The Journal of Neuroscience, July 15, 1999, 19(14):6191–6199 Interactions between Hippocampus and Medial Septum during Sharp Waves and Theta Oscillation in the Behaving Rat George Dragoi,1 Daniel Carpi,1 Michael Recce,2 Jozsef Csicsvari,1 and Gyo¨ rgy Buzsa´ki1 1Center for Molecular and Behavioral Neuroscience, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, and 2Department of Computer Science, New Jersey Institute of Technology, Newark, NJ 07102 The medial septal region and the hippocampus are connected Because both SPW and the negative peak of local theta waves reciprocally via GABAergic neurons, but the physiological role correspond to the maximum discharge probability of CA1 py- of this loop is still not well understood. In an attempt to reveal ramidal cells and interneuron classes, the findings indicate that the physiological effects of the hippocamposeptal GABAergic the activity of medial septal neurons can be negatively (during projection, we cross-correlated hippocampal sharp wave SPW) or positively (during theta waves) correlated with the (SPW) ripples or theta activity and extracellular units recorded activity of hippocampal interneurons. We hypothesize that the in the medial septum and diagonal band of Broca (MSDB) in functional coupling between medial septal neurons and hip- freely moving rats. The majority of single MSDB cells (60%) pocampal interneurons varies in a state-dependent manner. were significantly suppressed during SPWs. Most cells inhib- ited during SPW (80%) fired rhythmically and phase-locked to Key words: EEG; GABAergic neurons; interneurons; ripples; the negative peak of the CA1 pyramidal layer theta waves. cholinergic system; lateral septum The septal region and the hippocampus are connected recipro- early formulation, neurons in the MSDB have been assumed to cally (Raisman, 1966). The two major components of the septo- play the role of a pacemaker by providing a coherent and rhyth- hippocampal projection consist of cholinergic and GABAergic mic drive to the hippocampus (Green and Arduini, 1954; Petsche cells (Shute and Lewis, 1963; Lewis et al., 1967; Ko¨hler et al., et al., 1962; Stumpf et al., 1962; Winson, 1978; Andersen et al., 1984), residing in the medial septum and the diagonal band of 1979). Subsequent studies have shown that MSDB GABAergic Broca (MSDB). The cholinergic projection terminates on all cells have the intrinsic propensity to oscillate at theta frequency types of hippocampal cells (Shute and Lewis, 1966; Frotscher and (Serafin et al., 1996) and suggested that a coordinated output of Leranth, 1985; Freund and Antal, 1988), whereas septal the GABAergic and cholinergic populations is responsible for GABAergic neurons specifically innervate GABAergic interneu- phase-locking hippocampal neurons (Stewart and Fox, 1989; Lee rons (Freund and Antal, 1988; Gulya´s et al., 1991). A subpopu- et al., 1994; Brazhnik and Fox, 1997). In contrast, other studies lation of the target GABAergic cells, in turn, projects back to the could not confirm the hypothesis that either the putative cholin- MSDB and preferentially innervates the GABAergic population ergic or GABAergic MSDB neurons discharge coherently during (Alonso and Ko¨hler, 1982; Gaykema et al., 1991; To´th et al., the theta cycle (Gaztelu and Bun˜o, 1982; Stewart and Fox, 1989, 1993). The hippocamposeptally projecting neurons contain the 1990; King et al., 1998). Two major technical problems have calcium binding protein calbindin (To´th and Freund, 1992; To´th hampered progress in this area of research. The first is the lack of et al., 1993). Hippocampal pyramidal cells project only to the unambiguous criteria for the identification of neuronal types from lateral septum (Alonso and Ko¨hler, 1982; Leranth and Frotscher, the extracellularly recorded spike patterns and the lack of histo- 1989). The main projection of the lateral septum is subcortical, logical identification of intracellularly studied neurons in acute and axon collaterals to the MSDB are either absent or very sparse experiments. The second problem is to isolate the contribution of (Gulya´s et al., 1991; Staiger and Nu¨rnberger, 1991; Leranth et al., the participating members in multiple loops (Vertes and Kocsis, 1992). Therefore, the major reciprocal communication between 1997). the septum and hippocampus is mediated by the MSDB and Theta rhythm is generated by a consortium of various pathways hippocampal GABAergic cells. The functional role of this recip- and mechanisms. In contrast, SPW bursts are known to emerge in rocal hippocamposeptal GABAergic loop is not understood. the recurrent collateral system of CA3 pyramidal cells (Buzsa´ki et The hippocampal formation displays two behaviorally and al., 1983). The synchronous discharge of pyramidal cells induces physiologically distinct patterns: theta waves and sharp waves high frequency and coherent discharge of the target interneurons, (SPWs). The importance of the septum in hippocampal theta including the calbindin-immunoreactive subclass, with axonal generation has been documented by numerous experiments. In an projection to the MSDB (To´th and Freund, 1992). We reasoned, therefore, that by studying the correlation between hippocampal Received March 8, 1999; revised May 3, 1999; accepted May 5, 1999. SPWs and MSDB neuronal activity, we can assess the unidirec- This work was supported by National Institutes of Health Grants NS34994 and MH54671. We thank Z. Borhegyi, H. Hirase, C. King, and Z. Nada´sdy for help and tional effect of hippocampal interneurons on MSDB neuronal support and T. F. Freund for his comments on this manuscript. populations. In addition, previous research has established that Correspondence should be addressed to Gyo¨rgy Buzsa´ki, Center for Molecular both pyramidal neurons and various classes of interneurons dis- and Behavioral Neuroscience, Rutgers University, 197 University Avenue, Newark, NJ 07102. play their highest discharge probability around the negative peak Copyright © 1999 Society for Neuroscience 0270-6474/99/196191-09$05.00/0 of theta waves in the CA1 pyramidal layer (Fox et al., 1986; 6192 J. Neurosci., July 15, 1999, 19(14):6191–6199 Dragoi et al. • Hippocamposeptal Inhibition Skaggs et al., 1996; Csicsvari et al., 1999). Using both SPWs and theta oscillations as reference signals for the maximum discharge probability of interneurons, we examined their impact on the activity of MSDB neurons in these two respective states. MATERIALS AND METHODS Animals and surgery. Ten adult male Sprague Dawley rats (weighting 300–500 gm) were anesthetized with a mixture (4 ml/kg) of ketamine (25 mg/ml), xylazine (1.3 mg/ml), and acepromazine (0.25 mg/ml) adminis- tered intramuscularly, following National Institutes of Health guidelines. Tungsten high-impedance electrodes (0.5–1 MV; Micro Probe Inc., MD) were mounted on movable drives and used to record the unit activity in the MSDB area. Each complete turn of the microdrive moved the electrode 0.3 mm axially. A hole (1 mm in diameter) was drilled above the septal area in the midline. After cutting the dura mater, the sagittal sinus was gently moved aside, and the tungsten electrode was lowered into the midline, ;1 mm above the medial septum [anteroposterior (AP), 0–0.7; lateral (L), 0; ventral (V), 5], according to the atlas coordinates of Paxinos and Watson (1997). In one rat, two such microdrives were used, one for recording from the MSDB and the other one from the lateral septum. The hippocampal recording electrodes were constructed from 50-mm-diameter stainless steel wire coated with insulation (California Fine Wire, Grover Beach, CA). Two of these wires, with 250 mm vertical Figure 1. Location of the recorded units in MSDB. Lines indicate elec- and 300 mm horizontal distance between their tips, were mounted on the trode tracks. Asterisks, Estimated location of recording sites. Image, same type of microdrive as in the high-impedance electrode. These ChAT immunostaining illustrating the recording track. Inset, Parvalbumin electrodes were unilaterally implanted through holes drilled above the immunostaining of an adjacent section; the arrow points to the midline. dorsal hippocampus (AP, 4; L, 2.5; V, 1.5). In addition, a pair of stainless Arrowheads mark the tracks of the same electrode on diagram and steel wires (100 mm in diameter) with 0.5 mm vertical tip separation was histological sections. Numbers represent distance (in millimeters) from placed in the fimbria (AP, 21.3; L, 1; V, 3.8) to stimulate the commis- bregma. aca, Anterior commissure; cc, corpus callosum; 2n, optic nerve. sural afferents to the CA1 region. The microdrives, as well as the Anatomical diagrams adapted from Paxinos and Watson (1997). stimulating electrodes, were fixed to the skull with dental acrylic. Two stainless steel screws driven into the bone above the cerebellum served as reference and ground electrodes. Two additional screws in the frontal reference events. Thus, the histograms reflect discharge probabilities. bone acted as anchors. After the operation, the rats were kept one to a The method for constructing phase histograms have been described cage, weighed, and inspected daily. Recording began after 1 week of previously (Csicsvari et al., 1999). In short, each spike was assigned to a recovery. given phase (bin size of 20°) of the normalized field cycle (ripple–theta). Recording and data analysis. The wire electrodes were lowered into the To reduce bin-border variability, the action potential times were substi- hippocampus until the CA1 pyramidal layer was identified. Positioning of tuted with a Gaussian kernel function. Histograms with mean bin value the hippocampal electrodes was aided by the response evoked by com- of less than five spikes were excluded from the analysis. Theta and ripple missural stimulation and by the presence of field ripples (200 Hz oscil- averages were obtained by averaging filtered signals, using the positive lations) associated with unit activity (Ylinen et al., 1995). After stable peaks of the ripples waves and the negative peaks of the theta cycle as the recording from the pyramidal cell layer, the tungsten electrode was zero reference. moved into the MSDB until discriminable septal units were recorded.