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.