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Vasopressin and Oxytocin Excite Distinct Neuronal Populations in the Central Amygdala

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Science  08 Apr 2005:
Vol. 308, Issue 5719, pp. 245-248
DOI: 10.1126/science.1105636

Abstract

Vasopressin and oxytocin strongly modulate autonomic fear responses, through mechanisms that are still unclear. We describe how these neuropeptides excite distinct neuronal populations in the central amygdala, which provides the major output of the amygdaloid complex to the autonomic nervous system. We identified these two neuronal populations as part of an inhibitory network, through which vasopressin and oxytocin modulate the integration of excitatory information from the basolateral amygdala and cerebral cortex in opposite manners. Through this network, the expression and endogenous activation of vasopressin and oxytocin receptors may regulate the autonomic expression of fear.

The amygdala plays an important role in anxiety and fear behavior. Fear learning involves its lateral and basolateral parts, where the association between incoming fearful and neutral stimuli leads to potentiation of synaptic transmission. These parts project to the central amygdala (CeA), whose efferents to the hypothalamus and brainstem trigger the autonomic expression of fear (1). Selective gating of synaptic transmission through the CeA could therefore modulate the fear response (2, 3). Indeed, recent studies suggest that increased inhibition within the CeA could underlie the anxiolytic effects of benzodiazepines and alcohol (4, 5) and may also play a role in the extinction of conditioned fear through cortical afferents (6, 7).

The CeA expresses numerous neuropeptides and neuropeptide receptors, including high levels of receptors for vasopressin and oxytocin (8, 9). Activation of vasopressin and oxytocin receptors oppositely affects fear and anxiety-related behaviors. Vasopressin enhances aggressiveness, anxiety, and stress levels and the consolidation of fear memory (1013). Oxytocin decreases anxiety and stress and facilitates social encounters, maternal care, and the extinction of conditioned avoidance behavior (1317). At the cellular level, however, both neuropeptides increase neuronal excitability in various brain regions, including the CeA (1820), which raises the question of whether a local neuronal network could underlie their opposite behavioral effects.

We first determined the distribution of vasopressin and oxytocin receptors in the CeA using autoradiography on horizontal rat brain sections (21). Binding of 125I-labeled ligands revealed that expression of oxytocin receptors was restricted to the lateral and capsular division of the CeA (CeL/C) and vasopressin receptors in the medial part (CeM) (Fig. 1A). To determine the physiological effects of activating these receptors, we recorded spontaneous spiking activity extracellularly in acute brain slices of the CeA (21). Bath application of the highly specific oxytocin receptor agonist [Thr4,Gly7]-oxytocin (TGOT, 0.2 μM, for 30 s) (fig. S2) (21) increased spontaneous spike frequencies in 21% of 224 recorded neurons (to 284 ± 26% of the initial frequency) but decreased them in more than 50% (to 19 ± 2%). Both responses were fully reversible and repeatable (fig. S1) and could be blocked by the oxytocin receptor antagonist d(CH2)5[Tyr(Me)2,Thr4,Orn8,des-Gly-NH29]-vasotocin (OTA, 1 μM) [TGOT excitation: 301 ± 16%, P < 0.05, and OTA+TGOT: 93 ± 7%, P > 0.05, n = 6 experiments (21); TGOT inhibition: 6 ± 6%, P < 0.05, and OTA TGOT: 110 ± 24%, P > 0.05, n = 5 experiments; all relative values are expressed as percentages of the control frequency] (Fig. 1, B to D). Subsequent exposure of TGOT-excited cells to the general vasopressin receptor agonist [Arg8]-vasopressin (AVP, 0.02 to 0.2 μM, for 30 s) was only able to induce small increases in frequency (Fig. 1B), which were probably caused by a cross-reactivity of AVP on oxytocin receptors (fig. S2) (21). On the other hand, AVP potently excited more than 50% of TGOT-inhibited neurons (319 ± 9%, 24 out of 47 cells) (Fig. 1, C and D). This latter effect was fully reversible and repeatable (fig. S1) and appeared mediated by V1a receptors: It could be blocked by the V1 receptor antagonist d(CH2)5[Tyr(Me)2,Arg8]-vasopressin (TMA, 1 μM) (Fig. 1D) and could be mimicked neither by the V1b receptor agonist [1-deamino-4-cyclohexylalanine]-Arg-vasopressin (d[Cha]AVP, 1 μM) (Fig. 1D) nor by the V2 receptor agonist [deamino-Cys1,Val4,D-Arg8]-vasopressin (dVDAVP, 1 μM) (fig. S3). This was further confirmed by an additional combination of different vasopressin receptor agonists and antagonists (fig. S3). Thus, our findings suggest two groups of TGOT-responsive neurons in the CeA: one that is excited by oxytocin receptor activation and a second that is inhibited by activation of oxytocin receptors but excited by vasopressin V1a receptors.

Fig. 1.

Distributions and opposite actions of oxytocin and vasopressin receptors in the rat CeA. (A) Composite image of histoautoradiographs of adjacent horizontal sections, showing nonoverlapping binding-site areas for oxytocin (red) and vasopressin (green) in the CeA. La, lateral amygdala; Me, medial amygdala; CA2 and CA3, fields of the hippocampus. Scale bar, 400 μm. (B and C) Spike frequency examples of (B) a TGOT-excited, AVP-nonresponsive neuron with TGOT excitation blocked by OTA and (C) a TGOT-inhibited, AVP-excited neuron. Upper traces show examples of original recordings at the times as indicated. (D) Average excitatory (left) and inhibitory (middle) effects of TGOT that were blocked by OTA. Average excitation by AVP (right) can be blocked by TMA and is not mimicked by d[Cha]AVP (*P < 0.05, n = 5 experiments for all bars). Ctrl, control.

Earlier morphological studies have shown intense γ-aminobutyric acid (GABA)–positive staining in the CeA and projections from the CeL/C onto the CeM, which are thought to be GABAergic (8, 22). Hypothesizing that these could mediate the inhibitory effects of oxytocin receptor activation, we determined the precise position and projections of TGOT and AVP-excited cells using sharp-electrode intracellular current-clamp recordings. Neurons were held near the spiking threshold (–55 ± 4 mV), and excitation was measured as a rapid increase in spontaneous spike frequency accompanied by small depolarizations (3.4 ± 0.4 mV and 4.6 ± 0.8 mV, respectively; n = 6 experiments) that were resistant to tetrodotoxin (TTX, 1 μM) (Fig. 2A). Cells with excitatory responses were injected with biocytin from the intracellular recording pipette (n = 10 experiments). AVP-excited cells were restricted to the CeM and displayed moderately spiny dendrites and medium-sized cell bodies, with axon collaterals that projected in an anteromedial direction outside the CeA. TGOT-excited neurons were found in the CeL, were of the medium-sized spiny type, and contained several local axon collaterals, of which one or more typically projected toward the CeM (Fig. 2B). Confocal microscopy revealed these TGOT-excited cells to be immunopositive for GAD-67 (Fig. 2C), confirming that they were GABAergic.

Fig. 2.

Intracellular recordings and morphological properties of vasopressin and oxytocin excited neurons in the CeA. (A) Intracellular recordings of an AVP-excited neuron in the CeM (left) and a TGOT-excited neuron in the CeL/C (right) with depolarization that persisted in the presence of TTX but was blocked by OTA. (B) Morphology and projections of three oxytocin-excited (red axon collaterals) and three vasopressin-excited neurons (green axon collaterals), as revealed by biocytin injections after intracellular recordings (dendrites in black and somata marked by black arrows). Scale bar, 200 μm. (C) Neurochemical characterization of an oxytocin-excited neuron (OXT+, white arrow indicates soma) costained for GAD-67 (green) after biocytin injection (red) reveals costaining (yellow). Scale bar, 25 μm.

Would TGOT indeed affect GABAergic transmission in the CeM? We recorded postsynaptic currents in the CeM by the whole-cell voltage-clamp technique. Bath-applied TGOT evoked rapid increases in these currents that were blocked by OTA and completely disappeared in the presence of the GABA(A) receptor antagonist bicuculline (BIC, 20 μM) (Fig. 3A). Amplitudes and rise and decay times were not affected by 0.2 μM nor by 1 μM TGOT (Fig. 3B and table S1). Thus, TGOT appears to specifically enhance GABAergic transmission in the CeM through a rapid and reversible increase of the frequency of the inhibitory postsynaptic currents (IPSCs). Previous studies have shown that oxytocin is able to modulate synaptic transmission by a number of pre- and postsynaptic mechanisms (18). We therefore applied TGOT in the presence of TTX (1 μM) to cells that had previously responded to TGOT, but we found no significant effects on the miniature IPSC frequencies, amplitudes (Fig. 3, A and B), rise times, or decay times (table S1), which seems to exclude a postsynaptic effect by TGOT. A presynaptically mediated increase in IPSC frequency could result from an enhanced excitability of the cell body or from an increased release probability from the presynaptic site. We thus focally applied TGOT (1 μM) from a 1-μm patch pipette at the presynaptic site near the recorded neuron in the CeM, but this never caused a change in IPSC frequency (Fig. 3C, position A) (n = 5 experiments). Puffing of TGOT laterally, however, at distant sites in the CeL/C, was able to induce sharp increases in IPSC frequencies at specifically identified locations (Fig. 3C, position B, and table S1) (n = 5 experiments), which were blocked by subsequent application of TTX. These findings indicate that the inhibitory effects of TGOT are caused by an enhanced excitability of neurons in the CeL/C that leads to an increase of GABA release in the CeM.

Fig. 3.

Local effects of oxytocin on IPSCs in the CeA. (A) Examples of IPSC appearances in the presence of various treatments as indicated. (B) Average TGOT effects on IPSC frequency and amplitude in the absence and presence of TTX (left, n = 9 experiments); TGOT significantly enhanced mean IPSC frequency (*, P < 0.01) but did not affect amplitudes of IPSC or miniature IPSCs (P > 0.1, n = 5 experiments, middle and right) (table S1). Rel. Frequency, relative cumulative frequency. (C) Effects of local application of TGOT (1 μM) with a patch pipette in the CeM (position A) and in the CeL/C (position B) on IPSC frequency before and after TTX. R indicates the recording electrode.

Changes in the excitability of neurons in different subnuclei may be relevant to the behavioral function of the CeA, because they can modulate the integration of its distinct inputs (1). The CeL/C receives projections from cortical and subcortical areas (6, 8) and projects to the CeM, which also receives direct input from the basolateral amygdala (BLA) (Fig. 4A) (22). We indeed found that during stimulation of the excitatory afferents to the CeL/C, TGOT could enhance the probability of evoking postsynaptic action potentials (Fig. 4B). During stimulation of the excitatory afferents to the CeM, however, TGOT decreased the probability of evoking postsynaptic action potentials, but AVP increased it (Fig. 4C).

Fig. 4.

Effects of oxytocin and vasopressin on the gating of inputs from different afferent path-ways in the CeA. (A) Simplified model of local circuitry in the CeA, showing processing of different excitatory inputs (+) and GABAergic connections (–) between oxytocin and vasopressin receptor–expressing regions (CeL/C and CeM), stimulation electrode (S), and recording electrode (R). (B) Paired stimuli (S1 and S2, 50-ms separation, at 10-s intervals) applied to cortical afferents in the external capsule (6) resulted in excitatory potentials in CeL/C neurons under current-clamp. Stimuli were paired with postsynaptic current injections (D) such that the second stimulus (S2) regularly evoked action potentials. TGOT application (1 μM, for 1 min) caused a small depolarization of the membrane potential and increased probability of action potential generation after D, S1, or S2. The traces show superpositions of 15 sweeps, which were averaged per experiment in order to calculate the percentages of spikes evoked by each stimulus as indicated by the bar charts below (*, P < 0.05, n = 5 experiments). (C) Neurons in the CeM were stimulated through their afferents in the basolateral and basomedial nuclei (22). AVP (0.2 μM, for 1 min) caused similar effects as TGOT in the CeL, whereas subsequent administration of TGOT (1 μM, for 1 min) led instead to decreases in responses to S1 and S2 (*, P < 0.05, n = 5 experiments).

These findings reveal two major points. First, vasopressin and oxytocin modulate activity in CeM neurons in opposite ways through the activation of distinct elements of an inhibitory network (Fig. 4A). Second, through the activation of these distinct elements, vasopressin and oxytocin can differently affect the integration of distinct afferents to the CeA into a common output to the autonomic nervous system, thus providing a neurophysiological mechanism for their opposite effects on anxiety and fear behavior. As we have previously found a comparable distribution of oxytocin and vasopressin receptors throughout the central extended amygdala (9), this mechanism may also apply to regions that include the bed nucleus of the stria terminalis and parts of the nucleus accumbens. These latter structures are known to be involved in the control of anxiety, stress, motivation, and addiction (23) and are possibly regulated by vasopressin and oxytocin in a similar manner.

The results of this study suggest that the endogenous balance between oxytocin and vasopressin receptor expression and activation may set distinct, individually tuned levels for the activation of the autonomic fear response. The levels of these neuropeptides in the extracellular fluid of the CeA are increased during stress (12, 24), possibly through release from local vasopressinergic and oxytocinergic fibers (25, 26). Furthermore, variations in levels of receptor expression and injections of specific antagonists have been directly correlated with changes in anxiety and fear (12, 16, 17, 24, 27, 28). Together, these findings confirm the physiological and behavioral relevance of the proposed mechanism. Anxiety and fear can directly affect parental care, thereby modulating the expression of oxytocin and vasopressin receptors in offspring and establishing anxiety and fear traits that can be carried over several generations (16, 27). The elucidation of the opposite, modulatory mechanism of these two peptides in the CeA provides a solid rationale for the development of new, individually tailored treatments, working in concert with the more traditional GABAergic agonists (4, 5). Indeed, the vasopressin receptor could be a pharmacological target for the treatment of stress and anxiety-related disorders (10, 11).

Several recent lines of evidence suggest that fear extinction inhibits the expression of the conditioned reaction rather than erasing the memory (3). This inhibition is thought to be mediated by cortical afferents to the amygdala, originating in the medial prefrontal cortex (7). Our findings provide evidence for a functional link between cortical input in the CeL/C and inhibition of output from the CeA. The oxytocinergic modulation of the cortical input and the vasopressinergic effects on input from the BLA could implicate additional, opposing roles for these neuropeptides in fear extinction.

Supporting Online Material

www.sciencemag.org/cgi/content/full/308/5719/245/DC1

Materials and Methods

Figs. S1 to S3

Table S1

References and Notes

References and Notes

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