In preparations representing levels of biological organization from tissue through whole-organism, A1R activation depresses neuronal-network activity underlying respiratory rhythmogenesis [23, 24]. Although GABAAergic and glycinergic transmission affect the patterning and excitability of the respiratory network within in vitro preparations from neonatal mammals, the data presented herein show that the depression of preBötC rhythmogenesis by A1R activation is unaffected by reducing the overall level of synaptic input received by preBötC neurons, or by antagonizing GABAA or GABAA and glycine receptors. Thus, while the postsynaptic currents/potentials caused by GABAA/glycine receptor activation may integrate with the modulatory effects of A1R activation, the relative contribution of such integration to the combined effect is minor. The intracellular data presented herein are consistent with the notion that A1R-mediated depression of network activity may involve modulation of resting membrane properties as well as suppression of synaptic release.
The preBötC receives synaptic input from other regions within the slice preparation. Modulation of at least some of these inputs, such as the contralateral preBötC, can affect the pattern of preBötC output [10, 34]. Converting the slice to an island preparation removes the somata of many neurons sending axons to the preBötC. In excised preparations representing a variety of CNS regions, axon terminals severed from their somata continue to release messenger for a period of time after being cut away. In fact, such release may be affected by the application of modulatory substances to the cut terminals . However, converting the slice preparation to the island preparation reduces synaptic input to preBötC neurons (e.g., Fig. 6) suggesting that a large proportion of cut terminals in the island lose much, if not all, of their function shortly after being cut and/or that conversion leads to a decrease in intra-network communication. Activation of A1R depresses preBötC rhythmogenesis similarly in slice and island preparations, the latter representing a condition of reduced synaptic transmission. These findings, are consistent with the notion that the modulatory effects of A1R activation, although likely to integrate with concurrent synaptic inputs, are greater in their overall effect on preBötC activity than are A1R-induced changes in the net synaptic input to preBötC neurons from sources originating outside the preBötC.
Although not required for rhythmogenesis in the neonatal respiratory network GABAAergic and glycinergic transmission affect the pattern of respiratory network output [28–31, 33, 36] and blocking these forms of communication increases network excitability [31, 32]. Here antagonizing GABAA and glycine receptors induced seizure-like bursting in slice preparations. Within the island preparation antagonism of GABAA and glycine receptors appeared to synchronize preBötC bursting, increasing burst amplitude . Interestingly, this apparent synchronization during GABAA and glycine receptor antagonism occurred in islands from both very young (postnatal day 0- postnatal day 4), and older (postnatal day 4- postnatal day 7) mice. Although the Cl- equilibrium potential of preBötC neurons shifts from depolarizing to hyperpolarizing at around embryonic day 19  for mice, this shift is not apparent until after postnatal day 2  when medullary slice preparations are bathed in aCSF containing elevated K+ [30, 33].
As noted above, GABAAergic and glycinergic transmission affect respiratory network excitability and the pattern of respiratory network bursting. Accordingly, this study determined whether the blocking the postsynaptic currents/potentials evoked by activation of GABAA and glycine receptors may alter the extent of network depression observed during A1R activation. That is, do the postsynaptic potentials evoked by GABAA, or GABAA and glycine receptor activation integrate synergistically with the depressive modulatory effects of A1R activation? The lack of any noticeable difference in the response to A1R activation between slices in standard aCSF from those in aCSF with gabazine or with gabazine and strychnine suggests that the postsynaptic currents/potentials evoked by baseline GABAA/glycine receptor activation contribute little, if at all, to a potential combined effect.
In the medullary slice preparations used for this study baseline preparation-to-preparation variability in burst frequency was reasonable and similar between treatment groups. Had we evaluated the variability in burst-burst interval for slices (e.g., by calculating a regularity score), treatment with gabazine, or with combined gabazine and strychnine, which induced bursts of seizure-like activity, would likely have been shown to increase the variability in interburst interval (e.g., decrease burst regularity). By contrast to the frequency of bursting produced by slices, that produced by the island preparations used in this study tended to be somewhat more variable; whereas the slices from which islands were obtained burst at 0.2 – 0.5 Hz, the islands burst at 0.1 – 0.7 Hz. To minimize baseline island variability and the number of animals consumed to obtain island preparations we limited the islands used to only those bursting between 0.2 and 0.6 Hz. Given that the island preparation represents the most reduced preparation available for studying preBötC rhythmogenesis, it is perhaps not surprising that the frequency of bursting would be more variable in islands than in slices. In their initial description of the island preparation Johnson and colleagues  found that islands generated bursts at a higher frequency than slice preparations and that the SEM for preparation-to-preparation burst frequency was twice that in island preparations compared to slice preparations.
Although it did not do so in this study, using island preparations bursting over a wider range of baseline frequencies than slice preparations could contribute to a higher baseline frequency in islands than in slice preparations. However, such a difference would not, on its own, be likely to cause the differences in baseline burst frequency observed between island preparation treatment groups. Whereas islands used for testing the effects of NCPA in standard aCSF (Fig. 4A) burst at 0.37 ± 0.04 Hz, those used to examine the effects of NCPA in the presence of gabazine burst at 0.27 ± 0.04 Hz and those used to evaluate the effects of NCPA in the presence of gabazine and strychnine burst at 0.40 ± 0.06 Hz. This variability resulted from the distribution of baseline burst frequencies produced by island preparations in the various treatment groups. Whereas the burst frequencies generated by islands in the first (testing NCPA in standard aCSF) of these three groups were distributed fairly evenly between 0.2 and 0.6 Hz, those generated by islands in the second group (testing NCPA in the presence of gabazine) were distributed near the lower portion of the range with two of the islands bursting at the lower cutoff frequency. The frequency of bursts generated by island preparations in the third group (testing NCPA in the presence of gabazine and strychnine) clustered near the upper end of the allowed range with 2 of the preparations generating population-level bursts at the upper frequency limit. Although baseline firing frequency varied between island groups, the responses of those used to test the effects of NCPA and DPCPX in standard aCSF, or in aSCF containing gabazine were, as shown above, largely similar to those in the corresponding slice preparations. Both the increased baseline variability of islands and their responses to treatment may reflect the importance of modulatory input to preBötC neurons from other regions, such as the contralateral preBötC, and/or reduced intra-network communication.
Under normoxic baseline conditions it is unlikely that the preBötC would experience a substantial rise in extracellular adenosine concomitant with a substantial decrease in extracellular GABA and glycine concentration. However, hypoxic stress, after stimulating an initial augmentation of respiratory network activity, depresses respiratory network activity and decreases extracellular GABA levels  and glycinergic transmission  within the ventral respiratory group. These latter two effects are of interest since, as noted above, reducing GABAAergic and glycinergic transmission within the neonatal respiratory network tend to increase network activity [29–31]. However, while hypoxia decreases GABA and glycine-mediated transmission, it also increases extracellular adenosine and serotonin levels, depresses extracellular glutamate levels  and alters a variety of membrane properties [39–43]. Although adenosine represents only one of numerous variables that contribute to hypoxic depression of respiratory network output, the data presented here verify that A1R activation is sufficient to overcome potential increases in network excitability caused by reduction in GABA and glycine transmission and in so doing depress preBötC bursting [c.f. [22–24, 44]].
Throughout most of this study excitatory synaptic transmission between preBötC neurons was left intact so that population-level effects could be observed. By affecting presynaptic mechanisms of synaptic transmission, such as axon-terminal Ca++ conductances, A1R activation can directly affect synaptic transmission [45, 46]. In fact, our data show that NCPA decreased synaptic inputs to preBötC neurons. Thus, the data presented herein do not rule out the possibility that A1R activation may depress preBötC rhythmogenesis by directly inhibiting excitatory transmission between preBötC neurons. In fact, imunohistochemical data suggest that A1R are found at the axon terminals of interneurons within a variety of CNS regions, including the NTS where they may be involved in regulating transmitter release . However, A1R activation clearly decreases excitability of preBötC neurons, an effect that alone can decrease transmitter release.
During the present study A1R activation decreased the Rin of preBötC neurons regardless of whether or not those neurons were synaptically isolated from the rest of the network. Although not a quantitative measure of neuronal excitability due to its reliance on access resistance and seal resistance, holding current can reflect changes in membrane voltage that would occur, were the neuron not being subjected to voltage clamp. During the present study holding current increased (became more positive) in ~60% of the NCPA-treated neurons examined in synaptic isolation. By contrast, the holding current of control neurons (those examined in low Ca++/High Mg++ aCSF with TTX, but without NCPA) became more negative over time. In brainstem-spinal cord preparations Herlenius and Lagercrantz found that A1R activation decreased the Vm of expiratory neurons but did not affect Rin or Vm of inspiratory neurons . The difference between their study and the data presented herein may reflect the types of neurons from which data were obtained. Whereas Herlenius and Lagercrantz  defined inspiratory neurons in terms of discharge characteristics, here inspiratory neurons were defined as any that received a barrage of synaptic input during the population burst. Some neurons received concurrent barrages of EPSCs and IPSCs resulting in little or no net inward/outward current, suggesting that although defined as inspiratory per the criteria used herein, these may have actually been expiratory neurons.
Although NCPA affected resting membrane properties in this study, it did not affect whole-cell currents evoked by depolarizing voltage steps. However, depolarizing voltage steps activate multiple conductances in preBötC neurons, and different types of inspiration-related neuron express different combinations of voltage-sensitive ion channels . In other neurons A1R activation affects Ca++ conductance [17, 25, 48]. It is possible that one or more of types of these conductances were affected by A1R, but in combination with whole cell K+ conductances such changes were insufficient to affect total transmembrane current. Although beyond the scope of the present study, future work will provide a more detailed dissection of the effects of A1R activation on various membrane conductances. Rhythmogenesis within the preBötC of neonatal mice is thought to require synaptic interactions and the activity of pacemaker neurons [3, 5, 9, 10, 12, 32, 34, 49–53]. Upcoming research in our laboratory will examine whether A1R activation decreases the excitability and rhythmic production of action potential bursts by synaptically-isolated preBötC pacemaker neurons.