European Journal of Pharmacology, 192 (1991) 287-291 5:, 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0014-2999/91/$03.50 ADONIS 0014299991001066

287

FJP 51642

Somatostatin induced hyperpolarization of septal neurons is not blocked by pertussis toxin Michael J. Twery, L i n d a A. W o n g i a n d Joel P. G a l l a g h e r Department of Pharmacology and Toxwology. Unit,ersity of Texas Medical Branch. Gah,'eston. TX. L~S.A. Received 30 May 1990, revised MS received 8 August 1990, accepted 2 October 1990

The coupling of postsynaptic somatostatin receptors to pertussis toxin (PTX) sensitive guanine nucleotide regulatory proteins (G proteins) was investigated in dorsolateral septal nucleus (DLSN) neurons using a submerged brain slice preparation and intracellular recording techniques. Rats were pretreated with PTX i.c.v, and neuronal responsivity to somatostatin and baclofen, a selective GABA a receptor agonist, tested using a submerged brain slice preparation and intracellular recording techniques. In tissue obtained from rats pretreated with PTX (2.5 /tg) for 2-5 days, somatostatin applied by superfusion (0.1 ttM) produced membrane hyperpolarization and decreased the membrane resistance of DLSN neurons. Hyperpolarizing effects of somatostatin persisted in the presence of tetrodotoxin (0.3 ~M) blocking synaptic transmission. Current-voltage relations of the somatostatin-induced, PTX-resistant hyperpolarization indicated a reversal potential close to the equilibrium potential for potassium ions. Membrane hyperpolarizations in PTX treated tissue were similar to those recorded in tissue from vehicle control or untreated rats. Hyperpolarizing responses to the selective GABA B receptor agonist baclofen, however, were blocked by the PTX treatment used in the present study. Our findings suggest that the postsynaptic inhibitory effects of somatostatin in the DLSN is not mediated by a somatostatin receptor coupled to PTX-sensitive G proteins. These G proteins, however, appear to be an essential link in the postsynaptic GABA R receptor-mediated response of DLSN neurons. Somatostatin; Septum; G-proteins (guanine nucleotide-binding proteins); Pertussis toxin; K ÷ channels: GABA a receptors

I. I n t r o d u c t i o n

Somatostatin is known to be an inhibitor of hormone secretion and neuronal activity. Potassium and calcium currents have been implicated in this action and the somatostatin receptor appears to be linked to these membrane conductances through pertussis toxin (PTX) sensitive guanine nucleotide regulatory proteins (G proteins) (Koch, 1985; Lewis et al., 1986; Inoue et al., 1988; Pennefather et al., 1988; Wang et al., 1989). It has been suggested that these G proteins are crucial to the transmembrane signal transduction process since the toxin inactivates this subclass of G proteins (Ui et al., 1984; Stryer and Bourne, 1986) and blocks inhibition by somatostatin in a variety of tissues irreversibly. The presence of somatostatin-like immunoreactivity in nerve terminals and cell bodies of the lateral septum

1 Current address: Department of Neurology, The Children's Hospital, Boston, MA 02115, U.S.A. Correspondence to: M.J. Twery, National Institutes of Health, Building 10, Room 5C214, Bethesda, MD 20895, U.S.A.

suggests somatostatin may be a synaptic transmitter in this brain region. Consistent with this idea, we have recently recorded an inhibitory action of somatostatin on rat dorsolateral septal nucleus (DLSN) neurons intracellularly (Twery and Gailagher, 1989). Somatostatin increased the permeability of D L S N neuronal membranes to potassium at nanomolar concentrations and produced hyperpolarization sufficient to block spontaneous neuronal activity. In order to investigate whether postsynaptic somatostatin receptors (producing hyperpolarization) were coupled to PTX sensitive G proteins, the present study tested the responsivity of DLSN neurons to somatostatin and baclofen using tissue obtained from rats which had been pretreated i.c.v, with PTX. Baclofen is a selective agonist for GABA B receptors which are known to act through PTX-sensitive G proteins in postsynaptic DLSN neurons to produce a potassium-mediated hyperpolarization (Gailagher et al., 1989). The results indicate that somatostatin receptor-mediated inhibition of DLSN neurons is not blocked by a PTX treatment which eliminates inhibitory responses at postsynaptic G A B A a receptors in this nucleus. Portions of this work have appeared in abstract form (Twery et al., 1989).

288

2. Materials and methods

Male Sprague-l)awley rats (150-250 g) were prctreated i.c.v, with PTX (5 #1, 0.5 ffg/#l, n = 15) or saline vehicle (5 p.l, n = 3) 3-5 days prior to obtaining tissue for intracellular recording studies. The animals were anesthetized with Equithesin and the PTX/vehicle slowly injected over 10 min using a 26 g needle placed stereotaxically in the left ventricle at the level of the DLSN. Results obtained in tissue from saline control animals have been pooled with those obtained from untreated rats (n = 11) in the present report, since no differences in neuronal response to either somatostatin or baclofen, a G A B A u receptor agonist, were detected. Placement of the i.c.v, injection was verified during slice preparation by inspection of the needle track. For electrophysiological studies, brain tissue was obtained as previously described (Stevens et al., 1984) and placed in cold artificial cerebrospinal fluid (ACSF: pH 7.4) containing (in mM) NaCI 117, KCI 4.7, CaCI 2 2.5, MgCI e 1.2, N a H C O 3 25, N a H 2 P O 4 1.2, and D-glucose 11. ACSF was continuously bubbled with 95% 02 and 5% CO 2 gas mixture. Tissue slices containing septum = 500 ffm thick were cut using a Vibroslice (Campden Instruments). A single slice was transferred to the recording chamber and maintained submerged in A ( ' S F media at 32 + 1°('. Intracellular recordings obtained with a glass microelectrode containing 4 M potassium acetate (75-110 MI2) were amplified (Axoclamp 2A, Axon Instruments) and displayed with standard instruments. Brief hyperpolarizing current pulses (300 ms, 0.1 nA, 0.1 Hz) were injected to monitor bridge balance and apparent changes in input resistance. Estimates of slope resistance were obtained from current-voltage relationship data collected at similar baseline membrane potentials under control and treatment conditions. Current-voltage data were analyzed using a computer-controlled waveform analyzer (Data 6000, Analogies). In order to demonstrate the selectivity of the PTX treatment, each scptal neuron was tested with both somatostatin and baclofen. Somatostatin-14 (0.1-1 #M) and, in some cases, tetrodotoxin (TFX, 0.3 /~M) were added to the superfusion media for bath-application. Baclofen was applied by direct addition to the bath inlet (estimated dilution = 1/100). All drugs were obtained from Sigma Chemical Co. except baclofen which was from Research Biochemicals Inc.

3. Results

In tissue obtained from sham-operated (n = 3) or untreated (n = 11) rats, bath application of somatostatin to neurons in a submerged brain slice preparation of the rat DLSN produced a membrane hypcrpolarization

with slow onset and recovery (fig. 1A). This effect of somatostatin appeared to be the result of a direct action on postsynaptic I)LSN neurons since similar membrane

A Control o.1 pM SS-14

,,l/t~,,.f,IJl/Ji(ltrlll

....

'

:

' i l l ,'

I.TT..r~--~T-I.T.IT~TTTTFT1;TT{fTt''tl

"

:1

r

. ~

:;urrent (n~}. -02

-Ol

1C tt,v

3O '-

~o

a~

oi

&

.-q • -RO

~

-90

B

7?

PTX o.1 pM SS-14 i

-59

....,Ill}

1 '"q

17

(?ur'e"t (-~A) -C5

-04

. . . . . .

". . . . . .

.0"~

-07

-P 1

Or" r

"

,.

4

,,,,

t

~

. 4

.f

C1 ~ v

?'

i

?,

.

i

".S