Molecular characterization of Thy1 expressing fear-inhibiting neurons within the basolateral amygdala

Molecular characterization of neuron populations, particularly those controlling threat responses, is essential for understanding the cellular basis of behaviour and identifying pharmacological agents acting selectively on fear-controlling circuitry. Here we demonstrate a comprehensive workflow for identification of pharmacologically tractable markers of behaviourally characterized cell populations. Thy1-eNpHR-, Thy1-Cre- and Thy1-eYFP-labelled neurons of the BLA consistently act as fear inhibiting or ‘Fear-Off' neurons during behaviour. We use cell-type-specific optogenetics and chemogenetics (DREADDs) to modulate activity in this population during behaviour to block or enhance fear extinction. Dissociated Thy1-eYFP neurons are isolated using FACS. RNA sequencing identifies genes strongly upregulated in RNA of this population, including Ntsr2, Dkk3, Rspo2 and Wnt7a. Pharmacological manipulation of neurotensin receptor 2 confirms behavioural effects observed in optogenetic and chemogenetic experiments. These experiments identify and validate Ntsr2-expressing neurons within the BLA, as a putative ‘Fear-Off' population

Spike-Timing Precision and Neuronal Synchrony Are Enhanced by an Interaction between Synaptic Inhibition and Membrane Oscillations in the Amygdala

The basolateral complex of the amygdala (BLA) is a critical component of the neural circuit regulating fear learning. During fear learning and recall, the amygdala and other brain regions, including the hippocampus and prefrontal cortex, exhibit phase-locked oscillations in the high delta/low theta frequency band (∼2–6 Hz) that have been shown to contribute to the learning process. Network oscillations are commonly generated by inhibitory synaptic input that coordinates action potentials in groups of neurons. In the rat BLA, principal neurons spontaneously receive synchronized, inhibitory input in the form of compound, rhythmic, inhibitory postsynaptic potentials (IPSPs), likely originating from burst-firing parvalbumin interneurons. Here we investigated the role of compound IPSPs in the rat and rhesus macaque BLA in regulating action potential synchrony and spike-timing precision. Furthermore, because principal neurons exhibit intrinsic oscillatory properties and resonance between 4 and 5 Hz, in the same frequency band observed during fear, we investigated whether compound IPSPs and intrinsic oscillations interact to promote rhythmic activity in the BLA at this frequency. Using whole-cell patch clamp in brain slices, we demonstrate that compound IPSPs, which occur spontaneously and are synchronized across principal neurons in both the rat and primate BLA, significantly improve spike-timing precision in BLA principal neurons for a window of ∼300 ms following each IPSP. We also show that compound IPSPs coordinate the firing of pairs of BLA principal neurons, and significantly improve spike synchrony for a window of ∼130 ms. Compound IPSPs enhance a 5 Hz calcium-dependent membrane potential oscillation (MPO) in these neurons, likely contributing to the improvement in spike-timing precision and synchronization of spiking. Activation of the cAMP-PKA signaling cascade enhanced the MPO, and inhibition of this cascade blocked the MPO. We discuss these results in the context of spike-timing dependent plasticity and modulation by neurotransmitters important for fear learning, such as dopamine

Synergistic Activation of Dopamine D1 and TrkB Receptors Mediate Gain Control of Synaptic Plasticity in the Basolateral Amygdala

Fear memory formation is thought to require dopamine, brain-derived neurotrophic factor (BDNF) and zinc release in the basolateral amygdala (BLA), as well as the induction of long term potentiation (LTP) in BLA principal neurons. However, no study to date has shown any relationship between these processes in the BLA. Here, we have used in vitro whole-cell patch clamp recording from BLA principal neurons to investigate how dopamine, BDNF, and zinc release may interact to modulate the LTP induction in the BLA. LTP was induced by either theta burst stimulation (TBS) protocol or spaced 5 times high frequency stimulation (5xHFS). Significantly, both TBS and 5xHFS induced LTP was fully blocked by the dopamine D1 receptor antagonist, SCH23390. LTP induction was also blocked by the BDNF scavenger, TrkB-FC, the zinc chelator, DETC, as well as by an inhibitor of matrix metalloproteinases (MMPs), gallardin. Conversely, prior application of the dopamine reuptake inhibitor, GBR12783, or the D1 receptor agonist, SKF39393, induced robust and stable LTP in response to a sub-threshold HFS protocol (2xHFS), which does not normally induce LTP. Similarly, prior activation of TrkB receptors with either a TrkB receptor agonist, or BDNF, also reduced the threshold for LTP-induction, an effect that was blocked by the MEK inhibitor, but not by zinc chelation. Intriguingly, the TrkB receptor agonist-induced reduction of LTP threshold was fully blocked by prior application of SCH23390, and the reduction of LTP threshold induced by GBR12783 was blocked by prior application of TrkB-FC. Together, our results suggest a cellular mechanism whereby the threshold for LTP induction in BLA principal neurons is critically dependent on the level of dopamine in the extracellular milieu and the synergistic activation of postsynaptic D1 and TrkB receptors. Moreover, activation of TrkB receptors appears to be dependent on concurrent release of zinc and activation of MMPs

Factors influencing the aerobic respiration of Escherichia coli

Facultative anaerobic bacteria such as Escherichia coli are capable of obtaining energy from glycolysis, aerobic respiration, and anaerobic respiration. Although a considerable amount of information is available on the production of ATP in E. coli by glycolysis, very little is known about the production of energy by aerobic or anaerobic respiration. Since the properties of aerobic respiration were experimentally more readily attacked, an investigation of the factors influencing the aerobic respiration and respiratory chain-linked energy production of E. coli was undertaken. The principal technique utilized in these investigations was the polarographic determination of oxygen tension. Investigation of the properties of a commercially available, vibrating-reed, oxygen electrode revealed that silver ions were released from the silver anode of the uncoated oxygen electrode into the buffer solution surrounding the electrode. Loss of silver from the electrode was dependent on the buffer concentration and the type of buffer, and was relatively independent of the presence or absence of the polarizing voltage. It is postulated that the release of silver from the anode of the oxygen electrode involved chelation by the buffer ions. This problem was avoided subsequently by using a Clark oxygen electrode. The pH, buffer ion and the buffer concentration of the assay medium were observed to influence the rate of respiration of E. coli. In addition the buffer ion and the pH influenced the linearity of oxygen consumption with time. Glycylglycine-KOH buffer, pH 7.0, at a concentration of 300 mM was determined as "optimal" according to the criteria of: (i) supporting a high respiratory rate; (ii) supporting a constant rate of oxygen utilization; and (iii) maintenance of these characteristics of the E. coli cell suspension for a greater period of time than required to complete the experiment. The applicability of these criteria of classical enzyme kinetics to the determination of "optimal" conditions for the investigation of systems involved in energy conservation is questioned. During the simultaneous measurement of acid production and oxygen consumption, a 15 to 30 second lag in acid production was observed to occur during the transition from aerobic to anaerobic glucose utilization. This, observation is discussed with respect to the information currently available on the regulation of the amphibolic pathways of E. coli. Silver ions inhibited the oxidation of endogenous substrates, glucose, glycerol, D- and L-lactate, acetate, succinate and fumarate by intact-cell suspensions of E. coli. The oxidation of formate was only slightly inhibited under the conditions which resulted in complete inhibition of respiration on the previously indicated substrates. The oxidation of glucose and glycerol was more sensitive to silver ions than that of D- or L-lactate, fumarate or succinate. This was attributed to inhibition of glyceraldehyde-3-phosphate dehydrogenase. Before the onset of the inhibition by silver ions there was a period when respiration was stimulated. This effect was similar to that given by 2,4-dibromophenol. With both compounds the degree of stimulation was larger in iron-sufficient than in iron-deficient cells. It is postulated that silver ions uncouple respiratory chain-linked energy production as well as inhibiting the respiratory chain and glyceraldehyde-3-phosphate dehydrogenase in E. coli. Growth and cell respiration were affected when iron became limiting in batch cultures of E. coli growing on succinate. A decrease occurred in the efficiency with which succinate was converted to cell mass, in the respiratory control ratio, and in the levels of nonheme iron, cytochrome, and NADH and succinate oxidase activities. On addition of ferric citrate to the iron-limited cultures the above components returned at different rates to the levels found in iron-sufficient cells. The concentration of nonheme iron, the respiratory control ratio and the efficiency of conversion of succinate to cell mass recovered more rapidly than.the level of cytochrome b₁ and the oxidase activities. Succinate oxidase activity recovered more rapidly than either succinate dehydrogenase or cytochrome b₁ levels. It is postulated that nonheme iron is involved in respiratory chain-linked energy production in E. coli.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat