Study of the different stages of postnatal neurogenesis relies on using antigenic markers and transgenic mice. In particular, neural stem cells that express GFAP are studied using mice expressing GFP under the human GFAP promoter (GFAP-GFP). However, it remains unclear whether GFP and the commonly used progenitor markers label different cell populations in the neurogenic subventricular zone (SVZ) and its rostral extension into the olfactory bulb (i.e. rostral migratory stream, RMS). Here, we found that all GFP-fluorescent cells express GFAP, the radial glia marker brain lipid-binding protein (BLBP), Lewis X (LeX), and the astrocytic marker GLAST. Faint GFP fluorescence could be detected in a few cells expressing EGF receptors (EGFRs), Olig2, or S100, suggesting that GFAP-GFP cells generate these diverse cell types. GFP-fluorescent cells were slowly cycling, as shown by their long-term retention of BrdU, and less than 10% expressed the proliferative markers Ki67 and Mcm2. The majority of EGFR-expressing cells and Olig2-expressing cells were cycling. NG2 and EGFR identified distinct progenitor populations while Olig2 labeled a subset of EGFR-expressing cells. The entire neurogenic zone contained a mosaic of different cell types and was ensheathed by processes of GFAP-expressing cells and NG2 cells. Finally, using time-lapse imaging in acute slices, we show that GFP-fluorescent cells are stationary within the SVZ. Our findings collectively highlight the cellular mosaic of the neurogenic niche, show that the slowly-cycling GFAP-expressing cells are stationary and generate distinct intermediate progenitors.

Growing and regenerating axons need to interact with the molecules in the extracellular matrix as they traverse through their environment. An important group of receptors that serve this function is the integrin superfamily of cell surface receptors, which are evolutionarily conserved ␣␤ heterodimeric transmembrane proteins. The function of integrins is controlled by regulating the affinity for ligands (also called "integrin activation"). Previous results have shown that CNS inhibitory molecules inactivate axonal integrins, while enhancing integrin activation can promote axon growth from neurons cultured on inhibitory substrates. We tested two related molecules, kindlin-1 and kindlin-2 (Fermitin family members 1 and 2), that can activate ␤1, ␤2, and ␤3 integrins, for their effects on integrin signaling and integrin-mediated axon growth in rat sensory neurons. We determined that kindlin-2, but not kindlin-1, is endogenously expressed in the nervous system. Knocking down kindlin-2 levels in cultured sensory neurons impaired their ability to extend axons, but this was partially rescued by kindlin-1 expression. Overexpression of kindlin-1, but not kindlin-2, in cultured neurons increased axon growth on an inhibitory aggrecan substrate. This was found to be associated with enhanced integrin activation and signaling within the axons. Additionally, in an in vivo rat dorsal root injury model, transduction of dorsal root ganglion neurons to express kindlin-1 promoted axon regeneration across the dorsal root entry zone and into the spinal cord. These animals demonstrated improved recovery of thermal sensation following injury. Our results therefore suggest that kindlin-1 is a potential tool for improving axon regeneration after nervous system lesions.

In the postnatal subventricular zone (SVZ), neuroblasts migrate in chains along the lateral ventricle towards the olfactory bulb. AMPA/kainate receptors as well as metabotropic glutamate receptors subtype 5 (mGluR5) are expressed in SVZ cells. However, the cells expressing these receptors and the function of these receptors remain unexplored. We thus examined whether SVZ neuroblasts express mGluR5 and Ca 2+ -permeable kainate receptors in mouse slices. Doublecortin (DCX)-immunopositive cells (i.e. neuroblasts) immunostained positive for mGluR5 and GLU K5−7 -containing kainate receptors. RT-PCR from ∼10 GFP-fluorescent cell aspirates obtained in acute slices from transgenic mice expressing green fluorescent protein (GFP) under the DCX promoter showed mGluR5 and GLU K5 receptor mRNA in SVZ neuroblasts. Patch-clamp data suggest that ∼60% of neuroblasts express functional GLU K5 -containing receptors. Activation of mGluR5 and GLU K5 -containing receptors induced Ca 2+ increases in 50% and 60% of SVZ neuroblasts, respectively, while most neuroblasts displayed GABA A -mediated Ca 2+ responses. To examine the effects of these receptors on the speed of neuroblast migration, we developed a whole-mount preparation of the entire lateral ventricle from postnatal day (P) 20-25 DCX-GFP mice. The GABA A receptor (GABA A R) antagonist bicuculline increased the speed of neuroblast migration by 27%, as previously reported in acute slices. While the mGluR5 antagonist MPEP did not affect the speed of neuroblast migration, the homomeric and heteromeric GLU K5 receptor antagonists, NS3763 and UB302, respectively, increased the migration speed by 38%. These data show that although both GLU K5 receptor and mGluR5 activations increase Ca 2+ in neuroblasts, only GLU K5 receptors tonically reduce the speed of neuroblast migration along the lateral ventricle.

Animals selectively respond to environmental cues associated with food reward to optimize nutrient intake. Such appetitive conditioned stimulus-unconditioned stimulus (CS-US) associations are thought to be encoded in select, stable neuronal populations or neuronal ensembles, which undergo physiological modifications during appetitive conditioning. These ensembles in the medial prefrontal cortex (mPFC) control well-established, cue-evoked food seeking, but the mechanisms involved in the genesis of these ensembles are unclear. Here, we used male Fos-GFP mice that express green fluorescent protein (GFP) in recently behaviorally activated neurons, to reveal how dorsal mPFC neurons are recruited and modified to encode CS-US memory representations using an appetitive conditioning task. In the initial conditioning session, animals did not exhibit discriminated, cue-selective food seeking, but did so in later sessions indicating that a CS-US association was established. Using microprism-based in vivo 2-Photon imaging, we revealed that only a minority of neurons activated during the initial session was consistently activated throughout subsequent conditioning sessions and during cue-evoked memory recall. Notably, using ex vivo electrophysiology, we found that neurons activated following the initial session exhibited transient hyperexcitability. Chemogenetically enhancing the excitability of these neurons throughout subsequent conditioning sessions interfered with the development of reliable cue-selective food seeking, indicated by persistent, nondiscriminated performance. We demonstrate how appetitive learning consistently activates a subset of neurons to form a stable neuronal ensemble during the formation of a CS-US association. This ensemble may arise from a pool of hyperexcitable neurons activated during the initial conditioning session.

It is well established that cerebellar granule cell precursors (GCPs) initially derive from progenitors in the rhombic lip of the embryonic cerebellar primordium. GCPs proliferate and migrate tangentially across the cerebellum to form the external granule cell layer (EGL) in late embryogenesis and early postnatal development. It is unclear whether GCPs are specified exclusively in the embryonic rhombic lip or whether their precursor persists in the neonate. Using transgenic mice expressing DsRed under the human glial fibrillary acidic protein (hGFAP) promoter, we found 2 populations of DsRed + cells in the EGL in the first postnatal week defined by bright and faint DsRed fluorescent signal. Bright DsRed + cells have a protein expression profile and electrophysiological characteristics typical of astrocytes, but faint DsRed + cells in the EGL and internal granule cell layer (IGL) express markers and physiological properties of immature neurons. To determine if these astroglial cells gave rise to GCPs, we genetically tagged them with EGFP or βgal reporter genes at postnatal day (P)3-P5 using a hGFAP promoter driven inducible Cre recombinase. We found that GFAP promoter + cells in the EGL are proliferative and express glial and neural stem cell markers. In addition, immature granule cells (GCs) en route to the IGL at P12 as well as GCs in the mature cerebellum, 30 days after recombination, express the reporter protein, suggesting that GFAP promoter + cells in the EGL generate a subset of granule cells. The identification of glial cells which function as neuronal progenitor cells profoundly impacts our understanding of cellular plasticity in the developing cerebellum.