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Developmental Brain Resear

Research report

bIn vitroQ postnatal expression of 5-HT7 receptors in the rat hypothalamus:

an immunohistochemical analysis

Antonella Russoa,*, Rosalia Pellitterib, Sebastiana Monacoa, Rosa Romeoc, Stefania Stanzania

aDepartment of Physiological Sciences, University of Catania, Viale Andrea Doria 6, Catania 95025, ItalybInstitute of Neurological Sciences, National Research Council, Viale R. Margherita, 695123 Catania, Italy

cDepartment of Anatomy, Diagnostic Pathology, Phorens Medicine, Hygiene and Public Health, University of Catania, Via Santa Sofia, Catania, Italy

Accepted 10 November 2004

Available online 29 December 2004

Abstract

The neurotransmitter serotonin (5-HT) is involved in various physiological functions via multiple receptor subtypes. These have been

classified in seven receptor families (5-HT1–7) on the basis of their structures and functional characteristics. In this study, we examined the

expression of 5-HT7 receptors in the rat hypothalamic neurons cultured bin vitroQ during postnatal development. Neuronal cultures were

prepared from postnatal pups P2, P3 and P5, were treated with bFGF and processed by means of immunofluorescence technique using a

polyclonal 5-HT7 receptor antibody. In P2, we found a low density of 5-HT7 labeled hypothalamic bFGF-treated neurons and no 5-HT7

immunolabeling in control cultures; in P3, a moderate number of bFGF-treated neurons were observed but they were not bright. No 5-HT7

immunolabeling was found in controls. In P5, a heavy labeling of small sized bipolar neurons was seen in bFGF-treated neurons, while in

control cultures, few labeled neurons with a low stained density were observed. These results suggest that 5-HT7 receptors in hypothalamic

neurons begin to appear at P5 and then could be involved in many physiological implications that are not exerted at P2 and P3. This indicates

that 5-HT7 receptors have a potential significance in the rat hypothalamus during early postnatal development.

D 2004 Elsevier B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Serotonin receptors

Keywords: 5-HT7; Immunohistochemistry; Postnatal development; Rat; hypothalamic cultures

1. Introduction

Serotonin (5-HT) is a neurotransmitter implicated in the

regulation of behavioral, biological and physiological

functions of the central nervous system through a variety

of receptor subtypes. These were classified into seven

groups: 5-HT1–7, based on their gene structures, amino acid

sequences, functional and pharmacological characteristics

[3,5]. They belong to the G-protein-coupled receptor family,

except 5-HT3 being a ligand-gated channel [16]. Several

studies have shown the localization of different 5-HT

receptors in the rat brain. Very high concentration of 5-

0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.devbrainres.2004.11.002

* Corresponding author. Fax: +39 095330645.

E-mail address: antrusso@mbox.unict.it (A. Russo).

HT1 receptors was localized in choroid plexus, globus

pallidus, dentate gyrus, substantia nigra, reticular and

entorhinal cortex [27], while 5-HT2 receptors were localized

in olfactory tubercle, claustrum and layer IV of the neo-

cortex [28]. Moreover, 5-HT2 receptors were seen in

nucleus accumbens, in lateral amygdala and a moderate

number of 5-HT2 containing cells were found in hippo-

campus [21,43]. It was demonstrated that not only neurons

but also astrocytes express 5-HT2 receptors in various rat

brain areas and they are implicated in psychiatric disorders

[6,43].

Some authors studied the postnatal development of 5-

HT2A receptors in hypoglossal and facial motoneurons and

they showed that the percentage motoneurons expressing 5-

HT2Awas major on postnatal days 12–15 [41]. Furthermore,

ch 154 (2005) 211–216

A. Russo et al. / Developmental Brain Research 154 (2005) 211–216212

some data in the literature suggest that the 5-HT2C receptors

are more abundant than 5-HT2A receptors in the rat

suprachiasmatic nucleus [22] and are expressed in different

moment during postnatal development. 5-HT2A receptor

was at a low level at postnatal day 3 (P3) and increased

during the first 3 weeks; whereas the 5-HT2C receptor was

expressed at P3 at high level [12]. Recent studies

investigating the distribution of the 5-HT5, 5-HT6 and 5-

HT7 have been carried out using immunohistochemical

procedures. For example, 5-HT5 was localized in supra-

chiasmatic nucleus [26] and this suggests its possible role in

the regulation of physiological circadian rhythms.

During the last years, there were many studies regarding

the use of atypical antipsychotics for schizophrenia. Among

these, there is clozapine, a drug with high affinity to 5-HT6

and 5-HT7 [32]. Clozapine shows a divergent effect on the

regulation of 5-HT6 and 5-HT7 receptors, in fact it down-

regulates 5-HT6 and upregulates 5-HT7 receptors, this can

be important to know how they participate in the pharma-

cological treatment of mental diseases [45]. 5-HT7 receptors

were identified from a number of species as mouse, rat,

guinea pig and human [2,15,17,38]. Their expression is very

high within region of the thalamus, hypothalamus and

hippocampus, while it is low in the central cortex and

amygdala [7,37,38]. In rat tissue, four 5-HT7 receptor

isoforms were found, called 5-HT7A, 5-HT7B, 5-HT7C and

5-HT7D [8]. A number of reports suggests that 5-HT7

receptors, localized in suprachiasmatic nucleus [22,25,37],

are implicated in the regulation of circadian rhythms

[15,44].

The aim of the present study was to investigate bin vitroQ,the expression patterns of 5-HT7 receptors during postnatal

development (P2, P3, P5) in the rat hypothalamic cultures.

To delineate the localization of 5-HT receptors, we

performed immunohistochemical analysis using a polyclo-

nal antibody for 5-HT7 receptors.

Moreover, as Fibroblast Growth Factors-family repre-

sents a potential neurotrophins [42] whose actions may vary

at different points in development [31], we studied the effect

of basic Fibroblast Growth Factor (bFGF) on 5-HT7

expressing hypothalamic neuronal cultures.

2. Materials and methods

2.1. Neuronal cultures

Cell suspensions were prepared from postnatal rats (P2–

P3–P5); the day of birth was determined to be postnatal day

0 (P0). All animals (Wistar, Morini, Italy) were housed and

used in accordance with current institutional guidelines for

the laboratory animal care. Pups were anaesthetized in ether

and decapited, their brains were rapidly removed and

hypothalamic areas were isolated [33] and dissected out in

cold (+4 8C) Leibowitz L-15 medium. Then the pieces were

incubated for 6 min in 0.1% trypsin in a solution of 0.1 M

phosphate-buffered saline (PBS), pH 7.4, containing 0.02%

EDTA at 37 8C. Deoxyribonuclease (DNAse) was added

and the cells were centrifuged; the supernatant was removed

and the cells were resuspended and triturated through a fire-

polished Pasteur pipet in 1 ml of triturating solution (1%

bovine serum albumine, 0.001% DNAse, 0.1% soybean

trypsin inhibitor in PBS).

Dissociated cells were then plated on 16-mm diameter

poly-l-lysine (PLL, 10 Ag/ml) coated glass/coverslips at a

final density of 2 � 104 cells/coverslips and maintained in

Dulbecco’s modified Eagle’s medium (DMEM) supple-

mented with 10% fetal calf serum (FCS), 2 mM l-

glutamine, penicillin (50 Ag/ml) and streptomycin (50 U/

ml). Some wells were treated with bFGF (Human recombi-

nant, Boehringer Mannheim, 10 ng/ml), others were

maintained just DMEM without bFGF regarding them as

controls. Contaminating non-neuronal cells were reduced by

treatment with the antimitotic agent cytosine arabinoside

(10�5 M) added 24 h after the dissection. Cells were

incubated at 37 8C in DMEM for a week with two medium

changes.

2.2. Immunocytochemistry

In this study, the cells were identified by immunocyto-

chemical procedures. After washing coverslips twice in 37

8C Hank’s balanced salt solution, the cells were fixed for 30

min in ice-cold 4% paraformaldeyde in 0.1 M PBS; then

they were rinsed in PBS (3 times) and to permealize, the

membranes were incubated in 5% normal goat serum (NGS)

in PBS containing 0.1% Triton X-100 at room temperature

for 20 min. This step was followed by incubation overnight

at 4 8C with the primary antibody, a polyclonal antibody

raised against the serotonin7 (5-HT7) receptor (1:100,

Calbiochem) in PBS/Triton containing 1% NGS. After

washing in PBS, to visualize the primary antibody, cover-

slips were incubated in secondary antibody Fluoresceine

Isothiocyanate (FITC) labeled anti-rabbit antibody (1:100,

Chemicon) in PBS/Triton with 1% NGS, at room temper-

ature for 30 min. Coverslips were washed in PBS, mounted

in PBS/glycerol (50:50) on microscope slides.

To determine the negative immunofluorescence staining,

in some coverslips, the cells were incubated in PBS-Triton

without primary antibody.

3. Analysis

Coverslips were analyzed on a Reichert Jung fluorescent

microscope and 5-HT7 receptor positive neurons were

counted over the entire coverslip. For each culture condition

P2, P3 and P5, a minimum of two experiments were carried

out, utilizing for each experiment 10 cover slips, 5 as control

and 5 with bFGF. Data were assessed by statistical analysis

using Microsoft Excel (means F standard error) and

statistically significant differences were analyzed by t-test.

A. Russo et al. / Developmental Brain Research 154 (2005) 211–216 213

4. Results

We describe the postnatal expression (P2–P5) of 5-HT

receptors in the rat hypothalamus cultures by using

immunohistochemical procedure. Moreover, in this study,

we considered the effect of bFGF on hypothalamic neurons

expressing 5-HT7 receptors.

4.1. Expression of 5-HT7 in postnatal rats P2

Immunofluorescence histochemistry against 5-HT7 dem-

onstrated a low density of hypothalamic neurons in the

cultures treated with neurotrophic factor, bFGF (Fig. 1).

These positive neurons were very pale and it was possible to

note just few bipolar and small (20 Am) neurons (Fig. 2A).

No 5-HT7 immunolabeling was observed in the control

neurons, without bFGF.

4.2. Expression of 5-HT7 in postnatal rats P3

In P3 neurons stained for 5-HT7, a light positivity limited

only to treat cultures with bFGF began to appear (Fig. 1). In

fact, it was possible to see some small (20 Am) very labeled

neurons and 5-HT7 immunoreactivity was found both in

cytoplasm and in axons (Fig. 2B–C). In control cultures, no

5-HT7 labeling neuron was observed.

4.3. Expression of 5-HT7 in postnatal rats P5

5-HT7 reactivity was clearly present in postnatal rats P5

hypothalamus neurons and the number of positive neurons

was increased (Fig. 1). Neuronal cells appeared small (20–

25 Am), immunostaining was very intense and the immu-

Fig. 1. Histogram shows the increase in number of 5-HT7 positive neurons in

controls and in bFGF-treated cultures in P2, P3 and P5. Values are mean FS.E.M. per group. Results were analyzed using t-test: in all groups,

significantly difference was observed ( P b 0.001).

Fig. 2. Fluorescence photomicrograph showing anti-serotonin7 immunocy-

tochemical staining of neurons in different postnatal development. (A) 5-

HT7 positive neuron at P2 grown in the presence of bFGF. (B–C) 5-HT7

positive neuron at P3 grown in the presence of bFGF. (D) Group of neurons

observed at P5 in bFGF-treated cultures. (E) Single neuron at P5 in bFGF-

treated cultures. (F) Neurons observed at P5 in the absence of bFGF. Scale

bar = 50 Am.

noreactive dots were sparsely distributed in the whole

neuron. We noticed a clear difference between controls and

treated-bFGF neurons. In fact, in the control neurons

without neurotrophic factors, we observed a few labeled

neurons with a low stained density (2F); while in treated-

bFGF neurons, we noted a strong immunoreactivity of small

bipolar hypothalamic neurons (Fig. 2D–E).

In all experimental groups (P2–P3–P5), some coverslips

without primary antibody were performed showing a

negative immunofluorescence staining.

5. Discussion

The present study describes the expression patterns of 5-

HT7 serotonin receptor-like immunoreactivity in the rat

A. Russo et al. / Developmental Brain Research 154 (2005) 211–216214

hypothalamus during postnatal development based on

fluorescence immunohistochemistry. In particular, our

research was carried out on the appearance of the expression

of 5-HT7 in hypothalamic neurons under the effect of a

trophic factor (bFGF) at different postnatal ages (P2, P3,

P5). To date, no detailed investigation of the hypothalamic

localization of this receptor has been reported at postnatal

development P2–P5.

Serotonin (5-HT7) has been extensively implicated in

more behaviors, physiological mechanism, and in addition

to its role as neurotransmitter, it regulates many aspects of

development including neuronal growth and survival [10]

and its presence in early embryos of numerous species [11]

suggests that 5-HT may be evolved in synaptic transmission

and plays a crucial role during craniofacial morphogenesis

[36]. Furthermore, serotonin neurons are the first to emerge

during early development of the brain and spinal cord. Thus,

there is evidence that 5-HT is involved in a variety of

cellular processes regulating metabolism, proliferation and

morphology [1]. These events are mediated by several

receptor subtypes, classified into seven groups, 5-HT1–7 [3].

The 5-HT2 receptors are expressed in neurons relatively

late during postnatal development [21] and they may be

altered during psychoactive drug treatment, therefore neuro-

nal 5-HT2A receptors may have functional implications in

psychiatric disorders [43].

The presence of some 5-HT receptors, in particular 5-

HT2A, 5-HT3, 5-HT5A and 5-HT7, in the rat cerebellum,

may contribute to revealing the modulatory actions of

serotonin on cerebellar functions [6]. The 5-HT5A receptor

subtypes were localized in the rat suprachiasmatic nucleus

and this suggests its possible role in the regulation of

circadian rhythms [26].

A widespread distribution of 5-HT7 receptors in the

brain has been well documented and this asserted a

multiple physiological central roles [39]. Moreover, both

serotonin and serotonin receptors have also been impli-

cated in neuronal plasticity [13] and in memory formation

[19].

Previous studies reported a variety distribution of 5-HT7

receptors in the rat hypothalamus [34], including supra-

chiasmatic nucleus [4,7], medial preoptic area [25] and

periventricular hypothalamic [23]. Moreover, using both in

situ hybridization and immunocytochemistry of 5-HT7

receptor in rat hypothalamus, it was seen a high signal

localized especially in suprachiasmatic nucleus [25]. It is

known that suprachiasmatic nucleus regulates the circadian

system [9] and numerous physiological processes including

hormone secretion, activity and temperature rhythms and

regulation of sleep [30]. Its co-localization with vasopressin,

VIP and GABA-containing neurons [29] denotes multiple

neuronal roles of 5-HT7 receptor in suprachiasmatic nucleus

function. For this reason, the wide distribution of 5-HT7

receptors is involved in various functions particularly for

mediating effects of 5-HT on the activity of light-sensitive

suprachiasmatic nucleus neurons, which play a role in

regulating photic response of circadian rhythms [15,44].

Moreover, a recent study has demonstrated that 5-HT7

receptor subtypes play a role in the regulation of LH by

Zona Incerta in the brain [35] and are even involved in

nociceptor activation as it is expressed by rat primary

afferent nociceptors located in spinal cord [20].

Some authors have studied the time course and local-

ization of the 5-HT7 receptor mRNA in the diencephalon

and mesencephalon areas during postnatal development (P5,

P8, P15, P21), these data suggest a possible involvement of

5-HT7 receptor in directing development as promoting

branching, release of growth factors and an important role

as limbic functioning [40]. Considering this, in the present

study, we investigated bin vitroQ on the expression of 5-HT7

receptors in early postnatal rat hypothalamic cultures from

P2, P3 and P5.

A number of reports have indicated that growth factors

can promote the bin vitroQ survival of embryonic and

postnatal neurons. In particular, bFGF is effective promoter

of neuronal survival and neurite extension. We have

previously demonstrated that bFGF promotes the survival

of embryonic dopaminergic neurons bin vitroQ and it has a

positive effect on neurite outgrowth [18].

Furthermore, as 5-HT has been shown to synergize

with growth factors to stimulate cell proliferation [24] and

the neurite outgrowth [14], we carried out our study

administering to culture neurons bFGF. Our results have

reported that in the controls, the signal was absent at P2 at

P3 and it began to appear at P5 showing a strong

positivity. In bFGF-treated neurons, we found a low

density of neurons at P2, a light positivity at P3 and an

intense immunoreactivity in addition to an increased

number of neurons at P5. This means that postnatal age

P2–P3 is too early, and as 5-HT7 receptor plays an

important role in biobehavioral processes as sleep–wake

regulation, pain perception, consequently many physio-

logical actions are not exerted yet at this age. Moreover,

considering both the known synergize between growth

factors and serotonin and the positive effect of bFGF on

neurons cultured bin vitroQ and referring to the appearance

of 5-HT7 expression only in controls at P5 and no at P2

and P3, we could hypothesized that this synergize is more

decisive at P2 and P3 and that it could play a significative

role in many functions.

Therefore the present study clearly demonstrates that 5-

HT7 receptors have specific time course of their postnatal

development and these data suggest the functional involve-

ment of these receptors in developing brain acting by a

different mechanism.

Acknowledgments

This research was supported by grants from MIUR and

from Catania University. The authors wish to thank to Mr. S.

Bentivegna for expert photography work.

A. Russo et al. / Developmental Brain Research 154 (2005) 211–216 215

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