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The brain slice as a model for studying transmitter receptor interactions
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ASSESSMENT OF HIGH P(~ASSIUM-INDUC~ EPILEPTIFORM BURSTING IN IN VI'TRD HZP~r.AMPAL SLZCES
F. EL-SABBAN, C.B. SHIELDS, H.L. EDMO~)S,JR. and ~ , Departments of Surgery, Anesthesiology and Physiology & Biophysics, University of Louisville School of Medicine, Louisville, KY 40292, U.S.A. Hippocampal slices from adult Wistar rats were used to assess the presence and frequency of epileptiform bursting (EB) induced by high potassium levels in t~e bathing medium. ~ hippocampal slices were bathed initially in 3.5 mM K--artificial CSF and were then stabilized for 1 h. All slices were checked for evoked responses in region CA1 at the beginning and end of each 6-7 h experiment. Spontaneous EB was checked and recorded from regions CA3 and CA1. A branching logic design was used to define bursting, non-bursting and depolarized slices. Of 76 slices, 70 were responsive at the beginning and 68 at the end. Exposure to 8.0 n~4 K + produced EB in both CA1 (38/68 slices) and CA3 (35/68). Average time to first RR ~n c2%3 was 23 min. Bursting frequencies in CA1 and CA3 were highly correlated (r = 0.95, df 64). These results suggest that EB in CA1 is as reliable as that in CA3. Consequently, in screening large numbers of slices for EB, recording from CA1 is sufficient. THE BRAIN SLICE AS A MODEL FOR STUDYING TRANSMITI~R RECEPTOR INTERACTIONS S.J. ENNA and ~ j ~ , Nova Pharmaceutical Corporation, Centre, Baltimore, ~D 21224, U.S.A.
6200 Freeport
Receptor binding assays utilizing brain membrane fragments have generated a great deal of information on the biochemical and pharmacological properties of neurotransmitter receptor recognition site~. Unfortunately, it is difficult to study receptor function, especially second nnessenger production, in brain hc~ogenates since the response to receptor agonists is greatly attenuated in broken cells. Moreover, data from a variety of sources indicate that activation of one receptor system can influence the activity of another and that sometimes these interactions involve the participation of intracellular continuents. An in vitro assay system found to be particularly useful in studying such receptor interactions at the functional level is the analysis of transmitter-simulated cyclic AMP accumulation in brain slices (J. Neurochem. 16: 1609, 1969). Briefly, freshly dissected brain tissue is cut into 350 ~m slices using a McIlwain tissue chopper and suspended in oxygenated Krebs Ringer bicarbonate buffer (pH 7.4). Following a preincubation and several rinses with fresh buffer, the slices are incubated for 15 min at 37°C with neurotransmitters or drugs and the reaction terminated by the addition of 10% trichloroacetic acid. Total radioactivity is quantified using 50 ul portion of the acid supernatent and cyclic AMP content analyzed by the double column method of Salomon et al. (Anal. Biochem. 58: 541, 1974). Exposure of these brain slices to neurotransmitters and drugs known to stimulate receptors that are positively coupled to adenylate cyclase results in a concentration-dependent increase in the level of cyclic AMP that is many times greater than basal values. Moreover, using this preparation it was found that certain substances, such as gamma-aminobutyric and alpha-adrenergic agonists, which by themselves have no effect on cyclic AMP levels, greatly augment the response to transmitters that stimulate the production of this second messenger. ~dditional experiments revealed that this augmentation may be secondary to the activation of phospholipase A 2 and the generation of arachidonic acid. Thus, by using a brain slice preparation it has been possible to characterize an interaction between receptor systems that may represent an important mechanism for regulating synaptic activity.
ASSESSMENT OF HIGH P(~ASSIUM-INDUC~ EPILEPTIFORM BURSTING IN IN VI'TRD HZP~r.AMPAL SLZCES
F. EL-SABBAN, C.B. SHIELDS, H.L. EDMO~)S,JR. and ~ , Departments of Surgery, Anesthesiology and Physiology & Biophysics, University of Louisville School of Medicine, Louisville, KY 40292, U.S.A. Hippocampal slices from adult Wistar rats were used to assess the presence and frequency of epileptiform bursting (EB) induced by high potassium levels in t~e bathing medium. ~ hippocampal slices were bathed initially in 3.5 mM K--artificial CSF and were then stabilized for 1 h. All slices were checked for evoked responses in region CA1 at the beginning and end of each 6-7 h experiment. Spontaneous EB was checked and recorded from regions CA3 and CA1. A branching logic design was used to define bursting, non-bursting and depolarized slices. Of 76 slices, 70 were responsive at the beginning and 68 at the end. Exposure to 8.0 n~4 K + produced EB in both CA1 (38/68 slices) and CA3 (35/68). Average time to first RR ~n c2%3 was 23 min. Bursting frequencies in CA1 and CA3 were highly correlated (r = 0.95, df 64). These results suggest that EB in CA1 is as reliable as that in CA3. Consequently, in screening large numbers of slices for EB, recording from CA1 is sufficient. THE BRAIN SLICE AS A MODEL FOR STUDYING TRANSMITI~R RECEPTOR INTERACTIONS S.J. ENNA and ~ j ~ , Nova Pharmaceutical Corporation, Centre, Baltimore, ~D 21224, U.S.A.
6200 Freeport
Receptor binding assays utilizing brain membrane fragments have generated a great deal of information on the biochemical and pharmacological properties of neurotransmitter receptor recognition site~. Unfortunately, it is difficult to study receptor function, especially second nnessenger production, in brain hc~ogenates since the response to receptor agonists is greatly attenuated in broken cells. Moreover, data from a variety of sources indicate that activation of one receptor system can influence the activity of another and that sometimes these interactions involve the participation of intracellular continuents. An in vitro assay system found to be particularly useful in studying such receptor interactions at the functional level is the analysis of transmitter-simulated cyclic AMP accumulation in brain slices (J. Neurochem. 16: 1609, 1969). Briefly, freshly dissected brain tissue is cut into 350 ~m slices using a McIlwain tissue chopper and suspended in oxygenated Krebs Ringer bicarbonate buffer (pH 7.4). Following a preincubation and several rinses with fresh buffer, the slices are incubated for 15 min at 37°C with neurotransmitters or drugs and the reaction terminated by the addition of 10% trichloroacetic acid. Total radioactivity is quantified using 50 ul portion of the acid supernatent and cyclic AMP content analyzed by the double column method of Salomon et al. (Anal. Biochem. 58: 541, 1974). Exposure of these brain slices to neurotransmitters and drugs known to stimulate receptors that are positively coupled to adenylate cyclase results in a concentration-dependent increase in the level of cyclic AMP that is many times greater than basal values. Moreover, using this preparation it was found that certain substances, such as gamma-aminobutyric and alpha-adrenergic agonists, which by themselves have no effect on cyclic AMP levels, greatly augment the response to transmitters that stimulate the production of this second messenger. ~dditional experiments revealed that this augmentation may be secondary to the activation of phospholipase A 2 and the generation of arachidonic acid. Thus, by using a brain slice preparation it has been possible to characterize an interaction between receptor systems that may represent an important mechanism for regulating synaptic activity.