1. Neuroscience
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Spinal Shox2 interneuron interconnectivity related to function and development

  1. Ngoc T Ha
  2. Kimberly J Dougherty  Is a corresponding author
  1. Drexel University College of Medicine, United States
Research Article
Cite this article as: eLife 2018;7:e42519 doi: 10.7554/eLife.42519
8 figures, 1 table and 1 additional file

Figures

Unidirectional connections are present between Shox2 INs in neonatal mouse spinal cords.

(A and B) Examples of recordings from a pair of Shox2 INs that are unidirectionally connected. (Ai and Bi) Cartoon of stimulation and recording direction for 2 Shox2 INs. (Ai and ii) Current was injected in order to evoke five action potentials in Shox2 IN 1 (black). EPSCs (Ai) or EPSPs (Aii) were evident in Shox2 IN 2 (red) when recorded in voltage clamp or current clamp mode, respectively. Black arrows indicate direction of connectivity tested. (Bi and Bii) When the protocol was reversed and action potentials were evoked by current injections into Shox2 IN 2 (red), there was a lack of response in Shox2 IN 1 (black). All data shown in (A and B) were the average of 50 trials. (C and D) Examples of individual trials from two unidirectionally connected pairs demonstrating that postsynaptic responses did not occur for each presynaptic action potential. The voltage clamp recordings showing EPSCs in (C) and the current clamp recordings showing EPSPs in (D) are from Shox2 IN 2 in (A and B). In both (C and D), red arrows indicate action potential peaks in the presynaptic Shox2 IN.

https://doi.org/10.7554/eLife.42519.002
Bidirectional connections are present between Shox2 INs in neonatal spinal cord.

(A and B) Examples of recordings from a pair of bidirectionally connected Shox2 INs. (Ai and Bi) Cartoon corresponding to colors of traces in (A and B). (Ai and Aii) Current injections into Shox2 IN 1 evoked five action potentials (gray). Excitatory postsynaptic currents (Ai) or potentials (Aii) resulted in Shox2 IN 2 (blue) in voltage clamp or current clamp mode. Reversal of the protocol, current injection in Shox2 IN 2 (blue) also resulted in excitatory currents (Bi) or potentials (Bii) in Shox2 IN 1. All data in A and B were averages of 50 trials. (C and D) Examples of individual trials between bidirectionally connected pairs recorded in voltage clamp (C) and current clamp (D). Gray arrows signify the peaks of the presynaptic action potentials. Note the lack of failures and that responses appear nearly identical in each sweep. (Ei) Action potentials evoked in presynaptic unidirectionally (black) and bidirectionally (gray) connected pairs and examples of single postsynaptic currents (red, unidirectional; blue, bidirectional). Dotted lines highlight the beginning of the depolarization and action potential peak in the presynaptic cells, in order to visualize latency differences in the postsynaptic cells. (Eii) Mean latency of the EPSC peak, referenced to the peak of the evoked action potential between unidirectionally connected pairs (red) and bidirectionally connected pairs (blue). (Fi) Similar to Ei but current clamp recordings showing single EPSPs in postsynaptic unidirectional (red) and bidirectional (blue) Shox2 IN pairs in relation to the action potentials in their respective presynaptic Shox2 INs (black and gray). Dotted lines correspond to the start of the depolarization and the peak of the action potential in the presynaptic neurons to highlight the differences in the latency between the two types of connections. (Fii) Mean EPSP latency, peak of presynaptic action potential to start of postsynaptic depolarization, is shown for the unidirectional (red) and bidirectional (blue) Shox2 IN pairs. The depolarization in bidirectional pairs precedes the presynaptic action potential, resulting in a negative latency value. ** indicates p<0.01. Error bars represent SD.

https://doi.org/10.7554/eLife.42519.003
Figure 2—source data 1

Mean latency of EPSC source data for Figure 2Eii.

https://doi.org/10.7554/eLife.42519.004
Figure 2—source data 2

Mean latency of EPSP source data for Figure 2Fii.

https://doi.org/10.7554/eLife.42519.005
Bidirectional connectivity is due to electrical coupling.

(A and B) Examples of recordings from a pair of bidirectionally connected Shox2 INs. In (A), responses of Shox2 IN 1 (gray) and Shox2 IN 2 (blue) to 1 s long hyperpolarizing and depolarizing current steps injected into Shox2 IN one are shown. In (B), the protocol was reversed and responses to current steps in Shox2 IN2 are shown. As typical in bidirectionally connected Shox2 INs, both hyperpolarizing and depolarizing responses were evident in the non-injected cell. Additionally, spikelets in the non-injected neuron corresponded to action potentials generated in the neuron receiving the current steps. Darker shading corresponds to increasing current steps. (C) Coupling coefficients were highly variable with a mean of 13%, indicated by the green line. (D and E) Examples of recordings from a pair of unidirectionally connected Shox2 INs. In (D), 1 s long current steps were injected into Shox2 IN one while responses of Shox2 IN 1 (black) and Shox2 IN 2 (red) were recorded in current clamp mode. In (E), the same protocol was performed but current was injected into Shox2 IN 2. Darker shading corresponds to increasing current steps. (F) Distance between recorded neurons was not significantly different by connection type but connected cells were significantly closer together in slices than in dorsal horn-removed preparations. Empty bars for dorsal horn-removed preparations (dhr), filled bars for slices (s), unidirectional (red), bidirectional (blue), or not connected (gray) pairs, mean ±SD.

https://doi.org/10.7554/eLife.42519.006
Figure 3—source data 1

Coupling coefficients in neonates source data for Figure 3C.

https://doi.org/10.7554/eLife.42519.007
Figure 3—source data 2

Distance between recorded neurons source data for Figure 3F.

https://doi.org/10.7554/eLife.42519.008
Electrical coupling between Shox2 INs does not have a chemical component.

(A) Examples of recordings from a bidirectionally connected pair of Shox2 INs. Averaged recordings prior to (gray) and after the addition AMPA receptor and NMDA receptor antagonists, CNQX and APV or CPP (teal). Current injections into Shox2 IN 1 (bottom) elicited action potentials in the stimulated/presynaptic neuron (middle) and depolarizations in Shox2 IN2 (top). (B) There was no significant change to the amplitude of the first EPSP when fast glutamatergic transmission was blocked. (C) Examples of recordings from a bidirectional connected pair of Shox2 INs prior to (gray) and after the addition of gap junctional blocker, carbenoxolone (yellow). Traces are in the same order as in (A). (D) Bar graphs showed the amplitude of the first EPSP in the postsynaptic Shox2 IN in response to the first of five action potentials evoked in the presynaptic neuron. Carbenoxolone efficiently decreased the EPSPs in the responding Shox2 IN. * indicates p<0.05. Error bars represent SD.

https://doi.org/10.7554/eLife.42519.009
Figure 4—source data 1

EPSP amplitude pre- and post-glutamatergic antagonist source data for Figure 4B.

https://doi.org/10.7554/eLife.42519.010
Figure 4—source data 2

EPSP amplitude pre- and post-carbenoxolone source data for Figure 4D.

https://doi.org/10.7554/eLife.42519.011
Electrical synapses between Shox2 INs act as low-pass filters.

(A) Membrane oscillations in Shox2 IN 1 (gray) and Shox2 IN 2 (blue) resulting from subthreshold sinusoidal current injections (±20 pA) to Shox2 IN 1 at 2 Hz and 10 Hz frequencies. All traces are averages of 10 sweeps. (B) Coupling coefficients normalized to value at 2 Hz to demonstrate frequency-dependence. Coupling strength decreased with increasing frequency of injected current (0.2 Hz; n = 3; 1 Hz, n = 5; 2 Hz, n = 13; 5 Hz, n = 12; 10 Hz, n = 8; and 20 Hz, n = 7) Error bars represent SD. (C) Phase lag is frequency dependent. As the frequency of the injected current increased, phase lag increased (0.2 Hz, n = 2; 1 Hz, n = 5; 2 Hz, n = 13; 5 Hz, n = 12; 10 Hz, n = 9; and 20 Hz, n = 7).

https://doi.org/10.7554/eLife.42519.012
Figure 5—source data 1

Coupling coefficient source data for Figure 5B.

https://doi.org/10.7554/eLife.42519.013
Figure 5—source data 2

Phase lag source data for Figure 5C.

https://doi.org/10.7554/eLife.42519.014
Blocking gap junctions with carbenoxolone decreases locomotor frequency.

(A) Extracellular recordings from ventral roots at lumbar level 2 (L2)-flexor dominant root- and level 5 (L5)-extensor dominant root- on the right (r) and on the left (l) side of the spinal cord after application of NMDA (7 μM) and serotonin (8 μM). Alternation in ventral root bursts was present between the flexor and extensor root as well as between the right side and the left side of the spinal cord. (B) Addition of carbenoxolone (100 μM) decreased the frequency of locomotion (analyzed after 40 min of wash in) with little to no change in the pattern of locomotion. (C) Quantification of locomotor frequency shows that it is significantly reduced by the addition of carbenoxolone. *** indicates p<0.005. Error bar represents SD.

https://doi.org/10.7554/eLife.42519.015
Electrical coupling between Shox2 INs is age-dependent.

(A) Example of bidirectional electrical coupling detected between Shox2 INs in a P13 mouse. Hyperpolarizing and depolarizing current steps injected into either Shox2 IN 1 (left) or Shox2 IN 2 (right) resulted in hyperpolarizations and depolarizations in both neurons. Shading of lines is to better visualize separate sweeps of differing injected currents. Spikelets were observed in the connected Shox2 INs corresponding to action potentials generated in the IN depolarized by injected current. (B) Pie charts indicate the incidence of connectivity between Shox2 INs detected in P0-P5, P13-P17, and P23-P35 age groups. Darker colors in each represent bidirectional connections. Lighter wedges in P0-P5 and P23-P35 represent unidirectional connections. (C) Strength of coupling coefficient in bidirectionally connected Shox2 INs from different age groups as the mice mature. (D) Amplitude of the EPSP in the postsynaptic Shox2 IN in response to the first of five action potentials evoked in the presynaptic neuron in different age groups. * indicates p<0.05 and ** indicates p<0.01. Error bars represent SD.

https://doi.org/10.7554/eLife.42519.016
Figure 7—source data 1

Coupling coefficients by age group source data for Figure 7C.

https://doi.org/10.7554/eLife.42519.017
Figure 7—source data 2

EPSP amplitudes by age group source data for Figure 7D.

https://doi.org/10.7554/eLife.42519.018
Connectivity between Shox2 INs depends on function.

(A) Examples of recordings from a pair of Shox2+ non-V2a INs. Average of 50 responses of Shox2 IN 1 and Shox2 IN 2 to 5 action potentials evoked with current steps applied to the other IN. Action potential peaks are indicated with arrows. Pie graphs show the proportion of Shox2+ non-V2a pairs found to be bidirectionally connected (white) and not connected (gray). (B) Example recordings from a pair of Shox2+ V2a INs, as in (A). (C) Example recordings from a mixed pair consisting of one Shox2+ non-V2a INs and one Shox2+ V2a INs, displayed as in (A).

https://doi.org/10.7554/eLife.42519.019

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent
(M. musculus)
Shox2::CrePMID: 24267650
Genetic reagent
(M. musculus)
Rosa26-lsl-
tdTomato
Jackson
Laboratory
Stock #: 007909PMID: 20023653
Genetic reagent
(M. musculus)
Chx10GFPMutant Mouse
Regional
Resource Center
MMRRC Cat#:
011391-UCD
Now called
Vsx2-EGFP;
PMID: 14586460
Chemical
compound, drug
carbenoxolone
disodium salt
SigmaC4790
Chemical
compound, drug
5-HT, serotonin
creatinine sulfate
monohydrate
SigmaH7752
Chemical
compound, drug
NMDA, N-Methyl-
D-aspartic acid
SigmaM3262
Chemical
compound, drug
CNQX, 6-cyano-7-
nitroquinoxaline-2,3-
dione disodium salt
Tocris1045
Chemical
compound, drug
AP-5, 2-amino-5-
phosphopentanoic
acid
Tocris1234
Chemical
compound, drug
CPP, 3-((R)−2-
Carboxypiperazin
-4-yl)-propyl-1-
phosphonic
acid
Tocris2411

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