Neural circuit analysis of the dorsal nucleus of the lateral lemniscus and new viral approaches to neural circuit analysis in Mongolian gerbils

Auditory stimuli are processed by several parallel and serial neural circuits in the auditory brainstem. In the first part of this PhD thesis, synaptic integration of excitatory inputs in the neural network of the dorsal nucleus of the lateral lemniscus (DNLL) in Mongolian gerbils is investigated. The second part of this study analyses the feasibility of the use of viral vectors in Mongolian gerbils. This work aims to add to the available methods for neural circuit analysis in these animals by establishing tools for genetic manipulation. The DNLL receives excitatory inputs from the superior olivary complex (SOC) and provides GABAergic inhibition to its contralateral counterpart and both inferior colliculi (ICs). This GABAergic inhibition can outlast the triggering auditory stimulus by tens of milliseconds and thus differs substantially from the fast glycinergic inhibition prevailing in the SOC. It is thought that this persistent inhibition (PI) suppresses further processing of sound source information cues of lagging sounds, thereby providing the neuronal basis for sound localisation in reverberant environments. The mechanisms which PI is generated are still under debate. One hypothesized mechanism focuses on the output mechanism in DNLL neurons, favouring transmitter spillover or asynchronous release to evoke PI. A second mechanism states that integration of excitatory inputs leads to temporally extended activity in DNLL neurons, thereby prolonging the GABAergic output. Here, we tested in vitro the feasibility of the integration based mechanism in Mongolian gerbils. We analyzed the integration of excitatory inputs to DNLL neurons and found that five simultaneously stimulated excitatory fibres, each releasing on average ~18 vesicles are sufficient to trigger a single action potential (AP) in a DNLL neuron. A strong presynaptic stimulation pulse could trigger multiple APs. The input-output functions (IO-Fs) of DNLL neurons were dependent on NMDA receptor (NMDAR) mediated currents, which temporally extended the neuron's activity. The synaptic IO-Fs of DNLL neurons could also be modulated by voltage gated potassium, but not by calcium conductances.The NMDAR dependent activity amplification, which is maintained into adult stages, is shown to prolong the GABAergic output of DNLL neurons, thus contributing to PI generation. Viral vectors are widely used to alter the genetic content of a host organism. In Mongolian gerbils this approach may be suitable to compensate for the lack of genetic strategies in neural circuit analysis such as transgenic animal lines. Lentiviral and Semliki forest viral vectors were stereotactically injected into the IC or the medial nucleus of the trapezoid body (MNTB) in Mongolian gerbils. The lentiviral constructs were able to induce expression of the transgenic protein in the IC but not in MNTB principal neurons. The Semliki forest viral vector induced expression in both nuclei but also caused strong cytotoxic effects in the infected cells. In a further experiment, an eGFP expressing pseudorabies virus based on the attenuated Bartha strain (PRV-152) was stereotactically injected into the IC and was able to retrogradely infect the nuclei of the auditory brain stem in juvenile and adult Mongolian gerbils. PRV-152 spread synaptically to 2nd order neurons by ~20 hours after injection. Infection could also be started in the DNLL and showed a strongly pronounced neurotropism. The virus induced eGFP expression was high and allowed for a detailed visualization of the infected neurons, establishing PRV-152 as an effective tool for anatomical circuit analysis. The feasibility of using this virus in conjunction with electrophysiological investigations was also tested. 37% of 1st and 78% of 2nd order infected neurons show a significant decrease of excitability, which impedes the use of PRV-152 in combination with electrophysiological recordings for physiological analysis of neural circuits.