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Journal of Chemical Neuroanatomy 30 (2005) 17–26
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GABAergic parvalbumin-immunoreactive large calyciform presynaptic
complexes in the reticular nucleus of the rat thalamus
Bertalan Csillik a,*, András Mihály a, Beata Krisztin-Péva a, Zoltán Chadaide c,
Mohtasham Samsam b, Elizabeth Knyihár-Csillik c, Robert Fenyo a
a
Department of Anatomy, Albert Szent-Györgyi Medical and Pharmaceutical Center, University of Szeged,
40 Kossuth Lajos sgt, H-6701 Szeged, Hungary
b
Department of Neuroanatomy, University of Saba, Netherlands Antilles
c
Department of Neurology, Albert Szent-Györgyi Medical and Pharmaceutical Center, University of Szeged, Hungary
Received 1 December 2004; received in revised form 3 March 2005; accepted 25 March 2005
Available online 23 May 2005
Abstract
In the reticular thalamic nucleus of the rat, nearly all neurons are parvalbumin-immunoreactive. We found that in addition, though
superficially similar to large parvalbumin-immunoreactive neurons, also numerous peculiar parvalbumin-immunoreactive complexes are
present in the reticular thalamic nucleus which are not identical with parvalbumin-immunoreactive perikarya, as shown by nuclear variation
curves. Light and electron microscopic immunocytochemical studies revealed that these parvalbumin-immunoreactive complexes are brought
about by parvalbumin-immunoreactive calyciform terminals which establish synapses with large, parvalbumin-immunonegative dendritic
profiles. Transection of thalamo-reticular connections did not cause any alteration of calyciform terminals in the reticular thalamic nucleus.
Nuclear counterstaining revealed that parvalbumin-immunoreactive calyciform terminals originated from local parvalbumin-immunoreactive
interneuronal perikarya, which, depending of the length of the ‘‘neck’’ protruding from the perikaryon, establish somato-dendritic, axodendritic or dendro-dendritic synapses. Light and electron microscopic immunocytochemical investigations prove that the parvalbuminimmunoreactive calyciform complexes contain also GABA, that are likely to be inhibitory. In accordance with literature data, our results
suggest that parvalbumin-immunoreactive GABAergic calyciform terminals in the reticular thalamic nucleus may be instrumental in intrinsic
cell-to-cell communications and, as such, may be involved in synchronisation of thalamo-cortical oscillations, in the production of sleep
spindles and in attentional processes.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Thalamus; Reticular nucleus; Parvalbumin; GABA; Calyciform synapses; Somato-dendritc synapses; Dendro-dendritic synapses
1. Introduction
The reticular thalamic nucleus (RTN), the ‘‘guardian of
the gate’’ according to the controversial searchlight theory
(Crick, 1984), is a shell-shaped structure, semicircular in
coronal sections and hook-like in paramedian sagittal
sections of the brain (Koelliker, 1896; Cajal, 1911). The
RTN occupies a strategic position between the neocortex
and the thalamus (Pinault, 2004). By axon collaterals
innervating GABAergic RTN cells (De Biasi et al., 1986;
* Corresponding author. Tel.: +36 62 544 918/545 665 (secretary);
fax: +36 62 545 707.
E-mail address: [email protected] (B. Csillik).
0891-0618/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchemneu.2005.03.010
Houser et al., 1980), the RTN controls thalamo-cortical and
cortico-thalamic functions (Pinault, 2004).
Intrinsic cell-to-cell communications are instrumental in
the function of RTN (Pinault, 2004); these are supposed to
be mediated by dendro-dendritic synapses (Ahlsen and
Lindstom, 1982), postulated to be present in the RTN by the
Scheibels as early as 1972. Though the cell-to-cell
communications are generally thought to be involved in
lateral inhibition, some of them may be excitatory (Andersen
and Eccles, 1962; Alger and Nicoll, 1979; Spreafico et al.,
1988). It is assumed that dendro-dendritic synapses are
instrumental in thalamo-cortical oscillations (Llinás and
Jahnsen, 1982; Deschenes et al., 1984; Jahnsen and Llinás,
1984; Steriade and Deschenes, 1984), the RTN being the
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B. Csillik et al. / Journal of Chemical Neuroanatomy 30 (2005) 17–26
pacemaker of sleep spindles (Steriade et al., 1987); thus
GABAergic neurons may provide a key structure implicated
in normal and paroxysmal oscillatory activities of RTN
(Steriade, 2001; Jones, 2002).
The overwhelming majority of neurons in the RTN
display parvalbumin immunoreactivity (PV-IR; Celio,
1990). Several PV-IR structures, however, only seemingly
correspond to PV-IR large nerve cells: microstructural
analysis revealed that some of the large, PV-IR structures in
the RTN are, in reality, presynaptic terminals of somatodendritic synapses (Csillik et al., 2002). In the present paper,
we report on light and electron microscopic immunohistochemical studies, aiming to reveal whether or not, in
addition to somato-dendritic juunctions, the PV-IR large
calyciform complexes establish other types of synapses in
the RTN. GABA immunostaining was performed at the light
and electron microscopic levels, supplemented by fluorescence immunohistochemical studies in order to determine
whether the PV-IR calyciform complexes are identical with
similar structures expressing GABA. In order to estimate the
relative importance of GABAergic and PV-IR calyciform
complexes in the structure and in the function of RTN, the
numbers and sizes of calyciform terminals have been
determined by means of ocular micrometry and by
stereological methods.
2. Materials and methods
Investigations were carried out on 48 young adult Wistar
rats, obtained from the animal house of the Albert SzentGyörgyi Medical and Pharmaceutical Center, University of
Szeged. Care of the animals was in conformity with the
guidelines controlling experiments and procedures in live
animals, as described in the Principles of Laboratory Animal
Care (NIH Publication no. 85-23, revised 1985), and also
complied with the guidelines of the Hungarian Ministry of
Welfare. Experiments were carried out in accordance with
the European Communities Council Directive (24 November 1986; 86/609/EEC) and the Guidelines for Ethics in
Animal Experiments, of the Albert Szent-Györgyi Medical
and Pharmaceutical Center of the University of Szeged.
2.1. Immunocytochemistry
Animals were subjected to transcardial perfusion with
Zamboni’s picroaldehyde fixative in deep chloral hydrate
anesthesia. The brain was removed, post-fixed in the same
fixative overnight at 4 8C and sectioned with a cryostat either
in the coronal plane, 5.0–7.0 mm from the frontal pole ( 1.8
to 3.8 mm from the bregma), or in a paramedian sagittal
plane, 1.0–4.0 mm from the midline. Section thickness was
25 mm. For the demonstration of parvalbumin (PV), we used
a polyclonal anti-mouse PV antibody raised in rabbits
(Sigma, St. Louis, MO). Preliminary experiments showed
best staining results with antibody dilutions 1:2000–l:3500.
Endogenous peroxidase activity was blocked by the
application of 0.3% hydrogen peroxide diluted in methanol,
for 10 min, followed by three successive rinses in 0.1 M
phosphate buffer. Free-floating sections were pre-treated
with blocking serum (0–1.0 M phosphate buffered saline
[PBS]), 10% normal goat serum, l% bovine serum albumin
[BSA] and 0.3% Triton X-100) on a shaker plate at room
temperature for l h, and then transferred into the primary
antibody. Incubation was carried out at 4 8C on a shaker for
36 h, followed by three rinses in 0.1 M phosphate buffer. To
detect the bound primary antibody, we used the avidin–
biotin peroxidase method. Kits were obtained from Vector
Laboratories (Burlinghouse, CA, USA). The secondary
antibody, biotinylated anti-mouse immunoglobulin was
applied for 90 min at room temperature. Three more rinses
in 0.1 M phosphate buffer were followed by incubation in
the avidin–biotinylated-peroxidase complex for 60 min at
room temperature. After three rinses in 0.1 M phosphate
buffer, peroxidase activity was visualized by the histochemical reaction involving diamino-benzidine-tetrahydrochloride and hydrogen peroxide (3 ml of 30% H2O2 to
10 ml 1% DAB). After three rinses in 0.1 M phosphate
buffer, the free-floating sections were dehydrated in a graded
series of ethanols, cleared in xylene and coverslipped with
Permount.
The specificity of the immunohistochemical reaction was
assessed by means of the following treatments: (1) omission
of the first specific antiserum; (2) use of normal rabbit or
mouse serum instead of anti-PV antiserum; (3) treatment
according to the avidin–biotin complex method, from which
one of the steps had been omitted; (4) preabsorbtion of the
specific antibody with the blocking peptides sc7447P and
sc7448P at 4 8C for 24 h. None of these specimens showed
any reaction.
GABA-IR was detected at the light microscopic level by
means of a GABA antiserum (Chemicon, Temecula, CA,
USA), obtained from rabbit, dilution 1:500.
Staining of PV and GABA by means of immune
fluorescence was performed on 30 mm cryostat sections of
paraformaldehyde fixed rat brains, obtained in the paramedian sagittal plane. Sections were treated as free-floating
specimens. The simultaneous (‘‘cocktail’’) method was
employed. Blocking of endogeneous peroxidase was
performed with 2% H2O2. After rinsing in PBS, containing
0.01% H2O2 and 0.1% Triton X-100, a PV antiserum, raised
in mouse (Sigma) in a dilution of 1:2000, and GABA
antiserum, raised in rabbit (Chemicon) in a dilution of 1:500,
were used as primary antibodies. Incubation was performed
overnight at room temperature. After three times rinsing in
PBS, the sections were incubated in biotinylated anti-mouse
IgG made in horse (Vector Laboratories) for 90 min. After
rinsing in PBS, the incubation was followed in a mixture of
fluorescein iso-thio-cyanate (FITC) labeled anti-rabbit IgG,
raised in goat (Vector Laboratories), 1:200, and streptavidin
peroxidase-labeled CY3 (Jackson, West Grove, PA, USA),
1:100 for 2 h at room temperature. After three rinses, 15 min
B. Csillik et al. / Journal of Chemical Neuroanatomy 30 (2005) 17–26
each in PBS, the sections were mounted in polyvinyl alcohol
mounting medium (DABCO antifading, Sigma RBI). To
prove the specificity of the GABA antibody, pure GABA
(Tocris, Avonmouth, UK) was included in the cocktail
containing primary antibodies. In such specimens, GABAIR did not show up while that of PV persisted. Investigation
and photography of the sections was performed with a Nikon
Eclipse E600 microscope, equipped with B2A and G.2A
filters and a digital camera (SPOT RT Slider).
The intensity of the immunoreaction was measured by
densitometry, performed by digitalizing immunostained
sections with a SPOT RT Slider CCD camera
(1600 1200 pixels, 8 bits) attached to a Nikon Eclipse
E600 microscope using 40 objective and 10 eyepiece.
The captured images were analyzed by ImageProPlus v4.5
morphometric software (Media Cybernetics, Silver Sring,
MD, USA). Areae of interest were rectangular areae
measuring 0.008 mm2. Five rectangular fields were analyzed
per animal, each field chosen randomly in different sections
in a blinded manner. Gray values of the selected areas were
obtained using a ScionNIH image analysis software. The
images were captured directly from the microscopic slides,
using a 5 objective lens by means of a black and white
camera Cohu CCD and displayed on a computer monitor.
The software used was the ImageProPlus v.4.5 program
which automatically assigned the average gray value of the
screen pixels coined to the outlined area; a value of 0
indicated a white pixel and 225 indicated a black pixel.
Diameters of nuclei of PV-IR cells, and the diameters of
the dendrites surrounded by the calyciform endings were
determined by means of an ocular micrometer (Zeiss, Jena,
Germany), using a 100 oil immersion front lense.
Nuclear counterstaining of PV immunostained sections
was performed with a 0.001% cresyl violet solution.
Stereological determination of the numbers of PV-IR
calyciform terminals in the RTN was performed according
to the fractionator method described by West (1999) and by
Buckmaster and Dudek (1997). On the basis of PV-stained
serial sections, also the total PV-IR cell number in the RTN
was determined. The number of sections analyzed per RTN,
which extends 3.2 mm in the sagittal plane, was 13; total
section thickness for disector height was 30 mm.
For electron microscopic immunohistochemistry of PVIR, 50 mm thick free-floating sections were obtained on a
vibratome and processed like the samples for light
microscopy, with the only exception that Triton X-l00
was omitted from all of the solutions. Having visualized the
peroxidase activity, free-floating sections were post-fixed in
an osmic acid solution (2% OsO4 in phosphate buffer) for
1 h and dehydrated in a graded series of ethanol. Sections
were flat embedded in Durcupan on Liquid Release-treated
slides, and polymerized for 36 h at 56 8C. Selected
regions of the brain were excised and remounted. Ultrathin
serial sections were obtained on a Reichert Ultrotome, using
a diamond knife. Sections were stained with lead citrate
according to Reynolds, and analyzed and photographed
19
on a Zeiss Opton 902 electron microscope (Oberkochen,
Germany).
For electron microscopic immunohistochemistry of
GABA-IR, 50 mm thick free-floating sections were obtained
on a vibratome and processed like the samples for light
microscopy, with the only exception that Triton X-l00 was
omitted from all of the solutions. Having visualized the
peroxidase activity, free-floating sections were post-fixed in
an osmic acid solution (2% OsO4 in phosphate buffer) for
1 h and dehydrated in a graded series of ethanol. Sections
were flat embedded in Durcupan. Selected regions of the
brain were excised and remounted. Further treatment and
fine sectioning of the samples was identical with those of
PV-IR. In several cases, visualization of GABA-IR on PV
immunostained sections was attempted with l0 nm goldlabeled goat anti-mouse IgG (Amersham, UK), in a dilution
of 1:10, by means of the post-embedding technique.
Experimental surgery was performed in nembutal
anesthesia on six animals. RTN was isolated from the rest
of the thalamus by means of a 8 mm deep coronal incision,
6 mm anterior from the bregma, with a 3 mm-wide ‘glass
knife’ cut out from a histological coverslip (Palkovits et al.,
1982) through a 3-mm-wide line-shaped hole drilled on the
skull.
For the statistical evaluation of the results, the mean
values and the standard error (S.E.M.) were determined.
Student’s independent samples t-test was used; values
obtained from control and experimental animals were
compared according to the ‘‘one-way ANOVA’’ technique
(Bonferroni method or ‘‘post-hoc’’ test).
3. Results
In conventional coronal sections of the rat brain, RTN
appears as a PV-IR cap encircling the anterior portion of the
thalamus (Fig. 1). Most of the PV-IR elements are nerve cell
perikarya; some of the PV-immunopositive profiles in RTN,
however, while deceptively similar to PV-immunopositive
nerve cells equipped with large, immunonegative nuclei
were found to correspond to cytoplasmic elements which
embraced cross sections of large, immuno-negative structures (Fig. 2–6). More thorough scrutiny of the light
microscopic immunocytochemical specimens (Fig. 7)
revealed that the PV-IR profiles were not perikarya, but
identical with PV-IR calyciform nerve endings, surrounding
PV immunonegative large dendritic profiles. Nuclear
counterstaining (Fig. 6) excluded the possibility that the
PV immunonegative structures surrounded by the PV-IR
terminals could have been cell nuclei.
In the calyciform terminals, similar to those which
exhibited PV-IR, also GABA immunoreactivity could be
demonstrated (Figs. 8–10).
Diameters of nuclei and those of the dendritic bulbs
surrounded by PV-IR calyciform terminals were determined
at the immersion optical level by means of a Zeiss ocular
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Fig. 1–6. Fig. 1. Parvalbumin immunoreactivity (PV-IR) of the RTN (ret) at low power in a coronal section of the brain. Note strong PV-IR of RTN as contrasted
to the rest of the thalamus (asterisk). For orientation, see the symbol: S, superior; M, medial; L, lateral. Scale bar, 90 mm. Fig. 2. PV-IR of a terminal
(arrowhead), surrounding the cross section of a dendrite (arrow) in a paramedian sagittal section of the brain. Scale bar, 10 mm. Fig. 3. The calyciform PV
immunopositive complex (arrowhead) surrounds a dendrite (arrow). The pattern is deceptively similar to a PV-IR nerve cell equipped with a large
immunonegative nucleus. Scale bar, 10 mm. Fig. 4. The calyciform PV immunopositive complex (arrowhead) embraces the grazing section of a large
dendrite (arrow). Scale bar, 10 mm. Fig. 5. Three (partly incomplete) PV-IR axonal complexes (arrowheads), surrounding large, PV-immunonegative dendrites
(arrows). Arrowhead with asterisk denotes cross section of a nucleus. Scale bar, 10 mm. Fig. 6. Three calyciform PV-IR complexes (arrowheads) embrace three
immunonegative dendritic bulbs (arrows). Nuclear counterstaining with cresyl violet. Scale bar, 10 mm.
micrometer (Fig. 11). Accordingly, the spherical nuclei of
PV-IR neurons fall into four categories: (1) nuclei of
nondescript neurons, amounting to 20% of the whole
nuclear population; these nuclei are were very small,
measuring only 2–3 mm in diameter; (2) small fusiform cells
had nuclei with a diameter of 5–7 mm; this category
amounted to 24% of the whole PV-IR nuclear population;
(3) nuclei of large fusiform cells measured 6–8 mm; this
group made up 29% of the PV-IR nuclear population,
while (4) nuclei of large spherical cells had diameters of 8–
9 mm; this group amounted to 26% of the population of PVIR neurons in RTN. The diameters of the slightly ovoid
B. Csillik et al. / Journal of Chemical Neuroanatomy 30 (2005) 17–26
21
Fig. 7–10. Fig. 7. The PV-IR calyciform endings (arrowheads) surround a large dendritic profile (arrow). Scale bar, 10 mm. Fig. 8. A GABA-IR calyciform
ending (arrowhead) surrounds a GABA immunonegative dendritic profile (arrow). The whole complex is deceptively similar to a GABA-IR neuron. Scale bar,
10 mm. Fig. 9. Arrowhead indicates a GABA-IR terminal, surrounding a GABA-immunonegative dendritic profile. Scale bar, 10 mm. Fig. 10. Arrowhead
indicates a GABA-IR terminal, surrounding a GABA-immunonegative dendritic profile. Scale bar, 10 mm.
Fig. 11. Percentage values of variation curves of profiles of nuclei of PV-IR
neurons and of those of dendritic profiles surrounded by PV-IR calyciform
terminals in RTN of the normal rat. S, small nuclei of nondescript neurons;
SF, nuclei of small fusiform neurons; LF, nuclei of large fusiform neurons;
LS, nuclei of large spheroid neurons; D, profiles of PV immunonegative
dendritic bulbs.
profiles of dendritic bulbs, surrounded by PV-IR calyciform
elements, measured, in average, 10 mm 12 mm. The
dendritic profiles amounted to 12% of the total population
of PV-IR elements (nuclei and dendritic bulbs). Even more
characteristic is the comparison of the surface areas
occupied by the profiles of PV-immunonegative structures
(nuclei + dendritic bulbs): that of neuronal nuclei varied
between 5 mm2 and 84 mm2, while those of the dendritic
bulbs varied between 68 mm2 and 101 mm2. According to
stereological determinations, the number of PV-IR neurons
in the RTN in a young adult rat was 14,700, while that of PVIR calyciform endings was 2040.
In several cases it could be observed that perikarya of PVIR nerve cells were directly continuous with calyciform
terminals. In such cases, the perikarya of the PV-IR neurons
were equipped with neck-like protrusions, at the end of
which the calyciform terminal emerged, surrounding a large
dendritic profile. (Fig. 12a and b). According to low power
fluorescence microscopy (Fig. 12c–e), localization of PV-IR
structures in the RTN coincided with those which exerted
GABA-IR. Nuclear counterstaining of GABA-IR specimens
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B. Csillik et al. / Journal of Chemical Neuroanatomy 30 (2005) 17–26
Fig. 12. (a and b) PV-IR neuron equipped with a neck-like outgrowth (arrow), surrounding a dendritic bulb (D). (a) PV-IR without counterstaining; (b) PV-IR
after counterstaining with cresyl violet. Asterisk indicates nucleus of the PV-IR neuron; D, dendritic bulb. Scale bar, 10 mm. (c and d) Fluorescence microcopic
demonstration of PV (c), and GABA (d) of RTN at low power. Scale bar, 0.1 mm. (e–g) Fluorescence microcopic demonstration of GABA (e and g), and
superposition of GABA + PV (f) in the RTN at medium power. Scale bars, 40 mm. Arrows point at immuno-negative dendrites surrounded by GABA-IR
calyciform terminals. Note that the GABA-IR calyciform complexes (lower arrows) in e and f are strikingly similar to that depicted in h with the DAB method. (f
and g) GABA-IR in RTN after nuclear counterstaining with cresyl violet. (f) GABA-IR calyciform ending (arrowhead) surrounds dendritic bulb (D). (g) GABAIR nondescript cell in RTN with a very small nucleus (arrow with asterisk). Scale bar, 10 mm.
revealed identity of GABA-IR calyciform terminals with the
PV-IR ones (Fig. 12f). The small nuclei, which yielded the
first peak in the nuclear variation curves obtained from PVIR specimens, were found to belong to GABA-IR and PV-IR
nondescript cellular elements (Figs. 12g).
According to electron microscopic immunohistochemical investigations, the immunonegative profiles embraced
by the calyciform PV-IR structures showed every characteristics of large dendrites (Figs. 13 and 14); some of them
were even equipped with dendritic spines (Fig. 14). Within
the PV-IR calyciform presynaptic elements, synaptic
vesicles and mitochondria were found. GABA-IR could
be detected in the calyciform terminals also at the level of
electron microscopy (Figs. 15 and 16). In several cases,
dendro-dendritic attachment placques were observed
between vicinal PV- and GABA-immunonegative dendrites
(Fig. 17).
Transection of thalamo-reticular pathways did not induce
any alteration in the PV-IR and GABA-IR of the calyciform
terminals in RTN.
4. Discussion
The GABAergic RTN is, from the architectural,
immunocytochemical, electrophysiological and pharmacological viewpoints, a heterogenous structure, preferential
target of corticothalamic projections (Steriade, 2001).
B. Csillik et al. / Journal of Chemical Neuroanatomy 30 (2005) 17–26
Fig. 13–14. Fig. 13. Electron microscopic organization of a PV-immunopositive calyciform complex in the reticular nucleus of the rat thalamus (C),
full of synaptic vesicles. D, PV immunonegative dendritic profile. Fig. 14.
Dendritic profile in a virtually longitudinal section, equipped with two
dendritic spines (sp), surrounded by a calyciform PV-IR element (C)
containing numerous synaptic vesicles. The whole complex is in the
immediate vicinity of a blood vessel (BV), lined by a thin layer of
endothelial cytoplasm (asterisk).
Located at the intersection of thalamo-cortical and corticothalamic axons, the RTN occupies a strategic position
between the neocortex and the thalamus (Pinault, 2004). The
RTN is concerned with almost all functional modalities
represented by its auditory, gustatory, somatosensory, visual,
visceral, motor and limbic sectors. The body surface is
23
Fig. 15–16. Fig. 15. Dendritic profile (D) surrounded by three GABA-IR
elements of a calyiciform nerve ending (C1, C2, C3), loaded with synaptic
vesicles. Arrows indicate sites corresponding to presynaptic densities. Fig.
16. Two dendritic profiles (D1 and D2) surrounded by two GABA-IR
elements of a calyx (C), loaded with synaptic vesicles. Arrow points at
presynaptic density; er, endoplasmic reticulum in one of the dendrites.
somatotopically represented in the RTN (Shosaku et al.,
1989).
Spreafico et al. (1988, 1991) found three morphological
types of PV-IR neurons in the RTN of the adult rat, i.e. small
fusiform ‘‘f-type’’ cells, large fusiform ‘‘F-neurons’’ and
‘‘S-type cells’’ with round perikarya and multipolar
dendrites. In addition, we found several very small nuclei
that belonged to nondescript large PV-IR and/or GABA-IR
cellular elements.
Dendro-dendritic synapses were described in the RTN by
Deschenes et al. (1985), and by Pinault (2004) which were
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B. Csillik et al. / Journal of Chemical Neuroanatomy 30 (2005) 17–26
Fig. 17. Serial attachment plaqes (arrows) between two immunonegative
dendritic profiles (D1 and D2). Note part of a GABA-IR calyx (C) and a
GABA immunonegative axonal profile (A0) in the vicinity.
regarded to be relatively sparse as compared to the
overwhelming majority of axo-somatic and axo-dendritic
synapses. We found that in the rat RTN, some of the
synapses are brought about by large, PV-IR and GABA-IR
calyciform structures, which establish synapses with large
PV-immunonegative dendritic profiles. According to Cajal
(1911), RTN cells near the border of the anterior semilunar
and somatosensory nuclei are equipped with thick dendrites
that are oriented transversely. This peculiar anatomy of the
dendrites might be the reason why calyciform terminals can
most often be observed in paramedian sagittal sections of the
brain while their occurrence in coronal sections is limited.
According to Pinault et al. (1997,1998) dendro-dendritic
junctions might be a prominent anatomical substrate
underlying interneuronal communications in the RTN. In
addition, there occur close appositions between cell bodies
and dendrites in the RTN which are similar to gap junctions
(Ohara and Lieberman, 1985). Dendrites of RTN often
display signs of attachment placques and gap junctions,
observed also by us (Fig. 18), are characteristic of electrical
transmission which has been supposed to be involved in the
function of the RTN (Landisman et al., 2002).
The deceptive possibility that all the PV-IR and/or
GABA-IR structures in the RTN corresponded to nerve
cells equipped with large, immunonegative nuclei has been
ruled out on the basis of nuclear counterstaining and
electron microscopic immunocytochemical investigations.
Electron microscopic immunocytochemistry proved that
these structures are either parts of the perykarial cytoplasm
Fig. 18. Electron microscopy of a dendritic profile (D) surrounded by a
calyciform PV-IR terminal (asterisk) containing numerous synaptic vesicles. Arrows indicate postsynaptic densities. BV, blood vessel.
(Csillik et al., 2002) or calyciform terminals, as shown in
this paper.
Calcium-binding proteins are important molecules acting
like buffers to modulate dynamics of cytosolic Ca2+
transients (Baimbridge et al., 1992; Pinault, 2004). Presence
of calcium-binding proteins such as calbindin, parvalbumin
and calretinin, have been described in the RTN by Frassoni
et al. (1991), Spreafico et al. (1991), Clemence and
Mitrofanis (1992), Resibois and Rogers (1992), Winsky
et al. (1992), Lizier et al. (1997), Amadeo et al. (1998, 2001),
FitzGibbon et al. (2000) and Kakei et al. (2001). Calcium
binding proteins may exert significant action on membrane
potential, synaptic transmission and even on the firing
pattern of the related neurons (Baimbridge et al., 1992;
Caillard et al., 2000).
The fact that PV coexists with GABA in the central
nervous system is well documented (Lewis and Akil, 1998).
According to De Biasi et al. (1986, 1997), most of the PV-IR
structures in the RTN express also GABA. De Biasi et al.
(1988) reported that GABAergic terminals made synaptic
contact mainly with somata and proximal dendrites of nerve
cells. In our present experiments, we have proved by
simultaneous immunohistochemical reaction at the fluorescence microscopic level, that PV-IR presynaptic complexes
are identical with GABA-IR ones. Some of the GABA
immunoreactive structures described in the RTN and
believed to be neuronal perikarya (Houser et al., 1980)
are conspicuously similar to the presynaptic calyciform
complexes observed by us. Unfortunately, the fine structure
of these GABA-IR structures was not studied electron
microscopically; thus their cytological identity could not be
established.
B. Csillik et al. / Journal of Chemical Neuroanatomy 30 (2005) 17–26
On the other hand, according to Amadeo et al. (2001)
neurons of RTN are PV- and GABA-IR at all developmental
ages of the rat. Furthermore, ‘‘PV-positive cell bodies often
showed several somatic protrusions, sometimes surrounding
unlabelled vesicle-containing profiles or receiving presynaptically convex synaptic contacts from small terminals’’
(Amadeo et al., 2001). In our opinion, the ‘‘somatic
protrusions’’ may be identical with the cellular ‘‘necks’’
from which there emerged the presynaptic calyciform
complexes exhibiting PV-IR and GABA-IR, as described in
this paper. On the other hand, some of the electron
micrographs obtained from glutamic acid decarboxylase
stained feline RTN, published by Yen et al. (1985) are very
similar to parts of the electron microscopic pictures of our
PV-IR and GABA-IR calyciform terminals (see especially
Fig. 9D in Yen’s paper). Analogous patterns were found in
the monkey RTN (Williamson et al., 1994).
What can be the position of the peculiar calyciform
presynaptic complexes in the intrinsic circuitry of the RTN?
According to Carpenter and Sutin (1983), RTN does not
obtain afferents from the periphery; this has been proved
also by our earlier immunohistochemical studies (Csillik
et al., 1996). Furthermore, since transection of thalamoreticular pathways did not induce alterations in structure and
cytochemistry of calyciform complexes, it stands to reason
to assume the local origin of the PV-IR and GABA-IR
calyciform complexes. Indeed, though in small numbers, we
found PV-IR and GABA-IR interneurons in RTN, equipped
with a neck-like outgrowth from which the calyciform
complex originated. Thus, the calyciform complexes and the
neurons from which they derive, seem to be involved in
intrinsic cell-to-cell communication (Pinault, 2004) and thus
probably instrumental in synchronization of thalamocortical oscillations (Deschenes et al., 1984, Jahnsen and
Llinás, 1984; Steriade and Deschenes, 1984), in the
production of sleep spindles (Steriade et al., 1987) and in
paroxysmal oscillatory activities (Steriade, 2001; Jones,
2002).
Electrical stimulation of RTN (Knyihár-Csillik et al., in
press) results in activation of c-fos IR in the ipsilateral
retrosplenial cortex after bicuculline administration, while
stimulation of the retrosplenial cortex induces activation of
c-fos IR in the ipsilateral RTN. The results of electrical
stimulation support the well-known fact that RTN is in
synaptic contact with the cortex (Guillery et al., 1998;
Guillery and Harting, 2003; McAlonan and Brown, 2002)
which seem to be mediated by GABAergic structures.
According to Mihály et al. (1998) inhibitory neurons in the
RTN are primarily activated during focal neocortical
seizures.
RTN is supposed to be involved in attentional processes
(MacAlonen et al., 2000); recent studies of Llinás et al.
(2002) indicate a coincidence detector function of RTN.
Sensory systems are known to be subjected to modulation by
attention or distraction; the RTN is a key region in selective
attention (Stehberg et al., 2001), and in executive attention
25
(Kilmer, 2001). This seems to be brought about by activation
of RTN reflecting to attentional gating (MacAlonen et al.,
2000). It is up to further studies whether or not, the large
calyciform synapses play a part in this system.
Acknowledgements
These studies were supported by the Hungarian National
Research Foundation (OTKA), Grants No T-025033, T032039 and T-016046, and by Grants 40/2000 and 7/2003
from the Hungarian Medical Research Council (ETT). The
text has been kindly edited by Dr. Károly Balogh, Boston,
MA, USA.
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