Neuromodulatory inputs maintain expression of a lobster motor pattern-generating network in a modulation-dependent state: evidence from long-term decentralization in vitro

Abstract

Neuromodulatory inputs play a critical role in governing the expression of rhythmic motor output by the pyloric network in the crustacean stomatogastric ganglion (STG). When these inputs are removed by cutting the primarily afferent stomatogastric nerve (stn) to the STG, pyloric neurons rapidly lose their ability to burst spontaneously, and the network falls silent. By using extracellular motor nerve recordings from long-term organotypic preparations of the stomatogastric nervous system of the lobster Jasus lalandii, we are investigating whether modulatory inputs exert long-term regulatory influences on the pyloric network operation in addition to relatively short-term neuromodulation. When decentralized (stn cut), quiescent STGs are maintained in organ culture, pyloric rhythmicity gradually returns within 3-5 d and is similar to, albeit slower than, the triphasic motor pattern expressed when the stn is intact. This recovery of network activity still occurred after photoinactivation of axotomized input terminals in the isolated STG after migration of Lucifer yellow. The recovery does not depend on action potential generation, because it also occurred in STGs maintained in TTX-containing saline after decentralization. Resumption of rhythmicity was also not activity-dependent, because recovery still occurred in STGs that were chronically depolarized with elevated K+ saline or were maintained continuously active with the muscarinic agonist oxotremorine after decentralization. We conclude that the prolonged absence of extraganglionic modulatory inputs to the pyloric network allows expression of an inherent rhythmogenic capability that is normally maintained in a strictly conditional state when these extrinsic influences are present.

Fig. 1.

Expression of lobster pyloric network activity depends on descending modulatory inputs. A, Pyloric neural output from a combined STNS preparation (schemaat left) consisting of the STG connected via the stn to the CoG andOG. Motor nerves recorded are the lp–py nerve (lp–pyn), which carries LP andPY motoneuron axons, and the pd nerve (pdn), which contains PD neuron axons. B, Complete absence of spontaneous rhythmic activity in the same preparation 30 min after axonal conduction in thestn was blocked with tetrodotoxin (10⁻⁷m) placed in a Vaseline well around the stn (schema atleft). C, Spontaneous rhythmic activity reappearing minutes after the STG is functionally reconnected to the rostral ganglia by rinsing the toxin from the stn(schema at left).

Fig. 2.

Spontaneous pyloric output from an intact STNS during long-term organ culture in vitro.A, Day 1. Rhythmic pyloric network activity recorded extracellularly from the lateral ventricular nerve (lvn;top trace) and the pdn (bottom trace) of an intact isolated STNS (schema).B, Day 4. Same preparation and nerve recordings after 4 d in vitro. C, Day 7. Same preparation and nerve recordings after 7 d in vitro. Although the rhythm has slowed, the pyloric network is still spontaneously active.

Fig. 3.

Evolution of pyloric cycle frequency and phasing of motor bursts in long-term STNS preparations in which the STG remained connected to the rostral ganglia. A, Mean cycle frequency (±SE) of spontaneous pyloric rhythmicity in five combined STNS preparations at days 1, 5, and 7 in organ culture. The preparations were continuously active throughout the experiment, although cycle frequency gradually decreased. Each histogram was derived from at least 50 consecutive cycles per preparation.B, Phase relationships of the pyloric motoneurons on day 1 (clear boxes) and day 5 (shaded boxes)in vitro. The beginning and end of eachbox represent the mean (±SE) onset and offset phases of the burst of the indicated neuron; one cycle is shown. Results are from the same preparations used in A. Pyloric network phase relationships did not change significantly (paired Student’st test) in organ culture.

Fig. 4.

Pyloric network activity of a combined STNS remains conditional on functional stn inputs throughout long-term organ culture. A, Spontaneous rhythmic activity (right) was recorded extracellularly from pyloric motor nerves of an isolated STNS (left) in which the STG remained attached to the three rostral ganglia after 7 d in organ culture. B, Pyloric activity ceases (right) soon after disconnection of the STG from the three rostral ganglia by the application of 10⁻⁷m TTX to the stn (left). C, Pyloric activity returns (right) after the blockade of stn axonal conduction was removed (left).

Fig. 5.

Functional recovery of pyloric network activity during long-term organ culture of a decentralized STG.A, Pyloric rhythmicity in a freshly dissected, intact STNS (schema at left). Motor nerves recorded are the lp–pyn and the pdn.B, Same preparation and recordings made at daily intervals after cutting the stn (schema atleft). There is a complete absence of pyloric network activity on the 1st (i) and 2nd (ii) day after suppressing STG inputs. By the 3rd day (iii) after stn transection, a slow spontaneous rhythm emerges. By the 4th day after stn section (iv), the decentralized pyloric network expresses a more robust rhythm that, although still slower than the control pattern (compareA), consists of strongly coordinated bursting in the three pyloric motoneuron classes (LP, PY, and PD).

Fig. 6.

Evolution of pyloric cycle frequency and phasing in long-term decentralized STGs in vitro.A, Mean pyloric cycle frequency (±SE) of five STGs during the 7 d after decentralization. Each pointwas derived from at least 50 consecutive cycles (when rhythmicity was expressed) per preparation. B, Phase relationships of the same neurons on day 1 before decentralization (clear boxes) and in recovered rhythms 4 d after decentralization (black boxes). Data are from the same preparations used in A. Onset and offset phases before and after decentralization were compared using a paired Student’st test (*p < 0.05; **p < 0.01).

Fig. 7.

Recovery of pyloric network rhythm after photoinactivation of stn input terminals. A, Spontaneous pyloric output from a freshly dissected combined STNS is shown. Extracellular recordings are from the lvn and from distal branches carrying the PY and PD neuron axons (schema).B, Pyloric rhythm ceases after cutting the stn. A Vaseline well filled with Lucifer yellow was placed around the cut stn stump (schema) to dye-fill axons into the STG.C, After 18 hr of dye migration, STG illumination with blue light (schema) transiently activates the pyloric network during photoinactivation of dye-filled terminals.Di, The absence of any activity in the same preparation 2 hr after photoinactivation of stn inputs (schema) is shown. Dii–Div, Recordings from the same nerves shown in A–Di, at days 2 (Dii), 3 (Diii), and 4 (Div) after the original decentralization, show gradual recovery of spontaneous triphasic pyloric rhythmicity.

Fig. 8.

Test for photoinactivation of stn input terminals in the STG. A, Extracellular electrical stimulation (10 Hz for 1 sec at arrow) of the stn stump evokes pyloric activity in an otherwise quiescent STG, 2 hr after cutting the stn.B, Same preparation ≥18 hr after stn labeling with Lucifer yellow is shown. Stimulation (arrows) of the stained input nerve (now bathed in normal saline) again elicits pyloric rhythmicity (i) but has no effect ≈3 hr after illuminating the ganglion (ii). Increasing the stimulus intensity or resectioning the stn stump similarly had no effect.

Fig. 9.

Recovery of pyloric rhythmicity in a decentralized, long-term silent STG. A, Spontaneous pyloric pattern (right) recorded from thelp–pyn and the pdn of a freshly dissected combined STNS preparation (left) under normal saline. B, Total absence of activity in the same nerves (right) after the stn was cut in the presence of TTX (10⁻⁷m) in the bathing saline (left). The decentralized, silent preparation was maintained under these conditions during the following 4 d.C, Pyloric motor pattern (right) expressed on day 5, 6 hr after rinsing the toxin from the decentralized STG (left).

Fig. 10.

Recovery of pyloric rhythmicity in a decentralized, chronically depolarized STG. A, Absence of rhythm (right) in a freshly dissected STNS after cutting the stn in normal saline (left). Recordings are from the lp–pyn and the pdn.B, Activation of the rhythm (right) by superfusion of elevated (25 mm) K⁺saline (i, left), which was replenished daily over the following 4 d. Recordings are from the same nerves used in A on days 1 (i) and 5 (ii) of high K⁺ exposure.C, Robust spontaneous pyloric rhythm on day 5 (right), 1–2 hr after rinsing the preparation with normal (12 mm) K⁺ saline (left).

Fig. 11.

Recovery of pyloric rhythmicity in a continuously active decentralized STG. A, Absence of rhythm (right) in a freshly dissected STNS after stn transection (left). Recordings are from thelp–pyn and the pdn. B, Activation of the rhythm (right) by superfusion of 10⁻⁵m oxotremorine (oxo; left), which was replenished daily over the following 3 d. C, Spontaneous pyloric rhythm on day 5 (right), 24 hr after rinsing the muscarinic agonist from the preparation (left).