Adrenergic receptor characterization of CA1 hippocampal neurons using real time single cell RT-PCR.

The CA1 region of the rat hippocampus exhibits both alpha and beta adrenergic receptor (AR) responses, however, the specific AR subtypes involved and the neuronal expression patterns for these receptors are not well understood. We have employed single cell real time RT-PCR in conjunction with cell-specific immunohistochemical markers to determine the AR expression patterns for hippocampal neurons located in CA1, a region often implicated in learning and memory processes. Cytoplasmic samples were taken from 55 individual cells located in stratum oriens, pyramidale, or radiatum and reverse transcribed. All successfully amplified pyramidal neuron samples (n = 17) expressed mRNA for the beta2AR, with four cells additionally expressing mRNA for the beta1AR subtype. Positive interneurons from stratum oriens (n = 10) and stratum radiatum (n = 8) expressed mRNA for the alpha1A and/or alpha(1B)AR (n = 9/18) only when coexpressing transcripts for somatostatin. Interneurons containing neuropeptide Y or cholecystokinin (n = 9/18) were not positive for any of the nine AR subtypes, suggesting that CA1 interneuron AR expression is limited to a subset of somatostatin-positive cells. These findings suggest that only a select number of AR subtypes are transcriptionally expressed in CA1 and that these receptors are selective to specific neuronal cell types.

Beta1 adrenergic receptor-mediated enhancement of hippocampal CA3 network activity.

Norepinephrine is an endogenous neurotransmitter distributed throughout the mammalian brain. In higher cortical structures such as the hippocampus, norepinephrine, via beta adrenergic receptor (AR) activation, has been shown to reinforce the cognitive processes of attention and memory. In this study, we investigated the effect of beta1AR activation on hippocampal cornu ammonis 3 (CA3) network activity. AR expression was first determined using immunocytochemistry with antibodies against beta1ARs, which were found to be exceptionally dense in hippocampal CA3 pyramidal neurons. CA3 network activity was then examined in vitro using field potential recordings in rat brain slices. The selective betaAR agonist isoproterenol caused an enhancement of hippocampal CA3 network activity, as measured by an increase in frequency of spontaneous burst discharges recorded in the CA3 region. In the presence of alphaAR blockade, concentration-response curves for isoproterenol, norepinephrine, and epinephrine suggested that a beta1AR was involved in this response, and the rank order of potency was isoproterenol > norepinephrine = epinephrine. Finally, equilibrium dissociation constants (pK(b)) of subtype-selective betaAR antagonists were functionally determined to characterize the AR subtype modulating hippocampal CA3 activity. The selective beta1AR antagonists atenolol and metoprolol blocked isoproterenol-induced enhancement, with apparent K(b) values of 85 +/- 36 and 3.9 +/- 1.7 nM, respectively. In contrast, the selective beta2AR antagonists ICI-118,551 and butoxamine inhibited isoproterenol-mediated enhancement with apparent low affinities (K(b) of 222 +/- 61 and 9268 +/- 512 nM, respectively). Together, this pharmacological profile of subtype-selective betaAR antagonists indicates that in this model, beta1AR activation is responsible for the enhanced hippocampal CA3 network activity initiated by isoproterenol.