Selective neuronal expression of green fluorescent protein with cytomegalovirus promoter reveals entire neuronal arbor in transgenic mice

Abstract

In simple nervous systems, identified groups of neurons can be studied in depth. To allow the same advantage in the mammalian brain, we have generated green fluorescent protein (GFP) transgenic mice in which only a few types of neurons are strongly labeled with a fluorescent molecule, which the neurons synthesize internally, allowing the cells, their dendrites, filopodia, and axons to be identified in both living and fixed CNS, in slices and culture. The same neurons, with GFP expression controlled by part of the major immediate early promoter of human cytomegalovirus (CMV), show GFP beginning early in development, from one generation to the next, allowing cellular and physiological studies of axonal and dendritic growth, fate mapping, anatomical connections, and synapse formation in identified neurons. The human CMV promoter sequence we used was different from that used in previous work with other reporter genes and gave a dramatically different pattern of expression. Two transgenic lines with the same CMV promoter show similar anatomical patterns of expression in the present study. Strong GFP labeling was found in a subpopulation of mossy fibers that innervated parasagittal bands in the cerebellar cortex and olfactory axons that projected into the olfactory bulb, subsets of motoneurons and dorsal root ganglion cells, granule but not mitral cells of the olfactory bulb, and a group of neurons in the hypothalamic suprachiasmatic nucleus. A novel type of neuron was strongly labeled in the olfactory bulb external plexiform layer. In normal brains, CMV does not constitute a threat, but in the developing brain, CMV can cause debilitating neurodegeneration and death; studies using the CMV promoter aid in understanding the affinity of CMV that has been suggested for specific brain regions.

Fig. 1.

GFP transfected neurons. A, Two neurons with their neurites expressing GFP 2 d after transfection by electroporation. Scale bar, 12 μm. B, A cell with the appearance of a glial cell is seen after GFP transfection. Scale bar, 10 μm.

Fig. 2.

GFP in cell bodies and dendrites.A, Cells labeled with GFP immunoperoxidase in medulla. Scale bar, 20 μm. B, Long spine-like processes show GFP fluorescence, as shown in this example from the granule cell region of the olfactory bulb. Scale bar, 4 μm. C, Under the cerebellum (crb), neurons in the vestibular complex show GFP expression in cell bodies and dendrites. D, Higher magnification of C. Scale bar, 25 μm.E, In the spinal cord, motoneurons in the ventral horn of this sagittal section express GFP. Scale bar, 35 μm.

Fig. 3.

GFP in axons and their terminals.A, In the cochlear nuclei, some axons show strong GFP fluorescence and have terminal boutons. Scale bar, 2 μm.B, In the cerebellar cortex, GFP-expressing mossy fibers in the white matter enter the cerebellar cortex before spreading out to innervate the granule cell region. Scale bar, 10 μm.C–E, Mossy fiber terminals in the granule cell layer after GFP immunoperoxidase staining. Background visualization of granule cells is aided by DIC microscopy. C,Arrows show moss-like shape typical of these fibers.D, A later generation of transgenic mice shows a similar expression of GFP in the mossy fiber. E, A different line of transgenic mice with the same CMV promoter sequence as inC and D shows the same patterns of labeling, as shown here by the labeled mossy fiber. Scale bar (inC): C–E, 5 μm. F, In spinal cord, fibers make terminal boutons (arrows) in the gray matter (GM) after leaving the white matter (WM). Scale bar, 9 μm. G, In dorsolateral cervical spinal cord white matter, long axons can be followed for many millimeters. Scale bar, 5 μm.

Fig. 4.

Parasagittal bands of GFP in cerebellar mossy fibers. In this low-magnification micrograph, GFP-expressing mossy fibers are found in parasagittal bands (arrows) in this coronal section of the cerebellum. Only mossy fibers are labeled here. Scale bar, 35 μm.

Fig. 5.

GFP in axons. A, In a sagittal section of the midbrain, GFP-containing axons detected with immunoperoxidase have many labeled terminal boutons. No cell body labeling is seen in this area. B, In the living dorsal root, GFP fluorescent axons emerging from the DRG (right) are found (arrows). Scale bar, 4 μm. C, Five days after spinal cord dorsal column damage in the transgenic mouse, ascending GFP axons in the dorsal column begin to show small filopodia. GFP axon is immunostained with peroxidase in this horizontal section of midthoracic spinal cord. Scale bar, 4 μm.

Fig. 6.

Northern blot of GFP expression. RNA was extracted from various tissues of transgenic mice and probed on a Northern blot for GFP. The highest level of expression was in the olfactory bulb. Other brain regions showed lower levels of expression.Brain refers to the remaining brain after the other regions in the blot were removed. After stripping, as a control, the blot was probed with cyclophilin to demonstrate the general level of RNA loading in each lane.

Fig. 7.

Olfactory bulb GFP. A, In the olfactory bulb, two unusual neurons (large arrows) showing strong GFP expression were immunostained with GFP immunoperoxidase. Axons (small arrow) can sometimes be followed from the cell body down into the granule cell layer. GL, Glomerular layer; EPL, external plexiform layer; GCL, granule cell layer. Scale bar, 35 μm. B, In olfactory bulb, some glomeruli (long arrows) show strong GFP immunoperoxidase. Other glomeruli (short arrows) show little staining. Scale bar, 70 μm. C, In the olfactory mucosa of a developing mouse on the day of birth, some olfactory receptor cells, together with their sensory dendrite and efferent axons (small arrows), show GFP expression, consistent with the labeling of some glomeruli shown in B. Unlabeled background cells are seen with DIC microscopy. Scale bar, 15 μm.

Fig. 8.

GFP in cultured spinal cord neurons. After 4 d in vitro, a few neurons show strong GFP expression, even at their smallest filopodia. Inset,Arrow shows same small growth process. GFP fluorescence is found in cell bodies and all processes. Other cells in the same field show little fluorescence. Scale bar, 6 μm.