A technique for recording from spinal neurones in awake sheep

Journal of Neuroscience Methods, 46 (1993) 225-232

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© 1993 Elsevier Science Publishers B.V. All rights reserved (1165-0270/93/$06.00

NSM 01466

A technique for recording from spinal neurones in awake sheep J.F. H e r r e r o , T.W. Coates, M. Higgins, A. Livingston ~'*, A.E. W a t e r m a n 2 and P.M. H e a d l e y Departments of Physiology, 1 Pharmacology and 2 Veterinary Surgery, Unit'ersity of Bristol The School of Medical Sciences, University Walk, Bristol BS8 ITD (UK) (Received 19 August 1992) (Revised version received 18 November 1992) (Accepted 19 November 1992)

Key words: Spinal cord; Chronic recording; (Sheep) A technique is described for implanting a chamber on 1 or 2 vertebrae of the spinal column of the sheep. This chamber protrudes permanently through the dorsal skin of the back and is covered by a light bandage. Between recording sessions the chamber houses an inner cap sealing the hole that gives access to the cord. During recording sessions this cap is removed and a miniature manipulator inserted instead. This manipulator can accept a motor drive that holds a glass-coated tungsten microelectrode. The drive has a hole through which an arthroscope tube can be passed so that insertion of the electrode can be performed under visual control. Extracellular recordings have been made of single spinal neurones for up to 4 h in animals that are drug-free, untrained and only lightly restrained. Recording sessions can be repeated on a daily basis for several weeks until the dura a n d / o r arachnoid becomes too thickened to permit electrode penetrations. Animals remain healthy and their behaviour remains normal throughout this time.

Introduction

The spinal cord is a site of important sensory and motor processing, and consequently has been intensively studied for many years. The function of spinal neurones can only be fully investigated when the sensory input to the cord is unimpaired, i.e., in an intact animal. In vivo recordings, however, almost always require extensive preparatory surgery, and either the presence of a general anaesthetic or destruction of much of the central nervous system. There is now good evidence that both the preparatory surgery and general anaesthetics alter spinal function (see data and discussion by Hartell and Headley, 1991a,b) so that

* Current address: WCVM, University of Saskatoon, Saskatchewan S7K 0W0, Canada.

Correspondence: Dr. P.M. Headley, D e p a r t m e n t of Physiology, The School of Medical Sciences, University Walk, Bristol BS8 1TD, UK. Tel.: (0272) 303476; FAX: (0272) 303497.

results obtained under the usual in vivo recording conditions are undoubtedly distorted from the normal, conscious state. The only means for overcoming these problems is to prepare animals chronically so that recordings can be made in the absence of both recent surgery and anaesthetic agents. Experience has proved that such techniques are not easily applied to the spinal cord, although the trigeminal system of monkeys has been extensively investigated (e.g., Dubner et al., 1989). Spinal cord studies have been performed in rats (Wall et al., 1967) and cats (Collins, 1985; Sorkin et al., 1988) but only Collins' group has continued to generate results with this technique. There are, however, several problems associated with the use of cats. Firstly, the subjects have to be extensively trained to accept restraint and not to move during recording sessions. To be effective, this training must alter sensory processing at some level of the neuraxis, perhaps including the spinal cord. (In the primate trigeminal

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studies, extensive training is also required). Secondly, the preparatory surgery of cats necessarily involves the fusion of several lumbar vertebrae so as to provide a rigid platform for the implanted recording chamber. This extensive orthopaedic surgery is likely to alter both sensory processing (because of the nociceptive inputs generated) and motor function (because of the altered mechanics of the spinal column). Thirdly, the normal flexibility of the cat spinal column, together with the usual behaviour of this species, means that the animal is likely to access the external part of the implant between recording sessions and may interfere with it. Fourthly, the blood volume is rather small for taking repeated blood samples, as would be required for assessing the release of stress hormones or for relating plasma levels of anaesthetics or analgesics to neuronal responsiveness. There would consequently be advantages in choosing a larger species with a spinal column that is normally rigid and less accessible to interference, with vertebral bodies sufficiently strong to accept an implant without extensive fusion being necessary, and with a nature not requiring prolonged training prior to recording sessions. We considered that the sheep would meet these criteria, and here describe the methods we have developed to make chronic recordings from the lumbar spinal cord in this species.

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Fig. 1. The general arrangement of the lumbo-sacral spinal column of the sheep and of the position of a dental acrylic base attached to one lumbar vertebra. The titanium ba~plate and chamber are then fixed to the acrylic base. The chamber protudes permanently through the skin. Between recording sessions both the baseplate and the chamber are sealed with caps.

to each vertebra. Initial acute experiments were also performed to map the distribution of cord dorsum potentials in response to percutaneous electrical stimulation of the hindquarters, so that the hindlimb sensory inputs could also be related functionally to vertebral segments in both L6 and L7 sheep. In animals with 7 lumbar vertebrae the hindlimb sensory area is centred over the L6-L7 junction; in animals with 6 vertebrae over the rostral part of L6 vertebra.

Methods

The implant Anatomical considerations A complication of using sheep is that the number of lumbar vertebrae is variable between individuals. In most cases there are 6 or 7; we have used sheep with either number. This means that the relationship of the cord to the column varies between experiments. Although there is a report of the arrangement of the lumbosacral plexus in the sheep (Goshtal et al., 1971), there is no description of the anatomy, or of the funtional arrangement, of the neural entry to the lumbar cord. Anatomical dissections of cadaver material permitted the relationship of the lumbar root entries to be related

An initial stipulation was that the implant should be capable of being mounted on a single vertebra so that fusion of vertebrae was not an integral part of the procedure. In fact the anatomical considerations discussed above indicate a placement in sheep with 7 lumbar vertebrae requiring the fusion of L6 and L7 vertebrae. The general arrangement of the spinal column, and of the implant when on it, is shown in Fig. 1. Details of the titanium implant are shown in Fig. 2. The baseplate A is fixed by screws through 4 of the holes to the acrylic platform on the bone (see below and Fig. 1). Access to the cord is through the inner 18 mm hole in this baseplate, This hole

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Fig. 2. Scale drawing of the titanium implant. The baseplate A is attached to the acrylic base by screws through 4 countersunk holes (diameter 3 mm); the remaining holes are threaded and are used to mount the manipulator (Fig. 3). The cord can be sealed with screw cap B. The chamber C (wall thickness 1 mm) is screwed permanently onto the outer thread of the baseplate. Extension rings (not shown) can be attached to the top of the chamber to accommodate different sized sheep. A second, outer, seal is provided with cap D. is sealed between recording sessions by the stalked cap B. The outer thread of the baseplate is connected to the tube C which, together with optional extension rings (not shown) forms the chamber providing access from the skin surface to the cord. This chamber is sealed by cap D. The microelectrode is held by a miniature electrode drive assembly that controls electrode penetration. This drive is substantially the same as that used by Boissonade et al. (1991). It is, however, contructed from stainless steel with polytetrafluoroethylene (PTFE) insulation so that

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it can be autoclaved. At the bottom of the drive a 1 m m internal diameter stainless-steel tube 8 mm long is mounted concentric to the electrode, forming a guide tube (Fig. 4). A plug of dental impression material (Provil, Bayer Dental) in the end of the guide tube prevents the entry, by capillary action, of CSF which can otherwise short-circuit the electrode. The electrode driver is mounted onto a remotely controlled motor system which provides direct read-out of penetration depth (Coates et al., 1992). The electrode drive also incorporates a 2.8 mm hole along its length, through which a 2.7 mm arthroscope (Panoview, R.T. W o l d is inserted. By alternating arthroscopes with 25 ° and 70 ° angled views, observation of the whole area of the exposed spinal cord can be obtained, even when the manipulator system is in place within the chamber. This facility is used to guide electrode insertion sites in relation to the surface vasculature. The electrode drive is clamped in a manipulator (Fig. 3) which is screwed to the baseplate during recording sessions. The arrangement of baseplate mounting holes is such that several orientations are possible. The manipulator is constructed of stainless steel and brass components with a nylon locking screw that retains the electrode drive. The manipulator is sterilized by autoclave together with the electrode drive. The manipulator incorporates a worm-and-wheel that al-

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Prior to surgery, A-P radiographs are taken of the lumbar spinal column, the hooves are trimmed as necessary, the fleece sheared over the hindquarters and anthelmintic therapy provided. The sheep are kept in the animal house for about 1 week before the surgery is performed. At least 2 sheep are kept within sight of each other at all times since isolation is a considerable stress in this species. The animals are habituated during this period both to staying for up to 4 h in a slatted crate (0.7 × 1.6 m) and to being supported for shorter periods by means of a canvas sling; this is used to relieve the weight on the animal's hind legs, leaving the hooves either in contact with the floor or off it by a few centimetres. Food and water are available ad libitum at all times, including when in the crate. No other training is necessary.

Anaesthesia and peri-operative therapy Fig. 4. The complete assembly as used during recording sessions. lOWSthe drive to be tilted. A third mechanism for electrode orientation is provided by the electrode being 2 mm eccentric to the axis of the drive. The combination of orienting the manipulator on the baseplate, tilting with the worm-and-wheel, and rotating the drive about its axis permits access of the recording electrode to virtually the whole extent of the exposed spinal cord.

Selection and preparation of animals Different breeds of sheep vary greatly in body size; ewes of those readily available in the UK vary from approximately 30 to over 80 kg. Experience has shown that smaller sheep have fewer postoperative problems. Despite the weight difference, the vertebrae are not too much smaller in the lighter animals. Although we have used Cluns weighing up to 70 kg, we now use cross-bred ewes weighing 35-50 kg. The choice of breed appears not to be crucial, although animals of more placid lowland breeds do adapt more readily to the indoor housing and to the repeated handling. Obesity, however, is to be avoided since it predisposes to postoperative recumbency.

The animal is placed in ventral recumbency. Anaesthesia is induced by ketamine (10 mg/kg, i.v.) followed by halothane vaporized in O2/N20 (initially 3:2, reducing to 2: 1). The trachea is intubated with a cuffed tube under direct vision using a laryngoscope. The animal is ventilated at 15 breaths/min (but not paralyzed), with tidal volume adjusted to maintain end tidal CO 2 at about 4%. The heart rate is monitored and is used as an additional guide to anaesthetic depth. A saline drip is administered at a rate of 5-10 m l / k g / h . An oesophageal tube is inserted to protect against ruminal bloat. Prior to starting surgery the animal receives i.v. doses of the antibiotic ampicillin (4 mg/kg, Penbritin, SKB); the steroid anti-inflammatory agent dexamethasone (0.04 mg/kg, Dexadreson, Intervet); the non-steroidal analgesic flunixin (1 mg/kg, Finadyne, Schering-Plough); and the opioid analgesic buprenorphine (0.005 mg/kg, Temgesic, Reckitt and Colman). At the end of the period of surgery, usually 4-5 h later, a repeat dose of buprenorphine 0,005 mg/kg is given i.v. The exposed cord is covered with a solution containing 2 mg of dexamethasone and 5 mg of gentamicin (Cidomycin intrathecal, Roussel).

229 The topical treatment is continued on a daily basis throughout the recording period. Systemic administration is continued b.i.d, until the animal has fully recovered from the effects of the surgery. Ampicillin is given at the same doses as long as there seems to be any risk of infection. Dexamethasone is given at the same dose until postoperative swelling has subsided; thereafter the doses are reduced progressively over 6 days. Flunixin administration is limited to a maximum of 5 days, to reduce the risk of gastrointestinal ulceration. Buprenorphine is given at up to 0.005 mg/kg, b.i.d., as required to reduce any signs of postoperative discomfort; in general this is for no more than 1 week.

Surgical techniques One jugular vein is cannulated with a 'piggyback' indwelling catheter (Wallace 16 g x 20 cm in length). This cannula remains in place throughout the subsequent recording period and permits ready i.v. administration of drugs and venous blood sampling. It is flushed daily with heparinized saline. Sheep are placed in sternal recumbency with the hind legs extended. The back area is prepared for sterile operating. An incision of the skin is made over the last 3 lumbar vertebrae and rostral sacrum. The inter- and para-vertebral muscles are separated to expose the area of the intended recording site over either the last lumbar vertebra or the junction between the last 2 lumbar vertebrae (see above). The dorsal process of the last lumbar vertebra and the back half of the penultimate vertebra are removed. Using a dental drill, the dorsal surface of the appropriate vertebra(e) is then ground flat, until the inner cortical bone of the vertebra is exposed. A 1.8 cm cancellous bone screw is inserted through each lateral process of the last 2 vertebrae. Smaller screws (1.3 a n d / o r 0.6 cm, as can be fitted) are inserted into the rostral and caudal dorsolateral corners of the vertebra(e), taking care to avoid contact with the underlying dorsal roots and ganglia. The screws are then connected with 0.4 mm stainless-steel wire; together these make a firm anchor for the acrylic base. Three measures are

taken to control the spread of acrylic. Firstly the gaps around the lateral edges of the vertebrae are filled with quick-setting dental elastomer (Provil, Bayer Dental) which provides a soft filling for the crevices between bone and muscle and prevents the formation of sharp tongues of dental acrylic in this area. Secondly, shaped pieces of flexible plastic sheet are made into a roughly circular form that acts as an outer dam. Thirdly, a 20 mm P T F E ring is placed over the intended recording area. Dental acrylic is then poured outside this ring and around the screws and the wire; it is built up progressively to a depth of 7 - 8 mm (Fig. 1). This system provides a round hole, 20 mm in diameter, in the acrylic through which the bone is drilled out to expose the full width of the spinal cord. The surface of the cord is then cleaned carefully with warmed isotonic saline. A detailed diagram is made of the venous system as viewed by operating microscope through the dura mater; this will serve as a reference for positioning electrodes during recording sessions. The baseplate of the implant (see above and Fig. 2) is then threaded onto the outer tube, with bone wax sealing the thread, and is fixed onto the acrylic platform using screws through 4 of the 12 holes in the baseplate. To ensure fluid-tight seals, a thin layer of very liquid acrylic is used, firstly on the platform and then around the rim of the outer tube. Extension ring(s) are attached to the outer tube as required. Following the topical application of drugs (see above) the inner cap is threaded into place. The musculature and skin are repaired in multiple layers and a purse-string suture placed around the exposed implant which protrudes only a few mm through the skin. The animal is returned to its pen, which is fitted with a non-slip mat, is placed in sternal recumbency, and is observed until it regains consciousness (usually within 30 min) and has started to eat and drink (within 1 h).

Cuff electrodes About 1 week after the initial surgery, cuff electrodes are placed on a plantar nerve. The sheep is anaesthetized using the same protocol.

230

The medial plantar nerve is dissected free at 2 points about 10 cm apart along its path between the hock (heel) and fetlock (metatarsal-digit joint). At each site a pair of multi-stranded teflon-coated stainless-steel wires (Bromed Wire, AS 633, Cooner Wire Co.) are sewn into the perineurium. These electrodes and nerve are then surrounded by a cuff of dental elastomer (Provil, Bayer Dental). The wires are tracked subcutaneously to a connector box sewn to the skin close to the implant. The incisions on the skin are sutured and the skin is treated with local anaesthetic gel (xylocaine gel 2%).

Recording system For each recording session, the sheep is placed in the crate and is sometimes lightly restained with a sling (see above). At all times it has free access to food and water. Sterile precautions are taken when gaining access to the cord. The outer and inner caps of the implant are removed and the exposed cord is flushed with warm sterile isotonic saline. The miniature manipulator, autoclaved between each session, is screwed onto the baseplate in the desired orientation. A glass-coated tungsten electrode, presoaked in chlorhexidine solution, is

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Fig. 5. Sample record of a neurone recorded in an awake sheep. The recording site, estimated to be in lamina VII1, and the receptive field, are shown in the insert diagrams. The upper record is a spike discharge histogram of the same recording period as the spike record shown below. This continuous record shows r e s p o n s e s t o adequate activation of the receptive field, followed by low-intensity electrical stimulation via the cuff electrode previously implanted on the plantar nerve. T h e neurone responded to repeated light tapping of the skin (T), to rubbing the skin (R) and to repeated brief pinching of the skin (P; alternating a light grasp of the skin fold with transient stronger squeezes that were uncomfortable when tested on the experimenter, and which caused the sheep to flick its hindlimb). After this sequence the electrical stimulation was performed at low intensity (2 × the threshold for evoking a spike) at 3 different frequencies; t h e larger deflections are the stimulus artefacts. T h e lower trace shows a single response to one of the stimuli presented at 1 Hz: the arrowhead indicates the s t i m u l u s artefact (clipped at this magnification compared to the middle record). During this sequence the s h e e p looked at its hindlimb but showed no other signs of discomfort.

231 pushed into the electrode holder, the lower (non-motor) part of which has also been autoclaved. This assembly is clamped into the manipulator, at the desired rotation. An arthroscope tube is inserted through the electrode driver and the electrode advanced under visual control. The quality of vision varies and deteriorates as the dura thickens with repeated penetrations. It is often possible to watch the electrode beginning to penetrate the cord. On other occasions the surface has to be judged from the electrical characteristics of the recorded signal. Depth readings are taken from this point. The receptive field characteristics of each neurone are assessed by manual exploration of the hindquarters. In behavioural terms, the threshold for pinch stimuli becoming aversive is close to that in man. Low-intensity electrical stimulation via the cuff electrodes can be performed in the conscious state; as with manual exploration, the animal may turn to look at its hind legs, but shows no sign of distress. High-intensity electrical stimulation (sufficient to activate slowly conducting afferents) is only performed under anaesthesia. The sheep usually remain quiet during the recording session for 1-3 h, sometimes longer. During this time the animals usually eat, drink and chew the cud in a normal fashion. Cudding is considered of particular significance, since this behaviour is readily suppressed in any stressful situation. Once the animal becomes restless the session is terminated for that day. Using this technique, single-unit recordings have been maintained for up to 7 h in anaesthetized animals and up to 4 h in conscious animals. Recording sessions can be repeated daily until the d u r a / a r a c h n o i d thickens to the extent that electrodes will not penetrate. This happens some 3 - 6 weeks after the original implantation. An example of a recording obtained from a spinal neurone in an awake sheep is shown in Fig. 5. This neurone was estimated to lie in lamina VIII, as judged by electrode depth readings in conjunction with histology of electrolytic lesions made during the final recording session under anaesthesia. It can be seen that the signalto-noise ratio is acceptable and that spike height

remained relatively constant both during manual activation of the receptive field and during lowintensity electrical stimulation administered via the cuff electrode (note stimulus artefacts). This neurone displayed classic convergent (or wide dynamic range) properties, responding to lightly tapping or rubbing the skin, but discharging more vigorously with short pinch stimuli that were uncomfortable to the experimenter. As would be predicted with electrical stimulation at this intensity, the latency was short (16 ms) and there was no 'wind-up' (see Headley and Grillner, 1990). During such procedures sheep often look at the stimulated limb, but at these stimulus intensities do not show any aversive or escape responses. When recordings were made under anaesthesia. more intense stimulation could be utilized; under these conditions wind-up was frequently elicited.

Discussion

We have found that sheep recover from the initial surgery within a few days. This recovery is faster when lighter animals are used; postoperative stiffness causes the heavier animals more trouble. Sheep tolerate the implant very well. Contrary to popular belief, there are marked differences in the temperament between individual sheep, and it undoubtedly helps to select members of a more placid breed. With such animals training is not necessary, and only a few days is required for the animals to habituate to being lightly restrained in the crate. The general handling problems with sheep are, of course, greater than for small laboratory animals. As with any project involving recovery experiments, it is necessary to have veterinary input to the overall experimental regime; this is particularly important where scientific staff have no previous experience of the species. With these provisions the sheep has proved to be a suitable subject for this type of recording and fulfills the objectives of overcoming the limitations of using cats for this type of chronic preparation.

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Acknowledgements W e w i s h to t h a n k P r o f e s s o r s B. M a t t h e w s a n d D.M. Armstrong, and their colleagues, for much h e l p in s e t t i n g u p this t e c h n i q u e . W e also t h a n k the Wellcome Trust and the Spanish Ministry of Education for financial support.

References Boissonade, F.M., Banks, D. and Matthews, B. (1991) A technique for recording from brain-stem neurones in awake unrestrained cats. J. Neurosci. Methods, 38: 41-46. Coates, T.W., Harris, R.H. and Matthews, B. (1992) A miniature, remotely-controlled microelectrode driver for use in conscious, unrestrained animals. Med. Biol. Eng. Comp., 30: 248-250. Collins, J.G. (1985) A technique for chronic extracellular recording of neuronal activity in the dorsal horn of the lumbar spinal cord in drug-free, physiologically intact, cats. J. Neurosci. Methods, 12: 277-287.

Dubner, R., Kenshalo, D.R., Maixner, W., Bushnell, M.C. and Oliveras, J.-L. (1989) The correlation of monkey dorsal horn neuronal activity and the perceived intensity of noxious heat stimuli. J. Neurophysiol., 62: 450-457. Ghoshal, N.G. and Getty, R. (1971) The lumbosacral plexus (plexus lumbosacralis) of the sheep (Ovis aries). New Zealand Vet. J., 19: 85-90. Hartell, N.A. and Headley, P.M. (1991a) Spinal effects of four injectable anaesthetics on nociceptive reflexes in rats: a comparison of electrophysiological and behavioural measurements. Br. J. Pharmacol., 101: 563-568. Hartell, N.A. and Headley, P.M. (1991b) Preparative surgery enhances the direct spinal actions of three injectable anaesthetics in the anaesthetized rat. Pain, 46: 75-80. Headley, P.M. and Grillner, S. (1990) Excitatory amino acids and synaptic transmission: the evidence for a physiological function. Trends Pharmacol. Sci., 11: 205-211. Sorkin, L.S., Morrow, T.J. and Casey, K.L. (1988) Physiological identification of afferent fibers and postsynaptic sensory neurons in the spinal cord of the intact, awake cat. Exp. Neurol., 99: 412-427. Wall, P.D., Freeman, J. and Major, D. (1967) Dorsal horn cells in spinal and freely moving rats. Exp. Neurol., 19: 519-529.