Activation of 5HT1B receptors inhibits glycinergic synaptic inputs to mammalian motoneurons during postnatal development

Brain Research 956 (2002) 380–384 www.elsevier.com / locate / brainres

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Activation of 5HT 1B receptors inhibits glycinergic synaptic inputs to mammalian motoneurons during postnatal development Albert J. Berger*, Phan Huynh Department of Physiology and Biophysics, School of Medicine, University of Washington, Box 357290, Seattle, WA 98195 -7290, USA Accepted 8 August 2002

Abstract We investigated whether 5-HT 1B receptor-mediated inhibition of evoked glycinergic inhibitory postsynaptic currents (eIPSCs) in hypoglossal motoneurons (HMs) changed postnatally. In HMs from postnatal days 2–3 (P2–3, neonate) and P10–11 (juvenile) rats bath application of 5-HT (10 mM) caused a not significantly different large reduction in eIPSC amplitude to 35.0622.5% (mean6S.D.) and 35.4610.6% of control; respectively. The dose–response relationship for the 5-HT 1B receptor agonist, CP-93,129, revealed that the mean agonist concentration at half-maximal inhibition (IC 50 ) was similar, 1.6 and 2.0 nM, respectively. Additionally, strong antibody labeling of 5-HT 1B receptors in the hypoglossal motor nucleus was observed in neonates, juveniles and adults. These results demonstrate that over the postnatal period studied, 5-HT 1B receptor-mediated inhibition of glycinergic eIPSCs is not age dependent.  2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Serotonin Keywords: Synaptic transmission; Modulation; Presynaptic; Immunohistochemistry

Serotonin (5-HT) has been shown to presynaptically modulate fast ligand-gated synaptic inputs to motoneurons [9,11]. Dorsal-root evoked glutamatergic excitatory postsynaptic potentials (EPSPs) and glycinergic inhibitory postsynaptic potentials (IPSPs) in rat spinal motoneurons were both reduced by application of 5-HT [11]. Previously our laboratory has shown that glycinergic synaptic inputs to hypoglossal motoneurons (HMs) are inhibited by activation of presynaptic 5-HT 1B receptors [9]. The significance of 5-HT dependent modulation of synaptic inputs to motoneurons may be related to the role of 5-HT in physiologic functions including sleep and motor activity [10]. Serotonin is widely released from synaptic terminals arising from raphe neurons, the activity of raphe neurons is known to be dependent on the sleep– wake cycle [10], being highest in wakefulness and lowest in rapid eye movement (REM) sleep. Related to this is the finding that during carbachol-induced REM sleep there is a *Corresponding author. Tel.: 11-206-543-8196; fax: 11-206-6850619. E-mail address: [email protected] (A.J. Berger).

marked reduction in the concentration of 5-HT in the hypoglossal nucleus [3]. There exists little information on how the 5-HT dependent modulation of fast ligand-gated synaptic transmission may change during postnatal development. In the single study in which this issue was considered, 5-HT 1B receptormediated inhibition of thalamocortical EPSPs was dependent on postnatal developmental age [6]. Thus, in the present study we investigated whether 5-HT 1B receptormediated presynaptic inhibition of glycinergic synaptic inputs to HMs is dependent on postnatal age. We used whole-cell recording to measure electrically evoked glycinergic inhibitory postsynaptic currents (eIPSCs) in HMs. Two different age groups of rats were studied, neonate postnatal days 2–3 (P2–3) and juvenile (P10–11). A previous study of the rat visual cortex showed that over this age range the density of 5-HT 1B receptor binding sites significantly changed, it either increased or decreased and the change was dependent upon the precise cortex layer studied (i.e. superior, medial or inferior layers) [7]. Brainstem slices (200–300 mm thick) were prepared from rats anesthetized with halothane.

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03464-9

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During slicing, tissue was maintained in an ice-cold Ringer’s solution composed of (in mM): 120 NaCl, 2.5 KCl, 1 NaH 2 PO 4 , 26.2 NaHCO 3 , 25 glucose, 2.5 CaCl 2 and 1.0 MgSO 4 bubbled continuously with 95% O 2 and 5% CO 2 . Slices were incubated for 1 h at 37 8C and then stored and subsequently studied at room temperature in Ringer’s solution. Slices were viewed through a 403 water immersion objective utilizing infra-red differential interference contrast optics on a fixed-staged microscope (Zeiss), and HMs were identified as previously described [9]. Whole-cell recordings were made using glass pipettes containing (in mM): 140 CsCl, 10 NaCl, 10 HEPES, 10 ethylene glycolbis (b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid (EGTA), 4 ATP-Mg and 2 lidocaine N-ethyl bromide (QX-314), and the pH was adjusted to 7.30. HMs were voltage-clamped at 270 mV and series resistances during whole-cell recording were ,20 MV and were not compensated. To generate glycinergic eIPSCs in HMs we electrically stimulated afferent fibers lateral to the hypoglossal nucleus. To isolate glycinergic eIPSCs we added 6,7-dinitroquinoxaline (DNQX, 10 mM, Sigma), D(2)-2-amino-5phosphono-pentanoic acid (APV, 50 mM, Tocris) and bicuculline methiodide (5 mM, Sigma) to the Ringer’s

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solution to block AMPA, NMDA and GABAA receptormediated currents, respectively. At the end of each wholecell recording we added strychnine hydrochloride (2 mM, Sigma) to block the glycinergic eIPSCs. Following stable recording of isolated glycinergic eIPSCs in control conditions the slice was bathed for 7 min in either 5-HT (10 mM) or various concentrations of the selective 5-HT 1B agonist, CP-93,129. Following acquisition of these data, we bath applied strychnine to block glycine-receptor-mediated eIPSCs. In each condition 30 stimuli were applied and responses recorded and averaged using pClamp8 (Axon Instruments, Union City, CA, USA). For each average response the peak amplitude was measured after subtracting the residual average amplitude observed in strychnine. Only one HM from each slice was studied. Application of 5-HT (10 mM) resulted in a marked reduction in glycinergic eIPSCs (Fig. 1). Specifically, in nine neonate HMs the average eIPSC peak amplitude in the presence of 5-HT was 35.0622.5% (mean6S.D., range 6.2–64.8%) of control, while in eight juvenile HMs in of 5-HT it was 35.4610.6% (range 16.6–50.8%) of control (Fig. 1B); these mean values were not significantly different (P.0.05). As our previous experiments [9] showed that 5-HT

Fig. 1. Inhibition of glycinergic eIPSCs by 5-HT at two different postnatal ages. (A) Application of 5-HT (10 mM) causes a similar marked reduction in the eIPSC in both neonate (P2, lefthand traces) and juvenile (P11, righthand traces) HMs. Subsequent application of strychnine (2 mM) abolished the remaining eIPSC. All traces are the average response to 30 stimuli (stimuli applied at the downward arrow). (B) Average peak amplitude of eIPSCs in response application of 5-HT (10 mM) as a percentage of control response in a population of neonate (P2–3, n59) and juvenile (P10–11, n58) HMs. Data are calculated from the mean peak amplitude of 30 eIPSCs in control and during application of 5-HT. Bars show mean6S.D.

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dependent modulation of glycinergic synaptic transmission was dependent on activation of 5-HT 1B receptors, in the next series of experiments we utilized CP-93,129 to activate these receptors. We tested whether there was a shift in the dose–response relationship of the 5-HT 1B modulation of eIPSCs between neonatal and juvenile HMs. In a series of 4 HMs from each of the age groups, we found that the dose–response relationships were not different (Fig. 2). Specifically, based upon a logistic equation fit of the dose–response data, we found that IC 50 values were 1.6 and 2.0 nM in neonate and juvenile HMs, respectively. Further, the glycinergic eIPSCs were maximally inhibited by 82% in neonates compared to 78% in juveniles. In summary, these experiments revealed no developmentally correlated change in the degree to which activation of presynaptic 5-HT 1B receptors inhibits glycinergic synaptic transmission. We next studied whether expression of 5-HT 1B receptors in the hypoglossal nucleus changes with postnatal development. To do this we performed 5-HT 1B receptor antibody labeling in rats from three age groups: neonate (P2–3, n52 rats), juvenile (P13–16, n53 rats) and adult (2–3 months of age; 200–300 g, n56 rats). Rats were initially anesthetized with halothane in the case of neonates, and with sodium pentobarbital (i.p. 375 mg / kg) in the case of juveniles and adults. In the latter groups the animals were perfused transcardially with 4% paraformaldehyde. The whole brain was immediately dissected, removed, and post-fixed in 4% paraformaldehyde. In anesthetized neonates their brains were dissected, removed, and put into 4% paraformaldehyde overnight at 4 8C. For all ages, brains were transferred to a 30% sucrose / phosphate-buffered saline solution and then coronally sectioned (30 mm thick) on a freezing microtome. Free floating sections were placed in blocker solution for

1 h and then transferred to wells containing primary antibody (guinea pig anti-5HT 1B receptor polyclonal antibody, 1:4000; BD Pharmingen, San Diego, CA, USA) and incubated overnight. The sections were then incubated in secondary antibody (Alexa Fluorophore 488, 1:200; Molecular Probes, Eugene, OR, USA) for 1 h. Single-labeled sections were examined on a confocal that used 488 nm laser line for excitation of the Alexa Fluorphore 488. Digital images were analyzed using Adobe Photoshop 5.5 (Adobe, San Jose, CA) software. Positive control experiments using immunostaining of the substantia nigra compacta and reticulata (SNc / SNr) regions were carried out on adult rat coronal brain sections (n52 rats, data not shown), a region known to contain 5-HT 1B receptors [8]. We observed strong labeling for 5-HT 1B receptors in the SNc / SNr (data not shown). Negative control experiments were carried out by omitting the primary antibody step and following other procedural steps. For all age groups we found that within the hypoglossal motor nucleus strong antibody labeling of 5-HT 1B receptors occurred (Fig. 3). These anatomical results are in concordance with our electrophysiological and pharmacological studies. 5-HT 1B receptor labeling occurred both diffusely in the neuropil of the hypoglossal nucleus and within the cytoplasm of the large cells (presumably HMs). Although we do not know the function of the cytoplasmic 5-HT 1B receptor labeling, previous studies have demonstrated 5-HT 1B receptor protein and mRNA within the cytoplasm of neurons [1,2,5]. This study has demonstrated that glycinergic eIPSCs are inhibited by activation of 5-HT 1B receptors. Over the postnatal age range studied we did not observe any significant difference in the degree of eIPSC inhibition. This was true both at a fixed dose (10 mM) of 5-HT and

Fig. 2. The degree of inhibition of eIPSCs with application of the 5-HT 1B receptor agonist, CP-93,129, are dose-dependent and similar in neonate (P2, lefthand dose–response) and juvenile (P10, righthand dose–response) HMs. The mean antagonist concentration at half-maximal inhibition (IC 50 ) was similar (1.6 and 2.0 nM, respectively) as determined by nonlinear fitting of the data with a logistic function. In addition, the maximal inhibition was also similar (0.82 and 0.78, respectively). Data points are mean6S.D. from four HMs at each age.

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Fig. 3. Antibody labeling of the hypoglossal motor nucleus (XII) for serotonin 5-HT 1B receptor in neonate (P3, top row), juvenile (P16, middle row) and adult (|P60, bottom row) rats. Top row: In the neonate, strong 5-HT 1B receptor labeling was seen in XII. Staining was strongest over the cell bodies and diffuse throughout the remainder of the nucleus. Middle row: In the juvenile, as in the neonate, there was strong receptor labeling particularly over cell bodies in XII and diffuse elsewhere. Bottom row: In adult rat, strong labeling again was present in XII, particularly over cell bodies in XII and diffuse elsewhere. A higher magnification (right column) view of XII for all three ages shows labeling over the somas and dendrites. XII: Hypoglossal motor nucleus.

for the dose–response relationship using the selective 5HT 1B receptor agonist CP-93,129. Our results in adult rats, showing robust antibody labeling of 5-HT 1B receptors within the hypoglossal motor nucleus, are consistent with the previously published results of Manaker and Verderame [4] who used radioactively labeled cyanopindolol to bind to 5-HT 1B receptors. Our results extend this prior study to suggest that 5-HT 1B

receptors within the hypoglossal motor nucleus are similarly present at all age groups studied.

Acknowledgements This research was supported by research grant NIH-HL49657 to A.J. Berger. We thank Erika Eggers for her

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helpful comments on the manuscript, and Dr. Jennifer O’Brien for her help with the figures.

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