Neuroscience Research 41 (2001) 6770 www.elsevier.

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Subcellular localization of neuronal nitric oxide synthase in the
supercial gray layer of the rat superior colliculus
Claudia M.C. Batista
a,b
, Katia C. de Paula
b,1
, Leny A. Cavalcante
b
,
Rosalia Mendez-Otero
b,
*
a
Departamento de Histologia e Embriologia, ICB, Brazil
b
Instituto de Biosica Carlos Chagas Filho, Uni6ersidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil
Received 21 March 2001; accepted 18 June 2001
Abstract
The supercial layers of the rat superior colliculus (sSC) receive innervation from retina and include nitric oxide synthase
(NOS)-immunoreactive neurons. We used electron microscopic immunocytochemistry to assess the subcellular localization of
neuronal NOS (nNOS) in the sSC. nNOS immunoreactivity was detected on the external membrane of mitochondria, endoplasmic
reticulum, in pre- and postsynaptic proles and also diffusely distributed in the cytosol. Postsynaptic labeled regions were often
associated with presumptive retinal unlabeled terminals. Microtubules also appeared intensely labeled. These results show that
NOS immunoreactive neurons may be innervated by retinal terminals and suggest an association of nNOS with cytoskeletal
elements. 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
Keywords: Superior colliculus; Nitric oxide synthase; Immunocytochemistry; Cytoskeleton; Ultrastructure
The gas nitric oxide (NO) has been proposed to act
as an important biological messenger molecule in the
central nervous system and also to play a prominent
role in several neuropathological conditions (Brenman
and Bredt, 1997; Cudeiro and Rivadulla, 1999). NO can
be synthesized from L-arginine by various isoforms of
nitric oxide synthase (NOS). In the nervous system,
neuronal NOS (nNOS) accounts for the majority of the
physiological actions of NO (Garthwaite and Boulton,
1995). The mammalian superior colliculus (SC) is one
of the brain structures showing the highest numbers of
NOS-expressing neurons (Gonzalez-Hernandez et al.,
1992). Yet, virtually nothing is known about the func-
tion of this molecule in the SC and no detailed reports
have appeared which describe the intracellular distribu-
tion of this enzyme in this structure. The supercial
layers of the SC (sSC) have a visual function and
receive afferent bers from the retina, occipital cortex
and parabigeminal nucleus (Mendez-Otero et al., 1980;
Huerta and Harting, 1984). In previous light micro-
scopic studies, we have shown that in the adult rat
superior colliculus, all neuronal types described with
the Golgi technique are stained with NADPH-di-
aphorase and antibodies against nNOS, demonstrating
the presence of nNOS in these cells (Tenorio et al.,
1995, 1996; Soares-Mota et al., 2001). We have also
shown that removal of eye input to the SC results in
changes in the intracellular distribution of the enzyme
in SC neurons suggesting that retinal bers might be
involved in the control of the enzyme (Tenorio et al.,
1998). One of the purposes of this study was to deter-
mine the intracellular distribution of the nNOS in the
sSC of normal adult rats. The second purpose was to
investigate whether retinal afferents synapse onto NOS-
positive cells in the SC.
We have used Lister adult rats (n=4) obtained from
our breeding colony and all experimental protocols
were approved by the Committee for the Use of Exper-
imental Animals of our institution. All chemicals were
purchased from Sigma (St. Louis, MO) unless otherwise
* Corresponding author. Tel.: +55-21-562-6554; fax: +55-21-280-
8193.
E-mail address: rmotero@biof.ufrj.br (R. Mendez-Otero).
1
Present address: Departamento de Protozoologia, Fundacao Os-
waldo Cruz, RJ, Brazil.
0168-0102/01/$ - see front matter 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
PII: S0168- 0102( 01) 00268- 1
C.M.C. Batista et al. / Neuroscience Research 41 (2001) 6770 68
stated. Animals were anaesthetized with sodium pento-
barbital (50 mg/kg, i.p.) and intracardially perfused
with cold phosphate buffered saline (PBS), 10 mM (pH
7.4) for 1 min and then with 4% paraformaldehyde,
0.08% glutaraldehyde in 0.1 M phosphate buffer (PB),
pH 7.4 for 30 min. The brains were removed and
post-xed overnight at 4 C in the same xative. After
xation, the midbrain was vibratome-sectioned to 50
mm thickness in ice-cold 0.1 M PB (pH 7.4). Sections
were then transferred to plastic vials containing 30%
sucrose in 0.1 M PB. The vials were frozen by immer-
sion in liquid nitrogen and stored frozen until process-
ing for immunocytochemistry. For pre-embedding
immunocytochemistry, the solution for antibody incu-
bations contained 1% bovine serum albumin, 0.1%
glycine, 0.3% normal goat serum, in 0.1 M PB (pH 7.4).
Vibratome sections were incubated in 1:300 dilution of
monoclonal anti-nNOS (N-2280, Sigma) or rabbit poly-
clonal anti-nNOS (N31030, Transduction Laboratories)
for 48 h, rinsed and incubated overnight in biotinylated
secondary antibody, followed by incubation in avidin
peroxidase complex for 1 h. After rinsing, the sections
were incubated with 0.07% diaminobenzidine (Sigma
Fast DAB), 0.3% CoCl
2
(Metal Enhancer; Sigma) and
0.03% H
2
O
2
in 0.1 M PB (pH 7.6), for 812 min. The
tissue was then immersed in 1% osmium, 0.08% potas-
sium ferricyanide in 0.1 M PB (pH 7.4) for 15 min,
dehydrated, embedded in Spurr resin and mounted with
glass slides and coverslips pretreated with liquid-release
agent (Electron Microscopy Sciences, Fort Washington,
PA). Selected areas were cut out with a razor blade,
remounted and sectioned. After obtaining ultrathin sec-
tions, grids were left unstained or counterstained only
with lead citrate and viewed with a Zeiss 900 electron
microscope. Electron micrographs were scanned and
treatment of the image was performed with Adobe
Photoshop, using only the brightness and contrast com-
mands. Control sections were immunoprocessed with
the secondary antibodies in the absence of the primary
antibodies.
At the electron microscopic level, preembedding im-
munocytochemistry showed nNOS-immunoreactivity
(nNOS-IR) spread throughout the cytoplasm of neu-
ronal cell bodies and their processes in all samples
observed (Fig. 1A). nNOS immunoreaction products
were predominantly bound to the membranes, although
granular products were detected dispersed throughout
the cytoplasmatic matrix (Fig. 1A,B). The distribution
of nNOS-IR at the nuclear envelope is discontinuous
(Fig. 1B). There was also a conspicuous nNOS-IR on
the external membrane of mitochondria (Fig. 1AC)
and on ER proles in dendrites (Fig. 1C,G). In our
material, the Golgi apparatus was devoid of staining.
Both pre- and postsynaptic stained proles were found,
although nNOS-IR was observed predominantly in
postsynaptic regions (Fig. 1E). Stained mitochondria
were often seen in either pre- or postsynaptic elements.
At the labeled presynaptic regions, the reaction product
formed a heavy deposit associated to pleomorphic vesi-
cles (Fig. 1D). These vesicles were few in number and
loosely arranged, thus, having features reminiscent of
vesicles in presynaptic dendrites. More rarely, im-
munoreactive presynaptic axon terminals were seen
contacting immunoreactive dendritic shafts (Fig. 1F).
At the labeled postsynaptic sites, nNOS-IR was concen-
trated over paramembranous specializations of asym-
metric synapses (Fig. 1E). These very frequently labeled
areas were usually postsynaptic to unlabeled terminals
with pale mitochondria and round vesicles identied as
forming the bulk of retinofugal terminals (Behan, 1981;
Mize, 1983; Carter et al., 1991; Miguel-Hidalgo et al.,
1991). Occasionally, presynaptic proles containing
pleomorphic vesicles were seen making symmetrical
synapses with immunostained dendrites (Fig. 1F). It
has been proposed that the vast majority of retinal
terminals in SC are glutamatergic (Mize and Butler,
1996) and that antibody labeling to the NMDAR1
receptor subtype is present at postsynaptic densities
opposite to retinal terminals (Mize and Butler, 2000).
In this respect, it is important to mention that synaptic
localization of nNOS is regulated by its amino-terminal
PDZ domain that links the synthase to protein com-
plexes at postsynaptic densities containing NMDA re-
ceptors (Brenman and Bredt, 1997). More rarely,
unlabeled axon terminals forming axo-somatic synapses
with labeled cell bodies presented small pleomorphic
vesicles (data not shown) similar to those identied as
GABA-immunoreactive in the SC of other species
(Mize et al., 1994).
Our most remarkable nding was that both axons
and dendrites exhibited microtubules intensely labeled
in all stained neurons, especially when metal-enhanced
DAB was used (Fig. 1F,G). Association of NOS with
cytoskeletal elements may be involved in the process
proposed by Tenorio et al. (1998) in which local neural
activity from retinotectal cells would be coupled to
alterations of NOS protein levels within dendrites.
Physical interaction between the glutamate receptors
and the postsynaptic density protein-95 (PSD-95) is
supposed to be modulated by extensive synaptic activa-
tion. A population of labile microtubules, in addition to
contributing to morphological adaptations at the den-
dritic spine, might also allow for the transport of newly
synthesized proteins required during and for sustained
synaptic activity (Van Rossum and Hanisch, 1999). It is
possible to suggest that an association of NOS with
microtubules could favor the transport of this enzyme
during synaptic activity. A striking colocalization of
postsynaptic density-93 (PSD-93) and microtubule-as-
sociated protein 1A (MAP1A) has also been reported in
dendritic microtubules. PSD-93 and PSD-95 have three
PDZ domains and require micromolar concentrations
C.M.C. Batista et al. / Neuroscience Research 41 (2001) 6770 69
of other different PDZ binding peptides to potentiate
their binding activity to MAP1A in microtubules (Bren-
man et al., 1998). Based on our observations that
nNOS is associated with microtubules, we suggest that
this enzyme, which binds to the second PDZ domain of
PSD-95 (Brenman and Bredt, 1997), could be involved
in the binding activity of PSD-95 to MAP1A in
microtubules.
In summary, we have shown that immunoreactivity
to nNOS was detected in the rat superior colliculus
associated with endomembranes, microtubules, synaptic
vesicles and postsynaptic specializations of proles con-
nected to retinal terminals. Neurons immunostained for
NOS also receive connections from other regions (non-
retinal). In addition, we have veried a diffuse, granular
pattern of labeling throughout the cytoplasmic matrix.
Our results also show that nNOS may possibly be
transported by cytoskeletal-associated elements to distal
sites in the dendritic tree. NOS neurons, in addition to
other connections, may be innervated by retinal gluta-
matergic terminals. Retinal glutamatergic terminals
may innervate NOS neurons, in addition to other con-
nections, so that a participation of NO as a modulator
in glutamate release can be suggested.
Fig. 1. Neuronal nitric oxide synthase-immunoreactivity (nNOS-IR) in cell bodies and process proles in the supercial layers of the rat superior
colliculus. (A) nNOS-IR in a densely stained neuron. DAB reaction product is diffusely distributed throughout the cytosol and attached to
membranes of mitochondria and endoplasmic reticulum (arrows). The nucleus (N) is free of reaction product. (B) Nuclear envelope showing the
deposition of the reaction product (arrow). Note also the dark labeling in the cytosol. (C) A dendrite (d) transversally sectioned illustrating
nNOS-IR in the external membrane of mitochondria (m). (D) Heavy deposit of reaction product associated to pleomorphic vesicles (arrow) in a
presumptive presynaptic dendrite. (E) Immunoreactive postsynaptic prole of an asymmetric contact. The arrow points to the labeled postsynaptic
density. The pale mitochondria in the presynaptic prole suggest a retinal origin for this terminal. (F) Photomicrograph of a longitudinal section
containing microtubule proles within a dendrite. Arrowheads on the left point to immunoreactive microtubules. An unlabeled dendrite on the
right is shown for comparison in which a white arrow points to non-immunoreactive microtubules. Note also in this micrograph an
immunoreactive presynaptic axon terminal and an immuno-negative synaptic prole with pleomorphic vesicles making asymmetrical and
symmetrical (asterisk) synapses, respectively with the stained dendrite. (G) A transversal section of a labeled dendrite. Parallel immunoreactive
microtubules are distinguishable. The two arrowheads point to discernible immunoreactive microtubules. An unlabeled dendrite on the top of this
gure is shown for comparison, in which white arrows point to non-immunoreactive microtubules. Arrows point to endoplasmic reticulum proles
reactive for NOS in (C) and (G). Bar=560 nm for (A), 265 nm for (B,C), 230 nm for (D), 240 nm for (E), 460 nm for (F) and 430 nm for (G).
C.M.C. Batista et al. / Neuroscience Research 41 (2001) 6770 70
Acknowledgements
We thank Dr Elizabeth Debski for helpful comments
on earlier drafts of this manuscript, Dr John Smiley for
advice with the immunocytochemistry protocols and
Sergio Luiz de Carvalho and Felipe Marins for excel-
lent technical assistance. This study was supported by
FINEP, CNPq, FAPERJ, FUJB and PRONEX grants.
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