Adenosine-mediated presynaptic modulation of glutamatergic transmission in the laterodorsal tegmentum

Abstract

The laterodorsal tegmentum (LDT) neurons supply most of the cholinergic tone to the brainstem and diencephalon necessary for physiological arousal. It is known that application of adenosine in the LDT nucleus increases sleep in vivo (Portas et al., 1997) and directly inhibits LDT neurons in vitro by activating postsynaptic adenosine A(1) receptors (Rainnie et al., 1994). However, adenosine effects on synaptic inputs to LDT neurons has not been previously reported. We found that both evoked glutamatergic EPSCs and GABAergic IPSCs were reduced by adenosine (50 micrometer). A presynaptic site of action for adenosine A(1) receptors on glutamatergic afferents was suggested by the following: (1) adenosine did not affect exogenous glutamate-mediated current, (2) adenosine reduced glutamatergic miniature EPSC (mEPSC) frequency, without affecting the amplitude, and (3) inhibition of the evoked EPSC was mimicked by the A(1) agonist N6-cyclohexyladenosine (100 nm) but not by the A(2) agonist N6-[2-(3,5-dimethoxyphenyl)-2-(methylphenyl)-ethyl]-adenosine (10 nm). The A(1) receptor antagonist 8-cyclopentyltheophylline (CPT; 200 nm) potentiated the evoked EPSCs, suggesting the presence of a tonic activation of presynaptic A(1) receptors by endogenous adenosine. The adenosine kinase inhibitor, 5-iodotubercidin (10 micrometer), mimicked adenosine presynaptic and postsynaptic effects. These effects were antagonized by CPT or adenosine deaminase (0.8 IU/ml), suggesting mediation by increased extracellular endogenous adenosine. Together, these data suggest that the activity of LDT neurons is under inhibitory tone by endogenous adenosine through the activation of both presynaptic A(1) receptors on excitatory terminals and postsynaptic A(1) receptors. Furthermore, an alteration of adenosine kinase activity modifies the degree of this inhibitory tone.

Fig. 1.

Adenosine modulates both glutamatergic- and GABAergic-evoked synaptic transmissions. A, Glutamatergic evEPSCs are inhibited by adenosine (AD). The evEPSC recorded at V_h = −60 mV is reversibly reduced by adenosine (50 μm) and blocked by perfusion of glutamatergic receptor antagonists DNQX (20 μm) and APV (100 μm). B, In another neuron, a GABAergic evIPSC is inhibited by adenosine. The evIPSC recorded in the presence of DNQX and APV (V_h = −40 mV) is reversibly reduced by adenosine and blocked by GABA_A receptor antagonist BMI (20 μm). Each current trace is the average of 10 evEPSCs or evIPSCs.

Fig. 2.

Dose–response curve for adenosine inhibition of evEPSCs. The line represents the computer-generated fit to the data according to the equation: Y =min_resp + {(max_resp −min_resp)/(1 + 10 [(Log EC₅₀ − Log AD) × H])}, withmin_resp = 0%,max_resp = 77.3%, EC₅₀= 39.9 μm, AD = adenosine concentration in micromolar, and Hill slope,H = 0.98. The numbers indicate the number of cells recorded.

Fig. 3.

Modulation of glutamatergic transmission occurs independently of adenosine postsynaptic effects.A, Adenosine does not alter postsynaptic response to glutamate. Glutamate-evoked currents, elicited by exogenous glutamate (1 mm) pressure injected on the recording neuron (V_h = −60 mV), are unaffected by bath application of adenosine but are blocked by application of DNQX (20 μm) and APV (100 μm). The duration of each glutamate injection (1 sec) is indicated by the black bar above each current trace. B, In the same neuron, adenosine reversibly decreases evEPSC amplitude. Application of DNQX and APV eliminates the evEPSCs. C, Adenosine inhibition of the evEPSCs is independent of both presynaptic and postsynaptic GIRK activation. In another neuron, adenosine still reduces evEPSC amplitude in the presence of 2 mmBaCl₂. Each evEPSC current trace is the average of 10 evEPSCs (V_h = −60 mV).

Fig. 4.

Adenosine reduces glutamatergic mEPSC frequency without affecting mEPSC amplitude. A, Consecutive traces (2 sec each) recorded at V_h = −60 mV, showing typical mEPSC in control ACSF (left) and during the applications of adenosine (50 μm; middle) and DNQX (20 μm; right). Adenosine produces a decrease in frequency of mEPSC, and DNQX blocks the mEPSCs. B,C, Cumulative distribution plots of mEPSC amplitude and inter-event interval before (thick line) and during (thin line) adenosine application for the experiment shown in A. The cumulative plots show that adenosine does not induce a significant variation of the mEPSC amplitude (p > 0.1, K–S test; events analyzed = 685 in control and 232 with adenosine), whereas it does induce a shift toward the right in the distribution of inter-event intervals (p < 0.001, K–S test), indicating a decrease in mEPSC frequency. The mean mEPSC amplitude and frequency data pooled from nine neurons are represented in the two histograms (B, C, insets). Adenosine reduces mean mEPSC frequency (6.18 ± 0.79 Hz in control; 3.21 ± 0.34 Hz in adenosine; p = 0.0015, paired t test) without significant effect on the mean mEPSC amplitude (10.25 ± 1.44 pA in control; 9.35 ± 1.15 pA with adenosine; p = 0.066, paired ttest).

Fig. 5.

Exogenous and endogenous adenosine inhibit the evEPSCs through activation of A₁ receptors.A, Application of the A₁ receptor agonist CHA (100 nm) reduces the evEPSC amplitude.B, The evEPSC inhibition mediated by 50 μmadenosine (AD) is removed by the A₁ receptor antagonist CPT (200 nm). C, A neuron sensitive to adenosine (left) does not respond to the A₂ agonist DPMA (10 nm) but does respond to further application of CHA (right). D, Application of CPT alone induces an increase in the evEPSC amplitude.E, Summary of the effects on the evEPSC. The histogram shows the mean ± SE of the evEPSC peak amplitude during applications of CHA, DPMA, and CPT expressed as a percentage of their respective evEPSC amplitude in control. Only adenosine-sensitive cells were considered. The numbers indicate the number of cells recorded: *p < 0.01 versus evEPSC amplitude under control conditions; paired t test.

Fig. 6.

ITU induces postsynaptic activation of an inwardly rectifying K⁺ current and inhibition of the evoked glutamatergic transmission, similar to adenosine.A, Currents recorded atV_h = −60 mV. Downward deflections resulted from three different voltage protocol commands indicated by the numbers: 1, voltage pulses to −100 mV (200 msec; 0.2 Hz); 2, voltage ramps from −100 to −35 mV (10 mV/sec); 3, stimulation with a bipolar stimulating electrode (0.3 Hz). All protocols are performed in control ACSF and during applications of adenosine (AD; 50 μm), ITU (10 μm), and CPT (200 nm), indicated by black bars above the current traces. Both adenosine and ITU induce increases in the membrane conductance. The outward current elicited by ITU was rapidly blocked by application of CPT. B, evEPSCs recorded during control condition, adenosine, and ITU applications. The evEPSCs are inhibited by both adenosine and ITU. Each current trace is the average of 10 evEPSCs. C₁,C₂, Recordings from another neuron, obtained during the voltage-ramp protocols; each current trace displayed is the average of three consecutive voltage ramps. Adenosine (C₁) and ITU (C₂) increase the membrane slope conductance at all membrane potentials between −100 and −35 mV. The effect of ITU is antagonized by CPT (C₂). D, Current–voltage relationships of adenosine (I_AD) and ITU (I_ITU) evoked currents, calculated by digital subtraction (I_AD = AD − CON) and (I_ITU 61 ITU − CON) of the currents displayed in C₁ andC₂. I_AD andI_ITU show the same reversal potential (−81 mV) and the same voltage-dependent slope conductance, consistent with adenosine and ITU-mediated inwardly rectifying K⁺current activations. E, ITU chord conductance (G_ITU) as a function of membrane potential (E_m).G_ITU is obtained from the values ofI_ITU shown in D(G_ITU =I_ITU/(E_m− E_reversal) and is fit to the Boltzmann equation (line) G_ITU =G_ITU(min) + (G_ITU(max) −G_ITU(min))/{1 + exp [(E_m −E_1/2)/k]}, with G_ITU(max) = 56 nS,G_ITU(min) = 19.6 nS,E_1/2 = −87 mV, and k = 9.7 mV.

Fig. 7.

The inhibitory effect of ITU on the evEPSCs can be blocked by A₁ receptor antagonist CPT or by adenosine deaminase. A, CPT (200 nm) antagonized the inhibition of the evEPSC by ITU. B, In another neuron, adenosine deaminase (ADA) (0.8 IU/ml) partially removed the inhibitory effect of ITU. Each current trace is the average of 10 evEPSCs (V_h = −60 mV).