Materials and methods NR2A B, GluR1, GluR2, GluR2 3 and GluR4 in the rat

88 L in vitro [19,21,23,40,43]. Electrophysiological recording overnight at 48C. The brain stems were then serially cut shows that the monosynaptic excitatory postsynaptic po- into frontal sections 40 mm thickness with a freezing tential of second-order vestibular nuclear neurons in frog is microtome and then processed for the following immuno- mediated in part by NMDA receptors and in part by AMPA cytochemical reactions. receptors [40]. In addition, electrophysiological studies Double immunofluorescence was employed to demon- [2,7,15,23]; see review: [13] have demonstrated that strate the co-localization of different receptor subunits in glutamatergic input from the ipsilateral vestibular nerve is individual vestibular nuclear neurons in sections of eight mediated predominantly by AMPA receptors. A recent rats. Alternate sections of the brain stem that contained the study [31] has suggested that AMPA receptors are also vestibular nuclei were processed to double immunofluores- involved in non-primary vestibular excitatory transmission. cence. A mixture of one mouse antibody and one rabbit The AMPA receptors are therefore involved in both the antibody in 0.01 M phosphate-buffered saline PBS primary and non-primary vestibular transmission. Further- containing 3 normal goat serum and 0.1 Triton X-100 more, NMDA receptors, with the characteristics of high was used. The following primary antibodies that were 21 Ca permeability, play more roles in the long-term obtained from commercial source Chemicon International, modulation or long-term potentiation, LTP of synaptic Temcula, CA were employed in the present study: mouse- neurotransmission [3,6,11,32,42]. Recent evidence indi- anti-NR1 monoclonal antibody 1:1000, rabbit-anti- cates that functional AMPA receptors are important for the NR2A B polyclonal antibody 1:500, rabbit-anti-GluR1 maintenance of neuronal dendritic spines where most polyclonal antibody 1:500, mouse-anti-GluR2 monoclo- central excitatory synapses reside [1]. It is of interest that nal antibody 1:1000, rabbit anti-GluR2 3 polyclonal progressive co-expression of functional NMDA receptors antibody 1:500, and rabbit anti-GluR4 polyclonal anti- and AMPA receptors was observed in hippocampal neu- body 1:500. The following double immunofluorescence rons during postnatal development and LTP [24,28] We was then performed: NR1 and NR2A B written as NR1 hypothesize that the co-localization of NMDA receptors NR2A B, NR1 GluR1, NR1 GluR2 3, NR1 GluR4 and and AMPA receptors in individual vestibular nuclear NR2A B GluR2. The sections were incubated in the neurons provides a substrate for cross-talk between the mixtures of primary antibodies for 24–48 h at 48C. receptors during excitatory synaptic events of the vestibu- Subsequently, the sections were washed in PBS 33PBS lar nuclear neurons. The AMPA receptors GluR1, GluR2 and incubated for 2–4 h at room temperature in a mixture 3, and GluR4 subunits were previously reported in the of secondary antibodies, i.e., either donkey anti-mouse IgG chinchilla vestibular nuclei [31]. Several NMDA receptor conjugated with tetramethyl rhodamine isothiocyanate proteins and their mRNAs were also found in vestibular TRITC, 1:100–200; Chemicon and donkey anti-rabbit nuclear neurons [29,33,44]. However, it is not yet known IgG conjugated with dichlorotriazinyl aminofluroscein whether or not NMDA and AMPA receptors are co- DTAF; 1:100–200; Chemicon, or donkey anti-mouse expressed in individual vestibular nuclear neurons. The IgG conjugated with aminomethylcoumarin AMCA, co-localization of NMDA and AMPA receptor subunits, 1:100–200; Chemicon and donkey anti-rabbit IgG conju- viz. NR1, NR2A B, GluR1, GluR2, GluR2 3 and GluR4, gated with TRITC. After several washes in PBS, the was therefore investigated in individual vestibular nuclear sections were mounted in flurosave medium Calbiochem. neurons of the rat with the use of double immunofluores- The sections were examined both with fluorescence micro- cence. The present study provides the basis for elucidating scope Axioplan, Zeiss and a laser scan confocal micro- the interaction between NMDA receptors and AMPA scope LSM 510, Zeiss for DTAF-, AMCA- and TRITC- receptors in excitatory synaptic neurotransmission and labeled neurons. In addition, other sections of four rats synaptic plasticity within the vestibular nucleus. were processed with immunoperoxidase procedures using 3,39-diaminobenzidine DAB as the chromogen ABC kit, Vector Labs. to visualize the cellular localization of NR1,

2. Materials and methods NR2A B, GluR1, GluR2, GluR2 3 and GluR4 in the rat

vestibular nuclei. The sections were reacted in a single Eight adult male Sprague–Dawley rats weighing 230– immunoperoxidase reaction for comparison of the pattern 260 g were used in the present study. All procedures in of distribution and number of immunopositive neurons in each preparation conformed to the Principles of Labora- the regions of the vestibular nuclei. tory Animal Care and were approved by the University of For the control experiments of immunofluorescence and Hong Kong Committee on the Use of Live Animals in immunocytochemistry, the primary antibody was substi- Research. Briefly, the animals were anesthetized with tuted with normal mouse serum for NR1 and GluR2 or sodium pentobarbital 60–80 mg kg, i.p., and then per- normal rabbit serum for NR2A B, GluR1, GluR2 3 and fused transcardially with 100 ml of saline, followed by 500 GluR4 respectively. The sections were then processed ml of 0.1 M phosphate buffer PB; pH 7.4 containing 4 with the same double immunofluorescence or immuno- paraformaldehyde. The brain stems were removed immedi- peroxidase reaction sequence as described above. ately and placed in 0.1 M PB containing 30 sucrose In the present study, the perikaryal size of immuno- L .W. Chen et al. Brain Research 884 2000 87 –97 89 positive cells was measured under the light microscope NR1-IR neurons in the SpVe were triangle, oval, or multi- with the use of a morphometric micrometer. For semi- polar in shape, and their diameters ranged from 10 mm to quantification of immunopositive cells in representative 30 mm Fig. 1F. The NR1-IR neurons in groups x and y sections, double-labeled neurons in eight randomly select- subnuclei were mainly oval in shape and their diameters ed fields 403 objective of each of the following nuclei, ranged from 10 mm to 20 mm Fig. 1B. namely the superior vestibular nucleus SuVe, medial Immunoreactivity for NR2A B Fig. 2A–D, GluR2 vestibular vestibular nucleus MVe, lateral vestibular Figs. 3C–D, 4C–D and GluR2 3 Figs. 3E–F, 4E–F nucleus LVe, spinal vestibular nucleus SpVe, group x subunits was also found to be abundant in the vestbular and group y were counted. nuclei. The immunoreactive neurons were similar to the NR1-IR neurons in terms of cell number and distribution pattern in the SuVe, MVe, LVe, SpVe and group x, y

3. Results subnuclei. Immunoreactivity for GluR1 Figs. 3A–B, 4A–