Materials and methods cells directly in Nissl stained sections.

M . Funahashi et al. Brain Research 884 2000 104 –115 105 between conditioning and unconditioned stimuli is formed NaCl, 124; KCl, 5; CaCl , 2.0; MgCl , 1.6; NaHCO , 26; 2 2 3 [12]. These arise from several parts of the hippocampal glucose, 10; pH was maintained at 7.4 when exposed to formation, including area CA1 at the CA1-subiculum 95 O 5 CO . Thick slices of tissue about 2–4 mm 2 2 transition and the entorhinal cortex. The entorhinal cortex thick, which included portions of the lateral amygdala, and perirhinal cortex areas 35 and 36 have projections to hippocampus, entorhinal cortex and subicular complex, the lateral amygdala, whereas the other hippocampal were cut horizontally from the intact hemispheres. Thin afferents terminate in different amygdalar nuclei [11,30]. slices about 350 mm were cut from such blocks using a Our interest in the entorhinal inputs to the amygdala tissue slicer Microslicer DTK-3000, Dosaka, Japan and derives in part from an interest in hippocampal formation maintained in a holding chamber at room temperature inputs that may converge on cells receiving thalamic or 23–258C for at least 1 h. Single slices were transferred to association cortical inputs in the lateral nucleus of the a nylon mesh support in an interface recording chamber, amygdala. These entorhinal afferents may also mediate the where they were perfused with ACSF. The upper surfaces spread of synchronous population events that begin in the were exposed to a warmed, moistened atmosphere of 5 hippocampus or entorhinal cortex. Such events include CO in O . The temperature of the chamber was controlled 2 2 ‘abnormal’ synchronous discharges that can arise in the at 3560.18C. hippocampal formation such as interictal spikes [28], and ‘normal’ synchronous discharges such as sharp waves 2.2. Recording techniques [3,26]. Return projections from the amygdala to the entorhinal cortex have been shown to carry another Extracellular recording electrodes were stainless steel synchronous population discharge, called a sharp sleep acute conical tips; Roboz Microprobe, Rockville, MD potential, from the basolateral amygdala to the entorhinal with tip impedances at 1 kHz of 0.9 to 1.1 MV. Signals, cortex [21]. referred to the bath, were amplified DPA-100D, Dia One reason the hippocampal sharp wave has attracted Medical Systems, Tokyo, Japan, filtered 0.1 Hz to 10 attention is that the firing patterns of neurons participating kHz, 26 dB octave, and digitized Digidata 1200, Axon in the generation of a sharp wave resemble patterns of Instruments, Foster City, CA for off-line analysis. In- stimulation used to induce long term potentiation. This tracellular recording electrodes were pulled from 1 mm includes a relatively brief period of very high frequency diameter filament-containing glass capillary tubes and activity 200 Hz at the beginning of the event and a filled with 3 M potassium acetate tip resistances 80–120 trailing, longer duration period of gamma frequency 40– MV or a solution containing 2 Neurobiotin tracer 100 Hz activity. In the entorhinal cortex, distant sites can Vector Laboratories, Burlingame, CA, USA in 2 M be synchronous at zero phase lags during periods of potassium acetate tip resistances 100–180 MV [13]. gamma activity [8]. While it is unclear what information Intracellularly recorded signals were amplified by a high may be carried in a sharp wave, these events can lead to input impedance amplifier with facilities for current in- changes in synaptic efficacy themselves or they may jection using a bridge circuit and capacitance compensa- facilitate changes at other weaker inputs. tion Neurodata IR-183, New York, USA. Extracellular Sharp waves are believed to originate in intact animal stimulating electrodes were parallel bipolar 150 mm brains in area CA3 of the hippocampus [3]. From here, diameter stainless steel, 0.5 mm tip exposure, 0.19 mm tip these events propagate through successive hippocampal separation; FHC, Brunswick, ME, USA. Stimulus pulses formation regions to the entorhinal cortex [5]. The spread were put through constant-current isolation units Isolator- of activity beyond the entorhinal cortex has not been 10, Axon Instruments, Foster City, USA at 0.25 Hz, 50 ms defined. We sought to explore the propagation of syn- duration, 0.1–0.35 mA. chronous population discharges from the entorhinal cortex All electrodes were placed under direct visual guidance to the lateral nucleus of the amygdala and to identify the with a dissecting microscope according to measurements cellular targets of this activity. These data from brain slices from observable landmarks e.g. pial surface, angular may be a useful background for studies of sharp wave bundle, alveus, fimbria, ventricle. During experiments, the propagation in vivo. locations for all recording and stimulating electrode place- ments were drawn by hand using a dissecting microscope. Cell locations of labeled neurons were obtained by locating

2. Materials and methods cells directly in Nissl stained sections.

2.1. Slice preparation and maintenance 2.3. Pharmacological manipulations Male Sprague–Dawley albino rats 150–230 g were In some experiments CPP 3-[2-carboxypiperazin-4-yl]- anesthetized with halothane and decapitated. Each brain propyl-1-phosphonic acid; 10 mM, RBI, MA, USA and was removed from the skull, bisected, and placed briefly in CNQX 6-cyano-7-nitroquinoxaline-2,3-dione; 10 mM, ice cold artificial cerebrospinal fluid ACSF in mM RBI were added to the bathing medium to antagonize 106 M against NMDA and non-NMDA receptor mediated gluta- stellate neurons from our dataset are shown in Fig. 1. matergic transmission, respectively. Picrotoxin 100 mM, Pyramidal and stellate neurons were subclassified based on Sigma Chemical Co., St. Louis, MO, USA was used to their dendritic spine density spiny, slightly or sparsely block GABA receptor-mediated inhibitory transmission. spiny and aspiny. Spine density was actually a principal A characteristic in the classification scheme of McDonald 2.4. Neurobiotin-labeling of single cells [17]. Separately, recorded cells were classified based on their For cells recorded with Neurobiotin-containing elec- electrophysiological properties. As in a previous study trodes, after electrophysiological recording, Neurobiotin- [25], our principal characteristic was the appearance of tracer was injected into these cells using 2–4 nA depolariz- afterpotentials following action potentials Fig. 2. We ing rectangular current pulses 150 ms duration at 3.3 Hz distinguished two types of afterpotential that followed an for 20–30 min. Post-injection survival times ranged from action potential initiated by current injection. One was a 10 to 60 min. Slices with Neurobiotin-injected cells were hyperpolarizing afterpotential appearing in 63 of re- fixed in 4 paraformaldehyde and 0.2 picric acid in 0.1 corded cells. The other was a depolarizing afterpotential M phosphate buffer pH 7.4 from overnight to 10 days. appearing in 37 of recorded cells. Frozen sections 40–60 mm thick were cut from the fixed Neither of the two basic electrophysiological classes tissue and kept in phosphate buffered saline PBS, pH 7.4. corresponded to a single morphological cell class. The After rinses with PBS, these sections were treated with basic electrophysiological and morphological properties of 0.1 H O for 20 min and Triton-X100 0.4–0.5 in the cell classes are given in Tables 1 and 2. Cells with 2 2 PBS for 2 to 3 h. They were rinsed in PBS and then hyperpolarizing afterpotentials were found to be both incubated in the Vectastain ABC Reagent Vector Lab- pyramidal and stellate in shape, although most 19 22 oratories in PBS for 2 to 3 h. After rinses with PBS, were pyramidal cells. Cells with depolarizing afterpoten- sections were reacted with diaminobenzidine DAB and tials were also found to be both pyramidal 8 13 and H O 0.003 in PBS to visualize the injected cells. The stellate in shape 5 13, although the split was more 2 2 sections, which included the successfully stained cells, equitable. were counter stained by Nissl staining. 3.2. Intrinsic electrophysiological properties

3. Results Both electrophysiological classes of cells discharged