Endocannabinoid-Independent Retrograde Signaling at Inhibitory Synapses in Layer 2/3 of Neocortex: Involvement of Vesicular Glutamate Transporter 3

Abstract

Recent studies implicate dendritic endocannabinoid release from subsynaptic dendrites and subsequent inhibition of neurotransmitter release from nerve terminals as a means of retrograde signaling in multiple brain regions. Here we show that type 1 cannabinoid receptor-mediated endocannabinoid signaling is not involved in the retrograde control of synaptic efficacy at inhibitory synapses between fast-spiking interneurons and pyramidal cells in layer 2/3 of the neocortex.Vesicular neurotransmitter transporters, such as vesicular glutamate transporters (VGLUTs) 1 and 2, are localized to presynaptic terminals and accumulate neurotransmitters into synaptic vesicles. A third subtype of VGLUTs (VGLUT3) was recently identified and found localized to dendrites of various cell types. We demonstrate, using multiple immunofluorescence labeling and confocal laserscanning microscopy, that VGLUT3-like immunoreactivity is present in dendrites of layer 2/3 pyramidal neurons in the rat neocortex. Electron microscopy analysis confirmed that VGLUT3-like labeling is localized to vesicular structures, which show a tendency to accumulate in close proximity to postsynaptic specializations in dendritic shafts of pyramidal cells. Dual whole-cell recordings revealed that retrograde signaling between fast-spiking interneurons and pyramidal cells was enhanced under conditions of maximal efficacy of VGLUT3-mediated glutamate uptake, whereas it was reduced when glutamate uptake was inhibited by incrementing concentrations of the nonselective VGLUT inhibitor Evans blue (0.5-5.0 M) or intracellular Cl Ϫ concentrations (4 -145 mM). Our results present further evidence that dendritic vesicular glutamate release, controlled by novel VGLUT isoforms, provides fast negative feedback at inhibitory neocortical synapses, and demonstrate that glutamate can act as a retrograde messenger in the CNS.

Comparison of the ontogeny of the vesicular glutamate transporter 3 (VGLUT3) with VGLUT1 and VGLUT2 in the rat retina

“…Ultrastructural electron microscopic studies have also localized vesicle-like structures containing VGLUT3 in the cell bodies of granule cells in the cerebellum, which is consistent with the involvement of VGLUT3 in nonsynaptic modulation by glutamate [5,17]. Ectopic release of synaptic vesicles from glutamatergic neurons has been reported [31], and VGLUT3 has been implicated in retrograde signaling of neurons [19]. This form of perisomatic stimulation could modulate the synchronizing of the action potential firing in adjacent amacrine cells as described for granule cells in the cerebellum [43].…”

GABAergic Signaling at Mossy Fiber Synapses in Neonatal Rat Hippocampus

“…The following antisera were used: a rabbit polyclonal antibody against the N terminus of VGAT (Chaudhry et al, 1998); a guinea pig polyclonal antibody against synthetic peptide from rat VGAT protein (AB585; Chemicon, Temecula, CA) (Harkany et al, 2004); a guinea pig polyclonal antibody against synthetic peptide from rat vesicular glutamate transporter-1 (VGLUT1) protein (Chemicon, AB5905) (Todd et al, 2003); and a rabbit polyclonal antibody against the C-terminal 93 amino acid of zinc transporter 3 (ZnT3) (Palmiter et al, 1996) (kindly provided by Dr. T. B. Cole, University of Washington, Seattle, WA).…”

Wiring and firing neuronal networks: endocannabinoids take center stage

“…During the past decade, several retrograde messengers have been identified (Figure 1) that exhibit robust differences in their speed of affecting synaptic integration, temporal flexibility and efficiency, and spatial precision [2][3][4][5][6][7]. Endocannabinoid (eCB) signaling represents a key retrograde signaling pathway [8] for tuning both homosynaptic and heterosynaptic plasticity [9] in the postnatal brain.…”

“…The spatial confinement of eCB signaling to particular synapse populations supports the concept that neurons may simultaneously express molecular determinants of multiple retrograde signaling systems and possess the capacity for domain-specific recruitment of particular signaling machineries to subsynaptic microterritories along their dendrites. Accordingly, several spatially-segregated retrograde feed-back mechanisms have recently been proposed ( Figure 1): dendritic release of Wnt family ligands regulates neurotransmitter release at cerebellar mossy fiber-granule cell synapses [4]; quantal glutamate release suppresses inhibitory inputs originating from parvalbumin-containing GABAergic basket cells on cortical pyramidal cells [7]; while brain-derived neurotrophic factor (BDNF) released from secretory granules upon afferent stimulation enhances synaptic plasticity of both excitatory and inhibitory cortical synapses [5,6,24]. A striking similarity amongst the Wnt, glutamate and BDNF/TrkB signaling systems is that they play crucial roles during brain development through coordinated control of cellular positioning, identification, and presynaptic and postsynaptic differentiation [4,[25][26][27][28][29].…”