Although immunocytochemical studies (Bilak & Morest, 1998; Rubio & Wenthold, 1999) have demonstrated the presence and distribution of various types of glutamatergic receptors on the pyramidal cells of the cochlear nucleus, very few functional studies have been conducted on these receptors so far. To elucidate the possible involvement of the ionotropic glutamate receptors in the Ca²⁺ homeostasis of the pyramidal cells, animals 4-9 days old were decapitated and the dorsal part of the cochlear nucleus removed. The procedure was approved by the Animal Care Committee of the University of Debrecen. The tissue pieces underwent an enzymatic dissociation followed by mechanical trituration. The cells were then placed into Dulbecco’s modified Eagle’s medium containing 10 % heat-inactivated horse serum, and they were incubated at 37 °C in 5 % CO₂ atmosphere for 6 h. At the end of the incubation period 3 µM fura-2 AM was applied in the incubating medium. The [Ca²⁺]_i and its changes were determined using a PTI Deltascan system.

Extracellular application of 0.01-10 mM L-glutamate (3-20 s) resulted in transient increases of the [Ca²⁺]_i (the extracellular [Ca²⁺] was 5 mM). When 5 mM glutamate was used, the peak of the Ca²⁺ transient was 332 ± 139 % of the resting [Ca²⁺]_i, which was 11 ± 5 nM (n = 19; mean ± S.D.).

In the presence of 100 µM CNQX the peak of the Ca²⁺ transients was reduced to 8 ± 6 % of the control (n = 7), indicating that in the artificial cerebrospinal fluid (ACSF; containing 1 mM MgCl₂), the Ca²⁺ transients were produced by the activation of non-NMDA receptors mainly. Moreover, the majority of Ca²⁺ entering the cells originated from the extracellular space passing through the non-NMDA receptors, indicating a significant Ca²⁺ permeability of these channels. There are three lines of evidence supporting this conclusion: first, the inhibition of the voltage-gated Ca²⁺ channels with 200 µM CdCl₂ did not prevent the genesis of the Ca²⁺ transients, although the peak was somewhat reduced (to 76 ± 7 % of the control, n = 3). Second, in the absence of extracellular Na⁺, Ca²⁺ transients could still be evoked (n = 8). Third, when the extracellular Ca²⁺ concentration was reduced to 20 µM, the developing transients were instantly terminated, and the [Ca²⁺]_i returned to the resting level.

In the ACSF containing 1 mM MgCl₂ we could not observe a significant activation of the NMDA receptors, as AP-5 had a very slight effect on the amplitude of the developing transients. However, when 50 µM glycine was added to the extracellular solution, and the Mg²⁺ concentration was reduced to 10 µM, Ca²⁺ transients could be evoked even in the presence of CNQX. It is worth noting that the combination of glycine and glutamate alone was not able to evoke Ca²⁺ transients; the reduction of the extracellular Mg²⁺ was essential in evoking NMDA receptor-mediated responses.

These findings indicate that the pyramidal cells of the cochlear nucleus possess functioning ionotropic glutamatergic receptors even after our enzymatic isolation. The Ca²⁺ transients evoked by glutamate occurred mainly as the consequence of Ca²⁺ entry from the extracellular space through non-NMDA receptors. However, in the presence of glycine and low Mg²⁺, the activation of the NMDA receptors could also be demonstrated.

This work was supported by Hungarian Science Foundation (OTKA T31824) and Hungarian Ministry of Health (ETT 49/2000).

Bilak, S.R. & Morest, D.K. (1998). Synapse 28, 251-270.

Rubio, M.E. & Wenthold, R.J. (1999). J. Neurosci. 19, 5549-5562.