Figure 1 Monoclonal antibodies against agrin recognize molecules concentrated in and stably bound to the synaptic basal lamina at neuromuscular junctions. Top: normal neuromuscular junction in cutaneous pectoris muscle of frog (Rana pipiens). Bottom: site of a neuromuscular junction 3 weeks after damaging the cutaneous pectoris muscle by crushing it. Myofiber, axon terminal, and Schwann cell degenerated and were phagocytized in response to the trauma, but the basal lamina of the myofiber and Schwann cell persisted. Antibody binding in the damaged muscle is localized to the synaptic portion of the myofiber basal lamina and to the Schwann cell basal lamina, presenting a staining pattern identical to that at the normal neuromuscular junction. [The muscles were stained with a mixture of anti-agrin antibodies 3B5 and 5B1. Details of our staining procedures are in Magill-Solc and McMahan, submitted, and Reist et al. 1977.] Scale bar, 1 μm.

Figure 2 shows a frozen cross section from the spinal cord of an 18 d chick embryo that was incubated with an anti-agrin mAb and processed for immunohistochemistry. The motor neurons are clearly stained. Staining of motor neurons was also observed in the electric lobe of the Torpedo's brain, the part of the brain that innervates the electric organ and in the spinal cords of neonatal Torpedos and adult frogs. A search for staining in the lumbosacral region of the chick spinal cord at early developmental strages revealed that motor neurons stained as early as embryonic day 5, when motor neurons in

Figure 2 Anti-agrin antibodies stain the cell bodies of motor neurons. Ventral horn in the spinal cord of an 18 d chick embryo treated with mAb 5B1. Stain is distributed in patches in the motor neuron's cytoplasm and outlines capillaries (arrow). Scale bar, 50 μm.

this region begin to form neuromuscular junctions (Landmesser and Morris 1975).

Regardless of the age or species of animal, the stain in motor neurons, as observed by light microscopy, was concentrated in patches in the cytoplasm and excluded from the nucleus, suggesting that it was associated with cytoplasmic organelles (figure 2). Figure 3-an electron micrograph from a section of an adult frog spinal cord-reveals that the stain was concentrated in the motor neuron's Golgi apparatus.

We observed no neuronal staining with anti-agrin mAbs in regions of the brain and spinal cord that did not contain motor neurons (figure 4). On the other hand, capillaries throughout the CNS were outlined by the stain, as was the surface of the brain and spinal cord (figures 2, 4). Electron microscopy of the frog's spinal cord revealed that the stain associated with capillaries was concentrated in the basal lamina that lies between the capillaries and the endfoot processes of astrocytes. Thus, in the CNS, molecules antigenically similar to agrin are not confined to motor neurons, but motor neurons are distinct among neurons in that they have a high concentration of such molecules in their cell bodies.

We made extracts of the electric lobes of adult Torpedo brains and the spinal cords of adult Torpedos and frogs and 18 d chick embryos. As shown

Figure 3 Anti-agrin mAbs stain stacks of flattened cisternae in motor neurons characteristic of the Golgi apparatus. Cytoplasm of a motor neuron in the ventral horn of a frog spinal cord treated with a mixture of mAbs 3B5 and 5B1. Scale bar, 1 μm. in figure 5, these extracts caused the aggregation of AChRs on cultured chick myotubes. The extract from the electric lobe also caused AChE aggregation (data not shown).

To determine whether the AChR- and AChE-aggregating activities in the electric lobe extracts were antigenically related to agrin, we assayed the ability of anti-agrin antibodies to immunoprecipitate the activities. For these experiments, we used five mAbs, each of which was directed against a different epitope on agrin (Reist et al. 1987). Each of the mAbs immunoprecipitated nearly all of the AChR-aggregating activity from the extracts of electric lobe of Torpedo brain, as they did agrin from electric organ extracts (Reist et al. 1987). Two of these mAbs (the only ones tested) also immunoprecipitated electric lobe AChE-aggregating activity, as expected (data not shown). Most of the AChR-aggregating activity in extracts of the spinal cords of Torpedos, frogs, and chicks was also immunoprecipitated by anti-agrin antibodies. Thus, the AChR- and AChE-aggregating molecules in extracts of the electric lobe and spinal cord are antigenically similar to agrin.

Agrin-Like AChR-Aggregating Activity in Extracts of
Motor Neurons

To learn whether any of the AChR-aggregating activity detected in spinal cord extracts is derived from motor neurons, we separated motor neurons

Figure 4 Selective staining of motor neurons and nonneural structures by anti-agrin mAbs. Cross section of the lumbosacral region of a spinal cord from a 10 d chick embryo incubated with mAb 5B1. Motor neurons and the pial surface of the spinal cord are intensely stained. Capillaries (arrowhead) are lightly stained; compare with intense staining of capillaries at a later stage of development in figure 2. Glial cells and other neurons are not stained. The intensely stained structures outside of the spinal cord are ventral roots; much of the stain is probably in the Schwann cell basal lamina, which is known to stain intensely in the adult (Reist et al. 1987). The lightly stained region (arrow) of the spinal cord extending from the motor neurons to the ventral root was observed at higher magnification to be composed of narrow cell processes having a nearly uniform diameter, probably motor axons. Scale bar, 200 μm.

from other cellular components in the spinal cord of 6 d chick embryos according to the method described by Dohrmann et al. (1986). The separation procedure results in two cellular fractions, one in which more than 95 percent of the cells are motor neurons and the other with no or relatively few motor neurons (Dohrmann et al. 1986). When extracts of each fraction were made and tested for AChR-aggregating activity, we found that the specific activity of the motor neuron-containing fraction was sevenfold greater than that of the nonmotor neuron-containing fraction. Moreover, more than 60 percent of this activity was immunoprecipitated by anti-agrin antibodies. Thus, motor