Inhibitory collaterals in genetically identified medium spiny neurons in mouse primary corticostriatal cultures

Abstract

Inhibitory collaterals between striatal medium spiny neuron (MSN) subtypes have been shown to critically influence striatal output. However, the low rate of inhibitory collateral detection between striatal MSNs in conventional ex vivo slice recordings has made the study of these connections challenging. Furthermore, most studies on MSN collaterals have been conducted either blind or in models, in which only one MSN subtype can be distinguished. Here, we describe a dissociated culture system using striatal and cortical neurons harvested from genetically modified mice at postnatal day 0. These mice express tdTomato and enhanced green fluorescent protein (EGFP) downstream of the dopamine D1 and D2 receptor promoters, respectively, allowing for simultaneous distinction between the two major subtypes of MSNs. In vitro, these neurons develop spines, hyperpolarized resting membrane potentials and exhibit up-and-down states, while also maintaining expression of both fluorophores through time. Using paired whole-cell patch-clamp recordings from identified MSNs at 14 days in vitro, we are able to detect a much higher rate of inhibitory functional synapses than what has been previously reported in slice recordings. These collateral synapses release γ-Aminobutyric acid (GABA) and shape the firing patters of other MSNs. Although reduced in vitro models have a number of inherent limitations, the cultures described here provide a unique opportunity to study frequently observed functional collaterals between identifiable MSNs. Additionally, cultured neurons allow for control of the extracellular environment, with the potential to investigate pharmacological regulation of inhibitory MSNs collaterals.

GABAA receptor activity shapes the formation of inhibitory synapses between developing medium spiny neurons

“…The differences in functional properties of MSNs observed in dissociated primary culture can be attributed to the conditions in which neurons are grown in vitro , with empirically determined concentrations of growth factors and in the absence of endogenous active molecules. Nevertheless, functional properties and synaptic development of MSNs in vitro were similar to those described in more intact preparations as shown previously (Weiss et al, 1986 ; Kowalski et al, 1995 ; Falk et al, 2006 ; Lalchandani and Vicini, 2013 ), suggesting that these processes are largely driven by predetermined genetic programs (Yun et al, 2003 ).…”

Generation of Human Striatal Neurons by MicroRNA-Dependent Direct Conversion of Fibroblasts

“…The functional maturation of our cells from 6 to 12 weeks in monoculture (Figure S12) suggests that perhaps achieving a significantly hyperpolarized resting membrane potential requires longer culturing conditions. Consistently, MSNs from dissociated mouse striatal cultures have been shown to develop more negative membrane potentials with time in vitro (Lalchandani and Vicini, 2013). …”

Foxp2 loss of function increases striatal direct pathway inhibition via increased GABA release

“…However, corticostriatal GABAergic interneurons do not express Foxp2 (Chiu et al 2014; Hisaoka et al 2010). We, therefore, expect differences in inhibitory activity between wild-type and Foxp2 S321X /+ mice through changes in intra-striatal inhibition, which is regulated through MSNs and striatal interneurons (Taverna et al 2008; Lalchandani and Vicini 2013). We measured inhibitory activity only in D1R-MSNs, since unidirectional connections between D1R-MSNs are common, while connections between D1R-MSNs and D2R-MSNs are rare (6%) (Taverna et al 2008).…”