Distribution and densitometry mapping of L1-CAM immunoreactivity in the adult mouse brain--light microscopic observation

Abstract

Background: The importance of L1 expression in the matured brain is suggested by physiological and behavioral studies showing that L1 is related to hippocampal plasticity and fear conditioning. The distribution of L1 in mouse brain might provide a basis for understanding its role in the brain.

Results: We examined the overall distribution of L1 in the adult mouse brain by immunohistochemistry using two polyclonal antibodies against different epitopes for L1. Immunoreactive L1 was widely but unevenly distributed from the olfactory bulb to the upper cervical cord. The accumulation of immunoreactive L1 was greatest in a non-neuronal element of the major fibre bundles, i.e. the lateral olfactory tract, olfactory and temporal limb of the anterior commissure, corpus callosum, stria terminalis, globus pallidus, fornix, mammillothalamic tract, solitary tract, and spinal tract of the trigeminal nerve. High to highest levels of non-neuronal and neuronal L1 were found in the grey matter; i.e. the piriform and entorhinal cortices, hypothalamus, reticular part of the substantia nigra, periaqueductal grey, trigeminal spinal nucleus etc. High to moderate density of neuronal L1 was found in the olfactory bulb, layer V of the cerebral cortex, amygdala, pontine grey, superior colliculi, cerebellar cortex, solitary tract nucleus etc. Only low to lowest levels of neuronal L1 were found in the hippocampus, grey matter in the caudate-putamen, thalamus, cerebellar nuclei etc.

Conclusion: L1 is widely and unevenly distributed in the matured mouse brain, where immunoreactivity was present not only in neuronal elements; axons, synapses and cell soma, but also in non-neuronal elements.

Figure 1

Characterization of antibodies by western blotting, absorption testing and blocking with epidermal growth factor. a. The neuropil fraction (see Materials and Methods) obtained from mouse hippocampus (lanes 1, 2), the L1-transfected cell lysate (lane 3), and the mock-transfected cell lysate (lane 4) were western blotted using the antiFLL1 (lane 1) and antiCTL1 (lanes 2–4) antibodies. AntiCTL1 antibody could detect the full-length (200-kDa) recombinant L1 in the lysate of transfected High5 insect cells (lane 3), whereas no positive band was detectable in the mock-transfected (control) High5 lysate (lane 4). b. Omission of primary antibody (-antiCTL1) resulted in no immunostaining (substantia nigra). c. Blocking of antiCTL1 antibody with epidermal growth factor (EGF) did not interfere with the immunostaining in the neighboring section of (b). d-f,d'-f'. Absorption of the antibody with L1 gene-transfected membrane (L1m) completely eliminated the immunostaining, whereas absorption of the antibody with mock-transfected membrane (Mock) had no effect (d'-f'). d,d': stria terminalis; e,e': substantia nigra; f,f': cerebellar cortex. b-e,d',e': Bar= 200 μm; f,f': Bar = 50 μm.

Figure 2

L1 immunoreactive neurons and transection of stria terminalis. a,a' and b,b'. Transection of the stria terminalis resulted in no accumulation of L1 on the proximal side of the nerve (a'; L1 knife cut), but caused accumulation in the case of substance P axons (b', SP knife cut, open arrowheads). L1 control; control side of the same section immunostained with antiCTL1 antibody. SP control; control side of the same section immunostained with antiSP antibody. c,c' and d,d' No change was found on L1 in the distal side of the lesion of the bundle (c'; L1 knife cut), though substance P immunoreactivity was clearly diminished (d'; SP knife cut, open arrowheads). e and f, Bright-field photomicrograph showing L1 neurons in the lateral posterior thalamic nucleus of the upper brainstem. a,a',b,b': Bar = 25 μm; c,c',d,d',e: Bar = 200 μm; f: Bar = 50 μm.

Figure 3

Pseudocolor images of L1 immunoreactive structures in the forebrain and upper brainstem Pseudocolors were as shown in Fig. 4. The cytoarchitecture in the same areas of neighboring sections is presented on the right side as mirror images (a-i). The asterisk that appears in (i) shows a false positive that occurred in areas damaged by tissue processing. Abbreviations are given in the list of abbreviations used. Bar = 1 mm.

Figure 4

Pseudocolor images of L1 immunoreactive structures in the cerebellum, lower brainstem and upper cervical spinal cord. Pseudocolors were designated as red (highest), yellow (high), green (moderate), light blue (low), blue (lowest), and dark blue (none) in order of the density of brown oxidized DAB product. The real L1 densities of each pseudocolor were presented (right bottom). The cytoarchitecture in the same areas of neighboring sections is presented on the right side as mirror images (j-p q, q', r, r'). The asterisks show a false positive caused by damaged tissue. Bar = 1 mm.

Figure 5

L1 immunoreactive structures in the forebrain. a-j, Bright-field photomicrographs showing L1 structures in the olfactory bulb (a and b), corpus callosum (c), cerebral cortex (d), piriform cortex (e), entorhinal cortex (f), caudate putamen (g and h), and globus pallidus (i and j). A magnified view of the square in (a) is shown in the insert. b, h, and j represent higher magnification photomicrographs of the olfactory bulb, caudate putamen, and globus pallidus. a,c-f: Bar = 250 μm; insert in a: Bar = 50 μm; b: Bar = 200 μm; g,i: Bar = 500 μm; h,j: Bar = 50 μm.

Figure 6

L1 immunoreactive structures in the limbic areas. a-f. Bright-field photomicrographs showing the preoptic area of the hypothalamus (a and b), bed nucleus of stria terminalis (c), stria terminalis (c and d), hippocampus (e), and amygdaloid complex (f). A higher magnification photograph of CA3 which was immunoreacted with antiFLL1 antibody is shown in the insert in (e). Note that better staining for proximal axons was found in CA3 than with antiCTL1 antibody. a: Bar = 1 mm; b-f: Bar = 500 μm; insert in e: Bar = 100 μm.

Figure 7

L1 immunoreactive structures in the posterior hypothalamus, midbrain, and pons. a-h. Bright-field photomicrographs showing low power micrographs of the midbrain (a), posterior hypothalamus (b), ventral tegmental area (c), and reticulate part of substantia nigra (d), and higher magnification photographs of the substantia nigra (e), periaqueductal grey (f), central superior nucleus of the raphe (g), interpeduncular and pontine nucleus (h). a: Bar = 1 mm; b-d,f-h: Bar = 500 μm; e: Bar = 50 μm.

Figure 8

L1 immunoreactive structures in the cerebellum, lower brainstem, and upper cervical spinal cord. a-g. Bright-field photomicrographs showing low power micrographs of the cerebellum and lower brainstem (a), and cerebellar cortex (b), and higher magnification photographs of the cerebellar cortex (c), the solitary tract nucleus (d), caudal of photograph d (e), the trigeminal spinal nucleus (f), and the dorsal horn of cervical spinal cord (g). a: Bar = 1 mm; b,d,f: Bar = 500 μm; c: Bar = 100 μm; e,g: Bar = 200 μm.