Somatostatin inhibits excitatory transmission at rat hippocampal synapses via presynaptic receptors

Abstract

Somatostatin is one of the major peptides in interneurons of the hippocampus. It is believed to play a role in memory formation and to reduce the susceptibility of the hippocampus to seizure-like activity. However, at the cellular level, the actions of somatostatin on hippocampal neurons are still controversial, ranging from inhibition to excitation. In the present study, we measured autaptic currents of hippocampal neurons isolated in single-neuron microcultures. Somatostatin and the analogous peptides seglitide and octreotide reduced glutamatergic, but not GABAergic, autaptic currents via pertussis toxin-sensitive G-proteins. This effect was observed whether autaptic currents were mediated by NMDA or non-NMDA glutamate receptors. Furthermore, somatostatin did not affect currents evoked by the direct application of glutamate, but reduced the frequency of spontaneously occurring excitatory autaptic currents. These results show that presynaptic somatostatin receptors of the SRIF1 family inhibit glutamate release at hippocampal synapses. Somatostatin, seglitide, and octreotide also reduced the frequency of miniature excitatory postsynaptic currents in mass cultures without affecting their amplitudes. In addition, all three agonists inhibited voltage-activated Ca2+ currents at neuronal somata, but failed to alter K+ currents, effects that were also abolished by pertussis toxin. Thus, presynaptic somatostatin receptors in the hippocampus selectively inhibit excitatory transmission via G-proteins of the Gi/Go family and through at least two separate mechanisms, the modulation of Ca2+ channels and an effect downstream of Ca2+ entry. This presynaptic inhibition by somatostatin may provide a basis for its reportedly anticonvulsive action.

Fig. 1.

Autaptic currents of hippocampal microisland neurons and their modulation by somatostatin. A andB show EACs and IACs, respectively, recorded from two different neurons by the stimulation protocol shown inC. Currents in the bottom panels were recorded before (control), during, and after (wash) the application of 1 μm TTX (A, B), 10 μm CNQX (A), and 30 μm BMI (B), respectively. The time scale in C applies to the currents shown in A, B, D, and E. D, EACs measured at a holding potential of −70 mV before (control), during, and after (wash) the application of 1 μmsomatostatin. E shows the same sequence of recordings obtained in a neuron displaying IACs measured at a holding potential of −40 mV. F summarizes the effects of 1 μmsomatostatin on EACs and IACs. The boxes encompass the median 25th through 75th percentiles and contain a linemarking the median 50th percentile point. The caps of the error bars indicate the median 10th and 90th percentiles.

Fig. 2.

Pharmacological characterization of somatostatin receptors mediating the inhibition of EACs.A, Concentration–response curves for the reduction of EACs (measured as shown in Fig. 1D) by somatostatin (circles, n = 7–23, with the exception of 1 nm, for which n= 3), seglitide (squares, n = 5–9), and octreotide (triangles, n = 5–9). Data for the three agonists are pooled from different neurons, but not every neuron could be exposed to all agonist concentrations.B, Inhibition of EACs by 1 μmsomatostatin, seglitide, and octreotide in a common set of neurons (n = 5). C, Effects of 1 μm somatostatin on EACs in neurons treated with 200 ng/ml PTX for ≤ 24 hr (PTX), in neurons treated with heat-inactivated (95°C for 5 min) PTX (HI), and in control (untreated) neurons (ctl). Levels of significance for the difference between the results obtained in pretreated and in untreated neurons, respectively, are indicated above thebars.

Fig. 3.

Somatostatin acts at presynaptic receptors.A shows EACs recorded at −70 mV in the presence of 2 mm Mg²⁺, in the presence of 2 mmMg²⁺ + 10 μm CNQX, after replacement of Mg²⁺ by 10 μm glycine, and after addition of 50 μm AP5. B shows the inhibition of EACs by somatostatin in a different microisland neuron in the presence of 2 mm Mg²⁺ (top traces) and after replacement of Mg²⁺ by 10 μm glycine (bottom traces). Currents were obtained before, during, and after the application of 1 μm somatostatin.C, Currents evoked by 100 μm glutamate at −70 mV in a mass culture neuron in the presence of 1 μmsomatostatin, as well as before and after application of the peptide.

Fig. 4.

Somatostatin inhibits evoked as well as spontaneous EACs in microisland neurons. Recordings were obtained at −70 mV, and evoked EACs were elicited every 30 sec by 1 msec depolarizations to 0 mV. SEACs were recorded intermittently in sweeps of 2 sec. Current traces were obtained before (control), during, and after (washout) the application of 1 μmsomatostatin.

Fig. 5.

Somatostatin receptors inhibit mEPSCs in hippocampal mass culture neurons. A, MEPSCs were recorded in a mass culture neuron at −70 mV in 1 sec sweeps in the presence of 1 μm TTX and 30 μm BMI. More than 200 events were evaluated from recordings obtained before (control), during, and after (wash) the application of 1 μmsomatostatin. The distribution of mEPSC amplitudes (A₁) and inter-mEPSC intervals (A₂) is shown. B, Effects of 1 μm of somatostatin, seglitide, and octreotide on mean mEPSC amplitudes and mean inter-mEPSC intervals of five to six mass culture neurons. Results are shown as percentage of control. Changes in mean intervals (>50 events for each condition) were significant (p < 0.05) in each neuron.C, Effects of somatostatin on mEPSCs recorded as inA and B, but in the presence of 100 μm Cd²⁺ (n = 5). Changes in mean intervals were significant (p < 0.05) in each neuron. D, Effects of somatostatin on mEPSCs recorded as in A–C, but in neurons treated for ≥24 hr with 200 ng/ml PTX (n = 5). Note the loss of somatostatin effects.

Fig. 6.

Inhibition of voltage-activated Ca²⁺currents by somatostatin. A, Ca²⁺ currents were evoked in a mass culture neuron by depolarizations from −80 to 0 mV. Traces were recorded before (control), during, and after (wash) the application of 0.1 and 1 μm somatostatin. The inset shows rising phases of Ca²⁺ currents in the very same neuron under control conditions (a), in the presence of 1 μm somatostatin (b), and in the presence of somatostatin, but subsequent to a 50 msec predepolarization to +100 mV, followed by a 5 msec repolarization to −80 mV (c). Calibration, 50 pA, 10 msec. B, Current–voltage relationship of the Ca²⁺ currents in the same neuron as inA before (open circles) and during (solid circles) the application of 1 μmsomatostatin. C, The effect of somatostatin on the current–voltage relationship in B is shown as percentage of inhibition.

Fig. 7.

Lack of effect of somatostatin on voltage-dependent K⁺ currents. A, K⁺ currents in a mass culture neuron were evoked by depolarizations from −80 to 0 mV before (control), during, and after (wash) the application of 1 μmsomatostatin. B, Outward currents in a mass culture neuron were induced by a 0.2 sec ramp depolarization from −70 to +50 mV before, during, and after the application of 1 μmsomatostatin. Note that three traces are superimposed because of the lack of action of somatostatin.