Physiology & Behavior, Vol. 51, pp. 255-266. ©PergamonPress plc, 1992. Printedin the U.S.A.
0031-9384/92$5.00 + .00
Behavioural and Electrophysiological Studies of Entorhinal Cortex Lesions in the Rat J. J. H A G A N , t E. E. V E R H E I J C K , M. H. S P I G T A N D G. S. F. R U I G T CNS Pharmacology Laboratories, Organon International BV, P.O. Box 20, 5340 B H Oss, The Netherlands Received 14 February 1991 HAGAN, J. J., E. E. VERHEIJCK, M. H. SPIGT AND G. S. F. RUIGT. Behavioural and electrophysiological studies of entorhinal cortex lesions in the rat. PHYSIOL BEHAV 51(2) 255-266, 1992.--Bilateral ibotenic acid injections aimed at the entorhinal cortex (EC) lesioned the EC and subiculum in 30% of animals (group EC/S) and caused additional hippocampal damage in 50% (group RH). Both lesions increased acetylcholinesterase (ACHE) staining in the intermediate molecular layer of the dentate gyms. EC/S lesions increased diurnal deep sleep and the incidence of spindles but decreased REM sleep. RH lesions increased nocturnal deep sleep and decreased nocturnal quiet sleep. Both lesions reduced power over the theta frequency range from 6-10 Hz for epochs of REM sleep and quiet waking but not deep sleep. Peak frequency was unaffected. The RH group and a subset of the EC/S group were nocturnally, but not diurnally, hyperactive. Six weeks after the lesion there was no evidence for hyperactivity in a novel open field. The EC/S lesion impaired exploration as indicated by reduced motility and rearing in an open field and by the failure of EC/S-lesioned rats to increase contact time in response to a novel olfactory cue. Place navigation learning in a Morris maze was not affected by EC/S or RH lesions. However, when the spatial location of the hidden platform was shifted EC/S-lesioned rats were impaired. The sprouting response, reduced theta power and exploration deficits resemble those reported following electrolytic lesions, but the lack of effect on place navigation learning contrasts with reports of impaired spatial learning following electrolytic lesions. The data prompt a reexamination of the role which the EC projection to the hippocampus plays in spatial learning. Ibotenic acid Sleep/waking Theta rhythm Hyperactivity Learning Memory Entorhinal cortex/subiculum Rat
THE purpose of these experiments was to study the effects of bilateral ibotenic acid lesions of the entorhinal cortex on conditioned and unconditioned behaviour, sleep patterns and EEG. Theories concerning the role of the EC suggest an integrative function between the cortex and limbic system, a view partly based on neuroanatomical evidence. This demonstrates a substantial innervation by the cortex, amygdala, thalamus and olfactory bulb (18, 22, 52, 56) and the existence of a large projection, primarily glutamatergic or aspartergic (24, 31, 51, 57) and GABAergic (7) to the hippocampus (22, 52, 56), mammillary bodies (46) and the nucleus accumbens (19). Neurophysiological studies reinforce the conceptual link between hippocampal and entorhinal functions. Theta sources are found not only in the CA1 and dentate gyrus (54) but also in the medial EC (1, 2, 25). Furthermore, EC lesions abolish CA1 theta (54) and decrease theta amplitude in the dentate gyrus (26). Finally, only atropine-sensitive theta survives in the hippocampus after EC lesions (26, 54, 55.) Behavioural experiments underline the similarities between the effects of EC and hippocampal lesions. Deficits following EC lesions have been reported in 'T' mazes (20, 36-38, 43), radial mazes (14,34), place navigation (8, 9, 45), olfactory discrirnination learning (48) and other complex mazes (29,49). Learning or performance deficits are often persistent (9, 21, 45, 50), although partial functional recovery has been reported (8,
Open-field novelty
Place-navigation
37, 45). Hyperactivity is commonly reported (5, 17, 21, 29, 36, 39, 44, 45, 53), although the degree may depend upon lesion size (29,44) and postoperative period (5, 6, 21, 50). Activity eventually returns to control levels, with some evidence of residual nocturnal hyperactivity (6). Increased exploratory activity may cause the hyperactivity (17), although some data suggests decreased exploration despite hyperactivity (30), indicating that exploratory activity is actually reduced following EC lesions (30). The role of the EC in mediating the behavioural effects of lesions is equivocal for two reasons. First, the extent of damage to adjacent structures varies enormously between studies. In some, damage affects the subiculum (16, 17, 20, 36-38, 44, 45, 49, 50), amygdala and ventral hippocampus (5, 26, 54). Second is the uncertain contribution of damage caused by electrolytic or aspirative lesions to fibres of passage and adjacent fibre tracts. This is a long-standing issue in the interpretation of hippocampal lesion damage (11,57) in primates and in rats (12-14) and is emerging as a problem in the interpretation of EC functions. Despite extensive evidence that electrolytic damage to the area profoundly affects spatial learning tasks (8, 9, 14, 20, 29, 34, 36-38, 45, 49, 50), fibre-sparing excitotoxic lesions of the retrohippocampal area failed to impair the acquisition of spatial problem learning (3). To minimise the confounding influence of damage to the
lRequests for reprints should be addressed to J. J. Hagan at his present addres: SmithKline Beecham Medicinal Research Centre, Coldharbour Road, The Pinnacles, Harlow Essex CM19 5AD, England. 255
256
HAGAN, VERHEIJCK, SPIGT ANI:~ R!'l(i!
hippocampus and fibres of passage, a bilateral excitotoxic lesion was developed which avoided hippocampal penetration by the injection cannula. The first batch of animals was studied in an automated EEG system for analysing sleep/waking behaviour (40,41). Spectral EEG analysis was subsequently carried out on samples from quiet waking; REM sleep and deep sleep. Circadian variation in locomotion and eating was studied in the same animals over a period of 7 days. Open-field activity and responses to a novel olfactory cue were also evaluated (30). A second batch of animals was trained on place navigation in a Morris water maze (8, 9, 45) and were subsequently retrained with the escape platform shifted to the opposite side of the pool. Rats from the second batch were stained for acetylcholinesterase (ACHE) (15,35) to determine whether excitotoxic damage caused a similar sprouting response, as electrolytic lesions of the EC have been shown to stimulate sprouting of the septal, commissural and association pathways into the vacated dendritic zones of the dentate gyms (4, 23, 33, 47, 58) METHOD
Subjects Adult male Wistar rats (Cpb: Wu, TNO Zeist, The Netherlands), weighing 225-250 g on the day of operation, were used. They were individually housed in a temperature- (t8-23°C) and humidity- (60%) controlled environment under a 12-hour light/ dark cycle (lights on 7 a.m.). Food and water were available ad lib.
Surgery Two batches of animals were prepared in slightly different ways for the different experiments. Both were given excitotoxic lesions, but those in the first batch were also equipped with EEG recording electrodes. The animals were anaesthetised with Nembutal T M (60 mg/kg, IP) and mounted in a stereotaxic apparatus (Diosynth, Oss, The Netherlands) in the skull fiat position (35). Ibotenic acid (Cambridge Research Biochemicals) was dissolved in phosphate-buffered saline (pH 7.4) at a concentration of 10 mg/ml and stored ( - 2 0 ° C ) in 20 Ixl batches until used. Solutions were infused into the brain (0.1 ixl/min) using a blunt tipped stainless steel cannula (0.45 mm external diameter) connected via polyethylene tubing to a microinfusion pump (CMA 100). The cannula was positioned at an angle of 20 ° to the coronal plane with the tip pointing anterior, and 5 ° to the sagittal plane with the tip pointing to the midline. The first injection (0.3 txl) was made at the following coordinates relative to the interaural point: A P = 0 . 8 , L = 5 . 5 , D V = 4 . 2 . The cannula was then withdrawn to the second injection location (DV = 6.7) and a further 0.4 ixl injected. The process was repeated for the other side of the brain. The cannula was left in place for 5 minutes after completion of each injection to allow passive diffusion. Control rats were injected with phosphate-buffered saline. For both batches, 22 rats were lesioned with ibotenic acid and 10 were prepared as operated controls. Following the lesion procedure rats from the first batch (Experiments 1, 2, 3) were equipped with two epidural monopolar screw electrodes (stainless steel DIN 84) for registration of EEG (40). Briefly, three electrodes were screwed into holes drilled in the skull over the parieto-occipital cortex at the following coordinates relative to the interaural point: A P = 0 , L=O, and A P = 2, L = 2. A grounding electrode was placed over the frontal cortex AP = 13,4 L = 0). For electromyogram recordings two stainless steel Teflon coated electrodes (Narco 231-0010), insulated ex-
cept for the tip, were bilaterally implanted in the dorsal ned, musculature (m. platysma and m. acromio trapeziu,~ , All e!ec trodes were soldered to a connecter (type 8059-2G5, Augat l n c . Attlebora, USA). Two additional fixing screws were placed bilaterally ( A P = 6, L = 3) prior to embedding the connecter m the skull using dental cement. The animals were allowed to recover from surgery for at least l0 days. The second batch (Experiment 4) was prepared in the same way without electrodes. These a n i mals had their wounds stitched and dressed following the lesion procedure.
Histology Rats were deeply anaesthetised, decapitated, and their brains removed onto dry ice ( - 80°C). Coronal sections (22 I.tm) were cut on a freezing microtome (Minotome, Int. Equip. Corp.I and every 5th section through the lesioned area was saved for cresyl violet staining (35). With animals from the second batch thicker sections were cut (32 Ixm) and a parallel series of sections was saved for acetylcholinesterase staining (15.35). Following histological examination the first batch of animals. which were used in Experiments 1, 2 and 3. were divided into throe groups. Five had restricted or unilateral lesions and were excluded from further analysis. Seven rats ( E e l s lesions) had damage restricted to the entorhinal cortex and subiculum (see Fig. 11. At the caudal boundary of the lesion (level 0.7 in Fig. 1) both entorhinal and perirhinal cortex were damaged. At a more rostral level (level 1.37) lateral and medial EC were lesioned together with the greater part of the subiculum, but preand parasubiculum were undamaged. At its most rostral extent (level 2.7) damage was restricted to the medial EC and the lateral EC was largely spared. There was no damage to the hippocampus proper. Nine rats (RH lesions) also suffered quite extensive damage to the CA1 and dentate gyms in the posterior ventral hippocampus. Lesions in this group tended to be larger, extended more rostrally and affected adjacent cortical areas more than EC/S lesion, The same distribution was found in the second batch of rats, which were used in Experiment 4 on place navigation. AChE staining of the dorsal hippocampus revealed a marked increase in staining density along a narrow band in the intermediate zone of the molecular layer of the dentate gyms (see Fig. 2). EXPERIMENT 1: SLEEP/WAKINGBEHAVIOUR After a recovery period of at least 10 days EEG defined sleep-waking behaviour was measured (40). During recording the rats were individually housed for 26 hours in a silent room in stainless steel experimental cages (33 × 25 × 45 cm) placed on rubber pads for vibration isolation. Each rat was connected through a 6-wire fiat cable and a swivel connector (Air precision, Type APCL 13, Plessis Robinson, France) to ~m impedance transformer (gain 10 x ), located above the cage. The EEG signal was amplified (total gain of 25,000), bandpass filtered (3 dB at 0.5 and 100 Hz), sampled at 128 Hz and digitised by a 10-bit analog/digital (A/D) converter. The EMG signal was also amplified 25,000 times, bandpass filtered (3 dB at 100 and 300 Hz), positively rectified and integrated. The movement of each rat was detected by amplifying (total gain of 100) the capacitative movement artefacts generated by an open-ended wire in the fiat cable between the rat and swivel contact. The signal was bandpass filtered (3 dB at 0.5 and 100 Hz), rectified and integrated.
Data Analysis Epoch duration was set at 2 s by a data controller which transferred the data to a PDP 11/40 minicomputer system and in
ENTORHINAL CORTEX AND BEHAVIOUR
257
RH LESIONS
EC/S LESIONS
:
ill
0.~i ~'':-~;
2.7ram
- ~ "
ii
i
'i
J
3.2mm FIG. 1. Schematic reconstruction of the largest and smallest lesions in the entorhinal cortexand subiculum-lesioned group (EC/S) and in the group with additional damage to the posteroventral hippocampus (RH). Coronal levels are taken from the atlas of Paxinos and Watson (35)////--large lesion; \\\\--small lesion.
parallel to an Ampex tape unit. EEG power spectra were calculated per rat and per epoch and averaged power values within 5 predefined frequency bands were stored on disk for later off-line classification of sleep-waking stages. Classification was based on a set of EEG discriminant parameters which were derived for each rat from a discriminant analysis of visually scored polygraphic recordings of 100 representative epochs for each of six different classes of sleep-waking behaviour. A more detailed description of the calculation of discrimination parameters is given elsewhere (39,40). Active waking (A1). The EEG comprises low voltage, fast activity consisting predominantly of theta and superimposed high frequencies. The EMG and movement levels are high. Quiet waking (A2). This is the same as active waking but
with a low movement level. Quiet sleep (Q). The EEG contains lower frequencies against a background of nearly no theta. EEG spindles frequently occur which often last for several seconds and are nearly invariably preceded by a short movement of the whiskers. The amplitude is intermediate between waking and deep sleep. EMG is medium to high. Deep sleep (D) is characterised by high amplitude, low frequency EEG in the delta range concomitant with moderate to high EMG. Spindles may be present but are generally obscured by the low frequency activity. Pre-REM sleep (P). There is a clear theta rhythm in the EEG which is frequently interrupted by short-lasting (1-3 s) high-amplitude spindles. The EMG level is low.
258
H A G A N . V E R H E I J C K , S P I G T 4NI.) Rt!I(]~
HG. 2. AChE-stained coronal section through dorsal hippocampus of control (A) and EC/S-lesioned (BI rat. Note increased density of staining between outer (MO) and inner (MI) molecular layer of dentate gyms in EC/S-lesioned rat.
ENTORHINAL CORTEX AND BEHAVIOUR
Hours Epochs
259
Dark period 1-1 Control [ ] EC/S • RH
~ _ _
i1 ,_,,,n,,,ill 1000"1
rll l A1
A2
Q
oo.
O
P
n ~ m
~ ) m
,_,., ml o., R
S
FIG. 3. Average time spent in automatically classified sleep/wake stages during the dark and light periods of a 24-hour recording session. Sleep was based on 2-s epochs. A1 = active waking, A2 =quiet waking, Q = quiet sleep, D= deep sleep, P=Pre-REM sleep, S=Spindles. Shams N= 10; EC lesion N=6; RH lesion N=9. *p
0031-9384/92$5.00 + .00
Behavioural and Electrophysiological Studies of Entorhinal Cortex Lesions in the Rat J. J. H A G A N , t E. E. V E R H E I J C K , M. H. S P I G T A N D G. S. F. R U I G T CNS Pharmacology Laboratories, Organon International BV, P.O. Box 20, 5340 B H Oss, The Netherlands Received 14 February 1991 HAGAN, J. J., E. E. VERHEIJCK, M. H. SPIGT AND G. S. F. RUIGT. Behavioural and electrophysiological studies of entorhinal cortex lesions in the rat. PHYSIOL BEHAV 51(2) 255-266, 1992.--Bilateral ibotenic acid injections aimed at the entorhinal cortex (EC) lesioned the EC and subiculum in 30% of animals (group EC/S) and caused additional hippocampal damage in 50% (group RH). Both lesions increased acetylcholinesterase (ACHE) staining in the intermediate molecular layer of the dentate gyms. EC/S lesions increased diurnal deep sleep and the incidence of spindles but decreased REM sleep. RH lesions increased nocturnal deep sleep and decreased nocturnal quiet sleep. Both lesions reduced power over the theta frequency range from 6-10 Hz for epochs of REM sleep and quiet waking but not deep sleep. Peak frequency was unaffected. The RH group and a subset of the EC/S group were nocturnally, but not diurnally, hyperactive. Six weeks after the lesion there was no evidence for hyperactivity in a novel open field. The EC/S lesion impaired exploration as indicated by reduced motility and rearing in an open field and by the failure of EC/S-lesioned rats to increase contact time in response to a novel olfactory cue. Place navigation learning in a Morris maze was not affected by EC/S or RH lesions. However, when the spatial location of the hidden platform was shifted EC/S-lesioned rats were impaired. The sprouting response, reduced theta power and exploration deficits resemble those reported following electrolytic lesions, but the lack of effect on place navigation learning contrasts with reports of impaired spatial learning following electrolytic lesions. The data prompt a reexamination of the role which the EC projection to the hippocampus plays in spatial learning. Ibotenic acid Sleep/waking Theta rhythm Hyperactivity Learning Memory Entorhinal cortex/subiculum Rat
THE purpose of these experiments was to study the effects of bilateral ibotenic acid lesions of the entorhinal cortex on conditioned and unconditioned behaviour, sleep patterns and EEG. Theories concerning the role of the EC suggest an integrative function between the cortex and limbic system, a view partly based on neuroanatomical evidence. This demonstrates a substantial innervation by the cortex, amygdala, thalamus and olfactory bulb (18, 22, 52, 56) and the existence of a large projection, primarily glutamatergic or aspartergic (24, 31, 51, 57) and GABAergic (7) to the hippocampus (22, 52, 56), mammillary bodies (46) and the nucleus accumbens (19). Neurophysiological studies reinforce the conceptual link between hippocampal and entorhinal functions. Theta sources are found not only in the CA1 and dentate gyrus (54) but also in the medial EC (1, 2, 25). Furthermore, EC lesions abolish CA1 theta (54) and decrease theta amplitude in the dentate gyrus (26). Finally, only atropine-sensitive theta survives in the hippocampus after EC lesions (26, 54, 55.) Behavioural experiments underline the similarities between the effects of EC and hippocampal lesions. Deficits following EC lesions have been reported in 'T' mazes (20, 36-38, 43), radial mazes (14,34), place navigation (8, 9, 45), olfactory discrirnination learning (48) and other complex mazes (29,49). Learning or performance deficits are often persistent (9, 21, 45, 50), although partial functional recovery has been reported (8,
Open-field novelty
Place-navigation
37, 45). Hyperactivity is commonly reported (5, 17, 21, 29, 36, 39, 44, 45, 53), although the degree may depend upon lesion size (29,44) and postoperative period (5, 6, 21, 50). Activity eventually returns to control levels, with some evidence of residual nocturnal hyperactivity (6). Increased exploratory activity may cause the hyperactivity (17), although some data suggests decreased exploration despite hyperactivity (30), indicating that exploratory activity is actually reduced following EC lesions (30). The role of the EC in mediating the behavioural effects of lesions is equivocal for two reasons. First, the extent of damage to adjacent structures varies enormously between studies. In some, damage affects the subiculum (16, 17, 20, 36-38, 44, 45, 49, 50), amygdala and ventral hippocampus (5, 26, 54). Second is the uncertain contribution of damage caused by electrolytic or aspirative lesions to fibres of passage and adjacent fibre tracts. This is a long-standing issue in the interpretation of hippocampal lesion damage (11,57) in primates and in rats (12-14) and is emerging as a problem in the interpretation of EC functions. Despite extensive evidence that electrolytic damage to the area profoundly affects spatial learning tasks (8, 9, 14, 20, 29, 34, 36-38, 45, 49, 50), fibre-sparing excitotoxic lesions of the retrohippocampal area failed to impair the acquisition of spatial problem learning (3). To minimise the confounding influence of damage to the
lRequests for reprints should be addressed to J. J. Hagan at his present addres: SmithKline Beecham Medicinal Research Centre, Coldharbour Road, The Pinnacles, Harlow Essex CM19 5AD, England. 255
256
HAGAN, VERHEIJCK, SPIGT ANI:~ R!'l(i!
hippocampus and fibres of passage, a bilateral excitotoxic lesion was developed which avoided hippocampal penetration by the injection cannula. The first batch of animals was studied in an automated EEG system for analysing sleep/waking behaviour (40,41). Spectral EEG analysis was subsequently carried out on samples from quiet waking; REM sleep and deep sleep. Circadian variation in locomotion and eating was studied in the same animals over a period of 7 days. Open-field activity and responses to a novel olfactory cue were also evaluated (30). A second batch of animals was trained on place navigation in a Morris water maze (8, 9, 45) and were subsequently retrained with the escape platform shifted to the opposite side of the pool. Rats from the second batch were stained for acetylcholinesterase (ACHE) (15,35) to determine whether excitotoxic damage caused a similar sprouting response, as electrolytic lesions of the EC have been shown to stimulate sprouting of the septal, commissural and association pathways into the vacated dendritic zones of the dentate gyms (4, 23, 33, 47, 58) METHOD
Subjects Adult male Wistar rats (Cpb: Wu, TNO Zeist, The Netherlands), weighing 225-250 g on the day of operation, were used. They were individually housed in a temperature- (t8-23°C) and humidity- (60%) controlled environment under a 12-hour light/ dark cycle (lights on 7 a.m.). Food and water were available ad lib.
Surgery Two batches of animals were prepared in slightly different ways for the different experiments. Both were given excitotoxic lesions, but those in the first batch were also equipped with EEG recording electrodes. The animals were anaesthetised with Nembutal T M (60 mg/kg, IP) and mounted in a stereotaxic apparatus (Diosynth, Oss, The Netherlands) in the skull fiat position (35). Ibotenic acid (Cambridge Research Biochemicals) was dissolved in phosphate-buffered saline (pH 7.4) at a concentration of 10 mg/ml and stored ( - 2 0 ° C ) in 20 Ixl batches until used. Solutions were infused into the brain (0.1 ixl/min) using a blunt tipped stainless steel cannula (0.45 mm external diameter) connected via polyethylene tubing to a microinfusion pump (CMA 100). The cannula was positioned at an angle of 20 ° to the coronal plane with the tip pointing anterior, and 5 ° to the sagittal plane with the tip pointing to the midline. The first injection (0.3 txl) was made at the following coordinates relative to the interaural point: A P = 0 . 8 , L = 5 . 5 , D V = 4 . 2 . The cannula was then withdrawn to the second injection location (DV = 6.7) and a further 0.4 ixl injected. The process was repeated for the other side of the brain. The cannula was left in place for 5 minutes after completion of each injection to allow passive diffusion. Control rats were injected with phosphate-buffered saline. For both batches, 22 rats were lesioned with ibotenic acid and 10 were prepared as operated controls. Following the lesion procedure rats from the first batch (Experiments 1, 2, 3) were equipped with two epidural monopolar screw electrodes (stainless steel DIN 84) for registration of EEG (40). Briefly, three electrodes were screwed into holes drilled in the skull over the parieto-occipital cortex at the following coordinates relative to the interaural point: A P = 0 , L=O, and A P = 2, L = 2. A grounding electrode was placed over the frontal cortex AP = 13,4 L = 0). For electromyogram recordings two stainless steel Teflon coated electrodes (Narco 231-0010), insulated ex-
cept for the tip, were bilaterally implanted in the dorsal ned, musculature (m. platysma and m. acromio trapeziu,~ , All e!ec trodes were soldered to a connecter (type 8059-2G5, Augat l n c . Attlebora, USA). Two additional fixing screws were placed bilaterally ( A P = 6, L = 3) prior to embedding the connecter m the skull using dental cement. The animals were allowed to recover from surgery for at least l0 days. The second batch (Experiment 4) was prepared in the same way without electrodes. These a n i mals had their wounds stitched and dressed following the lesion procedure.
Histology Rats were deeply anaesthetised, decapitated, and their brains removed onto dry ice ( - 80°C). Coronal sections (22 I.tm) were cut on a freezing microtome (Minotome, Int. Equip. Corp.I and every 5th section through the lesioned area was saved for cresyl violet staining (35). With animals from the second batch thicker sections were cut (32 Ixm) and a parallel series of sections was saved for acetylcholinesterase staining (15.35). Following histological examination the first batch of animals. which were used in Experiments 1, 2 and 3. were divided into throe groups. Five had restricted or unilateral lesions and were excluded from further analysis. Seven rats ( E e l s lesions) had damage restricted to the entorhinal cortex and subiculum (see Fig. 11. At the caudal boundary of the lesion (level 0.7 in Fig. 1) both entorhinal and perirhinal cortex were damaged. At a more rostral level (level 1.37) lateral and medial EC were lesioned together with the greater part of the subiculum, but preand parasubiculum were undamaged. At its most rostral extent (level 2.7) damage was restricted to the medial EC and the lateral EC was largely spared. There was no damage to the hippocampus proper. Nine rats (RH lesions) also suffered quite extensive damage to the CA1 and dentate gyms in the posterior ventral hippocampus. Lesions in this group tended to be larger, extended more rostrally and affected adjacent cortical areas more than EC/S lesion, The same distribution was found in the second batch of rats, which were used in Experiment 4 on place navigation. AChE staining of the dorsal hippocampus revealed a marked increase in staining density along a narrow band in the intermediate zone of the molecular layer of the dentate gyms (see Fig. 2). EXPERIMENT 1: SLEEP/WAKINGBEHAVIOUR After a recovery period of at least 10 days EEG defined sleep-waking behaviour was measured (40). During recording the rats were individually housed for 26 hours in a silent room in stainless steel experimental cages (33 × 25 × 45 cm) placed on rubber pads for vibration isolation. Each rat was connected through a 6-wire fiat cable and a swivel connector (Air precision, Type APCL 13, Plessis Robinson, France) to ~m impedance transformer (gain 10 x ), located above the cage. The EEG signal was amplified (total gain of 25,000), bandpass filtered (3 dB at 0.5 and 100 Hz), sampled at 128 Hz and digitised by a 10-bit analog/digital (A/D) converter. The EMG signal was also amplified 25,000 times, bandpass filtered (3 dB at 100 and 300 Hz), positively rectified and integrated. The movement of each rat was detected by amplifying (total gain of 100) the capacitative movement artefacts generated by an open-ended wire in the fiat cable between the rat and swivel contact. The signal was bandpass filtered (3 dB at 0.5 and 100 Hz), rectified and integrated.
Data Analysis Epoch duration was set at 2 s by a data controller which transferred the data to a PDP 11/40 minicomputer system and in
ENTORHINAL CORTEX AND BEHAVIOUR
257
RH LESIONS
EC/S LESIONS
:
ill
0.~i ~'':-~;
2.7ram
- ~ "
ii
i
'i
J
3.2mm FIG. 1. Schematic reconstruction of the largest and smallest lesions in the entorhinal cortexand subiculum-lesioned group (EC/S) and in the group with additional damage to the posteroventral hippocampus (RH). Coronal levels are taken from the atlas of Paxinos and Watson (35)////--large lesion; \\\\--small lesion.
parallel to an Ampex tape unit. EEG power spectra were calculated per rat and per epoch and averaged power values within 5 predefined frequency bands were stored on disk for later off-line classification of sleep-waking stages. Classification was based on a set of EEG discriminant parameters which were derived for each rat from a discriminant analysis of visually scored polygraphic recordings of 100 representative epochs for each of six different classes of sleep-waking behaviour. A more detailed description of the calculation of discrimination parameters is given elsewhere (39,40). Active waking (A1). The EEG comprises low voltage, fast activity consisting predominantly of theta and superimposed high frequencies. The EMG and movement levels are high. Quiet waking (A2). This is the same as active waking but
with a low movement level. Quiet sleep (Q). The EEG contains lower frequencies against a background of nearly no theta. EEG spindles frequently occur which often last for several seconds and are nearly invariably preceded by a short movement of the whiskers. The amplitude is intermediate between waking and deep sleep. EMG is medium to high. Deep sleep (D) is characterised by high amplitude, low frequency EEG in the delta range concomitant with moderate to high EMG. Spindles may be present but are generally obscured by the low frequency activity. Pre-REM sleep (P). There is a clear theta rhythm in the EEG which is frequently interrupted by short-lasting (1-3 s) high-amplitude spindles. The EMG level is low.
258
H A G A N . V E R H E I J C K , S P I G T 4NI.) Rt!I(]~
HG. 2. AChE-stained coronal section through dorsal hippocampus of control (A) and EC/S-lesioned (BI rat. Note increased density of staining between outer (MO) and inner (MI) molecular layer of dentate gyms in EC/S-lesioned rat.
ENTORHINAL CORTEX AND BEHAVIOUR
Hours Epochs
259
Dark period 1-1 Control [ ] EC/S • RH
~ _ _
i1 ,_,,,n,,,ill 1000"1
rll l A1
A2
Q
oo.
O
P
n ~ m
~ ) m
,_,., ml o., R
S
FIG. 3. Average time spent in automatically classified sleep/wake stages during the dark and light periods of a 24-hour recording session. Sleep was based on 2-s epochs. A1 = active waking, A2 =quiet waking, Q = quiet sleep, D= deep sleep, P=Pre-REM sleep, S=Spindles. Shams N= 10; EC lesion N=6; RH lesion N=9. *p
