RAPID COMMUNICATION In Vitro Activation of the Medial Septum—Diagonal Band Complex Generates Atropine-Sensitive and Atropine-Resistant Hippocampal Theta Rhythm: An Investigation Using a Complete Septohippocampal Preparation Romain Goutagny, * Fre ´de ´ric Manseau, Jesse Jackson, Marc Danik, and Sylvain Williams ABSTRACT: The medial septum and diagonal band complex (MS-DB) is believed to play a key role in generating theta oscillations in the hip- pocampus, a phenomenon critical for learning and memory. Although the importance of the MS-DB in hippocampal theta rhythm generation is generally accepted, it remains to be determined whether the MS-DB alone can generate hippocampal oscillations or is only a transducer of rhythmic activity from other brain areas. Secondly, it is known that hip- pocampal theta rhythm can be separated into an atropine-sensitive and insensitive component. However, it remains to be established if the MS- DB can generate both types of rhythm. To answer these questions, we used a new in vitro rat septohippocampal preparation placed in a her- metically separated two side recording chamber. We showed that car- bachol activation of the MS-DB generated large theta oscillations in the CA1 and CA3 regions of the hippocampus. These oscillations were blocked by applying either the GABA A receptor antagonist bicuculline or the AMPA/kainate antagonist DNQX to the hippocampus. Interest- ingly, the application of the muscarinic receptor antagonist atropine produced only a partial decrease in the amplitude, without modification of the frequency, of theta. These results show for the first time, that upon optimal excitation, the MS-DB alone is able to generate hippocam- pal oscillations in the theta frequency band. Moreover, these MS-DB generated theta oscillations are mediated by muscarinic and nonmuscar- inic receptors and have a pharmacological profile similar to theta rhythm observed in awake animals. V V C 2008 Wiley-Liss, Inc. KEY WORDS: hippocampal theta oscillations; medial septum; atropine resistant theta; carbachol; in vitro oscillations Rhythmic patterns of electrical activity have been recorded from cortical areas at various frequencies including theta (3–12 Hz), beta (20– 30 Hz), gamma (40–80 Hz), and ultrafast (>80 Hz) frequencies. Field potential oscillations at theta frequencies are particularly prominent in the hippocampal formation in conscious or urethane-anesthetized ani- mals (Green and Arduini, 1954; Green and Rawlins, 1979) and are critical for memory formation (Winson, 1978; Bland, 1986; McNaughton et al., 2006). Since one of the most important inputs to the hippocampus originates from ascending fibers of neu- rons located in the medial septum and diagonal band complex (MS-DB; Chandler and Crutcher, 1983; Baisden et al., 1984; Amaral and Kurz, 1985), it has been postulated that the MS-DB acts as a pacemaker for the hippocampal theta oscillations. Lesion and stimulation studies suggested that the cholinergic and GABAergic components of the MS-DB, via the septo- hippocampal pathway, plays a pivotal role in the gen- eration of theta frequency oscillations in the hippo- campus (Stewart and Fox, 1990; Lee et al., 1994; Vinogradova et al., 1995; Yoder and Pang, 2005). A role in theta for the recently described glutamatergic neurons in the MS-DB still remains unknown (Sotty et al., 2003; Castaneda et al., 2005; Manseau et al., 2005). However, since the hippocampus receives im- portant rhythmic inputs from many brain areas such as the supramammillary nucleus (reviewed in Vertes and Kocsis, 1997), it remains to be established whether the MS/DB per se can generate hippocampal theta or is only a transducer of rhythmic activity origi- nating from other areas. Finally, two types of theta can be pharmacologically identified in the hippocam- pus; one that is sensitive to the muscarinic receptor blocker atropine and the other that is insensitive to this agent (Kramis et al., 1975). It has been proposed that the atropine-sensitive component is mediated by the MS-DB whereas the insensitive portion is medi- ated by other structures such as the entorhinal cortex (EC) (discussed in Buzsaki, 2002). Therefore, it remains to be determined if the MS-DB participates in the generation of atropine-insensitive theta. To address these questions, we used a new in vitro preparation preserving a half-septum and ipsilateral hippocampus still attached together (Khalilov et al., 1997; Manseau et al., 2005). To evaluate the specific role of the septum in the generation of hippocampal theta oscillations, we used a custom made dual cham- ber bath. In these experiments, a Teflon wall was Department of Psychiatry, Douglas Mental Health University Institute, McGill University, Montre ´al, Que ´bec, Canada Grant sponsor: Canadian Institute of Health Research (CIHR); Grant num- ber: MOP81111; Grant sponsor: Natural Sciences and Engineering Research Council of Canada (NSERC); Grant number: RGPIN238770. *Correspondence to: Romain Goutagny, Douglas Mental Health University Institute Research Center; McGill University 6875 Lasalle Blvd., Montre ´al, Que ´bec, H4H 1R3, Canada. E-mail: romain.goutagny@douglas.mcgill.ca Accepted for publication 9 January 2008 DOI 10.1002/hipo.20418 Published online 27 February 2008 in Wiley InterScience (www.interscience. wiley.com). HIPPOCAMPUS 18:531–535 (2008) V V C 2008 WILEY-LISS, INC.