Hibernation induces pentobarbital insensitivity in medulla but not cortex

Abstract

The 13-lined ground squirrel (Ictidomys tridecemlineatus), a hibernating species, is a natural model of physiological adoption to an extreme environment. During torpor, body temperature drops to 0-4 degrees C, and the cortex is electrically silent, yet the brain stem continues to regulate cardiorespiratory function. The mechanisms underlying selective inhibition in the brain during torpor are not known. To test whether altered GABAergic function is involved in regional and seasonal differences in neuronal activity, cortical and medullary slices from summer-active (SA) and interbout aroused (IBA) squirrels were placed in a standard in vitro recording chamber. Silicon multichannel electrodes were placed in cortex, ventral respiratory column (VRC), and nucleus tractus solitarius (NTS) to record spontaneous neuronal activity. In slices from IBA squirrels, bath-applied pentobarbital sodium (300 microM) nearly abolished cortical neuronal activity, but VRC and NTS neuronal activity was unaltered. In contrast, pentobarbital sodium (300 microM) nearly abolished all spontaneous cortical, VRC, and NTS neuronal activity in slices from SA squirrels. Muscimol (20 microM; GABA(A) receptor agonist) abolished all neuronal activity in cortical and medullary slices from both IBA and SA squirrels, thereby demonstrating the presence of functional GABA(A) receptors. Pretreatment of cortical slices from IBA squirrels with bicuculline (100 microM; GABA(A) receptor antagonist) blocked pentobarbital-dependent inhibition of spontaneous neuronal activity. We hypothesize that GABA(A) receptors undergo a seasonal modification in subunit composition, such that cardiorespiratory neurons are uniquely unaffected by surges of an endogenous positive allosteric modulator.

Fig. 1.

Multichannel recordings of spontaneous neuronal activity in cortical and medullary slices from interbout aroused (IBA) ground squirrels. A: cortical activity was recorded from neurons in the primary motor (M1), supplementary motor (M2), and primary somatosensory (S1) areas. Slices were cut approximately −1.30 mm caudal to bregma [anatomical images adapted from Paxinos and Watson (50)]. Shaded areas indicate electrode placement. B: medullary activity was recorded from the nucleus tractus solitarius (NTS) and ventral respiratory column (VRC). Slices were cut approximately −13.68 mm caudal to bregma. Shaded areas indicate electrode placement. C: representative cortical recordings are shown during baseline (top left) and after 60 min of pentobarbital sodium (300 μM) treatment (top right). Traces that cross the detection threshold (dashed white lines) are overlaid (bottom right). D: representative recordings from VRC neurons are shown during baseline (top left) and after 60 min of sodium pentobarbital (300 μM, top right). Traces that cross the detection threshold (dashed gray lines) are overlaid (bottom right).

Fig. 2.

Pentobarbital sodium alters spontaneous activity of NTS, VRC, and cortical neurons. A: mean normalized firing rate is shown for cortical neurons in slices from summer-active (SA) squirrels (n = 6; n = 77 neurons; open circles) and IBA squirrels (n = 6; n = 79 neurons, solid circles) in response to 300 μM pentobarbital sodium (applied at vertical line). At 120 min, neuronal activity from both SA and IBA squirrels was decreased significantly compared with time control (P < 0.001). IBA time controls are shown (n = 4; n = 22 neurons; gray circles). B: mean normalized firing rate is shown for VRC neurons in slices from SA squirrels (n = 6; n = 21 neurons; open squares) and IBA squirrels (n = 7; n = 54 neurons; solid squares). At 120 min, SA neuron activity was decreased compared with time controls (P < 0.001), while IBA neuron activity was not different from time controls (P > 0.05) IBA time controls are shown (n = 4; n = 40 neurons; gray squares). C: mean normalized firing rate is shown for NTS neurons in slices from SA squirrels (n = 6; n = 50 neurons, open triangles) and IBA squirrels (n = 7; n = 37 neurons; solid triangles). At 120 min, SA neuron activity was decreased compared with time controls (P < 0.01), while IBA neuron activity did not differ from time controls (P = 0.091). IBA time controls are shown (n = 4; n = 12 neurons; gray triangles). Error bars indicate means ± SE. †P < 0.05 compared with time controls.

Fig. 3.

Variability in neuronal activity at 60 min following bath-applied pentobarbital sodium. The activity of single neurons was normalized to the mean baseline activity for that neuron. Neurons were binned according to their normalized firing rates during the last 5 min of pentobarbital sodium (300 μM) application. A and B: number of neurons vs. mean normalized firing rate is shown for cortical neurons recorded in slices from IBA (n = 6; n = 79 neurons) and SA squirrels (n = 6; n = 77 neurons). C and D: number of neurons is shown for VRC neurons in brain stem slices from IBA (n = 7; n = 54 neurons) and SA squirrels (n = 6; n = 21 neurons). E and F: number of neurons is shown for NTS neurons in brain stem slices from IBA (n = 7; n = 37 neurons) and SA squirrels (n = 6; n = 21 neurons).

Fig. 4.

Pentobarbital sodium produces similar effects in lower bath [KCl]. A: responses are shown for VRC, NTS, and cortical neurons in slices from IBA squirrels exposed to 5 mM KCl and 150 μM pentobarbital sodium (open bars) compared with 9 mM KCl and 300 μM pentobarbital sodium (solid bars). The data show the mean normalized firing rate during the last 5 min of a 60-min drug application. B: dose-dependent effects of sodium pentobarbital on spontaneous activity of VRC, NTS, and cortical neurons in slices from IBA squirrels are shown. The pentobarbital sodium concentrations that were tested include 300 μM (solid bars), 200 μM (dark gray bars), and 100 μM (light gray bars). IBA time controls (open bars) are shown. Spontaneous activity was unaltered at all three concentrations in VRC neurons (300 μM, n = 54; 200 μM, n = 48; 100 μM, n = 53; time control, n = 40) and NTS neurons (300 μM, n = 29; 200 μM, n = 14; 100 μM, n = 13; time control, n = 12). In contrast, spontaneous activity in cortical neurons was unaltered at 100 μM (n = 46), decreased by 78 ± 6% at 200 μM (n = 43; †P < 0.001) and decreased by 89 ± 1% at 300 μM (n = 79; †P < 0.001) compared with time controls (n = 22). The mean normalized firing rate represents the average during the last 5 min of a 60-min drug application. Error bars indicate means ± SE.

Fig. 5.

Muscimol nearly abolishes spontaneous activity in cortical, VRC, and NTS neurons. A: mean normalized firing rate is shown for cortical neurons in slices from SA (n = 4; n = 52 neurons; open circles) and IBA squirrels (n = 4; n = 73 neurons, solid circles) in response to 20 μM muscimol (applied at vertical line). IBA time controls are shown (n = 4; n = 22 neurons, gray circles). B: mean normalized firing rate is shown for VRC neurons in slices from SA (n = 4; n = 12 neurons; open squares) and IBA squirrels (n = 4; n = 54 neurons; solid squares). IBA time controls are shown (n = 4; n = 40 neurons, gray squares). C: mean normalized firing rate is shown for NTS neurons in slices from SA (n = 4; n = 48 neurons, open triangles) and IBA squirrels (n = 4; n = 41 neurons; solid triangles). IBA time controls are shown (n = 4; n = 12 neurons, gray triangles). After a 60-min drug application, neuronal activity was nearly abolished in all neurons (†P < 0.05). Error bars indicate means ± SE.

Fig. 6.

Sodium pentobarbital effects in cortical neurons are blocked by bicuculline. In cortical slices from IBA squirrels (n = 4), mean normalized firing rate in 21 neurons was not altered by a 30-min exposure to bicuculline (100 μM). When bicuculline (100 μM) and pentobarbital sodium (300 μM) were bath applied simultaneously during the next 30 min, spontaneous activity in cortical neurons did not differ from time controls (P > 0.05) and was significantly greater than pentobarbital-treated cortical neurons without bicuculline pretreatment (P < 0.001; see Fig. 2A, solid circles). Error bars indicate means ± SE.