Neural coding in the mammalian central nervous system (CNS) relies on the ability of local inhibitory interneurons to filter information propagated by long-range excitatory pathways. Most inhibitory neurons release γ-aminobutyric acid (GABA), although this broad transmitter-based classification encompasses a diverse array of interneuron subtypes that display specialized synaptic features and form connections with distinct post-synaptic targets (Ascoli et al., 2008
). The existence of many GABAergic interneuron subtypes greatly expands the repertoire of inhibitory coding strategies available to CNS circuits (Gupta et al., 2000
; Jonas et al., 2004
). It also poses questions about the rules of specificity that govern the assembly of inhibitory circuits. Suggestions that GABAergic neurons seek out synaptic partners with high precision (Thomson and Morris, 2002
; Stepanyants et al., 2004
) have been countered by proposals that inhibitory networks can assemble in the absence of specificity cues (Li et al., 2007
The plight of GABAergic interneurons reflects a more general uncertainty as to whether synaptic specificity in the mammalian CNS is based on stringent or hierarchical rules of target recognition. The prevailing view is that synaptic specificity is built not upon absolutes that restrict neurons to a single class of synaptic targets, but rather on a hierarchy of synaptic preferences (Sotelo, 1990
; Shen, 2004
). In this view, apparent instances of extreme specificity would belie a neuron’s latent capacity to form connections with secondary targets. Genetic support for the hierarchical model has come primarily from studies in invertebrate nervous systems showing that removal of a preferred target invariably leads to the formation of ectopic synapses with new targets (Cash et al., 1992
; Shen and Bargmann, 2003
). In vertebrates, neurons grown in tissue culture typically form synapses with scant regard for target cell identity (Sanes and Poo, 1989
). Nevertheless in the intact mammalian CNS, it has not yet been possible to perform the invertebrate trick of eliminating a neuron’s preferred target with precision and selectivity. As a consequence, there has been no decisive test of the dominance of stringent or hierarchical rules of recognition specificity during the construction of GABAergic, or indeed, any other mammalian CNS circuits.
Furthermore, synaptic specificity involves more than just target selection. Individual synapses need to be fine-tuned to the demands of the circuits in which they operate (Glickfeld and Scanziani, 2006
). The diversity of GABAergic synapses is apparent in variant terminal morphologies, distinctions in the molecular machinery for exocytosis and, intriguingly, in the existence of dual pathways of GABA production (Kubota and Kawaguchi, 2000
; Monyer and Markram, 2004
). The synthesis of GABA is catalyzed by two distinct glutamic acid decarboxylase (GAD) enzymes. GAD67 is expressed in the cytosol of most GABAergic neurons, and is responsible for the bulk of neuronal GABA synthesis (Asada et al., 1997
). In contrast, GAD65 is expressed by a more restricted set of interneurons (Esclapez et al., 1994
), where it is bound to synaptic vesicles (Jin et al., 2003
). The activity of GAD65 underlies enhanced GABA release at high stimulation frequencies (Tian et al., 1999
), indicating that expression of this enzyme regulates the efficiency of inhibitory synaptic signaling. Defining rules of specificity that govern the construction of GABAergic synapses therefore also requires insight into mechanisms of pre-synaptic specialization and how they are coordinated with the events of target recognition.
The spinal circuits that control motor behavior have long provided a classical system for the exploration of inhibitory interneuron diversity and function. In the spinal cord, inhibitory interneurons assemble into feed-forward, feed-back, and reciprocal circuits that regulate sensory-motor reflexes (Jankowska and Puczynska, 2008
). Motor output is constrained by pre-synaptic inhibition of sensory transmitter release as well as by post-synaptic inhibition of motor neuron excitability (; Windhorst, 1996
; Rudomin, 2009
). The GABAergic synapses that participate in these two modes of inhibition are known to differ in their profile of GAD expression (Hughes et al., 2005
), yet both synaptic classes co-exist within the local microenvironment of the sensory-motor synapse (; Conradi, 1969
). The distinct connectivity and synaptic features of these two related classes of GABAergic interneurons suggested to us that they might provide an informative model system for probing two key issues in synapse assembly. Do neurons select their targets through stringent or hierarchical recognition systems? And what is the role, if any, of the target neuron in inducing the specialized pre-synaptic features that distinguish interneurons?
Two GABAergic inhibitory circuits in the spinal cord
To explore these issues we used molecular genetics to manipulate identified neurons in mouse spinal cord. We find that the connectivity and synaptic features of GABAergic interneurons that mediate pre-synaptic inhibitory control of proprioceptive sensory input are directed by signals provided, selectively, by their sensory terminal targets. In the absence of target sensory terminals, this set of GABAergic neurons refuses to form connections with other available neurons, fails to undergo pre-synaptic differentiation, and eventually retracts their axons from the ventral spinal cord. We also show that brain derived neurotrophic factor (BDNF), a secreted factor emanating from proprioceptive sensory terminals, induces the synaptic localization of GAD65 -- a defining feature of these GABAergic interneurons. Thus, the sensory terminal targets of this specialized set of CNS inhibitory interneurons promote the development of their pre-synaptic partners through stringent, rather than hierarchical, programs of cell recognition.