Chronic stress dampens excitatory synaptic gain in the paraventricular nucleus of the hypothalamus

Abstract

Key points: Glutamatergic synaptic inputs to corticotrophin-releasing hormone (CRH) secreting neurons in the paraventricular nucleus of the hypothalamus (PVN) are required for stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis. These synapses also undergo stress-induced plasticity, thereby influencing HPA axis stress adaptation. By using patch clamp electrophysiology, we show that, in adult non-stressed mice, action potentials at these glutamatergic afferents elicit multiquantal transmission to the postsynaptic PVN-CRH neurons (i.e. synaptic multiplicity). Mechanistically, synaptic multiplicity results from multivesicular release at common synaptic sites, which is facilitated upon elevation of release probability, effectively increasing the upper limit of the dynamic range of synaptic transmission. Following chronic variable stress, functional PVN glutamate synapse number increases, although its synaptic multiplicity paradoxically decreases. These two contrasting synaptic changes can, respectively, increase the baseline excitatory drive while also limiting the capacity for potentiation, and may preferentially increase the baseline excitatory drive onto PVN-CRH neurons.

Abstract: The activation of the hypothalamic-pituitary-adrenal (HPA) axis relies on excitation of neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVN) that secrete corticotrophin-releasing hormone (CRH). Afferent glutamate synapses onto these PVN-CRH neurons convey critical excitatory inputs during stress, and also undergo stress-induced plasticity, highlighting their roles in both stress activation and adaptation of the HPA axis. In the present study, using whole-cell patch clamp recordings from PVN-CRH neurons in brain slices from adult mice, we found that the amplitude of action potential-dependent spontaneous EPSCs (sEPSCs) was larger than that of action potential independent miniature EPSCs (mEPSCs), suggesting that action potentials at individual axons recruited multiquantal transmission onto the same postsynaptic neurons (i.e. synaptic multiplicity). The large, putative multiquantal sEPSCs had fast rise times similar to mEPSCs, and were abolished by replacing extracellular Ca²⁺ with Sr²⁺ , indicating Ca²⁺ -dependent synchronous release of multiple vesicles. Application of a low affinity, fast dissociating competitive AMPA receptor antagonist γ-d-glutamylglycine revealed that synaptic multiplicity resulted from multivesicular release targeting a common population of postsynaptic receptors. High-frequency afferent stimulation facilitated synaptic multiplicity, effectively increasing the upper limit of the dynamic range of synaptic transmission. Finally, we found that chronic variable stress (CVS), a stress model known to cause basal HPA axis hyperactivity, increased sEPSCs frequency but paradoxically decreased synaptic multiplicity. These results suggest that the CVS-induced synaptic changes may elevate the baseline excitatory drive at the same time as limiting the capacity for potentiation, and may contribute to the basal HPA axis hyperactivity.

Keywords: Neuroendocrine; patch clamp electrophysiology; synaptic plasticity.

Figure 1. Synaptic multiplicity of glutamate synapses onto PVN‐CRH neurons

A, schematic diagram illustrating synaptic modalities and their consequences for postsynaptic currents. In a synapse with multiplicity (left), an action potential and the ensuing Ca²⁺ influx triggers synchronous fusion of multiple synaptic vesicles that results in large, multiquantal EPSCs. Synaptic multiplicity can result from multisynapse contact or multivesicular release at a single synapse. In the presence of TTX and Cd²⁺, action potential independent vesicle fusion is asynchronous and causes small monoquantal EPSCs. In synapses without multiplicity (right), both action potential‐dependent and ‐independent vesicle fusion results in monoquantal EPSCs. B, summary of the mean frequency decrease from sEPSCs to mEPSCs. Recordings that showed a >15% decrease are shown in blue and ≤15% are shown in red. C, sample traces of sEPSCs and mEPSCs that showed a >15% frequency decrease. D, EPSC amplitude from the recording shown in (C). E, summary of the mean amplitude difference between sEPSCs and mEPSCs for the recordings that showed a >15% frequency decrease. ^*** P < 0.005.

Figure 2. Low concentration 4‐AP application probes synaptic multiplicity

A, sample traces of sEPSCs recorded in low Ca²⁺ aCSF during baseline, and after 4‐AP (30 μm) application, and subsequent TTX (0.5 μm) and Cd²⁺ (10 μm) application. B, sEPSC amplitude from the recording shown in (A). C and D, summary of the mean frequency (C) and amplitude (D) between baseline (grey), 4‐AP (red) and TTX +Cd²⁺ (blue). ^*** P < 0.005, ^** P < 0.01.

Figure 3. Synchronized multiquantal synaptic events underlie synaptic multiplicity

A, summary of a lower bound estimation of the probability of random summation of two sEPSCs compared to the proportion of events larger than twice the amplitude of mEPSCs (large event). A sample trace shown at the top illustrates the half‐width and interevent interval parameters to calculate the probability of random summation (average half‐width/average interevent interval). B, an example plot and linear regression line of 10–90% rise time vs. amplitude of individual sEPSCs. Sample traces illustrate that both large (>30 pA) and small (<15 pA) sEPSCs have fast 10–90% rise times. C, sample traces of sEPSCs recorded before and after replacing Ca²⁺ with Sr²⁺ in the aCSF. D, sEPSC amplitude distribution from the recording shown in (F). E, summary of mean sEPSC amplitude change by replacing Ca²⁺ with Sr²⁺ in the aCSF. ^*** P < 0.001, ^* P < 0.05. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 4. Synaptic multiplicity is due to multivesicular release

A, schematics illustrating two models of multiplicity. Left: temporal summation of monoquantal transmission that targets independent populations of postsynaptic receptors. Large and small EPSCs are achieved by a similar glutamate concentration in the synaptic cleft and therefore are equally sensitive to γ‐DGG. Right: multiquantal transmission that targets an overlapping population of postsynaptic receptors. Large EPSCs are caused by higher glutamate concentration in the cleft than smaller sEPSCs and are therefore less sensitive to γ‐DGG. Values shown at bottom of model are hypothetical relative amplitudes. B, sample traces from a recording in which there was an increase in mean sEPSC amplitude following 4‐AP application (4‐AP responder). C, cumulative plot for EPSC amplitude for the recording shown in (B). D, cumulative plot for normalized EPSC amplitude (EPSC/EPSC_MAX) before and after application of γ‐DGG from the recording shown in (B). E, sample traces from a recording where there was no change in the mean sEPSC amplitude following 4‐AP application (4‐AP non‐responder). F, cumulative EPSC amplitude for the recording shown in (E). G, cumulative EPSC/EPSC_MAX plot for the recording shown in (E). H, sample traces from a recording from baseline, as well as after 4‐AP and DNQX application. I, cumulative EPSC amplitude for the recording shown in (H). J, cumulative EPSC/EPSC_MAX plot for the recording shown in (H). K, summary of mean EPSC/EPSC_MAX after γ‐DGG (in 4‐AP responder and non‐responder groups) or DNQX application normalized to pre‐γ‐DGG/DNQX (i.e. post‐4‐AP). L, plots of post‐4‐AP mean EPSC amplitude (normalized to pre‐4‐AP) against post‐γ‐DGG/DNQX mean EPSC/EPSC_MAX (normalized to post‐4‐AP). ^*** P < 0.005.

Figure 5. Synaptic activity drives multiplicity transmission

A, sample traces of sEPSCs before and after afferent synaptic stimulation. B, plot of sEPSC amplitude before and after synaptic stimulation (20 Hz, 2 s) from the recording shown in (A). C and D, summary of sEPSC frequency (C) and amplitude (D) changes following synaptic stimulation. ^*** P < 0.001, ^* P < 0.05. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 6. CVS diminishes synaptic multiplicity

A, sample traces of sEPSCs before and after 4‐AP application in a slice from a CVS mouse. B and C, summary of sEPSC frequency (B) and amplitude (C) before and after 4‐AP application in slices from naïve (blue) and CVS mice (red). D and E, sample amplitude histograms of the recordings from naïve (D) and CVS (E) mice before (black) and after 4‐AP application (blue/red). Note that the number of events is normalized to the total event number. F and G, summary sEPSC amplitude from naïve (F) and CVS (G) mice. H, summary of the change in the percentage of large events (>40 pA, highlighted in F and G) before and after 4‐AP application in naïve (blue) and CVS (red) mice. ^** P < 0.01,^* P < 0.05.