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 G␣ q-11 dominant negative and is also markedly reduced in a phospholipase C ␤1 (PLC␤1) 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 G␣ q-11 /PLC␤1 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.

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 SK Ca 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 SK Ca 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 (I mAHP ) and the slow AHP current (I sAHP ). I mAHP is blocked by the SK Ca channel blockers apamin and bicuculline, whereas I sAHP is resistant to these agents but is inhibited by activation of muscarinic receptors. To test for a role for SK Ca channels, we overexpressed K Ca 2.1 (SK1) and K Ca 2.2 (SK2), the predominant SK Ca subunits expressed in the cortex, in pyramidal cells of cultured brain slices. Overexpression of K Ca 2.1 and K Ca 2.2 resulted in a fourfold to fivefold increase in the amplitude of I mAHP but had no detectable effect on I sAHP . As an additional test, we examined I sAHP in a transgenic mouse expressing a truncated SK Ca subunit (SK3-1B) capable of acting as a dominant negative for the entire family of SK Ca -IK Ca channels. Expression of SK3-1B profoundly inhibited I mAHP but again had no discernable effect on I sAHP . These results are inconsistent with the proposal that SK Ca channels mediate I sAHP in pyramidal cells and indicate that a different potassium channel mediates this current.

The mammalian superior colliculus (SC) is a sensorimotor midbrain structure responsible for orienting behaviors. Although many SC features are known, details of its intrinsic microcircuits are lacking. We used transgenic mice expressing reporter genes in parvalbumin-positive (PV+) and gamma aminobutyric acid-positive (GABA+) neurons to test the hypothesis that PV+ neurons co-localize GABA and form inhibitory circuits within the SC. We found more PV+ neurons in the superficial compared to the intermediate SC, although a larger percentage of PV+ neurons co-expressed GABA in the latter. Unlike PV+ neurons, PV+/GABA+ neurons showed predominantly rapidly inactivating spiking patterns. Optogenetic activation of PV+ neurons revealed direct and feedforward GABAergic inhibitory synaptic responses, as well as excitatory glutamatergic synapses. We propose that PV+ neurons in the SC may be specialized for a variety of circuit functions within the SC rather than forming a homogeneous, GABAergic neuronal subtype as they appear to in other regions of the brain.

In spite of a growing understanding of the actions of 5-hydroxytryptamine (5-HT) in the prefrontal cortex, the specific cellular mechanism used by 5-HT in this region remains poorly understood. Previous studies have shown that 5-HT inhibits the after hyper-polarization that follows a burst of spikes in pyramidal neurons. In the present study, we have used whole cell recordings in rat and mouse brain slices to re-examine this phenomenon with special emphasis on identifying the 5-HT receptor subtypes mediating this effect. Layer V pyramidal neurons display complex after hyper-polarizations that are mediated predominantly by calcium-activated potassium channels and involve two distinct currents known as medium after hyper-polarizating current and slow after hyper-polarizating current (I(sAHP)). Administration of 5-HT reduced the current underlying these after hyper-polarizations by selectively inhibiting I(sAHP). Pharmacological analysis of this response indicates that the main receptor responsible for this inhibition belongs to the 5-HT(2A) subtype. Thus, alpha-methyl-5-HT and 2,5-dimethoxy-4-bromoamphetamine (DOB) mimic the effect of 5-HT and the effect of these agonists is blocked by MDL 100 907. Similarly, administration of alpha-methyl-5-HT is without effect in slices derived from 5-HT(2A) receptor knockout mice. However, 5-HT(2A) receptor blockade only partially suppressed the ability of 5-HT to inhibit I(sAHP). This suggests the involvement of at least one more receptor subtype in this response. Consistent with this idea, administration of 5-carboxyamido-tryptamine, an agonist exhibiting no detectable affinity for 5-HT(2A) receptors, was also capable of suppressing I(sAHP). These results identify 5-HT(2A) receptors as being primarily involved in mediating the 5-HT-induced inhibition of I(sAHP) in prefrontal cortex, while also recognizing a contribution by an additional 5-HT receptor subtype.

1 The pharmacological pro®le of a series of (+)-2,5-dimethoxy-4-(X)-phenylisopropylamines (X=I, Br, NO 2 , CH 3 , or H) and corresponding phenylethylamines, was determined in Xenopus laevis oocytes injected with cRNA coding for rat 5-HT 2A or 5-HT 2C receptors. The e cacy and relative potency of these drugs were determined and compared to classical 5-HT 2 receptor agonists and antagonists. 2 The rank order of agonist potency at the 5-HT 2A receptor was: a-methyl-5-HT=5-HT4m-CPP4MK-212; at the 5-HT 2C receptor the order was: 5-HT4a-methyl-5-HT4MK-2124m-CPP. All these compounds were full agonists at the 5-HT 2C receptor, but a-methyl-5-HT and m-CPP showed lower e cacy at the 5-HT 2A receptor. 3 4-(4-Fluorobenzoyl)-1-(4-phenylbutyl)piperidine (4F 4PP) was 200 times more potent as a 5-HT 2A antagonist than at 5-HT 2C receptors. Conversely, RS 102221 was 100 times more potent as a 5-HT 2C antagonist, con®rming their relative receptor selectivities. 4 The phenylisopropylamines were partial agonists at the 5-HT 2A receptor, with I max relative to 5-HT in the 22+7 to 58+15% range; the corresponding phenylethylamines had lower or undetectable e cacies. All these drugs had higher e cacies at 5-HT 2C receptors; DOI was a full 5-HT 2C agonist. 2C-I and the other phenylethylamines examined showed relative e cacies at the 5-HT 2C receptor ranging from 44+10% to 76+16%. 5 2C-N was a 5-HT 2 receptor antagonist; the mechanism was competitive at the 5-HT 2A , but noncompetitive at the 5-HT 2C receptor. The antagonism was time-dependent at the 5-HT 2C receptor but independent of pre-incubation time at the 5-HT 2A receptor subtype. 6 The a-methyl group determines the e cacy of these phenylalkylamines at the 5-HT 2A and 5-HT 2C receptors.