Adenosine preferentially suppresses serotonin2A receptor-enhanced excitatory postsynaptic currents in layer V neurons of the rat medial prefrontal cortex.

Serotonin induces 'spontaneous' (non-electrically evoked) excitatory postsynaptic currents in layer V pyramidal neurons in the prefrontal cortex. This is likely due to a serotonin2A receptor-mediated focal release of glutamate onto apical dendrites. In addition, activation of the serotonin2A receptor selectively enhances late components of electrically evoked excitatory postsynaptic currents. In this study, using in vitro intracellular and whole-cell recording in rat brain slices, we examined the role of adenosine in modulating serotonin2A-enhanced 'spontaneous' and electrically evoked excitatory postsynaptic currents in layer V pyramidal neurons in the medial prefrontal cortex. Adenosine and N6-cyclopentyladenosine, an A1 adenosine agonist, markedly suppressed the serotonin2A-induced ('spontaneous') excitatory postsynaptic currents. However, adenosine had no effect on spontaneous miniature (tetrodotoxin-insensitive) postsynaptic potentials. Adenosine also blocked the late excitatory postsynaptic currents induced by the serotonin2A/2C agonist R(-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride. Surprisingly, in contrast to other regions, adenosine had a relatively small effect on electrically evoked fast excitatory postsynaptic currents. These findings represent a novel demonstration of adenosine's ability to preferentially modulate serotonin2A-mediated synaptic events in the medial prefrontal cortex. As the serotonin2A receptor is closely linked with the effects of atypical antipsychotics and hallucinogens, further understanding of the modulators of this receptor such as adenosine may provide useful therapeutic applications.

Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling.

DsRed, a recently cloned red fluorescent protein, has attracted great interest as an expression tracer and fusion partner for multicolor imaging. We report that three-photon excitation (lambda <760 nm) rapidly changes the fluorescence of DsRed from red to green when viewed subsequently by conventional (one-photon) epifluorescence. Mechanistically, three-photon excitation (lambda <760 nm) selectively bleaches the mature, red-emitting form of DsRed, thereby enhancing emission from the immature green form through reduction of fluorescence resonance energy transfer (FRET). The "greening" effect occurs in live mammalian cells at the cellular and subcellular levels, and the resultant color change persists for >30 h without affecting cell viability. This technique allows individual cells, organelles, and fusion proteins to be optically marked and has potential utility for studying cell lineage, organelle dynamics, and protein trafficking, as well as for selective retrieval of cells from a population. We describe optimal parameters to induce the color change of DsRed, and demonstrate applications that show the potential of this optical highlighter.

GABAergic antagonists block the inhibitory effects of serotonin in the lateral amygdala: a mechanism for modulation of sensory inputs related to fear conditioning.

Neurons in the lateral amygdala (LA) receive glutamatergic sensory input from the auditory thalamus and auditory cortex, and these inputs can be modulated by serotonin (5-HT). In the present study, we examined whether serotonergic inhibition of glutamatatergic excitation in the LA occurs via activation of GABAergic interneurons. Single-unit extracellular activity in the LA was recorded in response to iontophoretically applied glutamate. Concurrent application of 5-HT reduced the number of glutamate-evoked action potentials in the majority of neurons tested. GABA antagonists were then iontophoresed with both glutamate and 5-HT. Of the neurons that were inhibited by 5-HT, concurrent application of the GABA antagonists significantly reversed this effect. Application of the GABA antagonists alone had little or no effect on basal neuronal activity. We conclude that the 5-HT-induced inhibition of glutamatergic activity occurs in part through activation of serotonergic receptors on GABAergic interneurons.

Serotonin modulation of sensory inputs to the lateral amygdala: dependency on corticosterone.

The lateral nucleus of the amygdala (LA) receives excitatory (glutamatergic) inputs from thalamic and cortical sensory processing areas and is believed to be involved in evaluation of the affective significance of sensory events. We examined whether serotonin (5-HT) affects excitatory transmission in auditory afferents to the LA and, if so, whether this modulation of sensory transmission is regulated by the stress hormone corticosterone (CORT). Neuronal activity in the LA was elicited via iontophoretic ejection of L-glutamate or synaptically via electrical stimulation of auditory afferent pathways. In the intact rat, iontophoretically applied 5-HT inhibited both synaptically and glutamate-evoked action potentials in most neurons examined. However, after adrenalectomy (ADX), which eliminates endogenous CORT, 5-HT no longer inhibited evoked activity in the LA. High-CORT doses given to ADX animals reinstated the inhibition of excitatory transmission of 5-HT, whereas low-CORT doses had little effect. Immunocytochemical labeling of the glucocorticoid receptor in the intact rat demonstrated nuclear staining throughout several amygdala regions, including the LA. However, after ADX, no nuclear labeling was visible. With a high replacement dose of CORT (5 or 10 mg) after ADX, dense nuclear staining returned, but with a low replacement dose (1 mg/kg), there was only light nuclear staining. Thus, the ability of 5-HT to modulate glutamatergic activity in auditory pathways to the amygdala is dependent on the presence of CORT and possibly glucocorticoid activation. Via this mechanism, 5-HT modulates the processing of sensory information within the LA and thus may regulate amygdala-related functions.

Convergent but temporally separated inputs to lateral amygdala neurons from the auditory thalamus and auditory cortex use different postsynaptic receptors: in vivo intracellular and extracellular recordings in fear conditioning pathways.

The lateral nucleus of the amygdala (LA), a key component of the fear conditioning circuitry, receives a rapid but relatively impoverished auditory input from the auditory thalamus and a slower but richer input from the auditory cortex. We examined in urethane anesthetized rats whether individual cells in the LA receive convergent inputs from these two areas, and whether different postsynaptic receptors contribute to the temporally separated excitations over the two pathways. With both extracellular and intracellular recordings, individual cells could be activated by stimulation of each pathway. In extracellular recordings iontophoretic application of the N-methyl-D-aspartate (NMDA) receptor antagonist APV and the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor antagonist CNQX demonstrated that synaptic transmission in both pathways depends on AMPA receptors, whereas transmission in the thalamic pathway also depends on the involvement of NMDA receptors. The involvement of NMDA receptors in synaptic activation of the LA from the thalamus but not the cortex was confirmed in intracellular recordings using systemic injections of the NMDA antagonist MK-801. The slow time course of NMDA currents could provide LA cells with a mechanism to integrate the inputs arriving rapidly from the thalamus and somewhat later from the cortex, thus allowing the LA to integrate signals in the two pathways during the acquisition and expression of conditioned fear reactions.

The effect of the acute and chronic administration of CP 96,345, a selective neurokinin1 receptor antagonist, on midbrain dopamine neurons in the rat: a single unit, extracellular recording study.

In this study, we examined the effect of acute and chronic administration of the selective neurokinin1 receptor antagonist CP 96,345 on the basal activity of spontaneously active dopamine (DA) neurons in the substantia nigra pars compacta (SNC) and the ventral tegmental area (VTA). This was accomplished using the technique of in vivo, extracellular single unit recording in anesthetized rats. The intravenous (i.v.) administration of CP 96,345 (0.01-1.28 mg/kg) did not significantly alter the firing rate of spontaneously active DA neurons in the SNC and VTA areas. The acute administration of 5 or 10 mg/kg, i.p., of CP 96,345 produced a significant decrease in the number of spontaneously active SNC and VTA dopamine cells compared to vehicle-treated rats. In contrast to its effect on the number of spontaneously active DA neurons, the administration of 5 mg/kg, i.p., of CP 96,345 did not significantly alter the basal firing pattern of either SNC or VTA DA neurons. The acute administration of CP 96,345 (10 mg/kg, i.p.) significantly potentiated the suppressant action of (+)-apomorphine on the basal firing rate of spontaneously active SNC and VTA DA cells. The chronic administration of CP 96,345 (5 or 10 mg/kg, i.p.) for 21 days also produced a significant decrease in the number of spontaneously active SNC and VTA DA cells compared to vehicle controls. This effect was not reversed by the systemic administration of (+)-apomorphine (50 micrograms/kg, i.v.), suggesting that the reduction in the number of spontaneously active DA cells produced by CP 96,345 is probably not the result of depolarization inactivation. Overall, our results indicate that the tonic activation of NK1 receptors by substance P may be necessary to maintain the spontaneous activity of a proportion of midbrain DA neurons.