Proc. Natl. Acad. Sci. USA Vol. 85, pp. 948-952, February 1988 Neurobiology Somatostatin depresses excitability in neurons of the solitary tract complex through hyperpolarization and augmentation of IM, a non-inactivating voltage-dependent outward current blocked by muscarinic agonists (somatomedin-release-inhibitory factor/K+ conductance) T. JACQUIN*t, J. CHAMPAGNAT*, S. MADAMBAt, M. DENAVIT-SAUBIO*, AND G. R. SIGGINSt *Laboratoire de Physiologie Nervepse, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France; and tDivision of Preclinical Neuroscience and Endocrinology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037 Communicated by Floyd E. Bloom, October 15, 1987 ABSTRACT The synaptic function of somatostatin- disagreement on the primary effects of these somatostatin containing fibers in the nervous system is controversial. There- (SS) elements (see ref. 8 for review). Administration of SS fore, we used a slice preparation of the rat brain stem to test has been reported to inhibit (see, e.g., refs. 8-15) and to the electrophysiological effects of prosomatostatin-derived excite (8, 16-19) the discharge of central neurons in vivo and peptides on neurons of the solitary tract complex, which in vitro. In intracellular recordings from hippocampal slices contains an abundance of somatostatin-containing fibers and in vitro, depolarization with enhanced spiking (18, 19) and cell bodies. Superfusion of both somatostatin-14 and somato- hyperpolarization with depressed spiking (11, 12, 14, 15) statin-28 (the precursor for somatostatin-14), but not somato- have been recorded from the same cell type (CA1 pyramidal statin-28-(1-12) or -(1-10), predominantly inhibited spontane- cells) after SS administration. The hyperpolarizations are ous spike and subthreshold (probably synaptic) activity. In thought to be produced by an increase in K+ conductance intracellular recordings, somatostatin-14 and -28 hyperpo- (11, 15). It has been suggested that the SS-evoked excita- larized most neurons in association with a slight (10-35%) but tions observed might derive from enhancement by SS of reproducible decrease in input resistance. These hyperpolariz- acetylcholine-induced excitatory effects (13). ing responses were augmented in depolarized cells and per- A further complication is the suggestion that several sisted in cells in which spontaneous inhibitory postsynaptic different fragments of prosomatostatin-derived peptides ex- potentials became depolarizing after Cl injection. These data ist in nerve elements in, among other brain regions, the suggest that somatostatin receptors regulate a K+ conduc- hippocampus (20) and the STC (ref. 7 and L. Koda, R. Be- tance. In voltage-clamp studies, somatostatin-28 and -14 in- noit, N. Ling, C. Bakhit, S.M., and G.R.S., unpublished duced a steady outward current and augmented the voltage- data). We have reported data that two of these fragments, dependent, nonactivating outward K+ conductance (IM) SS14 and SS28, have potent inhibitory effects on neurons of shown to be blocked by activation of muscarinic cholinergic the STC (21). The present study (reported in abstract form, receptors. These results suggest (tl that somatostatin-contain- ref. 22) demonstrates the hyperpolarization and transmem- ing elements in the solitary tract complex play an inhibitory brane currents underlying these inhibitory effects and de- role through the activation of postsynaptic permeability to scribes one probable mechanism: an augmentation by SS14 potassium ions and (u) that the same ion channel type may be and SS28 (but not other prosomatostatin-derived peptides) coregulated by two neurotransmitter candidates, somatostatin of a voltage-dependent conductance probably to potassium and acetylcholine, through a reciprocal control mechanism. ions (the non-inactivating voltage-dependent outward cur- rent blocked by muscarinic agonists, IM). The solitary tract complex (STC), including the nuclei of the tractus solitarius and the dorsal vagal motor nucleus, is a METHODS dorsomedial brain stem structure regulating visceral func- tions. Excitatory and inhibitory synaptic activity occurs in Slices of rat brain stem were prepared as reported (1-3, 23, the STC (1-3). To date, the available information on inhibi- 24). Briefly, male Wistar or Sprague-Dawley rats weighing indi- 100-170 g were briefly anesthetized with halothane or ether, tory synaptic potentials seen in the medulla oblongata craniotomized, and decapitated at the upper cervical spinal cates that they are generated by large, rapid increases of cord. After transcollicular section, the brain stem was re- membrane conductance, mainly to Cl-, that can be mim- moved, immersed in cold (10°C) artificial cerebrospinal fluid icked by application of glycine and y-aminobutyric acid (2, (ACSF) and separated from the cerebellum. During these 3). In respiratory neurons in vivo, this Cl- mechanism surgical procedures, cold (10°C) ACSF was dripped onto mediates potent and fast suppression of neuronal activities exposed brain surfaces. (ACSF = 124 mM NaCl/5 mM following an afferent impulse or during certain phases of the KCI/2 mM MgSO4'7H20/1.25 mM KH2PO4/26 mM respiratory cycle (4, 5). NaHCO3/2 mM CaCI2/10 mM glucose.) The ACSF was Another potential inhibitory agonist found in the STC (6, gassed with 95% 02/5% CO2 in all cases. Coronal slices 7) is the tetradecapeptide somatostatin [somatostatin-14 (350-500 ,um thick) were cut on a tissue chopper, placed in (SS14) or somatomedin-release-inhibitory factor]. Despite warm ACSF in the considerable physiological research and abundant immuno- cold ACSF, and transferred to (31-37°C) histochemical data showing wide-spread somatostatin-con- cell bodies and fibers in several brain areas, there is Abbreviations: STC, solitary tract complex; I-V, current-voltage; taining IM, non-inactivating voltage-dependent outward current blocked by muscarinic agonists; IA, transient outward current; SS14, somato- The publication costs of this article were defrayed in part by page charge statin-14; SS28, somatostatin-28; SS, somatostatin; ACSF, artificial payment. This article must therefore be hereby marked "advertisement" cerebrospinal fluid. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 948 Downloaded by guest on October 6, 2021 Neurobiology: Jacquin et al. Proc. Natl. Acad. Sci. USA 85 (1988) 949 recording chamber. The upper surfaces of the slices were In intracellular recordings, neurons of the STC displayed first exposed to warm humidified 95% 02/5% CO2 for 10-45 membrane potentials of - 50 to - 70 mV, near or above the min, whereupon the slices were completely immersed and threshold for spontaneous discharge of action potentials continuously superfused at a constant rate (1-5 ml/min). (-45 to -55 mV; see refs. 1-3, 23). SS14 hyperpolarized 10 Solutions of the SS analogues (obtained from Jean Rivier of 16 cells (by 5-20 mV; Figs. 1 and 2), depolarized 1, and and Nick Ling at the Salk Institute) were made up immedi- had no measurable effect in 5 cells. SS28 hyperpolarized 7 ately before use in ACSF gassed with 95% 02/5% CO2 from cells and had no effect in 2 cells. The SS-induced hyperpo- frozen stock solutions (1 mM). Glass micropipettes contain- larizations were smallest in those cells with more negative ing 3 M KCl or 2 M KOAc of 50-80 Mil (KCl) or 80-150 Mfl membrane potentials ( - 55 to - 70 mV). Hyperpolarizations (KOAc) tip resistance, coupled to a Dagan 8100 (Minneap- were accompanied by decreases in the frequency of sponta- olis, MN) or Axon Instruments (Burlingame, CA) Axoclamp neous subthreshold potentials (presumed postsynaptic po- amplifier, were used to perform current-clamp and voltage- tentials). The hyperpolarizations appear to be a direct clamp recording. Single-electrode discontinuous voltage- postsynaptic effect, as they were still elicited during con- clamp of the neurons was performed as described (23), using comitant superfusion of 1 ,M tetrodotoxin to isolate the cell KCl pipettes and the high-frequency switching, current- from synaptic input (in 1 of 3 cells excited by SS14, tetro- injection and voltage-sampling method. The sampling fre- dotoxin blocked this response). Neither somatostatin-28- quency was set manually to 2.5-5 kHz with a duty cycle of (1-12) nor -(1-10) consistently hyperpolarized STC neurons 30 or 50%. Apparent input resistance and reversal or "null (at 0.5-5 ,M concentrations) in the same cells that showed potentials" for SS-induced responses were estimated in SS14- or SS28-evoked hyperpolarizations, suggesting that current-clamp mode by injection through the recording pi- specific SS receptors are involved in the inhibitory re- pette of steps of hyper- and depolarizing current steps, for sponses to SS28 and SS14 (see ref. 25). construction of current-voltage (I-V) curves. In voltage- The SS-evoked hyperpolarizations persisted in cells in clamp mode, voltage step commands of various amplitudes which sufficient Cl- was ejected from KCl-containing re- (0.8- to 1-sec duration) were delivered from various holding cording pipettes to invert presumed (spontaneous) inhibitory potentials, and instantaneous and steady-state I-V curves postsynaptic potentials to depolarizations (see refs. 1 and 26). were constructed before, during, and after SS superfusion. In these cells, the hyperpolarizations elicited by SS28 and SS14 were reduced when the membrane potential was hyper- RESULTS polarized by 10-20