Rhythm generation by the pre-Bötzinger complex in medullary slice and island preparations: effects of adenosine A(1) receptor activation

Abstract

Background: The pre-Bötzinger complex (preBötC) is a central pattern generator within the ventrolateral medulla oblongata's ventral respiratory group that is important for the generation of respiratory rhythm. Activation of adenosine A(1) receptors (A(1)R) depresses preBötC rhythmogenesis. Although it remains unclear whether A(1)R activation is important for organisms in a normal metabolic state, A(1)R activation is important to the response of the preBötC to metabolic stress, such as hypoxia. This study examined mechanisms linking A(1)R activation to depression of preBötC rhythmogenesis in medullary slice and island preparations from neonatal mice.

Results: Converting medullary slices to islands by cutting away much of the medullary tissue adjacent to the preBötC decreased the amplitude of action potential bursts generated by a population of neurons within the preBötC (recorded with an extracellular electrode, and integrated using a hardware integrator), without noticeably affecting burst frequency. The A(1)R agonist N6-Cyclopentyladenosine (NCPA) reduced population burst frequency in slices by ca. 33% and in islands by ca. 30%. As in normal (drug-free) artificial cerebrospinal fluid (aCSF), NCPA decreased burst frequency in slices when GABA(A)ergic or GABA(A)ergic and glycinergic transmission were blocked, and in islands when GABA(A)ergic transmission was antagonized. Converting slices to island preparations decreased synaptic input to inspiratory neurons. NCPA further decreased the frequency of synaptic inputs to neurons in island preparations and lowered the input resistance of inspiratory neurons, even when chemical communication between neurons and other cells was impeded.

Conclusion: Together these data support the suggestion that depression of preBötC activity by A(1)R activation involves both decreased neuronal excitability and diminished inter-neuronal communication.

Figure 1

Effects of A₁R activation on preBötC rhythmogenesis in medullary slice preparations. A. Representative effects of bath-applied NCPA (1 μM) alone, NCPA in the presence of gabazine (20 μM), or NCPA in combination with gabazine (20 μM) and strychnine (1 μM). B. Whether applied (i) alone, (ii) with gabazine, or (iii) with gabazine and strychnine, NCPA decreased burst frequency (a, value different from baseline at P < 0.05, Tukey post-hoc test; b, value different from step 2 of treatment – either continued aCSF, application of gabazine, or application of gabazine and strychnine – at P < 0.05, Tukey post-hoc test).

Figure 2

Block of chloride-mediated inhibition inducesseizure-like activity in medullary slice preparations. Three sequential sample recordings of integrated preBötC activity from a single medullary slice preparation. A. Activity recorded in recorded in drug free aCSF. B. Gabazine (20 μM) and Strychnine (1 μM) induce seizure-like bursting (brackets) characterized by increased burst frequency and elevated baseline, while slightly decreasing the frequency of population bursts generated between seizure-like bursts. C. Antagonism of A₁R with NCPA (1 μM) eliminated seizures for this slice and decreased population burst frequency.

Figure 3

Effects of slice to island conversion onpopulation burst parameters. A. The frequency of population bursts generated by the preBötC was unchanged by cutting away regions of the slice preparation adjacent to the preBötC (see text), thereby converting the section to an island preparation (frequency = 0.33 ± 0.02 Hz in slices vs. 0.37 ± 0.04 Hz in islands; n = 13 of each; paired t-test, P = 0.73). B. By contrast, the amplitude of integrated population bursts decreased by 30.1% with conversion of the slice to the island preparation (*, P < 0.05, paired t-test).

Figure 4

Effects of A₁R activation on preBötC rhythmogenesis in island preparations. A. Representative effects of NCPA (1 μM) alone, with gabazine (20 μM) and in combination with 20 μM gabazine and 1 μM strychnine. B. As with slice preparations, NCPA alone (i) or in combination with gabazine (ii) decreased burst frequency. By contrast to slices, NCPA applied in combination with gabazine and strychnine (iii) failed to affect burst frequency (a, value different from baseline at P < 0.05, Tukey post-hoc test; b, value different from the second step of treatment – either continued aCSF, application of gabazine, or application of gabazine and strychnine – at P < 0.05, Tukey post-hoc test).

Figure 5

Synaptic inputs to preBötC neurons. A. Spontaneous postsynaptic currents from an inspiratory neuron voltage clamped at -60 mV. Note both iPSCs from ePSCs appear as inward currents. B. The same neuron as in A, but voltage clamped at -35 mV. Note that chloride-mediated synaptic currents now appear as outward currents (arrows). Although it is impossible to accurately measure the amplitude or frequency of excitatory or inhibitory inputs during inspiration-related bursts of synaptic input it is possible to distinguish between excitatory and inhibitory inputs during the interburst interval. Vertical scale: 50 pA.

Figure 6

Manipulation of A₁R affects synaptic input to preBötC neurons. A. Representative current traces from slice (left) and island preparations (right). Vertical scale: 50 pA. B. Converting slices to island preparations reduced the frequency of total sPSCs and sEPSCs evoked in preBötC neurons (*P < 0.05, Two-sample t-test). This trend appeared to hold true for sIPSCs but did not attain statistical significance. C. NCPA (1 μM) decreased the frequency of sEPSCs and sIPSCs received by preBötC neurons within slice (i, ii) and island preparations (iii, iv) in the presence of strychnine (1 μM). The insets in (ii) and (iv) are magnified views of sIPSC frequencies in the presence of strychnine. Letters above columns show difference from mean frequency under baseline (a, P < 0.05) conditions and in the presence of strychnine (b, P < 0.05).

Figure 7

Effects of A₁R activation on individual inspiration-related preBötC neurons. A. NCPA (1 μM) decreased R_in of synaptically isolated neurons (n = 7; * P < 0.05). B. In this representative pair of whole cell current traces evoked by a voltage step from -60 to 20 mV I_Kd is similar during recording in normal aCSF (black trace) and in NCPA (grey trace). Vertical scale: 100 pA; time scale: 50 ms. C. Mean current-voltage relationship for inspiratory neurons (n = 5; error bars represent SEM).