होम Journal of Neurochemistry Development of brainstem 5-HT 1A receptor-binding sites in...

Development of brainstem 5-HT 1A receptor-binding sites in serotonin-deficient mice

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Journal of Neurochemistry
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10.1111/jnc.12311
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September, 2013
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Explicit formulas for Chebyshev impedance-matching networks, filters and interstages

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JOURNAL OF NEUROCHEMISTRY

| 2013

doi: 10.1111/jnc.12311

,
,

*Department of Anesthesiology, Boston Children’s Hospital and Harvard Medical School, Boston,
Massachusetts, USA
†Department of Physiology & Neurobiology, Geisel School of Medicine at Dartmouth, Lebanon,
New Hampshire, USA
‡Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston,
Massachusetts, USA
§Department of Biomedical Sciences, College of Veterinary Medicine, University of Missouri,
Columbia, Missouri, USA
¶Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
**Department of Neurology, University of Iowa, Iowa City, Iowa, USA
††Department of Neurology, Yale University, New Haven, Connecticut, USA

Abstract
The sudden infant death syndrome is associated with a
reduction in brainstem serotonin 5-hydroxytryptamine (5-HT)
and 5-HT1A receptor binding, yet it is unknown if and how
these findings are linked. In this study, we used quantitative
tissue autoradiography to determine if post-natal development
of brainstem 5-HT1A receptors is altered in two mouse models
where the development of 5-HT neurons is defective, the
Lmx1bf/f/p, and the Pet-1 / mouse. 5-HT1A receptor agonistbinding sites were examined in both 5-HT-source nuclei
(autoreceptors) and in sites that receive 5-HT innervation
(heteroreceptors). In control mice between post-natal day (P) 3
and 10, 5-HT1A receptor binding increased in several brainstem sites; by P25, there were region-specific increases and

decreases, refining the overall binding pattern. In the Lmx1bf/f/p
and Pet-1 / mice, 5-HT1A-autoreceptor binding was significantly lower than in control mice at P3, and remained low at
P10 and P25. In contrast, 5-HT1A heteroreceptor levels were
comparable between control and 5-HT-deficient mice. These
data define the post-natal development of 5-HT1A-receptor
binding in the mouse brainstem. Furthermore, the data
suggest that 5-HT1A-heteroreceptor deficits detected in
sudden infant death syndrome are not a direct consequence
of a 5-HT;  neuron dysfunction nor reduced brain 5-HT levels.
Keywords: autoreceptors, heteroreceptors, Lmx1b, Pet1,
raphe, sudden infant death syndrome.
J. Neurochem. (2013) 10.1111/jnc.12311

Serotonin 5-hydroxytryptamine (5-HT) is a major monoamine neurotransmitter that communicates with multiple
receptor subtypes throughout the neuroaxis to regulate key
physiological and behavioral functions. Importantly, it helps
regulate brainstem respiratory and autonomic responses to
homeostatic challenges, in part by virtue of actions at the 5HT1A receptor (Darnall et al. 2005; Curran and Leiter 2007;
Audero et al. 2008; Pham-Le et al. 2011). 5-HT1A receptors
are located somatodendritically on 5-HT neurons, where they
function as autoreceptors. In addition, 5-HT1A receptors are

located on non-5-HT neurons and act as heteroreceptors in
regions of the brainstem and forebrain that receive 5-HT
innervation. Autoreceptor and heteroreceptor populations are
Received April 26, 2013; revised manuscript received May 6, 2013;
accepted May 10, 2013.
Address correspondence and reprint requests to Kathryn G. Commons,
PhD, Boston Children’s Hospital, 300 Longwood Ave, EN 307, Boston,
MA 02115, USA. E-mail: kathryn.commons@childrens.harvard.edu
Abbreviations used: 5-HT, 5-hydroxytryptamine; SIDS, sudden infant
death syndrome.

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12311

1

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C. A. Massey et al.

known to exhibit region-specific adaptations to the over- or
under-availability of ligand [reviewed by (Hensler 2003)].
Alterations in brainstem 5-HT1A receptors are associated
with several human disorders (Kinney et al. 2011) including
the sudden infant death syndrome (SIDS) (Saito et al. 1999;
Panigrahy et al. 2000; Ozawa and Takashima 2002; Paterson
et al. 2006; Duncan et al. 2010; Waters 2010). The leading
cause of post-neonatal mortality in the United States today,
SIDS is defined by sleep-related death in the first post-natal
year of life that is unexplained by a complete autopsy and
death scene investigation (Kinney and Thach 2009). The
majority of SIDS deaths are associated with asphyxiagenerating circumstances that appear to trigger death, for
example, rebreathing exhaled gases in the face-down (prone)
sleep position (Pasquale-Styles et al. 2007; Kinney and
Thach 2009). Accordingly, a leading hypothesis in SIDS
research today is that SIDS is caused by a brainstem
abnormality that impairs the ability to generate protective
responses to life-threatening challenges (Kinney and Thach
2009; Kinney et al. 2009). Indeed, 5-HT1A receptors reductions have been observed in the brainstem of SIDS infants
that involve both auto- and heteroreceptor populations
(Kinney et al. 2003; Paterson et al. 2006; Machaalani et al.
2009; Duncan et al. 2010). Lower 5-HT1A-receptor binding
in SIDS cases is associated with decreased medullary tissue
content of 5-HT and tryptophan hydroxylase 2 (TPH2), the
rate-limiting biosynthetic enzyme for 5-HT, suggesting a 5HT-deficient disorder (Duncan et al. 2010).
Two murine models have been instrumental in examining
the developmental role of 5-HT on cardiorespiratory function
and thermoregulation, that is, homeostatic processes potentially relevant to the pathogenesis of SIDS (Erickson et al.
2007; Hodges et al. 2009; Kinney and Thach 2009;
Buchanan and Richerson 2010; Cummings et al. 2011a;
Richerson and Buchanan 2011). These 5-HT-deficient models include a conditional removal of the transcription factor
Lmx1b selectively in Pet1-expressing cells (Lmx1bflox/flox;
ePet-cre) resulting in the ‘Lmx1bf/f/p’ mouse in which there is
nearly a complete loss of 5-HT neurons by midgestation and
consequently 5-HT in the CNS (Zhao et al. 2006). The other
model is the Pet-1 / mouse in which 5-HT neurons persist
in the brain but the majority fail to differentiate appropriately
and do not produce 5-HT (Hendricks et al. 2003; Erickson
et al. 2007; Cummings et al. 2011a). Detectable 5-HT
synthesis appears to occur in only about 30% of the normal
number of 5-HT neurons, and these residual neurons innervate
a selective subset of brain regions, predominantly regions
associated with stress responses (Kiyasova et al. 2011).
In this study, we examined how 5-HT1A-receptors-binding
patterns develop in the brainstem in these two mouse models
with 5-HT deficiency. We applied the methodology of
quantitative tissue autoradiography, a technique used to
study SIDS tissue, in part to allow for comparisons to
5-HT1A-receptor binding in human brainstem disorders

(Paterson et al. 2006). We tested the hypothesis that
decreases in 5-HT1A-heteroreceptor binding in the brainstem
during development may be caused by 5-HT neuron dysfunction or associated 5-HT deficiency. Given the absence of 5-HT
neurons in the Lmx1bf/f/p mouse, and the demonstrated loss of
5-HT1A receptor gene expression in the dorsal and median
raphe nuclei of Pet-1 / mice (Liu et al. 2010; Jacobsen et al.
2011), we also anticipated finding a large decrease in
medullary 5-HT1A autoreceptors. As tissue autoradiography
does not reveal the cellular location of receptors, we
operationally defined 5-HT1A autoreceptors as those within
the 5-HT source nuclei and 5-HT1A heteroreceptors as those
localized to nuclei receiving 5-HT projections.

Materials and methods
Animals
Two different mouse strains were used in this study: Lmx1b
conditional knockout in Pet1-expressing cells [Lmx1bflox/flox;ePet-Cre/+
(Lmx1bf/f/p)] and Pet-1 / mice. Lmx1bf/f/p mice were bred at Yale
University in New Haven, Connecticut, and Pet-1 / mice at the
Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire. Animal protocols were approved by the Institutional Animal
Care and Use Committees at these institutions, and were consistent
with guidelines of the National Institutes of Health. For Lmx1bf/f/p
mice, controls were siblings lacking Cre recombinase on a C57BL/
6 background. For Pet-1 / mice, controls were heterozygote and
control siblings from a mixed C57BL/6 and 129 background;
heterozyotes have been shown to have normal number of 5-HT
immunolabeled neurons (Hendricks et al. 2003; Cummings et al.
2011a). Both male and female mice were included and were
analyzed at P3, P8-10, and P25. These ages were selected based on
the severe respiratory deficits in Lmx1bf/f/p mice at neonatal (< P4)
ages (Hodges et al. 2008) and vulnerability of Pet-1 / mice to
repeated hypoxic challenge at P8/10 (Cummings et al. 2011a).
Group size for the P3 and P8-10 groups averaged 7.5 and 7
individuals, respectively, with a minimum of five per group; for the
P25 age groups, the average group size was 4.75 individuals, with a
minimum of four individuals per group.
Tissue collection
Under deep anesthesia, mouse brains were removed, frozen, and
stored at 80°C until sectioned 20 lm thick with a cryostat.
Sections were collected serially onto sets of four slides such that
each slide contained every fourth section. Slides were stored at
80°C until processed for autoradiography.
Autoradiography
Slides were gradually warmed to 18–24°C before tissue incubations
for autoradiography, using a protocol adapted from human studies
(Paterson et al. 2006; Duncan et al. 2010). One quarter of the slides
collected for each mouse was used for total radioligand binding,
while another quarter containing the adjacent sections was processed
to determine non-specific-binding levels. Buffer solution consisted
of 0.17 M Tris with 0.01% ascorbic acid and 4 mM CaCl2 (pH 7.6).
At 18–24°C, slides were pre-incubated in buffer solution for
30 min. Then, sections were incubated in 4 nM 3H-8-hydroxy-2-

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12311

Brainstem 5-HT1A receptors in 5-HT mutants

(di-n-propylamino)tetralin and 10 lM pargyline diluted in fresh
buffer solution for 1 h for total specific binding. For non-specific
binding, 10 lM 5-HT was added to the 2-(di-n-propylamino)
tetralin-containing solution, and slides were incubated for 1 h.
Incubated slides were washed in ice-cold incubation buffer solution
twice for 5 min. All sections were then air dried for 15 min using a
cold air stream, and then placed on a slide warmer for 30 min to dry
completely.
Quantitative data analysis
Slides for total and non-specific binding were exposed on 3H-sensitive
film with one 3H-standard slide (Amersham, Pittsburgh, PA, USA) for
3 weeks. For quantification of receptor-binding density, NIH Image J
software (National Institutes of Health, Bethesda, MD, USA) was
used. To convert optical density into femtomoles/milligram tissue
(fmol/mg), a calibration curve was generated using the 3H-standards.
Seven sites were selected for analysis (Fig. 1) that were representative
of auto- and heteroreceptor sites: three nuclei that contain 5-HT source
neurons [raphe obscurus, dorsal raphe, and lateral paragigantocellular
nucleus (LPGi)], and 4 sites that receive 5-HT projections (spinal
trigeminal nucleus, inferior colliculus, interpeduncular nucleus, and
cerebellum). At least in adults, the majority of 5-HT1A receptors colocalize with markers of serotonin neurons in the raphe nuclei; thus,
we infer these areas largely represent autoreceptor populations (Czesak
et al. 2012). For each region, 5-HT1A-receptor-binding density was
measured in three sections/mouse and the values averaged to determine
total and non-specific binding. Non-specific-binding values were
subtracted from those of total binding to obtain specific binding.
Subsequently, individual values were averaged to yield group means and
standard error of the mean.
Statistical analysis
For each mutant group, data were first analyzed using gender as a
factor. When there was no significant effect of gender, males and
females were pooled and analyzed together. The data were compared
using a three-factor ANOVA for age (at three levels), genotype (at two
levels), and region, with region as a repeated measure. Following a
significant main effect of region, each region was analyzed
independently using slice ANOVAs for age and genotype with Tukey
or Student’s-t post hoc tests, depending on the number of comparison
groups. Mutant groups were not directly comparable to each other
because of differences in genetic background, rearing location, and
ages at harvesting, which were associated with some modest
differences between the two control groups in 5-HT1A-receptor
binding. Therefore, to evaluate the relative loss of receptor binding in
each mutant line (Fig. 5), the binding values were normalized to the
appropriate control values yielding a ‘percent retained binding’ value
(i.e., mutant binding/mean control binding expressed as a percent).
These values were averaged, and a standard error was determined for
each genotype and age. 5-HT1A-receptor-binding deficiencies in each
mutant in the dorsal raphe nucleus and the raphe obscurus were then
compared using ANOVA followed by Student’s t-test.

Results
Age effects
In control mice at P3, the dorsal raphe, inferior colliculus,
and spinal trigeminal nucleus had the highest levels of

3

5-HT1A binding (greater than 30 fmol/mg) (Figs 1–3).
Intermediate binding, in the range from 20 to 30 fmol/mg,
was found in the raphe obscurus and LPGi, and low binding
(< 20 fmol/mg) in the remaining sites analyzed, including
the cerebellum. Between p3 and P10, in every site measured
except the cerebellum, there were significant increases in 5HT1A-receptor binding (Figs 1, 2). Between P10 and P25,
control mice showed region-dependent changes in 5-HT1A–
receptor-binding levels (Figs 1, 3). Two sites exhibited
further increases in binding, including the interpeduncular
nucleus and the dorsal raphe nucleus. In four sites, the
inferior colliculus, LPGi, spinal trigeminal nucleus and raphe
obscurus, there were decreases in 5-HT1A-receptor binding.
These patterns of developmental changes in the control mice
for Lmx1bf/f/p were the same in the Pet-1 / control group
(Fig. 4).
Genotype Effects, Lmx1bf/f/p mice
At P3, in comparison to control mice, Lmx1bf/f/p mice
showed significantly reduced binding only in two sites, the
dorsal raphe nucleus and raphe obscurus (Figs 1–3), that is,
major sites of 5-HT source neurons in the brainstem. At P10,
5-HT1A binding was reduced in the dorsal raphe, inferior
colliculus and raphe obscurus in mutants compared to their
controls (Fig. 3). At P25, the magnitude of the reduction
of 5-HT1A binding in the dorsal raphe nucleus and raphe
obscurus was severe, with binding in the Lmx1bf/f/p
more than two-thirds reduced from control values (Fig. 3).
The maintenance of heteroreceptor binding in the quantified
areas appeared representative of additional heteroreceptor
populations, such as in the nucleus of the solitary tract
(Fig. 2).
Genotype effects, Pet-1 /
In Pet-1 / mice at P3, the only site with a significant
reduction compared to control was in the dorsal raphe
nucleus (Fig. 4). At P8, both the dorsal raphe and raphe
obscurus, however, demonstrated significant reductions in
binding compared to controls. At P25, the additional site of
the LPGi in Pet-1 / mice had significantly less binding then
control values (Fig. 4). In comparison to Lmx1bf/f/p mice
(Fig. 5), the loss of 5-HT1A binding in the dorsal raphe
nucleus and raphe obscurus was comparable at P3 and P8/
P10, however, at P25 5-HT1A-binding levels in Pet-1 /
mice were half that seen in their controls, a less severe loss
than that seen in Lmx1bf/f/p mice.

Discussion
Previous studies have reported an association between altered
brainstem 5-HT neurochemistry and SIDS. Specifically, there
is evidence for aberrant 5-HT neuron function including
decreases in tryptophan hydroxylase 2 and tissue 5-HT
content (Duncan et al. 2010). In addition, there are consistent

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12311

4

C. A. Massey et al.

(a)

(b)

(c)

(d)

(e)

Fig. 1 (a–d) Representative 5-hydroxytryptamine (5-HT)1A-receptor
binding at several levels through the hindbrain illustrate changes in
binding with age, and show that the overall pattern of 5-HT1A
binding in non-raphe areas in matched control (left columns) and
mutant mice (right columns) is similar. Higher magnification images
of bracketed regions including those showing raphe nuclei are
shown in Fig. 2. (a). In P3 control (left) and Lmx1bf/f/p (right)
sections, 5-HT1A binding is generally low (< 20 fmol/mg). (b) At P10,
5-HT1A binding increases are widespread. (c) By P25, changes in

5-HT1A binding are region dependent and a more distinctive pattern
of binding can be discerned. (d) Side-by-side sections from matched
control (left) and Pet1 / (right) at P25 show a similar pattern to
Lmx1bf/f/p and their controls. (e) Areas sampled for quantitative
analysis indicated by black ovals, IPN, interpeduncular nucleus;
DRN, dorsal raphe nucleus; IC, inferior colliculus; CB, cerebellum;
RO, raphe obscurus; LPGi, lateral paragigantocellular nucleus; Sp5,
spinal trigeminal nucleus. Scale bar in d = 4 mm, all images at
same scale.

reports of decreases in 5-HT1A-receptor binding (Paterson
et al. 2006; Machaalani et al. 2009; Duncan et al. 2010).
This not only involves 5-HT1A autoreceptors but also 5HT1A heteroreceptors in multiple medullary sites. The
question remains as to whether 5-HT1A-heteroreceptor
deficits can be explained by a primary dysfunction in 5-HT
neurons themselves. In this study, we examined the postnatal development of 5-HT1A receptors in two mouse models
with genetically induced deficits in 5-HT neuron function.
Our data indicate a decrease in 5-HT1A binding in both
mouse strains in areas that are normally enriched with 5-HT
neurons (autoreceptors), confirming and extending previous
observations to younger ages and medullary sites (Liu et al.
2010; Jacobsen et al. 2011). However, heteroreceptors, or

5-HT1A receptors located in areas containing non-5HT
neurons, were largely comparable to control values in the
brainstem at all post-natal ages examined.
Methodological considerations
5-HT1A-receptor autoradiography is a classic approach used
to quantify binding sites in the brain. Strengths of this
approach include the ability to compare results to studies in
human pathology cases, such as those employed in the study
of SIDS. In addition, many studies using animal models have
examined changes of 5-HT1A receptor levels in response to
manipulations of the 5-HT system or to drug exposure,
particularly in the case of serotonin-selective reuptake
inhibitors (Welner et al. 1989; Hensler et al. 1991; Hensler

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12311

Brainstem 5-HT1A receptors in 5-HT mutants

(a)

(a′)

(b)

(b′)

(c)

(c′)

(d)

(d′)

(e)

(e′)

(f)

(f′)

(g)

(g′)

(h)

(h′)

(i)

(i′)

5

Fig. 2 High magnification comparison of binding from sections indicated in Fig. 1. In controls for Lmx1bf/f/p, 5-hydroxytryptamine (5-HT)1A
binding is associated with the midline within the dorsal raphe nucleus
(DRN) at P3 (a), and increases at both P10 (b) and P25 (c). In Lmx1bf/f/p
mutants, loss of midline binding is evident (white arrowheads) at
each time-point (a, b, c). In controls 5-HT1A binding again visible in
the raphe obscurus (RO) (d, e, f) and nucleus of the solitary tract (g).
In Lmx1bf/f/p mutants, binding is much reduced along the midline at
each age in raphe obscurus (d, e, and f white arrowheads).
However, 5-HT1A receptor binding persists in the NTS (g, small
arrows). At P25, binding in Pet1-control dorsal raphe nucleus (h) and
raphe obscurus (i) is similar to Lmx1bf/f/p controls. Deficits in midline
binding are notable in sections from Pet1 / mice (h, i). All images at
the same scale, scale bar = 500 lm.

2002; Rossi et al. 2008). However, it should be noted that
there are potential adaptations in 5-HT1A-receptor function,
including changes in the ability to activate G-proteins and
subsequent intracellular signaling cascades that are not
detected using receptor autoradiography, and require alternative methods to evaluate (Fairchild et al. 2003; Hensler
2003). Indeed, receptor autoradiography provides view of
5-HT1A receptors that is distinct and complimentary to methods
that assay protein, mRNA levels, or intracellular signaling.
In both 5-HT-deficient mutant mouse lines, there were
decreases in 5-HT1A-autoreceptor levels. These reductions
are likely a direct consequence of the relevant genetic
mutations and associated cell loss or transfating rather than a
compensatory change in response to the loss of the
endogenous ligand, 5-HT. In Lmx1bf/f/p mice, the loss of
Pet1 expression and indeed, 5-HT neurons themselves would
be expected to produce a parallel loss of 5-HT1A autoreceptors (Ding et al. 2003; Zhao et al. 2006). Any residual
5-HT1A receptor binding in the raphe nuclei of Lmx1bf/f/p
mice could be associated with non-5-HT neurons or glia.
With respect to Pet-1 / mice, loss of autoreceptor binding
in the Pet-1 / mice is consistent with previously detected
decreases in mRNA expression in the dorsal raphe nucleus
and loss of agonist-evoked effects on currents (Liu et al.
2010; Jacobsen et al. 2011). Indeed, in 5-HT neurons Pet1 is
a direct transcriptional enhancer of the 5-HT1A receptor gene,
while 5-HT1A receptor transcription in non-5-HT neurons is
Pet1-independent (Jacobsen et al. 2011). Therefore, lack of
transcriptional activation of the 5-HT1A receptor gene would
be expected to result in a lower level of 5-HT1A receptor
expression in Pet1-dependent 5-HT neurons. However, in the
Pet-1 / mice, there is a residual population of 5-HT neurons
that appear Pet1-independent, and these neurons could
potentially express 5-HT1A receptors independent of Pet1
function (Hendricks et al. 2003; Kiyasova et al. 2011).
Alternatively, a very low level of expression could persist in
residual neurons within the area. These explanations could
account for the greater magnitude loss of 5-HT1A-receptor
binding in the Lmx1bf/f/p mouse compared to the Pet-1 /
mouse detected at P25.

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12311

6

C. A. Massey et al.

Interpeduncular nucleus

Control
LMX1b-ffp
Age effect/control
Age effect/LMX1b-ffp
Genotype effect

*

120

100

100

80

80

60

60

40

40

20

20

0

0
P3

Cerebellum

P10

40

20

0

100

80

80

60

60

60

40

40

20

20

*

*

P10

P25

P3

100

100

80

80

60

60

40

40

20

20

0

0

*

80

*

*

60
40
20
0

P3

P25

P8

P25

P3

LPGi
120

100

100

80

80

80

60

60

60

40

*

20

P3

P8

P25

P25

40
20
0

0

0

P8

Spinal trigeminal nucleus

120

*

P25

100

100

*

P10

Inferior colliculus

120

20
P25

P3

P25

120

Raphe obscurus

40

P8

P10

Dorsal raphe nucleus
120

Cerebellum

P25

5-HT1A-receptor binding increases between P3 and P10 at every site
except the cerebellum. Between P10 and P25, control mice show
region-dependent increases or decreases in binding. The dorsal raphe
nucleus and raphe obscurus are the only areas with consistent
genotype effects.

120

P8

P10

0

0
P3

P3

20

P3

Spinal trigeminal nucleus

80

Interpeduncular nucleus

40

P25

120

Fig. 3 Quantification of 5-hydroxytryptamine (5-HT)1A-receptor binding in control and Lmx1bf/f/p mice at each site and age. Significant
(p < 0.05) differences between consecutive ages are indicated below
the X-axis. Y-axis indicates 2-(di-n-propylamino)tetralin binding in fmol/
mg tissue. Significant differences between genotypes are indicated by
asterisk above the relevant measures. In control mice (black lines),

P3

0
P10

LPGi

0

60

40

100

*

*

60

120

P25

Control
Pet1-KO
Age effect/control
Age effect/Pet1-KO
Genotype effect

80

*

20
P3

P25

100

100

20

*

*

Inferior colliculus
120

120

40

P10

*

Raphe obscurus

60

P3

Dorsal raphe nucleus

120

P3

P8

P25

P3

P8

P25

Fig. 4 Quantification of 5-hydroxytryptamine (5-HT)1A-receptor binding in control and Pet-1 / mice by region and age. Significant
(p < 0.05) differences between consecutive ages are indicated below
the X-axis. Y-axis indicates 2-(di-n-propylamino)tetralin binding in fmol/
mg tissue. Significant differences between genotypes are indicated by

asterisk above the relevant measures. Between P3 and P8, 5-HT1Areceptor-binding levels increase at every site in control (black line), but
changes are region dependent between P8 and P25. A significant
effect of genotype was detected as early as P3 and primarily involved
the dorsal raphe nucleus and raphe obscurus.

Loss of the endogenous ligand, 5-HT, in both of these
mutants does not appear to consistently impact 5-HT1A–
heteroreceptor-binding levels in brainstem projection sites

during post-natal development. Adaptations in 5-HT1Areceptor binding in response to manipulations that increase
or decrease available 5-HT have primarily been studied in the

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12311

Brainstem 5-HT1A receptors in 5-HT mutants

Dorsal raphe nucelus

Raphe obscurus
90%

90%
LMX1b-ffp

LMX1b-ffp

Pet1–/–

60%

*

30%

Retained binding

Retained binding

7

Pet1–/–

*

60%

30%

0%

0%
P3

P8/P10

P25

P3

P8/P10

P25

Fig. 5 Percent retained binding (mutant binding/mean control binding, expressed as percent) in the dorsal raphe and raphe obscurus
in both genotypes with age. Each mutant is normalized to its
respective control group. At P3 and P8/10, differences between

genotypes are not significant. At P25, in both the dorsal raphe and
raphe obscurus the loss of 5-HT1A binding in Lmx1bf/f/p mice was
more profound then that seen in Pet-1 / mice. Mean  SEM,
*p < 0.05.

adult forebrain and in the primary sources of forebrain 5-HT,
the dorsal and median raphe nuclei [reviewed by (Frazer and
Hensler 1990; Hensler 2003)]. These studies show that
changes in 5-HT1A receptors can be complex and may not
only involve changes in receptor number or distribution but
also in signaling capacity, and furthermore, the specific
changes that occur appear to be region dependent (Hensler
2003). In the most relevant example, the adult Pet-1 /
mouse using Western blot, compensatory increases of 5HT1A receptors were detected in the hippocampus while
decreases were found in the striatum (Jacobsen et al. 2011).
Increases in hippocampal 5-HT1A receptors are also found in
cases where 5-HT projections were chemically lesioned, or
5-HT1A-receptor signaling was blocked by administration of
an antagonist (Patel et al. 1996; Abbas et al. 2007). Our
results would extend these observations using genetic models
with constitutive developmental losses of 5-HT and show
that at several brainstem sites no changes in 5-HT1A-receptor
binding are detected. Likewise, the results confirm that
transcriptional control of 5-HT1A receptors is region dependent in that heteroreceptors throughout the brainstem is Pet1-independent, as indeed, Pet1 expression is restricted to
serotonin neurons (Hendricks et al. 1999; Pfaar et al. 2002;
Albert et al. 2011; Jacobsen et al. 2011).
The maintenance of 5-HT1A heteroreceptors in the mutant
mouse lines also suggests that 5-HT1A-heteroreceptor levels
are unaffected by the severe physiological defects displayed
in these mouse lines. Specifically, both mouse lines have
lower weight gain during the first few weeks of life. In
addition, respiratory defects in both lines are likely to impact
brain oxygenation and pH: as neonates, Lmx1bf/f/p have
dramatic and prolonged (1–2 min) spontaneous apneas
(Hodges et al. 2009), and as adults, both Lmx1bf/f/p and
Pet-1 / have abnormally low respiratory responses to CO2
(Hodges and Richerson 2008; Hodges et al. 2011). Finally,
both lines are severely compromised in their ability to
thermoregulate during environmental cooling (Hodges and
Richerson 2010; Cummings et al. 2011b; Hodges et al.
2011). While these effects are interpreted as consequences of

the altered state of 5-HT neurons, the current results provide
evidence that these physiological deficits do not independently impact 5-HT1A heteroreceptor binding in these mice.
This study further describes the developmental time-course
of 5-HT1A-receptor binding in the brainstem during the early
post-natal period in mice. Analysis of the 5-HT1A-binding
sites in several brainstem areas revealed two phases of
development in the mouse. Before P8-10, there are broad
increases in 5-HT1A-receptor binding. After P8-10, there are
region-dependent increases or decreases that serve to refine
the overall pattern of 5-HT1A-receptor binding. Consistent
with the time-course of changes observed, the first three postnatal weeks are known to constitute an important period of
synaptic development and refinement in mouse (Bhatt et al.
2009). Indeed, P8, an age of apparent rapid changes in 5HT1A-receptor binding, corresponds to an age of peek
vulnerability to repeated hypoxic stress in Pet-1 / mice
(Cummings et al. 2011a). Likewise, an extended post-natal
development of 5-HT1A-receptor-binding sites has been
described in the forebrain (Miquel et al. 1994). In the rat
hypoglossal nucleus, a similar biphasic development of 5HT1A receptors is observed where mRNA and binding
increase until day 7 and decrease thereafter (Talley et al.
1997). However, it should be noted that our results differ
from those of (Liu and Wong-Riley 2010) who showed with
immunohistochemistry in rat that 5-HT1A receptor levels in
several brainstem nuclei are high at birth, only decreasing
after 2 weeks of age. These differences may be related to
species differences or the different methodology employed.

Conclusion
Our data suggest that the decreased 5-HT1A-heteroreceptor
binding in medullary regions of SIDS cases may not be
caused by dysfunction of 5-HT neurons nor the associated
reduced brain 5-HT levels. 5-HT1A receptors are known to be
regulated by multiple transcriptional enhancer and repressors,
including NF-kappa-B, Freud, REST, Deaf1, Hes proteins,
and glucocorticoid receptors (Albert et al. 2011). Future

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12311

8

C. A. Massey et al.

studies may hone in on the mechanism of dysregulation of 5HT receptors relevant for SIDS. Importantly it should be
noted that in these mutants there is a profound loss of ligand,
thus although present, 5-HT1A heteroreceptors may not
endogenously function as normal. Therefore, these mouse
lines continue to be useful to understand the importance of 5HT to homeostatic functions relevant to SIDS. The results
also reveal that the post-natal period in mouse development
is associated with rapid and dramatic changes of 5-HT1A
receptor binding that may be relevant to age-dependent
disorders in humans including SIDS.

Acknowledgements
Supported by NIH grant PO1 HD-036379. The authors declare no
conflicts of interest.

Authorship contribution
Study concept and design: KGC, HCK, EEN, GBR, SMD, KJC.
Acquisition of data: CAM, GK, AEC, RLH, DSP. Analysis and
interpretation of data: CAM, GK, KGC. Drafting of the manuscript:
CAM, GK, KGC. Critical Revision of the manuscript for important
intellectual content: HCK, KJC, EEN, AEC, RLH, DSP, GBR,
SMD. Statistical analysis: CAM, GK. Obtained funding: HCK,
EEN, GBR, SMD, KGC. Administrative, technical and material
support: CAM, GK, AEC, RLH, DSP. Study supervision: KGC.

References
Abbas S. Y., Nogueira M. I. and Azmitia E. C. (2007) Antagonistinduced increase in 5-HT1A-receptor expression in adult rat
hippocampus and cortex. Synapse 61, 531–539.
Albert P. R., Le Francois B. and Millar A. M. (2011) Transcriptional
dysregulation of 5-HT1A autoreceptors in mental illness. Mol.
Brain 4, 21.
Audero E., Coppi E., Mlinar B., Rossetti T., Caprioli A., Banchaabouchi
M. A., Corradetti R. and Gross C. (2008) Sporadic autonomic
dysregulation and death associated with excessive serotonin
autoinhibition. Science 321, 130–133.
Bhatt D. H., Zhang S. and Gan W. B. (2009) Dendritic spine dynamics.
Annu. Rev. Physiol. 71, 261–282.
Buchanan G. F. and Richerson G. B. (2010) Central serotonin neurons
are required for arousal to CO2. Proc. Natl Acad. Sci. USA 107,
16354–16359.
Cummings K. J., Commons K. G., Hewitt J. C., Daubenspeck J. A., Li
A., Kinney H. C. and Nattie E. E. (2011a) Failed heart rate
recovery at a critical age in 5-HT-deficient mice exposed to
episodic anoxia: implications for SIDS. J. Appl. Physiol. 111, 825–
833.
Cummings K. J., Li A. and Nattie E. E. (2011b) Brainstem serotonin
deficiency in the neonatal period: autonomic dysregulation during
mild cold stress. J. Physiol. 589, 2055–2064.
Curran A. K. and Leiter J. C. (2007) Baroreceptor-mediated inhibition of
respiration after peripheral and central administration of a 5-HT1A
receptor agonist in neonatal piglets. Exp. Physiol. 92, 757–767.
Czesak M., Le Francois B., Millar A. M., Deria M., Daigle M., Visvader
J. E., Anisman H. and Albert P. R. (2012) Increased serotonin-1A
(5-HT1A) autoreceptor expression and reduced raphe serotonin

levels in deformed epidermal autoregulatory factor-1 (Deaf-1) gene
knock-out mice. J. Biol. Chem. 287, 6615–6627.
Darnall R. A., Harris M. B., Gill W. H., Hoffman J. M., Brown J. W. and
Niblock M. M. (2005) Inhibition of serotonergic neurons in the
nucleus paragigantocellularis lateralis fragments sleep and
decreases rapid eye movement sleep in the piglet: implications
for sudden infant death syndrome. J. Neurosci. 25, 8322–8332.
Ding Y. Q., Marklund U., Yuan W., Yin J., Wegman L., Ericson J.,
Deneris E., Johnson R. L. and Chen Z. F. (2003) Lmx1b is
essential for the development of serotonergic neurons. Nat.
Neurosci. 6, 933–938.
Duncan J. R., Paterson D. S., Hoffman J. M. et al. (2010) Brainstem
serotonergic deficiency in sudden infant death syndrome. JAMA
303, 430–437.
Erickson J. T., Shafer G., Rossetti M. D., Wilson C. G. and Deneris E. S.
(2007) Arrest of 5HT neuron differentiation delays respiratory
maturation and impairs neonatal homeostatic responses to
environmental challenges. Respir. Physiol. Neurobiol. 159, 85–
101.
Fairchild G., Leitch M. M. and Ingram C. D. (2003) Acute and chronic
effects of corticosterone on 5-HT1A receptor-mediated
autoinhibition
in
the
rat
dorsal
raphe
nucleus.
Neuropharmacology 45, 925–934.
Frazer A. and Hensler J. G. (1990) 5-HT1A receptors and 5-HT1Amediated responses: effect of treatments that modify serotonergic
neurotransmission. Ann. N. Y. Acad. Sci. 600, 460–474; discussion
474-465.
Hendricks T., Francis N., Fyodorov D. and Deneris E. S. (1999) The
ETS domain factor Pet-1 is an early and precise marker of central
serotonin neurons and interacts with a conserved element in
serotonergic genes. J. Neurosci. 19, 10348–10356.
Hendricks T. J., Fyodorov D. V., Wegman L. J. et al. (2003) Pet-1 ETS
gene plays a critical role in 5-HT neuron development and is
required for normal anxiety-like and aggressive behavior. Neuron
37, 233–247.
Hensler J. G. (2002) Differential regulation of 5-HT1A receptor-G
protein interactions in brain following chronic antidepressant
administration. Neuropsychopharmacology 26, 565–573.
Hensler J. G. (2003) Regulation of 5-HT1A receptor function in brain
following agonist or antidepressant administration. Life Sci. 72,
1665–1682.
Hensler J. G., Kovachich G. B. and Frazer A. (1991) A quantitative
autoradiographic study of serotonin1A receptor regulation. Effect
of 5,7-dihydroxytryptamine and antidepressant treatments.
Neuropsychopharmacology 4, 131–144.
Hodges M. R. and Richerson G. B. (2008) Contributions of 5-HT
neurons to respiratory control: neuromodulatory and trophic
effects. Respir. Physiol. Neurobiol. 164, 222–232.
Hodges M. R. and Richerson G. B. (2010) The role of medullary
serotonin (5-HT) neurons in respiratory control: contributions to
eupneic ventilation, CO2 chemoreception, and thermoregulation.
J. Appl. Physiol. 108, 1425–1432.
Hodges M. R., Tattersall G. J., Harris M. B., McEvoy S. D., Richerson
D. N., Deneris E. S., Johnson R. L., Chen Z. F. and Richerson G.
B. (2008) Defects in breathing and thermoregulation in mice with
near-complete absence of central serotonin neurons. J. Neurosci.
28, 2495–2505.
Hodges M. R., Wehner M., Aungst J., Smith J. C. and Richerson G. B.
(2009) Transgenic mice lacking serotonin neurons have severe
apnea and high mortality during development. J. Neurosci. 29,
10341–10349.
Hodges M. R., Best S. and Richerson G. B. (2011) Altered ventilatory
and thermoregulatory control in male and female adult Pet-1 null
mice. Respir. Physiol. Neurobiol. 177, 133–140.

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12311

Brainstem 5-HT1A receptors in 5-HT mutants

Jacobsen K. X., Czesak M., Deria M., Le Francois B. and Albert P. R.
(2011) Region-specific regulation of 5-HT1A receptor expression
by Pet-1-dependent mechanisms in vivo. J. Neurochem. 116,
1066–1076.
Kinney H. C. and Thach B. T. (2009) The sudden infant death syndrome.
N. Engl. J. Med. 361, 795–805.
Kinney H. C., Randall L. L., Sleeper L. A. et al. (2003) Serotonergic
brainstem abnormalities in Northern Plains Indians with the sudden
infant death syndrome. J. Neuropathol. Exp. Neurol. 62, 1178–1191.
Kinney H. C., Richerson G. B., Dymecki S. M., Darnall R. A. and Nattie
E. E. (2009) The brainstem and serotonin in the sudden infant
death syndrome. Annu. Rev. Pathol. 4, 517–550.
Kinney H. C., Broadbelt K. G., Haynes R. L., Rognum I. J. and Paterson
D. S. (2011) The serotonergic anatomy of the developing human
medulla oblongata: implications for pediatric disorders of
homeostasis. J. Chem. Neuroanat. 41, 182–199.
Kiyasova V., Fernandez S. P., Laine J., Stankovski L., Muzerelle A.,
Doly S. and Gaspar P. (2011) A genetically defined
morphologically and functionally unique subset of 5-HT neurons
in the mouse raphe nuclei. J. Neurosci. 31, 2756–2768.
Liu Q. and Wong-Riley M. T. (2010) Postnatal changes in the
expressions of serotonin 1A, 1B, and 2A receptors in ten brain
stem nuclei of the rat: implication for a sensitive period.
Neuroscience 165, 61–78.
Liu C., Maejima T., Wyler S. C., Casadesus G., Herlitze S. and Deneris
E. S. (2010) Pet-1 is required across different stages of life to
regulate serotonergic function. Nat. Neurosci. 13, 1190–1198.
Machaalani R., Say M. and Waters K. A. (2009) Serotoninergic receptor 1A
in the sudden infant death syndrome brainstem medulla and
associations with clinical risk factors. Acta Neuropathol. 117, 257–265.
Miquel M. C., Kia H. K., Boni C., Doucet E., Daval G., Matthiessen L.,
Hamon M. and Verge D. (1994) Postnatal development and
localization of 5-HT1A receptor mRNA in rat forebrain and
cerebellum. Brain Res. Dev. Brain Res. 80, 149–157.
Ozawa Y. and Takashima S. (2002) Developmental neurotransmitter
pathology in the brainstem of sudden infant death syndrome: a
review and sleep position. Forensic Sci. Int. 130(Suppl), S53–59.
Panigrahy A., Filiano J., Sleeper L. A. et al. (2000) Decreased
serotonergic receptor binding in rhombic lip-derived regions of
the medulla oblongata in the sudden infant death syndrome.
J. Neuropathol. Exp. Neurol. 59, 377–384.
Pasquale-Styles M. A., Tackitt P. L. and Schmidt C. J. (2007) Infant
death scene investigation and the assessment of potential risk

9

factors for asphyxia: a review of 209 sudden unexpected infant
deaths. J. Forensic Sci. 52, 924–929.
Patel T. D., Azmitia E. C. and Zhou F. C. (1996) Increased 5-HT1A
receptor immunoreactivity in the rat hippocampus following 5,7dihydroxytryptamine lesions in the cingulum bundle and fimbriafornix. Behav. Brain Res. 73, 319–323.
Paterson D. S., Trachtenberg F. L., Thompson E. G., Belliveau R. A.,
Beggs A. H., Darnall R., Chadwick A. E., Krous H. F. and Kinney
H. C. (2006) Multiple serotonergic brainstem abnormalities in
sudden infant death syndrome. JAMA 296, 2124–2132.
Pfaar H., von Holst A., Vogt Weisenhorn D. M., Brodski C., Guimera J.
and Wurst W. (2002) mPet-1, a mouse ETS-domain transcription
factor, is expressed in central serotonergic neurons. Dev. Genes.
Evol. 212, 43–46.
Pham-Le N. M., Cockburn C., Nowell K. and Brown J. (2011)
Activation of GABAA or 5HT1A receptors in the raphe pallidus
abolish the cardiovascular responses to exogenous stress in
conscious rats. Brain Res. Bull. 86, 360–366.
Richerson G. B. and Buchanan G. F. (2011) The serotonin axis: Shared
mechanisms in seizures, depression, and SUDEP. Epilepsia 52
(Suppl 1), 28–38.
Rossi D. V., Burke T. F. and Hensler J. G. (2008) Differential regulation
of serotonin-1A receptor-stimulated [35S]GTP gamma S binding in
the dorsal raphe nucleus by citalopram and escitalopram. Eur. J.
Pharmacol. 583, 103–107.
Saito Y., Ito M., Ozawa Y. et al. (1999) Changes of neurotransmitters in
the brainstem of patients with respiratory-pattern disorders during
childhood. Neuropediatrics 30, 133–140.
Talley E. M., Sadr N. N. and Bayliss D. A. (1997) Postnatal
development of serotonergic innervation, 5-HT1A receptor
expression, and 5-HT responses in rat motoneurons. J. Neurosci.
17, 4473–4485.
Waters K. (2010) Serotonin in the sudden infant death syndrome. Drug
News Perspect. 23, 537–548.
Welner S. A., De Montigny C., Desroches J., Desjardins P. and SuranyiCadotte B. E. (1989) Autoradiographic quantification of
serotonin1A receptors in rat brain following antidepressant drug
treatment. Synapse 4, 347–352.
Zhao Z. Q., Scott M., Chiechio S., Wang J. S., Renner K. J., Gereau R.
W. t., Johnson R. L., Deneris E. S. and Chen Z. F. (2006) Lmx1b is
required for maintenance of central serotonergic neurons and mice
lacking central serotonergic system exhibit normal locomotor
activity. J. Neurosci. 26, 12781–12788.

© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12311