Neuroscience Letters, 141 (1992) 93-96

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© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Theta rhythms in the rat medial entorhinal cortex in vitro: evidence for involvement of muscarinic receptors Jan K o n o p a c k i and H e n r y k Go~¢biewski Department of Neurobiology, The University of L6d2, L6d2 (Poland) (Received 17 February 1992; Revised version received 6 April 1992; Accepted 6 April 1992)

Key words: Medial entorhinal cortex; Slice; Theta-like oscillation; Cholinergic Slice preparations obtained from the rat were used to study cholinergically induced field potentials in the medial entorhinal cortex. Perfusion of slices containing medial entorhinal cortex with acetylcholine, muscarine and eserine induced theta-like activity in a frequency range of 3-10 Hz and an amplitude of 200 300/tV. Nicotine, in contrast, did not produce any rhythmical slow waveforms. The cholinergically induced theta-like oscillations were abolished by perfusion of the muscarinic antagonists atropine sulphate and scopolamine but were unaffected by the nicotine blockers hexamethonium and mecamylamine.

Theta rhythms are electroencephalographic activities consisting of rhythmical sinusoidal slow waves. They are the largest (several mV), most prominent, and well synchronized waveforms generated by the mammalian brain [4]. Commonly, theta activity has been associated with the hippocampal formation since in this structure it is most conspicuous [4, 9]. However, a number of reports in the last decade have revealed the hippocampal formation not to be the only limbic region involved in the production of theta rhythms [1, 9-13]. Increasing attention has been paid to the role of the entorhinal cortex (EC) in the mechanisms responsible for generation of this EEG pattern [1-3, 10, 11, 13]. The EC region is a chief source of afferents to the hippocampal formation and receives multisynaptic projections from Ammon's horn field of the hippocampus [9, 14]. Only recently the medial entorhinal cortex (MEC) has been postulated to play a role in the generation of the hippocampal theta rhythms [10, 13]. Furthermore, the MEC per se was suggested to be a source of the in vivo theta activity [1, 11]. This suggestion was strongly supported by the experiments we recently conducted on rat MEC slice preparations [8]. Specifically, we demonstrated that in the in vitro conditions (deafferentation from the hippocampal formation and medial septum) the MEC neuronal network was capable of producing theta-like activity when the cholinergic agonist carbachol was added to the bath [8]. Correspondence: J. Konopacki, Department of Neurobiology, The University of L6dL Rewolucji 1905 Str. No. 66, 90-222 Ldd~', Poland.

In the present study, we extended our preliminary observations. We provide here evidence for the intrinsic MEC muscarinic receptors to be actively involved in the mechanism responsible for theta production. Forty-eight experiments were conducted on 44 MEC slices (350/~m) consisting of the entorhinal cortex, preand parasubicular region. The slices were obtained from male Wistar rats (100--150 g), which had been anesthetized with ether and decapitated. The brain was rapidly removed, halved and placed in cold (6°C), oxygenated (95% 02 + 5% CO2) artificial cerebrospinal fluid (CSF; composition in mM, see refs. 5-7). The right entorhinal cortex with adjacent hippocampal formation, pre- and parasubicular regions were dissected by separating the alveus from the part of overlying neocortex, along the lateral ventricle. The final dissection of entorhinal cortex with pre- and parasubiculum was made by cutting this area on the border between the perirhinal cortex and subiculum (see Fig. 1). Transverse slices of the entorhinal-pre-parasubicular area were cut perpendicularly to the long axis of this parahippocampal region. The slices were maintained at a gas- liquid interface in a tissue chamber as previously described [5-8]. Acetylcholine, muscarine, eserine, atropine sulphate, scopolamine, hexamethonium and mecamylamine were obtained from Sigma. The drug solutions were made fresh prior to each experiment using artificial CSF. Concentrations of acetylcholine (0.2-1.0 mM), muscarine (0.l-0.5 raM), eserine (0.054).2 mM), nicotine (0.2-1.0 raM) were studied to determine effective doses for generation of theta-

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Fig. 1. Schematic representation of a transverse section, i.e. section taken perpendicularly to the long axis of the hippocampal and parahippocampal regions of the rat. Broken line indicates plane where the combined entorhinal-pre-parasubicular slice preparation was dissected free. The diagram also shows a typical placement of electrode (R) for recording cholinergically induced theta in the medial entorhinal cortex (MEC).

like activity. Atropine sulphate and scopolamine were administered at a concentration of 0.001 mM: this dose was earlier found to antagonize the cholinergically induced theta within 6-10 min. Hexamethonium and mecamylamine were administered in concentrations from 0.5 to 1.0 mM. The glass recording electrodes (5 10 MI2, 2 M NaC1) were made from Kwik-Fil capillaries (W-P Instruments). The recording electrode was typically positioned along layers II-III of the MEC (Fig. 1). The recorded signals were filtered (0.001-10 kHz, band pass) and amplified (x 1000) using a Grass Instrument P-16 preamplifier operating in the differential mode. The EEG signals were simultaneously displayed and analyzed with the use of a digital storage oscilloscope (Kikusui, 7061A, Japan) and recorded on a polygraph (Am-l, Ormed, Poland). The concentrations of acetylcholine (0.6 mM), muscarine (0.5 mM) and eserine (0.1 mM) were found to produce an optimal EEG response (see Fig. 2A, middle panel). At higher concentrations, a brief period of high-

frequency seizure-like discharges, followed by electrical silence, was observed. This pattern of activity is typical for overdoses of the cholinergic agonists, when administered in vitro [5-7]. The cholinergic agonist acetylcholine (ACH) produced theta-like slow waves in a frequency (5~ _+ S.D.) of 4.11 + 0.33 Hz, n=6 and an amplitude of 203 _+ 68/IV, n--6. The frequency and amplitude of muscarine (MUSC) induced rhythmical slow waves were 7.60 + 2.4 Hz, n=5 and 222 +_ 107 ¢tV, n=5, respectively. The anticholinesterase agent, eserine (ESE), induced 'theta' rhythm in a frequency of 7.71 _+ 1.92 Hz, n=5 and amplitude of 277 _+ 66 ¢tV, n=5. Acetylcholine, muscarine and eserine induced theta-like activity was hardly synchronized. Typically, it could be recorded for a maximum of 20 30 min. However, when the slices containing MEC were perfused with eserine plus acetylcholine (ESE + ACH), the induced theta-like waves were well synchronized for 1 1.5 h. The frequency ofESE + ACH induced 'theta' was 7.23 _+ 3.86 Hz, n=15 and amplitude was 405 _+ 152 ¢tV, n=15. Of the cholinergic agents examined in the present study only nicotine was ineffective in producing rhythmical slow waves. The concentration of 1 mM nicotine usually induced irregular seizure activity. However, this activity never developed into regular, rhythmical waveforms, typically seen after perfusion with ESE + ACH (Fig. 2A, right panel). The cholinergically induced theta-like activity was found to be fully reversible; a wash-out with artificial CSF (without the cholinergic agents) required 5-10 min (Fig. 2A, right panel). To determine whether cholinergically induced thetalike activity is mediated by muscarinic receptors, we examined in separate experiments the blockade of ESE + ACH induced 'theta' with the use of scopolamine (SCOP), atropine sulphate (ATR), hexamethonium (HXM) and mecamylamine (MECA) (Fig. 2B). The nicotinic antagonists, hexamethonium and mecamylamine did not alter ESE + ACH induced theta-like oscillations. The muscarinic antagonists atropine sulphate and scopolamine, in contrast, completely blocked cholinergi-

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Fig. 2. Representative examples of the cholinergically induced theta-like activity in the medial entorhinal cortex in vitro. A: cholinergically induced theta (middle panel) recorded from separate experiments conducted on different slices in the presence of acetylcholine (ACH, 0.6 raM), muscarine (MUSC, 0.5 mM), eserine (ESE, 0.1 mM), eserine and acetylcholine (ESE + ACH, 0.1 + 0.1 mM). Note that in control bath conditions the slices did not manifest spontaneous EEG activity (CONTR, left panel). At 10 15 min following the washout (WASH, right panel) the recording returned to the control level. Of the cholinergic agents tested only nicotine (NICO, 1.0 mM) was found to be unaffected in producing slow rhythmical waves; however, the same slices exhibited EEG theta-like activity when perfused with eserine and acetylcholine (ESE + ACH, 0.1 + 0.1 mM, right panel). B: eserine and acetylcholine (ESE + ACH, 0.1 + 0. I raM) induced theta-like activity recorded from separate experiments conducted on different slices (middle panel). The top two recordings (right panel) show antagonism of cholinergically induced theta by scopolamine (SCOP, 0.001 mM) and atropine sulphate (ATR, 0.001 raM). The lower two recordings show lack of antagonism by hexamethonium (HXM, 0.1 mM) and mecamylamine (MECA, 0.1 mM). A, B: the samples of the cholinergic theta-like oscillations were taken 10 15 min from the onset of the perfusion; scopolamine and atropine samples were taken 10 min from the onset of the perfusion; hexamethonium and mecamylamine traces were taken 30 min after the perfusion onset. Calibration: l s and 200 ,uV.

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cally induced rhythmical slow waveforms. The present experiments demonstrated that the slice preparations containing MEC can be successfully used as a model to study neuronal mechanisms underlying theta rhythms in the medial entorhinal cortex. The above findings confirm our preliminary in vitro observations with use of carbachol [8] and provide evidence for the endogenous acetylcholine to be directly involved in theta production. Specifically, we demonstrated that the actions of exogenously applied acetylcholine or muscarine were mimicked by potentiating endogenous cholinergic transmission with eserine. The cholinergically-induced rhythmical slow waves closely resemble theta rhythm recorded from the same region in vivo [1, 11]. The basic question arises as to the source of 'theta' oscillations in the MEC maintained in vitro. It is generally agreed that in the intact animals the medial septal area constitutes the pacemaker for the generation of theta both in the hippocampal formation and EC. The lesions of the medial septum were found to permanently abolish the hippocampal and the entorhinal theta rhythm [4, 9, 12]. However, this does not mean that rhythmical slow waves cannot be generated in both these limbic structures when completely isolated from the medial septal rhythmical input. We have recently discovered that the rat hippocampal formation slices are capable of generating the cholinergically induced theta-like slow waveforms which closely resemble type 2 theta, recorded in the behaving and anesthetized rats [5 7]. Furthermore, the present study and our earlier experiments [8] revealed that well synchronized theta-like slow waves can be cholinergically induced in the in vitro maintained MEC as well. These results clearly demonstrate that the extrinsic rhythmical input from the medial septal area is not essential for generating theta oscillations in the MEC. The MEC neuronal network per se is capable of producing oscillations in the frequency of theta and generating theta-like field potentials. Our present results also strongly support the muscarinic nature of the cholinergically induced theta-like activity in the MEC maintained in vitro: (a) slices containing MEC manifested theta-like slow waves in the presence of muscarine, (b) nicotine perfusion has never induced rhythmical slow waveforms, (c) cholinergically induced 'theta' activity was abolished by the muscarinic antagonists atropine sulphate and scopolamine but was unaffected by the nicotinic blockers hexamethonium and mecamylamine.

In conclusion, we have presented evidence for the medial entorhinal muscarinic receptors to be actively involved in the oscillatory mechanism underlying theta slow waves in this region. The entorhinal oscillator can act in complete isolation from the rhythmical input of the medial septal region. This study was supported by the Ministry of National Education and The Committee of Scientific Research.

1 Alonso, A. and Garcia-Austt, E., Neuronal sources of theta rhythm in the entorhinal cortex of the rat. I. Laminar distribution of theta field potentials, Exp. Brain Res.. 67 (1987)493-501. 2 Alonso, A. and Kohler, C.. A study of the reciprocal connections between the septum and the entorhinal area using anterograde and retrograde axonal transport methods in the rat brain, J. Comp. Neurol., 275 (1984) 175 177. 3 Alonso, A. and Lilnas, R., Subthreshold Nat-dependent theta-like rhythmicity in stellate cells ofentorhinal cortex layer [I, Nature, 342 (1989) 175-177. 4 Bland, B.H., The physiology and pharmacology of hippocampal formation theta rhythms, Prog. Neurobiol., 26 (1986) 1 .54. 5 Konopacki, J., Bland, B.H., Maclver, B.H. and Roth, S.H., Cholinergic theta rhythm in transected hippocampal slices: independent CAI and dentate generators, Brain Res., 436 (1987) 217 222. 6 Konopacki, J., Bland, B.H. and Roth, S.H., Phase shifting of CA1 and dentate EEG ~theta" in hippocampal formation slices, Brain Res., 417 (1987) 399 402. 7 Konopacki, J., Bland, B.H. and Roth, S.H., Carbachol-induced EEG "theta' in hippocampal formation slices: evidence for a third generator of theta in CA4 area, Brain Res., 451 (1988) 33 42. 8 Konopacki, J., GolCbiewski, H. and Eckersdorf, B., Carbachol-induced theta-like activity in entorhinal cortex slices, Brain Res., 572 (1992) 76 80. 9 Lopes de Silva, F.H., Winer, M.P., Boeijnga, P.H. and Lohman, A.H.M., Anatomic organization and physiology of the limbic cortex, Physiol. Rev., 70 (1990) 453 510. 10 Montoya, C.P. and Sainsury, R.S., The effects of entorhinal cortex lesions on type 1 and type 2 theta, Physiol. Behav., 35 (1985) 121 126. 11 Mitchell, S.J. and Ranck, J.B.. Generation of theta rhythm in medial entorhinal cortex of freely moving rats, Brain Res., 189 (1980) 49 60. 12 Mitchell, S.J., Rawlins, J.N., Steward, O. and Olton, D.S., Medial septal area lesions disrupt theta rhythm and cholinergic staining in medial cntorhinal cortex and produce impaired radial arm maze behavior in rats, J. Neurosci., 2 (1982) 292 302. 13 Vanderwolf, C.M., Leung, L.W.S. and Cooley, R.K., Pathways through the cingulate, neo- and entorhinal cortices mediating atropine-resistant rhythmical slow activity, Brain Res., 347 (1985) 58 73. 14 Witter, M.P., Groenwegen, H.J., Lopes de Silva, F.M. and Lohman, A.H,M., Functional organization of the extrinsic and intrinsic circuitry of the parahippocampal region, Prog. Neurobiol., 33 (1989) 161 253.

Theta rhythms in the rat medial entorhinal cortex in vitro: evidence for involvement of muscarinic receptors.

Slice preparation obtained from the rat were used to study cholinergically induced field potentials in the medial entorhinal cortex. Perfusion of slic...
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