11. S: Small, Slow, Fatigue-Resistant, Type I
FF: Large, Fast, Fast-Fatigable, Type II
Nijssen et al. (2017)
Ruch TC, Patton HD (eds.) (1982):
Physiology and Biophysics, 20th ed
24. Astrocytes mediate Spatial Buffering
of K+ ions for Neurons via Kir4.1
Hibino et al. (2010)
1. Expression:
• Predominantly in glial cells of the
both brain and spinal cord
• Both perisynaptic processes and end feet
2. Function “Spatial buffering of K+ in the brain”:
• Maintain the ionic and osmotic environment in the
extracellular space, by transporting K+ from
high [K+]o to low [K+]o
• Prevent excessive [K+]o accumulation by
rapid clearance to avoid continuous depolarization
of neurons.High
Low
31. Fig. 1part1
To identify regions with the highest Kir4.1
expression levels
• mRNA profiling
• Histology
• Western Blot
• FACS(Aldh1l1-EGFP)
• Kir4.1 was enriched in ventral versus
dorsal AS.
32. Fig. 1part2
To identify regions with the highest Kir4.1
expression levels
• Histology
• MMP-9 as a marker of FaMNs
• VGLUT1 KO animals
• Kir4.1 expression:
FaMNs(MMP9+) > SaMNs(MMP9-)
• VGLUT1 L.O.F leads to AS Kir4.1
reduction
AS Kir4.1 expression levels show both
regional (i.e., ventral versus dorsal horn)
and subregional (i.e., FaMNs versus
SaMNs) differences and are regulated in a
VGLUT1-dependent manner in vivo.
33. Fig. 2part1
To investigate a requirement for AS-
encoded Kir4.1 for ventral horn MNs
• Histology
• Aldh1l1 x Kir fl/fl X ChAT-GFP
• FaMN (ChAT + /MMP-9 + /NeuN + )
SaMN (ChAT + /MMP-9 - / NeuN + )
gMN (ChAT + /MMP-9 - /NeuN - )
• No loss in FaMN, SaMN, or gMN
populations in the lumbar spinal cord of
AS-Kir4.1cKO mice.
AS Kir4.1 is dispensable for the
specification and survival of MNs.
34. To analyzed the morphological properties
of MN subpopulations
• Histology
• Aldh1l1 x Kir fl/fl X ChAT-GFP
• FaMN (ChAT + /MMP-9 + /NeuN + )
SaMN (ChAT + /MMP-9 - / NeuN + )
gMN (ChAT + /MMP-9 - /NeuN - )
• Selective decrease in size of FaMNs at
P30 and 6 months of age
• Whereas the size of SaMNs and gMNs
remained unaffected
Fig. 2part2
35. Fig. 2part3
To analyzed the morphological properties
of MN subpopulations
• Histology
• Aldh1l1 x Kir fl/fl X ChAT-GFP
• Cholera-toxin subunit B **
• TA muscle (FaMN major)
• ReducedFaMNsizein AS-Kir4.1cKO
animals compared to cre-negative
controls
The maintenance of large FaMN size has
a selective dependence on AS Kir4.1.
38. Fig. 3part1
To test weather loss of Kir4.1 would affect
K + homeostasis in the region and alter
physiologic function of all MN subtypes.
• Whole-cell patch-clamp recordings
• Aldh1l1 x Kir fl/fl X ChAT-GFP
• P12–P15 MNs
• Threshold (FF > S) **
Input resis-tance (FF < S)
Steady-State Firing (FF > S)
AHP Half Decay Time (FF < S)
• Kir4.1cKO animals had a significantly
lower activation threshold (rheobase),
larger input resistance, decreased
instantaneous and steady-state firing
frequency,and increased AHP half-
decay time
Maintenance of many FaMN biophysical
properties depends on intact AS Kir4.1
expression/function
39. Rheobase is a measure of membrane
potential. In neuroscience, rheobase
is the minimal current amplitude of
infinite duration (in a practical sense,
about 300 milliseconds) that results in
the depolarization threshold of the cell
membranes being reached, such as
an action potential or the contraction
of a muscle.
Rheobase is used for Threshold Detection
40. Fig. 3part2
To test relationship between MN area and
the size of the corresponding muscle fiber
subset
• Histology
• TA muscle (FaMN major)
• Myosin type 2
• Behavior**
• Grip Strength
• Gait Analysis
• Rotarod
• Reduced fiber areas of fast-twitch
muscle from AS-Kir4.1cKO at P30
• AS-Kir4.1cKO have
• decreased maximal peak force
• slower front and hindlimb movements
• basic movements in the open field
AS Kir4.1 is selectively required for
behavioral tasks involving strength or fast
movements.
43. Fig. 4part1
To ascertain whether SOD1 mutation is
sufficient to decrease KIR4.1 expression
in human AS
• Histology
• qPCR
• Western blot
• Human iPSC**
• Significantly decreased KIR4.1
expression in cultured human AS
carrying the SOD1D90A mutation.
Mutant SOD1 downregulates KIR4.1 in a
cell-autonomous manner.
45. Fig. 4part2
To rule out a requirement for AS Kir4.1 to
maintain MN survival in an animal model
of ALS
• Histology
• Aldh1l1 x Kir fl/fl x mSOD1 mice
• MN loss in mSOD1 mice at P80
• No evidence for exaggerated losses in
total MN or FaMN numbers in
mSOD1 : Aldh1l1 x Kir fl/fl x mSOD1
AS Kir4.1 is dispensable for MN survival
at P80 even in the setting of mutant
SOD1G93A mutation.
46. Fig. 5 To evaluate the effect of Kir4.1 on MN
size.
• AAV- gfa-ABC1D-Kir4.1-eGFP (GOF)
• AAV- gfa-ABC1D-tdTomato (CON)
• Neonatal ICV delivery to Spinal Cord**
• AS Kir4.1 overexpression increased the
size of FaMNs and SaMNs
• MNs abutting Kir4.1-eGFP-transduced
AS were significantly larger
AS Kir4.1 overexpression is sufficient to
increase the size of both FaMNs and
SaMNs via contact-mediated manner
47. Kim et al. (2016), Ayers et al. (2015), Kawasaki et al. (2016)
ICV Injection in the Neonatal Brian
48. Fig. 6 To test weather AS Kir4.1 regulates MN
size through the PI3K/mTOR/ pS6 **
pathway
• Histology
• Aldh1l1 x Kir fl/fl X ChAT-GFP(LOF)
• AAV- gfa-ABC1D-Kir4.1-eGFP (GOF)
AAV- gfa-ABC1D-tdTomato
• mTOR effector, pS6, levels were
decreased in FaMNs in P30 AS-
Kir4.1cKO mice and increased in P60
AAV-Kir4.1-injected mice.
mTOR signaling is a driver rather than
biomarker proxy of cell size
50. Fig. 6 To determine whether the PI3K/mTOR/
pS6 pathway was necessary for the
increase in MN size observed with GOF
• Histology
• AAV- gfa-ABC1D-Kir4.1-eGFP (GOF)
AAV- gfa-ABC1D-tdTomato
• Neonatal brain ICV inj.
• Rapamycin
• Rapamycin treatment prevented the
AAV-Kir4.1-mediated increase in MN
size
51. Fig. 6 To address whether extracellular K +
might directly regulate MN soma size
• Spinal cord slice incubation
• Histology
• ChAT-GFP
• Mannitol(hyperosmotic control)
• ChAT + MNs were smaller in the high K
+ condition compared with isotonic
ACSF and hyperosmotic mannitol ACSF
controls
Regulation of cell size via K+ ions
The Catwalk is an automated gait analysis system used to assess motor function and coordination in rodent models of CNS disorders. Subjects walk across an illuminated glass platform while a video camera records from below. Gait related parameters—such as stride pattern, individual paw swing speed, stance duration, and pressure—are reported for each animal. This test is used to phenotype transgenic strains of mice and evaluate novel chemical entities for their effect on motor performance.
