Susanne Meis

Identification of a Neuropeptide S Responsive Circuitry Shaping Amygdala Activity via the Endopiriform Nucleus

Neuropeptide S (NPS) and its receptor are thought to define a set of specific brain circuits involved in fear and anxiety. Here we provide evidence for a novel, NPS-responsive circuit that shapes neural activity in the mouse basolateral amygdala (BLA) via the endopiriform nucleus (EPN). Using slice preparations, we demonstrate that NPS directly activates an inward current in 20% of EPN neurons and evokes an increase of glutamatergic excitation in this nucleus. Excitation of the EPN is responsible for a modulation of BLA activity through NPS, characterized by a general increase of GABAergic inhibition and enhancement of spike activity in a subset of BLA projection neurons. Finally, local injection of NPS to the EPN interferes with the expression of contextual, but not auditory cued fear memory. Together, these data suggest the existence of a specific NPS-responsive circuitry between EPN and BLA, likely involved in contextual aspects of fear memory.

Control of glutamate and GABA release by nociceptin/orphanin FQ in the rat lateral amygdala

1 The actions of the heptadecapeptide termed nociceptin or orphanin FQ (N/OFQ) and the recently discovered putative precursor product nocistatin were examined on synaptic transmission in putative projection cells of the rat lateral amygdala using the whole‐cell patch‐clamp technique. 2 N/OFQ decreased evoked non‐NMDA receptor‐mediated excitatory postsynaptic current (EPSC) amplitudes in a concentration‐dependent manner, with a half‐maximal inhibitory effect elicited by 21.8 ± 7.5 nm and a Hill coefficient of 0.8 ± 0.2 (n= 22. Responses were maximally suppressed to 70.3 ± 1.7 % of the control value. The effect of N/OFQ was prevented by 1 μm[Phe1ψ(CH2‐NH)Gly2]NC(1‐13)NH2 (PheψN/OFQ), a substance known as an antagonist/partial agonist of the ORL receptor. 3 GABAA receptor‐mediated inhibitory postsynaptic currents (IPSCs) elicited through intra‐amygdaloid stimulation were reduced to 48.0 ± 6.8 % by 1 μm N/OFQ (n= 5. 4 Nocistatin had no measurable effect on evoked synaptic currents or membrane properties of recorded neurons. 5 N/OFQ reduced the frequency of spontaneous miniature EPSCs and IPSCs to 74.0 ± 2.6 % and 84.4 ± 1.1 %, respectively, without affecting the amplitudes. 6 The present findings indicate that N/OFQ, but not nocistatin, inhibits the release of glutamate and GABA in the lateral amygdala, presumably by acting on presynaptic release sites. These mechanisms may add to the role of N/OFQ in reducing stress vulnerability as recently proposed on the basis of behavioural and genetic approaches.

Postsynaptic BDNF signalling regulates long-term potentiation at thalamo-amygdala afferents

Non‐technical summary  Long‐term potentiation (LTP) in the lateral amygdala (LA) is a widely accepted cellular correlate of fear learning. In the present study we analysed the involvement of the neurotrophin brain‐derived neurotrophic factor (BDNF) in amygdala LTP by stimulating selectively thalamic or cortical sensory inputs into the LA. In heterozygous BDNF knockout mice we observed that LTP in the cortico‐amygdala pathway remained intact, whereas LTP in the thalamo‐amygdala pathway was abolished. Likewise, acute interference with BDNF/TrkB signalling in wild‐type mice by application of a BDNF scavenger (TrkB‐IgG) led to inhibition of LTP in this pathway. In addition, postsynaptic inhibition of TrkB receptors, which mediate BDNF effects, blocked LTP in the thalamic pathway. Thus, our data demonstrate for the first time a crucial role for postsynaptic BDNF signalling in mediating LTP selectively in the thalamo‐amygdala pathway in the LA.

Postsynaptic mechanisms underlying responsiveness of amygdaloid neurons to cholecystokinin are mediated by a transient receptor potential-like current

Projection neurons of mouse basolateral amygdala responded to CCK with an inward current at a holding potential of -70 mV. This response was mediated by CCK2 receptors as indicated by agonist and antagonist effectiveness, and conveyed via G-proteins of the G(q/11) family as it was abolished in gene knockout mice. Maximal current amplitude was insensitive to extracellular potassium, cesium, and calcium ions, respectively, whereas amplitude and reversal potential critically depended upon extracellular sodium concentration. The current reversed near -20 mV consistent with activation of a mixed cationic channel reminiscent of transient receptor potential (TRP) channels. Extracellular application of the non-selective TRP channel blockers 2-APB, flufenamic acid, Gd3+, and ruthenium red, respectively, inhibited CCK induced inward currents. Single cell PCR confirmed the expression of TRPC1,4,5 and coexpression of TRPC1 with TRPC4 or TRPC5 in some cells. CCK responses were associated with depolarization leading to an increase in cell excitability.

Mechanisms of somatostatin-evoked responses in neurons of the rat lateral amygdala.

The effects of somatostatin in the rat lateral amygdala (LA) in vitro were investigated through whole cell recording techniques. Somatostatin induced an inwardly rectifying K+ current in approximately 98% of LA projection neurons. Half‐maximal effects were obtained by 189 nm somatostatin. The effects of somatostatin were insensitive to tetrodotoxin, reduced by Ba2+, occluded or abolished by the presence of nonhydrolysable GTP or GDP analogues, respectively, and blocked or mimicked by a somatostatin receptor type 2 antagonist (BIM‐23627) or somatostatin receptor type 2 agonist (L‐779,976), respectively, while somatostatin receptor type 1, 3 and 4 agonists were ineffective (L‐797,591, L‐796,778, L‐803,087). Responses to somatostatin were associated with membrane hyperpolarization and decrease in input resistance, resulting in a dampening of cell excitability. It is suggested that these cellular mechanisms contribute to the role of somatostatin in decreasing anxiety behaviour as well as to anticonvulsant and antiepileptogenic actions of somatostatin or somatostatin agonists in the amygdala.

Developmental downregulation of GABAergic drive parallels formation of functional synapses in cultured mouse neocortical networks

Networks of cortical neurons in vitro spontaneously develop synchronous oscillatory electrical activity at around the second week in culture. However, the underlying mechanisms and in particular the role of GABAergic interneurons in initiation and synchronization of oscillatory activity in developing cortical networks remain elusive. Here, we examined the intrinsic properties and the development of GABAergic and glutamatergic input onto presumed projection neurons (PNs) and large interneurons (L‐INs) in cortical cultures of GAD67‐GFP mice. Cultures developed spontaneous synchronous activity already at 5–7 days in vitro (DIV), as revealed by imaging transient changes in Fluo‐3 fluorescence. Concurrently, spontaneous glutamate‐mediated and GABAA‐mediated postsynaptic currents (sPSCs) occured at 5 DIV. For both types of neurons the frequency of glutamatergic and GABAergic sPSCs increased with DIV, whereas the charge transfer of glutamatergic sPSCs increased and the charge transfer of GABAergic sPSCs decreased with cultivation time. The ratio between GABAergic and the overall charge transfer was significantly reduced with DIV for L‐INs and PNs, indicating an overall reduction in GABAergic synaptic drive with maturation of the network. In contrast, analysis of miniature PSCs (mPSCs) revealed no significant changes of charge transfer with DIV for both types of neurons, indicating that the reduction in GABAergic drive was not due to a decreased number of functional synapses. Our data suggest that the global reduction in GABAergic synaptic drive together with more synaptic input to PNs and L‐INs during maturation may enhance rhythmogenesis of the network and increase the synchronization at the level of population bursts.

Interaction between Low Voltage‐activated Currents in Reticular Thalamic Neurons in a Rat Model of Absence Epilepsy

A transient potassium (K+) outward current (IA) contributes to the distinctive patterns of low‐threshold spike firing observed in various classes of thalamic neurons through a functional interaction with a calcium (Ca2+)‐mediated inward current (IT). The present study was undertaken to investigate the properties of transient K+ currents and their interaction with IT in neurons of the reticular thalamic nucleus, and to compare these properties in reticular thalamic nucleus neurons from a rat model of absence epilepsy, designated the Genetic Absence Epilepsy Rat from Strasbourg (GAERS), with those from a Non‐epileptic Control strain (NEC). This comparative approach appeared to be particularly important in view of the recent finding of a selective increase in IT in reticular thalamic nucleus neurons from GAERS. Neurons were acutely isolated from the reticular thalamic nucleus through enzymatic procedures, and identified by morphological and immunocytochemical criteria. Ionic currents were analysed using whole‐cell patch‐clamp techniques. Transient K+ currents in reticular thalamic nucleus neurons with properties indicative of IA activated at ∼−55 mV (with half‐activation at −27 and −33 mV in NEC and GAERS respectively), declined rapidly with a voltage‐dependent time constant (τ= 4 ms at +45 mV), were 50% steady‐state‐inactivated at −81 and −86 mV in the two strains of rats respectively, and rapidly recovered from inactivation with a monoexponential time course (τ= 31 and 37 ms respectively). No significant differences in IA properties or densities were found between reticular thalamic nucleus neurons from GAERS and NEC rats. Analysis of the interaction between IA and IT indicated a shift in the balance between the two opposing membrane conductances towards the generation of a low‐voltage‐activated inward current in reticular thalamic nucleus neurons from GAERS compared with NEC, and a lack of IA to functionally compensate for this shift, which in turn may contribute to pathological forms of low‐threshold spike firing characterizing spike‐and‐wave discharges.

Antioscillatory Effects of Nociceptin/Orphanin FQ in Synaptic Networks of the Rat Thalamus

Postsynaptic and presynaptic effects of nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid-like orphan receptor, were investigated in an in vitro slice preparation of the rat thalamic reticular nucleus (NRT) and ventrobasal complex (VB). In NRT as well as VB, all tested neurons developed an outward current on application of 1 μm N/OFQ. Basic properties of the N/OFQ-induced current included inward rectification, dependence on extracellular K+, reduction by 100 μm Ba+, antagonistic effect of [Nphe1]nociceptin(1–13)NH2, and sensitivity to internal GDP-β-S. Miniature IPSCs (mIPSCs) mediated by GABAA receptors in VB neurons were not affected by 1 μm N/OFQ. In addition, paired-pulse depression of evoked IPSCs was unchanged, indicating a lack of presynaptic effects. By comparison, N/OFQ application resulted in a reduction in frequency of miniature EPSCs (mEPSCs) in a subpopulation of NRT neurons, whereas paired-pulse facilitation of evoked EPSCs was not altered. In either nucleus, current-clamp experiments revealed a hyperpolarization and associated decrease in input resistance in response to N/OFQ. Although N/OFQ had no measurable effect on calcium-mediated burst activity evoked by depolarizing steps from hyperpolarized values of the membrane potential, rebound bursts on relief of hyperpolarizing current steps were decreased. Slow thalamic oscillations induced in vitro by extracellular stimulation were dampened by N/OFQ in VB and NRT, as seen by delayed onset of rhythmic multiple-unit activity and reduction in amplitude and duration. We conclude that N/OFQ reduces the excitability of NRT and VB neurons predominantly through an increase of a G-protein-coupled inwardly rectifying K+ conductance.

Neuropeptide S-mediated facilitation of synaptic transmission enforces subthreshold theta oscillations within the lateral amygdala.

The neuropeptide S (NPS) receptor system modulates neuronal circuit activity in the amygdala in conjunction with fear, anxiety and the expression and extinction of previously acquired fear memories. Using in vitro brain slice preparations of transgenic GAD67-GFP (Δneo) mice, we investigated the effects of NPS on neural activity in the lateral amygdala as a key region for the formation and extinction of fear memories. We are able to demonstrate that NPS augments excitatory glutamatergic synaptic input onto both projection neurons and interneurons of the lateral amygdala, resulting in enhanced spike activity of both types of cells. These effects were at least in part mediated by presynaptic mechanisms. In turn, inhibition of projection neurons by local interneurons was augmented by NPS, and subthreshold oscillations were strengthened, leading to their shift into the theta frequency range. These data suggest that the multifaceted effects of NPS on amygdaloid circuitry may shape behavior-related network activity patterns in the amygdala and reflect the peptides potent activity in various forms of affective behavior and emotional memory.

Impaired transmission at corticothalamic excitatory inputs and intrathalamic GABAergic synapses in the ventrobasal thalamus of heterozygous BDNF knockout mice

Beside its role in development and maturation of synapses, brain-derived neurotrophic factor (BDNF) is suggested to play a critical role in modulation and plasticity of glutamatergic as well as GABAergic synaptic transmission. Here, we used heterozygous BDNF knockout (BDNF(+/-)) mice, which chronically lack approximately 50% of BDNF of wildtype (WT) animals, to investigate the role of BDNF in regulating synaptic transmission in the ventrobasal complex (VB) of the thalamus. Excitatory transmission was characterized at glutamatergic synapses onto relay (TC) neurons of the VB and intrathalamic inhibitory transmission was characterized at GABAergic synapses between neurons of the reticular thalamic nucleus (RTN) and TC neurons. Reduced expression of BDNF in BDNF(+/-) mice did not affect intrinsic membrane properties of TC neurons. Recordings in TC neurons, however, revealed a strong reduction in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in BDNF(+/-) mice, as compared to WT littermates, whereas mEPSC amplitudes were not significantly different between genotypes. A mainly presynaptic impairment of corticothalamic excitatory synapses in BDNF(+/-) mice was also indicated by a decreased paired-pulse ratio and faster synaptic fatigue upon prolonged repetitive stimulation at 40 Hz. For miniature inhibitory postsynaptic currents (mIPSCs) recorded in TC neurons, both, frequency and amplitude showed a significant reduction in knock-out animals, concurrent with a prolonged decay time constant, whereas paired-pulse depression and synaptic fatigue of inhibitory synapses were not significantly different between WT and BDNF(+/-) mice. Spontaneous IPSCs (sIPSCs) recorded in VB neurons of BDNF(+/-) animals showed a significantly reduced frequency. However, the glutamatergic drive onto RTN neurons, as revealed by the percentage reduction in frequency of sIPSCs after application of AMPA and NMDA receptor blockers, was not significantly different. Together, the present findings suggest that a chronically reduced level of BDNF to ∼50% of WT levels in heterozygous knock-out animals, strongly attenuates glutamatergic and GABAergic synaptic transmission in thalamic circuits. We hypothesize that this impairment of excitatory and inhibitory transmission may have profound consequences for the generation of rhythmical activity in the thalamocortical network.