Rodrigo Andrade

Novel anxiolytics discriminate between postsynaptic serotonin receptors mediating different physiological responses on single neurons of the rat hippocampus

SummaryThe effects of buspirone on hippocampal pyramidal cells of the CA1 region were examined by means of intracellular recordings in in vitro hippocampal brain slices. Bath administration of buspirone elicited a long lasting hyperpolarization which was mediated by an increase in potassium conductance and resembled the hyperpolarizing component of the response to 5-HT (5-hydroxytryptamine). Buspirone, however, failed to mimic the depolarizing action of 5-HT or to reduce the calcium-activated afterhyperpolarization. Quantitative comparisons of the hyperpolarizing responses of 5-HT and buspirone revealed that the maximal hyperpolarization induced by buspirone was significantly smaller than that induced by 5-HT. Since the buspirone induced hyperpolarization was also accompanied by a surmountable antagonism of 5-HT responses, these results indicate that buspirone behaves as a partial agonist at a subpopulation of 5-HT receptors in the CA1 region of the hippocampus. Administration of the buspirone congeners gepirone and isapirone also elicited a hyperpolarization and reduced 5-HT responses, although they lack anti-dopaminergic activity, indicating that the effects observed with buspirone are unlikely to be mediated through dopamine receptors. These results indicated that novel anxiolytics can discriminate between functional 5-HT receptors. In conjunction with previous biochemical and electrophysiological studies, the present results suggest that their administration might alter the balance of serotonergic actions on postsynaptic neurons.

Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex

Classic hallucinogens such as lysergic acid diethylamide are thought to elicit their psychotropic actions via serotonin receptors of the 5-hydroxytryptamine 2A subtype (5-HT2AR). One likely site for these effects is the prefrontal cortex (PFC). Previous studies have shown that activation of 5-HT2ARs in this region results in a robust increase in spontaneous glutamatergic synaptic activity, and these results have led to the widely held idea that hallucinogens elicit their effect by modulating synaptic transmission within the PFC. Here, we combine cellular and molecular biological approaches, including single-cell 5-HT2ARs inactivation and 5-HT2AR rescue over a 5-HT2AR knockout genetic background, to distinguish between competing hypotheses accounting for these effects. The results from these experiments do not support the idea that 5-HT2ARs elicit the release of an excitatory retrograde messenger nor that they activate thalamocortical afferents, the two dominant hypotheses. Rather, they suggest that 5-HT2ARs facilitate intrinsic networks within the PFC. Consistent with this idea, we locate a discrete subpopulation of pyramidal cells that is strongly excited by 5-HT2AR activation.

TRPC Channels Mediate a Muscarinic Receptor-Induced Afterdepolarization in Cerebral Cortex

Activation of muscarinic cholinergic receptors on pyramidal cells of the cerebral cortex induces the appearance of a slow afterdepolarization that can sustain autonomous spiking after a brief excitatory stimulus. Accordingly, this phenomenon has been hypothesized to allow for the transient storage of memory traces in neuronal networks. Here we investigated the molecular basis underlying the muscarinic receptor-induced afterdepolarization using molecular biological and electrophysiological strategies. We find that the ability of muscarinic receptors to induce the inward aftercurrent underlying the slow afterdepolarization is inhibited by expression of a Gαq-11 dominant negative and is also markedly reduced in a phospholipase C β1 (PLCβ1) knock-out mouse. Furthermore, we show, using a genetically encoded biosensor, that activation of muscarinic receptor induces the breakdown of phosphatidylinositol 4,5-bisphosphate in pyramidal cells. These results indicate that the Gαq-11/PLCβ1 cascade plays a key role in the ability of muscarinic receptors to signal the inward aftercurrent. We have shown previously that the muscarinic afterdepolarization is mediated by a calcium-activated nonselective cation current, suggesting the possible involvement of TRPC channels. We find that expression of a TRPC dominant negative inhibits, and overexpression of wild-type TRPC5 or TRPC6 enhances, the amplitude of the muscarinic receptor-induced inward aftercurrent. Furthermore, we find that coexpression of TRPC5 and T-type calcium channels is sufficient to reconstitute a muscarinic receptor-activated inward aftercurrent in human embryonic kidney HEK-293 cells. These results indicate that TRPC channels mediate the muscarinic receptor-induced slow afterdepolarization seen in pyramidal cells of the cerebral cortex and suggest a possible role for TRPC channels in mnemonic processes.

Enhancement of β-adrenergic responses by Gi-linked receptors in rat hippocampus

Abstract Many excitable cells express a class of neurotransmitter receptors functionally defined by their ability to increase potassium conductance through G proteins of the G i /G o class that directly activate the potassium channels. Biochemical studies have shown that these same receptors can also inhibit forskolin-stimulated adenylyl cyclase, although the functional significance of this effect remains unclear. In this study electrophysiological techniques were used to examine how activation of serotonin and γ-aminobutyric acid receptors belongingtothis class affect β-adrenergic responses signaled through adenylyl cyclase. Surprisingly, activation of these receptors not only failed to inhibit but actually enhanced β-adrenergic responses. These observations are consistent with evidence indicating that G i -linked receptors can enhance the ability of G s to stimulate certain adenylyl cyclases.

SKCa channels mediate the medium but not the slow calcium-activated afterhyperpolarization in cortical neurons

Many neurons, including pyramidal cells of the cortex, express a slow afterhyperpolarization (sAHP) that regulates their firing. Although initial findings suggested that the current underlying the sAHP could be carried through SKCa channels, recent work has uncovered anomalies that are not congruent with this idea. Here, we used overexpression and dominant-negative strategies to assess the involvement of SKCa channels in mediating the current underlying the sAHP in pyramidal cells of the cerebral cortex. Pyramidal cells of layer V exhibit robust AHP currents composed of two kinetically and pharmacologically distinguishable currents known as the medium AHP current (ImAHP) and the slow AHP current (IsAHP). ImAHP is blocked by the SKCa channel blockers apamin and bicuculline, whereas IsAHP is resistant to these agents but is inhibited by activation of muscarinic receptors. To test for a role for SKCa channels, we overexpressed KCa2.1 (SK1) and KCa2.2 (SK2), the predominant SKCa subunits expressed in the cortex, in pyramidal cells of cultured brain slices. Overexpression of KCa2.1 and KCa2.2 resulted in a fourfold to fivefold increase in the amplitude of ImAHP but had no detectable effect on IsAHP. As an additional test, we examined IsAHP in a transgenic mouse expressing a truncated SKCa subunit (SK3-1B) capable of acting as a dominant negative for the entire family of SKCa–IKCa channels. Expression of SK3-1B profoundly inhibited ImAHP but again had no discernable effect on IsAHP. These results are inconsistent with the proposal that SKCa channels mediate IsAHP in pyramidal cells and indicate that a different potassium channel mediates this current.

CELL EXCITATION ENHANCES MUSCARINIC CHOLINERGIC RESPONSES IN RAT ASSOCIATION CORTEX

The cerebral cortex receives a prominent cholinergic innervation which is thought to play an important role in regulating its normal function. Electrophysiological studies have shown that activation of cholinergic receptors results in a marked enhancement of excitatory stimuli onto cortical neurons and it has been suggested that this effect is secondary to the blockade of several voltage- and calcium-dependent potassium conductances in these cells. It is reported here that, in addition to these effects, activation of muscarinic receptors in the prefrontal cortex elicits the appearance of a slow calcium-dependent inward current in response to the generation of action potentials. This inward aftercurrent produces a slowly decaying depolarizing afterpotential which, when activated by stimulation of the cell, can summate with the carbachol-induced depolarization greatly increasing its magnitude. As a result the ability of muscarinic receptor to elicit a depolarization and excite cells in this region can be dramatically potentiated by evoked cell activation. This effect expands the range of mechanisms by which muscarinic receptors can facilitate excitatory inputs and provides a mechanism by which the association of brief excitatory stimuli to cholinergic stimulation can selectively enhance muscarinic responses among discrete cell populations in the cerebral cortex.

Blockade of neurotransmitter-activated K+ conductance by QX-314 in the rat hippocampus

Intracellular injection of QX-314 blocked the ability of baclofen and 5-carboxyamidotryptamine to hyperpolarize cells in the rat hippocampus. This effect was not associated with a reduction in the effects of norepinephrine on these cells nor a blockade of the potassium channels underlying the calcium-dependent afterhyperpolarization responsible or significant changes in membrane potential. These results suggest that QX-314 is an effective blocker of G-protein-gated potassium channels in this region.

Pharmacological and functional analysis of a novel serotonin receptor in the rat hippocampus

Administration of serotonin (5-hydroxytryptamine, 5-HT) to pyramidal cells of the CA1 region of the hippocampus results in a hyperpolarizing response which is followed by a rebound depolarization and a decrease in the calcium-activated afterhyperpolarization (AHP). While the hyperpolarizing response has been previously shown to be mediated by receptors of the 5-HT1A subtype, the identity of the receptor(s) involved in the depolarizing response and decrease of the AHP have not been identified. In the present study the effectiveness of a series of 5-HT receptor antagonists in blocking the membrane depolarization and reduction of the AHP was assessed. While a variety of 5-HT1 and 5-HT2 antagonists were found to be ineffective, the substituted benzamide BRL 24924 was found to be a potent and selective antagonist of the 5-HT-induced depolarization and decrease in the AHP in this region. This effect however appeared unrelated to the ability of this compound to block 5-HT3 receptors, as ICS 205-930 and MDL 72222 were markedly less efficacious in blocking these effects of 5-HT. Upon blockade of 5-HT1A receptors, 5-HT elicits a depolarization which is accompanied by a marked increase in excitability. These effects were also dose-dependently antagonized by BRL 24924. The present results thus suggest the presence in the CA1 region of the hippocampus of a novel 5-HT receptor at which BRL 24924 functions as a selective antagonist and which is capable of mediating slow excitatory responses in central neurons.

Htr2a Gene and 5-HT2A Receptor Expression in the Cerebral Cortex Studied Using Genetically Modified Mice

Serotonin receptors of the 5-HT2A subtype are robustly expressed in the cerebral cortex where they have been implicated in the pathophysiology and therapeutics of mental disorders and the actions of hallucinogens. Much less is known, however, about the specific cell types expressing 5-HT2A receptors in cortex. In the current study we use immunohistochemical and electrophysiological approaches in genetically modified mice to address the expression of the Htr2a gene and 5-HT2A receptors in cortex. We first use an EGFP-expressing BAC transgenic mice and identify three main Htr2A gene expressing neuronal populations in cortex. The largest of these cell populations corresponds to layer V pyramidal cells of the anterior cortex, followed by GABAergic interneurons of the middle layers, and non-pyramidal cells of the subplate/Layer VIb. We then use 5-HT2A receptor knockout mice to identify an antibody capable of localizing 5-HT2A receptors in brain and use it to map these receptors. We find strong laminar expression of 5-HT2A receptors in cortex, especially along a diffuse band overlaying layer Va. This band exhibits a strong anteroposterior gradient that closely matches the localization of Htr2A expressing pyramidal cells of layer V. Finally we use electrophysiological and immunohistochemical approaches to show that most, but not all, GABAergic interneurons of the middle layers are parvalbumin expressing Fast-spiking interneurons and that these cells are depolarized and excited by serotonin, most likely through the activation of 5-HT2A receptors. These results clarify and extend our understanding of the cellular distribution of 5-HT2A receptors in the cerebral cortex.

The calcium-activated slow AHP: cutting through the Gordian knot

The phenomenon known as the slow afterhyperpolarization (sAHP) was originally described more than 30 years ago in pyramidal cells as a slow, Ca2+-dependent afterpotential controlling spike frequency adaptation. Subsequent work showed that similar sAHPs were widely expressed in the brain and were mediated by a Ca2+-activated potassium current that was voltage-independent, insensitive to most potassium channel blockers, and strongly modulated by neurotransmitters. However, the molecular basis for this current has remained poorly understood. The sAHP was initially imagined to reflect the activation of a potassium channel directly gated by Ca2+ but recent studies have begun to question this idea. The sAHP is distinct from the Ca2+-dependent fast and medium AHPs in that it appears to sense cytoplasmic [Ca2+]i and recent evidence implicates proteins of the neuronal calcium sensor (NCS) family as diffusible cytoplasmic Ca2+ sensors for the sAHP. Translocation of Ca2+-bound sensor to the plasma membrane would then be an intermediate step between Ca2+ and the sAHP channels. Parallel studies strongly suggest that the sAHP current is carried by different potassium channel types depending on the cell type. Finally, the sAHP current is dependent on membrane PtdIns(4,5)P2 and Ca2+ appears to gate this current by increasing PtdIns(4,5)P2 levels. Because membrane PtdIns(4,5)P2 is essential for the activity of many potassium channels, these finding have led us to hypothesize that the sAHP reflects a transient Ca2+-induced increase in the local availability of PtdIns(4,5)P2 which then activates a variety of potassium channels. If this view is correct, the sAHP current would not represent a unitary ionic current but the embodiment of a generalized potassium channel gating mechanism. This model can potentially explain the cardinal features of the sAHP, including its cellular heterogeneity, slow kinetics, dependence on cytoplasmic [Ca2+], high temperature-dependence, and modulation.