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GABAergic mechanisms are involved in the control of tuberoinfundibular arcuate neurons by the accessory olfactory bulb
Neuroscience Letters, 120 (1990) 231-233
231
ElsevierScientificPublishers Ireland Ltd. NSL 07363
GABAergic mechanisms are involved in the control of tuberoinfundibular arcuate neurons by the accessory olfactory bulb C.-S. Li, H . K a b a , H . Saito a n d K. Seto Department of Physiology, Kochi Medical School, Kochi (Japan)
(Received 13 July 1990;Revisedxtersionreceived23 August 1990;Accepted27 August 1990) Key words: Accessoryolfactory bulb; Reciprocaldendrodendriticsynapse; ),-Aminobutyricacid; Bicuculline;Tuberoinfundibulararcuate neuron;
Unit recording i
In this study we examined electrophysiologicallythe involvementof the intrinsic GABAergic system of the accessoryolfactory bulb (AOB) in controlling the activity of tuberoinfundibular (TI) arcuate neurons in anaesthetized female mice. Local infusions of the ),-aminobutyricacid-A (GABAA)receptor antagonist, bicucullineinto the AOB enhanced the spontaneous firing activity of TI arcuate neurons with excitatory inputs from the AOB. This finding reveals a neural mechanism responsible for the pregnancy blocking effect of this drug in freely behaving female mice and, taken together with the cytoarchitectureof the AOB, suggests that the reciprocaldendrodendriticinteraction betweenmitral cells and GABAergic granule ceils in the AOB is critical to control of AOB output to TI arcuate neurons as part of the final common pathway of the accessoryolfactory system,
The accessory olfactory system originating in the vomeronasal organ is important in a variety of chemosensory primer effects including the acceleration of puberty, induction of oestrus and pregnancy block in female mice following exposure to male urinary odours (pheromones) [4]. Our electrophysiological studies [5, 6] have demonstrated that the projections of the accessory olfactory bulb (AOB) activate excitatory amino acid receptors within the amygdala and subsequently the stria terminalis route, thereby causing excitation of tuberoinfundibular (TI) dopaminergic arcuate neurons which are known to regulate prolactin secretion from the anterior pituitary. The basic synaptic organization in the AOB is the same as in the main olfactory bulb [1, 7, 10], where the mitral/tufted cell relay neurons form reciprocal dendrodendritic synapses with the intrinsic granule cells; the granule cells release inhibitory 7-aminobutyric acid (GABA) onto the mitral/tufted cell secondary dendrites at these synapses, whereas the mitral/tufted cells are excitatory to the granule cells [11]; the excitatory neurotransmitter is thought to be glutamate or a closely related substance [2]. One important difference between the two systems is that the reciprocal dendrodendritic synapses between AOB mitral and granule cells occur on the same dendrites as those receiving vomeronasal inputs Correspondence: H. Kaba, Department of Physiology,Kochi Medical School, Nankoku, Kochi 783, Japan.
0304-3940/90/$03.50 © 1990ElsevierScientificPublishers Ireland Ltd.
[1]. In the main olfactory bulb, there is strong evidence that the GABAergic granule cells act to control mitral/ tufted cell excitability [8, 9, 11]. The present study examines the effect of GABA transmitter blockade in the AOB on firing activity of TI arcuate neurons as part of the final common pathway of the accessory olfactory system. Eight Balb/c female mice, purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals (Hamamatsu, Japan) and reared as previously described [5], were used for the experiments. These animals were ovariectomized for at least four weeks prior to being implanted subcutaneously with silastic capsules containing 0.5/tg oestradiol. Unit recordings were made 5-14 days after capsule implantation, which has been shown to increase the percentage of TI arcuate neurons responding to AOB stimulation [5]. Anaesthesia, stimulation, recording, microinfusion and histological techniques have been previously described [5]. Briefly, animals were anaesthetized with chloral hydrate (400 mg/kg) and mounted in a stereotaxic instrument. A coaxial bipolar stimulating electrode was lowered into the left AOB (coordinates: 0 mm anterior to the rhinal fissure, 1.0 mm lateral to the midline, 1.3 mm ventral to the brain surface). A glass micropipette (tip diameter 30-50/~m) was then placed just anterior to the stimulating electrode. They were together held in place with dental cement and a stainless steel screw. Single unit extracellular recordings were made with glass
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Fig. 1. Effect of intra-accessory olfactory bulb (AOB) infusions of bicuculline (B) or saline (C) on firing activity of tuberoinfundibular (TI) arcuate neurons with excitatory inputs from the AOB. A: superimposed oscilloscope traces from the cell shown in B illustrate fulfillment of criteria for antidromic activation from the median eminence. Responses occur at a constant latency to the antidromic stimulus (arrow, top), follow closely paired stimulation pulses (middle), and are cancelled by collision with spontaneously generated potentials (star, bottom). Upper panels in B and C: peri-stimulus-time histograms are presented showing excitatory responses of TI arcuate neurons to electrical stimulation of the AOB. Arrows indicate time of stimulation. Lower panels in B and C: ratemeter records showing the effect of intra-AOB infusions of bicuculline (0.1 nmol in 0.25 ~tl) or saline on the same neurons from which peri-stimulus-time histograms were obtained. Arrows indicate time of infusion. Bicuculline, but not saline, caused an increase in spontaneous firing rate.
micropipettes filled with 0.5 M sodium acetate containing Pontamine Sky Blue dye. The left arcuate nucleus of the hypothalamus was systematically explored for the presence of cells which were stimulated antidromically from the median eminence and orthodromically from the AOB. Balanced negative-positive biphasic rectangular pulses (duration of each pulse = 0.2 ms, separation of rectangular pulses within each biphasic pair = 0.1 ms, intensity = 0.5 mA) were delivered to the AOB at 1/3 Hz. Bicuculline methiodide (Sigma), a GABAA receptor antagonist, was dissolved in saline and 0.1 nmol in 0.25 /tl was infused into the AOB from the glass micropipette. The effect of local infusions of bicuculline or saline into the AOB was tested on histologically verified arcuate neurons which were antidromically stimulated from the median eminence and also orthodromically sti-
Fig. 2. Average effect of bicuculline (solid circles) or saline (open circles) on spontaneous firing rate of TI arcuate neurons with excitatory inputs from the AOB as a function of time before or after intra-AOB infusions (arrow). Rates are expressed as a percentage of the mean preinfusion rate. Each point is the mean of five cells for bicuculline and three cells for saline; vertical lines indicate _+ S.D. Statistical significance was determined by Student's t-test; * P < 0 . 0 5 , **P<0.01, * * * P < 0.001, two-tailed test.
mulated from the AOB. Bicuculline (0.1 nmol) infused into the AOB increased spontaneous firing rates in all five T I neurons tested. An example of this effect is shown in Fig. I B (lower). Significant effect produced by this dose of bicuculline occurred immediately after the infusion and lasted for 14 min (Fig. 2). The mean maximum effect was 172 + 32% (S.D.) increase in spontaneous rate. Saline infused in the same manner was without effect (n = 3 cells, Fig. 1C, lower and Fig. 2). In freely behaving female mice, local infusions of bicuculline into the AOB following mating have been shown to cause a direct block to pregnancy [3]. This could be explained by the effect of the bicuculline antagonizing the GABA-mediated feedback inhibition on the mitral cells of the AOB, resulting in an over-excitation of the accessory olfactory system. This over-excitation would activate TI dopaminergic neurons as a final common pathway of the accessory olfactory system by way of the amygdala [5, 6], causing a return to oestrus. The present results provide direct support for this explanation and further suggest that GABAergic inhibitory feedback to the mitral cell of the AOB is crucial to close control of mitral cell output to TI arcuate neurons. C.-S.L. was in receipt of a Scholarship from the Ministry of Education, Science and Culture of Japan. The work was supported in part by a Grant-in-Aid for Scientific Research (No. 01570087) to H.K. from the Ministry.
233 I Barber, P.C., Parry, D.M., Field, P.M. and Raisman, G., Electron microscope autoradiographic evidence for specific transneuronal transport in the mouse accessory olfactory bulb, Brain Res., 152 (1978) 283-302. 2 Hal~,sz, N. and Shepherd, G.M., Neurochemistry of the vertebrate olfactory bulb, Neuroscience, 10 (1983) 579~19. 3 Kaba, H. and Keverne, E.B., The effect of microinfusions of drugs into the accessory olfactory bulb on the olfactory block to pregnancy, Neuroscience, 25 (1988) 1007-101 I. 4 Keverne, E.B., Pheromonal influences on the endocrine regulation of reproduction, Trends Neurosci., 6 (1983) 381-384. 5 Li, C.-S., Kaba, H., Saito, H. and Seto, K., Excitatory influence of the accessory olfactory bulb on tuberoinfundibular arcuate neurons of female mice and its modulation by oestrogen, Neuroscience, 29 (1989) 201-208. 6 Li, C.-S., Kaba, H., Saito, H. and Seto, K., Neural mechanisms underlying the action of primer pheromones in mice, Neuroscience, 36 (1990) 773-778.
7 Mori, K., Membrane and synaptic properties of identified neurons in the olfactory bulb, Prog. Neurobiol., 29 (1987) 275-320. 8 Mori, K. and Takagi, S., An intracellular study of dendrodendritic inhibitory synapses on mitral cells in the rabbit olfactory bulb, J. Physiol., 279 (1978) 569-588. 9 Nicoll, R.A., Pharmacological evidence for GABA as the transmitter in granule cell inhibition in the olfactory bulb, Brain Res., 35 (1971) 137-149. 10 Reinhardt, W., MacLeod, N.K., Ladewig, J. and Ellendorff, F., An electrophysiological study of the accessory olfactory bulb in the rabbit-II. Input-output relations as assessed from analysis of intraand extracellular unit recordings, Neuroscience, 10 (1983) 131- 139. 11 Shepherd, G.M., Synaptic organization of the mammalian olfactory bulb, Physiol. Rev., 52 (1972) 864-917.
231
ElsevierScientificPublishers Ireland Ltd. NSL 07363
GABAergic mechanisms are involved in the control of tuberoinfundibular arcuate neurons by the accessory olfactory bulb C.-S. Li, H . K a b a , H . Saito a n d K. Seto Department of Physiology, Kochi Medical School, Kochi (Japan)
(Received 13 July 1990;Revisedxtersionreceived23 August 1990;Accepted27 August 1990) Key words: Accessoryolfactory bulb; Reciprocaldendrodendriticsynapse; ),-Aminobutyricacid; Bicuculline;Tuberoinfundibulararcuate neuron;
Unit recording i
In this study we examined electrophysiologicallythe involvementof the intrinsic GABAergic system of the accessoryolfactory bulb (AOB) in controlling the activity of tuberoinfundibular (TI) arcuate neurons in anaesthetized female mice. Local infusions of the ),-aminobutyricacid-A (GABAA)receptor antagonist, bicucullineinto the AOB enhanced the spontaneous firing activity of TI arcuate neurons with excitatory inputs from the AOB. This finding reveals a neural mechanism responsible for the pregnancy blocking effect of this drug in freely behaving female mice and, taken together with the cytoarchitectureof the AOB, suggests that the reciprocaldendrodendriticinteraction betweenmitral cells and GABAergic granule ceils in the AOB is critical to control of AOB output to TI arcuate neurons as part of the final common pathway of the accessoryolfactory system,
The accessory olfactory system originating in the vomeronasal organ is important in a variety of chemosensory primer effects including the acceleration of puberty, induction of oestrus and pregnancy block in female mice following exposure to male urinary odours (pheromones) [4]. Our electrophysiological studies [5, 6] have demonstrated that the projections of the accessory olfactory bulb (AOB) activate excitatory amino acid receptors within the amygdala and subsequently the stria terminalis route, thereby causing excitation of tuberoinfundibular (TI) dopaminergic arcuate neurons which are known to regulate prolactin secretion from the anterior pituitary. The basic synaptic organization in the AOB is the same as in the main olfactory bulb [1, 7, 10], where the mitral/tufted cell relay neurons form reciprocal dendrodendritic synapses with the intrinsic granule cells; the granule cells release inhibitory 7-aminobutyric acid (GABA) onto the mitral/tufted cell secondary dendrites at these synapses, whereas the mitral/tufted cells are excitatory to the granule cells [11]; the excitatory neurotransmitter is thought to be glutamate or a closely related substance [2]. One important difference between the two systems is that the reciprocal dendrodendritic synapses between AOB mitral and granule cells occur on the same dendrites as those receiving vomeronasal inputs Correspondence: H. Kaba, Department of Physiology,Kochi Medical School, Nankoku, Kochi 783, Japan.
0304-3940/90/$03.50 © 1990ElsevierScientificPublishers Ireland Ltd.
[1]. In the main olfactory bulb, there is strong evidence that the GABAergic granule cells act to control mitral/ tufted cell excitability [8, 9, 11]. The present study examines the effect of GABA transmitter blockade in the AOB on firing activity of TI arcuate neurons as part of the final common pathway of the accessory olfactory system. Eight Balb/c female mice, purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals (Hamamatsu, Japan) and reared as previously described [5], were used for the experiments. These animals were ovariectomized for at least four weeks prior to being implanted subcutaneously with silastic capsules containing 0.5/tg oestradiol. Unit recordings were made 5-14 days after capsule implantation, which has been shown to increase the percentage of TI arcuate neurons responding to AOB stimulation [5]. Anaesthesia, stimulation, recording, microinfusion and histological techniques have been previously described [5]. Briefly, animals were anaesthetized with chloral hydrate (400 mg/kg) and mounted in a stereotaxic instrument. A coaxial bipolar stimulating electrode was lowered into the left AOB (coordinates: 0 mm anterior to the rhinal fissure, 1.0 mm lateral to the midline, 1.3 mm ventral to the brain surface). A glass micropipette (tip diameter 30-50/~m) was then placed just anterior to the stimulating electrode. They were together held in place with dental cement and a stainless steel screw. Single unit extracellular recordings were made with glass
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Fig. 1. Effect of intra-accessory olfactory bulb (AOB) infusions of bicuculline (B) or saline (C) on firing activity of tuberoinfundibular (TI) arcuate neurons with excitatory inputs from the AOB. A: superimposed oscilloscope traces from the cell shown in B illustrate fulfillment of criteria for antidromic activation from the median eminence. Responses occur at a constant latency to the antidromic stimulus (arrow, top), follow closely paired stimulation pulses (middle), and are cancelled by collision with spontaneously generated potentials (star, bottom). Upper panels in B and C: peri-stimulus-time histograms are presented showing excitatory responses of TI arcuate neurons to electrical stimulation of the AOB. Arrows indicate time of stimulation. Lower panels in B and C: ratemeter records showing the effect of intra-AOB infusions of bicuculline (0.1 nmol in 0.25 ~tl) or saline on the same neurons from which peri-stimulus-time histograms were obtained. Arrows indicate time of infusion. Bicuculline, but not saline, caused an increase in spontaneous firing rate.
micropipettes filled with 0.5 M sodium acetate containing Pontamine Sky Blue dye. The left arcuate nucleus of the hypothalamus was systematically explored for the presence of cells which were stimulated antidromically from the median eminence and orthodromically from the AOB. Balanced negative-positive biphasic rectangular pulses (duration of each pulse = 0.2 ms, separation of rectangular pulses within each biphasic pair = 0.1 ms, intensity = 0.5 mA) were delivered to the AOB at 1/3 Hz. Bicuculline methiodide (Sigma), a GABAA receptor antagonist, was dissolved in saline and 0.1 nmol in 0.25 /tl was infused into the AOB from the glass micropipette. The effect of local infusions of bicuculline or saline into the AOB was tested on histologically verified arcuate neurons which were antidromically stimulated from the median eminence and also orthodromically sti-
Fig. 2. Average effect of bicuculline (solid circles) or saline (open circles) on spontaneous firing rate of TI arcuate neurons with excitatory inputs from the AOB as a function of time before or after intra-AOB infusions (arrow). Rates are expressed as a percentage of the mean preinfusion rate. Each point is the mean of five cells for bicuculline and three cells for saline; vertical lines indicate _+ S.D. Statistical significance was determined by Student's t-test; * P < 0 . 0 5 , **P<0.01, * * * P < 0.001, two-tailed test.
mulated from the AOB. Bicuculline (0.1 nmol) infused into the AOB increased spontaneous firing rates in all five T I neurons tested. An example of this effect is shown in Fig. I B (lower). Significant effect produced by this dose of bicuculline occurred immediately after the infusion and lasted for 14 min (Fig. 2). The mean maximum effect was 172 + 32% (S.D.) increase in spontaneous rate. Saline infused in the same manner was without effect (n = 3 cells, Fig. 1C, lower and Fig. 2). In freely behaving female mice, local infusions of bicuculline into the AOB following mating have been shown to cause a direct block to pregnancy [3]. This could be explained by the effect of the bicuculline antagonizing the GABA-mediated feedback inhibition on the mitral cells of the AOB, resulting in an over-excitation of the accessory olfactory system. This over-excitation would activate TI dopaminergic neurons as a final common pathway of the accessory olfactory system by way of the amygdala [5, 6], causing a return to oestrus. The present results provide direct support for this explanation and further suggest that GABAergic inhibitory feedback to the mitral cell of the AOB is crucial to close control of mitral cell output to TI arcuate neurons. C.-S.L. was in receipt of a Scholarship from the Ministry of Education, Science and Culture of Japan. The work was supported in part by a Grant-in-Aid for Scientific Research (No. 01570087) to H.K. from the Ministry.
233 I Barber, P.C., Parry, D.M., Field, P.M. and Raisman, G., Electron microscope autoradiographic evidence for specific transneuronal transport in the mouse accessory olfactory bulb, Brain Res., 152 (1978) 283-302. 2 Hal~,sz, N. and Shepherd, G.M., Neurochemistry of the vertebrate olfactory bulb, Neuroscience, 10 (1983) 579~19. 3 Kaba, H. and Keverne, E.B., The effect of microinfusions of drugs into the accessory olfactory bulb on the olfactory block to pregnancy, Neuroscience, 25 (1988) 1007-101 I. 4 Keverne, E.B., Pheromonal influences on the endocrine regulation of reproduction, Trends Neurosci., 6 (1983) 381-384. 5 Li, C.-S., Kaba, H., Saito, H. and Seto, K., Excitatory influence of the accessory olfactory bulb on tuberoinfundibular arcuate neurons of female mice and its modulation by oestrogen, Neuroscience, 29 (1989) 201-208. 6 Li, C.-S., Kaba, H., Saito, H. and Seto, K., Neural mechanisms underlying the action of primer pheromones in mice, Neuroscience, 36 (1990) 773-778.
7 Mori, K., Membrane and synaptic properties of identified neurons in the olfactory bulb, Prog. Neurobiol., 29 (1987) 275-320. 8 Mori, K. and Takagi, S., An intracellular study of dendrodendritic inhibitory synapses on mitral cells in the rabbit olfactory bulb, J. Physiol., 279 (1978) 569-588. 9 Nicoll, R.A., Pharmacological evidence for GABA as the transmitter in granule cell inhibition in the olfactory bulb, Brain Res., 35 (1971) 137-149. 10 Reinhardt, W., MacLeod, N.K., Ladewig, J. and Ellendorff, F., An electrophysiological study of the accessory olfactory bulb in the rabbit-II. Input-output relations as assessed from analysis of intraand extracellular unit recordings, Neuroscience, 10 (1983) 131- 139. 11 Shepherd, G.M., Synaptic organization of the mammalian olfactory bulb, Physiol. Rev., 52 (1972) 864-917.