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. 2009 Dec 15;164(3):1210-23.
doi: 10.1016/j.neuroscience.2009.08.075. Epub 2009 Sep 10.

Fractalkine/CX3CL1 enhances GABA synaptic activity at serotonin neurons in the rat dorsal raphe nucleus

S Heinisch  1 , L G Kirby
Affiliations

Fractalkine/CX3CL1 enhances GABA synaptic activity at serotonin neurons in the rat dorsal raphe nucleus

S Heinisch et al. Neuroscience. .

Erratum in

  • Neuroscience. 2010 Feb 17;165(4):1559-60

Abstract

Serotonin (5-hydroxytryptamine; 5-HT) has an important role in mood regulation, and its dysfunction in the central nervous system (CNS) is associated with depression. Reports of mood and immune disorder co-morbidities indicate that immune-5-HT interactions may mediate depression present in immune compromised disease states including HIV/AIDS, multiple sclerosis, and Parkinson's disease. Chemokines, immune proteins that induce chemotaxis and cellular adhesion, and their G-protein coupled receptors distribute throughout the CNS, regulate neuronal patterning, and mediate neuropathology. The purpose of this study is to investigate the neuroanatomical and neurophysiological relationship between the chemokine fractalkine/CX3CL1 and its receptor CX3CR1 with 5-HT neurons in the rat midbrain raphe nuclei (RN). Immunohistochemistry was used to examine the colocalization of CX3CL1 or CX3CR1 with 5-HT in the RN, and whole-cell patch-clamp recordings in rat brain slices were used to determine the functional impact of CX3CL1 on 5-HT dorsal raphe nucleus (DRN) neurons. Greater than 70% of 5-HT neurons colocalize with CX3CL1 and CX3CR1 in the RN. CX3CL1 localizes as discrete puncta throughout the cytoplasm, whereas CX3CR1 concentrates to the perinuclear region of 5-HT neurons and exhibits microglial expression. CX3CL1 and CX3CR1 also colocalize with one another on individual RN cells. Electrophysiology studies indicate a CX3CL1-mediated enhancement of spontaneous inhibitory postsynaptic current (sIPSC) amplitude and dose-dependent increase of evoked IPSC (eIPSC) amplitude without affecting eIPSC paired-pulse ratio, a finding observed selectively in 5-HT neurons. CX3CL1's effect on eIPSC amplitude is blocked by pretreatment with an anti-CX3CL1 neutralizing antibody. Thus, CX3CL1 enhances postsynaptic GABA receptor number or sensitivity on 5-HT DRN neurons under conditions of both spontaneous and synaptically-evoked GABA release. CX3CL1 may indirectly inhibit 5-HT neurotransmission by increasing the sensitivity of 5-HT DRN neurons to GABA inputs. Therapies targeting CX3CL1 may treat serotonin related mood disorders, including depression experienced by patients with compromised immune systems.

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Figures

Figure 1
Figure 1
CX3CL1 or CX3CR1 colocalization with TPH in the DRN. Fluorescent photomicrographs of TPH with CX3CL1 (A–C) or CX3CR1 (D–F) containing cells in the DM (A, D) and VM (B, E) subdivisions of the DRN. The upper left panels show TPH-immunoreactivity in green, the upper right panels depict either CX3CL1- or CX3CR1-immunoreactivity in red, and the large panels show the merge image. Colocalization of CX3CL1 or CX3CR1 in serotonergic neurons is present throughout the DM- and VM-DRN (yellow arrows; A, B, D, E). Individual cells are also labeled only with TPH (green arrows; A, B, D), CX3CL1 (red arrows; B), or CX3CR1 (red arrows; D, E). Panels C and F show magnified images of CX3CL1 or CX3CR1 with TPH in the DRN. CX3CL1 localizes as discrete puncta within the cytoplasm and processes of TPH-positive neurons (white arrows, C merge), whereas CX3CR1 is expressed in a perinuclear pattern in TPH-positive neurons (white arrows, F merge) and other cells (dotted white arrows, F merge). Panel G depicts DRN cells co-expressing CX3CL1 and CX3CR1 (white arrows, merge). Panel H shows CX3CR1 expressing microglia (CD11b in green; white arrows) and neurons (anti-NeuN in blue; dotted white arrows) in DRN sections. Schematics from Paxinos and Watson (2005) are included to indicate the panel locations in the DRN. Scale Bars = 25μm (A, B, D, E); 10 μm (H), 5 μm (C, F, G).
Figure 2
Figure 2
Quantified colocalization of TPH with CX3CL1 or CX3CR1 in the raphe nuclei. Panels A and B show CX3CL1 and CX3CR1 expression in over 70% of serotonergic neurons throughout the DRN subdivisions, DM, LW, VM, and the MRN. Panel C indicates the rostrocaudal level (−8.00) and delineates the raphe subdivisions in which colocalization was quantified in a representative TPH-immunoreactive section of the raphe nuclei. * = p < 0.05 and ** = p < 0.01 by post-hoc Student-Newman-Keuls test. All values are mean ± SEM. Dorsal raphe nucleus (DRN); median raphe nucleus (MRN); dorsomedial (DM); lateral wing (LW); ventromedial (VM).
Figure 3
Figure 3
CX3CL1 stimulates sIPSC amplitude selectively in 5-HT DRN neurons. CX3CL1 (10 nM) enhances baseline sIPSC amplitude from 15.5 to 18.9 pA without affecting sIPSC frequency in a recorded 5-HT neuron, and bicuculline (20 μM) eliminates all sIPSC events (A, B white arrow). CX3CL1 significantly shifts the cumulative histogram for amplitude to the right, but does not impact the inter-event interval (C′ vs. C). Group data demonstrate a CX3CL1 selective increase in sIPSC amplitude (D′) with no change in sIPSC frequency (D) at 5-HT DRN neurons. * = p < 0.05 by paired Student’s t-test, # = p <0.05 by the Kolmogorov-Smirnov two sample test. Group data are represented as mean ± SEM.
Figure 4
Figure 4
CX3CL1 does not affect sIPSC frequency or amplitude in non-5-HT DRN neurons. CX3CL1 (10 nM) fails to modulate sIPSC frequency or amplitude in a recorded non-5-HT neuron, and bicuculline (20 μM) eliminates all sIPSC events (A, B white arrow). CX3CL1 does not impact the cumulative histogram for inter-event interval (C) or amplitude (C′). Group data show no effect of CX3CL1 on either sIPSC frequency (D) or amplitude (D′) parameters in non-5-HT DRN neurons. Group data are represented as mean ± SEM.
Figure 5
Figure 5
CX3CL1 selectively enhances eIPSC amplitude without modulating PPR in 5-HT DRN neurons. Panels A and B are representative traces (averaged from 60 events) showing eIPSC amplitude (A) and PPR (B) chart recordings before and after CX3CL1 (A, B) and bicuculline (A) treatment. CX3CL1 (10 nM) stimulates eIPSC amplitude (A, A′) and does not change baseline PPR (B, B′) in 5-HT neurons, and bicuculline (20 μM) completely eliminates all eIPSC events (A). * = p < 0.05 by paired Student’s t-test. Group data are represented as mean ± SEM.
Figure 6
Figure 6
CX3CL1’s concentration-dependent elevation of eIPSC amplitude with no effect on PPR in 5-HT DRN neurons. CX3CL1 enhances eIPSC amplitude at 10 nM but not at 1.0 nM (A). PPR is not changed by the 10 nM or 1.0 nM concentration of CX3CL1 (B). * = p < 0.05 by unpaired Student’s t-test. All data are represented as mean ± SEM.
Figure 7
Figure 7
Anti-CX3CL1 neutralizing antibody eliminates CX3CL1’s effects on evoked GABA synaptic activity in 5-HT DRN neurons. The increase in eIPSC amplitude produced by CX3CL1 (see Fig. 5) was blocked by CX3CL1-neutralizing antibody (2 μg/ml) pretreatment (A). Statistical analysis was performed by paired Student’s t-test. Group data are represented as mean ± SEM.
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