Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus)

Abstract

Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.

Fig 1. Examples of Cx43-like immunoreactivity in coronal sections of the mudpuppy spinal cord.

Anti-Cx43 produced punctuate staining (green) near nuclei in the ventral horn and strings of punta radiating outward to the sub-pial plexus (A-C). Confocal images showing the colocalization of Cx43-like (green) and GFAP-like (red) immunofluorescence patterns in coronal sections of the mudpuppy spinal cord (D-E). The wide-spread distribution of the Cx43-like immunoreactivity parallels the GFAP-like staining in the gray and white matters. The confocal images are from a single optical plane. The photos on the right are higher magnification images of the regions enclosed by the white boxes. Nuclei in all images were counter stained with DAPI (blue). The scale bar in A = 200 μM; in B and C = 50 μM; in D = 100 μM; and in E = 250 μM.

Fig 2. Depiction of the design for electrophysiology experiments.

The figure presents the EMG activity simultaneously recorded from the flexor and extensor muscles during a typical experiment. Walking-like activity was induced with 50 μM NMDA and 10 μM D-serine. The initiation and termination of FFA application (300 μM) is indicated with arrows. Segments used for analysis of the initial changes and tested intervals are shown.

Fig 3. CBX modulates the NMDA- and D-glutamate-induced walking-like activity.

A: Dose-response curves showing the effects of CBX on the cycle frequency. The mean frequency of D-glutamate- (●) and NMDA-induced (○) motor activity patterns were reduced by CBX in a dose-dependent manner. D-glutamate-induced activity was inhibited at lower concentrations than the NMDA-induced activity. B: Dose-response curves showing the effects of CBX on the peak EMG amplitude for D-glutamate-induced activity (●), NMDA-induced activity (○), and the dorsal root reflex (DRR, ▼). The amplitude of D-glutamate-induced activity was inhibited by CBX (concentrations > 100 μM) in a dose-dependent manner, whereas, the amplitude of NMDA-induced patterns were facilitated by CBX (concentrations > 1000 μM). In contrast, the amplitudes of the dorsal root reflexes (DRR) were not significantly affected by CBX. C: Paired recordings from the flexor and extensor muscles showing the effects of CBX on D-glutamate- (right) and NMDA-induced (left) motor activity patterns. Notice that the cycle frequency and EMG amplitude were reduced in the presence of 200 μM CBX (lower pair of records) compared to the control saline (upper pair of records). Values of the means and SEMs as well as their statistical significance are mentioned in the text. Data presented in all graphs are means ± SEMs.

Fig 4. FFA modulates the NMDA- and D-glutamate-induced walking-like activity.

A: Dose-response curves showing the effects of FFA on the cycle frequency. The mean frequency of D-glutamate- and NMDA-induced motor activity patterns were reduced by FFA concentrations > 100 μM in a dose-dependent manner. D-glutamate-induced activity was inhibited at lower concentrations than the NMDA-induced activity. B: Dose-response curves showing the effects of FFA on the peak EMG amplitude for D-glutamate-induced activity, NMDA-induced activity, and the dorsal root reflex. The amplitude of D-glutamate- and NMDA-induced activity was inhibited by FFA concentrations >100 μM in a dose-dependent manner. In contrast, the amplitudes of the dorsal root reflexes were not significantly affected by FFA at concentrations < 700 μM. C: Paired recordings from the flexor and extensor muscles showing the effects of FFA on D-glutamate- (right) and NMDA-induced (left) motor activity patterns. Notice that the cycle frequency and EMG amplitude were reduced in the presence of 500 μM FFA (lower pair of records) compared to the control saline (upper pair of records). Abbreviations, same as in Fig 3.

Fig 5. Heptanol modulates the NMDA- and D-glutamate-induced walking-like activity.

A: Dose-response curves showing the effects of Heptanol on the cycle frequency. The mean frequency of D-glutamate- and NMDA-induced (○) motor activity patterns was facilitated by Heptanol at low concentrations (100–300 μM) and reduced at concentrations >400 μM in a dose-dependent manner. D-glutamate-induced activity was inhibited at lower concentrations than the NMDA-induced activity. B: Dose-response curves showing the effects of Heptanol on the peak EMG amplitude for D-glutamate-induced activity, NMDA-induced activity, and the dorsal root reflex. The amplitude of D-glutamate- and NMDA-induced activity was facilitated by Heptanol at low concentrations (100–300 μM) and depressed at concentrations >400 100–300 μM. The amplitudes of the dorsal root reflexes were reduced (~20%) by Heptanol at 300 μM. C: Paired recordings from the flexor and extensor muscles showing the effects of Heptanol on D-glutamate- (right) and NMDA-induced (left) motor activity patterns. Notice that the cycle frequency and EMG amplitude were reduced in the presence of 700 μM Heptanol (lower pair of records) compared to the control saline (upper pair of records). Abbreviations, same as in Fig 3.

Fig 6. Octanol modulates the NMDA- and D-glutamate-induced walking-like activity.

A: Dose-response curves showing the effects of Octanol on the cycle frequency. The mean frequency of D-glutamate- and NMDA-induced motor activity patterns were reduced by Octanol concentrations >400 μM in a dose-dependent manner. B: Dose-response curves showing the effects of Octanol on the peak EMG amplitude for D-glutamate- and NMDA-induced activity. The amplitude of D-glutamate- and NMDA-induced activity was inhibited by Octanol concentrations >400 μM in a dose-dependent manner. C: Paired recordings from the flexor and extensor muscles showing the effects of Octanol on D-glutamate- (right) and NMDA-induced (left) motor activity patterns. Notice that the cycle frequency and EMG amplitude were reduced in the presence of 700 μM Octanol (lower pair of records) compared to the control saline (upper pair of records). Abbreviations, same as in Fig 3.