Vanilloid, purinergic, and CCK receptors activate glutamate release on single neurons of the nucleus tractus solitarius centralis

Abstract

Baroreceptor inputs to nucleus of the tractus solitarius medialis (mNTS) neurons can be differentiated, among other features, by their response to vanilloid or purinergic agonists, active only on C- or A-fibers, respectively. A major aim of this study was to examine whether neurons of NTS centralis (cNTS), a subnucleus dominated by esophageal inputs, exhibit a similar dichotomy. Since it has been suggested that cholecystokinin (CCK), exerts its gastrointestinal (GI)-related effects via paracrine activation of vagal afferent C-fibers, we tested whether CCK-sensitive fibers impinging upon cNTS neurons are responsive to vanilloid but not purinergic agonists. Using whole cell patch-clamp recordings from cNTS, we recorded miniature excitatory postsynaptic currents (mEPSCs) to test the effects of the vanilloid agonist capsaicin, the purinergic agonist α,β-methylene-ATP (α,β-Met-ATP), and/or CCK-octapeptide (CCK-8s). α,β-Met-ATP, capsaicin; and CCK-8s increased EPSC frequency in 37, 71, and 46% of cNTS neurons, respectively. Approximately 30% of cNTS neurons were responsive to both CCK-8s and α,β-Met-ATP, to CCK-8s and capsaicin, or to α,β-Met-ATP and capsaicin, while 32% of neurons were responsive to all three agonists. All neurons responding to either α,β-Met-ATP or CCK-8s were also responsive to capsaicin. Perivagal capsaicin, which is supposed to induce a selective degeneration of C-fibers, decreased the number of cNTS neurons responding to capsaicin or CCK-8s but not those responding to α,β-Met-ATP. In summary, GI inputs to cNTS neurons cannot be distinguished on the basis of their selective responses to α,β-Met-ATP or capsaicin. Our data also indicate that CCK-8s increases glutamate release from purinergic and vanilloid responsive fibers impinging on cNTS neurons.

Fig. 1.

Capsaicin (CAP) increases excitatory postsynaptic currents (EPSC) frequency in nucleus tractus solitarius centralis (cNTS) neurons. A: representative traces of spontaneous EPSCs (sEPSCs) recorded from a single cNTS neuron voltage clamped at −60 mV. Each figure represents 6 consecutive, overlapping traces. The magnitude of the increase in sEPSC frequency induced by the first application of CAP (100 nM; 1st CAP) was similar to that induced by a repeated application of CAP (100 nM; 2nd CAP) following a suitable period of washout and recovery. These data demonstrate a lack of tachyphylaxis in the response to CAP. B: representative traces of miniature EPSCs (mEPSCs) recorded in the presence of TTX (1 μM) from a single cNTS neuron voltage clamped at −60 mV. Each figure represents 6 consecutive, overlapping traces. Compared with control recordings (top trace), perfusion with CAP (100 nM) increased the frequency, but not the amplitude, of mEPSCs. Following washout (bottom trace) mEPSC frequency recovered to baseline levels. C: graphical representation of the effects of CAP (100 nM) on mESPC frequency (Ca), amplitude (Cb), rise-time (Cc) and decay-time (Cd). Note that CAP decreased mEPSC interevent interval (Ca), i.e., increased event frequency but had no effect on mEPSC amplitude or event kinetics. These data imply that CAP acts at presynaptic sites to increase mEPSC frequency.

Fig. 2.

Purinergic agonists increase sEPSC frequency in a subpopulation of cNTS neurons. A: representative traces from a cNTS neuron voltage clamped at −60 mV. Each figure represents 6 consecutive, overlapping traces. Compared with control recordings (top trace), perfusion with the nonselective agonist, ATP (100 μM) as well as the P2X receptor selective agonist, α,β-Met-ATP (10 μM) increased sEPSC frequency, but not amplitude. The ability of α,β-Met-ATP to increase sEPSC frequency was prevented by the P2X receptor selective antagonist, pyridoxal-phosphate-6-azophenyl-2-disulfonic acid (PPADS; 10 μM; bottom trace). B: graphical representation of the increase in sEPSC frequency, but not amplitude, induced by purinergic agonists. Note that both ATP and α,β-Met-ATP decreased the interevent interval, i.e., increased sEPSC frequency (left) but had no effect on sEPSC amplitude (right). In the presence of PPADS, however, α,β-Met-ATP no longer decreased the sEPSC interevent interval.

Fig. 3.

Differential effects of cholecystokinin-octapeptide (CCK-8s), CAP, and ATP on sEPSC frequency in cNTS neurons. A: representative traces from a single cNTS neuron voltage clamped at −60 mV. Each trace in A represents 6 consecutive, overlapping traces illustrating the increase in sEPSC frequency in response to α,β-Met-ATP, CCK-8s, and CAP, as well as the recovery following washout between drug treatments. B: graphic representation of the response of cNTS neurons to α,β-Met-ATP, CCK-8s, and CAP. Eight of the 28 cNTS neurons tested did not respond to any of the agonists and were not included in the graphic (see text for explanation of numbers). Note that all CCK-8s-responsive neurons were also CAP responsive, suggesting that CCK-8s may act upon vagal afferent CAP-sensitive C-fibers. In contrast, all α,β-Met-ATP-sensitive neurons were also CAP-sensitive, and a significant proportion of α,β-Met-ATP-sensitive neurons were also CCK-8s-sensitive. These results suggest that A- and C-afferent inputs onto cNTS neurons cannot be classified pharmacologically on the basis of their distinct responses to purinergic or vanilloid receptor agonists. In particular, all cNTS neurons receiving CAP-sensitive inputs (hence, presumably C-fibers) are also sensitive to purinergic agonists.

Fig. 4.

Effects of CCK-8s, CAP, and ATP on sEPSC frequency in cNTS neurons following perivagal CAP. Graphic representation of the proportion of cNTS neurons in which CAP (A), CCK-8s (B), and ATP (C) increase sEPSC frequency in brainstem slices from control rats and in brainstem slices from rats following perivagal CAP application. Note that perivagal CAP pretreatment decreases, but does not abolish, the ability of CAP and CCK-8s to increase sEPSC frequency in cNTS neurons. In contrast, perivagal CAP has no effect upon the proportion of cNTS neurons in which ATP increases sEPSC frequency. These results indicate that perivagal CAP: 1) does not induce a selective degeneration of vagal afferent C-fibers and 2) that CCK-8s does not act exclusively on CAP-sensitive C-fibers. P < 0.05. NS, not significant.