Distinct functions for cotransmitters mediating motor pattern selection

Abstract

Motor patterns are selected from multifunctional networks by selective activation of different projection neurons, many of which contain multiple transmitters. Little is known about how any individual projection neuron uses its cotransmitters to select a motor pattern. We address this issue by using the stomatogastric ganglion (STG) of the crab Cancer borealis, which contains a neuronal network that generates multiple versions of the pyloric and gastric mill motor patterns. The functional flexibility of this network results mainly from modulatory inputs it receives from projection neurons that originate in neighboring ganglia. We demonstrated previously that the STG motor pattern selected by activation of the modulatory proctolin neuron (MPN) results from direct MPN modulation of the pyloric rhythm and indirect MPN inhibition of the gastric mill rhythm. The latter action results from MPN inhibition of projection neurons that excite the gastric mill rhythm. These projection neurons are modulatory commissural neuron 1 (MCN1) and commissural projection neuron 2 (CPN2). MPN excitation of the pyloric rhythm is mimicked by bath application of proctolin, its peptide transmitter. Here, we show that MPN uses only its small molecule transmitter, GABA, to inhibit MCN1 and CPN2 within their ganglion of origin. We also demonstrate that MPN has no proctolin-mediated influence on MCN1 or CPN2, although exogenously applied proctolin directly excites these neurons. Thus, motor pattern selection occurs during MPN activation via proctolin actions on the STG network and GABA-mediated actions on projection neurons in the commissural ganglia, demonstrating a spatial and functional segregation of cotransmitter actions.

Fig. 1.

Schematics of the stomatogastric nervous system, including somata location and axonal pathways of the projection neurons MPN, MCN1, and CPN2, plus a summary of MPN actions on the gastric mill rhythm. A, There is a pair of MPN somata located either in the OG or in the nerve posterior to this ganglion. Each MPN projects an axon through eachson to the CoG and projects two axons through the stn to the STG. It also projects an axonal branch from each son through a peripheral nerve (dpon). For clarity, the complete projection of only one MPN is shown. B, There is a single MCN1 and CPN2 in each CoG. Each MCN1 projects through the ion and stn to theSTG. Each CPN2 projects through the sonand stn to the STG. For clarity, the complete projection of only one MCN1 and one CPN2 is shown. Ganglia:CoG, commissural ganglion; OG, oesophageal ganglion; STG, stomatogastric ganglion. Nerves: dgn, dorsal gastric nerve; dpon, dorsal posterior oesophageal nerve; ion, inferior oesophageal nerve; lgn, lateral gastric nerve;lvn, lateral ventricular nerve; mvn, medial ventricular nerve; pdn, pyloric dilator nerve;son, superior oesophageal nerve; stn, stomatogastric nerve. Neurons: CPN2, commissural projection neuron 2; MCN1, modulatory commissural neuron 1; MPN, modulatory proctolin neuron. Anterior is toward the top, and posterior is toward thebottom. C, Left, MPN inhibits an ongoing gastric mill rhythm. Activity in MCN1 (ion) and CPN2 (monitored with an intracellular recording of the CPN2 axon as it enters the STG, CPN2_SNAX) elicited a gastric mill rhythm, evident from the rhythmic bursting in the GM neuron and the gastric mill timed inhibition of the inferior cardiac (IC) and ventricular dilator (VD) neurons (mvn). MPN stimulation (betweenarrowheads) inhibited MCN1 and CPN2 in theCoGs, thus eliminating their excitation of the gastric mill rhythm (Modified from Blitz and Nusbaum, 1997a). C,Right, Schematic illustrating that MPN excites the pyloric rhythm directly (Nusbaum and Marder, 1989a) and inhibits the gastric mill rhythm via inhibition of MCN1 and CPN2 (Blitz and Nusbaum, 1997a). Darkened cell bodies and rhythm represent active neurons. Unfilled cell bodies and rhythm represent inactive neurons. T-junctions represent excitation, and filled circles represent inhibition.

Fig. 2.

The MPN inhibition of MCN1 and CPN2 appears to be monosynaptic. A, Left, In normal saline MPN stimulation (13 Hz) inhibited MCN1. This inhibition produced a smooth hyperpolarization and cessation of MCN1 action potentials. Note the depolarizing PSPs in MCN1 during MPN stimulation. These PSPs were not time-locked to each MPN action potential. A,Right, Despite maintaining approximately the same membrane potential as in normal saline, MCN1 was silent during superfusion of the CoGs with high divalent cation saline. This saline raises action potential threshold. Under this condition MPN stimulation (13 Hz) still inhibited MCN1, producing a smooth hyperpolarization. The depolarizing PSPs elicited during MPN stimulation in normal saline were eliminated. The persisting EPSPs are from an identified sensory neuron, AGR (see Results), for which the spike initiation zone is outside the region superfused with high divalent cation saline. B,Left, In normal saline MPN stimulation (11 Hz) inhibited CPN2, producing a smooth hyperpolarization and cessation of CPN2 action potentials. B, Right, When the CoGs were superfused with high divalent cation saline, MPN stimulation (8 Hz) still inhibited CPN2. The inhibition consisted of only a smooth hyperpolarization. The persisting PSPs are from the AGR neuron (see Results). Most hyperpolarized V_m: MPN, −63 mV. Action potentials are clipped in MCN1 and CPN2. Aand B are from different preparations.

Fig. 3.

Proctolin directly excites MCN1 and CPN2. A, In normal saline MCN1 and CPN2 were silent. Superfusion of proctolin (10⁻⁵m) to the CoGs caused a depolarization of the membrane potential of these neurons and elicited rhythmic bursts of action potentials in them. MCN1 and CPN2 recordings are from different preparations. B, With transmitter release suppressed in the CoGs because of the superfusion of low Ca²⁺ saline, MCN1 and CPN2 were weakly active. Superfusion of proctolin (10⁻⁵m) in this saline to the CoGs caused a depolarization of the MCN1 and CPN2 membrane potential and increased their firing frequency. A and B are from different preparations, as are the MCN1 and CPN2 recordings.

Fig. 4.

The proctolin excitation of MCN1 and CPN2 in the CoGs is sufficient to elicit a gastric mill rhythm from the STG.A, Left, In normal saline MCN1 was weakly active, and there was no gastric mill rhythm (note the lack of rhythmic bursting in the lgn and dgn).A, Right, Superfusion of proctolin (10⁻⁵m) selectively to the CoGs increased the MCN1 activity and elicited a gastric mill rhythm in the STG. This rhythm includes alternating bursting in the lateral gastric (LG; lgn) and dorsal gastric (DG; dgn) neurons. The additional presence of the GM neuron bursts (dgn) is characteristic of the MCN1/CPN2 version of the gastric mill rhythm (Norris et al., 1994; Blitz and Nusbaum, 1997a). InC. borealis the dgn contains the axons of the DG, GM, and AGR neurons. AGR is the tonically active unit in thedgn. B, Left, In normal saline CPN2 was silent, and there was no gastric mill rhythm. B,Right, Superfusion of proctolin (10⁻⁵m) selectively to the CoGs activated rhythmic CPN2 bursting that is time-locked to the elicited MCN1/CPN2 gastric mill rhythm. C, Summary schematic indicating that proctolin bath application to the CoGs activates MCN1 and CPN2 sufficiently for them to elicit a gastric mill rhythm from the STG network. Labeling is as in Figure 1.

Fig. 5.

GABA inhibits MCN1 and CPN2 directly, and this inhibition is blocked by picrotoxin. Left, With transmitter release suppressed by low Ca²⁺ saline, MCN1 and CPN2 are weakly active. Pressure application of GABA in low Ca²⁺ saline to the MCN1 and CPN2 CoG neuropil caused a hyperpolarization of their membrane potentials and a cessation of action potentials. Right, When picrotoxin (10⁻⁴m) in low Ca²⁺ saline was superfused to the CoGs, the same GABA puff had no influence on MCN1 or CPN2 at similar membrane potentials. MCN1 and CPN2 recordings are from different preparations.

Fig. 6.

GABA inhibition of MCN1 is sufficient to inhibit the gastric mill rhythm, and this inhibition is blocked by picrotoxin. Left, When the CoGs were superfused with low Ca²⁺ saline, MCN1 activity increased and a gastric mill rhythm was elicited (dgn). GABA (10⁻⁴m) puffed onto the MCN1 neuropil in the CoG inhibited MCN1 and terminated the gastric mill rhythm. The rhythm resumed after the end of the puff, coincident with the resumption of MCN1 activity. Right, When picrotoxin (10⁻⁴m) in low Ca²⁺saline was superfused to the CoGs, GABA no longer had any effect on MCN1 or the gastric mill rhythm.

Fig. 7.

Picrotoxin blocks the direct MPN inhibition of MCN1 and CPN2. A, Left,With the CoGs superfused with high divalent cation saline to reduce the possibility of MPN activating an intervening neuron, MPN stimulation (12.5 Hz) inhibited MCN1, producing a smooth hyperpolarization. MPN stimulation also excited the pyloric rhythm in the STG, which was superfused with normal saline. Excitation of the pyloric rhythm was evident from the increased activity in the IC and VD neurons (mvn). Most hyperpolarizedV_m: MPN, −46 mV. A,Right, When the CoGs were superfused with picrotoxin (10⁻⁴m) in high divalent cation saline, MCN1 displayed no response to MPN stimulation (10 Hz) despite maintaining the same membrane potential. However, the pyloric rhythm (mvn) was still excited by MPN stimulation under these conditions. Most hyperpolarized V_m: MPN, −33 mV. B, Left, MPN stimulation (7 Hz) inhibited CPN2, producing a smooth hyperpolarization when the CoGs were superfused with high divalent cation saline. MPN stimulation also excited the pyloric rhythm (mvn) in the STG, which was superfused with normal saline. Most hyperpolarizedV_m: MPN, −77 mV. B,Right: When the CoGs were superfused with picrotoxin (10⁻⁴m) in high divalent cation saline, MPN stimulation (7 Hz) had no influence on CPN2. The pyloric rhythm (mvn) was still excited by MPN stimulation under these conditions. Most hyperpolarized V_m: MPN, −78 mV.

Fig. 8.

MPN inhibition of the gastric mill rhythm is blocked by 10⁻⁴m picrotoxin, but not by 10⁻⁵m picrotoxin.Top, Superfusion of the CoGs with 10⁻⁵m picrotoxin in normal saline increased the activity of MCN1 and CPN2 (data not shown) and elicited gastric mill timed bursting in the LG neuron. MPN stimulation (13 Hz) terminated the gastric mill timed bursting in the LG neuron and the gastric mill timed inhibition of the VD neuron (mvn).Bottom, When the CoGs were superfused with 10⁻⁴m picrotoxin in normal saline, MPN stimulation (13 Hz) had no influence on the gastric mill rhythm. Most hyperpolarized V_m: LG, −68 mV; MPN, −74 mV. Scale bars are for top andbottom.

Fig. 9.

Schematic of the roles of the MPN transmitters in mediating selection of the MPN-elicited motor pattern. MPN uses proctolin to excite the pyloric rhythm within the STG (Nusbaum and Marder, 1989b). The role of GABA in the STG is unknown. MPN uses GABA in the CoGs to inhibit MCN1 and CPN2, removing their excitation of the gastric mill rhythm. MPN does not have a proctolin-mediated influence on these neurons. Labels are as in Figure 1.