Cellular and synaptic modulation underlying substance P-mediated plasticity of the lamprey locomotor network

Abstract

The tachykinin substance P modulates the lamprey locomotor network by increasing the frequency of NMDA-evoked ventral root bursts and by making the burst activity more regular. These effects can last in excess of 24 hr. In this paper, the effects of substance P on the synaptic and cellular properties of motor neurons and identified network interneurons have been examined. Substance P potentiated the amplitude of monosynaptic glutamatergic inputs from excitatory interneurons and reticulospinal axons. The amplitude and frequency of miniature EPSPs was increased, suggesting that the synaptic modulation was mediated presynaptically and postsynaptically. The postsynaptic modulation was caused by a specific effect of substance P on the NMDA component of the synaptic input, whereas the presynaptic component was calcium-independent. Substance P did not affect monosynaptic glycinergic inputs from lateral interneurons, crossed inhibitory interneurons, or ipsilateral segmental interneurons or postsynaptic GABAA or GABAB responses, suggesting that it has little effect on inhibitory synaptic transmission. At the cellular level, substance P increased synaptic inputs, resulting in membrane potential oscillations in motor neurons, crossed caudal interneurons, lateral interneurons, and excitatory interneurons. The spiking in response to depolarizing current pulses was increased in motor neurons, lateral interneurons, and excitatory interneurons, but usually was reduced in crossed inhibitory interneurons. Substance P reduced the calcium-dependent afterhyperpolarization after an action potential in motor neurons and lateral interneurons, but did not affect this conductance in excitatory or crossed inhibitory interneurons. The relevance of these cellular and synaptic changes to the modulation of the locomotor network is discussed.

Fig. 1.

Substance P potentiates excitatory synaptic transmission. Ai, Aii, A paired recording from an EIN and a motor neuron. The EIN input to the motor neuron was potentiated by substance P. One micromolar concentration of substance P applied for 10 min was used in this and all subsequent figures, unless stated otherwise. The onset and duration of substance P application is shown by the bar on the graphs in this and subsequent figures. Bi, Bii, Substance P also potentiated monosynaptic glutamatergic inputs in a motor neuron from a reticulospinal axon. Note that the initial electrical component of the EPSP was unaffected by substance P. Ci,Cii, A split-bath preparation (inset) was used to examine the effect of substance P on locomotor-related EPSPs. Fifty micromolar conentration of NMDA was added to the rostral bath to activate locomotor networks, and 5 μm strychnine was added to the caudal bath to block locomotor-related IPSPs. Substance P also potentiated the amplitude of these locomotor-related EPSPs. Ventral root recordings made in the rostral pool are shown above the intracellular traces. Effects of substance P on spontaneous spike-evoked EPSPs (D) (motor neurons,n = 3; LIN, n = 3; CC interneurons, n = 2) and IPSPs (E) (motor neurons, n = 4; LIN, n = 2; CC interneurons, n= 4) recorded in the absence of TTX.

Fig. 2.

Substance P increased the amplitude and frequency of mEPSPs. mEPSPs were recorded in the presence of TTX (1.5 μm) and strychnine (5 μm). The frequency was calculated by measuring the interval between successive mEPSPs.Ai, Aii, Sample traces of mEPSPs in control and in the presence of substance P. Histograms (Bi, Ci) and cumulative probability plots (Bii, Cii) are shown for the amplitude and frequency of the mEPSPs in control and in the presence of substance P. The shift in the amplitude cumulative probability to the right inBii indicates an increase in the number of large amplitude mEPSPs, whereas a shift in the curve to the left inCii indicates an increase in the number of shorter intervals between mEPSPs. The histograms and cumulative probability plots are from different representative motor neurons.

Fig. 3.

Properties of the increased amplitude and frequency of mEPSPs. A, The half-decay time of mEPSPs was increased in the presence of substance P. The insetshows averaged mEPSPs (n = 200) in control and in the presence of substance P. Calibration, inset: 0.25 mV, 3 msec. B, The increased amplitude of the mEPSPs was blocked in the presence of the NMDA receptor antagonist AP5 (100 μm). C, Blocking NMDA receptors with AP5 did not affect the increased frequency of the mEPSPs. D, The increase in mEPSP frequency was also not affected by the calcium channel antagonist cadmium (200 μm), suggesting that the increased glutamate release was not calcium-dependent. Data from one neuron are shown in B–D.

Fig. 4.

Substance P does not modulate monosynaptic glycinergic inhibitory inputs. Examples of paired recordings from a CCIN and a postsynaptic motor neuron (Ai,Aii), from an LIN and a CCIN (Bi,Bii), and from an SiIN and an LIN (Ci,Cii) are shown. In no case was the amplitude of the monosynaptic inhibitory potential affected by substance P. In each case, the inhibitory potentials were abolished by strychnine (5 μm). The bars on the graph indicate the onset and duration of substance P and strychnine application.

Fig. 5.

Substance P does not affect the amplitude or frequency of mIPSPs. mIPSPs were recorded in the presence of TTX (1.5 μm) and either 1 mm kynurenic acid or 100 μm AP5 and 10 μm CNQX, to block glutamatergic inputs. Ai, Aii, Sample traces showing mIPSPs in control and in the presence of substance P. Frequency histograms (Bi, Ci) and cumulative probability plots (Bii, Cii) are shown for the amplitude and frequency of the mIPSPs in control and in the presence of substance P. The frequency of mIPSPs was again calculated by measuring the interval between successive mIPSPs. Data from single representative neurons are shown, with separate neurons being used to provide histograms and cumulative probability plots.

Fig. 6.

Substance P does not modulate GABAergic responses.Ai, Trace showing the effect of pressure application of GABA (1 mm; 200 msec pulse duration) on a motor neuron. The GABA response had a fast and a slow component. Substance P did not affect either component of the GABA response. Aii, Trace showing the lack of an effect of substance P on responses to the GABA_A receptor agonist muscimol (200 μm).Aiii, Trace showing the lack of an effect of substance P on responses to the GABA_B receptor agonist baclofen (1 mm). Graphs showing the effect of substance P on the fast (B) and slow (C) GABA responses elicited in the neuron shown in Ai(1 and 2 refer to the fast and slow components, respectively). Substance P did not affect either component. The GABA_A receptor antagonist bicuculline blocked the fast component, whereas the slow component persisted. The reduction in the amplitude of the slow component may have been caused by it developing from the baseline, as opposed to a relatively hyperpolarized level when the bicuculline-sensitive component was present.

Fig. 7.

Substance P increases synaptic inputs and elicits membrane potential oscillations in network neurons. Representative effects of substance P on a motor neuron (A), an LIN (B), a CCIN (C), and an EIN (D). The EIN was examined in normal Ringer’s solution (Di) and in the presence of TTX (1.5 μm; Dii). E, The tachykinin agonist neurokinin A (1 μm) also induced membrane potential oscillations and spiking in unidentified neurons. The onset and duration (10 min) of drug application is shown by thebar underneath each figure. The spikes are clipped inA, D, and E.F, G, Graphs showing the effects of substance P on the amplitude and number of oscillations in motor neurons (n = 7), LINs (n = 5), CCINs (n = 4), and EINs (n = 5). The amplitude was measured from the baseline preceding the onset of the substance P effects to the peak depolarization (excluding spikes), and the frequency by the number of oscillations that occurred in the first 10 min after substance P application.

Fig. 8.

Substance P increases descending excitatory inputs. A split-bath preparation was used to examine the effects of substance P on descending excitatory neurons. A Vaseline barrier was built to separate the spinal cord into two pools (B,inset). Substance P was added to the rostral pool. Neurons were impaled in the caudal pool within five segments of the barrier. A, The effect of substance P application to the rostral pool when the caudal pool contained normal Ringer’s solution. The spikes have been clipped. B, Graph showing the potentiation of the amplitude of excitatory synaptic inputs by rostral application of substance P (n = 4).C, The effects of substance P when 5 μmstrychnine was added to the caudal pool. Recordings in Aand C are from unidentified gray matter neurons recorded in different experiments. D, Extracellular recording from the dorsal root and dorsal column in an isolated spinal cord preparation. A split-bath preparation was not used in these experiments. Substance P caused spiking in dorsal root and dorsal column axons, suggesting that it excites primary afferent axons. Thebar indicates the onset and duration (10 min) of substance P application.

Fig. 9.

Substance P does not significantly affect the input resistance of motor neurons (A), LINs (B), CCINs (C), or EINs (D). The input resistance was examined by injecting 100 msec hyperpolarizing current pulses using single electrode current clamp. The membrane potential was held at the same membrane potential (−70 mV) in control and in the presence of substance P. Kynurenic acid (1 mm) and strychnine (5 μm) were used to block synaptic inputs to the cells. Data from 14 motor neurons, 5 LINs, 8 CCINs, and 8 EINs are shown.

Fig. 10.

Substance P modulates the spiking in response to depolarizing current pulses in spinal neurons. Spiking was elicited by injecting 100 msec depolarizing current pulses into the somata under current clamp. The membrane potential in control and in the presence of substance P was set at −70 mV by current injection using single electrode current clamp. Substance P increased the spiking in motor neurons (A), LINs (B), and EINs (C) but usually reduced the spiking in CCINs (D). E, In addition to the effect on the number of spikes, substance P reduced the latency to the first spike in motor neurons and LINs, but increased it in CCINs. Each point for the different types of neurons is the latency to the first spike after the onset of the depolarizing current pulse in control, during substance P application, and after wash-off. The experiments shown here were performed in the presence of CNQX (10 μm), AP5 (100 μm), and strychnine (5 μm) to block synaptic inputs. Data from 14 motor neurons, 5 LINs, 8 CCINs, and 8 EINs are shown inA–D and from single representative cells of each type in E.

Fig. 11.

The effects of substance P on the action potential properties of spinal neurons. A, Substance P reduced the amplitude of the AHP_Ca in motor neurons and LINs but had no affect on the AHP_Ca in CCINs or EINs. Theinsets show an average of the AHP_Ca in four action potentials in control and in the presence of substance P in a motor neuron and a CCIN. Substance P had no significant effect on the early AHP (B), the spike amplitude (C), or the spike duration (D) in any type of neuron. Data from 14 motor neurons, 5 LINs, 8 CCINs, and 8 EINs are shown.

Fig. 12.

Schematic diagram summarizing the effects of substance P on the locomotor network. Excitatory connections are shown by solid bars, and inhibitory connections are shown byfilled circles. Darker shading indicates modulation of the AHP_Ca. Potentiated synaptic connections are shown by larger symbols. Increased excitability is shown by thicker lines, and reduced excitation is shown by a dashed line. MN, motor neuron;L, lateral interneuron; CC, CCIN;RS, reticulospinal input; E, EIN.