Substance P inhibits GABAB receptor signalling in the ventral tegmental area

Abstract

Substance P (SP) and its receptors are involved in anxiety-related behaviours and regulate the intake of drugs of abuse and alcohol. Within the midbrain ventral tegmental area (VTA), a region that is clearly involved in the control of these behaviours, SP is released by stress and has been shown to trigger relapse. SP activates neurokinin (NK) receptors, which excites midbrain dopamine (DA) neurons and leads to increased DA in target regions. In this study, we have investigated the mechanisms underlying SP actions in the VTA, specifically investigating interactions between SP and GABA(B) receptors. We show that in VTA neurons, NK receptor activation closes an inwardly rectifying potassium channel, and moreover inhibits GABA(B) receptor-mediated transmission through an interaction that depends upon phospholipase C (PLC), intracellular calcium and protein kinase C (PKC).

Figure 3. Current–voltage relationship for SP is inwardly rectifying

A, difference between current–voltage curves recording in SP and during a control period (n= 4, dark line). This current is blocked by 500 μm barium (n= 5, grey line). Shaded area indicates s.e.m. Membrane potential has been corrected for the liquid junction potential. B, bath application of SP produces an inward current in voltage-clamped neurons recorded using a caesium based internal solution (n= 7).

Figure 1. SP increases the firing rate of VTA neurons

A, representative traces from an individual VTA neuron before and after bath application of 1 μm SP. This neuron was filled with biocytin during recording (green), and immunocytochemically confirmed as TH⁺ (red). Scale bar, 50 μm. B, average of 12 I_h-expressing neurons shows significant increase in firing following bath application of SP.

Figure 2. SP actions depend upon PLC and intracellular calcium

A, the increase in firing rate due to SP is not blocked by the SRK inhibitor PP2 (1 μm, n= 6). B, SP does not alter the firing rate of I_h-expressing neurons following incubation with the PLC inhibitor U73122 (10 μm, n= 6). C, SP does not depolarize neurons when BAPTA (10 mm) is included in the whole-cell solution (n= 8).

Figure 4. SP inhibits GABA_B, but not GABA_A-mediated IPSCs

A, average of six neurons shows an SP-mediated inhibition of pharmacologically isolated GABA_B IPSCs. Upper left inset: trace showing that the GABA_B IPSC is completely blocked by the GABA_B antagonist CGP55845 (1 μm). Upper right inset: trace showing example IPSC in control and following SP application. B, average of five neurons showing that GABA_A IPSCs are not inhibited by SP. Insets: GABA_A IPSCs during control (left) and following SP application (right).

Figure 5. SP inhibits baclofen-mediated currents onto VTA neurons

A, average holding current change in response to 20 μm baclofen before and during bath application of SP (1 μm). Individual experiments are normalized so that the peak of the initial baclofen application is 100%. Responses are re-zeroed prior to 2nd baclofen application for comparison (hash marks on x-axis indicate the point of renormalization). B, application of baclofen 10 min after washout of SP shows that the inhibition of the baclofen current by SP reverses. C, control experiments showing that multiple applications of baclofen do not display desensitization when applications are separated by 20 min. D, summary data for control (n= 4), SP experiments (n= 5), and following washout of SP (n= 5). *P < 0.05 compared to control, one-way ANOVA with a Holm–Sidak post-hoc test.

Figure 6. Inhibition of GABA_B IPSCs requires PKC

Average of five neurons shows that GABA_B IPSCs are not inhibited by SP in slices that have been incubated in the PKC inhibitor Bis-1 (10 μm).

Figure 7. Inhibition of GABA_B is mediated through NK3 receptors

A, SP-mediated inhibition of evoked GABA_B IPSCs is unaffected by the NK1 antagonist L-732,138 (10 μm, n= 7). B, inhibition of GABA_B IPSCs by SP is blocked by the NK3 antagonist SB222200 (2 μm, n= 7).