The electrosensory system

The electrosensory lateral line lobe (ELL) of the gymnotiform fish, Apteronotus leptorhynchus, is a layered hindbrain nucleus receiving topographically organized input from electroreceptors on the fish body surface. The ELL contains, within separate laminae, GABAergic interneurons and pyramidal cells; some pyramidal cells have basal dendrites as well as an extensive spiny apical dendritic tree that ramifies in a molecular layer. Electroreceptor afferents terminate on the basal dendrites of pyramidal cells and granular interneurons. Electroreceptor afferents, as well as both direct and indirect feedback pathways, are glutamatergic; ELL pyramidal cells express high levels of the NR1 subunit of the NMDA receptor and its molecular layer contains high levels of NMDA receptor binding. The feedback pathways have a prominent NMDA receptor component and that EPSPs evoked by stimulation of this pathway are voltage-dependent.

NMDA receptors

Glutamate is the major excitatory neurotransmitter of the nervous system. There are four classes of glutamatergic receptors, three ionotropic called AMPA, NMDA and kainate recepdtors; and one class of metabotropic receptors which are coupled to protein G complexes. Among the ionotropic receptors AMPA and kainate receptors are responsible for the fast transient component of excitatory post-synaptic potential (EPSP), while NMDA receptors are responsible for the longer lasting, slower component. Among the ionotropic class, the NMDA receptors particularly critical for a variety of neuronal functions, including excitotoxicity, synaptic processing and synaptic plasticity.

NMDA receptors are permeable to calcium ions, blocked by magnesium ions in voltage-dependent manner, and can stay open for a relatively long period of time. NMDA receptors are considered coincidence detectors. Synaptic release of glutamate is usually not sufficient to open NMDA receptors unless the membrane has been depolarised as a result of coincident AMPA receptor activation. NMDA receptors are particularly important because they allow calcium to enter the post-synaptic region where it can activate pathways which modulate synaptic strength.

NMDA receptors are heterotetramers composed of two NR1 subunits and two NR2 subunits. The single gene encoding NR1 can produce 8 different NR1 polypeptides by alternative mRNA splicing of three different RNA cassettes. The amino terminal splicing cassette (also named exon 5) is regulates the pH sensitivity. The other cassettes are all at the carboxyl terminal. One of them (C1) is involved in membrane targeting of NMDA receptors and is also phosphorylated by protein kinase C (PKC). We have described the complete splicing patterns for the NR1 subunit from A. leptorhynchus.

The NR2 subunits are coded by four genes (NR2A-D). The NR2 subunits determine many of the critical properties of NMDA receptors. For example, the kinetics, are fast with NR2A containing receptors, and are increasingly slower for NR2B, NR2C and NR2D respectively. NR2B expressing NMDA receptors are the most permeable to calcium ions, followed by NR2A- and NR2C expressing receptors respectively. NR1/NR2A receptors are highly sensitive to Zn++ inhibition. NR1/NR2B is 400 times more sensitive to the antagonist ifenprodil than any other NMDA receptor subtype.

NMDA receptors and electrosensory system Recently, we have identified the homologues for the NMDA receptor subunits in A. leptorhynchus, .We have cloned and sequenced full length cDNAs for NR1-, NR2A- and NR2B- subunits. They all present strong homology with their mammalian homologues.. A key question for us is to understand how the regulated expression different the NR2 subunits contributes to the computational abilities of electrosensory neurons.

Figure 1: Immunohistochemical mapping of NMDA receptors on spines of electrosensory neurons



Another important question concerns the electrophysiological and pharmacological properties of the apteronotid NMDA receptors. Because fish and mammals diverged from their common ancestors approximately 400 million years ago, there are likely to be significant functional differences. We are addressing this issue by studies of recombinant NMDA receptors (NR1/NR2B and NR1/NR2A) expressed in cultured HEK cells. Any functional difference would be due to a change in the amino acid sequence. It would be then possible to use the differences as tools to track down functional motif in the receptor structure. Another goal for these studies is to relate the NMDA receptor properties seen in the invitro models with the in vivo properties of the ELL NMDA receptors. This should allow us to unravel the functional roles of the various NMDA receptor NR2 subunits.