Research ReportAnalysis of resurgent sodium-current expression in rat parahippocampal cortices and hippocampal formation
Introduction
Voltage-dependent Na+ currents active in a sub- or near-threshold range of membrane potentials have an important role in influencing the excitable properties and signal processing functions of central neurons. Such currents include the persistent Na+ current (INaP) and the resurgent Na+ current (INaR), which are generated by voltage-gated Na+ channels in addition to the classical, transient Na+ current (INaT) responsible for the action potential (AP). INaP is a small, non- or slowly-inactivating current that in a number of neurons has a major influence on membrane voltage in a subthreshold range, thereby promoting intrinsic subthreshold activities and/or specific patterns of discharge organization (discussed in Magistretti and Alonso, 2002). INaR has been identified more recently as a Na+-current component activated on repolarization after prominent, brief depolarizations that cause Na+-channel inactivation, like the AP itself (Raman and Bean, 1997). INaR is believed to result from Na+ channels transiently dwelling in the open state during recovery from inactivation. This atypical return path has been proposed to depend on a process of open-channel block proceeding in parallel, and in competition, with classical inactivation (Raman and Bean, 2001). According to this view, block is caused by an intracellular blocking particle binding to the Na+-channel open state, and preventing classical inactivation; whereas relief from block is promoted by repolarization, due to displacement of the blocking particle by Na+-ion inflow. The activation properties of INaR make this current suitable to provide a depolarizing drive early after the discharge of an AP. INaR may thus significantly enhance firing frequency during tonic firing (Khaliq et al., 2003, Akemann and Knöpfel, 2006, Magistretti et al., 2006) or promote discharge of AP doublets in response to brief above-threshold depolarizing stimuli (Raman and Bean, 1997). Originally, INaR was described in cerebellar Purkinje cells (Raman and Bean, 1997), then it was also found in other cerebellar neuron types (Raman et al., 2000, Afshari et al., 2004, Magistretti et al., 2006). So far, INaR has been observed in only a few central neuronal populations outside the cerebellum, namely subthalamic neurons (Do and Bean, 2003), mesencephalic trigeminal neurons (Enomoto et al., 2006), and neurons of the medial nucleus of the trapezoid body (Leão et al., 2006). No information is currently available on the possible expression and role of INaR in cortical structures, and, specifically, in parahippocampal cortices and hippocampal formation.
The parahippocampal region (PHR) and the hippocampus form, together, the so-called medial temporal-lobe memory system, which is well known to play a crucial role in various aspects of memory formation, sensory representation, and spatial orientation (Eichenbaum, 2000, Witter and Wouterlood, 2000). The PHR includes the perirhinal cortex (PRC) and entorhinal cortex (EC), which, according to cytoarchitectonic criteria, can further be divided into distinct subfields. The PRC consists of two subregions named area 35 and area 36, which form two narrow strips parallel to the rhinal sulcus. Area 36 is more dorsal and bordered laterally by the temporal cortex, area 35 is more ventral and bordered medially by the EC (Burwell, 2001, Suzuki and Amaral, 2003, Uva et al., 2004). The PHR is reciprocally connected with a variety of unimodal and multimodal association areas of the neocortex and with the hippocampal formation (HF), thus acting as an ‘interface’ between the hippocampus and the rest of the pallium (Witter and Wouterlood, 2000). Anatomical studies have shown that neocortical fibers directly contact area 36, from which efferent projections reach in sequence area 35, the EC, and the dentate gyrus in a cascade-like manner (Burwell and Amaral, 1998, Burwell, 2000, Burwell, 2001). Profuse, intrinsic associative projections also exist within the PRC and EC, suggesting that major signal integration and association processes take place in the PHR (Witter et al., 1986). Indeed, the PHR appears to play a role in specific memory functions independently of the hippocampus (Zola-Morgan et al., 1993, Young et al., 1997, Murray and Richmond, 2001). Intrinsic neuronal properties are likely to have major influence in the computational functions of the PHR and HF networks. In particular, the importance of Na+-dependent processes in contributing to intrinsic activity patterns has been widely recognized in the PRC (D’Antuono et al., 2001), EC (Klink and Alonso, 1993, Agrawal et al., 2001), and HF (Azouz et al., 1996). Na+-dependent events occurring during above-threshold activity include, in hippocampal CA1 pyramidal cells, depolarizing afterpotentials that follow the single AP and can promote burst firing (Azouz et al., 1996); and, in EC layer-V cells, the organization of the early phase of AP firing in burst-like doublets (Hamam et al., 2000), subtended by a depolarizing envelop that is most likely Na+-mediated (Agrawal et al., 2001).
In the present study we carried out a systematic analysis of INaR expression in various neuronal populations of rat PRC areas 36 and 35, medial EC (mEC), dentate gyrus (DG), and hippocampal CA3 and CA1 fields. We found that INaR is widely expressed in both superficial and deep layers of PRC and mEC. The current was observed in a smaller percentage of cells in the DG and even less frequently in CA1, whereas it appeared to be completely absent in CA3 neurons. These results provide a basis for further studies on the functional roles and molecular determinants of INaR in the various structures of the medial temporal-lobe memory system.
Section snippets
Results
Resurgent Na+ current (INaR) expression was examined in several neuronal populations of the parahippocampal region and hippocampal formation. The voltage-clamp protocol applied to evoke INaR is shown in Fig. 2A1, and consisted of a 19-ms depolarizing step at 0 mV followed by 100-ms step repolarizations at − 15 to − 85 mV in − 5-mV increments. A protocol dedicated to INaT activation, consisting of 19-ms step depolarizations at − 75 to +20 mV in + 5-mV increments, was also routinely applied to
Discussion
The present work is among the first reports of INaR expression in central neurons outside the cerebellum, and demonstrates that INaR is widely present in the cortical regions of the medial temporal lobe memory system.
INaR was found in a vast majority of neurons in parahippocampal cortices (PRC areas 36 and 35, and mEC), in both superficial and deep layers. The highest levels of INaR expression, in terms of both percent of cells and current peak amplitude, were observed in stellate neurons of
Slice preparation
Young Wistar rats (18–23 days old) were anaesthetized by inhalation of halothane (Sigma-Aldrich S.r.l., Milan, Italy) and decapitated. The brain was quickly extracted under hypothermic conditions and submerged in an ice-cold artificial cerebrospinal fluid (ACSF) composed of (in mmol/l): 125 NaCl, 3 KCl, 24 NaHCO3, 1.25 KH2PO4, 1.2 MgSO4, 2 CaCl2, 10 d-glucose (pH 7.4 by saturation with 95% O2, 5% CO2). Brain dissection was carried out accordingly to the slicing plane chosen and the structure to
Acknowledgments
This study was supported by a grant (PRIN 2005 no. 2005059453_003) from the Italian Ministry of Education, University and Research (MIUR) to J.M.
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