COMPARACIÓN DE LAS METODOLOGÍAS

The purpose o f the experiments described in this chapter was to dissociate the roles of different cerebellar components in the formation of a motor memory. The work featured here focuses on the classically conditioned nictitating membrane response (NMR) in the rabbit as an example of a motor memory. There has been a long­ standing debate as to whether a primary site of information storage pertaining to this task exists within the cerebellum (McCormick et al., 1982a) (Welsh and Harvey,

1989) (Yeo et al., 1985c). The debate has further encompassed the possibility that such storage may occur at one of two cerebellar locations. The cortex and underlying deep nuclei of the cerebellum have both been considered strong candidate sites for the occurrence of the physiological changes involved in motor learning. Influential

models (Marr, 1969) (Mauk and Donegan, 1997) and empirical studies (Gilbert and Thach, 1977) (McCormick and Thompson, 1984b) of cerebellar function have implicated both structures in learning.

The vast majority of research conducted into the involvement of cerebellar

components in motor learning has centred on classical eyeblink conditioning and, in particular, classical conditioning of the nictitating membrane response (NMR). This behaviour is regarded as an extremely well defined model of generic motor learning (Gormezano et al., 1962). Lesion studies have identified both lobule HVI in the cerebellar cortex (Yeo et al., 1985b) and one of the deep nuclei, the anterior interpositus nucleus (AIP) (Lavond et al., 1984) (Yeo et al., 1985c), to be essential structures in the performance of the conditioned NMR. Localised reversible

inactivation of these structures using pharmacological agents (Attwell et al., 2001) has confirmed that deficits in NMR conditioning incurred by lesions are due to a failure in learning rather than performance. Such studies have not been able to dissociate

between effects in HVI, the AIP and key afferent sites such as the pontine nuclei and inferior olive in the brainstem. This may be due to a distribution o f the plasticity required for NMR conditioning across the system. However, it may alternatively be the case that plasticity is discretely localised at one o f these sites, although their

connectivity may result in a failure to dissoeiate the effects o f reversible inactivation at these various sites.

In support o f this interpretation, lobule HVI, the AIP and the dorsal accessory com ponent o f the inferior olive (DAO) have been shown to form a functional loop within cerebellar eyeblink circuitry (Hesslow and Ivarsson, 1994) (H esslow and Ivarsson, 1996). A consequence o f this arrangem ent is that pharm acological intervention at any one o f these loci could interfere with physiological changes

occurring at another. Reversible inactivation during the training process may therefore exert its deleterious effect on NM R conditioning either because o f a primary effect on storage processes or because o f a secondary effect on the input that is necessary for encoding information during learning (Yeo et al., 1997) (Ramnani and Yeo, 1996).

CNQX Picro- toxln & £

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Figure 2.01: Primary, secondary and tertiary effects of reversible inactivation -

This figure depicts a supposed set o f consequences for 3 cerebellar inactivation experiments that impair eyeblink conditioning. A) Infusion o f AM PA receptor blocker CNQX into the cerebellar cortex (Attwell et al., 2001). B) Infusion o f

G A B Aareceptor antagonist picrotoxin into the deep nuclei (Garcia and Mauk, 1998). C) Infusion o f sodium channel blocker lidocaine into the inferior olive (W elsh and Harvey, 1998). Assuming a certain amount o f tonic activity at each site, the arrangem ent o f these structures into a loop means that any one o f these different ‘inactivations’ putatively results in a similar effect on the activity o f each component o f the system, as indicated by the arrows. In the case o f both B and C the

consequences for cortical activity may not be so straightforward. Reduced climbing fibre-m ediated complex spiking may actually lead to increased simple spiking due to a reduction in the time spent by the Purkinje cell m embrane in the refractory state.

The principal concern of the studies presented in this first experimental chapter is to circumvent this problem. Here reversible inactivation is restricted to the period in which memory consolidation is thought to occur. The inactivating agent is delivered to select sites within the cerebellum after NMR conditioning has finished in order to target just potential consolidation processes. It is considered likely that some o f the processes of memory consolidation are related to information storage rather than perpetuated information encoding. Post-training inactivation could therefore exclude the possibility that encoding is prevented by interference with information

transmission.

Systemic post-training infusions of various drugs have been shown to affect the consolidation of NMR conditioning in rabbits (Scavio et al., 1992) (Hernandez and Powell, 1983). 1 have attempted to interfere with the consolidation of this task with localised infusions and thereby isolate a site of memory storage. These experiments have used the GABAa receptor agonist muscimol as an inactivating agent. This

substance affects somatically generated action potentials without blocking action potentials in fibres o f passage. It can therefore be used to target select loci within the brain. Within the cerebellum, muscimol has previously been infused pre-training to investigate eyeblink conditioning (Hardiman et al., 1996) (Krupa et al., 1993).

Localised muscimol infusions impinge upon NMR conditioning when applied during training to key areas of the previously described olivo-cortico-nuclear loop.

Muscimol has not thus far been infused after NMR conditioning. It has, however, already been shown to impair some other forms of memory when applied after

training. Post-training systemic injections prevent the acquisition of passive avoidance in the mouse (Ammassari-Teule et al., 1991). Inhibitory avoidance in rats is impaired by localised infusions of muscimol into the amygdala (Brioni et al., 1989) (Castellano and McGaugh, 1990) (Izquierdo et al., 1997) (Zanatta et al., 1997). My experiments adopt a similar approach to these studies in evaluating the neural substrate of

consolidation in NMR conditioning. In these experiments guide cannulae are used to direct muscimol infusions towards either lobule HVI of the cerebellar cortex or the AIP ipsilateral to the eye being trained.

M ethods

All subjects included in this chapter were Male Dutch belted rabbits (2.0-2.2kg). All procedures using these subjects conformed to Home Offiee licensing regulations.

Surgery

The cannulation procedure was conducted with the animals under general anaesthesia. Initially animals were intubated under fentanyl/fluanisone anaesthesia (Hypnorm, Janssen; 0.1/5.0 mg/kg, i.m.). Supplementary benzodiazepam (Valium, Roehe; 0.5 mg/kg, i.v.) was supplied as a muscle relaxant. Subsequently each subject was infused intravenously with 20% mannitol (20ml, Iml/min) in order to reduee the size of the brain and ease the avoidance of major sinuses during surgery. Enrofloxacin antibiotic was also administered (20 mg, i.p.). Anaesthesia was maintained during surgery with halothane (1.5-2.5%) in a nitrous oxide/oxygen mixture (1:3). The head was placed in a stereotaxie instrument (Kopf) so as to fix its position and the scalp was incised and drawn back. All surgical equipment was sterilised in an autoclave and washed in Hibitane (0.5% hexachlorophene in 70% alcohol) when re-used. Aseptic conditions were maintained throughout the surgery. Both skull and dura were removed in order to expose the right cerebellar cortex. 11mm long 26G stainless steel cannula guides were then implanted targeting either HVI in the cortex or the deep nuclei. HVI implantations were placed according to visual inspection of the cerebellar surface. Deep nuelear implants were placed stereotaxically. Initial placements used

coordinates that had been determined by previous pilot studies. Adherence to these eoordinates resulted in an overly dorsal cannula placement (see DNR group in the Results section). They were therefore modified accordingly. With the head positioned such that the skull landmark lambda was positioned 4.1mm above the more anterior landmark bregma, the suceessful eoordinates were AP = -5.2mm (relative to lambda), ML = -4.5mm and V = -11mm from skull surface. Following introduction of the cannula, the exposed brain was covered with sterile absorbable gelatin foam. The cannula guide was then cemented to the skull with dental acrylic and, once the acrylie had set, the sealp was sutured around the cannula guide. A stylet was inserted into the guide in order to prevent blockade of the guide or invasion by potentially infective

agents. Each animal was treated with buprenorphine (Vetergesic, lOOmg/day) analgesia and the antibiotic enrofloxacin (Baytril, 30mg/day) for 3 days post- operatively. All animals were provided with food and water ad libitum and

maintained on a 12hr light/dark cycle in individual cages. They were allowed at least 1 full week’s recovery before behavioural testing began.

This same surgery protocol was followed for the two subjects in study 2 but cannulae were implanted bilaterally in the paramedian lobes.

Behaviour

The NMR conditioning experiments presented here were based on those first developed by Gormezano et al. (Gormezano et al., 1962). Both the apparatus and the techniques used were very similar. Subjects were first restrained in a specially designed Perspex stock (see flgni e 1.01). A nylon monofilament loop was then sutured into the right nictitating membrane (NM) of each subject under local anaesthesia (proxymetacaine hydrochloride; 0.5% w/v). A low-torque potentiometer was bracketed around the ears and muzzle o f the subject and secured so as to rest on top o f the head. A hook at the end of a lever coupled to the potentiometer shaft was inserted into the monafilament loop in the NM and trapped with a plastic collar. The potentiometer was isotonically coupled to the movement via the lever and a universal joint to transduce the movement of the NM. This mechanism did not require a restoring force to transduce the movement (see (Gruart and Yeo, 1995)).

Each subject was then placed into a sound-attenuating chamber facing a centrally mounted loudspeaker. Conditioned and unconditioned stimuli were delivered in this ventilated enclosure. The unconditioned stimulus (US) consisted of 2mA periorbital electrical stimulation. This stimulus comprised a 60ms train of 3 biphasic pulses delivered through stainless steel clips attached to the skin. One clip was placed directly below the lower eye-lid in a central position and the other just behind the temporal canthus of the eye. The conditioned stimulus (CS) was an 81dB tone, 410 msec duration 1 kHz sine wave. It was delivered against a 57dB background noise produced by the ventilation fans. On paired trials the interstimulus interval (ISI) between the

onsets of the CS and the US was 350 ms and the intertrial interval was randomly selected between 25 and 35 seconds.

Subjects received a habituation session on the day before the beginning of experimental training. Each subject was restrained within the conditioning chamber over this period with the NM transducer in place. All subjects were then allowed a full 25-min session in which to adapt to the novel experience of being restrained and enclosed within this environment. The only difference between the habituation session and the training sessions was that the CS and US were not presented.

The two animals that were included in study 2 did not receive any training before sacrifice.

Reversible inactivation

Either muscimol (Sigma) (3.5mM, 2pl in 0.0IM phosphate buffered saline (PBS), pH7.4) or vehicle (0.0IM PBS, 2|il) was infused over two minutes into the right

cerebellar cortex. Initial muscimol infusions into the deep nuclei (see DNR group in the Results section) were of the same molarity as cortical infusions but of a smaller volume (l|xl). This dosage of muscimol had been determined in a previous dose-response study (Ramnani, 1997). Animals receiving this dose of muscimol were not completely prevented from performing previously acquired CRs by pre-training infusions of muscimol (See Phase 5 performance testing). This lack of complete effect could be attributed to an overly dorsal guide cannula placement (see Surgery section), but it was also considered that the dosage of muscimol should match that used in the cortex (7nmoles) for the sake of later comparisons between the groups. Therefore, the

molarity, but not the volume of infusions was accordingly modified. Muscimol (7mM, l|uil in 0.0IM PBS) was infused into the deep nuclei over one minute. All infusions were delivered through 12mm long (1mm below guide) 31G stainless steel injection cannulae (Plastics One, Roanoke VA, USA) using microsyringes (Hamilton, Sigma). The injection cannula was removed from the guide five minutes after injection.

Histology

Before sacrificing, perfusing and fixing subjects, ^H-muscimol in PBS was infused through the injection cannulae at the same position at which infusions had been delivered during the experiment. These infusions matched the experimental infusions in both dosage and volume. Tnmoles of ^H-muscimol was delivered in each final infusion. For subjects receiving deep nuclear infusions, this dose was in l|il PBS. For the cortically implanted subjects the volume was 2pl. Again drug was gradually infused at a rate of 1 pl/min. Muscimol contained 1 |xCi/|Lil of radioactive drug.

Two hours after the end of this final muscimol infusion each subject was given heparin sodium (500U/kg, i.v.) in order to prevent blood coagulation during perfusion and then sacrificed with an overdose of pentobarbitone sodium (90mg/kg, i.v.). This time was chosen for sacrifice because it was the point at which, for all subjects included in the experiment, the drug had a complete effect on the performance of previously acquired CRs when delivered pre-training (see Phase 5 performance testing). It was deemed necessary to have a consistency in the time allowed for radioactive drug spread across subjects. Transcardial perfusions with IL of 0.9% saline followed by 2L of 4% formaldehyde solution cleared and fixed the brain tissue. The brain was then removed with rongeurs and post-fixed in 4% formaldehyde. Subsequently, it was embedded in gelatin and cryoprotected in 20%

sucrose/formaldehyde solution. 50|am coronal sections o f the brain were taken in series after completion of fixation and cryoprotection using a sledge microtome. Sections were then organised into six interleaved series, in which sections were 300|tim apart, and mounted on gelatin-coated slides. One o f these series was immediately Nissl stained with cresyl violet for histological analysis, another was used for autoradiography and the rest were kept as spare series. Cannulation damage was assessed from Nissl stained sections by an experimenter blind to both

' - à # '

Figure 2.02: Criteria for damage - The middle panel shows a caudal coronal section o f cortical