The rapid-freeze technique in neurobiology

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7 Hen'nstein, R. J. (1974)J. Exp. Anal. Behav. 21, 159-164 8 Heyman, G. M. (1979)J. Exp. Anal. Behav. 31, 41-51 9 Hinde, R. A. (1970) Animal Behaviour, A

Symhesis of Ethology and Comparative Psychology, McGraw-Hill, New York 10 Houston, A.J. Theor. BioL (in press) 11 Krantz, D. H., Luce, R. and Suppes, P. (eds) (1974) Learning, Memory, and Thinking, W. H. Freeman, San Francisco 12 Krebs, J. R., Kacelnik, A. and Taylor, P. (1978) Nature (London) 275, 27-31

13 Maynard Smith, J. (1972) On Evolution, Edinburgh University Press, Edinburgh 14 Maynard Smith, J. (1974)J. Theor. Biol. 47, 209-221 15 Maynard Smith, J. (1978)Sci. Am. 239, 176.-193 16 Maynard Smith, J. (1979)Proc. R. Soc. London, Ser. B 205,475--488 17 Maynard Smith, J. andPrice, G. R. (1973)Nature (London) 246, 15-18 18 Mazur, J. E. (1981)Science 214, 823-825 19 McFarland, P. J. (I 974) inAdvances in the Study of Behaviour (Lehrman, D., Rosenblatt, J. S., Hinde. R A. and Shae, E., eds), Vol. 5, pp.

The rapid-freeze techr', neurobiology

in

John E. Rash The rapid-freeze technique is currently touted as a revolutionary new tool for the analysis o f nerve structure and function 69. However, supercooling for instantaneous vitrification o f water has been known for over 1 O0years. For example, in the popular literature o f 1877, Jules Verne described just such instantaneous freezing o f the oceans o f an entire planetoid a2. While rapid freezing on such a scale has not proven possible, freeze preservation o f cell structure has met with increasing success since the early days o f light t tl"7° and electron microscopies 7t.n. ". Likewise, the "new' concept o f contact freezing o f nerve cells against a liquid.nitrogen- or liquid-helium-cooled metal block tt ~s.=,.N.88was first described over 40years ago TOand adapted for electron microscopy nearly 20 years ago u-~. However, the rapid-freeze technique could not be utilized fully in neurobiology until the techniques of freeze substitution, freeze fracture and freeze etch were developed x7,um and the kinetics o f rapid freezing were analysed U~,u.~. This report summarizes the unique opportunities and limitations now afforded by the rapid-freezing technique in neurobiology. Introduction For the neuroscientist attempting to establish the fmer details of nerve cell structure and function, the primary method for discriminating 'reality' from 'artefact' has been by the visualization of physiological events in living cells using light microscopy, combined with time-lapse or highspeed cinematographies. However, for components smaller than 0.2 ~ m (the practical limits of resolution of the light microscope), the correlation of structure, function and composition has required the examination of chemically preserved tissues by electron optical techniques, despite the knowledge that there are at least four potential sources of sample preparation artefact: (1) chemical fixatives cannot preserve all biological macromolecules; (2) many diffusible substances are not immobilized by chemical fixation and are extracted or redistributed during sample preparation74-~s;

(3) during fixation, suheefiular diffusion barriers are altered unpredictably, resulting in uncertainty about preservation of the volume and content of intracellular and extracellular~ n t s S ' - s s ; and (4) after chemical =fixation, enzymes may retain at least partial activity~, thereby remaining capable of altering cell composition and structure. In addition to these considerations, nerves and their supporting tissues are especially difficult to preserve for morphological analysis because: (a) many events of synaptic tra_nsmission occur in milliseconds or less, which is at least three orders of magnitude more ~ t ~ the penetration and/or action of chemical fixatives~S; (b) the central nervous systems and many ganglia are much too complex to permit ~ of individual ceils for ins/m fixation without s t i m ~ i n g or mima~ tially altering the target ueumns; and (c) most neurons are protected by blood-brain and blood-nerve barriers which further

1983, Elsevier Science Publishers B .V . , Amsterdam 0378 - 5912/83/$01.00

201-225, Academic Press, New York 20 Milinski, M. (1979)Z. Tierpsychol. 51,36-40 21 Neimark, E. D. and Estes, W. K (1967)Stimulus Sampling Theory, Holden-Day, London 22 Rose, R. M. and Benjamin. P. RJ. Exp. Biol. 92. 197-201 CALVIN B. HARLEY

Departments of Biochemistry and Medicine, McMaster University, Hamilton, Ontario, Canada LSN 3Z5. JOHN MAYNARD SMITH

School of Biological Sciences, University of Sussex, Falmer, Brighton BNI 9QG, UK

.~ penetration and action of most Consequently, many inveshave turned to the rapid-freeze tecnmque to obtain near instantaneous preservation of nerve cell ultrastructure.

Comparison of conventional and rapid freezin~

A. Crystallization vs. vitrification The terms 'rapid freezing', 'fast freezing', 'ultra-rapid freezing', 'quick freezing', 'quench cooling', 'cryoquenching" and'freeze vitrification' refer to a (theoretical) process of 'cryofixation' in which sample-cooling rates are so rapid (104-106 °C s -t) that most water molecules pass from the relatively ~ u s liquid state (water) to the relatively amorphous solid state (vitreous ice) without forming detectable crystalline arraysg'~2-1U~,~. In contrast, at slower freezing rates (10-4-104 °C s-X), water molecules to and deposit in ordered arrays, forming ice crystals. The growing ice crystals may destructively compress or displace suheellular components~,~'4a`4s or perforate cytomembranes=°. Between the forming ice crystals, dilute solutes are progressively concentrated, ultimately to freeze at their eutectic temperatures~,St. Although such freezing effects often lead to cell destructionet ~, the effects of high local concentrations of various salts on cytoplasmic and membrane proteins and nucleic acids are not established, nor are the effects of ~prefracture' shearing stresse# 7,s° or lipid phase separations s~4z adequately described. The rapid-freeze technique, in contrast to slower freezing mothods, is based on the ability of a very cold substance (the 'heat sink') to dissipate heat from the specimen so rapidly ~ the water molecules are cooled at an average rate greater than 104°C s -t over the range of +30°C to -100°C (i.e. 303° K to 173° K)a,9'm~s,27. During rapid freezing, water is transformed into the vitreous solid state without releasing (all of) the conventional 80 cal g-~ (heat of crystallization). Consequently, rapid freezing requires the removal of less heat than conventional freezing to the same tempexature. An important corollary is that the 80

209

TINS -June 1 983 cal g-1 'latent heat of crystallization' is available for instantaneous release upon ice-crystal formation, thereby accounting for 'incipient melting'8 and substantial heat release during 'devitfification'8.4L Thus, as vitrified water is slowly raised to and above the glass transition temperature (circa - 140°C37,s9), the release of the 'latent heat of fusion' may trigger an exothermic wave of 'recrystallization' to pass through the tissue, quickly raising the temperature towards 0°C 3.87 and resulting in the formation of large ice crystals. When vitrified water or glycerol solutions of 40-55% are rewarmed over the range of -115°C to -40°C, a relatively large amount of heat is released4°. Moreover, if the sample temperature is raised approximately 500(2 above the glass transition temperature of the glycerol concentration within the sample, substantial recrystallization is reported to occur within 2.5 min 87. (Although no reliable recrystallization data are available for glycerinated cytoplasm, many investigators report ice-crystal damage to cell ultrastructure if the temperature of glycerinated samples is raised even briefly to -90°C (H. Moor, T. Reese and R. L. Steere, unpublished observations). It should be noted, however, that alterations in subcellular morphology by internal recrystallization are difficult to document unless complementary freeze-fracture/freeze-etch preparations are examined',TL) On the other hand, cell ultrastructure is not detectably altered by the short-order translations of water molecules which may occur during long-term storage of samples in liquid nitrogenaa.

acceptable cryofixation to about 10-15 tim, regardless of freezing methods or cryogen used U'~4,~9. (Exception: See Appendix for a description of 'highpressure' freezing45.9~.) Rapid freezing in n e u r o b i o l o g y Attempts to obtain high-quality cell preservation by rapid-freezing techniques have been made since the early days of light and electron microscopies2'xs'~'sg'79,7~.88,~. However, the explosive development of rapid-freeze technologies may be traced directly to the particular needs of neurobiology and to the availability of high-speed analytical methods commonly employed by neurobiologists.

A. Estimating the volume of the extracellular space In 1964, Van Harreveld and co-workers revived the rapid-freezing and freezesubstitution methods of Simpson 7° and Feder and Sidman t7 to analyse changes in the volume of the extracellular space (ECS) in the CNS (a) during perfusion fixation89, (b) following brief asphyxiation ~, and (c) during spreading depressio# 5. In these studies, they obtained the first ultrastructural estimates of the volume of the ECS that corresponded to values obtained by biochemical methods 1.~9. Glutaraldehyde fixation was shown to produce a marked loss of ECS, which was at least partially reversed by post-fixation with osmium tetroxide 89. Thus, they showed that the volume of ECS is subject to large and relatively rapid changes. Despite these data concerning the necessity for non-chemical methods of preparing nervous tissues, a renewal of interest in rapid freezing did not develop until (1) its value was recognized for the preparation of samples for freeze-fracture and freeze-etch analysis :~,89 and (2) high-speed monitoring equipment was used to synchronize the instant of freezing with the extremely transient events of synaptic transmission27'~'sT-sL

B. Heat conduction through water and ice The insulating properties of water and of the developing layer of vitreous ice limit heat transfer from deeper within the sampies'9,~'~s'2~'s2. Maximum heat transfer occurs at the sample interface with the heat sink and diminishes rapidly with increasing depth. (Calculations suggest that true vitrification is limited to a thickness of only 1 B. Analysis o f transmitter release at the /xm92.) Unfortunately, a reliable value is neuromuscular junction not available for the rate of heat transfer By 1974, it had been firmly established through the initial layer of true vitreous ice. that the membrane fusion events of transNevertheless, under optimum freezing mitter release could not be investigated conditions, the first measurable ice crystals adequately by conventional techniques of (i.e. those larger than 10 nm) ~9.~4,2~.49,e2 aldehyde fixationaS. Consequently, Landis appear at a depth of about 10-15/xm. Still and Reese38 and Heuser et al. ~9 modified deeper, the heat of fusion released by inci- the rapid-freeze device of Van Harrepient ice-crystal formation becomes greater veldss,a4'8~to allow (a) direct nerve stimulathan the rate of heat loss through ice to the tion and intracellular recording, (b) the use heat sink, thereby reducing the rate of of liquid helium as a coolant, and (c) specimen cooling and resulting in the improved shock-absorbing devices in an development of still larger ice crystals. initially unsuccessful attempt to determine Thus, the limit on heat transfer through the time course of transmitter release at the frozen tissues limits the useful depth of amphibian neuromuscular junction.

Because of technical problems related to premature specimen cooling and a concomitant reduction in nerve propagation velocity, they initially postulated that exocytosis was too transient for rapidfreeze techniques to catch in progress (i.e. that the entire time course of synaptic vesicle (SV) attachment, membrane fusion, and SV incorporation into the nerveterminal plasma membrane occurred in less than 10 ms) ~. To resolve the issue, detailed experiments were initiated in several laboratories using high-speed electrical recording techniques combined with freeze substitution, freeze fracture, freeze etch, and conventional thin-section electron microscopy ~7,8~.s8. By compensating for the reduced propagation velocity caused by premature specimen cooling, Heuser, Reese and co-workers were able to capture the middle and later sequences of SV fusion and showed that SV remnants were recognizable 100 ms after transmitter release2L As a result of those detailed studies, other models of quantal transmitter release at the neuromuscular junction are no longer tenable. On the other hand, early fears that membrane fusion events were artefactually created or altered by the compressional wave (improperly called 'shock wave' ) caused by specimen contact at 1-3 m s-' with the heat sinks4 have proven to be unfounded 49,82 (i.e. insufficient energy is imparted per unit area to produce large-scale phase changes and rearrangements in membrane lipids sufficient to cause, reverse or mimic the normal sequence of membrane fusion events). Nevertheless, an easily recognized crushing or shearing artefact49 has been detected in metal-contact-frozen samples and has been attributed to mechanical irv stability ('bounce '5) at initial contact or to ' splash' 49.

C. Mechanisms o f membrane fusion At about the same time as the membrane fusion studies of Heuser, Reese and coworkers 27'~'s8 and of Van Harreveldaa, rapid-freeze preparations revealed that many of the most cherished images of membrane fusion eventss° could be traced to artefacts of ghitaraldehyde fixations.7.2a. Consequently, many investigators have concluded that models of membrane fusion with long-lived particle-free intermediates3 5 .4.8. 4. 9. .5 2 5 4 5 5 6 6 probably represent artefacts of lipid instability in glutaraldehyde-fixed membranes 8.~.2a,49, rather than naturally occurring 'liposomelike' fusion eventssl. Despite the elegance of these data concerning the time course of membrane fusion events in neuronal and non-neuronal tissues, it is important to note that no one has

210 captured or recognized the initial events of membrane fusion (here defined as membrane attachment, establishment of initial intermembrane continuity, and the formation of the initial transmembranepore). Nor is it established whether intramembrane particles (IMPs) or IMP-free areas are required for directed membrane fusion. Moreover, mechanisms for incorporating lipid reservoirs into new membrane (for example, growing nerve growth cones) are not yet establisheds2. Thus, membrane fusion mechanisms remain among the most important unresolved problems in neurobiology.

D. Analysis of matrix components in freeze-etch replicas The fourth major contribution of the rapid-freeze technique to neurobiology stems from its ability to provide highresolution, three-dimensional freeze-etch images of the cytoplasmic and extracellular matrices. The beautifully detailed images of coated pits and coated vesicles2. provide compelling support for a general model of

I'tNS -June 1 t),'L", 'endocytosis' during membrane internalization26,s6,64. Likewise, equally detailed freeze-etch images of cytoplasmic microfilaments and microtubulesxS'zS's6.~7 provide new data concerning molecular organization of the intracelhilar contractile apparatus and appear to support the 'microtrabeculae' hypothesis of Wolosewick and Porteff;.

E. Localization of diffusible substances

layers immediately beneath the fi-actare plane1~,8s. These difficulties appear to be overcome by the use of rapid freezing and cryoultramicrotomy in combination with high-resolution electron-probe microa n a l y s i s 74-7'~. These techniques permit relatively high-resolution localization of diffusible substances that are present at high local concentrations (for example calcium/magnesium phosphate granules in mitochondria), as well as determination of relative concentrations of elements within larger areas 74-76. Improvements in rapidfreezing technology, cryo-ultramicrotomy, X-ray spectroscopy, and computerized data analysis should permit much higher spatial and temporal resolution, and provide information concerning the normal distributions and movements of ions within and between intracellular and extracellular compartments74-7~.

Diffusible substances cannot be localized in living tissues using conventional fixation and embedding procedures because the substances may he extracted or displaced. Rapid-freeze techniques provide a means for in-situ localization and quantification of diffusible substances by immobilizing them within the ice matrix. Early attempts to localize diffusible substances using rapid-freezing techniques combined with autoradiography6a were severely limited by the deposition of emul- F. In-situlabelling of membrane sions and their prolonged exposure on frozen macromolecules One of the most promising features of the samples, as well as by the difficulty in identifying 'cryptic' sources of radiation in the rapid-freeze technique is the opportunity it

Fig. 1. Rapidly frozen, minimally etched nerve axon. Neurofilamenta and cross bridges were revealed by rotary replication. Note the single mlerombule (left center) and the cross-fractured myelin sheath. ( × 110 000). (Courtesy of Drs Bruce Schnapp and Tom Reese. )

211

T I N S - J u n e 1 983

Fig, 2, In high-magniftcation stereo images, the 100 ft neurofilaments exhibit distinctive patterns of globular protrusions (left center). Between the neurofilaments, a cross-bridging filamentous network is observed, consisting of both transverse and longitudinal elements (right center). Rapid freezing of uncryoprotected specimens allows minimal etching for high-resolution imaging of cytoplasmic components. (× 85 000). (Courtesyof Drs Bruce Schnappand Tom Reese.) affords for high-resolution in-situ labelling of both fixed and unfixed subcellular constituents in freeze-fracture and freeze-etch replicas. Several major versions of this powerful new labelling technique have been demonstrated: (1) pre-fracture labellingS3,sg; (2) post-fracture label ling3O.SS.57,s9 61; and (3) post-shadow labelling 5a,61. These new in-situ labelling techniques extend the capabilities of histochemistry and cytochemistry to the identification of individual intramembrane particles ~° and cytoplasmic macromolecules3° in freeze-fracture and freeze-etch replicas.

Summary Rapid freezing for electron microscopy has several unique advantages over conventional chemical fixation procedures, including (a) the morphology, volume and composition of cell constituents are preserved almost instantaneously, (b) morphological changes such as those at transmitter release may be analysed with a resolution of 1-2 ms, (c) the natural sequences of membrane fusion phenomena may be differentiated from artefacts of conventional chemical fixation procedures, (d) short-lived chemical intermediates of complex reaction sequences may be isolated and analysed, (e) diffusion processes arc suspended, allowing precise localization and in-situ quantification of ions and other soluble substances, (f) temperature-dependent phase transitions of membrane iipids may be examined without the use of chemical fixatives, and (g) deep etching of uncryoprotected specimens (fixed or unfixed) corn-

bined with pre- and post-shadow labelling techniques permits individual molecular components of membranes, cytoplasm and extracellular matrix to be identified in situ. The major hurdles in obtaining highquality images from rapidly frozen cells have been overcome; the major limitations to the rapid-freeze technique have been recognized; new methods for viewing have been devised; and new techniques are being developed which take advantage of the high temporal resolution and molecular preservation afforded by rapid freezing. Thus, the rapid-freeze technique combined with high-speed intracellular and extracellular recording techniques is rightfully taking its place as one of the most important analytical tools in neurobiology.

Acknowledgements I thank Drs Bruce Schnapp and Tom Reese for the beautiful electron micrograph of a rapidly frozen, minimally etched nerve axon, shown in Figs 1 and 2.

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H., McClellan, G. and Somlyo, A. P. (1981) J. Cell BIOL 90, 577--594 Somlyo, A. V., Shuman, H. and Somlyo, A. P. (1977)J. Cell BIOL 74, 828-857 Somlyo, A. V., Shuman, H. and Sorrdyo, A. P. (1977) J. Cell Biol. 81, 316-335 Steere,R. L. (1957)J. Biophys. Biochem. Cytol. 3, 45---60 Steere, R. L. (1969) Cryobiology 6, 137-150 Steere, R. L. and Erbe, E. F. (1979)J. Microsc. (Oxford) 117, 211-218 Steere. R. L.. Erbe. E. F. and Moselely, J. M. (1980) J. Cell Biol. 86, 113-122 Tanner,J. E. (1975)Cryobiology 12, 353-363 Terracio, 1., Bankson, P. W. and McAteer, J. A. (1981) Cryobiology 18, 55-71 Van Harreveld; A. and Crowell, J. (1964) Anat. Rec. 149, 381-386 Van Harrcveld, A., Crowell, J. and Malholtra, S. K. (1965)J. CellBiol. 25, 1t7-137

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John E. Rash is Associate Professor at the Department of Anatomy, Colorado State University, Fort Collins, Colorado, CO 80523, USA.

Appendix. Rapid-freezing methods and devices A. Contact freezing Samples plunged against a liquickmtrogen- or liquid-helium, cooled metal blocid ,27.~.3s:°.". Especially useful for freezing cells exposed at natural interfaces and for freezing freshly cut surfaces. Not useful for structures deep within tissues. Designed for Balzers 300 and 400 series devices. Commercial models available through: 1. The 'Slammer', Polaron lnstnanents, UK and USA (Cost: about $7 000); 2. The 'Gentleman Jim', Prof. A. Boyue, Quick Freezing Devices, 112 East Gittings Street, Baltimore, MD 22230, USA (Cost: about $1 800); 3. Prof. J. Escaig, 105 Boulevard Raspali, 75006 Paris, France (Price available on request).

B. Plunge freezing Samples are plunged into a liquid 'quenchant's.",1~.".7'-7~. Under optimum conditions, freezing rates and freezing depths comparable to' contact' freezing methods. Especially useful for very thin samples, metal clad 'sandwiches', and surfaces of very small, incgulatly shaped tissues (for example small ganglia and isolated neurons). Commercial versions available through: 1. Prof. J. Escalg, 105 Boulevard Raspail, 75006 Paris, France (Price available on request). C. Jet freezing

Tissues sandwiched between thin metal sheets are frozen in apposed sprays of liquid quenclumt (usaally propane). Maximum thickness devoid of detectable ice crystals is about 20-30 btm. Samples thicker than 100/zm develop very large ice crystals in their centers, leading to ~ +prefracturing 's°, and a higher tendency to fi'acna~ within the poorly frozen layer. 'Sandwich' specimen holders (also see above) are especially useful for producing double replicas ('complementary' freeze-etch replicas). Commercial models available through: 1. Original version available from Balzers COrlxntion (worldwide); 2. Blueprints for an improved version are available from Prof. L. A. Staehelin, Department MCDB, University of Colorado, Boulder, CO 80302, USA (Cost of blueprints approximately $5).

D. Spray freezing Suspensions of ceils or cell homogenates are aspirated as tiny droplets (t0-20/~m) intoa liquid

quenchanP. Frozen droplets 'glued' at low tempenmne into c o n ~ freeze.~ supports. Small droplet size and shearing and ~ i v e forces exerted d ~ aspiration ~ u d e use for freezing intact nerve cells or tissues. May be useful for e x m i ~ ~ w . a l ; subcelMat end non-cellular preparations. Easily consmlot~ from common laboratory supplies.

E. High-pressure freezing Very large samples (1 nmP) frozen ~__nrJer__veay high ptcssuxe (2 000 attn.; 30 000 P.S.I; or 2 × liP Pa) ....... . Vitrification requires 10-300 ms ( d ~ oft s a ~ size)° , which is far too long to preserve events of synaptic transmiaalonS',n. Extreme wessu~ may cause s u u c t t ~ Cheages in lipid protein components d t h e cell N e ~ , a~e,m tobe ~ ~ i ~ o ~ ultra-

structure within very large tissue samples, May laeve useful for analysing~ : ~ ultra, struauralchanges deep withinIm'getissuesamples.Comm~cial model available~ from B ~ Corporation. F. Cry o p rotectant freezing (not a rapid-freeze technique) One or more chemical altars ~ 1 , ethylene glycol, dime~yisulfoxide, s w , dextran, polyvinylpyrolidone, ethanol, methanol) iattodueed into ~ i ~ to ~ with or

ice-crys~ lattice formation' t " , thereby redeeiag the coaiag rate ~ f ~ r ~ . =t'rhe slight reduction in the atra3untof heat of crystalltTationreleased d ~ vitrification does not substantially increase the freezing rate.)Because of the extended time gequired f o r ~ , many cryowotectants are thought to induce artefacts, especially in unfixed tissues~t.9°.