Published: 1 July 2022| Version 1 | DOI: 10.17632/dwr96htx3j.1
Neil McNaughton


Anxiety and panic are both elicited by threat and co-occur clinically. But, at the neural level, anxiety appears to inhibit the generation of panic; and vice versa. Panic and anxiety are thought to engage more posterior/medial (m) and anterior (a) parts of the periaqueductal gray (PAG), respectively. Anxiety also engages the hippocampus and medial prefrontal cortex. Here we tested if mPAG but not aPAG stimulation would suppress prefrontal and hippocampal theta rhythm as do anxiolytic drugs. Twelve male rats with implanted electrodes were stimulated alternately (30 s interval) in the left PAG or right reticular formation (RPO - as a positive control) with recording in the left prelimbic cortex and left and right hippocampus. PAG stimulation was set to produce freezing and RPO to produce 7-8 Hz theta rhythm before tests lasting 10 minutes on each of 5 days. mPAG stimulation decreased, and aPAG increased, theta power at all sites during elicited freezing. mPAG, but not aPAG, stimulation decreased prefrontal theta frequency. Stimulation did not substantially change circuit dynamics (pairwise phase consistency and partial directed coherence). Together with previous reports, our data suggest that panic- and anxiety-control systems are mutually inhibitory, and neural separation of anxiety and panic extends down to the aPAG and mPAG, respectively. Our findings are consistent with recent proposals that fear and anxiety are controlled by parallel neural hierarchies extending from PAG to the prefrontal cortex. The data are provided as Spike2 files. Trigger coding is: StimOnRPO = onset of 1s RPO train; StimOnPAG = onset of 8s PAG train; Inject (downward) = pump on; StimOff = offset of train; StimOn = onset of train; Stim = stimulation pulses. Channel coding is: P = prefrontal; HR = right hippocampus; HL = left hippocampus; digits are electrode numbers. Note that recording was against a common reference, but analysis was of electrode pairs (e.g. the difference of HR2 and HR5 for calculation of a nominal bipolar right hippocampal recording). Voltage values are after amplification x5,000. Sampling rate is 512Hz.


Steps to reproduce

Rats were implanted with one of two different types of recording electrode arrays targeting the left prelimbic cortex (PrL) and the left and right hippocampus (HPC). All had bipolar stimulating electrodes aimed at the right reticularis pontis oralis (RPO) and left periaqueductal grey (PAG) and a guide cannula aimed at the midline nucleus incertus. Recordings were referenced to a common screw electrode behind lambda and, during post-processing, subtraction of an adjacent pair within an array delivered a local "bipolar” signal. Ground was a bare silver wire wrapped around the outside of the implant. Both RPO and PAG stimulating electrodes were 0.005” bipolar twisted stainless-steel wires with a 0.5 mm tip separation. One type of recording array was multipolar (up to 11), with 0.001” diameter nichrome wires. For HPC, 8 tips were spaced 200 µm apart; for PrL, 11 tips were spaced 500 µm apart. For further details see The second type was bipolar or tripolar and built from twisted, PFA-insulated 0.005” stainless steel wires. For the HPC arrays, 2 twisted wires with a tip separation of 0.6mm were aimed at the hippocampal fissure and stratum oriens of CA1. For PrL, 3 twisted wires with a tip separation of 1mm were aimed to span the dorso-ventral extent of the PrL. Stimulating currents for the PAG and RPO electrodes were delivered by a custom-built, constant current isolated stimulator controlled by custom software programmed in Visual Basic 6. Stimulation was set at 100 Hz with monophasic pulses with a width of 0.1 ms. Trains for the RPO stimulation were set to 1 s duration, and PAG trains were set to 8 s maximum duration.First, the current for appropriate RPO responses was established. When animals were immobile, an initial current of 10 µA was delivered in a 1-s train. High-intensity currents can provoke undesired motor reactions in free-moving animals, so during the delivery of the current, the animal’s behavior and real-time LFPs were observed. We defined the ideal response as one where repeated stimulation elicited clear HPC theta during the stimulation train with no evoked motor response. If no induced theta or motor responses were observed, the stimulating current was increased in 5 µA steps until the highest current that caused no motor responses was reached. Following the determination of RPO-stimulation values, the animals were tested for PAG-evoked responses. The goal for the PAG stimulation was to establish the minimum current intensity necessary to induce the emotional response of freezing. Stimulation trains were 8 s long, and the first current was set at 10 A, with increases in 5 µA steps until the desired freezing behavior was evoked. Rats were tested for 10 minutes at the beginning of a single recording session. A 1-s RPO train was delivered, followed 30 s later by an 8-s PAG train and then 30s later the next RPO train, so there were 10 stimulations of each type in total.


University of Otago


Animal Defence, Anxiety, Electroencephalography, Rodent, Research on the Nature of Panic Disorder