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Carbachol-induced EEG ‘theta’ activity in hippocampal brain slices
196
BraiJt Rew,rc/t. 41)5f ]~)~7)it)t~ ]t).~
BRE 22076
Carbachol.indu
EEG 'theta' activity in hippocampal brain slices
Jan Konopacki 1, M. Bruce Maclver 2, Brian H. Bland 3 and Sheldon H. Roth: t Department of Animal Physiology, The University of Lodz, Lodz (Poland), 2Department of Pharmacology and Therapeutics and 3Department of Psychology, The University of Calgary, Calgary (Canada)
(Accepted 28 October 1986) Key words: 0; Hippocampal brain slice; Carbachol; Muscarinic; Electro encephalogram(EEG)
Application of the cholinergic agonist carbachol (50 pM) produced 0-like rhythmical waveforms, recorded in the stratum moleculare of the dentate gyrus. Atropine sulfate (50 pM) antagonized the carbachol-induced0-like activity, consistent with this action of atropine in vivo. These results provide the first direct evidence that hippocampalneurons are capable of producingsynchronizedslowwave activitywhen isolated from pulsed rhythmic inputs of the medial septum and other brain regions. Theta rhythms are electroencephalographic activities consisting of rhythmical sinusoidal-like slow waves. They are the largest (several millivolts), most prominent, and well-characterized rhythmical waveforms generated by the mammalian brain 12'17. Topographic studies of 0 have shown that it is localized to the stratum oriens of the hippocampal formation and the stratum moleculare of the dentate gyrus a'2. Behavioral and pharmacological studies suggest there are two types of 0; one has been termed type 1 and is correlated with voluntary motor behavior 3'7'15 and the other, termed type 2 0, can occur independently during immobility and is thought to be correlated with sensory processing specific to the organization of voluntary motor behaviors 4. Type 2 0 appears to be mediated by a cholinergic septohippocampal pathway, since lesions of the medial septum completely abolish hippocampal 0, and septal neuron rhythmic discharge activity is phase-locked to the hippocampal formation 0 rhythm 6'8'9A°'13. Type 2 0 can be produced by systemic injections of anticholinesterase agents like eserine and blocked by muscarinic antagonists such as atropine sulfate or scopolamine 16. Recently, Rowntree and Bland le demonstrated that type 2 0 could be produced in vivo by direct microinfusion of carbachol into the hippocampal formation, and that this 0 could be antagonized by subse-
quent infusions of atropine sulfate. These results suggested that the hippocampal formation can generate 0 activity given a non-rhythmic (continuous) cholinergic drive; however, rhythmic inputs from the medial septum could have contributed to 0 generation in these in vivo experiments. The question remains whether hippocampal neurons are capable of generating rhythmic activity in isolation. In the present study we report that carbachol can induce hippocampal 0-like activity in a preparation completely isolated from pulsed afferent inputs, and this response can also be antagonized by atropine sulfate. These resuits provide direct evidence for an intrinsic mechanism of 0 generation localized to neurons within the hippocampal formation. Experiments were performed on 41 hippocampal slices prepared from Sprague-Dawley rats weighing 100-150 g. Slices were prepared as previously described 11, placed in a tissue chamber on nylon mesh screens, and superfused with artificial cerebrospinal fluid (CSF) 35 °C, at a flow rate of 1-2 ml/min. The upper surface of slices were exposed to an atmosphere of prehumidified and warmed 95% 02/5% CO2. The artificial CSF had the following composition (mM): NaCI 124, KC1 5, CaCi 2 2, NaH~PO 4 1.25, MgSO4 2, NaHCO 3 26, glucose 1014. All solutions were made fresh prior to each experiment using
Correspondence: S.H. Roth, Department of Pharmacologyand Therapeutics, The Universityof Calgary, Calgary, Canada.
197 prefiltered and deionized water (Millipore Super Q system). Chemicals were reagent grade or better and obtained from Fisher Scientific, Canada. All drugs were obtained from Sigma Chemicals St. Louis, M O U.S.A.). Stimulating electrodes were made from parallel nichrome wires (22 gauge), microetched and insulated to within 0.5 m m of the tips. Stimulus pulses (0.01 ms, 1-12 V) were delivered from a Grass S 88 stimulator via a SIU 5 isolation unit. Glass recording electrodes (5-10 M~2; 2 M NaC1) were made from Kwik-Fil capillaries (W-P Instruments, CT, U.S.A.). Recorded signals were filtered (0.001-10 kHz, band pass) and amplified ( x l 0 0 0 ) using a Grass Instruments P-15 preamplifier operating in the differential mode. Signals were displayed on digital oscilloscopes (Gould OS 4020) and plotted on a Gould strip chart recorder. The stimulating electrode was placed on perforant path fibers to produce monosynaptically evoked field potentials recorded with glass electrodes in the molecular or granular layers of the dentate gyrus (Fig. 1A). Field potentials were used to monitor the viability of slices (a population spike amplitude of at least 8 mV and field excitatory postsynaptic potentials (EPSP) of 4 mV was found to be associated with slices capable of producing 0-like slow-wave activity); however, electrical stimulation was not required to produce slow-wave activity in the presence of carbachol. Carbachol-induced 0 recorded from the molecular layer of hippocampal slice is shown in Fig. 1B (in vitro) compared with carbachol-induced 0 activity from the same location in vivo (unpublished data from Bland, see also 12). Frequencies of slow-wave activity recorded from the slice preparations were similar to the normal physiological range for 0 in rats (2-12 Hz), and amplitudes were also comparable to in vivo recordings (e.g. 0.2-1.4 mV) 2-4. Concentrations of carbachol from 10 to 300 p M were studied. Slowwave activity was observed in approximately 25% of slices at concentrations of 1 0 - 5 0 p M (n = 8). At concentrations above 50 pM, P-like activity was observed in 85% of slices (n = 20). Concentrations above 200 p M produced a brief period of high-frequency seizure-like discharge, followed by electrical silence which lasted for several minutes prior to 8like activity. This pattern of seizure activity followed by 0 is similar to that produced by high concentra-
A
B IN VIVO
~:': if,' ~ :?,i ,r i(i ,f
=, 6t
IN VITRO
C
5 15
20
30
C
"
CARB
W5
w/o I
d-TUBO + CARB
Fig. 1. Carbachoi-induced 0 rhythm and antagonism by atropine sulfate. A: a diagram of the dentate region of the hippocampal formation showing the location of a stimulating electrode on perforant path (pp) fibers in the molecular layer (ML), used to monitor slice viability. A recording electrode (R) was placed in the granule cell layer (GL) or in the molecular layer just below the terminal field of perforant-path fibers. The optimal locations for recording slow-wave 0 activity are indicated by the bar on the right side of the diagram. The locations of afferent cholinergic fibers from the septum (sep) and efferent axons forming the mossy fiber (mr) pathways are also shown. B: comparison of carbachol-induced 0 recorded from the molecular layer in urethane-anesthetized rat (in vivo) and hippocampal slice (in vitro). C: the time course of carbachol-induced (50 pM) 0 recorded from the molecular layer of a hippocampal slice. 0-Like activity was observed 10 rain following the start of perfusion with carbachol and both amplitude and frequency varied with time. The first recording is a control (C), the numbers indicate min from start of carbachol perfusion and the last two records are following washout (W) with artificial cerebrospinal fluid. Note that 10 min following the washout the recording returned to the control level. D: 0 activity recorded from 3 separate experiments on different slices. The top recordings illustrate carbachol-induced (CARB; 50 pM) 0 and reversal with wash (W). Antagonism of carbachol-induced 0 by atropine sulfate (ATR, 50 pM) and lack of antagonism by D-tubocurarine (D-TUBO, 50 pM) are also shown. Calibration bars: 1 s and 0.5 mV; calibration in C for C and D.
tions of carbachol in vivo 12. The time course of carbachol-induced 0 in a singleslice experiment is shown in Fig. 1C. The first trace
198 (top left) shows the control baseline activity. After 5 min of perfusing with carbachol, small-amplitude arrhythmic activity was recorded; 0-like activity was observed after approximately 10 min. 0 Activity could be recorded for at least 1 h in the presence of carbachol. In most experiments, the amplitude and frequency varied with time, and optimal activity occurred between 10 and 30 min of exposure. Five min after washout with control solution (W5 in Fig. 1C) 0like activity was no longer apparent and 10 min later (Wl0) the recording returned to control levels. Fig. 1D shows the results of 3 separate experiments to illustrate that the cholinergic response was atropine sensitive. In each experiment, 50/~M of carbachol was perfused following the control (C) recordings on the left. The middle recordings illustrate the carbachoi-induced 0 for each slice. Note the variability in the frequency and amplitude of the 0like activity recorded after 20 min of carbachol exposure in each preparation. The upper right trace of Fig. 1D shows the reversal of carbachol-induced 0 after several minutes of washout (W) with control solution. The middle recording on the right illustrates the
block of carbachol-induced 0 by the muscarinic antagonist atropine sulfate (50 ktM) in all experiments (n = 8). The lower right recording demonstrates that 50/~M of D-tubocurarine (nicotinic antagonist) did not alter 0 (5 experiments). These results are consistent with previous in vivo investigations 4,6'12, providing further evidence for a muscarinic component in the generation of 0. The results of the present study do not support the septal 'pacemaker' hypothesis s-l°'13 which states that rhythmic cell discharges, originating in the medial septal region and transmitted via the septohippocampal pathway, are responsible for the appearance of 0 in the hippocampal formation. The present study clearly demonstrates that a rhythmic input is not critical for generating hippocampal formation 0 (at least type 2 0) since pulsed discharge activity from extrinsic afferents was eliminated in the slice preparation. Generator mechanisms intrinsic to the hippocampal formation can produce rhythmical slow waves in response to a continuous (non-rhythmic) cholinergic stimulus.
1 Bland, B.H., Andersen, P. and Ganes, T., Two generators of hippocampal 0 activity in rabbits, Brain Research, 94 (1975) 199-218. 2 Bland, B.H. and Whishaw, J.Q., Generators and topography of hippocampal 0 (RSA) in the anaesthetized and freely moving rat, Brain Research, 118 (1976) 259-280. 3 Bland, B.H., Sainsbury, R.S., Seto, M., Sinclair, B,R. and Whishaw, J.Q., The use of sodium pentobarbital for the study of immobility-related (type 2) hippocampal theta, Physiol. Behav., 27 (1981) 363-368. 4 Bland, B.H., The physiology and pharmacology of hippocampal formation theta rhythms, Prog. Neurobiol., 26 (1986) 1-54. 5 Kerkut, G.A. and Wheal, H.V., Electrophysiology of isolated mammalian CNS preparations, Academic, New York, 1981, pp. 1-732. 6 Kolb, B. and Whishaw, J.Q., Effects of brain lesions and atropine on hippocampal and neocortical electroencephalograms in the rat, Exp. Neurol., 56 (1977) 1-22. 7 Kramis, R., Vanderwolf, C.H. and Bland, B.H., Two types of hippocampal rhythmical slow activity in both the rabbit and the rat: relations to behavior and effects of atropine, diethyl ether, urethane, and pentobarbital, Exp. Neurol., 49 (1975) 58-85. 8 Petsche, H. and Stumpf, C., Topographic and toposcopic study of origin and spread of the regular synchronized arousal pattern in the rabbit, Electroencephalogr. Clin. Neurophysiol., 12 (1960) 589-600. 9 Petsche, H., Stumpf, C. and Gogolak, G., The significance of the rabbit's septum as a relay station between the midbrain and hippocampus I. The control of hippocampus
arousal activity by the septum cells, Electroencephalogr. Clin. Neurophysiol., 14 (1962) 202-211. 10 Rawlins, J.N.P., Feldon, T. and Gray, J.A., Septo-hippocampal connections and the hippocampal theta rhythm, Exp. Brain Res., 37 (1979) 49-63. 11 Roth, S.H., Tan, K.S. and Maclver, M.B., Selective and differential effects of barbiturates on neuronal activity. In S.H. Roth and K.W. Miller (Eds.), Molecular and Cellular Mechanisms of Anaesthetics, Plenum, New York, 1986, pp. 43-56. 12 Rowntree, C.J. and Bland, B.H., An analysis of chotinoceptive neurons in the hippocampal formation by direct microinfusion, Brain Research, 362 (1986) 98-113. 13 Sainsbury, R.S. and Bland, B.H., The effects of selective septal lesions on 0 production in CA1 and the dentate gyrus of the hippocampus, Physiol. Behav., 26 (1981) 1097-1101. 14 Schwartzkroin, P.A. and Altschuler, Development of kitten hippocampal neurons, Brain Research, 134 (1977) 429-444. 15 Vanderwolf, C.H., Neocortical and hippocampal activation in relation to behavior: effects of atropine, eserine, phenothiazines and amphetamine, J. Comp. Physiol. Psychol., 88 (1975) 306-323. 16 Whishaw, J.Q., Bland, B.H., Robinson, T.E. and Vanderwolf, C.H., Neuromuscular blockade: the effects on two hippocampal RSA (theta) systems and neocortical desynchronization, Brain Res. Bull., 1 (1976) 573-581. 17 Winson, J., Patterns of hippocampal theta rhythm in the freely moving rat, Electroencephalogr, Clin. Neurophysiol., 36 (1974) 291-30l.
BraiJt Rew,rc/t. 41)5f ]~)~7)it)t~ ]t).~
BRE 22076
Carbachol.indu
EEG 'theta' activity in hippocampal brain slices
Jan Konopacki 1, M. Bruce Maclver 2, Brian H. Bland 3 and Sheldon H. Roth: t Department of Animal Physiology, The University of Lodz, Lodz (Poland), 2Department of Pharmacology and Therapeutics and 3Department of Psychology, The University of Calgary, Calgary (Canada)
(Accepted 28 October 1986) Key words: 0; Hippocampal brain slice; Carbachol; Muscarinic; Electro encephalogram(EEG)
Application of the cholinergic agonist carbachol (50 pM) produced 0-like rhythmical waveforms, recorded in the stratum moleculare of the dentate gyrus. Atropine sulfate (50 pM) antagonized the carbachol-induced0-like activity, consistent with this action of atropine in vivo. These results provide the first direct evidence that hippocampalneurons are capable of producingsynchronizedslowwave activitywhen isolated from pulsed rhythmic inputs of the medial septum and other brain regions. Theta rhythms are electroencephalographic activities consisting of rhythmical sinusoidal-like slow waves. They are the largest (several millivolts), most prominent, and well-characterized rhythmical waveforms generated by the mammalian brain 12'17. Topographic studies of 0 have shown that it is localized to the stratum oriens of the hippocampal formation and the stratum moleculare of the dentate gyrus a'2. Behavioral and pharmacological studies suggest there are two types of 0; one has been termed type 1 and is correlated with voluntary motor behavior 3'7'15 and the other, termed type 2 0, can occur independently during immobility and is thought to be correlated with sensory processing specific to the organization of voluntary motor behaviors 4. Type 2 0 appears to be mediated by a cholinergic septohippocampal pathway, since lesions of the medial septum completely abolish hippocampal 0, and septal neuron rhythmic discharge activity is phase-locked to the hippocampal formation 0 rhythm 6'8'9A°'13. Type 2 0 can be produced by systemic injections of anticholinesterase agents like eserine and blocked by muscarinic antagonists such as atropine sulfate or scopolamine 16. Recently, Rowntree and Bland le demonstrated that type 2 0 could be produced in vivo by direct microinfusion of carbachol into the hippocampal formation, and that this 0 could be antagonized by subse-
quent infusions of atropine sulfate. These results suggested that the hippocampal formation can generate 0 activity given a non-rhythmic (continuous) cholinergic drive; however, rhythmic inputs from the medial septum could have contributed to 0 generation in these in vivo experiments. The question remains whether hippocampal neurons are capable of generating rhythmic activity in isolation. In the present study we report that carbachol can induce hippocampal 0-like activity in a preparation completely isolated from pulsed afferent inputs, and this response can also be antagonized by atropine sulfate. These resuits provide direct evidence for an intrinsic mechanism of 0 generation localized to neurons within the hippocampal formation. Experiments were performed on 41 hippocampal slices prepared from Sprague-Dawley rats weighing 100-150 g. Slices were prepared as previously described 11, placed in a tissue chamber on nylon mesh screens, and superfused with artificial cerebrospinal fluid (CSF) 35 °C, at a flow rate of 1-2 ml/min. The upper surface of slices were exposed to an atmosphere of prehumidified and warmed 95% 02/5% CO2. The artificial CSF had the following composition (mM): NaCI 124, KC1 5, CaCi 2 2, NaH~PO 4 1.25, MgSO4 2, NaHCO 3 26, glucose 1014. All solutions were made fresh prior to each experiment using
Correspondence: S.H. Roth, Department of Pharmacologyand Therapeutics, The Universityof Calgary, Calgary, Canada.
197 prefiltered and deionized water (Millipore Super Q system). Chemicals were reagent grade or better and obtained from Fisher Scientific, Canada. All drugs were obtained from Sigma Chemicals St. Louis, M O U.S.A.). Stimulating electrodes were made from parallel nichrome wires (22 gauge), microetched and insulated to within 0.5 m m of the tips. Stimulus pulses (0.01 ms, 1-12 V) were delivered from a Grass S 88 stimulator via a SIU 5 isolation unit. Glass recording electrodes (5-10 M~2; 2 M NaC1) were made from Kwik-Fil capillaries (W-P Instruments, CT, U.S.A.). Recorded signals were filtered (0.001-10 kHz, band pass) and amplified ( x l 0 0 0 ) using a Grass Instruments P-15 preamplifier operating in the differential mode. Signals were displayed on digital oscilloscopes (Gould OS 4020) and plotted on a Gould strip chart recorder. The stimulating electrode was placed on perforant path fibers to produce monosynaptically evoked field potentials recorded with glass electrodes in the molecular or granular layers of the dentate gyrus (Fig. 1A). Field potentials were used to monitor the viability of slices (a population spike amplitude of at least 8 mV and field excitatory postsynaptic potentials (EPSP) of 4 mV was found to be associated with slices capable of producing 0-like slow-wave activity); however, electrical stimulation was not required to produce slow-wave activity in the presence of carbachol. Carbachol-induced 0 recorded from the molecular layer of hippocampal slice is shown in Fig. 1B (in vitro) compared with carbachol-induced 0 activity from the same location in vivo (unpublished data from Bland, see also 12). Frequencies of slow-wave activity recorded from the slice preparations were similar to the normal physiological range for 0 in rats (2-12 Hz), and amplitudes were also comparable to in vivo recordings (e.g. 0.2-1.4 mV) 2-4. Concentrations of carbachol from 10 to 300 p M were studied. Slowwave activity was observed in approximately 25% of slices at concentrations of 1 0 - 5 0 p M (n = 8). At concentrations above 50 pM, P-like activity was observed in 85% of slices (n = 20). Concentrations above 200 p M produced a brief period of high-frequency seizure-like discharge, followed by electrical silence which lasted for several minutes prior to 8like activity. This pattern of seizure activity followed by 0 is similar to that produced by high concentra-
A
B IN VIVO
~:': if,' ~ :?,i ,r i(i ,f
=, 6t
IN VITRO
C
5 15
20
30
C
"
CARB
W5
w/o I
d-TUBO + CARB
Fig. 1. Carbachoi-induced 0 rhythm and antagonism by atropine sulfate. A: a diagram of the dentate region of the hippocampal formation showing the location of a stimulating electrode on perforant path (pp) fibers in the molecular layer (ML), used to monitor slice viability. A recording electrode (R) was placed in the granule cell layer (GL) or in the molecular layer just below the terminal field of perforant-path fibers. The optimal locations for recording slow-wave 0 activity are indicated by the bar on the right side of the diagram. The locations of afferent cholinergic fibers from the septum (sep) and efferent axons forming the mossy fiber (mr) pathways are also shown. B: comparison of carbachol-induced 0 recorded from the molecular layer in urethane-anesthetized rat (in vivo) and hippocampal slice (in vitro). C: the time course of carbachol-induced (50 pM) 0 recorded from the molecular layer of a hippocampal slice. 0-Like activity was observed 10 rain following the start of perfusion with carbachol and both amplitude and frequency varied with time. The first recording is a control (C), the numbers indicate min from start of carbachol perfusion and the last two records are following washout (W) with artificial cerebrospinal fluid. Note that 10 min following the washout the recording returned to the control level. D: 0 activity recorded from 3 separate experiments on different slices. The top recordings illustrate carbachol-induced (CARB; 50 pM) 0 and reversal with wash (W). Antagonism of carbachol-induced 0 by atropine sulfate (ATR, 50 pM) and lack of antagonism by D-tubocurarine (D-TUBO, 50 pM) are also shown. Calibration bars: 1 s and 0.5 mV; calibration in C for C and D.
tions of carbachol in vivo 12. The time course of carbachol-induced 0 in a singleslice experiment is shown in Fig. 1C. The first trace
198 (top left) shows the control baseline activity. After 5 min of perfusing with carbachol, small-amplitude arrhythmic activity was recorded; 0-like activity was observed after approximately 10 min. 0 Activity could be recorded for at least 1 h in the presence of carbachol. In most experiments, the amplitude and frequency varied with time, and optimal activity occurred between 10 and 30 min of exposure. Five min after washout with control solution (W5 in Fig. 1C) 0like activity was no longer apparent and 10 min later (Wl0) the recording returned to control levels. Fig. 1D shows the results of 3 separate experiments to illustrate that the cholinergic response was atropine sensitive. In each experiment, 50/~M of carbachol was perfused following the control (C) recordings on the left. The middle recordings illustrate the carbachoi-induced 0 for each slice. Note the variability in the frequency and amplitude of the 0like activity recorded after 20 min of carbachol exposure in each preparation. The upper right trace of Fig. 1D shows the reversal of carbachol-induced 0 after several minutes of washout (W) with control solution. The middle recording on the right illustrates the
block of carbachol-induced 0 by the muscarinic antagonist atropine sulfate (50 ktM) in all experiments (n = 8). The lower right recording demonstrates that 50/~M of D-tubocurarine (nicotinic antagonist) did not alter 0 (5 experiments). These results are consistent with previous in vivo investigations 4,6'12, providing further evidence for a muscarinic component in the generation of 0. The results of the present study do not support the septal 'pacemaker' hypothesis s-l°'13 which states that rhythmic cell discharges, originating in the medial septal region and transmitted via the septohippocampal pathway, are responsible for the appearance of 0 in the hippocampal formation. The present study clearly demonstrates that a rhythmic input is not critical for generating hippocampal formation 0 (at least type 2 0) since pulsed discharge activity from extrinsic afferents was eliminated in the slice preparation. Generator mechanisms intrinsic to the hippocampal formation can produce rhythmical slow waves in response to a continuous (non-rhythmic) cholinergic stimulus.
1 Bland, B.H., Andersen, P. and Ganes, T., Two generators of hippocampal 0 activity in rabbits, Brain Research, 94 (1975) 199-218. 2 Bland, B.H. and Whishaw, J.Q., Generators and topography of hippocampal 0 (RSA) in the anaesthetized and freely moving rat, Brain Research, 118 (1976) 259-280. 3 Bland, B.H., Sainsbury, R.S., Seto, M., Sinclair, B,R. and Whishaw, J.Q., The use of sodium pentobarbital for the study of immobility-related (type 2) hippocampal theta, Physiol. Behav., 27 (1981) 363-368. 4 Bland, B.H., The physiology and pharmacology of hippocampal formation theta rhythms, Prog. Neurobiol., 26 (1986) 1-54. 5 Kerkut, G.A. and Wheal, H.V., Electrophysiology of isolated mammalian CNS preparations, Academic, New York, 1981, pp. 1-732. 6 Kolb, B. and Whishaw, J.Q., Effects of brain lesions and atropine on hippocampal and neocortical electroencephalograms in the rat, Exp. Neurol., 56 (1977) 1-22. 7 Kramis, R., Vanderwolf, C.H. and Bland, B.H., Two types of hippocampal rhythmical slow activity in both the rabbit and the rat: relations to behavior and effects of atropine, diethyl ether, urethane, and pentobarbital, Exp. Neurol., 49 (1975) 58-85. 8 Petsche, H. and Stumpf, C., Topographic and toposcopic study of origin and spread of the regular synchronized arousal pattern in the rabbit, Electroencephalogr. Clin. Neurophysiol., 12 (1960) 589-600. 9 Petsche, H., Stumpf, C. and Gogolak, G., The significance of the rabbit's septum as a relay station between the midbrain and hippocampus I. The control of hippocampus
arousal activity by the septum cells, Electroencephalogr. Clin. Neurophysiol., 14 (1962) 202-211. 10 Rawlins, J.N.P., Feldon, T. and Gray, J.A., Septo-hippocampal connections and the hippocampal theta rhythm, Exp. Brain Res., 37 (1979) 49-63. 11 Roth, S.H., Tan, K.S. and Maclver, M.B., Selective and differential effects of barbiturates on neuronal activity. In S.H. Roth and K.W. Miller (Eds.), Molecular and Cellular Mechanisms of Anaesthetics, Plenum, New York, 1986, pp. 43-56. 12 Rowntree, C.J. and Bland, B.H., An analysis of chotinoceptive neurons in the hippocampal formation by direct microinfusion, Brain Research, 362 (1986) 98-113. 13 Sainsbury, R.S. and Bland, B.H., The effects of selective septal lesions on 0 production in CA1 and the dentate gyrus of the hippocampus, Physiol. Behav., 26 (1981) 1097-1101. 14 Schwartzkroin, P.A. and Altschuler, Development of kitten hippocampal neurons, Brain Research, 134 (1977) 429-444. 15 Vanderwolf, C.H., Neocortical and hippocampal activation in relation to behavior: effects of atropine, eserine, phenothiazines and amphetamine, J. Comp. Physiol. Psychol., 88 (1975) 306-323. 16 Whishaw, J.Q., Bland, B.H., Robinson, T.E. and Vanderwolf, C.H., Neuromuscular blockade: the effects on two hippocampal RSA (theta) systems and neocortical desynchronization, Brain Res. Bull., 1 (1976) 573-581. 17 Winson, J., Patterns of hippocampal theta rhythm in the freely moving rat, Electroencephalogr, Clin. Neurophysiol., 36 (1974) 291-30l.