Nuclear magnetic resonance study of squid giant axon

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131 Biochimica et Biophysica Acta, 630(1980) 131--136 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 21528

N U C L E A R MAGNETIC R E S O N A N C E S T U D Y OF SQUID GIANT A X O N

DONALD C. CHANG and CARLTON F. HAZLEWOOD Departments of Physiology and Pediatrics, Baylor College of Medicine, Houston TX 77030 and Department of Physics, Rice University, Houston, TX 77001 (U.S.A.) (Received January 3rd, 1980)

Key words: NMR; Water state; Axopiasm; (Giant axon)

Summary Using a spin-echo technique, the spin-lattice and spin-spin relaxation times (T1 and T2 ) of water protons in a single nerve fiber (giant axon of squid) were determined. Similar measurements were also carried out on axoplasm extruded from these nerve fibers. It was found that the relaxation times of water protons of b o t h the intact fiber and the extruded axoplasm are approximately equal (and much less than those of a free solution), suggesting that the relaxation times of cellular water are shortened mainly b y water-protein interactions rather than b y water-membrane interactions. The primary purpose of this paper is to report the results of a nuclear magnetic resonance (NMR) s t u d y on the relaxation times of water protons in squid (Loligo pealei) giant axon. As far as we know, this is the first NMR study of water protons in any isolated, single nerve axon. Pulsed NMR techniques have been used extensively during the past few years to study the dynamic molecular motion of water and ions in many biological systems [ 1--5 ]. The general findings are that the relaxation times of water in tissues are significantly shorter than those of bulk water or physiological solutions such as serum. Shortening of the relaxation times can be attributed to ion-water-macromolecular interaction [2]. Other interpretations, however, also have been proposed. It has been suggested that the shortening of relaxation times of cytoplasmic water can be caused b y water-membrane interactions. For instance, Brownstein and Tart [6] suggested that cytoplasmic water is relaxed mainly b y diffusion toward the membrane, the surface of which acts as a relaxation center. It has also been suggested by others that the shortening o f relaxation times is caused b y the inhomogeneity of the magnetic field which arises from the difference in magnetic sus-

132 ceptibility b e t w e e n the membranes and the cytoplasmic water [7--9]. Since the morphologies of most of the biological systems that have been studied previously are complicated, it has been difficult to test the effect of the membrane-water interaction. The squid giant axon offers some advantages as a biological model to evaluate the importance o f the membrane effect. It has t w o features that are particularly important for this study: (a) The morphology of the axon is simple in comparison with other biological cells and tissues [10], it is relatively free of organeUes and the internal membrane systems, such as the endoplasmic reticulum, are sparse [ 1 1 ] . For example, the mitochondria occupies only approx. 1% of total volume and the endoplasmic reticulum, which appears to be concentrated near the axon membrane, occupies only approx. 4 - 5 % of the volume [ 1 1 ] , and (b) the cytoplasm of the axon can be removed from the axon b y the use of a roller technique [12] and this permits one to study the cytoplasm isolated from the surface membrane. In this study, the spin-lattice relaxation time (T1) and the spin-spin relaxation time (T2) of water protons in squid giant axons and axoplasm were measured with a pulsed NMR (spin-echo) technique [13]. TI was determined b y the use o f a magnetization inversion m e t h o d and T2 was determined b y the use of the Carr-Purcell-Meiboom-GiU technique [13--15]. The measurements were made on a portable Spin-Lock CPS2 spectrometer (SpinLock Electronics Co., Port Credit, Ontario, Canada), which is equipped with a permanent magnet. The signal is obtained by using phase-sensitive detection. The resonance frequency was 32 MHz. The squid used in this study were supplied b y the Marine Biological Laboratory of Woods Hole, MA. The axon was cleaned carefully by cutting off the connective tissues and the small fibers that were attached. Care was taken not to scratch the surface of the axon. The axon was blotted gently on a low-ash filter paper to remove the extraceUular water before it was placed in the sample holder inside the NMR spectrometer. The average diameter of the giant axons that were used was approx. 0.5 ram, and the weight of the excised axons was approx. 9 rag. Often it was necessary to pool two axons to make a sample. Even with t w o axons in the sample tube, it was necessary to develop some procedure to enhance the signal-to-noise ratio. Ordinarily, a signal-averaging c o m p u t e r would have been used b u t none was available to us at the Marine Biological Laboratory. In an a t t e m p t to circumvent this problem, a photo-averaging technique was developed. A typical record of the spin~cho measurements, obtained b y multiple exposures, is shown in Fig. 1. Each band in the record consists of a collection of traces which represent the echo signal that was recorded in repeated measurements. Different bands represent the average echos at different time delay, t, which is defined as the time between the 90 ° pulse and the echo. The difference between t in each band is 54 ms. By measuring the rate of decay of the peak amplitude o f each echo as a function of t, one can obtain the spin-spin relaxation time, T2. A sample plot for the measurements of T2 is presented in Fig. 2. The ordinate is the height of each photographically averaged echo on a log scale, and the abscissa is the time delay (t) in seconds. A sample graph of the T~

133

Fig. 1. A s a m p l e r e c o r d o f t h e s p i n - e c h o m e a s u r e m e n t o n w a t e r p r o t o n s o f a g i a n t a x o n . T h e a m p l i r u d e o f t h e t r a n s v e r s e m a g n e t i z a t i o n is d i s p l a y e d as a f u n c t i o n o f t i m e ( 0 . 2 5 m s p e r divis/on). E a c h b a n d is a c o l l e c t i o n o f r e p e a t e d e c h o t r a c e s w h i c h h a v e t h e s a m e t i m e d e l a y . T h e d i f f e r e n c e in t i m e d e l a y b e t w e e n e a c h n e i g h b o r i n g b a n d is 54 m s (see t e x t ) . T h e m e a s u r e m e n t w a s m a d e a t r o o m ternperature.

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Fig. 2. A s a m p l e p l o t o f t h e T I a n d T 2 d a t a o f s q u i d g i a n t a x o n . T h e o r d i n a t e is t h e l o n g l t u d l n a l m a g n e t i z a t i o n (in t h e ease o f T I ) o r t h e t r a n s v e r s e m a g n e t i z a t i o n (in t h e case o f T 2 ) in a s e m i - l o g f a s h i o n w i t h a n a r b i t r a r y scala. T h e abscissa is t i m e in s e c o n d s . T h e s p i n - e c h o e s o f a n a x o p l a s m s a m p l e a l w a y s f o l l o w a s i m p l e e x p o n e n t i a l d e c a y w h i l e t h e e c h o e s o f an a x o n s a m p l e o f t e n d o n o t . Since t h e fast d e c a y i n g p r o t o n signal f r o m t h e c o n n e c t i v e tissue is i l k e y t o i n t e r f e r e w i t h t h e a x o n r e l a x a t i o n t i m e m e a s u r e m e n t , w e c h o s e t o i g n o r e t h e fizst e c h o in t h e d e t e r m i n a t i o n of T~. U s i n g s u c h a p r o c e d u r e , w e o f t e n f o u n d t h e d e c a y of t h e a x o n fits a single e x p o n e n t i a l f u n c t i o n .

134 TABLE I SUMMARY OF NMR MEASUREMENTS All v a l u e s are m e a n + S.D. w h e r e m o r e t h a n o n e d e t e m z i n a t i o n w a s m a d e . N u m b e r o f s a m p l e s s t u d i e d is g i v e n in t h e p a r e n t h e s i s f o l l o w i n g t h e s a m p l e i d e n t i f i c a t i o n . T h e m e a n i n g o f t h e R e m a r k s is discuss e d in t h e t e x t . Sample

T 1 (s)

A x o n (6) A x o n (2) A x o n (2) Axoplasm (3) A x o p l a s m (1) ' M e m b r a n e ' (2) Sea w a t e r (3)

1.49 1.64 1.48 1.50 1.29 0.178 2.95

T 2 (s) + + + +

0.05 0.04 0.01 0.14

+ 0.026 ± 0.15

0.348 0.390 0.322 0.326 0.301 0.014 1.83

Remarks + + + +

0.036 0.003 0.020 0.025

Fresh sample Non-fresh sample 'Depolarized' axon Fresh s a m p l e 4-days-old

+ 0.005 + 0.09

measurement also is shown in Fig. 2. The relaxation times, as determined on the different samples, are summarized in Table I. The axon samples that are labelled 'fresh' were those in which the TI and T2 measurements were made immediately after the dissection. The axon samples labelled 'depolarized' were p u t into a high potassium (240 mM tC) artificial sea water for a few minutes before the NMR experiments were conducted. It is known that a high concentration of potassium outside can depolarize the axon [ 1 6 ] . However, it is uncertain whether the axons remained depolarized when most of the external solution was removed. The axon samples labelled 'non-fresh' were inexcitable axons which had been stored in sea water at 2°C for 1 or 2 days. The axoplasm sample which is labelled '4days-old' consisted of axoplasm extruded from several axons and then stored in a glass NMR t u b e at 2°C for 4 days. The samples labelled 'membrane' actually were axon sheaths, which c o n s i s t e d of the axon membrane plus the Schwann cells and a thin layer of connective tissue. The signal from these latter samples was extremely weak and the uncertainty in the data was relatively large. It m a y be concluded, from the results of this study, that causes other than the water-membrane interactions are important to the reduction of relaxation times o f water protons in biological cells. Firstly, although the axon contains very little infolding membrane systems, such as endoplasmic reticulum, the relaxation times of the water protons are still significantly shortened in comparison to those of bulk water. The spin-lattice relaxation time o f the giant axon is reduced b y a factor of two, while the spin-spin relaxation time is reduced by a factor o f six. It appears unlikely that the reduction of the T2 can be caused b y local magnetic field gradients generated at the membrane/water interfaces. This conclusion is consistent witl~ an earlier NMR study of the magnetic field dependency of T2 in muscle [2]. Secondly, the relaxation times of the water protons in extruded axoplasm are practically identical to those of the intact axon. Since the extruded axoplasm contains practically no membranes, it is apparant that the shortening o f the relaxation times of axoplasmic water cannot be predominantly caused b y the membrane-water interactions. It seems more plausible to propose that the observed shortening of T1 and T2 is caused b y interactions between water molecules and the fibrous protein matrix which is

135 found inside the axoplasm [ 1 7 - - 1 9 ] . It has been proposed that the T1 could be shortened through a cross relaxation between the water protons and the protein protons [20] ; and that the T2 o f cellular water may be reduced due to a hydrogen exchange between water and macromolecules [21]. It has also been suggested that macromolecules can affect the structure of the cellular water near the protein surface and, therefore, induce a faster relaxation rate [2,22,23]. The possibility of these mechanisms being operative are being currently actively investigated. It should be pointed o u t that the density of the protein matrix inside the axon is much less than that in skeletal muscle. This may explain w h y the relaxation times of muscle water are reduced to a degree far greater than that o f axoplasmic water [1,2]. The data listed in Table I indicate that the relaxation times may partially reflect the physiological state of the biological sample. For example, the relaxation times o f inexcitable axons that had been kept at 2°C in natural sea water for more than 24 h are longer than those of a fresh, excitable axon. This fact implies that, after the death of the axon, the structure of the axoplasm becomes more 'liquid-like' due to chemical decomposition. Indeed, a visual inspection confirmed that the axoplasm inside the non-fresh axon was liquid-like while the axoplasm inside a fresh axon appeared as a gel of certain rigidity. On the other hand, the T2 of the 4-day-old axoplasm sample stored inside a glass t u b e retained the gel form and gave a T2 similar to that of a fresh axoplasm sample. The relaxation times, therefore, are related to the physical structure of the axoplasm. We feel that the difference between the effects of aging on the axon and the axoplasm may be due to the different ways in which the samples were handled. The non-fresh axon was kept in sea water, while the axoplasm was kept in a glass t u b e so that no exchange of electrolyte with sea water was possible. The NMR measurements indicate that axons kept in sea water degrade quickly, while the structure of the isolated axoplasm can remain intact much longer. This result suggests that the change in the structure of the axoplasm depends on the availability of additional ions, such as Na÷ and/or Ca 2÷. It has been shown that, when small amounts of Ca 2÷ are introduced inside an axon, the fibrous structure of the axoplasm degenerates [ 1 7 ] . Our NMR study seems to be consistent with this observation. In conclusion, we have demonstrated in this study of squid giant axon that the cell membrane does n o t affect significantly the measured relaxation times of cytoplasmic water. It is suggested that it is the water-protein interactions instead of the water-membrane interactions which are mainly responsible for the shortening of relaxation times of water in axons. Supported b y grants O N R N00014-77-C0092 and N00014-76-C0100, USDA 58-7B30-9-60, and U.S.P.H.S. GM-20154 and R R 0 0 1 8 8 and The R o b e r t A. Welch Foundation Q-390. We thank Dr. I. Tasaki for the use of his facilities and Carolyn Edwards and Carolyn Aylward for their secretarial assistance.

136 References 1 2 3 4 5 6 7 S 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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