The involvement of hypothalamic sleep pathways in general anesthesia: testing the hypothesis using the GABAA receptor beta3N265M knock-in mouse

Abstract

The GABA(A) receptor has been identified as the single most important target for the intravenous anesthetic propofol. How effects at this receptor are then translated into a loss of consciousness, however, remains a mystery. One possibility is that anesthetics act on natural sleep pathways. Here, we test this hypothesis by exploring the anesthetic sensitivities of GABAergic synaptic currents in three specific brain nuclei that are known to be involved in sleep. Using whole-cell electrophysiology, we have recorded GABAergic IPSCs from the tuberomammillary nucleus (TMN), the perifornical area (Pef), and the locus ceruleus (LC) in brain slices from both wild-type mice and mice that carry a specific mutation in the GABA(A) receptor beta(3) subunit (N265M), which greatly reduces their sensitivity to propofol, but not to the neurosteroid alphaxalone. We find that this in vivo pattern of anesthetic sensitivity is mirrored in the hypothalamic TMN and Pef nuclei, consistent with their role as direct anesthetic targets. In contrast, anesthetic sensitivity in the LC was unaffected by the beta(3)N265M mutation, ruling out this nucleus as a major target for propofol. In support of the hypothesis that orexinergic neurons in the Pef are involved in propofol anesthesia, we further show that these neurons are selectively inhibited by GABAergic drugs in vivo during anesthesia, and that a modulation in the activity of Pef neurons alone can affect loss of righting reflex. Overall, our results support the idea that GABAergic anesthetics such as propofol exert their effects, at least in part, by modulating hypothalamic sleep pathways.

Figure 1.

Characterization of GABAergic IPSCs in identified orexinergic Pef neurons. A, The coronal slice shows the location of the perifornical region (highlighted in red) with respect to the fornix (fx). We used slices corresponding to ∼−1.7 mm from the Bregma. B, The left panel shows an example of a tonically firing (∼11 Hz) orexinergic neuron with a depolarized membrane potential (approximately −46 mV) compared with a relatively hyperpolarized (resting at approximately −60 mV), silent MCH-containing neuron shown on the right. C, Continuous current trace recorded from a WT orexinergic Pef neuron under voltage clamp (−60 mV). In this example, IPSCs occurred at an average frequency of 1.6 Hz and were confirmed as GABAergic due to their bicuculline sensitivity (data not shown; n = 10). Individual events (i–iii) highlighted in red were representative of three IPSC groups. D, Expanded trace for each example highlighted in C. The first trace (i) shows superimposition of IPSCs interrupting the decay phase. The second trace (ii) shows superimposition of IPSCs interrupting the rising phase. Finally, the third trace (iii) shows a single, isolated IPSC. To get an accurate representation of the IPSC parameters, only events falling into category (iii) were included in the analysis. E, Isolated IPSCs from a typical orexinergic neuron. The isolated events (77 events, shown in gray) were used to construct the average IPSC trace (shown in black) of the neuron. This example average IPSC trace had a 10–90% rise time of 1.3 ms, a peak amplitude of −69.2 pA and a decay time of 15.9 ms. The histogram shows the distribution of peak amplitudes recorded from all wild-type orexinergic neurons. The coefficient of variation was 59.4 ± 7.0% (n = 9).

Figure 2.

Characterization of GABAergic IPSCs in identified histaminergic neurons in the TMN. A, The coronal slice shows the location of the TMN (highlighted in red) as ventrolateral to the fornix (fx) at the level of the mammillary recess. We used slices corresponding to ∼−2.1 mm from the Bregma. B, A tonically firing (4.4 Hz) TMN neuron is shown on the left. On the right is an example of mRNA expression for the histaminergic neuronal marker ADA and β_1–3 subunit of the GABA_A receptor in a single neuron using single cell RT-PCR. Tonic firing and expression of ADA was typical of large cells in this region. C, A typical histaminergic TMN neuron. The left panel shows a neurobiotin-filled neuron following whole-cell recording, the center panel shows the staining pattern for histamine and the right panel is the merged image. D, GABAergic IPSCs recorded in the whole-cell configuration from a typical histaminergic TMN neuron. IPSCs occurred at a frequency of ∼1.5 Hz. E, A representative average trace (shown in black) was constructed from monotonic IPSCs (49 events, shown in gray). This example average IPSC had a 10–90% rise time of 1.8 ms, a peak amplitude of −56.0 pA and a decay time of 23.3 ms. The histogram shows the distribution of peak amplitudes recorded from all wild-type histaminergic neurons. The coefficient of variation was 60.0 ± 7.8% (n = 12).

Figure 3.

Characterization of GABAergic IPSCs in identified noradrenergic neurons in the LC. A, A coronal slice shows the LC (highlighted in red) as being at the floor of the fourth ventricle (4 V) and ventral to the cerebellum. We used slices corresponding to ∼−5.9 mm from the Bregma. B, Tonic firing (∼1.8 Hz) of an LC neuron recorded in current clamp, which hyperpolarized (from approximately −55 mV to −75 mV) and ceased firing after exposure to 30 μm noradrenaline (NA). This reversible autoreceptor response is characteristic of noradrenergic neurons and was seen in all recorded cells. C, A continuous trace showing GABAergic IPSCs recorded in voltage clamp from a typical LC neuron. D, Isolated IPSCs (37 events, shown in gray) were selected to construct a representative trace (shown in black) of individual IPSC properties. This example average IPSC had a 10–90% rise time of 3.5 ms, a peak amplitude of −28.2 pA and a decay time of 28.5 ms. The histogram shows the distribution of peak amplitudes recorded from all wild-type noradrenergic neurons. The coefficient of variation was 50.2 ± 4.3% (n = 12).

Figure 4.

The β₃N265M mutation does not affect control IPSC properties in Pef, TMN and LC neurons. A–C, The average IPSCs constructed from each recorded WT neuron (black traces) are shown together with the average β₃N265M IPSCs (shown in red), the Pef (A; WT n = 9, β₃N265M n = 8), the TMN (B; WT n = 12, β₃N265M n = 12), and the LC (C; WT n = 12, β₃N265M n = 10). D, Average 10–90% rise times for WT and knock-in Pef neurons (1.2 ± 0.1 ms; 1.3 ± 0.1 ms; means ± SEMs), TMN neurons (1.9 ± 0.2 ms; 2.1 ± 0.2 ms) and LC neurons (3.7 ± 0.4 ms; 3.3 ± 0.4 ms) respectively are shown in the top panel. E, Decay times for WT and β₃N265M Pef neurons (21.3 ± 1.5 ms; 24.6 ± 2.3 ms), TMN neurons (30.3 ± 2.8 ms; 31.0 ± 2.1 ms) and LC neurons (37.1 ± 2.5 ms; 32.6 ± 2.2 ms). F, Average peak amplitudes for WT and β₃N265M Pef neurons (−50.2 ± 6.6 pA; 35.3 ± 4.5 pA), TMN neurons (−37.4 ± 5.2 pA; 51.4 ± 9.2 pA), LC neurons (−19.0 ± 1.6 pA; −19.6 ± 1.5 pA). There were no differences in IPSC parameters between WT and β₃N265M knock-in animals in any of the brain regions studied.

Figure 5.

The neurosteroid alphaxalone markedly increases GABAergic IPSC decay times in both WT and β₃N265M knock-in Pef, TMN, and LC neurons. The main traces in each panel shows the effects of alphaxalone on the cumulative probability distributions of IPSC decay times averaged across all recorded neurons under control conditions (black curve) and during exposure to 2 μm alphaxalone (red curve). The lighter lines show the SEMs. The insets show representative average IPSCs (normalized to peak amplitude) from individual neurons. A, The cumulative decay time distributions for Pef neurons from both WT and β₃N265M knock-in mice show clear, and comparable, rightward shifts in the presence of alphaxalone (for WT neurons, τ increased by 175 ± 30%, n = 5, and for β₃N265M neurons, τ increased by 159 ± 30%, n = 4). Data from a typical WT Pef neuron (left inset), shows a large increase in the average IPSC decay time from 17 ms (n = 51) for control, to 53 ms (n = 81) in the presence of alphaxalone. Data from a typical β₃N265M Pef neuron (right inset) shows a similar increase from 16 ms (n = 51) to 40 ms (n = 105). B, The cumulative decay time distributions for TMN neurons from both WT and β₃N265M knock-in mice show clear, and comparable, rightward shifts (for WT neurons, τ increased by 188 ± 30%, n = 5, and for β₃N265M neurons, τ increased by 178 ± 41%, n = 4). Data from a typical WT TMN neuron (left inset), shows a large increase in the average IPSC decay time from 24 ms (n = 63) for control to 71 ms (n = 49) in the presence of alphaxalone. Data from a typical β₃N265M TMN neuron (right inset) shows a similar increase from 21 ms (n = 50) to 82 ms (n = 48). C, The cumulative decay time distributions for LC neurons from both WT and β₃N265M knock-in mice show clear, and comparable, rightward shifts (for WT neurons, τ increased by 149 ± 15%, n = 8, and for β₃N265M neurons, τ increased by 154 ± 26%, n = 6). Data from a typical WT LC neuron (left inset), shows a large increase in the average IPSC decay time from 27 ms (n = 127) for control to 52 ms (n = 71) in the presence of alphaxalone. Data from a typical β₃N265M LC neuron (right inset) shows a similar increase from 29 ms (n = 59) to 78 ms (n = 47).

Figure 6.

The contrasting effects of propofol on IPSC decay times in WT and β₃N265M knock-in Pef, TMN and LC. Each panel shows the effect of propofol on the cumulative frequency distribution of IPSC decay times averaged across all recorded neurons before (black curve) and during exposure to 1.5 μm propofol (red curve). The lighter lines show the SEMs. The insets show the average IPSC decay phase from a representative neuron. A, For WT Pef neurons the cumulative frequency distributions show that, on average, propofol causes a 100 ± 14% (n = 5) increase in median decay time. The left inset shows data from a typical WT Pef neuron. The average IPSC decay time under control conditions was 21 ms (n = 51), which increased to 50 ms (n = 81) in the presence of propofol. In contrast, propofol caused an increase of only 16 ± 4% (n = 4) in β₃N265M knock-in neurons. The right inset shows data from a typical β₃N265M Pef neuron. The average IPSC decay time was 19 ms (n = 51) under control conditions and did not change significantly in the presence of propofol (20 ms; n = 105). B, For WT TMN neurons the cumulative frequency distributions show that, on average, propofol causes an 124 ± 18% (n = 7) increase in median decay time. The left inset shows data from a typical WT TMN neuron. The average decay time was 19 ms (n = 63), which increased to 48 ms (n = 49) in the presence of propofol. However, propofol caused an increase of only 23 ± 11% (n = 8) in β₃N265M knock-in neurons. The right inset shows data for a typical β₃N265M TMN neuron. Propofol minimally affected the IPSC decay time, changing it from 20 ms (n = 50) to 21 ms (n = 48). C, In the LC, cumulative frequency distributions show that, on average, propofol causes a similar increase in decay times for WT and β₃N265M knock-in neurons (79 ± 29%, n = 4, increase in median decay time in WT LC neurons and 89 ± 23%, n = 4, in β₃N265M knock-in neurons). The left inset shows data from a typical WT LC neuron. Propofol caused an increase in IPSC decay time from 27 ms (n = 127) to 57 ms (n = 71). The right inset shows data from a typical LC neuron from the β₃N265M knock-in. Propofol caused an increase in IPSC decay time from 35 ms (n = 59) to 51 ms (n = 47).

Figure 7.

Evidence for the involvement of orexinergic neurons in anesthetic-induced LORR. A, The schematic coronal slice (top left) shows the location of Pef orexinergic neurons (red) close to the fornix. The image top right shows 3,3-diaminobenzidine tetrahydrochloride-visualized neurons, double-stained for cytosolic orexin-A (brown) and nuclear c-Fos (black). The red arrow points to a single-labeled orexinergic-positive, but c-Fos-negative neuron and the black arrow indicates a double-labeled c-Fos-positive orexinergic neuron. The lower images show (left) a level of c-Fos expression in orexinergic neurons following systemic dexmedetomidine (Dex; 150 μg/kg) that is similar to control levels, in contrast to the marked depression of c-Fos expression observed (right) following pentobarbital administration (Pento; 50 mg/kg). B, C-Fos-positive neurons following systemic administration of dexmedetomidine (Dex; 150 μg/kg, i.p.), muscimol (Mus; 5 mg/kg, i.p.), propofol (Prop; 100 mg/kg, i.p.) and pentobarbital (Pento; 50 mg/kg, i.p.) expressed as a percentage of those observed following saline administration. Following saline injection, 47.8 ± 2.9% of orexinergic neurons were c-Fos positive. C, When administered directly into the Pef (bilateral injections, 0.2 μg/0.2 μl/side), the GABA_A receptor antagonist gabazine increases c-Fos expression in orexinergic neurons, whereas the GABA_A receptor agonist muscimol decreases expression compared with the injection of control saline (normalized to 100%; 45.5 ± 4.4% of neurons were c-Fos positive), showing that the activity of Pef neurons can be modulated by GABAergic agents. D, When administered directly into the Pef (bilateral injections, 0.2 μg/0.2 μl/side), gabazine antagonizes Dex-induced LORR (150 μg/kg, s.c.), whereas muscimol significantly increases Dex-induced LORR compared with the injection of control saline (normalized to 100%; control sleep time was 286 min), showing that the modulation of Pef neurons can directly influence LORR. Significance was tested using an unpaired Student's t test. E, The intracerebroventricular administration of orexin-A significantly antagonizes propofol and dexmedetomidine anesthesia, but has no effect on ketamine anesthesia compared with the intracerebroventricular injection of saline. In each case the mean control sleep times have been normalized to 100% (control sleep times were 8.2 min for Prop, 26.2 min for Dex and 3.6 min for ketamine). Significance was tested using a paired Student's t test with respect to control saline injections on the same animal. The data are means ± SEMs, and the asterisks denote the level of significance (*p < 0.05, **p < 0.01, ***p < 0.001).