Confocal laser scanning microscopy and ultrastructural study of VGLUT2 thalamic input to striatal projection neurons in rats

Abstract

We examined thalamic input to striatum in rats using immunolabeling for the vesicular glutamate transporter (VGLUT2). Double immunofluorescence viewed with confocal laser scanning microscopy (CLSM) revealed that VGLUT2+ terminals are distinct from VGLUT1+ terminals. CLSM of Phaseolus vulgaris-leucoagglutinin (PHAL)-labeled cortical or thalamic terminals revealed that VGLUT2 is rare in corticostriatal terminals but nearly always present in thalamostriatal terminals. Electron microscopy revealed that VGLUT2+ terminals made up 39.4% of excitatory terminals in striatum (with VGLUT1+ corticostriatal terminals constituting the rest), and 66.8% of VGLUT2+ terminals synapsed on spines and the remainder on dendrites. VGLUT2+ axo-spinous terminals had a mean diameter of 0.624 lm, while VGLUT2+ axodendritic terminals a mean diameter of 0.698 µm. In tissue in which we simultaneously immunolabeled thalamostriatal terminals for VGLUT2 and striatal neurons for D1 (with about half of spines immunolabeled for D1), 54.6% of VGLUT2+ terminals targeted D1+ spines (i.e., direct pathway striatal neurons), and 37.3% of D1+ spines received VGLUT2+ synaptic contacts. By contrast, 45.4% of VGLUT2+ terminals targeted D1-negative spines (i.e., indirect pathway striatal neurons), and only 25.8% of D1-negative spines received VGLUT2+ synaptic contacts. Similarly, among VGLUT2+ axodendritic synaptic terminals, 59.1% contacted D1+ dendrites, and 40.9% contacted D1-negative dendrites. VGLUT2+ terminals on D1+ spines and dendrites tended to be slightly smaller than those on D1-negative spines and dendrites. Thus, thala-mostriatal terminals contact both direct and indirect pathway striatal neurons, with a slight preference for direct. These results are consistent with physiological studies indicating slightly different effects of thalamic input on the two types of striatal projection neurons.

Convergence of cortical and thalamic input to direct and indirect pathway medium spiny neurons in the striatum

“…Our data demonstrates that MSNs receive a higher proportion of their glutamatergic innervation from cortical as opposed to thalamic terminals (see also Lei et al 2013). However, there was no difference between the pattern of innervation of the two subtypes of MSNs.…”

Effect of early embryonic deletion of huntingtin from pyramidal neurons on the development and long-term survival of neurons in cerebral cortex and striatum

“…To study the laminar organization of cerebral cortex, we used immunolabeling with a mouse monoclonal antibody (Sigma-Aldrich, C89848) to detect the lightly labeled calbindinergic neurons defining layers 2–3 (Van Brederode et al, 1991; Kondo et al, 1997; Fauser et al, 2013), a rabbit polyclonal antibody (Sigma/Aldrich, V2514) to detect VGLUT2+ fibers in layer 4 (Deng et al, 2013), the SMI-32 mouse monoclonal antibody (Covance, SMI-32R) and a rat monoclonal antibody against Ctip2 (Abcam, AB18465) to define neurons in layer 5 (Özdinler et al, 2011; Fauser et al, 2013), and a rabbit polyclonal antibody against FoxP2 (Abcam, AB16046) to define neurons in layer 6 (Özdinler et al, 2011). The specificity of these antibodies for their target antigens has been demonstrated by the manufacturer by Western blot and in published studies (Stillman et al, 2009; Hirano et al, 2011; Hashimoto et al, 2012; Huang et al, 2012; Lei et al, 2013). …”

The ins and outs of the striatum: Role in drug addiction

“…The second largest source of striatal glutamate originates in the thalamus (Lei et al, 2013, Wall et al, 2013). Midline and intralaminar thalamic structures, including the paraventricular nucleus, mediodorsal, central median, and parafasicular nuclei, send dense glutamatergic projections to the striatum and synapse directly onto both dMSNs and iMSNs (Berendse and Groenewegen, 1990, Li and Kirouac, 2008, Haber and Calzavara, 2009, Lei et al, 2013, Wall et al, 2013).…”

“…Midline and intralaminar thalamic structures, including the paraventricular nucleus, mediodorsal, central median, and parafasicular nuclei, send dense glutamatergic projections to the striatum and synapse directly onto both dMSNs and iMSNs (Berendse and Groenewegen, 1990, Li and Kirouac, 2008, Haber and Calzavara, 2009, Lei et al, 2013, Wall et al, 2013). Finally, the amygdala and hippocampus provide glutamatergic inputs mostly to ventral striatum and both of these projections appear to synapse primarily on dMSNs (Groenewegen and Trimble, 2007, Britt et al, 2012, MacAskill et al, 2012, Pascoli et al, 2012, Wall et al, 2013, MacAskill et al, 2014).…”