Development of Locomotion Flashcards
what do intrinsic spinal motor circuits control?
rhtyhmic and coordinated muscle contractions that mediate locootor behaviours such as swimming and walking
are intrinsic motor circuits found in all vertebrates?
yes
what are CPGs?
central pattern generators. They are neural circus that can generate rhythmic motor patterns autonomously i the absence of phasic inputs
why do CPGs receive sensory feedback?
to modulate the locomotor programme and allow animals to adapt to environmental perturbations such as walking n un even terrain.
where do the sensory inputs come from which feed to CPGs?
proprioceptive aferent input from muscle spindles and golgi tendon organs
what allows spinal circuits to detect and respond to external obstacles?
exteroreceptors
where are the spinal circuits that control the forelimbs and hind limbs found in rats and man?
- for the forelimbs they are found in the cervical enlargement
- for the hindlimbsthey are found in the thoracic and lumbar regions
where are the spinal circuits found in fish?
sinal CPGs are repeated along the length of the funk and tail
what are the key things that CPGs are able to control during locomotion?
timing and coordination of the muscle contracts
what does it mean that CPGs control the ‘timing of locomotion’?
- that they control the frequency of tail or leg movements, which translates to differences in swimming or walking speed
what does it mean that CPGs control the ‘coordination’ of muscle contractions?
- 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
what is the role of commisural interneurons?
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.
in what animals has the spinal locomotor circuit been eel studied?
the lamprey
where are the spinal cord motor circuits found in the lamprey
along the entire body axis nearly
how does the lamprey mediate the progressive activation of the CPGs from anterior to posterior in order to mediate an undulatory wave of contraction?
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.
in walking circuits, what are the two major components of the intrasgemental CIN system involved in left-right coordination?
- 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)
within the lamprey, how to the CINs function?
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.
how have genetic experiments sought to shed light on the function of interneurons within CPG circuits?
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
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?
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?
what controls the flexor and extensor alternations in limbed animals?
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.
how are CPGS thought to mediate rhythm generation?
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.
how was optogenetics used to investigate the role of excitatory interneurons in rhythm generation?
- 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.
during development what seems to be involved in initiating rhythmic CPG activity, how does this differ to adults?
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.
in post natal mammals, what seem to trigger locomotor activity?
MNs release glutamate, in addition to acetlycholine, from their central recurrent terminals; this appears to trigger locomotor activity.
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.
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).
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.
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.
at early stages in development, what is the GABA/glycine function?
excitatory!
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
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.
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.
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.
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.
what receptor is involved in the high concentrations of chloride during the start of development?
NKCC2
what receptor is involved in the low concentration of chloride during development
KCC2
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
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.
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.
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.
specifically which axons appear to be needed for locooor networks to generate L-R alternating locomotor pattern?
5HT
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)
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.
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!
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This in vitro prep is especially amenable to electrophysiology!
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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)!
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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
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.
how do you record excitatory action on the spine?
EMG recordings
what cells are thought to be sufficient to generate the rhythm of movement?
ipsilaterally excitatory interneurons
what do the CIMi cells inhibit?
all of the CPG cells on the right
what neurotransitted is released from the commisural interneuron?
glycine- inhibitory
what does the reciprocal inhibition ensure?
that only one side of the animal can be activated at one time.
what does the IINe work like?
a pacemaker
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
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
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)
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
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
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
where do the dual inhibitory interneurons come from?
V0 - dbx1 expressing
what happens when you KO dbx1?
lose alternation and get periods of synchronous activity
if you express a toxin in VO neurons what happens?
the mice hop rather than walk
where do the synchrony neurons comes from?
V3
where do the excitatory but inhibitory via inhibitory intenreuorns come from?
ventral V0 and V3
at E15 is the LR alternation present?
no
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
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
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.!
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Consequently, the reversal potential for chloride (ECl) becomes more negative and GABA/glycine-receptor mediated currents change from depolarising to hyperpolarising.!
