Stress preconditioning of spreading depression in the locust CNS

Abstract

Cortical spreading depression (CSD) is closely associated with important pathologies including stroke, seizures and migraine. The mechanisms underlying SD in its various forms are still incompletely understood. Here we describe SD-like events in an invertebrate model, the ventilatory central pattern generator (CPG) of locusts. Using K(+) -sensitive microelectrodes, we measured extracellular K(+) concentration ([K(+)](o)) in the metathoracic neuropile of the CPG while monitoring CPG output electromyographically from muscle 161 in the second abdominal segment to investigate the role K(+) in failure of neural circuit operation induced by various stressors. Failure of ventilation in response to different stressors (hyperthermia, anoxia, ATP depletion, Na(+)/K(+) ATPase impairment, K(+) injection) was associated with a disturbance of CNS ion homeostasis that shares the characteristics of CSD and SD-like events in vertebrates. Hyperthermic failure was preconditioned by prior heat shock (3 h, 45 degrees C) and induced-thermotolerance was associated with an increase in the rate of clearance of extracellular K(+) that was not linked to changes in ATP levels or total Na(+)/K(+) ATPase activity. Our findings suggest that SD-like events in locusts are adaptive to terminate neural network operation and conserve energy during stress and that they can be preconditioned by experience. We propose that they share mechanisms with CSD in mammals suggesting a common evolutionary origin.

Figure 1. Stress-induced motor pattern failure is associated with surges of [K⁺]_o.

Simultaneous recordings of the ventilatory motor pattern (Vent), the temperature of the superfusing saline at the MTG (Temp in A) and the extracellular potassium concentration ([K⁺]_o). A. An abrupt increase in [K⁺]_o was reliably associated with heat-induced failure of the ventilatory motor pattern, which occurred in 100% of preparations (N = 17). [K⁺]_o was restored to normal baseline levels if heat was removed and this was associated with recovery of ventilatory motor patterning. B. 10⁻³ M NaN₃ was bath-applied until 1 minute post-failure (N = 6). [K⁺]_o gradually decreased and the motor pattern recovered upon superfusion of standard locust saline. C. N₂ was bubbled into the superfusing saline for 5 minutes, then blown over the preparation until 1 minute post-failure (N = 18). Re-oxygenation resulted in [K⁺]_o clearance and recovery of the motor pattern. i and ii show expansions of the motor pattern trace pre- and post-stress to show more clearly the ventilatory motor pattern. In B and C the temperature was constant at room temperature (∼22°C).

Figure 2. Characteristics of [K⁺]_o surges.

Simultaneous recordings of the ventilatory motor pattern (Vent), a monitor of pressure-injection of a bolus of K⁺ within the MTG (Trig in B) and the extracellular potassium concentration ([K⁺]_o). A. Continuous bath application of 10⁻⁴ M ouabain elicited multiple surges in [K⁺]_o (N = 13). In the experiment shown here it took 434 seconds for 10⁻⁴ M ouabain to penetrate the MTG and induce failure of motor pattern generation. B. A 35 nl pressure-injection of locust saline containing a 15-fold higher [K⁺] (150 mM compared to 10 mM) into the MTG neuropile was sufficient to bring [K⁺]_o to threshold and induce an abrupt surge (N = 18). C. Two K⁺ -sensitive microelectrodes were inserted in different regions of the MTG (a and b) to illustrate propagation (dotted line). In this experiment the propagation speed was 1.9 mm/min.

Figure 3. The hyperthermic [K⁺]_o event is not representative of complete collapse of the K⁺ gradient.

A. During a continuous increase of temperature [K⁺]_o increased sharply at ∼40°C coincident with motor pattern failure and continued to rise until a second plateau was reached at ∼60°C. When the heater was turned off [K⁺]_o remained elevated and motor pattern generation failed to recover (N = 11). B. Only the second plateau was evident during temperature increase to ∼60°C following prior anoxic arrest of motor pattern generation. When the N₂ was turned off and internal temperature returned to room temperature [K⁺]_o remained elevated and motor pattern generation failed to recover (N = 12).

Figure 4. The hyperthermic [K⁺]_o event is delayed by blocking Na⁺ channels and neural activity using TTX and is diminished by blocking K⁺ channels using TEA. A. 10⁻⁶ M TTX abolished electrical activity within minutes while [K⁺]_o remained stable.

The abrupt rise in [K⁺]_o was not significantly diminished by TTX. The hyperthermic [K⁺]_o event occurred at a significantly higher temperature (measured at dotted line) in the absence of electrical activity (46±1°C) compared to CON locusts (39±1°C) (C) (N_CON = 17; N_TTX = 15). B. 10⁻¹ M TEA significantly reduced the hyperthermic [K⁺]_o event (N_CON = 17; N_TEA = 6) (D). [K⁺]_o increased with continued temperature increase to ∼60°C.

Figure 5. Failure and recovery of the CPG are not dependent on MTG ATP and HS preconditioning does not confer protection by improving ATP availability.

A. ATP levels did not differ from the pre-stress level (Normoxia at room temperature; N = 16) in locusts treated with 10⁻⁴ M ouabain or hyperthermia (HS) at failure or recovery, or in all other groups at recovery following stress-induced failure with the exception of NaN₃-treated locusts. There was a main effect of treatment driven by significant differences among groups within failure and recovery (not indicated by symbols). There was no effect of HS pre-treatment on ATP levels at failure or recovery of motor pattern generation. Asterisks indicate significant differences from the pre-stress level and daggers indicate significantly different ATP levels at failure and recovery in ganglia of NaN₃-treated locusts. Sample sizes (failure, recovery): N_Ouabain = 10,11; N_{HS-hyperthermia} = 8,8; N_Anoxia = 9,8; N_{CON-hyperthermia} = 7,7; N_{Sodium azide} = 8,9. B. CON and HS locusts did not have significantly different ATP levels during anoxia-induced coma or subsequent recovery. ATP levels dropped in both CON and HS locusts after 30 minutes of anoxia and remained stable until locusts were removed from the anoxic environment. ATP levels increased to around pre-anoxia values after one hour in normally oxygenated air. Sample sizes (CON, HS): N_0min = 16,16; N_15min = 12,11; N_30min = 11,11; N_60min = 9,9; N_120min = 12,11; N_150min = 11,12; N_180min = 10,10. Asterisks indicate significant differences from 0 and 15 minutes and daggers indicate significant differences from 180 minutes (or 60 minutes recovery) according to post hoc Tukey tests (P<0.05).

Figure 6. HS and ouabain affect [K⁺]_o.

A. Heat shock preconditioning resulted in a significantly higher [K⁺]_o in HS locusts compared to CON locusts. All animals pooled: N_CON = 25; N_HS = 26. B. CON-OUA and HS-OUA locusts had a significantly greater increase in [K⁺]_o after 15 minutes of bath application with 10⁻⁵ M ouabain compared to CON and HS locusts. HS pre-treatment did not have an effect on Δ[K⁺]_o over this 15 minute period (N_CON = 12; N_CON-OUA = 10; N_HS = 17; N_HS-OUA = 8). C. CON and HS locusts had a markedly different Δ[K⁺]_o in response to a 5°C increase in temperature. Δ[K⁺]_o was positive in CON locusts and negative in HS locusts after only one minute of temperature increase. There was no effect of 10⁻⁵ M ouabain treatment on Δ[K⁺]_o one minute into the temperature ramp (N_CON = 12; N_CON-OUA = 10; N_HS = 17; N_HS-OUA = 8). Asterisks indicate a significant effect of ouabain and daggers indicate a significant effect of pre-treatment according to t-test (A) and post hoc Tukey tests (P<0.05) (B,C).

Figure 7. HS delays the [K⁺]_o event and speeds recovery by increasing the rate of [K⁺]_o clearance.

A. Time to recovery was positively correlated with failure temperature in CON and HS locusts (N_CON = 13; N_HS = 15). Animals whose ventilatory motor pattern failed at a higher temperature had a longer time to recovery when temperature returned to normal levels. B. The correlations seen in CON and HS animals were shifted in animals treated with 10⁻⁵ M ouabain (N_CON-OUA =9; N_HS-OUA =8). The relationship between failure temperature and time to recovery in HS-OUA animals resembled CON animals. Data points for each group were fitted with exponential curves that rise to a plateau (A, B). C. Ventilatory motor pattern generation in HS locusts failed at a significantly higher temperature than in CON locusts with and without 10⁻⁵ M ouabain treatment. There was a significant effect of ouabain on the ability to withstand high temperature stress in CON locusts but not in HS locusts (N_CON = 15; N_CON-OUA = 9; N_HS = 17; N_HS-OUA = 8). D. Ventilatory motor pattern generation in HS locusts took significantly less time to recover following heat-induced failure than in CON locusts with and without ouabain treatment. CON-OUA locusts took significantly more time to recover than CON locusts, however there was no significant effect of ouabain treatment on time to recovery in HS locusts (N_CON = 13; N_CON-OUA = 10; N_HS = 15; N_HS-OUA = 8). E. Overall there were no main effects of HS pre-treatment or ouabain treatment on the rate of [K⁺]_o increase (mM/s) associated with failure of the motor pattern (N_CON = 15; N_CON-OUA = 10; N_HS = 18; N_HS-OUA = 7). F. The rate of [K⁺]_o decrease (mM/s) associated with recovery of the motor pattern was significantly greater in HS locusts than in CON locusts and this effect of pre-treatment was conserved in ouabain-treated locusts. Ouabain treatment significantly decreased the rate of [K⁺]_o clearance in CON locusts but not in HS locusts (N_CON = 15; N_CON-OUA = 10; N_HS = 18; N_HS-OUA = 8). Asterisks indicate a significant effect of ouabain and daggers indicate a significant effect of pre-treatment according to post hoc Tukey tests (P<0.05) (C–F).