Introduction The amygdala has been subdivided into multiple nuclei

Brain Research 884 2000 104–115 www.elsevier.com locate bres Research report Propagation of synchronous burst discharges from entorhinal cortex to morphologically and electrophysiologically identified neurons of rat lateral amygdala a , a b Makoto Funahashi , Ryuji Matsuo , Mark Stewart a Department of Physiology , Okayama University Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japan b Department of Physiology and Pharmacology , SUNY Health Science Center, Brooklyn, NY 11203, USA Accepted 15 August 2000 Abstract Intracellular and field potential recordings were taken from the lateral nucleus of the amygdala in a rat horizontal brain slice preparation that included hippocampal formation. Pyramidal cells comprised the majority of labeled cells 77. Electrophysiological classification based on hyperpolarizing or depolarizing afterpotentials subdivided both the pyramidal and non-pyramidal cell classes, although pyramidal cells tended to have hyperpolarizing afterpotentials 70 and non-pyramidal cells tended to have depolarizing afterpotentials 63. Synchronous population bursts were triggered with single extracellular stimuli in the deep layers of entorhinal cortex. These events propagated from deep layers of entorhinal cortex into the lateral nucleus of the amygdala. Latencies were consistent with a direct entorhinal to amygdala projection. Individual lateral nucleus neurons exhibited responses ranging from a long burst response that included an initial period of 200 Hz firing and a tail of gamma frequency firing lasting over 100 ms grade 1 to an epsp with no firing grade 4. Half of pyramidal cells responding to events initiated in entorhinal cortex were found to receive epsps strong enough to trigger firing. Only one stellate neuron fired in response to entorhinal stimulation. Excitatory postsynaptic responses included NMDA and non-NMDA receptor mediated components. We demonstrate that synchronous population events can propagate from entorhinal cortex to the lateral nucleus of the amygdala and that pyramidal neurons of the lateral nucleus are more common targets than stellate neurons. We conclude that other synchronous events such as sharp waves and interictal spikes can spread from entorhinal cortex to amygdala in the same manner.  2000 Elsevier Science B.V. All rights reserved. Theme : Other systems of the CNS Topic : Limbic system and hypothalamus Keywords : Amygdala; Retrohippocampal area; Sharp wave; Rat

1. Introduction The amygdala has been subdivided into multiple nuclei

and subnuclei with some variability in nomenclature The amygdala is a subcortical component of the limbic [11,23], but much is known about its organization [22]. In system that is critical for the expression of emotion. Basic particular, the lateral nucleus of the amygdala is considered neuroanatomical studies [1,11,24,30] and experiments with a principal target for the cortical and subcortical projec- classically conditioned fear responses [6,14,16] are being tions that carry information about a conditioning stimulus used to define the cortical and subcortical pathways into that animals learn to fear [6,14,16]. For example, direct the amygdala that mediate emotional learning and memory projections from the auditory thalamus and relayed projec- [4]. tions from the auditory association cortex have been shown to converge on the lateral nucleus of the amygdala with information about a tone that signals a foot shock. Corresponding author. Tel.: 181-86-235-6642; fax: 181-86-235- Another system of inputs to the amygdala may encode 6644. E-mail address : mfunadent.okayama-u.ac.jp M. Funahashi. information about the context within which an association 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 5 4 - 7 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.