AEP elicited with 400/~A stimulation and before any ADs were triggered is presented in
64 Fig. 1A. For descriptive purposes, the A E P is divided into 4 components (C1-C4). At low current intensities (i.e. less than 400/~A) two components are seen, an initial surface-negative wave (C1) and a later surface-positive wave (C2). At higher current intensities, a second surface-negative wave (C3) and a later surface-positive wave (C4) are elicited (see Fig.
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The first and second A D s were short in duration (11.8 + 1.4 and 14.0 + 1.0 s, respectively; mean + S.E.M.) and did not produce any behavioral signs of seizure generalization such as head nodding, chewing or forelimb clonus. Small A D spikes could be recorded in the PC within 2 s after stimulation offset and grew in amplitude as the discharge continued. The A D threshold at the first A D was 229 + 41/~A. Fig. 2A and C illustrates the effect of a focal A D on the A E P for a single subject and mean values for all subjects, respectively. Short-term effects were similar at 5 and 30 min post A D although they were more prominent at 30 min. Two effects were consistently significant across animals, an increase in the amplitude of C1 (6 of 8 rats exhibited this at 5 and 30 min) and a prolongation of C4 (8 of 8 and 7 of 8 rats at 5 and 30 min, respectively). The effects on C1 and C4 persisted for 24 h in 5 of 8 rats and for 48-72 h in 4 of 8 rats.
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Fig. 1. A: typical averaged evoked potential (AEP) calculated from 10 potentials evoked in the pyriform cortex by stimulation of the olfactory bulb. The arrow indicates the stimulation artifact. The numbers 1-4 are used to label the different components (C1-C4) of the EP for descriptive purposes. Note that positive is up in this and all subsequent figures. B: statistical comparison of two AEPs, each consisting of 500 points (100 ms). The thickness at a point in AEP 2, which is the same AEP as in A, represents the critical interval for a t-test (P < 0.01) between that point and the corresponding point of AEP 1. The line (3) beneath the superimposed AEPs represents regions where the two AEPs differ significantly (i.e. 1 and 2 do not overlap). C: AEPs recorded above the superficial pyramidal cell layer at various current intensities (No. 828). Current intensities are 100,200,400 and 800/zA for 1-4, respectively. D: AEPs recorded below the superficial pyramidal cell layer at various current intensities (No. 823). Current intensities are the same as in C.
1C, D). The functional significance of these components is discussed below (see Discussion). The A E P recorded dorsal to the superficial pyramidal cell layer of the PC (Fig. 1C) is an approximate mirror image of the A E P recorded ventral to the cell layer (Fig. 1D), which demonstrates that the EPs are generated within the PC and not volume conducted from a more distant site.
Kindled animals required 11.6 + 1.8 (range: 8-17) A D s to reach kindling criterion. For the Kindled group, the A D s at kindling criterion lasted 74.2 + 4.4 s. The onset of forelimb clonus occurred 19.6 + 3.3 s after the termination of OB stimulation. Seizure generalization, as reflected by forelimb clonus, lasted 51.2 + 3.6 s. After the two A D s at the start, the Non-Kindled group received 10,0 + 2.6 daily control stimulations (range: 6-15). These stimulations never elicited an AD. An example of the effect of a kindled seizure on the A E P elicited by 400 ~ A is presented in Fig. 2B. Elicitation of a kindled seizure (Kindled group) produced an increase in the amplitude and duration of C4 which was consistently significant across animals
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TIME AFTER 1st AFTERDISCHARGE Fig. 2. A: the effect of a focal olfactory bulb epileptiform afterdischarge on the pyriform cortex AEP (No. 823). The lines under the various AEPs signify regions where those AEPs differ significantly from the AEP (shown on the left) recorded immediately prior to the afterdischarge. All AEPs were elicited with 400/~A stimulation. B: short-term effects of an olfactory bulb kindled seizure on the pyriform cortex AEP (No. 823). This Kindled rat received high-frequency stimulation (50 pulses/s) of the olfactory bulb which elicited its 5th generalized afterdischarge. The lines under the various AEPs signify regions where those AEPs differ significantly from the AEP (shown on the left) recorded immediately prior to the 5th generalized afterdischarge. All AEPs were elicited with 400/~A stimulation. C: time course of the effect of a focal OB afterdischarge on various measures of the pyriform cortex AEP. C1, peak amplitude of C1. C2, peak amplitude of C2. C3, amplitude of C3 (measured from C2 peak amplitude to amplitude at 26 ms). C4, duration of C4 (measured from stimulation artifact to zero cross after C4). These data are based on mean values for 8 rats.
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(5 of 5 rats). This was a transient postictal effect which was absent 24 h after the seizure. Another consistent effect was an increase in the size of C3, which was most evident at 30 min post A D (significant in 5 of 5 rats). The 1 pulse/s stimulation (Non-Kindled
Fig. 3. Long-term effects of kindling on the pyriform cortex AEP. The lines under the various AEPs signify regions where those AEPs differ significantly from the AEP recorded immediately prior to the first afterdischarge (shown on the left). The delay intervals represent time since the last afterdischarge in kindling (Kindled rats) or the last 1 pulse/s stimulation (NonKindled rats). All AEPs were elicited with 400 #A stimulation.
66 should be noted that these rats had experienced two ADs 48-72 h apart at the beginning of the experiment in addition to subsequent daily 1 pulse/s stimulation. Therefore, these rats may be best considered as partially kindled. At 1 and 3 days following completion of 1 pulse/s stimulation, all of the Non-
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Kindled rats exhibited a significant increase in the amplitude of CI and an increase or prolongation of C4. Examination of Fig. 4 indicates that this longterm potentiation of C1 amplitude is observed over a wide range of current intensities. Three of 5 Kindled rats demonstrated a significant increase in C1 amplitude at 1 and 3 days after kindling criterion. Fig. 4 indicates that this effect can be observed over a wide range of current intensities. The AEPs of two kindled animals (No, 823 and 829) appear qualitatively different after kindling. C2 is absent and C3 is much larger in relation to the size of the other components. Although one of the NonKindled rats (No. 834) demonstrated an increase in C3, it was small by comparison. DISCUSSION
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Fig. 4. Effect of kindling on I/O relations for C1 amplitude. A: I/O curves for Kindled rats were measured 7 days before the first AD and after kindling (48 h following a generalized AD). Data points are averages for 5 rats. B: I/O curves for NonKindled rats. The first I/O curve was measured 7 days before the first AD. The second I/O curve was measured 11-20 days after the second AD. After the second AD, these rats received daily 1 pulse/s stimulation until 5 days prior to measurement of the second I/O curve followed by one additional train 48 h before its measurement. Data points are averages for 3 rats.
The waveform of the pyriform cortex EP observed in the present experiment is similar to the EP elicited with OB stimulation in the rat a and rabbit 34 and with L O T stimulation in the rat3L cat t,s, rabbit 34 and opossum~5, ~6. Previous research has produced reasonable agreement, based upon both anatomical and physiological data, concerning the neuronal events which occur in the PC in response to L O T stimulation 1.8,9A4-16,3435. C1 (see Fig. 1A) is a reflection of two distinct events, a monosynaptic and disynaptic population excitatory postsynaptic potential (EPSP) in PC pyramidal cells. These two EPSPs (termed A l and B 1, respectively, by Haberly 15) are differentially affected by conditioning shocks and pharmacological manipulation16,35. The subsequent surface-positive waves (C2 and C4 in Fig. 1A) are temporally associated with inhibitory postsynaptic potentials (IPSPs) and inhibition of firing in PC pyramidal cells following their activationl,S, 9A6 and may be a reflection of feedforward and/or recurrent inhibition mediated via inhibitory interneurons ~6. C3 is typically not seen in vivo with L O T stimulationl,S,16.34,35; however, a component of similar polarity and latency has been reported for in vivo stimulation of the OB in the rabbit 34 and rat 4. The neuronal elements which generate this component are unknown at present. It does not appear to be dependent upon L O T stimulation because Satou et at. 34 report that it can still be evoked by OB stimula-
67 tion after section of the LOT, which abolishes all other components of the EP.
Seizure-induced changes in the evoked potential A single focal AD increased C1 amplitude as early as 5 min after the AD, and this effect persisted for 48-72 h. At present we cannot determine with certainty whether this change recorded in the PC represents a functional change in the OB (e.g. altered mitral cell responsiveness to the stimulation), the PC (e.g. increased transmitter release, increased postsynaptic response) or a combination of the above. Nevertheless, this effect appears similar to results obtained in the hippocampal formation. Douglas and Goddard 5 reported that the first kindling stimulus applied to the entorhinal cortex increased the amplitude of the monosynaptic EPSP and population spike recorded in the dentate gyrus, and these effects persisted at least 5 days. In addition, Maru et al. xl reported that the first AD elicited in the perforant path produced an increase in the dentate gyrus EPSP as early as 7 min after the AD and this effect persisted for at least 24 h. These data suggest that a single episode of relatively localized and brief epileptiform activity in a set of neurons can produce a rapid and persistent effect on neuronal communication within the seizure's path. The observation that a focal AD produces a qualitatively similar effect with similarly rapid onset in two separate limbic pathways suggests that this phenomenon may occur in other forebrain sites as well. A highly consistent effect observed in the present experiment was an increase in the amplitude and duration of C4 following seizure activity. This effect appeared within 5 min of a focal AD and was still evident 48-72 h later. In Non-Kindled animals, this effect was still present 9-18 days after the last of two focal ADs (3 days following their last I pulse/s stimulation). These data suggest that one or two ADs produce a relatively permanent increase in C4. As indicated above, such an effect might be indicative of enhanced inhibitory mechanisms in the PC. However, this effect may be secondary to the increase in C1 amplitude if C4 is a response to excitation of pyramidal cells, as it would be if C4 reflects recurrent inhibition. In addition, the changes which occurred in C4 may not have been due entirely to seizure activity. Recent evidence indicates that high frequency electrical
stimulation of the OB below AD threshold produces a large increase in C2 or C4 without any corresponding effect on C1, and that a small effect can be produced even by 1 pulse/s stimulation 41. Animals in the present experiment received single- and paired-pulse stimulation during EP testing and either 50 pulse/s (Kindled) or 1 pulse/s (Non-Kindled) stimulation as the experimental treatment. Therefore, at present we cannot specify the relative contributions of seizure activity and electrical stimulation to the changes in C4 seen in this experiment. In Kindled rats, an increase in the amplitude and duration of C4 was also consistently observed 5-30 min after a generalized AD, but the effect had disappeared by 24 h. This transient postictal change in the EP may have implications concerning the mechanisms responsible for seizure-inhibitory effects which are seen shortly following a previous kindled seizure7,13,17,23,33. However, based on reasoning similar to the above, we cannot specify the relative contributions of the generalized seizure and high-frequency electrical stimulation to this effect. The large increase in C3 in Kindled rats appears to have been caused specifically by the kindling process. Racine et al. 3° have reported that kindling produced by LOT stimulation elicits a spike-type component in the pyriform cortex EP during periods when spontaneous interictal spikes are present. The AEPs from the two animals which showed a large increase in C3 (No. 823 and 829 in Fig. 3) resemble the data of Racine et al. 30. The results from the present experiment indicate that the increase in C3 is long-lasting but exhibits some decline from 1 to 3 days following a generalized AD. If this component reflects extraLOT input 34, then its increase following kindling indicates that seizure-induced changes in PC function are not confined to LOT input. A phenomenon of interest related to kindling is long-term potentiation (LTP) 2,3,5,6,11,29,which refers to an increase in the initial EPSP component of the EP following repeated electrical stimulation. Highfrequency stimulation is most effective for producing LTP 6 and eliciting an AD 13. Research on LTP in the dentate gyrus indicates clearly that elicitation of an AD is not necessary to initiate LTP 5. In this respect, the projection from the OB to the PC may differ from the perforant path. For example, Racine et al. 30 produced little or no LTP via high-frequency LOT stimu-
68 lation below A D threshold, even though similar stimulation of other limbic forebrain sites produced prominent LTP effects. In contrast, kindling via stim-
study indicates that a single A D triggered by OB stimulation produces a rapid and long-term potentiation of the EPSP component of the EP.
ulation of the L O T reliably produced an increase in response amplitude 30. We have observed a similar pattern of results for OB stimulation. High-frequen-
ACKNOWLEDGEMENT
cy OB stimulation below A D threshold does not increase C1 (although it does increase the amplitude and duration of C2 or C4) 41. In contrast, the present
This research was supported by a grant from the Marie Wilson Howells Memorial Fund.
REFERENCES
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1 Biedenbach, M.A. and Stevens, C.F., Synaptic organization of the cat olfactory cortex as revealed by intracellular recording, J. Neurophysiol., 32 (1969) 204-214. 2 Bliss, T.V.P. and Gardner-Medwin, A.R., Long-lasting potentiation of synaptic transmission in the dentate area of unanesthetized rabbits following stimulation of the perforant path, J. Physiol. (London), 232 (1973) 357-374. 3 Bliss, T.V.P. and Lomo, T., Long-lasting potentiation of synaptic transmission in the dentate area of the anesthetized rabbit following stimulation of the perforant path, J. Physiol. (London), 232 (1973) 331-356. 4 Cragg, B.G., Responses of the hippocampus to stimulation of the olfactory bulb and of various afferent nerves in five mammals, Exp. Neurol., 2 (1960) 547-572. 5 Douglas, R.M. and Goddard, G.V., Long-term potentiation of the perforant path-granule cell synapse in the rat hippocampus, Brain Research, 86 (1975) 205-215. 6 Dunwiddie, T. and Lynch, G., Long-term potentiation and depression of synaptic responses in the rat hippocampus: localization and frequency dependency, J. Physiol. (London), 276 (1978) 353-367. 7 Freeman, F.G. and Jarvis, M.F., The effect ofinterstimulation interval on the assessment and stability of kindled seizure thresholds, Brain Res. Bull., 7 (1981) 629-633. 8 Freeman, W.J., Relation between unit activity and evoked potentials in prepyriform cortex of cats, J. Neurophysiol., 31 (1968) 337-348. 9 Freeman, W.J., Effects of surgical isolation and tetanization on prepyriform cortex in cats, J. Neurophysiol., 31 (1968) 349-357. 10 Giacchino, J.L., Somjen, G.G., Frush, D.P. and McNamara, J.O., Lateral entorhinal cortical kindling can be established without potentiation of the entorhinal-granule cell synapse, Exp. Neurol., 86 (1984) 483-492. 11 Goddard, G.V., The continuing search for mechanism. In J. Wada (Ed.), Kindling 2, Raven Press, New York, 1981, pp. 1-10. 12 Goddard, G.V. and Douglas, R.M., Does the engram of kindling model the engram of normal long term memory? In J.A. Wada (Ed.), Kindling 1, Raven Press, New York, 1976, pp. 1-18. 13 Goddard, G.V., Mclntyre, D.C. and Leech, C.K., A permanent change in brain function resulting from daily electrical stimulation, Exp. Neurol., 25 (1969) 295-330. 14 Haberly, L.B., Unitary analysis of opossum prepyriform cortex, J. Neurophysiol., 36 (1973) 762-774. 15 Haberly, L.B., Summed potentials evoked in opossum prepyriform cortex, J. Neurophysiol., 36 (1973) 775-788.
69
cephalogr. Clin. Neurophysiol., 39 (1975) 261-271. 32 Rossetto, M.A. and Vandercar, D.H., Lightweight FET circuit for differential or single-ended recording in freemoving animals, Physiol. Behav., 9 (1972) 105-106. 33 Sainsbury, R.S., Bland, B.H. and Buchan, D.H., Electrically induced seizure activity in the hippocampus: time course for postseizure inhibition of subsequent kindled seizures, Behav. Biol., 22 (1978) 479-488. 34 Satou, M., Mori, K., Tazawa, Y. and Takagi, S.F., Monosynaptic and disynaptic activation of pyriform cortex by synchronous lateral olfactory tract volleys in the rabbit, Exp. Neurol., 81 (1983) 571-585. 35 Schwob, J.E., Haberly, L.B. and Price, J.L., The development of physiological responses of the piriform cortex in rats to stimulation of the lateral olfactory tract, J. Comp. Neurol., 223 (1984) 223-237. 36 Shepherd, G.M., The Synaptic Organization of the Brain, 2nd edn., Oxford University Press, New York, 1979, 436 pp. 37 Sutula, T. and Steward, O., Quantitative analysis of synaptic potentiation during kindling, Neurology, Suppl. 2, 33 (1983) 188. 38 Stasheff, S.F., Wilson, W.A. and Bragdon, A.C., Induction of epileptiform activity in hippocampal slices by the presentation of a kindling-like stimulus, Soc. Neurosci.
Abstr., 10 (1984) 549. 39 Stripling, J.S. and Hendricks, C., Facilitation of kindling by convulsions induced by cocaine or lidocaine but not pentylenetetrazol, Pharmacol. Biochem. Behav., 15 (1981) 793-798. 40 Stripling, J.S. and Hendricks, C., Effect of cocaine and lidocaine on the expression of kindled seizures in the rat, Pharmacol. Biochem. Behav., 14 (1981)397-403. 41 Stripling, J.S., Patneau, D.K. and Gramlich, C.A., Longterm changes in the pyriform cortex evoked potential produced by stimulation of the olfactory bulb, Soc. Neurosci. Abstr., 10 (1984) 76. 42 Tanaka, T., Modification of amygdalo-cortical evoked potentials by kindling and pentetrazol-induced generalized convulsions in cat, Electroencephalogr. Clin. Neurophysiol., 43 (1977) 675-678. 43 Tuff, L.P., Racine, R.J. and Adamec, R., The effects of kindling on GABA-mediated inhibition in the dentate gyrus of the rat. I. Paired-pulse depression, Brain Research, 277 (1983) 79-90. 44 Wadman, W.J., Lopes Da Silva, F.H. and Leung, L.S., Two types of interictal transients of reversed polarity in rat hippocampus during kindling, Electroencephalogr. Clin. Neurophysiol., 55 (1983) 314-319.
61
BRE 11289
Effect of Olfactory Bulb Kindling on Evoked Potentials in the Pyriform Cortex R.D. RUSSELL and J.S. STRIPLING
Department of Psychology, Universityof Arkansas, Fayetteville, AR 72701 (U.S.A.) (Accepted April 5th, 1985)
Key words: evoked potential - - kindling - - olfactory bulb - - long-term potentiation - pyriform cortex - - rat - - post-seizure inhibition
The potential evoked in the pyriform cortex by single-pulse stimulation of the olfactory bulb was examined before and after single and repeated elieitation of an epileptiform afterdiseharge produced by stimulation of the olfactory bulb. A single afterdiseharge (AD) produced a rapid (i.e. within 5 min) increase in the amplitude of an early surface-negative wave and duration of a later surface-positive wave. These effects persisted at least 48-72 h. Repeated elicitation of ADs resulted in kindling. A large increase in the amplitude of a later surface-negative wave (approximately 25 ms latency) occurred during kindling. This wave remained significantly elevated for at least 72 h after the last AD. Long-term potentiation of the early surface-negative wave was produced by kindling or two focal ADs. A short-term effect which was consistently observed following a focal or generalized AD was a prolongation of a late surface-positive wave. These effects are discussed in relation to long-term potentiation, postseizure inhibition, and kindling development.
INTRODUCTION Sufficiently intense electrical stimulation of limbic system structures can readily trigger an epileptiform afterdischarge ( A D ) which does not propagate far from the site of stimulation. As A D s are elicited repeatedly, they gradually intensify. This p h e n o m e n o n has been termed 'kindling' and can be elicited by repeated stimulation of most, and possibly all, limbic structurest3,22, 26. Kindling development is evident electrophysiologically as an increase in the duration and amplitude of the A D , especially in secondary structures to which the seizure does not propagate strongly in the earliest stages of kindling 25. Kindling development is also evident behaviorally. The first few A D s produce no apparent behavioral manifestations whereas later ADs, although initially focal, propagate to distant sites and produce a behavioral convulsion characterized by forelimb clonus, rearing, and falling 25. This behavioral index of seizure generalization correlates with a large increase in A D amplitude recorded in sites distant from the area of stimulation 25. Racine et al. 28 were the first to demonstrate that kindling produced subsequent alterations in evoked
potentials (EPs) elicited via stimulation of the kindled site. Subsequent research has studied the effects of kindling on the E P both in viv05,10,2°, 21,30,31,37,43,44 and in vitro19,24,38 in the rat, and in vivo in other species such as the cat 42, rabbit is and monkey 12. The majority of these studies have examined changes in the EPs recorded in the hippocampal formation. However, Racine et al. 30 have demonstrated that kindling of numerous other limbic sites results in changes in non-hippocampal EPs elicited via stimulation of the kindled sites. One of the limbic circuits which Racine et al. 30 examined was the input to the pyriform cortex (PC) from the lateral olfactory tract (LOT), which consists of olfactory bulb (OB) mitral cell axons which synapse on pyramidal cells in the PC 36. Kindling stimulation applied to the L O T appeared to elicit a late spike-type component in the PC evoked potentiaP o. The present experiment further explores the effect of kindling on the PC evoked potential. However, the OB was chosen for electrical stimulation as opposed to the L O T because OB kindling has been of special interest in this laboratory for several years (e.g. refs. 39, 40). The projection from the OB to the PC appears to be well suited for studying the effect of kin-
Correspondence: J.S. Stripling, Department of Psychology, University of Arkansas, Fayetteville, AR 72701, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
62 dling on the EP for several reasons. First, the OB and PC are among the fastest kindling sites in the brain26, 27. Second, the highly laminar organization of the PC makes this paleocortical structure ideal for the recording and analysis of field potentials. In addition, the pyriform cortex EP is relatively well understood in terms of neuronal activity in the pC~,8, 9.14-16,34,35.
The present experiment assessed the effects of OB kindling on the PC potential evoked by OB stimulation. Changes in the EP were examined following both focal (i.e. localized) seizures triggered by OB stimulation at the start of kindling and generalized seizures triggered by OB stimulation at the end of kindling. The long-term effects of focal and generalized seizures were assessed by the comparison of animals which experienced only two focal seizures with others which were kindled to a criterion of 5 generalized seizures (as indicated by the presence of forelimb clonus). MATERIALS AND METHODS
Subjects The subjects were 9 male Long-Evans rats which weighed 333-367 g at the time of surgery. The rats were housed individually in clear plastic cages and given free access to Purina Rat Chow and tap water. The colony room was maintained on a 12/12 h lightdark cycle and all data were collected during the light phase.
Surgery Rats were injected with sodium pentobarbital (42.5 mg/kg, i.p.), chloral hydrate (100 mg/kg, i.p.), and atropine sulfate (4 mg/kg, i.p.) and placed in a Kopf stereotaxic instrument with the incisor bar positioned 5 mm above the plane of the interaural line. A single bipolar stimulation electrode was implanted in the left OB (8.9 mm anterior and 1.2 mm lateral to bregma, and 1.8 mm ventral to the dura) of each animal. The electrode was made by joining a 125¢tm and a 200 ~m diameter wire with Epoxylite varnish so that the 125/~m Wireextended 0.5 m m beyond the tip of the 200/~m wire. A single monopolar electrode (200 #m diameter) was lowered toward the left pyriform cortex (2.4 mm anterior and 2.8 mm lateral to bregma at the dura with a 14° lateral angle) and
placed at a depth dependent upon the amplitude and polarity of the pyriform cortex EP. Four rats received placements dorsal to the superficial pyramidal cell layer of the PC and the other 5 received ventral placements. These placements resulted in a positive or negative polarity, respectively, for the initial wave of the EP. The recording reference was a stainless steel screw inserted in the bone overlying the right anterior neocortex. The electrodes were cemented in place and the rats were allowed at least 3 weeks postoperative recovery before kindling began.
Electrical stimulation All electrical stimulation (for EPs and ADs) was delivered via the OB electrode. Constant-current stimulation was provided by a Grass $48 stimulator coupled to a Grass PSIU6 stimulus isolation unit. All stimulation consisted of monophasic square-wave pulses which were 0.2 ms in duration. The I25 #m lead of the OB electrode was the cathode and was positioned at surgery so that it was posterior to the 200/~m anode. EPs were elicited by single- or pairedpulse stimulation, while ADs were triggered by 2 s trains of 100 pulses delivered at a frequency of 50 pulses/s.
Electrophysiological recording All electrophysiological activity (EPs and ADs) was recorded via the PC electrode. The animal was placed in a Faraday cage during recording, and a field-effect transistor circuit was used to minimize cable movement artifacts 32. The EP signal was amplified by a Model 461D preamplifier and 411 power amplifier in a Beckman R 6 t l polygraph, using a 0.5-2500 Hz bandpass. Amplified EPs were digitized by a Tecmar Lab Master analog-to-digital converter sampling at 5 kHz. An IBM Personal Computer was used to average the digitized evoked potentials, either on-line or from evoked potentials stored previously on floppy diskette, and calculate the S.E.M. associated with each point of the averaged EP (AEP). A Hewlett Packard 7470A digital plotter was used to plot the AEPs and results of statistical analyses. ADs were recorded with the Beckman R611 polygraph.
Kindling procedure On the first day of kindling the AD threshold was
63 determined for all animals using an ascending staircase procedure. Stimulation intensity began at 100/~A and was delivered at 60 s intervals with 100/~A current increments until an AD was elicited. The animals were then divided into Kindled (n = 5) and Non-Kindled (n = 4) groups matched for AD threshold and EP polarity and amplitude. Two to three days after the first AD, a second AD was elicited in all animals by delivering a single train at a current intensity of 150% of the individual's AD threshold. Animals in the Kindled group were then kindled to criterion by daily stimulation at this current intensity. The kindling criterion was 5 consecutive ADs accompanied by bilateral forelimb clonus. Each animal in the Non-Kindled group experienced no further ADs but instead received a matched number of daily stimulations at a frequency (1 pulse/s) which did not elicit ADs. Thus, at the end of the experimental treatment, animals in the Kindled group had been kindled to the point of exhibiting generalized seizures, while animals in the Non-Kindled group had received the same total number of stimulation pulses, but administered in such a way that only two localized seizures were elicited at the beginning of the treatment.
Evoked potential tests Each AEP was calculated from 10 EPs elicited at 1 s intervals. During kindling, EPs were elicited by stimulating with 10 single pulses followed by 10 paired pulses (20 ms inter-pulse interval) at current intensities of 100,200,400 and 800/~A, in that order, with a 60 s interval separating each current intensity tested. For determining the effect of a focal OB seizure, EPs were collected from all animals immediately before and 5, 15, 30 min, 24 and 48-72 h after the first AD. For determining the effect of kindling, EPs were collected immediately before and 5, 15, 30 min, 24 and 72 h after the 5th AD accompanied by forelimb clonus (Kindled group) or matched control stimulation (Non-Kindled group). Immediately after the 72 h EP test, an AD was triggered in the Kindled group and the Non-Kindle d group received 1 pulse/s stimulation. Fo~y-eight h later, input/output (I/O) curves were measured for current intensities ranging from 25 to 1000/zA. These curves were compared with I/O curves measured 7 days before each animal
experienced its first AD.
Histology Electrode placement was verified histologically using the Prussian blue technique. All animals had good OB and PC placements. Examination of tissue slices with a light microscope revealed that one animal (No. 833) in the Non-Kindled group had a lesion along the OB electrode tract. This animal also required excessive current (originally 800/~A and subsequently greater than 2000 pA compared to 400 p A maximum for the other animals) to trigger an AD. Therefore, this animal's data were excluded from analysis. Data analysis Regions of statistically significant differences between two AEPs from the same animal were determined by conducting a t-test, with P < 0.01, between each of the corresponding points of the two AEPs (see Fig. 1A, B). A total of 100 ms (500 digitized points), which included 5 ms of baseline activity, was analyzed for each comparison. For determining short-term (5-30 min) and long-term (1-3 days) effects of a focal AD, each rat's AEP immediately preceding the first AD was compared to each of its AEPs collected at various times after that AD (5, 15, 30 min, 24 and 48-72 h). This AEP was also used as the basis for comparison in assessing long-term (1-3 days) effects of kindling (Kindled group) or two focal ADs followed by daily 1 pulse/s stimulation (NonKindled group). The AEP recorded immediately before the 5th AD with clonus (Kindled group) was used as the basis for comparison in assessing shortterm (i.e. 5-30 min) effects of a kindled AD. Comparable short-term comparisons were made in nonkindled animals for assessing the effects of 1 pulse/s stimulation. Paired-pulse EPs were not analyzed because kindled animals showed a large increase in a late component (C3, discussed below) of the EP elicited by the first pulse which obscured the response to the second pulse. RESULTS
The pyriform cortex evoked potential A typical AEP elicited with 400/~A stimulation and before any ADs were triggered is presented in
64 Fig. 1A. For descriptive purposes, the A E P is divided into 4 components (C1-C4). At low current intensities (i.e. less than 400/~A) two components are seen, an initial surface-negative wave (C1) and a later surface-positive wave (C2). At higher current intensities, a second surface-negative wave (C3) and a later surface-positive wave (C4) are elicited (see Fig.
2
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25
The first and second A D s were short in duration (11.8 + 1.4 and 14.0 + 1.0 s, respectively; mean + S.E.M.) and did not produce any behavioral signs of seizure generalization such as head nodding, chewing or forelimb clonus. Small A D spikes could be recorded in the PC within 2 s after stimulation offset and grew in amplitude as the discharge continued. The A D threshold at the first A D was 229 + 41/~A. Fig. 2A and C illustrates the effect of a focal A D on the A E P for a single subject and mean values for all subjects, respectively. Short-term effects were similar at 5 and 30 min post A D although they were more prominent at 30 min. Two effects were consistently significant across animals, an increase in the amplitude of C1 (6 of 8 rats exhibited this at 5 and 30 min) and a prolongation of C4 (8 of 8 and 7 of 8 rats at 5 and 30 min, respectively). The effects on C1 and C4 persisted for 24 h in 5 of 8 rats and for 48-72 h in 4 of 8 rats.
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Fig. 1. A: typical averaged evoked potential (AEP) calculated from 10 potentials evoked in the pyriform cortex by stimulation of the olfactory bulb. The arrow indicates the stimulation artifact. The numbers 1-4 are used to label the different components (C1-C4) of the EP for descriptive purposes. Note that positive is up in this and all subsequent figures. B: statistical comparison of two AEPs, each consisting of 500 points (100 ms). The thickness at a point in AEP 2, which is the same AEP as in A, represents the critical interval for a t-test (P < 0.01) between that point and the corresponding point of AEP 1. The line (3) beneath the superimposed AEPs represents regions where the two AEPs differ significantly (i.e. 1 and 2 do not overlap). C: AEPs recorded above the superficial pyramidal cell layer at various current intensities (No. 828). Current intensities are 100,200,400 and 800/zA for 1-4, respectively. D: AEPs recorded below the superficial pyramidal cell layer at various current intensities (No. 823). Current intensities are the same as in C.
1C, D). The functional significance of these components is discussed below (see Discussion). The A E P recorded dorsal to the superficial pyramidal cell layer of the PC (Fig. 1C) is an approximate mirror image of the A E P recorded ventral to the cell layer (Fig. 1D), which demonstrates that the EPs are generated within the PC and not volume conducted from a more distant site.
Kindled animals required 11.6 + 1.8 (range: 8-17) A D s to reach kindling criterion. For the Kindled group, the A D s at kindling criterion lasted 74.2 + 4.4 s. The onset of forelimb clonus occurred 19.6 + 3.3 s after the termination of OB stimulation. Seizure generalization, as reflected by forelimb clonus, lasted 51.2 + 3.6 s. After the two A D s at the start, the Non-Kindled group received 10,0 + 2.6 daily control stimulations (range: 6-15). These stimulations never elicited an AD. An example of the effect of a kindled seizure on the A E P elicited by 400 ~ A is presented in Fig. 2B. Elicitation of a kindled seizure (Kindled group) produced an increase in the amplitude and duration of C4 which was consistently significant across animals
65 5 sin
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TIME AFTER 1st AFTERDISCHARGE Fig. 2. A: the effect of a focal olfactory bulb epileptiform afterdischarge on the pyriform cortex AEP (No. 823). The lines under the various AEPs signify regions where those AEPs differ significantly from the AEP (shown on the left) recorded immediately prior to the afterdischarge. All AEPs were elicited with 400/~A stimulation. B: short-term effects of an olfactory bulb kindled seizure on the pyriform cortex AEP (No. 823). This Kindled rat received high-frequency stimulation (50 pulses/s) of the olfactory bulb which elicited its 5th generalized afterdischarge. The lines under the various AEPs signify regions where those AEPs differ significantly from the AEP (shown on the left) recorded immediately prior to the 5th generalized afterdischarge. All AEPs were elicited with 400/~A stimulation. C: time course of the effect of a focal OB afterdischarge on various measures of the pyriform cortex AEP. C1, peak amplitude of C1. C2, peak amplitude of C2. C3, amplitude of C3 (measured from C2 peak amplitude to amplitude at 26 ms). C4, duration of C4 (measured from stimulation artifact to zero cross after C4). These data are based on mean values for 8 rats.
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(5 of 5 rats). This was a transient postictal effect which was absent 24 h after the seizure. Another consistent effect was an increase in the size of C3, which was most evident at 30 min post A D (significant in 5 of 5 rats). The 1 pulse/s stimulation (Non-Kindled
Fig. 3. Long-term effects of kindling on the pyriform cortex AEP. The lines under the various AEPs signify regions where those AEPs differ significantly from the AEP recorded immediately prior to the first afterdischarge (shown on the left). The delay intervals represent time since the last afterdischarge in kindling (Kindled rats) or the last 1 pulse/s stimulation (NonKindled rats). All AEPs were elicited with 400 #A stimulation.
66 should be noted that these rats had experienced two ADs 48-72 h apart at the beginning of the experiment in addition to subsequent daily 1 pulse/s stimulation. Therefore, these rats may be best considered as partially kindled. At 1 and 3 days following completion of 1 pulse/s stimulation, all of the Non-
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Kindled rats exhibited a significant increase in the amplitude of CI and an increase or prolongation of C4. Examination of Fig. 4 indicates that this longterm potentiation of C1 amplitude is observed over a wide range of current intensities. Three of 5 Kindled rats demonstrated a significant increase in C1 amplitude at 1 and 3 days after kindling criterion. Fig. 4 indicates that this effect can be observed over a wide range of current intensities. The AEPs of two kindled animals (No, 823 and 829) appear qualitatively different after kindling. C2 is absent and C3 is much larger in relation to the size of the other components. Although one of the NonKindled rats (No. 834) demonstrated an increase in C3, it was small by comparison. DISCUSSION
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Fig. 4. Effect of kindling on I/O relations for C1 amplitude. A: I/O curves for Kindled rats were measured 7 days before the first AD and after kindling (48 h following a generalized AD). Data points are averages for 5 rats. B: I/O curves for NonKindled rats. The first I/O curve was measured 7 days before the first AD. The second I/O curve was measured 11-20 days after the second AD. After the second AD, these rats received daily 1 pulse/s stimulation until 5 days prior to measurement of the second I/O curve followed by one additional train 48 h before its measurement. Data points are averages for 3 rats.
The waveform of the pyriform cortex EP observed in the present experiment is similar to the EP elicited with OB stimulation in the rat a and rabbit 34 and with L O T stimulation in the rat3L cat t,s, rabbit 34 and opossum~5, ~6. Previous research has produced reasonable agreement, based upon both anatomical and physiological data, concerning the neuronal events which occur in the PC in response to L O T stimulation 1.8,9A4-16,3435. C1 (see Fig. 1A) is a reflection of two distinct events, a monosynaptic and disynaptic population excitatory postsynaptic potential (EPSP) in PC pyramidal cells. These two EPSPs (termed A l and B 1, respectively, by Haberly 15) are differentially affected by conditioning shocks and pharmacological manipulation16,35. The subsequent surface-positive waves (C2 and C4 in Fig. 1A) are temporally associated with inhibitory postsynaptic potentials (IPSPs) and inhibition of firing in PC pyramidal cells following their activationl,S, 9A6 and may be a reflection of feedforward and/or recurrent inhibition mediated via inhibitory interneurons ~6. C3 is typically not seen in vivo with L O T stimulationl,S,16.34,35; however, a component of similar polarity and latency has been reported for in vivo stimulation of the OB in the rabbit 34 and rat 4. The neuronal elements which generate this component are unknown at present. It does not appear to be dependent upon L O T stimulation because Satou et at. 34 report that it can still be evoked by OB stimula-
67 tion after section of the LOT, which abolishes all other components of the EP.
Seizure-induced changes in the evoked potential A single focal AD increased C1 amplitude as early as 5 min after the AD, and this effect persisted for 48-72 h. At present we cannot determine with certainty whether this change recorded in the PC represents a functional change in the OB (e.g. altered mitral cell responsiveness to the stimulation), the PC (e.g. increased transmitter release, increased postsynaptic response) or a combination of the above. Nevertheless, this effect appears similar to results obtained in the hippocampal formation. Douglas and Goddard 5 reported that the first kindling stimulus applied to the entorhinal cortex increased the amplitude of the monosynaptic EPSP and population spike recorded in the dentate gyrus, and these effects persisted at least 5 days. In addition, Maru et al. xl reported that the first AD elicited in the perforant path produced an increase in the dentate gyrus EPSP as early as 7 min after the AD and this effect persisted for at least 24 h. These data suggest that a single episode of relatively localized and brief epileptiform activity in a set of neurons can produce a rapid and persistent effect on neuronal communication within the seizure's path. The observation that a focal AD produces a qualitatively similar effect with similarly rapid onset in two separate limbic pathways suggests that this phenomenon may occur in other forebrain sites as well. A highly consistent effect observed in the present experiment was an increase in the amplitude and duration of C4 following seizure activity. This effect appeared within 5 min of a focal AD and was still evident 48-72 h later. In Non-Kindled animals, this effect was still present 9-18 days after the last of two focal ADs (3 days following their last I pulse/s stimulation). These data suggest that one or two ADs produce a relatively permanent increase in C4. As indicated above, such an effect might be indicative of enhanced inhibitory mechanisms in the PC. However, this effect may be secondary to the increase in C1 amplitude if C4 is a response to excitation of pyramidal cells, as it would be if C4 reflects recurrent inhibition. In addition, the changes which occurred in C4 may not have been due entirely to seizure activity. Recent evidence indicates that high frequency electrical
stimulation of the OB below AD threshold produces a large increase in C2 or C4 without any corresponding effect on C1, and that a small effect can be produced even by 1 pulse/s stimulation 41. Animals in the present experiment received single- and paired-pulse stimulation during EP testing and either 50 pulse/s (Kindled) or 1 pulse/s (Non-Kindled) stimulation as the experimental treatment. Therefore, at present we cannot specify the relative contributions of seizure activity and electrical stimulation to the changes in C4 seen in this experiment. In Kindled rats, an increase in the amplitude and duration of C4 was also consistently observed 5-30 min after a generalized AD, but the effect had disappeared by 24 h. This transient postictal change in the EP may have implications concerning the mechanisms responsible for seizure-inhibitory effects which are seen shortly following a previous kindled seizure7,13,17,23,33. However, based on reasoning similar to the above, we cannot specify the relative contributions of the generalized seizure and high-frequency electrical stimulation to this effect. The large increase in C3 in Kindled rats appears to have been caused specifically by the kindling process. Racine et al. 3° have reported that kindling produced by LOT stimulation elicits a spike-type component in the pyriform cortex EP during periods when spontaneous interictal spikes are present. The AEPs from the two animals which showed a large increase in C3 (No. 823 and 829 in Fig. 3) resemble the data of Racine et al. 30. The results from the present experiment indicate that the increase in C3 is long-lasting but exhibits some decline from 1 to 3 days following a generalized AD. If this component reflects extraLOT input 34, then its increase following kindling indicates that seizure-induced changes in PC function are not confined to LOT input. A phenomenon of interest related to kindling is long-term potentiation (LTP) 2,3,5,6,11,29,which refers to an increase in the initial EPSP component of the EP following repeated electrical stimulation. Highfrequency stimulation is most effective for producing LTP 6 and eliciting an AD 13. Research on LTP in the dentate gyrus indicates clearly that elicitation of an AD is not necessary to initiate LTP 5. In this respect, the projection from the OB to the PC may differ from the perforant path. For example, Racine et al. 30 produced little or no LTP via high-frequency LOT stimu-
68 lation below A D threshold, even though similar stimulation of other limbic forebrain sites produced prominent LTP effects. In contrast, kindling via stim-
study indicates that a single A D triggered by OB stimulation produces a rapid and long-term potentiation of the EPSP component of the EP.
ulation of the L O T reliably produced an increase in response amplitude 30. We have observed a similar pattern of results for OB stimulation. High-frequen-
ACKNOWLEDGEMENT
cy OB stimulation below A D threshold does not increase C1 (although it does increase the amplitude and duration of C2 or C4) 41. In contrast, the present
This research was supported by a grant from the Marie Wilson Howells Memorial Fund.
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