Electroencephalography and Clinical Neurophysiology, 1977, 4 2 : 3 8 9 - - 3 9 6
389
© E l s e v i e r / N o r t h - H o l l a n d Scientific Publishers Ltd.
HIPPOCAMPAL RSA (THETA), APNEA, B R A D Y C A R D I A AND EFFECTS OF ATROPINE U N D E R W A T E R SWIMMING IN THE R A T *
DURING
IAN Q. WHISHAW ** a n d T I M O T H Y S C H A L L E R T
Department of Psychology, University of Lethbridge, Lethbridge, Alberta (Canada) ( A c c e p t e d for p u b l i c a t i o n : J u n e 17, 1 9 7 6 )
Many electroenceph alographic (EEG) studies have shown that the rhythmical slow activity (RSA or theta rhythm) which can be recorded from the hippocampus proper and dentate gyrus in the rat (Winson 1974; Bland and Whishaw 1976) is closely related to concurrent voluntary or Type I m o t o r activity (Vanderwolf 1969, 1975; Whishaw and Vanderwolf 1973). Amplitude and frequency may also be related to the details of movement; amplitude increases as the size of movement increases, while frequency increases with the force or vigor with which a movement is initiated (Whishaw and Vanderwolf 1973). However, during continuous l o c o m o t o r activity such as walking, trotting, or swimming, RSA frequency remains constant (Bland and Vanderwolf 1972; Whishaw and Vanderwolf 1973). The significance of the RSA relation to m o v e m e n t is n o t known b u t these studies have suggested that RSA may be a sign that the hippocampus is involved in some way with the organization and execution of movement. The attention of researchers has been drawn to the possibility that RSA may be related to other rhythmical physiological or behavioral activity such as respiration (or sniffing), heart rate, and vibrissae movements (Komisaruk 1970; Kurtz and Adler 1973; Macrides 1975). Theoretically, RSA could be an artifact, a physiological sign, or, as sug* This research was s u p p o r t e d by the N a t i o n a l Research C o u n c i l of Canada, G r a n t No. A 8 2 7 3 . ** T o w h o m r e p r i n t s s h o u l d be addressed.
gested ~by Komisaruk (1970), a direct pacemaker of such rhythmical peripheral events. Finally, RSA could have a modulatory action such as has been suggested for sniffing (Macrides 1975). The purpose of the present experiment was to vary respiration, heart rate, and vibrissae movement and observe concomitant change in hippocampal RSA. A naturally occurring behavior was chosen for observation since physiological manipulations, such as brain stimulation which has been used to dissociate heart rate and RSA (Whishaw et al. 1972), may not accurately reflect normal relations. One way to arrest respiration, sniffing, and vibrissae movement is to have a rat swim underwater. If these events are related to RSA it would be expected that RSA might change or be absent during the apnea (respiratory arrest) of underwater swimming. If, on the other hand, RSA is related solely to movement it would be expected that RSA could remain relatively unchanged. It was expected that heart rate might change in frequency since all diving vertebrates examined show slowing of the heart (bradycardia) during underwater swimming (Andersen 1966). Electrocardiograph (EKG) records have not been taken in the rat during diving. Therefore, we recorded EKG activity and related it to RSA during underwater and surface swimming. In addition to describing physiological changes this paper describes simple techniques for obtaining EEG and EKG during surface and underwater swimming.
390 Methods
A nim a ls Twelve adult ( 4 0 0 - - 5 0 0 g) male S p r a g u e - D a w l e y rats were used in the e x p e r i m e n t s . Surgical procedure Animals were a n e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l (50 m g / k g , i n t r a p e r i t o n e a l ) and E E G and E K G e l e c t r o d e s i m p l a n t e d . Bipolar r e c o r d i n g e l e c t r o d e s were i m p l a n t e d in each dorsal h i p p o c a m p u s and s e n s o r i - m o t o r c o r t e x . Coordinates for hippocampal placements were: 4.0 m m p o s t e r i o r and 2.5 m m lateral to b r e g m a , and 3.5 m m ventral f r o m the skull surface. C o o r d i n a t e s f o r n e o c o r t i c a l elect r o d e s were: 1.0 m m anterior, 1.0 m m lateral and 1.5 m m ventral. T h e skull was aligned with b r e g m a and l a m b d a on the h o r i z o n t a l plane. E l e c t r o d e s consisted o f t w o 250 p N i c h r o m e wires insulated to t h e cross-section o f the tips. T h e tips were staggered 0 . 5 - 1.0 m m . Male c o n n e c t o r s w e r e soldered to the wires a n d the assemblies w e r e fixed in place with d e n t a l c e m e n t a n c h o r e d t o j e w e l e r ' s screws inserted in the skull. A m a l e c o n n e c t o r soldered to a j e w e l e r ' s screw in the f r o n t a l b o n e served as a g r o u n d c o n n e c t i o n during recording. E l e c t r o d e s for E K G r e c o r d i n g w e r e 1 cm d i a m e t e r gold plated discs sewed into cutane o u s m a x i m u s muscles over the ventral segm e n t of the 6 t h rib. (A n u m b e r of E K G rec o r d i n g e l e c t r o d e s were tested b u t only the discs w e r e f o u n d to p r o d u c e artifact-free r e c o r d i n g during v i g o r o u s m o v e m e n t . ) T h e wires f r o m the discs w e r e d r a w n up u n d e r the skin and soldered to m a l e c o n n e c t o r s which were fixed to the skull w i t h dental c e m e n t . Recording and data analysis T h e rats were c o n n e c t e d to a p o l y g r a p h w i t h shielded p h o n o - a r m cable. C o n n e c t i o n s on the head a s s e m b l y were w a t e r - p r o o f e d b y c o a t i n g t h e m with m e l t e d p a r a f f i n w a x (congealing p o i n t 49°C). When t h e w a x h a r d e n e d the c o n n e c t i o n s w e r e shielded f r o m w a t e r and f i r m l y s u p p o r t e d so t h a t m o v e m e n t
I.Q. WIfISI-tAW, T. SCttALLERT a r t i f a c t was eliminated. EEG activity was r e c o r d e d with h a l f - a m p l i t u d e filters set at 1 and 75 c/see; E K G with filters at 10 and 75 c/see. A m a n u a l l y activated m a r k e r was used to indicate b e h a v i o r on the chart. R e c o r d s were a n a l y z e d by m e a s u r i n g f r e q u e n c y and a m p l i t u d e of individual waves with a clear plastic ruler (ram scale) as p r e v i o u s l y described (Whishaw and V a n d e r w o l f 1973).
Drugs A t r o p i n e sulfate (50 m g / k g ) was dissolved in a physiological saline vehicle and given via the i n t r a p e r i t o n e a l route. Procedure R e c o r d i n g s were m a d e w h e n the rats were s w i m m i n g on the surface or u n d e r w a t e r in a 108 × 108 c m t a n k filled to a d e p t h o f 30 cm with 38°C water. Animals were forced to swim u n d e r w a t e r b y placing t h e m at the b o t t o m of t h e tank. T h e y s w a m spont a n e o u s l y to the surface or, if light pressure was app]ied to their backs, t h e y s w a m across t h e t a n k and t h e n s w a m to t h e surface. Each rat was required to swim across the t a n k b o t h on the surface and u n d e r w a t e r 6 t i m e s with surface and u n d e r w a t e r trials given in r a n d o m order. Core t e m p e r a t u r e was m o n i t o r e d before and a f t e r the s w i m m i n g tests with a rectal p r o b e inserted 6.5 cm. E E G activity was r e c o r d e d f r o m 7 rats d u r i n g u n d e r w a t e r and surface s w i m m i n g . E E G and E K G were r e c o r d e d f r o m 5 additional animals b e f o r e and a f t e r 50 m g / k g a t r o p i n e sulfate.
Results The p a t t e r n o f s w i m m i n g in t h e r a t has been previously described (Whishaw and Vanderwolf 1971). Usually the animals p a d d l e d using their hind legs with the f r o n t p a w s t u c k e d u p u n d e r their chins. T h e f r o n t paws o c c a s i o n a l l y were used f o r s w i m m i n g , p a r t i c u l a r l y w h e n the animals were turning. Vibrissae m o v e m e n t s did n o t occur. T h e
HIPPOCAMPAL RSA AND UNDERWATER SWIMMING
pattern of swimming underwater appeared identical to that displayed during surface swimming except that respiration was arrested and the nostrils were closed. The EEG recordings showed that hippocampal RSA and neocortical desynchronization occurred during both surface and underwater swimming. An example of hippocampal and neocortical EEG during surface and underwater swimming is shown in Fig. 1. Measures of RSA frequency during surface swimming showed that the mean frequency and standard errors were 8.3 + 0.11 c/sec (range, 7.7--9.0 c/sec; measure based on 10 sec samples/condition/rat). RSA frequency during underwater swimming was reduced in every animal resulting in an overall mean of 7.7 +- 0.10 c/sec (range, 6.6--8.8 c/sec). This difference was significant; t (6) = 5.5, P
391
< 0.01. The distribution of RSA frequency based on measures of individual waves is shown in Fig. 2. It can be seen in Fig. 2 that the entire frequency spectrum was slower during underwater as compared to surface swimming.
Effects of atropine Hippocampal RSA and neocortical desynchronization were recorded during surface and underwater swimming after rats were given atropine sulfate (50 mg/kg). Mean RSA frequency during surface swimming was 8.4 +0.7 c/sec (range, 7.3--8.6 c/sec). During underwater swimming, RSA frequency was reduced in every animal and the overall mean reduction {0.60 c/sec) was significant; t (4) = 4.4, P < 0.05. Thus, the mean RSA frequency during the
AC
surface swimming
underwoter swimming ,
~
1 Second
Fig. 1. EEG activity of the neocortex and hippocampus during surface (top) and underwater (boltom) swimming in the rat. Note that the neocortical desynchronized pattern of EEG and hippocampal RSA are present during both surface and underwater swimming. AC, anterior sensori-motor cortex; RH, right hippocampus.
392
I.Q. WHISHAW, T. SCHALLERT 5--
A
t w o swimming conditions, surface and underwater, in atropinized rats was similar to RSA frequency recorded from non-atropinized rats. This can be seen in the f r e q u e n c y distrib u t i o n shown in Fig. 2.
4CONTROl.
R S A amplitude Measures of RSA a m p l i t u d e indicated no significant differences b e t w e e n surface and u n d e r w a t e r swimming in n o r m a l (mean a m p l i t u d e , 1.5 ± 0.3 vs. 1.5 ± 0.2 mV; n = 7}, or in atropinized rats (1.7 + 0.3 vs. 1.7 ± 0.4 mV; n = 5).
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389
© E l s e v i e r / N o r t h - H o l l a n d Scientific Publishers Ltd.
HIPPOCAMPAL RSA (THETA), APNEA, B R A D Y C A R D I A AND EFFECTS OF ATROPINE U N D E R W A T E R SWIMMING IN THE R A T *
DURING
IAN Q. WHISHAW ** a n d T I M O T H Y S C H A L L E R T
Department of Psychology, University of Lethbridge, Lethbridge, Alberta (Canada) ( A c c e p t e d for p u b l i c a t i o n : J u n e 17, 1 9 7 6 )
Many electroenceph alographic (EEG) studies have shown that the rhythmical slow activity (RSA or theta rhythm) which can be recorded from the hippocampus proper and dentate gyrus in the rat (Winson 1974; Bland and Whishaw 1976) is closely related to concurrent voluntary or Type I m o t o r activity (Vanderwolf 1969, 1975; Whishaw and Vanderwolf 1973). Amplitude and frequency may also be related to the details of movement; amplitude increases as the size of movement increases, while frequency increases with the force or vigor with which a movement is initiated (Whishaw and Vanderwolf 1973). However, during continuous l o c o m o t o r activity such as walking, trotting, or swimming, RSA frequency remains constant (Bland and Vanderwolf 1972; Whishaw and Vanderwolf 1973). The significance of the RSA relation to m o v e m e n t is n o t known b u t these studies have suggested that RSA may be a sign that the hippocampus is involved in some way with the organization and execution of movement. The attention of researchers has been drawn to the possibility that RSA may be related to other rhythmical physiological or behavioral activity such as respiration (or sniffing), heart rate, and vibrissae movements (Komisaruk 1970; Kurtz and Adler 1973; Macrides 1975). Theoretically, RSA could be an artifact, a physiological sign, or, as sug* This research was s u p p o r t e d by the N a t i o n a l Research C o u n c i l of Canada, G r a n t No. A 8 2 7 3 . ** T o w h o m r e p r i n t s s h o u l d be addressed.
gested ~by Komisaruk (1970), a direct pacemaker of such rhythmical peripheral events. Finally, RSA could have a modulatory action such as has been suggested for sniffing (Macrides 1975). The purpose of the present experiment was to vary respiration, heart rate, and vibrissae movement and observe concomitant change in hippocampal RSA. A naturally occurring behavior was chosen for observation since physiological manipulations, such as brain stimulation which has been used to dissociate heart rate and RSA (Whishaw et al. 1972), may not accurately reflect normal relations. One way to arrest respiration, sniffing, and vibrissae movement is to have a rat swim underwater. If these events are related to RSA it would be expected that RSA might change or be absent during the apnea (respiratory arrest) of underwater swimming. If, on the other hand, RSA is related solely to movement it would be expected that RSA could remain relatively unchanged. It was expected that heart rate might change in frequency since all diving vertebrates examined show slowing of the heart (bradycardia) during underwater swimming (Andersen 1966). Electrocardiograph (EKG) records have not been taken in the rat during diving. Therefore, we recorded EKG activity and related it to RSA during underwater and surface swimming. In addition to describing physiological changes this paper describes simple techniques for obtaining EEG and EKG during surface and underwater swimming.
390 Methods
A nim a ls Twelve adult ( 4 0 0 - - 5 0 0 g) male S p r a g u e - D a w l e y rats were used in the e x p e r i m e n t s . Surgical procedure Animals were a n e s t h e t i z e d w i t h s o d i u m p e n t o b a r b i t a l (50 m g / k g , i n t r a p e r i t o n e a l ) and E E G and E K G e l e c t r o d e s i m p l a n t e d . Bipolar r e c o r d i n g e l e c t r o d e s were i m p l a n t e d in each dorsal h i p p o c a m p u s and s e n s o r i - m o t o r c o r t e x . Coordinates for hippocampal placements were: 4.0 m m p o s t e r i o r and 2.5 m m lateral to b r e g m a , and 3.5 m m ventral f r o m the skull surface. C o o r d i n a t e s f o r n e o c o r t i c a l elect r o d e s were: 1.0 m m anterior, 1.0 m m lateral and 1.5 m m ventral. T h e skull was aligned with b r e g m a and l a m b d a on the h o r i z o n t a l plane. E l e c t r o d e s consisted o f t w o 250 p N i c h r o m e wires insulated to t h e cross-section o f the tips. T h e tips were staggered 0 . 5 - 1.0 m m . Male c o n n e c t o r s w e r e soldered to the wires a n d the assemblies w e r e fixed in place with d e n t a l c e m e n t a n c h o r e d t o j e w e l e r ' s screws inserted in the skull. A m a l e c o n n e c t o r soldered to a j e w e l e r ' s screw in the f r o n t a l b o n e served as a g r o u n d c o n n e c t i o n during recording. E l e c t r o d e s for E K G r e c o r d i n g w e r e 1 cm d i a m e t e r gold plated discs sewed into cutane o u s m a x i m u s muscles over the ventral segm e n t of the 6 t h rib. (A n u m b e r of E K G rec o r d i n g e l e c t r o d e s were tested b u t only the discs w e r e f o u n d to p r o d u c e artifact-free r e c o r d i n g during v i g o r o u s m o v e m e n t . ) T h e wires f r o m the discs w e r e d r a w n up u n d e r the skin and soldered to m a l e c o n n e c t o r s which were fixed to the skull w i t h dental c e m e n t . Recording and data analysis T h e rats were c o n n e c t e d to a p o l y g r a p h w i t h shielded p h o n o - a r m cable. C o n n e c t i o n s on the head a s s e m b l y were w a t e r - p r o o f e d b y c o a t i n g t h e m with m e l t e d p a r a f f i n w a x (congealing p o i n t 49°C). When t h e w a x h a r d e n e d the c o n n e c t i o n s w e r e shielded f r o m w a t e r and f i r m l y s u p p o r t e d so t h a t m o v e m e n t
I.Q. WIfISI-tAW, T. SCttALLERT a r t i f a c t was eliminated. EEG activity was r e c o r d e d with h a l f - a m p l i t u d e filters set at 1 and 75 c/see; E K G with filters at 10 and 75 c/see. A m a n u a l l y activated m a r k e r was used to indicate b e h a v i o r on the chart. R e c o r d s were a n a l y z e d by m e a s u r i n g f r e q u e n c y and a m p l i t u d e of individual waves with a clear plastic ruler (ram scale) as p r e v i o u s l y described (Whishaw and V a n d e r w o l f 1973).
Drugs A t r o p i n e sulfate (50 m g / k g ) was dissolved in a physiological saline vehicle and given via the i n t r a p e r i t o n e a l route. Procedure R e c o r d i n g s were m a d e w h e n the rats were s w i m m i n g on the surface or u n d e r w a t e r in a 108 × 108 c m t a n k filled to a d e p t h o f 30 cm with 38°C water. Animals were forced to swim u n d e r w a t e r b y placing t h e m at the b o t t o m of t h e tank. T h e y s w a m spont a n e o u s l y to the surface or, if light pressure was app]ied to their backs, t h e y s w a m across t h e t a n k and t h e n s w a m to t h e surface. Each rat was required to swim across the t a n k b o t h on the surface and u n d e r w a t e r 6 t i m e s with surface and u n d e r w a t e r trials given in r a n d o m order. Core t e m p e r a t u r e was m o n i t o r e d before and a f t e r the s w i m m i n g tests with a rectal p r o b e inserted 6.5 cm. E E G activity was r e c o r d e d f r o m 7 rats d u r i n g u n d e r w a t e r and surface s w i m m i n g . E E G and E K G were r e c o r d e d f r o m 5 additional animals b e f o r e and a f t e r 50 m g / k g a t r o p i n e sulfate.
Results The p a t t e r n o f s w i m m i n g in t h e r a t has been previously described (Whishaw and Vanderwolf 1971). Usually the animals p a d d l e d using their hind legs with the f r o n t p a w s t u c k e d u p u n d e r their chins. T h e f r o n t paws o c c a s i o n a l l y were used f o r s w i m m i n g , p a r t i c u l a r l y w h e n the animals were turning. Vibrissae m o v e m e n t s did n o t occur. T h e
HIPPOCAMPAL RSA AND UNDERWATER SWIMMING
pattern of swimming underwater appeared identical to that displayed during surface swimming except that respiration was arrested and the nostrils were closed. The EEG recordings showed that hippocampal RSA and neocortical desynchronization occurred during both surface and underwater swimming. An example of hippocampal and neocortical EEG during surface and underwater swimming is shown in Fig. 1. Measures of RSA frequency during surface swimming showed that the mean frequency and standard errors were 8.3 + 0.11 c/sec (range, 7.7--9.0 c/sec; measure based on 10 sec samples/condition/rat). RSA frequency during underwater swimming was reduced in every animal resulting in an overall mean of 7.7 +- 0.10 c/sec (range, 6.6--8.8 c/sec). This difference was significant; t (6) = 5.5, P
391
< 0.01. The distribution of RSA frequency based on measures of individual waves is shown in Fig. 2. It can be seen in Fig. 2 that the entire frequency spectrum was slower during underwater as compared to surface swimming.
Effects of atropine Hippocampal RSA and neocortical desynchronization were recorded during surface and underwater swimming after rats were given atropine sulfate (50 mg/kg). Mean RSA frequency during surface swimming was 8.4 +0.7 c/sec (range, 7.3--8.6 c/sec). During underwater swimming, RSA frequency was reduced in every animal and the overall mean reduction {0.60 c/sec) was significant; t (4) = 4.4, P < 0.05. Thus, the mean RSA frequency during the
AC
surface swimming
underwoter swimming ,
~
1 Second
Fig. 1. EEG activity of the neocortex and hippocampus during surface (top) and underwater (boltom) swimming in the rat. Note that the neocortical desynchronized pattern of EEG and hippocampal RSA are present during both surface and underwater swimming. AC, anterior sensori-motor cortex; RH, right hippocampus.
392
I.Q. WHISHAW, T. SCHALLERT 5--
A
t w o swimming conditions, surface and underwater, in atropinized rats was similar to RSA frequency recorded from non-atropinized rats. This can be seen in the f r e q u e n c y distrib u t i o n shown in Fig. 2.
4CONTROl.
R S A amplitude Measures of RSA a m p l i t u d e indicated no significant differences b e t w e e n surface and u n d e r w a t e r swimming in n o r m a l (mean a m p l i t u d e , 1.5 ± 0.3 vs. 1.5 ± 0.2 mV; n = 7}, or in atropinized rats (1.7 + 0.3 vs. 1.7 ± 0.4 mV; n = 5).
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