The role of medial amygdala inhibitory neurons in regulating social behavior

Aggression is an evolutionary conserved social behavior that is necessary for an animalÂŽs survival. The neural substrates coordinating aggression are known to exist in the amygdala and the hypothalamus. While the medial amygdala (MeA) and the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) have been implicated in aggression control and are known to be anatomically inter-connected, it has not yet been described at the circuit-level whether an interaction between these nuclei plays a role in aggression control.Posteriodorsal MeA (MeApd) is a majorly GABAergic nucleus (~70%), however the role of heterogeneous sub-populations of the MeA-GABA neurons in aggression is poorly understood. Since we were interested to identify the inhibitory cell types in the MeApd that control aggression, we first attempted to stimulate all the GABA neurons in the MeApd. Using a fast variant of channelrhodopsin-2E123T,H134R (ChETA) in VGATCre mice, we found that optogenetic stimulation of MeA-GABA neurons in the resident males interrupted intruder-directed aggression, contrary to the previous findings by Hong et al., where stimulation of MeApd-GABA neurons using channelrhodopsin-2H134R (ChR2) evoked aggression. An in-depth investigation of this discrepancy, using two AAV serotypes, revealed that optogenetic stimulation with ChETA suppressed aggression, whereas optogenetic stimulation with ChR2 increased aggression, irrespective of the AAV serotype. Recordings of the membrane potential changes reported larger plateau depolarizations, smaller action potential amplitudes, and larger local inhibition when using ChR2 as compared to ChETA. Thus, in the first part I showed that optogenetic stimulation of the brain areas with heterogenous population of interconnected GABA neurons such as the MeApd, can strongly depend on the properties of optogenetic tools.In the second part, we further explored how the MeApd-GABA neurons control aggression via long-range projections to their target nuclei in hypothalamus. Anterograde tracing revealed synapses from the MeApd-GABA neurons in the ventral premammillary (PMv) and the lateral ventromedial hypothalamic nuclei (VMHvl); both nuclei were previously identified in positive control of aggression (Lin et al., 2011; Stagkourakis et al., 2018). Ex-vivo optogenetic mapping of synaptic outputs from the MeApd-GABA projecting axons revealed evoked IPSCs in glutamatergic neurons in the VMHvl and the PMv, suggesting likely mechanism for inhibiting aggression. In-vivo optogenetic activation of the MeApd-GABA axonal terminals above the VMHvl or the PMv had no effect on aggression and instead led to an increased mounting, likely caused by optic fiber implantation in the hypothalamus. To avoid this artifact, we have combined retrograde AAV with Cre ON/Flip ON approach and specifically stimulated the VMHvl projecting MeApd-GABA neurons. This approach revealed that in aggressive mice, stimulation of this pathway tends to reduce aggression. Finally, we performed in-vivo Ca2+ imaging of the VMHvl projecting MeApd-GABA neurons during the RI test, which showed that these neurons are largely multimodal and get activated during different sub-types of social behaviours including aggression.This study shows that direct inhibitory path from the MeApd-GABA neurons to the VMHvl might suppress aggressive behaviour. Altogether, this work provides a step forward in our understanding of the neural substrates underlying aggression.

Motor Control in the Mouse Whisker System

Varun Sreenivasan

The motor systems of the mammalian brain are a remarkable product of many millions of years of evolution. It is the motor systems that make us and other mammals mobile, that guide our actions in response to sensory information and conversely modulate the very nature of the sensory input that our brain receives. The mystacial vibrissae or whiskers present on the face of mice are important sensors that provide high resolution tactile information to the brain. Whereas the pathways that are involved in signaling and processing sensory information from the whiskers have been the subject of intense investigation for many years, we know relatively little about how whisker movements are controlled by the brain. In the first part of the thesis, I present evidence for two parallel pathways originating from the neocortex that control antagonistic movements of the whiskers. I first describe whisker movements elicited upon Channelrhodopsin-2 (ChR2) stimulation of whisker somatosensory cortex (wS1) and whisker motor cortex (wM1). At short latencies, optogenetic stimulation of wS1 drove whisker retraction while optogenetic stimulation of wM1 drove rhythmic whisker protraction. Next, using recent developments in Rabies virus retrograde tracing, I mapped the location of the motor neurons in the facial nucleus, finding two distinct neuron pools for the extrinsic (retractor) muscle Nasolabialis and the intrinsic (protractor) follicular muscle. These distinct motor neuron populations received premotor input from different sources in the brainstem. While the premotor neurons of the extrinsic muscle were located more densely in the spinal trigeminal nucleus (Sp5), the premotor neurons of the intrinsic muscle were located more densely in the reticular formation (Rt). Anterograde tracing from wS1 and wM1 cortex revealed a complementary distribution of axons in the brainstem. Whereas wS1 strongly innervated Sp5, wM1 innervated the Rt. These data therefore begin to unravel the circuitry from the neocortex to the whisker motor neurons. In the second part of the thesis, I investigated the subthreshold and spiking correlates of whisker movements using whole-cell current clamp recordings of individual excitatory neurons in wM1. Brief 1 ms optogenetic stimulation of wS1 elicited rhythmic whisking after long latencies which was abolished upon pharmacological inactivation of wM1. Whole-cell recordings in wM1 during wS1 stimulation showed that the long-range synaptic input from wS1 to wM1 is encoded as a bi-phasic membrane potential response with an early depolarizing post-synaptic potential (Dep. PSP), followed by a hyperpolarizing post-synaptic potential (Hyp. PSP) in individual wM1 neurons. Approximately 150 ms after the wS1 stimulus, the Vm showed a rebound depolarization, which, on most trials, was followed by late rhythmic whisking. Whole-cell recordings during self-generated whisker movements showed that upon whisking, wM1 undergoes a profound cortical state change, where the mean Vm is more depolarized, slow fluctuations of the Vm are reduced and spike rates in layer 5 increase. Aligning the membrane potential trajectories to the onset of whisker movement epochs revealed that changes in Vm occurred almost 100 ms before movement onset. Finally, an examination of the Vm dynamics during rhythmic whisking showed that the Vm of individual wM1 neurons is modulated by the phase of the whisk cycle. This modulation is stronger in layer 2/3 compared to layer 5.

EPFL2015

Target-specific membrane potential dynamics of neocortical projection neurons during goal-directed behavior

Carl Petersen, Takayuki Yamashita

Goal-directed behavior involves distributed neuronal circuits in the mammalian brain, including diverse regions of neocortex. However, the cellular basis of long-range cortico-cortical signaling during goal-directed behavior is poorly understood. Here, we recorded membrane potential of excitatory layer 2/3 pyramidal neurons in primary somatosensory barrel cortex (S1) projecting to either primary motor cortex (M1) or secondary somatosensory cortex (S2) during a whisker detection task, in which thirsty mice learn to lick for water reward in response to a whisker deflection. Whisker stimulation in 'Good performer' mice, but not 'Naive' mice, evoked long-lasting biphasic depolarization correlated with task performance in S2-projecting (S2-p) neurons, but not M1-projecting (M1-p) neurons. Furthermore, S2-p neurons, but not M1-p neurons, became excited during spontaneous unrewarded licking in 'Good performer' mice, but not in 'Naive' mice. Thus, a learning-induced, projection-specific signal from S1 to S2 may contribute to goal-directed sensorimotor transformation of whisker sensation into licking motor output.

Elife Sciences Publications Ltd2016

Motor Control in the Mouse Whisker System

Varun Sreenivasan

EPFL2015