In 5 experiments, in which a robust U l and U2 response could be recorded, the optic tract was tetanically stimulated at a frequency of lOOHz for a total period of 2 seconds. This tetanus regime consisted of 4 groups of 50 stimuli delivered at a frequency of lOOHz with each burst separated by 500msec. These stimulus
parameters would usually induce long-term potentiation (LTP) in the perforant path to dentate gyrus pathway of the hippocampus (Bliss and Colingridge, 1993). In this preparation the effect of this high frequency stimulation paradigm was monitored on the peak amplitude of the U l and U2 response which was recorded at a constant
150/xm below the tectal surface. First, stable responses were recorded from the
tectum (at a stimulation rate of 1/50 sec) for at least 30 minutes before the tetanus was delivered. The stimulus intensity was at threshold for the U2 response and so did not elicit the maximal peak amplitude for this response (figure 6.7 A). The same stimulus strength was employed during the tetanus as that used during the control period. The peak amplitude of the U l and U2 response was then calculated for up to one hour post tetanus during which the optic tract was once again stimulated at the control frequency of 1/50 sec.
In 1 preparation we did observe a change in the magnitude of the U2 response (figure 6.7 A & B). Immediately following the lOOHz train a 50% increase in the peak amplitude of the U2 response was observed with no corresponding increase in the peak amplitude of the U l response. However, in the remaining 4 experiments both the U l and U2 response was depressed immediately following the tetanus and both responses recovered to basal levels within 10 stimuli. When all 5 experiments were normalised and averaged we could not detect any significant difference in the Ul and U2 peak amplitude following high frequency stimulation (figure 6.7 C).
A 2 0 0 - 1 5 0 - ( n = 1 ) X 1 5 0 - 1 0 0 H z , 2 s e c ( n = 5 ) 3 0 6 0 S T I M U L U S ( 1 / 5 0 s e c ) F i g u r e 6 . 7 E f f e c t o f h ig h f r e q u e n c y s t i m u l a t i o n o n t h e p e a k a m p l i t u d e o f t h e U l a n d U 2 r e s p o n s e .
A. T w o traces recorded 150/rm b e lo w th e tectal su rface fo llo w in g o p tic tract s tim u la tio n . T h e left h a n d re c o r d w as obta ined d u rin g the co n tro l p e rio d im m e d ia te ly b efo r e a h ig h freq u en cy tr ain w as d e liv e r e d to the optic tract. Stimuli w e re d e li v e re d at a rate o f 1/5 0 s e c w h ic h w e r e m a x im a l fo r the U l r e s p o n s e (filed circle) but at threshold for the U 2 re s p o n s e (o pen circle). T h e right h a n d tr a c e w as r e c o r d e d f r o m the te c tu m fo llow ing the lOOHz tetanus. In this e x a m p le th e p e a k a m p litu d e o f th e U I r e s p o n s e has n o t altere d but the peak a m p litu d e o f the U 2 re s p o n se has n o ticeab ly in creased. Scale bar = 10msec, I m V
B. T h is g ra p h depicts the peak a m p litu d e s fo r the U l re s p o n ses (filled circles) a nd U 2 r e s p o n s e s (o p en circles) calc ulated for all stim uli d e li v e re d d u r in g e x p e rim e n t A. T h e s tim u lu s tim e is o n th e ab s c i s s a w ith the n o rm alised da ta o n the o rd in a te . T h e p e a k a m p litu d e valu es for th e U 1 a nd U 2 r e s p o n s e h a v e been no rm alised to their 100% level relative to the first 10 re s p o n se o f the e x p e r i m e n t . F o r o v e r 30 m in u te s b efore the tetanus was d e liv e re d the peak a m p litu d e o f the U l a nd U 2 re s p o n se r em ain at a p p r o x im a te ly this 100% level. T h e filled b o x illu strates the d u r a tio n o f the h igh fr equency train. D u r in g th e tetanus period the peak a m p lit u d e s o f the U l a nd U 2 re s p o n se s have not b een c alc u la te d . F o llo w in g this tetan us the peak a m p litu d e o f the U l r e s p o n se re m a in s at co n tro l levels but the peak a m p l it u d e o f the U2 re sponse has increased by 5 0 % . T h e U 2 peak a m p litu d e r e m a in s at this ele va te d level for o v e r 30 minutes post-tetanus.
C . T h e a v erag e o f 5 separate e x p e rim e n t s in w h ic h the U l (filled circle) and U2 peak a m p lit u d e s (o p en circle) w ere calculated b efore and after hig h frequency s tim u la tio n (filled b o x ). T h e p eak a m p l it u d e for the U l and U2 re sponse d o e s no t c h a n g e fr o m its 100% control level f o llo w in g the tetanus.
II. Pharmacology
lïxtracellular fîehi pcüemüals were recKHxied aü a cÆwistant degyüiof twdkyw
the pial suifacxsimd the pedc anipliüide of the Ul auid U2 response was calcidadxxl
following supra-threshold stimulation of the optic tract at a repetition rate o f l/50sec.
TThe pwsak ampiUtuck: of die Ul arxi U2 respcMise was morritcmxl for a control period of ait leat 3()nnriutes beixxne the aiüaijgonist was adcksd to the txidihig maliurn at a dcdiiKxi
concentration. One-way analysis of variance (ANOVA) demonstrated that there was
110 stadsticai &i9nifk%uice(]):>().()5) txehvaen die avismge pwsak arnpiituckss nscorcked in the (hffercait (sxpNerhiwental groups. In eauch expierininetal gitmp ()ne cxmceiitmdcHi of antagonist was tested on a naive preparation. The effect o f each drug was allowed to
stalxUise for ait least 3()iidriutes anclllwsn the tiawe was returiKxi to ncimial fmg ring;er. Thi(5 96 dümgeiii die ptxik arnpilituck:4)f die III arid IJ2 resqponœ was calcukikxi for a given pKsrhirbailicMi arui arrp süiüstical sigrûficarice in this cliang;is was dekmmineci using;
a paired non-parametric two-tailed t-test.
6.2.4. Effect of non-NMDA-type glutamate receptor antagonists
The selective non-NMDA-type glutamate receptor competitive antagonist CNQX was added to the bathing medium in a total o f 23 preparations at specific concentrations ranging from 50nM to 50^M. Figure 6.8 shows the results o f one
ecpKanumient in whrcdi SO/iA/I CISK):X wa&iuidai. In this exjperinieritaiisüdile Ul aund U:2
response had been recorded for 30 minutes and the addition of CNQX caused a very rapid decrease in the peak amplitude of the U l response. The decrease in the U2
resiiomx; v/as nc)t()bseivred imdl 5 minihes siibsexiuent to (ZNQOC apqplkxidcm bqf
which time the U l response had been reduced by -3 0 9 6 its original value. It was noted that the U l and U2 responses were transiently elevated during the initial inclusion o f the antagonist into the bathing medium. However, after 30 minutes of drug application the U l and U2 response had completely disappeared. At this
cxincxsntration (if antag;ornst itiwas ncK possUble to re\nsrse this blcxzk.
The effect o f CNQX was dose dependent (Table 6.1) but was not reversible in all experiments. At a lOjuM concentration a significant suppression was observed in
both the U l peak amplitude (47.396 ± 5.196; p < 0.001) and the U2 peak amplitudes
(61.496 ± 6.996; p < 0.001). At a 50fiM concentration a 71.496 + 5.796 suppression
in the U l peak amplitude and a 70.496 ± 9.096 suppression in the U2 peak amplitude
response was increased by an average o f 30% , ranging from a 16% decrease in one preparation to an 81% increase in another. The U l response did not exhibit such a m arked fluctuations in the presence o f 50nM CN Q X .
CNQX > E LU Q Z ) < LU Q_ 2. 5 2.0 1. 5 1.0 0 . 5 0 2. 5 -1 2.0 - 1. 5 - 1. 0 - 0 . 5 - 0 - .0- " O' o • I m V 2 0 m s e c - # — — K . T T 3 0 6 0 STIMULI ( 1 / 5 0 s e c : 9 0 120
Figure 6.8 Effect of a non-NM DA-type glutam ate receptor antagonist on posLsynaptic field potentials recorded from the tectal neuropil.
T h i s figure describ es the re sults o f a typical e x p e rim e n t in w h ic h the effect o f 20/xM C N Q X w a s a s s e s s e d on tectal field potentials recorded in vitro. T h e o p tic tract w a s stim u lated at a rate o f 0 . 0 2 Hz. T h e pe a k a m p litu d e s, plo tted as positi ve v alu es, for the U l r e s p o n se s (filled circles) a n d U 2 r e s p o n s e s ( o p e n circles) w e re calc ulated for each stim ula tio n. R e p re s e n t a tiv e traces a re s h o w n fo r r e s p o n s e s b e fo r e , d u rin g and after the inclusion o f antagonist into th e b a th in g m e d iu m . T h e solid b ar in d ic a te s the period for w h ic h C N Q X w as pre sent. Both the U l and U 2 peak a m p lit u d e s w e re s ig n ific a n tl y re duced by C N Q X . In this e x p e rim e n t the effect w a s ir re v e rs ib le but this w a s no t th e ca se to r all e x p e r im e n ts . N o te h o w the U l res ponse w a s reduced by C N Q X b e fo r e the later U 2 re s p o n s e w a s a ff e c te d . In so m e case s a transient e n h a n c e m e n t o f bo th peak a m p litu d e s w as o b s e r v e d w h e n C N Q X c o n ta i n in g m e d iu m w as a dded but this result was not consistent.