PRELIMINARY NOTES 231 PN 4 1 0 0 8

Extracellular pathways in the peripheral nerve fibres: Schwann-cell-layer permeability to thorium dioxide

The structural and functional individuality of the neuronal and glial cell plasma membranes, axon and Schwann cell included, have been demonstrated in previous works 1-3. The functional significance of the space between adjacent plasma mem- branes in the nerve tissue, especially in the squid giant nerve fibre, has been studied 4. Any ion or molecule entering or leaving a cell passes through an extracellular space. The light space observed between adj acent plasma membranes in electron micrographs of osmium- and permanganate-fixed nerve tissue represents a real extracellular space. The role of these spaces as communicat ing pathways in the nervous system, between

Fig. 1. E lec t ron mic rog raph of a cross sect ion of the squid g i a n t nerve fibre shows the endo- n e u r i u m (E), the Schwann-ce l l l aye r (SC) and the axon (A). The e n d o n e u r i u m represen ts the b u l k of the ex t r ace l lu l a r space. Tho r ium d iox ide pa r t i c l e s are observed congrega ted a t the endo- neur ium, becoming scarce across the t h i c k base- m e n t m e m b r a n e (bm). Par t i c les are also seen in the l u m e n of the channels (ch) t h a t cross the Schwann-ce l l l aye r f rom i ts ou te r surface to the inner surface, and in the space s epa ra t i ng the a x o l e m m a (ax) f rom the Schwann-ce l l inner p l a s m a membrane . Some of the t h o r i u m d iox ide pa r t i c l e s are i nd i ca t ed by arrows. Scale ~ o. i /z.

Fig. 2. E lec t ron mic rog raph of a cross sect ion of a smal l squ id nerve fibre shows the endo- ne u r i um (E), the sa te l l i t e Schwann cell (SC) and the axon (A). The mesaxon (mx) is made by the appos i t ion of the p l a s m a m e m b r a n e s b o u n d i n g the two ends of the s h e a t h i n g Schwann cell. T h o r i u m d iox ide par t ic les are seen filling the mesaxon gap and the space s epa ra t i ng the Schwann-ce l l p l a s m a m e m b r a n e

from the a x o l e m m a (ax). Scale = o.~ u.

Biochim. Biophys. Acta, 88 (1964) 231-233

232 PRELIMINARY NOTES

Fig. 3. Electron micrograph of par t of a node of Ranvier in a myelinated fibre of the frog sciatic nerve. A typical pa t te rn of myelin (my) is indicated. The axon (A) is shown covered by the Schwann-cell (SC) layer except at the region seen in the upper r ight corner of the micrograph where only the axo lemma (ax) separates the axoplasm from the endoneur ium (E). The endo- neur ium represents the bulk of the extracellu- lar space. At this region the dense particles of t ho r ium dioxide penetra te among the finger- like projections of the Schwann cell to reach

the axolemlnal surface. Scale = o.i tt.

Fig. 4. Electron micrograph of pa r t of a bundle of unmyel inated fibres of the frog sciatic nerve wi th the endoneur ium (E), Schwann cell (SC) and axon (A). Thor ium dioxide particles are seen congregated in the endoneurium. Particles are also seen in the mesaxon (rex) gap and in the space separat ing the Schwann-cell p lasma membrane from the axolemma (ax). Scale =

0.I ~.

the bulk of the extracellular fluid and the cells, has been established in the squid nerve fibre.

The present communication deals with the permeability to thorium dioxide of the spaces separating adjacent plasma membranes in the peripheral nerve fibres of the squid and the flog.

Isolated stellar nerves, including the giant fibre, of the squid Dorvteuthis plei and sciatic nerves of the frog Rana pipiens deprived of epineurium, were incubated for 2 or 3 h in their respective physiological solutions, to which Thorotrast (a sus- pension of thorium dioxide containing 25 % of ThO2) was added. Artificial sea water 5 was used for the squid nerves and Ringer-Conway medium 6 for the frog nerves. The final concentrations of thorium dioxide in the artificial sea water and Ringer-Conway solution were 9 and 8 °o respectively. The NaC1 concentration of the solutions was adjusted to maintain normal osmolarity. At the end of the incubation period the

Biochim. Bioph3,s..4cla, 88 (1064) 23i-233

PRELIMINARY NOTES 233

nerves were fixed in ice-cold, buffered, I o / o s m i u m tetroxide prepared in the respec- t ive physiological solution, and embedded in methacrylate. Thin sections were made in a LKB Ultratome and observed in a Siemens Elmiskop I.

The results are shown in Figs. 1- 4. As observed in the electron micrographs the thorium dioxide particles are found in (a) the Schwann cell channels and the a x o n - Schwann-cell space of the squid giant nerve fibres, Fig. r; (b) the mesaxon gap and the axon-Schwann-cel l space of the medium and small size unmyelinated fibres of the squid, Fig. 2; (c) the mesaxon gap and the axon-Schwann-cel l space of the un- myelinated fibres of the flog, Fig. 3; and (d) the Ranvier-node gap of the frog myelinated fibre, Fig. 4. The restriction offered by the basement membrane of the squid giant fibre to the diffusion of thorium dioxide is clearly shown in Fig. I.

Besides confirming our earlier observations on the structure and permeabili ty of the Schwann cell channels and the axon-Schwann-cel l space of the giant nerve fibre of the squid, the present results permit us to generalize on the basic aspects of those findings to the mesaxon gaps and the axon-Schwann-cell space of other unmyelinated fibres and the Ranvier-node gaps of the myelinated nerve fibres.

Departamento de Bioflsica, Instituto Venezolano de Investigaciones Cientificas,

Caracas (Venezuela)

GLORIA M. VILLEGAS

RAIMUNDO VILLEGAS

1 R. VILLEGAS AND G. M. VILLEGAS, J. Gen. Physiol., Suppl. 5, 43 (196o) 73- 2 a . M. VILLEGAS AND R. VILLEGAS, J. Ullrastructure Res., 8 (1963) 89. 3 R. VILLEGAS, L. VILLEGAS, M. GIM/;NEZ AND G. M. VILLEGAS, J. Gen. Physiol., 46 (1963) lO47. 4 R. ¥ILLI~GAS, C. CAPUTO AND L. VILLI;GAS, J . Gen. Physiol., 46 (1962) 245. 5 A. L. HODGKIN AND B. KATZ, J. Physiol., lO8 (1949) 37. 6 p. j . BOYLE AND E. J. CONWAY, J. Physiol., ioo (1941) i.

Received April I6th, 1964 Biochim. Biophys. Acta, 88 (1964) 231-233

r N 410o 9

Electric moments and cholinesterase inhibitory properties of selected N-alkyl substituted amides

The mechanism by which a molecule functions as an enzyme inhibitor is clearly related to the at tract ion which the enzyme surface and inhibitor molecule have for each other 1. These attractions are electrostatic in nature regardless of their classification (van der Waals forces, ionic charge attraction, etc.). Furthermore, the at tractive force is analogous to the force, F, between two point charges e 1 and e 2 separated by a distance r (Coulomb's Law, 1784; Eqn. I where k is a constant). These attractive

F = kele~/r ~ (I)

forces, therefore, are a quanti tat ive function of the charge distributions at the enzyme site (e.g., anionic, esteratic 2) and at the corresponding location (e.g., amide group, ester group, quaternary nitrogen, etc.) of a t tachment of the inhibitor molecule. Such charge distributions may be studied by electric-dipole-moment analyses and, there-

Biochim. Biophys. Acta, 88 (1964) 233-235