2 9 Chapter 1 General Introduction
6.A im s of th is w o rk an d tech n iq u es used.
Epilepsies com prise som e of the com m onest neurological conditions
enco u n tered in m edicine. Epilepsy affects betw een 0.04% a n d 0.1% of the
global p o p u latio n an d in m any cases, continues to be h ighly debilitating
desp ite the ad v en t of m o d ern p h arm acotherapy an d n eu ro su rg ery . For this
reason, it is im p o rtan t th at anticonvulsant th erap y is continually im p ro v ed in
an a tte m p t to control incidence of refractory seizures an d im p ro v e often
unacceptable d ru g side effect profiles. Inform atiom reg ard in g the m an n er in
w h ich existing anticonvulsant d ru g s such as VPA alter brain n eurochem istry
is therefore of fu n d am en tal im portance an d m ay aid in fu tu re d esig n of novel
com pounds.
In the preceding sections of this introduction, experim ental a n d clinical
evidence for the involvem ent of central excitatory an d inhibitory
n eu ro tran sm issio n in the pathophysiology of epilepsies has b een p resen ted ,
to g eth er w ith a brief review of the know n pharm acodynam ic actions of VPA
o n these system s. Pharm acological agents th at alter brain biochem istry m ig h t
be expected to exert a pro p o rtio n of their action at the cellular level by altering
extracellular concentrations of neurotransm itters. D em onstration of such p h en o m en a w o u ld have im portant im plications for the assu m ed m ode of
action attrib u ted to such agents since it w o u ld pro v id e a likely indication of
changes in neu ro tran sm itter activity at the synaptic level. Such d ata w o u ld
therefore p ro v id e a 'm issing link' in a chain of events from p rim ary
biochem ical changes induced by the d ru g th ro u g h to its ultim ate
3 0 Chapter 1 General Introduction
G iven the lack of data currently available reg ard in g the effect of VPA (and
oth er anticonvulsants) on central neurochem istry in vivo, it w as decid ed th at
this sh o u ld be investigated in relation to the kno w n neurochem ical actions of
the drug. The w ork p resented in this thesis falls into tw o m ajor Categories,
b ased em pirically on the techniques used: Thus in chapters 3-6, the effects of
acute an d prolonged VPA adm inistration on extracellular neu ro tran sm itter an d
m etabolite levels in ra t brain w ere extensively stu d ied using the techniques of
in vivo m icrodialysis an d high perform ance liquid ch ro m ato g rap h y (HPLC).
In the pen u ltim ate chapter (8), interactions betw een VPA an d m onoam inergic n eu ro tran sm itters w ere stu d ied inferentially usin g an established m odel of
intractable epilepsy (pilocarpine-induced lim bic seizures; Turski et al, 1989).
These tw o in viv o p harm acologicahpproaches w ere b o u g h t together in chapter
7, in w hich the effects of VPA a n d a chem oconvulsant on extracellular
excitatory an d inhibitory am ino acid n eu ro tran sm itters w ere m o n ito red
concurrently w ith behavioural changes.
6.1 In vivo m icrodialvsis studies.
The relatively n ew technique of in vivo m icrodialysis has becom e an
established a n d reliable m ethod of m onitoring extracellular levels of a variety
of end o g en o u s an d exogenous substances. The brain is p articu larly am enable it
to stu d y usin g this technique (review ed by Benveniste, 1989) and^is n o w often
the m eth o d of choice for in vivo m onitoring of the extracellular (EC) space.
The technique has a num ber of advantages over other system s u sed for
stu d y in g the b rain EC space an d these are sum m arised in table 3. D etailed
3 1 Chapter 1 General Introduction
A s indicated in sections 4.1 an d 4.2, the hip p o cam p u s a n d striatu m are
co n sid ered to play crucial roles in the generation an d sp re a d of seizure
activity. In addition, both structures are richly inn erv ated by m onoam inergic
a n d am ino acid n eurotransm itter pathw ays, allow ing the collection an d
analysis of a large n u m b er of neurotransm itters an d m etabolites, u sin g the
m icrodialysis technique. Since m any of these substances share roles in
controlling n eu ro n al excitability via interaction w ith receptors, activation or
blocking of w hich can p rofoundly alter the character, d u ra tio n an d intensity
of seizures, it seem ed logical to examine the neurochem ical effects of VPA in
these structures. In addition, VPA m ay be selectively accum ulated in the
h ip p o cam p u s, com pared to the cortex and striatu m (Loscher an d N au , 1983)
su g gesting th at the d ru g m ig h t effect these structures differently. F urtherm ore,
there is som e evidence to suggest that the hip p o cam p u s is a specific site for
the an ticonvulsant action of VPA. In rabbits, subjected to electrically in d u ced
focal seizures in neocortex, visual cortex an d h ip pocam pus, VPA (but n o t
carbam azepine or phénobarbital) strongly su p p ressed seizure after-discharges
(AD) recorded in the hippocam pus, b u t not cortical regions (K ubota et al,
1990). The converse w as ap p aren t for the other anticonvulsants. These findings
do n o t exclude the possibility that the striatu m m ay be an im p o rta n t site for
the an tico n v u lsan t m echanism of action of VPA. In the pilocarpine m odel of
lim bic m otor seizures, the striatum appears to play a critical role in
m o d u la tin g seizure threshold an d intensity (Turski et al, 1988; Al-Tajir an d
Starr, 1990) a n d VPA is highly effective in p reventing these seizures (Turski
3 2 Chapter 1 General Introduction A dvantages D isadvantages In situ I o n -s e le c tiv e m ic r o e le c tr o d e s a. A ll b ra in r e g io n s c a n b e e x a m in e d . b . T im e r e s o lu tio n < l s . c. T ip o f e le c t io d e < l p m . a. O n ly d e te c ts io n s C a rb o n fib re m ic r o e le c tr o d e a. A ll b ra in r e g io n s c a n b e e x a m in e d . b . C a n b e u s e d in c o n s c io u s a n im a ls. c. T im e r e s o lu tio n < I m in . d . T ip o f e le c tr o d e < 1 0 0 p m a. O n ly d e te c ts o x id is a b le c o m p o u n d s . b . S e le c tiv ity p o o r in a b se n c e o f p r e v io u s H P L C a n a ly s is . c. D r a in a g e d e p le t io n . d . S o m e e le c tr o d e s h a v e sh o r t w o r k in g life in v iv o E x situ P u s h - p u ll c a n n u la a. A ll b ra in r e g io n s c a n b e e x a m in e d . b . C a n b e u s e d in c o n s c io u s a n im a ls. c. BBB^ in ta c t f o llo w in g im p la n ta tio n . a. T im e r e s o lu tio n > 1 0 m in b . D r a in a g e d e p le t io n . c. E n z y m a tic d e g r a d a t io n o f c o lle c te d c o m p o u n s . d . D e p r o te in is a tio n o f sa m p le p rior to H P L C . e. T is s u e tra u m a f o llo w in g a n d d u r in g im p la n ta tio n . f. C a n n u la d ia m e te r > l m m M ic r o d ia ly s is p r o b e a. A ll b ra in r e g io n s c a n b e e x a m in e d . b . C a n b e u s e d in c o n s c io u s a n im a ls. c. BBB in ta c t f o llo w in g im p la n ta tio n . d . M in u te t is s u e tra u m a w it h in th e first 4 8 h o u r s . e . N o n e e d for d e p r o te in is a tio n o f s a m p le s . g . N o e n z y m a tic d e g r a d a tio n o f s a m p le s . a. T im e r e s o lu tio n > 5 m in b . D r a in a g e d e p le t io n . c. P r o b e d ia m e te r a p p r o x im a te ly 0 .6 m m f" B lo o d b r a in b arrier.
Table 3. A d v an tag es a n d d isad v an tag es of v ario u s in v iv o m o n ito rin g techniques.
3 3 Chapter 1 General Introduction
effect b y enhancing GABAergic neurotransm ission in the su b stan tia nigra
(Loscher, 1989), a structure intim ately associated w ith striatal function
(G raybiel an d Ragsdale, 1979). M ethodologically, the h ip p o cam p u s an d
striatu m in the ra t lend them selves to in vivo m icrodialysis. Reference to figure
2 (chapter 2) illustrates the relative ease w ith w hich m icrodialysis p ro b es can
be be im p lan ted into these structures. All m icrodialysis experim ents p resen ted
in successive chapters used conscious anim als, since anaesthesia is th o u g h t to
interfere w ith the effect of VP A on brain EC m onoam ine levels, as estim ated
u sin g in vivo differential pulse voltam etry (Crespi et al, 1986).
6.2 Pilocarpine seizure m odel.
The pilocarpine m odel of lim bic m otor seizures w as chosen to investigate the
role of m onoam inergic neurotransm ission in the m echanism of action of VP A
on the basis of findings p resen ted in chapter 3. and d u e to the established role
of m onoam ines in the aetiology of seizures in duced by the cholinom im etic
(Turski et al, 1988; Janusz an d Kleinrok, 1989) The neurochem ical
consequences of pilocarpine adm inistration a n d su b seq u en t seizures in ro d en ts
are discussed in chapter 8. The reader is referred to Turski et al (1989) for a