Table 1.1 Classification of Serotonin Receptors.
II. Materials & Methods
2.1 Animals.
M ale W istar rats (in - b re d strain, Bioresources Unit, Trinity College Dublin) w eig h in g betw een 2 4 0 - 3 6 0 g and w ere used for the acute stress experim ents. This w eight range corre sp o n d s to an age o f 7-11 weeks. W istar rats w ere ho u se d 2 p e r cage (u n le ss oth erw ise stated) in a Scantainer (D e n m a rk ) with food (standard rodent c h o w ) and w ater available ad libitum.
Male Congenital Learned Helpless (cLH, cN L H ) and control anim als (outbred Sprague- Daw ley rats) w eighing betw een 300-400g were obtained from B. Vollmayr, M annheim , G erm any. M ale Flinders Sensitive Line (FSL ) and Flinders Resistant Line (F R L ) rats (S prague D aw ley strain) w e ig h in g betw een 2 0 0 -3 0 0 g w ere obtained from A. Mathe, K arolinska Institute, Stockholm , Sweden. Sprague Daw ley rats were housed 4 per cage in a Scantainer with food and water available ad libitum.
Imported anim als w ere rested following arrival at the laboratory for a period o f at least one week. T he a nim als w ere housed under a tw e lve hour lig h t-d a rk cycle with the room tem pe ra ture being m ainta ine d betw'een 19-2 2 d egrees Celcius. A n im als w ere w eighed before use lo determ ine dose o f anaesthetic required.
2.2 Anaesthesia.
Prior to surgery a nim als w ere anaesthetised with urethane (ethyl carbam ate; 2.1 g /k g i.p.). The anim al usually rem ained stably anaesthetized for at least 4 - 5 hours w ithout requiring further a naesthetic. A heating blanket (H a rv a rd A pparatus H om eotherm ic B lanket Control Unit) w as used to m onitor and m aintain a tem p e ra tu re o f b e tw e en 3 6 - 3 8 ° C in the a naesthetised rat. Follow ing com pletion o f the experim ental protocol the animal was killed by cervical dislocation. The experimental protocols were licensed by the Departm ent o f Health &. Children, Ireland.
2.3 Surgery.
A fter 10 m inutes had lapsed follow ing delivery o f anaesthetic the a nim als paw was pinched to test for any m uscle response to the stim ulus. This w as used to assess the depth o f a n a esth esia in the anim al and to see i f a n a esth etic s u p p le m e n ta tio n was nec essa ry. Local anaesthetic (N o ro ca in e ) (2m l) c o n ta in in g lidocaine 2 0 m g /m l and a d re n a lin e 0 .0 1 2 5 m g /m l w as injected s u b c u ta n e o u s ly to the skull reg io n w h e re electrodes were to be later implanted. A scalpel incision was m ade from betw een the ears to b e tw e e n the eyes. T h e p e rio ste u m w as r e m o v e d using a c u rv e d scissors e xposing the skull surface. The skull was wiped clean and allowed to dry.
2.4 Position of Electrodes.
M on o p lo a r recording electrodes and bipolar stim ulating electrodes w ere used in each e xperim ent. The electrodes w ere m ade in the laboratory using two lengths o f teflon coated tungsten wire (625|.im tungsten diameter, 750^im total external diameter. A dvent Research Materials Ltd.) the ends o f which having had the teflon rem oved. Each o f the two wires were soldered to an individual pin o f a tw o pin connector and then twisted together. The wires w ere then glued together with cyanoacrylate to ensure strength and stability, allow ed to dry and fixed in place by dental (acrylic) cem ent. The electrical c o n tin u ity o f each wire was c h e ck e d before use to e n su re that the e le c tro d e was w orking. The ends o f the wires were cut at an angle exposing the tips so that one was m arginally below the other. The reference and ground electrodes c onsisted o f small stainless steel screws (Bilaney, G erm any) to which single pins had been soldered to the screw.
T he c o o rd in a te s for both the r e fe re n c e and earth s c re w s and the re c o r d in g and stim ulating electrodes w ere noted on the skull surface using a w a te rp r o o f marker. The recording electrode was positioned 3.4 m m posterior to b reg m a and 2 .5m m lateral to the midline. The stim ulating electrode was positioned 4.2 m m posterior to bregm a and 3.8 m m lateral to the m idline. A sc re w w h ic h acted as a refe re n c e ele c tro d e was
p o s itio n e d 8.0 m m a n te r io r to b r e g m a a n d lateral on th e o p p o s ite h e m is p h e r e (left) to th at u sed fo r e le c tr o d e im p la n ta tio n . T h e ea rth s c r e w w h ic h a c te d as g r o u n d e le c tro d e w a s p o s i t i o n e d 7 .0 m m p o s t e r i o r to b r e g m a a n d 5 m m la te r a l to th e m i d li n e . C o o r d i n a t e s fo r th e e l e c tr o d e s p o s itio n w a s o b t a in e d b y re f e r r in g to a rat b ra in atlas ( P e llig rin o et al. 1979) a n d w o rk c a rried out p re v io u s ly in o u r laboratory.
A d e n ta l drill u s in g drill bits (1.5 m m a n d 1 m m ) m a d e b u r r h o le s o v e r th e e le c tr o d e im p la n ta tio n sites. T h e drill w a s no t a llo w e d to p e n e tr a te th e skull in su ch a m a n n e r as to d is tu r b th e u n d e r ly in g d u r a m a tte r o r c o rtic a l h e m is p h e r e s . T h e s c r e w s w e r e then im p la n te d a n d s e c u r e d in p la c e w ith a sin g le d r o p o f glu e. T h e d u ra w a s th e n b u rs t u sin g a sterile s y rin g e ne e d le . T h is w a s d o n e to a llo w e a s y in se rtio n o f the e le c tro d e s. T h e rat w a s th en p l a c e d in th e s te r e o ta x ic r e c o r d i n g a p p a r a t u s ( A S l I n s tr u m e n ts ) in p r e p a r a t i o n fo r th e i m p l a n t a t i o n o f e l e c t r o d e s in to th e C A l a r e a o f th e d o r s a l h ip p o c a m p u s . F o ll o w i n g s a tis f a c to r y l o c a lis a tio n o f th e e l e c t r o d e s ( F i g u r e 2 . 1 ) th e e le c tro d e a s s e m b ly w a s fixed in p la c e fo r th e d u ra tio n o f the e x p e r im e n t u sin g g lu e an d dental ce m e n t.
2.5 C a n n u l a I m p l a n t a t i o n .
D r u g s t h a t w e r e n o t a b l e to p a s s t h e b l o o d b r a i n b a r r i e r w e r e d e l i v e r e d i n tr a c e r e b r o v e n tr ic u la r ly u sin g a c a n n u la . C a n n u l a e w'ere m a d e from a s ta in le s s steel h y p o d e r m i c n e e d le (2 2 g a u g e , 0.7 m m o u t e r d i a m e t e r ) w h i c h w a s c u t to 13m m in len g th . T h e b e v e lle d e n d o f th e n e e d le w a s g r o u n d d o w n to a le n g th o f 1.5 m m to r e d u c e th e a n g le o f th e e x p o s e d tip. W h e n th e c a n n u la w a s no t in use an in ternal plug c o n s is tin g o f sta in le s s steel w ire (2 8 g a u g e , 0 .3 6 m m d i a m e t e r ) w a s u s e d to p r e v e n t b l o c k a g e s fro m o c c u rrin g . T h e c a n n u la w a s in s e rte d 1.5 m m a n te r io r to b r e g m a , 0.5 m m lateral to the m id lin e a n d 3.55 m m b e lo w the d u r a ’s surfa ce. T h e s e c o o r d in a te s h ad b ee n s h o w n p re v io u s ly by o u r lab o ra to ry to b e e f fe c tiv e fo r in je c tin g into the v entric le s. V e rific a tio n o f the lo cation o f the c a n n u la w a s c a rrie d out p o s t- m o r te m b y c h e c k in g the s p r e a d o f d y e (I n d ia n ink) a f te r i.c.v. in je c tio n . F o ll o w i n g i m p la n ta t io n in th e right lateral c e re b r a l v e n tric le the c a n n u la w a s s e a le d w ith g lu e a n d den tal c e m e n t. A lO^l H a m ilto n sy rin g e to w h ic h an internal c a n n u la (28 g a u g e , 0 .3 6 m m o u te r d ia m e te r ) w a s
attached was used to deliver drug solutions i.c.v. T he internal cannula was fixed so as to p r o tr u d e 1.8 m m b e lo w the end o f the c a n n u la a s se m b ly . T h e d ru g s w e re a dm inistered in a 5|il volum e o v e r a 5 m inute period. Follow ing injection the injector w'as slowly rem oved and the stainless steel plug replaced.
2.6 Recording Apparatus.
T h e e le c tro p h y sio lo g ica l e q u ip m e n t was su rro u n d e d by a Faraday cage to rem ove environm ental electrical interference. In addition all electrical e quipm ent was grounded to a central point to e lim inate 50 Hertz noise. T he e le c tro p h y sio lo g ica l e q u ip m e n t c o m p rise d o f a co n sta n t c urre nt isolation unit (G rass Instrum ent Co. photoelectric stim ulus isolation unit) linked to the stim ulating electrode. The e v oked response was transmitted via a pre-am plifier (gain 11) to an analogue-to-digital coverter (M acL ab 4S, A nalog Digital Instruments) operated by Scope Program versions 3.28 and 3.5 using an Apple Machintosh Pow er PC G3.
2.7 Location o f Recording and Stimulating Electrodes during Surgery.
The rat w'as placed in the stereotaxic and the electrodes low ered to the surface o f the brain. This was taken as point ‘O’. T he position o f the electrodes m oving through the cortical and hippocam pal layers to the dendrites o f stratum radiatum w as constantly m onitored as they were lowered through the tissue. Evoked responses (0.1 ms duration, 2 m s delay, 4V pulse through the stim ulating electrode at a frequency o f 0.1 Hz) were displayed on the c om puter screen as the electrodes w ere lowered into place in the C A l a rea. Both the c e re b ra l c o rte x a n d the h i p p o c a m p a l f o rm a tio n p o s s e s s lam in a r structures. W hen a local depolarisation such as an e xcitatory post-synaptic potential (E PSP) was created, a current was set up along a vertical superficial-deep axis. A phase reversal was encountered when this dipole was crossed, indicating that this was the area generating electrom otive forces and the response recorded was not from a distal site by volta g e co n d u c tio n . By this m e th o d it w as possible to d e te rm in e w h ic h layer the
electrodes w ere in by referring to the electrophysiological criteria determ ined for each region o f the hippocam pus as defined by Leung (1980).
T he m onopolar recording electrode was positioned initially to a depth o f 2.5 m m below the surface o f the dura. The bipolar stim ulating electrode was then positioned initially to a depth o f 1.6 m m below the surface o f the dura. The stim ulating electrode was then low ered in increm ents o f 0.05m m to approxim ately 2.5 m m b e lo w the dura. T he first p otentia l rec o rd e d w a s the e v o k e d p ositive re sp o n se as the s tim u la tin g e le c tro d e penetrated the alveus. A larger positive response was seen as the stim ulating electrode penetrated the stratum oriens. Upon approaching the cell body layer the am plitude o f the e v o k e d r e s p o n s e b e c a m e s m a lle r and re v e rs e d as the s tim u la tin g e le c tro d e penetrated through from the cell body layer o f stratum radiatum into the dendritic layer. Further small a djustm ents (increm ents o f 20jj,m.) w ere m ade in the positioning o f the s tim u la tin g e le c tro d e to m a x im iz e the E P S P a m p litu d e . O n c e s a tisfie d m in o r adjustm ents (10 !_u t i) were m ade to the position o f the recording electrode to further m axim iz e EPSP am plitude. Electrode position w ere verified post-m orte m by m anual dissection.
2.8 Input/O utput curves.
O n c e found, evoked responses (0.033 Hz) w ere recorded continuously for the duration o f the experiment. The anim al was allow ed a recovery period o f 1 hour post electrode implantation after which an 1/0 curve was recorded. The 1/0 curve w as constructed by recording the average am plitude o f four evoked responses induced by each o f a series o f stim ulating intensities (2-10 V at 1 V intervals).