Corticotropin-releasing factor and Urocortin I modulate excitatory glutamatergic synaptic transmission

Abstract

Corticotropin-releasing factor (CRF)-related peptides serve as hormones and neuromodulators of the stress response and play a role in affective disorders. These peptides are known to alter complex behaviors and neuronal properties, but their receptor-mediated effects at CNS synapses are not well described. Here we show that excitatory glutamatergic transmission is modulated by two endogenous CRF-related peptide ligands, corticotropin-releasing factor [CRF rat/human (r/h)] and Urocortin I (Ucn I), within the central nucleus of the amygdala (CeA) and the lateral septum mediolateral nucleus (LSMLN). These limbic nuclei are reciprocally innervated, are involved in stress and affective disorders, and have high densities of the CRF receptors CRF1 and CRF2. Activation of these receptors exerts diametrically opposed actions on glutamatergic transmission in these nuclei. In the CeA, CRF(r/h) depressed excitatory glutamatergic transmission through a CRF1-mediated postsynaptic action, whereas Ucn I facilitated synaptic responses through presynaptic and postsynaptic CRF2-mediated mechanisms. Conversely, in the LSMLN, CRF caused a CRF1-mediated facilitation of glutamatergic transmission via postsynaptic mechanisms, whereas Ucn I depressed EPSCs by postsynaptic and presynaptic CRF2-mediated actions. Furthermore, antagonists of these receptors also affected glutamatergic neurotransmission, indicating that endogenous ligands tonically modulated synoptic activity at these synapses. These data show that CRF receptors in CeA and LSMLN synapses exert and maintain a significant synaptic tone and thereby regulate excitatory glutamatergic transmission. The results also suggest that CRF receptors may provide novel targets in affective disorders and stress.

Figure 1.

Illustration of brain slices containing the amygdala (left) and septum (right) showing recording and stimulationsites. Slices (30 μm) were stained with cresyl violet and with drawing applied to denote positioning of stimulating and recording electrodes. Single recording electrode is shown within the CeA, with stimulating electrode 1 positioned to activate the VAP-CeA pathway and electrode 2 positioned to activate the BLA-CeA pathway. CeA, Central amygdala nucleus; LA, lateral amygdala nucleus; BLA, basolateral anterior amygdala nucleus; VAP, ventral amygdala pathway; BLP, basolateral posterior amygdala nucleus; ec, external capsule; ic, internal capsule; opt, optic tract. Single stimulating electrode is shown within the ventral LSMLN with the recording electrode positioned in the LSMLN. LSMLN, Lateral septum mediolateral nucleus; MS, medial septum; DLSN, dorsolateral septal nucleus.

Figure 2.

Pharmacological antagonists identified glutamate as the mediator of excitatory transmission within the CeA and LSMLN. Left, EPSC evoked by stimulation of the VAP-CeA pathway (comparable responses obtained at BLA-CeA pathway not depicted); right, LSMLN. (1) Evoked postsynaptic currents in control ACSF solution. (2) EPSC isolated initially from fast and slow inhibitory postsynaptic currents with picrotoxin (PTX), bicuculline methiodide (BIM), and CGP55845 (CGP).(3) The remaining glutamatergic EPSC is then blocked with the addition of DNQX and d-APV.

Figure 3.

CRF(r/h) and Ucn I affect excitatory glutamatergic transmission in opposite directions in the CeA and LSMLN. A, B, Concentration-response curves for depression or facilitation of EPSCs by CRF or Ucn I at two CeA pathways. C, D, Concentration-response curves show opposite effects of CRF and Ucn I on EPSCs in LSMLN. Each point represents the mean ± SEM. Traces in A-D depict EPSCs in control ACSF with GABA receptor antagonists (left), in the presence of CRF(r/h) or Ucn I (middle), respectively, and after a 30 min wash (right). Calibration: 50 pA, 20 msec.

Figure 4.

Different effects of CRF receptor nonpeptide (NBI 27914, antalarmin) and peptide (astressin, α-helical-CRF_(9-41), and astressin₂-B) antagonists observed at two CeA excitatory glutamatergic synapses. A, At VAP- and BLA-CeA synapses, NBI 27914 (100 nm) and antalarmin (100 nm) resulted in facilitation of EPSCs. B, Astressin (100 nm) and α-helical CRF_(9-41) (1 μm) depressed EPSC amplitude. C, Astressin₂-B (100 nm), a selective CRF₂ antagonist, depressed EPSCs more effectively in the VAP-compared with the BLA-CeA synapse. Note also a greater degree of depression in the VAP-CeA compared with the BLA-CeA (A-C). D, Ucn II (100 nm), a selective CRF₂ agonist, facilitated VAP-CeA, whereas it depressed BLA-CeA EPSCs. E, After 10 min pretreatment with NBI 27914 (100 nm) and in its continued presence, NBI 27914 blocked the depressant action of CRF(r/h) (50 nm) at both synapses but did not affect facilitation by Ucn I at either synapse. Similar treatment with astressin₂-B (100 nm) blocked the facilitation by Ucn II (100 nm) at the VAP-CeA and also blocked Ucn II (100 nm) depression of EPSCs at the BLA-CeA synapse.

Figure 5.

CRF₁ and CRF₂ receptor activation tonically regulate EPSC amplitude in the LSMLN. A, Top, CRF(r/h, 50 nm) facilitated whereas Ucn I (100 nm) depressed LSMLN EPSCs. Middle, NBI 27914 (100 nm) resulted in depression of baseline EPSCs and prevented a 50 nm CRF-induced facilitation. Bottom, NBI 27914 (100 nm) resulted in depression of EPSC and further added to the Ucn I (100 nm)-induced depression. B, Selective CRF₁ agonist, stressin₁ (100 nm), similar to CRF(r/h) (50 nm) (A, top left), facilitated EPSC. C, Top, Ast₂-B(100 nm) resulted in facilitated EPSC, whereas in its presence CRF(r/h) (50 nm) further enhanced the EPSC. Bottom, Ast₂-B(100nm) resulted in EPSC facilitation, whereas in its presence Ucn I (100 nm) now facilitated, rather than depressed, the EPSC. D, Graphic summary of effects of NBI (100 nm), CRF(r/h) (50 nm), and Ucn I (100 nm) on LSMLN EPSCs, and NBI (100 nm) block of CRF(r/h) (50 nm) facilitation, but not Ucn I (100 nm) depression. E, Graphic summary of EPSC facilitation by stressin₁ (100 nm; CRF₁ agonist) and Ast₂-B (100 nm; CRF₂ antagonist). Ast₂-B (100 nm) did not block facilitation by CRF(r/h) (50 nm), whereas Ast₂-B reversed Ucn I (50 nm) depression to facilitation.

Figure 6.

CRF(r/h) and Ucn I affected mEPSCs differently in the CeA. A, Typical traces of mEPSCs before and after CRF(r/h) (50 nm). B, Graph of cumulative fraction of mEPSCs from single cell depicted in A and plotted as a function of amplitude (p < 0.005; maximum amplitude difference was 0.11 by K-S test). Interevent interval (p < 0.005; maximum difference was 0.11 by K-S test). C, Typical traces of mEPSCs before and after UcnI (200 nm). D, Graph of cumulative fraction of mEPSCs from single cell depicted in C as a function of amplitude (p<0.0001; maximum amplitude difference was 0.11 by K-S test). Interevent interval (p < 0.0001; maximum interevent interval difference was 0.36 by K-S test). Insets depict average changes increase from control at 1.0 (n = 6).

Figure 8.

Diagrams depicting presynaptic and postsynaptic distribution and function of CRF₁ and CRF₂ within two limbic pathways. Tonic and phasic activation of CRF₁ or CRF₂ receptors regulates excitatory glutamatergic transmission positively by facilitation (green) or negatively by depression (red). Endogenous CRF peptides may be co-released from nerve terminals (intracellularly; CRF Peptides enclosed inside yellow retangle on nerve terminals) or be circulated and maintained in the extracellular space (CRF Peptides as unbound yellow rectangle and free in synaptic spaces), or both. Left (at CeA), CRF₁ located postsynaptically; activation leads to depression of EPSC amplitude. CRF₂ located presynaptically and postsynaptically; activation may lead to enhanced glutamate release and facilitation of EPSC amplitude, but end result also depends on level of CRF₁ activation. Right (at LSMLN), CRF₁ located postsynaptically; activation leads to facilitation of EPSC amplitude. CRF₂ located postsynaptically and presynaptically; activation leads to depression of EPSC amplitude, but result depends on net CRF₁ and CRF₂ effects. Tables below diagram (Left, CeA; right, LSMLN) summarize mEPSC and evoked EPSC data and suggest CRF receptor types mediating the respective results.

Figure 7.

CRF(r/h) and Ucn I have different effects on mEPSCs within the LSMLN. A, Typical traces of mEPSCs before and after CRF(r/h) (50 nm). B, Graph of cumulative fraction of mEPSCs from single cell depicted in A and plotted as a function of amplitude (p < 0.001; maximum amplitude difference was 0.29 by K-S test). Interevent interval (p < 0.401 by K-S test). C, Ucn I (200 nm) depressed the amplitude (p < 0.0001; maximum amplitude difference was 0.36 by K-S test). Maximum difference of the interevent interval was 0.42 by K-S test). Insets depict decrease from control at 1.0 (n = 5) of mEPSCs.D,Graphic plot of the effect of Ucn Ion the cumulative fraction of mEPSCs as a function of mEPSC amplitude and interevent interval. E, Plot of EPSC₂/EPSC₁ ratio versus interstimulus intervals of EPSCs before (control) and in the presence of CRF(r/h); CRF did not affect this ratio. F, Ucn I increased the EPSC₂/EPSC₁ ratio significantly (^*p < 0.05; n = 5), especially at the shorter intervals.