5-HT1B receptor-mediated presynaptic inhibition of retinal input to the suprachiasmatic nucleus

Abstract

The suprachiasmatic nucleus (SCN) receives glutamatergic afferents from the retina and serotonergic afferents from the midbrain, and serotonin (5-HT) can modify the response of the SCN circadian oscillator to light. 5-HT1B receptor-mediated presynaptic inhibition has been proposed as one mechanism by which 5-HT modifies retinal input to the SCN (Pickard et al., 1996). This hypothesis was tested by examining the subcellular localization of 5-HT1B receptors in the mouse SCN using electron microscopic immunocytochemical analysis with 5-HT1B receptor antibodies and whole-cell patch-clamp recordings from SCN neurons in hamster hypothalamic slices. 5-HT1B receptor immunostaining was observed associated with the plasma membrane of retinal terminals in the SCN. 1-[3-(Trifluoromethyl)phenyl]-piperazine HCl (TFMPP), a 5-HT1B receptor agonist, reduced in a dose-related manner the amplitude of glutamatergic EPSCs evoked by stimulating selectively the optic nerve. Selective 5-HT1A or 5-HT7 receptor antagonists did not block this effect. Moreover, in cells demonstrating an evoked EPSC in response to optic nerve stimulation, TFMPP had no effect on the amplitude of inward currents generated by local application of glutamate. The effect of TFMPP on light-induced phase shifts was also examined using 5-HT1B receptor knock-out mice. TFMPP inhibited behavioral responses to light in wild-type mice but was ineffective in inhibiting light-induced phase shifts in 5-HT1B receptor knock-out mice. The results indicate that 5-HT can reduce retinal input to the circadian system by acting at presynaptic 5-HT1B receptors located on retinal axons in the SCN.

Fig. 1.

Light micrographs of coronal sections through the mouse anterior hypothalamus illustrating the 5-HT-immunoreactive process in the mid-caudal SCN (a), the HRP-labeled retinal processes in the mid-caudal SCN viewed using dark-field optics (b), 5-HT_1Breceptor immunoreactivity in the mid-caudal SCN (c), and the absence of 5-HT_1Breceptor immunoreactivity in the SCN treated with 5-HT_1Breceptor antiserum preabsorbed with the peptide used for raising the antiserum (d). OC, Optic chiasm;III, third ventricle. Scale bars, 100 μm.

Fig. 2.

Electron micrographs of the mouse SCN showing immunoperoxidase-labeled 5-HT_1B receptors in fibers (arrows) in the ventral region of the SCN close to myelinated fibers of the optic chiasm (OC) (a) and a 5-HT_1B-immunopositive axonal profile judged to be optic in origin based on the ultrastructure of the mitochondria (b). Note that the immunoperoxidase reaction product (arrow inb) is associated with the plasma membrane. Scale bars, 0.5 μm.

Fig. 3.

Electron micrographs illustrating immunoperoxidase-labeled 5-HT_1B receptors in nerve terminals in the mouse SCN. a, In a nerve terminal containing numerous electron lucent synaptic vesicles and a few dense core vesicles, the immunoreaction product is located on the plasma membrane. b, Two optic profiles containing large pale mitochondria are shown. One of them (1), presumably a preterminal, is immunopositive for 5-HT_1Breceptor peptide, whereas the other (2), making synaptic contact with a dendrite (D), is unlabeled. Scale bars, 0.5 μm.

Fig. 4.

The effect of 5-HT and TFMPP on optic nerve-evoked EPSCs in the hamster SCN. a, 5-HT reduced the amplitude of optic nerve–evoked EPSCs. The average of 5–10 consecutive responses is shown for each condition. b, Cumulative responses from four neurons are graphed relative to the control response. At a concentration of 100 μm, 5-HT reduced the amplitude of the evoked EPSC by ∼60%. c,Trace 1, Electrical stimulation of the optic nerve resulted in an EPSC of relatively constant latency in this SCN neuron.Trace 2, Bath application of the 5-HT_1Breceptor agonist TFMPP (30 μm) reduced the EPSC amplitude by ∼20%. Trace 3, The EPSC amplitude recovered after an ∼15 min wash to control recording conditions. Trace 4, A higher concentration of TFMPP (100 μm) further reduced the EPSC amplitude. Trace 5, The EPSC amplitude recovered after an ∼20 min wash. The average of five to eight consecutive responses, not including failures, is shown.d, Dose-dependent effect of TFMPP (10–300 μm) on optic nerve-evoked EPSC amplitude is shown. Data represent the mean ± SEM of three to six cells per dose. Thenumber of cells is indicated in parentheses above each concentration.

Fig. 5.

The effect of TFMPP in the presence of 5-HT_1A and 5-HT₇ receptor antagonists. The effect of TFMPP (a 5-HT_1B receptor agonist) on the optic nerve-evoked EPSC was not blocked in the presence of the 5-HT_1A and 5-HT₇ receptor antagonists WAY-100635 and ritanserin, respectively. The average of five to eight responses is shown for each condition.

Fig. 6.

TFMPP reduced the amplitude of evoked EPSCs but had no effect on the inward current evoked by direct application of glutamate. a, TFMPP reversibly reduced the amplitude of the optic nerve-evoked EPSC. The average of eight events is shown.b, In the same neuron, direct application of glutamate (20 mm) to the surface of the slice evoked an inward current that was not reduced by TFMPP. c, The effect of 100 μm TFMPP on the optic nerve-evoked EPSC and on the glutamate-evoked inward current in several neurons is shown. Thenumber of neurons examined is indicatedabove each column set.

Fig. 7.

Location of an SCN neuron that responded to optic nerve stimulation and to TFMPP. a, This SCN neuron was filled with biocytin during a whole-cell patch-clamp recording and visualized with an avidin–rhodamine conjugate. b, The same tissue was labeled for 5-HT immunoreactivity. Immunopositive fibers are present near the position of the recorded neuron (asterisk). c, The same neuron was rereacted with avidin–biotin–horseradish peroxidase complex and visualized with DAB. d, The neuron was reconstructed digitally using Neurolucida (MicroBrightField). Inset, The position of the neuron relative to the SCN borders in thehorizontal plane of view is shown. OC, Optic chiasm.

Fig. 8.

The effect of systemic administration of TFMPP on light-induced phase delays of the circadian rhythm of wheel-running activity in wild-type and 5-HT_1B receptor knock-out (KO) mice is illustrated in representative actograms. Mice were maintained in DD throughout the experiment and received injections of vehicle (top) or TFMPP (25 mg/kg, i.p.) (bottom) at CT 15.5 followed by light exposure (10 min at 20 lux) at CT 16 to elicit phase delays. TFMPP reduced light-induced phase shifts in wild-type mice (left) but had little effect in the 5-HT_1B receptor knock-out mouse (right). The approximate time of light stimulation is indicated by the inverted triangles.

Fig. 9.

Effect of systemic administration of TFMPP on light-induced phase delays of the free-running activity rhythm. Data represent the mean ± SEM of six to seven animals per group (numbers shown on each bar). Light-induced phase shifts were significantly smaller in TFMPP-treated wild-type mice compared with that in vehicle-injected animals (p < 0.001), whereas TFMPP produced no inhibition of light-induced phase shifts in 5-HT_1B receptor knock-out (KO) mice (p > 0.05).