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Receptor site » « Glycine 2+ - binding / site

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Figure 37

A: In the absence o f glutamate the NMDA receptor is closed.

B: Channel opening requires binding o f glutamate and the co-agonist glycine. The receptor for glutamate is located on the NR2B subunit (Laube et al 1997), and die glycine receptor is on the N R l subunit (Lynch et al 1994). At normal resting potential the pore o f the receptor channel is blocked by a magnesium ion (Mayer et al 1984).

C: Depolarization o f the postsynaptic membrane causes the magnesium ion to move into the extracellular space, opening the channel and allowing calcium influx (Nowak et al 1984).

Chapter 4. Postnatal development o f spinal cord connectivity.

developing animals, this is clearly impractical, and many previous experiments have relied upon daily intraperitoneal injections o f MK-801 (Kalb 1994; Lawson and Lowrie 1998). This method is unsatisfactory for three reasons; firstly, high concentrations o f MK-801 must be administered (typically 0.5-1 mg/kg) to ensure an assumed effective level o f drug is maintained in vivo until the next injection. Secondly, such high levels result in ataxia in the pups leading to poor feeding and subsequent weight loss and high mortality. Large reductions in size and weight o f rat pups receiving daily MK-801 injections have been reported, with a concomitant reduction in cortical size (O ’Donoghue et al 1993). Thirdly, doses o f MK-801 in this range have been shown to induce apoptosis in postnatal cortex (Ikonomidou at al 1999).

A viable alternative has proved to be the use o f implantable sustained release polymers (Langer 1990). An example o f such a polymer, EL VAX, has been successfully utilised in a number o f neuroscience applications (Schnupp et al 1995; Jablonska et al 1995; Mooney et al 1998; Penschuck et al 1999). There are several advantages to its use in the current application;

O Easily manipulated - allows for small discrete implants.

0 Reduced disturbance to the CNS area being studied. © Restricted local release minimises effects in other areas. O Reduced toxicity.

© Greater length o f time o f drug delivery. © Constant low level release.

EL VAX is an ethylene-vinyl acetate copolymer that has been successfully used to deliver a number o f compounds to CNS regions, eg TTX (Chiaia et al 1992), NT-3 (Doughty et al 1998) and 5H-T (Mooney et al 1998). Its presence has been shown to have less deleterious effects, particularly on cytoarchitecture, than other implantable substances, eg gelfoam (Persico 1997). O f particular interest have been NMDA receptor antagonists (Cline and Constantine-Paton 1990; Schlagger at al 1993) and their release properties in EL VAX preparations have now been well characterised, with sustained release at a constant level maintained for at least 60 days (Smith et al 1995).

It is possible to draw an analogy between the two systems described above, and the postnatal changes that occur in the dorsal horn o f the spinal cord;

1. Each provide a pathway to transmit sensory information from the periphery to higher CNS areas (Kaas et al 1983; Killackey et al 1990; Cellerino and Maffei 1996).

Chapter 4. Postnatal development o f spinal cord connectivity.

3. Initially diffuse projections are ‘fine-tuned’ postnatally (Fitzgerald et al 1994; Fitzgerald and Jennings 1999).

4. In all cases, layer/laminar specific changes occur.

1.4: Development of primary afferent innervation of the mammalian dorsal horn

The central terminals o f Ap cutaneous afferents have a wider dorsoventral distribution within the spinal cord dorsal horn during the immediate postnatal period than in the adult. This was originally described by Cajal in 1909. Figures 38 shows one o f Cajal’s original drawings and transverse sections o f Dil labelled neonatal spinal cords (see methods section 2.8). The primary afferent bundles projecting into the superficial laminae can be clearly seen. Figure 39 shows a further example. The final pattern o f laminar organisation is not finally established until after 3 weeks o f age (Fitzgerald et al 1994; Mimics and Koerber 1995b; Park et al 1999). During this time A and C-fibres occupy the same territory until the A-fibres gradually withdrawal. The developmental profile o f this withdrawal is shown in figure 40. Sciatic nerves o f rats at the ages shown were injected with BHRP (as described in the methods 2.6). At early ages labelling is clearly evident in the entire dorsoventral extent o f the superficial dorsal horn. By P21 some thinning o f labelling has occurred in SG and is gone by P28. This is in agreement with previous anatomical and electrophysiological reports (Fitzgerald et al 1994; Park et al 1999).

In the rat, dorsal roots first reach the lumbar spinal cord at E l 2, where they bifurcate, projecting rostrocaudally along the dorsal columns before sending collaterals in to the grey matter at E l 5 (Fitzgerald et al 1991). The first o f these fibres to innervate the dorsal horn

1.5: Possible mechanisms for the postnatal withdrawal of A-fihres from lamina II.

Several hypotheses have been proposed to explain this phenomenon. From E l 5 until E l 9, cutaneous A-fibres are the only afferents terminating in the dorsal horn. While inhibitory factors prevent them growing into the ventral horn during this time (Fitzgerald et al 1993; Messersmith et al 1995), no such restrictions are apparent in the dorsal horn and therefore they are free to terminate throughout all the superficial laminae. At E l 9, C-fibres start to enter the dorsal horn and proceed to terminate specifically in SG (Fitzgerald 1987). This is possibly due to all postsynaptic sites in deeper laminae being occupied by the earlier projecting Ap-fibres. From this time the two populations co-exist until A-fibre withdrawal is complete 3 weeks later. In the spinal cord, SG is the last region to differentiate and

Primary afferent projections into the superficial