Development of Locomotion Flashcards
1
Q
what do intrinsic spinal motor circuits control?
A
rhtyhmic and coordinated muscle contractions that mediate locootor behaviours such as swimming and walking
2
Q
are intrinsic motor circuits found in all vertebrates?
A
yes
3
Q
what are CPGs?
A
central pattern generators. They are neural circus that can generate rhythmic motor patterns autonomously i the absence of phasic inputs
4
Q
why do CPGs receive sensory feedback?
A
to modulate the locomotor programme and allow animals to adapt to environmental perturbations such as walking n un even terrain.
5
Q
where do the sensory inputs come from which feed to CPGs?
A
proprioceptive aferent input from muscle spindles and golgi tendon organs
6
Q
what allows spinal circuits to detect and respond to external obstacles?
A
exteroreceptors
7
Q
where are the spinal circuits that control the forelimbs and hind limbs found in rats and man?
A
- for the forelimbs they are found in the cervical enlargement
- for the hindlimbsthey are found in the thoracic and lumbar regions
8
Q
where are the spinal circuits found in fish?
A
sinal CPGs are repeated along the length of the funk and tail
9
Q
what are the key things that CPGs are able to control during locomotion?
A
timing and coordination of the muscle contracts
10
Q
what does it mean that CPGs control the ‘timing of locomotion’?
A
- that they control the frequency of tail or leg movements, which translates to differences in swimming or walking speed
11
Q
what does it mean that CPGs control the ‘coordination’ of muscle contractions?
A
- it involves mediating the appropriate sequential activation of motonuerons. For example Mns on the left and right sides fire in a left-right alteration during swimming and walking. In limbed animals, flexor-exensor alteration is a second form of coordination
12
Q
what is the role of commisural interneurons?
A
L-R alternation requires the communication between CPG circuits on either side of a spinal for segment. Their axons cross the midline in order to do this.
13
Q
in what animals has the spinal locomotor circuit been eel studied?
A
the lamprey
14
Q
where are the spinal cord motor circuits found in the lamprey
A
along the entire body axis nearly
15
Q
how does the lamprey mediate the progressive activation of the CPGs from anterior to posterior in order to mediate an undulatory wave of contraction?
A
this is achieved by generating a small time delay (phase lag) between spinal cord segments, such that segments at more caudal locations are activated later.
16
Q
in walking circuits, what are the two major components of the intrasgemental CIN system involved in left-right coordination?
A
- inhibiitory: both direct and indirect inhibition of contralateral Mns. This CIN component is probably active during walking and mediates L-R alternation
- excitatory- glutamatergic CINS that project to contralateral MNs. This component might be active during synchronous L-R locomotor behaviour (hopping, galloping)
17
Q
within the lamprey, how to the CINs function?
A
CINs mediate L-R alternation between CPGs in each segment along the anterior-posterior axis. In lampreys, as well as frog tadpoles, the CINs mediating L-R alternation are inhibitory and project to MNs and other CPG neurons on the opposite side of the spinal cord segment.
18
Q
how have genetic experiments sought to shed light on the function of interneurons within CPG circuits?
A
within the ventral spinal cord, interneurons are driver from four main domains: v1, v2, v3 and V0 (the other region is the MN region). Progenitor cells in each of these domains have specific patterns of transcription factor expression and the post-mitotic neurons that differentiate from each domain also have specific gene expression profiles. SO you can KO in mice different interneuron sup populations. You can then look at the locomotor activity, with periods where L-R alteration break down. genetically modified mice lacking V0 CINs show a rabbit-like hopping gait, possibly due to unopposed activity in the excitatory pathway
19
Q
what gene is only expressed in VO domain interneurons and how can this be used to look at the role of interneurons in the CPGs action in locomotion?
A
dbx-1 is expressed in the V0 domain- you can create KO mice for this gene and then look at the effect on locomotion- does it alter LR alternation?
20
Q
what controls the flexor and extensor alternations in limbed animals?
A
it is thought that there are commsiral interneruons that project to the extensor on one side and the flexor on the other side and teh same for the opposite. this allows the movments to be coordinated, however, the flexor extensor CPG is not well understood.
21
Q
how are CPGS thought to mediate rhythm generation?
A
The circuitry of the rhythm-generating core is not well understood. However, for both walking and swimming CPGs, ipsilaterally-projecting excitatory (glutamatergic) neurons are required and specific activation of these neurons is sufficient to activate rhythm generation. Some of the excitatory neurons thought to be involved in rhythm generation make direct connections to MNs.
22
Q
how was optogenetics used to investigate the role of excitatory interneurons in rhythm generation?
A
- made trasngenic mice that expressed channelrhodopsin-2 under the control of the vesicular glutamate transrpoter 2 promoter which labels all glutamatergic neurone in the CPG region of the spinal cord. stimulating the isolated cord with blue light (which activates CHR-2 and depolarises the neurone expressing it) was sufficient to initiate and sustain locomotor-like activate, similar to tha produced with drugs. This suppers the idea that excitatory interneurons are involved in rhythm generation circuitry.
23
Q
during development what seems to be involved in initiating rhythmic CPG activity, how does this differ to adults?
A
during development, intraspinal mechanisms seem to be involved in initiating activity. Spontaneous rhythmic activity is a pronounced feature of the developing nervous system and appears to be required for normal circuit formation.Rhythmic CPG activity is normally initiated by descending signals from supra spinal circuits in adults.
24
Q
in post natal mammals, what seem to trigger locomotor activity?
A
MNs release glutamate, in addition to acetlycholine, from their central recurrent terminals; this appears to trigger locomotor activity.
25
what is the role of radial cells in early mouse development?
In early mouse development, radial cells (embryonic progenitor cells) release glycine which appears necessary for normal spontaneous activity in the spinal
cord.
26
in zebrafish larvae, what is an example of spontaneous movements being activated from non supra spinal circuits?
In zebrafish larvae, (optogenetic) activation of GABAergic Kolmer-Agduhr cells can drive swimming episodes and optical silencing of KA cells reduces spontaneous swim frequency. At this developmental stage, GABA is excitatory (see Section B).
27
what demonstrates that CPG LR alternation has to develop over time in the post natal stage?
• During mammalian development, activity of the spinal pattern generation network also changes as development progresses. Initially, all hindlimb muscle groups are synchronously active, later L-R alternation appears and later still flexor-extensor alternation emerges.
28
how does the locomotor pattern of movement change as animal develop?
in post natal spinal cords L-R alteration is observed, which is dependent on reciprocal inhibition between left and goth sides, mediated by inhibitory CINs. However, at early embryonic stage this is not the case: left and right sides show synchronous activity with motorneurons on both sides active in phase with each other.
29
at early stages in development, what is the GABA/glycine function?
excitatory!
30
how does the switch from GABA/signals being excitatory, to them being inhibitory, occur?
- these changes occur as a result of changes to chloride homeostasis
31
explain the differences in chloride ion concentration in the early embryonic neurons compared to the mature enrols and how this results in the transit from excitatory to inhibitory signals
In mature neurons, [Cl–]i is low and its reversal potential is around -70 mV. Thus, activation of GABAA and glycine-receptor gated Cl– channels results in an influx of chloride. The inhibitory action of glycine and GABA consists of both shunting incoming excitatory currents and hyperpolarising the membrane potential, moving it away from the action potential threshold.
By contrast, at early embryonic stages [Cl–]i is high and ECl- can exceed both resting membrane potential and spike threshold. Thus, activation of Cl– conductances in immature embryonic neurons causes excitatory membrane depolarisations, which can cause the neurons to fire.
32
how does the concentration of high chloride change to low chloride in maturing neurons
- they occur due to changes in transmembrane chloride transport.
- in immature neurons, the incward-directed Cl- cotransorted NKCC2 has ben proposed to be important in the active accumulation of intracellular Cl-. NKKC1 is probably down regulated during development. Conversely, up regulation of the outward-directed CL- cotransporter KCC2 is widely accepted to underlie the decrease in intracellular chloride concentration and therefore the shift from GABA/glcyine-induced depolarisation to hyper polarisation in several regions of the CNS.
33
what is generally the progression from excitatory to inhibitory GABA function and the effect on the LR alternation?
The early excitatory function of GABAergic/glycinergic "inhibitory" CINs is thought to synchronise CPG activity on left and right sides of the spinal cord and thus produce the synchronous pattern of motoneuron firing observed in fetal rats. Shortly before birth, changes in chloride homeostasis mean that GABA/glycinergic CINs become functionally inhibitory. Their action can now mediate reciprocal inhibition between L and R sides and therefore alternating limb movements.
34
describe examples of the GABA excitatory to inhibitory being conserved.
The early excitatory actions of GABA appear to be conserved across different regions of the CNS and between different species. GABA-mediated excitation develops before glutamatergic excitation and appears to be involved in generating early patterns of network activity that shape the functional architecture of the developing brain through activity-dependent processes. This activity includes “Giant Depolarising Potentials” (GDPs), which are patterns of slow network activity associated with large changes in intracellular calcium concentration. GABA-mediated activity is important for neuronal migration, synapse formation and plasticity as well as the upregulation of KCC2, which brings the excitatory functions of GABA to an end as the neuronal network matures.
35
what receptor is involved in the high concentrations of chloride during the start of development?
NKCC2
36
what receptor is involved in the low concentration of chloride during development
KCC2
37
how is locomotion initiated in the lamprey?
the basal ganglia integrates inputs from the pallium and thalamus and selects a locomotor programme. Command systems in the diencephaln and midbrains re recruited which activate glutamatergic reticulospinal neurons that in turn activate spinal cord CPG
38
how is locomotion in mammals initiated?
locomotor regions in the brainstem, including the midbrain locomotor region (MLR), are involved in initiating locomotion and controlling speed and gait. Electrical stimulation of the MLR at low intensity evokes walking and as stimulation intensity increases, so too does walking speed. At higher intensities locomotion switches to trotting and then galloping. Thus, the MLR may
control locomotor output via a ʻsimpleʼ descending command that is modulated only in intensity.
39
how are signals from the midbrain locomotor region relayed to the spinal cord?
glutamatergic reticulospinal neurons (like in lamprey). Monoaminergic neurons also project from the brainstem to the spinal cord. These include catecholaminergic neurons (noradrenergic and dopaminergic) and serotonergic neurons.
40
describe an experiment which demonstrated that descending pathways promote the developmental maturation of inhibitory neurotransmission in the penal cord
5HT axons from the raphe are amongst the first descending axons to reach the lumbar spinal cord in rats at E17. 5HT appears to be necessary for spinal locomotor networks to generate the L-R alternating locomotor pattern. If the spinal cord is transected (at thoracic level) on the day of birth (postnatal day 0, P0), rats display normal L-R alternation for the first few postnatal days (P1-3), but by P6-7 L-R alternation deteriorates. Application of 5HT2-receptor agonists restores normal L-R alternation. The developmental upregulation of KCC2 in the lumbar spinal cord during the first postnatal week is greatly reduced following spinal cord transection.
41
specifically which axons appear to be needed for locooor networks to generate L-R alternating locomotor pattern?
5HT
42
how was it first shown that the descending motor tracts from the brain(suprapsinal) were not needed for locomotion and that instead there were intrinsic locomotor circuits ? (2)
- the hind leg of cats will walk on a treadmill after complete transection of the spinal cord
- walking can be evoked by non-phasic electrical stimulation of the cut cord, great currents evoke faster stepping frequencies (hence no sensory input is being involved)
43
how was it shown that intrinsic motor circuits could control both the timing and the coordination of locomotor movements?
The hind legs of cats will walk on a treadmill after complete transection of the spinal cord.!
Walking can be evoked by non-phasic electrical stimulation of the cut cord; greater currents evoke faster stepping frequencies.
The left and right legs move in alternation with appropriate flexor-extensor alternation within each limb.
44
how was it shown that the intrinsic circuits for LR alternation are present in embryonic rodents?
The spinal cord can be isolated from embryonic and neonatal rodents and kept alive in vitro for several days!
!
This in vitro prep is especially amenable to electrophysiology!
!
The isolated spinal cord will generate a locomotor pattern when stimulated with neuroactive substances that simulate descending inputs from supraspinal circuits (eg serotonin and/or dopamine)!
!
The isolated cord from neonatal rat pups produces the key features of the locomotor pattern include rhythm generation, L-R alternation and flexor-extensor alternation- you measure this by placing electrodes at lumbar level 2 and 5 and also at the flexor extension alternation
45
describe the reciprocal inhibition networks in the lamprey that that mediate L-R alterations.
- ipsilaterally excitatory interneurons activate themselves and also the ipsilateral commisural interneuron and the motornueron which simulates contraction . When they activate the commisural interneuron this inhibits the contralateral commisural interneuron. When IINe neurons stop firing during its relative firing phase then the left stops inhibiting the right and then the right can fire.
46
how do you record excitatory action on the spine?
EMG recordings
47
what cells are thought to be sufficient to generate the rhythm of movement?
ipsilaterally excitatory interneurons
48
what do the CIMi cells inhibit?
all of the CPG cells on the right
49
what neurotransitted is released from the commisural interneuron?
glycine- inhibitory
50
what does the reciprocal inhibition ensure?
that only one side of the animal can be activated at one time.
51
what does the IINe work like?
a pacemaker
52
how was it shown that the glutamatergic excitatory interneurons were sufficient to generate the CPG action?
they expressed ChR2 driven by vglut2 to label all glutamategric neurons in the PCG region of the spinal cord and when they activated these will constant blue light,they got rhythmic, well controlled locomotion in the lamprey
53
how would you test whether these LR CPGs are in place win the embryo?
you can isolate the spinal cord and stimulate it- with neuro activators- and see where you get the same movemento or electrical activity with LR alternations etc - at E15 - but it is synchronous
54
how was it shown that the chick develops the CPG for LR or synchronous very early?
grafted one region of spinal cord into another area- wing to limb- if you let the chick develop you find that if you have wing CPG rather than leg CPG in there legs then they hop (because wings flap)
55
what did the chick experiment prove about the CPGs? (3)?
- tell us that these intrinsic spinal cord circuits control the movement programme
- these circuits are determine early in development ( so genetic)
- in terms of left right alternation, it isn't always LR
56
what is the arrangement of the CPG within mammals?
- there is a core of exc interneurons which generate the rhythm and they activate CINs which are either mediating inhibition (reciprocal) and these are composed of direct inhibitory neurons (CINi) which project to the right hand side and inhibit everything on the right and there are also excitatory neurons which project to the righ but they also activate inhibitory interneurons which again inhibit MNs on the right - this is the dual inhibitory pathway
- in parralele with this tree are excitatory commisural neurons which also excite the neurons on the riht- these are the synchronous pathways
57
what is the dual inhibitory pathway in mammals?
- LR alternation
here is a core of exc interneurons which generate the rhythm and they activate CINs which are either mediating inhibition (reciprocal) and these are composed of direct inhibitory neurons (CINi) which project to the right hand side and inhibit everything on the right and there are also excitatory neurons which project to the righ but they also activate inhibitory interneurons which again inhibit MNs on the right - this is the
58
where do the dual inhibitory interneurons come from?
V0 - dbx1 expressing
59
what happens when you KO dbx1?
lose alternation and get periods of synchronous activity
60
if you express a toxin in VO neurons what happens?
the mice hop rather than walk
61
where do the synchrony neurons comes from?
V3
62
where do the excitatory but inhibitory via inhibitory intenreuorns come from?
ventral V0 and V3
63
at E15 is the LR alternation present?
no
64
what happens when you cut the ventral commissure to the synchrony at E15? what does this show?
the synchrony breaks down, tho shows that you still need the communication between the two dies
65
how was the early synchrony explored
a piece of early spinal cord was removed and put in a dish- and the left and right sides are separated by a membrane. You can then stimulate one side and record the activatty on boths sides in synchorny- but when you look at the neurons mediating this (if you provide gaba inhibitors) then the synchrony disappears. surprising because they are mediating the excitatory coupling between the left and right sides at E15
66
why do GABA/glycine signalling induce an excitatory actin potential?
- because intracellular chloride concentration is high and E cl is positive to the resting membrane potential and spike threshold In immature neurons, [Cl-]i is high, but during development it decreases to approximately 25% of its former concentration.!
!
Consequently, the reversal potential for chloride (ECl) becomes more negative and GABA/glycine-receptor mediated currents change from depolarising to hyperpolarising.!
67
what two main regions initiated locomotion and control speed? in the lamprey
the MLR and the DLR (diencephalic)
68
do spinal cord CPGs have to switched on?
no they have to be activated?
69
how did they show that the sensory input into the cat was not needed?
??
70
what is involved in steering and posture in the lamprey
signals for steering and posterior
come from the tectum (steering) in the lamprey if more activation on the left side of the reticluospinal system there is longer ventral root bursts on the left and causes the animal to steer to the left
vestibular input then help maintain the lamprey to maintain dorsal posture
71
what controls the CPGs in humans?
the cortex is not required for locomotion but is required for navigating complex terrain or climbing a ladder or sight driven movement- it takes control via corticospinal projections
- the cerebellum also generates rapid projection to correct errors
- the MLR activate is what is needed to initiate locomotion
72
what experiment was done to show how the MLR controls speed of movement?
put an electrode int he MLR of a cat and stimulate it then the cat will start walking- at low level the cat will wak slowly and as you turn up the current the frequency of movement increases- also when you get to a threshold intensity you get synchrony during galloping- likely reflects switched from the dual inhibitory pathway and then you transition to the V3 synchronous pathway.
73
how was optogenetics used to show that activation of the hindbrain neurones which descend the commands to activate locomotion
express ChR2 in the hindbrain and if you shine blue late the fish goes from not swimming to swimming, you can do the same with inhibitory genetics of the neuron- this show sthatthese neurons aren't active all the time- they have to be turned on
74
at P0, where are the corticospinal neurons?
- just developing past the hid brain - but you don't need a cortex for locomotion
75
describe the experiment which demonstrated that demonstrated the role of the dopaminergic encephalon tract?
An evolutionarily conserved dopaminergic tract originates in the diencephalon and projects to the spinal cord (the “dopaminergic diencephalospinal tract”).!
!
This descending pathway controls a developmental transition in swimming behaviour in larval zebrafish.!
!
At 3 days post-fertilisation (dpf), zebrafish spend most of their time lying immobile on their side, but occasionally perform long, rapid swims.!
!
By 4 dpf their behaviour changes. Larvae inflate their swim bladder -- enabling postural control -- and show increased locomotion and begin foraging behaviour. At 4 dpf, swims are shorter and more frequent.!
!
Treating 3 dpf larvae with D4R receptor antagonists prevents the developmental switch from long to short swim bouts. Furthermore, treating 4 dpf larvae with the antagonist reverts swimming behaviour to the immature pattern of long bouts (data not shown). Note that in these experiments, the whole larval animal is treated with the drug, which is added to the tank water.!
!
To demonstrate that the site of action of DA is within the spinal cord, larvae were spinalised at 4 dpf to remove all descending inputs. Treatment with NMDA alone evokes immature long swim bouts, but NMDA + DA (or D4R receptor agonists) evokes the mature pattern of short swims.!
!
Thus DA, supplied by descending axons, appears to regulate a developmental switch in locomotor behaviour in larval zebrafish.!
76
what is a prerequisite for mice to be able to move? what gives this?
they need postural support which is give by the serotinergic fibres
77
when do the serotinergic nerves reach the lumbar region?
at E17 but not actually fully functional until P7
78
while the serotinergic axons are developing in the lumbar region, what else are they thought to be doing?
they are thought to be needed for LR patterning- Serotonin appears to influence the developmental upregulation of KCC2, which results in the maturation of inhibitory neurotransmission
79
what must happen before the rat pup can carry out voluntary movement?
Glutamatergic reticulospinal axons are involved in initiation of locomotion and control of locomotor speed, by relaying signals from brainstem command centres.!
!
Corticospinal axons reach spinal cord targets later: they are still growing through the hindbrain at birth and only reach the hind limb circuits by P6, so no “voluntary” movements are possible before then.
80
which type of spinal axons are involved in posture as well as activation and modulation of spinal CPGs?
Reticulospinal axons are involved in control of posture as well as activation and modulation of spinal locomotor networks.! The serotinergic, and catecholamine axons arrive around E17 but the seratonergic dont function until P7. These the rteiculospinal tract provides postural support.
Postural control is a prerequisite for locomotion. Reticulospinal axons, descending in the medial reticulospinal tract, facilitate motor neurons innervating leg extensors and axial muscles, allowing the legs to support the body
81
even though serotinergic neurons reach the lumbar region at E17 why can they function
Although descending catecholamine/serotoninergic fibres reach the lumbar cord before birth, they remain non-functional for the hindlimb circuits until P7, although their function can be mimicked by intradural L-Dopa at P5.
82
what are slow muscle fibres?
Slow muscle fibres produce less force but sustain it longer. They contain many mitochondria for aerobic respiration and so fatigue very slowly (purple traces). They are redder in color.
83
what are fast muscle fibres?
Fast muscle fibres produce strong fast twitches but operate anaerobically, so fatigue rapidly (blue traces). They are pale in color.!
84
how od fast and slow muscle fibres change over development?
Muscles start life with pale fast-type fibres -- the default condition. With time, these fibres switch where needed to the slow type needed for postural support, as shown by immunostaining for the slow isoform of myosin ATPase (lower panels).
!Postural support is a precondition for walking but slow muscle fibres take more than 3 weeks after birth in the rat to mature. This process is activity-dependent.
!
85
how can neuronal activity control the differentiated character of muscle fibres?
In the adult rat, phasic motor neurons innervate fast muscles such the extensor digitorum longus (EDL). Tonic motor neurons connect to slow postural muscles, eg, the SOLEUS!
!
However, even in the adult the EDL can be turned slow and the SOLEUS turned fast by cross- innervating them. This was achieved in the 1960s by cutting each nerve and letting it regenerate down the distal stump of the other.!
!
During development the early polyneuronal innervation of single muscle fibres withdraws into a more focused pattern between P0 and P14.!
Correlated with this, the muscle fibres develop the slow (postural) or fast (locomotor) properties determined by the activity patterns of their motor neurons.!
86
how can the movement of a cats hind paw be separated?
F = flexion
| then E1, E2 and E3
87
through what phase of movement of the cat hind leg does flexion occur?
F- first stage
88
what can be added to spinal preparations to stimulate the neurons?
NMDA or serotonin
89
what was the original theory of walking and how was it disproved?
“An early view of the neural control of locomotion was that it involved a “chaining” of reflexes: Successive stretch reflexes in flexor and extensor muscles were thought to produce the basic rhythm of walking. This view was disproved by Graham Brown, who showed that rhythmic locomotor patterns were generated even after complete removal of all sensory input (deafferenta-tion) from the moving limbs.- This is carried out by removing the dorsal toots that inner the limbs because the dorsal roots carry only sensory axons
90
as well as removing the sensory neurons via removing DRG, what is a more sophisticated way of testing whether sensory input or stretching of the muscle contributes to walking?
- you can paralyse the muscle using d-tubocurarine
- you then stimulate the motor neurons and measure whether the motor nerves to the flexor and extensor muscles still fire alternatively and you find that they do so can't be to do with th muscle
91
what are CPGs which activated spontaneously called and what are these involved in?
Bursters- respiration
92
what time of potentials mediate rhythms?
plateau potentilas
93
describe how the plateau potential occurs in the contralateral side of neurons?
“One important mechanism in the initiation of activity is the opening of NMDA-type glutamate receptor-channels. Once the inhibition from contralateral commissural interneurons is terminated, the NMDA-type receptor-channels in all ipsilateral neurons are opened by a brief depolarization (post-inhibitory rebound) and the voltage-dependency of the channels leads to plateau potentials.
Activation of low-voltage Ca2+ channels further strengthens the depolarization. The influx of Ca2+ through these channels and the NMDA-type receptor-channels activates calcium-dependent K+ channels. The resultant increase in K+ conductance terminates the plateau potentials and so contributes to the termination of activity.”
94
as well as the plateau potential, what other mechanism exists to mediate the inhibition of the signalling CIN at the time?
“ delayed excitation of the local inhibitory interneurons. When excited, these interneurons inhibit the commissural interneurons (Figure 36-6), thereby removing inhibition from the contralateral half of the network and enabling it to become active.- these are exictatory neurons
95
what key experiment showed that there is afferent input into the CPGs?
“One of the clearest indications that somatosensory signals from the limbs regulate the step cycle is that the rate of stepping in spinal and decerebrate cats matches the speed of the motorized treadmill belt on which they tread. Specifically, afferent input regulates the duration of the stance phase. As the stepping rate increases, the stance phase becomes shorter while the swing phase remains relatively constant. This observation suggests that some form of sensory input signals the end of stance and thus leads to the initiation of swing.”
96
where are the afferents though to control the regulation of the length of stance phase?
in the hip - the stretch flexor
97
what are the three supra spinal regulation pathways of stepping?
1. activates the spinal locomotor system, initiates walking and controls the overall speed of locomotion
2. refines the motor pattern in response to feedback from the limbs
3. visually guides limb moment
98
what is the role of the MLR?
mesencephalic locomotor region- controls the rhythm of the locomotor pattern via an increase of decrease in signal intensity
99
as well as the MLR, which other two regions of the brain can stimulate locomotion when stimulated
- subthalamic locomotor region and the pontine reticular formation
100
what does the MLR connect to to stimulate the CPGs?
- they connect with neurons in the medulllary reticular formation, whose axons descend in the ventrolateral region of the spine - these should be glutamteric
101
how has the motor cortex been implicated in movements?
“Experimental lesions of the motor cortex do not prevent animals from walking on a smooth floor or even on smooth inclines, but they severely impair tasks requiring a high degree of visuomotor coordination, such as walking on the rungs of a horizontal ladder, stepping over a series of barriers, and stepping over single objects placed on a treadmill belt. Such “skilled walking” is associated with considerable modulation in the activity of a large number of neurons in the motor cortex”
“Many of these neurons project directly to the spinal cord and thus may regulate the activity of interneurons in the central pattern generator for locomotion, thereby adapting the timing and magnitude of motor activity to a specific task. ”
102
what human evidence is there for CPGs?
- In one striking case a patient with nearly complete transection of the spinal cord showed uncontrollable, spontaneous, rhythmic movements of the legs when the hips were extended. This behavior closely parallels the rhythmic stepping movements in chronic spinal cats.
- In another study on a few patients with severe spinal cord injury, stepping on a treadmill was improved by clonidine, a drug influencing biogenic amines.
- “infants make rhythmic stepping movements immediately after birth if held upright and moved over a horizontal surface. This strongly suggests that some of the basic neuronal circuits for locomotion are innate”
- “stepping can occur in infants who lack cerebral hemispheres (anen-cephaly), these circuits must be located at or below the brain stem, perhaps entirely within the spinal cord.”
103
what evidence is there against humans having spinal CPGs?
“ humans with spinal cord transection generally are not able to walk spontaneously.”
104
what will definitely be the differences in walking with two legs a humans in terms of CPGs as opposed to quadrupeds?
“Nevertheless, human bipedal locomotion differs from most animal locomotion in that it places significantly greater demands on descending systems that control balance during walking. Indeed, maturation of the systems that control balance and stepping patterns is necessary for the infant to begin walking independently at the end of the first year. In contrast, horses can stand and walk within hours after birth.”
“spinal networks in humans are more dependent on supraspinal centers than those in quadrupeds. ”
105
why is it hard to study CPGs in mammals?
large number of cells in the spinal cord
106
what are rental cells?
the inhib inter neurons that are activated by contralateral CINs
107
what gene do V3 neurons express
sim1
108
what percentage of dbx1 expressing V0 cells become GABA neurons?
70%
109
what do the V3 neurons become?
all excitatory and predominantly contralaterrally projecting
110
what are the fates of V0 neurons and what does this say about the likely contribution to the dual inhibitory pathway?
- 70% GABA and 25% glutamatergic- when these are both deleted in mice there is synchronous moment so both involved in LR- the gaba are the direct and the 25% are the indirect probably
111
how was the role of V3 inter neurons tested and what was found?
Sim1 neurons were permanently blocked or the neuronal activity was reduced by genetically driven expression systems.
- this resulted in locomotor activity being disrupted with large variability and imbalance of left right motor activity
- they were shown to have anatomical connections to the renshaw cells inhibitory Ia inter neurons and motor neurons.
- these suggest that the V3 are involved in regulating the coordination of precision and regularity of motor neuron activity across the cord although their role in left right alterations is minor compared to the V0 cmmisural inter neurons.
- a sub population is likely involved in the indirect pathway - deletion mean not enough to upset LR completely but perturb it
112
what did ablation experiments on V2a neurons reveal?
- they are glutamatergic and project ipislaterally
- ablation studies showed the locomotor frequency and motor burst amplitude of drug induced locomotion like activity became more variable and left right was disrupted
- suggested they project only to the indirect pathway and are activated by the rhythm cells
113
what do V2a neurons express
chx10
114
what is the rhythm cell thought to be, what is the evidence?
- elimination of V0-V2 classes does not perturb rhythm
- not homeobox 9 posiitve neurons even though they posses rhythmogenic properties but not at the right time
- maybe they span several classes
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where do the cells migrate if they are going to form neural crest cells?
ventrally between the dermamoyotome and the neural tube
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what must occur within the cell in order for the NC to migrate?
it must down regulate N-cadherin and cadherin 6
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what happens to the basal lamina around the neural tube when NCs migrate?
it dissolves transiently
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what % of the migrating neural crest cells to form DRG are early migrating?
a third
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where do the early migrating NC, DRG-destined cells migrate to? how many neurons do they give rise to on average? where do they reside?
they form the machano and proprioceptors, they produce on average 3.1 neurons each and they reside in the ventrolateral region of the DRG
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how many neurons on average to the early migrating DRG fated NCs give rise to?
3.1
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a third of the Ncs are early migrating and reside in the DRG, what do the remainder form and what are their characteristics?
they generate on average 53.9 neurons each and neurogenesis continues for a longer time. they reside in the dorsomedial region of the DRGand in the ventrolateral region
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how is the timing of elimination linked to the neuronal fate?
those that delaminate early will become the mechano and proprio and reside in the ventrolateral region of the DRG and those that delaminate late will commit after migration and DRG condensation and these are lily the nociceptive neurons
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how does the expression of neurogenins influence the fate of NCs?
it biases them NCCs to the sensory neuron fate as opposed to an autonomic lineage
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what initiates the first wave of neurogenesis?
nrg2
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where in ngn1 expressed?
in a subset of migratory NCCs that are derived from a location close to the dorsal neural tube and continues to be expressed until they reach the DRG
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how does the expression of the two neurogenins influence the fate of the NCC?
the first NGn2 mediated wave produced TrkB/TrkC mechanoreceptive and proprioceptive neural subtypes., that might all arise from a TrkC + population
- the second wave ngn-2 positive neurones is expressed by NCCs that go on to form small TrkA + neurons and large TrkB/TrkC neurons within the DRG
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what supports the claim that NGN1 can give rise to large TrkB and C expression neurons as well as TrkA neurons?
NGN2 null mice still have the NGN1 driven large TrkB and TrkC neurons
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what evidence is there for NCCs requiring WNT for NGN activity? (2)
- cultures NCCs with disrupted b-catenin, sox10, multipoint NCCs fail to express neurogeneins and to express differentiate
into sensory neurons
- when you express a CA form of b-catenin in NCCs, they terminate their migratory pathway early and aggregate into ectopic ganglion like structures which express ngn2.
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where is WNT expressed in the neural tube during NCC delamination?
drosaln
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what factors direct sensory vs autonomic neurons from NCCS?
WNt direct sensory and BMP in autonomic - shown by culturing in the presence and checking markers
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what has WNT been shown to induce and how does this suggest a role for it ind development?
it has been shown to induce the expression of Ngn2 which suggest it controls the first wave of migration of NCCs and their fate
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can ngn expression mediate sensory neuron subtype differentiation?
no - Trk receptors do this
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how was the strong influence that TrkC expression has over sensory neuron after demonstrated?
the genetic replacement of TrkC with TrkA expression in a KI animal resulted in a change in fate of small neurons to a proprioceptive phenotype
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what drives the expression of TrkA?
runx1 and Brn3a (targeted deletions of Brna1 result in cell death as a consequence of of failure of neurotorphins to support a cell by binding trek receipt
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how does Brna3a direct the expression of TrkA?
it binds directly to the TrkA promoter
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what is the role of KLF7, how was this shown?
- KO results in a loss of TrkA expression - it cooperates with DNA binding proteins such as Brm3a to drive full TrkA transcription
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what is the phenotype of a runx3 KO?
severe limb ataxia as a result of disruption of the monosynaptic circuitry between 1A muscle spindle afferents and alpha motor neurons and have a loss of TrkC expression, a loss of proprioception
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what does runx3 stimulate the expression of and then what separates these two subtypes?
initates TrkC expression and then the TrkB neurons arise from this population and so does Ret expression. these all act in combination
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once TrkC has been expression, what cells types can arise?
TrkC+ TrkB, TrkB+ret, TrkB, Ret, TrkC+ Runx3
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what are the four expression patterns of mechanoreceptors?
TrkC+ TrkB, TrkB+ret, TrkB, Ret,
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what is the expression pattern of propriocptor ?
TrkC and runx3
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without Runx1 and runx3, what happens?
all subtypes of sensory neurons die by apoptosis
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what is runx1 required for? how does it do this
the expression of trKA, it binds directly to the promoter
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what is the role of runx1 in further diversification? how has this been demonstrated?
it acts in the further diversification of the TrkA neurons into different types of nociceptors during the late embryonic and postnatal stages. It seems to act as a repressor. Initially all embryonic small neurons initially express TrkA but then some stop and begin to express Ret. In mice that selectively lack Runx1 in the PNS, the transition from TrkA to Ret expression is impaired, resulting in an increase in TrkA expressing neuron and decrease in ret expressing neurons.
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as well as TrkA expression, what else is RUnx1 required for?
- the repression of CGRP, the DRG acid sensing channel DRASIC and u-class opioid receptors, as well as the upregulation of several ion channels and seven transmembrane domain receptors.
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what is the role of semaphoring
between E6 and E10 in the chick, when sema3a is expressed only in the ventral spinal cord, nt-3 dependent muscle axons express progressively lower nrp1 and lose their responsiveness to sema3a, whereas NGF dependent axons show the opposite pattern. SO from E7 in the chick SEMA3a repels NGF responsive neurons that ruminate in the dorsal horn but not NT3 responsive axons, thereby contributing to the correct patterning of sensory projections in the spinal cord - elimination of SEMA3A or NRP1 results in misprojection of cutaneous nociceptive CGRP and trek afferents along the spinal cord midline into the deeper spinal cord laminae - by acting on NRP1 receptors, SEMA3A prevent cutaneous TrkA axons from projecting to deeper laminae
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how is runx1 implicated in the segregation of of axon terminations?
runx1 continues to be expressed in non peptinergic neurons and this s accompanied by the down regulation of TRka and the segregation of central terminations into he deeper lamina IIa. Loss of unction of runx1 in mice results in failure of the non-pep axons to terminate in the lamina IIa, instead they terminate in more superfical laminae with the axons from pep neurons.
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what is it called when neurons are arranged according to the spatial regions that they innervate
topographic map
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what is the third wave of NCCS?
later wave of nociceptors that arises from the boundary cap cells - the nc derivatives that migrate down the root from the dorsal root entry zone
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what are the ngns?
basic helix loop helix TFs
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which sub group of nociceptive neurons are generated first?
- peptidergic
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what are the A fibres?
mechano and proprio
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what are the C fibres?
noci
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which nerve fibres reach the the skin first? which penetrates the grey matter first
A fibres for both (E15-17 for latter)
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by what time have the A afferents withdrawn from the dorsal laminae? what type of A neurons are they thought to be?
3 weeks post total - hair follicle or maybe A fibres pressure receptors
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what is an experimental example of intersegmental reflexes occurring neonatally?
a prick on the foot can cause all 4 limbs to move in a at pup
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what can explain the bad ciirdination of the tail flick response?
- the receptive fields of hindlimb flexors muscle are large and disorganised in young animals such that noxious stimulus evoked limb withdrawal is not always appropriate to the stimulus
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what are gaba receptors permeable to?
anions
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what is the reversal potential for chloride in early gaba synapses? what does this indicate?
at a more depolarised level than the resting membrane potential. this indicates that the concentration of chloride is higher in neonatal neurons
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what are the two receptors which show a developmental shift from inhibitory to stimulatory?
glycine and gaba
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in what animals has the developmental shift in actions of GABA been observed?
xenopus, zebrafish, chick
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what techniques have been used in what neurons to show that chloride ions are higher in immature as opposed to adult neurons?
chloride imaging technqiues- perforated patch recordings- in the inferior olive
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when GABA receptor anion channels are opened in the immature neuron, what happens? what does this mean for the membrane potential?
chloride ions move out- it increases- brings it towards the threshold for a.p - so excitatory
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what evidence is there that KCC2 stimulates the conversion of gaba to inhibitory? (2)
First, changes in the levels of KCC2 messenger RNA in hippocampal cultures and slices correlate with the modification of GABA actions. Second, the transfection of KCC2 into hippocampal neurons converts the actions of GABA from excitatory to inhibitory, and GABA is excitatory in mice that lack KCC2.
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what evidence is there for the activity of GABA being required for the switch to take place?
blocking GABAa receptors with bicuculline and picrotoxin prevented the shift from taking place- the KCC2 transported was not expressed and GABA continued to exert a depolarising action- blocking glutamate receptors did not have this effect.
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what was the piece of evidence which showed that all that is needed for the transition to occur is ongoing release of gaba?
blocking all ongoing activity by continuous applications of the sodium channel blocker tetrodotoxin did not prevent the shift from excitation to inhibition. This implies that the presence of miniature postsynaptic currents (PSCs), which are generated by the action-potential-independent quantal release of GABA, is sufficient to trigger the expression of KCC2 and a reduction in [Cl–]i. In other words, all that is needed to produce the shift is an ongoing release of GABA, even when all the network activity is blocked and the terminals are disconnected from their parent soma.
