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–
B or GluR4 Figs. 3G–H, 4G–H, however, was less 3.1. Control experiments for immunocytochemistry
abundant than that for NR1. The GluR1-IR or GluR4-IR neurons were also fewer in number than NR1-IR neurons
In those sections that were incubated in normal mouse in different regions of the vestibular nuclei.
or rabbit serum in place of the primary antibodies, no In addition, astrocyte-like immunoreacitve cells are
immunoreactivity was detected data not shown. For prominent in NMDA2A B antibody-incubated sections.
double labeling procedures, only single immunoreactivity Weakly stained astrocytes were also seen in GluR1 and
was detected when one of the two primary antibodies was GluR2 3-stained sections. GluR4-like immunoreactive as-
substituted by normal serum during the reaction sequence. trocytes were hardly detected in our experiment data not
When two primary antibodies were substituted, no im- show. These results were consistent with previous report
munoreactivity was detected data not shown. [30].
3.2. Immunoreactivity for NMDA and AMPA receptor 3.3. Co-localization of NMDA and AMPA receptor
subunits in different regions of the vestibular nuclei subunits
Immunoreactivity for NR1, NR2A B, GluR1, GluR2, Co-expression of immunoreactivity for NMDA receptor
GluR2 3 and GluR4 was identified by the presence of subunits, NR1 and NR2A B, was observed in individual
DAB immunoreaction products in the sections of the rat vestibular nuclear neurons Fig. 5. Double labeled neu-
brain stem. The nomenclature and boundaries defined in rons were found throughout all subdivisions of the vestibu-
the rat brain atlas of Paxinos and Watson [27] were utilized lar nucleus including the SuVe, MVe, LVe, SpVe and group
in this study. In the vestibular nuclei, immunoreactivity for x, y subnuclei. Virtually all the NR1-IR neurons were
NR1 Fig. 1, NR2A B Fig. 2, GluR1, GluR2, GluR2 3 found to exhibit NR2A B immunoreactivity Table 1.
or GluR4 Figs. 3 and 4 was predominantly observed in Fig. 5A, A9 illustrates individual SpVe neurons displaying
neuronal somata and in their proximal dendrites. The co-localization of NR1 and NR2A B immunoreactivity in
immunopositive neurons were primarily distributed in the the somata and dendritic processes. Similarly, co-expres-
superior vestibular nucleus SuVe, medial vestibular nu- sion of immunoreactivity for NR2A B and GluR2, NR1
cleus MVe, lateral vestibular nucleus LVe, spinal ves- and GluR1, NR1 and GluR2 3, or NR1 and GluR4, was
tibular nucleus SpVe and group x, y subnucleus Figs. also found in individual vestibular nuclear neurons. Double
1–4. NR1 immunoreactivity was highly expressed in the labeled neurons were found in the SuVe, MVe, LVe, SpVe
LVe Fig. 1C–D, SpVe Figs. 1E–F and MVe Figs. and group x, y subnuclei. In addition, all the vestibular
1G–H. In SuVe, however, only low NR1-immunoreactivi- neurons that expressed GluR1 Fig. 5 or GluR4 Fig. 5
ty was observed data not shown. Moderate expression of immunoreactivity were found to express NR1 immuno-
NR1 immunoreactivity was found in the groups x and y reactivity. However, the NR1 GluR1 and NR1 GluR4
subnuclei see Fig. 1A–B for y subnucleus. Within the double-labeled neurons were fewer in number. This was
MVe, a majority of NR1-immunoreactive IR neurons was particularly evident in the SuVe, MVe and group x, y
spherical, oval or fusiform in shape Fig. 1 H. The size of subnuclei. Representative NR1 GluR1 and NR2A B
these neurons ranged from small to medium with a GluR2 double labeled neurons in the SpVe are shown in
diameter between 10 mm to 20 mm. The LVe was Fig. 5B, B9, C, C9.
characterized with the presence of giant NR1-IR neurons, which were multi-polar with diameters of 30 mm to 50 mm
3.4. Semi-quantification Fig. 1D. A number of small and medium-sized NR1-IR
neurons were also found in the LVe Fig. 1D. The NR1-IR Results of the semi-quantification analyses of the im-
neurons in the SuVe were similar to those of MVe in their munopositive cell numbers in the vestibular nuclei are
shape and size but most of them were weakly labeled. The shown in Table 1. All of the NR1-IR neurons were found
90 L
Fig. 1. Photomicrographs showing the immunoreactivity for NMDA receptor subunit NR1 in subdivisions of the vestibular nuclei. The photomicrographs in the right panel are high magnification views of those in the left panel A1B, C1D, E1F and G1H. NR1-immunoreactive neurons are shown in regions
including the group y subnucleus Y, lateral vestibular nucleus LVe, medial vestibular nucleus MVe, spinal vestibular nucleus SpVe. Abbreviations :
DC, dorsal cochlear nucleus; icp, inferior cerebellar peduncle; Int, interpositus nucleus of the cerebellum. Scale bars: 300 mm in G for A, C and E; 80 mm in H for B, D and F.
L .W. Chen et al. Brain Research 884 2000 87 –97
91
Fig. 2. Photomicrographs showing the immunoreactivity for NR2A B in the vestibular nuclei. The NR2A B-immunoreactive neurons are shown in lateral vestibular nucleus LVe A1B and spinal vestibular nucleus SpVe C1D. Abbreviations as in Fig. 1. Scale bars: 300 mm in C for A; 80 mm in D for
B.
to display NR2A B and GluR2 3 immunoreactivity. Simi- subunits in individual vestibular nuclear neurons, and c
larly, all of the NR2A B-IR neurons displayed GluR2 NMDA receptors and AMPA receptors are co-localized in
immunoreactivity. Only a proportion of NR1-IR neurons, a large proportion of vestibular nuclear neurons.
however, exhibited GluR1 or GluR4 immunoreactivity. About 52 of the NR1-IR neurons displayed GluR1
4.1. Glutamate and neurotransmission between the immunoreactivity, while 46 of NR1-IR neurons dis-
vestibular afferents and vestibular nuclear neurons played GluR4 immunoreactivity.
Glutamate-containing axonal terminals have been shown to make synaptic contacts with the somata and the de-
4. Discussion ndrites of vestibular nuclear neurons [35]. Pharmacological
