Download Neural Control of Movement: Motor Neuron Subtypes

List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
II
III
IV
Enjin A, Rabe N, Nakanishi ST, Vallstedt A, Gezelius H, Memic F, Lind M, Hjalt T, Tourtellotte WG, Bruder C, Eichele G,
Whelan PJ, Kullander K (2010) Identification of novel spinal
cholinergic genetic subtypes disclose Chodl and Pitx2 as markers for fast motor neurons and partition cells. J Comp Neurol
518:2284-2304.
Wootz H, Enjin A, Wallen-Mackenzie Å, Lindholm D, Kullander K (2010) Reduced VGLUT2 expression increases motor
neuron viability in Sod1G93A mice. Neurobiol Dis 37:58-66
Enjin A, Leao KE, Mikulovic S, Le Merre P, Tourtellotte WG,
Kullander K. 5-ht1d marks gamma motor neurons and regulates
development of sensorimotor connections Manuscript
Enjin A, Leao KE, Eriksson A, Larhammar M, Gezelius H,
Lamotte d’Incamps B, Nagaraja C, Kullander K. Development
of spinal motor circuits in the absence of VIAAT-mediated
Renshaw cell signaling Manuscript
Reprints were made with permission from the respective publishers.
Cover illustration
Carousel by Sasha Svensson
Contents
Introduction.....................................................................................................9
Background ...................................................................................................11
Neural control of movement.....................................................................11
The motor neuron .....................................................................................12
Organization of motor neurons............................................................14
Motor neuron subtypes ........................................................................14
Development of motor neurons ...........................................................16
Degeneration of motor neurons in ALS...............................................17
Proprioception ..........................................................................................18
Anatomy and physiology of the muscle spindle..................................18
Proprioceptive circuits .........................................................................20
Development of the monosynaptic stretch reflex ................................21
Recurrent inhibition..................................................................................22
Development of recurrent inhibition ...................................................23
Aims..............................................................................................................24
Methodological considerations .....................................................................25
Mice..........................................................................................................25
Transgenic mice...................................................................................25
Mouse lines..........................................................................................26
Histological staining procedures ..............................................................26
In situ hybridization.............................................................................26
Immunofluorescence............................................................................27
Quantification of stainings...................................................................28
Ventral root electrophysiology.................................................................28
Stretch reflex measurements................................................................29
Fictive locomotion ...............................................................................30
Behavioral testing.....................................................................................30
Rotarod ................................................................................................31
Beam Walking .....................................................................................31
Hanging wire .......................................................................................31
Grip strength ........................................................................................32
Digigait ................................................................................................32
Result summary ............................................................................................33
Paper I ......................................................................................................33
Paper II .....................................................................................................34
Paper III....................................................................................................34
Paper IV ...................................................................................................35
Discussion .....................................................................................................37
Novel markers for subtypes of motor neurons and premotor interneurons
..................................................................................................................37
Fast and slow motor neurons ...............................................................38
Gamma motor neurons ........................................................................39
Renshaw cells ......................................................................................40
Partition cells .......................................................................................40
Excitotoxicity and motor neuron subtypes in ALS.....................................40
The influence of 5-ht1d and VIAAT-mediated Renshaw cell signaling on
the development of spinal motor circuits .................................................42
Development of the stretch reflex in 5-ht1d-/- knockout mice .............42
Development of spinal motor circuits in the absence of VIAATmediated Renshaw cell signaling.........................................................44
Motor behavior in 5-ht1d-/- and Chrna2::Cre;Viaatlx/lx mice ...................46
The stretch reflex amplitude in locomotion.........................................46
Recurrent inhibition in locomotion......................................................47
Acknowledgements.......................................................................................49
References.....................................................................................................51
Abbreviations
5-HT
5-ht1d
AHP
ALS
Calca
ChAT
Chodl
Chrna2
CNS
CPG
DRG
Egr3
En1
EPSP/EPSC
ERR
FF
FR
GPCR
GTO
IPSP/IPSC
LMC
MMC
NMJ
Pitx2
S
SMA
TrkC
VAChT
Serotonin
Serotonin receptor 1d
After hyperpolarization
Amyotrophic lateral sclerosis
Calcitonin/Calcitonin-related polypeptide alpha
Choline acetyltransferase
Chondrolectin
Cholinergic receptor alpha 2
Central nervous system
Central pattern generator
Dorsal root ganglia
Early growth response 3
Engrailed 1
Excitatory postsynaptic potential/current
Estrogen-related receptor beta
Fast fatigable motor unit
Fatigue-resistant motor unit
G protein-coupled receptor
Golgi tendon organ
Inhibitory postsynaptic potential/current
Lateral motor column
Medial motor column
Neuromuscular junction
Paired-like homeodomain transcription factor 2
Slow motor unit
Spinal muscular atrophy
Neurotrophic tyrosine receptor kinase C .
Vesicular acetylcholine transporter
Introduction
Movement is central for the life of animals. From the relatively simple but
vital breathing movements to the subtler act of communicating an emotion
through facial expression, in essence what is happening is a movement - a
muscle contraction. Most of us perform these behaviors every day without
any apparent effort, and they may seem plain, but in fact, correct movement
is a delicate interplay between the brain, muscles and the environment.
Consider for example the routine act picking up a coffee cup. What you
do is to stretch out your arm in a straight trajectory, grab the ear of the coffee
cup and lift it to your mouth to have a sip. Consider that movement again:
moving your arm in a straight trajectory requires precise activation of just
the right ones of the approximately 50 muscles in your upper arm in the correct sequence. Depending on the starting position of your arm and the location of the cup, different sets muscles are activated, still performing the same
straight trajectory. Grabbing and lifting the coffee cup to your mouth involves activating even more muscles of the arm and digits, again in the right
sequence with just the right force. When the cup reaches your mouth, you
need to form a tight seal with your lips around the cup and then activate
muscles in your mouth and throat to swallow the coffee. In view of the complexity of this simple act, it is not surprising that most parts of the brain and
much of the brains computational power in fact are involved in creating and
tuning movements
The structures in the central nervous system (CNS) directly involved in
movements are the motor cortex, basal ganglia, cerebellum, parts of the
brain stem and spinal cord. Lesions to any of these structures in humans or
experimental animals leads to deficiencies in movement, from paralysis after
a complete transection of the spinal cord to the tremor and difficulties of
initiating movements in a patient with Parkinson’s disease caused by a lesion
of the substantia nigra of the basal ganglia. Correct and automatic movements also require the integration of information from sensory systems and
other parts of the brain. Consider that cup of coffee again. We use visual
information to determine the distance to the cup and to estimate the force we
need to apply to lift it. When grabbing it we use tactile information to correct
for the actual weight. Maybe the cup is unexpectedly hot! That will activate
yet another motor program that instead of lifting the cup causes withdrawal
of the arm. This type of sensory information is processed in other parts of the
brain such as the visual cortices and thalamus, and from these brain regions,
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the information is fed to the structures regulating movement. When considering such indirect input as a part of the neural correlates of movement it can
be proposed that the great majority of the brain is involved in shaping
movements.
For this thesis on neural control of movement, focus is on the structures in
this schema that are in direct contact with muscles. These are the motor neurons, which activate muscles, and proprioceptive sensory neurons, which
provide the CNS with feedback on muscle activity.
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Background
Neural control of movement
Before going in to the details of the spinal circuits that are the focus of this
thesis, the principles and circuits that underlie movement in animals will be
briefly introduced to put the function of spinal circuits into context. Numerous neural circuits in the brain are required to generate the full repertoire of
movements present in an individual (Figure 1). This neural correlate of
movement is organized in a hierarchal system where each level contributes
with information necessary for a specific movement. This has been reviewed
extensively and is only summarized here [1-3].
The foundation of neural control of movement is formed by the motor
neurons. Motor neurons are the only cells in the CNS capable of directly
activating muscles. Thus, the motor neurons forms them the final common
path of neural activity, as proposed by the English neuroscientist Sir Charles
Sherrington [4]. To generate movement, motor neurons integrate information
from the supraspinal and sensory structures and transform it into a precise
temporal and magnitudal activation of muscles.
In direct contact with motor neurons are ensembles of neurons, termed
central pattern generators (CPGs), which are capable of generating a rhythmic and patterned output from a tonic input. The pattern provides the
framework for motor neuron activation that coordinates muscles for stereotypic movements such as breathing, chewing and swimming. Each stereotypic movement pattern is generated by a specific CPG that is located in the
brain stem or spinal cord. Of particular interest for this thesis is the CPG that
creates the pattern for hindlimb activation during locomotion. This CPG is
located in the ventral spinal cord at lumbar levels.
Although the activity generated by CPGs sets the basic rules for motor
neuron activation during movements, to adapt to a changing environment
this activity must be modulated. Sensory structures and descending pathways
from the brain stem are therefore in direct and indirect contact with motor
neurons and CPGs to modulate its activity. The modulation entails both correcting for external perturbations, setting the speed and steering and well as
initiation of CPG activity.
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Figure 1. Schematic illustration of a cross-section of the rodent brain. Shaded
areas indicate regions involved in motor control.
In direct contact with the spinal cord is also the motor cortex. This structure
is involved in the conscious control of movement. The direct connection to
the spinal cord is used to directly modulate movements. The motor cortex is
also involved in the planning of movements. To accomplish these functions,
inputs from most areas of the brain converge in the motor cortex and the
motor cortex in turn project to many brain areas.
Two such areas are the basal ganglia and cerebellum. Basal ganglia is involved in the selection of motor programs by balancing a permissive and
inhibitory effect on motor programs. When this balance shifts toward increased inhibitory influence, it is manifested as trouble initiating movements
in a Parkinson’s patient. In contrast, when the balance is shifted toward permissive function, it can be illustrated by the constant involuntary movements
of a patient suffering from Huntington’s disease. Both are neurodegenerative
diseases that affect different parts of the basal ganglia. Sensory information
from all senses are integrated in the cerebellum which creates an output that
modulates ongoing activity to control the timing, duration and amplitude of
movements.
Thus, the neural basis of movement is a flow of motor and sensory information that is processed at several levels in many structures at the same
time. However, all this information must inevitably end up on motor neurons
to generate an effect on the motor behavior of the organism.
The motor neuron
Several cell types in the brain are called motor neurons. These include the
upper motor neurons that transmit motor information within the brain and
the preganglionic motor neurons of the autonomic nervous system. However,
for this thesis a motor neuron is defined as a neuron with the cell body in the
central nervous system that projects its axon to skeletal muscles. This somatic motor neuron is a classical experimental model in neuroscience. It was
from motor neurons that the first demonstration of chemical neurotransmission was made [5] and that neurotransmitter release is quantal and vesicular
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[6]. It was also by studying sensorimotor synapses the molecular principles
of memory storage was described, and it was by recording from motor neurons that Sir John Eccles could propose the existence of the axon initial segment and demonstrate the first chemical synapse in the central nervous system on the Renshaw cell [4, 7].
A motor neuron innervates dozens to hundreds of muscle fibers in one
muscle. The motor neuron and all the muscle fibers it innervates is called a
motor unit. A train of action potentials in a motor neuron causes release of
acetylcholine at all muscle fibers it innervates with high fidelity. The motor
neuron synapse on the muscle fiber is called the neuromuscular junction
(NMJ). Release of acetylcholine at the NMJ activates nicotinic receptors on
the muscle fiber, which will start a cascade of signaling events that leads to
the contraction of the muscle fiber. This is the essential function of the motor
neuron: to evoke muscle fiber contraction.
Figure 2. Schematic illustration of the organization of motor neurons in the
spinal cord. (A) A spinal cord with cervical, thoracic, lumbar and sacral parts
marked. MMC motor neurons are located at all levels of the spinal cord, LMC motor
neurons are located only at cervical and lumbar levels. Motor pools are subgroups of
the MMC and LMC that innervate one muscle (B) Organization of motor columns in
the spinal cord at cervical, thoracic and lumbar levels. Stippled line in (A) denotes
level of cross section (C) LMC motor neurons innervate muscles in the limb (left)
and MMC motor neurons innervate muscles in the trunk (right). (D) Within the
motor columns, individual motor pools innervate different muscles.
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Organization of motor neurons
Motor neurons are located along the entire length of the spinal cord and in
discrete nuclei in the brain stem (Figure 2). The spinal motor neurons innervate the muscles of the body while the brain stem motor neurons innervate
the muscles of the eye, face, neck, mouth and throat. In the spinal cord, motor neurons are organized in columns based on the location of their target
muscles. Motor neurons in the lateral motor column (LMC) innervate the
limbs and motor neurons in the median motor column (MMC) innervate the
trunk. The LMC innervating the arms or forelimbs are located on cervical
levels, while the LMC innervating legs or hindlimbs are located on lumbar
levels and the MMC is present at most levels of the spinal cord.
Within each column, the motor neurons innervating one specific muscle are
clustered in motor pools. The motor pool forms a cigar-shaped entity that
spans a couple of segments of the spinal cord. One motor pool contains 20300 motor neurons depending on the muscle [8].
Motor neuron subtypes
Within each motor pool, three subtypes of motor neurons exist: alpha, beta
and gamma. They are classified based on which type of muscle fiber they
innervate (Figure 3). Alpha motor neurons are the archetypal motor neurons.
They innervate the force-generating muscle fibers and their activity leads to
contraction of muscles. In contrast, activity in gamma motor neurons causes
no change in the total length of the muscle. Instead, gamma motor neurons
innervate a specialized type of muscle fiber called the muscle spindle, which
serves as a muscle length sensor. Beta motor neurons form synapses on both
force generating muscle fibers and muscle spindle fiber.
Figure 3. Schematic illustration of subtypes of motor neurons. Alpha motor neuron innervating extrafusal force-generating muscle fiber, gamma motor neuron innervating intrafusal muscle spindle fiber and beta motor neuron innervating both
extra- and intrafusal muscle fiber.
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Alpha, beta and gamma motor neurons lie intermingled with each other in
the motor pool [9]. Morphologically, both alpha and gamma motor neurons
have large dendritic trees [10]. However, looking at the soma area, gamma
motor neurons are smaller than alpha motor neurons and have a thinner axon, which is reflected in a slower conduction velocity. Alpha and gamma
motor neurons also receive different synaptic inputs. Alphas receive socalled C-boutons and M-boutons contacting their soma, while gammas do
not [11, 12]. C-boutons are cholinergic terminals derived from partition cells
(see discussion) and M-boutons are from Ia sensory neurons [13, 14].
Slow and fast motor units
Alpha motor neurons can be further subdivided based on the type of muscle
fiber it innervates. Mammalian muscles are composed of two types of muscle
fiber: slow-twitch and fast-twitch. The basis for this classification is the duration of the twitch contraction time [15]. Slow fibers (S) also generate a
smaller force and are more resistant to fatigue than fast fibers. Fast motor
neurons can be further subdivided into fatigable (FF) and fatigue-resistant
(FR) based on the duration they can sustain contraction. Most mammalian
muscles consist of a mix of slow and fast fibers in different ratios depending
on how the muscle is used. This can be illustrated by two muscles in the
calves: gastrocnemicus and soleus. Gastrocnemicus, which is used for example for jumping, has a low slow/fast ratio while soleus, which is used for postural control, has a higher slow/fast ratio [16].
The motor neurons innervating slow and fast muscle fibers exhibit discrete properties. This was first shown by Eccles et al for afterhyperpolarization (AHP) duration [17]. AHP is the phenomena where the
membrane potential after an action potential undershoots the resting membrane potential. Slow motor neurons have a longer after-hyperpolarization
time than fast motor neurons. The functional consequence of this is that slow
motor neurons have a longer “waiting period” between action potentials and
thus cannot fire in the same frequency as fast motor neurons.
Subsequent recordings from both cat and rat have shown that fast and slow
motor neurons differ in other electrical properties [18, 19]. These include input
resistance, a measure of the resistance over the plasma membrane, and rheobase, a measure of the current needed to generate an action potential. Slow
motor neurons have a higher input resistance, which is one of the underpinnings of Henneman’s size principle of motor unit recruitment. Henneman’s
size principle dictates that slow (smaller) motor units will be recruited first
when activating a muscle, after that (larger) fast fatigue-resistant and then fast
fatigable motor units will follow [20, 21]. Thus, a slow movement generating
smaller force will include only slow motor units, while increasing the speed or
force of the movement will include fast motor units as well.
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Development of motor neurons
The CNS contains a great diversity of different cell types. Just in the spinal
cord, it is estimated that hundreds of different cell types are formed during
development, each with a different character [22]. As has already been outlined, motor neurons are categorized as alpha, beta and gamma. Alphas exist
as slow, fast fatigue-resistant and fast fatigable, and gammas exist as dynamic and static (description of subtypes of gamma motor neurons will follow in next section). In addition, motor neurons are located in different columns and in motor pools, each with a specific target muscle.
Neural diversity is created in the spinal cord by the generation of distinct
progenitor domains in the dorsoventral axis. The timing and concentration of
morphogen factors such as sonic hedgehog, fibroblast growth factors and
retinoic acid induces the expression of transcription factors that acts mutually exclusive to define specific progenitor domains [23-25]. The progenitor
domain that generates all motor neurons is designated pMN and is defined
by the expression of basic helix-loop-helix protein Olig2 and the homeodomain transcription factors Pax6, Nkx6.1 and Nkx6.2 [26, 27]. When progenitor cells in the pMN exit the cell cycle, they start expressing the homeodomain transcription factors Hb9, Isl1 and Isl2 that defines the generic motor
neuron fate [28-30]. The development of columnar and motor pool identity
is acquired in a similar way where a extracellular morphogen gradient is
translated into a code of in this case Hox transcription factors that act mutually exclusively to define boundaries for the motor columns and motor pools
in a process that requires FoxP1 [31-33]. Encoded in these transcription factor networks is the information on where to settle in the spinal cord, what
neurotransmitter to use and how to project the axon in the periphery.
In contrast to the well-defined extrinsic and intrinsic signals that are directing the subtype-specification of motor neurons into columns and pool the
molecular mechanisms that specifies alpha, beta and gamma motor neurons
have not been described. In the adult spinal cord, gamma motor neurons
express Hb9 and Isl1, but if that is also true in the embryonic spinal cord is
not known [34]. However, gamma motor neurons appear to develop later in
embryogenesis than alpha motor neurons as gamma axons reach the muscle
spindles as late as E18.5 in the extensor digitorum longus muscle while alpha motor axons form NMJs at E14.5 [35]. In addition, small putative gamma motor neurons are generated later in development than large putative
alpha motor neurons [36].
Similar to for alpha, beta and gamma motor neuron character, the slow
and fast character of alpha motor neurons has not been described molecularly in the embryo. However, extensive studies on chick embryos has shown
that when given the choice, fast motor axons preferentially innervate fast
motor fibers and vice versa, suggesting that fast and slow motor neurons
develop properties specific for its subtypes to find and synapse with the cor16
responding muscle fiber in the embryo [37-40]. It should be noted though
that although fast and slow motor neurons likely are specified early in spinal
cord development, the adult fast and slow motor units are plastic. Motor
units can be converted from a fast to slow and vice versa by alterations in the
firing frequency of the innervating motor neuron [41, 42]. Thus, the fast and
slow motor neuron character of a motor neuron appear to be specified genetically in the embryo, but can adapt its character to match requirements of
changed activity in the adult.
Degeneration of motor neurons in ALS
Neurodegenerative diseases are characterized by a progressive loss of neural
function. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease
that affects the motor neurons. People suffering from ALS typically experience muscle weakness that progresses over time and eventually lead to complete paralysis. Although most cases of ALS cannot be attributed to genetic
traits, 5% of the cases are inherited. Of these, 20% have a mutation in the
enzyme superoxide dismutase 1 (Sod1) [43]. Clinically, both sporadic and
inherited ALS has indistinguishable characteristics and mice expressing mutant Sod1 develop a disease similar to human ALS [44].
In ALS, both the upper and lower motor neurons degenerate by unknown
mechanisms. A number of pathologies have been identified in both humans
and mouse models of the disease including protein aggregates, excitotoxicity
and deficiencies in retrograde transport [44]. However, it is not established
whether these pathologies are causing disease or are the result of disease. Of
particular interest for this thesis is the excitotoxicity observed in motor neurons during disease. Excitotoxicity is caused by an excessive glutamatereceptor activation leading to a massive Ca2+ influx that will cause damage
to the mitochondria and eventually apoptosis. In ALS patients and the
Sod1G85A mouse model of ALS, diminished glutamate transport has been
reported as well as a reduced expression of the glutamate transporter EAAT2 in the motor cortex and spinal cord [45, 46]. In addition, mice heterozygous for EAAT-2 expression develop disease earlier than littermates while
drugs that enhance EAAT2 activity prolong life span [47-49].
Another feature of degeneration of motor neurons in ALS of interest for
this thesis is the selective degeneration of subtypes of motor neurons. In
ALS patients as well as mouse models of ALS, the degeneration of motor
axons is predictable and follows a scheme of decay [50, 51]. In this scheme,
FF motor units degenerate first at P50, FR follows at P80 and S units remain
until death [51]. Thus, selective vulnerability of motor neuron subtypes appears to be present in ALS.
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Proprioception
We constantly have a sense of where our limbs and body are located in relation to each other in the three-dimensional space. Even with our eyes closed,
and even those blind from birth, can tell if their arm hanging along the side
of the body or extended into the air. The sense that makes this possible is
called proprioception. Unlike the classical senses like vision and olfaction
that transmits signals about the state of the external world to the brain, proprioceptive sensors reports how the limbs, trunk and head are positioned and
moving in relation to each other.
Proprioception is not necessary for movement, but is required for a
smooth and automatic movement. This can be illustrated by the story of Ian
Waterman, who suffered an unusual degeneration of proprioceptive sensory
neurons [52]. The degeneration, likely caused by a viral infection, led to a
specific loss of proprioceptive signaling below the neck while sparing other
somatosensory modalities such as pain, temperature and deep pressure. Just
after the loss of proprioception, Waterman was unable to maintain his posture or to initiate voluntary movements suggesting that proprioceptive information is an integral part of the neural activity that generates an upright
position. Through practice, he would learn to move and eventually walk
again, although his gait lacks the fluidity and dynamics of normal gait [53].
The most striking thing with his newly developed gait though, is that every
step is a conscious act that requires constant visual feedback for him to control his limbs.
Proprioception is dependent on information from joints, skin and muscles.
The modalities signaled by these structures include joint extension/flexion,
skin stretch, muscle stretch and muscle force [54, 55]. This information is
signaled from specialized structures in the target organs to the spinal cord
where it is integrated in spinal circuits that relay the signal to the brain or
create a motor output. For this thesis, the proprioceptive information on
muscle stretch is under focus. This information comes from the muscle spindles.
Anatomy and physiology of the muscle spindle
The muscle spindle is a fusiform encapsulated organ that is embedded within
muscles [56]. It is composed of a specialized type of intrafusal muscle fiber.
Muscle spindles are positioned in parallel with the force-generating extrafusal muscle fibers. They are predominantly found within the deep belly
region of the muscle, where slow muscle fibers predominate. In cats, the
number of spindles per muscle range from 10 in a small muscle to over 100
in a large muscle and the total amount in all human muscles is estimated to
be 25 000 muscle spindles [57, 58].
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Figure 4. Schematic illustration of muscle spindle innervation. Ia afferents forms
annular terminations in the central domain. Group II afferents form “flower-spray”
terminations adjacent to Ia afferent and gamma and beta motor neurons forms synapses at the polar ends.
When a muscle is stretched, the muscle spindle is also stretched. This activates sensory neurons in contact with the muscle spindle. Information from
the muscle spindle is transmitted to the spinal cord by two types of sensory
neurons: group Ia and group II. Group Ia afferents form annular terminations
around the central domain of the muscle spindle, while group afferents form
so-called flower spray terminations (Figure 4).
Group Ia and II afferents signal different overlapping modalities of muscle stretch. When a passive muscle at length x is stretched to a new length y,
Group Ia afferents fire action potentials as the muscle is being stretched. The
frequency of this signal is dependent on the velocity of the stretch. Thus, Ia
afferents provide the CNS with information on the velocity of length change.
Group II afferents on the other hand signals at one frequency at length x and
at a higher frequency at length y. Thus group II afferents will provide the
CNS with information on how stretched a muscle is.
As previously mentioned, muscle spindles also receive motor innervation
from beta and gamma motor neurons. A signal from a gamma or beta motor
neuron contracts the muscle spindle fiber. This contraction is so small that it
does not generate a contraction of the whole muscle, but merely lead to contraction of the muscle spindle. This contraction leads to increased sensitivity
of group Ia and II sensory afferents. Subtypes of gamma motor neuron have
been described that relate to the modality of muscle spindle stretch sensitivity they regulate. Dynamic gamma motor neurons increase the sensitivity of
primarily Ia afferents to dynamic changes in muscle spindle length and static
gamma motor neurons increase the sensitivity of primarily group II afferents
[59]. This motor innervation provides a system for the CNS to adjust proprioceptive feedback depending on the task. For example walking on level
ground requires less sensitive proprioceptive feedback than balancing on a
plank. Thus, gamma motor neurons are more active in the balancing act than
when walking on the floor.
The motor innervation to muscle spindles also makes sure that muscle
spindles move in concert with extrafusal fibers. If a muscle would contract
while the muscle spindle don’t, then it would slacken and group Ia and II
sensory neurons wouldn’t signal during muscle contraction. Therefore, alpha
19
and gamma motor neurons in one motor pool are often active at the same
time in the locomotor cycle, which is referred to as alpha-gamma linkage.
Proprioceptive circuits
The sensory signals are transmitted to the spinal cord where they are integrated into spinal circuits to modulate ongoing movement and relayed to the
brain. In the spinal cord, group Ia afferent makes contacts with Ia inhibitory
interneurons, dorsal spinocerebellar tract neurons and alpha motor neurons.
Group II afferents contact group II interneurons in the intermediate and deep
dorsal horn and presumably gamma motor neurons (Figure 5) [60-62].
The connection between alpha motor neurons and Ia afferents provides
alpha motor neurons in one motor pool with monosynaptic feedback from
muscle spindles in the same muscle it innervates. This circuit is activated
when testing the knee jerk reflex (also called stretch reflex). Ia afferent information from that same muscle signal to Ia inhibitory interneurons that
inhibit motor neurons innervating antagonist muscles. The third central connection of Ia afferents is to spinocerebellar projection neurons. The spinocerebellar neurons relay proprioceptive signals to the cerebellum.
The group II interneurons are a loosely defined group of interneurons that
innervate alpha and gamma motor neurons and other interneurons in the
spinal cord [63, 64]. The role of the group II interneurons is not well defined,
but given their monosynaptic input from group II afferents about the length
of muscles and close contact with motor neurons they likely have a role in
coordinating muscle activity [64].
Figure 5. Schematic illustration of central terminations in the spinal cord
formed by muscle spindle afferents in the lumbar spinal cord. Group Ia afferents
terminate dorsal to the central canal on Clarke’s column neurons, in the intermediate
horn of Ia inhibitory interneurons and on alpha motor neurons. Group II afferents
terminate in the deep dorsal horn on group II interneurons and possibly in the ventral
horn on gamma motor neurons.
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Group Ia and II afferents are both subject to presynaptic inhibition from local interneurons in the spinal cord as well as descending tracts [65, 66]. Presynaptic inhibition is a mechanism to centrally inhibit sensory signaling
when it is required. Presynaptic inhibition can also serve to change the relative importance of a certain proprioceptive synapse, e.g. by silencing the
synapse between group Ia-alpha motor neurons contacts while Ia-Ia interneuron contacts signal at normal rates.
Development of the monosynaptic stretch reflex
Similar to the development of motor neurons, sensory neurons in the DRG
are specified by the action of morphogens that activate transcriptional programs unique for each sensory neuron subtype [67]. Upon acquiring its character, proprioceptive sensory neurons can be identified by the expression of
the RUNT-domain transcription factor Runx3 and the neurotrophic tyrosine
receptor kinase-C (TrkC). Runx3 is required for proprioceptive neuron survival and acts in a concentration-dependent manner to determine the laminar
termination of sensory axons in the spinal cord [68, 69].
Proprioceptive neurons follow motor axons out to the muscle [70]. The
proprioceptive neurons require the motor neurons, as ablation of motor neurons leads to an absence of muscle innervation of sensory neurons [71].
When entering the muscle, the Ia afferent neuron induces the formation of a
muscle spindle from primary myocytes by signaling through neuregulin 1
[72]. This activates expression of the zinc-finger transcription factor Egr3,
and ETS transcription factors Er81 and Pea3 that serve to differentiate the
muscle spindle [72-74].
Figure 6. Activity-dependent and retrograde influence on development of the
stretch reflex. Reducing the stretch reflex activity leads to increased synaptic
strength in the Ia-motor neurons synapse (left). Removing Nt-3 production from the
muscle spindle lead to reduced synaptic strength in the Ia-motor neurons synapse
(right).
In the spinal cord, Ia afferents project with high precision to the correct motor pool in an activity-independent manner using genetically specified axon
guidance molecules [75-77]. Maturation of the circuit, on the other hand,
forms in an activity-dependent manner involving target-derived molecules
21
from the muscle spindle (Figure 6). Reducing stretch reflex activity by tetrodotoxin application to the nerve, d-tubocurare treatment or axotomy lead
to increased synaptic input from Ia afferents to motor neurons [76, 78, 79].
In muscle-specific ErbB2 knockout and Egr3 knockouts where muscle spindle develop abnormally, appropriate connections form with the correct motor
pool but the dorsal root-evoked EPSP is decreased in amplitude and the
number of proprioceptive synapses are reduced [80]. Muscle spindle-specific
deletion of Nt-3 also leads to reduced reflex amplitude, however it is delayed
compared to spindle-specific ErbB2 knockouts [80]. Thus, Nt-3 is one of the
muscle spindle-derived factors that regulate the amplitude of the monosynaptic stretch reflex, but likely, other factors are involved as well.
Recurrent inhibition
Birdsey Renshaw observed that upon antidromic stimulation of the ventral
root, excitation of motor neurons by Ia afferents were reduced [81, 82]. John
Eccles and colleagues showed that this effect was caused by cholinergic
activation of an inhibitory cell type in the ventral horn, which he named the
Renshaw cell in honor of its discoverer [83].
Figure 7. Schematic illustration of the recurrent inhibition circuit. RC, Renshaw
cell. MN, motor neuron.
The Renshaw cell-Motor neuron circuit is as simple as it is vexing (Figure
7). Concomitantly with sending action potentials to the muscle, alpha motor
neurons signal via an axon collateral to Renshaw cells in the spinal cord.
This activates the Renshaw cell, which respond with a high-frequency burst
that inhibits the same motor neuron. The location of the Renshaw cell close
to the motor neuron, “eavesdropping” on all motor neuron activity, puts it at
a central position for motor output. Still, there is no conclusive data on what
function the Renshaw cell has in movement.
22
The motor neuron uses acetylcholine and another unknown neurotransmitter that activates NMDA receptors to signal to Renshaw cells [84-86].
This generates a high-frequency burst of Renshaw cells that in turn use glycine and GABA to signal to motor neurons [87]. Only alpha motor neurons
activate Renshaw cells, as gamma motor neurons do not have intraspinal
axon collaterals [10]. Different motor unit types activate Renshaw cells with
different intensity, where FF motor neurons generate a larger recurrent IPSP
than S motor neurons, however, the response to this IPSP is equally large in
all motor unit types [88].
Development of recurrent inhibition
Renshaw cells develop from the progenitor (P)1 progenitor domain [89]. As
P1 neurons mature into ventral (V)1 neuron they start expressing the homeodomain transcription factor engrailed 1 (En1) and send axons to motor
neurons in a guidance process involving Netrin [90].
The functional development of Renshaw cells has mainly been studied in
chick embryos where Renshaw cells are called R-interneuron [91]. These
studies have shown that the first axonal connections is likely formed by Rinterneurons on motor neurons and later in development motor neurons send
axonal collaterals to innervate R-interneurons [92]. The connection between
R-interneurons and motor neurons has been proposed as the source of episodes of rhythmic spontaneous activity [93, 94]. In the embryonic nervous
system, such spontaneous activity in developing circuits is believed to shape
the connectivity, synaptic strength and excitability as the circuits mature
[95]. This has been observed in the chick spinal cord where blocking spontaneous activity leads to changes in excitability and synaptic strength [96-98].
In the mouse spinal cord, where this spontaneous activity starts around E12.5
and is mediated by acetylcholine, glycine and GABA [99] disruption of
spontaneous activity by knockout of ChAT leads to abnormal development
of spinal motor circuits and motor axons makes guidance errors in the developing limb [100, 101].
In addition to the suggested role in spontaneous episodes of rhythmic activity in the spinal cord, Renshaw cells have transient features during development. GABA and glycine are excitatory during development, which
means Renshaw cells will exert recurrent facilitation on motor neurons [92].
In addition, Renshaw cells receive synaptic contacts from Ia afferents during
development [102]. The excitatory action of Renshaw cells reverses as chloride homeostasis change in the spinal cord (E15.5 in mouse) and the Ia afferent input to Renshaw cells wane [102, 103]. Thus, it is possible that
Renshaw cells have a specific role during development.
23
Aims
The aims of the work in this thesis are:
To find and characterize novel molecular markers expressed in subpopulations of neurons involved in locomotion
To determine how expression of the fast and slow motor neuron markers
Chodl, Calca and ERR are affected in the Sod1G93A mouse model of
ALS
To determine the role of 5-ht1d in development of proprioceptive circuits
To determine the contribution of VIAAT-mediated Renshaw cell signaling to development of spinal locomotor circuits
24
Methodological considerations
This section provides a short discussion of the principles and validity of the
methods used in the experiments for this thesis. Detailed information of the
procedures can be found in the respective paper. Only methods performed by
the author of this thesis are presented.
Mice
The experiments presented in this thesis were conducted on mice. All procedures were approved by the local Swedish ethical committee (permit C147/7
and C79/9) or by the University of Calgary Animal Care Committee. Mice
were chosen as the model system because they are available for transgenic
use and they are an established model system for locomotion with welldescribed histological, electrophysiological and behavioral protocols. Although other species are equally well suited for transgenic use, such as Drosophila melanogaster, C. elegans and zebrafish, these systems are not as
developed for studies of motor behavior. It can be argued that the rat is the
model organism of choice for motor behavior, however it was only until
recently that rats were available for transgenic use [104]. Therefore, to study
neural circuit development and function and to relate this information to
motor behavior the mouse was chosen as the model organism.
Transgenic mice
Transgenic animals have been used in all papers included in this thesis. The
genome of a transgenic mouse has been modified by genetic engineering.
Such modification can be the deletion of a gene (knockout) or insertion of a
foreign gene such as Cre to target floxed alleles or mutant Sod1 to create a
disease model.
Knockout animals are valuable tools that can be used to study gene function. Removal of a gene of interest and subsequent studies of the cells, circuits and behavior may give information on the function of the gene. However, since most genes are expressed in more than one cell type, a knockout
of a gene may affect several cell types other the one being studied. By default, the gene is also knocked out already from conception, which means
that eventual compensations for the knockout during embryonic develop25
ment always need to be considered. In these instances, the Cre/lox-system is
useful. Cre is a site-specific recombinase that recognizes a 34 bp sequence
called loxP. If loxP-sites are introduced flanking a gene of interest and Cre is
expressed from a promoter specific for the cell of interest, it will generate a
conditional knockout of the gene only in cells where both promoters are
active at the same time. In addition to creating cell-specific knockout, the
Cre/lox system can also be used to drive expression of reporter molecules
such as GFP that facilitates finding a cell type for patch-clamp recordings or
to visualize cell bodies and neurites.
Mouse lines
For in situ hybridizations, immunohistochemistry, retrograde tracings and
microarray experiments of wild type tissue, the inbred C57/BL6 line was
used. For electrophysiology in paper I Swiss Webster mice was used. The
transgenic mouse lines used in the thesis are described in Table 1. Transgenic animals were genotyped using PCR on isolated genomic DNA using
the primers described in papers I-IV.
Histological staining procedures
In situ hybridization
In situ hybridization is a technique for labeling nucleic acids by taking advantage of their ability to bind to a complementary strand of nucleic acid
[105, 106]. The complementary nucleic acid, called a probe, is labeled with
an epitope tag. An antibody coupled to an enzyme binds to the tag and when
a substrate for the enzyme is added, the location where the
probe/antibody/enzyme-complex has bound is visualized. For the experiments in this thesis, both chromogen and fluorescent substrates have been
used interchangeably without any noticeable difference in sensitivity. The
fluorescent staining works better to combine with immunohistochemistry.
In Paper I, double in situ hybridization was used. In double in situ hybridization, two transcripts can be located on the same tissue sample and
stained in different colors. First, an antibody complex binding to one of the
probes is developed in one color. Then the enzyme is inactivated and the
antibody/enzyme complex recognizing the second probe is developed in
another color. If inactivation is incomplete, the first probe will also develop
together with the second probe, which means careful controls are required
for double in situ hybridization.
26
Table 1. Transgenic mouse lines used in this thesis
Name
Locus
Transgene description
Obtained from
Reference
Targeted mutation of the
zinc-finger DNA-binding
domain and carboxy terminal of Egr3
Warren Tourtellotte
(Northwestern
Egr3-/Egr3
University)
[74]
James F Martin
(Texas A&M System Health Science
Center)
Pitx2-Cre
Pitx2
[107]
Targeted integration of a
Silvia Arber (Frielox-STOP-lox-mGFP-IRES- drich Miescher
NLS-LacZ-pA cassette into Institute for BioMapt
exon 2 of the Mapt gene.
medical Research)
[108]
TaumGFP-nlslacZ
Exons 4, 5 and 6 were
Vglut2lx/lx
Slc17a6
flanked with loxP sites
Generated in our lab [109]
A plasmid with Sod1G93A
driven by the human Sod1
Jackson labs, Bar
[110]
Sod1G93A
Random
promoter
Harbour, ME
A plasmid with Cre driven
by the PGK promoter
PGK-Cre
Random
[111]
Targeted mutation of the
MMRRC/Lexicon
coding exon of Htr1d.
genetics
5-ht1d-/Htr1d
[112]
In a BAC containing the
htr1d gene, htr1d was re5-ht1d::GFP
Random
placed with GFP
MMRRC/GENSAT [113]
In a BAC containing the
Chrna2 gene, Chrna2 was
Chrna2::Cre
Random
replaced with Cre
Generated in our lab
Slc32a1
Exon 2 of the Slc32a1 gene Bradford B. Lowell [114]
Viaatlx/lx
was flanked with loxP sites (Harvard Medical
School)
R26::lsl.Tomato Gt(ROSA)2 A CAG-lox-stop-loxAllen brain institute [115]
6Sor
tdTomato-WPRE-pA plasmid was inserted into the
Gt(ROSA)26Sor locus
Immunofluorescence
Immunofluorescence takes advantage of the specific binding of an antibody
to its epitope. The technique is useful to determine the localization of proteins in a tissue sample.
In this thesis, immunofluorescence has been used to co-localize proteins
in synapses and cell body and to visualize axons in the muscle. Immunofluorescence has also been combined with retrograde tracings, in situ hybridization and fluorescent protein expression from transgenic mice.
When using antibodies it is important to be careful in the interpretation of
stainings [116]. Antibodies are notorious for producing background and unspecific staining. In this thesis, precaution has been made to make sure stain-
27
ing is representative by comparing them to previously reported expression
pattern and with matching in situ hybridizations.
Quantification of stainings
Quantification of stainings in the current thesis has been performed manually
after in situ hybridization or immunofluorescence.
To make confident estimates of the number of cells expressing a certain
gene after in situ hybridization, only cells with a complete staining around
the nucleus was counted. When possible, we only counted sections from the
same spinal segment (e.g. L1-L3), however some quantification was made
from the same column (e.g. lumbar LMC).
The number of synapses in contact with motor neurons or Renshaw cells
was quantified in Paper III and IV. A synapse was in most stainings defined
by the expression of the synaptic protein synaptophysin [117]. If co-staining
with synaptophysin was not performed, a synapse was defined as a bulging
of a stained axon in close contact with a ChAT-stained motor neuron (e.g.
Calbindin and Tomato) or by strong expression of a subtype-specific vesicular transporter (VAChT). To obtain comparable results, the synapses quantified were from the same level of the spinal cord and the confocal images
were of the same optical thickness and number. The same criteria were used
for the quantification of muscle spindle innervation.
In paper I and III, soma area of motor neurons were measured after in situ
hybridization with a probe binding VAChT mRNA. Care was taken not to
overdevelop the staining as this could lead to an overestimation of soma size.
Soma areas were measured by manually drawing the outline of the stained
area as illustrated in figure 3L in paper I. Soma area quantifications were
performed using either Volocity software (Paper I) or ImageJ software (Paper III).
Ventral root electrophysiology
With electrophysiology, the flow of ions across the cell membrane that constitute the basis for neural activity is measured. By attaching an electrode to
the ventral root, the population activity of motor neurons projecting through
that root can be measured. For the experiments in this thesis, the spinal cord
was dissected from the animal and kept alive for several hours in artificial
cerebrospinal fluid (aCSF).
28
Stretch reflex measurements
Figure 8. Schematic illustration of experimental setup for ventral root electrophysiology. (A) For measurement of stretch reflexes a stimulus electrode is attached
to the dorsal root and recording electrode to the ventral root. The response is recorded in the ventral root to dorsal root stimulation. (B) For measurement of druginduced fictive locomotion electrodes are attached to left and right L2 and L5 ventral
roots. The spinal cord is perfused with oxygenated aCSF containing 5 m NMDA
and 10 m 5-HT. This induces a CPG rhythm of left-right and flexion extension
coordination.
To measure sensorimotor connections, a recording electrode is attached to
the ventral root and a stimulus electrode is attached to the dorsal root (Figure
8). Experiments were performed in hemisected spinal cords to ensure oxygenation and to avoid presynaptic inhibition from contralateral sources
[118]. A 0.2 ms stimulus pulse of increasing intensity was applied until the
amplitude of the response in the ventral root did not increase any more. This
was defined as the maximal amplitude. For recordings, a stimulus 1.5x of the
lowest stimulus that generated maximal amplitude was applied to ensure that
all Ia synapses were activated.
Stimulation of the dorsal root with this intensity activates motor neurons
and many interneurons in the spinal cord. The recorded response in the ventral root is seen as a short first deflection and a longer second deflection. The
first deflection with an onset 6 ms after stimulation, mainly corresponds to
a monosynaptic activation. The second deflection corresponds to polysynaptic activation.
The response in the ventral root depends on several factors: synaptic
strength, number of Ia synapses, number of motor neurons, membrane prop-
29
erties of motor neurons, number of afferents activated (tightness of dorsal
electrode) and recording of response (tightness of ventral electrode).
Two stimulus protocols have been used. In paper III stimulus pulses were
given at 1 Hz and in paper IV stimulus pulses were given at 0.033 Hz. A
0.1 Hz stimulus induces a short-term depression at the Ia-MN synapse,
which means that the maximal response will be underestimated and the synaptic response recorded in the ventral root will be affected, by such shortterm depression [119].
Fictive locomotion
The ability of the locomotor CPG in the spinal cord to evoke rhythmic activity in the absence of sensory input or descending drive is taken advantage of
in this method. Electrodes are attached to the ventral roots of the isolated
spinal cord to measure the signals of motor axons (Figure 8). Usually, lumbar level (L)2 and L5 are used. L2 innervates mainly hip flexor muscles and
L5 innervates hip and ankle extensor as well as some hip flexor muscles [8,
120]. When serotonin and NMDA are added, the activity in the ventral roots
is similar to the activity that is seen during locomotion with an alternation of
left-right and flexion-extension [121].
The output from the ventral roots is a rhythmic activity that is coordinated. To analyze the frequency of the rhythm, the phase relationship and the
coherence we used continuous wavelet transform. This gives a timefrequency representation of the recorded rhythm that can analyze long
stretches of data [122, 123]. Before this type of analysis, the raw data was
rectified. Analysis was performed in the Matlab-based software Spinalcore
[123].
To analyze individual burst parameters we applied a time-series analysis
using a custom-written program in Matlab [124]. To calculate the burst duration data was high-pass filtered at 0.1 Hz, rectified and low-pass filtered at 5
Hz. An algorithm was used to determine the length of time between the increase in ventral root discharge rate to half-maximum and the following
decrease to half-maximum. This defined the number of bursts and the burst
duration and from this data, interburst duration and cycle period was calculated.
Behavioral testing
To study behavior is to measure the product of neural activity in the complete organism. To unambiguously relate activity in a specific set of neurons
to behavior should provide a good estimate of the function of that set of neurons for the organism. In this thesis, various motor tests have been per30
formed to test how alterations in proprioceptive signaling and recurrent inhibition translate into deficiencies in movements.
Rotarod
The rotarod was designed to measure neurological deficits in rats and mice
[125]. The mouse is placed on a steady rod ( 1¼ ) and then the rod starts
rotating. The rotation speed is either constant (Paper IV) or linearly accelerating from 0-45 rpm over 60 seconds (Paper III and paper IV). The latency
until the mouse falls down is measured.
To stay on the rotating rod the mouse has to coordinate its body and balance itself while compensating for the rotation of the rod. In the accelerating
rotarod, the mouse has to compensate for the acceleration as well. Deficiencies in rotarod performance have been observed in mice with connectivity
deficiencies of En1+ cell including Renshaw cells and in TrkC mice lacking
proprioceptive sensory neurons [126, 127].
Beam Walking
The beam walking test was designed to measure motor cortex deficiencies in
rats but have later been shown to measure locomotor deficit in mice as well
[128, 129]. The mouse is put in one end of a one meter long round wooden
stick. The starting point is brightly lit and the other end is darker with and
enclosed space containing food pellets. Each mouse was given three trials to
complete the task in 15 seconds. If the mouse was not crossing the beam, it
was counted as a failed trial. While the mouse runs to the other end of the
beam, it is videotaped from behind. The videotaped is analyzed for foot slips
of the hindlegs. Data is presented as slips per successful trial. If a mouse
didn’t have any successful trials, it was removed from analysis.
To be able to cross the beam, the mouse has to maintain its equilibrium,
with a narrow base of support. In addition, correct foot placement is constrained to the narrow beam. Impaired behavior in beam walking have been
observed in rats treated with pyridoxine, which cause degeneration of proprioceptive sensory neurons [130].
Hanging wire
Hanging wire is a test of motor strength. The mouse is put on a horizontal thin
steel wire with its forepaws and is carefully released by the tail. Scoring is
made by judging if the mouse manages to pull itself up with at least one of its
hind paws onto the steel wire directly ( 1 second) of if it struggles ( 1 second). Each mouse is tested three times and the best trial is used for analysis.
To accomplish this task the mouse has to pull its own weight and coordinate
its body to grab the steel wire with the hindpaws.
31
Grip strength
To measure maximal grip strength the mouse grips a wire mesh with its
forepaws and is pulled back horizontally by a steady slow movement. The
force detector records the force when the mouse releases the grip. Each
mouse was tested 5 times and the average value was used for analysis.
Digigait
The Digigait system (Mouse specifics) was used to analyze gait [131]. Each
animal was allowed to run on a treadmill with a clear belt while being videotaped from underneath with a high-speed camera. 1000 frames (10 seconds)
of video were analyzed using the Treadscan software (Mouse specifics) to
detect stance and swing phase for all limbs in each frame. The software determines the time each paw is placed on the belt and from this extracts basic
gait parameters such as stride length, swing and stance phases, and stepping
frequency.
32
Result summary
Paper I
Paper I describes a screen for genes expressed in subpopulations of cells in
the ventral spinal cord. By comparing gene expression in the ventral compared to the dorsal lumbar spinal cord using a microarray hybridization approach and by selecting interesting candidates from the digital gene expression atlas Genepaint, a shortlist of 341 candidate genes was generated. The
specific expression of these genes was analyzed using in situ hybridization.
The in situ hybridizations revealed that among the 341 genes, 57 were expressed in a pattern reminiscent of cholinergic neurons (including motor
neurons) and 102 were expressed in a pattern suggesting expression in both
motor neurons and non-cholinergic interneurons.
Three of these genes, Chondrolectin (Chodl), Calcitonin/Calcitoninrelated polypeptide alpha (Calca) and estrogen-related receptor beta (ERR )
were expressed in a pattern suggesting they were specific to subpopulations
of motor neurons and one gene, paired-like homeodomain transcription factor (Pitx2) in cholinergic interneurons. All these genes were co-expressed
with the vesicular acetylcholine transporter (VAChT), which suggest that
they use acetylcholine as their neurotransmitter.
Double in situ hybridizations revealed that Calca and Chodl were expressed in the same motor neurons whereas ERR was expressed in a complementary population of motor neurons. The Calca+/Chodl+ motor neurons
had a larger soma area than the ERR + motor neurons and whole-cell patch
clamp-recordings revealed that Chodl+ motor neurons had a shorter afterhyperpolarization half-decay and a larger rheobase current. These data suggest that the Calca+/Chodl+ motor neurons represent a population of fast
motor neurons. The complementary expression pattern of ERR in small
motor neurons taken together with an observation that ERR -expression
remains in early growth factor 3 (Egr3) mutant mouse which lacks gamma
motor neurons suggest that ERR is expressed by slow motor neurons. Expression of Chodl started in the motor neuron region at E10.5 and ERR was
expressed from E11.5.
Pitx2 was expressed in cholinergic interneurons close to the central canal.
Expression started at E14.5. At this stage only 20% Pitx2 was expressed in
cholinergic cells. The remaining non-cholinergic cells did not express vesicular inhibitory amino acid transporter (Viaat) and therefore wasn’t inhibi33
tory either. From P11 however, all Pitx2 cells in the lumbar spinal cord were
cholinergic. Genetic tracing of Pitx2-cells using a Pitx2-Cre mouse revealed
that the Pitx2 cells formed cholinergic synapses on motor neurons suggesting they could be the origin of the C-bouton.
Paper II
In paper II, the Sod1G93A mouse model of ALS was crossed with mice heterozygous for vesicular glutamate transporter 2 (Vglut2) to generate
Sod1G93A;Vglut2+/- mice. In Sod1G93A;Vglut2+/- mice, VGLUT2-expression is
reduced and presumably, excitotoxic load on motor neurons is reduced as
well.
In Sod1G93A;Vglut2+/- mice, both survival and disease onset followed a
similar time-course as Sod1G93A;Vglut2+/+. However, motor neuron survival
was increased in the Sod1G93A;Vglut2+/- mice measured after in situ hybridization and immunofluorescence. Tibialis anterior also contained more innervated NMJs in Sod1G93A;Vglut2+/- mice. In the brainstem, neurodegeneration
of the facial nucleus was rescued by the Vglut2 mutation whereas motor
neurons in the hypoglossus nucleus were unaffected in both
Sod1G93A;Vglut2+/- and Sod1G93A;Vglut2+/+.
Analysis for the expression of the markers for fast and slow motor neurons described in paper I was also performed. Expression of Chodl was
completely abolished in Sod1G93A;Vglut2+/- and Sod1G93A;Vglut2+/+ whereas
expression of Calca and ERR was reduced but not absent in both mutants.
Paper III
In parallel with the screen described in paper I, another database search was
performed using the GENSAT database. This yielded another marker for a
subpopulation of motor neurons, the serotonin receptor 1d (5-ht1d). 5-ht1d
was co-expressed with VAChT and soma area estimation showed that 5-ht1d
was expressed in the smallest motor neurons. In early growth response 3
(Egr3) knockout mice, the number of motor neurons expressing 5-ht1d was
reduced by 90%. These data suggests that 5-ht1d is expressed by gamma
motor neurons. In addition to this, 5-ht1d was expressed in proprioceptive
sensory neurons.
To characterize the electrical properties of gamma motor neurons wholecell patch clamp recordings was conducted on motor neurons in the 5ht1d::GFP transgenic mouse. No differences were seen between 5ht1d::GFP+ (gamma) and 5-ht1d::GFP- (alpha) motor neurons. However,
when the alpha motor neurons were subdivided into fast and slow motor
34
neurons using hierarchal clustering method gamma motor neurons could be
clustered as a group.
To study the contribution of 5-ht1d to development of proprioceptive circuits 5-ht1d-/- knockout mice was analyzed. In 5-ht1d-/- mice, gamma motor
neurons survived and muscle spindles showed normal innervation by both
gamma motor neurons and sensory neurons.
In 5-ht1d-/- mutants, the monosynaptic activation of motor neurons by Ia
afferents had reduced amplitude. The number of Ia synapses per motor neurons was unaltered, suggesting that the proprioceptive synapses were weaker
in the mutant. Despite the altered stretch reflex amplitude, the adult 5-ht1d-/mice showed normal behavior on an accelerating rotarod. Remarkably, in a
beam walking test 5-ht1d-/- mice had fewer foot faults than 5-ht1d+/+ mice
suggesting improved coordination abilities.
Paper IV
Some markers with specific expression in the ventral spinal cord identified
in paper I were not expressed in motor neurons. In paper IV, one of these
molecular markers was further analyzed. Cholinergic receptor alpha 2
(Chrna2) was expressed in small interneurons in the ventral-most spinal cord
that co-expressed the Ca2+-binding protein Calbindin, a previously described
Renshaw cell marker [132]. A Chrna2::Cre mouse was created to further
study the Chrna2-expressing cells.
Patch-clamp recording from
Chrna2::Cre;lsl.Tomato-expressing cells showed that these cells responded
with a monosynaptic latency to antidromic stimulation of the ventral root. In
addition, Chrna2::Cre;lsl.Tomato-expressing axons contacted motor neurons
with synapses expressing vesicular inhibitory amino acid transporter (Viaat).
These observations suggested that Chrna2 was specific for Renshaw cells.
To examine the role of Renshaw cells in development of spinal motor circuits the Chrna2::Cre mouse was crossed with a mouse with a floxed allele
for Viaat. The VIAAT protein transports GABA and glycine into synaptic
vesicles so tentatively in Chrna2::Cre;Viaatlx/lx mice, Renshaw cells have
deficient neurotransmission. In these mutants, whole-cell patch clamp recordings showed that motor neurons developed with increased action potential threshold and decreased action potential amplitude. Motor neurons also
had reduced capacity to adapt to increasing current injections with a lower
slope of frequency. Other electrophysiological characters such as input resistance, rheobase and AHP half-decay were normal. In addition, motor neurons were also contacted by an increased number of Calbindin+, ChAT+ and
VGLUT1+ synapses. However, measurement of the physiology of VGLUT1+
synapses by recording the stretch reflex response showed that despite the
increased synaptic number, the stretch reflex amplitude was normal in
Chrna2::Cre;Viaatlx/lx mice.
35
Drug-induced fictive locomotion was also normal in the mutant and application of nicotinic antagonist Mecamylamine, which blocks the activation
of Renshaw cells by motor neurons, had similar effects in both mutant and
control. In addition, analysis of motor behavior of the adult
Chrna2::Cre;Viaatlx/lx mice showed no difference including the ability to
maintain balance at different speeds (steady rotarod), ability to maintain
balance as speed increases (accelerating rotarod), and ability for correct foot
placement balance (beam walking). No difference was seen in stride length,
stance and swing phase, steeping frequency or other gait parameters either.
36
Discussion
Novel markers for subtypes of motor neurons and
premotor interneurons
Figure 9. Novel molecular markers for subtypes of motor neurons and premotor interneurons presented in this thesis. RC, Renshaw cell. F, fast alpha motor
neurons. S?, putative slow alpha motor neuron. , gamma motor neuron. P, partition
cell. PN, proprioceptive afferent neuron
In this thesis, novel markers are presented for fast alpha motor neurons,
gamma motor neurons, Renshaw cells, putative slow motor neurons and
medial partition cells (Figure 9). The identification of these genes forms the
foundation on which the rest of the work in this thesis stands on. Identification of genes expressed by specific cell populations are valuable for studies
37
of the function of a cell, both as a tool to drive expression of transgenic
tools, as a marker of a cell population in histology and by studying the function of the protein.
Fast and slow motor neurons
In paper I, it is presented that Chodl is expressed in a subtype of large motor
neurons. Patch-clamp recordings from CHODL-expressing cells show that
they have a shorter AHP half-decay and a larger rheobase current. These are
features suggesting that Chodl is expressed in fast motor neurons [18, 19,
133, 134]. Data is also presented that Calcitonin/Calcitonin gene related
polypeptide alpha (Calca) is expressed in the same motor neurons as Chodl.
The Calca mRNA is spliced into the neuropeptide CGRP in the brain [135].
Observation has been made that CGRP is expressed at NMJs on fast muscle
fibers in rats, and is specifically enriched in NMJs innervating fast fatigable
muscle fibers [136]. From the data presented in paper I no conclusion on the
subtype-specificity of Calca/Chodl-expression in fast motor neurons can be
drawn. Thus, Calca and Chodl mark a population of fast motor neurons in
the mouse spinal cord.
ERR is expressed in a complementary motor neuron population to Chodl
and Calca with a small soma area. Expression of ERR mRNA persists in
Egr3-/- suggesting that ERR is not expressed by gamma motor neurons.
Based on these observation it is suggested in paper I that ERR is expressed
by slow motor neurons. However, in light of the recent finding that beta
motor neurons persist in the Egr3-/- knockout it cannot be ruled out that
ERR is expressed by this motor neuron cell population as well.
Expression of ERR is detected in motor neurons already from E11.5 and
Chodl is detected in the motor neuron region already from E10.5. Despite
extensive studies on motor neuron development over the past two decades,
the mechanisms inducing the fast and slow character of motor neurons are
largely unknown. The identification of ERR and Chodl expressed in this
pattern at this time point of development may provide clues into fast/slow
differentiation. ERR is an orphan nuclear receptor that has a role in the
differentiation of rod photoreceptors and endolymph-producing epithelia in
the inner ear [137, 138]. Notably, in the photoreceptors of ERR knockouts,
expression of several genes involved in energy metabolism as well as in ion
transport is downregulated, and in the inner ear ion channels and transporters
are downregulated. These genes encode two neural characters, membrane
properties and metabolic load, where fast and slow motor neurons are different and would be expected to express a different set of genes [139, 140].
The function of the CHODL protein is currently unknown. Chodl mRNA
is alternatively spliced into transmembrane or secreted isoforms that all carry
a C-type lectin domain [141, 142]. Proteins with C-type lectin domains have
been implicated in cell migration and cell-cell interaction [143], and CLEC38
38, a Caenorhabditis elegans with a c-type lectin, modulates unc-40/DCCmediated axon outgrowth and presynaptic patterning [144]. Thus, one potential function of CHODL could be the axon guidance of motor neurons.
Gamma motor neurons
5-ht1d is proposed to be specifically expressed by gamma motor neurons
based on the following criteria of known gamma motor neuron character
[145]: 1. Expression in the smallest motor neurons 2. Expression at all levels
of the spinal cord, 3. Loss of expression in Egr3-/- spinal cord. 4. 5-ht1d is
expressed by 28% of motor neurons, which is the percentage of motor neurons previously suggested to have a gamma identity [146, 147].
Recently, ERR (also known as ERR3) has been suggested to be specific
for gamma motor neurons based on soma size, diminished expression in a
mutant mouse which lacks muscle spindles, similar to Egr3-/- mutants and
lack of VGLUT1 synapses on the cell body [146]. In addition, through a
combination of transgenic mice expressing tau-LacZ from the locus of
GDNF receptor GFR 1 (GFR 1-TLZ) and GFP from the Hb9-promoter
(Hb9::GFP) gamma motor neurons can be identified as those expressing
GFR 1-TLZ while not expressing Hb9::GFP. Again, this was based on the
expression in small motor neurons, the diminished expression in Egr3-/- spinal cord and the lack of VGLUT1 input [147].
In contrast to ERR and GFR 1-TLZ/Hb9::GFP, 5-ht1d appears to be
specifically expressed by gamma motor neurons throughout postnatal development. ERR is expressed by most motor neurons until the second postnatal week when it becomes specific for gamma motor neurons and GFR 1TLZ is expressed in beta motor neurons and alpha motor neurons as well. 5ht1d is specific for gamma motor neurons at P11 and 5-ht1d::GFP is expressed in small motor neurons from P0 and onwards. 5-ht1d mRNA could
not be detected in the spinal cord embryonically or at P0 while a robust expression was seen in the DRGs. Nevertheless, 5-ht1d has not been detected
in alpha motor neurons at any stage analyzed, which makes it a more suitable marker for transgenic studies of gamma motor neurons. This is illustrated in paper III where the specific expression of 5-ht1d::GFP allows
patch-clamp recordings from gamma motor neurons.
Both alpha and gamma motor neurons receive serotonergic input and at
least alpha motor neurons express many subtypes of serotonin receptors
[148, 149]. Depending on the subtype, activation of serotonin receptors lead
to distinct downstream signaling events. Activation of 5-HT1D leads to signaling using the Gi/Go system of G-protein coupled receptors (GPCR) that
lead to a reduced excitation through reduced levels of cAMP. However, if
this is the effect of 5-HT1D activation on gamma motor neurons remains to
be experimentally determined.
39
Renshaw cells
Chrna2 is proposed to be specific for Renshaw cells based on the following
criteria for a Renshaw cell character [150]: 1. Expression in the ventral grey
matter of the spinal cord close to the white matter border, 2. Co-expression
with Calbindin, Viaat and large Gephyrin clusters, 3. A mecamylaminesensitive, short-latency monosynaptic response to ventral root stimulation, 4.
VIAAT- and Calbindin-expressing synapses originating from Chrna2:Creexpressing cells on motor neurons.
Previously, Calbindin and lineage tracing in En1-Cre mice have been the
most specific ways to genetically mark Renshaw cell, but these two approaches will also label many non-Renshaw interneurons, likely with integral functions for locomotor control. Therefore, the identification of Chrna2
provides a novel specific way of targeting Renshaw cells for studies of their
function. This is illustrated in paper IV in this thesis, where the specific expression of Chrna2 is used to specifically remove expression of the vesicular
inhibitory amino acid transporter (Viaat) from Renshaw cells. VIAAT
pumps GABA and glycine into synaptic vesicles of inhibitory neurons [151].
Cultured neurons from spinal cord of a full knockout and from hypothalamus
in a hypothalamus-specific knockout fail to produce IPSCs suggesting that a
knockout of VIAAT is enough to diminish neurotransmission with GABA or
glycine [114, 151]. Whether Renshaw cells are deficient in inhibitory neurotransmission has not been assessed by the current study, but extending from
the described studies, we consider it likely that Renshaw cell do not signal
with inhibitory neurotransmitters in Chrna2::Cre;Viaatlx/lx mice [114, 151].
Partition cells
Partition cells are a cell population of cholinergic cells adjacent to the central
canal [152]. Pitx2 is proposed to be specifically expressed in partition cells
based on the following observations: 1. Large cholinergic neurons close to
the central canal that does not co-stain with a retrograde tracer from the ventral root, 2. They form synapses on motor neurons.
Another study was published when paper I was under review, came to the
same conclusion about Pitx2 neuron identity [22]. Zagoraiou et al. establish
that Pitx2 neurons are the source of the C boutons on alpha motor neurons
and that this input is required for the task-dependent modulation of muscle
activation amplitude.
Excitotoxicity and motor neuron subtypes in ALS
Reduced Vglut2 expression did not alter the time of disease onset or death in
the Sod1G93A mouse model of ALS. However, motor neurons and their con40
nection to the NMJ were partially rescued in the spinal cord and fully rescued in the facial nucleus. These observations are similar to what has been
observed previously when extracellular glutamate uptake by astrocytes was
increased by overexpressing the plasma membrane transporter of glutamate,
EAAT-2 [153]. Thus, reducing glutamate affects motor neuron survival, but
does not have a major influence on the general features of disease progression.
In the spinal cord of Sod1G93A mice and Sod1G93A;Vglut2+/ mice, expression of fast motor neuron marker Chodl was not detected, while putative
slow motor neuron marker ERR was reduced in similar ratios as the general
motor neurons markers VAChT and Cht-1. In contrast to the total absence of
Chodl expression, few cells in the motor neuron region expressed Calca in
both mutants. Since Calca and Chodl are expressed by the same subtype of
fast motor neurons, the selective susceptibility of fast motor axon is not directly translated to gene expression in the spinal cord.
Both Chodl and Calca mRNA are subject to alternative splicing [135,
142]. Mutations in RNA-editing enzymes TDP-43 and FUS/TLS has been
described in cases of familial ALS, suggesting that aberrant mRNA processing may be a common link in the pathogenesis of different mutations in ALS
[154]. Notably, aberrant mRNA processing of Chodl mRNA is also seen in
motor neurons of a mouse model of another motor neuron degenerative disease, spinal muscular atrophy [155]. Thus, the absence of Chodl mRNA in
Sod1G93A mice may be a reflection of hypersensitivity of this mRNA to deficiencies in mRNA processing.
Motor neurons in the brain stem appear to be differently affected by disease in Sod1G93A mice [156]. In Sod1G93A;Vglut2+/ mice, the loss of motor
neurons in the facial nucleus was completely rescued. The facial motor neurons innervate muscles responsible for facial expressions and do not have
muscle spindles. Therefore, they also likely lack the Ia afferents, which provide monosynaptic feedback on muscle stretch directly to the motor neurons
[157]. In the rat spinal cord, monosynaptic synapses use VGLUT1 for synaptic transmission and most VGLUT1 input to motor neurons comes from
monosynaptic afferents [158]. If facial motor neurons lack this glutamatergic
monosynaptic input, it is plausible that reducing expression of VGLUT2 will
have a greater impact on reducing glutamate-induced excitotoxicity, which
could explain why we see a complete rescue of these motor neurons.
41
The influence of 5-ht1d and VIAAT-mediated Renshaw
cell signaling on the development of spinal motor
circuits
Apart from their role as signaling molecules in neurotransmission in the
adult CNS, neurotransmitters are involved in development of neural circuits
[159, 160]. The roles neurotransmitter have during development range from
developmentally early events such as stem cell proliferation [161] and axon
guidance [162, 163] to later roles such as the activity-dependent refinement
of neural circuits [95]. In paper III and paper IV we interfere with neurotransmitter signaling in two different ways: in paper III a serotonin receptor
is removed from all cells that express it and in paper IV we remove vesicular
packaging of GABA and glycine in a small defined population. Both these
deletions have implications on motor circuits that suggest a role for them
during development.
Development of the stretch reflex in 5-ht1d-/- knockout mice
In paper III, we demonstrate that full knockouts of 5-ht1d have an almost
50% reduction of the maximal amplitude in the stretch reflex circuit. In 5ht1d-/- mice, Ia afferents form normal annular innervation on muscle spindles
in tibialis anterior and have a normal number of VGLUT1+ Ia synapses on
the soma and proximal dendrites of motor neurons. Thus, in the absence of
5-ht1d, the stretch reflex circuit is anatomically normal while it is defective
in its physiology.
Reduced amplitude of the stretch reflex despite normal amounts of Iamotor neuron synapses can have many causes. These include a lower number of motor neurons, hypoexcitable motor neurons, increased short-term
depression and reduced synaptic strength in the Ia-motor neuron synapse.
The exact number of motor neurons in 5-ht1d mutant mice has not been
counted. However, if a 50% reduction in amplitude were caused by a matching 50% reduction in number of motor neurons, it would be clearly noticeable, as the number of motor neurons per LMC in the segments stained is
approximately 20-30 in a 60 m section. In addition, in soma area estimations we did not find a depletion of any specific subtype, as the histogram of
soma area distributions was the same between 5-ht1d-/- and 5-ht1d+/+ mice.
Therefore, it is improbable that a reduced number of motor neurons are the
cause of the observed reduction of amplitude.
Another possible explanation for the reduced amplitude is that motor neurons develop into a hypoexcitable state in 5-HT1D knockouts. Depletion of
serotonin with PCPA for a short period during postnatal development lead to
an increased input conductance and an increased rheobase, which are signs
of hypoexcitability [164]. The effect of PCPA was only observed in some
42
motor pools as others developed normally. However, in contrast to many
other serotonin receptors, 5-HT1D is not expressed on alpha motor neurons.
Therefore, if alpha motor neurons were hypoexcitable in 5-ht1d-/- mice, it
would be by an indirect mechanism, which makes it less likely.
Figure 10. Two hypotheses of reduced stretch reflex amplitude in 5-ht1d-/- mice.
(A) In peripheral 5-HT hypothesis, serotonin released by muscle spindles regulates
the development of the stretch reflex by signaling via 5-HT1D receptors expressed
by Ia proprioceptive neurons. In 5-ht1d-/- mice, this signaling is absent leading to
altered development of stretch reflex amplitude. (B) In short-term depression hypothesis, a train of action potentials in Ia afferents (>0.1 Hz) activates a short-term
depression of the Ia afferent-motor neuron synapse. This depression mechanism acts
via descending 5-HT neuron that presynaptically signals to Ia afferents. In 5-ht1d-/mice, this signaling is absent leading to increased depression of stretch reflex amplitude.
The monosynaptic EPSP generated in motor neurons upon dorsal rootstimulation is short-term depressed when stimulated at >0.1 Hz [119]. The
exact mechanism behind this depression has not been well described although it involves presynaptic inhibition of Ia afferents and does not involve
signaling with GABA [119]. In the stretch reflex experiments in paper III,
dorsal root afferents were stimulated at 0.5 Hz, which means this short-term
depression mechanism was likely in effect (Figure 10). In the age groups
used (P2-P6), stimulus at 0.5 Hz depresses the amplitude by 50% [119].
Serotonin is known to presynaptically inhibit glutamate-release onto motor
neurons and depress the stretch reflex [165-168]. This depression is blocked
when adding BRL-15572, a selective 5-HT1D antagonist, while BRL-15572
alone has no effect on the amplitude [169]. These results taken together with
43
the observed expression pattern of 5-ht1d in proprioceptive sensory neurons
suggest that serotonin could depress the stretch reflex by acting presynaptically on Ia afferents. From these observations, it is possible that the reduced
amplitude observed in 5-ht1d-/- was an effect of altered short-term depression
in the Ia afferent-motor neurons synapse (Figure 10).
A fourth possible explanation is that peripheral serotonin released from
the muscle spindle might be involved in synapse development of the Iamotor neuron synapse (Figure 10). Synapse development of the Ia-motor
neuron synapse involves signaling both centrally in the CNS and peripherally in the muscle. Muscle spindle-derived signals are necessary for tuning
the synaptic strength of the Ia-MN synapse [170, 171]. Conditional deletion
of ErbB2 from muscle spindles and full knockout of Egr3 lead to abnormal
development of muscle spindles and a reduced stretch reflex amplitude [80,
172]. Muscle spindle-specific deletion of Nt-3 has similar but delayed reduction in stretch reflex amplitude [80]. These observations have led to the suggestion that other muscle spindle-derived factors are involved in tuning the
stretch reflex circuit. In paper III, expression of Tph1, Aadc and VMAT2 in
muscle spindles at postnatal day 2 is presented. Tph1 and Aadc encode the
enzymes that synthesize serotonin from L-Tryptophan. Thus, muscle spindles express the components necessary to produce and release serotonin at
the time when Ia-motor neuron synapses are shaped. While it is possible that
serotonin contribute to the development of the proprioceptive circuitry, the
data presented in this thesis does not distinguish between the proposed hypotheses and they are not mutually exclusive. Future experiments are needed
to address these questions.
Development of spinal motor circuits in the absence of VIAATmediated Renshaw cell signaling
In Chrna2::Cre;Viaatlx/lx mice, the output of both the stretch reflex and locomotor CPG is normal in the isolated spinal cord. In contrast to the normal
activity in motor circuits, motor neurons develop with an altered character in
Chrna2::Cre;Viaatlx/lx mice. Patch-clamp recordings reveal that the difference between membrane potential and action potential threshold is larger
than in Chrna2::Cre;Viaatlx/lx mice compared to controls. This means that a
larger depolarization of the membrane potential is required of the motor
neuron to generate an action potential. This observation suggests that motor
neurons in Chrna2::Cre;Viaatlx/lx mice are less excitable. In addition, the
synaptic density of glutamatergic, cholinergic and Calbindin+ synaptic contacts is increased on soma and proximal dendrite of motor neurons, which
means that more possible synaptic release sites are in contact with motor
neurons Chrna2::Viaatlx/lx mice. These two observations taken together, the
hypoexcitability and increased synaptic density, are two factors that could
44
compensate for each other and enables the motor neuron output to be normal
in Chrna2::Viaatlx/lx mice (Figure 11). In paper IV, we observed an unchanged stretch reflex amplitude, despite increased number of the VGLUT1+
synapses that are specific for the stretch reflex [146, 173].
Figure 11. Model of development of stretch reflex circuit in absence of VIAATmediated Renshaw cell signaling. (Left) Circuit in Viaatlx/lx mouse. RC, Renshaw
cell. MN, motor neuron. (Right) Absence of VIAAT-mediated Renshaw cell signaling lead to increased Ia afferent synapses and reduced excitability. These two parameters compensate for each other and create a normal output of the stretch reflex.
Blocking GABAA receptors in the chick embryo by in ovo application of
Gabazine transiently reduces spontaneous movements that later recover
within 12 hours [98]. Notably, when spontaneous neural activity in the spinal
cord is perturbed by addition of gabazine, motor neurons respond to the perturbation with a change in cellular excitability and matching increase in
quantal mEPSC [97, 98]. Whether the increase in quantal mEPSC is caused
by increased number of synaptic contacts is not established by these studies,
but it is a plausible mechanism [174]. This is similar to what we observe in
Chrna2::Cre;Viaatlx/lx mice. Thus, it is possible that the hypoexcitability of
motor neurons and an increased synaptic input are factors that have been
modulated by homeostatic plasticity signaling mechanisms to ensure that
motor neuron output activity is normal in the absence of VIAAT-mediated
Renshaw cell signaling.
45
Motor behavior in 5-ht1d-/- and Chrna2::Cre;Viaatlx/lx
mice
Experiments performed in reduced preparations such as fictive locomotion
and stretch reflex measurements are useful to determine the physiology of
small entities of neural circuits. However, in the intact animal many more
circuits are participating to generate motor behavior. In paper III and IV,
behavioral assays were applied to assess how the molecular and physiological deficiencies were reflected in the behaving animal.
The stretch reflex amplitude in locomotion
Egr3-/- knockouts and muscle-spindle specific knockouts of Nt-3 and Erbb2
have been described to have a reduced extracellular or EPSP amplitude of
the monosynaptic stretch reflex [74, 80, 172]. These mutants all display an
ataxic gait and abnormal posture [74, 80, 172]. In light of this data, it is surprising that 5-ht1d mutants, who also have reduced stretch reflex amplitude,
do not display any deficiencies in motor coordination. Even when challenged
on a rotarod, these mice behave as control animals, and strikingly, when
balancing on a beam walking task, they have fewer hindpaw slips compared
to controls.
However, while 5-ht1d-/- mice have normal muscle spindle innervation
and number of proprioceptive synapses on motor neurons, Egr3-/- knockouts,
and muscle-spindle specific knockouts of Nt-3 and Erbb2 have abnormal
muscle spindle development and/or reduced number of VGLUT1 synapses
on motor neurons [74, 80, 172]. In addition, the reduced amplitude reported
for these latter mutants is 80-90%, and it is thus plausible that the proprioceptive feedback to motor neurons is severely impaired causing the ataxic
gait. In contrast, in 5-ht1d-/- mice, the amplitude reduction is 50% and above
severe phenotypic effect threshold suggesting that muscle spindle feedback
remains largely functional.
Reduced stretch reflex amplitude suggests deficient afferent feedback
from muscle spindles. It is surprising that 5-ht1d-/- mice perform better than
controls when crossing a narrow beam even though they have reduced
stretch reflex amplitude. However, studies in humans of the H-reflex, an
indirect measure of the stretch reflex, have shown that the amplitude of this
reflex is modulated during different gaits [175]. Notably, while walking over
a narrow beam, the H-reflex amplitude is smaller than when walking on a
level treadmill [176]. This may seem counter-intuitive, especially since it is
known that gamma motor neurons and Ia afferent are more active during
beam walking tasks, which would be expected to cause an increased Hreflex [177]. However, the stretch reflex feedback to motor neurons is
known to have a destabilizing effect if overly excited [178, 179]. Therefore,
Llewellyn et al. suggested that the Ia afferent-motor neurons connection is
46
presynaptically inhibited during difficult tasks such as beam walking to not
overexcite motor neurons, biasing the signaling of Ia afferents to active Ia
inhibitory interneurons or spinocerebellar neurons instead [176]. Therefore,
it is possible that 5-ht1d-/- mutants are “primed” for skilled walking on a
balancing beam by having reduced reflex amplitude. It should be noted however, that given the widespread expression of 5-ht1d in the brain, impact on
behavior from such areas needs to be considered when interpreting the improved behavior of 5-ht1d-/- mice.
Recurrent inhibition in locomotion
Even if changes in excitability and synapse number compensate for the absence of Renshaw-cell signaling to generate a functional output of the stretch
reflex, Renshaw cells nevertheless do not express the vesicular transporter of
GABA and glycine in Chrna2::Cre;Viaatlx/lx mice. Thus, unless they acquire
another signaling mechanism in the absence of Viaat, Renshaw cells are not
participating in the activity of motor circuits in Chrna2::Cre;Viaatlx/lx mice.
It is unlikely that the role of an entire class of interneurons can be compensated for by alterations in excitability and synaptology of motor neurons.
The normal fictive locomotion and motor behaviors are for that reason probable to occur in the absence of VIAAT-mediated Renshaw cell signaling.
Thus, the activity of Renshaw cells is not an integral part of locomotor circuits generating locomotion. Moreover, their activity is not needed to generate normal gait, motor coordination and generate maximal grip strength. It is
still possible that Renshaw cells have a function in a more specific motor
behavior not measured in paper IV.
The close physiological proximity of Renshaw cells to motor neurons has
led to the idea that they have a central role in movement and locomotion.
Various roles of Renshaw cells have been proposed from being important in
flexion-extension [180] to regulating force generation [150]. It has however
also been suggested that Renshaw cells may have a small role in motor control. This latter suggestion is mainly based on the observation that Renshaw
cells make weak connections to motor neurons mainly located to the dendrites [181-184]. In light of this, perhaps it is not surprising that Renshaw
cells do not appear to have a major function for motor behavior.
47
Acknowledgements
It is with deep sincerity that I wish to thank my colleagues at the department
and elsewhere. The five years I have spent at the neuroscience department
have been great and I am glad I had such great colleagues to share them
with. A special thanks to the following people:
My supervisor Klas Kullander. Thank you for creating such a great environment to do research in. That perfect mix of a relaxed and friendly attitude
with taking research seriously is an unbeatable combination. I have learned a
lot about being a scientist from you.
The original gang that was in the lab from the beginning. I will always remember those first years with sentimental nostalgia. Nadine, the lab has not
been the same since you left. I think that says it all. Anna, you are the smartest person I know and truly an inspiration. Henrik B, your impressive integrity and fairness will forever have an influence on me. Karin, your friendship has made spending time in the lab so much better. Henrik G, thank you
for all the fun and great collaborations over the years. Malin, it was always
fun to share an office with you. I miss that. Åsa Mackenzie, you are an inspiration as a scientist.
Co-authors in the lab: Kia, for bringing invaluable skills to the lab and being
such a great person to work with. Chetan, for having the enthusiasm of a
child in a candy store whenever new experiments are suggested. Martin, for
all the great times both in the lab and elsewhere. Hanna, for your absurdly
efficient pragmatism that always gets things done and for that contagious
happiness. Sanja, for all that useful knowledge of statistics that suddenly
raised the level on our analysis and for never thinking twice about helping
out. Co-authors from other labs that have done all those essential experiments we couldn’t do: Patrick Whelan, Stan Nakanishi, Boris Lamotte
d’Incamps, Arild Njå, Warren Tourtellotte, Bayle Shanks and Gregor
Eichele.
Other people in the lab and at the department: Kasia, for being such a genuinely friendly and kind person. Björn, for your genuine interest in science
that always results in great conversations and for you immense help with this
thesis. Fatima, Lina, Christiane and all the rest of the Kullander, Mac49
kenzie and Leao labs for five great years. Nicole, Greta and others at the
department for being great friends.
All students that have helped me over the years: Daniel, Smitha, Henrik,
Kali, Tobias, Ingrid, Elodie, Emma and Ida. A special thanks to Pierre
who finally managed to get in situs working on muscles and Anders who,
apart from contributing vital experiments to paper IV made all schematic
illustrations in this thesis.
Birgitta for doing all that you do and always taking the time to help. Susanne, Jenny, Sussie and Eva for taking care of the mice. Emma, Ulla and
Maria, and previous and current Deans at the department Lars Oreland,
Håkan Aldskogius and Ted Ebendal for handling administration. A special
thanks to Håkan for being my co-supervisor.
A big thank you to Sasha Svensson for making the beautiful cover drawing
for this thesis.
Thank you to my family and friends for being there. A special thank you to
Olof for being a great friend and Elin for making my life so much better.
50
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