Comparison of baroreceptive to other afferent synaptic transmission to the medial solitary tract nucleus

Abstract

Cranial nerve visceral afferents enter the brain stem to synapse on neurons within the solitary tract nucleus (NTS). The broad heterogeneity of both visceral afferents and NTS neurons makes understanding afferent synaptic transmission particularly challenging. To study a specific subgroup of second-order neurons in medial NTS, we anterogradely labeled arterial baroreceptor afferents of the aortic depressor nerve (ADN) with lipophilic fluorescent tracer (i.e., ADN+) and measured synaptic responses to solitary tract (ST) activation recorded from dye-identified neurons in medial NTS in horizontal brain stem slices. Every ADN+ NTS neuron received constant-latency ST-evoked excitatory postsynaptic currents (EPSCs) (jitter < 192 micros, SD of latency). Stimulus-recruitment profiles showed single thresholds and no suprathreshold recruitment, findings consistent with EPSCs arising from a single, branched afferent axon. Frequency-dependent depression of ADN+ EPSCs averaged approximately 70% for five shocks at 50 Hz, but single-shock failure rates did not exceed 4%. Whether adjacent ADN- or those from unlabeled animals, other second-order NTS neurons (jitters < 200 micros) had ST transmission properties indistinguishable from ADN+. Capsaicin (CAP; 100 nM) blocked ST transmission in some neurons. CAP-sensitive ST-EPSCs were smaller and failed over five times more frequently than CAP-resistant responses, whether ADN+ or from unlabeled animals. Variance-mean analysis of ST-EPSCs suggested uniformly high probabilities for quantal glutamate release across second-order neurons. While amplitude differences may reflect different numbers of contacts, higher frequency-dependent failure rates in CAP-sensitive ST-EPSCs may arise from subtype-specific differences in afferent axon properties. Thus afferent transmission within medial NTS differed by axon class (e.g., CAP sensitive) but was indistinguishable by source of axon (e.g., baroreceptor vs. nonbaroreceptor).

Fig. 1.

The presence of fluorescent, anterogradely transported dye from the aortic depressor nerve (ADN) allowed anatomical identification of solitary tract nucleus (NTS) neurons directly receiving aortic baroreceptor terminals (ADN+) in vitro. A–C are from a single representative ADN+ neuron. A: under infrared differential interference contrast (IR DIC) microscopy, numerous cell bodies were apparent (top micrograph, black arrows) in thin brain stem slices used for recordings. Using fluorescence excitation, bright puncta of DiI label were revealed along the surface of some cell bodies and their proximal dendrites (bottom micrograph, right, white arrow, ADN+), and these were considered anatomically identified second-order baroreceptive NTS neurons. Note that adjacent dye-negative neurons (white arrow, left, ADN−) had similar appearance under IR DIC but lacked DiI puncta. Scale bar is 10 μm. B: shocks delivered to the solitary tract (ST) evoked large low-jitter excitatory postsynaptic currents (EPSCs) in this ADN+ neuron (original traces recorded from right ADN+ neuron depicted in A). Amplitudes depressed with shocks (1–5, solid circles above traces) were repeated at a 20-ms interval in a burst of 5 stimuli repeated each 3 s (7 trials displayed). C: expanded analysis of EPSC1 (dashed line box in B) examined the timing of the onset of the EPSC, latency (dashed line and arrow to bottom left), and the amplitude (dashed line and arrow to bottom plot). Examination of the timing of EPSC1 (open oval) showed that the latency of this ADN+ neuron varied minimally (bottom plot, left) with ∼9 ms and a jitter (SD of latency) of 106 μs (n = 38 trials), well below the 200-μs cutoff for monosynaptic pathways directly from ST. The amplitudes of EPSC1 (shaded oval) characteristically varied from shock to shock, but increasing shock intensity indicated a sharp threshold profile for evoking the EPSC and no change in amplitude at suprathreshold intensities (bottom plot, right). Shaded squares are individual values, and solid squares are means ± SE. Original synaptic response traces displayed in B and C were recorded using tests at twice the threshold intensity, but note that many trials are not displayed to increase clarity. Similar results were found in all ADN+ neurons. ADN− neurons were tested using identical protocols and analyses.

Fig. 2.

Dye-identified second-order NTS neurons (ADN+, n = 32) had comparable ST synaptic characteristics to adjacent ADN− NTS neurons, meeting the 200-μs cutoff for monosynaptic ST pathways (n = 13). A: using the procedures for study outlined in Fig. 1, latency-jitter paired values for ADN+ and ADN− second-order NTS neurons overlapped. The ADN+ neuron whose recordings were displayed in Fig. 1 is marked by an arrow. B: frequency-dependent depression during a burst of 5 shocks to ST (20-ms interval) were similar between ADN+ and ADN− neurons, and synaptic failure rates increased similarly as the stimulus burst progressed (P > 0.05).

Fig. 3.

Capsaicin (CAP) exposure (100 nM) identified CAP-sensitive and CAP-resistant NTS neurons with ADN+ puncta. CAP rapidly and reversibly suppressed ST-evoked EPSCs in CAP-sensitive neurons (A, shaded box) but had no effect on CAP-resistant ST transmission (B). Note that ADN+ CAP-sensitive neurons tended to have much smaller amplitude ST-EPSCs than CAP-resistant NTS neurons. Representative traces shown in the top portions of A and B include 10 control and 7 CAP trials for the CAP-sensitive neuron and include 11 control and 11 CAP trials for the CAP-resistant neuron. This presynaptic action of CAP reflects the presence of transient receptor potential vanilloid type 1 receptors of ST afferent terminals that arise from unmyelinated afferent axons (32). Note that, during CAP block of ST synced EPSCs, spontaneous synaptic events continue to occur.

Fig. 4.

A: across all ADN+ neurons tested with CAP (n = 18), latency-jitter values for ST-EPSCs had considerable overlap between CAP sensitive (shaded squares, n = 10) and CAP resistant (open circles, n = 8). The CAP-sensitive point marked by the arrow corresponds to the representative ADN+ neuron presented in detail in Fig. 1. B: the mean ST-EPSC amplitudes of CAP sensitive (left, shaded bars), however, were significantly smaller (*P < 0.02) than for CAP resistant (open bars) to the first as well as subsequent ST shocks (ST-EPSC #) during the burst of 5 stimuli. Failures of CAP-resistant ST-EPSCs (right, open bars) were quite low at all ST shock positions and remained <5% throughout. Failures for CAP-sensitive neurons were similar to CAP-resistant neurons at ST-EPSC1 (right, shaded bars), but increased substantially for later responses within the burst (*P < 0.001). C: a plot of the individual mean failure rates against the mean amplitude for successful, nonfailure events shows that failures occurred at event amplitudes well above the detection threshold of 5–15 pA. Failure rates were calculated from a minimum of 30 trials for EPSC1–EPSC5 for each neuron. Small solid points indicate zero failure points (dashed horizontal line), whereas larger points indicate mean event amplitudes at corresponding mean failure rates. The mean reliable level of detection of single events is represented by dotted vertical line, although this was adjusted in each neuron (range 5–20 pA). Note that no failures occurred in CAP-resistant EPSCs (open circles) until amplitudes were quite depressed (corresponding to EPSC3–EPSC5), whereas recurring failures were found in CAP-sensitive EPSCs (shaded squares) at amplitudes as high as 145 pA.

Fig. 5.

In a larger cohort of NTS neurons from naive animals, synaptically identified second-order neurons had similar synaptic response profiles to ADN+ neurons. A: CAP-sensitive (shaded squares, n = 19) and CAP-resistant (open squares, n = 28) ST-EPSCs generally overlapped in latency and jitter distributions. B: CAP-sensitive ST-EPSCs had significantly smaller amplitudes than CAP-resistant responses (P = 0.01) and failed more often as the burst of 5 shocks progressed (P < 0.001), suggesting a use-dependent increase in failures only in the CAP-sensitive transmission. *Post hoc testing significant differences between CAP-sensitive and -resistant values, respectively. The general patterns are consistent with those for ADN+ neurons (Fig. 4), suggesting that the differences identified by CAP are similar for all ST afferents and not peculiar to ADN baroreceptive NTS neurons.

Fig. 6.

Variance-mean analysis of glutamate release for ST-EPSCs from ADN+ and naive NTS neurons. Within a single, representative ADN+ neuron, the variance of the ST-EPSC amplitudes increased as the extracellular Ca²⁺ concentration was reduced, while the mean amplitude declined in a characteristic fashion (A and B). The relationship between the variance and the mean of the ST-EPSC amplitude from A and B at various Ca²⁺ conditions was well described by a parabolic model (r² values always exceeded 0.9). C: data were fit with a least squares method for each neuron using the equation: y = A + K₁x + K₂x². In this case, r² = 0.997 and A = −0.478, K₁ = 16.95, and K₂ = −23.69. This fit estimated the probability of glutamate release (P_R) as 0.895 in this ADN+ neuron at 2 mM Ca²⁺. D: group averages of variance-mean relationships show that the average of three ADN+ neurons (solid line), r² = 0.997, A = 0.081 ± 0.130, K₁ = 20.40 ± 0.80, K₂ = −20.43 ± 0.76, was similar to the relationship for the average of 6 naive neurons (dashed line), r² = 0.999, A = 0.005 ± 0.111, K₁ = 27.93 ± 0.80, K₂ = −27.84 ± 0.57. The mean P_R was similar for both groups of neurons at ∼0.91. In some long-lasting recordings (4 cases), neurons were exposed to CAP, and all three ADN+ neurons were CAP resistant and their curve overlapped with data from one naive neuron that was CAP sensitive (replotted as a shaded trace with shaded squares).