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A) Scientific background
Dendritic spines, the postsynaptic protrusions onto which excitatory afferents impinge in central neurons, were
described a hundred years ago by Ramon y Cajal, but their role in neuronal functions is still an enigma. Early
morphological studies, using high power optical or electron microscopy, found that density of spines increases
in the developing neuron, that they vary in shape and size, and that they are lost during later stages of brain
maturation and in relation to learning, experience and aging (17, 42, Huttenlocher, Burgess and Coss, Bock and
Braun, Braun et al). The rules governing these variations are still debated and the mechanisms underlying these
changes are unknown. Being the postsynaptic component of a synapse, it is apparent that the spine is a complex
device, where a number of receptors and ion channels, second messengers and effector systems crowd to allow
synaptic transmission and plasticity (3,18). The presence of a cytoskeleton within the spine head as well as the
presence of a nearby protein synthetic machinery encourage the proposition that the spine may respond
mechanically to ambient demand, to the degree that it may even twitch (6). The functional significance of the
variations in shape and size of the spines is totally unknown. The small size of the dendritic spine, at the limit of
optical resolution, restricted its accessibility to labor-intensive ultrastructural and high-resolution optical
analysis conducted in fixed tissue (18). The lack of appropriate research tools resulted in an extremely slow and
inconsistent progress in this field, which is undoubtedly one of the most important ones in neuroscience. This
restriction has been lifted in recent years, with the advent of sensitive, high resolution imaging methodologies of
living neurons. Several most powerful advances in this context involve (i) imaging of free intracellular calcium
concentration in living dendrites and spines, (1, 16,32), (ii) 3-D, light-microscopic reconstruction of dendritic
spines using the confocal laser scanning microscope (30,36, Sommerkorn et al, Herzog et al), (iii) the
photoactivation of caged neurotransmitter molecules in restricted spheres using two photon systems, and (iv) the
fluorescent tagging of key molecules in the neurons. Linked to tremendous advances in molecular biology, these
technological breakthroughs promise a rapid progress in century old issues in neuroscience (21).
Regulation of spine plasticity: endogenous vs. exogenous factors, different or common molecular mechanisms?
Conditions under which the number and size of spines are modified include a) neuronal activity and hormonal
regulation (exogenous regulation) and b) genetic factors (endogenous regulation). We propose that variations in
dendritic spine density are related to ongoing neuronal activity. Early EM studies describe several changes in
the structure of the spine and the postsynaptic density following extensive plasticity-related stimulation,
including a change in the shape and length of postsynaptic membrane and a change in the shape of the spine
(12,28). Recent studies using light and confocal microscope analysis of spines indicate that a major
consequence of excessive synaptic activity is the formation of new dendritic spines. The synaptic stimulation
reported to produce new spines ranges from recurrent epileptiform activity in the rat hippocampus (5), blockade
of inhibition in cultured neurons (MS), to an enriched environmental stimulation (Comery et al 1995), maternal
separation (Helmeke, Braun) or even to a single experience in the life of a young chick (29). Regressive
synaptic changes appear to be common events during functional brain maturation (Huttenlocher, Wolff,
Zekevic) and in relation to juvenile learing events (lit). Regressive changes of spine synapses also require
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synaptic activation and are induced by strong emotional experience such as sexual activity (Rollenhagen and
Bischof zebra finch), filial imprinting (Bock and Braun, Braun et al) and exposure to extensive social contact
(Burgess and Coss). The induction and regulatory mechanisms of proliferative and regressive changes of spine
synapses are mostly unclear. Since both, experimentally and environmentally evoked synaptic activity involves
the release of neurotransmitters (e.g. glutamate), peptides (e.g. endorphins) and hormones (e.g. estrogen,
gluococorticoids) leading to the synthesis of effector proteins (e.g. cytoskeleton proteins, FMRP) that interact
with postsynaptic targets, these systems are likely candidates for being involved in spine plasticity.
B) Objectives and integrated research plan
The objectives of the proposed study are to analyze the nature of the stimuli and the underlying cellular and
molecular mechanisms, which cause proliferative or regressive changes in spine number. A corollary
question is whether and in which way these factors also modify the spatial arrangement of spines along the
dendritic tree and the maturation of spine synapses, which may be reflected in parameters such as spine size
and shape. And finally, we will address the question of the functional significance of spine shape and size. A
corollary question is whether novel spines or spines with pathological appearance differ functionally from
mature ones. Our working hypothesis states that the modification of spines and their synaptic connections
within functional pathways, is guided by genetic and environmental factors, and follows universal
principles that involve common cellular and molecular mechanisms. In support of this hypothesis are the
strikingly similar alterations of neuronal and synaptic architecture, which have recently been found by our team
in mouse mutants lacking the protein FMRP, in the experience-induced (maternal separation) and in the drug
induced forms of synaptic plasticity in culture, which indicates that common molecular and biochemical
cascades may be involved. We will analyze the chain of events from stimulus-induced,
neuromodulator-regulated neuronal activity through intracellular second messenger to synaptic effector
molecules. In particular we will analyze the role of glutamate and of 5HT that we propose to be responsible for
the formation, stabilization, maintenance and elimination of synaptic contacts. As effectors whose expression
are differentially up- or downregulated by neurotransmitters and/or modulators in relation to synaptic activity
we will analyze the role of FMRP, a recently identified protein that appears to be essential for spine elimination
and maturation (Comery et al 1997). To compile a general concept of spine plasticity, investigations at the
levels of brain systems, single cells, synapses and molecular systems are required, which will be achieved
through extensive collaborations among the three teams (see also item 5). Our models for spine plasticity are
1) a genetic model (see part 1. WG), the fragile X mouse mutant, which lacks FMRP, that appears to be the first
identified molecular factor that regulates synaptic selection processes and spine maturation. These mutants, as
well as human FraX patients display abnormally high spine densities and shapes; 2) maternal separation in
newborn pups, as model for experience-induced regulation of spines (see part 2. KB). Maternal separation
induces changes of spine densities and morphology in the anterior cingulate cortex (Helmeke, Braun), which are
strikingly similar to those observed in the FraX mutant mouse; 3) tissue cultured neurons from hippocampus and
anterior cingulate cortex (see part 3. MS), in which changes of spine densities and size are induced by chronic
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and acute drug treatments (33, 37). Our aim is to compile a general functional concept of spine plasticity
during brain development and in relation to experience, learning and memory formation.
C) Necessity for international collaboration
As outlined in more detail under item 5, a close collaboration among the three groups is essential and will
benefit from the complementary theoretical backgrounds and technological expertise of the team
members, who share a long standing interest in dendritic spines. The three groups have established different
methodologies and the most advanced technologies to be applied in this field. At a conceptual level, ideas and
strategies for testing hypotheses will be exchanged rapidly between the participating groups and will contribute
to the rapid progress in the quest for the comprehension of the spine enigma. Having access to new and
advanced technologies, we have now reached a turning point where, for the first time, it is possible to address
these issues simultaneously and cooperatively in our established models for spine plasticity.
D) Description of research project of each collaborating team member
1) USA, William T. Greenough (WG)
The Role of Synapse Elimination in Development and Memory
Synapse overproduction and elimination is a key process development, and may also operate in adult
learning and memory. We propose to test the hypothesis that fragile X mental retardation protein (FMRP)
synthesis at the synapse is critical to the synapse maturation and elimination process. The work proposed
is intended to be basic (i.e., not clinical) in orientation but is based on data from human patients and
knockout [KO] mice that suggest a role of FMRP in synapse maturation and elimination.
Hypothesis I: Synapse elimination is a key process in development and is modulated by experience.
Studies of peripheral and central nervous system development have for years suggested that the synaptic
organization pattern is in part generated by overproduction and systematic loss and preservation of
synapses. The first evidence for a learning-induced reduction of spine synapses was shown after speech
learning in mynah birds (Rausch and Scheich 82) and after auditory filial imprinting in chicks (Scheich et
al, 91, Scheich and Braun 89; Bock and Braun, 98, Braun et al 98). Similar spine reductions have been
reported after sexual experience and song-learning in male zebra finches (Rollenhagen and Bischof 1994,
Wallh‫ה‬ußer et al 1995). There is little evidence for very substantial cerebral cortical overproduction and
loss in rodents, possibly because the developmental timecourse is compressed such that elimination
begins before synaptogenesis is complete. Evidence for this includes the (Greenough & Chang, 1988)
report that, in somatosensory barrel cortex, the development of highly oriented dendritic fields occurs as a
result of the loss of branches extending in the ”wrong” direction, which occurs in the same time frame as
the embellishment of branches going in the ”right” direction.
Hypothesis II: Synapse elimination is a key process in adult learning and memory Synapse
overproduction and loss probably occurs in adults. This is suggested by the rapidity of dendritic and
synaptic responses to complex environment experience, much of which is evident after 4 or 10 days
exposure (Wallace et al., 1992; Sirevaag et al.). The best evidence for overshoot and loss of synapses and
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dendrites is seen in the intact sensory-motor forelimb cortex in response to damage of its contralateral
homologue; a clear overshoot in the number of synapses per neuron occurs, followed by a return to levels
above the pre-injury baseline. (Jones et al., 1996; Jones & Schallert, 1994). The overshoot may be evident
here because synaptogenesis is triggered synchronously, whereas synaptogenesis resulting from learning
occurs gradually over time obscuring individual localized peaks and pruning (e.g., Kleim et al., 1996).
Hypothesis III: Synthesis of FMRP at the synapse is involved in synapse elimination. We (Hwang &
Greenough 1984) found that polyribosomal aggregates (PRAs) in spines typically appeared only after
spines received innervation. This suggests that postsynaptic protein synthesis may be involved in the late
stages of synaptogenesis. We also found more PRAs in spines in animals housed in a complex
environment (Greenough et al., 1985). Subsequently, we found that the Fragile X Mental Retardation
Protein (FMRP) is synthesized at the synapse, in a synaptoneurosome preparation (Weiler et al., 1997).
The inability to synthesize FMRP, usually due to an abnormally long CGG repeat in the promoter region
leading to hypermethylation and silencing of the FMR1 gene, is the leading cause of genetically inherited
mental retardation (Hagerman & Chronister, 1996). A FMR1 knockout mouse (KO) (Consortium, 1994)
exhibits impaired learning and neuroanatomical abnormalities (see below) compared to wild type (WT)
mice of the same strain. Synthesis of FMRP occurs in synaptoneurosomes in response to glutamate,
KCl, and Class I metabotropic glutamate receptor agonists. A phospholipase C--protein kinase C cascade
leads from receptor activation to protein synthesis. The PKC inhibitor calphostin prevents the protein
synthesis (Weiler & Greenough, 1993; Weiler et al., 1995, 1996, 1997). Several lines of evidence from
our unpublished work suggest that FMRP expession at the synapse could be involved in the synapse
maturation, stabilization and elimination process. FMRP expression is upregulated in development at
times corresponding to synapse elimination. In adulthood, FMR1 mRNA remains upregulated in "plastic"
brain regions such as cerebellar cortex, cerebral cortex, hippocampus and olfactory bulb. FMR1 mRNA
is downregulated in adult thalamic, hypothalamic and brain stem structures (Cohen et al., in preparation).
These findings suggest a continuing role of FMRP expression in adult synaptic plasticity. Synapses in
KO adults also retain an immature appearance (Boyer et al., 1998), being longer and thinner than normal
(Comery et al., 1997). Similar morphology and a higher density of spines also occurs in FraXs patients
(Irwin & Greenough, in preparation). There is an overabundance of spines and of synapses in KO mice,
as if normal elimination has not taken place (Comery et al., 1997; Dyson & Greenough; Irwin &
Greenough, unpublished. Taken together, these data suggest that FMRP synthesis at synapses may be a
part of a synapse stabilization and elimination process that is involved both in developmental brain
organization and in adult learning and memory.
FMRP expression is upregulated in weanling and adult brain in response to experience and learning
(Irwin et al., submitted). This occurs well after the onset of the experience. In rats reared in a complex
environment, statistically significant upregulation, relative to individually housed rats, was seen in visual
cortex after 20, but not after 10 days of exposure to the environment. In 2-3 month old rats learning a
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complicated set of motor skills, upregulation was seen in motor cortex after 7 but not after 3 (statistically)
days of training. Finally, FMRP appears to participate in synaptic protein synthesis. It binds to the
translation complex, dissociating at 0.5M KCl and DHPG-initiated polyribosomal aggregation and protein
synthesis, which occurs in WT mice, does not occur in KO mice (our unpublished observations). In EM,
there is about a 40% reduction in PRA-associated spine synapses in the KO mice at 15 days of age, when
synapse association with PRA is very high (our unpublished observations). Thus the presence of FMRP at
the synapse may be necessary for, or facilitative of, synapse-associated protein synthesis.
Plan of Operation
The experiments address the function of FMRP synthesis (and possibly other proteins) at the synapse.
a) To examine effects of the absence of FMRP on synaptic development and function, a developmental
study in WT and KO mouse visual cortex will i) examine the relationship of PRAs in spines to stages of
synaptic development in the WT mice (also noting whether they are associated with particular structural
components of the spine), will ii) determine if and how this relationship differs in KO mice, will also iii)
examine the process of synaptic development in KO compared to WT mice and will iv) examine PRA
location and size in KO vs. WT mice. The study will use serial section reconstruction EM and the
disector method to examine numbers of synapses per neuron. For studies of individual synapses, we will
use synapses on the apical dendrites of layer V pyramids as they pass through layer IV, as these synapses
are re-identifiable across stages of development, and they exhibit the morphological characteristics
described above (Comery et al., 1997). The reconstruction study will, for this one type of spine synapse
type and location, complement the confocal microscopic 3-D analysis of spine morphology, which will be
carried out in collaboration with KB (see part 2.). Depending upon the outcome of this study and the
next, we may extend this analysis to the synapses of the middle or outer third of the dentate gyrus
molecular layer, also a quite homogeneous population of entorhinal cortical afferent synapses. This
study will provide detailed information that can be compared with morphology and expression patterns in
the tissue culture studies headed by MS (see part 3.). b) To assess the relationship of FMR1mRNA and
FMRP expression to synaptic development we will examine their expression using in situ hybridization
and immunocytochemistry in dendritic/synaptic regions vs somatic regions in WT mice at ages ranging
from birth to adulthood. This study will focus on the hippocampal formation because of the clear
stratification of somatic and dendritic-synaptic regions. In collaboration with KB we will test whether
experience modulates the developmental regulation of FRM1mRNA and FMRP expression and compare
the normal patterns with those seen in maternally deprived pups. We will compare serial sections from the
same animals prepared for ISH and ICC to see if the relative expression in RNA and protein vary, as
might be expected if translation is regulated by synaptic activity. These studies will provide detailed in
vivo comparison data for the tissue culture studies of MS. c) In collaboration with MS, we will conduct
quantitative examinations of 1, 2, 3, and 4 week tissue culture preparations from WT and KO mice.
Baseline morphological studies will determine whether there are differences in spine density and in
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dendritic branching patterns. Functional studies to be conducted by MS, will examine the formation of
functional synapses among the cells in culture (see below). d) As a direct test of the synapse elimination
hypothesis, we will examine two long distance connections that are normally eliminated during
development to see if they persist in the KO mouse. First, projections from primary visual cortex to
spinal cord are established in early postnatal development and subsequently withdrawn (O'Leary &
Terashima, 1988). Using dye injection above and below the pyramidal decussation across postnatal ages,
as done by O’Leary and colleagues, we can determine this. Second, cerebellar Purkinje cells are
innervated by multiple climbing fibers in development and this is removed by axon collateral withdrawal
in development, a process that does not occur in PKC knockout mice (Kano et al., 1995). e) We will
perform Golgi study of the orientation of spiny stellate cell dendrites in the whisker barrels of
somatosensory cortex in KO and normal mice. The rationale for this is that, if the KO mouse can't
eliminate synapses and if synapse elimination is needed for dendrite elimination, then these cells should
have less oriented dendritic fields, which are normally highly polarized into the hollow of the barrel.
2. Germany, A. Katharina Braun (KB)
Aims Taking advantage of the pronounced plasticity of the juvenile brain we will investigate the
dynamics, the regulating factors and underlying second messenger and effector systems which mediate
proliferative and regressive changes of spines following an emotional experience of maternal separation.
The role of the neuromodulator serotonin (5-HT) in experience-induced spine plasticity will be studied
on the systems and the cellular/synaptic level. Possible links between this modulator of neuronal activity
and a recently discovered factor for spine stabilization and maturation, the FMRP protein (see part 1),
will be studied during experience-induced spine plasticity.
Mechanisms of learning- and experience induced spine proliferation and elimination
As outlined in part 1. (WG) the organization of higher associative circuits by experience and learning is
achieved by synaptic selection processes, which involve synaptic proliferation, potentiation stabilization
and elimination. These morphological changes are guided by distinct synaptic activity patterns (see part
3. MS), which are required for the proliferation (37) but also for the selective pruning of spines (Scheich
et al, Braun et al, Bock et al). Synaptic activity patterns are modified by neuromodulators such as 5-HT
which is up- or downregulated in response to changes in the emotional environment (Gruss, Bickerdike,).
Our previous results in newborn animals indicate that the emotional component is the most critical factor
for the induction of spine changes (Bock and Braun) and that 5HT is acutely and permanently altered
during and after socio-emotional deprivation, respectively (Bickerdike, Lange et al, Gruss and Braun).
Thus, our first working hypothesis is that serotonergic mechanisms play a key role in the regulation
of experience-induced spine plasticity. A role in neuronal differentiation, synapse formation and
plasticity, long before its function as neurotransmitter emerges, has been proposed for 5HT (Lavdas et al
1997), since it is one of the first transmitter systems to innervate cortical areas, and fiber densities peak
around the first two postnatal weeks (Morilak et al 1994; Lange et al). Serotonergic influences on
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dendritic growth and spine density have been described (Chubakov et al 1986; Gonzalez-Burgos 1996;
Yan et al 1997a+b; ), and 5HT1A receptors have been localized in dendrites and spines (Kin et al 1996)
however the cellular and molecular mechanisms of its role in spine plasticity are yet unclear. Our second
hypothesis concerning these mechanisms is that synaptic proteins may represent effector systems that
regulate spine plasticity. A candidate may be the recently identified protein FMRP (Comery et al, Weiler
et al 1998) may serve as effector of spine stabilization and maturation (see part 1. WG). FMRP and its
FMR1mRNA is located in dendrites and somata of a variety of neuron populations in vivo and in vitro
(Weiler et al 1997; KB and MS unpubl), it is associated with polyribosomes (Feng et al) and synthesized
at synapses (Weiler et al 1997). As outlined in part 1 (WG) expression of FMRP is highest during early
stages of development, during which synaptic reorganisation takes place (Cohen et al SFN abstract).
Plan of Operation
Factors that mediate proliferative and regressive changes of dendritic spines
Neuromodulators. Pharmacological manipulation of 5HT release and receptor activation or blockade will
test the involvement of this neuromodulator system in the experience-induced changes of spines. The
upregulation of the 5HT system, which we observed in our deprived animals may be one factor that
contributes to their increased spine densities (Helmeke, Braun). We found elevated 5HT levels and
enhanced serotonergic fiber innervation in the mPFC during acute social isolation (Gruss and Braun),
which are maintained after chronic social deprivation (Bickerdike et al, KB unpublished observations)
Thus, the blockade of 5HT-receptors during maternal separation should suppress spine proliferation.
Drugs will be delivered either via implanted drug-loaded microspheres (ref), implanted microcannulas or
microdialysis probes. Pyramidal neurons in the mPFC (anterior cingulate cortex, infralimbic cortex) will
be selectively prelabelled by in vivo injections of retrograde tracers into a) the nucleus accumbens (tracer
A e.g. Fast blue) and in the dorsomedial thalamus (tracer B e.g. fluorescent beads) or other afferent
regions of interest, and then in vitro intracellularly injected with Lucifer yellow. In collaboration with MS
further analysis on the single cell level will be performed in cultured neurons from mPFC, where
pharmacological manipulation can be performed under controlled conditions. In addition to quantitative
morphological analysis of spine density, shape, size and spatial distribution along the dendritic tree (see
below) in DiI labelled neurons we will analyze changes of neuronal activity patterns in cultured neurons
after acute (hours) or chronical (days) treatment with agonists or antagonists for 5HT.
Effectors: If FMRP is essential for experience-induced spine proliferation, elimination and maturation,
does acute or chronic maternal deprivation induce changes of FMR1mRNA expression or FMRP
synthesis? Few week old normal pups display high densities of FMRP-positive neurons in the mPFC and
other cortical regions (unpubl.). In collaboration with WG we will analyze changes of FMRP and
FMR1mRNA after maternal separation using western and northern blot techniques and quantitative in situ
hybridisation and immunocytochemistry. Is FMRP an effector of 5HT-mediated spine plasticity? These
links can be tested in vivo and in vitro using pharmacological stimulation of the 5HT system and
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measuring changes of FMRPmRNA expression. Another approach will use FMRP suppression in vitro
using antisense oligonucleotides (see part 3. MS) during pharmacologial stimulation of the 5HT system to
reveal possible functional links during spine proliferation and maturation. Quantitative analysis of spine
plasticity will be performed in using newly developed pattern recognition algorithms for
3D-reconstruction from confocal image stacks of fluorescent (Lucifer Yellow, DiI, calcein) labelled
neurons, dendrites and spines (Sommerkorn et al, Herzog et al, Watzel et al). This automated image
analysis system allows, in addition to conventional counting of spines, the quantitative analysis of spine
size (volume), shape categorization via Kohonen maps, and it includes algorithms for the analysis of the
spatial distribution of spines along the dendritic tree. These algorithms are currently extended for
analogous measurements on 3D reconstructed Golgi-impregnated or biocytin-injected neurons that are
imaged with a new 3D-computer microscope (Schwertner GbR). Compared to time and labor intensive
EM quantification of only few spines, this system allows the analysis of a large number of spines, which
improves the chances of obtaining statistically significant data. To ensure compatible results from the
experiments of the three teams their morphological data will be analyzed with this system.
3. Israel, Menahem Segal (MS)
The objectives of the proposed research are to contribute to the understanding of the nature of the stimuli
which cause formation of new dendritic spines in cultured neurons, and to study the roles of spine
morphology in regulation of calcium dynamics in the spine and its parent dendrite. Our first working
hypothesis states that ongoing synaptic activity regulates density and shape of dendritic spines in a
dynamic fashion such that an increase in activity causes formation of new dendritic spines, while a
decrease in activity causes elimination of spines. We further hypothesize that an increase in synaptic
activity causes an elevation of intracellular calcium concentration, which activates a cascade of second
messengers and nuclear factors, synthesis of new synaptic proteins and formation of spines in the region
of enhanced activity. Our long-term goal is to track down the sequence of events leading from the
activation of the excitatory receptor, through the activation of the nucleus to the formation of spines.
Many studies have already identified molecular elements of synaptic memory, but their involvement in
spine formation remain largely unknown.
Our second working hypothesis states that-the morphology of the spine determines the efficacy of
spine function. As indicated above, it is by now well established that dendritic spines can behave quite
independently of their parent dendrites, in their ability to raise [Ca]i in response to afferent stimulation.
However, none of the studies reported thus far addressed the heterogeneity of spine shape, size and
density in relation to their independence of and interaction with the parent dendrites. We have only
recently begun to address this issue by causing release of calcium from stores (Korkotian and Segal,
1998). We plan to extend these initial studies on the involvement of spine morphology in its function.
Plan Of Operation
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What makes dendritic spines External stimuli: In previous studies we found that blockade of
inhibition by bicuculline, a GABA-A antagonist, or application of estradiol, cause a 40-100% increase in
dendritic spine density. If indeed estradiol and bicuculline act by blockade of inhibition, leading to
enhanced glutamate excitation, then facilitated glutamate excitation should mimic this effect. We will
expose cultures to one of several stimuli that facilitate glutamate excitation, which will act to enhance the
efficacy of the synaptic excitation, and observe changes in dendritic spines. In collaboration with KB,
we will examine effects of serotonin on formation and elimination of spines in cultures. The cultures will
be treated, fixed and stained with DiI and analyzed to detect changes in dendritic spines. Parallel
experiments will verify that the drugs did indeed modify the properties of synaptic currents, as seen
before (Murphy et. al. 1998).
Second messengers, transcription factors and immediate early
genes. Previously, we found that CREB is essential for estradiol-induced spine formation (34). We will
examine if CREB is also critical for the glutamate or 5-HT-induced spine formation. We will determine
if protein serine/threonine kinases are also involved in spine formation. For example, CamKinase II has
been implicated in synaptic memory (LTP induction, (3)). Using a selective CamKinase II antagonist
(KN62) we will test its effect on glutamate-induced spine formation. The culture will be exposed to the
drug either before and/or during exposure to the tested spine-producing agent, and the effect on spine
morphology is analyzed. Effector proteins: In collaboration with KB and WG we will examine the
involvement of FMRP in the responses of the cultured neurons to the stimuli which cause formation or
elimination of dendritic spines. This will be conducted either with cultures taken from FMRP-KO mice
or with cultures treated with an antisense oligonucleotide for FMRP. In either case we will examine if the
cells that do not have normal FMRP respond to plasticity producing stimuli the same as normal cells. We
will use time lapse imaging for detection of novel dendritic spines, and addressing basic questions
related to spine formation and elimination in normal and FMRP-KO cells: Can we predict where will a
novel spine, responding to an enhanced synaptic activity, be formed and which one is likely to be
eliminated? How is the process of synapse formation related to spine formation, is the novel spine
growing off an existing synapse? Once they are formed, are the novel spines different from the ‘old’
ones? Earlier work, restricted to several hours of observation could detect formation of filopodia, which
have a much more dynamic behavior and are far less stable (48). We will stain individual cells with DiI
or another fluorescent tag (e.g. green fluorescent protein, GFP) and automatically scan several fields with
submicrometer resolution across hours of observation, in a controlled environmental chamber at the
confocal microscope. We will introduce the stimulus (e.g. bicuculline), and monitor possible
morphological changes in the neurons. If indeed we detect reliably novel spines, we will proceed with a
series of subsequent questions. For example, we will stain presynaptic terminals with the dye FM1-43,
which is taken up by active boutons and ask if the presence of a presynaptic terminal is a predictor of
formation of a spine. Once we identify a novel spine, we can study its calcium handling ability in
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comparison to ‘old’ spines. Do the novel spines have a calcium store? This can be addressed by
measuring responses to caffeine, as detailed earlier (26a).
Functions of dendritic spines The following studies will focus on the roles of dendritic spine shape and
size in passage of synaptic information into the dendrite, the interactions among adjacent spines, and the
roles of calcium stores in synaptic plasticity. We will load an individual cell in the culture with a calcium
dye (calcium green 1) and stain presynaptic terminals with FM1-43 under the confocal microscope. We
will bath the cell with caged glutamate and locate spines which are touched by an FM1-43-stained
terminal. We will apply a pulsed UV laser light beam onto the spine, and record changes in [Ca]i in the
spine and its adjacent dendrite as well as electrical activity recorded from the soma. We will scan the
region near the spine to identify ‘hot spots’ effective in producing the [Ca]i change. We will examine the
possible interactions among adjacent spines: We will stimulate one terminal with the UV laser and
expect to see a rise in [Ca]i in postsynaptic spine, and observe changes in adjacent spines. We will
correlate the distance between spines, the density and the dimensions of each spine, with the magnitude
of rise of [Ca]i. We will supplement these studies with recording of synaptic activity in individual patch
clamped neurons, to detect changes in functional synapses in drug treated as well as KO-derived cultures.
Collaborations among the three teams:
In collaboration with WG and KB, MS will grow cells taken from FMRP KO mice in culture and study
their physiological and morphological properties, their reactivity to chronic stimulation which produce or
remove spines, their spontaneous and evoked synaptic activity, and their ability to handle calcium loads.
In collaboration with WG, KB will compare spine density, shape and spatial distribution in FraXKO
mice and WT controls using their image analysis system. WG in collaboration with KB will analyze
experience- or serotonin-induced changes of FMR1mRNA and FMRP synthesis in the limbic system
using quantitative biochemical assays, in situ hybridization and immunocytochemistry. These studies will
contribute to the understanding of the role of FMRP in spine morphology and functions. In collaboration
with KB, MS will grow cells from mPFC in culture, and examine, using the parameters listed above, the
role of the of serotonergic system in spine morphology and functions. In collaboration with KB, cultures
studied and treated in MS´s lab will be analyzed morphologically using their automated image analysis
system . These studies will serve to analyze the contribution of the glutamatergic and the serotonergic
transmitter and receptor systems in spine formation and maturation.
Questions from KB to WG and MS:
1) Is is better to have a separate paragraph with a brief list/description of the experiments that will be
done in collaboration and trash these parts in the individual sectionss???? May reduce redundancies
and may save space and may look more coherent? (MS: I suggest to keep a separate section for
collaboration, and refer to it in the main text for each of us. this way it will look like we are both
doing separate experiments but also collaborate.) (I AGREE WITH THIS—WG)
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2) Related to this question is: Do our proposed experiments clearly lead to our aim, as stated in the
general part, to find the principal/universal mechanism/concept of spine/synapse plasticity? Should
we add a final few sentences on how we will incorporate our data into a common concept
(DIFFICULT(!!) but I think important) (MS: Yes, we should try to write a few sentences at the
end saying that what we thought is right, and that there are some general principles, such as such
and such.
3) We still need to shrink our parts (or the general part) a bit, even reducing the space for
referencecs (which will be numbered) may not be enough, Bill´s experimental part is still missing.
(MS: I will shring my part later tonight)(I AM NOW AT 2.5 PAGES—WG)
6. References:
1. Alford, S. et al. (1993) J. Physiol. 469, 693-716.
Bickerdike M, et al (1993) Behav. Pharmacol. 4, 231-236
3. Bliss, T.V.P. and Collingridge, G.L. (1993) Nature 361, 31-39.
Bock J, Braun K (1998) Neural Plasticity, in presss
Bock J, Braun K (1998) Eur. J. Neurosci. Suppl., 150
Braun K, Michaelis B, Herzog A, Schütze W, Wang J, Scheich H (1997) Soc. Neurosci. 23, 98.8.
Braun K et al (1998) Behav. Brain Res, in press
Braun K et al (1998) Soc. Neurosci. 24, in press
5. Bundman, M.C. and Gall, C.M. (1994) Hippocampus 4, 611-622.
BurgessJ. W. and Coss R.G. (1980) Neuroscience Lett. 17, 277-281.
Chubakov AR,et al (1986) Brain Res. 369:285-297
6. Crick, F. (1982) Trends Neurosci. 5, 44-46.
Comery TA, et al (1997) PNAS 94, 5401-5404
Comery TA et al (1995) Neurobiol. Learn. Mem. 1995; 63: 217-219.
12. Fifkova, E. and Van Harreveld, A. (1977) J. Neurocytol. 6, 211-230.
Gonzales-Burgos I, et al (1996) Int. J. Devel. Neurosci. 14, 673-679
Gruss M, Braun K (1997) Neuroscience 76, 891-899
16. Guthrie, P.B. Segal, M. and Kater, S.B. (1991) Nature 354, 76-80.
17. Harris, K.M. and Kater, S.B.(1994) Ann. Rev. Neurosci. 17, 341-371.
18. Harris, K. M. and Stevens, J.K. (1989) J. Neurosci.9, 2982-2997.
Helmeke C, Braun K, Poeggel G (1998) Eur. J. Neurosci. Suppl., 219
Herzog A et al (1997) In: G. Sommer et al (Eds.) 7'th Int. Conf., CAIP'97, Kiel, Germany, Proc. Lect.
Notes of Comp. Sci. 1296, pp. 114-121 Springer
11
Huttenlocher PR (1979) Brain Res. 163: 195-205.
Jungnickel J et al (1998) Eur. J. Neurosci. Suppl., 219
21. Katz LC; Dalva MB. (1994) J Neurosci Methods 54:205-18
Kia H.K. et al (1996) J. Comp. Neurol. 365, 289-305
26a. Korkotian E. and Segal M. (1998) Eur. J. Neuroscience 10, 2076-2084.
Lange E, Metzger M, Poeggel G., Braun K (1998) 91. Ann. Meeting of the DZG, 49
Lavdas AA, et al (1997) J. Neurosci. 17:7872-7880
28. Lee, K.S. et al. (1980) J. Neurophysiol. 44, 413-422.
29. Loundes, M. and Stewart, M. G. (1994) Brain Res. 654 129-136.
30. Moser, M.B et al (1994) PNAS. 91, 12673-12675.
Morilak DA et al (1994) Neuropsychopharmacol. 11, 157-166
32. Murphy, T.H. et al. (1994) Science 263, 529-532.
33. Murphy D. D. and Segal M. (1996) J. Neuroscience 16, 4059-4068
34. Murphy D. D. and Segal M. (1997) Proc. Natl. Acad. Sci. USA 94 1482-1487.
Murphy D.D. et al (1998) J. Neuroscience 18:2550-2559.
36. Papa, M. et al (1995) J. Neurosci.15, 1-11.
37. Papa M. and Segal M. Neurosci., 71, 1005-1011 (1996).
Poeggel, G, Braun K (1997) Brain Res. 743, 162-170
Rausch G, Scheich H (1982) Neurosci. Lett. 29, 129-133
Rollenhagen A, Bischof HJ (1994) Behav. Brain Res. 65, 83-88
Rollenhagen A. and Bischof HJ (1998) NeuroReport 9, 2325-2329
Scheich H, Braun K (1988) In: Barth F (ed) Proc. German Zool.Soc. G. Fischer V. pp 77-95
Scheich H, et al (1991) In: Memory: Organization and Locus of Change. (Ed: LR Squire et al), Oxford
Univ. Press, pp114-159
Sommerkorn, G, et al (1996) In G. D. Smith, et al (Eds) Proc. Int. Conf. of Artif. Neur. Netw. and Gen.
Algor. (ICANNGA97), Springer V., 129-133
42. Swindale, N.V. (1981) Trends Neurosci. 4, 220-224.
Wallh‫ה‬ußer-Franke et al (1995) Neurobiol. Of Learn and Mem
Watzel, R et al (1995) DAGM, Bielefeld, Springer Verlag, p160-167
Weiler IJ, et al (1997) PNAS 94, 5395-5400
Wolff JR, Missler M (1993) APMIS Suppl. 40, 9-23.
Zecevic N, et al (1989) Dev. Brain Res. 1989; 50: 11-31.
Yan W, et al (1997) Dev.Brain Res. 98:17-184
Yan W, et al (1997) Dev. Brain Res. 98:185-190
48. Ziv NE and Smith SJ. Neuron (1996) 17:91-102.
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