Download Drosophila GABA, short neuropeptide F and their receptors

Chemical signalling in the Drosophila brain:
GABA, short neuropeptide F and their receptors
Doctoral dissertation 2011
Lina E. Enell
Department of Zoology
Stockholm University
106 91 Stockholm
ABSTRACT
γ-aminobutyric acid (GABA) and short neuropeptide F (sNPF) are widespread
signalling molecules in the brain of insects. In order to understand more about
the signalling and to some extent start to unravel the functional roles of these
two substances, this study has examined the locations of the transmitters and
their receptors in the brain of the fruit fly Drosophila melanogaster using
immunocytochemistry in combination with Gal4/UAS technique. The main
focus is GABA and sNPF in feeding circuits and in the olfactory system. We
found both GABA receptor types in neurons in many important areas of the
Drosophila brain including the antennal lobe, mushroom body and the central
body complex. The metabotropic GABAB receptor (GABABR) is expressed in
a pattern similar to the ionotropic GABAAR, but some distribution differences
can be distinguished (paper I). The insulin-producing cells contain only
GABABR, whereas the GABAAR is localized on neighbouring neurons. We
found that GABA regulates the production and release of insulin-like peptides
via GABABRs (paper II). The roles of sNPFs in feeding and growth have
previously been established, but the mechanisms behind this are unclear. We
mapped the distribution of sNPF with antisera to the sNPF precursor and
found the peptide in a large variety of interneurons, including the Kenyon cells
of the mushroom bodies, as well as in olfactory sensory neurons that send
axons to the antennal lobe (paper III). We also mapped the distribution of the
sNPF receptor in larval tissues and found localization in six median
neurosecretory cells that are not insulin-producing cells, in neuronal branches
in the larval antennal lobe and in processes innervating the mushroom bodies
(paper IV).
In summary, we have studied two different signal substances in the
Drosophila brain (GABA and sNPF) in some detail. We found that these
substances and their receptors are widespread, that both sNPF and GABA
act in very diverse systems and that they presumably play roles in feeding,
metabolism and olfaction.
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Table of contents
______________________________________________________________
List of papers
3
1. Introduction
1.1. The nervous system of insects
1.1.1. Antennal lobe
1.1.2. Mushroom body
1.1.3. Other neurons and circuits of interest
1.2. Neurotransmitters and neuropeptides
1.2.1 Receptors
1.2.2. GABA and GABA receptors
1.2.2.1. Ionotropic GABA receptors
1.2.2.2. Metabotropic GABA receptors
1.2.3. Short neuropeptide F
1.2.3.1. Short neuropeptide receptor
1.2.4. Insulin, insulin-like peptides and insulin receptor
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4
5
5
6
6
7
8
9
10
11
12
13
2. Aims of the thesis
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3. Methods
3.1. The Gal4/UAS system and RNA interference
3.2. Fly stocks
3.3. Antisera, immunocytochemistry and antisera characterization
3.4. In situ hybridization
3.5. Quantification of immunofluorescence
3.6. Survival assays
3.7. Measurements of trehalose, lipids and growth
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16
17
19
21
21
21
21
4. Results and discussion
4.1. Paper I
4.2. Paper II
4.3. Paper III
4.4. Paper IV
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22
23
25
26
5. General discussion
27
6. Conclusions and future perspectives
29
7. References
31
8. Acknowledgements/tack
39
2
LIST OF PAPERS
This thesis is based on the following papers and will be referred to by their
Roman numerals.
______________________________________________________________
I.
Enell LE, Hamasaka Y, Kolodziejczyk A, Nässel DR. (2007) GammaAminobutyric acid (GABA) signalling components in Drosophila:
immunocytochemical localization of GABA(B) receptors in relation to
the GABA(A) receptor subunit RDL and a vesicular GABA transporter.
J Comp Neurol. 505:18-31.
II.
Enell LE, Kapan N, Söderberg JAE, Kahsai L, Nässel DR. (2010)
Insulin signaling, lifespan and stress resistance are modulated by
metabotropic GABA receptors on insulin producing cells in the brain of
Drosophila. PLoS One. 30;5:e15780.
III.
Nässel DR, Enell LE, Santos JG, Wegener C, Johard HA. (2008) A
large population of diverse neurons in the Drosophila central nervous
system expresses short neuropeptide F, suggesting multiple distributed
peptide functions. BMC Neurosci. 19;9:90.
IV.
Enell LE, Carlsson MA, Aumann D, Nässel DR. (2011) Distribution of
the short neuropeptide F receptor and its ligands in the chemosensory
and hormonal systems of larval Drosophila. (Manuscript)
______________________________________________________________
Publications not included in the thesis
Root CM, Masuyama K, Green DS, Enell LE, Nässel DR, Lee CH, Wang JW.
(2008) A presynaptic gain control mechanism fine-tunes olfactory behavior.
Neuron 59:311-21.
Johard HA, Enell LE, Gustafsson E, Trifilieff P, Veenstra JA, Nässel DR.
(2008) Intrinsic neurons of Drosophila mushroom bodies express short
neuropeptide F: relations to extrinsic neurons expressing different
neurotransmitters. J Comp Neurol. 507:1479-96.
Nässel DR, Enell LE, Hamasaka Y, Johard H. (2005) Neurotransmitters and
their receptors in circadian clock circuits of Drosophila. Pesticides 3:133-140.
3
1. INTRODUCTION
1.1. The nervous system of insects
The insect nervous system consists of a dorsal brain (Fig. 1) and a ventral
nerve cord. The brain is divided into three lobes or neuromeres that are in fact
three fused ganglia. The first neuromere is called protocerebrum and receives
inputs from the compound eye. The mushroom body and central body
complex are also part of the protocerebrum, as well as neurosecretory cells
projecting to neurohemal organs of the corpora cardiaca and corpora allata.
The deutocerebrum is the middle neuromere that receives inputs from the
antennae and processes sensory information like odors, taste and tactile
sensation. The antennal lobe is located in this region. The third neuromere is
the tritocerebrum that contains neurons that connect to the labrum and
anterior digestive canal. The subesophageal ganglion is located just below the
brain and is primarily in control of the mouthparts and a relay for connections
to the ventral nerve cord. The ventral nerve cord consists of segmentally
arranged ganglia running along the midline of the thorax and abdomen. There
are three thoracic ganglia and these are mainly in control of locomotion,
whereas the abdominal ganglia control reproduction and other functions of the
abdomen. In Drosophila, the abdominal and thoracic ganglia are fused into
one thoracic-abdominal ganglion (Strausfeld, 1976).
PI
A
α-lo
eye
β,γ-lo
AL
SOG
B
PI
Lo p
Me
Ca
l ho
Me
PB
l deu
La
oe
Lo
SOG
Figure 1. The adult Drosophila melanogaster brain. A. Frontal view. Labelled are the pars
intercerebralis (PI), the antennal lobe (AL), the subesophageal ganglion (SOG), the lobes of
the mushroom body (α-lo and β,γ-lo) and the compound eye (eye). The unlabelled arrows
indicate parts of the protocerebrum. B. The calyx (Ca) of the mushroom body, protocerebral
bridge (PB), the parts of the optic lobes: lamina (La), medulla (Me), lobular plate (Lo p) and
lobula (Lo), lateral horn (l ho), lateral deutocerebrum (l deu) and the esophageal opening (oe)
are labelled. Pictures modified from flybrain.org.
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1.1.1. Antennal lobe
The olfactory pathway in insects starts in the antennae, where olfactory
sensory neurons (OSNs) express one type of olfactory receptor (Or). Each
OSN sends information about an odor to the antennal lobe. The antennal lobe
is the insect homolog of the olfactory bulb in vertebrates and both are
organized similarly in spherical packed neuropils called glomeruli. Adult
Drosophila have 43-50 glomeruli and larvae only 21 (Vosshall and Stocker,
2007), compared to the over 1500 in mammals (Kosaka et al., 1998). This
renders Drosophila a simple, but yet adequate model for olfaction studies.
Each glomerulus in the antennal lobe receives information from one or
sometimes two types of olfactory receptors (Vosshall and Stocker, 2007).
OSNs synapse with local interneurons and projection neurons in the
glomeruli. The local interneurons interconnect glomeruli and each local
interneuron sends branches to almost all glomeruli (Stocker et al., 1997).
Many local interneurons contain GABA, but also acetylcholine, tachykinins
and other neuropeptides have been associated to these cells (Carlsson et al.,
2010; Roy et al., 2007; Wilson and Laurent, 2005; Winther et al., 2003).
Projection neurons send axons to higher protocerebral brains centres such as
the mushroom bodies and lateral horn.
1.1.2. Mushroom body
The mushroom bodies in insects have a role in olfactory memory and
learning. They are composed of the thousands of Kenyon cells, the small but
numerous intrinsic neurons of the mushroom bodies. Kenyon cells send their
axons through the peduncle (or stalk) from which they bifurcate into a dorsal
α-lobe and a medial β-lobe. Some axons do not bifurcate and form a medial γlobe. A third morphological subdivision deriving from the Kenyon cells can be
distinguished, the α´- and β´-lobe (Crittenden et al., 1998). Just below the cell
bodies of the Kenyon cells is the calyx, which is the input (dendritic) region of
the mushroom body. The calyces receive information mainly from the
antennal lobes (Vosshall and Stocker, 2007), but neurons of the optic lobes
can in some insects send axons to the calyces (Strausfeld and Li, 1999). The
mushroom body derives from four neuroblasts that give raise to all Kenyon
cells and supporting glial cells. One neuroblast contributes to most, if not all,
types of cells within the mushroom body and all are practically identical in
structure and gene expression patterns (Ito et al., 1997). In other words, the
mushroom body is composed of four identical clonal units.
Extrinsic cells associated with the mushroom bodies seem to be
important for the functional role of the mushroom bodies. Two large dorsal
paired medial neurons (DPM) are innervating the mushroom bodies and
express the amnesiac gene. Mutations in amnesiac results in a defect
memory consolidation (Feany and Quinn, 1995). The predicted peptides
produced by the amnesiac gene have however not been found in Drosophila
(Nässel and Winther, 2010). DPM neurons also seem to express
acetylcholine (Yu et al., 2005) that might be the major transmitter of these
cells. Six dopaminergic neurons that innervate the mushroom bodies have
been shown to have a role in appetitive memory performance (Krashes et al.,
2007). Food deprivation is normally a motivator for olfactory learning, and
stimulation of the dopaminergic neurons proved to decrease olfactory learning
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in starved flies, whereas blocking them increased learning in fed flies. These
cells express receptors to neuropeptide F (NPF) and were demonstrated to be
under control of NPF (Krashes et al., 2007).
1.1.3. Other neurons and circuits of interest
Gustatory and olfactory inputs signal about presence if food and about food
quality. However, in order to monitor nutritional needs and maintain
homeostasis, the organism utilizes internal cues. For example, there are 20
neurons in the subesophageal ganglion (SOG) that contain the neuropeptide
gene hugin. The hugin expressing cells connect to higher brain circuits that
regulate feeding behaviour. Branches of the hugin expressing cells are found
in the SOG, in the protocerebrum where insulin-producing cells are located, in
the ventral nerve cord, in the pharyngeal muscles and in the ring gland
(Melcher and Pankratz, 2005). The ring gland is a larval endocrine organ
involved in metabolism and growth and is producing ecdysteroids and juvenile
hormone. It is composed of the corpus cardiacum, the corpus allatum and the
prothoracid gland. Cells of the corpus cardiacum produce adipokinetic
hormone (AKH), the insect equivalent to glucagon that raises circulating
glucose levels (Kim and Rulifson, 2004). The insulin-producing cells (IPCs) in
the pars intercerebralis project to the ring gland and several other sets of
peptidergic neurosecretory cells have axon terminations there (Cao and
Brown, 2001; Siegmund and Korge, 2001; Wegener et al., 2006).
The central body complex is positioned between the peduncles of the
mushroom body and is comprised of the ellipsoid body, fan shaped body,
noduli and the protocerebral bridge and is believed to serve as integration
centre for motor and sensory functions (Hanesch et al., 1989; Homberg,
2008). Flies with mutations in the central body have defective walking activity
and learning behavior [see (Davis, 1996)].
1.2. Neurotransmitters and neuropeptides
Neurons are communicating with each other by chemical and occasionally
electrical signalling. The chemical transmission is based on various types of
substances such as neuropeptides or different kinds of neurotransmitters.
Classical neurotransmitters include γ-aminobutyric acid (GABA), glutamate,
acetylcholine (ACh) and biogenic amines such as serotonin, octopamine,
histamine and dopamine. These are small molecules that can bind to and
activate both ion channels and G-protein-coupled receptors (GPCRs) (Squire,
2003). Classical transmitters are synthesized and stored in small vesicles at
the axon terminal. When the presynaptic membrane gets depolarized, calcium
channels open that will activate calcium-sensitive proteins on the vesicles.
These proteins (SNAREs) change shape, making the vesicles fuse with the
presynaptic cell membrane and release their content into the synaptic cleft
(Chen and Scheller, 2001).
Neuropeptides are by far the most structurally and functionally diverse
signalling molecules and exist in all animals with a nervous system. They
consist of chains of 3-200 amino acids that are produced in neuronal cell
bodies and transported in vesicles to the presynaptic terminal. Transcription
and translation of neuropeptides in the rough endoplasmic reticulum first
6
produce precursor proteins (prepropeptides). The prepropeptides are
thereafter transported to the Golgi apparatus where they are modified and
packed into large dense-core vesicles. The vesicles are then transported
along microtubules to the presynaptic terminal (Nässel. 2002).
There are only about ten classical neurotransmitters, whereas there is
a huge diversity of neuropeptides. It is not really clear how many
neuropeptides mammals possess, but the number is considerably higher than
in invertebrates. With the entire Drosophila genome sequenced we know from
database mining or peptidomic analysis that there are beyond 30 genes
encoding neuropeptide precursors plus an additional seven genes encoding
insulin-like peptide precursors (Hewes and Taghert, 2001; Nässel, 2009; Yew
et al., 2009). Neuropeptides act on G-protein-coupled receptors and there are
at least 44 putative peptide-activated GPCRs identified in Drosophila (Hewes
and Taghert, 2001; Nässel, 2009). Neuropeptides can act as neuromodulators
or as neurohormones. A neuromodulator in this sense means that it is
released within or outside the synaptic cleft, mediating many different effects,
normally by acting on G-protein-coupled receptors. A neurohormone on the
other hand is released into the circulation and acts on targets far away from
the release site. Neuropeptides are often colocalized with classical
transmitters with the advantage that different functional messages can be
signalled to the target cell. Classical transmitters are often released in
response to smaller changes in Ca2+ levels, whereas peptides are released in
response to much higher Ca2+ levels. Neuropeptides can often diffuse longer
distances and therefore activate other target cells than the classical
transmitter [reviewed in (Nässel, 2009)].
This thesis mainly focuses on GABA (a classical transmitter), as well
as sNPF and insulin (both neuropeptides) and their receptors.
1.2.1. Receptors
As mentioned earlier, classical transmitters (with few exceptions) act on both
ion channel receptors (ionotropic receptors) and G-protein-coupled receptors
(GPCRs or metabotropic receptors), whereas neuropeptides are believed to
act on GPCRs and in some cases tyrosine kinase receptors (e. g. insulin-like
peptides).
Ion channels consists of proteins that form pores and regulate ion
transport across the plasma membrane (Fig. 3A). When a transmitter (ligand)
bind to an ion channel on the postsynaptic cell a conformational change of the
receptor opens the channel and ions can pass and change the membrane
potential within milliseconds. Ionotropic receptors are usually very selective to
one or a few ions, such as K+, Cl-, Ca2+ or Na+, and the flow of ions can be
inwardly or outwardly directed. Thus, a ligand can cause different responses
of the postsynaptic cell depending on what type of receptor it activates. An
excitatory receptor depolarizes the postsynaptic membrane, whereas an
inhibitory receptor causes a hyperpolarization (Squire, 2003).
GPCRs are composed of a polypeptide chain that spans the
membrane seven times (Fig 3B). The hydrophilic regions between the
transmembrane domains form an extracellular N-terminal, three intracellular
loops, three extracellular loops and an intracellular C-terminal. Binding of a
ligand to a GPCR can be accomplished in various ways. Small ligands, like
7
acetylcholine, bind to a pocket formed by a part of the extracellular regions of
the polypeptide chain. Neuropeptides bind with higher affinity, since it involves
both extracellular loops and transmembrane domains. Whereas binding of a
ligand to an ion channel causes a fast response, GPCR mediated activation is
a slightly slower process, but usually with a longer response as a result. The
G protein complex is composed of three subunits, Gα, Gβ and Gγ. In an
inactive form of G-proteins, the Gα binds to GDP (guanosine diphosphate).
When a ligand binds to a GPCR, a change occurs in the orientation of the
transmembrane domains that involves the second and the third intracellular
loops. This conformational change results in an exchange of GDP for a GTP
(guanosine triphosphate), and thereby activates the G-protein. Gβ and Gγ stay
on the membrane, whereas the Gα- and GTP-complex is released into the
cytoplasm where it can start a signalling cascade by influencing ion channels
directly or by affecting the adenylate cyclase pathway. This pathway includes
the conversion of ATP to cyclic AMP (cAMP) that functions as a second
messenger. Second messengers can influence ion channels or affect nuclear
or transcriptional activity. There are various kinds of Gα subunits, for example
Gi (inhibitory), Gs (stimulatory) and Gq/0 (Vanden Broeck, 1996). The β/γ
subunits can also vary in cellular responses they are causing upon GPCR
activation. They can for example activate the phospholipase C pathway.
Phospholipase C mediates the production of inositol 1,4,5-trisphosphate (IP3)
and diacyl glycerol (DAG) through cleavage of phosphatidylinositol 4,5bisphosphate (PIP2). IP3 diffuses through the cytosol and binds to and opens
Ca2+ channels particularly on the endoplasmatic reticulum, thus increasing the
cytosolic levels of calcium. Calcium and DAG can activate protein kinase C
that phosphorylates other molecules. Another function of the β/γ subunits is to
act on G-protein coupled inward rectifying potassium channels (GIRKs)
(Squire, 2003).
1.2.2. GABA and GABA receptors
Gamma-aminobutyric acid (GABA) is a major inhibitory transmitter in the brain
of many animals (Fig. 2). It is produced by a large number of neurons in the
central nervous system (CNS), but there is no GABA containing axons
emerging to the periphery in Drosophila. In mammals, many conditions are
caused by an imbalanced amount of GABA in the brain. Epilepsy, Parkinson’s
disease, stress, sleep disorders, depression, addiction, pain, anxiety and
Huntington’s disease are examples of disorders correlated with decreased
GABA activity, whereas increased GABA levels can cause schizophrenia
(Albin and Gilman, 1989; Benes and Berretta, 2001; Bettler et al., 2004). In
insects, GABA has roles in olfaction memory and learning, vision and the
circadian clock (Cayre et al., 1999; Hamasaka et al., 2005; Stopfer et al.,
1997; Wilson and Laurent, 2005).
A major difference between GABA and glutamate (amino acid
transmitters) on one hand and other transmitters on the other is that the
former are derived from glucose metabolism. Synthesis of GABA occurs in
cells with glutamic acid decarboxylase (GAD), a cytosolic enzyme that
converts GABA from glutamate (Squire, 2003). GAD is activated by inorganic
phosphatases and inactivated by ATP, aspartate and GABA. Increased GABA
8
levels in the brain will thus inactivate GAD and reduce GABA synthesis [for a
review, see (Petroff, 2002)].
GABA is loaded into vesicles at the synapse by vesicular GABA
transporters, vGAT (Owens and Kriegstein, 2002). These transport GABA
against its concentration gradient by a vacuolar ATPase that exchange one
H+ for one neutral amino acid [reviewed in (Gasnier, 2000)]. GABA is released
into the synaptic cleft upon vesicle fusion with the membrane (described in
more detail in section 1.2), and can then bind to receptors on the postsynaptic
cell. Reuptake of GABA is achieved with different GABA transporters placed
on both neurons and glia. There are also autoreceptors (GABABR) located on
the presynaptic cell that serve as a feedback regulators (Squire, 2003).
O
N2H
OH
Figure 2. Structure of gamma-aminobutyric acid, GABA.
1.2.2.1. Ionotropic GABA receptors
There are two types of GABA receptors, the ionotropic (GABAAR) and
metabotropic receptors (GABABR). The GABAAR is a pentameric subunit
complex (Fig. 3A) that forms a pore in the membrane (Hosie et al., 1997).
Each subunit has an extracellular N-terminus, four transmembrane regions
and an extracellular C-terminal (Buckingham et al., 2005). When GABA binds
to this type of receptor a conformational change will occur, the channel opens
and ions (particularly Cl- ions) flow through down a concentration gradient.
This causes hyperpolarization of the postsynaptic membrane (Darlison et al.,
2005).
Vertebrates have two major classes of ionotropic GABA receptors,
GABAA and GABAC receptors. GABAA receptors are more widespread in the
CNS than GABAC receptors and the latter are not antagonized by bicuculline
as GABAA receptors are. Both are, however, blocked by the plant toxin
picrotoxin (Hosie et al., 1997). Five subunits of the ionotropic GABA receptor
have been identified in mammals: α, β, γ, δ and ρ. GABAA receptors are
formed as heteromers of α, β and γ or δ subunits whereas GABAC receptors
are composed of ρ subunits (Hosie et al., 1997).
To date three subunits have been cloned and characterized in
Drosophila; RDL (resistant to dieldrin) (Ffrench-Constant et al., 1991), GRD
(GABA- and glycine-like receptor of Drosophila) (Harvey et al., 1994) and
LCCH3 (ligand-gated chloride channel homologue 3) (Henderson et al.,
1993). RDL can form functional homomultimers, whereas LCCH3 and GRD
cannot. This suggests that these subunits form heteromers with RDL or other
subunits (Hosie et al., 1997), giving rise to GABA receptors with different
pharmacological and kinetic properties (Gisselmann et al., 2004). The most
studied subunit in insects is RDL, since it is a suitable model of insect
ionotropic GABA receptors. It is located throughout the entire nervous system
9
in many different insect orders of both embryos and adults and has been
found both at synapses as well as on neuronal cell bodies (Buckingham et al.,
2005).
1.2.2.2. Metabotropic GABA receptors
The last major neurotransmitter receptors to be cloned in mammals were the
G-protein-coupled GABAB receptors (Kaupmann et al., 1997), and some
years later they were cloned in Drosophila (Mezler et al., 2001).
In accordance with other GPCRs, the GABAB receptor is composed of
a single polypeptide that spans the membrane seven times (fig 3B). It has a
large domain in the extracellular N-terminal called the venus flytrap module
(VFTM) that is responsible for binding the ligand. The intracellular C-terminal
activates G-proteins (Gi, see section 1.2.1) and contains a coiled-coil region,
making it possible for the receptors to form dimers. As will be outlined below
GABAB receptors must form dimers to be functional.
Presynaptic GABAB receptors can inhibit release of more transmitter by
downregulating voltage-gated Ca2+ channels via Gβ/γ or inhibit adenylate
cyclase via Gi that results in decreased cAMP levels and protein kinase A
(PKA) activity (Kaupmann et al., 1998; Kubota et al., 2003). Postsynaptic
GABABRs can affect the adenylate cyclase pathway and commonly, via Gβ/γ,
act on G-protein-coupled inwardly rectifying K+ channels (GIRKs), that induce
hyperpolarization (Kaupmann et al., 1998).
There are three subtypes of GABABRs in Drosophila (and presumably
other insects), namely Dm-GABABR1-3 (Bettler et al., 2004). In mammals,
however, only two subtypes has been identified, m-GABABR1-2. DmGABABR1 and Dm-GABABR2 are similar to the mammalian GABABR1 and
R2. Dm-GABABR3 seem to be insect specific (Bettler et al., 2004) with a
possible role in the circadian clock (Dahdal et al., 2010), and will be discussed
in more detail below.
A functional GABABR requires formation of a dimer of GABABR1 and
R2 subunits. The ligand binding part is situated in the GABABR1, with its
venus flytrap module (Kaupmann et al., 1997), and the presumed ligand
binding sequence is not conserved in GABABR2. The GABABR2 on the other
hand activate G-proteins, increase GABABR1 agonist affinity (Pin et al., 2003)
and assist GABABR1 to reach the cell surface. GABABR1 stays in the
endoplasmatic reticulum (ER) after its biosynthesis due to an ER retention
signal. This signal will be masked upon interaction of the coiled-coil regions of
the two subunits, making GABABR1 reach the surface together with R2.
GABABR1 with mutations in the ER retention signal can reach the surface
alone, form homodimers, but is not able to activate G-proteins (Pin et al.,
2004). GABABR2 with a mutation in the part of the intracellular loop that
contact the G-protein can logically not couple to G-protein, whereas the same
mutation in GABABR1 has no effect (Pin et al., 2003). GABABR1-deficient
mice have no detectable pre- and postsynaptic responses to known GABABR
agonists and the GABABR2 subunit is strongly downregulated in these mice
(Bowery et al., 2002).
The third, insect specific GABABR3 subunit also has a coiled-coil
region that might suggest that it form dimers (Mezler et al., 2001). Whether it
forms heterodimers with other subunits or if it is acting as homodimer is not
10
known, but the possibility that it forms dimers with GABABR1 or R2 is small,
since the distribution of GABABR3 in embryos, observed with in situ
hybridization is rather different from these subunits. GABABR1 and R2
however, seem to be co-localized in neurons both in insects and mammals
(Mezler et al., 2001). The functional roles for GABAB receptors are not fully
unraveled. One possible function seems to be a role in addiction. GABABRdeficient flies appear to be more tolerant to ethanol sedation than wildtype
flies (Dzitoyeva et al., 2003) and nicotine-resistant flies have decreased levels
of GABABR transcripts (Passador-Gurgel et al., 2007). Lower levels of
GABABR1 also affect growth negatively and can be lethal (Dzitoyeva et al.,
2005). The receptor also seems to be involved in the circadian clock
(Hamasaka et al., 2005), as it is expressed in a subset of the clock cells (sLNv) and responses to GABA is prevented when blocked with GABABR
antagonist (but not with GABAAR antagonist). A recent study (Dahdal et al.,
2010) suggest that also GABABR3 is expressed in the s-LNvs and helps
generating 24 hr rhythms via GABA-mediated inhibition of acetylcholineinduced Ca2+ responses. GABABR3 RNA levels and the response to GABA
were reduced in isolated larval LNvs (known to develop into adult s-LNvs) in
Drosophila expressing GABABR3-RNAi specifically in the clock neurons.
Furthermore, these flies showed a lengthened 24 hr rhythm. This is the first
report on a possible function of the GABABR3 subunit (Dahdal et al., 2010).
N
A
B
C
Figure 3. Schematic drawings of receptors. A. An ionotropic receptor is composed of five
subunits and forms a pore in the membrane. B. A metabotropic receptor with an extracellular
N-terminal, seven transmembrane domains, three extra- and three intracellular loops and an
intracellular C-terminal.
1.2.3. Short neuropeptide F
The mammalian neuropeptide Y (NPY) plays an important role in food-intake
regulation, metabolism as well as in memory and learning [see (Lee et al.,
2004)]. The invertebrate equivalent is named neuropeptide F (NPF) and the
name refers to a phenylalanine (F) residue in the C-terminal instead of a
tyrosine (Y) of the vertebrate peptide. NPF is involved in larval feeding,
foraging and social behavior (Wu et al., 2003). The levels of NPFs are
decreased upon the behavioral switch from the continuous feeding of the 3rd
instar larva to the wandering stage when the larva search for a puparation site
11
outside the food source (Wu et al., 2003). Whereas NPF seem to be a
motivator for food intake, another peptide is regulating feeding rate and
interact with insulin to regulate growth. This peptide, encoded by a different
precursor gene, is called short neuropeptide F (sNPF). The snpf gene was
first discovered in Drosophila (Vanden Broeck, 2001) and has up to date only
been found in arthropods (Nässel and Wegener, 2011). As the name implies,
its products are only 6-11 amino acids (Vanden Broeck, 2001), whereas NPF
consist of 36 amino acids in Drosophila and are sometimes referred to as long
NPF. This name is however misleading since the active peptide in some
insects are even shorter than the sNPFs (Nässel and Wegener, 2011). The
one snpf precursor gene gives rise to four sNPFs (sNPF1-4), whereas the npf
precursor gene only produces one NPF in Drosophila (Lee et al., 2006;
Mertens et al., 2002). The number of sNPF peptides varies in different insects
and it has been suggested that the multiple forms of sNPFs have been
generated by intragenic duplication, whereas NPFs are multiplied by gene
duplication (Nässel and Wegener, 2011). Even though the functions of these
peptides to some extent are similar and the names imply close relationship,
sNPF does not seem to be related to NPF nor to the vertebrate NPY (Nässel
and Wegener, 2011). The only “relationship” is the similarity in receptors
these peptides activate (discussed more detailed in next section).
The expression of the snpf gene in the CNS occurs from late stage of
the embryo and in the adult brain it is widely distributed (Lee et al., 2004). The
four different forms of sNPFs in Drosophila vary to some extent in size and
sequence. sNPF1 and sNPF2 have an RLRFamide sequence at the Cterminal, whereas sNPF3 and sNPF4 have an RLRWamide in the same
position (Nässel, 2002).
One possible function of sNPF seems to be control of feeding and thus
body size. Overexpression of the peptide results in bigger and heavier flies
due to increased food consumption, whereas knockdown of the snpf gene
produces smaller flies (Lee et al., 2004) and is believed to be caused by
interaction with insulin signalling (Lee et al., 2009; Lee et al., 2008). Mass
spectrometry and HPLC has shown presence of sNPFs in the hemolymph of
Drosophila (Garczynski et al., 2006), which might suggest a possible
neuroendocrine role. This is further supported by immunocytochemistry and in
situ hybridization, showing localization in the neurohemal organs but not in the
gut (Lee and Park, 2004). The peptide is expressed in a subset of the clock
neurons, the LNd cells (Johard et al., 2009), indicating a role in the circadian
rhythm. In Locusta migratoria, sNPF stimulates ovarian development
(Cerstiaens et al., 1999) and might therefore have a role in reproduction in
these animals. Since the peptide is so abundant in the brain, one would
suspect many different functional roles, but these are still far from understood.
1.2.3.1. Short neuropeptide receptor
As mentioned earlier, the mammalian NPY and its invertebrate homolog NPF
are not related to sNPF, but the receptors of these peptides seem more
related (Feng et al., 2003; Mertens et al., 2002; Reale et al., 2004). The
mammalian NPY receptor, Y2, and the Drosophila sNPF receptor, sNPFR,
show 33% identity and 49% homology (Mertens et al., 2002). As all
neuropeptide receptors, the sNPFR is a G-protein-coupled receptor (Mertens
12
et al., 2002). The only known ligands to sNPFR are sNPFs, since they are the
only of tested peptides (including FMRFamides and other peptides with an
RFamide C-terminus) that can activate the receptor when expressed in
Xenopus oocytes or mammalian Chinese hamster ovary cells (Feng et al.,
2003; Mertens et al., 2002).
sNPFR transcript has been identified in CNS of embryos with in situ
hybridization and in adult Drosophila with Northern blot (Feng et al., 2003),
but its localization in the periphery is not at all established. RT-PCR have
revealed presence of the receptor in the brain, gut, fat body, and Malpighian
tubules of larvae and in whole bodies and ovaries of adults (Mertens et al.,
2002). The sNPFR can activate inwardly rectifying K+ channels (GIRKs)
(Reale et al., 2004) and Ca2+-dependent inward chloride currents (Mertens et
al., 2002) when expressed in Xenopus oocytes. The G-proteins Gi and G0 are
sensitive to pertussis toxin, and applying this to the oocytes decreases the
activation of GIRK currents, which might indicate that Gi or G0 are involved in
this process (Reale et al., 2004). Taken together, these results suggest that
activation of the receptor is controlling neuronal excitability or induce
hyperpolarization of the cell. This does not exclude, however, that sNPFR is
activating other systems or function differently in vivo.
1.2.4. Insulin, insulin-like peptides and insulin receptor
Insulin is the major hormone that regulates carbohydrate homeostasis, control
fat and cellular uptake of amino acids. In humans, insulin is produced by βcells of the pancreas and released into the circulation as a response to high
sugar levels. Circulating insulin initiates uptake of glucose from the blood to
store it as glycogen in the muscle and liver. The insulin propeptide includes a
carboxyl A-chain and a terminal B-chain that is connected by a C-chain. The
C-chain is cleaved off in the Golgi apparatus to produce the mature and
functional insulin peptide. The presumed function of the C-chain is not
completely established. The insulin receptor is a tyrosine-kinase and upon
binding of insulin, the tyrosine kinase domains phosphorylate, which triggers a
signalling cascade (Squire, 2003).
Drosophila is a satisfactory model for insulin signalling because the
pathway is well conserved between invertebrates and vertebrates. A
simplified comparison of the insulin-signalling pathway in three different
animals (C.elegans, Drosophila and mammals) is seen in Figure 4. Drosophila
insulin-like peptides (DILPs), like mammalian insulin, regulate circulating
sugar (glucose and trehalose) levels and store excess energy as glycogen
and lipids [reviewed in (Teleman, 2010)]. Adipokinetic hormone, AKH, plays a
role similar to mammalian glucagon and act as an antagonistic peptide to
insulin-like peptide (Kim and Rulifson, 2004). Glucagon and AKH elevate
circulating blood sugar and they function together with insulin to maintain
glucose homeostasis.
There are seven Insulin-like peptides in Drosophila, DILP1-7, but only
one known receptor, dInR (Brogiolo et al., 2001). DILP1-5 are encoded on the
3rd chromosome and DILP6-7 on the X-chromosome, but all in different loci
with their own promoter regions. All DILPs, except DILP6, have similar
structure (Grönke et al., 2010; Teleman, 2010; Teleman et al., 2008), yet
presumably diverse functions based on their dissimilar distribution patterns.
13
DILP 2, 3 and 5 are found in a specific cluster of the median neurosecretory
cells, the insulin producing cells, IPCs. The IPCs send axons to the larval ring
gland and aorta and adult corpora cardiaca and aorta where insulin is
released into the hemolymph. DILP 2 is also expressed in the imaginal discs
and salivary glands. DILP 4 and 5 are expressed in the midgut (Brogiolo et al.,
2001; Broughton et al., 2005; Ikeya et al., 2002). DILP 6 is produced in fat
bodies (Slaidina et al., 2009) and show sequence similarities to mammalian
insulin-like growth factors, IGFs. DILP 7 is expressed in ten neurons in the
abdominal ganglia, both during larval stages and in the adult fly (Brogiolo et
al., 2001; Leevers, 2001; Miguel-Aliaga et al., 2008). One pair of DILP 7expressing neurons in the ventral nerve cord sends axons that terminate on
the MNCs in the protocerebrum in larvae (Miguel-Aliaga et al., 2008). Relaxin
is an important reproductive hormone in mammals and DILP 7 is the assumed
ortholog of relaxin due to its effect on female reproduction (Yang et al., 2008).
DILP 1 has not yet been detected in tissues.
Mammals
C. elegans
Insulin
InR
Drosophila
Insulin
DILPs
daf-2
dInR
IRS1-4
chico
PI3K
PTEN
age-1
Daf-18
dPI3K
dPTEN
PDK1
PDK1
dPDK1
Akt
Akt
dAkt
FOXO
mTOR
p27Kip1
4EBP
Growth
Daf-16
dFOXO
dTOR
d4EBP
Lifespan
Growth
Figure 4. The insulin-signalling pathway is well conserved among different animals. Picture
modified from (Puig et al., 2003).
Overexpression of any of the DILPs during development results in
increased body size, whereas the same experiment in adult causes
trehalosemia, but without altered body size (Ikeya et al., 2002). Growth is
limited to the larval stages in Drosophila, since adult flies do not increase in
size (Teleman, 2010). This means that genetic manipulation of insulin
pathway components in adults only alter metabolism and stress responses,
whereas also growth is affected in larvae. Deletion of the IPCs leads to
decreased larval growth and also an altered lifespan is observed in adult flies;
these flies live longer and are more resistant to metabolic stress. Increased
14
glucose levels in the circulation and more fat accumulation are also observed
(Broughton et al., 2005; Ikeya et al., 2002). Upon larval fasting, DILP 2, 3 and
5 levels decrease (Ikeya et al., 2002; Teleman et al., 2008), but expression of
DILP 6 and 7 is upregulated (Teleman et al., 2008). Knocking down only DILP
2 in IPCs produces flies that have higher trehalose levels, but lack the other
changes in phenotypes, presumably due to a compensatory effect of the other
DILPs (Broughton et al., 2008).
The Drosophila insulin receptor (dInR) is, like the mammalian InR, a
receptor tyrosine kinase and shows homology with insulin receptors in other
organisms. In fact, human insulin can activate the dInR with almost the same
affinity (Nasonkin et al., 2002).
2. AIMS OF THE THESIS
Little is known about GABA- and sNPF signalling and functions in Drosophila.
The aim of this thesis is to learn more about sNPF and GABA signalling and
to unravel some of their functional roles. Our main focus has been the role of
sNPF and GABA in feeding circuits and in the olfactory system, including the
mushroom bodies. We first decided to investigate the localizations of these
transmitters and their other signaling components in the brain. For this we
raised some new antisera and tested out various Gal4 drivers. The next step
was to perform experimental work on sNPF and GABAB signalling by applying
Gal4-UAS technology to cell specifically interfere with the peptide and
receptor expression and then test behavior and physiology in various assays.
PAPER I
GABA is the major inhibitory transmitter in vertebrates as well as in
invertebrates. The ionotropic receptor of GABA, GABAAR, used for fast,
inhibitory action of GABA, has been extensively studied in insects. Little is
however known about the metabotropic GABA receptor, GABABR, used for
slow and modulatory actions of GABA. In paper I we therefore determined the
distribution of GABABRs in relation to ionotropic GABA receptors and other
markers for GABA-signalling in larval and adult Drosophila brains. This would
give us an idea what circuits are involving GABA signalling via the GABABRs
and facilitate future behavioral and physiological experiments.
PAPER II
Insulin-like peptides are important hormones regulating several physiological
processes in an animal. Many studies regarding insulin have been carried out
with main focus of the effects downstream of the insulin receptor. However,
little is known about what regulates production and release of insulin. In
mammals, insulin release depends on glucose/ATP via glucose transporters
(GLUTs) (Zierler, 1999), whereas functional GLUTs or nutrient sensors have
been less investigated in invertebrates. What are the factors controlling insulin
release in Drosophila? We found that the GABABR is localized on the insulin
producing cells, IPCs, and tested whether GABA might be one of the factors
involved in the regulation of insulin. Previous studies have shown that insulin
has roles in lifespan, growth, stress resistance, regulation of lipid and
carbohydrate levels (Rulifson et al., 2002), so we tested these parameters
after knocking down the GABABR in the IPCs.
15
PAPER III and IV
The role of sNPF in feeding and growth has been established (Lee et al.,
2008; Lee et al., 2004). To investigate the presence of sNPF signalling in
feeding circuits and other relevant brain regions we mapped the distribution of
sNPF and its receptor in the CNS and in the intestine of Drosophila in relation
to different neuronal markers. In paper III we determined the localization of
the mRNA (snpf) and the peptide precursor (sNPF) and asked whether the
peptide has a global function or if its functions are dependent on the neurons
releasing the peptide (i.e. multiple distributed functions). Paper IV focuses on
the sNPF receptor in order to find out action sites for the peptide. Are the
receptors postsynaptic on neurons involved in feeding?
3. METHODS
3.1 The Gal4/UAS system and RNA interference
In all four studies of this thesis we have used immunocytochemical methods
combined with the Gal4-UAS technique [developed by (Brand and Perrimon,
1993)]. This technique is based on two transgenic fly strains. One expresses
Gal4, a yeast transcription factor (from Saccharomyces cerevisiae) that is not
normally found in Drosophila. The gal4 gene is coupled to an endogenous
Drosophila promoter of interest and is only expressed in those cells where the
promoter usually is activated. The other fly stain contains a yeast-specific
upstream activating sequence (UAS) coupled to a Drosophila gene of interest.
When the two transgenic fly strains are crossed, Gal4 binds to UAS in the
progeny and induce transcription of the gene it controls in specified cells only
(Fig. 4).
P
X
GAL4
UAS
Gene A
A
F1
Gene A
UAS
Figure 5. The UAS-Gal4 technique involves two parental transgenic fly stains (P), one
bearing the yeast transcription activator protein Gal4 and the other the promoter region UAS
(Upstream Activation Sequence) to which Gal4 bind to activate transcription. The parental
flies are not affected, however, the progeny (F1) having both UAS and Gal4 will express the
gene of interest (Gene A).
16
We used this technique for two purposes. One was to drive expression
of green fluorescent protein (GFP) in specific neurons of interest, often in
combination with different antibodies. We also used this technique to cellspecifically downregulate the expression of different gene transcripts by RNA
interference (RNAi). RNAi involves a double stranded RNA (dsRNA) with a
complementary sequence to the target mRNA. The enzyme DICER cleaves
the dsRNA into 20-25 nucleotide small interfering RNA (siRNA) that
assembles to large complexes (RNA-induced silencing complexes, RISCs).
The siRNA then guide the RISCs to the complementary, target mRNA and the
catalytic component of the complex cleaves the target mRNA and prevents
translation (Fire et al., 1998).
3.2. Fly stocks
All transgenic strains of Drosophila melanogaster used in this thesis are listed
in Table 1 and 2. In addition to the Gal4 and UAS strains, we used Oregon R
and W1118 as wild type flies in all four studies. Some of the Gal4 and UAS
strains were used in more than one study, whereas others were used in one
only.
OK107-Gal4 was used in all four studies. It is expressed in the Kenyon
cells of the mushroom bodies as well as in median neurosecretory cells
(MNC) in the pars intercerebralis, including the insulin producing cells, IPCs.
Except to localize the mushroom body neurons and MNCs, this strain was
used in paper II to knock down the GABABR in these cells with UAS-GABABRRNAi. UAS-GABABR-RNAi was also crossed with MB247-Gal4 that is
expressed specifically in the intrinsic neurons of the mushroom body.
GH146-Gal4 and GH298-Gal4 were used in paper I to identify
projection neurons and local interneurons of the antennal lobes, respectively.
OK6-Gal4 for motorneurons and 21D-Gal4 for L2 type monopolar cells of the
lamina were also used in paper I. Glutamic acid decarboxylase 1 (GAD1) is
the biosynthetic enzyme for GABA, and thus a gad1-Gal4 strain was used in
paper I, II and III to identify putative GABA-producing neurons.
In paper III we used many Gal4 lines as markers; Cha-Gal4 (choline
acetyltransferase) for cholinergic extrinsic neurons, th-Gal4 (tyrosinehydroxylase) for dopamine, tdc-Gal4 (tyrosine decarboxylase) for octopamine
and tyramine producing neurons, OK6-Gal4 for motorneurons, OK371-Gal4
(vesicular glutamate transporter) for glutamatergic neurons, npf-Gal4 for
neurons expressing long neuropeptide F and snpf-Gal4 for short neuropeptide
F. The latter was also used in paper IV. C929-Gal4 was used in paper III and
IV to visualize large peptidergic neurons, since it is expressed in DIMMpositive cells. DIMM is required for differentiation of large peptidergic neurons
and endocrine cells (Hewes et al., 2003).
In paper II and IV we used different markers for insulin-like peptides,
Dilp2-Gal4 and Dilp3-Gal4. Dilp7-Gal4 in paper IV showed ten large cells in
the ventral ganglia of the larvae. For overexpression of Dilp2, we used UASDilp2 in paper III. Rdl-Gal4, UAS-Rdl-RNAi, GABABR-Gal4 and UASGABABR-RNAi for different types of GABA receptors were used in paper II.
A new snfpr-Gal4 (unpublished) was tested in paper IV, but the
expression pattern could not be confirmed when used together with sNPFR
antisera.
17
To visualize Gal4-expression with green fluorescent protein, the Gal4
strains in all studies were crossed with transgenic flies expressing UAS-mcd8GFP or UAS-GFP.S65t.
All flies have been fed standard Drosophila food and raised under
12h:12h light-dark conditions at 18°C or 25°C. Crosses of Gal4-UAS flies
were made at 25°C. At least 10 larval or adult fly brains were used in each
experiment.
Table 1. Gal4 strains used.
Gal4 lines
Description
21D
L2 monopolar cells in the lamina of the
optic lobe
c929
Large peptidergic neurons, endocrine
cells
Cha
Choline acetyltransferase promoterGal4-GFP fusion
Dilp2
Insulin-like peptide 2
Reference
(Gorska-Andrzejak et al.,
2005)
(Hewes et al., 2003)
Paper
I
(Salvaterra and Kitamoto,
2001)
(Wu et al., 2005)
III
III, IV
II, IV
Dilp3
Insulin-like peptide 3
(Buch et al., 2008)
II, IV
Dilp7
Insulin-like peptide 7
(Yang et al., 2008)
IV
GABABR2
Metabotropic GABA receptor
(Root et al., 2008)
II
gad1
GABAergic neurons
(Ng et al., 2002)
I, II, III
GH146
Projection neurons in the antennal lobe
(Stocker et al., 1997)
I
GH298
Local interneurons in the antennal lobe
(Stocker et al., 1997)
I
Hug
Hugin producing cells in the
subesophageal ganglion
Intrinsic neurons of the mushroom
bodies
Neuropeptide F
(Melcher and Pankratz,
2005)
(Aso et al., 2009)
IV
(Wu et al., 2003)
III
(Lee et al., 1999)
OK371
Mushroom body and median
neurosecretory cells (MNC)
Vesicular glutamate transporter
(Mahr and Aberle, 2006)
I, II, III,
IV
III
OK6
Motorneurons
(Aberle et al., 2002)
I
Or83b
Olfactory receptor neurons
(Larsson et al., 2004)
IV
rdl
GABAAR subunit
(Kolodziejczyk et al., 2008)
II
snpf
Short neuropeptide F
(Nässel et al., 2008)
III, IV
Snpfr
Short neuropeptide F receptor
Unpublished
tdc
Tyrosine decarboxylase
(Cole et al., 2005)
III
th
Tyrosine-hydroxylase
(Friggi-Grelin et al., 2003)
III
MB247
npf
OK107
18
II
Table 2. A list of UAS strains used.
UAS-lines
Description
GABABR2-RNAi Metabotropic GABA receptor
Reference
(Root et al., 2008)
Paper
II
rdl-RNAi
Ionotropic GABA receptor subunit
(Liu et al., 2009)
II
Irk3-RNAi
Inwardly rectifying K channels
(Dietzl et al., 2007)
II
Dilp2
Insulin-like peptide 2
(Wu et al., 2005)
II
gfp.S65T
Green fluorescent protein
mcd8-gfp
Green fluorescent protein
+
I, II, III,
IV
I, II, III,
IV
3.3. Antisera, immunocytochemistry and antisera characterization
All antisera used are listed in table 3 together with the fixative, dilution and
reference where they were first used and characterized.
We designed antigens and had antisera produced for several proteins
of interest. Three new antisera were thus used for the work of this thesis. In
paper I we raised two rabbit polyclonal antisera against peptide sequences of
each of the genes rdl and vgat. Anti-RDL was produced against a peptide
sequence (CLHVSDVVADDLVLLGEE) of the C-terminal of the GABAA
receptor subunit. The synthesized peptide was coupled to Limulus
hemocyanin as a carrier. Anti-vGAT was produced against a peptide
sequence (CQTARQQIPERKDYEQamide) of the N-terminal of the transporter
and coupled to keyhole limpet hemocyanin. Both were injected into two
rabbits each.
For detection of the sNPFR in paper IV, we produced four rabbit and
four guinea pig polyclonal antisera against two different peptide sequences of
the sNPFR gene. One was a peptide sequence of the N-terminal
(CILADVAASDEDRSGGIIHNQ) and the other a portion of the C-terminal
(ETDGDYLDSGDEQTVEVRC) of the receptor. The synthesized peptides were
coupled to maleimid-coupled thyroglobulin.
All three types of antisera were characterized in similar fashion.
Preimmune sera were collected prior to the first injection of the antigen. After
three booster injections, the antisera were collected. The antisera were tested
for specificity on CNS tissue by immunocytochemistry and Western blots with
antisera, preimmune sera and antisera preabsorbed with the antigen.
Preabsorbed antisera were prepared by incubation of 50 nmol/ml antigen in
diluted antisera for 24 hours at 4°C. The procedure for Western blot is
described in detail in paper I and IV.
To detect the products of the snpf gene, we tried three different
antibodies (produced by Drs J. Veenstra, Bordeaux, France). The most
frequently used was raised against 20 amino acids of the snpf precursor
protein. The other two were raised against short sequences of the C-terminals
of the peptides sNPF1 and 3 and would recognize sNPF1-2 or sNPF3-4
respectively. The antisera produced to sNPF1 are likely to cross-react also
with other RFamides.
To amplify the GFP signal we applied a mouse monoclonal GFPantibody or a rat monoclonal anti-cd8 in some specimens. Bouin´s fixative
19
weakens the GFP signal drastically and anti-GFP is not compatible with
Bouin´s. Therefore, anti-cd8 proved to be a better choice in cases where
Bouin´s fixation was required.
The procedure for immunocytochemistry is described in detail in
material and methods in the papers, but briefly brains of 3rd instar larvae were
fixed on ice for 2 hours in 4% paraformaldehyde (PFA) in 0,1M phosphate
buffer (PB; pH 7.4) or in Bouin´s fixative for 30 minutes on ice. Adult flies were
decapitated in PBS-TX and fixated for 4 hours in 4% PFA or 1 hour in Bouin´s
fixative on ice. Larval brains were used as whole mounts, whereas adult
brains were sectioned on a cryostat or used as whole mounts. The tissues
were incubated in primary antiserum 12-72 hours (depending on the
antiserum used) in 4°C, washed in PBS-TX (phosphate-buffered saline
containing Triton X) and incubated with secondary antiserum conjugated to a
florophore for 2 hours in room temperature or over night at 4°C. After washing
in PBS-TX and PBS, the tissues were mounted in 80% glycerol in PBS.
Specimens were imaged using a Zeiss LSM 510 microscope or a motorized
Zeiss Axioplan 2 microscope with a CCD-camera. The images were
processed with Zeiss LSM software and edited in Adobe Photoshop 7.0 or
later version.
Table 3. Primary antisera used for immunocytochemistry. R, rabbit. GP, guinea pig. M,
mouse.
Antibody Host Dilution Fixative Recognize part of
Reference
GABABR
R
1:16000 PFA
C-terminus
(Hamasaka et al., 2005)
RDL
R
1:40000
Bouin´s
GABAAR subunit
This thesis (Paper I)
vGAT
R
1:1000
PFA
This thesis (Paper I)
GAD1
R
GABA
R
1:5001000
1:1000
PFA or
Bouin´s
PFA
Vesicular GABA
transporter
Glutamic acid
decarboxylase
γ-aminobutyric acid
sNPFp
R
1:4000
PFA
sNPF precursor
This thesis (Paper III)
sNPFR
R/GP
1:40000
Bouin´s
N-terminus
This thesis (Paper IV)
sNPFR
R/GP
1:40000
Bouin´s
T-terminus
This thesis (Paper IV)
DILP2
R
1:1000
PFA
A-chain
(Cao and Brown, 2001)
DILP7
R
1:5000
PFA
B-chain
(Miguel-Aliaga et al., 2008)
Fas
M
1:75
PFA
Fasciclin-2
(Hummel et al., 2000)
CRZ
R
1:2000
PFA
Corazonin
(Cantera et al., 1994)
GFP
M
1:1000
PFA
Invitrogen
CD8
Rat
1:100
Bouin´s
Green fluorescent
protein
Membrane protein
20
(Featherstone et al., 2000)
(Wilson and Laurent, 2005)
Invitrogen
3.4. In situ hybridization
For distribution pattern of snpf transcript we performed in situ hybridization
with a digoxigenin (Dig)-labeled snpf riboprobe on whole mount larval or adult
brains. Probe production and in situ protocol is described detailed in paper III.
Briefly, heads were fixed in 4% PFA and made permeable with proteinase K.
After another postfixation in 4% PFA and prehybridization in hybridization
buffer, the brains were incubated with the probes for 48 hours at 55°C. The
probe was either hydrolyzed or used intact. The brain tissues were incubated
in alkaline phosphatase (AP) tagged anti-Dig antiserum for 2 hours at room
temperature. AP histochemistry was carried out in NBT/BCIP solution for
about an hour, whereafter the tissues were mounted in 80% glycerol.
3.5. Quantification of immunofluorescence
To determine the difference in insulin levels between starved and fed flies and
between control flies and flies lacking the GABABR on insulin producing cells
(paper II), we measured the intensity of immunofluorescence in these cells.
This was performed in the confocal microscope with the same settings and
exposure time for all specimens. The images were analysed and the intensity
of immunofluorescence was quantified in each cell with ImageJ. 20-30 cells
were analyzed for each genotype.
3.6. Survival assays
We performed three different survival assays; lifespan under normal feeding
conditions and during starvation or desiccation.
To measure longevity under normal feeding conditions, male flies (4-6
days old) of different genotypes were counted each day for survival and
transferred to new bottles with fresh food. Two replicates with at least 80 flies
of each genotype per replicate were counted.
Survival during starvation was measured by placing individual flies in
small bottles containing 0,5% aqueous agarose, making water accessible but
not food. Dead flies were counted every 12 hours until all flies were dead.
Three replicates with at least 40 flies per genotype in each replicate were
counted. Male flies, 4-6 days old, were used.
Survival during desiccation was carried out similar to the starvation
assay, with the difference that the bottles were empty with no access to either
food or water and the flies were counted each hour.
The flies were kept in a 25°C incubator with 12:12 light:dark conditions
and controlled humidity.
3.7. Measurements of trehalose, lipids and growth
The trehalose levels were measured in normally fed flies as well as after 5 or
12 hours of starvation in different genotypes according to (Isabel et al., 2005).
4-8 days old male flies were kept in tubes with different food conditions and
their wet weight was measured. The flies were then incubated in ethanol and
sonicated. Yellow anthrone that turns green when it binds to sugar was
added, and the color intensity was measured in an ELISA plate reader against
a standard curve.
21
Levels of lipids were measured in whole bodies of different genotypes
after 0, 12 and 24 hours of starvation. Wet weight of male flies was
determined and thereafter dry weight, by placing the flies in 65ºC for 24 hours.
Lipids were then extracted in dry flies by diethyl ether. When the ether was
removed and the flies had been exposed to another drying, the lean dry
weight was obtained. The total lipid content was considered as the difference
between dry weight and lean dry weight.
To investigate growth rate, we weighed either third instar larvae or 3-6
days old male or female flies. The length of the pupae was measured in a
Zeiss Axioplan 2 microscope with a CCD-camera and analyzed in AxioVision
4.6.3.0.
4. RESULTS AND DISCUSSION
4.1. PAPER I
We mapped the distribution of the metabotropic GABA receptor (GABABR) in
Drosophila larvae and adults and analyzed the relations to known structures
in the brain, to the ionotropic GABA receptor (GABAAR) and other markers for
GABA signaling. GABA and both types of GABA receptors are abundant in
the entire CNS of Drosophila and are present in the antennal lobe, mushroom
bodies, parts of the central body complex and optic lobes. The GABAAR
subunit RDL and GABABR2 have similar distribution in the Drosophila brain,
but yet distinct patterns of the two can be observed in many brain regions.
We used an antiserum against the GABABR2 subunit, but this
immunolabeling probably reflects the distribution of functional metabotropic
GABA receptors since the two subunits (R1 and R2) have to dimerize to
function (Kaupmann et al., 1997; Pin et al., 2003). Unfortunately, antiGABABR2 only labeled neuronal fibers and commonly not cell bodies, making
it difficult to directly identify specific neurons. However, double labeling with
GABABR2-Gal4 driven GFP together with anti-GABABR2 revealed overlapping
distribution patterns and we thus have a reliable marker for both GABABR
positive cell bodies and fibers.
Vesicular GABA transporters are supposed to be located in the
synaptic terminations of GABAergic neurons and staining with anti-vGAT
probably corresponds to these sites. The antiserum to the GABAAR subunit
RDL probably labels most GABAAR action sites. This antiserum also did not
stain cell bodies, but labelled fiber branches that are most likely to be on the
postsynaptic side of the neuron. The GABABR can be both pre- and/or
postsynaptic, and that is reflected in the immunolabeling with anti-GABABR2.
In the antennal lobe, we found GABAergic local interneurons expressing
GABABR but not RDL. This could suggest that the metabotropic receptors are
both pre- and postsynaptic and probably function as autoreceptors at these
locations. Another location where GABABR-IR fibers are presynaptic is in cells
of the lamina in the optic lobe. These are most likely the GABAergic
centrifugal C2 neurons, and thus show presynaptic GABAB receptors also in
this region. Another study (Kolodziejczyk et al., 2008) has shown GABABR2
immunoreactivity (IR) in C2 and C3 neurons (both GABAergic), but not the
GABAAR subunit RDL. A later study revealed that the olfactory sensory
neurons (OSNs) express the GABABR (Root et al., 2011). This presynaptic
22
GABABR was shown to modulate sensitivity in OSN to specific odors in a gain
control mechanism.
Other regions where the two receptor types differed were in the
mushroom bodies, the ellipsoid body in the central body complex and in some
of the clock neurons. In the peduncles and lobes of the mushroom body we
only observed RDL-IR and not GABABR-IR. We found RDL and vGAT
immunostaining in the entire ellipsoid body, whereas anti-GABABR2 only
stained the outer layer. The protocerebral bridge and the fan-shaped body
were weakly stained with all three antibodies. We found GABABR
immunoreactivity in a subset of clock neurons where there where was no RDL
staining. These results agree with a previous study (Hamasaka et al., 2005)
that showed that GABA application decreased intracellular calcium levels in
one group of clock neurons. Furthermore, this response was blocked by the
GABAB-R antagonist CGP54626 and not by the GABAA-R antagonist
picrotoxin.
4.2. PAPER II
Insulin and insulin-like peptides (ILPs) are important hormones that are
involved in many physiological processes. The ILP signaling pathway is well
conserved among mammals and invertebrates, and consequently makes
Drosophila a good model organism to understand functions and signalling
mechanisms. Little is actually known about regulation of the production and
release of insulin-like peptides in insects. Mammalian insulin is released when
glucose enter the β-cells in the islets of Langerhans in the pancreas via
glucose transporters (GLUT2), but such transporters have not been
characterized in insects. When investigating GABA signalling with
immunocytochemistry in combination with UAS-GAL4, we found that the
metabotropic GABABRs are expressed in IPCs in the brain, whereas the
ionotropic GABAARs (RDL) are not. This made us suspect that GABA is
somehow affecting production and/or release of Drosophila insulin-like
peptides (DILPs) via GABABRs. It was clear that most, possibly all, of the
IPCs express GABABRs. In close vicinity to the IPCs are some other neurons
containing GABABRs, but not DILPs. These cells are most likely other median
neurosecretory cells (MNCs). We also tried to reveal the identity of
GABAergic inputs on IPCs. This proved to be difficult because of the
numerous neurons containing GABA, making tracing of individual neurons
almost impossible. However, both GABA and GAD1 immunoreactivity
displayed branching in close association with the IPCs. We show that none of
the IPCs are GABAergic and therefore the GABABRs located in these sites
are postsynaptic.
The mechanism behind the regulation of IPCs by GABA was further
investigated by knocking down the expression of GABABRs in the IPCs by
RNAi. Since GABA is inhibitory, we hypothesized that knocking down the
GABA receptor would lead to increased production and/or release of DILPs
and in consequence the flies would display phenotypes of increased DILP
signalling. Insulin has reported effects on lifespan, stress resistance and
growth (Broughton et al., 2005; Ikeya et al., 2002), so we tested these
parameters in flies with diminished GABABR expression on the IPCs. Indeed
lifespan and stress resistance were affected in the same manner as when
23
insulin is overexpressed. Normally fed Dilp2-Gal4 x UAS-GABABR-RNAi flies
had a significantly reduced lifespan compared to controls, although the effect
was not so drastic. When the same flies where tested in stress resistance
assays (starvation and desiccation), these flies showed decreased resistance
compared to the controls. Overexpression of DILP2 in IPCs produced the
same phenotype, which would further support the idea that GABA is inhibiting
insulin release or production. Also one Dilp3-Gal4 (for insulin-like peptide 3)
and one OK107-Gal4 strain that include the mushroom bodies as well as most
IPCs crossed with the same GABABR-RNAi gave the same phenotype:
decreased survival at metabolic stress conditions. To make sure that the
mushroom body is not involved in this process, we used a mushroom bodyspecific GAL4 line (MB247-Gal4) crossed with GABABR-RNAi. These flies did
not display significantly altered survival at metabolic stress compared to their
controls, and thus confirming that mushroom bodies are not involved.
We have found that GABA regulates insulin via its metabotropic
GABABR, but not via the ionotropic GABAAR. The GABAAR subunit RDL-Gal4
expression was found in the neighboring cells, but not on the IPCs. This result
was further supported by the lack of effect of starvation and desiccation on
survival when knocking down RDL in IPCs.
Irk3 is a putative subunit of a G-protein-coupled inwardly rectifying K+
channel, GIRK, likely to be downstream of the GABABR (Kaupmann et al.,
1998). Knocking down this subunit in IPCs decreased the survival at
starvation, the same phenotype as when knocking down GABABRs.
We also measured relative DILP fluorescence in starved and fed flies
lacking the GABABR on the IPCs and compared them to controls. The results
indicate that DILP storage is increased and release is decreased in starved
control flies as shown previously (Geminard et al., 2009), as DILP
immunoreactivity is more intense than in fed control flies. Fed transgenic flies
displayed even higher immunofluorescence compared to controls, indicating
increased DILP production or reduced release. The highest relative
immunofluorescence was seen in starved flies with diminished GABABR on
IPCs. Taken together, these results indicate that GABA via GABABRs inhibits
DILP signalling from IPCs. The fact that even fed transgenic flies had higher
immunofluorescence than starved controls suggests that GABA is affecting
both production and release. More release of DILPs is perhaps compensated
by more production. Another study (Broughton et al., 2010) has shown that
restricted diet can affect the different DILPs in various ways, one can be
upregulated whereas another is downregulated. This variability might be
masked in our assay, since the antiserum against DILP 2 used is most likely
to crossreact also with DILP 3 and 5, and the immunofluorescence we see
could be a total level of all three DILPs.
Since DILPs are known to regulate growth during development
(Broughton et al., 2005; Ikeya et al., 2002), we determined weights of third
instar larvae and length of pupae and adult flies lacking GABABR on IPCs. We
could not see an effect on either weight or length. This might support the idea
that GABA is released as a response to stress and is not affecting insulin
signalling under normal conditions (also noted in weak affect on healthy
lifespan).
GABA decreases insulin release via both GABAA and GABABRs in the
pancreas in mammals in response to glucose (Dong et al., 2006). The
24
Drosophila fat body has nutrient sensors, but the IPCs in the brain do not
seem to. Thus many questions remain. How do GABAergic neurons sense
nutrients? What is the identity of GABAergic neurons with outputs on IPCs?
We know from immunocytochemistry that the IPCs are not GABAergic and
thus we know that GABA is released from other neurons and that the
receptors are postsynaptic to these cells.
4.3. PAPER III
We mapped the distribution of sNPF (both expression of mRNA with in situ
hybridization
and
the
peptide
precursor
and
peptides
with
immunocytochemistry) in larvae and adult Drosophila and found expression in
a large number of diverse neurons. The widespread distribution of sNPF in a
considerable variety of interneurons as well as in olfactory sensory neurons
(OSNs) of the antennae indicates multiple roles as neuromodulator and
probably cotransmitters.
First we investigated whether the peptides are likely to be released into
the circulation and act as hormonal peptides. In order to do so we looked for
Large cells that Episodically release Amidated Peptides, LEAP neurons, by
using a c929-Gal4 line as a marker. These large cells express the
transcription factor DIMM together with PHM which is an enzyme required to
α-amidate neuropeptides (Park et al., 2008). We found no coexpression of
c929-Gal4 driven GFP and anti-sNPF precursor in the larva, indicating that
sNPF is not released as a hormone but instead signals locally and act as
cotransmitter or neuromodulator. We did however find sNPF immunoreactivity
in three pairs of large cells (LNCs) that also contained corazonin that have
axons terminating in the anterior aorta and near the AKH-producing cells in
the ring gland. We found punctuate sNPF immunoreactivity close to the AKHcells, which could suggest that sNPF regulates AKH production, but we
cannot eliminate the possibility that they are released into the circulation. The
same sNPF/corazonin-immunolabelled cells arborize close to insulin
producing MNCs. Thus it remains open whether these LNCs regulate AKH or
DILP producing neurons (or none).
We also investigated whether sNPFs may be released as
cotransmitters with some of the classical neurotransmitters, since this is often
the case for small peptides in mammals. We screened for colocalization with
GABA, glutamate, acetylcholine, dopamine and octopamine/tyrosine and
found sNPF to be present in some neurons containing GABA, glutamate and
acetylcholine. However, most of the sNPF-IR neurons did not contain any of
the tested classical transmitters.
In the antennae we found sNPF-IR in a set of olfactory sensory
neurons (OSNs) and their terminations in the antennal lobe. In the OSNs
sNPF is colocalized with choline acetyltransferase, Cha-Gal4, that is the
enzyme for synthesis of acetylcholine and is an indicator for cholinergic
neurons. The projection neurons of the antennal lobe did not express sNPF,
but Cha. A description of the detailed expression of sNPF in OSNs has been
carried out (Carlsson et al., 2010). In this study, sNPF was found to be
expressed in a subpopulation of OSNs in both the antennae and maxillary
palps and their axons were detected in thirteen glomeruli of 50.
25
One pair of dorsal median (DP) neurons in the first abdominal
neuromere expressed both Cha and DILP7 together with sNPF. These
neurons have branches in the vicinity of the DILP producing MNC dendrites in
the pars intercerebralis.
In a previous study we found snpf and sNPF to be expressed in the
majority of the mushroom body intrinsic Kenyon cells both in larvae and adults
(Johard et al., 2008). The intrinsic neurons of the mushroom bodies are part
of the central circuitry for olfactory learning and memory and we investigated
whether sNPF was expressed together with any other transmitters in these
cells. However, none of the tested transmitters labeled the Kenyon cells.
4.4. PAPER IV
Short neuropeptide F seems to be involved in feeding, growth and regulation
of insulin-like peptides, but other functions and sites of actions are not entirely
established. In order to understand more about sNPF signalling, the sNPF
receptor (sNPFR) needs to be mapped. The distribution of the receptor has
not been revealed, except in Drosophila embryos using in situ hybridization
(Feng et al., 2003). We therefore produced antisera against sNPFR, mapped
the distribution in larvae and compared to its peptide ligands and other known
markers, especially markers associated with feeding. The receptor was found
in roughly 100 cells in the larval brain and ventral nerve cord. These cells do
not produce sNPF, but branches of sNPF cells seem to contact fibers of
sNPFR expressing neurons. It has been claimed that sNPFR is located on the
IPCs (Lee et al., 2008), but our antisera did not label these cells. Instead, we
found sNPFR immunoreactivity in three neighbouring MNCs in each
hemisphere. These cells are included in OK107-Gal4 and c929-Gal4 and may
produce diuretic hormone, DH31 and/or DH44 (see Park et al., 2008). Further
sNPFR immunoreactivity was seen in AKH-producing cells in the ring gland
and in 8-10 cells in the intestine.
We found fibers of both sNPF and sNPFR expressing neurons in the
glomeruli of the larval antennal lobes. sNPF-IR in neuronal processes in
glomeruli colocalized with Or83b-Gal4 driven GFP and are therefore likely to
derive from OSNs similar to adult flies (Carlsson et al., 2010). There was no
colocalization of Or83b-Gal4 and anti-sNPFR, but the antiserum labelled
processes in the larval antennal lobe (LAL). This suggests that the sNPFR is
not presynaptic in the larval OSNs, as it is in adult OSNs (Root et al., 2011).
These authors also showed that sNPFR is upregulated presynaptically on the
OSNs upon starvation and insulin proved to be the regulating factor. When
the flies are fed and the level of insulin-like peptides in the circulation is high,
the sNPFR is downregulated in OSNs. Upon starvation, and lower levels of
circulating insulin-like peptides, there is an upregulation of sNPFRs (Root et
al., 2011). If this is a generally occurring phenomenon in Drosophila, perhaps
the levels of sNPFR in OSNs of the larvae are quite low. The larvae hatch in
the food and they are continuous feeders submerged in food. This would
result in continuously high insulin levels in feeding larvae and perhaps as a
consequence lower levels of sNPFRs in OSNs (and maybe elsewhere in the
brain). It could also be that larvae do not express sNPFR in the same pattern
as adults do. Perhaps sNPF is not as important in feeding behaviours in
larvae as it is in adults. That would for example explain why the Hugin
26
producing cells (Bader et al., 2007; Meng et al., 2002) express sNPF in
adults, but not in larvae.
We had substantial difficulties with double labeling with receptor
antiserum and Gal4 driven GFP. Bouin´s fixative was required to obtain
labelling with the antisera to sNPFR and this harsh treatment repressed the
GFP signal. One way to get around weak GFP signal is to use anti-GFP, but
this antiserum required PFA as fixative and is not compatible with Bouin´s.
When we finally thought about using anti-cd8 to increase the green signal in
our specimens, the different sNPFR antisera had already passed their
expiration date and did not function any longer. We also tried to apply the
antisera to adult specimens, but the staining was not satisfactory. In
cryosections or paraffin sections sometimes a few cells labelled weakly, but
no staining was observed in the majority of the specimens. Whole mount
specimens did not work at all for adults.
We also tested a new sNPFR promoter Gal4 line to visualize sNPFR
expressing neurons (kindly provided by Dr K. Yu, Korea Research Institute of
Bioscience and Biotechnology, Korea). However, even after considerable
efforts, we could not confirm that this Gal4 line was accurate in visualizing the
sNPFR. The expression pattern of the antiserum against sNPFR and the Gal4
line did not match at all and therefore we did not use the Gal4 line in paper IV.
The antiserum seemed to be reliable, since we designed two sequences
specific for the sNPFR N-terminal and C-terminal respectively and injected
into four rabbits and four guinea pigs. All antisera marked more or less the
same cells and Western blot revealed a band at predicted size that was not
visible when applying preimmune sera or antisera preincubated with the
peptide used for immunization.
5. GENERAL DISCUSSION
We wanted to learn more about sNPF and GABA signalling in the CNS of
Drosophila and to unravel some of the functional roles of these molecules.
This study has mainly focused on the role of sNPF and GABA in feeding and
the olfactory system, including the mushroom bodies. First we mapped the
distribution of these two transmitters and their receptors and compared their
localization with other signalling components in the brain. For this we raised
some new antisera and tried out various Gal4 drivers.
Regulated food intake is essential for survival and healthy life. For food
search external signals, such as taste and olfaction, are needed. Internal
signals are required to maintain homeostasis to initiate hunger and to signal
satiety. Both sNPF and GABA seem to have roles in feeding and olfaction and
presumably also in olfactory memory and learning. We found the GABABR to
be localized on the IPCs in the pars intercerebralis and interfered with GABA
receptors specifically in these cells. Our results indicate that GABA regulates
production and/or release of DILPs via GABABR. The sNPFs have also been
suggested regulators of DILP production in Drosophila (Lee et al., 2008),
since overexpression of sNPFRs in the IPCs resulted in 10% heavier flies,
and knocking down sNPFR produced smaller flies. Knocking down sNPFs in
sensory neurons downregulated Akt in the fat body, dFOXO was
translocalized to the nucleus, and the translational inhibitor 4E-BP was
upregulated. All of these substrates are known to be signals downstream of
27
the insulin receptor. Furthermore, sNPF knockdown reduced cell size,
elevated circulating glucose levels and extended lifespan in adult flies (Lee et
al., 2008), indicating that DILP signalling is reduced in these flies. Taken
together, these data suggest that both sNPFR and GABABR are expressed on
the adult IPCs. Earlier serotonin has been implicated in DILP regulation
(Kaplan et al., 2008). It is not surprising that several factors are involved in the
regulation of the DILPs since they are very important hormones. Interestingly,
insulin has been shown to regulate the sNPFR levels on a subset of OSNs
(Root et al., 2011).
It was stated that sNPFR immunoreactivity (IR) is displayed on the
IPCs also in the larval brain (Lee et al., 2008). However, the images are
somewhat ambiguous and the sNPFR-IR neurons could just as well be cells
adjacent to the IPCs. We did not observe sNPFR-IR on the IPCs in the larval
MNCs, but sNPFR-IR neurons were localized nearby IPCs in the same
cluster. We suggest that these cells coexpress Diuretic hormone 31 or 44
(DH31 or DH44) or possibly myosuppressin (Park et al., 2008). It is possible
that sNPFR is not expressed on the IPCs in larval brains, but the expression
pattern might change during metamorphosis and be expressed on IPCs in
adults. The GABAAR subunit, RDL, was also not detected in the IPCs, but in
adjacent neurons in the same cluster and in close vicinity. Thus, we suggest
that GABAAR and sNPFR are expressed in the same neurosecretory cells in
the larva (but this needs to be confirmed).
We have not been able to determine the identity of the neurons
containing GABA and sNPF that innervate the IPCs. Both transmitters are
very abundant in the brain and individual neurons are difficult to trace. Two
abdominal neurons containing DILP7 known to arborize close to the IPCs
(Miguel-Aliaga et al., 2008) also contain sNPF and are likely candidates for
DILP regulation at least in the larva.
We found sNPF and GABABR2 in the antennal OSNs of Drosophila
and we expected to find sNPFR-IR axon terminals from the OSNs in the larval
antennal lobe, since this was detected in the adult (Root et al., 2011).
However, we could not confirm this, but cannot entirely rule out the possibility
that sNPFR is present in low levels in OSNs in larvae. Perhaps the sNPFR
antiserum did not penetrate the tissue well enough because of the deep
location of the larval antennal lobe. Another possibility is that the expression
of the receptor changes during metamorphosis. The levels of sNPFR in adult
OSNs increase as a result of starvation and lower levels of circulating insulin
(Root et al., 2011). The larvae in our experiments are fed and therefore the
levels of sNPFR might be lower and not detectable with the antisera.
The mushroom bodies are the main brain centres for learning and
olfactory memory. We found intense GABABR staining in the calyces, but not
in the peduncle and lobes. This is expected, since extrinsic GABAergic
neurons terminate in the calyx (Keene and Waddell, 2007; Yasuyama et al.,
2002). However, RDL has previously been shown to be present in the
mushroom body lobes and peduncles (Cayre et al., 1999; Strambi et al.,
1998). We also found that the intrinsic Kenyon cells of the mushroom bodies
contain sNPF. The Kenyon cells do not express the receptor to sNPF, but we
found sNPFR-IR neuron processes innervating the lobes of the mushroom
bodies in larvae. We assume that sNPF probably acts as a cotransmitter in
the intrinsic neurons of the mushroom bodies, but that another substance will
28
be identified as a primary transmitter. There is no evidence that Kenyon cells
contain GABA, neither in the cell bodies nor axons or dendrites. We can
probably also exclude glutamate, monoamines and acetylcholine as
transmitters in the same cells. These transmitters, however, seem to be used
by extrinsic neurons innervating the mushroom body. It is of interest to
determine the neurotransmitters of the Kenyon cells lacking sNPF and also to
screen for an additional classical transmitter in the sNPF-expressing cells.
6. CONCLUSIONS AND FUTURE PERSPECTIVES
In paper I we found that the ionotropic and metabotropic GABA receptors
have similar distribution in the Drosophila CNS apart from a few regions. Parts
of the mushroom bodies and ellipsoid body contain only GABAAR. A fraction
of the clock neurons only contain GABABR as well as some neurons in the
optic lobe and in the olfactory receptor neurons (Root et al., 2008) where the
receptor presumably acts presynaptically. In paper II we found another
location where the two receptor types differ, namely in the median
neurosecretory cells in the pars intercerebralis where GABABRs are located
on the insulin producing cells and GABAARs are located on adjacent neurons
in the same cell cluster. We also found that GABA is regulating production
and/or release of insulin-like peptides via GABABRs and not via GABAARs.
The functional roles for the metabotropic GABABR need to be further
investigated. Drug addiction is a relevant condition to study. In mammals,
GABABR agonists can reduce cravings for different drugs, e.g. cocaine,
heroine, alcohol and nicotine. This occurs in the brain region involved in
reward and reinforcement, where GABABRs are blocking the increase in
dopamine release that otherwise would be induced by the drug [see (Bettler
et al., 2003)]. A role for GABABRs in alcohol tolerance and nicotine-resistance
in Drosophila has been suggested (Dzitoyeva et al., 2003; Passador-Gurgel et
al., 2007), but the mechanisms remain unclear. Other addictive drugs would
also be interesting to test in Drosophila. The functional roles of GABABRs in
locomotion behaviors would be of interest since the receptor has been
displayed in the central body complex. The relevance of GABABRs in
circadian rhythm regulation also needs further investigation.
Paper III and IV focus on the locations of short neuropeptide F and its
receptor. We found a widespread distribution of sNPF in many interneurons
and in a subset of antennal olfactory sensory neurons, suggesting multiple
roles as cotransmitters or neuromodulators. The receptor distribution in the
larvae indicates roles in feeding and olfaction as we found the receptor in
some cells of the midgut, in some neurosecretory cells and in the larval
antennal lobe.
The finding of sNPFs in the Kenyon cells of the mushroom
bodies is exciting, since it is the first and so far only neurotransmitter found in
these cells. We believe that sNPF acts as a cotransmitter in these cells, and
therefore it would be of interest to determine the classical or primary
transmitter used by the Kenyon cells. In addition, we only found sNPF
expression in a subset of the Kenyon cells. The transmitter utilized by the
remaining cells is still unknown. It would also be of interest to study the role of
sNPF in olfactory associative learning.
29
The sNPFs have been shown to stimulate ovarian development in
locusts (Cerstiaens et al., 1999) and therefore it would be of interest to
investigate if this is also the case in Drosophila and investigate the roles of the
peptide in reproduction.
Based on distributional studies of the peptide, the next step would be to
study the function of sNPF in locomotor behaviors, hormonal release and in
the circadian clock. The role of sNPF in locomotor behaviors has been
investigated previously (Kahsai et al., 2010), but more experiments are
needed. Another aspect is to further study the modulation of chemosensory
inputs to the antennal lobe. Both sNPF and sNPFR have been found in the
adult OSNs (Carlsson et al., 2010; Root et al., 2011) and the levels of sNPFR
were increased in starved flies due to decreased levels of insulin in the
circulation (Root et al., 2011). However, we did not find sNPFR in the axons of
the larval OSNs. Perhaps this is because the larvae are continuous feeders
and have constant high levels of circulating insulin. Therefore it would be
interesting to starve larvae to see if sNPFR is upregulated in the OSNs. It is
also possible that larvae do not possess sNPFRs in the OSNs and that the
expression starts at a later stage during development. This is our suggestion
for the lack of sNPFR in the larval IPCs, since the receptor most likely is
present in the adult IPCs (Lee et al., 2009). We only studied third instar
larvae, but perhaps a mapping throughout all development stages is
necessary.
We have started to understand a little more about GABA and sNPF
signalling in the brain of Drosophila, but further experiments on the role of the
two substances are needed to unravel further functions of these substances
and mechanisms of signalling.
30
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8. ACKNOWLEDGEMENT/TACK
Finally I am here! There are so many people that made this work possible and
that I would like to thank!
First of all I would like to thank my supervisor Dick Nässel. You helped
me to keep my motivation during these years, with your incredible ability to
always say the right things at the right moment. Thank you for always keeping
your door open, for your advices and expertise. My co-supervisor Åsa
Winther, you have been a great help in the lab. I would not have been able to
cross flies if it wasn’t for you. You have also been a good friend outside the
lab. Thank you for trying to keep me fit by dragging me to the gym.
My roomies deserve a special thanks. Jeannette ”Miss Google”
Söderberg, thank you for all the laughters and tears that we have shared and
for all the good talks about everything and nothing. Your curiosity and interest
in other people is admirable and I learned a lot about myself thanks to you.
Jiangnan Lou, thank you for interesting discussions and for teaching me
about proper chinese food. My former roomies: Yasutaka Hamasaka, thank
you for teaching me about immunocytochemistry and Ryan Birse for not
giving up on my attempts to make that %&#€%”&# Gal4-line (I am sorry to tell
you that I gave up on it when you left). Kerstin Mehnert and Ellan
Gustafsson, I really miss you guys!
I would also like to thank people at the department for making the
environment pleasant to be and work in. Agata Kolodziejczyk, you are such
a lovable and crazy person, thank you for cheering me up. Always! Neval
Kapan, thank you for good cooperation and interesting conversations. Micke
Carlsson, sorry that I didn’t always understand your skånska and forced you
to speak stockholmska. Kristin Eriksson, you are such a ”frisk fläkt” at the
department, keep up the good work. Bertil Borg, thank you for reading and
commenting manuscripts and for introducing me to the weird world of Albert
Engström. Count me in for the yearly glögg in the kitchen. Erik Hoffmann,
you do a good job down in the basement, but we miss you upstairs. Heinrich
Dircksen, thank you for valuable discussions and for always repairing broken
apparatus in the lab. Yi Ta Shao, thank you for the sake and for the nice party
after your lic. Good luck with your thesis! Rafael Cantera, you made the
lectures about neurology interesting and perhaps I would not have continued
in the field if it wasn’t for you. Minna Miettinen, thank you for always helping
out with a smile on your face. Anette Lorents, Berit Strand and Siw
Gustafsson, tack för trevliga diskussioner och korsordslösningar i
lunchrummet och för all hjälp med allt runtomkring forskningen. Erland
Dannerlid, tack för dina små teckningar, för dina underfundiga kommentarer
och för hjälpen till rummet på Tjärnö (du vet vad jag menar).
I would also like to thank former people at the department: Lily “Miss
Ouri” Kahsai, you are always so nice and I wish you all the best! Helena
Johard, my travel buddy. Whatever happens in Zakopane stays in
Zakopane... Farideh Rezaei, thanks a lot for taking such good care of me
when I was new and lost in the lab. Johannes ”Bert” Strauss, you somehow
managed to make the lab more interesting with your fun and quirky ideas.
Ana Beramendi and Helena Elofsson, ni var i slutfasen när jag började, men
välkomnade mig och lärde mig mycket om hur det är att vara doktorand.
39
To all other colleagues at Zootis, present and former, thanks for
helping out in various ways. Ulf, thanks for all your help with the computers.
Lus-Lina och Fjärils-Lina, tack för att jag fick bli Flug-Lina. ;) Jessica,
Aleksandra, Marina, Marianne, Helena, Anna, Magne and everyone
involved in doktorandrådet for standing up for our rights. Maria Lissåker, I
forgive you for calling me juvenile, and I am sorry about your shoes... Sören
Nylin, thank you for always listening to us PhD students and taking us
seriously.
“Biologbrudarna”, Ida von Scheele, Åsa Andersson, Yvonne
Sundström, Agnes Karlson, Julia Borg och Anna Davidsson, tack för att
ni gjorde pluggåren sååå mycket roligare och för att ni fortfarande gör åren så
mycket roligare.
There is also a world outside the walls of the university. I would like to
thank all my family and friends for always being there for me. Min extrafamilj,
Birgitta, Chrille och Peter, tack för att ni har låtit mig bli en del av er härliga
familj! Tack till alla i min egen familj. Mormor och Teta, ni är underbara! Sara
”Kajan”, ”sis”, ”Inga”, du är den bästa syster och vän som går att få! Tack för
att du alltid lyssnar, stöttar och finns där. Love u!! Mamma och pappa, tack
för att ni alltid har stöttat och trott på mig. Ni ställer upp i vått och torrt, och för
det är jag evigt tacksam! Tack för att ni finns! Bud, dig har jag så mycket att
tacka för. Du har fått utstå mycket under dessa år, men du har stått vid min
sida och väglett mig i rätt riktning. Jag älskar dig! Parp. Slutligen vill jag tacka
min älskade dotter Lea, min lilla solstråle, du är en sån häftig liten människa.
Du har öppnat mina ögon och fått mig att uppskatta det enkla och vackra i
livet.
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